Sample records for glucuronidase

  1. Phenoxyacetohydrazide Schiff Bases: β-Glucuronidase Inhibitors

    Waqas Jamil; Shagufta Perveen; Syed Adnan Ali Shah; Muhammad Taha; Nor Hadiani Ismail; Shahnaz Perveen; Nida Ambreen; Khan, Khalid M.; Choudhary, Muhammad I.


    Phenoxyacetohydrazide Schiff base analogs 1–28 have been synthesized and their in vitro β-glucouoronidase inhibition potential studied. Compounds 1 (IC50 = 9.20 ± 0.32 µM), 5 (IC50 = 9.47 ± 0.16 µM), 7 (IC50 = 14.7 ± 0.19 µM), 8 (IC50 = 15.4 ± 1.56 µM), 11 (IC50 = 19.6 ± 0.62 µM), 12 (IC50 = 30.7 ± 1.49 µM), 15 (IC50 = 12.0 ± 0.16 µM), 21 (IC50 = 13.7 ± 0.40 µM) and 22 (IC50 = 22.0 ± 0.14 µM) showed promising β-glucuronidase inhibition activity, better than the standard (D-saccharic acid-1...

  2. Phenoxyacetohydrazide Schiff Bases: β-Glucuronidase Inhibitors

    Waqas Jamil


    Full Text Available Phenoxyacetohydrazide Schiff base analogs 1–28 have been synthesized and their in vitro β-glucouoronidase inhibition potential studied. Compounds 1 (IC50 = 9.20 ± 0.32 µM, 5 (IC50 = 9.47 ± 0.16 µM, 7 (IC50 = 14.7 ± 0.19 µM, 8 (IC50 = 15.4 ± 1.56 µM, 11 (IC50 = 19.6 ± 0.62 µM, 12 (IC50 = 30.7 ± 1.49 µM, 15 (IC50 = 12.0 ± 0.16 µM, 21 (IC50 = 13.7 ± 0.40 µM and 22 (IC50 = 22.0 ± 0.14 µM showed promising β-glucuronidase inhibition activity, better than the standard (D-saccharic acid-1,4-lactone, IC50 = 48.4 ± 1.25 µM.

  3. Beta-glucuronidase of family-2 glycosyl hydrolase: a missing member in plants.

    Arul, Loganathan; Benita, George; Sudhakar, Duraialagaraja; Thayumanavan, Balsamy; Balasubramanian, Ponnusamy


    Glycosyl hydrolases hydrolyze the glycosidic bond in carbohydrates or between a carbohydrate and a non-carbohydrate moiety. beta-glucuronidase (GUS) is classified under two glycosyl hydrolase families (2 and 79) and the family-2 beta-glucuronidase is reported in a wide range of organisms, but not in plants. The family-79 endo-beta-glucuronidase (heparanase) is reported in microorganisms, vertebrates and plants. The E. coli family-2 beta-glucuronidase (uidA) had been successfully devised as a reporter gene in plant transformation on the basis that plants do not have homologous GUS activity. On the contrary, histochemical staining with X-Gluc was reported in wild type (non-transgenic) plants. Data shows that, family-2 beta-glucuronidase homologous sequence is not found in plants. Further, beta-glucuronidases of family-2 and 79 lack appreciable sequence similarity. However, the catalytic site residues, glutamic acid and tyrosine of the family-2 beta-glucuronidase are found to be conserved in family-79 beta-glucuronidase of plants. This led to propose that the GUS staining reported in wild type plants is largely because of the broad substrate specificity of family-79 beta-glucuronidase on X-Gluc and not due to the family-2 beta-glucuronidase, as the latter has been found to be missing in plants.

  4. Implications of alpha-amylase production and beta-glucuronidase expression in Escherichia coli strains.

    Caldini, G; Strappini, C; Trotta, F; Cenci, G


    Two Escherichia coli strains in which alpha-amylase production differed were used to study in depth some characteristics related to beta-glucuronidase induction by starch. The beta-glucuronidase background activity in Luria broth medium was comparable for the two isolates, but only amylase positive S1 was able to grow on starch molecules supplied as the sole carbon source. In this case growth resulted at higher beta-glucuronidase levels (p beta-glucuronidase activity of amylase negative M94 remained unchanged during starvation on starch medium, but an induced response was observed with methylumbelliferyl-glucuronide. These results further support the hypothesis that starch metabolism is involved in the complex beta-glucuronidase regulation of E. coli strains. This is relevant not only for basic research but also to investigating gut microbial enzymology.

  5. β-Glucuronidase-coupled assays of glucuronoyl esterases.

    Fraňová, Lucia; Puchart, Vladimír; Biely, Peter


    Glucuronoyl esterases (GEs) are microbial enzymes with potential to cleave the ester bonds between lignin alcohols and xylan-bound 4-O-methyl-d-glucuronic acid in plant cell walls. This activity renders GEs attractive research targets for biotechnological applications. One of the factors impeding the progress in GE research is the lack of suitable substrates. In this work, we report a facile preparation of methyl esters of chromogenic 4-nitrophenyl and 5-bromo-4-chloro-3-indolyl β-D-glucuronides for qualitative and quantitative GE assay coupled with β-glucuronidase as the auxiliary enzyme. The indolyl derivative affording a blue indigo-type product is suitable for rapid and sensitive assay of GE in commercial preparations as well as for high throughput screening of microorganisms and genomic and metagenomic libraries.

  6. Thiadiazole derivatives as New Class of β-glucuronidase inhibitors.

    Salar, Uzma; Taha, Muhammad; Ismail, Nor Hadiani; Khan, Khalid Mohammed; Imran, Syahrul; Perveen, Shahnaz; Wadood, Abdul; Riaz, Muhammad


    Thiadiazole derivatives 1-24 were synthesized via a single step reaction and screened for in vitro β-glucuronidase inhibitory activity. All the synthetic compounds displayed good inhibitory activity in the range of IC50=2.16 ± 0.01-58.06 ± 1.60 μM as compare to standard d-saccharic acid 1,4-lactone (IC50=48.4 ± 1.25 μM). Molecular docking study was conducted in order to establish the structure-activity relationship (SAR) which demonstrated that thiadiazole as well as both aryl moieties (aryl and N-aryl) involved to exhibit the inhibitory potential. All the synthetic compounds were characterized by spectroscopic techniques (1)H, (13)C NMR, and EIMS.

  7. Structure and Inhibition of Microbiome β-Glucuronidases Essential to the Alleviation of Cancer Drug Toxicity

    Wallace, Bret D.; Roberts, Adam B.; Pollet, Rebecca M.; Ingle, James D.; Biernat, Kristen A.; Pellock, Samuel J.; Venkatesh, Madhu Kumar; Guthrie, Leah; O’Neal, Sara K.; Robinson, Sara J.; Dollinger, Makani; Figueroa, Esteban; McShane, Sarah R.; Cohen, Rachel D.; Jin, Jian; Frye, Stephen V.; Zamboni, William C.; Pepe-Ranney, Charles; Mani, Sridhar; Kelly, Libusha; Redinbo, Matthew R.


    The selective inhibition of bacterial β-glucuronidases was recently shown to alleviate drug-induced gastrointestinal toxicity in mice, including the damage caused by the widely used anticancer drug irinotecan. Here, we report crystal structures of representative β-glucuronidases from the Firmicutes Streptococcus agalactiae and Clostridium perfringens and the Proteobacterium Escherichia coli, and the characterization of a β-glucuronidase from the Bacteroidetes Bacteroides fragilis. While largely similar in structure, these enzymes exhibit marked differences in catalytic properties and propensities for inhibition, indicating that the microbiome maintains functional diversity in orthologous enzymes. Small changes in the structure of designed inhibitors can induce significant conformational changes in the β-glucuronidase active site. Finally, we establish that β-glucuronidase inhibition does not alter the serum pharmacokinetics of irinotecan or its metabolites in mice. Together, the data presented advance our in vitro and in vivo understanding of the microbial β-glucuronidases, a promising new set of targets for controlling drug-induced gastrointestinal toxicity.

  8. Genetic diversity of b-glucuronidase activity among 14 strains of the dominant human gut anaerobe Ruminococcus gnavus

    Diane Beaud


    Full Text Available Bacterial beta-glucuronidase activity in the gut increases the enterohepatic circulation of toxic compounds and plays a major role in the etiology of colon cancer. Previously, we had found that the gus gene, which codes for beta-glucuronidase in a dominant anaerobic species of the gut microbiota, Ruminococcus gnavus strain E1, is transcribed as part of an operon that includes three ORFs that code for beta-glucoside permeases of the phosphotransferase systems. This genetic organization had never been described. We have now compared beta-glucuronidase activity and the genetic environment of the gus gene in 14 strains of Ruminococcus gnavus.We found that five out of the seven glucuronidase-positive R. gnavus strains possessed another glucuronidase gene different from the gusA operon of R. gnavus E1. This dominant commensal intestinal species appears to have a high degree of genetic diversity in the genes that control beta-glucuronidase activity.

  9. Characterization of inhibitor(s) of β-glucuronidase enzyme activity in GUS-transgenic wheat

    Ramadan, Ahmed M Ali


    The uidA gene, encoding for β-glucuronidase (GUS), is the most frequently used reporter gene in plants. As a reporter enzyme, GUS can be assayed both qualitatively and quantitatively. In wheat, there are numerous reports of failure in detecting GUS enzyme activity in tissues of transgenic plants, while other reports have suggested presence of β-glucuronidase inhibitor(s) in wheat tissues. In the present study, we show that the β-glucuronidase enzyme activity is not only tissue-specific but also genotype-dependent. Our data demonstrate that the glucuronic acid could be the candidate inhibitor for β-glucuronidase enzyme activity in wheat leaves and roots. It should be noted that the assays to detect β-glucuronidase enzyme activity in wheat should be interpreted carefully. Based on the data of our present study, we recommend studying the chemical pathways, the unintended effects and the possible loss-of-function of any candidate transgene prior to transformation experiments. © 2011 Springer Science+Business Media B.V.

  10. Correction of murine mucopolysaccharidosis VII by a human. beta. -glucuronidase transgene

    Kyle, J.W.; Vogler, C.; Hoffmann, J.W.; Sly, W.S. (St. Louis Univ. School of Medicine, MO (USA)); Birkenmeier, E.H.; Gwynn, B. (Jackson Laboratory, Bar Harbor, ME (USA))


    The authors recently described a murine model for mucopolysaccharidosis VII in mice that have an inherited deficiency of {beta}-glucuronidase. Affected mice, of genotype gus{sup mps}/gus{sup mps}, present clinical manifestations similar to those of humans with mucopolysaccharidosis VII (Sly syndrome) and are shown here to have secondary elevations of other lysosomal enzymes. The mucopolysaccharidosis VII phenotype in both species includes dwarfism, skeletal deformities, and premature death. Lysosome storage is visualized within enlarged vesicles and correlates biochemically with accumulation of undegraded and partially degraded glycosaminoglycans. In this report they describe the consequences of introducing the human {beta}-glucuronidase gene, GUSB, into gus{sup mps}/gus{sup mps} mice that produce virtually no murine {beta}-glucuronidase. Transgenic mice homozygous for the mucopolysaccharidosis VII mutation expressed high levels of human {beta}-glucuronidase activity in all tissues examined and were phenotypically normal. Biochemically, both the intralysosomal storage of glycosaminoglycans and the secondary elevation of other acid hydrolases were corrected. These findings demonstrate that the GUSB transgene is expressed in gus{sup mps}/gus{sup mps} mice and that human {beta}-glucuronidase corrects the murine mucopolysaccharidosis storage disease.

  11. Synthesis of novel disulfide and sulfone hybrid scaffolds as potent β-glucuronidase inhibitor.

    Taha, Muhammad; Ismail, Nor Hadiani; Imran, Syahrul; Wadood, Abdul; Rahim, Fazal; Al Muqarrabin, Laode Muhammad Ramadhan; Zaki, Hamizah Mohd; Ahmat, Norizan; Nasir, Abdul; Khan, Fahad


    Novel series of disulfide and sulfone hybrid analogs (1-20) were synthesized and characterized through EI-MS and (1)H NMR and evaluated for β-glucuronidase inhibitory potential. All synthesized analogs except 13 and 15 showed excellent β-glucuronidase inhibitory potential with IC50 value ranging in between 2.20-88.16μM as compared to standard d-saccharic acid 1,4 lactone (48.4±1.25μM). Analogs 19, 16, 4, 1, 17, 6, 10, 3, 18, 2, 11, 14 and 5 showed many fold potent activity against β-glucuronidase inhibitor. Structure activity relationship showed that substitution of electron withdrawing groups at ortho as well as para position on phenyl ring increase potency. Electron withdrawing groups at meta position on phenyl ring showed slightly low potency as compared to ortho and para position. The binding interactions were confirmed through molecular docking studies.

  12. 40 CFR 174.525 - E. coli B-D-glucuronidase enzyme as a plant-incorporated protectant inert ingredient; exemption...


    ... 40 Protection of Environment 23 2010-07-01 2010-07-01 false E. coli B-D-glucuronidase enzyme as a... E. coli B-D-glucuronidase enzyme as a plant-incorporated protectant inert ingredient; exemption from the requirement of a tolerance. Residues of E. coli B-D-glucuronidase enzyme are exempt from...

  13. Deconjugation Kinetics of Glucuronidated Phase II Flavonoid Metabolites by B-glucuronidase from Neutrophils

    Bartholomé, R.; Haenen, G.; Hollman, P.C.H.; Bast, A.; Dagnelie, P.C.; Roos, D.; Keijer, J.; Kroon, P.A.; Needs, P.W.; Arts, I.C.W.


    Flavonoids are inactivated by phase II metabolism and occur in the body as glucuronides. Mammalian ß-glucuronidase released from neutrophils at inflammatory sites may be able to deconjugate and thus activate flavonoid glucuronides. We have studied deconjugation kinetics and pH optimum for four sourc

  14. Synthesis and biological evaluation of novel N-arylidenequinoline-3-carbohydrazides as potent β-glucuronidase inhibitors.

    Taha, Muhammad; Sultan, Sadia; Nuzar, Herizal Ali; Rahim, Fazal; Imran, Syahrul; Ismail, Nor Hadiani; Naz, Humera; Ullah, Hayat


    Thirty N-arylidenequinoline-3-carbohydrazides (1-30) have been synthesized and evaluated against β-glucuronidase inhibitory potential. Twenty four analogs showed outstanding β-glucuronidase activity having IC50 values ranging between 2.11±0.05 and 46.14±0.95 than standard d-saccharic acid 1,4 lactone (IC50=48.4±1.25μM). Six analogs showed good β-glucuronidase activity having IC50 values ranging between 49.38±0.90 and 80.10±1.80. Structure activity relationship and the interaction of the active compounds and enzyme active site with the help of docking studies were established. Our study identifies novel series of potent β-glucuronidase inhibitors for further investigation.

  15. Separation of Recombinant β-Glucuronidase from Transgenic Tobacco by Aqueous Two-Phase Extraction


    Separation of Recombinant à -Glucuronidase from Transgenic Tobacco by Aqueous Two-Phase Extraction Kristin Coby Ross Abstract Biopharmaceutical manufacturing is a rigorous and expensive process. Due to the medicinal nature of the product, a high purity level is required and several expensive purification steps must be utilized. Cost-effective production and purification is essential for any biopharmaceutical product to be successful and development of the fastest, most economical, ...

  16. Kinetic and equilibrium studies of adsorption of β-glucuronidase by clinoptilolite-rich minerals

    Demirbüker Kavak, Dilek; Ülkü, Semra


    The adsorption of the bacterial β-glucuronidase (GUS) enzyme, which is thought to be responsible for the production of reactive metabolites related to some diseases and cancer development, by clinoptilolite-rich mineral was investigated. Batch experiments were performed to analyze of the effects of the clinoptilolite amount and particle size, initial GUS concentration, shaking rate, pH and temperature on the adsorption equilibrium and kinetics. Adsorption equilibrium data were interpreted in ...

  17. Serum Zinc and β D Glucuronidase Enzyme Level in Type 2 Diabetes Mellitus with Periodontitis.

    Pushparani, D S


    The exact mechanism by which nutritional deficiency and lysosomal enzyme change, modify periodontal destruction has not yet been precisely defined. The study aimed to determine the serum zinc and β D glucuronidase enzyme level in the selected groups and how its increase or decrease levels are related to type 2 diabetes mellitus (T2DM) with periodontitis when compared to other groups. Six hundred subjects were selected and are categorized into four groups as Group I (control healthy subjects, n=150), Group II (T2DM with periodontitis, n=150), Group III (T2DM without periodontitis, n=150) and Group IV (Non-DM with periodontitis, n=150). The lab investigations included measuring fasting blood glucose, serum zinc and β D- glucuronidase levels. In the results, the level of serum zinc was found to be lesser in group III subjects and the activity of serum β D glucuronidase was found to be elevated nine times in group III (T2DM with periodontitis) and two times elevated in group II (T2DM without periodontitis) and group IV (Non-DM with periodontitis), when compared to control. Zinc has been reported to reduce the stabilization of lysosomal membranes. Periodontitis has been taken as the prime condition in this study and categorized as experimental groups. Perturbations in mineral metabolism are more pronounced in diabetic populations. When the level of zinc is decreased, the structural integrity of lysosomal membrane has been lost and it would have caused for the increased release of β D glucuronidase in T2DM with periodontitis.

  18. Synthesis, β-glucuronidase inhibition and molecular docking studies of hybrid bisindole-thiosemicarbazides analogs.

    Taha, Muhammad; Ismail, Nor Hadiani; Imran, Syahrul; Rahim, Fazal; Wadood, Abdul; Khan, Huma; Ullah, Hayat; Salar, Uzma; Khan, Khalid Mohammed


    Hybrid bisindole-thiosemicarbazides analogs (1-18) were synthesized and screened for β-glucuronidase activity. All compounds showed varied degree of β-glucuronidase inhibitory potential when compared with standard d-saccharic acid 1,4-lactone (IC50=48.4±1.25μM). Compounds 4, 7, 9, 6, 5, 12, 17 and 18 showed exceptional β-glucuronidase inhibition with IC50 values ranging from 0.1 to 5.7μM. Compounds 1, 3, 8, 16, 13, 2 and 14 also showed better activities than standard with IC50 values ranging from 7.12 to 15.0μM. The remaining compounds 10, 11, and 15 showed good inhibitory potential with IC50 values 33.2±0.75, 21.4±0.30 and 28.12±0.25μM respectively. Molecular docking studies were carried out to confirm the binding interaction of the compounds.

  19. Impediments to Enhancement of CPT-11 Anticancer Activity by E. coli Directed Beta-Glucuronidase Therapy

    Hsieh, Yuan-Ting; Chen, Kai-Chuan; Cheng, Chiu-Min; Cheng, Tian-Lu; Tao, Mi-Hua; Roffler, Steve R.


    CPT-11 is a camptothecin analog used for the clinical treatment of colorectal adenocarcinoma. CPT-11 is converted into the therapeutic anti-cancer agent SN-38 by liver enzymes and can be further metabolized to a non-toxic glucuronide SN-38G, resulting in low SN-38 but high SN-38G concentrations in the circulation. We previously demonstrated that adenoviral expression of membrane-anchored beta-glucuronidase could promote conversion of SN-38G to SN-38 in tumors and increase the anticancer activity of CPT-11. Here, we identified impediments to effective tumor therapy with E. coli that were engineered to constitutively express highly active E. coli beta-glucuronidase intracellularly to enhance the anticancer activity of CPT-11. The engineered bacteria, E. coli (lux/βG), could hydrolyze SN-38G to SN-38, increased the sensitivity of cultured tumor cells to SN-38G by about 100 fold and selectively accumulated in tumors. However, E. coli (lux/βG) did not more effectively increase CPT-11 anticancer activity in human tumor xenografts as compared to non-engineered E. coli. SN-38G conversion to SN-38 by E. coli (lux/βG) appeared to be limited by slow uptake into bacteria as well as by segregation of E. coli in necrotic regions of tumors that may be relatively inaccessible to systemically-administered drug molecules. Studies using a fluorescent glucuronide probe showed that significantly greater glucuronide hydrolysis could be achieved in mice pretreated with E. coli (lux/βG) by direct intratumoral injection of the glucuronide probe or by intratumoral lysis of bacteria to release intracellular beta-glucuronidase. Our study suggests that the distribution of beta-glucuronidase, and possibly other therapeutic proteins, in the tumor microenvironment might be an important barrier for effective bacterial-based tumor therapy. Expression of secreted therapeutic proteins or induction of therapeutic protein release from bacteria might therefore be a promising strategy to enhance anti

  20. A note on starch hydrolysis and beta-glucuronidase activity among flavobacteria.

    Petzel, J P; Hartman, P A


    Most flavobacteria tested with the fluorogenic substrate 4-methylumbelliferyl-beta-D-glucuronide possessed beta-glucuronidase (GUD), but when some of the same strains were tested with the API ZYM gallery, all were negative for GUD. Conflicting reports also appear in the literature about starch hydrolysis among flavobacteria. We observed that the results obtained can depend on the medium used and the length of incubation. Our results indicate that GUD activity and starch hydrolysis are more widely distributed in the genus Flavobacterium than previously reported.

  1. Impediments to enhancement of CPT-11 anticancer activity by E. coli directed beta-glucuronidase therapy.

    Hsieh, Yuan-Ting; Chen, Kai-Chuan; Cheng, Chiu-Min; Cheng, Tian-Lu; Tao, Mi-Hua; Roffler, Steve R


    CPT-11 is a camptothecin analog used for the clinical treatment of colorectal adenocarcinoma. CPT-11 is converted into the therapeutic anti-cancer agent SN-38 by liver enzymes and can be further metabolized to a non-toxic glucuronide SN-38G, resulting in low SN-38 but high SN-38G concentrations in the circulation. We previously demonstrated that adenoviral expression of membrane-anchored beta-glucuronidase could promote conversion of SN-38G to SN-38 in tumors and increase the anticancer activity of CPT-11. Here, we identified impediments to effective tumor therapy with E. coli that were engineered to constitutively express highly active E. coli beta-glucuronidase intracellularly to enhance the anticancer activity of CPT-11. The engineered bacteria, E. coli (lux/βG), could hydrolyze SN-38G to SN-38, increased the sensitivity of cultured tumor cells to SN-38G by about 100 fold and selectively accumulated in tumors. However, E. coli (lux/βG) did not more effectively increase CPT-11 anticancer activity in human tumor xenografts as compared to non-engineered E. coli. SN-38G conversion to SN-38 by E. coli (lux/βG) appeared to be limited by slow uptake into bacteria as well as by segregation of E. coli in necrotic regions of tumors that may be relatively inaccessible to systemically-administered drug molecules. Studies using a fluorescent glucuronide probe showed that significantly greater glucuronide hydrolysis could be achieved in mice pretreated with E. coli (lux/βG) by direct intratumoral injection of the glucuronide probe or by intratumoral lysis of bacteria to release intracellular beta-glucuronidase. Our study suggests that the distribution of beta-glucuronidase, and possibly other therapeutic proteins, in the tumor microenvironment might be an important barrier for effective bacterial-based tumor therapy. Expression of secreted therapeutic proteins or induction of therapeutic protein release from bacteria might therefore be a promising strategy to enhance anti

  2. Assessment of beta-glucuronidase levels in goat's milk as an indicator of mastitis: comparison with other mastitis detection methods.

    Oliszewski, R; Kairúz de Núñez, M S; Elias de González, S N; Oliver, G


    The use of somatic cell counts (SCCs) for the diagnosis of mastitis is not a well-established procedure for the caprine species, because nonleucocytic cell-like particles are normally observed as a result of the apocrine secretion process of the goat mammary gland. The infection levels of 124 goats were measured by the beta-glucuronidase test, which was compared with the SCC method and the California mastitis test (CMT). Seventy-nine of 124 samples (63.7%) showed SCCs lower than 1.3 x 10(3) cells per ml. Of these samples, 93% showed low levels of beta-glucuronidase activity (CMT gave similar results. In this study, the beta-glucuronidase test was standardized for goat milk and shown to be reliable, enabling one to count only the somatic enzyme cells in milk and avoiding the interference encountered with the SCC method.

  3. The effects of the presence of the corneal epithelium and supplemental hydrocortisone on ß-glucuronidase levels with corneal preservation

    Mauger TF


    Full Text Available Thomas Mauger, Eric QuartettiHavener Eye Institute, Department of Ophthalmology, The Ohio State University, OH, USAPurpose: To analyze the levels of ß-glucuronidase during prolonged (14 day corneal preservation with epithelialized (EPI and deepithelialized (DEP corneas and the effect of supplemental hydrocortisone (HCT on these levels.Methods: Thirty-six freshly excised bovine corneas were preserved in Optisol solution (4°C for 14 days with the following conditions EPI/no supplemental HCT, DEP no supplemental HCT, EPI/10-8 M HCT, and EPI 10-4 M HCT. ß-glucuronidase activity levels were measured at the end of this period.Results: ß-glucuronidase levels (nmol/mL/h for each group were found to be: EPI – No HCT: 4.302, SEM 0.586; DEP – No HCT: 2.178, SEM 0.271; EPI – 10-8 M HCT: 4.472, SEM 0.435; EPI – 10-4 M HCT: 2.072, SEM 0.437. The EPI – No HCT and EPI – 10-8 M HCT were not significantly different as were the DEP – No HCT and EPI – 10-4 M HCT groups. The EPI – No HCT group and the EPI – 10-8 M HCT group were independently significantly different from the DEP – No HCT and the EPI – 10-4 M HCT groups.Conclusions: The corneal epithelium contributes significantly to the formation of the lysosomal enzyme, ß-glucuronidase, during preservation. The addition of 10-4 M HCT decreases the production of ß-glucuronidase during corneal preservation in this model.Keywords: ß-glucuronidase, preservation 

  4. Identification and Cloning of gusA, Encoding a New β-Glucuronidase from Lactobacillus gasseri ADH†

    Russell, W. M.; Klaenhammer, T. R.


    The gusA gene, encoding a new β-glucuronidase enzyme, has been cloned from Lactobacillus gasseri ADH. This is the first report of a β-glucuronidase gene cloned from a bacterial source other than Escherichia coli. A plasmid library of L. gasseri chromosomal DNA was screened for complementation of an E. coli gus mutant. Two overlapping clones that restored β-glucuronidase activity in the mutant strain were sequenced and revealed three complete and two partial open reading frames. The largest open reading frame, spanning 1,797 bp, encodes a 597-amino-acid protein that shows 39% identity to β-glucuronidase (GusA) of E. coli K-12 (EC The other two complete open reading frames, which are arranged to be separately transcribed, encode a putative bile salt hydrolase and a putative protein of unknown function with similarities to MerR-type regulatory proteins. Overexpression of GusA was achieved in a β-glucuronidase-negative L. gasseri strain by expressing the gusA gene, subcloned onto a low-copy-number shuttle vector, from the strong Lactobacillus P6 promoter. GusA was also expressed in E. coli from a pET expression system. Preliminary characterization of the GusA protein from crude cell extracts revealed that the enzyme was active across an acidic pH range and a broad temperature range. An analysis of other lactobacilli identified β-glucuronidase activity and gusA homologs in other L. gasseri isolates but not in other Lactobacillus species tested. PMID:11229918

  5. Isolation and characterization of an endo-beta-D-glucuronidase from the fungus Kobayasia nipponica.

    Tsuchihashi, H; Yadomae, T; Miyazaki, T


    An endo-beta-D-glucuronidase was isolated and characterized from Kobayasia nipponica. The enzyme was purified by ammonium sulfate fractionation, CM-Sephadex chromatography, gel filtration with Sephacryl S-200, and heparin-Sepharose chromatography. The enzyme shows the following properties: optimum pH 5.0, thermal stability below 37 degrees C, pH stability 5-6, optimum temperature 45-55 degrees C, and Km 0.12% for L-idurono-D-glucuronan (protuberic acid (PA), L-IdUA:D-GlcUA = 1:2) from Kobayasia nipponica, 0.19% for PF (L-IdUA:D-GlcUA = 1:3) from Pseudocolus fusiformis, and 0.23% for (1-4)-beta-D-glucuronan(mucoric acid) from Mucor mucedo as determined from Hofstee plots. The molecular weight values estimated by gel filtration through Sephacryl S-200 and Sephadex G-50 and by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate were 10,500 and 10,200, respectively. The endo-beta-D-glucuronidase was inactive towards several glycosaminoglycans.

  6. Histochemical studies on genetical control of hormonal enzyme inducibility in the mouse. III. Beta-glucuronidase distribution pattern of epididymis in different genotypes

    Blecher, S R; Kirkeby, S


    This article reports the application of Hayashi's histochemical technique for beta-glucuronidase to mouse epididymis. A methodological study, which established optimal conditions for demonstrating the enzyme in this organ, is reported. The distribution pattern of beta-glucuronidase is described a...


    Genotypic and phenotypic assays for glutamate decarboxylase (GAD) and B-D-glucuronidase (GUD) were compared for their abilities to detect various strains of Escherichia coli and to discriminate among other bacterial species. Test strains included nonpathogenic E.coli, three major...

  8. Activity of neutrophil β-glucuronidase in diabetic and nondiabetic patients with chronic generalized periodontitis and healthy subjects.

    Surna, Algimantas; Sakalauskienė, Jurgina; Gleiznys, Alvydas; Ivanauskienė, Eglė; Saferis, Viktoras


    OBJECTIVE. The aim of the study was to establish the dynamics of β-glucuronidase activity in subjects suffering from type 1 diabetes and chronic untreated generalized periodontitis, subjects suffering from chronic untreated generalized periodontitis only, and control subjects not suffering from generic diseases with healthy periodontal tissue. MATERIAL AND METHODS. The study involved 165 19-50-year-old subjects who were divided into three groups: healthy subjects (n=55), subjects with chronic untreated generalized periodontitis (n=55), and subjects with type 1 diabetes and chronic untreated generalized periodontitis (n=55). Neutrophilic leukocytes of peripheral venous blood were exposed to bacterial stimuli: opsonized zymosan, nonopsonized Staphylococcus aureus, and prodigiosan. The activity of β-glucuronidase was determined by the spectrofluorimetry method. RESULTS. The diagnostic value of changes in β-glucuronidase activity of neutrophilic leukocytes markedly increased in all study groups after stimulation of neutrophilic leukocytes by opsonized zymosan, nonopsonized Staphylococcus aureus, and prodigiosan as compared to control media not exposed to any stimulus (Pperiodontal pathology markers and the activity of β-glucuronidase of neutrophilic leukocytes in incubated media in patients with type 1 diabetes mellitus and periodontitis was found under the effect of nonopsonized Staphylococcus aureus. CONCLUSIONS. If periodontal impairment is severe, diabetes mellitus possibly causes a faster destruction of the periodontal tissue and presents a higher risk of periodontitis for patients with diabetes.

  9. Histochemical demonstration of activity of acid phosphatase and beta-glucuronidase in bovine incisor tooth germs

    Kirkeby, S; Salling, E; Moe, D


    Activity of acid phosphatase and beta-glucuronidase was shown in bovine preodontoblasts and preameloblasts prior to the onset of secretion. In the preameloblasts the rather weak reaction consisted of small discrete granules dispersed in the cytoplasm apical, lateral, and proximal to the nucleus....... After initiation of enamel formation, a change in localization and intensity of the colored reaction product was observed in the ameloblasts. The activity appeared stronger and was restricted to a narrow zone just apical to the nucleus. It is proposed that the acid hydrolases in the tooth forming cells...... are located to the Golgi complex. The differences in activity of acid hydrolases between bone and tooth forming cells are expounded....

  10. Biotransformation of dehydroepiandrosterone with Macrophomina phaseolina and β-glucuronidase inhibitory activity of transformed products.

    Choudhary, M Iqbal; Zafar, Salman; Khan, Naik Tameen; Ahmad, Saeed; Noreen, Shagufta; Marasini, Bishnu P; Al-Khedhairy, Abdulaziz A; Atta-Ur-Rahman


    The biotransformation of dehydroepiandrosterone (1) with Macrophomina phaseolina was investigated. A total of eight metabolites were obtained which were characterized as androstane-3,17-dione (2), androst-4-ene-3,17-dione (3), androst-4-ene-17β-ol-3-one (4), androst-4,6-diene-17β-ol-3-one (5), androst-5-ene-3β,17β-diol (6), androst-4-ene-3β-ol-6,17-dione (7), androst-4-ene-3β,7β,17β-triol (8), and androst-5-ene-3β,7α,17β-triol (9). All the transformed products were screened for enzyme inhibition, among which four were found to inhibit the β-glucuronidase enzyme, while none inhibited the α-chymotrypsin enzyme.

  11. Biotransformation of dianabol with the filamentous fungi and β-glucuronidase inhibitory activity of resulting metabolites.

    Khan, Naik T; Zafar, Salman; Noreen, Shagufta; Al Majid, Abdullah M; Al Othman, Zeid A; Al-Resayes, Saud Ibrahim; Atta-ur-Rahman; Choudhary, M Iqbal


    Biotransformation of the anabolic steroid dianabol (1) by suspended-cell cultures of the filamentous fungi Cunninghamella elegans and Macrophomina phaseolina was studied. Incubation of 1 with C. elegans yielded five hydroxylated metabolites 2-6, while M. phaseolina transformed compound 1 into polar metabolites 7-11. These metabolites were identified as 6β,17β-dihydroxy-17α-methylandrost-1,4-dien-3-one (2), 15α,17β-dihydroxy-17α-methylandrost-1,4-dien-3-one (3), 11α,17β-dihydroxy-17α-methylandrost-1,4-dien-3-one (4), 6β,12β,17β-trihydroxy-17α-methylandrost-1,4-dien-3-one (5), 6β,15α,17β-trihydroxy-17α-methylandrost-1,4-dien-3-one (6), 17β-hydroxy-17α-methylandrost-1,4-dien-3,6-dione (7), 7β,17β,-dihydroxy-17α-methylandrost-1,4-dien-3-one (8), 15β,17β-dihydroxy-17α-methylandrost-1,4-dien-3-one (9), 17β-hydroxy-17α-methylandrost-1,4-dien-3,11-dione (10), and 11β,17β-dihydroxy-17α-methylandrost-1,4-dien-3-one (11). Metabolite 3 was also transformed chemically into diketone 12 and oximes 13, and 14. Compounds 6 and 12-14 were identified as new derivatives of dianabol (1). The structures of all transformed products were deduced on the basis of spectral analyses. Compounds 1-14 were evaluated for β-glucuronidase enzyme inhibitory activity. Compounds 7, 13, and 14 showed a strong inhibition of β-glucuronidase enzyme, with IC50 values between 49.0 and 84.9 μM. Copyright © 2014 Elsevier Inc. All rights reserved.

  12. Bioactivity-guided fractionation to identify β-glucuronidase inhibitors in Nymphaea pubescens flower extract

    Jayashree Acharya


    Full Text Available The plant Nymphaea pubescens Willd. (Family: Nymphaeaceae is edible having medicinal importance. The objective of the study was to analyze the potential hepatoprotective properties of the flowers and pedicels of N. pubescens by inhibiting the enzyme β-glucuronidase. Crude methanol extracts of flower and pedicel as well as chloroform, ethyl acetate, and aqueous fractions of the flower extract were tested for their activities against the enzyme in vitro. The extracts and the fractions were analyzed by GC–MS to identify metabolites present in them. Flower (IC50 value = 270.27 ± 4.67 μg/ml and pedicel (IC50 value = 868.46 ± 28.21 μg/ml extracts have shown to inhibit the β-glucuronidase activity. Chloroform (IC50 value = 147.16 ± 6.68 μg/ml, ethyl acetate (IC50 value = 183.94 ± 2.37 μg/ml, and aqueous (IC50 value = 339.43 ± 5.34 μg/ml fractions showed significantly stronger activity than that of silymarin (IC50 value = 792.62 ± 10.01 μg/ml, the known inhibitor of the enzyme. GC–MS-based analysis of the flower extract and solvent fractions led to the identification of kaempferol having 79-fold stronger activity than that of silymarin, IC50 value of kaempferol being 10.44 ± 0.084 μg/ml or 0.0037 mM ± 0.0001.

  13. Effect of the beta-glucuronidase inhibitor saccharolactone on glucuronidation by human tissue microsomes and recombinant UDP-glucuronosyltransferases.

    Oleson, Lauren; Court, Michael H


    Glucuronidation studies using microsomes and recombinant uridine diphosphoglucuronosyltransferases (UGTs) can be complicated by the presence of endogenous beta-glucuronidases, leading to underestimation of glucuronide formation rates. Saccharolactone is the most frequently used beta-glucuronidase inhibitor, although it is not clear whether this reagent should be added routinely to glucuronidation incubations. Here we have determined the effect of saccharolactone on eight different UGT probe activities using pooled human liver microsomes (pHLMs) and recombinant UGTs (rUGTs). Despite the use of buffered incubation solutions, it was necessary to adjust the pH of saccharolactone solutions to avoid effects (enhancement or inhibition) of lowered pH on UGT activity. Saccharolactone at concentrations ranging from 1 to 20 mM did not enhance any of the glucuronidation activities evaluated that could be considered consistent with inhibition of beta-glucuronidase. However, for most activities, higher saccharolactone concentrations resulted in a modest degree of inhibition. The greatest inhibitory effect was observed for glucuronidation of 5-hydroxytryptamine and estradiol by pHLMs, with a 35% decrease at 20 mM saccharolactone concentration. Endogenous beta-glucuronidase activities were also measured using various human tissue microsomes and rUGTs with estradiol-3-glucuronide and estradiol-17-glucuronide as substrates. Glucuronide hydrolysis was observed for pHLMs, lung microsomes and insect-cell expressed rUGTs, but not for kidney, intestinal or human embryonic kidney HEK293 microsomes. However, the extent of hydrolysis was relatively small, representing only 9-19% of the glucuronide formation rate measured in the same preparations. Consequently, these data do not support the routine inclusion of saccharolactone in glucuronidation incubations. If saccharolactone is used, concentrations should be titrated to achieve activity enhancement without inhibition.

  14. Regulation of the alpha-glucuronidase-encoding gene ( aguA) from Aspergillus niger.

    de Vries, R P; van de Vondervoort, P J I; Hendriks, L; van de Belt, M; Visser, J


    The alpha-glucuronidase gene aguA from Aspergillus niger was cloned and characterised. Analysis of the promoter region of aguA revealed the presence of four putative binding sites for the major carbon catabolite repressor protein CREA and one putative binding site for the transcriptional activator XLNR. In addition, a sequence motif was detected which differed only in the last nucleotide from the XLNR consensus site. A construct in which part of the aguA coding region was deleted still resulted in production of a stable mRNA upon transformation of A. niger. The putative XLNR binding sites and two of the putative CREA binding sites were mutated individually in this construct and the effects on expression were examined in A. niger transformants. Northern analysis of the transformants revealed that the consensus XLNR site is not actually functional in the aguA promoter, whereas the sequence that diverges from the consensus at a single position is functional. This indicates that XLNR is also able to bind to the sequence GGCTAG, and the XLNR binding site consensus should therefore be changed to GGCTAR. Both CREA sites are functional, indicating that CREA has a strong influence on aguA expression. A detailed expression analysis of aguA in four genetic backgrounds revealed a second regulatory system involved in activation of aguA gene expression. This system responds to the presence of glucuronic and galacturonic acids, and is not dependent on XLNR.

  15. Serum β-glucuronidase as a potential colon cancer marker: a preliminary study.

    Waszkiewicz, Napoleon; Szajda, Sławomir Dariusz; Konarzewska-Duchnowska, Emilia; Zalewska-Szajda, Beata; Gałązkowski, Robert; Sawko, Anna; Nammous, Halim; Buko, Vyacheslav; Szulc, Agata; Zwierz, Krzysztof; Ładny, Jerzy Robert


    Colorectal cancer is characterized by high morbidity and mortality in developed countries. The lack of low-cost, easy-to-use screening diagnostic methods is one of the causes of late diagnosis of colorectal cancer. Beta-glucuronidase (GLU) is a lysosomal exoglycosidase involved in degradation of glycosaminoglycans of the cell membranes and extracellular matrix of normal and cancerous colon tissues. The aim of our research was to evaluate the activity of GLU in the serum of colorectal cancer and estimate its potential value in the diagnosis of colorectal cancer. Blood samples were collected from 21 patients with colorectal adenocarcinoma and 17 healthy subjects. GLU activity was determined by the colorimetric method of Marciniak et al. by measuring the amount of p-nitrophenol released from 4-nitrophenyl-beta-D-glucuronide, at λ = 405 nm. We found significantly greater activity of GLU (p<0.0001) in the serum of patients with colorectal cancer, as compared to the healthy subjects. The serum GLU activity significantly differentiates patients with colorectal cancer from healthy individuals. Serum GLU activity has diagnostic value and may be used in the diagnosis of colon adenocarcinoma.

  16. Serum β-glucuronidase as a potential colon cancer marker: a preliminary study

    Napoleon Waszkiewicz


    Full Text Available Colorectal cancer is characterized by high morbidity and mortality in developed countries. The lack of low-cost, easy-to-use screening diagnostic methods is one of the causes of late diagnosis of colorectal cancer. Beta-glucuronidase (GLU is a lysosomal exoglycosidase involved in degradation of glycosaminoglycans of the cell membranes and extracellular matrix of normal and cancerous colon tissues. The aim of our research was to evaluate the activity of GLU in the serum of colorectal cancer and estimate its potential value in the diagnosis of colorectal cancer.Blood samples were collected from 21 patients with colorectal adenocarcinoma and 17 healthy subjects. GLU activity was determined by the colorimetric method of Marciniak et al. by measuring the amount of p-nitrophenol released from 4-nitrophenyl-beta-D-glucuronide, at λ = 405 nm.We found significantly greater activity of GLU (p<0.0001 in the serum of patients with colorectal cancer, as compared to the healthy subjects. The serum GLU activity significantly differentiates patients with colorectal cancer from healthy individuals.Serum GLU activity has diagnostic value and may be used in the diagnosis of colon adenocarcinoma.

  17. Reversible Heat-Induced Inactivation of Chimeric β-Glucuronidase in Transgenic Plants1

    Almoguera, Concepción; Rojas, Anabel; Jordano, Juan


    We compared the expression patterns in transgenic tobacco (Nicotiana tabacum) of two chimeric genes: a translational fusion to β-glucuronidase (GUS) and a transcriptional fusion, both with the same promoter and 5′-flanking sequences of Ha hsp17.7 G4, a small heat shock protein (sHSP) gene from sunflower (Helianthus annuus). We found that immediately after heat shock, the induced expression from the two fusions in seedlings was similar, considering chimeric mRNA or GUS protein accumulation. Surprisingly, we discovered that the chimeric GUS protein encoded by the translational fusion was mostly inactive in such conditions. We also found that this inactivation was fully reversible. Thus, after returning to control temperature, the GUS activity was fully recovered without substantial changes in GUS protein accumulation. In contrast, we did not find differences in the in vitro heat inactivation of the respective GUS proteins. Insolubilization of the chimeric GUS protein correlated with its inactivation, as indicated by immunoprecipitation analyses. The inclusion in another chimeric gene of the 21 amino-terminal amino acids from a different sHSP lead to a comparable reversible inactivation. That effect not only illustrates unexpected post-translational problems, but may also point to sequences involved in interactions specific to sHSPs and in vivo heat stress conditions. PMID:12011363

  18. High resolution crystal structure of human β-glucuronidase reveals structural basis of lysosome targeting.

    Md Imtaiyaz Hassan

    Full Text Available Human β-glucuronidase (GUS cleaves β-D-glucuronic acid residues from the non-reducing termini of glycosaminoglycan and its deficiency leads to mucopolysaccharidosis type VII (MPSVII. Here we report a high resolution crystal structure of human GUS at 1.7 Å resolution and present an extensive analysis of the structural features, unifying recent findings in the field of lysosome targeting and glycosyl hydrolases. The structure revealed several new details including a new glycan chain at Asn272, in addition to that previously observed at Asn173, and coordination of the glycan chain at Asn173 with Lys197 of the lysosomal targeting motif which is essential for phosphotransferase recognition. Analysis of the high resolution structure not only provided new insights into the structural basis for lysosomal targeting but showed significant differences between human GUS, which is medically important in its own right, and E. coli GUS, which can be selectively inhibited in the human gut to prevent prodrug activation and is also widely used as a reporter gene by plant biologists. Despite these differences, both human and E. coli GUS share a high structure homology in all three domains with most of the glycosyl hydrolases, suggesting that they all evolved from a common ancestral gene.

  19. Improved buprenorphine immunoassay performance after urine treatment with β-glucuronidase.

    Snyder, Marion L; Darragh, Alicia; Flood, James G; Jones, Jenny; Ropar, Kaitlin; Jarolim, Petr; Melanson, Stacy E F


    Buprenorphine (BUP), a semi-synthetic opioid analgesic, is increasingly prescribed for the treatment of chronic pain and opioid dependence. Urine immunoassay screening methods are available for monitoring BUP compliance and misuse; however, these screens may have poor sensitivity or specificity. We evaluated whether the pretreatment of urine with β-glucuronidase (BG) improves the sensitivity and overall accuracy of three BUP enzyme immunoassays when compared with liquid chromatography-tandem mass spectrometry (LC-MS-MS). Urine samples sent to our laboratories for BUP testing (n = 114) were analyzed before and after BG pretreatment by cloned enzyme donor immunoassay (CEDIA), enzyme immunoassay (EIA) and homogenous EIA (HEIA) immunoassays using a common 5 ng/mL cutoff. Total BUP and norbuprenorphine (NBUP) concentrations were measured by LC-MS-MS as the reference method. Urine BG pretreatment improved EIA, HEIA and CEDIA sensitivities from 70, 82 and 94%, respectively, to 97% for each of the three methods, when compared with LC-MS-MS. While the specificity of the EIA and HEIA remained 100% after BG pretreatment, the specificity of the CEDIA decreased from 74 to 67%. Urine pretreatment with BG is recommended to improve sensitivity of the EIA and HEIA BUP screening methods. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email:

  20. Controlled Aggregation and Increased Stability of β-Glucuronidase by Cellulose Binding Domain Fusion

    Kim, Moonjung; Kwon, Kil Koang; Fu, Yaoyao; Kim, Haseong; Lee, Hyewon; Lee, Dae-Hee; Jung, Heungchae; Lee, Seung-Goo


    Cellulose-binding domains (CBDs) are protein domains with cellulose-binding activity, and some act as leaders in the localization of cellulosomal scaffoldin proteins to the hydrophobic surface of crystalline cellulose. In this study, we found that a CBD fusion enhanced and improved soluble β-glucuronidase (GusA) enzyme properties through the formation of an artificially oligomeric state. First, a soluble CBD fused to the C-terminus of GusA (GusA-CBD) was obtained and characterized. Interestingly, the soluble GusA-CBD showed maximum activity at higher temperatures (65°C) and more acidic pH values (pH 6.0) than free GusA did (60°C and pH 7.5). Moreover, the GusA-CBD enzyme showed higher thermal and pH stabilities than the free GusA enzyme did. Additionally, GusA-CBD showed higher enzymatic activity in the presence of methanol than free GusA did. Evaluation of the protease accessibility of both enzymes revealed that GusA-CBD retained 100% of its activity after 1 h incubation in 0.5 mg/ml protease K, while free GusA completely lost its activity. Simple fusion of CBD as a single domain may be useful for tunable enzyme states to improve enzyme stability in industrial applications. PMID:28099480

  1. Serum hexosaminidase and ß-glucuronidase activities in infants: effects of age and sex

    Mabe P.


    Full Text Available We investigated the effect of age and sex on the serum activity of hexosaminidase (HEX and ß-glucuronidase (BGLU in 275 normal term infants aged 12 h to 12 months. Up to six weeks of life, HEX was significantly higher in boys (P<=0.023. During the age period of 1-26 weeks, BGLU was also higher in boys, but differences were significant only at 2-6 and 7-15 weeks (P<=0.016. The developmental pattern of HEX and BGLU was sex dependent. HEX activity increased in both sexes from 4-7 days of life, reaching a maximum of 1.4-fold the birth value at 2-6 weeks of age in boys (P<0.001 and a maximum of 1.6-fold at 7-15 weeks in girls (P<0.001. HEX activity gradually decreased thereafter, reaching significantly lower levels at 27-53 weeks than during the first three days of life in boys (P = 0.002 and the same level of this age interval in girls. BGLU increased in both sexes from 4-7 days of age, showing a maximum increase at 7-15 weeks (3.3-fold in boys and 2.9-fold in girls, both P<0.001. Then BGLU decreased in boys to a value similar to that observed at 4-7 days of age. In girls, BGLU remained elevated until the end of the first year of life. These results indicate a variation of HEX and BGLU activities during the first year of life and a sex influence on their developmental pattern. This observation should be considered in the diagnosis of GM2 gangliosidosis and mucopolysaccharidosis type VII.

  2. The GH67 α-glucuronidase of Paenibacillus curdlanolyticus B-6 removes hexenuronic acid groups and facilitates biodegradation of the model xylooligosaccharide hexenuronosyl xylotriose.

    Septiningrum, Krisna; Ohi, Hiroshi; Waeonukul, Rattiya; Pason, Patthra; Tachaapaikoon, Chakrit; Ratanakhanokchai, Khanok; Sermsathanaswadi, Junjarus; Deng, Lan; Prawitwong, Panida; Kosugi, Akihiko


    4-O-Methylglucuronic acid (MeGlcA) side groups attached to the xylan backbone through α-1,2 linkages are converted to hexenuronic acid (HexA) during alkaline pulping. α-Glucuronidase (EC hydrolyzes 1,2-linked MeGlcA from xylooligosaccharides. To determine whether α-glucuronidase can also hydrolyze HexA-decorated xylooligosaccharides, a gene encoding α-glucuronidase (AguA) was cloned from Paenibacillus curdlanolyticus B-6. The purified protein degraded hexenuronosyl xylotriose (ΔX3), a model substrate prepared from kraft pulp. AguA released xylotriose and HexA from ΔX3, but the Vmax and kcat values for ΔX3 were lower than those for MeGlcA, indicating that HexA side groups may affect the hydrolytic activity. To explore the potential for biological bleaching, ΔX3 degradation was performed using intracellular extract from P. curdlanolyticus B-6. The intracellular extract, with synergistic α-glucuronidase and β-xylosidase activities, degraded ΔX3 to xylose and HexA. These results indicate that α-glucuronidase can be used to remove HexA from ΔX3 derived from pulp, reducing the need for chemical treatments in the pulping process.

  3. Effect of dietary fiber on the induction of colorectal tumors and fecal beta-glucuronidase activity in the rat.

    Bauer, H G; Asp, N G; Oste, R; Dahlqvist, A; Fredlund, P E


    The purpose of the present study was to investigate whether three different types of dietary fiber, wheat bran, carrot fiber, and citrus pectin, influenced the induction of colorectal tumors produced by 1,2-dimethylhydrazine in rats. In all groups, the tumor yield was high (87 to 97%). In the wheat bran and carrot fiber groups, the incidence of colorectal tumors was not significantly different from that of the group fed on the fiber-free basic diet. The citrus pectin group, however, had a significantly higher incidence of colorectal tumors (p less than 0.001). An increased number of auditory duct tumors was also noted in this group. In a separate experiment, dietary pectin induced a 10-fold increase in fecal beta-glucuronidase activity but did not alter this activity in the bowel wall. It has been suggested that dietary fiber protects against the induction of colorectal tumors, but this was not the case in the experiment. It is possible that the high tumor yield made the demonstration of a weak protective effect of wheat bran impossible. The reason for the increased occurrence of tumors in the citrus pectin group is obscure and will be subjected to further investigation. Fecal beta-glucuronidase activity might be one factor of importance in the activation of the carcinogen.

  4. Probiotic effects of beta-glucuronidase on the peach-potato aphid Myzus persicae (Aphididae).

    Cherqui, A; Alla, S; Saguez, J; Doury, G; Sangwan-Norreel, B S; Giordanengo, P


    beta-glucuronidase (GUS) is a reporter protein commonly expressed in transgenic plants allowing the visualization of the transformed individuals. In our recent work, we showed that consumption of transformed potato plants expressing this GUS enzyme improves performance of the phloem feeding aphid Myzus persicae. Those results led us to the conclusion that the expression of GUS in potato plants might be responsible for the probiotic effect measured in feeding aphids. In the present paper, artificial diets were used to provide active GUS (10 and 500 microg ml(-1)), inactivated heated GUS (500 microg ml(-1)), glucuronic acid (10, 100 and 500 microg ml(-1)), and bovine serum albumin (500 microg ml(-1)) to M. persicae. Our results reveal that these chemicals provided as food intake might influence the biological parameters of this aphid. Experiments showed a probiotic effect of 500 microg ml(-1) GUS diet, resulting in reduced larval mortality, and increased adult reproduction period and fecundity, which led to an increased population growth potential (r(m)=0.17+/-0.01 versus r(m)=0.12+/-0.03 for aphids fed on control diet). A lower amount of added GUS led to fewer variations, biological parameters being only slightly altered (r(m)=0.14+/-0.03). Statistically similar alterations of the biological parameters were obtained when comparing aphids fed on the diet added with inactivated GUS or the non-structural bovine serum albumin protein (r(m)=0.15+/-0.02 and 0.14+/-0.03, respectively). Feeding assays conducted with glucuronic acid supplemented diets enhanced longevity and nymph production of the adult aphids and reduced larval mortality, resulting in r(m)=0.15+/-0.02 for the highest dose (500 microg ml(-1)). Although 100 microg ml(-1) glucuronate diet did not induce any effect on M. persicae (r(m)=0.12+/-0.03), aphids fed on 10 microg ml(-1) glucuronate diet exhibited unexpected reduced demographic parameters (r(m)=0.10+/-0.03). Immuno-histological analysis showed GUS

  5. A combination of PhP typing and β-d-glucuronidase gene sequence variation analysis for differentiation of Escherichia coli from humans and animals.

    Masters, N; Christie, M; Katouli, M; Stratton, H


    We investigated the usefulness of the β-d-glucuronidase gene variance in Escherichia coli as a microbial source tracking tool using a novel algorithm for comparison of sequences from a prescreened set of host-specific isolates using a high-resolution PhP typing method. A total of 65 common biochemical phenotypes belonging to 318 E. coli strains isolated from humans and domestic and wild animals were analysed for nucleotide variations at 10 loci along a 518 bp fragment of the 1812 bp β-d-glucuronidase gene. Neighbour-joining analysis of loci variations revealed 86 (76.8%) human isolates and 91.2% of animal isolates were correctly identified. Pairwise hierarchical clustering improved assignment; where 92 (82.1%) human and 204 (99%) animal strains were assigned to their respective cluster. Our data show that initial typing of isolates and selection of common types from different hosts prior to analysis of the β-d-glucuronidase gene sequence improves source identification. We also concluded that numerical profiling of the nucleotide variations can be used as a valuable approach to differentiate human from animal E. coli. This study signifies the usefulness of the β-d-glucuronidase gene as a marker for differentiating human faecal pollution from animal sources.

  6. Isolation and analysis of a gene encoding alpha-glucuronidase, an enzyme with a novel primary structure involved in the breakdown of xylan.

    Ruile, P; Winterhalter, C; Liebl, W


    This is the first report describing the analysis of a gene encoding an alpha-glucuronidase, an enzyme essential for the complete breakdown of substituted xylans. A DNA fragment that carries the gene for alpha-glucuronidase was isolated from chromosomal DNA of the hyperthermophilic bacterium Thermotoga maritima MSB8. The alpha-glucuronidase gene (aguA) was identified and characterized with the aid of nucleotide sequence analysis, deletion experiments and expression studies in Escherichia coli, and the start of the coding region was defined by amino-terminal sequencing of the purified recombinant enzyme. The aguA gene encodes a 674-amino-acid, largely hydrophilic polypeptide with a calculated molecular mass of 78593 Da. The alpha-glucuronidase of T. maritima has a novel primary structure with no significant similarity to any other known amino acid sequence. The recombinant enzyme was purified to homogeneity as judged by SDS-PAGE. Gel filtration analysis at low salt concentrations revealed a high apparent molecular mass (> 630 kDa) for the recombinant enzyme, but the oligomeric structure changed upon variation of the ionic strength or the pH, yielding hexameric and/or dimeric forms which were also enzymatically active. The enzyme hydrolysed 2-O-(4-O-methyl-alpha-D-glucopyranosyluronic acid)-D-xylobiose (MeGlcAX2) to xylobiose and 4-O-methylglucuronic acid. The K(m) for MeGlcAX2 was 0.95 mM. The pH optimum was 6.3. Maximum activity was measured at 85 degrees C, about 25 degrees C or more above the values reported for all other alpha-glucuronidases known to date. When incubated at 55-75 degrees C, the enzyme suffered partial inactivation, but thereafter the residual activity remained nearly constant for several days.

  7. aguA, the gene encoding an extracellular alpha-glucuronidase from Aspergillus tubingensis, is specifically induced on xylose and not on glucuronic acid.

    de Vries, R P; Poulsen, C H; Madrid, S; Visser, J


    An extracellular alpha-glucuronidase was purified and characterized from a commercial Aspergillus preparation and from culture filtrate of Aspergillus tubingensis. The enzyme has a molecular mass of 107 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 112 kDa as determined by mass spectrometry, has a determined pI just below 5.2, and is stable at pH 6.0 for prolonged times. The pH optimum for the enzyme is between 4.5 and 6.0, and the temperature optimum is 70 degrees C. The alpha-glucuronidase is active mainly on small substituted xylo-oligomers but is also able to release a small amount of 4-O-methylglucuronic acid from birchwood xylan. The enzyme acts synergistically with endoxylanases and beta-xylosidase in the hydrolysis of xylan. The enzyme is N glycosylated and contains 14 putative N-glycosylation sites. The gene encoding this alpha-glucuronidase (aguA) was cloned from A. tubingensis. It consists of an open reading frame of 2,523 bp and contains no introns. The gene codes for a protein of 841 amino acids, containing a eukaryotic signal sequence of 20 amino acids. The mature protein has a predicted molecular mass of 91,790 Da and a calculated pI of 5.13. Multiple copies of the gene were introduced in A. tubingensis, and expression was studied in a highly overproducing transformant. The aguA gene was expressed on xylose, xylobiose, and xylan, similarly to genes encoding endoxylanases, suggesting a coordinate regulation of expression of xylanases and alpha-glucuronidase. Glucuronic acid did not induce the expression of aguA and also did not modulate the expression on xylose. Addition of glucose prevented expression of aguA on xylan but only reduced the expression on xylose.

  8. aguA, the Gene Encoding an Extracellular α-Glucuronidase from Aspergillus tubingensis, Is Specifically Induced on Xylose and Not on Glucuronic Acid

    de Vries, Ronald P.; Poulsen, Charlotte H.; Madrid, Susan; Visser, Jaap


    An extracellular α-glucuronidase was purified and characterized from a commercial Aspergillus preparation and from culture filtrate of Aspergillus tubingensis. The enzyme has a molecular mass of 107 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 112 kDa as determined by mass spectrometry, has a determined pI just below 5.2, and is stable at pH 6.0 for prolonged times. The pH optimum for the enzyme is between 4.5 and 6.0, and the temperature optimum is 70°C. The α-glucuronidase is active mainly on small substituted xylo-oligomers but is also able to release a small amount of 4-O-methylglucuronic acid from birchwood xylan. The enzyme acts synergistically with endoxylanases and β-xylosidase in the hydrolysis of xylan. The enzyme is N glycosylated and contains 14 putative N-glycosylation sites. The gene encoding this α-glucuronidase (aguA) was cloned from A. tubingensis. It consists of an open reading frame of 2,523 bp and contains no introns. The gene codes for a protein of 841 amino acids, containing a eukaryotic signal sequence of 20 amino acids. The mature protein has a predicted molecular mass of 91,790 Da and a calculated pI of 5.13. Multiple copies of the gene were introduced in A. tubingensis, and expression was studied in a highly overproducing transformant. The aguA gene was expressed on xylose, xylobiose, and xylan, similarly to genes encoding endoxylanases, suggesting a coordinate regulation of expression of xylanases and α-glucuronidase. Glucuronic acid did not induce the expression of aguA and also did not modulate the expression on xylose. Addition of glucose prevented expression of aguA on xylan but only reduced the expression on xylose. PMID:9440512

  9. Ciprofloxacin blocked enterohepatic circulation of diclofenac and alleviated NSAID-induced enteropathy in rats partly by inhibiting intestinal β-glucuronidase activity.

    Zhong, Ze-Yu; Sun, Bin-Bin; Shu, Nan; Xie, Qiu-Shi; Tang, Xian-Ge; Ling, Zhao-Li; Wang, Fan; Zhao, Kai-Jing; Xu, Ping; Zhang, Mian; Li, Ying; Chen, Yang; Liu, Li; Xia, Lun-Zhu; Liu, Xiao-Dong


    Diclofenac is a non-steroidal anti-inflammatory drug (NSAID), which may cause serious intestinal adverse reactions (enteropathy). In this study we investigated whether co-administration of ciprofloxacin affected the pharmacokinetics of diclofenac and diclofenac-induced enteropathy in rats. The pharmacokinetics of diclofenac was assessed in rats after receiving diclofenac (10 mg/kg, ig, or 5 mg/kg, iv), with or without ciprofloxacin (20 mg/kg, ig) co-administered. After receiving 6 oral doses or 15 intravenous doses of diclofenac, the rats were sacrificed, and small intestine was removed to examine diclofenac-induced enteropathy. β-Glucuronidase activity in intestinal content, bovine liver and E coli was evaluated. Following oral or intravenous administration, the pharmacokinetic profile of diclofenac displayed typical enterohepatic circulation, and co-administration of ciprofloxacin abolished the enterohepatic circulation, resulted in significant reduction in the plasma content of diclofenac. In control rats, β-glucuronidase activity in small intestinal content was region-dependent: proximal intestineciprofloxacin (10-2000 μmol/L) dose-dependently inhibited β-glucuronidase activity in distal small intestine content or E coli incubated in vitro, but did not affect that in proximal small intestine content or bovine liver incubated in vitro. After receiving 6 oral doses or 15 intravenous doses of diclofenac, typical enteropathy was developed with severe enteropathy occurred in distal small intestine. Co-administration of ciprofloxacin significantly alleviated diclofenac-induced enteropathy. Co-administration of ciprofloxacin attenuated enterohepatic circulation of diclofenac and alleviated diclofenac-induced enteropathy in rats, partly via the inhibition of intestinal β-glucuronidase activity.

  10. Effects of dietary bran and the colon carcinogen 1,2-dimethylhydrazine on faecal. beta. -glucuronidase activity in mice

    London, J.F.; Clapp, N.K.; Henke, M.A.


    To evaluate the physiological changes that might provide either mechanistic or prognostic information in colon carcinogenesis, we treated 8-wk-old male BALB/c mice with semisynthetic bran diets containing 20% by weight of either soya bean, winter wheat, or corn bran and gave ten weekly injections (from age 11 to 21 wk) of 20 mg 1,2-dimethylhydrazine (DMH)/kg body weight. Appropriate controls were included. Faecal samples were collected 11 to 13 wk after completion of DMH treatment and after 24 to 26 wk of continuous bran diet feeding. Daily faecal ..beta..-glucuronidase activity fluctuations were observed, but these were not statistically significant. Faecal enzyme activity varied depending upon the type of bran diet (control > wheat > soya bean > corn). DMH generally depressed faecal enzyme activity to a statistically significant degree (P < 0.03). These results demonstrate the persistent enzyme alterations produced by DMH and the assay's sensitivity for detecting such change. Further, the effects of diet suggest that dietary brans cause physiological changes that have the potential to modify colon carcinogenesis.

  11. Effect of Dimer Dissociation on Activity and Thermostability of the α-Glucuronidase from Geobacillus stearothermophilus: Dissecting the Different Oligomeric Forms of Family 67 Glycoside Hydrolases

    Shallom, Dalia; Golan, Gali; Shoham, Gil; Shoham, Yuval


    The oligomeric organization of enzymes plays an important role in many biological processes, such as allosteric regulation, conformational stability and thermal stability. α-Glucuronidases are family 67 glycosidases that cleave the α-1,2-glycosidic bond between 4-O-methyl-d-glucuronic acid and xylose units as part of an array of hemicellulose-hydrolyzing enzymes. Currently, two crystal structures of α-glucuronidases are available, those from Geobacillus stearothermophilus (AguA) and from Cellvibrio japonicus (GlcA67A). Both enzymes are homodimeric, but surprisingly their dimeric organization is different, raising questions regarding the significance of dimerization for the enzymes' activity and stability. Structural comparison of the two enzymes suggests several elements that are responsible for the different dimerization organization. Phylogenetic analysis shows that the α-glucuronidases AguA and GlcA67A can be classified into two distinct subfamilies of bacterial α-glucuronidases, where the dimer-forming residues of each enzyme are conserved only within its own subfamily. It seems that the different dimeric forms of AguA and GlcA67A represent the two alternative dimeric organizations of these subfamilies. To study the biological significance of the dimerization in α-glucuronidases, we have constructed a monomeric form of AguA by mutating three of its interface residues (W328E, R329T, and R665N). The activity of the monomer was significantly lower than the activity of the wild-type dimeric AguA, and the optimal temperature for activity of the monomer was around 35°C, compared to 65°C of the wild-type enzyme. Nevertheless, the melting temperature of the monomeric protein, 72.9°C, was almost identical to that of the wild-type, 73.4°C. It appears that the dimerization of AguA is essential for efficient catalysis and that the dissociation into monomers results in subtle conformational changes in the structure which indirectly influence the active site region

  12. Discovery of Specific Inhibitors for Intestinal E. coli  β-Glucuronidase through In Silico Virtual Screening

    Ta-Chun Cheng


    Full Text Available Glucuronidation is a major metabolism process of detoxification for carcinogens, 4-(methylnitrosamino-1-(3-pyridy-1-butanone (NNK and 1,2-dimethylhydrazine (DMH, of reactive oxygen species (ROS. However, intestinal E. coli   β-glucuronidase (eβG has been considered pivotal to colorectal carcinogenesis. Specific inhibition of eβG may prevent reactivating the glucuronide-carcinogen and protect the intestine from ROS-mediated carcinogenesis. In order to develop specific eβG inhibitors, we found that 59 candidate compounds obtained from the initial virtual screening had high inhibition specificity against eβG but not human βG. In particular, we found that compounds 7145 and 4041 with naphthalenylidene-benzenesulfonamide (NYBS are highly effective and selective to inhibit eβG activity. Compound 4041  (IC50=2.8 μM shows a higher inhibiting ability than compound 7145  (IC50=31.6 μM against eβG. Furthermore, the molecular docking analysis indicates that compound 4041 has two hydrophobic contacts to residues L361 and I363 in the bacterial loop, but 7145 has one contact to L361. Only compound 4041 can bind to key residue (E413 at active site of eβG via hydrogen-bonding interactions. These novel NYBS-based eβG specific inhibitors may provide as novel candidate compounds, which specifically inhibit eβG to reduce eβG-based carcinogenesis and intestinal injury.

  13. Activity of alpha-fucosidase and beta-glucuronidase in serum and urine of patients administered parenteral nutrition.

    Raczkowska, Katarzyna; Szajda, Slawomir Dariusz; Raczkowski, Krzysztof; Zasadowska, Wioletta; Chojnowska, Sylwia; Kepka, Alina; Zalewska-Szajda, Beata; Waszkiewicz, Napoleon; Knaś, Malgorzata; Snarska, Jadwiga; Zwierz, Krzysztof; Ladny, Jerzy Robert


    In hospital patients suffering from adverse clinical and biochemical symptoms of malnutrition, it is often necessary to employ parenteral nutrition to avoid the body's tissue becoming broken down by being metabolised. Thus, the patient's welfare and survival can be supported throughout any periods of medical crisis. Two of the enzymes responsible for metabolising glycoconjugates are alpha-fucosidase (FUC) and beta-glucuronidase (GLU), present in lysosomes. They release fucose or glucuronic acid from the non-reducing end of oligosaccharide chains. To determine the effect of parenteral nutrition administered to ill patients, on glycoconjugate metabolism, by measuring serum and urinary activities of FUC and GLU. Material and methods. Blood samples and the daily urine collection were taken from 23 patients' who had been undergoing parenteral nutrition for either 5 or 10 days, as well as from a baseline sample. Enzyme activities in serum and urine were determined by the method of Zwierz et al. Serum FUC activities were significantly lower after 10 days compared to 5, (p< 0.0172), whereas GLU activities were significantly lower after both 5 and 10 days, (p< 0.0007 and p< 0.0208 respectively), compared to levels before starting parenteral nutrition. GLU activities were however higher after 10 days than those after 5 days, (p< 0.0023). In urine, FUC activities were significantly decreased after 10 days compared to 5 days after starting parenteral nutrition, (p< 0.0245). Urine GLU activities were unaffected by parenteral nutrition nor was any effect seen on FUC or GLU activities when calculated per 1mg creatinine. Serum FUC and GLU activities can be used for assessing the effect of parenteral nutrition on glycoconjugate metabolism. The significant decreases of serum GLU activity observed after 5 and 10 days, may serve to indicate that the components of parental nutrition are appropriate and that the body has become suitably adapted to this form of nutrition.

  14. Fluorescent antibody-viability staining and beta-glucuronidase assay as rapid methods for monitoring Escherichia coli viability in coastal marine waters.

    Caruso, G; De Pasquale, F; Mancuso, M; Zampino, D; Crisafi, E


    A faecal pollution monitoring of coastal Messina waters was performed by comparing three (microscopic, enzyme, and culture) methods. Evidence of Escherichia coli cells (29.99 to 96.79% of the total enteropathogenic serotypes) retaining their viability into the marine environment was shown. beta-Glucuronidase activity rates suggested that living cells were also metabolically active. Heavily polluted sites were detected, where improperly treated urban wastes were discharged. Significant relationships between microscopic and enzymatic data proved both methods to be suitable alternatives to the culture method for E. coli detection, improving environmental quality assessment.

  15. Histochemical studies on genetical control of hormonal enzyme inducibility in the mouse. V. Histochemical evidence for androgen inducibility of beta-glucuronidase in the epididymis

    Blecher, S R; Kirkeby, S


    The enzyme beta-glucuronidase (beta G) is shown, by histochemical methods, to be androgen inducible in the mouse epididymis. This trait has previously been believed to exist only in the kidney. Its presence in the genital tract constitutes a valuable tool in study of the developmental genetics of...... of the reproductive system. Data presented here and previously imply co-ordinated genetic control of heterogeneous lysosomal populations. The results reported also provide a system for study of X-chromosomal activation, and of the developmental androgen dependence of the epididymis....

  16. Measurement of beta-glucuronidase in effluent of perifused alveolar macrophages challenged with chemically modified chrysotile asbestos.

    Forget, G; Lacroix, M J; Calvert, R; Sirois, P


    Chrysotile asbestos has been implicated with lung disorders, notably fibrotic lesions and cancer. In vitro, chrysotile fibers are cytotoxic to lung macrophages and stimulate the release of inflammatory mediators. Reports to the effect that chemical modifications of asbestos fibers reduce their cytotoxic and inflammatory potential initiated the present study of three fiber modifications. The cytotoxic and inflammatory effects of magnesium-leached chrysotile, POCL3-treated chrysotile, and CaO-treated chrysotile were studied in a perifused rat alveolar macrophage culture system, relative to untreated fibers. Natural Canadian chrysotile (UICC "B") caused dose-dependent cell mortality and clumping. The release of beta-glucuronidase (beta-Glu), a lysosomal enzyme, was also dose dependent. Rhodesian chrysotile (UICC "A") caused similar cytotoxic and inflammatory effects. However, magnesium-leached chrysotile was less cytotoxic (39% less) and had a lesser clumping capacity (31% less) than untreated chrysotile. Total secretion of beta-Glu elicited by magnesium-leached chrysotile was reduced by 43% from the untreated sample, but kinetic monitoring indicates that this reduction in inflammatory potential is only significant during the first 12 h of an 18-h culture period. POCl3 treatment of chrysotile fibers produced differing effects depending on the length of the fibers under study. Treating fibers with a mean length of 8 micron produced a secretion pattern similar to that produced by acid leaching. The total output for the treated sample was 44% lower than with untreated chrysotile; the difference was only significant during the first 12 h of perifusion. Cell mortality and aggregation were not reduced in any important way with POCl3 treatment of these longer fibers. When ultra-short fibers (mean length = 0.8 micron) were treated with POCl3, the total decrease in beta-Glu output was equal to 41%, and the release of enzyme was significantly lower during the whole 18-h

  17. Construction of a fusion gene comprising the Taka-amylase A promoter and the Escherichia coli beta-glucuronidase gene and analysis of its expression in Aspergillus oryzae.

    Tada, S; Gomi, K; Kitamoto, K; Takahashi, K; Tamura, G; Hara, S


    Northern blot analysis of glucose-grown and starch-grown mycelia of Aspergillus oryzae RIB40 was conducted using the cloned Taka-amylase A (TAA) gene as a probe. The amount of mRNA homologous to the TAA gene was increased when this fungus was grown with starch as a sole carbon source. In order to analyze the induction mechanism, we inserted the Escherichia coli uidA gene encoding beta-glucuronidase (GUS) down-stream of the TAA promoter and introduced the resultant fusion gene into the A. oryzae genome. Production of a functional GUS protein was induced by starch, but not by glucose. When the effects of various sugars on expression of the fusion gene were examined, the results suggested that the expression of the fusion gene was under control of the TAA gene promoter.

  18. The activity of N-acetyl-β-d-hexosaminidase A and B and β-glucuronidase in nasal polyps and hypertrophic nasal concha.

    Chojnowska, Sylwia; Minarowska, Alina; Waszkiewicz, Napoleon; Kępka, Alina; Zalewska-Szajda, Beata; Gościk, Elżbieta; Kowal, Krzysztof; Olszewska, Ewa; Konarzewska-Duchnowska, Emilia; Minarowski, Łukasz; Zwierz, Krzysztof; Ładny, Jerzy Robert; Szajda, Sławomir Dariusz


    Nasal polyps and hypertrophic lower nasal conchae are common disorders of nasal cavity. The majority of etiopathogenetic theories indicate inflammatory background of polyps and hypertrophic concha. N-acetyl-β-D-hexosaminidase and β-glucuronidase are lysosomal exoglycosidases revealing accelerated activity in inflammatory processes. The aim of the study was to evaluate the catabolism of glycoconjugates in nasal polyps and hypertrophic nasal concha basing on the activity of N-acetyl-β-D-hexosaminidase (HEX) and β-glucuronidase (GLU). Material consisted of nasal polyps taken from 40 patients during polypectomy in patients with chronic rhinosinusitis with nasal polyps (CRSwNP) and hypertrophic lower nasal conchae taken from 20 patients during mucotomy. The activity of HEX, HEX A, HEX B and GLU in supernatant of homogenates of nasal polyps and hypertrophic lower nasal concha tissues has been estimated using colorimetric method. Statistically significant decrease has been observed in concentration of the activity (per 1mg of tissue) of HEX (p<0.05), HEX B (p<0.001) and specific activity (per 1mg of protein) of HEX B (p<0.001) in nasal polyps tissue in comparison to hypertrophic lower nasal conchae tissue. Decrease in the activity and specific activity concentration of the majority of examined lysosomal exoglycosidases (increasing in inflammations) in comparison to hypertrophic lower nasal conchae suggests electrolytes disorders and questions the inflammatory background of nasal polyps. Copyright © 2013 Polish Otorhinolaryngology - Head and Neck Surgery Society. Published by Elsevier Urban & Partner Sp. z.o.o. All rights reserved.

  19. Synthesis, in vitro β-glucuronidase inhibitory activity and in silico studies of novel (E)-4-Aryl-2-(2-(pyren-1-ylmethylene)hydrazinyl)thiazoles.

    Salar, Uzma; Khan, Khalid Mohammed; Syed, Shazia; Taha, Muhammad; Ali, Farman; Ismail, Nor Hadiani; Perveen, Shahnaz; Wadood, Abdul; Ghufran, Mehreen


    Current research is based on the synthesis of novel (E)-4-aryl-2-(2-(pyren-1-ylmethylene)hydrazinyl)thiazole derivatives (3-15) by adopting two steps route. First step was the condensation between the pyrene-1-carbaldehyde (1) with the thiosemicarbazide to afford pyrene-1-thiosemicarbazone intermediate (2). While in second step, cyclization between the intermediate (2) and phenacyl bromide derivatives or 2-bromo ethyl acetate was carried out. Synthetic derivatives were structurally characterized by spectroscopic techniques such as EI-MS, (1)H NMR and (13)C NMR. Stereochemistry of the iminic double bond was confirmed by NOESY analysis. All pure compounds 2-15 were subjected for in vitro β-glucuronidase inhibitory activity. All molecules were exhibited excellent inhibition in the range of IC50=3.10±0.10-40.10±0.90μM and found to be even more potent than the standard d-saccharic acid 1,4-lactone (IC50=48.38±1.05μM). Molecular docking studies were carried out to verify the structure-activity relationship. A good correlation was perceived between the docking study and biological evaluation of active compounds.

  20. A comparative analysis of green fluorescent protein and -glucuronidase protein-encoding genes as a reporter system for studying the temporal expression profiles of promoters

    P Kavita; Pradeep Kumar Burma


    The assessment of activity of promoters has been greatly facilitated by the use of reporter genes. However, the activity as assessed by reporter gene is a reflection of not only promoter strength, but also that of the stability of the mRNA and the protein encoded by the reporter gene. While a stable reporter gene product is an advantage in analysing activities of weak promoters, it becomes a major limitation for understanding temporal expression patterns of a promoter, as the reporter product persists even after the activity of the promoter ceases. In the present study we undertook a comparative analysis of two reporter genes, -glucuronidase (gus) and green fluorescent protein (sgfp), for studying the temporal expression pattern of tapetum-specific promoters A9 (Arabidopsis thaliana) and TA29 (Nicotiana tabacum). The activity of A9 and TA29 promoters as assessed by transcript profiles of the reporter genes (gus or sgfp) remained the same irrespective of the reporter gene used. However, while the deduced promoter activity using gus was extended temporally beyond the actual activity of the promoter, sgfp as recorded through its fluorescence correlated better with the transcription profile. Our results thus demonstrate that sgfp is a better reporter gene compared to gus for assessment of temporal activity of promoters. Although several earlier reports have commented on the possible errors in deducing temporal activities of promoters using GUS as a reporter protein, we experimentally demonstrate the advantage of using reporter genes such as gfp for analysis of temporal expression patterns.

  1. Facile and ultrasensitive fluorescence sensor platform for tumor invasive biomaker β-glucuronidase detection and inhibitor evaluation with carbon quantum dots based on inner-filter effect.

    Lu, Shuaimin; Li, Guoliang; Lv, Zhengxian; Qiu, Nannan; Kong, Weiheng; Gong, Peiwei; Chen, Guang; Xia, Lian; Guo, Xiaoxi; You, Jinmao; Wu, Yongning


    Early detection and diagnosis have great practical significances for the effective prevention and treatment of cancer. In this study, we developed a novel, facile and ultra-sensitive fluorescence assay for the determination of tumor invasive biomarker β-glucuronidase (GLU) based on the inner-filter effect (IFE). The nitrogen-doped carbon quantum dots (N-CQDs) with green photoluminescence were employed as the fluorophore in IFE, and 4-nitrophenyl-β-D-glucuronide (PNPG) was used to act as GLU substrate, and GLU catalytic product (p-nitrophenol (PNP)) was capable of acting as the robust absorber in IFE to turn off the fluorescence of N-CQDs due to the complementary overlap between the absorption of PNP and the excitation of N-CQDs. Thus, signal of GLU activity could be recorded by the fluorescence intensity of N-CQDs. Unlike other fluorescence sensing mechanism such as fluorescence resonance energy transfer (FRET) or photoinduced electron transfer (PET), IFE has no requirement for electron or energy transfer process or any chemical modification of fluorophore, which makes our assay more flexible and simple. The proposed method exhibited a good linear relationship from 1UL(-1) to 60UL(-1) (R(2)=0.9967) with a low detection limit of 0.3UL(-1). This method was also successfully applied to the analysis of serum samples and the inhibitor screening from natural product. The developed sensor platform was proven to be reliable, facile, sensitive, and selective, making it promising as a candidate for GLU activity detection in clinic tumor diagnose and anti-tumor drug screening.

  2. Facile high-throughput forward chemical genetic screening by in situ monitoring of glucuronidase-based reporter gene expression in Arabidopsis thaliana

    Vivek eHalder


    Full Text Available The use of biologically active small molecules to perturb biological functions holds enormous potential for investigating complex signaling networks. However, in contrast to animal systems, the search for and application of chemical tools for basic discovery in the plant sciences, generally referred to as ‘chemical genetics’, has only recently gained momentum. In addition to cultured cells, the well-characterized, small-sized model plant Arabidopsis thaliana is suitable for cultivation in microplates, which allows employing diverse cell- or phenotype-based chemical screens. In such screens, a chemical’s bioactivity is typically assessed either through scoring its impact on morphological traits or quantifying molecular attributes such as enzyme or reporter activities. Here, we describe a facile forward chemical screening methodology for intact Arabidopsis seedlings harboring the β-glucuronidase (GUS reporter by directly quantifying GUS activity in situ with 4-methylumbelliferyl-β-D-glucuronide (4-MUG as substrate. The quantitative nature of this screening assay has an obvious advantage over the also convenient histochemical GUS staining method, as it allows application of statistical procedures and unbiased hit selection based on threshold values as well as distinction between compounds with strong or weak bioactivity. At the same time, the in situ bioassay is very convenient requiring less effort and time for sample handling in comparison to the conventional quantitative in vitro GUS assay using 4-MUG, as validated with several Arabidopsis lines harboring different GUS reporter constructs. To demonstrate that the developed assays is particularly suitable for large-scale screening projects, we performed a pilot screen for chemical activators or inhibitors of salicylic acid-mediated defense signaling using the Arabidopsis PR1p::GUS line. Importantly, the screening methodology provided here can be adopted for any inducible GUS reporter line.

  3. Effects of abomasal infusion of flaxseed (Linum usitatissimum) oil on microbial β-glucuronidase activity and concentration of the mammalian lignan enterolactone in ruminal fluid, plasma, urine and milk of dairy cows.

    Côrtes, Cristiano; da Silva-Kazama, Daniele; Kazama, Ricardo; Benchaar, Chaouki; dos Santos, Geraldo; Zeoula, Lucia M; Gagnon, N; Petit, Hélène V


    Ruminal microbiota plays an important role in the conversion of plant lignans into mammalian lignans. The main mammalian lignan present in the milk of dairy cows fed flax products is enterolactone (EL). The objectives of the present study were to investigate the effects of abomasal infusion of flax oil on the metabolism of flax lignans and concentrations of EL in biological fluids of dairy cows. A total of six rumen-cannulated dairy cows were assigned within a 2 × 3 factorial arrangement of six treatments utilising flax hulls (0 and 15·9 % of DM) and abomasal infusion of flax oil (0, 250 and 500 g/d). There were six periods of 21 d each. Samples were collected during the last 7 d of each period and subjected to chemical analysis. Flax hull supplementation increased concentrations of EL in ruminal fluid, plasma, urine and milk, while flax oil infusion had no effect. Post-feeding, β-glucuronidase activity in the ruminal fluid of cows infused with 250 g flax oil was significantly lower for cows fed hulls than for those fed the control diet. The present study demonstrated that the presence of a rich source of n-3 fatty acids such as flax oil in the small intestine does not interfere with the absorption of the mammalian lignan EL and that lower ruminal β-glucuronidase activity had no effect on the conversion of flax lignans into EL in the rumen of dairy cows.

  4. Expressão eficiente do gene reporter beta-glucuronidase nos tecidos vasculares de batata (Solanum tuberosum L. utilizando de um promotor específico (BRA3 de Agrobacterium rhizogenes Efficient expression of beta-glucuronidase reporter gene in vascular tissue of potato (Solanum tuberosum L. utilizing a specific promoter (BRA3 from Agrobacterium rhizogenes

    Antonio Carlos Torres


    Full Text Available Promotores tecido-específico controlam a transcrição de genes em diferentes tecidos vegetais bem como em diferentes estádios de desenvolvimento da planta, levando à indução de distintos níveis de atividade transiente e/ou estável do gene. Tais promotores podem ser empregados para a expressão seletiva de genes de interesse. O promotor rol A de Agrobacterium rhizogenes, por exemplo, é floema-específico, sugerindo que possa ser empregado em estratégias de defesa de plantas que são infectadas por vírus com replicação restrita ao floema. A expressão do gene marcador da ß-glucuronidase (gus dirigido pelo promotor rol A (pBRA3 foi observada em plantas transgênicas de batata (cvs. Macaca e Baronesa. Entrenós e secções de folhas foram submetidos ao cocultivo com A. tumefaciens. A atividade do gene gus avaliada em brotações resistentes à canamicina não se restringiu ao floema (alto nível de expressão do gene, mas também se manifestou no xilema dos caules. As expressões transiente e estável são, no entanto, tecido-específicas, localizadas sobretudo no sistema vascular de entrenós e ausente em raízes e folhas. As plantas gus positivas foram micropropagadas, plantadas em casa de vegetação e avaliadas por PCR, utilizando-se 'primers' específicos para o gene npt II. Nenhuma alteração fenotípica foi observada em plantas transgênicas, em relação às não transformadas.Tissue-especific promoters allow the modulation of gene transcription in different tissue types as well as in different stages of plant development, leading different levels of transient and stable activity of the gene product. These promoters have been employed for selective gene expression. The Agrobacterium rhizogenes rol A gene promoter (BRA3 controls phloem-specific expression indicating that this promoter might have an important role in plant defense strategies against virus which replicated only in the phloem. The expression of ß-glucuronidase

  5. Assays of dioxins and dioxin-like compounds in actually contaminated soils using transgenic tobacco plants carrying a recombinant mouse aryl hydrocarbon receptor-mediated β-glucuronidase reporter gene expression system.

    Inui, Hideyuki; Gion, Keiko; Utani, Yasushi; Wakai, Taketo; Kodama, Susumu; Eun, Heesoo; Kim, Yun-Seok; Ohkawa, Hideo


    The transgenic tobacco plant XD4V-26 carrying the recombinant mouse aryl hydrocarbon receptor XD4V-mediated β-glucuronidase (GUS) reporter gene expression system was used for assay of dioxins and dioxin-like compounds consisting of polychlorinated dibenzeno-p-dioxins, polychlorinated dibenzofurans, and coplanar polychlorinated biphenyls (Co-PCBs) in actually contaminated soils. The transgenic tobacco plant XD4V-26 showed a significant dose-dependent induced GUS activity when cultured on MS medium containing PCB126 [toxic equivalency factor (TEF) = 0.1]. In contrast, PCB169 and PCB180, which have 0.03 of TEF and unassigned TEF values, respectively, did not significantly induce GUS activity under the same conditions as with PCB126. When the tobacco plants were cultivated for up to 5 weeks on actually contaminated soils with dioxins and dioxin-like compounds collected from the periphery of an incinerator used for disposal of residential and industrial wastes, GUS activity in the leaves was dose-dependently increased. The plants clearly detected 360 pg-TEQ g(-1) of dioxins and dioxin-like compounds in this assay. There was a positive correlation between GUS activity and TEQ value of dioxins and dioxin-like compounds in the plants. This assay does not require any extraction and purification processes for the actually contaminated soil samples.

  6. Biochemical and Structural Characterization of a Five-domain GH115 α-Glucuronidase from the Marine Bacterium Saccharophagus degradans 2-40 T

    Wang, Weijun [Univ. of Toronto, ON (Canada); Yan, Ruoyu [Univ. of Toronto, ON (Canada); Nocek, Boguslaw P. [Argonne National Lab. (ANL), Argonne, IL (United States); Vuong, Thu V. [Univ. of Toronto, ON (Canada); Di Leo, Rosa [Univ. of Toronto, ON (Canada); Xu, Xiaohui [Univ. of Toronto, ON (Canada); Cui, Hong [Univ. of Toronto, ON (Canada); Gatenholm, Paul [Chalmers Univ. of Technology, Gothenburg (Sweden); Toriz, Guillermo [Chalmers Univ. of Technology, Gothenburg (Sweden); Univ. of Guadalajara (Mexico); Tenkanen, Maija [Univ. of Helsinki (Finland); Savchenko, Alexei [Univ. of Toronto, ON (Canada); Master, Emma R. [Univ. of Toronto, ON (Canada)


    Glucuronic acid (GlcAp) and/or methylglucuronic acid (MeGlcAp) decorate the major forms of xylan in hardwood and coniferous softwoods as well as many cereal grains. Accordingly, the complete utilization of glucuronoxylans or conversion to sugar precursors requires the action of main chain xylanases as well as -glucuronidases that release the - (132)-linked (Me)GlcAp side groups. Herein, a family GH115 enzyme from the marine bacterium Saccharophagus degradans 2-40T, SdeAgu115A, demonstrated activity toward glucuronoxylan and oligomers thereof with preference toward MeGlcAp linked to internal xylopyranosyl residues. Unique biochemical characteristics of NaCl activation were also observed. The crystal structure of SdeAgu115A revealed a five-domain architecture, with an additional insertion C domain that had significant impact on the domain arrangement of SdeAgu115A monomer and its dimerization. The participation of domain C in substrate binding was supported by reduced substrate inhibition upon introducing W773A, W689A, and F696A substitutions within this domain. In addition to Asp-335, the catalytic essentiality of Glu-216 was revealed by site-specific mutagenesis. A primary sequence analysis suggested that the SdeAgu115A architecture is shared by more than half of GH115 members, thus defining a distinct archetype for GH115 enzymes.

  7. Assays of polychlorinated biphenyl congeners and co-contaminated heavy metals in the transgenic Arabidopsis plants carrying the recombinant guinea pig aryl hydrocarbon receptor-mediated β-glucuronidase reporter gene expression system.

    Shimazu, Sayuri; Ohta, Masaya; Ohkawa, Hideo; Ashida, Hitoshi


    The transgenic Arabidopsis plant XgD2V11-6 carrying the recombinant guinea pig (g) aryl hydrocarbon receptor (AhR)-mediated β-glucuronidase (GUS) reporter gene expression system was examined for assay of polychlorinated biphenyl (PCB) congeners and co-contaminated heavy metals. When the transgenic Arabidopsis plants were treated with PCB126 (toxic equivalency factor; TEF: 0.1) and PCB169 (TEF: 0.03), the GUS activity of the whole plants was increased significantly. After treatment with PCB80 (TEF: 0), the GUS activity was nearly the same level as that treated with 0.1% dimethylsulfoxide (DMSO) as a vehicle control. After exposure to a 1:1 mixture of PCB126 and PCB169, the GUS activity was increased additively. However, after exposure to a mixture of PCB126 and PCB80, the GUS activity was lower than that of the treatment with PCB126 alone. Thus, PCB80 seemed to be an antagonist towards AhR. When the transgenic plants were treated with each of the heavy metals Fe, Cu, Zn, Cd and Pb together with PCB126, Cd and Pb increased the PCB126-induced GUS activity. On the other hand, Fe, Cu and Zn did not affect the PCB126-induced GUS activity. In the presence of the biosurfactant mannosylerythritol lipid-B (MEL-B) and the carrier protein bovine serum albumin (BSA), the PCB126-induced GUS activity was increased, but the Cd-assisted PCB126-induced GUS activity was not affected. Thus, MEL-B and BSA seemed to increase uptake and transport of PCB126, respectively.

  8. Biology-oriented drug synthesis (BIODS): In vitro β-glucuronidase inhibitory and in silico studies on 2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl aryl carboxylate derivatives.

    Salar, Uzma; Khan, Khalid Mohammed; Taha, Muhammad; Ismail, Nor Hadiani; Ali, Basharat; Qurat-Ul-Ain; Perveen, Shahnaz; Ghufran, Mehreen; Wadood, Abdul


    Current study is based on the biology-oriented drug synthesis (BIODS) of 2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl aryl carboxylate derivatives 1-26, by treating metronidazole with different aryl and hetero-aryl carboxylic acids in the presence of 1,1'-carbonyl diimidazole (CDI) as a coupling agent. Structures of all synthetic derivatives were confirmed with the help of various spectroscopic techniques such as EI-MS, (1)H -NMR and (13)C NMR. CHN elemental analyses were also found in agreement with the calculated values. Synthetic derivatives were evaluated to check their β-glucuronidase inhibitory activity which revealed that except few derivatives, all demonstrated good inhibition in the range of IC50 = 1.20 ± 0.01-60.30 ± 1.40 μM as compared to the standard d-saccharic acid 1,4-lactone (IC50 = 48.38 ± 1.05 μM). Compounds 1, 3, 4, 6, 9-19, and 21-24 were found to be potent analogs and showed superior activity than standard. Limited structure-activity relationship is suggested that the molecules having electron withdrawing groups like NO2, F, Cl, and Br, were displayed better activity than the compounds with electron donating groups such as Me, OMe and BuO. To verify these interpretations, in silico study was also performed, a good correlation was observed between bioactivities and docking studies. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  9. Induction of β-glucuronidase in Penicillium purpurogenum Li-3%产紫青霉中β-葡萄糖醛酸苷酶的诱导表达

    郭晓晓; 王小艳; 冯世江; 李春


    针对产紫青霉(Penicillium purpurogenum)Li-3发酵生产β-葡萄糖醛酸苷酶存在的碳代谢阻遏现象,研究β-葡萄糖醛酸苷酶的高效诱导表达策略.在发酵条件优化的基础上,建立了新的产酶诱导工艺:葡萄糖的初始质量浓度5 g/L,在葡萄糖耗尽时加入20%诱导剂(10 g/L GL+ 1.2% Tween80)进行诱导,每24h添加1次诱导剂,诱导72 h后立即转到40℃摇床发酵48 h.采用该工艺进行发酵,菌体出现了“二次生长”现象,比酶活从647.99 U/mL提高至2 356 U/mL,提高了近3倍.%Carbon catabolite repression was found when Penicillium purpurogenum Li-3 grew in medium containing other carbon sources than glycyrrhizin. The induction strategy of β-glucuronidase was studied and a new technology for the enzyme producing induction was established, which based on the optimization of fermentation conditions. The inducer was added after obtaining a large mycelium biomass in nutrient medium with glucose as the sole carbon source. When glucose was completely consumed in the nutrient medium containing 5 g/L of glucose, 20% inducer (10% glycyrrhizin + 1. 2% Tween80) was added every 24 h. Then the culture temperature was moved to 40 ℃ before fermentation lasted for 48 h. The phenomenon of " second production" of P. purpurogenum Li-3 was found after the procedure. The enzyme activity was enhanced from 647. 99 U/mL to 2 356 U/mL, thus it raised about three fold.

  10. Beta-glucuronidase in physiology and disease.

    Basińska, Agnieszka; Floriańczyk, Bolesław


    beta-glucoronidase (EC is a lysosomal enzyme catylysing the decomposition of beta-D-glucoronides--compounds arising as a result of the combination of beta-D-glucoronic acid and a number of compounds both exo- and endogenous, containing hydroxylic, carboxylic, amine, imine or thiol groups. The most common test evaluating the activity of the enzyme is that using phenolphtalein glucoronide as a biosynthetic substrate. The freed aglycons are colorimetrically assayed. The activity of beta-glucoronidase increases in many pathological conditions: liver infammations, cirrhosis of the liver, inflammations of other organs, cholestatic jaundice, tuberculosis, sarcoidosis and also in neoplasms. Many authors point to beta-glucoronidase as a sensitive indicator signalling cell damage.

  11. Improvement of thermostability ofβ-glucuronidase through rational design%理性设计提高β-葡萄糖醛酸苷酶的热稳定性

    汤恒; 黄申; 冯旭东; 李春


    采用同源序列比对策略和脯氨酸效应的设计策略,以同源建模的三维结构为基础,结合定点突变技术,对重组产紫青霉β-葡萄糖醛酸苷酶(PGUS-E)进行理性设计,获得了2个热稳定性明显提高的突变体PGUS-E I130V和PGUS-E G280P,再将突变位点进行组合获得突变体PGUS-E I130V+G280P。相比PGUS-E,PGUS-E I130V、PGUS-E G280P和PGUS-E I130V+G280P在60℃下的半衰期T1/2分别比原始酶的23 min 提高3.5倍,5倍和5.5倍,达到82 min,117 min和128min。突变体的动力学参数Kcat/Km值分别为1.534×107 mol−1·L·min−1,1.368×107 mol−1·L·min−1和1.283×107 mol−1·L·min−1,与原始酶(1.316×107 mol−1·L·min−1)接近,对底物的亲和力基本不变。结果表明在蛋白质构象不稳定的区段中引入脯氨酸,以及在相应位置引入嗜热菌的氨基酸,均可提高蛋白质热稳定性。%The rational design for enhancing protein thermostability has become a hot issue in ennzyme engineering. A three-dimensional structure was modeled by the SWISS-MODEL, which was very helpful for the rational design to engineer the recombinantβ-glucuronidase from Penicillium purpurogenum Li-3 expressed in E. coli (PGUS-E). By using the design strategy of homologous sequence alignment and introducing proline mutation at appropriate sites, a simple site-directed mutagenesis protocol was developed to enhance thermostability of PGUS-E. Two mutant enzymes with higher thermostability were obtained:PGUS-E I130V and PGUS-E G280P. Then, these two sites were combined and mutant PGUS-E I130V+G280P was obtained. Further analysis of their thermostability at 60℃ and kinetics were performed. Compared to PGUS-E, thermostability of mutants was significantly improved, and the halftime (T1/2, 60℃) of mutants I130V, G280P and I130V+G280P increased by 3.5 times ,5 times and 5.5 times, respectively, while Kcat/Km of mutant enzyme remained nearly unchanged. This

  12. The effect of calculus bovis cultivated by glucuronidase on anti-convulsion and anti-inflammation in mice%酶促牛黄对小鼠的抗惊厥及抗炎作用

    许贵斌; 高允生


    Objective To observe the effects of calculus bovis cultivated by glucuronidase (CBCG )on anti-convulsion and anti-inflammation in mice, evaluate the pharmaceutical value of CBCG.Methods ( 1 )Anti-convulsion experiment.Appl ying nikethamide( NTM )-induced convulsion model and maximal electroshock convulsion ( MES ) model to observe the effect of CBCG on anti-convulsion in mice.( 2 )Anti-inflammation experiment.To apply carrageenan( CGN )-induced foot inflammation model in mice and dimethyl benzene( DMB )-induced inflammation model in mice to observe the effect of CBCG on antiinflammation.Results ( 1 )The outcome of anti-convulsion experiment showed that the CBCG and pentobarbital sodium ( PBS ) could prolong the NTM-induced convulsion latent phase in mice, raised the convulsion threshold value of maximal electroshock mice and reduce convulsion cases induced by maximal electroshock( P < 0.05, P < 0.01 ).The effect of PBS was better than CBCG( P <0.01 ),no difference was found among different CBCG groups( P >0.05 ).( 2 )The results of anti-inflammatory experiments showed that the CBCG and the aspirin( ASP ) could inhibit DMB-induced ear swell and CGN induced foot swell in mice( P < 0.05, P < 0.01 ).The CBCG had dose depend trend against DMB-induced ear swell in mice, the effect was highest in CBCG high-dose group( P < 0.05, P < 0.01 ), compared with NS gorup, ASP gorup.NCB group and CBCG group could inhibit foot swell in mice 3 ~5 h after medication( P < 0.05, P < 0.01 ), the effect was highest in NCB and CBCG high-dose group.Conclusion It proved that the CBCG had the effect of anti-convulsion and anti-inflammation in mice.%目的 观察酶促牛黄(CBCG)对小鼠的抗惊厥、抗炎作用,初步评价CBCG的药用价值.方法 (1)抗惊厥实验:分2个子实验,各取50只小鼠随机分为生理盐水组(NS组)、戊巴比妥钠组(PBS组)及CBCG高、中、低剂量组各10只,分别采用尼可刹米和最大电休克法制备小鼠惊厥

  13. Lentiviral-mediated gene therapy results in sustained expression of β-glucuronidase for up to 12 months in the gus(mps/mps) and up to 18 months in the gus(tm(L175F)Sly) mouse models of mucopolysaccharidosis type VII.

    Derrick-Roberts, Ainslie L K; Pyragius, Carmen E; Kaidonis, Xenia M; Jackson, Matilda R; Anson, Donald S; Byers, Sharon


    A number of mucopolysaccharidosis type VII (MPS VII) mouse models with different levels of residual enzyme activity have been created replicating the range of clinical phenotypes observed in human MPS VII patients. In this study, a lentivirus encoding murine β-glucuronidase was administered intravenously at birth to both the severe (Gus(mps/mps) strain) and attenuated (Gus(tm(L175F)Sly) strain) mouse models of MPS VII. Circulating enzyme levels were normalized in the Gus(mps/mps) mice and were 3.5-fold higher than normal in the Gus(tm(L175F)Sly) mouse 12 and 18 months after administration. Tissue β-glucuronidase activity increased over untreated levels in all tissues evaluated in both strains at 12 months, and the elevated level was maintained in Gus(tm(L175F)Sly) tissues at 18 months. These elevated enzyme levels reduced glycosaminoglycan storage in the liver, spleen, kidney, and heart in both models. Bone mineral volume decreased toward normal in both models after 12 months of therapy and after 18 months in the Gus(tm(L175F)Sly) mouse. Open-field exploration was improved in 18-month-old treated Gus(tm(L175F)Sly) mice, while spatial learning improved in both 12- and 18-month-old treated Gus(tm(L175F)Sly) mice. Overall, neonatal administration of lentiviral gene therapy resulted in sustained enzyme expression for up to 18 months in murine models of MPS VII. Significant improvements in biochemistry and enzymology as well as functional improvement of bone and behavior deficits in the Gus(tm(L175F)Sly) model were observed. Therapy significantly increased the lifespan of Gus(mps/mps) mice, with 12 months being the longest reported lentiviral treatment for this strain. It is important to assess the long-term outcome on enzyme levels and effect on pathology for lentiviral gene therapy to be a potential therapy for MPS patients.

  14. Transient expression of β-glucuronidase reporter gene in ...



    May 18, 2009 ... control treatment. Inoculation and ... of teak shoots to the negative pressure treatment was tested. In ... used as representatives to test the shoot sensitivity to the vacuum- ... distilled water, immersed in 200 mg/l carbenicillin solution for 5-10 ... microscope and recorded with a Nikon photograph camera.

  15. Transient gene expression of b-glucuronidase in citrus thin epicotyl transversal sections using particle bombardment

    Bespalhok Filho João C.


    Full Text Available Studies were carried out to optimize the conditions for transient gene expression through particle bombardment on Carrizo citrange (Citrus sinensis x Poncirus trifoliata thin epicotyl sections. The best conditions for transient GUS expression were: M-25 tungsten particles, 1550 psi helium pressure, 9 cm distance between specimen and DNA/particle holder and culture of explants in a high osmolarity medium (0.2 M mannitol + 0.2 M sorbitol 4 h prior and 20 h after bombardment. Under these conditions, an average of 102 blue spots per bombardment (20 explants/plate were achieved. This protocol is currently being used for transformation of Carrizo citrange and sweet orange (Citrus sinensis.

  16. High resolution crystal structure of human β-glucuronidase reveals structural basis of lysosome targeting

    Hassan, Md Imtaiyaz; Waheed, Abdul; Grubb, Jeffery H; Klei, Herbert E; Korolev, Sergey; Sly, William S


    ...). Here we report a high resolution crystal structure of human GUS at 1.7 Å resolution and present an extensive analysis of the structural features, unifying recent findings in the field of lysosome targeting and glycosyl hydrolases...

  17. High Resolution Crystal Structure of Human [beta]-Glucuronidase Reveals Structural Basis of Lysosome Targeting

    Hassan, Md; Waheed, Abdul; Grubb, Jeffery; Klei, Herbert; Korolev, Sergey; Sly, William


    ...). Here we report a high resolution crystal structure of human GUS at 1.7 Å resolution and present an extensive analysis of the structural features, unifying recent findings in the field of lysosome targeting and glycosyl hydrolases...

  18. Effect of β-Glucuronidase on Extraction Efficiency of Silymarin from ...


    Validated HPLC-UV Analysis. Muhammad ... Silybum marianum (milk thistle), has hepato- protective and .... transferred into a clean sample test tube and ... The validation run comprised a set of .... The rapid and simple analysis of compound.




    Evidence is accumulating that cigarette smoking plays an important role in the protease-antiprotease imbalance in alpha-1-antitrypsin-sufficient emphysema. Since most smokers, however, do not develop emphysema, it has to be presumed that other factors in addition to smoking contribute to the origin




    The selectivity of anticancer agents may be improved by antibody-directed enzyme prodrug therapy (ADEPT), The immunogenicity of antibody-enzyme conjugates and the low tumor to normal tissue ratio calls for the use of a human enzyme and the development of a monoclonal antibody (MAb) against that enzy

  1. High Resolution Crystal Structure of Human [beta]-Glucuronidase Reveals Structural Basis of Lysosome Targeting: e79687

    Md Imtaiyaz Hassan; Abdul Waheed; Jeffery H Grubb; Herbert E Klei; Sergey Korolev; William S Sly


    ...). Here we report a high resolution crystal structure of human GUS at 1.7 Å resolution and present an extensive analysis of the structural features, unifying recent findings in the field of lysosome targeting and glycosyl hydrolases...

  2. [Clinical application of beta-glucuronidase (beta-GCD) assay in the diagnosis of malignant tumors--analysis of 743 cases].

    Wang, W Z


    Chromatometric assay of beta-GCD in the urine, gastric juice and saliva was conducted in 743 subjects. In the urine of 104 normal persons, 5 (4.8%) were beta-GCD positive. In the urine of 35 urinary bladder cancer patients, 20 (57.1%) were positive. beta-GCD content was very low in the gastric juice and saliva of normal subjects, giving a positivity of 0%. But it was high in the gastric juice of stomach cancer patients, giving a positive rate of 83.3%. The positivities in the saliva of stomach cancer and malignant ovarian tumor patients were both 62.2% which was higher than that of patients suffering from other malignancies or benign tumors. beta-GCD assay is simple, stable and is valid in the diagnosis of stomach cancer and ovarian malignancies. The mechanism of beta-GCD elevation and its relation to metastasis are discussed.

  3. Diagnostic significance of serum β-glucuronidase in metastatic cancer%血清β-葡萄糖醛酸酶诊断转移癌31例

    于笑难; 杨波; 秦海明; 郑伟


    @@ 0 引言恶性肿瘤常见有多种酶的改变[1-3],近年发现[1,4]肿瘤细胞含有大量的β-葡萄糖醛酸酶,β- GCD的水平与存活的细胞数有相关性. 采用ELISA法测定健康人35例,良性肿瘤患者30例及恶性肿瘤患者67例血清β-GCD及CEA,观察β-GCD治疗前后的变化如下.

  4. Coexpression and Application of Thermostable Xylanase and Glucuronidase%耐热木聚糖酶和葡萄糖醛酸苷酶的共表达及应用

    沈艺红; 薛业敏; 侯静静; 许家兴; 李相前


    目的:提高海栖热袍菌(Thermotoga maritima MSB8)来源的木聚糖酶XynB和α-葡萄糖醛酸苷酶AguA生产效率并降低生产成本.方法:利用基因重组技术将海栖热袍菌的XynB和AguA基因置于不同表达盒下构建共表达载体pET-20b-xynB-aguA和pET-28a-xynB-aguA,分别转化大肠杆菌Escherichia coli JM109 (DE3)进行诱导表达,获得双酶混合物进而水解桦木木聚糖及玉米芯.结果:重组菌E.coli JM109 (DE3) /pET-28a-xynB-aguA比E.coli JM109 (DE3) /pET-20b-xynB-aguA产酶更具优势,在LB培养基中诱导培养8h,XynB和AguA的产量分别达到7.6 U/mL和0.5 U/mL,在TB培养基中培养,XynB可达到10.27 U/mL,AguA为1.5 U/mL.在80℃水解条件下,双酶比单一木聚糖酶能够更彻底降解桦木木聚糖,所得酶解液中木二糖的含量和纯度更高;从还原糖释放量及电子显微镜观察可以看出双酶液对农副产品玉米芯具有良好的降解作用.结论:XynB和AguA基因(T.maritima)的克隆共表达具有可行性,并且在生物转化、食品工业和饲料生产等领域具有潜在应用前景.

  5. 经密码子优化的葡萄糖醛酸酶在E.coli中的表达%The Effect of mRNA Secondary Structure on α-glucuronidase Expression

    杜兴兰; 邵蔚蓝



  6. The two Rasamsonia emersonii α-glucuronidases, ReGH67 and ReGH115, show a different mode-of-action towards glucuronoxylan and glucuronoxylo-oligosaccharides

    Murciano Martínez, Patricia; Appeldoorn, Maaike M.; Gruppen, Harry; Kabel, Mirjam A.


    Background: The production of biofuels and biochemicals from grass-type plant biomass requires a complete utilisation of the plant cellulose and hemicellulosic xylan via enzymatic degradation to their constituent monosaccharides. Generally, physical and/or thermochemical pretreatments are perform

  7. Effects of ulinastatin on plasma β-glucuronidase,granulocyte elastase and fibronectin during cardiopulmonary bypass%心内直视手术中应用乌司他丁对β-GCD、Gel及Fn的影响

    余剑波; 姚尚龙; 袁世荧



  8. β-葡萄糖醛酸酶测定对肺癌鉴别诊断的意义%Clinical significance of measurement of Beta-glucuronidase in patients with lung cancer

    于笑难; 杨波; 薛文成; 郑威



  9. 抗β-葡萄糖苷酸酶(β-GUS)兔单克隆抗体的制备和鉴定%Preparation and Identification of Anti-β-glucuronidase (β-GUS) Protein Rabbit Monoclonal Antibodies

    沈金儿; 倪庚; 郑晓冬


    通过免疫兔子、细胞融合、筛选杂交瘤细胞、构建重组载体、抗体纯化等步骤,成功制备出抗β-葡萄糖苷酸酶(β-GUS)兔单克隆抗体.运用间接ELISA法测定该兔单克隆抗体的相关特性,结果表明该兔单克隆抗体的效价为64000左右,亲和常数为2.13×109 L/mol.间接ELISA检测β-GUS蛋白时,其最低检测限为50 ng/mL.本次试验为研制定性或定量检测β-GUS蛋白的ELISA试剂盒奠定了基础.%In this research, anti-(3-GUS monoclonal antibodies (McAb) of rabbits were obtained after several procedures. These procedures included injecting the β-GUS protein into rabbits, cells fusion, hybridoma cell screening, plas-mid DNA recombination, monoclonal antibody purity and so on. After that, several relation characters were detected by indirect ELISA, the results showed that the affinity constant was 2.13×109L/mol, the titer of McAb was about 64 000 and the detection limit of β-GUS protein by indirect ELISA was about 50 ng/mL. This paper lays material foundation for ELISA kits which can detect β-GUS qualitatively or quantitatively.

  10. Inhibition of cholesterol crystallization under bilirubin deconjugation: partial characterization of mechanisms whereby infected bile accelerates pigment stone formation.

    Nakai, Kuniharu; Tazuma, Susumu; Nishioka, Tomoji; Chayama, Kazuaki


    Pigment gallstones have been reported to be closely associated with biliary tract infection. We previously reported that addition of unconjugated bilirubin (UCB), which is deconjugated by beta-glucuronidase in infected bile, could enhance cholesterol crystal formation in supersaturated model bile (MB). The present study evaluated the effect of beta-glucuronidase on the processes of pigment gallstone formation and cholesterol crystallization. Supersaturated MB (taurocholate/lecithin/cholesterol at 71:18:11, a total lipid concentration of 10.0 g/dl and a cholesterol saturation index (CSI) of 2.0) and native rat bile were mixed at a ratio of 3:1. Then, mixed bile was incubated with or without beta-glucuronidase and changes of the following parameters were investigated over time: (1) the UCB/total bilirubin ratio; (2) cholesterol crystal formation; (3) the precipitate weight and the cholesterol concentration in the precipitate and supernatant; and (4) the lipid distribution of vesicles in the supernatant. Compared with beta-glucuronidase-free bile, (1) beta-glucuronidase-containing bile showed a significant increase of the UCB/total bilirubin ratio, (2) as well as a significantly longer nucleation time (96+/-17.0 vs. 114+/-20.0) and fewer cholesterol crystals. (3) The precipitate weight and the cholesterol concentration in the precipitate were significantly increased, while the cholesterol concentration in supernatant was decreased. (4) When mixed bile was incubated with beta-glucuronidase, the cholesterol concentration in the vesicles was lower than in bile without beta-glucuronidase. The precipitate weight and the cholesterol concentration in the precipitate was increased by incubation with beta-glucuronidase, while cholesterol concentration was decreased in the supernatant (especially in the vesicles). This means that bile vesicles were more stable and it was more difficult for cholesterol crystals to form. Thus, the presence of beta-glucuronidase may inhibit the

  11. Disease: H00132 [KEGG MEDICUS

    Full Text Available ; beta-glucuronidase [HSA:2990] [KO:K01195] Urine glycosaminoglycans Heparan sulfate [CPD:C00925] Dermatan sulfate [CPD:C00426] Chond...roitin sulfate [CPD:C00607] Hematopoietic stem cell transplantation (limited experi

  12. A highly efficient method for Agrobacterium mediated transformation ...



    Aug 23, 2010 ... order to introduce target DNA into the rice cell, of which,. Agrobacterium .... Histochemical assay of -glucuronidase and measurement of GUS activity by ..... Genetic transformation and hybridization: Genomic changes in ...

  13. Expression pattern of the CsPK3 auxin-responsive protein kinase gene.

    Chono, M; Suzuki, Y; Nemoto, K; Yamane, H; Murofushi, N; Yamaguchi, I


    We have previously cloned a cDNA of a putative serine/threonine protein kinase gene named CsPK3 from cucumber, the mRNA level of which was up-regulated by auxin and down-regulated by light irradiation. To examine the CsPK3 gene expression in detail, we cloned a genomic DNA of CsPK3 gene and made transgenic tobacco (Nicotiana tabacum L. cv. Petit Havana SR1) plants containing the fused CsPK3 promoter-beta-glucuronidase gene. The beta-glucuronidase expression was detected in the shoot apex, vascular tissues, and the outermost layer of cortex. The histological distribution of CsPK3 mRNA in cucumber seedlings was supported by in situ hybridization, where the positive signals were observed in similar tissues as those observed by beta-glucuronidase staining. The responsiveness of the CsPK3 gene to auxin and light was also confirmed for beta-glucuronidase activity. The pattern of beta-glucuronidase staining changed during the development of the tobacco seedlings. The results of our experiment showed that CsPK3 was expressed in a wide variety of tissues and cells in which the developmental and growth controls by auxin are suggested.

  14. Evaluation of butyrate-induced production of a mannose-6-phosphorylated therapeutic enzyme using parallel bioreactors.

    Madhavarao, Chikkathur N; Agarabi, Cyrus D; Wong, Lily; Müller-Loennies, Sven; Braulke, Thomas; Khan, Mansoor; Anderson, Howard; Johnson, Gibbes R


    Bioreactor process changes can have a profound effect on the yield and quality of biotechnology products. Mannose-6-phosphate (M6P) glycan content and the enzymatic catalytic kinetic parameters are critical quality attributes (CQAs) of many therapeutic enzymes used to treat lysosomal storage diseases (LSDs). Here, we have evaluated the effect of adding butyrate to bioreactor production cultures of human recombinant β-glucuronidase produced from CHO-K1 cells, with an emphasis on CQAs. The β-glucuronidase produced in parallel bioreactors was quantified by capillary electrophoresis, the catalytic kinetic parameters were measured using steady-state analysis, and mannose-6-phosphorylation status was assessed using an M6P-specific single-chain antibody fragment. Using this approach, we found that butyrate treatment increased β-glucuronidase production up to approximately threefold without significantly affecting the catalytic properties of the enzyme. However, M6P content in β-glucuronidase was inversely correlated with the increased enzyme production induced by butyrate treatment. This assessment demonstrated that although butyrate dramatically increased β-glucuronidase production in bioreactors, it adversely impacted the mannose-6-phosphorylation of this LSD therapeutic enzyme. This strategy may have utility in evaluating manufacturing process changes to improve therapeutic enzyme yields and CQAs.

  15. Preventive use of Lactobacillus plantarum LS/07 and inulin to relieve symptoms of acute colitis.

    Hijová, Emília; Šoltésová, Alena; Salaj, Rastislav; Kuzma, Jozef; Strojný, Ladislav; Bomba, Alojz; Gregová, Kristína


    The aim of presented study was to investigate the influence of Lactobacillus plantarum LS/07 and inulin on the activity of β-glucuronidase enzyme, and counts of coliform and lactobacilli in fresh caecal digesta, cytokine levels (IL-6, IL-8), and trancription nuclear factor kappa beta (NFκB) activities in colon tissue and blood samples of rats with dextran sulphate sodium (DSS) induced acute colitis. The rats were randomly divided into four groups - CG, AC, AC+PRE and AC+PRO. Colitis was induced using of 5% DSS in drinking water for 7d. DSS application increased activity of β-glucuronidase (P LS/07 decreased β-glucuronidase activity (P LS/07 and inulin suppressed expression observed markers, which play an important role in the inflammatory process, which predisposes their use in prevention or treatment of acute colitis.

  16. Optimizing the fluorometric B-glucoronidase assay in ruminant milk for at more precise determination of

    Larsen, Torben; Aulrich, Karen


    variables (r=0·25–0·43; n=825). Simple indices based on β-glucuronidase and LDH or NAGase activity were tested as indicators of mastitis (SCC), but were not found to improve the diagnostic value. Future studies may further verify whether β-glucuronidase can compete with well-established indicators......Activity of the enzyme β-glucuronidase (EC is found in milk from ruminants with mastitis. However, the use of this enzymic activity as an indicator of mastitis has gained little attention possibly because of its low activity when compared with other mastitis indicators. The determination...... it into large scale analyses. The assay performance is satisfactory regarding precision, linearity etc., and it appears comparable to analogous fluorometric assays for mastitis indicators in milk. From a local dairy herd, 825 milk samples were analysed for potential mastitis indicators, i.e. β...

  17. Alleviating Cancer Drug Toxicity by Inhibiting a Bacterial Enzyme

    Wallace, Bret D.; Wang, Hongwei; Lane, Kimberly T.; Scott, John E.; Orans, Jillian; Koo, Ja Seol; Venkatesh, Madhukumar; Jobin, Christian; Yeh, Li-An; Mani, Sridhar; Redinbo, Matthew R. (Einstein); (UNC); (North Carolina Central University)


    The dose-limiting side effect of the common colon cancer chemotherapeutic CPT-11 is severe diarrhea caused by symbiotic bacterial {beta}-glucuronidases that reactivate the drug in the gut. We sought to target these enzymes without killing the commensal bacteria essential for human health. Potent bacterial {beta}-glucuronidase inhibitors were identified by high-throughput screening and shown to have no effect on the orthologous mammalian enzyme. Crystal structures established that selectivity was based on a loop unique to bacterial {beta}-glucuronidases. Inhibitors were highly effective against the enzyme target in living aerobic and anaerobic bacteria, but did not kill the bacteria or harm mammalian cells. Finally, oral administration of an inhibitor protected mice from CPT-11-induced toxicity. Thus, drugs may be designed to inhibit undesirable enzyme activities in essential microbial symbiotes to enhance chemotherapeutic efficacy.

  18. The glyceraldehyde-3-phosphate dehydrogenase promoter of the food yeast Candida utilis strain NRRL Y-660 is functional in Agrobacterium tumefaciens.

    González, Tania; Eng, Felipe; Fraga, Reinaldo; Fonseca, Jennifer; Amores, Isis


    The glyceraldehyde-3-phosphate dehydrogenase promoter of the food yeast Candida utilis strain NRRL Y-660 was cloned to create a novel integrative vector for Agrobacterium tumefaciens-mediated transformation. The new binary vector harbors β-glucuronidase activity as reporter and kanamicin/geneticin resistance as selection marker. Recombinant clones of A. tumefaciens show kanamycin resistance and high β-glucuronidase activity under the control of the C. utilis promoter. This finding can be explained by the presence of a prokaryotic core in the yeast promoter, predicted by in silico analysis of the sequence. This is the first report about functionality of a yeast promoter in A. tumefaciens.

  19. Reference: 516 [Arabidopsis Phenome Database[Archive

    Full Text Available in219 mutant under all light conditions, except cFR. Furthermore, promoter activi...velopmentally regulated. Subcellular localization studies revealed that the beta-glucuronidase-FIP1 fusion p...icate that FIP1 may interact with FIN219 to regulate cell elongation and flowering in response to light. Glu

  20. Selection of Arabidopsis mutants overexpressing genes driven by the promoter of an auxin-inducible glutathione S-transferase gene

    Kop, D.A.M. van der; Schuyer, M.; Pinas, J.E.; Zaal, B.J. van der; Hooykaas, P.J.J.


    Transgenic arabidopsis plants were isolated that contained a T-DNA construct in which the promoter of an auxin-inducible glutathione S-transferase (GST) gene from tobacco was fused to the kanamycin resistance (nptII) as well as to the β-glucuronidase (gusA) reporter gene. Subsequently, seeds were tr

  1. Diversity and enzymatic characterization of Bacillus species isolated ...

    traditional cassava starters used for attiéké production ... Fermentation plays an important role in the production of cassava-based foods in West Africa. ..... β-GAL: Béta-galactosidase (lactase) β-GLUC : Béta-glucuronidase .... Solid-state (solid-.

  2. The barley Jip23b gene

    Müller-Uri, Frieder; Cameron-Mills, Verena; Mundy, John


    The barley gene (Jip23) encoding a 23,000-Da protein of unknown function was isolated and shown to be induced by jasmonate methyl ester (MeJA) in leaves. 5'upstream Jip23 sequence was isolated and fused to the beta-glucuronidase gene (GUS), and this reporter was introduced by particle bombardment...

  3. Agrobacterium-mediated transformation of apple (Malus x domestica Borkh.): an assessment of factors affecting gene transfer efficiency during early transformation steps.

    De Bondt, A; Eggermont, K; Druart, P; De Vil, M; Goderis, I; Vanderleyden, J; Broekaert, W F


    The factors influencing transfer of an intron - containing β-glucuronidase gene to apple leaf explants were studied during early steps of an Agrobacterium tumefaciens-mediated transformation procedure. The gene transfer process was evaluated by counting the number of β-glucuronidase expressing leaf zones immediately after cocultivation, as well as by counting the number of β-glucuronidase expressing calli developing on the explants after 6 weeks of postcultivation in the presence of 50 mg/l kanamycin. Of three different tested disarmed A. tumefaciens strains, EHA101(pEHA101) was the most effective for apple transformation. Cocultivation of leaf explants with A. tumefaciens on a medium with a high cytokinin level was more conducive to gene transfer than cocultivation on media with high auxin concentrations. Precultivation of leaf explants, prior to cocultivation, slightly increased the number of β-glucuronidase expressing zones measured immediately after cocultivation, but it drastically decreased the number of transformed calli appearing on the explants 6 weeks after infection. Other factors examined were: Agrobacterium cell density during infection, bacterial growth phase, nature of the carbon source, explant age, and explant genotype.

  4. Functional Analysis of Promoters in the Nisin Gene Cluster of Lactococcus lactis

    Ruyter, Pascalle G.G.A. de; Kuipers, Oscar P.; Beerthuyzen, Marke M.; Alen-Boerrigter, Ingrid van; Vos, Willem M. de


    The promoters in the nisin gene cluster nisABTCIPRKFEG of Lactococcus lactis were characterized by primer extension and transcriptional fusions to the Escherichia coli promoterless β-glucuronidase gene (gusA). Three promoters preceding the nisA, nisR, and nisF genes, which all give rise to gusA expr

  5. Enzymatic activity toward poly(L-lactic acid) implants

    Schakenraad, J.M.; Hardonk, M.J.; Feijen, J.; Molenaar, I.; Nieuwenhuis, P.


    Tissue reactions toward biodegradable poly(L-lactic acid) implants were monitored by studying the activity pattern of seven enzymes as a function of time: alkaline phosphatase, acid phosphatase, -naphthyl acetyl esterase, -glucuronidase, ATP-ase, NADH-reductase, and lactate dehydrogenase. Cell types

  6. Immunoliposomes as enzyme-carriers (immuno-enzymosomes) for antibody-directed enzyme prodrug therapy (ADEPT) : Optimization of prodrug activating capacity

    Vingerhoeds, MH; Haisma, HJ; Belliot, SO; Smit, RHP; Crommelin, DJA; Storm, G

    Purpose. Immuno-enzymosomes are tumor-specific immunoliposomes bearing enzymes on their surface. These enzymes are capable of converting relatively nontoxic prodrugs into active cytostatic agents. The enzyme beta-glucuronidase (GUS)(4) was coupled to the external surface of immunoliposomes directed

  7. Conserved regulation of the soybean early nodulin ENOD2 gene promoter in determinate and indeterminate transgenic root nodules.

    Lauridsen, P.; Franssen, H.; Stougaard, J.; Bisseling, T.; Marcker, K.A.


    The beta-glucuronidase (GUS) activity expressed from the soybean early nodulin ENOD2(B) gene promoter was localized histochemically in nodules of Lotus corniculatus and Trifolium repens. In both the determinate Lotus nodules and the indeterminate Trifolium nodules, activity was found in the parenchy

  8. An expression system based on the promoter region of the Aspergillus awamori 1,4-bèta-endoxylanase A gene

    Gouka, R.J.; Hessing, J.G.M.; Punt, P.J.; Stam, H.; Musters, W.; Hondel, C.A.M.J.J. van den


    A new, highly inducible fungal promoter derived from the Aspergillus awamori 1,4-β-endoxylanase A (exlA) gene is described. Induction analysis, carried out with the wild-type strain in shake flasks, showed that exlA expression is regulated at the transcriptional level. Using a β-glucuronidase (uidA)

  9. Integration of C4-specific ppdk gene of Echinochloa to C3 upland ...



    Mar 18, 2008 ... expression of Echinochloa C4 type PPDK in upland rice with the enzyme activity being elevated 1 - 11 folds. However, no ... glucuronidase gene; Efpdk, coding region of pdk gene from Echinochloa fruma. MATERIALS AND ... A transgenesis vector was constructed on the basis of .... Molecular cloning.




    Chimaeric genes of promoter sequences from the potato gene encoding granule-bound starch synthase (GBSS) and the beta-glucuronidase (GUS) reporter gene were used to study GBSS expression and regulation. Analysis of stable transformants revealed that a GBSS promoter sequence of 0.4 kb was sufficient




    Activities of the beta-glucuronidase (GUS) reporter enzyme were evaluated in transgenic plants, protoplasts, and intertypic heterokaryons of Solanum tuberosum and Nicotiana tabacum. With GUS under control of the promoter of the cauliflower-mosaicvirus 35S RNA gene (CaMV), activities of the enzyme we

  12. A single hemoglobin gene in Myrica gale retains both symbiotic and non-symbiotic specificity

    Heckmann, Anne B.; Hebelstrup, Kim Henrik; Larsen, Knud


    and protein were also detected in leaves of M. gale. In Arabidopsis thaliana the MgHb promoter, linked to a beta-glucuronidase coding region, directed expression in the vascular tissue, in shoot meristem and at root branch point--a pattern very similar to the combined expression pattern of the two non...

  13. Design of immuno-enzymosomes with maximum enzyme targeting capability : effect of the enzyme density on the enzyme targeting capability and cell binding properties

    Fonseca, MJ; Haisma, HJ; Klaasen, S; Vingerhoeds, MH; Storm, G


    Immuno-enzymosomes have been proposed for the targeting of enzymes to cancer cells to achieve site specific activation of anticancer prodrugs. Previously, we reported that the enzyme beta-glucuronidase (GUS), capable of activating anthracycline-glucuronide prodrugs, can be coupled to the surface of

  14. Characterization, subcellular localization and nuclear targeting of casein kinase 2 from Zea mays

    Peracchia, G; Jensen, A B; Culiáñez-Macià, F A


    by using in-frame fusions of the maize CK2alpha subunit to the reporter gene encoding beta-glucuronidase (GUS) which were assayed in transiently transformed onion epidermal cells. Analysis of chimeric constructs identified one region containing a nuclear localization signal (NLS) that is highly conserved...

  15. Evaluation and comparison of the GUS, LUC and GFP reporter system for gene expression studies in plants

    Ruijter, de N.C.A.; Verhees, J.A.; Leeuwen, van W.; Krol, van der A.R.


    The detailed analysis of the expression pattern of a plant gene can give important clues about its function in plant development, cell differentiation and defence reactions. Gene expression studies have been greatly facilitated by the employment of proteins like beta-glucuronidase (GUS), green fluor

  16. Hormonal Interference with Pheromone Systems in Parasitic Acarines, Especially Ixodid Ticks.


    hydroxyecdysone by treatment of frations in Locusta migratoria extracts with the enzymie glucuronidase. A second fraction, which did not respond to this enzyne...products of ecdysone in Locusta migratoria . Hoppe-Ze.l er’Is ;.. Z. Physiol. Chem. Bd. 354: 1043-1048.. Obenchain, F.D. and J.H. Oliver. 1975

  17. Characterization, subcellular localization and nuclear targeting of casein kinase 2 from Zea mays

    Peracchia, G; Jensen, A B; Culiáñez-Macià, F A


    by using in-frame fusions of the maize CK2alpha subunit to the reporter gene encoding beta-glucuronidase (GUS) which were assayed in transiently transformed onion epidermal cells. Analysis of chimeric constructs identified one region containing a nuclear localization signal (NLS) that is highly conserved...

  18. Screening and bioconversion of glycyrrhizin of Glycyrrhiza glabra root extract to 18β-glycyrrhetinic acid by different microbial strains

    Makhmur Ahmad


    Full Text Available Objective: The objective of the present study is to perform screening of different microorganisms (7 bacteria and 14 fungi for conversion of glycyrrhizin (GL to 18β-glycyrrhetinic acid (GA. Penicillium chrysogenum produced the highest concentration of β-glucuronidase enzyme (61 U/mL and produced GA of 52 μg/mL while E. coli produced the highest β-glucuronidase of 376 U/mL with GA concentration of 2.1 μg/mL. Materials and Methods: Submerged and solid state biotransformation of GL was carried out. To 9.0 mL of bacterial supernatant, 1.0 mL 0.2% w/v of aqueous Glycyrrhiza glabra root extract was added and incubated at 37°C for 24 h. β-glucuronidase activity was measured and high-performance liquid chromatography analysis was carried out. Results and Discussion: Induced-Escherichia coli produces 2.1 μg/mL of GA with an enzyme activity of 376 U/mL which shows that the enzyme has a potential biotransformation capability. Rhizopus oryzae and P. chrysogenum have the potential ability to biotransform GL to GA with 2.6 μg/mL and 61 μg/mL of GA with enzyme activity of 569 U/mL and 61 U/mL, respectively. Conclusions: G. glabra roots containing GL can be hydrolyzed by microbial β-glucuronidase enzyme under sub-merged fermentation (SmF. β-glucuronidase, an enzyme of E. coli, was found to be the best microbial source of enzyme which biocatalyzed the reaction than fungal strain under SmF.

  19. Membrane receptor-initiated signaling in 1,25(OH)2D3-stimulated calcium uptake in intestinal epithelial cells.

    Khanal, Ramesh C; Peters, Tremaine M Sterling; Smith, Nathan M; Nemere, Ilka


    Demonstrating 1,25(OH)2D3-stimulated calcium uptake in isolated chick intestinal epithelial cells has been complicated by simultaneous enhancement of both uptake and efflux. We now report that in intestinal cells of adult birds, or those of young birds cultured for 72 h, 1,25(OH)2D3-stimulates 45Ca uptake to greater than 140% of corresponding controls within 3 min of addition. Such cells have lost hormone-stimulated protein kinase C (PKC) activity, believed to mediate calcium efflux. To further test this hypothesis, freshly isolated cells were preincubated with calphostin C, and calcium uptake monitored in the presence or absence of steroid. Only cells treated with the PKC inhibitor demonstrated a significant increase in 45Ca uptake in response to 1,25(OH)2D3, relative to corresponding controls. In addition, phorbol ester was shown to stimulate efflux, while forskolin stimulated uptake. To further investigate the mechanisms involved in calcium uptake, we assessed the role of TRPV6 and its activation by beta-glucuronidase. beta-Glucuronidase secretion from isolated intestinal epithelial cells was significantly increased by treatment with 1,25(OH)2D3, PTH, or forskolin, but not by phorbol ester. Treatment of cells with beta-glucuronidase, in turn, stimulated 45Ca uptake. Finally, transfection of cells with siRNA to either beta-glucuronidase or TRPV6 abolished 1,25(OH)2D3-enhanced calcium uptake relative to controls transfected with scrambled siRNA. Confocal microscopy further indicated rapid redistribution of enzyme and calcium channel after steroid. 1,25(OH)2D3 and PTH increase calcium uptake by stimulating the PKA pathway to release beta-glucuronidase, which in turn activates TRPV6. 1,25(OH)2D3-enhanced calcium efflux is mediated by the PKC pathway.

  20. Effect of dairy products on initiation of precursor lesions of colon cancer in rats.

    Abdelali, H; Cassand, P; Soussotte, V; Daubeze, M; Bouley, C; Narbonne, J F


    This study reports the modulating effect of some dairy products on initiation of putative preneoplasic lesions in rat colon (aberrant crypts) by 1,2-dimethylhydrazine dihydrochloride. Uninoculated skim milk, skim milk fermented with Bifidobacterium sp Bio (Danone strain 173010), and a suspension of the same lactic acid bacteria were incorporated in the animals' diet. The tested diets significantly reduced the incidence of aberrant crypts compared with the control diet by 51%, 49%, and 61%, respectively. The effects of the diets on cecal pH, hepatic UDP-glucuronyltransferase activity, and cecal microflora enzyme beta-glucuronidase were also studied. There was no significant difference in cecal pH between rats fed experimental diets and control rat. The diet supplemented with the Bifidobacterium strain suspension significantly decreased only the cecal beta-glucuronidase activity. Both enzyme activities were reduced in rats fed fermented skim milk- or uninoculated skim milk-supplemented diets compared with control animals.

  1. Effects of apples and specific apple components on the cecal environment of conventional rats: Role of apple pectin

    Licht, Tine Rask; Hansen, Max; Bergström, Anders


    Background: Our study was part of the large European project ISAFRUIT aiming to reveal the biological explanations for the epidemiologically well-established health effects of fruits. The objective was to identify effects of apple and apple product consumption on the composition of the cecal...... study (14 weeks), while no effects of apple juice, puree or pomace on microbial composition in cecum were observed. Administration of either 0.33 or 3.3% apple pectin in the diet resulted in considerable changes in the DGGE profiles. A 2-fold increase in the activity of beta-glucuronidase was observed......-glucuronidase producing Clostridiales, and decreases the population of specific species within the Bacteroidetes group in the rat gut. Similar changes were not caused by consumption of whole apples, apple juice, puree or pomace....

  2. Lotus hairy roots expressing inducible arginine decarboxylase activity.

    Chiesa, María A; Ruiz, Oscar A; Sánchez, Diego H


    Biotechnological uses of plant cell-tissue culture usually rely on constitutive transgene expression. However, such expression of transgenes may not always be desirable. In those cases, the use of an inducible promoter could be an alternative approach. To test this hypothesis, we developed two binary vectors harboring a stress-inducible promoter from Arabidopsis thaliana, driving the beta-glucuronidase reporter gene and the oat arginine decarboxylase. Transgenic hairy roots of Lotus corniculatus were obtained with osmotic- and cold-inducible beta-glucuronidase and arginine decarboxylase activities. The increase in the activity of the latter was accompanied by a significant rise in total free polyamines level. Through an organogenesis process, we obtained L. corniculatus transgenic plants avoiding deleterious phenotypes frequently associated with the constitutive over-expression of arginine decarboxylation and putrescine accumulation.

  3. Exoglycosidase markers of diseases.

    Chojnowska, Sylwia; Kępka, Alina; Szajda, Sławomir Dariusz; Waszkiewicz, Napoleon; Bierć, Marcin; Zwierz, Krzysztof


    Exoglycosidases are hydrolases involved in lysosomal degradation of oligosaccharide chains of glycoconjugates (glycoproteins, glycolipids and proteoglycans). In tissues and body fluids, a higher exoglycosidase specific activity is found in N-acetyl-β-hexosaminidase, than β-glucuronidase, α-L-fucosidase, β-galactosidase, α-mannosidase and α-glucosidase. Determination of exoglycosidases (especially N-acetyl-β-hexosaminidase and β-glucuronidase) in body fluids could be an inexpensive, easy to perform and sensitive test for pathological evaluation, as well as in screening and monitoring many diseases, including alcohol abuse, risk of arteriosclerosis, bacterial infections (e.g. Lyme borreliosis), chronic inflammatory processes, such as rheumatoid arthritis and juvenile idiopathic arthritis, asthma, autoimmune hepatitis and primary biliary cirrhosis, as well as cancers.

  4. Agrobacterium-mediated transformation of Ruta graveolens L.

    Lièvre, Karine; Tran, Thi Lê Minh; Doerper, Sébastien; Hehn, Alain; Lacoste, Paul; Thomasset, Brigitte; Bourgaud, Frédéric; Gontier, Eric


    Agrobacterium tumefaciens is used to develop a genetic transformation method for a medicinal plant Ruta graveolens. The direct plant regeneration strategy is preferred to callus line establishment. In vitro seedlings, 2- -to 3-wk-old, are used to excise hypocotyls and co-cultivated for 3 d with A. tumefaciens strain C58C1Rif containing plasmid pTDE4 harbouring neomycin phosphotransferase (npt II, kanamycin resistance) and beta-glucuronidase encoding genes. The Southern blot analysis has shown that 78% kanamycin resistant plants contain gene encoding beta-glucuronidase. The GUS histochemical assay shows that 67% transgenic plants exhibit the corresponding enzymatic activity. Routine transformation efficiency of R. graveolens L. is 11% and could reach up to 22%. Transgenic plants are grown in the greenhouse within 4 months after the initial seedlings.

  5. Preventive effect of phytic acid on lysosomal hydrolases in normal and isoproterenol-induced myocardial infarction in Wistar rats.

    Brindha, E; Rajasekapandiyan, M


    This study was aimed to evaluate the preventive role of phytic acid on lysosomal enzymes in isoproterenol (ISO)-induced myocardial infarction (MI) in male Wistar rats. Rats subcutaneously injected with ISO (85 mg/kg) at an interval of 24 h for two days showed a significant increase in the activities of lysosomal enzymes (glucuronidase, N-acetyl glucosaminidase, galactosidase, cathepsin-B and cathepsin-D) were increased significantly in serum and the heart of ISO-induced rats, but the activities of glucuronidase and cathepsin-D were decreased significantly in the lysosomal fraction of the heart. Pretreatment with phytic acid (25 and 50 mg/kg) daily for a period of 56 d positively altered activities of lysosomal hydrolases in ISO-induced rats. Thus, phytic acid possesses a cardioprotective effect in ISO-induced MI in rats.

  6. Optimised deconjugation of androgenic steroid conjugates in bovine urine.

    Pedersen, Mikael; Frandsen, Henrik L; Andersen, Jens H


    After administration of steroids to animals the steroids are partially metabolised in the liver and kidney to phase 2 metabolites, i.e., glucuronic acid or sulphate conjugates. During analysis these conjugated metabolites are normally deconjugated enzymatically with aryl sulphatase and glucuronidase resulting in free steroids in the extract. It is well known that some sulphates are not deconjugated using aryl sulphatase; instead, for example, solvolysis can be used for deconjugation of these aliphatic sulphates. The effectiveness of solvolysis on androgenic steroid sulphates was tested with selected aliphatic steroid sulphates (boldenone sulphate, nortestosteron sulphate and testosterone sulphate), and the method was validated for analysis of androgenic steroids in bovine urine using free steroids, steroid sulphates and steroid glucuronides as standards. Glucuronidase and sulphuric acid in ethyl acetate were used for deconjugation and the extract was purified by solid-phase extraction. The final extract was evaporated to dryness, re-dissolved and analysed by LC-MS/MS.

  7. Influence of udder infection status on milk enzyme activities and somatic cell count throughout early lactation in goats

    Stuhr, T; Aulrich, K; Barth, K;


    . A total of 60 dairy goats were sampled at weekly intervals over a period of 6 weeks after kidding and the bacteriological status, milk SCC and the activity of N-acetyl-β-d-glucosaminidase (NAGase), β-glucuronidase and lactate dehydrogenase (LDH) of udder halves were analysed. Infections with minor...... after parturition, the study revealed a highly significant influence of lactation week on log10 SCC (F4,255 = 11.63, p LDH (F4,285 = 5.02, p ...-glucuronidase activity were not influenced by the stage of lactation. The infection status of udder halves had a highly significant effect on log10 SCC (F3,528 = 18.88, p LDH (F3,534 = 12.39, p

  8. Histochemical analysis of experimental granulomatous uveitis.

    Kristeva, M; Biswas, J; Pararajasegaram, G; Sevanian, A; Rao, N A


    We have previously demonstrated the effects of various inhibitors of arachidonic acid metabolism on experimental lens-induced granulomatous uveitis. In the present study, we investigated the effect of these same inhibitors on the expression of lysosomal enzymes at different stages of choroidal inflammation in experimental lens-induced granulomatous uveitis and compared this to the inflammation observed at each stage examined. Lysosomal enzymes such as acid phosphatase, beta-glucuronidase and succinate dehydrogenase are known to be liberated during the maturation of mononuclear phagocytes to epithelioid cell granulomas. Although animals treated with nordihydroguaiaretic acid showed less severe inflammation than did indomethacin-treated or control animals, none of these agents appeared to affect the expression of acid phosphatase and beta-glucuronidase, as determined histochemically. Succinate dehydrogenase could not be detected in any of the eyes examined, even though sections of liver and kidney from these same animals were positive for this enzyme.

  9. Liposomal dry powders as aerosols for pulmonary delivery of proteins

    Lu, Dongmei; Hickey, Anthony J.


    The purpose of this research was to develop liposomal dry powder aerosols for protein delivery. The delivery of stable protein formulations is essential for protein subunit vaccine delivery, which requires local delivery to macrophages in the lungs. β-Glucuronidase (GUS) was used as a model protein to evaluate dry powder liposomes as inhaled delivery vehicles. Dimyristoyl phosphatylcholine:cholesterol (7∶3) was selected as the liposome composition. The lyophilization of liposomes, micronizati...

  10. RNase protection assays and RNA gel blots: a direct comparison of sensitivity.

    Higgs, D C; Colbert, J T


    RNase protection assays are commonly thought to be a more sensitive means of detecting and quantitating specific mRNAs than are RNA gel blots (Northern blots). We have directly compared the sensitivity of these two approaches by assaying for known amounts of in vitro synthesized beta-glucuronidase mRNA. With the probes and protocols employed here, the ability to detect a specific mRNA was similar whether RNase protection or RNA gel blot analyses were performed.

  11. Nonhemocyte sources of selected lysosomal enzymes in Biomphalaria glabrata, B. tenagophila and B. straminea (Mollusca: Pulmonata

    Gary E. Rodrick


    Full Text Available The specific activities of acid phosphatase, alkaline phosphatase, β-glucuronidase, lysozymes, glutamate-oxalacetate transaminase and glutamate-pyruvate transaminate were determined in the head-foot and digestive gland of Brazilian Biomphalaria glabrata (Touros, B. tenagophila (Caçapava and B. straminea (Monsenhor Gil. All six enzymes were detected inthe 3000g supernatant. Both cytoplasmic enzymes, glutamate-oxalacetate and glutamate-pyruvate transaminase exhibited the highest specific activities. In the case of the four hydrolytic enzymes assayed, β-glucuronidase exhibited the highest specific activity while lysozyme showed the lowest activity. All six enzymes are thought to be produced by cells within the head-foot and digestive gland of B. glabrata, B. tenagophila and B. straminea.Foram determinadas, na massa cefalopedal e na glândula digestiva de Biomphalaria glabrata de Touros (Rio Grande do Norte B. tenagophila de Cacapava (Sao Paulo e B. straminea de Monsenhor Gil (Piauí, as atividades específicas das seguintes enzimas: fosfatase acida, fosfatase alcalina, beta-glucuronidase, lisozima, transaminase glutâmico-oxalacetica e transaminase glutâmico-piruvica. As seis enzimas referidas foram detectadas no sobrenadante a 3000g. Ambas as enzimas citoplasmaticas - transaminases glutamico-oxalacetica e glutamico-piruvica - mostraram as atividades específicas mais altas. No caso das quatro enzimas hidrolíticas, a beta-glucuronidase revelou a mais alta atividade específica, enquanto a lisozima revelou a mais baixa. E admitido que todas as seis enzimas sao produzidas por celulas presentes na massa cefalopedal e na glândula digestiva das tres especies de moluscos examinadas.

  12. Fluorescence Assay for Evaluating Microbicidal Activity of Hand Antiseptics

    Lopez-Gigosos, Rosa M.; Mariscal, Alberto; Mariscal-Lopez, Eloisa; Gutierrez-Bedmar, Mario; Fernandez, Joaquin


    We developed a fluorescent β-d-glucuronidase activity (BGA)-based assay for detecting and quantifying Escherichia coli in samples to assess the biocide efficacy of hand antiseptics. The fluorescence level is proportional to the number of viable E. coli organisms present. We compared our assay results to those of the E. coli plate count method specified by the European standard for testing hygienic hand rub disinfectant products (EN1500). The plate count method requires excessive handling and ...

  13. Determination of Intestinal Enzyme Activities During Infancy Period

    Emel Örün


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

  14. Biochemical changes in polymorphonuclear leucocytes in diabetic patients.


    A study on the functional ability of polymorphonuclear leucocytes (PMNL) indicates that the total lysosomal enzyme levels viz. Beta-glucuronidase, lysozyme, acid phosphatase and alkaline phosphatase were not altered in diabetics, compared to that in control subjects. However, the findings also reveal that the release of these lysosomal enzymes in response to a particulate stimulus is impaired in diabetics. This suggests that the bactericidal capacity of these cells, which are involved in phag...

  15. Salivary and serum inflammatory mediators among pre-conception women with periodontal disease.

    Jiang, Hong; Zhang, Yiming; Xiong, Xu; Harville, Emily W; O, Karmin; Qian, Xu


    There have been inconsistent conclusions regarding the levels of inflammatory mediators in saliva and serum among people with or without periodontal disease. Although pre-conception has been put forward as the optimal time for the periodontal treatment in order to improving pregnancy outcomes, few studies have been conducted to examine inflammatory mediators in saliva and serum among pre-conception women. Pre-conception women were recruited between January 2012 and December 2014. Women were provided with an oral health examination to detect periodontal disease. Salivary and serum samples were collected at the same of examination. Inflammatory mediators includinginterleukin-1 beta (IL-1β), IL-6, tumor necrosis factor alpha (TNF-α) and beta-glucuronidase (β-glucuronidase) were tested and analyzed among women with overall periodontal disease (n = 442) or moderate/severe periodontal disease (n = 247). Results were compared to that in women with a healthy periodontium (n = 91). Significantly increased concentrations of inflammatory mediators of IL-1β, IL-6, TNF-α and β-glucuronidase in saliva and IL-1β, β-glucuronidase and TNF-α in serum were found among pre-conception women with moderate/severe periodontal disease, compared with women without periodontal disease. Significantly increased levels were also found in all the above saliva inflammatory mediators and in serum IL-1β and TNF-α among women with overall periodontal disease. The levels of all inflammatory mediators in saliva and almost all inflammatory mediators except IL-6 in serum significantly increased with severity of periodontal disease. Periodontal disease is highly associated with the elevated levels of inflammatory mediators in saliva and some mediators in serum among pre-conception women.

  16. Biomonitoring of industrial dusts on animals. II. Bioindication on alveolar macrophages.

    Kaváciková, Z


    Rats and rabbits were exposed through the respiratory system to industrial dusts (magnesite emissions, solid wastes from nickel refinery dump, cement emissions) at biomonitory stations or in experimental chamber. Following exposure the animals were killed, the alveolar macrophages isolated and acid phosphatase and beta-glucuronidase estimated in the isolated cells. The activity of both enzymes was enhanced in the exposed animals in all cases. The enhancement was dependent on the length of exposure and amount of inhaled particles.

  17. Optimised deconjugation of androgenic steroid conjugates in bovine urine

    Pedersen, Mikael; Frandsen, Henrik Lauritz; Andersen, Jens Hinge


    with selected aliphatic steroid sulphates (boldenone sulphate, nortestosteron sulphate and testosterone sulphate), and the method was validated for analysis of androgenic steroids in bovine urine using free steroids, steroid sulphates and steroid glucuronides as standards. Glucuronidase and sulphuric acid......After administration of steroids to animals the steroids are partially metabolised in the liver and kidney to phase 2 metabolites, i.e., glucuronic acid or sulphate conjugates. During analysis these conjugated metabolites are normally deconjugated enzymatically with aryl sulphatase...

  18. A new cyclopamine glucuronide prodrug with improved kinetics of drug release.

    Renoux, Brigitte; Legigan, Thibaut; Bensalma, Souheyla; Chadéneau, Corinne; Muller, Jean-Marc; Papot, Sébastien


    We prepared a new glucuronide prodrug of cyclopamine designed to target selectively the Hedgehog signalling pathway of cancer cells. This prodrug includes a novel self-immolative linker bearing a hydrophilic side chain that can be easily introduced via"click chemistry". With this design, the prodrug exhibits reduced toxicity compared to the free drug on U87 glioblastoma cells. However, in the presence of β-glucuronidase, the prodrug conducts to the quick release of cyclopamine thereby restoring its antiproliferative activity.

  19. Activation of Zoosporogenesis-Specific Genes in Phytophthora infestans Involves a 7-Nucleotide Promoter Motif and Cold-Induced Membrane Rigidity

    Tani, Shuji; Judelson, Howard


    Infections of plants by the oomycete Phytophthora infestans typically result from zoospores, which develop from sporangia at cold temperatures. To help understand the relevant cold-induced signaling pathway, factors regulating the transcription of the zoosporogenesis-specific NIF (nuclear LIM-interactor-interacting factor) gene family were examined. Sequences required for inducing PinifC3 were identified by analyzing truncated and mutated promoters using the β-glucuronidase reporter in stable...

  20. Assembly of Two Transgenes in an Artificial Chromatin Domain Gives Highly Coordinated Expression in Tobacco


    The chromatin loop model predicts that genes within the same chromatin domain exhibit coordinated regulation. We here present the first direct experimental support for this model in plants. Two reporter genes, the E. coli beta-glucuronidase gene and the firefly luciferase gene, driven by different promoters, were placed between copies of the chicken lysozyme A element, a member of the matrix-associated region (MAR) group of chromatin boundary elements, and introduced in tobacco (Nicotiana tab...


    Charmaine Joy Simeonovic; Andrew eZiolkowski; Zuopeng eWu; Fui Jiun eChoong; Craig eFreeman; Christopher eParish


    Heparanase (Hpse) is the only known mammalian endo-β-D-glucuronidase that degrades the glycosaminoglycan heparan sulfate (HS), found attached to the core proteins of heparan sulfate proteoglycans (HSPGs). Hpse plays a homeostatic role in regulating the turnover of cell-associated HS and also degrades extracellular HS in basement membranes (BMs) and the extracellular matrix (ECM), where HSPGs function as a barrier to cell migration. Secreted Hpse is harnessed by leukocytes to facilitate their ...

  2. Pharmacokinetic Modeling of Trivalent and Hexavalent Chromium Based on Ingestion and Inhalation of Soluble Chromium Compounds.


    be largely Cr(III) although some Cr(VI) exposure probably also occurs. Stainless-steel welders are exposed to nickel as well as to chromium compounds...welders are equivocal with respect to involvement of chromium, particularly since nickel in some chemical forms is an established lung carcinogen (Stern...microglobulin (Lindberg and Vesterberg, 1983), retinol-binding protein (Franchini and Mutti , 1988), B-glucuronidase ( Mutti et al., 1979), and kidney brush border

  3. Effects of ammonia on processing and secretion of precursor and mature lysosomal enzyme from macrophages of normal and pale ear mice: evidence for two distinct pathways


    Lysosomal enzymes have been shown to be synthesized as microsomal precursors, which are processed to mature enzymes located in lysosomes. We examined the effect of ammonium chloride on the intracellular processing and secretion of two lysosomal enzymes, beta-glucuronidase and beta-galactosidase, in mouse macrophages. This lysosomotropic drug caused extensive secretion of both precursor and mature enzyme forms within a few hours, as documented by pulse radiolabeling and molecular weight analys...

  4. Protective effect of squalene on certain lysosomal hydrolases and free amino acids in isoprenaline-induced myocardial infarction in rats

    Farvin, Sabeena; Surendraraj, A.; Anandan, R.


    This study was aimed to evaluate the preventive role of squalene on free amino acids and lysosomal alterations in experimentally induced myocardial infarction in rats. The levels of lysosomal enzymes (beta-glucuronidase, beta-galactosidase, beta-glucosidase, acid phosphatase and cathepsin D......) in plasma and lysosomal fractions, hydroxyproline content and free amino acids in heart tissue were determined. Isoprenaline administration to rats resulted in decreased stability of the membranes which was reflected by significantly (p...

  5. Genetic Differences among the a/J x C57bl/6j Recombinant Inbred Mouse Lines and Their Degree of Association with Glucocorticoid-Induced Cleft Palate

    Liu, Sharon L.; Erickson, Robert P.


    Hydrocortisone sodium phosphate was injected intramuscularly into A/J, C57BL/6J and recombinant inbred lines from these two parental lines to study the genetics of steroid-induced cleft palate in a situation of identical maternal and fetal genotypes. The strains were typed for H-2 (the major histocompatibility locus), β-glucuronidase and β2-microglobulin, which served as markers on chromosomes 17, 5 and 2, respectively. Hepatic glucocorticoid binding capacity had been previously measured in H...

  6. [Enhancement of photoassimilate utilization by manipulation of ADP-glucose pyrophosphorylase gene]. Final progress report

    Okita, T.W.


    Part 1 of this research focuses on patterns of gene expression of ADPG-pyrophosphorylase in native and transgenic potato plants. To elucidate the mechanism controlling AGP expression during plant development, the expression of the potato tuber AGP small subunit (sAGP) gene was analyzed in transgenic potato plants using a promoter-{beta}-glucuronidase expression system. Part II evaluated the structure-function relationships of AGP.

  7. Level of Dietary Fat Does Not Affect Fuel Oxidation or Endurance Exercise Performance of Soldiers


    METHODS Eight male military volunteers with normal fsting blood cholesterol and triglyceride levels and no history of diabetes gave their informed consent...Sustainment Module) on the intestinal microbiota and the bacterial P-glucuronidase activity was studied in young healthy male military subjects. the microbiota of the feces of young active military subjects maintained on model NSM rations that were either high fat/3300 kcal or lower fat

  8. A fluorescent assay amenable to measuring production of beta-D-glucuronides produced from recombinant UDP-glycosyl transferase enzymes.

    Trubetskoy, O V; Shaw, P M


    Beta-glucuronidase cleavage of 4-methylumbelliferyl beta-D-glucuronide generates the highly fluorescent compound, 4-methylumbelliferone. We show that other beta-D-glucuronide compounds act as competitors in this assay. The 4-methylumbelliferyl beta-D-glucuronide cleavage assay can easily be adapted to high throughput formats to detect the presence of beta-D glucuronides generated using recombinant glycosyl transferase preparations.

  9. Measuring Faecal Epi-Androsterone as an Indicator of Gonadal Activity in Spotted Hyenas (Crocuta crocuta.

    Susanne Pribbenow

    Full Text Available Enzyme immunoassays (EIA that measure faecal testosterone metabolites (fTM are useful tools to monitor gonadal activity. The aim of this study was to validate an "in-house" epiandrosterone EIA to monitor fTM in spotted hyenas. FTM were characterised in a male and a female hyena that each received an injection of 3H-testosterone. High-performance liquid chromatography (HPLC analyses revealed a cluster of highly polar enzyme-hydrolysable hormone metabolite conjugates. We performed hydrolysis using β-glucuronidase to deconjugate metabolites and improve sensitivity of the assay. Because β-glucuronidase from Helix pomatia has been reported to bias testosterone measurements in some species, we compared the enzymatic activity of the commonly used β-glucuronidase extracted from H. pomatia with the same enzyme from Escherichia coli. Our results showed that β-glucuronidases from both sources produced similar results from spotted hyena faeces. We therefore hydrolysed samples with H. pomatia enzymes. HPLC analyses also demonstrated that following hydrolysis the epiandrosterone EIA measured significant amounts of immunoreactive metabolites corresponding to radiolabelled metabolites in both sexes. Additionally, HPLC and GC-MS analyses confirmed the presence of epiandrosterone in faeces of spotted hyenas. The biological relevance of the epiandrosterone EIA was validated by demonstrating (1 a significant increase in fTM levels in response to a testosterone injection within 16 h, (2 no biological responsiveness to an adrenocorticotropic hormone (ACTH injection and (3 significant differences in fTM levels between juvenile males and adult immigrant males in a free-ranging wild population. Our results clearly demonstrate that the epiandrosterone EIA is a reliable non-invasive method to monitor gonadal activity in spotted hyenas.

  10. Variations of lysosomal enzymes in different parts of rabbit Fallopian tube during ovum transport.

    Puri, R K; Roy, S K


    Some lysosomal enzymes (viz., acid DNase, acid RNase and beta-glucuronidase) were estimated in different parts of the rabbit Fallopian tube during different hours post coitum (p. c.). At estrus, alterations of acid RNase and beta-glucuronidase were observed in different anatomical segments of the Fallopian tube but acid DNase was undetectable. When these enzymes were compared at different hours p.c., it was noticed that when the ovum reaches ampullary (A), ampullary-isthmic junction (AIJ) and isthmic (I) segments of the Fallopian tube at the respective hours 14, 24 and 70, the acid DNase activity showed increased value in these parts when compared to their preceding groups. Acid RNase also showed similar type of pattern except that it was not altered at 14 hr p. c. At 144 hr p. c. both the enzymes had no significant alteration over 70 hr value, beta-glucuronidase, however, did not show this type of pattern in all the segments till 144 hr p. c. The increased activity of acid RNase and DNase in AIJ and I segments of the tube till 70 hr p. c. suggests the increased lysosomal activity in the tubal fluid produced by secretory cells. The possible involvement of these lysomal factors in the process of fertilization and preparation of ovum prior to implantation is suggested.

  11. [Metabolites of R, S-1-(2-methoxyphenyl)-4-[3-(naphtha-1-yl-oxy)-2-hydroxypropyl]-piperazin in rat plasma].

    Li, Li; Zhou, Xin; Yuan, Mu; Zhou, Hong; Wang, Dao-Ping


    To study the metabolites of R, S-1-(2-methoxypheyl)-4-[3-(naphthal-yl-oxy)-2-hydroxypropyl] -piperazine, (naftopidil, NAF), a novel antihypertensive drug in rat plasma. The rat plasma samples were analyzed by LC/MS after oral administration of NAF. According to MS relativity of metabolites and parent compound (NAF) and metabolic rule of compound with similar structure, the structure of potential metabolites were postulated. Phase I metabolites were identified by HPLC/MS and by comparison with authentic standards, phase II conjugates were indirectly identified with beta-D-glucuronidase in presence or absence of glucuronidase selective inhibitor D-saccharric acid beta-1,4-Lactone. Phase I metabolites desmethyl-naftopidil (DMN), (phenyl) hydroxynaftopidil (PHN), (naphthyl) -hydroxy-naftopidil (NHN) were separated and identified in rat plasma by comparison with reference substances, phase II conjugates, NAF and NHN glucuronide conjugates were separated and tentatively identified by hydrolysis with glucuronidase, the aglycones, NAF and NHN, were identified in rat plasma. The major metabolic pathway of NAF in rat plasma should be the hydroxylation of the phenyl or nephthyl moiety of NAF and demethylation of NAF. Therefore, (naphthyl) hydroxyl-metabolite and NAF followed by conjugation with beta-glueuronic acid.

  12. Evaluation of cytotoxic properties of a cyclopamine glucuronide prodrug in rat glioblastoma cells and tumors.

    Bensalma, Souheyla; Chadeneau, Corinne; Legigan, Thibaut; Renoux, Brigitte; Gaillard, Afsaneh; de Boisvilliers, Madryssa; Pinet-Charvet, Caroline; Papot, Sébastien; Muller, Jean Marc


    Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor. Activation of the developmental hedgehog (Hh) pathway is observed in GBM, particularly in the so-called glioma stem cells (GSCs). An inhibitor of this pathway is the steroidal alkaloid cyclopamine, an antagonist of the Hh coreceptor Smoothened (SMO). To limit the toxicity of cyclopamine toward Hh-dependent non-tumor cells, our group previously reported the synthesis of a prodrug (called 1b), designed to deliver cyclopamine in the presence of β-glucuronidase, an enzyme found in the necrotic area of GBM. Here, we aimed to analyze the in vitro, ex vivo, and in vivo cytotoxic properties of this prodrug in the C6 rat GBM cells. In the presence of β-glucuronidase, the activated prodrug 1b was toxic and downregulated expression of Gli1, a Hh target gene, in C6 cells and C6-GSCs, but not in normal rat astrocytes in which the Hh pathway is weakly activated. In the absence of β-glucuronidase, prodrug 1b displayed no obvious toxicity toward rat brain tissue explants while cyclopamine clearly affected brain tissue viability. When administered to rats bearing fluorescent C6-derived GBM, the prodrug 1b reduced the tumor density more efficiently than cyclopamine. Prodrug 1b thus appears as a promising concept to optimize confinement of cyclopamine cytotoxicity within the tumors, with more limited effects in the surrounding normal brain tissue.

  13. Two glutamine synthetase genes from Phaseolus vulgaris L. display contrasting developmental and spatial patterns of expression in transgenic Lotus corniculatus plants.

    Forde, B G; Day, H M; Turton, J F; Shen, W J; Cullimore, J V; Oliver, J E


    The gln-gamma gene, which specifies the gamma subunit of glutamine synthetase in Phaseolus vulgaris L., has been isolated and the regulatory properties of its promoter region analyzed in transgenic Lotus corniculatus plants. A 2-kilobase fragment from the 5'-flanking region of gln-gamma conferred a strongly nodule-enhanced pattern of expression on the beta-glucuronidase reporter gene. Parallel studies on the promoter of another glutamine synthetase gene (gln-beta) showed that a 1.7-kilobase fragment directed 20-fold to 140-fold higher levels of beta-glucuronidase expression in roots than in shoots. Histochemical localization of beta-glucuronidase activity in nodules of the transgenic plants indicated that the chimeric gln-gamma gene was expressed specifically in the rhizobially infected cells; expression of the gln-beta construct was detected in both cortical and infected regions of young nodules, and became restricted to the vascular tissue as the nodule matured. We conclude that gln-beta and gln-gamma genes are differentially expressed both temporally and spatially in plant development and that the cis-acting regulatory elements responsible for conferring these contrasting expression patterns are located within a 2-kilobase region upstream of their coding sequences.

  14. The profile of lysosomal exoglycosidases in replicative and stress-induced senescence in early passage human fibroblasts

    Małgorzata Knaś


    Full Text Available The aim of the present study was to assess the profiles of the exoglycosidases: N-acetyl-β-hexosoaminidase, β glucuronidase and β galactosidase, α mannosidase and α fucosidase in fibroblast culture undergoing replicative and stress-induced senescence. Half of the cell culture was grown in normal conditions, without the stressor, and the other half of the cell was treated with 0.15 mM tert-butylhydroperoxide. The activities of total N-acetyl-β-hexosoaminidase as well as β glucuronidase in the cell lysate were determined in duplicates using the method of Marciniak et al. The activities of β galactosidase, α mannosidase and α fucosidase in the cell lysate were determined in duplicates using the method of Chatteriee et al. with the modification by Zwierz et al. The activities of the exoglycosidases examined, with the exception of β glucuronidase, showed a significant increase between individual days of the experiment in both non-stressed and stressed fibroblast cell culture. On each day of the experiment, in the cell lysate of stressed fibroblasts, the activities of exoglycosidases were significantly higher compared to the non-stressed cells. There were very strong correlations between SA-β-GAL staining and b galactosidase activity on individual days of the experiment in both non-stressed and stressed fibroblast cell culture. Replicative and stress-induced senescence results in significant changes to the level of lysosomal exoglycosidases, and results in enhanced lysosomal degradative capacity.

  15. Irinotecan-induced mucositis manifesting as diarrhoea corresponds with an amended intestinal flora and mucin profile.

    Stringer, Andrea M; Gibson, Rachel J; Bowen, Joanne M; Logan, Richard M; Ashton, Kimberly; Yeoh, Ann S J; Al-Dasooqi, Noor; Keefe, Dorothy M K


    Chemotherapy-induced diarrhoea is a major oncological problem, caused by the cytotoxic effects of cancer chemotherapy. Irinotecan is linked with severe mucositis and diarrhoea, the mechanisms of which remain poorly understood. Bacterial beta-glucuronidase is thought to be involved in the metabolism of irinotecan, implicating the intestinal flora. Intestinal mucins may also be implicated in the development of chemotherapy-induced diarrhoea. Rats were treated with 200 mg/kg of irinotecan and killed at 96, 120 and 144 h. The rats were monitored for diarrhoea. Pathology and immunohistochemical staining was performed. The samples were cultured and faecal DNA was analysed using real-time polymerase chain reaction. Severe diarrhoea was observed from 72 to 96 h. A decrease in body mass was also observed after treatment. Significant changes in goblet cell numbers (both complete and cavitated cells) were observed in the small and large intestines. Changes in MUC gene expression were observed in the small intestine only. Modifications were observed to the intestinal flora profile, especially Escherichia coli, and an increase in the expression of beta-glucuronidase was detected. In conclusion, irinotecan-induced diarrhoea may be caused by an increase in some beta-glucuronidase-producing bacteria, especially E. coli, exacerbating the toxicity of active metabolites. Accelerated mucous secretion and mucin release may also contribute to the delayed onset of diarrhoea.

  16. Effect of collection, transport, processing and storage of blood specimens on the activity of lysosomal enzymes in plasma and leukocytes

    Burin M.


    Full Text Available This study was designed to evaluate the effect of different conditions of collection, transport and storage on the quality of blood samples from normal individuals in terms of the activity of the enzymes ß-glucuronidase, total hexosaminidase, hexosaminidase A, arylsulfatase A and ß-galactosidase. The enzyme activities were not affected by the different materials used for collection (plastic syringes or vacuum glass tubes. In the evaluation of different heparin concentrations (10% heparin, 5% heparin, and heparinized syringe in the syringes, it was observed that higher doses resulted in an increase of at least 1-fold in the activities of ß-galactosidase, total hexosaminidase and hexosaminidase A in leukocytes, and ß-glucuronidase in plasma. When the effects of time and means of transportation were studied, samples that had been kept at room temperature showed higher deterioration with time (72 and 96 h before processing, and in this case it was impossible to isolate leukocytes from most samples. Comparison of heparin and acid citrate-dextrose (ACD as anticoagulants revealed that ß-glucuronidase and hexosaminidase activities in plasma reached levels near the lower normal limits when ACD was used. In conclusion, we observed that heparin should be used as the preferable anticoagulant when measuring these lysosomal enzyme activities, and we recommend that, when transport time is more than 24 h, samples should be shipped by air in a styrofoam box containing wet ice.

  17. Preventive Effects of a Chinese Herbal Formula, Shengjiang Xiexin Decoction, on Irinotecan-Induced Delayed-Onset Diarrhea in Rats

    Zhang, Pan; Liu, Sida; Pan, Lin


    Irinotecan is a well-known chemotherapy drug for the treatment of various cancers. However, delayed-onset diarrhea is a common adverse reaction, limiting the application of the drug. The study presented was designed to evaluate the preventive effects of Shengjiang Xiexin decoction (SXD) on irinotecan-induced diarrhea and to explore the possible mechanisms of this action. We established a diarrhea rat model. The condition of the rats was observed. The proliferation and apoptosis of intestinal cells were measured using immunohistochemical assays and a caspase-3 activity assay, respectively. The expression of Lgr5 and CD44 staining were used to observe intestinal stem cells (ISCs). In addition, the activity of β-glucuronidase in the rats' feces was measured. Our results showed that the number of proliferating intestinal cells in the SXD groups was obviously higher, while the activity of caspase-3 was lower. The expression of Lgr5 and the integrated option density (IOD) of CD44 stain were increased significantly by SXD. Additionally, SXD decreased the activity of β-glucuronidase after irinotecan administration. In conclusion, SXD exhibited preventive effects on irinotecan-induced diarrhea, and this action was associated with an inhibitory effect on intestinal apoptosis and β-glucuronidase and a promotive effect on intestinal cell proliferation due to increased maintenance of ISCs. PMID:28167974

  18. Preventive Effects of a Chinese Herbal Formula, Shengjiang Xiexin Decoction, on Irinotecan-Induced Delayed-Onset Diarrhea in Rats

    Chao Deng


    Full Text Available Irinotecan is a well-known chemotherapy drug for the treatment of various cancers. However, delayed-onset diarrhea is a common adverse reaction, limiting the application of the drug. The study presented was designed to evaluate the preventive effects of Shengjiang Xiexin decoction (SXD on irinotecan-induced diarrhea and to explore the possible mechanisms of this action. We established a diarrhea rat model. The condition of the rats was observed. The proliferation and apoptosis of intestinal cells were measured using immunohistochemical assays and a caspase-3 activity assay, respectively. The expression of Lgr5 and CD44 staining were used to observe intestinal stem cells (ISCs. In addition, the activity of β-glucuronidase in the rats’ feces was measured. Our results showed that the number of proliferating intestinal cells in the SXD groups was obviously higher, while the activity of caspase-3 was lower. The expression of Lgr5 and the integrated option density (IOD of CD44 stain were increased significantly by SXD. Additionally, SXD decreased the activity of β-glucuronidase after irinotecan administration. In conclusion, SXD exhibited preventive effects on irinotecan-induced diarrhea, and this action was associated with an inhibitory effect on intestinal apoptosis and β-glucuronidase and a promotive effect on intestinal cell proliferation due to increased maintenance of ISCs.

  19. Fecal microbial determinants of fecal and systemic estrogens and estrogen metabolites: a cross-sectional study

    Flores Roberto


    Full Text Available Abstract Background High systemic estrogen levels contribute to breast cancer risk for postmenopausal women, whereas low levels contribute to osteoporosis risk. Except for obesity, determinants of non-ovarian systemic estrogen levels are undefined. We sought to identify members and functions of the intestinal microbial community associated with estrogen levels via enterohepatic recirculation. Methods Fifty-one epidemiologists at the National Institutes of Health, including 25 men, 7 postmenopausal women, and 19 premenopausal women, provided urine and aliquots of feces, using methods proven to yield accurate and reproducible results. Estradiol, estrone, 13 estrogen metabolites (EM, and their sum (total estrogens were quantified in urine and feces by liquid chromatography/tandem mass spectrometry. In feces, β-glucuronidase and β-glucosidase activities were determined by realtime kinetics, and microbiome diversity and taxonomy were estimated by pyrosequencing 16S rRNA amplicons. Pearson correlations were computed for each loge estrogen level, loge enzymatic activity level, and microbiome alpha diversity estimate. For the 55 taxa with mean relative abundance of at least 0.1%, ordinal levels were created [zero, low (below median of detected sequences, high] and compared to loge estrogens, β-glucuronidase and β-glucosidase enzymatic activity levels by linear regression. Significance was based on two-sided tests with α=0.05. Results In men and postmenopausal women, levels of total urinary estrogens (as well as most individual EM were very strongly and directly associated with all measures of fecal microbiome richness and alpha diversity (R≥0.50, P≤0.003. These non-ovarian systemic estrogens also were strongly and significantly associated with fecal Clostridia taxa, including non-Clostridiales and three genera in the Ruminococcaceae family (R=0.57−0.70, P=0.03−0.002. Estrone, but not other EM, in urine correlated significantly with

  20. High-Yield Expression of M2e Peptide of Avian Influenza Virus H5N1 in Transgenic Duckweed Plants.

    Firsov, Aleksey; Tarasenko, Irina; Mitiouchkina, Tatiana; Ismailova, Natalya; Shaloiko, Lyubov; Vainstein, Alexander; Dolgov, Sergey


    Avian influenza is a major viral disease in poultry. Antigenic variation of this virus hinders vaccine development. However, the extracellular domain of the virus-encoded M2 protein (peptide M2e) is nearly invariant in all influenza A strains, enabling the development of a broad-range vaccine against them. Antigen expression in transgenic plants is becoming a popular alternative to classical expression methods. Here we expressed M2e from avian influenza virus A/chicken/Kurgan/5/2005(H5N1) in nuclear-transformed duckweed plants for further development of avian influenza vaccine. The N-terminal fragment of M2, including M2e, was selected for expression. The M2e DNA sequence fused in-frame to the 5' end of β-glucuronidase was cloned into pBI121 under the control of CaMV 35S promoter. The resulting plasmid was successfully used for duckweed transformation, and western analysis with anti-β-glucuronidase and anti-M2e antibodies confirmed accumulation of the target protein (M130) in 17 independent transgenic lines. Quantitative ELISA of crude protein extracts from these lines showed M130-β-glucuronidase accumulation ranging from 0.09-0.97 mg/g FW (0.12-1.96 % of total soluble protein), equivalent to yields of up to 40 μg M2e/g plant FW. This relatively high yield holds promise for the development of a duckweed-based expression system to produce an edible vaccine against avian influenza.

  1. Abiotic stress leads to somatic and heritable changes in homologous recombination frequency, point mutation frequency and microsatellite stability in Arabidopsis plants

    Yao Youli, E-mail: [Department of Biological Sciences, University of Lethbridge, Lethbridge, T1K 3M4 Alberta (Canada); Kovalchuk, Igor, E-mail: [Department of Biological Sciences, University of Lethbridge, Lethbridge, T1K 3M4 Alberta (Canada)


    In earlier studies, we showed that abiotic stresses, such as ionizing radiation, heavy metals, temperature and water, trigger an increase in homologous recombination frequency (HRF). We also demonstrated that many of these stresses led to inheritance of high-frequency homologous recombination, HRF. Although an increase in recombination frequency is an important indicator of genome rearrangements, it only represents a minor portion of possible stress-induced mutations. Here, we analyzed the influence of heat, cold, drought, flood and UVC abiotic stresses on two major types of mutations in the genome, point mutations and small deletions/insertions. We used two transgenic lines of Arabidopsis thaliana, one allowing an analysis of reversions in a stop codon-containing inactivated {beta}-glucuronidase transgene and another one allowing an analysis of repeat stability in a microsatellite-interrupted {beta}-glucuronidase transgene. The transgenic Arabidopsis line carrying the {beta}-glucuronidase-based homologous recombination substrate was used as a positive control. We showed that the majority of stresses increased the frequency of point mutations, homologous recombination and microsatellite instability in somatic cells, with the frequency of homologous recombination being affected the most. The analysis of transgenerational changes showed an increase in HRF to be the most prominent effect observed in progeny. Significant changes in recombination frequency were observed upon exposure to all types of stress except drought, whereas changes in microsatellite instability were observed upon exposure to UVC, heat and cold. The frequency of point mutations in the progeny of stress-exposed plants was the least affected; an increase in mutation frequency was observed only in the progeny of plants exposed to UVC. We thus conclude that transgenerational changes in genome stability in response to stress primarily involve an increase in recombination frequency.

  2. Upregulation of the Creatine Transporter Slc6A8 by Klotho

    Ahmad Almilaji


    Full Text Available Background/Aims: The transmembrane Klotho protein contributes to inhibition of 1,25(OH2D3 formation. The extracellular domain of Klotho protein could function as an enzyme with e.g. β-glucuronidase activity, be cleaved off and be released into blood and cerebrospinal fluid. Klotho regulates several cellular transporters. Klotho protein deficiency accelerates the appearance of age related disorders including neurodegeneration and muscle wasting and eventually leads to premature death. The main site of Klotho protein expression is the kidney. Klotho protein is also appreciably expressed in other tissues including chorioid plexus. The present study explored the effect of Klotho protein on the creatine transporter CreaT (Slc6A8, which participates in the maintenance of neuronal function and survival. Methods: To this end cRNA encoding Slc6A8 was injected into Xenopus oocytes with and without additional injection of cRNA encoding Klotho protein. Creatine transporter CreaT (Slc6A8 activity was estimated from creatine induced current determined by two-electrode voltage-clamp. Results: Coexpression of Klotho protein significantly increased creatine-induced current in Slc6A8 expressing Xenopus oocytes. Coexpression of Klotho protein delayed the decline of creatine induced current following inhibition of carrier insertion into the cell membrane by brefeldin A (5 µM. The increase of creatine induced current by coexpression of Klotho protein in Slc6A8 expressing Xenopus oocytes was reversed by β-glucuronidase inhibitor (DSAL. Similarly, treatment of Slc6A8 expressing Xenopus oocytes with recombinant human alpha Klotho protein significantly increased creatine induced current. Conclusion: Klotho protein up-regulates the activity of creatine transporter CreaT (Slc6A8 by stabilizing the carrier protein in the cell membrane, an effect requiring β-glucuronidase activity of Klotho protein.

  3. An efficient method for Agrobacterium-mediated genetic transformation and plant regeneration in cumin (Cuminum cyminum L.).

    Pandey, Sonika; Mishra, Avinash; Patel, Manish Kumar; Jha, Bhavanath


    Cumin is an annual herbaceous medicinally important plant having diverse applications. An efficient and reproducible method of Agrobacterium-mediated genetic transformation was herein established for the first time. A direct regeneration method without callus induction was optimised using embryos as explant material in Gamborg's B5 medium supplemented with 0.5-μM 6-benzyladenine and 2.0-μM α-naphthalene acetic acid. About 1,020 embryos (a mean of 255 embryos per batch) were used for the optimisation of transformation conditions. These conditions were an Agrobacterium cell suspension of 0.6 OD600, a co-cultivation time of 72 h, 300-μM acetosyringone and wounding of explants using a razor blade. Pre-cultured elongated embryos were treated using optimised conditions. About 720 embryos (a mean of 180 embryos per batch) were used for transformation and 95 % embryos showed transient β-glucuronidase expression after co-cultivation. Putative transformed embryos were cultured on B5 medium for shoot proliferation and 21 regenerated plants were obtained after selection and allowed to root. T0 plantlets showed β-glucuronidase expression and gene integration was confirmed via PCR amplification of 0.96 and 1.28 kb fragments of the hygromycin-phosphotransferase II and β-glucuronidase genes, respectively. In this study, a transformation efficiency of 1.5 % was demonstrated and a total of 11 transgenic plants were obtained at the hardening stage, however, only four plants acclimatised during hardening. Gene copy number was analysed by Southern blot analysis of hardened plants and single-copy gene integration was observed. This is the first successful attempt of Agrobacterium-mediated genetic transformation of cumin.

  4. Abiotic stress leads to somatic and heritable changes in homologous recombination frequency, point mutation frequency and microsatellite stability in Arabidopsis plants.

    Yao, Youli; Kovalchuk, Igor


    In earlier studies, we showed that abiotic stresses, such as ionizing radiation, heavy metals, temperature and water, trigger an increase in homologous recombination frequency (HRF). We also demonstrated that many of these stresses led to inheritance of high-frequency homologous recombination, HRF. Although an increase in recombination frequency is an important indicator of genome rearrangements, it only represents a minor portion of possible stress-induced mutations. Here, we analyzed the influence of heat, cold, drought, flood and UVC abiotic stresses on two major types of mutations in the genome, point mutations and small deletions/insertions. We used two transgenic lines of Arabidopsis thaliana, one allowing an analysis of reversions in a stop codon-containing inactivated β-glucuronidase transgene and another one allowing an analysis of repeat stability in a microsatellite-interrupted β-glucuronidase transgene. The transgenic Arabidopsis line carrying the β-glucuronidase-based homologous recombination substrate was used as a positive control. We showed that the majority of stresses increased the frequency of point mutations, homologous recombination and microsatellite instability in somatic cells, with the frequency of homologous recombination being affected the most. The analysis of transgenerational changes showed an increase in HRF to be the most prominent effect observed in progeny. Significant changes in recombination frequency were observed upon exposure to all types of stress except drought, whereas changes in microsatellite instability were observed upon exposure to UVC, heat and cold. The frequency of point mutations in the progeny of stress-exposed plants was the least affected; an increase in mutation frequency was observed only in the progeny of plants exposed to UVC. We thus conclude that transgenerational changes in genome stability in response to stress primarily involve an increase in recombination frequency.

  5. Development of A Flexible System for the Simultaneous Conversion of Biomass to Industrial Chemicals and the Production of Industrial Biocatalysts

    Gao, Johnway; Hooker, Brian S.; Skeen, R S.; Anderson, D B.; Lankey, R. L.; Anastas, P. T.


    A flexible system was developed for the simultaneous conversion of biomass to industrial chemicals and the production of industrial biocatalysts. In particular, the expression of a bacterial enzyme, beta-glucuronidase (GUS), was investigated using a genetically modified starch-degrading Saccharomyces strain in suspension cultures in starch media. Different sources of starch including corn and waste potato starch were used for yeast biomass accumulation and GUS expression studies under controls of inducible and constitutive promoters. A thermostable bacterial cellulase, Acidothermus cellulolyticus E1 endoglucanase gene was also cloned into an episomal plasmid expression vector and expressed in the starch-degrading Saccharomyces strain.

  6. Anti-ulcer and membrane stabilizing actions of zinc acexamate.

    Pfeiffer, C J; Bulbena, O; Esplugues, J V; Escolar, G; Navarro, C; Esplugues, J


    The effects of zinc acexamate on stress and reserpine ulcers as well as on gastric mast cells degranulation and membrane stability were evaluated in the rat. Zinc acexamate (100 mg/kg) has demonstrated an inhibitory effect on cold-restraint stress and reserpine-induced ulcer in a dose-dependent manner. Pretreatment of rats, prior to cold restraint stress, reduced gastric mast cell degranulation. Zinc acexamate (10(-4) M) inhibits Triton X-100 release of beta-glucuronidase in isolated hepatic lysosomes. These observations suggest that ulcer protective actions of zinc acexamate may be exerted in part through enhancing gastric mucosal resistance by stabilizing biological membrane integrity.

  7. Mutations in the Histone-like Nucleoid Structuring Regulatory Gene (hns) Decrease the Adherence of Shiga Toxin-producing Escherichia coli 091:H21 Strain B2F1 to Human Colonic Epithelial Cells and Increase the Production of Hemolysin


    long wave ultra- violet light (Chapman et aI., 1991; Zadik et al., 1993). Because 0157:H7 strains are negative for p-glucuronidase this cross-linking with ultra violet (UV) light (Church and Gilbert, 1984). The blot was blocked at least 4 hours before addition of labeled probe (see...with a trans-blot SO semi-dry apparatus (Bio-Rad). The membranes were blocked with 5% nonfat dried milk ( Carnation Co., Los Angeles, CAl in Tris

  8. Urinary metabolites of daidzin orally administered in rats.

    Yasuda, T; Ohsawa, K


    In a study on the metabolism of flavonoids, the isoflavone glycoside daidzin was orally administered to rats. Urine samples were collected and treated with beta-glucuronidase and arylsulfatase. Aglycone daidzein (M3) and other three metabolites, 3',4',7-trihydroxyisoflavone (M1), 4',7-dihydroxyisoflavanone (M2) and 4',7-dihydroxyisoflavan (M4) were isolated from the urine following treatment with enzymes. The structures of M1, M2 and M4 were determined on the basis of chemical and spectral data.

  9. Osmotic stress-regulated the expression of glutathione peroxidase 3 in Arabidopsis

    MIAO YuChen; GUO JingGong; LIU ErTao; LI Kun; DAI Jie; WANG PengCheng; CHEN Jia; SONG ChunPeng


    Gene expression of glutathione peroxidase 3 (ATGPX3) in response to osmotic stress was analyzed in Arabidopsis using ATGPX3 promoter-glucuronidase (GUS) transgenic plants. High levels of GUS expression were detected under osmotic stress in ATGPX3 promoter-GUS transgenic plants. Compared with the wild type, the growth and development of ATGPX3 mutants (atgpx3-1) were more sensitive to mannitol. In addition, the expression of RD29A, ABI1, ABI2 and RbohD in atgpx3-1 was induced by ABA stress. These results suggest that ATGPX3 might be involved in the signal transduction of osmotic stress.

  10. 含有MAR序列的植物表达载体pBI121-MARs的构建%Construction of Plant Expression Vector pBI121-MARs

    武冬梅; 陈创夫; 史芳芳; 祝建波


    将MAR(Matrix Attachment Region)构建到外源基因表达框的两侧,可以促进外源基因的表达,减少转基因沉默.实验利用PCR方法从普通烟草基因组中分离到一段MAR序列,将其正向重复插入到植物表达载体pBI121 β-葡糖醛酸酶(β-glucuronidase,GUS)基因表达框的两侧,形成一个新的植物表达载体.

  11. Disparate sequence characteristics of the Erysiphe graminis f.sp. hordei glyceraldehyde-3-phosphate dehydrogenase gene

    Christiansen, S.K.; Justesen, A.F.; Giese, H.


    to be similar for all four genes. The results of the codon-usage analysis suggest that Egh is more flexible than other fungi in the choice of nucleotides at the wobble position. Codon-usage preferences in Egh and barley genes indicate a level of difference which may be exploited to discriminate between fungal...... and plant genes in sequence mixtures. The Egh gpd promoter appears to be superior to that of the Egh beta-tubulin gene (tub2) for driving the E. coli beta-glucuronidase (GUS) gene in transformation experiments....

  12. Expression of the 1-Aminocyclopropane-1-Carboxylic Acid Deaminase Gene Requires Symbiotic Nitrogen-Fixing Regulator Gene nifA2 in Mesorhizobium loti MAFF303099

    Nukui, Noriyuki; MINAMISAWA, KIWAMU; Ayabe, Shin-Ichi; Aoki, Toshio


    Many soil bacteria contain 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase, which degrades ACC, a precursor of the phytohormone ethylene. In order to examine the regulation of the acdS gene encoding ACC deaminase in Mesorhizobium loti MAFF303099 during symbiosis with the host legume Lotus japonicus, we introduced the β-glucuronidase (GUS) gene into acdS so that GUS was expressed under control of the acdS promoter, and we also generated disruption mutants with mutations in a nitrogen fix...

  13. Mitochondrial dysfunction leads to deconjugation of quercetin glucuronides in inflammatory macrophages.

    Akari Ishisaka

    Full Text Available Dietary flavonoids, such as quercetin, have long been recognized to protect blood vessels from atherogenic inflammation by yet unknown mechanisms. We have previously discovered the specific localization of quercetin-3-O-glucuronide (Q3GA, a phase II metabolite of quercetin, in macrophage cells in the human atherosclerotic lesions, but the biological significance is poorly understood. We have now demonstrated the molecular basis of the interaction between quercetin glucuronides and macrophages, leading to deconjugation of the glucuronides into the active aglycone. In vitro experiments showed that Q3GA was bound to the cell surface proteins of macrophages through anion binding and was readily deconjugated into the aglycone. It is of interest that the macrophage-mediated deconjugation of Q3GA was significantly enhanced upon inflammatory activation by lipopolysaccharide (LPS. Zymography and immunoblotting analysis revealed that β-glucuronidase is the major enzyme responsible for the deglucuronidation, whereas the secretion rate was not affected after LPS treatment. We found that extracellular acidification, which is required for the activity of β-glucuronidase, was significantly induced upon LPS treatment and was due to the increased lactate secretion associated with mitochondrial dysfunction. In addition, the β-glucuronidase secretion, which is triggered by intracellular calcium ions, was also induced by mitochondria dysfunction characterized using antimycin-A (a mitochondrial inhibitor and siRNA-knockdown of Atg7 (an essential gene for autophagy. The deconjugated aglycone, quercetin, acts as an anti-inflammatory agent in the stimulated macrophages by inhibiting the c-Jun N-terminal kinase activation, whereas Q3GA acts only in the presence of extracellular β-glucuronidase activity. Finally, we demonstrated the deconjugation of quercetin glucuronides including the sulfoglucuronides in vivo in the spleen of mice challenged with LPS. These results

  14. Assessment of the microbiological quality of the poultry products from convenience food group

    Bożena Danyluk


    Full Text Available The objective of this study was to evaluate the microbiological quality of chosen poultry products from convenience food group, taking into account the seasons. There were assessed products after heat treatment (marinated, roasted leg; two-phase method breaded, fried and roasted piquant wings; three-phase method breaded, fried and roasted filet and frozen (marinated: leg, wings and filet. Microbiological examination involved determining the total number of mesophilic aerobic bacteria, coagulase-positive staphylococci and glucuronidase-positive E. coli and presence of Salmonella and Listeria monocytogenes in 25 g. The obtained results showed, that the level of microbiological contamination of the samples was without reservation.

  15. Effect of cooling method on the microbiological quality of raw materials meant for the convenience food production

    Bożena Danyluk


    Full Text Available The objective of this study was to evaluate the microbiological status of poultry carcass cuts, in chosen manufacturing plants, taking into account the seasons. Eviscerated carcasses were chilled using two methods: cold-air blast with water sprays and water immersion. Microbiological examination of carcass cuts: filet, thighs and wings involved determining the total number of aerobic bacteria, coagulase-positive staphylococci and glucuronidase-positive E. coli. There was no effect of the type of carcass, cooling method, and the time of the year on the microbiological quality of the samples.

  16. Agrobacterium tumefaciens-mediated GUS gene transfer to Sophora japonica L.

    Zhang Xiao-ying; Wang Hua-fang; Yin Wei-lun; Zhu Zhen


    Agrobacterium-mediated genetic transformation of Sophorajaponica was standardized using the Agrobacterium tumefaciens strain LBA4404 that harbored the binary vector pBI121 containing genes for β-glucuronidase (GUS) and neomycin phosphotransterase (npt Ⅱ). S. japonica transformants were selected by the ability of the leaf explants to produce kanamycin-resistant calli that regenerated into kanamycin-resistant plantlets. Successful transformation was confirmed by histochemical assay for GUS activity, PCR analysis and Southern blot. The period of nearly two months was required for the regeneration of transgenic plantlets from the explants. The transformed plants resembled their parents in morphology.

  17. Molecular farming of industrial proteins from transgenic maize.

    Hood, E E; Kusnadi, A; Nikolov, Z; Howard, J A


    Recombinant egg white avidin and bacterial B-glucuronidase (GUS) from transgenic maize have been commercially produced. High levels of expression were obtained in seed by employing the ubiquitin promoter from maize. The recombinant proteins had activities that were indistinguishable from their native counterparts. We have illustrated that down-stream activities in the production of these recombinant proteins, such as stabilizing the germplasm and processing for purification, were accomplished without any major obstacles. Avidin (A8706) and GUS (G2035) are currently marketed by Sigma Chemical Co.

  18. Increase of Expression Levels of Reporter Gene in Transgenic Tobaccos by Matrix Attachment Regions


    The matrix attachment region (MAR) located downstream of Plastocyanin gene was isolated from the genome of pea. To study the effect of MARs on foreign gene expression in transgenic plants, T-DNA vector was constructed in which MARs flanked bothβ-glucuronidase(GUS) gene and selectable marker neomycin phosphotransferase (NPT-II) gene. The plant expression vectors were transferred into leaf discs via Agrobacterium-mediated transformation procedure. The result of GUS measurement showed that pea MAR could increase transgene expression level. The mean expression levels of GUS gene expression in population containing MARs could be increased twofold when compared with that of population without MARs.

  19. Reference: 625 [Arabidopsis Phenome Database[Archive

    Full Text Available he ABA-insensitive phenotype of the sdir1-1 mutant, whereas SDIR1 could not rescue the abi5-1 mutant. This suggests that SDI...ansduction. We show that the RING finger E3 ligase, Arabidopsis thaliana SALT- AND DROUGHT-INDUCED RING FINGER1 (SDI...R1), is involved in abscisic acid (ABA)-related stress signal transduction. SDI...y ABA. Plants expressing the ProSDIR1-beta-glucuronidase (GUS) reporter construct confirmed strong induction... stress. The green fluorescent protein-SDIR1 fusion protein is colocalized with intracellular membranes. We demonstrate that SDI

  20. Effect of subinhibitory concentrations of four commonly used biocides on the conjugative transfer of Tn916 in Bacillus subtilis

    Seier-Petersen, Maria Amalie; Jasni, A.; Aarestrup, Frank Møller


    Objectives Large amounts of biocides are used to reduce and control bacterial growth in the healthcare sector, food production and agriculture. This work explores the effect of subinhibitory concentrations of four commonly used biocides (ethanol, hydrogen peroxide, chlorhexidine digluconate...... was determined by an analysis of the transcriptional response of the promoter upstream of tet(M) using β-glucuronidase reporter assays.Results Ethanol significantly (P = 0.01) increased the transfer of Tn916 by 5-fold, whereas hydrogen peroxide, chlorhexidine digluconate and sodium hypochlorite did...

  1. Fluorogenic assay for rapid detection of Escherichia coli in food.


    An assay procedure to screen for Escherichia coli in foods by using 4-methylumbelliferyl-beta-D-glucuronide (MUG) incorporated into lauryl tryptose (LST) broth was evaluated. The beta-glucuronidase produced by E. coli cleaves the MUG substrate to yield a fluorescent end product. E. coli-negative samples can be identified by lack of fluorescence in LST-MUG within 24 h. MUG was not inhibitory to coliforms and E. coli. Over 1,400 food and dairy samples were tested to compare the standard three-t...

  2. Fluorescence assay for evaluating microbicidal activity of hand antiseptics.

    Lopez-Gigosos, Rosa M; Mariscal, Alberto; Mariscal-Lopez, Eloisa; Gutierrez-Bedmar, Mario; Fernandez, Joaquin


    We developed a fluorescent β-d-glucuronidase activity (BGA)-based assay for detecting and quantifying Escherichia coli in samples to assess the biocide efficacy of hand antiseptics. The fluorescence level is proportional to the number of viable E. coli organisms present. We compared our assay results to those of the E. coli plate count method specified by the European standard for testing hygienic hand rub disinfectant products (EN1500). The plate count method requires excessive handling and materials and is not valid if the number of organisms per plate is too low or high for counting in many of the samples. We optimized the fluorescent assay based on the cleavage of 4-methylumbelliferyl-β-d-glucuronide by adding 4-nitrophenyl-β-d-glucuronide, a nonfluorogenic BGA substrate, to induce glucuronidase activity and reduce assay time. Furthermore, our method can be automated and eliminates the need for multiple dilutions. Fluorescence was temporally monitored, and the time required to reach a specific value of fluorescence was correlated with the initial number of viable E. coli organisms on the samples. There was a positive correlation (P counts by plate count and fluorescence methods. Reported effects in fluorescent BGA were compared to the EN1500 plate count method with five hand disinfectants. We found our method more advantageous, because it was as sensitive as the EN1500 method, requires less time to complete, and is less expensive and less laborious than conventional plating techniques.

  3. Operation of an efficient site-specific recombination system of Zygosaccharomyces rouxii in tobacco cells.

    Onouchi, H; Yokoi, K; Machida, C; Matsuzaki, H; Oshima, Y; Matsuoka, K; Nakamura, K; Machida, Y


    Recombinase encoded by the R gene of pSR1 of Zygosaccharomyces rouxii mediates reciprocal recombination between two specific recombination sites (RSs) to induce excision or inversion of the DNA segment that is flanked by the RSs. We report here that site-specific recombination mediated by this system takes place effeciently in tobacco cells. To monitor the recombination events in tobacco cells, we have constructed two types of cryptic beta-glucuronidase reporter gene in such a way that recombination such as inversion of the construct or excision of the intervening sequence results in their expression. When these cryptic reporter constructs were transiently introduced together with the R gene by electroporation into protoplasts of tobacco cells, beta-glucuronidase activity was detected. The cryptic reporter genes, when stably resident in the chromosome of tobacco cells, were also activated by the R gene. Structural analyses of the genomic DNA isolated from these tobacco cells showed that the R protein did in fact catalyze precise recombination between two copies of RSs in tobacco cells, with resultant activation of the cryptic reporter genes. This observation provides the basis for development of a DNA technology whereby large regions of DNA can be manipulated in plant chromosomes. Potential uses of this recombination system are discussed.

  4. Alkyl and phenolic glycosides from Saussurea stella.

    Wang, Tian-Min; Wang, Ru-Feng; Chen, Hu-Biao; Shang, Ming-Ying; Cai, Shao-Qing


    One alkyl glycoside, saussurostelloside A (1), two phenolic glycosides, saussurostellosides B1 (2) and B2 (3), and 27 known compounds, including eleven flavonoids, seven phenolics, six lignans, one neolignan, one phenethyl glucoside and one fatty acid, were isolated from an ethanol extract of Saussurea stella (Asteraceae). Their structures were elucidated by NMR, MS, UV, and IR spectroscopic analysis. Of the known compounds, (+)-medioresinol-di-O-β-D-glucoside (7), picraquassioside C (10), and diosmetin-3'-O-β-D-glucoside (27) were isolated from the Asteraceae family for the first time, while (+)-pinoresinol-di-O-β-D-glucoside (6), di-O-methylcrenatin (11), protocatechuic acid (14), 1,5-di-O-caffeoylquinic acid (17), formononetin (28), and phenethyl glucoside (29) were isolated from the Saussurea genus for the first time. The anti-inflammatory activities of three new compounds (1-3), five lignans ((-)-arctiin (4), (+)-pinoresinol-4-O-β-D-glucoside (5), (+)-pinoresinol-di-O-β-D-glucoside (6), (+)-medioresinol-di-O-β-D-glucoside (7) and (+)-syringaresinol-4-O-β-D-glucoside (8)), one neolignan (picraquassioside C (10)), and one phenolic glycoside (di-O-methylcrenatin (11)) were evaluated by testing their inhibition of the release of β-glucuronidase from PAF-stimulated neutrophils. Only compound 5 showed moderate inhibition of the release of β-glucuronidase, with an inhibition ratio of 39.1%.

  5. Release of lysosomal enzymes in Candida albicans phagocytosis by rat peritoneal macrophages.

    Fontenla de Petrino, S E; Sirena, A


    The present paper reports the in vitro release of lysosomal enzymes in the supernatant of cultures of rat peritoneal macrophages, with the addition of Candida albicans cells. Macrophages were taken from the rat peritoneal cavity 72 hr after non-specific activation with Brain-Heart-Infusion (B.H.I.) broth containing 10% proteose-peptone No. 3. They were then cultured in Parker medium No. 199 (TC 199). After 24 hr a suspension of Candida albicans cells, in a determined concentration, was added to the peritoneal macrophage cultures. At that time, and during pre-determined periods, the following enzymes in the culture supernatants were studied using colorimetric methods: beta-glucuronidase, beta-galactosidase and acid phosphatase. It is concluded that, under identical conditions, the release of beta-galactosidase and acid phosphatase is higher than for beta-glucuronidase. The release rate of all three enzymes is the highest at a 6 hr incubation period, after which, a gradual decrease leads to the rate down to 50% at 24 hr.

  6. Role of intestinal barrier in pathogenesis of pigment gallstone in a guinea pig model

    Yang Su; Shuo-Dong Wu; Jun-Zhe Jin; Zhen-Hai Zhang; Ying Fan


    BACKGROUND:The function of the intestinal barrier has drawn more and more attention from researchers in recent years for its important role in many diseases such as burns, wounds, and pancreatitis. In our experimental studies on pigment gallstone, we found potential relationships between the function of the intestinal barrier and pigment gallstone formation. This study was undertaken to investigate the possible action and mechanism of the function of the intestinal barrier in the pathogenesis of pigment gallstone. METHODS:Eighty guinea pigs were divided into a normal group (CON), a pigment gallstone group (PS) and an intestinal mucosa protection group (GLN). Normal forage, pigment gallstone-forming forage and pigment gallstone-forming forage with supplemental intestinal mucosa protector (glutamine) were given to each group. In the gallstone-forming rate, morphology of intestinal mucosa, intestinal permeability, serum endotoxin and biliaryβ-glucuronidase were assessed after 8 weeks. RESULTS: The rate of gallstone-formation was 73.9% in the PS group. Damage of intestinal mucosa, endotoxemia (from 77±43×10-6 EU/L to 1367±525×10-6 EU/L, P CONCLUSIONS: The function of the intestinal barrier is correlated with pigment gallstone formation. Dysfunction of the intestinal barrier function may promote pigment gallstone formation through bacterial translocation, endotoxemia, and biliaryβ-glucuronidase.

  7. Cholesterol gallstones and bile host diverse bacterial communities with potential to promote the formation of gallstones.

    Peng, Yuhong; Yang, Yang; Liu, Yongkang; Nie, Yuanyang; Xu, Peilun; Xia, Baixue; Tian, Fuzhou; Sun, Qun


    The prevalence of cholesterol gallstones has increased in recent years. Bacterial infection correlates with the formation of gallstones. We studied the composition and function of bacterial communities in cholesterol gallstones and bile from 22 cholesterol gallstone patients using culture-dependent and culture-independent methods. Altogether fourteen and eight bacterial genera were detected in cholesterol gallstones and bile, respectively. Pseudomonas spp. were the dominant bacteria in both cholesterol gallstones and bile. As judged by diversity indices, hierarchical clustering and principal component analysis, the bacterial communities in gallstones were different from those in bile. The gallstone microbiome was considered more stable than that of bile. The different microbial communities may be partially explained by differences in their habitats. We found that 30% of the culturable strains from cholesterol gallstones secreted β-glucuronidase and phospholipase A2. Pseudomonas aeruginosa strains showed the highest β-glucuronidase activity and produced the highest concentration of phospholipase A2, indicating that Ps. aeruginosa may be a major agent in the formation of cholesterol gallstones. Copyright © 2015 Elsevier Ltd. All rights reserved.

  8. Catabolism of salivary glycoconjugates in acute ethanol intoxication.

    Waszkiewicz, Napoleon; Szajda, Sławomir D; Jankowska, Anna; Waszkiewicz, Magdalena; Kepka, Alina; Konarzewska, Beata; Szulc, Agata; Snarska, Jadwiga; Zwierz, Krzysztof


    The aim was to study the effects of a single large dose of ethanol (approximately 2.0 g/kg of body weight, as 40% vodka) on the specific activities of alpha-mannosidase, alpha-fucosidase, beta-glucuronidase, and beta-galactosidase as well as on the total protein concentration in saliva in eight healthy young volunteers. Resting whole saliva samples were collected 12 hours prior to and 36 and 108 hours after alcohol consumption. Exoglycosidase activities were assayed in the supernatants by the colorimetric method. Protein content was determined by the Lowry method. Thirty-six hours after alcohol consumption the specific activities of alpha-fucosidase and beta-glucuronidase were significantly higher than before drinking. The specific activity of beta-galactosidase showed a greater tendency to increase than alpha-mannosidase after the drinking session. The total protein concentration was significantly lower after alcohol consumption than at baseline, even at 108 hr. Significant inverse correlations between total protein content and the specific activities of the exoglycosidases in saliva were found after the drinking session. Acute ingestion of a large dose of ethanol increased the activity of salivary exoglycosidases, which might be followed by subsequent degradation of proteins in saliva. The observed changes might contribute to salivary defense system malfunction as well as to oral malodor production.

  9. Construction of phosphomannose isomerase (PMI) transformation vectors and evaluation of the effectiveness of vectors in tobacco (Nicotiana tabacum L).

    Bahariah, Bohari; Parveez, Ghulam Kadir Ahmad; Masani, Mat Yunus Abdul; Khalid, Norzulaani


    Phosphomannose isomerase (pmi) gene isolated from Escherichia coli allows transgenic plants carrying it to convert mannose-6- phosphate (from mannose), a carbon source that could not be naturally utilized by plants into fructose-6-phosphate which can be utilized by plants as a carbon source. This conversion ability provides energy source to allow the transformed cells to survive on the medium containing mannose. In this study, four transformation vectors carrying the pmi gene alone or in combination with the β-glucuronidase (gusA) gene were constructed and driven by either the maize ubiquitin (Ubi1) or the cauliflower mosaic virus (CaMV35S) promoter. Restriction digestion, PCR amplification and sequencing were carried out to ensure sequence integrity and orientation. Tobacco was used as a model system to study the effectiveness of the constructs and selection system. PMI11G and pMI3G, which carry gusA gene, were used to study the gene transient expression in tobacco. PMI3 construct, which only carries the pmi gene driven by CaMV35S promoter, was stably transformed into tobacco using biolistics after selection on 30 g 1(-1) mannose without sucrose. Transgenic plants were verified using PCR analysis. PMI/pmi - Phosphomannose isomerase, Ubi1 - Maize ubiquitin promoter, CaMV35S - Cauliflower mosaic virus 35S promoter, gusA - β-glucuronidase GUS reporter gene.

  10. Genetic transformation of loblolly pine using mature zygotic embryo explants by Agrobacterium tumefaciens


    Agrobacterium tumefaciens strain LBA 4404 carrying pBI121 plasmid was used to transform mature zygotic embryos of three genotypes (E-Hb, E-Ma, and E-Mc) of loblolly pine. The results demonstrated that the expression frequency of b-glucuronidase reporter gene (GUS) varied among genotypes after mature zygotic em-bryos were infected with Agrobacterium tumefaciens cultures. The highest frequency (27.8%) of GUS expressing embryos was obtained from genotype E-Mc with mean number of 21.9 blue GUS spots per embryo. Expression of b-glucuronidase reporter gene was observed on cotyledons, hypocotyls, and radicles of transformed mature zy-gotic embryos, as well as on organogenic callus and regenerated shoots derived from co-cultivated mature zygotic embryos. Nineteen regenerated transgenic plants were obtained from GUS expression and kanamycin resistant calli. The presence and integration of the GUS gene was confirmed by polymerase chain reaction (PCR) and Southern blot analysis. These results suggested that an efficient Agrobacterium tumefaciens-mediated transfor-mation protocol for stable integration of foreign genes into loblolly pine has been developed and that this transfor-mation system could be useful for the future studies on transferring economically important genes to loblolly pine.

  11. A novel inducible protein production system and neomycin resistance as selection marker for Methanosarcina mazei.

    Mondorf, Sebastian; Deppenmeier, Uwe; Welte, Cornelia


    Methanosarcina mazei is one of the model organisms for the methanogenic order Methanosarcinales whose metabolism has been studied in detail. However, the genetic toolbox is still limited. This study was aimed at widening the scope of utilizable methods in this group of organisms. (i) Proteins specific to methanogens are oftentimes difficult to produce in E. coli. However, a protein production system is not available for methanogens. Here we present an inducible system to produce Strep-tagged proteins in Ms. mazei. The promoter p1687, which directs the transcription of methyl transferases that demethylate methylamines, was cloned into plasmid pWM321 and its activity was determined by monitoring β-glucuronidase production. The promoter was inactive during growth on methanol but was rapidly activated when trimethylamine was added to the medium. The gene encoding the β-glucuronidase from E. coli was fused to a Strep-tag and was cloned downstream of the p1687 promoter. The protein was overproduced in Ms. mazei and was purified in an active form by affinity chromatography. (ii) Puromycin is currently the only antibiotic used as a selectable marker in Ms. mazei and its relatives. We established neomycin resistance as a second selectable marker by designing a plasmid that confers neomycin resistance in Ms. mazei.

  12. A Novel Inducible Protein Production System and Neomycin Resistance as Selection Marker for Methanosarcina mazei

    Sebastian Mondorf


    Full Text Available Methanosarcina mazei is one of the model organisms for the methanogenic order Methanosarcinales whose metabolism has been studied in detail. However, the genetic toolbox is still limited. This study was aimed at widening the scope of utilizable methods in this group of organisms. (i Proteins specific to methanogens are oftentimes difficult to produce in E. coli. However, a protein production system is not available for methanogens. Here we present an inducible system to produce Strep-tagged proteins in Ms. mazei. The promoter p1687, which directs the transcription of methyl transferases that demethylate methylamines, was cloned into plasmid pWM321 and its activity was determined by monitoring β-glucuronidase production. The promoter was inactive during growth on methanol but was rapidly activated when trimethylamine was added to the medium. The gene encoding the β-glucuronidase from E. coli was fused to a Strep-tag and was cloned downstream of the p1687 promoter. The protein was overproduced in Ms. mazei and was purified in an active form by affinity chromatography. (ii Puromycin is currently the only antibiotic used as a selectable marker in Ms. mazei and its relatives. We established neomycin resistance as a second selectable marker by designing a plasmid that confers neomycin resistance in Ms. mazei.

  13. Identification of metabolites in plasma and urine of Uruguayan propolis-treated rats.

    Kumazawa, Shigenori; Shimoi, Kayoko; Hayashi, Katsumi; Ishii, Takeshi; Hamasaka, Tomoko; Nakayama, Tsutomu


    Propolis is a resinous substance collected by honeybees from various plant sources. It is extensively used in food and beverages to improve health and prevent diseases such as heart disease, diabetes, and cancer. To investigate the absorption and metabolism of the components in propolis, in the present study, we administered ethanol extracts of Uruguayan propolis (poplar type propolis) orally to rats and analyzed their plasma and urine by high-performance liquid chromatography with photodiode array and mass spectrometric detection. After deconjugation of the components by beta-glucuronidase/sulfatase treatment of the specimen, pinobanksin 5-methyl ether, pinobanksin, kaempferol, chrysin, pinocembrin, and galangin were detected in plasma of rats orally administered propolis. These compounds were detected also in urine after beta-glucuronidase/sulfatase treatment. Furthermore, pinobanksin 5-methyl ether, pinobanksin, chrysin, pinocembrin, and galangin were present in the urine also in free form. These results suggest that flavonoids in propolis are metabolized and circulate in the body after oral administration of propolis.

  14. The salubrious effect of tamoxifen [correction of Tamaxifen] on serum marker enzymes, glycoproteins, and lysosomal enzymes level in breast cancer woman.

    Thangaraju, M; Rameshbabu, J; Vasavi, H; Ilanchezhian, S; Vinitha, R; Sachdanandam, P


    Tumour markers correlate strongly with prognosis based on tumour burden and surgical resectability. If chemotherapy is extremely effective in certain stage of the disease, the sensitive marker may be of great use in monitoring disease response and drug treatment. Hence, this study was launched to evaluate the changes in tumour marker enzymes like lactate dehydrogenase (LDH), glutamate oxaloacetate transaminase (SGOT), glutamate pyruvate transaminase (SGPT), alkaline phosphatase, and acid phosphatase in before and after 3 and 6 months tamoxifen treated breast cancer patients. In addition, the changes in serum glycoproteins viz., hexose, hexosamine, and sialic acid and lysosomal enzymes such as N-acetyl-beta-D-glucosaminidase, beta-D-galactosidase, and beta-D-glucuronidase were analysed in these patients. These values were compared with their age matched healthy control subjects. At 6 months evaluation, the tamoxifen treated postmenopausal breast cancer women showed a statistically significant decreased (p sialic acid and N-acetyl-beta-D-glucosaminidase, beta-D-galactosidase, and beta-D-glucuronidase were decreased significantly (p breast cancer as well as the bone demineralisation in postmenopausal breast cancer women. Thus, tamoxifen may also have its antitumour activity through its beneficial effects on tumour marker enzymes and serum proteins in breast cancer women.

  15. Protective effects of sinapic acid on lysosomal dysfunction in isoproterenol induced myocardial infarcted rats.

    Roy, Subhro Jyoti; Stanely Mainzen Prince, Ponnian


    In the pathology of myocardial infarction, lysosomal lipid peroxidation and resulting enzyme release play an important role. We evaluated the protective effects of sinapic acid on lysosomal dysfunction in isoproterenol induced myocardial infarcted rats. Male Wistar rats were treated with sinapic acid (12 mg/kg body weight) orally daily for 10 days and isoproterenol (100 mg/kg body weight) was injected twice at an interval of 24 h (9th and 10th day). Then, lysosomal lipid peroxidation, lysosomal enzymes in serum, heart homogenate, lysosomal fraction and myocardial infarct size were measured. Isoproterenol induced myocardial infarcted rats showed a significant increase in serum creatine kinase-MB and lysosomal lipid peroxidation. The activities of β-glucuronidase, β-galactosidase, cathepsin-B and D were significantly increased in serum, heart and the activities of β-glucuronidase and cathepsin-D were significantly decreased in lysosomal fraction of myocardial infarcted rats. Pre-and-co-treatment with sinapic acid normalized all the biochemical parameters and reduced myocardial infarct size in myocardial infarcted rats. In vitro studies confirmed the free radical scavenging effects of sinapic acid. The possible mechanisms for the observed effects are attributed to sinapic acid's free radical scavenging and membrane stabilizing properties. Thus, sinapic acid has protective effects on lysosomal dysfunction in isoproterenol induced myocardial infarcted rats.

  16. Efficacy of boswellic acid on lysosomal acid hydrolases, lipid peroxidation and anti-oxidant status in gouty arthritic mice

    Evan Prince Sabina; Haridas Indu; Mahaboobkhan Rasool


    Objective:To evaluate the efficacy of boswellic acid against monosodium urate crystal-induced inflammation in mice. Methods:The mice were divided into four experimental groups. Group I served as control;mice in group II were injected with monosodium urate crystal;group III consisted of monosodium urate crystal-induced mice who were treated with boswellic acid (30 mg/kg/b.w.);group IV comprised monosodium urate crystal-induced mice who were treated with indomethacin (3 mg/kg/b.w.). Paw volume and levels/activities of lysosomal enzymes, lipid peroxidation, anti-oxidant status and inflammatory mediator TNF-αwere determined in control and monosodium urate crystal-induced mice. In addition, the levels of β-glucuronidase and lactate dehydrogenase were also measured in monosodium urate crystal-incubated polymorphonuclear leucocytes (PMNL) in vitro. Results:The activities of lysosomal enzymes, lipid peroxidation, and tumour necrosis factor-αlevels and paw volume were increased significantly in monosodium urate crystal-induced mice, whereas the activities of antioxidant status were in turn decreased. However, these changes were modulated to near normal levels upon boswellic acid administration. In vitro, boswellic acid reduced the level of β-glucuronidase and lactate dehydrogenase in monosodium urate crystal-incubated PMNL in concentration dependent manner when compared with control cells. Conclusions: The results obtained in this study further strengthen the anti-inflammatory/antiarthritic effect of boswellic acid, which was already well established by several investigators.

  17. [The blood-brain barrier and neurodegenerative lysosomal storage diseases].

    Urayama, Akihiko


    Enzyme replacement therapy has been a very effective treatment for several lysosomal storage diseases. However, correcting central nervous system (CNS) storage has been challenging due to the presence of the blood-brain barrier (BBB), which hampers the entry of circulating lysosomal enzymes into the brain. In our previous studies, we discovered that luminally expressed cation-independent mannose 6-phosphate (M6P) receptor is a universal transporter for lysosomal enzymes that contain M6P moieties on the enzyme molecule. This receptor-mediated transport of lysosomal enzymes showed developmental down-regulation that resulted in a failure of delivery of lysosomal enzymes across the BBB in the adult brain. Conceptually, if one can re-induce M6P receptor-mediated transport of lysosomal enzymes in adult BBB, this could provide a novel brain targeting approach for treating abnormal storage in the CNS, regardless of the age of subjects. We found that systemic adrenergic stimuli restored functional transport of β-glucuronidase across the adult BBB. The concept of manipulating BBB transport activity by endogenous characteristics has also been demonstrated by another group who showed effective treatment in a Pompe disease model animal in vivo. It is intriguing that lysosomal enzymes utilize multiple mechanisms for their transport across the BBB. This review explores pharmacological manipulations for the delivery of lysosomal enzymes into the CNS, and the mechanisms of their transport across the BBB, based on existing evidence from studies of β-glucuronidase, sulfamidase, acid α-glucosidase, and arylsulfatase A.

  18. In vitro anti-inflammatory and xanthine oxidase inhibitory activity of Tephrosia purpurea shoot extract.

    Nile, Shivraj H; Khobragade, Chandrahasy N


    The methanolic extract of Tephrosia purpurea (Leguminosae) shoots was evaluated in-vitro for its anti-inflammatory and xanthine oxidase inhibitory activity. Anti-inflammatory activity was measured by the Diene-conjugate, HET-CAM and beta-glucuronidase methods. The enzyme inhibitory activity was tested against isolated cow milk xanthine oxidase. The average anti-inflammatory activity of T. purpurea shoot extract in the concentration range of 1-2 microg/mL in the reacting system revealed significant anti-inflammatory activities, which, as recorded by the Diene-conjugate, HET-CAM and beta-glucuronidase assay methods, were 45.4, 10.5, and 70.5%, respectively. Screening of the xanthine oxidase inhibitory activity of the extract in terms of kinetic parameters revealed a mixed type of inhibition, wherein the Km and Vmax values in the presence of 25 to 100 microg/mL shoot extract was 0.20 mM/mL and 0.035, 0.026, 0.023 and 0.020 microg/min, while, for the positive control, the Km and Vmax values were 0.21 mM/mL and 0.043 microg/min, respectively. These findings suggest that T. purpurea shoot extract may possess constituents with good medicinal properties that could be exploited to treat the diseases associated with oxidative stress, xanthine oxidase enzyme activity and inflammation.

  19. Equivalency testing of TTC Tergitol 7 agar (ISO 9308-1:2000) with five culture media for the detection of E. coli in water samples in Greece.

    Mavridou, A; Smeti, E; Mandilara, G; Mandilara, G; Boufa, P; Vagiona-Arvanitidou, M; Vantarakis, A; Vassilandonopoulou, G; Pappa, O; Roussia, V; Tzouanopoulos, A; Livadara, M; Aisopou, I; Maraka, V; Nikolaou, E; Mandilara, G


    In this study ten laboratories in Greece compared the performance of reference method TTC Tergitol 7 Agar (with the additional test of beta-glucuronidase production) with five alternative methods, to detect E. coli in water, in line with European Water Directive recommendations. The samples were prepared by spiking drinking water with sewage effluent following a standard protocol. Chlorinated and non-chlorinated samples were used. The statistical analysis was based on the mean relative difference of confirmed counts and was performed in line with ISO 17994. The results showed that in total, three of the alternative methods (Chromocult Coliform agar, Membrane Lauryl Sulfate agar and Trypton Bilex-glucuronidase medium) were not different from TTC Tergitol 7 agar (TTC Tergitol 7 agar vs Chromocult Coliform agar, 294 samples, mean RD% 5.55; vs MLSA, 302 samples, mean RD% 1; vs TBX, 297 samples, mean RD% -2.78). The other two alternative methods (Membrane Faecal coliform medium and Colilert 18/ Quantitray) gave significantly higher counts than TTC Tergitol 7 agar (TTC Tergitol 7 agar vs MFc, 303 samples, mean RD% 8.81; vs Colilert-18/Quantitray, 76 samples, mean RD% 18.91). In other words, the alternative methods generated performance that was as reliable as, or even better than, the reference method. This study will help laboratories in Greece overcome culture and counting problems deriving from the EU reference method for E. coli counts in water samples.

  20. Effects of amoxicillin/clavulanic acid on the pharmacokinetics of valproic acid.

    Lee, Soo-Yun; Huh, Wooseong; Jung, Jin Ah; Yoo, Hye Min; Ko, Jae-Wook; Kim, Jung-Ryul


    Valproic acid (VPA) is mainly metabolized via glucuronide, which is hydrolyzed by β-glucuronidase and undergoes enterohepatic circulation. Amoxicillin/clavulanic acid (AMC) administration leads to decreased levels of β-glucuronidase-producing bacteria, suggesting that these antibiotics could interrupt enterohepatic circulation and thereby alter the pharmacokinetics of VPA. This study aimed to evaluate the effects of AMC on the pharmacokinetics of VPA. This was an open-label, two-treatment, one-sequence study in 16 healthy volunteers. Two treatments were evaluated; treatment VPA, in which a single dose of VPA 500 mg was administered, and treatment AMC + VPA, in which multiple doses of AMC 500/125 mg were administered three times daily for 7 days and then a single dose of VPA was administered. Blood samples were collected up to 48 hours. Pharmacokinetic parameters were calculated using noncompartmental methods. Fifteen subjects completed the study. Systemic exposures and peak concentrations of VPA were slightly lower with treatment AMC + VPA than with treatment VPA (AUClast, 851.0 h·mg/L vs 889.6 h·mg/L; C max, 52.1 mg/L vs 53.0 mg/L). There were no significant between-treatment effects on pharmacokinetics (95% confidence interval [CI]) of AUClast and C max (95.7 [85.9-106.5] and 98.3 [91.6-105.6], respectively). Multiple doses of AMC had no significant effects on the pharmacokinetics of VPA; thus, no dose adjustment is necessary.

  1. Avaliação de ensaios analíticos para detecção de coliformes fecais em queijo Minas

    Pereira M.L.


    Full Text Available Foram submetidas à pesquisa de coliformes fecais, utilizando-se a técnica do número mais provável (NMP/g, 168 amostras de variedades de queijo Minas (20 frescal, 48 canastra e 100 padronizado coletadas em Belo Horizonte. Para a comparação de diferentes ensaios em temperatura elevada, utilizou-se o caldo EC isoladamente, e caldos EC e triptofano em paralelo. Visando à pesquisa de indol foi realizado ensaio para confirmação de produção de beta-D-glucuronidase e indol em caldo fluorocult lauril sulfato. Os resultados demonstraram não haver diferença estatística significativa entre as três metodologias utilizadas para a pesquisa de coliformes fecais, considerando os índices de aceitação definidos pelos padrões legais de inspeção de queijo Minas. A facilidade de execução do ensaio da beta-D-glucuronidase em caldo fluorocult lauril sulfato, associada à confiabilidade dos resultados e demanda de tempo (redução de 96 para até 48h, permitem sugeri-lo como método de escolha para enumeração de coliformes em queijo Minas.

  2. Tissue-specific expression patterns of Arabidopsis NF-Y transcription factors suggest potential for extensive combinatorial complexity.

    Siefers, Nicholas; Dang, Kristen K; Kumimoto, Roderick W; Bynum, William Edwards; Tayrose, Gregory; Holt, Ben F


    All aspects of plant and animal development are controlled by complex networks of transcription factors. Transcription factors are essential for converting signaling inputs, such as changes in daylength, into complex gene regulatory outputs. While some transcription factors control gene expression by binding to cis-regulatory elements as individual subunits, others function in a combinatorial fashion. How individual subunits of combinatorial transcription factors are spatially and temporally deployed (e.g. expression-level, posttranslational modifications and subcellular localization) has profound effects on their control of gene expression. In the model plant Arabidopsis (Arabidopsis thaliana), we have identified 36 Nuclear Factor Y (NF-Y) transcription factor subunits (10 NF-YA, 13 NF-YB, and 13 NF-YC subunits) that can theoretically combine to form 1,690 unique complexes. Individual plant subunits have functions in flowering time, embryo maturation, and meristem development, but how they combine to control these processes is unknown. To assist in the process of defining unique NF-Y complexes, we have created promoter:beta-glucuronidase fusion lines for all 36 Arabidopsis genes. Here, we show NF-Y expression patterns inferred from these promoter:beta-glucuronidase lines for roots, light- versus dark-grown seedlings, rosettes, and flowers. Additionally, we review the phylogenetic relationships and examine protein alignments for each NF-Y subunit family. The results are discussed with a special emphasis on potential roles for NF-Y subunits in photoperiod-controlled flowering time.

  3. Quantitative and qualitative morphologic, cytochemical and ultrastructural characteristics of blood cells in the Crested Serpent eagle and Shikra.

    Salakij, Chaleow; Kasorndorkbua, Chaiyan; Salakij, Jarernsak; Suwannasaeng, Pimsuda; Jakthong, Pattarapong


    The Crested Serpent eagle (Spilornis cheela) is a bird of prey found in the tropical rain forest in Thailand. The Shikra (Accipiter badius) is a sparrow hawk and common resident in Thailand. Blood samples from 9 Crested Serpent eagles and 12 Shikras were obtained from September 2010 to November 2014. They were clinically healthy and negative for blood parasites detectable by light microscopy and molecular techniques (partial cytochrome b gene for avian malaria and partial 18S rRNA gene for trypanosome). Cytochemical staining (Sudan black B, peroxidase, α-naphthyl acetate esterase, and β-glucuronidase) and transmission electron microscopy were performed. Hematological results were reported as the mean ± standard deviation and median. Heterophils were the most prevalent leukocytes in the Crested Serpent eagle, but in the Shikra, lymphocytes were the most prevalent leukocytes. In the Shikra, some vacuoles were observed in the cytoplasm of the eosinophils. All blood cells in both types of raptors stained positively for β-glucuronidase but negatively for peroxidase. The ultrastructure of heterophils showed more clearly differentiate long rod granules in Crested Serpent eagle and spindle-shaped granules in Shikra. The ultrastructure of the eosinophils in the Crested Serpent eagle revealed varied electron-dense, round-shaped granules with round, different electron-dense areas in the centers of some granules, which differed from the structure reported for other raptors. These quantitative results may be useful for clinical evaluations of Crested Serpent eagles and Shikras that are undergoing rehabilitation for release.

  4. Biological measurement of estrogenic activity in urine and bile conjugates with the in vitro ER-CALUX reporter gene assay.

    Legler, Juliette; Jonas, Arjen; Lahr, Joost; Vethaak, A Dick; Brouwer, Abraham; Murk, Albertinka J


    Although estrogens are excreted as biologically inactive conjugates, they can be reconverted to an active form, possibly by bacteria. A simple method was developed to deconjugate estrogen metabolites present in human urine and fish bile back to active estrogens by enzymatic hydrolysis with beta-glucuronidase or live Escherichia coli cells. Deconjugated extracts were tested for estrogenic activity in the in vitro stable estrogen receptor-mediated chemical-activated luciferase gene expression (ER-CALUX) assay. Estrogen glucuronides in urine obtained from human males and females were effectively converted to active forms after incubation with beta-glucuronidase or E. coli. The highest estrogenic activity was found in deconjugated metabolites from urine of a pregnant woman, in which levels up to 3,000 nmol estradiol equivalents per liter of urine were found after overnight incubation of urine with E. coli. Bile sampled from male bream and flounder from various freshwater and marine locations was also deconjugated and a good correlation was found between high biliary estrogenic activity and elevated levels of xenoestrogenic activity in surface water as well as in plasma vitellogenin. Therefore, the measurement of deconjugated bile could form a useful (indirect) biomarker for internal dose of xenoestrogens in male fish.

  5. Analysis of Gastric Juice in Acid Peptic Diseases

    Prasad V. Khodke , , ,


    Full Text Available Background: Peptic ulcer disease is an imbalance between offensive and defensive gastric factors. A bacterium called Helicobacter pylori has been considered a major causative agent for gastric and duodenal ulcers. This is a major cause of mortality in developing countries. Aims and Objectives:The aim of this study was to assess the biochemical parameters in gastric juice of acid peptic disease patients (Study Group and normal healthy individuals (Control Group in humans. Material and Methods: A total of 70 patients suffering peptic ulcer disease with H. pylori infection and 15 non-infected individuals were chosen as control group. Results: We observed an increased significant level (p<0.05 in pH, b-glucuronidase activity, malondialdehyde (MDA and superoxide dismutase (SOD as well as catalase(CAT levels in gastric juice and decrease significant level (p<0.05 in pepsin activity, urea, mucin and nitric oxide level. Conclusion: The present study showed variations in levels of MDA, b-glucuronidase, SOD, CAT activities, pepsin, mucin urea and nitric oxide. Therefore these parameters can be used as additional parameters for diagnosis and prognosis of acid peptic diseases. These can be used by clinicians to adopt treatment strategies in betterment of acid peptic disease patients.

  6. Fluorogenic assay for rapid detection of Escherichia coli in food.

    Moberg, L J


    An assay procedure to screen for Escherichia coli in foods by using 4-methylumbelliferyl-beta-D-glucuronide (MUG) incorporated into lauryl tryptose (LST) broth was evaluated. The beta-glucuronidase produced by E. coli cleaves the MUG substrate to yield a fluorescent end product. E. coli-negative samples can be identified by lack of fluorescence in LST-MUG within 24 h. MUG was not inhibitory to coliforms and E. coli. Over 1,400 food and dairy samples were tested to compare the standard three-tube most-probable-number procedure with the MUG-containing or non-MUG-containing LST procedure. LST-MUG testing detected a greater number of E. coli, with a lower false-positive rate (1.4%) and in a shorter time, than did the standard procedure. All false-positive results in the LST-MUG testing were attributable to beta-glucuronidase-producing staphylococci. No false-negative result was encountered. Use of MUG in LST broth obviates the EC broth step, allowing a 2.5-day procedure to a completed E. coli test versus the present 4- to 6-day standard most-probable-number method.

  7. HT-2 toxin 4-glucuronide as new T-2 toxin metabolite: enzymatic synthesis, analysis, and species specific formation of T-2 and HT-2 toxin glucuronides by rat, mouse, pig, and human liver microsomes.

    Welsch, Tanja; Humpf, Hans-Ulrich


    Glucuronides of the mycotoxin T-2 toxin and its phase I metabolite HT-2 toxin are important phase II metabolites under in vivo and in vitro conditions. Since standard substances are essential for the direct quantitation of these glucuronides, a method for the enzymatic synthesis of T-2 and HT-2 toxin glucuronides employing liver microsomes was optimized. Structure elucidation by nuclear magnetic resonance spectroscopy (NMR) and mass spectrometry revealed that besides T-2 toxin glucuronide and HT-2 toxin 3-glucuronide also the newly identified isomer HT-2 toxin 4-glucuronide was formed. Glucuronidation of T-2 and HT-2 toxin in liver microsomes of rat, mouse, pig, and human was compared and metabolites were analyzed directly by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). A distinct, species specific pattern of glucuronidation of T-2 and HT-2 toxin was observed with interesting interindividual differences. Until recently, glucuronides have frequently been analyzed indirectly by quantitation of the aglycone after enzymatic cleavage of the glucuronides by β-glucuronidase. Therefore, the hydrolysis efficiencies of T-2 and HT-2 toxin glucuronides using β-glucuronidases from Helix pomatia, bovine liver, and Escherichia coli were compared.

  8. Inhibitory effects of Spirulina in zymosan-induced arthritis in mice

    Diadelis Remirez


    Full Text Available The anti-inflammatory effect of microalgae Spirulina was studied in zymosan-induced arthritis in mice. Four days after the intra-articular injection of zymosan (15 mg/ml, Spirulina (100 and 400 mg/kg per-orally was administered to animals for 8 days. The mice were than killed and β-glucuronidase was measured in the synovial fluid. Each knee joint was totally removed for histopathological studies. Spirulina significantly reduced the levels of β-glucuronidase that had been increased by zymosan. Histopathological and ultrastructural studies showed inhibition of the inflammatory reaction, whereas no destruction of cartilage, well-preserved chondrocytes, and normal rough endoplasmic reticulum and mitochondria were seen. The anti-arthritic effect exerted by Spirulina as shown in this model may be at least partly due to the previously reported anti-inflammatory and antioxidative properties of its constituent, phycocyanin. To our knowledge, this is the first report on the anti-inflammatory effect of Spirulina in an experimental model of arthritis.

  9. A rapid and stable Agrobacterium-mediated transformation method of a medicinal plant Chelone glabra L.

    Gao, Zhenrui; Li, Ying; Chen, Jinhua; Chen, Zhixing; Cui, Min-Long


    Transformation approach is a useful tool for the study of gene function, the mechanism of molecular regulation, and increase usefulness of components by reverse genetic approach in plants. In this study, we developed a stable and rapid method for Agrobacterium-mediated transformation of a medicinal plant Chelone glabra L. using leaf explants. Stable transformants were obtained using Agrobacterium tumefaciens strains GV2260 and GV3101 that harbored the binary vector pBI121 and contained the neomycin phosphotransferase gene (NPT II) as a selectable marker and a reporter gene β-glucuronidase (GUS). Putative transformants were identified by kanamycin selection and a histochemical assay. PCR and Southern blot analysis confirmed the integration of the GUS gene into transformed genomes as well as detected stable expression of the β-glucuronidase gene (GUS) by RT-PCR. Resulting transformed plants had morphologically normal phenotypes. This method requires two changes of medium and few leaf explants as well as the transformation efficiency of 2-8 % after 2-3 months of inoculation. This method can provide a quick and economical transformation method for reverse genetic approach to change the secondary metabolic pathway to increase useful components in C. glabra.

  10. Do selenium and glutathione inhibit the toxic effects of mercury in marine lamellibranchs?

    Patel, B; Chandy, J P; Patel, S


    The effect of selenium (SeO2) and glutathione (GSH) on the bioaccumulation of mercury (HgCl2) and on the activities of lysosomal enzymes in four species of tropical estuarine lamellibranchs is reported. A definite correlation between mercury levels in the external medium and tissue uptake and physiological behaviour--opening and closing of shell valves, response to mechanical stimulus, mucus secretion, and incidence of bleeding--was evident. In the clams exposed to Hg (range 0.1-5.0 mg l-1), bioaccumulation was dependent on the ambient concentration of Hg. The highest bioaccumulation of Hg occurred during the initial 24 h exposure period. Further exposure of up to 7 days did not increase the body burden of Hg. Of the four bivalve species exposed to 0.1 mg Hg l-1, Perna viridis showed the highest levels of Hg (approximately 47 ppm) followed by Anadara granosa, A. rhombea (approximately 25 ppm) and Meretrix casta (approximately 9 ppm). The uptake of Hg by A. granosa was greatly reduced by GSH, whereas Se enhanced it by 50% when administered in combination with Hg. However, the presence of Hg did not influence the uptake of Se. Exposure to combined GSH and Hg resulted in almost complete inhibition of Hg uptake in all four bivalve species. Prior exposure to GSH, however, did not have the same influence on their uptake of Hg. Nevertheless, exposure of clams to GSH following initial exposure to Hg resulted in complete depuration of accumulated Hg. The activities of lysosomal enzymes--arylsulfatase, acid phosphatase, beta-galactosidase and beta-glucuronidase--varied considerably. Treatment with Hg and GSH, separately and in combination, significantly enhanced the levels of beta-galactosidase (P less than 0.05) and beta-glucuronidase (P less than 0.001) in the digestive gland after 96 h exposure. Although Se increased beta-glucuronidase activity (P less than 0.001), it had no effect on beta-galactosidase. On exposure to Hg + Se the activity of both enzymes decreased

  11. Transformation of Lesquerella Fendleri with the New Binary Vector pGPro4-35S

    Grace Q. Chen


    Full Text Available Problem statement: Crop genetic engineering requires the use of various promoters to control the expression of introduced transgenes. Some of the binary vectors currently available for promoter characterization in dicotyledonous plants have pitfalls due to their construction, such as containing a selectable marker cassette with enhancer sequences that can potentially interfere with the expression specificity of nearby promoters. Also, many binary vectors are quite large in size and contain few useful restriction sites making their in vitro manipulation technically challenging. Approach: A small (7698 bp and flexible binary vector named pGPro4 was constructed to possess unique features favorable for promoter analysis in dicot plants. A nopaline synthase (nos promoter was used to control the expression of the selectable marker of pGPro4 to prevent the problem of interference with the neighboring promoter-reporter fusion. In pGPro4, the nos promoter and hygromycin phosphotransferase II (hptII sequences are flanked by loxP sites, which allow for Cre recombinase-mediated removal when hygromycin resistance is no longer desired. pGPro4 also contains a bifunctional β-glucuronidase-enhanced Green Fluorescent Protein (gusA-eGFP reporter gene that provides visual detection of reporter gene expression using either fluorescence in live cells or histochemical detection of β-glucuronidase activity. Results and Conclusion: To demonstrate the usefulness of the pGPro4 vector, a CaMV35S promoter was fused to gusA-eGFP and the resulting plasmid, pGPro4-35S, was used to transform Lesquerella fendleri. Primary shoots were generated from explants at an expected frequency of 10-27.5%, indicating that the nos promoter drove sufficient hptII expression to generate hygromycin resistant plants. Six independent transgenic L. fendleri lines were grown to maturity and generated T1 seeds. The bifunctionality of the gusA-eGFP reporter gene was verified by detecting both green

  12. Flunitrazepam excretion patterns using the Abuscreen OnTrak and OnLine immunoassays: comparison with GC-MS.

    Salamone, S J; Honasoge, S; Brenner, C; McNally, A J; Passarelli, J; Goc-Szkutnicka, K; Brenneisen, R; elSohly, M A; Feng, S


    A study was conducted to compare the performance of the OnLine and OnTrak immunoassays for benzodiazepines with gas chromatographic-mass spectrometric (GC-MS) analysis in detecting flunitrazepam (FNP) and its metabolites in human urine. Urine was collected over a 72-h period from six individuals (four male and two female) who had taken a single oral dose of either 1 or 4 mg of FNP. The OnTrak assay was run at a 100-ng/mL cutoff of nordiazepam (NDP), and the OnLine assay was run with a standard curve from zero to 200 ng/mL of NDP with and without beta-glucuronidase treatment. Each sample was analyzed by GC-MS using FNP, 7-amino-FNP, 3-hydroxy-FNP, desmethyl-FNP, 7-amino-3-hydroxy-FNP, and desmethyl-3-hydroxy-FNP as standards with beta-glucuronidase treatment. The specimens from the 1-mg dose did not yield a positive result by immunoassay over the 72-h collection period. Specimens from the 4-mg dose did yield positive results in both immunoassays. The time of the first positive result ranged from 4 to 12 h, and the time to the last positive result ranged from 18 to 60 h. Treatment of the samples with beta-glucuronidase increased the OnLine values between 20 and 60%, but it did not appreciably increase the detection time. GC-MS analysis showed no detectable levels of FNP, 3-hydroxy-FNP, desmethyl-FNP, 7-amino-3-hydroxy-FNP, and desmethyl-3-hydroxy-FNP. However, all samples collected past time zero showed detectable levels of 7-amino-FNP (> 2 ng/mL) with peak concentrations at 12-36 h. The peak levels of 7-amino-FNP by GC-MS paralleled the peak levels of the immunoassay response. The amount of 7-amino-FNP metabolite quantitated by GC-MS, however, accounted for only 15-20% of the total immunoassay crossreactive FNP metabolites.

  13. The influence of Lactobacillus casei DN 114 001 on the activity of faecal enzymes and genotoxicity of faecal water in the presence of heterocyclic aromatic amines.

    Nowak, Adriana; Śliżewska, Katarzyna; Błasiak, Janusz; Libudzisz, Zdzisława


    High activity of bacterial enzymes in human colon and genotoxicity of faecal water (FW) are biomarkers of the harmful action of microbiota. The aim of the present study was to assess the activity of β-glucuronidase and β-glucosidase and the genotoxicity of FW in vitro after incubation with 2-amino-3-methyl-3H-imidazo[4,5-f]quinoline (IQ) or 2-amino-1-methyl-6-phenyl-1H-imidazo[4,5-b]pyridine (PhIP) and probiotic Lactobacillus casei DN 114 001 (Actimel). Our results indicate, that IQ and PhIP greatly increased the activity of faecal enzymes (it was up to four times higher, as measured by spectrophotometric methods) and the genotoxicity of FW (% DNA in the tail was up to 3.2 times higher, as evaluated by the comet assay on Caco-2 cells) in 15 individuals from three age-dependent groups (breast-fed children, adults aged 30-40 years, elderly aged 75-85 years). Lb. casei DN 114 001 decreased the activity of faecal enzymes and the genotoxicity of FW exposed to PhIP and IQ mostly to control values. The activity of faecal enzymes after incubation with IQ was reduced by 71.8% in the FW of children, 37.5% in adults and 64.2% in elderly (β-glucuronidase); as well as by 59.9% in children and 87.9% in elderly (β-glucosidase). For PhIP the reduction was by 59.0% in the FW of children, 50.0% in adults and 81.2% in elderly (β-glucuronidase) and by 20.2% in children, 20.7% in adults and 84.1% in elderly (β-glucosidase). Lb. casei DN 114 001 also decreased the genotoxicity of FW to the greatest extent in adults after incubation with IQ (by 65.4%) and PhIP (by 69.6%) and it was found to correlate positively with the decrease in faecal enzymes activity. In conclusion, Lb. casei DN 114 001 may exert the protective effects against genotoxic and possibly pro-carcinogenic effects of food processing-derived chemicals present in faecal water. Copyright © 2014. Published by Elsevier Ltd.

  14. The reversed terminator of octopine synthase gene on the Agrobacterium Ti plasmid has a weak promoter activity in prokaryotes.

    Shao, Jun-Li; Long, Yue-Sheng; Chen, Gu; Xie, Jun; Xu, Zeng-Fu


    Agrobacterium tumefaciens transfers DNA from its Ti plasmid to plant host cells. The genes located within the transferred DNA of Ti plasmid including the octopine synthase gene (OCS) are expressed in plant host cells. The 3'-flanking region of OCS gene, known as OCS terminator, is widely used as a transcriptional terminator of the transgenes in plant expression vectors. In this study, we found the reversed OCS terminator (3'-OCS-r) could drive expression of hygromycin phosphotransferase II gene (hpt II) and beta-glucuronidase gene in Escherichia coli, and expression of hpt II in A. tumefaciens. Furthermore, reverse transcription-polymerase chain reaction analysis revealed that an open reading frame (ORF12) that is located downstream to the 3'-OCS-r was transcribed in A. tumefaciens, which overlaps in reverse with the coding region of the OCS gene in octopine Ti plasmid.

  15. Improved heterologous gene expression in Trichoderma reesei by cellobiohydrolase I gene (cbh1) promoter optimization

    Ti Liu; Tianhong Wang; Xian Li; Xuan Liu


    To improve heterologous gene expression in Trichoderma reesei, a set of optimal artificial cellobiohydrolase I gene (cbh1) promoters was obtained. The region from-677 to -724 with three potential glucose repressor binding sites was deleted. Then the region from-620 to-820 of the modified cbh1 promoter, including the CCAAT box and the Ace2 binding site, was repeatedly inserted into the modified cbh1 promoter, obtaining promoters with copy numbers 2, 4,and 6. The results showed that the glucose repression effects were abolished and the expression level of the glucuronidase (gus) reporter gene regulated by these multi-copy promoters was markedly enhanced as the copy number increased simultaneously. The data showed the great promise of using the promoter artificial modification strategy to increase heterologous gene expression in filamentous fungi and provided a set of optional high-expression vectors for gene function investigation and strain modification.

  16. Isolation of a pollen-specific promoter in tritordeum

    Tu Zhiming; Zhang Jiangzhou; Chen Lin; Chuan Qin; Yang Guangxiao; He Guangyuan


    The promoter is a cis-acting element in regulating gene expression. A promoterless plasmid containing UidA gene was transformed into tritordeum by barmbadment. Histochemical analysis of various tissues in transgenic tritordeum was carried to examine tissue-specific expression of GUS(beta-glucuronidase) activity. The pollen-specific promoter was trapped and identified successfully in a transformant line. PCR(polymerase chain reaction) method was used to isolate this pollen-specific promoter. By sequencing and analyzing the amplified fragment from PCR, a part of UidA gene and a flanking sequence were obtained. Some essential elements of plant promoters were found in the sequence. To determine the function of it, the cloned fragment was fused with UidA gene, then cloned and transformed into Triticum durum. The transgenic plant transformed by this vector showed GUS expression only in pollen. Therefore a pollen-specific promoter was isolated successfully.

  17. Analysis of tomato gene promoters activated in syncytia induced in tomato and potato hairy roots by Globodera rostochiensis.

    Wiśniewska, A; Dąbrowska-Bronk, J; Szafrański, K; Fudali, S; Święcicka, M; Czarny, M; Wilkowska, A; Morgiewicz, K; Matusiak, J; Sobczak, M; Filipecki, M


    The potato cyst nematode (Globodera rostochiensis) induces feeding sites (syncytia) in tomato and potato roots. In a previous study, 135 tomato genes up-regulated during G. rostochiensis migration and syncytium development were identified. Five genes (CYP97A29, DFR, FLS, NIK and PMEI) were chosen for further study to examine their roles in plant-nematode interactions. The promoters of these genes were isolated and potential cis regulatory elements in their sequences were characterized using bioinformatics tools. Promoter fusions with the β-glucuronidase gene were constructed and introduced into tomato and potato genomes via transformation with Agrobacterium rhizogenes to produce hairy roots. The analysed promoters displayed different activity patterns in nematode-infected and uninfected transgenic hairy roots.

  18. Specimen block counter-staining for localization of GUS expression in transgenic arabidopsis and tobacco

    Kim, M. K.; Choi, J-W; Jeon, J-H; Franceschi, V. R.; Davin, L. B.; Lewis, N. G.


    A simple counter-staining procedure has been developed for comparative beta-glucuronidase (GUS) expression and anatomical localization in transgenic herbaceous arabidopsis and tobacco. This protocol provides good anatomical visualization for monitoring chimeric gene expression at both the organ and tissue levels. It can be used with different histochemical stains and can be extended to the study of woody species. The specimens are paraffin-embedded, the block is trimmed to reveal internal structure, safranin-O staining solution is briefly applied to the surface of the block, then washed off and, after drying, a drop of immersion oil is placed on the stained surface for subsequent photographic work. This gives tissue counter-staining with good structural preservation without loss of GUS staining product; moreover, sample observation is rapid and efficient compared to existing procedures.

  19. Three new compounds from Morus nigra L.

    Wang, Lei; Yang, Yan; Liu, Chao; Chen, Ruo-Yun


    A new 2-arylbenzofuran derivative, mornigrol D (1), along with two new flavones, mornigrol G (2) and mornigrol H (3), and six known compounds, norartocarpetin (4), dihydrokaempferol (5), albanin A (6), albanin E (7), moracin M (8), and albafuran C (9), were isolated from the barks of Morus nigra. Their structures were elucidated by spectroscopic analysis. Compounds 1 and 9 showed antioxidative activities in vitro with inhibition ratios of 98 and 99% at the concentration of 10(-4) mol/l, and of 74 and 75% at the concentration of 10(-5) mol/l. In addition, compounds 1 and 4 showed potent anti-inflammatory activities (inhibition of release of beta-glucuronidase from rat polymorphonuclear leucocytes induced by platelet activating factor) with inhibitory ratios of 65.9% (P < 0.01) and 67.7% (P < 0.01) at a concentration of 10(-5) mol/l.

  20. Specific degradation of 3' regions of GUS mRNA in posttranscriptionally silenced tobacco lines may be related to 5'-3' spreading of silencing

    Braunstein, Thomas Hartig; Moury, Benoit; Johannessen, Marina;


    Target regions for posttranscriptional silencing of transgenes often reside in the 3' region of the coding sequence, although there are exceptions. To resolve if the target region is determined by the gene undergoing silencing rather than by the structure of the transgene loci or the plant genetic...... background, we have performed detailed analyses of target regions in three spontaneously beta-glucuronidase (GUS) silencing tobacco lines of different origin. From quantitative cosuppression experiments, we show that the main target region in all three tobacco lines is found within the 3' half of the GUS...... coding region but upstream of the last 200 nt. The quantities of small (21-25 nt) RNAs homologous to 5' or 3' regions of the GUS coding sequence were found to correlate approximately with the target strength of the corresponding regions. These results suggest that transgene locus structure and plant...

  1. Selection of Arabidopsis mutants overexpressing genes driven by the promoter of an auxin-inducible glutathione S-transferase gene.

    van der Kop, D A; Schuyer, M; Pinas, J E; van der Zaal, B J; Hooykaas, P J


    Transgenic arabidopsis plants were isolated that contained a T-DNA construct in which the promoter of an auxin-inducible glutathione S-transferase (GST) gene from tobacco was fused to the kanamycin resistance (nptII) as well as to the beta-glucuronidase (gusA) reporter gene. Subsequently, seeds were treated with EMS to obtain mutants in which both reporter gene fusions were up-regulated. Northern analysis showed that the mRNA level of a related, endogenous auxin-inducible GST gene of Arabidopsis was increased in some of these mutants as well. Two of the gup (GST up-regulated) mutants were characterized in more detail and roughly mapped. Both had epinastic cotyledons and leaves, a phenotype that turned out to be linked to the gup mutation.

  2. The ABNORMAL GAMETOPHYTES (AGM) gene product of Arabidopsis demonstrates a role in mitosis during gamete development.

    Sorensen, Anna-Marie; Kroeber, Sandra; Saedler, Heinz


    Screening a T-DNA mutagenized population of Arabidopsis thaliana for reduced seed set and segregation distortion led to the isolation of the ABNORMAL GAMETOPHYTES (AGM) mutant. Homozygous plants were never recovered, but heterozygous plants showed mitotic defects during gametogenesis resulting in approximately 50% abortion of both the male and female gametes. Isolation of the genomic sequence flanking the co-segregating T-DNA element led to the identification of a gene located on chromosome 5, predicted to encode a transmembrane protein. BLAST homology searches identified two homologous proteins that are not redundant, as is clear from the existence of the agm mutant. Unexpectedly, expression studies using the beta-glucuronidase reporter gene suggest that AGM and its closest Arabidopsis homolog are mostly expressed in cells undergoing mitosis. Thus, AGM is not a gametophytic gene as originally speculated on the basis of segregation distortion, but rather classified as an essential gene crucial to the process of mitosis in plants.

  3. Effect of Liposome Size on Internal RNA Replication Coupled with Replicase Translation.

    Sunami, Takeshi; Ichihashi, Norikazu; Nishikawa, Takehiro; Kazuta, Yasuaki; Yomo, Tetsuya


    Cell membranes inhibit the diffusion of intracellular materials, and compartment size can strongly affect the intracellular biochemical reactions. To assess the effect of the size of microcompartments on intracellular reactions, we constructed a primitive cell model consisting of giant liposomes and a translation-coupled RNA replication (TcRR) system. The RNA was replicated by Qβ replicase, which was translated from the RNA in giant liposomes encapsulating the cell-free translation system. A reporter RNA encoding the antisense strand of β-glucuronidase was introduced into the system to yield a TcRR read-out (green fluorescence). We demonstrate that TcRR was hardly detectable in larger liposomes (230 fL) but was more effective in smaller (7.7 fL) liposomes. Our experimental and theoretical results show that smaller microcompartments considerably enhance TcRR because the synthesized molecules, such as RNA and replicases, are more concentrated in smaller liposomes.

  4. Agrobacterium-mediated transformation and assessment of factors influencing transgene expression in loblolly pine (Pinus taeda L.)


    This investigation reports a protocol for transfer and expression of foreign chimeric genes in loblolly pine (Pinus taeda L.). Transformation was achieved by co-cultivation of mature zygotic embryos with Agrobacterium tumefaciens strain LBA4404 which harbored a binary vector (pBI121) including genes for 3-glucuronidase (GUS) and neomycin phosphotransferase (NPTI1). Factors influencing transgene expression including seed sources of loblolly pine, concentration of bacteria, and the wounding procedures of target explants were investigated. The expression of foreign gene was confirmed by the ability of mature zygotic embryos to produce calli in the presence of kanamycin, by histochemical assays of GUS activity, by PCR analysis, and by Southern blot. The successful expression of the GUS gene in different families of loblolly pine suggests that this transformation system is probably useful for the production of the genetically modified conifers.

  5. The abnormal isoform of the prion protein accumulates in late-endosome-like organelles in scrapie-infected mouse brain.

    Arnold, J E; Tipler, C; Laszlo, L; Hope, J; Landon, M; Mayer, R J


    The prion encephalopathies are characterized by accumulation in the brain of the abnormal form PrPsc of a normal host gene product PrPc. The mechanism and site of formation of PrPsc from PrPc are currently unknown. In this study, ME7 scrapie-infected mouse brain was used to show, both biochemically and by double-labelled immunogold electron microscopy, that proteinase K-resistant PrPsc is enriched in subcellular structures which contain the cation-independent mannose 6-phosphate receptor, ubiquitin-protein conjugates, beta-glucuronidase, and cathepsin B, termed late endosome-like organelles. The glycosylinositol phospholipid membrane-anchored PrPc will enter such compartment for normal degradation and the organelles may therefore act as chambers for the conversion of PrPc into infectious PrPsc in this murine model of scrapie.

  6. Identification of seed-specific promoter nap300 and its comparison with 7S promoter


    By fusing seed-specific promoter nap300 with β-glucuronidase gene, it was found that this about 300bp DNA fragment was sufficient to direct seed-specific gene expression. The substitution mutation in both distB and proxB elements had a little effect on the expression efficiency and almost no effect on the organ-specific expression pattern. In the experiment designed to compare nap300 with 7S promoter, the result showed that tissue specificity for nap300 was higher than that for 7S, and its expression level was lower than 7S's. There was no big difference in their expression pattern, and the maximal activity stage for the two promoters was identical, which indicated they could be used simultaneously for expressing different foreign genes in seeds.

  7. Evaluation on Inducible Effect of pOp6 Promoter in Transgenic Rice

    Rouyi CHEN; Changxiang ZHENG; Jiang CHENG; Minna PAN; Mariena KETUDAT-CAIRNS


    In order to analyze the Os1bglu4 phenotype, the inducible promoter of the transgenic rice which knock-down the Os1bglu4 expression was assessed. The re-sult showed that 30 μM dexamethasone(DEX) had the stronger induction effect than 10 μM DEX by β-Glucuronidase (GUS) staining. qRT-PCR further verified the Os1bglu4 gene deletion. The effect of DEX and its solvent absolute ethanol on seed development was measured, and no significant effect was observed. The con-clusion is that final concentration of DEX at 30 μM is suitable for pOp6 promoter in-duction.

  8. A simple and efficient Agrobacterium-mediated procedure for transformation of tomato.

    Sharma, Manoj K; Solanke, Amolkumar U; Jani, Dewal; Singh, Yogendra; Sharma, Arun K


    We describe a highly efficient and reproducible Agrobacterium-mediated transformation protocol applicable to several varieties of tomato (Solanum lycopersicum, earlier known as Lycopersicum esculentum). Conditions such as co-cultivation period, bacterial concentration, concentration of benzyl amino purine (BAP), zeatin and indole acetic acid (IAA) were optimized. Co-cultivation of explants with a bacterial concentration of 108 cells/ml for three days on 2 mg/l BAP, followed by regeneration on a medium containing 1 mg/ml zeatin resulted in a transformation frequency of 41.4%. Transformation of tomato plants was confirmed by Southern blot analysis and beta-glucuronidase (GUS) assay. The protocol developed showed very high efficiency of transformation for tomato varieties Pusa Ruby, Arka Vikas and Sioux. The optimized transformation procedure is simple, efficient and does not require tobacco, Petunia, tomato suspension feeder layer or acetosyringone.

  9. 2-Amino-1-methyl-6-(5-hydroxy-)phenylimidazo[4,5-b]pyridine (5-OH-PhIP), a biomarker for the genotoxic dose of the heterocyclic amine, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)

    Frandsen, H; Frederiksen, H; Alexander, J


    resulting in formation of adducts, and with proteins and other cellular constituents resulting in unstable products which are degraded to 5-hydroxy-PhIP. Rats were dosed orally with PhIP and urine and faeces were collected to 24, 48 and 72 h. Urine and faeces samples were hydrolysed with glucuronidase......IP. In a preliminary study, using molecular imprinted polymer a specific sorbent for purification, after enzymatic hydrolysis and purification on a C18 column, we have identified 5-OH-PhIP in a 24-h urine sample from a male volunteer who had ingested a fried beef. This indicates that urinary 5-OH-PhIP could be used...

  10. Hydrolysis of soy isoflavone conjugates using enzyme may underestimate isoflavone concentrations in tissue

    Hebron C. Chang; Myriam Laly; Melody Harrison; Thomas M. Badger


    Objective: To investigate the differences of using enzymatic hydrolysis and acid hydrolysis for identification and quantification of isoflavone aglycones from biomatrices. Methods: β-glucuronidase/sulfatase isolated from Helix pomatia for routine enzymatic hydrolysis or 6N HCl was used to release glucuronide and sulfate conjugates in the serum, urine and tissue samples. Profiles of soy isoflavones after enzymatic hydrolysis or acid hydrolysis in several tissues of rat fed with diets containing soy protein isolate were also compared using LC/MS and HPLC-ECD. Results: Acid hydrolysis released more aglycone than enzymatic digestion ( P <0.05) in liver tissue. The total genistein, daidzein and other metabolites were 20% to 60% lower in samples from enzymatic hydrolysis than in acid hydrolysis. Conclusion: These results indicated that unknown factors in tissues reduced the enzymatic hydrolytic efficiency for releasing isoflavone aglycones even in optimized condition. This would underestimate isoflavone tissue concentrations up to 60%.

  11. Agrobacterium tumefaciens-mediated GUS gene transformation of Robinia pseudoacacia 'Idaho'

    Sun Hai-jun; Li Min; Chen Shou-yi; He Si-jie; Wang Hua-fang


    Based on the plant regeneration system, a GUS gene transformation system to Idaho locust (Robinia pseudoacacia 'Idaho')mediated by Agrobacterium tumefaciens was established. The successful transformation was confirmed by regenerating the shoots from the infected leaves in the presence of hygromysin; by histochemical X-gluc assays of β-glucuronidase (GUS) and by PCR and PCR-Southern blotting analysis. The ratio of positive transgenic plants is 5.8% (5 out of 86 plants). With this system, the target gene DREB was introduced into the leaves of Idaho locust. The transgenic plants regenerated, which was verified by PCR-Southem blotting. It is suggested that the transformation system could be a new, simple, reliable and practical route to gene transformation of R.pseudoacacia 'Idaho' mediated with A. tumefaciens.

  12. [Isolation and physico-chemical characteristics of human cancerocerebral antigen].

    Prokopenko, P G; Borisenko, S A; Tatarinov, Iu S


    During gel filtration on Sephadex G-200 human cancerocerebral antigen (CCA) was eluted as two protein fractions with molecular mass of 135,000 and 270.000 daltons. Only one band of protein with molecular mass of about 15,000 daltons was noted after electrophoresis in 10% polyacrylamide gel containing SDS. As characteristic properties of CCA were recognized an electrophoretic polymorphism and a distinct trend to polymerization and isomeria. The antigen was not stained with dyes designed for staining base proteins, lipo-,glyco- and ferroproteins; CCA was thermostable (5 min at 80 degrees), it was inactivated by trypsin and protease but was resistant to pronase, hexokinase, alpha-amylase and beta-glucuronidase. A procedure was developed for isolation of CCA from brain, including fractionation with ammonium sulfate, ion exchange chromatography on DEAE-Sephadex A-50. The procedure enabled to obtain the CCA preparations suitable for radioimmunological, immunobiological assays and amino acid analyses.

  13. Arabidopsis DNA methyltransferase AtDNMT2 associates with histone deacetylase AtHD2s activity

    Song, Yuan [Key Laboratory of Arid and Grassland Agroecology, Ministry of Education, School of Life Science, Lanzhou University, Lanzhou 730000 (China); Southern Crop Protection and Food Research Centre, Agriculture and Agri-Food Canada, 1391 Sandford Street, London, ON, Canada N5V4T3 (Canada); Wu, Keqiang [Institute of Plant Biology, National Taiwan University, Taipei 106, Taiwan (China); Dhaubhadel, Sangeeta [Southern Crop Protection and Food Research Centre, Agriculture and Agri-Food Canada, 1391 Sandford Street, London, ON, Canada N5V4T3 (Canada); An, Lizhe, E-mail: [Key Laboratory of Arid and Grassland Agroecology, Ministry of Education, School of Life Science, Lanzhou University, Lanzhou 730000 (China); Tian, Lining, E-mail: [Southern Crop Protection and Food Research Centre, Agriculture and Agri-Food Canada, 1391 Sandford Street, London, ON, Canada N5V4T3 (Canada)


    DNA methyltransferase2 (DNMT2) is always deemed to be enigmatic, because it contains highly conserved DNA methyltransferase motifs but lacks the DNA methylation catalytic capability. Here we show that Arabidopsis DNA methyltransferase2 (AtDNMT2) is localized in nucleus and associates with histone deacetylation. Bimolecular fluorescence complementation and pull-down assays show AtDNMT2 interacts with type-2 histone deacetylases (AtHD2s), a unique type of histone deacetylase family in plants. Through analyzing the expression of AtDNMT2: ss-glucuronidase (GUS) fusion protein, we demonstrate that AtDNMT2 has the ability to repress gene expression at transcription level. Meanwhile, the expression of AtDNMT2 gene is altered in athd2c mutant plants. We propose that AtDNMT2 possibly involves in the activity of histone deacetylation and plant epigenetic regulatory network.

  14. In vitro glucuronidation of ochratoxin a by rat liver microsomes.

    Han, Zheng; Tangni, Emmanuel K; Di Mavungu, José Diana; Vanhaecke, Lynn; De Saeger, Sarah; Wu, Aibo; Callebaut, Alfons


    Ochratoxin A (OTA), one of the most toxic mycotoxins, can contaminate a wide range of food and feedstuff. To date, the data on its conjugates via glucuronidation request clarification and consolidation. In the present study, the combined approaches of ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS), UHPLC-Orbitrap-high resolution mass spectrometry (HRMS) and liquid chromatography-multiple stage mass spectrometry (LC-MS(n)) were utilized to investigate the metabolic profile of OTA in rat liver microsomes. Three conjugated products of OTA corresponding to amino-, phenol- and acyl-glucuronides were identified, and the related structures were confirmed by hydrolysis with β-glucuronidase. Moreover, OTA methyl ester, OTα and OTα-glucuronide were also found in the reaction solution. Based on these results, an in vitro metabolic pathway of OTA has been proposed for the first time.

  15. 核基质结合区在转基因烟草中提高报导基因的表达水平%Matrix Attachment Regions(MARs) Can Increase Expression Levels of Reporter Gene in Transgenic Tobaccos

    李旭刚; 朱祯; 徐军望; 徐鸿林; 肖桂芳


    从豌豆基因组中分离了位于质体蓝素(Plastocyanin)基因下游的核基质结合区(Matrix attachment region,MAR).为进一步研究此段MAR序列在转基因植物中对外源基因表达水平的影响,将MAR序列构建在报导基因β-葡糖醛酸酶(β-glucuronidase, GUS)基因和植物选择标记新霉素磷酸转移酶(Neomycin phosphotransferase, NPT-II)基因的两侧形成T-DNA结构.采用叶盘法,经农杆菌介导转化烟草.GUS定量检测表明,MAR的存在使GUS基因的平均表达水平提高了2倍.

  16. Guanidinylated neomycin mediates heparan sulfate-dependent transport of active enzymes to lysosomes.

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


    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, beta-D-glucuronidase (GUS) and alpha-L-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.

  17. Identification of a root-specific glycosyltransferase from Arabidopsis and characterization of its promoter

    Virupapuram Vijaybhaskar; Veeraputhiran Subbiah; Jagreet Kaur; Pagadala Vijayakumari; Imran Siddiqi


    A set of Ds-element enhancer trap lines of Arabidopsis thaliana was generated and screened for expression patterns leading to the identification of a line that showed root-specific expression of the bacterial uidA reporter gene encoding -glucuronidase (GUS). The insertion of the Ds element was found to be immediately downstream to a glycosyltransferase gene At1g73160. Analysis of At1g73160 expression showed that it is highly root-specific. Isolation and characterization of the upstream region of the At1g73160 gene led to the definition of a 218 bp fragment that is sufficient to confer root-specific expression. Sequence analysis revealed that several regulatory elements were implicated in expression in root tissue. The promoter identified and characterized in this study has the potential to be applied in crop biotechnology for directing the root-specific expression of transgenes.

  18. [Characterization of first sorbitol-fermenting shiga toxin-producing Escherichia coli O157:H- strain isolated in Poland].

    Jakubczak, Aleksandra; Szych, Jolanta; Januszkiewicz, Kamil


    Sorbitol-fermenting shiga toxin-producing E. coli O157:H- strains have emerged as a cause of human disease in many European and non-European countries. The role of SF VTEC O157:H- in the etiology of pediatric HUS and diarrhea is significant. We characterized the first SF VTEC O157:H- strain isolated from 9 year old patient in Poland. Strain possessed many traits characteristics for SF VTEC O157:H-. It fermented sorbitol after overnight incubation and produced beta-glucuronidase. It possessed the stx2, eae-gamma, EhlyA and sfpA genes and did not harbour plasmid-encoded katP and espP genes. Motility was not expressed but the strain possessed the chromosomal fliC locus for H7 antigen. The spread of SF VTEC O157:H- strains demonstrates the need for appropriate procedures for their microbiological diagnosis in Poland.

  19. Accumulation of recalcitrant xylan in mushroom-compost is due to a lack of xylan substituent removing enzyme activities of Agaricus bisporus.

    Jurak, Edita; Patyshakuliyeva, Aleksandrina; Kapsokalyvas, Dimitris; Xing, Lia; van Zandvoort, Marc A M J; de Vries, Ronald P; Gruppen, Harry; Kabel, Mirjam A


    The ability of Agaricus bisporus to degrade xylan in wheat straw based compost during mushroom formation is unclear. In this paper, xylan was extracted from the compost with water, 1M and 4M alkali. Over the phases analyzed, the remaining xylan was increasingly substituted with (4-O-methyl-)glucuronic acid and arabinosyl residues, both one and two arabinosyl residues per xylosyl residue remained. In the 1M and 4M KOH soluble solids of spent compost, 33 and 49 out of 100 xylosyl residues, respectively, were substituted. The accumulation of glucuronic acid substituents matched with the analysis that the two A. bisporus genes encoding for α-glucuronidase activity (both GH115) were not expressed in the A. bisporus mycelium in the compost during fruiting. Also, in a maximum likelihood tree it was shown that it is not likely that A. bisporus possesses genes encoding for the activity to remove arabinose from xylosyl residues having two arabinosyl residues.

  20. Development of genetic transformation methodologies for an industrially-promising microalga: Scenedesmus almeriensis.

    Dautor, Yasmeen; Úbeda-Mínguez, Patricia; Chileh, Tarik; García-Maroto, Federico; Alonso, Diego López


    The development of the microalgal industry requires advances in every aspect of microalgal biotechnology. In this regard, the availability of genetic engineering tools for industrially-promising species is key. As Scenedesmus almeriensis has promise for industrial use, we describe here an Agrobacterium-based methodology that allows stable genetic transformation of it for the first time, thus opening the way to its genetic manipulation. Transformation was accomplished using two different antibiotic resistance genes [hygromicine phophotransferase (hpt) and Shble] and it is credited by PCR amplification of both hpt/Shble and GUS genes and by the β-glucuronidase activity of transformed cells. Nevertheless, the single 35S promoter seems unable to direct gene expression to a convenient level in S. almeriensis as suggested by the low GUS enzymatic activity. Temperature was critical for the transformation efficiency.

  1. Bioavailability of (-)-epicatechin upon intake of chocolate and cocoa in human volunteers.

    Baba, S; Osakabe, N; Yasuda, A; Natsume, M; Takizawa, T; Nakamura, T; Terao, J


    We evaluated the levels of (-)-epicatechin (EC) and its metabolites in plasma and urine after intake of chocolate or cocoa by male volunteers. EC metabolites were analyzed by HPLC and LC/MS after glucuronidase and/or sulfatase treatment. The maximum levels of total EC metabolites in plasma were reached 2 hours after either chocolate or cocoa intake. Sulfate, glucuronide, and sulfoglucuronide (mixture of sulfate and glucuronide) conjugates of nonmethylated EC were the main metabolites present in plasma rather than methylated forms. Urinary excretion of total EC metabolites within 24 hours after chocolate or cocoa intake was 29.8+/-5.3%'; and 25.3+/-8.1% of total EC intake. EC in chocolate and cocoa was partly absorbed and was found to be present as a component of various conjugates in plasma, and these were rapidly excreted in urine.

  2. Optimization of the uidA Gene Transfer of Rosa hybrida via Agrobacterium tumefaciens:an Assessment of Factors Influencing the Efficiency of Gene Transfer

    Gao Liping; Bao Manzhu


    To develop a transformation protocol of Rosa hybrida 'Samantha' via Agrobacterium tumefaciens, the authors examined the effect of different factors on T-DNA transfer by measuring transient expression levels of an intron-containing β-glucuronidase gene. The results indicate that explant, light condition, salt concentration and acetosyringone (AS) concentration in co-culture medium are the most important factors, and factors like co-culture temperature, co-culture period and bacteria density have a strong effect on the growth of bacteria and then T-DNA transfer. Optimized co-cultivation was performed by inoculation of embryogenic callus with bacteria at a density of OD600= 0.5-0.8 for 20 min and co-culture in darkness under 23 °C on medium with 1/2 MS salts and 300 μmol·L-1 AS for 3 d.

  3. In Vitro Glucuronidation of Ochratoxin A by Rat Liver Microsomes

    Zheng Han


    Full Text Available Ochratoxin A (OTA, one of the most toxic mycotoxins, can contaminate a wide range of food and feedstuff. To date, the data on its conjugates via glucuronidation request clarification and consolidation. In the present study, the combined approaches of ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS, UHPLC-Orbitrap-high resolution mass spectrometry (HRMS and liquid chromatography-multiple stage mass spectrometry (LC-MSn were utilized to investigate the metabolic profile of OTA in rat liver microsomes. Three conjugated products of OTA corresponding to amino-, phenol- and acyl-glucuronides were identified, and the related structures were confirmed by hydrolysis with β-glucuronidase. Moreover, OTA methyl ester, OTα and OTα-glucuronide were also found in the reaction solution. Based on these results, an in vitro metabolic pathway of OTA has been proposed for the first time.

  4. Studies on the metabolism of gomisin A (TJN-101). II. Structure determination of biliary and urinary metabolites in rat.

    Ikeya, Y; Mitsuhashi, H; Sasaki, H; Matsuzaki, Y; Matsuzaki, T; Hosoya, E


    After oral administration of gomisin A (1) to rats, the bile and urine were collected and treated with beta-glucuronidase and arylsulfatase. Seven metabolites, met B (2), met A-III (3), met E (4), met D (5), met F (6), met G (7), and met H (8) were isolated from the bile treated with the enzymes. Eight metabolites 2-8, and met A-II (9) were isolated from the urine treated with the enzymes. A major metabolite 2, and two minor metabolites 3 and 9 were identified as met B, met A-III, and met A-II, respectively, which are oxidative products of 1 formed by rat liver S9 mix. The structures of five new metabolites 4-7, and 8 were determined on the basis of chemical and spectral studies.

  5. Co-suppression in transgenic Petunia hybrida expressing chalcone synthase A (chsA)

    李艳; 惠有为; 张仲凯; 黄兴奇; 李毅


    Chalcone synthase A is a key enzyme in the anthocyanin biosynthesis pathway. Expression of chsA gene in transgenic Petunia hybrida resulted in flower color alterations and co-suppression of transgenes and endogenous genes. We fused the β-glucuronidase (uidA) gene to the C-terminal of chsA gene, and transferred the fusion gene into Petunia hybrida via Agrobacterium tumefaciens. GUS histochemical staining analysis showed that co-suppression occurred specifically during the development of flowers and co-suppression required the mutual interaction of endogenous genes and transgenes. RNA in situ hybridization analysis suggested that co-suppression occurred in the entire plant, and RNA degradation occurred in the cytoplasm.

  6. Acid-Base and the Skeleton

    Bushinsky, David A.


    Chronic metabolic acidosis increases urine calcium (Ca) excretion in the absence of a concomitant increase in intestinal Ca absorption resulting in a net loss of total body. The source of this additional urine Ca is almost certainly the skeleton, the primary reservoir of body Ca. In vitro metabolic acidosis, modeled as a primary reduction in medium bicarbonate concentration, acutely (24 h) cell-mediated mechanisms predominate. In cultured neonatal mouse calvariae, acidosis-induced, cell-mediated Ca efflux is mediated by effects on both osteoblasts and osteoclasts. Metabolic acidosis inhibits extracellular matrix production by osteoblasts, as determined by measurement of collagen levels and levels for the non-collagenous matrix proteins osteopontin and matrix gla protein. Metabolic acidosis upregulates osteoblastic expression of RANKL (Receptor Activator of NFκB Ligand), an important osteoclastogenic and osteoclast-activating factor. Acidosis also increases osteoclastic activity as measured by release of β-glucuronidase, an enzyme whose secretion correlates with osteoclast-mediated bone resorption.

  7. Expressional Analysis of an EREBP Transcription Factor Gene OsEBP-89 in Rice

    HuiSHEN; Zong-YangWANG


    OsEBP-89 gene encodes an ethylene responsive element binding protein (EREBP) transcription factor from rice (Oryza sativa). Northern blot analysis revealed that OsEBP-89 was expressed in root, stem,seeds, flowers and leaves. Histochemical assay showed that β-glucuronidase (GUS) was expressed mainly in phloem of vascular tissues of the root and stem transition region (RST), basal part of sheath roots, stem node and basal part of adventitious roots, also in endosperm of seeds in transgenic rice harboring OsEBP-89/GUS construct (pNSG). A sequence region from -279 to -97 was found to play an important role for OsEBP-89 gene expression through promoter deletion assay. The possible function of OsEBP-89 gene was discussed.

  8. Expressional Analysis of an EREBP Transcription Factor Gene OsEBP-89 in Rice

    Hui SHEN; Zong-Yang WANG


    OsEBP-89 gene encodes an ethylene responsive element binding protein (EREBP) transcription factor from rice (Oryza sativa). Northern blot analysis revealed that OsEBP-89 was expressed in root, stem,seeds, flowers and leaves. Histochemical assay showed that β-glucuronidase (GUS) was expressed mainly in phloem of vascular tissues of the root and stem transition region (RST), basal part of sheath roots, stem node and basal part of adventitious roots, also in endosperm of seeds in transgenic rice harboring OsEBP-89/GUS construct (pNSG). A sequence region from -279 to -97 was found to play an important role for OsEBP-89gene expression through promoter deletion assay. The possible function of OsEBP-89 gene was discussed.

  9. Extracellular enzymatic activities of Bipolaris sorokiniana isolates.

    Geimba, Mercedes P; Brandelli, Adriano


    Several enzymatic activities were investigated in six isolates of the fungus Bipolaris sorokiniana, originating from different areas of Brazil. Among the glycosidases studied, beta-glucosidase, beta-N-acetylglucosaminidase, beta-xylosidase, cellobiohydrolase, and chitobiohydrolase were the major activities. In some isolates, beta-glucuronidase, beta-galactosidase, and alpha-mannosidase activities were also present. Polysaccharide-hydrolyzing enzymes, such as pectin lyase and carboxymethyl cellulase were detected in significant amounts, and their activities were variable among the different isolates. Other enzymes, namely phosphatases, proteinases and phenol oxidase, were also examined, showing variable amounts depending on the isolate. The pH dependence of all enzymes tested was investigated. Endoproteinase, carboxymethyl cellulase, and phenoloxidase had maximum activity in the pH range of 6-8, whilst all other enzymes showed maximum activity at pH 4-6.

  10. Functional analysis of cis-acting sequences regulating root-specific expression in transgenic tobacco


    Two different length fragments, RSF1 and RSF2 which contained the cis-acting sequences of root-spe- cific gene TobRB7, were isolated from tobacco genome. The abilities of these fragments to direct root-specific expression were studied by fusing them to the β-glucuronidase (GUS) report gene with different directions. After the recombined vectors were transformed into tobacco, the expression pattern was performed by histochemical staining and the quantitative analysis of GUS activity. The data suggested that the cis-acting element of TobRB7 gene direct GUS expression not only as root-specific but also as bidirectional. In our studies, the short fragment, RSF2, performed stronger activity than RSF1 with any direction. The stronger activity of GUS expression was determined by reverse inserting of RSF1 or RSF2 than positive inserting.

  11. Heterologous expression of the Aspergillus nidulans alcR-alcA system in Aspergillus niger.

    Nikolaev, I; Mathieu, M; van de Vondervoort, P; Visser, J; Felenbok, B


    The inducible and strongly expressed alcA gene encoding alcohol dehydrogenase I from Aspergillus nidulans was transferred together with the activator gene alcR, in the industrial fungus Aspergillus niger. This latter organism does not possess an inducible alc system but has an endogenously constitutive lowly expressed alcohol dehydrogenase activity. The overall induced expression of the alcA gene was of the same order in both fungi, as monitored by alcA transcription, alcohol dehydrogenase activity and heterologous expression of the reporter enzyme, beta-glucuronidase. However, important differences in the pattern of alcA regulation were observed between the two fungi. A high basal level of alcA transcription was observed in A. niger resulting in a lower ratio of alcA inducibility. This may be due to higher levels of the physiological inducer of the alc regulon, acetaldehyde, from general metabolism in A. niger which differs from that of A. nidulans.

  12. Evaluation of the effect of triterpenes on urinary risk factors of stone formation in pyridoxine deficient hyperoxaluric rats.

    Vidya, Lakshminarasimhan; Lenin, Mahimainathan; Varalakshmi, Palaninathan


    Investigations were carried out to evaluate the efficacy of the pentacyclic triterpene, lupeol and its ester, lupeol linoleate, against calcium oxalate urolithiasis in rats. Administration of a pyridoxine deficient diet containing 3% glycollic acid for 21 days led to increased excretion of stone forming constituents such as calcium, oxalate and uric acid. Crystal deposition and subsequent renal tubular damage resulted in increased excretion of the tubular enzymes alkaline phosphatase, lactate dehydrogenase, gamma glutamyl transferase, beta glucuronidase and N-acetyl glucosaminidase along with reduced fibrinolytic enzymes. A reduction in the urinary inhibitory factors magnesium and glycosaminoglycans was also observed. Treatment with lupeol and lupeol linoleate reduced the extent of tubular damage as evidenced from reduced enzymuria and minimized the excretion of stone forming constituents.

  13. Activity of the truncated promoter of pollen-specific G9 gene of cotton and the transgenic recessive nuclei-sterile tobacco


    A 557 bp fragment from the translation initiation site of the G9 gene expressed in maturing pollens of cotton was isolated from genomic DNA of upland cotton (Gossypium hirsutum L.) cv. “Zhongkang 17”, and two expression vectors for plant transformation were constructed via fusing this fragment with β-glucuronidase gene (Gus) and cytotoxin gene Barnase. The promoter activity of this fragment was demonstrated via transient expression of Gus gene in cotton and by the integrated expression of Barnase gene in tobacco. This promoter can initiate the expression of exogenous gene specifically and efficiently in plant pollen. The transgenic tobacco plant containing G9-Barnase fusion gene showed the characteristics of recessive nuclei-sterility.

  14. Plant regeneration of transgenic China Rose (Rosa chinensis Jacq.) from organogenic callus

    Chen Ji-ren; Liu Rong; Chen Shou-yi; Wang Hua-fang


    Different types of explants of China Rose (Rosa chinensis Jacq.) were placed on a Schenk and Hildebrandi (SH) medium containing L-proline and 2,4-dichlorophenoxyacefic acid (2,4-D). Organogenesis was observed on callus induced from both whole leaf and petiole and the high frequency of organogenesis was observed on the whole leaf. Shoot regeneration was obtained via organogenesis. The effects of pH and concentrations of antibiotics on maintenance of organogenesis capacity were investigated in subsequent subcultures. The pH value was found to play a critical role in retaining organogenesis capacity. The binary vector pBI121,carrying the gus gene coding for β-glucuronidase (GUS) and the npt Ⅱ gene mediated by Agrobacterium tumefaciens, was used for transformation of organogenic callus using 50 mg·L-1 geneficin for selection. Six regenerated lines showed GUS activity, of which five were verified for the presence of npt Ⅱ gene by PCR.

  15. Pharmacokinetic Profile of Eight Phenolic Compounds and Their Conjugated Metabolites after Oral Administration of Rhus verniciflua Extracts in Rats.

    Jin, Ming Ji; Kim, In Sook; Park, Jong Suk; Dong, Mi-Sook; Na, Chun-Soo; Yoo, Hye Hyun


    Rhus verniciflua (Toxicodendron vernicifluum) is a medicinal tree popularly used in Asian countries such as China, Japan, and Korea as a food additive or herbal medicine because of its beneficial effects. R. verniciflua extract (RVE) contains diverse phenolic compounds, such as flavonoids, as its major biological active constituents. In this study, the pharmacokinetic profiles of eight phenolic compounds were investigated following oral administration of RVE to rats. The eight phenolic compounds were 2,4-dihydroxybenzoic acid, 3,4-dihydroxybenzoic acid, fisetin, fustin, butin, sulfuretin, taxifolin, and garbanzol. The plasma concentrations of the eight compounds were determined by using a liquid chromatography-triple-quadrupole mass spectrometer before and after treatment with β-glucuronidase. When 1.5 g/kg RVE was administered, the eight compounds were all detected in plasma, mainly as conjugated forms. These pharmacokinetic data would be useful for understanding the pharmacological effects of RVE.

  16. Regeneration of transgenic plants of Prunus armeniaca containing the coat protein gene of Plum Pox Virus.

    da Câmara Machado, M L; da Câmara Machado, A; Hanzer, V; Weiss, H; Regner, F; Steinkellner, H; Mattanovich, D; Plail, R; Knapp, E; Kalthoff, B; Katinger, H


    A system was developed which allows the transfer of foreign genes into apricot cultivars. We report the transformation and regeneration of Prunus armeniaca plants with Agrobacterium tumefaciens strain LBA 4404 containing various binary plasmids, pBinGUSint, carrying the marker gene ß-glucuronidase (GUS) and pBinPPVm, carrying the coat protein gene of Plum Pox Virus (PPV). The marker gene GUS was used for optical evaluation of the efficiency of the transformation system. The coat protein gene of PPV was used to introduce coat protein mediated resistance against one of the most important pathogens of stone fruit trees in Europe and the whole Mediterranean area. This is the first report of the successful integration of a viral coat protein gene into a fruit tree species, opening a new perspective on the control of the disease.

  17. The requirement of multiple defense genes in soybean Rsv1-mediated extreme resistance to soybean mosaic virus.

    Zhang, Chunquan; Grosic, Sehiza; Whitham, Steven A; Hill, John H


    Soybean mosaic virus (SMV) is a major viral pathogen of soybean. Among the three SMV resistance genes, Rsv1 mediates extreme resistance (ER) against most SMV strains, including the β-glucuronidase-tagged G2 isolate that was previously used in studies of Rsv1. Using virus-induced gene silencing (VIGS), we screened 82 VIGS constructs to identify genes that play a role in Rsv1-mediated ER to SMV infection. The target genes included putative Rsv1 candidate genes, soybean orthologs to known defense-signaling genes, and 62 WRKY transcription factors. We identified eight VIGS constructs that compromised Rsv1-mediated resistance when the target genes were silenced, including GmEDR1, GmEDS1, GmHSP90, GmJAR1, GmPAD4, and two WRKY transcription factors. Together, our results provide new insight into the soybean signaling network required for ER against SMV.

  18. Composite potato plants with transgenic roots on non-transgenic shoots: a model system for studying gene silencing in roots

    Horn, Patricia; Santala, Johanna; Nielsen, Steen Lykke;


    Composite plants, with transgenic roots on a non-transgenic shoot, can be obtained by shoot explant transformation with Agrobacterium rhizogenes. The aim of this study was to generate composite potato plants (Solanum tuberosum) to be used as a model system in future studies on root...... of composite potato plants expressed significantly higher amounts of β-glucuronidase (GUS) than the roots of a GUS-transgenic potato line event. Silencing of the uidA transgene (GUS) was tested by inducing roots on the GUS-transgenic cv. Albatros event with strains of A. rhizogenes over-expressing either......-mediated silencing (co-suppression) was not functional in roots. The results suggest that composite plants offer a useful experimental system for potato research, which has gained little previous attention....

  19. The influence of co-cultivation on expression of the antifungal protein in Aspergillus giganteus.

    Meyer, Vera; Stahl, Ulf


    The afp gene of Aspergillus giganteus encodes a small, highly basic polypeptide with antifungal activity, named Antifungal Protein (AFP). The protein is secreted by the mould and inhibits the growth of various filamentous fungi. In this paper we report that co-cultivation of A. giganteus with various microorganisms alters afp expression. It was found that co-cultivation modulates afp expression on the level of transcription, using a reporter system based on the beta-glucuronidase gene. The presence of Fusarium oxysporum triggered afp transcription whereas dual cultures of A. giganteus and A. niger resulted in suppression of afp transcription. Growth tests performed with several carbon and nitrogen sources, revealed that the influence of co-cultivation is strongly dependent on the medium composition.

  20. Hairy cell leukemia: enzyme-histochemical and ultrastructural investigation of one case.

    Pilotti, S; Carbone, A; Lombardi, L; Tavolato, C; Rilke, F


    The investigation was carried out on blood smears, bone marrow aspirates, one lymph node biopsy, and the surgically removed spleen of a 53-year-old man with hairy cell leukemia. In the blood smears stained with May-Grünwald-Giemsa, 60 to 70% of the hairy cells contained tubular inclusions that corresponded to the ribosome-lamella complexes demonstrated at electron microscopy. In blood smears, imprints and cryostatic sections of the lymph node and of the spleen, hairy cells revealed tartrate-resistant acid phosphatase, beta-glucuronidase and adenosine-triphosphatase activity. In the spleen neutral esterase and alkaline phosphatase demonstrated the numerical increase of the histiocytes, which ultrastructurally displayed phagocytic activity. The presence in the spleen of pseudosinuses lined by hairy cells was confirmed by electron microscopy as well as by cytoenzymology.

  1. Genetic transformation of selected mature cork oak (Quercus suber L.) trees.

    Alvarez, R; Alonso, P; Cortizo, M; Celestino, C; Hernández, I; Toribio, M; Ordás, R J


    A transformation system for selected mature cork oak (Quercus suber L.) trees using Agrobacterium tumefaciens has been established. Embryos obtained from recurrent proliferating embryogenic masses were inoculated with A. tumefaciens strains EHA105, LBA4404 or AGL1 harbouring the plasmid pBINUbiGUSint [carrying the neomycin phosphotransferase II (nptII) and beta-glucuronidase (uidA) genes]. The highest transformation efficiency (4%) was obtained when freshly isolated explants were inoculated with A. tumefaciens strain AGL1. Evidence of stable transgene integration was obtained by PCR for the nptII and uidA genes, Southern blotting and expression of the uidA gene. The transgenic embryos were germinated and successfully transferred to soil.

  2. Two differentially regulated Arabidopsis genes define a new branch of the DFR superfamily

    Østergaard, L; Lauvergeat, V; Naested, H


    Two tandem genes were identified on Arabidopsis chromosome II (AtCRL1 and AtCRL2) encoding proteins with homology to members of the dihydroflavonol-4-reductase (DFR) superfamily. The encoded CRL1 and CRL2 proteins share 87% mutual amino acid sequence identity whereas their promoter regions...... resembling the expression pattern of late embryogenic abundant ABA-responsive genes. Differential expression of the two genes during plant development was confirmed in plants expressing transcriptional fusions between the two promoters and the Escherichia coli beta-glucuronidase reporter gene. This showed...... that, whereas high expression of AtCRL1 in mature seeds declines during subsequent vegetative growth, transcriptional activity from the AtCRL2 promoter increases during vegetative growth. Expression of both genes is restricted to vascular tissue. Based upon their homology to proteins involved in lignin...

  3. Modulation of gene expression made easy

    Solem, Christian; Jensen, Peter Ruhdal


    A new approach for modulating gene expression, based on randomization of promoter (spacer) sequences, was developed. The method was applied to chromosomal genes in Lactococcus lactis and shown to generate libraries of clones with broad ranges of expression levels of target genes. In one example...... beta-glucuronidase, resulting in an operon structure in which both genes are transcribed from a common promoter. We show that there is a linear correlation between the expressions of the two genes, which facilitates screening for mutants with suitable enzyme activities. In a second example, we show......, overexpression was achieved by introducing an additional gene copy into a phage attachment site on the chromosome. This resulted in a series of strains with phosphofructokinase activities from 1.4 to 11 times the wild-type activity level. In this example, the pfk gene was cloned upstream of a gusA gene encoding...

  4. Multivariate data analysis of enzyme production for hydrolysis purposes

    Schmidt, A.S.; Suhr, K.I.


    of the structure in the data - possibly combined with analysis of variance (ANOVA). Partial least squares regression (PLSR) showed a clear connection between the two differentdata matrices (the fermentation variables and the hydrolysis variables). Hence, PLSR was suitable for prediction purposes. The hydrolysis......Data from enzyme production experiments were analysed using different multivariate methods. The data set comprised of 12 objects (3 fungi (¤Aspergillus oryzae, Aspergillus fumigatur, Trichoderma reesei¤) grown on 4 substrates (lenzing and/or wet-oxidisedzylan)) and 12 variables (pH, biomass, 7...... enzyme activities (xylanase, zylosidase, arabinosidase, cellulase, acetyl zylan esterase, glucuronidase, feroyl esterase) and 3 hydrolysis efficiencies (reducing suggars at 3 different enzyme loadings)). Principalcomponent analysis (PCA) proved to be an efficient method to obtain an overview...

  5. Heparanase expression upregulates platelet adhesion activity and thrombogenicity

    Österholm, Cecilia; Zhang, Xiao; Hedin, Ulf; Vlodavsky, Israel; Li, Jin-Ping


    Heparanase is an endo-glucuronidase that specifically cleaves heparan sulfate (HS) and heparin polysaccharides. The enzyme is expressed at low levels in normal tissues, but is often upregulated under pathological conditions such as cancer and inflammation. Normal human platelets express exceptionally high levels of heparanase, but the functional consequences of this feature remain unknown. We investigated functional roles of heparanase by comparing the properties of platelets expressing high (Hpa-tg) or low (Ctr) levels of heparanase. Upon activation, Hpa-tg platelets exhibited a much stronger adhesion activity as compared to Ctr platelets, likely contributing to a higher thrombotic activity in a carotid thrombosis model. Furthermore, we found concomitant upregulated expression of both heparanase and CD62P (P-selectin) upon activation of mouse and human platelets. As platelets play important roles in tumor metastasis, these findings indicate contribution of the platelet heparanase to hyper-thrombotic conditions often seen in patients with metastatic cancer. PMID:27129145

  6. A common deletion in the uridine diphosphate glucuronyltransferase (UGT) 2B17 gene is a strong determinant of androgen excretion in healthy pubertal boys

    Juul, A; Sørensen, K; Aksglaede, L


    BACKGROUND: Testosterone (T) is excreted in urine as water-soluble glucuronidated and sulfated conjugates. The ability to glucuronidate T and other steroids depends on a number of different glucuronidases (UGT) of which UGT2B17 is essential. The aim of the study was to evaluate the influence of UGT......2B17 genotypes on urinary excretion of androgen metabolites in pubertal boys. STUDY DESIGN: A clinical study of 116 healthy boys aged 8-19 yr. UGT2B17 genotyping was performed using quantitative PCR. Serum FSH, LH, T, estradiol (E2), and SHBG were analyzed by immunoassays, and urinary levels...... of androgen metabolites were quantitated by gas chromatography/mass spectrometry in all subjects. RESULTS: Ten of 116 subjects (9%) presented with a homozygote deletion of the UGT2B17 gene (del/del), whereas 52 and 54 boys were hetero- and homozygous carriers of the UGT2B17 gene (del/ins and ins...

  7. Effects of Bos taurus autosome 9-located quantitative trait loci haplotypes on enzymatic mastitis indicators of milk from dairy cows experimentally inoculated with Escherichia coli

    Sørensen, Lars Peter; Engberg, Ricarda Greuel; Løvendahl, Peter;


    . In particular, we investigated the suitability of β-glucuronidase (GLU) as an early indicator of E. coli mastitis. Besides GLU, the enzymes l-lactate dehydrogenase (LDH), N-acetyl-β-d-glucosaminidase (NAGase), and alkaline phosphatase were included. The study was conducted in an experimental setup with 31....... The enzymes LDH, NAGase, and alkaline phosphatase showed similar responses, with a significantly increased activity and higher peak values for the HH than the HL cows. Significant differences between HH and HL cows were detected at different time points for these 3 enzymes, but not after adjusting P...... infection but with differences in the size and profile of the activity of the 4 enzymes. The enzyme GLU was an equally good indicator of E. coli mastitis compared with the other studied enzymes, although it showed a slower response compared with LDH and NAGase....

  8. Enzymatic treatment of estrogens and estrogen glucuronide

    Takaaki Tanaka; Toshiyuki Tamura; Yuuichi Ishizaki; Akito Kawasaki; Tomokazu Kawase; Masahiro Teraguchi; Masayuki Taniguchi


    Natural and synthetic estrogens from sewage treatment systems are suspected to influence the reproductive health of the animals in the rivers.In this article we investigated the enzymatic treatment of three estrogens (estrone,17β-estradiol,and 17α-ethynyletstradiol) by a fungal laccase which oxidize phenolic compounds with dissolved oxygen.The elimination of the estrogenic activities by enzymatic oxidation was demonstrated by medaka vitellogenin assay.In addition,we developed an enzymatic treatment system comprised of β-D-glucuronidase and the laccase for 17β-estradiol 3-(β-D-glucuronide) degradation.The two enzymes eliminated 17β-estradiol 3-(β-D-glucuronide) and the intermediate,17β-estradiol,efficiently.

  9. A simple and efficient Agrobacterium-mediated procedure for transformation of tomato

    Manoj K Sharma; Amolkumar U Solanke; Dewal Jani; Yogendra Singh; Arun K Sharma


    We describe a highly efficient and reproducible Agrobacterium-mediated transformation protocol applicable to several varieties of tomato (Solanum lycopersicum, earlier known as Lycopersicum esculentum). Conditions such as co-cultivation period, bacterial concentration, concentration of benzyl amino purine (BAP), zeatin and indole acetic acid (IAA) were optimized. Co-cultivation of explants with a bacterial concentration of 108 cells/ml for three days on 2 mg/l BAP, followed by regeneration on a medium containing 1 mg/ml zeatin resulted in a transformation frequency of 41.4%. Transformation of tomato plants was confirmed by Southern blot analysis and -glucuronidase (GUS) assay. The protocol developed showed very high efficiency of transformation for tomato varieties Pusa Ruby, Arka Vikas and Sioux. The optimized transformation procedure is simple, efficient and does not require tobacco, Petunia, tomato suspension feeder layer or acetosyringone.

  10. Agrobacterium-mediated transformation of tomato (Solanum lycopersicum L.) using the expansin 10 (CsEXP10) gene.

    Sun, Y D; Luo, W R; Sun, S Y; Ni, L


    The cucumber expansin 10 (CsEXP10) gene was previously cloned from young cucumber fruits but its role has not been defined. To determine the role of this gene in plant growth and development, a CsEXP10 gene transformation system was established. The open reading frame of the gene was inserted behind the CaMV35S promoter of vector pCAMBIA1301, and the construct was introduced into tomato plants by Agrobacterium-mediated transformation. In total, 19 kanamycin-positive lines were produced and nine independent transgenic lines were identified by β-glucuronidase and polymerase chain reaction (PCR) analysis. Quantitative real-time PCR analysis showed that levels of the CsEXP10 transcript were higher in transgenic lines than in a non-transgenic line.

  11. Cinnamic acid, coumarin and vanillin: Alternative phenolic compounds for efficient Agrobacterium-mediated transformation of the unicellular green alga, Nannochloropsis sp.

    Cha, Thye-San; Chen, Chin-Fong; Yee, Willy; Aziz, Ahmad; Loh, Saw-Hong


    The use of acetosyringone in Agrobacterium-mediated gene transfer into plant hosts has been favored for the past few decades. The influence of other phenolic compounds and their effectiveness in Agrobacterium-mediated plant transformation systems has been neglected. In this study, the efficacy of four phenolic compounds on Agrobacterium-mediated transformation of the unicellular green alga Nannochloropsis sp. (Strain UMT-M3) was assessed by using β-glucuronidase (GUS) assay. We found that cinnamic acid, vanillin and coumarin produced higher percentages of GUS positive cells as compared to acetosyringone. These results also show that the presence of methoxy group in the phenolic compounds may not be necessary for Agrobacterium vir gene induction and receptor binding as suggested by previous studies. These findings provide possible alternative Agrobacterium vir gene inducers that are more potent as compared to the commonly used acetosyringone in achieving high efficiency of Agrobacterium-mediated transformation in microalgae and possibly for other plants.

  12. Effect of dietary honey on intestinal microflora and toxicity of mycotoxins in mice

    Hegazy Eman M


    Full Text Available Abstract Background Bee honey is a functional food which has a unique composition, antimicrobial properties and bifidogenic effect. In order to assess whether honey can inhibit the toxic effect of mycotoxins, the present study was undertaken. Methods Production of biomass and toxins by Aspergillus parasiticus and Aspergillus ochraceus were followed in media without and with honey. Although aflatoxins and ochratoxin A. were administrated to male Swiss albino mice up to 1 μg and 10 ng/kg body weight/day respectively. The experimental animals were fed diets without our with 10% honey for two months. The changes in colonic probiotic bacteria, determintal colon enzyme glucuronidases, and genotoxicity were followed. Results Addition of 32% in its media increased the biomass of A parasiticus, while the biomass of A. ochraceus decreased and Ochratoxin A. was not produced. When the honey was added at the ratio of 32 and 48% in the medium. No relationship was found between mycelium weight and production of mycotoxins. Oral administration of aflatoxins (mixture of B1, B2, G1 and G2 and Ochratoxin A. induced structural and numerical chromosomal aberrations in bone marrow and germ cells of male mice, whereas, honey treatment reduced the genotoxicity of mycotoxins. Also both toxins induced histopathological changes in liver and kidney. Feeding on diet supplemented with honey improved the histopathological changes in case of aflatoxin group, but not in the case of ochratoxin A. group (except of kidney in two cases. No significant differences were found in the activity of colon β-glucuronidase between group fed diet with or without honey. On the other hand, the colon bifido bacteria and lactobacilli counts were increased markedly in group receiving diet supplemented with honey. Conclusion Substituting sugars with honey in processed food can inhibit the harmful and genotoxic effects of mycotoxins, and improve the gut microflora.

  13. Comparative effects of cellulose and soluble fibers (pectin, konjac glucomannan, inulin) on fecal water toxicity toward Caco-2 cells, fecal bacteria enzymes, bile acid, and short-chain fatty acids.

    Chen, Hsiao-Ling; Lin, You-Mei; Wang, Yi-Chun


    The aim of this study was to compare the effects of cellulose and three soluble dietary fibers, pectin, konjac glucomannan (KGM), and inulin, on the cytotoxicity and DNA damage of fecal water-treated Caco-2 cells, a human colon adenocarcinoma cell line, and to investigate the fecal components that potentially modulate the fecal toxicity, that is, bacterial enzymes, bile acids, and short-chain fatty acids. Six-week-old BALB/cJ mice were randomly allocated to consume an AIN-93 diet that contained no dietary fiber (fiber-free) or 5% (w/w) cellulose, pectin, KGM, and inulin for 3 weeks. Feces were collected during days 18-21. Fecal waters were co-incubated with Caco-2 cells to determine the cytotoxicity and DNA damage. In addition, the fecal bacterial enzymes, bile acids, and short-chain fatty acids were determined. Results indicated that all fiber diets similarly increased the survival rate (%) of fecal water-treated Caco-2 cells as compared with the fiber-free diet. The inhibition of fecal water-induced DNA damage in Caco-2 cells was greater for the pectin and inulin diets than for the cellulose and KGM diets. In contrast, cellulose exerted the greatest inhibitory effect on the fecal β-glucuronidase activity. Cellulose and all soluble dietary fibers reduced the secondary bile acid concentrations in the fecal water, but only soluble fibers increased the fecal concentrations of short-chain fatty acids, as compared with no fiber. Therefore, this study suggests that all dietary fibers substantially reduced the fecal water toxicity, which is associated with decreased secondary bile acid levels by all fibers, reduced fecal β-glucuronidase activity by cellulose, and increased short-chain fatty acid levels by soluble dietary fibers.

  14. Genome sequencing reveals diversification of virulence factor content and possible host adaptation in distinct subpopulations of Salmonella enterica

    Rodriguez-Rivera Lorraine D


    Full Text Available Abstract Background Divergence of bacterial populations into distinct subpopulations is often the result of ecological isolation. While some studies have suggested the existence of Salmonella enterica subsp. enterica subclades, evidence for these subdivisions has been ambiguous. Here we used a comparative genomics approach to define the population structure of Salmonella enterica subsp. enterica, and identify clade-specific genes that may be the result of ecological specialization. Results Multi-locus sequence analysis (MLSA and single nucleotide polymorphisms (SNPs data for 16 newly sequenced and 30 publicly available genomes showed an unambiguous subdivision of S. enterica subsp. enterica into at least two subpopulations, which we refer to as clade A and clade B. Clade B strains contain several clade-specific genes or operons, including a β-glucuronidase operon, a S-fimbrial operon, and cell surface related genes, which strongly suggests niche specialization of this subpopulation. An additional set of 123 isolates was assigned to clades A and B by using qPCR assays targeting subpopulation-specific SNPs and genes of interest. Among 98 serovars examined, approximately 20% belonged to clade B. All clade B isolates contained two pathogenicity related genomic islands, SPI-18 and a cytolethal distending toxin islet; a combination of these two islands was previously thought to be exclusive to serovars Typhi and Paratyphi A. Presence of β-glucuronidase in clade B isolates specifically suggests an adaptation of this clade to the vertebrate gastrointestinal environment. Conclusions S. enterica subsp. enterica consists of at least two subpopulations that differ specifically in genes involved in host and tissue tropism, utilization of host specific carbon and nitrogen sources and are therefore likely to differ in ecology and transmission characteristics.

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

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


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

  16. Cell-specific expression of the promoters of two nonlegume hemoglobin genes in a transgenic legume, Lotus corniculatus.

    Andersson, C R; Llewellyn, D J; Peacock, W J; Dennis, E S


    The promoters of the hemoglobin genes from the nitrogen-fixing tree Parasponia andersonii and the related nonnitrogen-fixing Trema tomentosa both confer beta-glucuronidase reporter gene expression to the central zone of the nodules of a transgenic legume, Lotus corniculatus. beta-Glucuronidase expression was high in the uninfected interstitial cells and parenchyma of the surrounding boundary layer and was low in the Rhizobium-infected cells. This contrasts with the expression of both the P. andersonii hemoglobin protein in P. andersonii nodules and the endogenous Lotus leghemoglobins that are expressed in the infected cells at very high levels. The expression pattern of the P. andersonii and T. tomentosa hemoglobin promoters in L. corniculatus resembles that of a nonsymbiotic hemoglobin gene from Casuarina glauca, which was introduced into this legume, and suggests that only the nonsymbiotic functions of the P. andersonii promoter are being recognized. Deletion of the distal segments of both the P. andersonii and T. tomentosa promoters identified regions important for the control of their tissue-specific and temporal activity in Lotus. Potential regulatory elements, which enhance nodule expression and suppress nonnodule expression, were also identified and localized to a distal promoter segment. A proximal AAGAG motif is present in the P. andersonii, T. tomentosa, and nonsymbiotic Casuarina hemoglobin genes. Mutation of this motif in the P. andersonii promoter resulted in a significant reduction in both the nodule and root expression levels in L. corniculatus. Some of the regulatory motifs characterized are similar to, but different from, the nodulin motifs of the leghemoglobins.

  17. Multiple NSAID-induced hits injure the small intestine: underlying mechanisms and novel strategies.

    Boelsterli, Urs A; Redinbo, Matthew R; Saitta, Kyle S


    Nonsteroidal anti-inflammatory drugs (NSAIDs) can cause serious gastrointestinal (GI) injury including jejunal/ileal mucosal ulceration, bleeding, and even perforation in susceptible patients. The underlying mechanisms are largely unknown, but they are distinct from those related to gastric injury. Based on recent insights from experimental models, including genetics and pharmacology in rodents typically exposed to diclofenac, indomethacin, or naproxen, we propose a multiple-hit pathogenesis of NSAID enteropathy. The multiple hits start with an initial pharmacokinetic determinant caused by vectorial hepatobiliary excretion and delivery of glucuronidated NSAID or oxidative metabolite conjugates to the distal small intestinal lumen, where bacterial β-glucuronidase produces critical aglycones. The released aglycones are then taken up by enterocytes and further metabolized by intestinal cytochrome P450s to potentially reactive intermediates. The "first hit" is caused by the NSAID and/or oxidative metabolites that induce severe endoplasmic reticulum stress or mitochondrial stress and lead to cell death. The "second hit" is created by the significant subsequent inflammatory response that would follow such a first-hit injury. Based on these putative mechanisms, strategies have been developed to protect the enterocytes from being exposed to the parent NSAID and/or oxidative metabolites. Among these, a novel strategy already demonstrated in a murine model is the selective disruption of bacteria-specific β-glucuronidases with a novel small molecule inhibitor that does not harm the bacteria and that alleviates NSAID-induced enteropathy. Such mechanism-based strategies require further investigation but provide potential avenues for the alleviation of the GI toxicity caused by multiple NSAID hits.

  18. Triple Recycling Processes Impact Systemic and Local Bioavailability of Orally Administered Flavonoids.

    Dai, Peimin; Zhu, Lijun; Luo, Feifei; Lu, Linlin; Li, Qiang; Wang, Liping; Wang, Ying; Wang, Xinchun; Hu, Ming; Liu, Zhongqiu


    Triple recycling (i.e., enterohepatic, enteric and local recycling) plays a central role in governing the disposition of phenolics such as flavonoids, resulting in low systemic bioavailability but higher gut bioavailability and longer than expected apparent half-life. The present study aims to investigate the coexistence of these recycling schemes using model bioactive flavonoid tilianin and a four-site perfused rat intestinal model in the presence or absence of a lactase phlorizin hydrolase (LPH) inhibitor gluconolactone and/or a glucuronidase inhibitor saccharolactone. The result showed that tilianin could be metabolized into tilianin glucuronide, acacetin, and acacetin glucuronide, which are excreted into the bile and luminal perfusate (highest in the duodenum and lowest in the colon). Gluconolactone (20 mM) significantly reduced the absorption of tilianin and the enteric and biliary excretion of acacetin glucuronide. Saccharolactone (0.1 mM) alone or in combination of gluconolactone also remarkably reduced the biliary and intestinal excretion of acacetin glucuronide. Acacetin glucuronides from bile or perfusate were rapidly hydrolyzed by bacterial β-glucuronidases to acacetin, enabling enterohepatic and enteric recycling. Moreover, saccharolactone-sensitive tilianin disposition and glucuronide deconjugation, which was more active in the small intestine than the colon, points to the small intestinal origin of the deconjugation enzyme and supports the presence of local recycling scheme. In conclusion, our studies have demonstrated triple recycling of a bioactive phenolic (i.e., a model flavonoid), and this recycling may have an impact on the site and duration of polyphenols pharmacokinetics in vivo.

  19. Impact of obesity on accumulation of the toxic irinotecan metabolite, SN-38, in mice.

    Mallick, Pankajini; Shah, Pranav; Gandhi, Adarsh; Ghose, Romi


    Our aim is to investigate the impact of high fat diet-induced obesity on plasma concentrations of the toxic irinotecan metabolite, SN-38, in mice. Diet-induced obese (DIO, 60% kcal fed) and lean mice (10% kcal fed) were treated orally with a single dose of 10mg/kg irinotecan to determine pharmacokinetic (PK) parameters. Feces and livers were collected for quantification of irinotecan and its metabolites (SN-38 & SN-38G). SN-38G formation by Ugt1a1 enzyme was analyzed in liver S9 fractions. Expression of the pro-inflammatory cytokine, TNF-α was determined in liver and plasma. Hepatic β-glucuronidase and carboxylesterase enzymes (CES) were also determined. AUC0-8 and Cmax of SN-38 increased by 2-fold in DIO mice compared to their lean controls. This was accompanied by a~2-fold reduction in AUC0-8 and Cmax of SN-38G in DIO mice. There were no differences in the PK parameters of irinotecan in DIO or lean mice. Conversion of SN-38 to SN-38G by Ugt1a1 enzyme was reduced by ~2-fold in liver S9 fractions in DIO mice. Furthermore, in DIO mice, β-glucuronidase activity increased by 2-fold, whereas there was no change in CES activity. TNF-α mRNA expression was 3 fold higher in DIO mice. Our study demonstrates that reduced hepatic Ugt1a activity during obesity likely contributes to reduced glucuronidation, and results in higher levels of the toxic metabolite, SN-38. Thus, irinotecan dosage should be closely monitored for effective and safe chemotherapy in obese cancer patients who are at a higher risk of developing liver toxicity. Copyright © 2015 Elsevier Inc. All rights reserved.

  20. Permeability of Rosmarinic acid in Prunella vulgaris and Ursolic acid in Salvia officinalis Extracts across Caco-2 Cell Monolayers

    Qiang, Zhiyi; Ye, Zhong; Hauck, Cathy; Murphy, Patricia A.; McCoy, Joe-Ann; Widrlechner, Mark P.; Reddy, Manju B.; Hendrich, Suzanne


    Ethnopharmacological relevance Rosmarinic acid (RA), a caffeic acid-related compound found in high concentrations in Prunella vulgaris (self-heal), and ursolic acid (UA), a pentacyclic triterpene acid concentrated in Salvia officinalis (sage), have been traditionally used to treat inflammation in the mouth, and may also be beneficial for gastrointestinal health in general. Aim of the study To investigate the permeabilities of RA and UA as pure compounds and in P. vulgaris and S. officinalis ethanol extracts across human intestinal epithelial Caco-2 cell monolayers. Materials and methods The permeabilities and Phase II biotransformation of RA and UA as pure compounds and in herbal extracts were compared using Caco-2 cells with HPLC detection. Results The apparent permeability coefficient (Papp) for RA and RA in P. vulgaris extracts was 0.2 ± 0.05 × 10−6 cm/s, significantly increased to 0.9 ± 0.2 × 10−6 cm/s after β-glucuronidase/sulfatase treatment. Papp for UA and UA in S. officinalis extract was 2.7 ± 0.3 × 10−6 cm/s and 2.3 ± 0.5 × 10−6 cm/s before and after β-glucuronidase/sulfatase treatment, respectively. Neither compound was affected in permeability by the herbal extract matrix. Conclusion RA and UA in herbal extracts had similar uptake as that found using the pure compounds, which may simplify the prediction of compound efficacy, but the apparent lack of intestinal glucuronidation/sulfation of UA is likely to further enhance the bioavailability of that compound compared with RA. PMID:21798330

  1. In Vitro and In Vivo Human Metabolism of Synthetic Cannabinoids FDU-PB-22 and FUB-PB-22.

    Diao, Xingxing; Scheidweiler, Karl B; Wohlfarth, Ariane; Pang, Shaokun; Kronstrand, Robert; Huestis, Marilyn A


    In 2014, FDU-PB-22 and FUB-PB-22, two novel synthetic cannabinoids, were detected in herbal blends in Japan, Russia, and Germany and were quickly added to their scheduled drugs list. Unfortunately, no human metabolism data are currently available, making it challenging to confirm their intake. The present study aims to identify appropriate analytical markers by investigating FDU-PB-22 and FUB-PB-22 metabolism in human hepatocytes and confirm the results in authentic urine specimens. For metabolic stability, 1 μM FDU-PB-22 and FUB-PB-22 was incubated with human liver microsomes for up to 1 h; for metabolite profiling, 10 μM was incubated with human hepatocytes for 3 h. Two authentic urine specimens from FDU-PB-22 and FUB-PB-22 positive cases were analyzed after β-glucuronidase hydrolysis. Metabolite identification in hepatocyte samples and urine specimens was accomplished by high-resolution mass spectrometry using information-dependent acquisition. Both FDU-PB-22 and FUB-PB-22 were rapidly metabolized in HLM with half-lives of 12.4 and 11.5 min, respectively. In human hepatocyte samples, we identified seven metabolites for both compounds, generated by ester hydrolysis and further hydroxylation and/or glucuronidation. After ester hydrolysis, FDU-PB-22 and FUB-PB-22 yielded the same metabolite M7, fluorobenzylindole-3-carboxylic acid (FBI-COOH). M7 and M6 (hydroxylated FBI-COOH) were the major metabolites. In authentic urine specimens after β-glucuronidase hydrolysis, M6 and M7 also were the predominant metabolites. Based on our study, we recommend M6 (hydroxylated FBI-COOH) and M7 (FBI-COOH) as suitable urinary markers for documenting FDU-PB-22 and/or FUB-PB-22 intake.

  2. Transformation of indica rice (Oryza sativa L. cv. RD6 mediated by Agrobacterium tumefaciens

    Manit Kosittrakul


    Full Text Available High percentage of callus induction at 97% was obtained when seeds of rice (Oryza sativa L. cv. RD6 were cultured on modified N6 medium supplemented with 3% (w/v sucrose, 22.5 μM 2,4-D and 0.8% agar under light condition. The suitable regeneration medium was N6 medium supplemented with 3% (w/v sucrose, 2.5 μM IAA, 18 μM BA and 0.8% agar. A test had been performed to determine the effect of antibiotics on the regeneration of rice cv. RD6. It was found that kanamycin concentration up to 150 mg l-1 and hygromycin concentration at 10 mg l-1 were effective for selection of transformants. Cefotaxime and carbenicillin concentration up to 250 mg l-1 had the highest phytotoxicity to plant regeneration. Agrobacterium-mediated gene transfer protocols for rice cv. RD6 were performed using A. tumefaciens strain LBA4404, which harbored the plasmid pBI121 containing genes for β- glucuronidase (GUS and kanamycin resistance (nptII, and strain EHA105, which harbored plasmid pCAMBIA1301 containing genes for β-glucuronidase (GUS and hygromycin resistance (hptII. GUS activities were found in rice calli after co-cultivation. A number of morphologically normal fertile transgenic rice plants were obtained. Stable integration, expression and inheritance of transgenes were demonstrated by molecular and genetic analysis of transgenic plants in T0 and T1 generation. Mendelian segregation was observed in T1 progeny.

  3. Expression and genomic integration of transgenes after Agrobacterium-mediated transformation of mature barley embryos.

    Uçarlı, C; Tufan, F; Gürel, F


    Mature embryos in tissue cultures are advantageous because of their abundance and rapid germination, which reduces genomic instability problems. In this study, 2-day-old isolated mature barley embryos were infected with 2 Agrobacterium hypervirulent strains (AGL1 and EHA105), followed by a 3-day period of co-cultivation in the presence of L-cystein amino acid. Chimeric expression of the b-glucuronidase gene (gusA) directed by a viral promoter of strawberry vein banding virus was observed in coleoptile epidermal cells and seminal roots in 5-day-old germinated seedlings. In addition to varying infectivity patterns in different strains, there was a higher ratio of transient b-glucuronidase expression in developing coleoptiles than in embryonic roots, indicating the high competency of shoot apical meristem cells in the mature embryo. A total of 548 explants were transformed and 156 plants developed to maturity on G418 media after 18-25 days. We detected transgenes in 74% of the screened plant leaves by polymerase chain reaction, and 49% of these expressed neomycin phosphotransferase II gene following AGL1 transformation. Ten randomly selected T0 transformants were analyzed using thermal asymmetric interlaced polymerase chain reaction and 24 fragments ranged between 200-600 base pairs were sequenced. Three of the sequences flanked with transferred-DNA showed high similarity to coding regions of the barley genome, including alpha tubulin5, homeobox 1, and mitochondrial 16S genes. We observed 70-200-base pair filler sequences only in the coding regions of barley in this study.

  4. Engineering the cell surface display of cohesins for assembly of cellulosome-inspired enzyme complexes on Lactococcus lactis

    Wieczorek Andrew S


    Full Text Available Abstract Background The assembly and spatial organization of enzymes in naturally occurring multi-protein complexes is of paramount importance for the efficient degradation of complex polymers and biosynthesis of valuable products. The degradation of cellulose into fermentable sugars by Clostridium thermocellum is achieved by means of a multi-protein "cellulosome" complex. Assembled via dockerin-cohesin interactions, the cellulosome is associated with the cell surface during cellulose hydrolysis, forming ternary cellulose-enzyme-microbe complexes for enhanced activity and synergy. The assembly of recombinant cell surface displayed cellulosome-inspired complexes in surrogate microbes is highly desirable. The model organism Lactococcus lactis is of particular interest as it has been metabolically engineered to produce a variety of commodity chemicals including lactic acid and bioactive compounds, and can efficiently secrete an array of recombinant proteins and enzymes of varying sizes. Results Fragments of the scaffoldin protein CipA were functionally displayed on the cell surface of Lactococcus lactis. Scaffolds were engineered to contain a single cohesin module, two cohesin modules, one cohesin and a cellulose-binding module, or only a cellulose-binding module. Cell toxicity from over-expression of the proteins was circumvented by use of the nisA inducible promoter, and incorporation of the C-terminal anchor motif of the streptococcal M6 protein resulted in the successful surface-display of the scaffolds. The facilitated detection of successfully secreted scaffolds was achieved by fusion with the export-specific reporter staphylococcal nuclease (NucA. Scaffolds retained their ability to associate in vivo with an engineered hybrid reporter enzyme, E. coli β-glucuronidase fused to the type 1 dockerin motif of the cellulosomal enzyme CelS. Surface-anchored complexes exhibited dual enzyme activities (nuclease and β-glucuronidase, and were

  5. Effects of amoxicillin/clavulanic acid on the pharmacokinetics of valproic acid

    Lee SY


    Full Text Available Soo-Yun Lee,1 Wooseong Huh,2 Jin Ah Jung,3 Hye Min Yoo,2 Jae-Wook Ko,1,2 Jung-Ryul Kim2,4 1Department of Health Sciences and Technology, SAIHST, Sungkyunkwan University, 2Department of Clinical Pharmacology and Therapeutics, Samsung Medical Center, Seoul, 3Department of Clinical Pharmacology, Inje University, Busan Paik Hospital, Busan, 4Department of Clinical Research and Evaluation, SAIHST, Sungkyunkwan University, Seoul, Republic of Korea Abstract: Valproic acid (VPA is mainly metabolized via glucuronide, which is hydrolyzed by β-glucuronidase and undergoes enterohepatic circulation. Amoxicillin/clavulanic acid (AMC administration leads to decreased levels of β-glucuronidase-producing bacteria, suggesting that these antibiotics could interrupt enterohepatic circulation and thereby alter the pharmacokinetics of VPA. This study aimed to evaluate the effects of AMC on the pharmacokinetics of VPA. This was an open-label, two-treatment, one-sequence study in 16 healthy volunteers. Two treatments were evaluated; treatment VPA, in which a single dose of VPA 500 mg was administered, and treatment AMC + VPA, in which multiple doses of AMC 500/125 mg were administered three times daily for 7 days and then a single dose of VPA was administered. Blood samples were collected up to 48 hours. Pharmacokinetic parameters were calculated using noncompartmental methods. Fifteen subjects completed the study. Systemic exposures and peak concentrations of VPA were slightly lower with treatment AMC + VPA than with treatment VPA (AUClast, 851.0 h·mg/L vs 889.6 h·mg/L; Cmax, 52.1 mg/L vs 53.0 mg/L. There were no significant between-treatment effects on pharmacokinetics (95% confidence interval [CI] of AUClast and Cmax (95.7 [85.9–106.5] and 98.3 [91.6–105.6], respectively. Multiple doses of AMC had no significant effects on the pharmacokinetics of VPA; thus, no dose adjustment is necessary. Keywords: drug–drug interaction, pharmacokinetics

  6. The 5′UTR-intron of the Gladiolus polyubiquitin promoter GUBQ1 enhances translation efficiency in Gladiolus and Arabidopsis

    Kamo Kathryn


    Full Text Available Abstract Background There are many non-cereal monocots of agronomic, horticultural, and biofuel importance. Successful transformation of these species requires an understanding of factors controlling expression of their genes. Introns have been known to affect both the level and tissue-specific expression of genes in dicots and cereal monocots, but there have been no studies on an intron isolated from a non-cereal monocot. This study characterizes the levels of GUS expression and levels of uidA mRNA that code for β-glucuronidase (GUS expression in leaves of Gladiolus and Arabidopsis using GUBQ1, a polyubiquitin promoter with a 1.234 kb intron, isolated from the non-cereal monocot Gladiolus, and an intronless version of this promoter. Results Gladiolus and Arabidopsis were verified by Southern hybridization to be transformed with the uidA gene that was under control of either the GUBQ1 promoter (1.9 kb, a 5′ GUBQ1 promoter missing its 1.234 kb intron (0.68 kb, or the CaMV 35 S promoter. Histochemical staining showed that GUS was expressed throughout leaves and roots of Gladiolus and Arabidopsis with the 1.9 kb GUBQ1 promoter. GUS expression was significantly decreased in Gladiolus and abolished in Arabidopsis when the 5′UTR-intron was absent. In Arabidopsis and Gladiolus, the presence of uidA mRNA was independent of the presence of the 5′UTR-intron. The 5′-UTR intron enhanced translation efficiency for both Gladiolus and Arabidopsis. Conclusions The GUBQ1 promoter directs high levels of GUS expression in young leaves of both Gladiolus and Arabidopsis. The 5′UTR-intron from GUBQ1 resulted in a similar pattern of β-glucuronidase translation efficiency for both species even though the intron resulted in different patterns of uidA mRNA accumulation for each species.

  7. Pharmacodynamics, pharmacokinetics and biodistribution of recombinant human N-acetylgalactosamine 4-sulfatase after 6months of therapy in cats using different IV infusion durations.

    Ruane, Therese; Haskins, Mark; Cheng, Alphonsus; Wang, Ping; Aguirre, Gustavo; Knox, Van W Rafe; Qi, Yulan; Tompkins, Troy; O'Neill, Charles A


    Mucopolysaccharidosis VI (MPS VI) is a lysosomal storage disease characterized by an absence or marked reduction of lysosomal N-acetylgalactosamine-4-sulfatase activity. Affected individuals have widespread accumulation of unmetabolized glycosaminoglycan substrates leading to detrimental effects. Recombinant human N-acetylgalactosamine 4-sulfatase (rhASB) is an approved enzyme replacement therapy for patients with MPS VI. Despite the known efficacy of weekly 4-h rhASB infusions, some clinicians wish to treat patients using reduced infusion times. This study compared the pharmacodynamics, pharmacokinetics, and tissue biodistribution of rhASB when administered as 2- and 4-h intravenous infusions using a feline model of MPS VI. Study animals were MPS VI-affected cats that demonstrate clinical signs and biochemical derangements similar to human MPS VI patients. Beginning at age 4weeks, animals received weekly 2-h (N=6) or 4-h (N=6) IV infusions of rhASB for 26weeks (Naglazyme® [galsulfase] Solution for Intravenous Infusion; BioMarin Pharmaceutical, Inc.). The control group consisted of untreated MPS VI-affected cats (N=6). The pharmacokinetic parameters of plasma rhASB and urinary glycosaminoglycan were determined at weeks 13 and 26. Animals were euthanized 48h after the last infusion and tissue concentration of ASB, GAG and β-glucuronidase were measured in the liver, spleen, aorta, and kidney. Skeletal and ophthalmological evaluations were performed within 2weeks of euthanasia. At week 13, the mean AUC0-t in animals treated with 4-h infusions was similar to 2-h infusions while the Cmax of the 4-h infusion was 50% of the 2-h infusion. By week 26, the mean AUC0-t of the 4-h infusion was 1.3-fold higher than the 2-h infusion (p<0.05) while Cmax of the 4-h infusion was 70% of the 2-h infusion (p<0.05). Among animals treated with 2- and 4-h infusions, there was no difference in urinary GAG excretion, tissue GAG storage, tissue galsulfase activity, and β-glucuronidase

  8. Composition of Chicory Root, Peel, Seed and Leaf Ethanol Extracts and Biological Properties of Their Non-Inulin Fractions

    Joanna Milala


    Full Text Available The chemical composition of the ethanol extracts of chicory root, peel, seed and leaf has been determined, in particular their inulin and phenolic fractions. The root and peel extracts were characterized by large mass fractions of inulin (60.1 and 46.8 g per 100 g of fresh mass, respectively, predominantly with degree of polymerization in the range from 3 to 10, while phenolics, determined as caffeoylquinic acids, made up 0.5 and 1.7 g per 100 g of fresh mass, respectively. The leaf and seed extracts had decidedly lower mass fractions of inulin (1.7 and 3.2 g per 100 g of fresh mass, respectively and higher mass fractions of phenolics (9.6 and 4.22 g per 100 g of fresh mass, respectively recognized as caffeoylquinic acids, chicoric acid and quercetin glucuronide. The biological properties of a non-inulin fraction from each extract were determined on Wistar rats fed with diets rich in fructose and saturated fat, as a model of metabolic changes related to westernization of human eating habits. The diets contained the same amount of inulin (6 % with various phenolic fractions. Some changes were noted in the microbial enzymatic activity of the caecum after feeding for 4 weeks with the diet containing the highest mass fraction of phenolics (0.208 %, derived from the mixture of peel and seed extracts (decreased activity of β-galactosidase and β-glucuronidase, as well as with the diet containing leaf extract (decreased β-glucuronidase activity. All the diets showed no essential influence on the caecal concentration and profile of short-chain fatty acids, except acetate, whose concentration decreased significantly in rats fed with the diet enriched with root extract. The addition of peel and leaf extracts to the fructose diets significantly increased the serum antioxidant capacity of lipophilic substances. The study indicates that parts of chicory and its byproducts might be a source of valuable compounds to improve the physiological activity of

  9. Aplicação de genes marcadores em estudos de ecologia microbiana com ênfase no sistema GUS Applications of markers genes on ecologic microbial studies with enphasis on GUS system

    Fábio Martins Mercante


    Full Text Available Muitos aspectos ecológicos envolvidos nas interações entre espécies leguminosas e estirpes de rizóbio têm sido facilmente entendidos com o emprego de técnicas que utilizam genes marcadores. A introdução de um gene marcador específico tem se mostrado altamente viável para análises dessas interações. Os genes marcadores são capazes de codificar para produtos que podem ser facilmente identificados ou medidos, especialmente, enzimas que podem atuar em diferentes substratos, fornecendo produtos coloridos ou fluorescentes facilmente detectáveis. De uma maneira geral, os genes marcadores têm sido utilizados em diferentes aspectos da ecologia microbiana, como nos estudos de competição entre estirpes de rizóbio, expressão de genes simbióticos, colonização da rizosfera e raízes, entre outros. Em todos esses estudos, os genes repórteres precisam ser introduzidos no genoma alvo através de um plasmídeo ou por inserção cromossomal. Nesta revisão, são enfatizados, principalmente, os diversos usos e aplicações de genes marcadores nos estudos de ecologia microbiana, com ênfase no sistema GUS (b-glucuronidase.Many of the ecological aspects involved with the interactions between legume species and rhizobia strains have been made easily to understood with the use of reporter gene techniques. The introduction of a specific reporter gene in an organism has shown to be highly efficient to analyze such interactions. These reporter genes generally code for products that can be easily identified or measured, mainly enzymes that can act on a variety of substrates, supplying colored or fluorescent detectable products. In general, the marker genes have been used in different aspects of microbial ecology, as in the competition studies among rhizobia strains, symbiotic gene expression, rhizosphere and root colonization, among others. In all studies, the marker genes need to be introduced into the genome by a plasmid or through a chromosomal

  10. Repression vs. activation of MOX, FMD, MPP1 and MAL1 promoters by sugars in Hansenula polymorpha: the outcome depends on cell's ability to phosphorylate sugar.

    Suppi, Sandra; Michelson, Tiina; Viigand, Katrin; Alamäe, Tiina


    A high-throughput approach was used to assess the effect of mono- and disaccharides on MOX, FMD, MPP1 and MAL1 promoters in Hansenula polymorpha. Site-specifically designed strains deficient for (1) hexokinase, (2) hexokinase and glucokinase, (3) maltose permease or (4) maltase were used as hosts for reporter plasmids in which β-glucuronidase (Gus) expression was controlled by these promoters. The reporter strains were grown on agar plates containing varied carbon sources and Gus activity was measured in permeabilized cells on microtitre plates. We report that monosaccharides (glucose, fructose) repress studied promoters only if phosphorylated in the cell. Glucose-6-phosphate was proposed as a sugar repression signalling metabolite for H. polymorpha. Intriguingly, glucose and fructose strongly activated expression from these promoters in strains lacking both hexokinase and glucokinase, indicating that unphosphorylated monosaccharides have promoter-derepressing effect. We also show that maltose and sucrose must be internalized and split into monosaccharides to exert repression on MOX promoter. We demonstrate that at yeast growth on glucose-containing agar medium, glucose-limitation is rapidly created that promotes derepression of methanol-specific promoters and that derepression is specifically enhanced in hexokinase-negative strain. We recommend double kinase-negative and hexokinase-negative mutants as hosts for heterologous protein production from MOX and FMD promoters.

  11. NtSCP1 from Tobacco Is an Extracellular Serine Carboxypeptidase III That Has an Impact on Cell Elongation1[C][W][OA

    Bienert, Manuela Désirée; Delannoy, Mélanie; Navarre, Catherine; Boutry, Marc


    The leaf extracellular space contains several peptidases, most of which are of unknown function. We isolated cDNAs for two extracellular serine carboxypeptidase III genes from tobacco (Nicotiana tabacum), NtSCP1 and NtSCP2, belonging to a phylogenetic clade not yet functionally characterized in plants. NtSCP1 and NtSCP2 are orthologs derived from the two ancestors of tobacco. Reverse transcription-polymerase chain reaction analysis showed that NtSCP1 and NtSCP2 are expressed in root, stem, leaf, and flower tissues. Expression analysis of the β-glucuronidase reporter gene fused to the NtSCP1 transcription promoter region confirmed this expression profile. Western blotting of NtSCP1 and expression of an NtSCP1-green fluorescent protein fusion protein showed that the protein is located in the extracellular space of tobacco leaves and culture cells. Purified His-tagged NtSCP1 had carboxypeptidase activity in vitro. Transgenic tobacco plants overexpressing NtSCP1 showed a reduced flower length due to a decrease in cell size. Etiolated seedlings of these transgenic plants had shorter hypocotyls. These data provide support for a role of an extracellular type III carboxypeptidase in the control of cell elongation. PMID:22214816

  12. NtSCP1 from tobacco is an extracellular serine carboxypeptidase III that has an impact on cell elongation.

    Bienert, Manuela Désirée; Delannoy, Mélanie; Navarre, Catherine; Boutry, Marc


    The leaf extracellular space contains several peptidases, most of which are of unknown function. We isolated cDNAs for two extracellular serine carboxypeptidase III genes from tobacco (Nicotiana tabacum), NtSCP1 and NtSCP2, belonging to a phylogenetic clade not yet functionally characterized in plants. NtSCP1 and NtSCP2 are orthologs derived from the two ancestors of tobacco. Reverse transcription-polymerase chain reaction analysis showed that NtSCP1 and NtSCP2 are expressed in root, stem, leaf, and flower tissues. Expression analysis of the β-glucuronidase reporter gene fused to the NtSCP1 transcription promoter region confirmed this expression profile. Western blotting of NtSCP1 and expression of an NtSCP1-green fluorescent protein fusion protein showed that the protein is located in the extracellular space of tobacco leaves and culture cells. Purified His-tagged NtSCP1 had carboxypeptidase activity in vitro. Transgenic tobacco plants overexpressing NtSCP1 showed a reduced flower length due to a decrease in cell size. Etiolated seedlings of these transgenic plants had shorter hypocotyls. These data provide support for a role of an extracellular type III carboxypeptidase in the control of cell elongation.

  13. Arabidopsis NIP3;1 Plays an Important Role in Arsenic Uptake and Root-to-Shoot Translocation under Arsenite Stress Conditions.

    Xu, Wenzhong; Dai, Wentao; Yan, Huili; Li, Sheng; Shen, Hongling; Chen, Yanshan; Xu, Hua; Sun, Yangyang; He, Zhenyan; Ma, Mi


    In Arabidopsis, the nodulin 26-like intrinsic protein (NIP) subfamily of aquaporin proteins consists of nine members, five of which (NIP1;1, NIP1;2, NIP5;1, NIP6;1, and NIP7;1) were previously identified to be permeable to arsenite. However, the roles of NIPs in the root-to-shoot translocation of arsenite in plants remain poorly understood. In this study, using reverse genetic strategies, Arabidopsis NIP3;1 was identified to play an important role in both the arsenic uptake and root-to-shoot distribution under arsenite stress conditions. The nip3;1 loss-of-function mutants displayed obvious improvements in arsenite tolerance for aboveground growth and accumulated less arsenic in shoots than those of the wild-type plants, whereas the nip3;1 nip1;1 double mutant showed strong arsenite tolerance and improved growth of both roots and shoots under arsenite stress conditions. A promoter-β-glucuronidase analysis revealed that NIP3;1 was expressed almost exclusively in roots (with the exception of the root tips), and heterologous expression in the yeast Saccharomyces cerevisiae demonstrated that NIP3;1 was able to mediate arsenite transport. Taken together, our results suggest that NIP3;1 is involved in arsenite uptake and root-to-shoot translocation in Arabidopsis, probably as a passive and bidirectional arsenite transporter.

  14. In situ enzyme aided adsorption of soluble xylan biopolymers onto cellulosic material.

    Chimphango, Annie F A; Görgens, J F; van Zyl, W H


    The functional properties of cellulose fibers can be modified by adsorption of xylan biopolymers. The adsorption is improved when the degree of biopolymers substitution with arabinose and 4-O-methyl-glucuronic acid (MeGlcA) side groups, is reduced. α-l-Arabinofuranosidase (AbfB) and α-d-glucuronidase (AguA) enzymes were applied for side group removal, to increase adsorption of xylan from sugarcane (Saccharum officinarum L) bagasse (BH), bamboo (Bambusa balcooa) (BM), Pinus patula (PP) and Eucalyptus grandis (EH) onto cotton lint. The AguA treatment increased the adsorption of all xylans by up to 334%, whereas, the AbfB increased the adsorption of the BM and PP by 31% and 44%, respectively. A combination of AguA and AbfB treatment increased the adsorption, but to a lesser extent than achieved with AguA treatment. This indicated that the removal of the glucuronic acid side groups provided the most significant increase in xylan adsorption to cellulose, in particular through enzymatic treatment.

  15. An alternative approach for gene transfer in trees using wild-type Agrobacterium strains.

    Brasileiro, A C; Leplé, J C; Muzzin, J; Ounnoughi, D; Michel, M F; Jouanin, L


    Micropropagated shoots of three forest tree species, poplar (Populus tremula x P. alba), wild cherry (Prunus avium L.) and walnut (Juglans nigra x J. regia), were inoculated each with six different wild-type Agrobacterium strains. Poplar and wild cherry developed tumors that grew hormone-independently, whereas on walnut, gall formation was weak. On poplar and wild cherry, tumors induced by nopaline strains developed spontaneously shoots that had a normal phenotype and did not carry oncogenic T-DNA. From these observations, we have established a co-inoculation method to transform plants, using poplar as an experimental model. The method is based on inoculation of stem internodes with an Agrobacterium suspension containing both an oncogenic strain that induces shoot differentiation and a disarmed strain that provides the suitable genes in a binary vector. We used the vector pBI121 carrying neo (kanamycin resistance) and uidA (beta-glucuronidase) genes to facilitate early selection and screening. Poplar plants derived from kanamycin-resistant shoots that did not carry oncogenic T-DNA, were shown to contain and to express neo and uidA genes. These results suggest that wild-type Agrobacterium strains that induce shoot formation directly from tumors can be used as a general tool for gene transfer, avoiding difficult regeneration procedures.

  16. Prebiotic effects of almonds and almond skins on intestinal microbiota in healthy adult humans.

    Liu, Zhibin; Lin, Xiuchun; Huang, Guangwei; Zhang, Wen; Rao, Pingfan; Ni, Li


    Almonds and almond skins are rich in fiber and other components that have potential prebiotic properties. In this study we investigated the prebiotic effects of almond and almond skin intake in healthy humans. A total of 48 healthy adult volunteers consumed a daily dose of roasted almonds (56 g), almond skins (10 g), or commercial fructooligosaccharides (8 g) (as positive control) for 6 weeks. Fecal samples were collected at defined time points and analyzed for microbiota composition and selected indicators of microbial activity. Different strains of intestinal bacteria had varying degrees of growth sensitivity to almonds or almond skins. Significant increases in the populations of Bifidobacterium spp. and Lactobacillus spp. were observed in fecal samples as a consequence of almond or almond skin supplementation. However, the populations of Escherichia coli did not change significantly, while the growth of the pathogen Clostridum perfringens was significantly repressed. Modification of the intestinal microbiota composition induced changes in bacterial enzyme activities, specifically a significant increase in fecal β-galactosidase activity and decreases in fecal β-glucuronidase, nitroreductase and azoreductase activities. Our observations suggest that almond and almond skin ingestion may lead to an improvement in the intestinal microbiota profile and a modification of the intestinal bacterial activities, which would induce the promotion of health beneficial factors and the inhibition of harmful factors. Thus we believe that almonds and almond skins possess potential prebiotic properties.

  17. Expression pattern and core region analysis of AtMPK3 promoter in response to environmental stresses


    The protein kinase AtMPK3,a component of the MAP kinase cascade,plays an important role in stress signal transduction in plant cells. To clarify how AtMPK3 is regulated at the transcriptional level in response to various environmental factors, the 1016-bp promoter sequence upstream of the transcription start site of the AtMPK3 gene was isolated. Analyses of the promoter sequence using plant promoter databases revealed that the AtMPK3 promoter contains many potential cis-acting elements involved in environmental stress responses. We constructed four deletion mutants of the AtMPK3 promoter, and introduced the intact and truncated promoter sequences fused to the β-glucuronidase (GUS) gene into Arabidopsis. GUS histochemical staining and quantitative fluorometric GUS assays were performed to visualize and compare the expression patterns in response to different environmental stimuli. The region between-188 and-62 upstream of the transcription start site was identified as the essential DNA sequence of the AtMPK3 promoter for responses to drought, high salinity, low temperature, and wounding. These results advance our understanding of the molecular mechanisms controlling AtMPK3 expression in response to different environmental stimuli.

  18. Arabidopsis alcohol dehydrogenase expression in both shoots and roots is conditioned by root growth environment

    Chung, H. J.; Ferl, R. J.


    It is widely accepted that the Arabidopsis Adh (alcohol dehydrogenase) gene is constitutively expressed at low levels in the roots of young plants grown on agar media, and that the expression level is greatly induced by anoxic or hypoxic stresses. We questioned whether the agar medium itself created an anaerobic environment for the roots upon their growing into the gel. beta-Glucuronidase (GUS) expression driven by the Adh promoter was examined by growing transgenic Arabidopsis plants in different growing systems. Whereas roots grown on horizontal-positioned plates showed high Adh/GUS expression levels, roots from vertical-positioned plates had no Adh/GUS expression. Additional results indicate that growth on vertical plates closely mimics the Adh/GUS expression observed for soil-grown seedlings, and that growth on horizontal plates results in induction of high Adh/GUS expression that is consistent with hypoxic or anoxic conditions within the agar of the root zone. Adh/GUS expression in the shoot apex is also highly induced by root penetration of the agar medium. This induction of Adh/GUS in shoot apex and roots is due, at least in part, to mechanisms involving Ca2+ signal transduction.

  19. Development of a bipartite ecdysone-responsive gene switch for the oomycete Phytophthora infestans and its use to manipulate transcription during axenic culture and plant infection.

    Gamboa-Meléndez, Heber; Judelson, Howard S


    Conditional expression systems have been proven to be useful tools for the elucidation of gene function in many taxa. Here, we report the development of the first useful inducible promoter system for an oomycete, based on an ecdysone receptor (EcR) and the ecdysone analogue methoxyfenozide. In Phytophthora infestans, the potato late blight pathogen, a monopartite transactivator containing the VP16 activation domain from herpes simplex virus, the GAL4 DNA-binding domain from yeast and the EcR receptor domain from the spruce budworm enabled high levels of expression of a β-glucuronidase (GUS) reporter gene, but unacceptable basal activity in the absence of the methoxyfenozide inducer. Greatly improved performance was obtained using a bipartite system in which transcription is activated by a heterodimer between a chimera of VP16 and the migratory locust retinoid X receptor, and a separate EcR-DNA-binding domain chimera. Transformants were obtained that exhibited >100-fold activation of the reporter by methoxyfenozide, with low basal levels of expression and induced activity approaching that of the strong ham34 promoter. Performance varied between transformants, probably as a result of position effects. The addition of methoxyfenozide enabled strong induction during hyphal growth, zoosporogenesis and colonization of tomato. No significant effects of the inducer or transactivators on growth, development or pathogenicity were observed. The technology should therefore be a useful addition to the arsenal of methods for the study of oomycete plant pathogens.

  20. Transferring cucumber mosaic virus-white leaf strain coat protein gene into Cucumis melo L. and evaluating transgenic plants for protection against infections

    Gonsalves, C.; Xue, B.; Yepes, M.; Fuchs, M.; Ling, K.; Namba, S. (Cornell Univ., Geneva, NY (United States). Dept. of Plant Pathology)


    A single regeneration procedure using cotyledon examples effectively regenerated five commercially grown muskmelon cultivars. This regeneration scheme was used to facilitate gene transfers using either Agrobacterium tumefaciens or microprojectile bombardment methods. In both cases, the transferred genes were from the T-DNA region of the binary vector plasmid pGA482GG/cp cucumber mosaic virus-white leaf strain (CMV-WL), which contains genes that encode neomycin phosphotransferase II (NPT II), [beta]-glucuronidase (GUS), and the CMV-WL coat protein (CP). Explants treated with pGA482GG/cpCMV-WL regenerated shoots on Murashige and Skoog medium containing 4.4 [mu]m 6-benzylaminopurine (BA), kanamycin (Km) at 150 mg[center dot]liter[sup [minus]1] and carbenicillin (Cb) at 500 mg[center dot]liter[sup [minus]1]. The authors' comparison of A. tumefaciens- and microprojectile-mediated gene transfer procedures shows that both methods effectively produce nearly the same percentage of transgenic plants. R[sub 0] plants were first tested for GUS or NPT II expression, then the polymerase chain reaction (PCR) and other tests were used to verify the transfer of the NPT II, GUS, and CMV-WL CP genes.

  1. Microbial short-chain fatty acid production and extracellular enzymes activities during in vitro fermentation of polysaccharides from the seeds of Plantago asiatica L. treated with microwave irradiation.

    Hu, Jie-Lun; Nie, Shao-Ping; Li, Chang; Fu, Zhi-Hong; Xie, Ming-Yong


    Effects of microwave irradiation on microbial short-chain fatty acid production and the activites of extracellular enzymes during in vitro fermentation of the polysaccharide from Plantago asiatica L. were investigated in this study. It was found that the apparent viscosity, average molecular weight, and particle size of the polysaccharide decreased after microwave irradiation. Reducing sugar amount increased with molecular weight decrease, suggesting the degradation may derive from glycosidic bond rupture. The polysaccharide surface topography was changed from large flakelike structure to smaller chips. FT-IR showed that microwave irradiation did not alter the primary functional groups in the polysaccharide. However, short-chain fatty acid productions of the polysaccharide during in vitro fermentation significantly increased after microwave irradiation. Activities of microbial extracellular enzymes xylanase, arabinofuranosidase, xylosidase, and glucuronidase in fermentation cultures supplemented with microwave irradiation treated polysaccharide were also generally higher than those of untreated polysaccharide. This showed that microwave irradiation could be a promising degradation method for the production of value-added polysaccharides.

  2. Expression of Five Endopolygalacturonase Genes and Demonstration that MfPG1 Overexpression Diminishes Virulence in the Brown Rot Pathogen Monilinia fructicola.

    Chien-Ming Chou

    Full Text Available Monilinia fructicola is a devastating pathogen on stone fruits, causing blossom blight and fruit rot. Little is known about pathogenic mechanisms in M. fructicola and related Monilinia species. In this study, five endopolygalacturonase (endo-PG genes were cloned and functionally characterized in M. fructicola. Quantitative reverse-transcriptase PCR (qRT-PCR revealed that the five MfPG genes are differentially expressed during pathogenesis and in culture under various pH regimes and carbon and nitrogen sources. MfPG1 encodes the major endo-PG and is expressed to significantly higher levels compared to the other four MfPGs in culture and in planta. MfPG1 function during pathogenesis was evaluated by examining the disease phenotypes and gene expression patterns in M. fructicola MfPG1-overexpressing strains and in strains carrying the β-glucuronidase (GUS reporter gene fused with MfPG1 (MfPG1-GUS. The MFPG1-GUS reporter was expressed in situ in conidia and hyphae following inoculation of flower petals, and qRT-PCR analysis confirmed MfPG1 expression during pathogenesis. MfPG1-overexpressing strains produced smaller lesions and higher levels of reactive oxygen species (ROS on the petals of peach and rose flowers than the wild-type strain, suggesting that MfPG1 affecting fungal virulence might be in part resulted from the increase of ROS in the Prunus-M. fructicola interactions.

  3. High-efficient transgenic hairy roots induction in chicory: re-dawn of a traditional herb

    Sara Kabirnataj


    Full Text Available Plant roots can be manipulated by Agrobacterium rhizogenes to stimulate the production of heterologous proteins for pharmaceutical applications as green cell-factories. During the present study, four bacterial strains (A4, ATCC15834, ATCC11325 and A13 in combination with three co-cultivation media (MS, B5, LS were examined to establish an efficient and reliable transformation system for chicory (Cichorium intybus L. using A. rhizogenes. The maximum chicory hairy roots induction was achieved using A13 strain. The observation confirmed that MS medium was more effective on hairy root growth. Dried biomass accumulation of hairy roots infected by A13 strain was 1.10 g l-1 in MS medium which was significantly higher than those grown in LS and B5 medium (0.88 and 0.72 g l-1, respectively. Beta-glucuronidase (GUS gene was introduced by A13 strain carrying the pCAMBIA1304 binary vector. The results showed that the highest frequency of transformation (63.15 % was achieved using A13 strain and MS cultivation medium. Detection of GUS and hptII genes by PCR and GUS histochemical localization confirmed the integrative transformation in hairy roots. In conclusion, the whole process was successfully optimized as a pre-step to manipulate the chicory hairy root cells to improve the unique potential of secondary metabolite production.

  4. Transgenic rice homozygous lines expressing GNA showed enhanced resistance to rice brown planthopper


    Mature seed-derived calli from two elite Chinese japonica rice (Oryza sativa L.) cultivars Eyi 105 and Ewan 5 were co-transformed with two plasmids, pWRG1515 and pRSSGNA1, containing the selectable marker hygromycin phosphotransferase gene (hpt), the reporter β-glucuronidase gene (gusA) and the snowdrop (Galanthus nivalis)lectin gene (gna) via particle bombardment. 61 independent transgenic rice plants were regenerated from 329 bombarded calli. 79% transgenic plants contained all the three genes, revealed by PCR/Southern blot analysis. Western blot analysis revealed that 36 out of 48 gna-containing transgenic plants expressed GNA (75 %) at various levels with the highest expression being approximately 0.5% of total soluble protein. Genetic analysis confirmed Mendelian segregation of transgenes in progeny. From the R2 generations whose R1 parent plants showing 3:1 Mendelian segregation patterns,we identified five independent homozygous lines containing and expressing all the three transgenes. Insect bioassay and feeding tests showed that these homozygous lines had significant inhibition to rice brown planthopper (Nilaparvata lugens, BPH) by decreasing BPH survival and overall fecundity, retarding BPH development and declining BPH feeding.These BPH-resistant lines have been incorporated into rice insect resistance breeding program. This is the first report that homozygous transgenic rice lines expressing GNA, developed by genetic transformation and through genetic analysis-based selection, conferred enhanced resistance to BPH, one of the most damaging insect pests in rice.

  5. Evaluation of somatic embryos of alfalfa for recombinant protein expression.

    Fu, Guohua; Grbic, Vojislava; Ma, Shengwu; Tian, Lining


    Somatic embryos of alfalfa can accumulate higher levels of recombinant proteins comparing to vegetative organs. Somatic embryos may be explored as a new system for new protein production for plants. Plants have been explored via genetic engineering as an inexpensive system for recombinant protein production. However, protein expression levels in vegetative tissues have been low, which limits the commercial utilization of plant expression systems. Somatic embryos resemble zygotic embryos in many aspects and may accumulate higher levels of proteins as true seed. In this study, somatic embryo of alfalfa (Medicago sativa L.) was investigated for the expression of recombinant proteins. Three heterologous genes, including the standard scientific reporter uid that codes for β-glucuronidase and two genes of interest: ctb coding for cholera toxin B subunit (CTB), and hIL-13 coding for human interleukin 13, were independently introduced into alfalfa via Agrobacterium-mediated transformation. Somatic embryos were subsequently induced from transgenic plants carrying these genes. Somatic embryos accumulated approximately twofold more recombinant proteins than vegetative organs including roots, stems, and leaves. The recombinant proteins of CTB and hIL-13 accumulated up to 0.15 and 0.18 % of total soluble protein in alfalfa somatic embryos, respectively. The recombinant proteins expressed in somatic embryos also exhibited biological activities. As somatic embryos can be induced in many plant species and their production can be scaled up via different avenues, somatic embryos may be developed as an efficient expression system for recombinant protein production.

  6. Expression of NAC1 up-stream regulatory region and its relationship to the lateral root initiation induced by gibberellins and auxins.

    Wang, Youhua; Duan, Liusheng; Lu, Mengzhu; Li, Zhaohu; Wang, Minjie; Zhai, Zhixi


    A 1050 bp up-stream regulatory fragment of the transcription factor gene NAC1 in Arabidopsis thaliana was isolated using polymerase chain reaction (PCR) based techniques. The fragment was used to substitute the 35S promoter of the pBI121 plasmid to construct a beta-glucuronidase gene (GUS) expression system. The construct was introduced into tobacco (Nicotiana tabaccum) plants by the Agrobacterium-mediated transferring method. GUS expression pattern was studied by using the transgenic lines. The results showed that the GUS driven by the NAC1 up-stream regulatory region was specifically expressed in the root meristem region, basal areas of the lateral root primordium and the lateral roots. The GUS expression was induced by 3-indolebutyric acid (IBA) and gibberellins (GA3 and GA4+7). The results indicated that the up-stream regulatory fragment of NAC1 responded to plant hormones. The fragment might be involved in both auxins and gibberellins signaling in promoting the development of lateral roots.

  7. Arabidopsis gene expression patterns during spaceflight

    Paul, A.-L.; Ferl, R. J.

    The exposure of Arabidopsis thaliana (Arabidopsis) plants to spaceflight environments resulted in the differential expression of hundreds of genes. A 5 day mission on orbiter Columbia in 1999 (STS-93) carried transgenic Arabidopsis plants engineered with a transgene composed of the alcohol dehydrogenase (Adh) gene promoter linked to the β -Glucuronidase (GUS) reporter gene. The plants were used to evaluate the effects of spaceflight on two fronts. First, expression patterns visualized with the Adh/GUS transgene were used to address specifically the possibility that spaceflight induces a hypoxic stress response, and to assess whether any spaceflight response was similar to control terrestrial hypoxia-induced gene expression patterns. (Paul et al., Plant Physiol. 2001, 126:613). Second, genome-wide patterns of native gene expression were evaluated utilizing the Affymetrix ATH1 GeneChip? array of 8,000 Arabidopsis genes. As a control for the veracity of the array analyses, a selection of genes identified with the arrays was further characterized with quantitative Real-Time RT PCR (ABI - TaqmanTM). Comparison of the patterns of expression for arrays of hybridized with RNA isolated from plants exposed to spaceflight compared to the control arrays revealed hundreds of genes that were differentially expressed in response to spaceflight, yet most genes that are hallmarks of hypoxic stress were unaffected. These results will be discussed in light of current models for plant responses to the spaceflight environment, and with regard to potential future flight opportunities.

  8. Isolation of Persicaria minor sesquiterpene synthase promoter and its deletions for transgenic Arabidopsis thaliana

    Omar, Aimi Farehah; Ismail, Ismanizan


    Sesquiterpene synthase (SS) catalyzes the formation of sesquiterpenes from farnesyl diphosphate (FDP) via carbocation intermediates. In this study, the promoter region of sesquiterpene synthase was isolated from Persicaria minor to identify possible cis-acting elements in the promoter. The full-length PmSS promoter of P. minor is 1824-bp sequences. The sequence was analyzed and several putative cis-acting regulatory elements were identified. Three cis-acting regulatory elements were selected for deletion analysis which are cis-acting element involved in wound responsiveness (WUN), cis - acting element involved in defense and stress responsiveness (TC) and cis-acting element involved in ABA responsiveness (ABRE). Series of deletions were conducted to assess the promoter activity producing three truncated fragments promoter; Prom 2 1606-bp, Prom 3 1144- bp, and Prom 4 921-bp. The full-length promoter and its deletion series were cloned into the pBGWFS7 vector which contain β-glucuronidase (GUS) gene and green fluorescent protein (GFP) as the reporter gene. All constructs were successfully transformed into Arabidopsis thaliana based on PCR of positive BASTA resistance plants.

  9. Antiobesity and lipid-lowering effects of Bifidobacterium spp. in high fat diet-induced obese rats

    Lee Si


    Full Text Available Abstract Background Recent studies have reported the preventive effects of probiotics on obesity. Among commensal bacteria, bifidobacteria is one of the most numerous probiotics in the mammalian gut and are a type of lactic acid bacteria. The aim of this study was to assess the antiobesity and lipid-lowering effects of Bifidobacterium spp. isolated from healthy Korean on high fat diet-induced obese rats. Methods Thirty-six male Sprague-Dawley rats were divided into three groups as follows: (1 SD group, fed standard diet; (2 HFD group, fed high fat diet; and (3 HFD-LAB group, fed high fat diet supplemented with LAB supplement (B. pseudocatenulatum SPM 1204, B. longum SPM 1205, and B. longum SPM 1207; 108 ~ 109 CFU. After 7 weeks, the body, organ, and fat weights, food intake, blood serum levels, fecal LAB counts, and harmful enzyme activities were measured. Results Administration of LAB reduced body and fat weights, blood serum levels (TC, HDL-C, LDL-C, triglyceride, glucose, leptin, AST, ALT, and lipase levels, and harmful enzyme activities (β-glucosidase, β-glucuronidase, and tryptophanase, and significantly increased fecal LAB counts. Conclusion These data suggest that Bifidobacterium spp. used in this study may have beneficial antiobesity effects.

  10. Silencing of the gibberellin receptor homolog, CsGID1a, affects locule formation in cucumber (Cucumis sativus) fruit.

    Liu, Bin; Liu, Xingwang; Yang, Sen; Chen, Chunhua; Xue, Shudan; Cai, Yanling; Wang, Dandan; Yin, Shuai; Gai, Xinshuang; Ren, Huazhong


    Gibberellins are phytohormones with many roles, including the regulation of fruit development. However, little is known about the relationship between GA perception and fleshy fruit ontogeny, and particularly locule formation. We characterized the expression of cucumber (Cucumis sativus) GA receptor gene (CsGID1a) using quantitative real-time PCR, in situ hybridization and a promoter::β-glucuronidase (GUS) assay. CsGID1a-RNAi cucumber fruits were observed by dissecting microscope, scanning electron microscopy and transmission electron microscopy. Finally, genome-wide gene expression in young fruits from a control and the RNAi line was compared using a digital gene expression (DGE) analysis approach. The expression pattern of CsGID1a was found to be closely correlated with fruit locule formation, and silencing CsGID1a in cucumber resulted in fruits with abnormal carpels and locules. Overexpression of CsGID1a in the Arabidopsis thaliana double mutant (gid1a gid1c) resulted in 'cucumber locule-like' fruits. The DGE analysis suggested that expression of genes related to auxin synthesis and transport, as well as the cell cycle, was altered in CsGID1a-RNAi fruits, a result that was supported by comparing the auxin content and cellular structures of the control and transgenic fruits. This study demonstrates a previously uncharacterized GA signaling pathway that is essential for cucumber fruit locule formation.

  11. Two different approaches in skin cancer therapy: using a photosensitizer/a natural product

    Abraham, Annie; Gayathri, Devi D.; Cibin, T. R.; Ramaiah, D.


    This paper deals with two potential modes for the treatment of skin cancer-one a novel approach using a squaraine dye and the other using a natural product- the flavonoid fraction of Saraca asoka. Squaraine dye is a photosensitizing agent, which is preferentially taken up and retained by the tumor cells and when irradiated with high power visible light results in the selective destruction of the tumor cells by photodynamic therapy. The uniqueness of this mode of treatment lies in the selective destruction of tumor cells without affecting the neighbouring normal cells, which is much advantageous over radiation therapy now frequently used. The chemopreventive and therapeutic effects of the plant component are explored as well. The experimental models were Swiss albino mice in which skin tumor was induced by DMBA. Marked reduction in tumor volume and burden in the treated groups were observed. The reversal of biochemical enzyme markers like rhodanese, myeloperoxidase, β-D glucuronidase, lactate dehydrogenase, hexokinase and sialic acid to near normal levels were observed in the PDT and flavonoid fraction treated groups. The live photographs of the experimental animals and histopathological data further support the obtained results. The study assumes importance as it combines a traditional treatment mode and a novel aspect in cancer therapy using the same experimental models. Also this is the first report on PDT using a squaraine dye for skin cancer therapy in vivo.

  12. Zearalenone, deoxynivalenol and aflatoxin B1 and their metabolites in pig urine as biomarkers for mycotoxin exposure.

    Thieu, N Q; Pettersson, H


    Methods to determine zearalenone (ZEA), deoxynivalenol (DON), aflatoxins (AF) and their metabolites in pig urine were developed as biomarkers for pig exposure to the mycotoxins in feed. Urine samples were incubated with β-glucuronidase to cleave conjugates, extracted and cleaned-up with solid phase and immunoaffinity columns, followed by HPLC with UV and fluorescence detection. Good recoveries (83-130%), low variation (2-10%), and low detection limits (0.3-9.9 ng/ml) were obtained. The results of controlled AFB1 feeding trials found no difference in urine concentrations of AFB1 or AFM1 from pigs fed three different levels (127, 227, 327 µg/kg) of AFB1 in diets. The excretion of AFB1 and AFM1 in urine was on average 30% of the oral dose and the ratio AFB1 to AFM1 was around 23%. The analysis of 15 Vietnamese pig urine samples indicate a relatively high exposure of ZEA, DON and AF, which were found as toxin or metabolites in 47, 73, and 80% of the urine samples, respectively.

  13. Therapeutic effects of Semecarpus anacardium Linn. nut milk extract on the changes associated with collagen and glycosaminoglycan metabolism in adjuvant arthritic Wistar rats.

    Ramprasath, Vanu Ramkumar; Shanthi, Palanivelu; Sachdanandam, Panchanatham


    The effect of milk extract of Semecarpus anacardium Linn. nut milk extract (SA) was studied to gain some insight into this intriguing disease with reference to collagen metabolism. Arthritis was induced in rats by injecting Freund's complete adjuvant containing 10mg of heat killed mycobacterium tuberculosis in 1 ml paraffin oil (0.1 ml) into the left hind paw of the rat intradermally. After 14 days of induction, SA (150 mg/kg body weight/day) was administered orally by gastric intubations for 14 days. Decreased levels of collagen and glycosaminoglycans (GAGS) components (chondroitin sulphate, heparan sulphate, hyaluronic acid) and increase in the levels of connective tissue degrading lysosomal glycohydrolases such as acid phosphatase, beta-glucuronidase, beta-N-acetyl glucosaminidase and cathepsin-D observed in arthritic animals were reverted back to near normal levels upon treatment with SA. The drug effectively regulated the uriniray markers of collagen metabolism namely hexosamine, hexuronic acid, hydroxyproline and total GAGS. Electron microscopic studies also revealed the protective effect of SA. Hence, it can be suggested that SA very effectively regulate the collagen metabolism that derange during arthritic condition.

  14. Potent chemopreventive effect of mangiferin on lung carcinogenesis in experimental Swiss albino mice

    Peramaiyan Rajendran


    Full Text Available Aim of the Study: In the present study the effects of mangiferin were tested against lung cancer-bearing mice in both the pre-initiation and post-initiation periods. Materials and Methods: Healthy male Swiss albino mice (6-8 weeks old were used throughout the study. The animals were treated with mangiferin (100 mg/kg body weight dissolved in corn oil two weeks before (pre-initiation and the twelfth week after (post-initiation the establishment of B (a P (50 mg/kg body weight-induced lung carcinoma. Results: The body weight decreased and the lung weight and levels of xenobiotic and liver marker enzymes markedly increased in the carcinogen-administered animals; and mangiferin treatment brought the values of these parameters back to the near-normal ones. The activities of lysosomal enzymes in the animals with B (a P-induced experimental lung carcinogenesis were also assessed. In these animals there was an increase in the activities of lysosomal enzymes such as acidphosphatase, β-glucuronidase, N-acetyl glucosaminidase, and β-galactosidase. Conclusion: Supplementation with mangiferin attenuated all these alterations, thus indicating its anticancer effect. Overall, the above data showed that the anticancer effect of mangiferin as a chemopreventive agent was pronounced.

  15. Satellite RNAs interfere with the function of viral RNA silencing suppressors

    Wanxia eShen


    Full Text Available Viral satellite RNAs (satRNAs are small subviral RNAs and depend on the helper virus for replication and spread. satRNAs can attenuate helper virus-induced symptoms, the mechanism of which remains unclear. Here, we show that two virus-encoded suppressors of RNA silencing (VSRs, Cucumber mosaic virus (CMV 2b and Tombusvirus P19, suppress hairpin RNA (hpRNA-induced silencing of a β-glucuronidase (GUS gene in Nicotiana benthamiana. This suppression can be overcome by CMV Y-satellite RNA (Y-Sat via the Y-Sat-derived small interfering RNAs (siRNAs, which bind to the VSRs and displace the bound hpGUS-derived siRNAs. We also show that microRNA target gene expression in N. tabacum was elevated by CMV infection, presumably due to function of the 2b VSR, but this upregulation of microRNA target genes was reversed in the presence of Y-Sat. These results suggest that satRNA infection minimizes the effect of VSRs on host siRNA and microRNA-directed silencing. Our results suggest that the high abundance of satRNA-derived siRNAs contributes to symptom attenuation by binding helper virus-encoded VSRs, minimizing the capacity of the VSRs to bind host siRNA and miRNA and interfere with their function.

  16. Introduction of cryIB-cryIAa Hybrid Gene Into Rice (Oryza sativa Genom cv. Rojolele using Agrobacterium-Mediated Transformation



    Full Text Available Rojolele is one of Indonesian local variety from Javanica group that susceptible to yellow stem borer (Scirpophaga incertulas. Previous study showed that Rojolele can be cultured and regenerated in vitro. Two cry genes, cryIB-cryIAa were fused and introduced into rice cv. Rojolele in an attempt to improve resistance and to obtain durable resistance rice against the yellow stem borer. Two-week old embryogenic calli of Rojolele rice were inoculated with Agrobacterium tumefaciens harbored with binary vector pCAMBIA 1301, 1303, or 1304 carrying cryIB-cryIAa hybrid gene, hygromycin resistant gene (hpt, and -glucuronidase (gus gene interrupted with an intron. The transformed calli were selected gradually on medium containing hygromycin (50, 100 mg/l and regenerated on medium containing 0.5 mg/l IAA and 0.3 mg/l BAP. GUS activity in infected calli was detected by histochemical assay 3 days after inoculation. The highest (100% transformation efficiency were obtained from calli transformed with pCAMBIA 1303 and 1304. Thirty four out of 77 transformed shoots were tested positive for the cryIB-cryIAa gene using PCR analysis. These shoots were grown in the soil to maturity and to collect the seeds. PCR analysis of the T1 progeny revealed that two out of six lines tested showed a Mendelian segregation pattern. These two lines were also potentially resistant to yellow stem borer based on bioassay in planta.

  17. Effect of soybean, Vicia faba, and vitamin C on the carcinogenicity of DMBA.

    el-Aaser, A A; Zakhary, N I; el-Guindy, S M; Hafiez, A R; Halawa, F; Mokhtar, N


    A single dose of 10 mg of 7,12-dimethylbenz[a]anthracene (DMBA), administered to rats through intragastric intubation, was sufficient to induce many biochemical and histopathological changes in their mammary tissue. Significant increases were observed in the activity levels of the enzymes acid ribonuclease, 5-nucleotidase, alkaline phosphatase, and beta-glucuronidase in mammary tissue homogenates of DMBA-treated rats after an experimental period of five months. Histopathological studies of the mammary tissue also revealed malignant epithelial tumors (cribriform carcinoma) induced among 85% of the treated rats, with an incidence of 4 tumors in 12 mammary glands. Nevertheless, administration of 30% soybean in the diet of rats or 5,000 ppm ascorbic acid in their drinking water in addition to DMBA revealed a significant chemoprotective effect against the carcinogenesis induced by DMBA alone. This chemoprotective effect was demonstrated by the normalization of the activity levels of the enzymes studied in mammary tissue homogenates, because most of the enzymes were maintained at near the levels in the control animals. The incidence and number of tumors were also decreased. Cribriform carcinoma was observed in 50% of the rats, and the incidence of the affected glands was 2 in 12 mammary glands among both groups. On the other hand, a less chemoprotective effect was observed due to Vicia faba administration.

  18. Detection of the misuse of steroids in doping control.

    Parr, Maria Kristina; Schänzer, Wilhelm


    The list of prohibited substances of the World Anti-Doping Agency (WADA) classifies the administration of several steroids in sports as doping. Their analysis is generally performed using urine specimen as matrix. Lots of the steroids are extensively metabolised in the human body. Thus, knowledge of urinary excretion is extremely important for the sensitive detection of steroid misuse in doping control. The methods routinely used in steroid screening mainly focus on substances, that are excreted unconjugated or as glucuronides. Common procedures include deconjugation using a beta-glucuronidase enzyme. Following extraction and concentration the analytes are submitted to LC-MS(/MS) analysis and/or GC-MS(/MS) analyses. Besides the classical steroids, more and more products appear on the market for "dietary supplements" containing steroids that have never been marketed as approved drugs, mostly without proper labelling of the contents. To cover the whole range of potential products comprehensive screening tools have to be utilised in addition to the classical methods. Endogenous steroids, e.g. testosterone, represent a special group of compounds. As classical chemical methodology is incapable of discriminating synthetic hormones from the biosynthesised congeners, the method of steroid profiling is used for screening purpose. Additionally, based on isotope signatures a discrimination of synthetic and natural hormones can be achieved.

  19. A Casparian strip domain-like gene, CASPL, negatively alters growth and cold tolerance.

    Yang, Jinghua; Ding, Changqing; Xu, Baochen; Chen, Cuiting; Narsai, Reena; Whelan, Jim; Hu, Zhongyuan; Zhang, Mingfang


    A cold-induced transcript encoding a Casparian strip membrane domain (CASP)-like protein (ClCASPL) was identified in watermelon (Citrullus lanatus). Fluorescence microscopy analysis showed that ClCASPL-GFP is localized in the plasma membrane. The orthologous gene in Arabidopsis thaliana (AtCASPL4C1) was also found to play an important role in cold tolerance. Expression analysis using a β-glucuronidase (GUS) reporter reveals that AtCASPL4C1 is widely expressed in a variety of organs and is cold inducible. Analysis of AtCASPL4C1 T-DNA knock-out plants showed altered growth dynamics, faster growth, increased biomass (dry weight) and earlier flowering compared to wild type (Col-0) and ClCASPL overexpressing plants. AtCASPL4C1 knock-out plants showed elevated tolerance to cold stress, while overexpressing CICASPL resulted in increased sensitivity to cold stress in Arabidopsis. Interestingly, AtCASPL4C1 knock-out plants did not display significant alterations in the Casparian strip formation in roots. Thus, the combination of these results suggests a role for CICASPL and AtCASPL4C1 beyond Casparian strip formation in roots, possibly indicating a more fundamental role in vascular tissue.

  20. Expression in Arabidopsis of a nucellus-specific promoter from watermelon (Citrullus lanatus).

    Dwivedi, Krishna K; Roche, Dominique; Carman, John G


    Though many tissue-specific promoters have been identified, few have been associated specifically with the angiospermous megasporangium (nucellus). In the present study the 2000-bp regulatory region upstream to the watermelon, Citrullus lanatus (Thunb.) Matsum & Nakai, gene WM403 (GenBank accession no. AF008925), which shows nucellus-specific expression, was cloned from watermelon gDNA and fused to the β-glucuronidase reporter gene (GUS). The resulting plasmid, WM403 Prom::GUS(+), which also contained NPTII, was transformed into Arabidopsis thaliana ecotype Co1-0. Seedlings were selected on kanamycin-containing medium, and transformants were confirmed by PCR. GUS assays of T(3) transformants revealed weak promoter activation in epidermal layers of the placenta and locule septum during premeiotic ovule development but strong activation in the nucellus, embryo sac and early embryo, from early embryo sac formation to early globular embryo formation. Expression in seeds was absent thereafter. These results indicate that the WM403 promoter may be useful in driving nucellus-specific gene expression in plants including candidate genes for important nucellus-specific traits such as apospory or adventitious embryony. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.

  1. Transformation of pecan and regeneration of transgenic plants.

    McGranahan, G H; Leslie, C A; Dandekar, A M; Uratsu, S L; Yates, I E


    A gene transfer system developed for walnut (Juglans regia L.) was successfully applied to pecan (Carya illinoensis [Wang] K. Koch). Repetitively embryogenic somatic embryos derived from open-pollinated seed of 'Elliott', 'Wichita', and 'Schley' were co-cultivated with Agrobacterium strain EHA 101/pCGN 7001, which contains marker genes for beta-glucuronidase activity and resistance to kanamycin. Several modifications of the standard walnut transformation techniques were tested, including a lower concentration of kanamycin and a modified induction medium, but these treatments had no measurable effect on efficiency of transformation. Nineteen of the 764 viable inoculated embryos produced transgenic subclones; 13 of these were from the line 'Elliott'6, 3 from 'Schley'5/3, and 3 from 'Wichita'9. Transgenic embryos of 'Wichita'9 germinated most readily and three subclones were successfully micropropagated. Three transgenic plants of one of these subclones were obtained by grafting the tissue cultured shoots to seedling pecan rootstock in the greenhouse. Gene insertion, initially detected by GUS activity, was confirmed by detection of integrated T-DNA sequences using Southern analysis.

  2. Evaluation of four phloem-specific promoters in vegetative tissues of transgenic citrus plants.

    Dutt, M; Ananthakrishnan, G; Jaromin, M K; Brlansky, R H; Grosser, J W


    'Mexican' lime (Citrus aurantifolia Swingle) was transformed with constructs that contained chimeric promoter-gus gene fusions of phloem-specific rolC promoter of Agrobacterium rhizogenes, Arabidopsis thaliana sucrose-H(+) symporter (AtSUC2) gene promoter of Arabidopsis thaliana, rice tungro bacilliform virus (RTBV) promoter and sucrose synthase l (RSs1) gene promoter of Oryza sativa (rice). Histochemical β-glucuronidase (GUS) analysis revealed vascular-specific expression of the GUS protein in citrus. The RTBV promoter was the most efficient promoter in this study while the RSs1 promoter could drive low levels of gus gene expression in citrus. These results were further validated by reverse transcription real-time polymerase chain reaction and northern blotting. Southern blot analysis confirmed stable transgene integration, which ranged from a single insertion to four copies per genome. The use of phloem-specific promoters in citrus will allow targeted transgene expression of antibacterial constructs designed to battle huanglongbing disease (HLB or citrus greening disease), associated with a phloem-limited Gram-negative bacterium.

  3. Expression of the dspA/E gene of Erwinia amylovora in non-host plant Arabidopsis thaliana

    Hasan Murat Aksoy


    Full Text Available In the Erwinia amylovora genome, the hrp gene cluster containing the dspA/E/EB/F operon plays a crucial role in mediating the pathogenicity and the hypersensitive response (HR in the host plant. The role of the dspA/E gene derived from E. amylovora was investigated by monitoring the expression of the β-glucuronidase (GUS reporter system in transgenic Arabidopsis thaliana cv. Pri-Gus seedlings. A mutant ΔdspA/E strain of E. amylovora was generated to contain a deletion of the dspA/E gene for the purpose of this study. Two-week-old seedlings of GUS transgenic Arabidopsis were vacuum-infiltrated with the wild-type and the mutant (ΔdspA/E E. amylovora strains. The Arabidopsis seedlings were fixed and stained for GUS activity after 3–5 days following infiltration. The appearance of dense spots with blue staining on the Arabidopsis leaves indicated the typical characteristic of GUS activity. This observation indicated that the wild-type E. amylovora strain had induced a successful and efficient infection on the A. thaliana Pri-Gus leaves. In contrast, there was no visible GUS expression on leaf tissues which were inoculated with the ΔdspA/E mutant E. amylovora strain. These results indicate that the dspA/E gene is required by the bacterial cells to induce HR in non-host plants.

  4. Elevated urine heparanase levels are associated with proteinuria and decreased renal allograft function.

    Itay Shafat

    Full Text Available Heparanase is an endo-β-glucuronidase that cleaves heparan sulfate side chains, leading to structural modifications that loosen the extracellular matrix barrier and associated with tumor metastasis, inflammation and angiogenesis. In addition, the highly sulfated heparan sulfate proteoglycans are important constituents of the glomerular basement membrane and its permselective properties. Recent studies suggest a role for heparanase in several experimental and human glomerular diseases associated with proteinuria such as diabetes, minimal change disease, and membranous nephropathy. Here, we quantified blood and urine heparanase levels in renal transplant recipients and patients with chronic kidney disease (CKD, and assessed whether alterations in heparanase levels correlate with proteinuria and renal function. We report that in transplanted patients, urinary heparanase was markedly elevated, inversely associated with estimated glomerular filtration rate (eGFR, suggesting a relationship between heparanase and graft function. In CKD patients, urinary heparanase was markedly elevated and associated with proteinuria, but not with eGFR. In addition, urinary heparanase correlated significantly with plasma heparanase in transplanted patients. Such a systemic spread of heparanase may lead to damage of cells and tissues alongside the kidney.The newly described association between heparanase, proteinuria and decreased renal function is expected to pave the way for new therapeutic options aimed at attenuating chronic renal allograft nephropathy, leading to improved graft survival and patient outcome.

  5. Optimization of Agrobacterium mediated genetic transformation of cotyledonary node explants of Vigna radiata.

    Yadav, Sushil Kumar; Katikala, Sweety; Yellisetty, Varalaxmi; Kannepalle, Annapurna; Narayana, Jyothi Lakshmi; Maddi, Vanaja; Mandapaka, Maheswari; Shanker, Arun Kumar; Bandi, Venkateswarlu; Bharadwaja, Kirti Pulugurtha


    A reproducible and highly efficient protocol for genetic transformation mediated by Agrobacterium has been established for greengram (Vigna radiata L. Wilczek). Double cotyledonary node (DCN) explants were inoculated with Agrobacterium tumefaciens strain LBA 4404 harboring a binary vector pCAMBIA 2301 containing neomycin phosphotransferase (npt II) gene as selectable marker, β-glucuronidase (GUS) as a reporter (uidA) gene and annexin 1 bj gene. Important parameters like optical density of Agrobacterium culture, culture quantity, infection medium, infection and co-cultivation time and acetosyringone concentration were standardized to optimize the transformation frequency. Kanamycin at a concentration of 100 mg/l was used to select transformed cells. Transient and stable GUS expressions were studied in transformed explants and regenerated putative plants, respectively. Transformed shoot were produced on regeneration medium containing 100 mg/l kanamycin and 250 mg/l cefotaxime and rooted on ½ MS medium. Transient and constitutive GUS expression was observed in DCN explants and different tissues of T(0) and T(1) plants. Rooted T(0) and T(1) shoots confirming Polymerase Chain Reaction (PCR) positive for npt II and annexin 1bj genes were taken to maturity to collect the seeds. Integration of annexin gene into the greengram genome was confirmed by Southern blotting.

  6. Xcc-facilitated agroinfiltration of citrus leaves: a tool for rapid functional analysis of transgenes in citrus leaves.

    Jia, Hongge; Wang, Nian


    Xanthomonas citri subsp. citri pretreatment before agroinfiltration could significantly promote transient expression in citrus leaves which were previously recalcitrant to agroinfiltration. Transient expression via agroinfiltration is widely used in biotechnology but remains problematic in many economically important plants. Xanthomonas citri subsp. citri (Xcc)-facilitated agroinfiltration was employed to promote transient protein expression in Valencia sweet orange leaves, which are recalcitrant to agroinfiltration. However, it is unclear whether Xcc-facilitated agroinfiltration has broad application, i.e., whether Xcc-facilitated agroinfiltration could be used on other citrus varieties. In addition, we intended to investigate whether Xcc-facilitated agroinfiltration could be used to hasten transgene function assays, e.g., Cre/lox system and Cas9/sgRNA system. In this report, Xcc-facilitated agroinfiltration was further exploited to enhance β-glucuronidase (GUS) expression in five citrus varieties. Xcc-facilitated agroinfiltration also significantly increased GFP expression in six citrus varieties tested. Both GUS and GFP assays indicated that Xcc-facilitated agroinfiltration had the best performance in grapefruit. After Xcc-facilitated agroinfiltration was carried out in grapefruit, protoplast analysis of the transformed cells indicated that there were more than 20 % leaf cells expressing GFP. In grapefruit, usefulness of Xcc-facilitated agroinfiltration was assayed in three case studies: (1) fast functional analysis of Cre/lox system, (2) the heat shock regulation of HSP70B promoter derived from Arabidopsis, and (3) Cas9/sgRNA-mediated genome modification.

  7. Disruption of OsYSL15 leads to iron inefficiency in rice plants.

    Lee, Sichul; Chiecko, Jeff C; Kim, Sun A; Walker, Elsbeth L; Lee, Youngsook; Guerinot, Mary Lou; An, Gynheung


    Uptake and translocation of metal nutrients are essential processes for plant growth. Graminaceous species release phytosiderophores that bind to Fe(3+); these complexes are then transported across the plasma membrane. We have characterized OsYSL15, one of the rice (Oryza sativa) YS1-like (YSL) genes that are strongly induced by iron (Fe) deficiency. The OsYSL15 promoter fusion to beta-glucuronidase showed that it was expressed in all root tissues when Fe was limited. In low-Fe leaves, the promoter became active in all tissues except epidermal cells. This activity was also detected in flowers and seeds. The OsYSL15:green fluorescent protein fusion was localized to the plasma membrane. OsYSL15 functionally complemented yeast strains defective in Fe uptake on media containing Fe(3+)-deoxymugineic acid and Fe(2+)-nicotianamine. Two insertional osysl15 mutants exhibited chlorotic phenotypes under Fe deficiency and had reduced Fe concentrations in their shoots, roots, and seeds. Nitric oxide treatment reversed this chlorosis under Fe-limiting conditions. Overexpression of OsYSL15 increased the Fe concentration in leaves and seeds from transgenic plants. Altogether, these results demonstrate roles for OsYSL15 in Fe uptake and distribution in rice plants.

  8. A Meloidogyne incognita effector is imported into the nucleus and exhibits transcriptional activation activity in planta.

    Zhang, Lei; Davies, Laura J; Elling, Axel A


    Root-knot nematodes are sedentary biotrophic endoparasites that maintain a complex interaction with their host plants. Nematode effector proteins are synthesized in the oesophageal glands of nematodes and secreted into plant tissue through a needle-like stylet. Effectors characterized to date have been shown to mediate processes essential for nematode pathogenesis. To gain an insight into their site of action and putative function, the subcellular localization of 13 previously isolated Meloidogyne incognita effectors was determined. Translational fusions were created between effectors and EGFP-GUS (enhanced green fluorescent protein-β-glucuronidase) reporter genes, which were transiently expressed in tobacco leaf cells. The majority of effectors localized to the cytoplasm, with one effector, 7H08, imported into the nuclei of plant cells. Deletion analysis revealed that the nuclear localization of 7H08 was mediated by two novel independent nuclear localization domains. As a result of the nuclear localization of the effector, 7H08 was tested for the ability to activate gene transcription. 7H08 was found to activate the expression of reporter genes in both yeast and plant systems. This is the first report of a plant-parasitic nematode effector with transcriptional activation activity.

  9. Regulation of Motility and Phenazine Pigment Production by FliA Is Cyclic-di-GMP Dependent in Pseudomonas aeruginosa PAO1.

    Lo, Yi-Ling; Shen, Lunda; Chang, Chih-Hsuan; Bhuwan, Manish; Chiu, Cheng-Hsun; Chang, Hwan-You


    The transcription factor FliA, also called sigma 28, is a major regulator of bacterial flagellar biosynthesis genes. Growing evidence suggest that in addition to motility, FliA is involved in controlling numerous bacterial behaviors, even though the underlying regulatory mechanism remains unclear. By using a transcriptional fusion to gfp that responds to cyclic (c)-di-GMP, this study revealed a higher c-di-GMP concentration in the fliA deletion mutant of Pseudomonas aeruginosa than in its wild-type strain PAO1. A comparative analysis of transcriptome profiles of P. aeruginosa PAO1 and its fliA deletion mutant revealed an altered expression of several c-di-GMP-modulating enzyme-encoding genes in the fliA deletion mutant. Moreover, the downregulation of PA4367 (bifA), a Glu-Ala-Leu motif-containing phosphodiesterase, in the fliA deletion mutant was confirmed using the β-glucuronidase reporter gene assay. FliA also altered pyocyanin and pyorubin production by modulating the c-di-GMP concentration. Complementing the fliA mutant strain with bifA restored the motility defect and pigment overproduction of the fliA mutant. Our results indicate that in addition to regulating flagellar gene transcription, FliA can modulate the c-di-GMP concentration to regulate the swarming motility and phenazine pigment production in P. aeruginosa.

  10. Expression and affinity purification of recombinant proteins from plants

    Desai, Urvee A.; Sur, Gargi; Daunert, Sylvia; Babbitt, Ruth; Li, Qingshun


    With recent advances in plant biotechnology, transgenic plants have been targeted as an inexpensive means for the mass production of proteins for biopharmaceutical and industrial uses. However, the current plant purification techniques lack a generally applicable, economic, large-scale strategy. In this study, we demonstrate the purification of a model protein, beta-glucuronidase (GUS), by employing the protein calmodulin (CaM) as an affinity tag. In the proposed system, CaM is fused to GUS. In the presence of calcium, the calmodulin fusion protein binds specifically to a phenothiazine-modified surface of an affinity column. When calcium is removed with a complexing agent, e.g., EDTA, calmodulin undergoes a conformational change allowing the dissociation of the calmodulin-phenothiazine complex and, therefore, permitting the elution of the GUS-CaM fusion protein. The advantages of this approach are the fast, efficient, and economical isolation of the target protein under mild elution conditions, thus preserving the activity of the target protein. Two types of transformation methods were used in this study, namely, the Agrobacterium-mediated system and the viral-vector-mediated transformation system. Copyright 2002 Elsevier Science (USA).

  11. Simultaneous determination of five free and total flavonoids in rat plasma by ultra HPLC-MS/MS and its application to a comparative pharmacokinetic study in normal and hyperlipidemic rats.

    Wang, Xiaofan; Zhao, Xu; Gu, Liqiang; Lv, Chunxiao; He, Bosai; Liu, Zhenzhen; Hou, Pengyi; Bi, Kaishun; Chen, Xiaohui


    A simple and rapid ultra-high performance liquid chromatography-tandem mass spectrometry (uHPLC-MS/MS) method has been developed for the simultaneous determination of five free flavonoids (amentoflavone, isorhamnetin, naringenin, kaempferol and quercetin) and their total (free and conjugated) forms, and to compare the pharmacokinetics of these active ingredients in normal and hyperlipidemic rats. The free and total forms of these flavonoids were extracted by liquid-liquid extraction with ethyl acetate. The conjugated flavonoids were deconjugated by the enzyme β-Glucuronidase and Sulfatase. Chromatographic separation was accomplished on a ZORBAX Eclipse XDB-C8 USP L7 column using gradient elution. Detection was performed on a 4000Q uHPLC-MS/MS system from AB Sciex using negative ion mode in the multiple reaction monitoring (MRM) mode. The lower limits of quantification were 2.0-5.0ng/mL for all the analytes. Intra-day and inter-day precision were less than 15% and accuracy ranged from -9.3% to 11.0%, and the mean extraction recoveries of analytes and internal standard (IS) from rat plasma were all more than 81.7%. The validated method was successfully applied to a comparative pharmacokinetic study of five free and total analytes in rat plasma. The results indicated that the absorption of five total flavonoids in hyperlipidemia group were significantly higher than those in normal group with similar concentration-time curves.

  12. GhMPK7, a novel multiple stress-responsive cotton group C MAPK gene, has a role in broad spectrum disease resistance and plant development.

    Shi, Jing; An, Hai-Long; Zhang, Liang; Gao, Zheng; Guo, Xing-Qi


    Mitogen-activated protein kinase (MAPK) cascades play a pivotal role in environmental responses and developmental processes in plants. Previous researches mainly focus on the MAPKs in groups A and B, and little is known on group C. In this study, we isolated and characterized GhMPK7, which is a novel gene from cotton belonging to the group C MAPK. RNA blot analysis indicated that GhMPK7 transcript was induced by pathogen infection and multiple defense-related signal molecules. Transgenic Nicotina benthamiana overexpressing GhMPK7 displayed significant resistance to fungus Colletotrichum nicotianae and virus PVY, and the transcript levels of SA pathway genes were more rapidly and strongly induced. Furthermore, the transgenic N. benthamiana showed reduced ROS-mediated injuries by upregulating expression of oxidative stress-related genes. Interestingly, the transgenic plants germinated earlier and grew faster in comparison to wild-type plants. beta-glucuronidase activity driven by the GhMPK7 promoter was detected in the apical meristem at the vegetative stage, and it was enhanced by treatments with signal molecules and phytohormones. These results suggest that GhMPK7 might play an important role in SA-regulated broad-spectrum resistance to pathogen infection, and that it is also involved in regulation of plant growth and development.

  13. Anthocyanins in Strawberry Polyphenolic Extract Enhance the Beneficial Effects of Diets with Fructooligosaccharides in the Rat Cecal Environment.

    Bartosz Fotschki

    Full Text Available The administration of fructooligosaccharides (FOS beneficially modulates gastrointestinal functions and may enhance the metabolism of polyphenols. However, different polyphenolic components in the diet may have different influences on the activities of the digestive enzymes and microbiota in the gastrointestinal tract. Therefore, a 4-week study of forty-eight male Wistar rats was conducted to investigate the physiological response of the rat cecal environment to diets without and with FOS that contained two different strawberry polyphenolic extracts, specifically EP (polyphenolic profile 60, 35, 5, and 0% ellagitannins, proanthocyanidins, flavonols, anthocyanins, respectively and EPA (polyphenolic profile: 50, 35, 6, and 9%, respectively. When combined with FOS, both extracts beneficially enhanced the acidification of the cecal digesta (P≤0.05 vs the groups without extracts, but the dietary combination of EPA and FOS elicited the greatest reduction in putrefactive short-chain fatty acid production and the lowest fecal β-glucuronidase activity in the cecum (P≤0.05 vs group EP. Moreover, the addition of dietary FOS elevated the metabolism of the examined strawberry extracts in the cecum and thereby increased the concentrations of the metabolites in the cecal digesta and urine (P≤0.05 vs the group with cellulose. Overall, both strawberry extracts modulated the effects of FOS in the gastrointestinal tract; however, the combination with EPA extract that contained anthocyanins exhibited greater beneficial effects in the lower gut environment than the EP extract.

  14. Enhancing effect of lipids and emulsifiers on the accumulation of quercetin metabolites in blood plasma after the short-term ingestion of onion by rats.

    Azuma, Keiko; Ippoushi, Katsunari; Ito, Hidekazu; Horie, Hideki; Terao, Junji


    The effects of co-ingested lipids and emulsifiers on the accumulation of quercetin metabolites in blood plasma after the short-term ingestion of onion by rats were investigated. Plasma extracts of rats that had been fed onion-containing diets for one and two weeks were analyzed by HPLC with electrochemical detection after a treatment with sulfatase/beta-glucuronidase. Almost all of the quercetin metabolites in the plasma were sulfate/glucuronide conjugates of quercetin and isorhamnetin. More than 4.6% (w/w) of soybean oil in the diets significantly enhanced the accumulation of quercetin metabolites in the plasma. Fish oil and beef tallow increased this to an extent similar to that with soybean oil, and lecithin was more effective than the other three lipids. Two emulsifiers, sodium caseinate and sucrose fatty acid ester, also showed an enhancing effect on the accumulation of quercetin metabolites. These results indicate that co-ingested lipids and emulsifiers could enhance the bioavailability of quercetin glucosides in onion.

  15. Advanced glycation end-products induce heparanase expression in endothelial cells by the receptor for advanced glycation end products and through activation of the FOXO4 transcription factor.

    An, Xiao-Fei; Zhou, Lei; Jiang, Peng-Jun; Yan, Ming; Huang, Yu-Jun; Zhang, Su-Na; Niu, Yun-Fei; Ten, Shi-Chao; Yu, Jiang-Yi


    As an endo-β (1-4)-D: -glucuronidase, heparanase can specifically cleave carbohydrate chains of heparan sulfate (HS) and has been implicated in development of endothelial cells dsyfunction. The advanced glycation end products (AGEs) play a pivotal role in the pathology of diabetic complications. In the present study, we investigated the effect of AGE-bovine serum albumin (AGE-BSA) on heparanase expression in human microvascular endothelial cells (HMVECs) and the underlying molecular mechanisms. The results indicated that in vitro direct exposure of HMVECs to AGE-BSA (300, 1000, and 3000 μg/ml) could increase heparanase mRNA and protein expression in a dose and time-dependent manner. The effect of 1000 μg/ml AGE-BSA could be abolished by neutralization with antibody of the receptor for advanced glycation end products (RAGE). Moreover, pretreatment with inhibitors of nuclear factor-κB (NF-κB) or PI3-kinase did not affect heparanase expression induced by AGE-BSA. Nevertheless, small interference RNA (siRNA) for transcriptional factor FOXO4 could reduce the increase of heparanase expression in HMVECs induced by 1000 μg/ml AGE-BSA. These results suggest that AGEs could induce heparanase expression in HMVECs by RAGE and predominantly through activation of the FOXO4 transcription factor.

  16. Importance of surface characteristics of QUARTZ DQ 12 for acute inflammation

    Albrecht, C.; Becher, A.; Scins, R.P.F.; Hoehr, D.; Unfried, K.; Knaapen, A.M.; Borm, P.J.A. [Institut fuer medizinische Forschung (IUF), Duesseldorf (Germany)


    Although quartz is known to induce inflammation in rat lungs, mechanisms are not yet fully understood. The importance of particle surface characteristics was investigated in vivo after intratracheal instillation of different preparations of quartz in rat lungs. Three days after instillation of 2 mg DQ12 quartz, or DQ12 coated with polyvinylpyridine-N-oxide (PVNO) or Aluminium lactate (AL), lungs of female Wistar rats were lavaged in situ to determine markers of inflammation. Control rats received saline or the coating substances alone. DQ12 induced a marked inflammatory response, as indicated by a significant increase in the number of neutrophils and macrophages, as well as in the levels of b-glucuronidase and myeloperoxidase. None of these inflammatory markers was increased for both coated quartz preparations, with the exception of neutrophil influx which was also increased after treatment with AL quartz. Our results indicate that surface characteristics are important in the onset of quartz-induced lung inflammation which could imply a different development of persistent inflammation. This will be investigated in later follow-up time points of the same animal study. (orig.)

  17. Pharmacokinetics of the estrogen receptor subtype-selective ligands, PPT and DPN: quantification using UPLC-ES/MS/MS.

    Sepehr, Estatira; Lebl-Rinnova, Marketa; Mann, Meagan K; Pisani, Samantha L; Churchwell, Mona I; Korol, Donna L; Katzenellenbogen, John A; Doerge, Daniel R


    Estrogen receptor (ER) subtype specific agonists, diarylpropionitrile (DPN) for ERβ and propylpyrazoletriol (PPT) for ERα, are pharmacological probes used frequently to define mechanisms for estrogen actions in vitro and in vivo. Quantitative analytical methodology was developed and validated for DPN and PPT, based on synthetic stable labeled analogs (DPN-d(4) and PPT-d(5)) using isotope dilution liquid chromatographic tandem electrospray mass spectrometric detection. The validated method produced high sensitivity, with detection limits of 0.04-0.07ng/ml serum. Serum pharmacokinetics were evaluated in Long-Evans rats following a single subcutaneous injection (2mg/kg bw) of both compounds. The role of Phase II metabolism was evaluated using β-glucuronidase and arylsulfatase hydrolysis to measure total DPN and PPT in addition to the parent compounds. The pharmacokinetic properties of DPN and PPT reported could facilitate experimental designs requiring specified levels of receptor occupancy for quantitative comparisons of ER subtype specificities for natural and synthetic estrogens in vivo. Published by Elsevier B.V.

  18. Relationship among Shigella spp. and enteroinvasive Escherichia coli (EIEC) and their differentiation.

    Ud-Din, Abu; Wahid, Syeda


    Shigellosis produces inflammatory reactions and ulceration on the intestinal epithelium followed by bloody or mucoid diarrhea. It is caused by enteroinvasive E. coli (EIEC) as well as any species of the genus Shigella, namely, S. dysenteriae, S. flexneri, S. boydii, and S. sonnei. This current species designation of Shigella does not specify genetic similarity. Shigella spp. could be easily differentiated from E. coli, but difficulties observed for the EIEC-Shigella differentiation as both show similar biochemical traits and can cause dysentery using the same mode of invasion. Sequencing of multiple housekeeping genes indicates that Shigella has derived on several different occasions via acquisition of the transferable forms of ancestral virulence plasmids within commensal E. coli and form a Shigella-EIEC pathovar. EIEC showed lower expression of virulence genes compared to Shigella, hence EIEC produce less severe disease than Shigella spp. Conventional microbiological techniques often lead to confusing results concerning the discrimination between EIEC and Shigella spp. The lactose permease gene (lacY) is present in all E. coli strains but absent in Shigella spp., whereas β-glucuronidase gene (uidA) is present in both E. coli and Shigella spp. Thus uidA gene and lacY gene based duplex real-time PCR assay could be used for easy identification and differentiation of Shigella spp. from E. coli and in particular EIEC.

  19. Opposite Stereoselectivities of Dirigent Proteins in Arabidopsis and Schizandra Species

    Kim, Kye-Won; Moinuddin, Syed G. A.; Atwell, Kathleen M.; Costa, Michael A.; Davin, Laurence B.; Lewis, Norman G.


    How stereoselective monolignol-derived phenoxy radical-radical coupling reactions are differentially biochemically orchestrated in planta, whereby for example they afford (+)- and (-)-pinoresinols, respectively, is both a fascinating mechanistic and evolutionary question. In earlier work, biochemical control of (+)-pinoresinol formation had been established to be engendered by a (+)-pinoresinol-forming dirigent protein in Forsythia intermedia, whereas the presence of a (-)-pinoresinol-forming dirigent protein was indirectly deduced based on the enantiospecificity of downstream pinoresinol reductases (AtPrRs) in Arabidopsis thaliana root tissue. In this study of 16 putative dirigent protein homologs in Arabidopsis, AtDIR6, AtDIR10, and AtDIR13 were established to be root-specific using a β-glucuronidase reporter gene strategy. Of these three, in vitro analyses established that only recombinant AtDIR6 was a (-)-pinoresinol-forming dirigent protein, whose physiological role was further confirmed using overexpression and RNAi strategies in vivo. Interestingly, its closest homolog, AtDIR5, was also established to be a (-)-pinoresinol-forming dirigent protein based on in vitro biochemical analyses. Both of these were compared in terms of properties with a (+)-pinoresinol-forming dirigent protein from Schizandra chinensis. In this context, sequence analyses, site-directed mutagenesis, and region swapping resulted in identification of putative substrate binding sites/regions and candidate residues controlling distinct stereoselectivities of coupling modes.

  20. Optimization of a liquid chromatography-tandem mass spectrometry method for quantification of the plant lignans secoisolariciresinol, matairesinol, lariciresinol, and pinoresinol in foods.

    Milder, Ivon E J; Arts, Ilja C W; Venema, Dini P; Lasaroms, Johan J P; Wähälä, Kristina; Hollman, Peter C H


    A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the quantification of the four major enterolignan precursors [secoisolariciresinol, matairesinol, lariciresinol, and pinoresinol] in foods. The method consists of alkaline methanolic extraction, followed by enzymatic hydrolysis using Helix pomatia (H. pomatia) beta-glucuronidase/sulfatase. H. pomatia was selected from several enzymes based on its ability to hydrolyze isolated lignan glucosides. After ether extraction samples were analyzed and quantified against secoisolariciresinol-d8 and matairesinol-d6. The method was optimized using model products: broccoli, bread, flaxseed, and tea. The yield of methanolic extraction increased up to 81%, when it was combined with alkaline hydrolysis. Detection limits were 4-10 microg/(100 g dry weight) for solid foods and 0.2-0.4 microg/(100 mL) for beverages. Within- and between-run coefficients of variation were 6-21 and 6-33%, respectively. Recovery of lignans added to model products was satisfactory (73-123%), except for matairesinol added to bread (51-55%).

  1. Opposite stereoselectivities of dirigent proteins in Arabidopsis and schizandra species.

    Kim, Kye-Won; Moinuddin, Syed G A; Atwell, Kathleen M; Costa, Michael A; Davin, Laurence B; Lewis, Norman G


    How stereoselective monolignol-derived phenoxy radical-radical coupling reactions are differentially biochemically orchestrated in planta, whereby for example they afford (+)- and (-)-pinoresinols, respectively, is both a fascinating mechanistic and evolutionary question. In earlier work, biochemical control of (+)-pinoresinol formation had been established to be engendered by a (+)-pinoresinol-forming dirigent protein in Forsythia intermedia, whereas the presence of a (-)-pinoresinol-forming dirigent protein was indirectly deduced based on the enantiospecificity of downstream pinoresinol reductases (AtPrRs) in Arabidopsis thaliana root tissue. In this study of 16 putative dirigent protein homologs in Arabidopsis, AtDIR6, AtDIR10, and AtDIR13 were established to be root-specific using a β-glucuronidase reporter gene strategy. Of these three, in vitro analyses established that only recombinant AtDIR6 was a (-)-pinoresinol-forming dirigent protein, whose physiological role was further confirmed using overexpression and RNAi strategies in vivo. Interestingly, its closest homolog, AtDIR5, was also established to be a (-)-pinoresinol-forming dirigent protein based on in vitro biochemical analyses. Both of these were compared in terms of properties with a (+)-pinoresinol-forming dirigent protein from Schizandra chinensis. In this context, sequence analyses, site-directed mutagenesis, and region swapping resulted in identification of putative substrate binding sites/regions and candidate residues controlling distinct stereoselectivities of coupling modes.

  2. Compost Grown Agaricus bisporus Lacks the Ability to Degrade and Consume Highly Substituted Xylan Fragments

    de Vries, Ronald P.; Gruppen, Harry; Kabel, Mirjam A.


    The fungus Agaricus bisporus is commercially grown for the production of edible mushrooms. This cultivation occurs on compost, but not all of this substrate is consumed by the fungus. To determine why certain fractions remain unused, carbohydrate degrading enzymes, water-extracted from mushroom-grown compost at different stages of mycelium growth and fruiting body formation, were analyzed for their ability to degrade a range of polysaccharides. Mainly endo-xylanase, endo-glucanase, β-xylosidase and β-glucanase activities were determined in the compost extracts obtained during mushroom growth. Interestingly, arabinofuranosidase activity able to remove arabinosyl residues from doubly substituted xylose residues and α-glucuronidase activity were not detected in the compost enzyme extracts. This correlates with the observed accumulation of arabinosyl and glucuronic acid substituents on the xylan backbone in the compost towards the end of the cultivation. Hence, it was concluded that compost grown A. bisporus lacks the ability to degrade and consume highly substituted xylan fragments. PMID:26237450

  3. Comparative activation states of tumor-associated and peritoneal macrophages from mice bearing an induced fibrosarcoma.

    Valdez, J C; de Alderete, N; Meson, O E; Sirena, A; Perdigon, G


    Balb/c mice bearing a methylcholanthrene-induced fibrosarcoma were used to compare the activation levels of tumor-associated and peritoneal macrophages. Two stages of tumor growth were examined, namely "small" and "large" tumors, with average diameters of 10 and 30 mm, respectively. The activation state, determined by measurement of both phagocytic index and beta-glucuronidase content, was found to be markedly higher in tumor-associated macrophages than in their peritoneal counterparts and it was, in addition, independent of tumor progression. The percentage of tumor-associated macrophages, which were detected on the basis of Fc receptor expression, remained constant in the growing neoplasm, at approximately 23% of total cell population. None of these parameters were affected by inoculation with an immunopotentiating dose of heat-killed Candida albicans which, on the other hand, seemed not to alter the course of the tumor. These data suggest that within the tumor microenvironment macrophages would somehow be maintained at a constant proportion and at a highly activated state, while outside the tumor they would be at a lower activation level. Our results also suggest that TAM would not possess antitumor activity in vivo, although we have found this activity in vitro.

  4. Identification of fungus-responsive cis-acting element in the promoter of Brassica juncea chitinase gene, BjCHI1.

    Gao, Ying; Zan, Xin-Li; Wu, Xue-Feng; Yao, Lei; Chen, Yu-Ling; Jia, Shuang-Wei; Zhao, Kai-Jun


    Chitinases are a group of pathogenesis-related proteins. The Brassica juncea chitinase gene BjCHI1 is highly inducible by pathogenic fungal infection, suggesting that the promoter of BjCHI1 might contain specific cis-acting element responsive to fungal attack. To identify the fungus-responsive element in BjCHI1 promoter (BjC-P), a series of binary plant transformation vectors were constructed by fusing the BjC-P or its deletion-derivatives to β-glucuronidase (GUS) reporter gene. Expression of the GUS reporter gene was systematically assayed by a transient gene expression system in Nicotiana benthamiana leaves treated with fungal elicitor Hexa-N-Acetyl-Chitohexaose, as well as in transgenic Arabidopsis plants inoculated with fungus Botrytis cinerea. The histochemical and quantitative GUS assays showed that the W-box-like element (GTAGTGACTCAT) in the region (-668 to -657) was necessary for the fungus-response, although there were another five W-box-like elements in BjC-P. In addition, gain-of-function analysis demonstrated that the fragment (-409 to -337) coupled to the W-box-like element was needed for full magnitude of the fungal induction. These results revealed the existence of a novel regulation mechanism of W-box-like element involved in plant pathogenic resistance, and will benefit the potential application of BjC-P in engineering crops.

  5. Renal peroxidative changes mediated by oxalate: the protective role of fucoidan.

    Veena, Coothan Kandaswamy; Josephine, Anthony; Preetha, Sreenivasan P; Varalakshmi, Palaninathan; Sundarapandiyan, Rajaguru


    Oxalate, one of the major constituents of renal stones is known to induce free radicals which damage the renal membrane. Damaged epithelia might act as nidi for stone formation aggravating calcium oxalate precipitation during hyperoxaluria. In the present study, the beneficial effects of fucoidan on oxalate-induced free radical injury were investigated. Male Wistar rats were divided into four groups. Hyperoxaluria was induced in two groups by administration of 0.75% ethylene glycol in drinking water for 28 days and one of them was treated with fucoidan from Fucus vesiculosus at a dose of 5 mg/kg b.wt subcutaneously commencing from the 8th day of induction. A control and drug control (fucoidan alone) was also included in the study. The extent of renal injury in hyperoxaluria was evident from the increased activities of alkaline phosphatase, gamma-glutamyl transferase, beta-glucuronidase, N-acetyl-beta-D-glucosaminidase in urine. There was a positive correlation between plasma malondialdehyde levels and renal membrane damage indicating a striking relation between free radical formation and cellular injury. Increased protein carbonyl and decreased thiols further exemplified the oxidative milieu prevailing during hyperoxaluria. Decreased renal membrane ATPases accentuated the renal membrane damage induced by oxalate. Renal microscopic analysis showed abnormal findings in histology as an evidence of oxalate damage. The above biochemical and histopathological discrepancies were abrogated with fucoidan administration, indicating its protective role in oxalate mediated peroxidative injury.

  6. Rice OsRAD21-2 is Expressed in Actively Dividing Tissues and its Ectopic Expression in Yeast Results in Aberrant Cell Division and Growth

    Chunyan Gong; Tang Li; Qi Li; Longfeng Yan; Tai Wang


    Rad21 and its meiotic counterpart Rec8,the key components of the cohesin complex,are essential for sister chromatid cohesion and chromosome segregation in mitosis and meiosis,respectively.In contrast to yeast and vertebrates,which have only two RAD21/REC8 genes,the rice genome encodes four Rad21/Rec8 proteins.Here,we report on the cloning and characterization of OsRAD21-2 from rice (Oryza sativa L.).Phylogenetic analysis of the full-length amino acids showed that OsRad21-2 was grouped into the plant-specific Rad21 subfamily.Semi-quantitative reverse transcription-polymerase chain reaction revealed OsRAD21-2 preferentially expressed in premeiotic flowers.Further RNA in situ hybridization analysis and promoter::β-glucuronidase staining indicated that OsRAD21-2 was mainly expressed in actively dividing tissues including premeiotic stamen,stem intercalary meristem,leaf meristem,and root pericycle.Ectopic expression of OsRAD21-2 in fission yeast resulted in cell growth delay and morphological abnormality.Flow cytometric analysis revealed that the OsRAD21-2-expressed cells were arrested in G2 phase.Our results suggest that OsRad21-2 functions in regulation of cell division and growth.

  7. Regulation of Motility and Phenazine Pigment Production by FliA Is Cyclic-di-GMP Dependent in Pseudomonas aeruginosa PAO1.

    Yi-Ling Lo

    Full Text Available The transcription factor FliA, also called sigma 28, is a major regulator of bacterial flagellar biosynthesis genes. Growing evidence suggest that in addition to motility, FliA is involved in controlling numerous bacterial behaviors, even though the underlying regulatory mechanism remains unclear. By using a transcriptional fusion to gfp that responds to cyclic (c-di-GMP, this study revealed a higher c-di-GMP concentration in the fliA deletion mutant of Pseudomonas aeruginosa than in its wild-type strain PAO1. A comparative analysis of transcriptome profiles of P. aeruginosa PAO1 and its fliA deletion mutant revealed an altered expression of several c-di-GMP-modulating enzyme-encoding genes in the fliA deletion mutant. Moreover, the downregulation of PA4367 (bifA, a Glu-Ala-Leu motif-containing phosphodiesterase, in the fliA deletion mutant was confirmed using the β-glucuronidase reporter gene assay. FliA also altered pyocyanin and pyorubin production by modulating the c-di-GMP concentration. Complementing the fliA mutant strain with bifA restored the motility defect and pigment overproduction of the fliA mutant. Our results indicate that in addition to regulating flagellar gene transcription, FliA can modulate the c-di-GMP concentration to regulate the swarming motility and phenazine pigment production in P. aeruginosa.

  8. Arabidopsis VASCULAR-RELATED UNKNOWN PROTEIN1 regulates xylem development and growth by a conserved mechanism that modulates hormone signaling.

    Grienenberger, Etienne; Douglas, Carl J


    Despite a strict conservation of the vascular tissues in vascular plants (tracheophytes), our understanding of the genetic basis underlying the differentiation of secondary cell wall-containing cells in the xylem of tracheophytes is still far from complete. Using coexpression analysis and phylogenetic conservation across sequenced tracheophyte genomes, we identified a number of Arabidopsis (Arabidopsis thaliana) genes of unknown function whose expression is correlated with secondary cell wall deposition. Among these, the Arabidopsis VASCULAR-RELATED UNKNOWN PROTEIN1 (VUP1) gene encodes a predicted protein of 24 kD with no annotated functional domains but containing domains that are highly conserved in tracheophytes. Here, we show that the VUP1 expression pattern, determined by promoter-β-glucuronidase reporter gene expression, is associated with vascular tissues, while vup1 loss-of-function mutants exhibit collapsed morphology of xylem vessel cells. Constitutive overexpression of VUP1 caused dramatic and pleiotropic developmental defects, including severe dwarfism, dark green leaves, reduced apical dominance, and altered photomorphogenesis, resembling brassinosteroid-deficient mutants. Constitutive overexpression of VUP homologs from multiple tracheophyte species induced similar defects. Whole-genome transcriptome analysis revealed that overexpression of VUP1 represses the expression of many brassinosteroid- and auxin-responsive genes. Additionally, deletion constructs and site-directed mutagenesis were used to identify critical domains and amino acids required for VUP1 function. Altogether, our data suggest a conserved role for VUP1 in regulating secondary wall formation during vascular development by tissue- or cell-specific modulation of hormone signaling pathways.

  9. Bioaccumulation of 14C-17alpha-ethinylestradiol by the aquatic oligochaete Lumbriculus variegatus in spiked artificial sediment.

    Liebig, Markus; Egeler, Philipp; Oehlmann, Jörg; Knacker, Thomas


    A bioaccumulation study was performed with the endobenthic freshwater oligochaete Lumbriculus variegatus MULLER exposed to the radiolabelled synthetic steroid 17alpha-ethinylestradiol (14C-EE2) in a spiked artificial sediment. Concentration of total radioactivity increased constantly and almost linearly during 35 days of exposure. The accumulation factor normalised to worm lipid content and sediment TOC (AFlipid/OC) was 75 at the end of the uptake period, but a steady state was not reached. Uptake kinetics were calculated fitting the measured AFs to a kinetic rate equation for constant uptake from sediment using iterative non-linear regression analysis. After 10 days of elimination in contaminant-free sediment 50% of the accumulated total radioactivity was excreted by the worms. Extracts from L. variegatus sampled at the end of the uptake phase were analysed by thin layer chromatography (TLC). The results showed that 6% of the total radioactivity incorporated by the worms was 14C-EE2. After treatment of extracts with beta-glucuronidase the amount of 14C-EE2 increased to 84%. These results suggest that L. variegatus has the potency to accumulate high amounts of conjugated EE2. Hence, a transfer of EE2 to benthivores and subsequent secondary poisoning of predators might be possible.

  10. Cloning the bacterial bphC gene into Nicotiana tabacum to improve the efficiency of phytoremediation of polychlorinated biphenyls

    Novakova, Martina; Mackova, Martina; Antosova, Zuzana; Viktorova, Jitka; Szekeres, Miklos; Demnerova, Katerina


    The aim of this work was to construct transgenic plants with increased capabilities to degrade organic pollutants, such as polychlorinated biphenyls. The environmentally important gene of bacterial dioxygenase, the bphC gene, was chosen to clone into a plant of Nicotiana tabacum. The chosen bphC gene encodes 2,3-dihydroxybiphenyl-1,2-dioxygenase, which cleaves the aromatic ring of dihydroxybiphenyl, and we cloned it in fusion with the gene for β-glucuronidase (GUS), luciferase (LUC) or with a histidine tail. Several genetic constructs were designed and prepared and the possible expression of desired proteins in tobacco plants was studied by transient expression. We used genetic constructs successfully expressing dioxygenase's genes we used for preparation of transgenic tobacco plants by agrobacterial infection. The presence of transgenic DNA , mRNA and protein was determined in parental and the first filial generation of transgenic plants with the bphC gene. Properties of prepared transgenic plants will be further studied. PMID:21468210

  11. Regulation of eight avr genes by hrpG and hrpX in Xanthomonas campestris pv. campestris and their role in pathogenicity

    XU Rongqi; LI Xianzhen; WEI Hongyu; JIANG Bole; LI Kai; HE Yongqiang; FENG Jiaxun; TANG Jiliang


    Eight putative avirulence genes in Xanthomonas campestris pv. campestris (Xcc) strain 8004 were characterized by Tn5gusA5 mutagenesis and gene expression analysis. The virulence test of mutants on Chinese radish showed that all mutants in individual avr genes except avrBs2 mutant were not significantly different from the wild type in virulence. The avrBs2 mutant showed reduced virulence and bacterial growth in planta. Gene expression analysis using β-glucuronidase as reporter indicated that avrBs1 . 1, avrBs1,avrXccB, avrXccC, avrXccE1 were regulated by hrpG, whereas avrXccA1, avrXccA2 and avrBs2 were not. RT-PCR analysis showed that all hrpG-regulated genes except avrBs1 were also regulated by hrpX. In addition, it was demonstrated that avrBs1 was responsible for elicitation of a type Ⅲ dependent hypersensitive reaction (HR) on nonhost plant pepper ECW-10R, and wild type Xcc 8004 was unable to cause HR on pepper ECW-20R.

  12. Impact of ubiquitous inhibitors on the GUS gene reporter system: evidence from the model plants Arabidopsis, tobacco and rice and correction methods for quantitative assays of transgenic and endogenous GUS

    Gerola Paolo D


    Full Text Available Abstract Background The β-glucuronidase (GUS gene reporter system is one of the most effective and employed techniques in the study of gene regulation in plant molecular biology. Improving protocols for GUS assays have rendered the original method described by Jefferson amenable to various requirements and conditions, but the serious limitation caused by inhibitors of the enzyme activity in plant tissues has thus far been underestimated. Results We report that inhibitors of GUS activity are ubiquitous in organ tissues of Arabidopsis, tobacco and rice, and significantly bias quantitative assessment of GUS activity in plant transformation experiments. Combined with previous literature reports on non-model species, our findings suggest that inhibitors may be common components of plant cells, with variable affinity towards the E. coli enzyme. The reduced inhibitory capacity towards the plant endogenous GUS discredits the hypothesis of a regulatory role of these compounds in plant cells, and their effect on the bacterial enzyme is better interpreted as a side effect due to their interaction with GUS during the assay. This is likely to have a bearing also on histochemical analyses, leading to inaccurate evaluations of GUS expression. Conclusions In order to achieve reliable results, inhibitor activity should be routinely tested during quantitative GUS assays. Two separate methods to correct the measured activity of the transgenic and endogenous GUS are presented.

  13. BN 52021 (a platelet activating factor-receptor antagonist decreases alveolar macrophage-mediated lung injury in experimental extrinsic allergic alveolitis

    J-L. Pérez-Arellano


    Full Text Available Several lines of research indirectly suggest that platelet activating factor (PAF may intervene in the pathogenesis of extrinsic allergic alveolitis (EAA. The specific aim of our study was to evaluate the participation of PAF on macrophage activation during the acute phase of EAA in an experimental model of this disease developed in guinea pigs. Initially we measured the concentration of PAF in bronchoalvedar lavage fluid, blood and lung tissue. In a second phase we evaluate the participation of PAF on alveolar macrophage activation and parenchymal lung injury. The effect of PAF on parenchymal lung injury was evaluated by m easuring several lung parenchymatous lesion indices (lung index, bronchoalvedar lavage fluid (BALF lactic hydrogenase activity and BALF alkaline phosphatase activity and parameters of systemic response to the challenge (acute phase reagents. We observed that induction of the experimental EAA gave rise to an increase in the concentration of PAF in blood and in lung tissue. The use of the PAF-receptor antagonist BN52021 decreases the release of lysosomal enzymes (β-glucuronidase and tartrate-sensitive acid phosphatase to the extracellular environment both in vivo and in vitro. Furthermore, antagonism of the PAF receptors notably decreases pulmonary parenchymatous lesion. These data suggest that lung lesions from acute EAA are partly mediated by local production of PAF.

  14. DGP1, a drought-induced guard cell-specific promoter and its function analysis in tobacco plants

    LI; Jun; GONG; Ximing; LIN; Huiqiong; SONG; Quanbo; CHEN; J


    The genetic regulation of stomatal movement mainly depends on an efficient control system of gene expression, and guard cell-specific promoter is becoming the best choice. Here we combined the dehydration responsive element (DRE) with guard cell specific element (GCSE) to construct a novel promoter, DGP1. Histochemical assays in transgenic tobacco carryingβ-glucuronidase (gus) gene fused to DGP1 demonstrated that GUS activity was found to be highly inducible by drought treatment and specifically restricted to guard cells. No GUS activity was detected in roots, stems or flowers after treatment. Further quantitative analysis showed that GUS activity in the epidermal strips was apparently induced by dehydration and dramatically increased with the elongation of treatment. The GUS activity after 8 h treatment was 179 times that of those without treatment. Although GUS activity in roots, stems or mesophyll increased after treatment, no great changes were observed. These results suggested that DGP1 could drive target gene expressed in guard cells when plant is subjected to drought stress. And this gets us prepared to control opening and closing of stomata through plant gene engineering.

  15. Establishing a Gene Trap System Mediated by T-DNA(GUS) in Rice

    Shi-Yan Chen; Ai-Min Wang; Wei Li; Zong-Yang Wang; Xiu-Ling Cai


    Two plasmids, p13GUS and p13GUS2, were constructed to create a gene trap system containing the promoterless β-glucuronidase (GUS) reporter gene in the T-DNA region. Transformation of these two plasmids into the rice variety Zhonghua 11 (Oryza sativa ssp. japonica cv.), mediated by Agrobacterium tumefaciens, resulted in 942 independent transgenic lines. Histochemical GUS assays revealed that 31 To plants had various patterns of the reporter gene expression, including expression in only one tissue, and simultaneously in two or more tissues. Hygromycin-resistant (hygr) homozygotes were screened and the copy number of the T-DNA inserts was determined in the GUS-positivs transgenic plants. The flanking sequences of the T-DNA were isolated by inverse-polymerase chain reaction and the insert positions on the rice genome of T-DNA were determined by a basic local alignment search tool in the GUS-positive transgenic plants transformed with plasmid p13GUS. Moreover, calii induced from the seeds of the T1 generation of 911 GUS-negative transgenic lines were subjected to stress and hormone treatments. Histochemical GUS assays were carried out on the calli before and after treatment. The results revealed that calli from 21 lines displayed differential GUS expression after treatment. All of these data demonstrated that this trap system is suitable for identifying rice genes, including those that are sensitive to induction.

  16. Heterologous expression of mannanase and developing a new Reporter gene system in Lactobacillus casei and Escherichia coli

    Lin, Jinzhong; Zou, Yexia; Ma, Chengjie


    from the S-layer of L. acidophilus (pELWH). The secretion of ManB was detected in the supernatant of the pELSH-ManB transformants and in the S-layer of the cell surface of the pELWH-ManB transformants. This is the first report demonstrating that the B. pumilus manB gene is a useful reporter gene in L......Reporter gene systems are useful for studying bacterial molecular biology, including the regulation of gene expression and the histochemical analysis of protein products. Here, two genes, β-1,4-mannanase (manB) from Bacillus pumilus and β-glucuronidase (gusA) from Escherichia coli K12, were cloned...... into the expression vector pELX1. The expression patterns of these reporter genes in Lactobacillus casei were investigated by measuring their enzymatic activities and estimating their recombinant protein yields using western blot analysis. Whereas mannanase activity was positively correlated with the accumulation...

  17. Limits and perspective of oral therapy with statins and aspirin for the prevention of symptomatic cholesterol gallstone disease.

    Cariati, Andrea; Piromalli, Elisa


    The prevalence of gallstones disease in Western countries is 10 - 15%. Gallstones can be one of two types - cholesterol or pigment - with cholesterol gallstones representing nearly the 80% of the total. Cholesterol and pigment gallstones have different predisposing factors: cholesterol gallstones are related to supersaturated bile in cholesterol, whereas black pigment gallstones are related to hyperbilirubinbilia factors (hemolysis, etc.); these are necessary, but not sufficient, factors to produce gallstones in vivo. Gall bladder mucosa factors (gall bladder secretion of mucin, local bile stasis and production of endogenous biliary β-glucuronidase) may coexist with the aforementioned factors and facilitate gallstone nucleation and growth. The gold-standard treatment for symptomatic gallstones is laparoscopic cholecystectomy. Several studies have reported a significant reduction in the onset of symptomatic gallstones disease in patients undergoing chronic therapy with statins, which can reduce bile cholesterol saturation. Aspirin, which has been shown to reduce the local production of gall bladder mucins (mucosal or parietal factors of gallstone formation) in animal experimental models, does not appear to reduce the risk of symptomatic gallstones disease when tested alone. The new horizon of oral therapy for the prevention of symptomatic gallstone disease needs to evaluate the long-term effect of statins and chronic aspirin administration in patients with dyslipidemia and/or atherosclerosis.

  18. Physcomitrella patens auxin conjugate synthetase (GH3) double knockout mutants are more resistant to Pythium infection than wild type.

    Mittag, Jennifer; Šola, Ivana; Rusak, Gordana; Ludwig-Müller, Jutta


    Auxin homeostasis is involved in many different plant developmental and stress responses. The auxin amino acid conjugate synthetases belonging to the GH3 family play major roles in the regulation of free indole-3-acetic acid (IAA) levels and the moss Physcomitrella patens has two GH3 genes in its genome. A role for IAA in several angiosperm--pathogen interactions was reported, however, in a moss--oomycete pathosystem it had not been published so far. Using GH3 double knockout lines we have investigated the role of auxin homeostasis during the infection of P. patens with the two oomycete species, Pythium debaryanum and Pythium irregulare. We show that infection with P. debaryanum caused stronger disease symptoms than with P. irregulare. Also, P. patens lines harboring fusion constructs of an auxin-inducible promoter from soybean (GmGH3) with a reporter (ß-glucuronidase) showed higher promoter induction after P. debaryanum infection than after P. irregulare, indicating a differential induction of the auxin response. Free IAA was induced upon P. debaryanum infection in wild type by 1.6-fold and in two GH3 double knockout (GH3-doKO) mutants by 4- to 5-fold. All GH3-doKO lines showed a reduced disease symptom progression compared to wild type. Since P. debaryanum can be inhibited in growth on medium containing IAA, these data might indicate that endogenous high auxin levels in P. patens GH3-doKO mutants lead to higher resistance against the oomycete.

  19. Mature Luffa Leaves (Luffa cylindrica L.) as a Tool for Gene Expression Analysis by Agroinfiltration.

    Błażejewska, Kamila; Kapusta, Małgorzata; Zielińska, Elżbieta; Tukaj, Zbigniew; Chincinska, Izabela A


    We exploited the potential of cucurbits for ectopic gene expression. Agroinfiltration is a simple and commonly used method to obtain transient expression of foreign genes in plants. In contrast to in vitro transformation techniques, agroinfiltration can be used for genetic modification of mature plant tissues. Although the cucurbits are commonly used as model plants for molecular biology and biotechnology studies, to date there are no literature sources on the possibility of transient gene expression in mature cucurbit tissues. Our research has shown that mature leaves of Luffa cylindrica L. (luffa), in contrast to other cucurbit species, can be successfully transiently transformed with Agrobacterium tumefaciens. We efficiently transformed luffa leaves with a reporter gene encoding β-glucuronidase (GUS). The GUS activity in transiently transformed leaf tissues was detected within 24 h after the infiltration with bacteria. Additionally, we have shown that the activity of a transiently expressed the GUS gene can be monitored directly in the EDTA-exudates collected from the cut petioles of the agroinfiltrated leaves. The results suggest that luffa leaves can be useful as a plant expression system for studies of physiological and biochemical processes in cucurbits.

  20. Quantitative Determination of Common Urinary Odorants and Their Glucuronide Conjugates in Human Urine

    Maria Wagenstaller


    Full Text Available Our previous study on the identification of common odorants and their conjugates in human urine demonstrated that this substance fraction is a little-understood but nonetheless a promising medium for analysis and diagnostics in this easily accessible physiological medium. Smell as an indicator for diseases, or volatile excretion in the course of dietary processes bares high potential for a series of physiological insights. Still, little is known today about the quantitative composition of odorous or volatile targets, as well as their non-volatile conjugates, both with regard to their common occurrence in urine of healthy subjects, as well as in that of individuals suffering from diseases or other physiological misbalancing. Accordingly, the aim of our study was to develop a highly sensitive and selective approach to determine the common quantitative composition of selected odorant markers in healthy human subjects, as well as their corresponding glucuronide conjugates. We used one- and two-dimensional high resolution gas chromatography-mass spectrometry in combination with stable isotope dilution assays to quantify commonly occurring and potent odorants in human urine. The studies were carried out on both native urine and on urine that had been treated by glucuronidase assays, with analysis of the liberated odor-active compounds using the same techniques. Analytical data are discussed with regard to their potential translation as future diagnostic tool.

  1. Serum concentrations of anthraquinones after intake of Folium Sennae and potential modulation on P-glycoprotein.

    Peng, Yu-Hsuan; Lin, Shiuan-Pey; Yu, Chung-Ping; Tsai, Shang-Yuan; Chen, Min-Yu; Hou, Yu-Chi; Chao, Pei-Dawn Lee


    Folium Sennae (leaves of Cassia angustifolia or senna) is a laxative and a component in diets for weight control. It contains a variety of anthranoids such as sennosides, aloe-emodin, and rhein. In order to measure the serum concentrations of senna anthranoids, Sprague-Dawley rats were orally administered with single dose and multiple doses of Folium Sennae. The concentrations of anthranoids in serum were determined by HPLC method before and after hydrolysis with sulfatase and β-glucuronidase. The results showed that in the serum, aloe-emodin glucuronides and rhein glucuronides were the major metabolites. Traces of rhein free form were present transiently during the early phase, whereas the free form of aloe-emodin was not detected. We also evaluated the modulation effect of Folium Sennae on P-glycoprotein by using the LS 180 cell model which showed that it significantly inhibited P-glycoprotein by 16-46 %. In conclusion, senna anthranoids were rapidly and extensively metabolized to rhein glucuronides and aloe-emodin glucuronides in rats. Folium Sennae ingestion inhibited the efflux function of P-glycoprotein in the intestine.

  2. Agrobacterium-mediated transformation of apple (Malus x domestica Borkh.): an assessment of factors affecting regeneration of transgenic plants.

    De Bondt, A; Eggermont, K; Penninckx, I; Goderis, I; Broekaert, W F


    We have previously developed a protocol for efficient gene transfer and regeneration of transgenic calli following cocultivation of apple (cv. Jonagold) explants with Agrobacterium tumefaciens (De Bondt et al. 1994, Plant Cell Reports 13: 587-593). Now we report on the optimization of postcultivation conditions for efficient and reproducible regeneration of transgenic shoots from the apple cultivar Jonagold. Factors which were found to be essential for efficient shoot regeneration were the use of gelrite as a gelling agent and the use of the cytokinin-mimicing thidiazuron in the selective postcultivation medium. Improved transformation efficiencies were obtained by combining the hormones thidiazuron and zeatin and by using leaf explants from in vitro grown shoots not older than 4 weeks after multiplication. Attempts to use phosphinothricin acetyl transferase as a selectable marker were not successful. Using selection on kanamycin under optimal postcultivation conditions, about 2% of the leaf explants developed transgenic shoots or shoot clusters. The presence and expression of the transferred genes was verified by β-glucuronidase assays and Southern analysis. The transformation procedure has also been succesfully applied to several other apple cultivars.

  3. Phytoestrogens in milk: Overestimations caused by contamination of the hydrolytic enzyme used during sample extraction.

    Bláhová, L; Kohoutek, J; Procházková, T; Prudíková, M; Bláha, L


    Isoflavones are natural phytoestrogens with antioxidant and endocrine-disrupting potencies. Monitoring of their levels is important to ensure the high quality and safety of food, milk, and dairy products. The efficiency and accuracy of phytoestrogen analyses in complex matrices such as milk depend on the extraction procedure, which often uses hydrolysis by means of the β-glucuronidase/sulfatase enzyme originating from Helix pomatia. The present study reveals that the commercially available hydrolytic enzyme is contaminated by several phytoestrogen isoflavones (genistein, daidzein, formononetin, and biochanin A) and their metabolite equol, as well as flavones (naringenin and apigenin) and coumestrol. We show that the concentrations of daidzein and genistein in the enzyme could have impaired the results of analyses of the main isoflavones in several previously published studies. Of 8 analyzed compounds, only equol was confirmed in the present study and it serves as a reliable marker of phytoestrogens originating from cow feed. Critical reassessment of phytoestrogen concentrations in milk is needed because several previously published studies might have overestimated the concentrations depending on the extraction procedure used.

  4. Extracellular ATP inhibits root gravitropism at concentrations that inhibit polar auxin transport

    Tang, Wenqiang; Brady, Shari R.; Sun, Yu; Muday, Gloria K.; Roux, Stanley J.


    Raising the level of extracellular ATP to mM concentrations similar to those found inside cells can block gravitropism of Arabidopsis roots. When plants are grown in Murashige and Skoog medium supplied with 1 mM ATP, their roots grow horizontally instead of growing straight down. Medium with 2 mM ATP induces root curling, and 3 mM ATP stimulates lateral root growth. When plants are transferred to medium containing exogenous ATP, the gravity response is reduced or in some cases completely blocked by ATP. Equivalent concentrations of ADP or inorganic phosphate have slight but usually statistically insignificant effects, suggesting the specificity of ATP in these responses. The ATP effects may be attributable to the disturbance of auxin distribution in roots by exogenously applied ATP, because extracellular ATP can alter the pattern of auxin-induced gene expression in DR5-beta-glucuronidase transgenic plants and increase the response sensitivity of plant roots to exogenously added auxin. The presence of extracellular ATP also decreases basipetal auxin transport in a dose-dependent fashion in both maize (Zea mays) and Arabidopsis roots and increases the retention of [(3)H]indole-3-acetic acid in root tips of maize. Taken together, these results suggest that the inhibitory effects of extracellular ATP on auxin distribution may happen at the level of auxin export. The potential role of the trans-plasma membrane ATP gradient in auxin export and plant root gravitropism is discussed.

  5. Identification of flunixin glucuronide and depletion of flunixin and its marker residue in bovine milk.

    Jedziniak, P; Olejnik, M; Szprengier-Juszkiewicz, T; Smulski, S; Kaczmarowski, M; Żmudzki, J


    Residues of flunixin [and its marker residue 5-hydroxyflunixin (5OHFLU)] were determined in milk from cows that intravenously received therapeutic doses of the drug. The samples were collected during each milking (every 12 h) for six consecutive days, and concentrations of flunixin and its metabolites were determined by the method with and without enzymatic hydrolysis (beta-glucuronidase). The highest flunixin concentration in milk was observed 12 h after dosing (2.4 ± 1.42 μg/kg, mean ± SD). Flunixin concentrations in the samples determined with enzymatic hydrolysis were significantly higher (P flunixin glucuronide to the milk. Additionally, unambiguous identification of flunixin glucuronide in the bovine milk was performed with linear ion-trap mass spectrometry. The 5OHFLU concentrations analyzed without enzymatic hydrolysis (22.3 ± 16.04 μg/kg) were similar to this obtained with enzymatic hydrolysis. Flunixin and 5OHFLU concentrations dropped below the limits of detection at 48 h after last dosing.

  6. Effects of flunixin, tolfenamic acid, R(-) and S(+) ketoprofen on the response of equine synoviocytes to lipopolysaccharide stimulation.

    Landoni, M F; Foot, R; Frean, S; Lees, P


    The objective of this study was to analyse the effects of 4 nonsteroidal anti-inflammatory drugs (NSAIDs) on the production of beta-glucuronidase (beta-glu), tumour necrosis factor alpha (TNF alpha), interleukin-6 (IL-6), interleukin-1 (IL-1) and prostaglandin E2 (PGE2) by lipopolysaccharide (LPS)-stimulated equine synoviocytes. The agents studied were flunixin, tolfenamic acid, S(+)ketoprofen (KTP) and R(-)ketoprofen. LPS-induced release of beta-glu from synoviocytes was inhibited in a concentration dependent manner by all 4 compounds, tolfenamic acid being the most potent. Of the 2 KTP enantiomers, S(+)KTP exerted the greatest inhibitory effect. Tolfenamic acid and flunixin increased the production of IL-6-like activity by LPS-stimulated synoviocytes only at the highest concentration studied (1000 mumol/l). Lower concentrations produced no effect on IL-6. Flunixin, tolfenamic acid and S(+)KTP produced statistically significant and concentration related increases in the release of IL-1-like activity by LPS-stimulated synoviocytes. Prostaglandin E2 synthesis was markedly inhibited in a concentration dependent manner by the 4 NSAIDs. However, R(-)KTP was effective only at the highest concentrations investigated (1000 and 100 mumol/l). The present findings are compatible with the possibility that longterm use of NSAIDs in arthropathies, by removing the regulator role of PGE2 on IL-1 synthesis, might enhance the pathological process of cartilage degeneration.

  7. Heterologous radioimmunoassay of 19-nortestosterone and trenbolone

    Vogt, K.

    Using /sup 3/H-methyltrienolone (86 Ci/mmol) in a heterologous radioimmunoassay instead of /sup 3/H-19-nortestosterone (19 Ci/mmol) in the generally used homologous system the sensitivity of the test could be improved 7-fold (50% relative binding by 15 picograms of 19-nortestosterone). Due to the relatively high cross reactivity for trenbolone of about 35% (with a different antiserum 50% have been found for trenbolone-17..cap alpha..) it is possible to have a simultaneous control of that illegal anabolic. In case of suspicion the substances responsible for the positive radioimmunoassay can be verified with a very specific assay for trenbolone. A preliminary examination of the findings via determination of 'internal blanc values' has proved to be a good measure again. With the test using the previously described extraction and clean up procedures prosecution limits of 0.1 ng/g muscle, 1 ng/ml urine and 10 ng/ml bile might be reasonable. The blanc values depend significantly on the colour of urine samples and on the enzyme used for hydrolysis (glucuronidase from Escherichia coli gave no additional blanc in contrast to succ. helix pomatioa).

  8. Analysis of Domain Architecture and Phylogenetics of Family 2 Glycoside Hydrolases (GH2).

    Talens-Perales, David; Górska, Anna; Huson, Daniel H; Polaina, Julio; Marín-Navarro, Julia


    In this work we report a detailed analysis of the topology and phylogenetics of family 2 glycoside hydrolases (GH2). We distinguish five topologies or domain architectures based on the presence and distribution of protein domains defined in Pfam and Interpro databases. All of them share a central TIM barrel (catalytic module) with two β-sandwich domains (non-catalytic) at the N-terminal end, but differ in the occurrence and nature of additional non-catalytic modules at the C-terminal region. Phylogenetic analysis was based on the sequence of the Pfam Glyco_hydro_2_C catalytic module present in most GH2 proteins. Our results led us to propose a model in which evolutionary diversity of GH2 enzymes is driven by the addition of different non-catalytic domains at the C-terminal region. This model accounts for the divergence of β-galactosidases from β-glucuronidases, the diversification of β-galactosidases with different transglycosylation specificities, and the emergence of bicistronic β-galactosidases. This study also allows the identification of groups of functionally uncharacterized protein sequences with potential biotechnological interest.

  9. Development and Validation of a Fluorescent Multiplexed Immunoassay for Measurement of Transgenic Proteins in Cotton (Gossypium hirsutum).

    Yeaman, Grant R; Paul, Sudakshina; Nahirna, Iryna; Wang, Yongcheng; Deffenbaugh, Andrew E; Liu, Zi Lucy; Glenn, Kevin C


    In order to provide farmers with better and more customized alternatives to improve yields, combining multiple genetically modified (GM) traits into a single product (called stacked trait crops) is becoming prevalent. Trait protein expression levels are used to characterize new GM products and establish exposure limits, two important components of safety assessment. Developing a multiplexed immunoassay capable of measuring all trait proteins in the same sample allows for higher sample throughput and savings in both time and expense. Fluorescent (bead-based) multiplexed immunoassays (FMI) have gained wide acceptance in mammalian research and in clinical applications. In order to facilitate the measurement of stacked GM traits, we have developed and validated an FMI assay that can measure five different proteins (β-glucuronidase, neomycin phosphotransferase II, Cry1Ac, Cry2Ab2, and CP4 5-enolpyruvyl-shikimate-3-phosphate synthase) present in cotton leaf from a stacked trait product. Expression levels of the five proteins determined by FMI in cotton leaf tissues have been evaluated relative to expression levels determined by enzyme-linked immunosorbent assays (ELISAs) of the individual proteins and shown to be comparable. The FMI met characterization requirements similar to those used for ELISA. Therefore, it is reasonable to conclude that FMI results are equivalent to those determined by conventional individual ELISAs to measure GM protein expression levels in stacked trait products but with significantly higher throughput, reduced time, and more efficient use of resources.

  10. Rikkunshito Ameliorates Cancer Cachexia Partly through Elevation of Glucarate in Plasma

    Katsuya Ohbuchi


    Full Text Available Cancer cachexia, which is characterized by decreased food intake, weight loss and systemic inflammation, increases patient’s morbidity and mortality. We previously showed that rikkunshito (RKT, a Japanese traditional herbal medicine (Kampo, ameliorated the symptoms of cancer cachexia through ghrelin signaling-dependent and independent pathways. To investigate other mechanisms of RKT action in cancer cachexia, we performed metabolome analysis of plasma in a rat model bearing the Yoshida AH-130 hepatoma. A total of 110 metabolites were detected in plasma and RKT treatment significantly altered levels of 23 of those metabolites in cachexia model rats. Among them, glucarate, which is known to have anticarcinogenic activity through detoxification of carcinogens via inhibition of β-glucuronidase, was increased in plasma following administration of RKT. In our AH-130 ascites-induced cachexia rat model, administration of glucarate delayed onset of weight loss, improved muscle atrophy, and reduced ascites content. Additionally, glucarate reduced levels of plasma interferon-γ (IFN-γ in tumor-bearing rats and was also found to suppress LPS-induced IFN-γ expression in splenocytes in vitro. These results suggest that glucarate has anti-inflammatory activity via a direct effect on immune host cells and suggest that RKT may also ameliorate inflammation partly through the elevation of glucarate in plasma.

  11. [Simultaneous determination of zeranols and chloramphenicol in foodstuffs of animal origin by combination immunoaffinity column clean-up and liquid chromatography-tandem mass spectrometry].

    Wang, Qing; Wang, Guomin; Xi, Cunxian; Li, Xianliang; Chen, Dongdong; Tang, Bobin; Zhang, Lei; Zhao, Hua


    A combination immunoaffinity column (IAC-CZ) clean-up and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analytical method was successfully developed for zearalenol, beta-zearalenol and zearalenone) and chloramphenicol (CAP) in foodstuffs of animal origin. The samples (fish, liver, milk and honey) were enzymatically digested by beta-glucuronidase/sulfatase for about 16 h and then extracted with ether. The extracts were evaporated to dryness and then the residues were dissolved by 1.0 mL of 50% acetonitrile solution. After filtered and diluted with PBS buffer, the reconstituted solution were cleaned-up with a IAC-CZ and then analyzed by LC-MS/MS in multiple reaction monitoring (MRM) mode. The chromatographic separation was performed on a Shimadzu Shim-pack VP-ODS column with gradient elution by acetonitrile and 2 mmol/L ammonium acetate solution. The detection was carried out by electrospray negative ionization mass spectrometry in MRM mode. The proposed method was validated by the limit of detection (0.04-0.10 microg/kg), linearity (R2 > or = 0.999 0), average recoveries (70.9%-95.6%) and precisions (2.0% - 11.8%). The developed method is reliable, sensitive and has good applicability. The combination immunoaffinity column was proved to be an effective pretreatment technique to decrease the matrix effect, and it met the requirements of residue analysis of co-occurring zeranols and chloramphenicol.

  12. Stereoselective glucuronidation of carvedilol by Chinese liver microsomes


    Objective: To study the stereoselective glucuronidation of carvedilol (CARV) by three Chinese liver microsomes.Methods: The metabolites of CARV were identified by a hydrolysis reaction with β-glucuronidase and HPLC-MS/MS. The enzyme kinetics for CARV enantiomers glucuronidation was determined by a reversed phase-high pressure liquid chromatography (RP-HPLC) assay using (S)-propafenone as internal standard after precolumn derivatization with 2,3,4,6-tetra-O-acetyl-β-D-glucopyranosylisothiocyanate. Results: Two CARV glucuronides were found in three Chinese liver microsomes incubated with CARV. The non-linear regression analysis showed that the values of Km and Vmax for (S)-CARV and (R)-CARV enantiomers were (118±44) μmol/L, (2 500±833) pmol/( protein) and (24±7) μmol/L, (953+399) pmol/( protein),respectively. Conclusion: These results suggested that there was a significant (P<0.05) stereoselective glucuronidation of CARV enantiomers in three Chinese liver microsomes, which might partly explain the enantioselective pharmacokinetics of CARV.

  13. Urinary mutagenicity, CYP1A2 and NAT2 activity in textile industry workers.

    Fanlo, Ana; Sinuès, Blanca; Mayayo, Esteban; Bernal, Luisa; Soriano, Antonia; Martínez-Jarreta, Begoña; Martínez-Ballarín, Enrique


    The two major causes of bladder cancer have been recognised to be cigarette smoke and occupational exposure to arylamines. These compounds are present both in tobacco smoke and in the dyes used in textile production. Aromatic amines suffer oxidative metabolism via P450 cytochrome CYP1A2, and detoxification by the polymorphic NAT2. The aim of the present work was to assess the association between occupational-derived exposure to mutagens and CYP1A2 or NAT2 activity. This cross-sectional study included 117 textile workers exposed to dyes and 117 healthy controls. The urinary mutagenicity was determined in 24 h urine using TA98 Salmonella typhimurium strain with microsomal activation S9 (MIS9) or incubation with beta-glucuronidase (MIbeta). Urinary caffeine metabolite ratios: AFMU+1X+1U/17U, and AFMU/AFMU+1X+1U were calculated to assess CYP1A2 and NAT2 activities, respectively. The results show that workers present a strikingly higher urine mutagenicity than controls (p0.05) was compared, and the urinary mutagenicity was not significantly associated with the CYP1A2 activity marker (r=0.04 and r=-0.01 for MIS9 and MIbeta, respectively). This study clearly indicates the need for further protective policies to minimise exposure to the lowest feasible limit in order to avoid unnecessary risks.

  14. Chemopreventive effect of myrtenal on bacterial enzyme activity and the development of 1,2-dimethyl hydrazine-induced aberrant crypt foci in Wistar Rats

    Lokesh Kumar Booupathy


    Full Text Available Colon cancer remains as a serious health problem around the world despite advances in diagnosis and treatment. Dietary fibers are considered to reduce the risk of colon cancer as they are converted to short chain fatty acids by the presence of anaerobic bacteria in the intestine, but imbalanced diet and high fat consumption may promote tumor formation at different sites, including the large bowel via increased bacterial enzymes activity. The present study was conducted to characterize the inhibitory action of myrtenal on bacterial enzymes and aberrant crypt foci (ACF. Experimental colon carcinogenesis induced by 1,2-dimethylhydrazine is histologically, morphologically, and anatomically similar to human colonic epithelial neoplasm. Discrete microscopic mucosal lesions such as ACF and malignant tumors function as important biomarkers in the diagnosis of colon cancer. Methylene blue staining was carried out to visualize the impact of 1,2-dimethylhydrazine and myrtenal. Myrtenal-treated animals showed decreased levels of bacterial enzymes such as β-glucuronidase, β-glucosidase, and mucinase. Characteristic changes in the colon were noticed by inhibiting ACF formation in the colon. In conclusion, treatment with myrtenal provided altered pathophysiological condition in colon cancer-bearing animals with evidence of decreased crypt multiplicity and tumor progression.

  15. Role of the plant-specific endoplasmic reticulum stress-inducible gene TIN1 in the formation of pollen surface structure in Arabidopsis thaliana

    Iwata, Yuji


    Accumulation of unfolded proteins in the endoplasmic reticulum (ER) of eukaryotic cells triggers the transcriptional activation of ER-resident molecular chaperones and folding enzymes to maintain cellular homeostasis. This process is known as the ER stress response or the unfolded protein response. We have identified tunicamycin induced 1 (TIN1), a plant-specific ER stress-inducible Arabidopsis thaliana gene. The TIN1 protein is localized in the ER; however, its molecular function has yet to be clarified. In this study, we performed functional analysis of TIN1 in planta. RT-PCR analysis showed that TIN1 is highly expressed in pollen. Analysis using the β-glucuronidase reporter gene demonstrated that the TIN1 promoter is active throughout pollen development, peaking at the time of flowering and in an ovule of an open flower. Although a T-DNA insertion mutant of TIN1 grows normally under ambient laboratory conditions, abnormal pollen surface morphology was observed under a scanning electron microscope. Based on the current and previous observations, a possible physiological function of TIN1 during pollen development is discussed. © 2012 The Japanese Society for Plant Cell and Molecular Biology.

  16. A novel, biodegradable and reversible polyelectrolyte platform for topical-colonic delivery of pentosan polysulphate.

    Shah, Hardik K; Conkie, Jim A; Tait, Robert C; Johnson, James R; Wilson, Clive G


    The goal of the present work was to develop a swellable hydrogel colonic delivery system, which would maximise the availability of the therapeutic agent at a site of inflammation, especially where the water is scarce. A novel method was developed to manufacture a biodegradable and reversible polyelectrolyte complex (PEC) containing chitosan and poly acrylic-acid (PAA). The PEC was analysed using FTIR and DSC, which confirmed the formation of non-permanent swollen gel-network at an alkaline pH. Pentosan polysulphate (PPS) was incorporated in a PEC and an activated partial thromboplastin time assay was developed to measure the release of PPS from PEC. In vitro studies suggested that the release of PPS was dependent on the initial drug loading and the composition of the PEC. The gel strength of the swollen network, determined using a texture analyser, was dependent on polymer composition and the amount of PPS incorporated. Bacterial enzymes were collected from the rat caecum and colon for the digestion studies and characterised for glucosidase activity, glucuronidase activity and protein content. The digestion of the reversible polyelectrolyte complexes was measured using a dinitro salicylic acid assay and an increased release of drug was also confirmed in the presence of bacterial enzymes.

  17. The Cotton WRKY Gene GhWRKY41 Positively Regulates Salt and Drought Stress Tolerance in Transgenic Nicotiana benthamiana.

    Xiaoqian Chu

    Full Text Available WRKY transcription factors constitute a very large family of proteins in plants and participate in modulating plant biological processes, such as growth, development and stress responses. However, the exact roles of WRKY proteins are unclear, particularly in non-model plants. In this study, Gossypium hirsutum WRKY41 (GhWRKY41 was isolated and transformed into Nicotiana benthamiana. Our results showed that overexpression of GhWRKY41 enhanced the drought and salt stress tolerance of transgenic Nicotiana benthamiana. The transgenic plants exhibited lower malondialdehyde content and higher antioxidant enzyme activity, and the expression of antioxidant genes was upregulated in transgenic plants exposed to osmotic stress. A β-glucuronidase (GUS staining assay showed that GhWRKY41 was highly expressed in the stomata when plants were exposed to osmotic stress, and plants overexpressing GhWRKY41 exhibited enhanced stomatal closure when they were exposed to osmotic stress. Taken together, our findings demonstrate that GhWRKY41 may enhance plant tolerance to stress by functioning as a positive regulator of stoma closure and by regulating reactive oxygen species (ROS scavenging and the expression of antioxidant genes.

  18. Isolation of pea matrix attachment region and study on its function in transgenic tobaccos

    李旭刚; 朱祯; 徐军望; 吴茜; 徐鸿林


    A DNA fragment containing consensus sequence of matrix attachment region (MAR) has been isolated from pea genome. Compared with original DNA sequence, one 115 bp-long repeat sequence is deleted in the obtained DNA sequence. DNA fragments located upstream and down-stream of repeat DNA sequence respectively share 84% and 93% homology to the corresponding original sequence, and contain A-box or T-box and TATAA sequence, which is characteristics short sequence of MARs. To test the function of the DNA sequence, the plant expression vectors in which b-glucuronidase gene (GUS, uidA) was used as reporter gene were constructed and transferred into tobaccos via Agrobacterium-mediated transformation procedure. Quantitative GUS assay showed that the average level of uidA expression was increased twofold for the presence of MAR, and the highest level of GUS activity of transgenic plants could be increased six times. The results cited above suggest that the isolated DNA sequence contains consensus sequence of MARs and has capability to increase expression level of gene in transgenic plants.

  19. Heterogenic expression of genes encoding secreted proteins at the periphery of Aspergillus niger colonies.

    Vinck, Arman; de Bekker, Charissa; Ossin, Adam; Ohm, Robin A; de Vries, Ronald P; Wösten, Han A B


    Colonization of a substrate by fungi starts with the invasion of exploring hyphae. These hyphae secrete enzymes that degrade the organic material into small molecules that can be taken up by the fungus to serve as nutrients. We previously showed that only part of the exploring hyphae of Aspergillus niger highly express the glucoamylase gene glaA. This was an unexpected finding since all exploring hyphae are exposed to the same environmental conditions. Using GFP as a reporter, we here demonstrate that the acid amylase gene aamA, the α-glucuronidase gene aguA, and the feruloyl esterase gene faeA of A. niger are also subject to heterogenic expression within the exploring mycelium. Coexpression studies using GFP and dTomato as reporters showed that hyphae that highly express one of these genes also highly express the other genes encoding secreted proteins. Moreover, these hyphae also highly express the amylolytic regulatory gene amyR, and the glyceraldehyde-3-phosphate dehydrogenase gene gpdA. In situ hybridization demonstrated that the high expressers are characterized by a high 18S rRNA content. Taken together, it is concluded that two subpopulations of hyphae can be distinguished within the exploring mycelium of A. niger. The experimental data indicate that these subpopulations differ in their transcriptional and translational activity.

  20. Agrobacterium-transformed rice plants expressing synthetic cryIA(b) and cryIA(c) genes are highly toxic to striped stem borer and yellow stem borer.

    Cheng, X; Sardana, R; Kaplan, H; Altosaar, I


    Over 2,600 transgenic rice plants in nine strains were regenerated from >500 independently selected hygromycin-resistant calli after Agrobacterium-mediated transformation. The plants were transformed with fully modified (plant codon optimized) versions of two synthetic cryIA(b) and cryIA(c) coding sequences from Bacillus thuringiensis as well as the hph and gus genes, coding for hygromycin phosphotransferase and beta-glucuronidase, respectively. These sequences were placed under control of the maize ubiquitin promoter, the CaMV35S promoter, and the Brassica Bp10 gene promoter to achieve high and tissue-specific expression of the lepidopteran-specific delta-endotoxins. The integration, expression, and inheritance of these genes were demonstrated in R0 and R1 generations by Southern, Northern, and Western analyses and by other techniques. Accumulation of high levels (up to 3% of soluble proteins) of CryIA(b) and CryIA(c) proteins was detected in R0 plants. Bioassays with R1 transgenic plants indicated that the transgenic plants were highly toxic to two major rice insect pests, striped stem borer (Chilo suppressalis) and yellow stem borer (Scirpophaga incertulas), with mortalities of 97-100% within 5 days after infestation, thus offering a potential for effective insect resistance in transgenic rice plants.

  1. Quantitative clinical characteristics of 53 patients with MPS VII: a cross-sectional analysis.

    Zielonka, Matthias; Garbade, Sven F; Kölker, Stefan; Hoffmann, Georg F; Ries, Markus


    The main purpose of the study was to provide quantitative data regarding survival and diagnostic delay. Mucopolysaccharidosis (MPS) type VII (OMIM 253220) is a progressive neurometabolic disorder caused by deficiency of the lysosomal enzyme β-glucuronidase (GUS). Hard clinical end points have not been quantitatedMethods:We quantitatively analyzed published cases with MPS VII (N = 53/88 with sufficient data). Main outcome measures were onset of disease and survival. The role of biomarkers such as GUS residual enzyme activity and levels of storage material assessed as urinary excretion of glucosaminoglycans (GAG) as potential predictors of clinical outcomes were investigated. The analysis was conducted according to STROBE criteria. Median survival of the postnatally diagnosed population was 42 months. Median age of disease onset was the first day of life; median age at diagnosis was 11 months. Hydrops fetalis was frequent. Patients with residual GUS activity in fibroblasts more than 1.4% or urinary GAG excretion less than 602% of normal survived longer than patients with GUS enzyme activity below or GAG excretion above these thresholds. MPS VII has its disease onset prenatally. In the absence of a prenatal diagnosis, most cases are clinically apparent at birth. Our data corroborate a phenotype-biomarker association in MPS VII. The survival data characterize the natural history with important implications for therapeutic studies.Genet Med advance online publication 06 April 2017Genetics in Medicine (2017); doi:10.1038/gim.2017.10.

  2. A simple method suitable to study de novo root organogenesis

    Xiaodong eChen


    Full Text Available De novo root organogenesis is the process in which adventitious roots regenerate from detached or wounded plant tissues or organs. In tissue culture, appropriate types and concentrations of plant hormones in the medium are critical for inducing adventitious roots. However, in natural conditions, regeneration from detached organs is likely to rely on endogenous hormones. To investigate the actions of endogenous hormones and the molecular mechanisms guiding de novo root organogenesis, we developed a simple method to imitate natural conditions for adventitious root formation by culturing Arabidopsis thaliana leaf explants on B5 medium without additive hormones. Here we show that the ability of the leaf explants to regenerate roots depends on the age of the leaf and on certain nutrients in the medium. Based on these observations, we provide examples of how this method can be used in different situations, and how it can be optimized. This simple method could be used to investigate the effects of various physiological and molecular changes on the regeneration of adventitious roots. It is also useful for tracing cell lineage during the regeneration process by differential interference contrast observation of -glucuronidase staining, and by live imaging of proteins labeled with fluorescent tags.

  3. Freshwater molluscs from volcanic areas as model organisms to assess adaptation to metal chronic pollution

    Zaldibar, Benat [Biologia Zelularra eta Histologia laborategia, Zoologia eta Animalien Biologia Zelularra Saila, Zientzi Fakultatea, Euskal Herriko Unibertsitatea, 644 P.K. E-48080 Bilbo, Basque Country (Spain); Rodrigues, Armindo [Cirn e Departamento Biologia, Universidade dos Acores, R. Mae de Deus, APT 1422, PT-9501-855 Ponta Delgada (Portugal); Lopes, Marco [Cirn e Departamento Biologia, Universidade dos Acores, R. Mae de Deus, APT 1422, PT-9501-855 Ponta Delgada (Portugal); Amaral, Andre [Cirn e Departamento Biologia, Universidade dos Acores, R. Mae de Deus, APT 1422, PT-9501-855 Ponta Delgada (Portugal); Marigomez, Ionan [Biologia Zelularra eta Histologia laborategia, Zoologia eta Animalien Biologia Zelularra Saila, Zientzi Fakultatea, Euskal Herriko Unibertsitatea, 644 P.K. E-48080 Bilbo, Basque Country (Spain); Soto, Manu [Biologia Zelularra eta Histologia laborategia, Zoologia eta Animalien Biologia Zelularra Saila, Zientzi Fakultatea, Euskal Herriko Unibertsitatea, 644 P.K. E-48080 Bilbo, Basque Country (Spain)]. E-mail:


    Cellular biomarkers of exposure and biological effects were measured in digestive gland of snails (Physa acuta) sampled in sites with and without active volcanism in Sao Miguel Island (Azores). Metal content in digestive cell lysosomes was determined by image analysis after autometallography (AMG) as volume density of autometallographed black silver deposits (Vv{sub BSD}). Lysosomal structural changes (lysosomal volume, surface and numerical densities - Vv{sub LYS,} Sv{sub LYS} and Nv{sub LYS-}, and surface-to-volume ratio - S/V{sub LYS}-) were quantified by image analysis, after demonstration of {beta}-glucuronidase activity, on digestive gland cryotome sections. Additional chemical analyses (atomic absorption spectrophotometry) were done in the digestive gland of snails. The highest metal concentrations were found in snails from the active volcanic site, which agreed with high intralysomal Vv{sub BSD}. Digestive cell lysosomes in snails inhabiting sites with active volcanism resembled a typical stress situation (enlarged and less numerous lysosomes). In conclusion, the biomarkers used in this work can be applied to detect changes in metal bioavailability due to chronic exposure to metals (volcanism), in combination with chemical analyses.

  4. Efficient genetic transformation of Lotus corniculatus L. using a direct shoot regeneration protocol, stepwise hygromycin B selection, and a super-binary Agrobacterium tumefaciens vector

    Nikolić Radomirka


    Full Text Available Cotyledons from 6-day-old Lotus corniculatus cv. Bokor seedlings, transversally cut into two halves, were capa­ble of regenerating buds without intervening callus formation. The explants were co-cultivated with the Agrobacterium tumefaciens LBA4404/pTOK233 superbinary vector carrying the uidA-intron gene and the genes hpt and nptII. They were cultured for 14 days on a regeneration medium, then subjected to a stepwise hygromycin B selection procedure consisting of gradually increasing antibiotic concentrations (5-15 mg L-1 over 21 weeks. Transformed shoots were obtained within 5 months after co-cultivation. Out of 124 initially co-cultivated explants, 52 (42% plants survived hygromycin B selection. The presence of transgenes in regenerated plants was verified by β-glucuronidase histochemical assays and PCR analysis for the presence of uidA gene sequences. Hygromycin B-resistant and PCR-positive T0 plants were cultured in the greenhouse to produce flowers and seeds. The obtained data demonstrate that the reported transformation protocol could be useful for introducing agriculturally important genes into the new L. corniculatus cultivar Bokor.

  5. Measuring gene flow from two birdsfoot trefoil (Lotus corniculatus) field trials using transgenes as tracer markers.

    De Marchis, F; Bellucci, M; Arcioni, S


    Genetic engineering is becoming a useful tool in the improvement of plants but concern has been expressed about the potential environmental risks of releasing genetically modified (GM) organisms into the environment. Attention has focused on pollen dispersal as a major issue in the risk assessment of transgenic crop plants. In this study, pollen-mediated dispersal of transgenes via cross-fertilization was examined. Plants of Lotus corniculatus L. transformed with either the Escherichia coli asparagine synthetase gene asnA or the beta-glucuronidase gene uidA, were used as the pollen donor. Nontransgenic plants belonging to the species L. corniculatus L., L. tenuis Waldst. and Kit. ex Willd, and L. pedunculatus Cav., were utilized as recipients. Two experimental fields were established in two areas of central Italy. Plants carrying the uidA gene were partially sterile, therefore only the asnA gene was used as a tracer marker. No transgene flow between L. corniculatus transformants and the nontransgenic L. tenuis and L. pedunculatus plants was detected. As regards nontransgenic L. corniculatus plants, in one location flow of asnA transgene was detected up to 18 m from the 1.8 m2 donor plot. In the other location, pollen dispersal occurred up to 120 m from the 14 m2 pollinating plot.

  6. In vitro safety assessments and antimicrobial activities of Lactobacillus rhamnosus strains isolated from a fermented mare's milk.

    Aryantini, Ni Putu Desy; Yamasaki, Eiki; Kurazono, Hisao; Sujaya, I Nengah; Urashima, Tadasu; Fukuda, Kenji


    Safety and probiotic characteristics such as antimicrobial activities of three Lactobacillus rhamnosus strains, FSMM15, FSMM22 and FSMM26, previously isolated as potential probiotics from fermented mare's milk were investigated. The three FSMM strains were susceptible to ampicillin, gentamycin, kanamycin, streptomycin, tetracycline and chloramphenicol, whereas they were resistant to erythromycin (minimal inhibitory concentration (MIC) = 4-8 µg/mL) and clindamycin (MIC = 4 µg/mL); bioconversion of bile salts, hemolytic activity and mucin degradation activity were negative; enzymatic activities of α-chymotrypsin and β-glucosidase were detected, but those of α-galactosidase, β-glucuronidase and N-acetyl-β-glucosaminidase, were undetectable. Among the strains, strain FSMM15 was chosen as a safer probiotic candidate due mainly to the lack of plasminogen binding ability. Despite lower acid production of strain FSMM15 than others, its cell-free culture supernatant inhibited growths of Salmonella Typhimurium LT-2, Shigella sonnei, Listeria monocytogenes, and Escherichia coli O157 with comparable levels of ampicillin, suggesting a favorable aspect of strain FSMM15 as a probiotic strain. © 2016 Japanese Society of Animal Science.

  7. Applying unconventional secretion of the endochitinase Cts1 to export heterologous proteins in Ustilago maydis.

    Stock, Janpeter; Sarkari, Parveen; Kreibich, Saskia; Brefort, Thomas; Feldbrügge, Michael; Schipper, Kerstin


    The demand on the biotechnological production of proteins for pharmaceutical, medical and industrial applications is steadily growing. For the production of challenging proteins, we aim to establish a novel expression platform in the well characterized eukaryotic microorganism Ustilago maydis. In filaments of this fungus, secretion of the endochitinase Cts1 depends on mRNA transport along microtubules, which is mediated by the key RNA-binding protein Rrm4. Here, we report two important findings: (i) Cts1 secretion occurs via a novel unconventional route and (ii) this secretory mechanism can be exploited for the export of active heterologous proteins. Initially, we used β-glucuronidase (Gus) as a reporter for unconventional secretion. This bacterial enzyme is inactivated by N-glycosylation during its passage through the conventional eukaryotic secretory pathway. By contrast, in our system Gus was exported in its active form by fusion to Cts1 confirming its secretion by an unconventional route. As a proof-of-principle for economically important biopharmaceuticals we expressed an active single-chain antibody. Importantly, the novel protein export pathway circumvents N-glycosylation which is advantageous in many applications, e.g., to avoid undesired immune reactions in humans. Thus, the unconventional Cts1 secretion machinery has a high potential for the production of biotechnologically relevant proteins.

  8. Toxicity of corexit 9527 and Nigerian crude oil in vivo and in vitro

    George, S.; Nelson, G.; Brooks, L. [Environmental Protection Agency, Research Triangle Park, NC (United States)] [and others


    When oil is spilled into aquatic systems, chemical dispersants frequently are applied to enhance emulsification and biological availability. In this study, toxicity of Corexit 9527 (Cx), a widely used dispersant, was evaluated with oil both in vivo and in vitro. Initially, both Cx and oil were bioassayed in a micro-suspension modification of the Salmonella reversion bioassay. Next, rats were dosed 5 weeks with oil, Cx or Cx/oil. Body and tissue weights, urine mutagenicity, and effect on intestinal enzymes and microbiota were determined. Cx (1:1000 dilution) was toxic in the bioassay and oil was negative (TA98 & TA100 +/-S9). Similar results were observed for urinary metabolites. Body and tissue weights were unaffected, but a significant reduction in small intestinal azo reductase (AR) and/or {beta}-glucuronidase (BG) was observed in animals that received Cx, oil, or Cx/oil. Effects on cecal AR were observed in the 3 treatment groups. One week nitroreductase (NR) activity was elevated, and 3 and 5 week BG activities were significantly different from controls. This correspond to an elimination of enterobacteria and enterococci in oil-treated rats and enterobacteria fluctuations in Cx- and Cx/oil-treated animals. Even though treatment of rats with Cx has no direct effect on the production of mutagenic urine metabolites from oil-treated animals, significant changes are observed in intestinal microbiota and their enzymes responsible for promutagen and procarcinogen activation.

  9. Long-term changes of salivary exoglycosidases and their applicability as chronic alcohol-drinking and dependence markers.

    Waszkiewicz, Napoleon; Kratz, Ewa Maria; Chojnowska, Sylwia; Zalewska, Anna; Zwierz, Krzysztof; Szulc, Agata; Szajda, Sławomir Dariusz; Nestsiarovich, Anastasiya; Kapitau, Andrei; Kępka, Alina; Ostrowska, Lucyna; Ferens-Sieczkowska, Mirosława


    Investigation of long-term dynamic changes of salivary activity/output of exoglycosidases, deglycosylation processes and their applicability as alcohol markers. Exoglycosidase (α-fucosidase (FUC), β-galactosidase (GAL), β-glucuronidase (GLU), β-hexosaminidase (HEX, HEX A and HEX B isoenzymes) and α-mannosidase (MAN)) activities were measured in the saliva of healthy social drinking controls (C), alcohol-dependent non-smokers (ANS) and alcohol-dependent smokers (AS) at the 1st, 15th, 30th and 50th day of abstinence after chronic alcohol drinking. The activity of exoglycosidases was 2-3-fold (MAN), 2-6 fold (FUC), 8-25-fold (HEX A) and 19-40-fold (GLU) higher in the ANS and AS groups than in controls, and had good/excellent sensitivity, specificity and accuracy. The higher outputs of exoglycosidases were in the AS and ANS groups than in controls at the 1st day (GLU, HEX A) and at the 50th day (GLU, FUC, MAN) of abstinence. We found numerous correlations between alcohol-drinking days with GLU and HEX A, alcohol amounts with HEX A and duration of alcohol dependence with FUC and MAN activity/output. Salivary exoglycosidases/deglycosylation processes were still very high up to 50 days after the end of alcohol consumption. We found markers of chronic alcohol consumption (HEX A), alcohol dependence (FUC and MAN) and chronic alcohol consumption and dependence (GLU).

  10. Activity of lysosomal exoglycosidases in serum and synovial fluid in patients with chronic Lyme and rheumatoid arthritis.

    Pancewicz, Slawomir; Popko, Janusz; Rutkowski, Ryszard; Knaś, Malgorzata; Grygorczuk, Sambor; Guszczyn, Tomasz; Bruczko, Marta; Szajda, Slawomir; Zajkowska, Joanna; Kondrusik, Maciej; Sierakowski, Stanislaw; Zwierz, Krzysztof


    Lysosomal exoglycosidases participate in the destruction of the articular cartilage by cleaving glycoside bonds in glycoproteins and proteoglycans. The aim of the study was to determine the activity of exoglycosidases: hexosaminidase, beta-glucuronidase, beta-galactosidase, alpha-mannosidase and alpha-fucosidase in serum and synovial fluid of patients with Lyme and rheumatoid arthritis. The study group consisted of 10 patients with chronic Lyme arthritis (age 18 - 74 y), 13 with rheumatoid arthritis (age 32 - 70 y) and 10 with juvenile idiopathic arthritis (age 8 - 17 y). The control group consisted of 9 healthy volunteers (age 24 - 62 y). The activity of the exoglycosidases was determined with the p-nitrophenyl derivatives of sugars as substrates. A significant increase of the activity of all the exoglycosidases in serum and in synovial fluid of the patients with different forms of arthritis was found. The ratio of synovial fluid/serum activity of exoglycosidases was above 2.0 in LA but not in JIA and RA patients. As the main source of exoglycosidases in the joint is the synovial membrane, this result supports the appropriateness of therapeutic synovectomy in chronic Lyme arthritis with knee effusion. The serum activity of hexosaminidase may be used in monitoring the course of Lyme arthritis and the efficiency of treatment.

  11. The activity of lysosomal exoglycosidases in serum of alcohol-dependent men supplemented with borage oil enriched with vitamin E.

    Zaniewska, Agnieszka; Borzym-Kluczyk, Malgorzata; Szajda, Slawomir D; Romatowski, Jacek; Gil, Andrzej; Knas, Malgorzata; Dobryniewski, Jacek; Zwierz, Krzysztof


    The aim of this study was to determine the activity of the lysosomal exoglycosidases: alpha-mannosidase (MAN), alpha-fucosidase (FUC), and beta-glucuronidase (GLUCUR) in serum of alcohol-dependent men supplemented and not supplemented with borage oil enriched with vitamin E. Serum was collected from eight social drinkers and 16 alcohol-dependent men after a drinking period. The activity of exoglycosidases and the concentration of protein in serum were determined. The increase in specific activity of MAN and GLUCUR was significant in serum of alcohol-dependent men both not supplemented and supplemented with borage oil enriched with vitamin E, in comparison with the specific activity in serum of social drinkers. In serum of alcohol-dependent men treated with borage oil enriched with vitamin E, specific activity of MAN and GLUCUR fluctuated in comparison with alcohol-dependent men not supplemented. Specific activity of FUC in serum of alcohol-dependent men both not supplemented and supplemented with borage oil enriched with vitamin E showed a tendency to increase, in comparison with social drinkers. Specific activity of FUC had a tendency to decrease in serum of alcohol-dependent men supplemented with borage oil enriched with vitamin E, in comparison with alcohol-dependent men not supplemented. Thus, supplementation of alcohol-dependent men after a long-lasting drinking period with borage oil and vitamin E did not change the rate of catabolism of the oligosaccharide chains of glycoconjugates, as evaluated by serum activity of exoglycosidases.

  12. Effects of a mixture of fatty acids from sugar cane (Saccharum officinarum L.) wax oil in two models of inflammation: zymosan-induced arthritis and mice tail test of psoriasis.

    Ledón, N; Casacó, A; Remirez, D; González, A; Cruz, J; González, R; Capote, A; Tolón, Z; Rojas, E; Rodríguez, V J; Merino, N; Rodríguez, S; Ancheta, O; Cano, M C


    A mixture of fatty acids obtained from sugar cane (Saccharum officinarum L.) wax oil (FAM), in which the main constituents are palmitic, oleic, linoleic, and linolenic acids, was evaluated in two models of inflammation: zymosan-induced arthritis and in the tail test for psoriasis, both on mice. In the first model, FAM significantly reduced zymozan-induced increase of beta glucuronidase (DE(50) 90+/-7 mg/kg). Histopathological studies showed inhibition in cellular infiltration and reduction of synovial hyperplasia and synovitis, whereas in the second test, histopathological and ultrastructural studies showed that topical application of FAM induced orthokeratosis with the presence of keratohyalin granules in the previously parakeratotic adult mouse tail, and without effects on epidermal thickness. The ED(50) of FAM in this model was 155+/-10 mg. The results of our studies showed that topical application of FAM exerts an important anti-inflammatory activity in both tests without evidence of irritant effects. The anti-inflamatory effects exerted by FAM may be due to its inhibitory effects on arachidonic acid metabolism. To our knowledge, this is the first report on the anti-inflammatory effect of sugar cane by-products in experimental models of arthritis and psoriasis.

  13. Iron-binding E3 ligase mediates iron response in plants by targeting basic helix-loop-helix transcription factors.

    Selote, Devarshi; Samira, Rozalynne; Matthiadis, Anna; Gillikin, Jeffrey W; Long, Terri A


    Iron uptake and metabolism are tightly regulated in both plants and animals. In Arabidopsis (Arabidopsis thaliana), BRUTUS (BTS), which contains three hemerythrin (HHE) domains and a Really Interesting New Gene (RING) domain, interacts with basic helix-loop-helix transcription factors that are capable of forming heterodimers with POPEYE (PYE), a positive regulator of the iron deficiency response. BTS has been shown to have E3 ligase capacity and to play a role in root growth, rhizosphere acidification, and iron reductase activity in response to iron deprivation. To further characterize the function of this protein, we examined the expression pattern of recombinant ProBTS::β-GLUCURONIDASE and found that it is expressed in developing embryos and other reproductive tissues, corresponding with its apparent role in reproductive growth and development. Our findings also indicate that the interactions between BTS and PYE-like (PYEL) basic helix-loop-helix transcription factors occur within the nucleus and are dependent on the presence of the RING domain. We provide evidence that BTS facilitates 26S proteasome-mediated degradation of PYEL proteins in the absence of iron. We also determined that, upon binding iron at the HHE domains, BTS is destabilized and that this destabilization relies on specific residues within the HHE domains. This study reveals an important and unique mechanism for plant iron homeostasis whereby an E3 ubiquitin ligase may posttranslationally control components of the transcriptional regulatory network involved in the iron deficiency response.

  14. Effects of a Lactobacillus salivarius mixture on performance, intestinal health and serum lipids of broiler chickens.

    Shokryazdan, Parisa; Faseleh Jahromi, Mohammad; Liang, Juan Boo; Ramasamy, Kalavathy; Sieo, Chin Chin; Ho, Yin Wan


    The ban or severe restriction on the use of antibiotics in poultry feeds to promote growth has led to considerable interest to find alternative approaches. Probiotics have been considered as such alternatives. In the present study, the effects of a Lactobacillus mixture composed from three previously isolated Lactobacillus salivarius strains (CI1, CI2 and CI3) from chicken intestines on performance, intestinal health status and serum lipids of broiler chickens has been evaluated. Supplementation of the mixture at a concentration of 0.5 or 1 g kg-1 of diet to broilers for 42 days improved body weight, body weight gain and FCR, reduced total cholesterol, LDL-cholesterol and triglycerides, increased populations of beneficial bacteria such as lactobacilli and bifidobacteria, decreased harmful bacteria such as E. coli and total aerobes, reduced harmful cecal bacterial enzymes such as β-glucosidase and β-glucuronidase, and improved intestinal histomorphology of broilers. Because of its remarkable efficacy on broiler chickens, the L. salivarius mixture could be considered as a good potential probiotic for chickens, and its benefits should be further evaluated on a commercial scale.

  15. Stable genetic transformation of Vigna mungo L. Hepper via Agrobacterium tumefaciens.

    Saini, R; Sonia; Jaiwal, P K; Jaiwal, S


    Vigna mungo is one of the large-seeded grain legumes that has not yet been transformed. We report here for the first time the production of morphologically normal and fertile transgenic plants from cotyledonary-node explants inoculated with Agrobacterium tumefaciens carrying binary vector pCAMBIA2301, the latter of which contains a neomycin phosphotransferase ( nptII) gene and a beta-glucuronidase (GUS) gene ( uidA) interrupted with an intron. The transformed green shoots, selected and rooted on medium containing kanamycin, tested positive for nptII and uidA genes by polymerase chain reaction (PCR) analysis. These shoots were established in soil and grown to maturity to collect the seeds. Mechanical wounding of the explants prior to inoculation with Agrobacterium, time lag in regeneration due to removal of the cotyledons from explants and a second round of selection at the rooting stage were found to be critical for transformation. Analysis of T(0) plants showed the expression and integration of uidA into the plant genome. GUS activity in leaves, roots, flowers, anthers and pollen grains was detected by histochemical assay. PCR analysis of T(1) progeny revealed a Mendelian transgene inheritance pattern. The transformation frequency was 1%, and 6-8 weeks were required for the generation of transgenics.

  16. Transformation of a recalcitrant grain legume, Vigna mungo L. Hepper, using Agrobacterium tumefaciens-mediated gene transfer to shoot apical meristem cultures.

    Saini, Raman; Jaiwal, Pawan K


    The efficiency of Vigna mungo L. Hepper transformation was significantly increased from an average of 1% to 6.5% by using shoot apices excised from embryonic axes precultured on 10 microM benzyl-6-aminopurine (BAP) for 3 days and wounded prior to inoculation in Agrobacterium tumefaciens strain EHA105 carrying the binary vector pCAMBIA2301, which contains a neomycin phosphotransferase gene (nptII) and a beta-glucuronidase (GUS) gene (gusA) interrupted by an intron. The transformed green shoots that were selected and rooted on medium containing kanamycin, and which tested positive for nptII gene by polymerase chain reaction, were established in soil to collect seeds. GUS activity was detected in whole T(0) shoots and T(1) seedlings. All T(0) plants were morphologically normal, fertile and the majority of them transmitted transgenes in a 3:1 ratio to their progenies. Southern analysis of T(1) plants showed integration of nptII into the plant genome.

  17. Expression pattern of diacylglycerol acyltransferase-1, an enzyme involved in triacylglycerol biosynthesis, in Arabidopsis thaliana.

    Lu, Chaofu Lu; de Noyer, Shen Bayon; Hobbs, Douglas H; Kang, Jinling; Wen, Yancheng; Krachtus, Dieter; Hills, Matthew J


    Triacylglycerol (TAG) is the major carbon storage reserve in oilseeds such as Arabidopsis. Acyl-CoA:diacylglycerol acyltransferase (DGAT) catalyses the final step of the TAG synthesis pathway. Although TAG is mainly accumulated during seed development, and DGAT has presumably the highest activity in developing seeds, we show here that TAG synthesis is also actively taking place during germination and seedling development in Arabidopsis. The expression pattern of the DGAT1 gene was studied in transgenic plants containing the reporter gene beta-glucuronidase (GUS) fused with DNA sequences flanking the DGAT1 coding region. GUS activity was not only detected in developing seeds and pollen, which normally accumulate storage TAG, but also in germinating seeds and seedlings. Western blots showed that DGAT1 protein is present in several tissues, though is most abundant in developing seeds. In seedlings, DGAT1 is expressed in shoot and root apical regions, correlating with rapid cell division and growth. The expression of GUS in seedlings was consistent with the results of RNA gel blot analyses, precursor feeding and DGAT assay. In addition, DGAT1 gene expression is up-regulated by glucose and associated with glucose-induced changes in seedling development.

  18. Liquid chromatography-high resolution mass spectrometry with immunoaffinity clean-up for the determination of the oxidative stress biomarker 8-iso-prostaglandin F2alpha in wastewater.

    Ryu, Yeonsuk; Reid, Malcolm J; Thomas, Kevin V


    A reliable oxidative stress biomarker, 8-iso-prostaglandin F2α (8-iso-PGF2α), was for the first time quantitatively analysed in wastewater using an analytical method consisting of liquid chromatography-high resolution mass spectrometry coupled to immunoaffinity clean-up (IAC-LC-HRMS). Factors influencing the method's robustness were investigated, including analyte stability in sewage and enzymatic deconjugation with β-glucuronidase. The IAC-LC-HRMS method was linear over the range of 0.1-100ng/mL with correlation coefficient (R(2)) of 0.999. The quantification limits were sufficiently low to detect 8-iso-PGF2α in sewage (method quantification limit of 0.3ng/L) and precision, expressed as relative standard deviation was less than 7% and the accuracy expressed as relative recovery was in the 103-113% range. As a result, the application of the method to 24-h composite wastewater samples from Oslo showed 8-iso-PGF2α concentrations of 18.9-23.3ng/L for 8 days in March 2015. This study demonstrates a standard method to analyse 8-iso-PGF2α in sewage that will contribute to the further investigation of the potential use of 8-iso-PGF2α as a sewage biomarker for assessing the status of community health. Copyright © 2015 Elsevier B.V. All rights reserved.

  19. A Cre/loxP-mediated self-activating gene excision system to produce marker gene free transgenic soybean plants.

    Li, Zhongsen; Xing, Aiqiu; Moon, Bryan P; Burgoyne, Susan A; Guida, Anthony D; Liang, Huiling; Lee, Catharina; Caster, Cheryl S; Barton, Joanne E; Klein, Theodore M; Falco, Saverio C


    Marker-gene-free transgenic soybean plants were produced by isolating a developmentally regulated embryo-specific gene promoter, app1, from Arabidopsis and developing a self-activating gene excision system using the P1 bacteriophage Cre/loxP recombination system. To accomplish this, the Cre recombinase gene was placed under control of the app1 promoter and, together with a selectable marker gene (hygromycin phosphotransferase), were cloned between two loxP recombination sites. This entire sequence was then placed between a constitutive promoter and a coding region for either beta-glucuronidase (Gus) or glyphosate acetyltransferase (Gat). Gene excision would remove the entire sequence between the two loxP sites and bring the coding region to the constitutive promoter for expression. Using this system marker gene excision occurred in over 30% of the stable transgenic events as indicated by the activation of the gus reporter gene or the gat gene in separate experiments. Transgenic plants with 1 or 2 copies of a functional excision-activated gat transgene and without any marker gene were obtained in T0 or T1 generation. This demonstrates the feasibility of using developmentally controlled promoters to mediate marker excision in soybean.

  20. Cold tolerance of potato plants transformed with yeast invertase gene

    Alexander N. Deryaabin


    Full Text Available Our study was carried out with potato plants (Solanum tuberosun L.,cv. Désirée transformed with the yeast invertase gene under the control of the B33 class I patatin promoter and with the proteinase inhibitor II leader peptide sequence providing for the apoplastic enzyme localization (B33-inv plants and with the plants transformed with the reporter gene encoding bb-glucuronidase under the control of the 35S CaMV promoter (control plants. Exposure to 5°C during 6 days caused an increase in invertase activity and sugar content in B33-inv leaves in comparison with the control plants. Cell membranes of B33-inv plant cells showed greater cold tolerance under low temperature conditions than control plants that was recorded by electrolyte release. We supposed that higher cold tolerance of B33-inv plants was caused by stabilizing effect of sugar on the membranes, because B33-inv plants differ from the control plants in higher invertase activity, induced by expression of yeast invertase gene, and high content of sugars.

  1. Amino acid substitution in NPC1 that abolishes cholesterol binding reproduces phenotype of complete NPC1 deficiency in mice

    Xie, Xuefen; Brown, Michael S.; Shelton, John M.; Richardson, James A.; Goldstein, Joseph L.; Liang, Guosheng


    Substitution mutations in adjacent amino acids of the N-terminal domain of NPC1, a lysosomal membrane protein, abolish its cholesterol binding activity and impair its ability to export cholesterol from lysosomes of cultured cells lacking npc1 [Kwon HJ, et al. (2009) Cell 137:1213–1224]. Here, we show that the same two mutations (proline-202 and phenylalanine-203, both changed to alanine) reproduce the phenotype of complete NPC1 deficiency when knocked into the mouse npc1 gene by homologous recombination. Homozygous npc1pf/pf mice exhibited neurodegeneration beginning at day 49 and died at a median age of 84 d, as previously reported for mice that lack npc1. Liver and other organs of the npc1pf/pf mice accumulated excess cholesterol in lysosomes. In liver, mRNAs encoding several lysosomal proteins were elevated, including NPC1 and NPC2 and several digestive enzymes (acid lipase, β-glucuronidase, and cathepsins B and D). Weekly treatment with hydroxypropyl-β-cyclodextrin (HPCD) beginning at 7 wk reduced hepatic cholesterol accumulation and diminished the lysosomal mRNAs. We conclude that the cholesterol binding site in the N-terminal domain of NPC1 is essential for cholesterol export from lysosomes in living animals as it is in cultured cells. The HPCD-mediated reduction of excess lysosomal enzymes may contribute to the ability of this drug to delay the progression of NPC disease in mice. PMID:21896731

  2. In plant activation: an inducible, hyperexpression platform for recombinant protein production in plants.

    Dugdale, Benjamin; Mortimer, Cara L; Kato, Maiko; James, Tess A; Harding, Robert M; Dale, James L


    In this study, we describe a novel protein production platform that provides both activation and amplification of transgene expression in planta. The In Plant Activation (INPACT) system is based on the replication machinery of tobacco yellow dwarf mastrevirus (TYDV) and is essentially transient gene expression from a stably transformed plant, thus combining the advantages of both means of expression. The INPACT cassette is uniquely arranged such that the gene of interest is split and only reconstituted in the presence of the TYDV-encoded Rep/RepA proteins. Rep/RepA expression is placed under the control of the AlcA:AlcR gene switch, which is responsive to trace levels of ethanol. Transgenic tobacco (Nicotiana tabacum cv Samsun) plants containing an INPACT cassette encoding the β-glucuronidase (GUS) reporter had negligible background expression but accumulated very high GUS levels (up to 10% total soluble protein) throughout the plant, within 3 d of a 1% ethanol application. The GUS reporter was replaced with a gene encoding a lethal ribonuclease, barnase, demonstrating that the INPACT system provides exquisite control of transgene expression and can be adapted to potentially toxic or inhibitory compounds. The INPACT gene expression platform is scalable, not host-limited, and has been used to express both a therapeutic and an industrial protein.

  3. Overlap of proteome changes in Medicago truncatula in response to auxin and Sinorhizobium meliloti.

    van Noorden, Giel E; Kerim, Tursun; Goffard, Nicolas; Wiblin, Robert; Pellerone, Flavia I; Rolfe, Barry G; Mathesius, Ulrike


    We used proteome analysis to identify proteins induced during nodule initiation and in response to auxin in Medicago truncatula. From previous experiments, which found a positive correlation between auxin levels and nodule numbers in the M. truncatula supernodulation mutant sunn (supernumerary nodules), we hypothesized (1) that auxin mediates protein changes during nodulation and (2) that auxin responses might differ between the wild type and the supernodulating sunn mutant during nodule initiation. Increased expression of the auxin response gene GH3:beta-glucuronidase was found during nodule initiation in M. truncatula, similar to treatment of roots with auxin. We then used difference gel electrophoresis and tandem mass spectrometry to compare proteomes of wild-type and sunn mutant roots after 24 h of treatment with Sinorhizobium meliloti, auxin, or a control. We identified 131 of 270 proteins responding to treatment with S. meliloti and/or auxin, and 39 of 89 proteins differentially displayed between the wild type and sunn. The majority of proteins changed similarly in response to auxin and S. meliloti after 24 h in both genotypes, supporting hypothesis 1. Proteins differentially accumulated between untreated wild-type and sunn roots also showed changes in auxin response, consistent with altered auxin levels in sunn. However, differences between the genotypes after S. meliloti inoculation were largely not due to differential auxin responses. The role of the identified candidate proteins in nodule initiation and the requirement for their induction by auxin could be tested in future functional studies.

  4. (Homo)glutathione depletion modulates host gene expression during the symbiotic interaction between Medicago truncatula and Sinorhizobium meliloti.

    Pucciariello, Chiara; Innocenti, Gilles; Van de Velde, Willem; Lambert, Annie; Hopkins, Julie; Clément, Mathilde; Ponchet, Michel; Pauly, Nicolas; Goormachtig, Sofie; Holsters, Marcelle; Puppo, Alain; Frendo, Pierre


    Under nitrogen-limiting conditions, legumes interact with symbiotic rhizobia to produce nitrogen-fixing root nodules. We have previously shown that glutathione and homoglutathione [(h)GSH] deficiencies impaired Medicago truncatula symbiosis efficiency, showing the importance of the low M(r) thiols during the nodulation process in the model legume M. truncatula. In this study, the plant transcriptomic response to Sinorhizobium meliloti infection under (h)GSH depletion was investigated using cDNA-amplified fragment length polymorphism analysis. Among 6,149 expression tags monitored, 181 genes displayed significant differential expression between inoculated control and inoculated (h)GSH depleted roots. Quantitative reverse transcription polymerase chain reaction analysis confirmed the changes in mRNA levels. This transcriptomic analysis shows a down-regulation of genes involved in meristem formation and a modulation of the expression of stress-related genes in (h)GSH-depleted plants. Promoter-beta-glucuronidase histochemical analysis showed that the putative MtPIP2 aquaporin might be up-regulated during nodule meristem formation and that this up-regulation is inhibited under (h)GSH depletion. (h)GSH depletion enhances the expression of salicylic acid (SA)-regulated genes after S. meliloti infection and the expression of SA-regulated genes after exogenous SA treatment. Modification of water transport and SA signaling pathway observed under (h)GSH deficiency contribute to explain how (h)GSH depletion alters the proper development of the symbiotic interaction.

  5. Genomic analysis and gene structure of the plant carotenoid dioxygenase 4 family: a deeper study in Crocus sativus and its allies.

    Ahrazem, Oussama; Trapero, Almudena; Gómez, M Dolores; Rubio-Moraga, Angela; Gómez-Gómez, Lourdes


    The plastoglobule-targeted enzyme carotenoid cleavage dioxygenase (CCD4) mediates the formation of volatile C13 ketones, such as β-ionone, by cleaving the C9-C10 and C9'-C10' double bonds of cyclic carotenoids. Here, we report the isolation and analysis of CCD4 genomic DNA regions in Crocus sativus. Different CCD4 alleles have been identified: CsCCD4a which is found with and without an intron and CsCCD4b that showed the presence of a unique intron. The presence of different CCD4 alleles was also observed in other Crocus species. Furthermore, comparison of the locations of CCD4 introns within the coding region with CCD4 genes from other plant species suggests that independent gain/losses have occurred. The comparison of the promoter region of CsCCD4a and CsCCD4b with available CCD4 gene promoters from other plant species highlighted the conservation of cis-elements involved in light response, heat stress, as well as the absence and unique presence of cis-elements involved in circadian regulation and low temperature responses, respectively. Functional characterization of the Crocus sativus CCD4a promoter using Arabidopsis plants stably transformed with a DNA fragment of 1400 base pairs (P-CsCCD4a) fused to the β-glucuronidase (GUS) reporter gene showed that this sequence was sufficient to drive GUS expression in the flower, in particular high levels were detected in pollen.

  6. Production of oleanolic acid glycosides by hairy root established cultures of Calendula officinalis L.

    Długosz, Marek; Wiktorowska, Ewa; Wiśniewska, Anita; Pączkowski, Cezary


    In order to initiate hairy root culture initiation cotyledons and hypocotyls of Calendula officinalis L. were infected with Agrobacterium rhizogenes strain ATCC 15834 or the same strain containing pCAMBIA 1381Z vector with β-glucuronidase reporter gene under control of promoter of NIK (Nematode Induced Kinase) gene. The efficiency of induction of hairy roots reached 33.8% for cotyledons and 66.6% for hypocotyls together for both transformation experiments. Finally, eight control and nine modified lines were established as a long-term culture. The hairy root cultures showed the ability to synthesize oleanolic acid mainly (97%) as glycosides; control lines contained it at the average 8.42 mg · g(-1) dry weight in tissue and 0.23 mg · dm(-3) in medium; modified lines: 4.59 mg · g(-1) for the tissue, and 0.48 mg · dm(-3) for the medium. Additionally lines showed high positive correlation between dry/fresh weight and oleanolic acid concentration in tissue. Using the Killiani mixture in acidic hydrolysis of oleanolic acid glycosides released free aglycones that were partially acetylated in such conditions.

  7. Physicochemical and biofunctional properties of crab chitosan nanoparticles.

    Nguyen, The Han; Kwak, Hae Soo; Kim, Sang Moo


    The physicochemical and biofunctional properties of crab chitosan nanoparticles of two different sizes (Nano A and B) manufactured by dry milling method were evaluated for commercialization. The deacetylation degrees (DD) of Nano A, B and the control chitosan were 90.9, 93.0, and 92.7% respectively whereas their molecular weights (M(w)) were 43.9, 44.7 and 208.8 kDa. The average sizes of the dispersed Nano A, B and the control chitosan in cetyltrimethylammonium chloride were 735.9, 849.4 and 2,382.4 nm, respectively, which were lower than 1441.7, 2935.6 and 6832.9 nm of the intact chitosans. Chitosan nanoparticles had mild tyrosinase, antioxidant and angiotensin I converting enzyme (ACE), but weak collagenase, elastase and beta-glucuronidase inhibitory activity. However, Nano A had strong alpha-glucosidase inhibitory activity, which was comparable to that of acarbose, a commercial alpha-glucosidase inhibitor. In addition, the minimum inhibitory concentrations (MICs) of chitosan and its nanoparticles ranged from 30 to > 200 microg/mL against each four gram-positive and gram-negative bacteria. Therefore, crab chitosan nanoparticles could be used as a nutraceutical, cosmeceutical or pharmaceutical product.

  8. Secreted and Transmembrane αKlotho Isoforms Have Different Spatio-Temporal Profiles in the Brain during Aging and Alzheimer's Disease Progression

    Massó, Anna; Sánchez, Angela; Gimenez-Llort, Lydia; Lizcano, Jose Miguel; Cañete, Manuel; García, Belen; Torres-Lista, Virginia; Puig, Meritxell; Bosch, Assumpció; Chillon, Miguel


    The Klotho protein is a β-glucuronidase, and its overexpression is associated with life extension. Its mechanism of action is not fully understood, although it has been recently reported that αKlotho improves synaptic and cognitive functions, and it may also influence a variety of structures and functions during CNS maturation and aging. The αKlotho gene has two transcripts, one encoding a transmembrane isoform (m-KL), and the other a putative secreted isoform (s-KL). Unfortunately, little is known about the secreted αKlotho isoform, since available antibodies cannot discriminate s-KL from the KL1 domain cleaved from the transmembrane isoform. This study shows, for the first time, that the klotho transcript produced by alternative splicing generates a stable protein (70 kDa), and that in contrast to the transmembrane Klotho isoform, it is ten times more abundant in the brain than in the kidney suggesting that the two isoforms may have different functions. We also studied whether klotho expression in the CNS was influenced by aging, Alzheimer's disease (AD), or a healthy lifestyle, such as voluntary moderate continuous exercise. We observed a strong correlation between high expression levels of the two klotho transcripts and the healthy status of the animals. Expression of Klotho in brain areas decayed more rapidly in the 3xTg-AD model of AD than in healthy animals, whilst moderate continuous exercise in adulthood prevents the decline in expression of both klotho transcripts. PMID:26599613

  9. Environmental exposure to organophosphate pesticides: assessment of endocrine disruption and hepatotoxicity in pregnant women.

    Cecchi, A; Rovedatti, M G; Sabino, G; Magnarelli, G G


    In utero exposure is the first point of contact with environmental xenobiotics that may affect the maternal-placental-fetal balance. Considering that maternal pathophysiological changes affect intrauterine development, this pilot study was conducted to address how environmental exposure to organophosphate pesticides (OPs) during pregnancy may contribute to maternal endocrine disruption and disturbed hepatic function. A prospective study was carried out with pregnant women (n=97) living in a rural area of the Rio Negro province where OPs are intensively applied throughout 6 months of the year. Blood samples were obtained and biomarkers of OPs exposure (cholinesterases and β-glucuronidase), cortisol (CT) and progesterone (PG) levels, as well as glycemia, were determined. Parameters of liver injury were assayed by measuring aspartate aminotransferase (AST) and alanine aminotransferase (ALT); liver function was assayed by measuring albumin. Biomonitoring carried out during the pre-spraying period (PreS) and spraying period (SP) showed that the population studied was exposed to OPs, proven by the fact that plasma (PCh) and erythrocyte cholinesterase (AChE) decreased very significantly (pnormal values for the general population, the increase in CT in the maternal compartment may lead to impaired newborn health later in life.

  10. Agrobacterium Mediated Transformation of Fld and GUS Genes into Canola for Salinity Stress

    Niapour, Nazila


    Full Text Available Salinity is one of the major abiotic stress which limits wide spread canola cultivation. One way to overcome this problem could be transfection, to produce tolerable species. Cotyledonary and hypocotyls explants obtained from 4 and 7 days old seedling of Elite and RJS003 varieties were utilized in this study. Genetic transformation was implemented through Agrobacterium tumefaciens LBA4404 containing PBI121 plasmid and Agrobacterium tumefaciens C58, LBA4404, AGL0 and EHA 101 strains which contain P6u- ubi- fvt1 construct. The T-DNA region of P6u- Ubi- Fvt1 plasmid included HPT (Hygromycin phosphotransferase plant selectable marker and Fld (flavodoxin gene. PBI121 plasmid had NptII (Neomycin phosphotransferase plant Selectable marker and β-glucuronidase (GUS reporter genes. Transfected explants were analyzed by PCR and histochemical assay for Fld and Gus genes, respectively. Our data indicated that the cotyledonary explants of both cultivars were incompetent to be infected with Fld gens. However, the transformation in Elite hypocotyls explants with Agrobacterium tumefaciens C58 and LBA 4404 strains were confirmed through PCR product and histochemical evaluation for Fld and GUS genes, respectively. Therefore, the result of this manuscript may to certain degree fulfill the endeavor appointed to this oilseed.

  11. High-efficiency stable transformation of the model fern species Ceratopteris richardii via microparticle bombardment.

    Plackett, Andrew R G; Huang, Liandong; Sanders, Heather L; Langdale, Jane A


    Ferns represent the most closely related extant lineage to seed plants. The aquatic fern Ceratopteris richardii has been subject to research for a considerable period of time, but analyses of the genetic programs underpinning developmental processes have been hampered by a large genome size, a lack of available mutants, and an inability to create stable transgenic lines. In this paper, we report a protocol for efficient stable genetic transformation of C. richardii and a closely related species Ceratopteris thalictroides using microparticle bombardment. Indeterminate callus was generated and maintained from the sporophytes of both species using cytokinin treatment. In proof-of-principle experiments, a 35S::β-glucuronidase (GUS) expression cassette was introduced into callus cells via tungsten microparticles, and stable transformants were selected via a linked hygromycin B resistance marker. The presence of the transgene in regenerated plants and in subsequent generations was validated using DNA-blot analysis, reverse transcription-polymerase chain reaction, and GUS staining. GUS staining patterns in most vegetative tissues corresponded with constitutive gene expression. The protocol described in this paper yields transformation efficiencies far greater than those previously published and represents a significant step toward the establishment of a tractable fern genetic model.

  12. Factors affecting gene transformation in mangosteen

    Sompong Te-chato


    Full Text Available Factors affecting gene transformation in mangosteen (Garcinia mangostana L. were investigated. Types of explants, strains and densities of Agrobacterium tumefaciens, and co-culture methods were examined to optimize gene transformation. The results showed that among strains of Agrobacterium tumefaciens tested, LBA 4404 containing pBI 121 gave the calli with the highest resistance to kanamycin. Kanamycin at the concentration of 50-100 mg/l was the best range for selection of transformants. Higher density of agrobacteria tended to promote higher frequency of transformation. The best co-culture method was dipping the explant in a solution of agrobacteria for 10 minutes, followed by culturing onto co-culture medium without antibiotic for 48 hours. Among the explants used to co- culture with bacteria, half leaf treatment gave the best result for transformation; however, callus proliferation and plantlet regeneration were inferior to whole leaf treatment. Activity of β-Glucuronidase (GUS could not be detected, thus resistance to kanamycin was used for detecting transformability. Shoot primordia could be induced from kanamycin-resistant calli grown in regeneration medium. After maintenance by subculturing to the same medium 2 to 3 times in 2-3 months, the developed shoots turned brown and finally died. Hence, the transformed plant of mangosteen was not obtained from this experiment.

  13. Functional characterisation and cell specificity of BvSUT1, the transporter that loads sucrose into the phloem of sugar beet (Beta vulgaris L.) source leaves.

    Nieberl, P; Ehrl, C; Pommerrenig, B; Graus, D; Marten, I; Jung, B; Ludewig, F; Koch, W; Harms, K; Flügge, U-I; Neuhaus, H E; Hedrich, R; Sauer, N


    Sugar beet (Beta vulgaris L.) is one of the most important sugar-producing plants worldwide and provides about one third of the sugar consumed by humans. Here we report on molecular characterisation of the BvSUT1 gene and on the functional characterisation of the encoded transporter. In contrast to the recently identified tonoplast-localised sucrose transporter BvTST2.1 from sugar beet taproots, which evolved within the monosaccharide transporter (MST) superfamily, BvSUT1 represents a classical sucrose transporter and is a typical member of the disaccharide transporter (DST) superfamily. Transgenic Arabidopsis plants expressing the β-GLUCURONIDASE (GUS) reporter gene under control of the BvSUT1-promoter showed GUS histochemical staining of their phloem; an anti-BvSUT1-antiserum identified the BvSUT1 transporter specifically in phloem companion cells. After expression of BvSUT1 cDNA in bakers' yeasts (Saccharomyces cerevisiae) uptake characteristics of the BvSUT1 protein were studied. Moreover, the sugar beet transporter was characterised as a proton-coupled sucrose symporter in Xenopus laevis oocytes. Our findings indicate that BvSUT1 is the sucrose transporter that is responsible for loading of sucrose into the phloem of sugar beet source leaves delivering sucrose to the storage tissue in sugar beet taproot sinks. © 2017 German Botanical Society and The Royal Botanical Society of the Netherlands.

  14. The role of heparanase in pulmonary cell recruitment in response to an allergic but not non-allergic stimulus.

    Morris, Abigail; Wang, Bo; Waern, Ida; Venkatasamy, Radhakrishnan; Page, Clive; Schmidt, Eric P; Wernersson, Sara; Li, Jin-Ping; Spina, Domenico


    Heparanase is an endo-β-glucuronidase that specifically cleaves heparan sulfate proteoglycans in the extracellular matrix. Expression of this enzyme is increased in several pathological conditions including inflammation. We have investigated the role of heparanase in pulmonary inflammation in the context of allergic and non-allergic pulmonary cell recruitment using heparanase knockout (Hpa-/-) mice as a model. Following local delivery of LPS or zymosan, no significant difference was found in the recruitment of neutrophils to the lung between Hpa-/- and wild type (WT) control. Similarly neutrophil recruitment was not inhibited in WT mice treated with a heparanase inhibitor. However, in allergic inflammatory models, Hpa-/- mice displayed a significantly reduced eosinophil (but not neutrophil) recruitment to the airways and this was also associated with a reduction in allergen-induced bronchial hyperresponsiveness, indicating that heparanase expression is associated with allergic reactions. This was further demonstrated by pharmacological treatment with a heparanase inhibitor in the WT allergic mice. Examination of lung specimens from patients with different severity of chronic obstructive pulmonary disease (COPD) found increased heparanase expression. Thus, it is established that heparanase contributes to allergen-induced eosinophil recruitment to the lung and could provide a novel therapeutic target for the development of anti-inflammatory drugs for the treatment of asthma and other allergic diseases.

  15. Identification and properties of insect resistance-associated maize anionic peroxidases.

    Dowd, Patrick F; Johnson, Eric T; Pinkerton, T Scott


    Previous studies with transgenic plants have indicated a tobacco anionic peroxidase can confer enhanced resistance to a variety of insects when expressed in different plant species. Tissue that expresses high levels of this enzyme often browns rapidly when damaged. Maize roots damaged under sterile conditions browned and had an anionic peroxidase induced. When introduced biolistically, maize callus transformants expressing a maize peroxidase gene with a predicted isoelectric point of ca. 5.1 produced browner callus compared to a corresponding beta-glucuronidase (GUS) transformant as callus aged. Higher production of only one isozyme of ca. pI 4.5 was noted. When the callus was fed to two maize pest caterpillar species, growth rates were slower (as reflected by weights) relative to the GUS callus. Based on examination of published information and electrophoretic properties, this gene appears to code for Px11, a peroxidase isozyme that is primarily produced in root tissue and callus. When sequence of the gene in several inbreds was examined, coding variations were noted, and abilities to utilize ferulic and p-coumaric acids differed. These coding differences may influence the ability of corresponding forms of the peroxidase to promote resistance. In addition to potential use in marker assisted breeding, enhanced expression of this anionic peroxidase through breeding or genetic engineering may lead to enhanced insect or disease resistance. Published by Elsevier Ltd.

  16. Fruit preferential activity of the tomato RIP1 gene promoter in transgenic tomato and Arabidopsis.

    Agarwal, Priyanka; Kumar, Rahul; Pareek, Amit; Sharma, Arun K


    Isolation and functional characterization of tissue- and stage-specific gene promoters is beneficial for genetic improvement of economically important crops. Here, we have characterized a putative promoter of a ripening-induced gene RIP1 (Ripening induced protein 1) in tomato. Quantification of the transcript level of RIP1 showed that its expression is fruit preferential, with maximum accumulation in red ripe fruits. To test the promoter activity, we made a reporter construct by cloning 1450 bp putative RIP1 promoter driving the GUS (ß-glucuronidase) gene expression and generated stable transgenic lines in tomato and Arabidopsis. Histochemical and fluorometric assays validated the fruit-specific expression of RIP1 as the highest GUS activity was found in red ripe tomatoes. Similarly, we detected high levels of GUS activity in the siliques of Arabidopsis. On the contrary, weak GUS activity was found in the flower buds in both tomato and Arabidopsis. To characterize the specific regions of the RIP1 promoter that might be essential for its maximum activity and specificity in fruits, we made stable transgenic lines of tomato and Arabidopsis with 5'-deletion constructs. Characterization of these transgenic plants showed that the full length promoter is essential for its function. Overall, we report the identification and characterization of a ripening-induced promoter of tomato, which would be useful for the controlled manipulation of the ripening-related agronomic traits in genetic manipulation studies in future.

  17. Improved Therapeutic Efficacy in Bone and Joint Disorders by Targeted Drug Delivery to Bone.

    Takahashi, Tatsuo


     Site-specific drug delivery to bone is considered achievable using acidic amino acid (L-Asp or L-Glu) homopeptides known as acidic oligopeptides. We found that fluorescence-labeled acidic oligopeptides containing six or more residues bound strongly to hydroxyapatite, which is a major component of bone, and were selectively delivered to and retained in bone after systemic administration. We explored the applicability of this result for drug delivery by conjugation of estradiol and levofloxacin with an L-Asp hexapeptide. We also similarly tagged enzymes (tissue-nonspecific alkaline phosphatase, β-glucuronidase, and N-acetylgalactosamine-6-sulfate sulfatase) and decoy receptors (endogenous secretory receptor for advanced glycation end products and etanercept) to assess whether these would improve therapeutic efficacy. The L-Asp hexapeptide-tagged drugs, including enzymes and decoy receptors, were efficiently delivered to bone in comparison with the untagged drugs. An in vivo experiment confirmed the efficacy of L-Asp hexapeptide-tagged drugs on bone and joint disorders, although there was some loss of bioactivity of estradiol and levofloxacin in vitro, suggesting that the acidic hexapeptide was partly removed by hydrolysis in the body after delivery to bone. It was expected that the ester linkage to the hexapeptide would be susceptible to hydrolysis in situ, releasing the drug from the acidic oligopeptide. These results support the usefulness of acidic oligopeptides as bone-targeting carriers for therapeutic agents. We present some pharmacokinetic and pharmacological properties of the L-Asp hexapeptide-tagged drugs.

  18. Two phloem nitrate transporters, NRT1.11 and NRT1.12, are important for redistributing xylem-borne nitrate to enhance plant growth.

    Hsu, Po-Kai; Tsay, Yi-Fang


    This study of the Arabidopsis (Arabidopsis thaliana) nitrate transporters NRT1.11 and NRT1.12 reveals how the interplay between xylem and phloem transport of nitrate ensures optimal nitrate distribution in leaves for plant growth. Functional analysis in Xenopus laevis oocytes showed that both NRT1.11 and NRT1.12 are low-affinity nitrate transporters. Quantitative reverse transcription-polymerase chain reaction and immunoblot analysis showed higher expression of these two genes in larger expanded leaves. Green fluorescent protein and β-glucuronidase reporter analyses indicated that NRT1.11 and NRT1.12 are plasma membrane transporters expressed in the companion cells of the major vein. In nrt1.11 nrt1.12 double mutants, more root-fed (15)NO3(-) was translocated to mature and larger expanded leaves but less to the youngest tissues, suggesting that NRT1.11 and NRT1.12 are required for transferring root-derived nitrate into phloem in the major veins of mature and larger expanded leaves for redistributing to the youngest tissues. Distinct from the wild type, nrt1.11 nrt1.12 double mutants show no increase of plant growth at high nitrate supply. These data suggested that NRT1.11 and NRT1.12 are involved in xylem-to-phloem transfer for redistributing nitrate into developing leaves, and such nitrate redistribution is a critical step for optimal plant growth enhanced by increasing external nitrate.

  19. Identification of stable reference genes for quantitative PCR in cells derived from chicken lymphoid organs.

    Borowska, D; Rothwell, L; Bailey, R A; Watson, K; Kaiser, P


    Quantitative polymerase chain reaction (qPCR) is a powerful technique for quantification of gene expression, especially genes involved in immune responses. Although qPCR is a very efficient and sensitive tool, variations in the enzymatic efficiency, quality of RNA and the presence of inhibitors can lead to errors. Therefore, qPCR needs to be normalised to obtain reliable results and allow comparison. The most common approach is to use reference genes as internal controls in qPCR analyses. In this study, expression of seven genes, including β-actin (ACTB), β-2-microglobulin (B2M), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), β-glucuronidase (GUSB), TATA box binding protein (TBP), α-tubulin (TUBAT) and 28S ribosomal RNA (r28S), was determined in cells isolated from chicken lymphoid tissues and stimulated with three different mitogens. The stability of the genes was measured using geNorm, NormFinder and BestKeeper software. The results from both geNorm and NormFinder were that the three most stably expressed genes in this panel were TBP, GAPDH and r28S. BestKeeper did not generate clear answers because of the highly heterogeneous sample set. Based on these data we will include TBP in future qPCR normalisation. The study shows the importance of appropriate reference gene normalisation in other tissues before qPCR analysis.

  20. In planta transformation of sorghum (Sorghum bicolor (L.) Moench) using TPS1 gene for enhancing tolerance to abiotic stresses

    Varalaxmi Yellisetty; L. A. Reddy; Maheswari Mandapaka


    An in planta transformation protocol for sorghum (Sorghum bicolor (L.) Moench) using shoot apical meristem of germinating seedlings is reported in this study. Agrobacterium tumefaciens strain, LBA4404 with pCAMBIA1303 vector and construct pCAMBIA1303TPS1 were individually used for transformation. Since, the transgene is integrated into the cells of already differentiated tissues, the T0 plants were chimeric and stable integration was observed in T1 generation. -Glucuronidase (GUS) expression in the seedlings and spikelets of emerging cob was the first indication of transformability in T0 generation which was further confirmed by PCR analysis using hpt and TPS1 gene-specific primers. Screening on 25 mg/L hygromycin combined with PCR analysis was used for selection of transformants in the T1 generation. Transformation efficiencies ranged between 34–38% and 26–34% using pCAMBIA1303 vector and construct pCAMBIA1303TPS1, respectively. Molecular characterization of the T2 transgenics using PCR, RT-PCR and Southern blot analyses further revealed the integration, expression and inheritance of the transgene. These results indicate the feasibility of the method to generate transgenics with pCAM-BIA1303 vector and construct pCAMBIA1303TPS1. The abiotic stress tolerance of TPS1 transgenics developed in the present study was evident by the ability of the transformants to tolerate 200 mM NaCl as well as higher root growth and biomass.

  1. Functional characterisation of wheat Pgip genes reveals their involvement in the local response to wounding.

    Janni, M; Bozzini, T; Moscetti, I; Volpi, C; D'Ovidio, R


    Polygalacturonase-inhibiting proteins (PGIPs) are cell wall leucine-rich repeat (LRR) proteins involved in plant defence. The hexaploid wheat (Triticum aestivum, genome AABBDD) genome contains one Pgip gene per genome. Tapgip1 (B genome) and Tapgip2 (D genome) are expressed in all tissues, whereas Tapgip3 (A genome) is inactive because of a long terminal repeat, Copia retrotransposon insertion within the coding region. To verify whether Tapgip1 and Tapgip2 encode active PGIPs and are involved in the wheat defence response, we expressed them transiently and analysed their expression under stress conditions. Neither TaPGIP1 nor TaPGIP2 showed inhibition activity in vitro against fungal polygalacturonases. Moreover, a wheat genotype (T. turgidum ssp. dicoccoides) lacking active homologues of Tapgip1 or Tapgip2 possesses PGIP activity. At transcript level, Tapgip1 and Tapgip2 were both up-regulated after fungal infection and strongly induced following wounding. This latter result has been confirmed in transgenic wheat plants expressing the β-glucuronidase (GUS) gene under control of the 5'-flanking region of Tdpgip1, a homologue of Tapgip1 with an identical sequence. Strong and transient GUS staining was mainly restricted to the damaged tissues and was not observed in adjacent tissues. Taken together, these results suggest that Tapgips and their homologues are involved in the wheat defence response by acting at the site of the lesion caused by pathogen infection.

  2. Development of a Biomarker for Penconazole: A Human Oral Dosing Study and a Survey of UK Residents’ Exposure

    Craig Sams


    Full Text Available Penconazole is a widely used fungicide in the UK; however, to date, there have been no peer-reviewed publications reporting human metabolism, excretion or biological monitoring data. The objectives of this study were to i develop a robust analytical method, ii determine biomarker levels in volunteers exposed to penconazole, and, finally, to iii measure the metabolites in samples collected as part of a large investigation of rural residents’ exposure. An LC-MS/MS method was developed for penconazole and two oxidative metabolites. Three volunteers received a single oral dose of 0.03 mg/kg body weight and timed urine samples were collected and analysed. The volunteer study demonstrated that both penconazole-OH and penconazole-COOH are excreted in humans following an oral dose and are viable biomarkers. Excretion is rapid with a half-life of less than four hours. Mean recovery of the administered dose was 47% (range 33%–54% in urine treated with glucuronidase to hydrolyse any conjugates. The results from the residents’ study showed that levels of penconazole-COOH in this population were low with >80% below the limit of detection. Future sampling strategies that include both end of exposure and next day urine samples, as well as contextual data about the route and time of exposure, are recommended.

  3. Stable transformation of ferns using spores as targets: Pteris vittata and Ceratopteris thalictroides.

    Muthukumar, Balasubramaniam; Joyce, Blake L; Elless, Mark P; Stewart, C Neal


    Ferns (Pteridophyta) are very important members of the plant kingdom that lag behind other taxa with regards to our understanding of their genetics, genomics, and molecular biology. We report here, to our knowledge, the first instance of stable transformation of fern with recovery of transgenic sporophytes. Spores of the arsenic hyperaccumulating fern Pteris vittata and tetraploid 'C-fern Express' (Ceratopteris thalictroides) were stably transformed by Agrobacterium tumefaciens with constructs containing the P. vittata actin promoter driving a GUSPlus reporter gene. Reporter gene expression assays were performed on multiple tissues and growth stages of gametophytes and sporophytes. Southern-blot analysis confirmed stable transgene integration in recovered sporophytes and also confirmed that no plasmid from A. tumefaciens was present in the sporophyte tissues. We recovered seven independent transformants of P. vittata and four independent C. thalictroides transgenics. Inheritance analyses using β-glucuronidase (GUS) histochemical staining revealed that the GUS transgene was stably expressed in second generation C. thalictroides sporophytic tissues. In an independent experiment, the gusA gene that was driven by the 2× Cauliflower mosaic virus 35S promoter was bombarded into P. vittata spores using biolistics, in which putatively stable transgenic gametophytes were recovered. Transformation procedures required no tissue culture or selectable marker genes. However, we did attempt to use hygromycin selection, which was ineffective for recovering transgenic ferns. This simple stable transformation method should help facilitate functional genomics studies in ferns.

  4. Transformation of the monocotyledonous Alstroemeria by Agrobacterium tumefaciens.

    Akutsu, M; Ishizaki, T; Sato, H


    An efficient procedure is described for the transformation of the monocotyledonous Alstroemeria by Agrobacterium tumefaciens via callus regeneration. Calli derived from ovules were co-cultivated with A. tumefaciens strains EHA101 and LBA4404, which harbored the binary vector plasmids pIG121Hm and pTOK233, respectively. These plasmids contain the beta-glucuronidase gene ( gusA) as a reporter gene and the hygromycin phosphotransferase and neomycin phosphotransferase II ( nptII) genes as selective markers. Inoculated calli were first plated for 4 weeks on medium containing cefotaxime to eliminate bacteria, following which time transformed cells were selected on medium that contained 20 mg/l hygromycin. A histochemical assay for GUS activity revealed that hygromycin-based selection was completed after 8 weeks. The integration of the T-DNA of pIG121Hm and pTOK233 into the genome of the cells was confirmed by PCR analysis. Efficient shoot regeneration from the transformed calli was observed on half-strength MS medium supplemented with 0.5 mg/l naphthaleneacetic acid and 0.5 mg/l benzyladenine after about 5 months of culture. The presence of the gusA and nptII genes in the genomic DNA of regenerated plants was detected by means of PCR and PCR-Southern hybridization, and the expression of these transgenes was verified by reverse transcription-PCR.

  5. HopX1 in Erwinia amylovora functions as an avirulence protein in apple and is regulated by HrpL.

    Bocsanczy, A M; Schneider, D J; DeClerck, G A; Cartinhour, S; Beer, S V


    Fire blight is a devastating disease of rosaceous plants caused by the Gram-negative bacterium Erwinia amylovora. This pathogen delivers virulence proteins into host cells utilizing the type III secretion system (T3SS). Expression of the T3SS and of translocated and secreted substrates is activated by the alternative sigma factor HrpL, which recognizes hrp box promoters upstream of regulated genes. A collection of hidden Markov model (HMM) profiles was used to identify putative hrp boxes in the genome sequence of Ea273, a highly virulent strain of E. amylovora. Among potential virulence factors preceded by putative hrp boxes, two genes previously known as Eop3 and Eop2 were characterized. The presence of functionally active hrp boxes upstream of these two genes was confirmed by β-glucuronidase (GUS) assays. Deletion mutants of the latter candidate genes, renamed hopX1(Ea) and hopAK1(Ea), respectively, did not differ in virulence from the wild-type strain when assayed in pear fruit and apple shoots. The hopX1(Ea) deletion mutant of Ea273, complemented with a plasmid overexpressing hopX1(E)(a), suppressed the development of the hypersensitivity response (HR) when inoculated into Nicotiana benthamiana; however, it contributed to HR in Nicotiana tabacum and significantly reduced the progress of disease in apple shoots, suggesting that HopX1(Ea) may act as an avirulence protein in apple shoots.

  6. Insertion DNA Accelerates Meiotic Interchromosomal Recombination in Arabidopsis thaliana.

    Sun, Xiao-Qin; Li, Ding-Hong; Xue, Jia-Yu; Yang, Si-Hai; Zhang, Yan-Mei; Li, Mi-Mi; Hang, Yue-Yu


    Nucleotide insertions/deletions are ubiquitous in eukaryotic genomes, and the resulting hemizygous (unpaired) DNA has significant, heritable effects on adjacent DNA. However, little is known about the genetic behavior of insertion DNA. Here, we describe a binary transgenic system to study the behavior of insertion DNA during meiosis. Transgenic Arabidopsis lines were generated to carry two different defective reporter genes on nonhomologous chromosomes, designated as "recipient" and "donor" lines. Double hemizygous plants (harboring unpaired DNA) were produced by crossing between the recipient and the donor, and double homozygous lines (harboring paired DNA) via self-pollination. The transfer of the donor's unmutated sequence to the recipient generated a functional β-glucuronidase gene, which could be visualized by histochemical staining and corroborated by polymerase chain reaction amplification and sequencing. More than 673 million seedlings were screened, and the results showed that meiotic ectopic recombination in the hemizygous lines occurred at a frequency  >6.49-fold higher than that in the homozygous lines. Gene conversion might have been exclusively or predominantly responsible for the gene correction events. The direct measurement of ectopic recombination events provided evidence that an insertion, in the absence of an allelic counterpart, could scan the entire genome for homologous counterparts with which to pair. Furthermore, the unpaired (hemizygous) architectures could accelerate ectopic recombination between itself and interchromosomal counterparts. We suggest that the ectopic recombination accelerated by hemizygous architectures may be a general mechanism for interchromosomal recombination through ubiquitously dispersed repeat sequences in plants, ultimately contributing to genetic renovation and eukaryotic evolution.

  7. Prebiotics and bioactive natural substances induce changes of composition and metabolic activities of the colonic microflora in cancerous rats.

    Hijová, Emília; Bomba, Alojz; Bertková, Izabela; Strojný, Ladislav; Szabadosová, Viktória; Šoltésová, Alena


    Prebiotics are defined as selectively fermented food ingredients that induce specific changes in the composition and/or activity in the gastrointestinal microbiota beneficial to the host well-being and health. The aim of the presented experiment was to investigate the effect of a prebiotic applied alone or in combination with Hyppocastani extractum siccum, and Lini oleum virginale in rats with dimethylhydrazine induced colon cancer. Wistar albino rats were fed high fat diet supplemented with the prebiotic alone or in combination with Horse chestnut and flaxseed oil. The activity of faecal glycolytic enzymes, lipid parameters, bile acids, short chain fatty acids and counts of coliforms and lactobacilli were determined. Treatment with the prebiotic alone and in combination with selected substances significantly decreased the activity of glycolytic bacterial enzyme β-glucuronidase (Pprebiotic with Horse chestnut. The prebiotic alone decreased the lipid parameters (Pprebiotic and bioactive natural substances significantly reduced number of coliforms (PPrebiotic alone significantly increased the count of lactobacilli (Pprebiotics have a protective effect and may be the useful for colon cancer prevention and treatment.


    Bernheimer, Alan W.; Schwartz, Lois L.


    Bernheimer, Alan W. (New York University School of Medicine, New York), and Lois L. Schwartz. Lysosomal disruption by bacterial toxins. J. Bacteriol. 87:1100–1104. 1964.—Seventeen bacterial toxins were examined for capacity (i) to disrupt rabbit leukocyte lysosomes as indicated by decrease in turbidity of lysosomal suspensions, and (ii) to alter rabbit liver lysosomes as measured by release of β-glucuronidase and acid phosphatase. Staphylococcal α-toxin, Clostridium perfringens α-toxin, and streptolysins O and S affected lysosomes in both systems. Staphylococcal β-toxin, leucocidin and enterotoxin, Shiga neurotoxin, Serratia endotoxin, diphtheria toxin, tetanus neurotoxin, C. botulinum type A toxin, and C. perfringens ε-toxin were not active in either system. Staphylococcal δ-toxin, C. histolyticum collagenase, crude C. perfringens β-toxin, and crude anthrax toxin caused lysosomal damage in only one of the test systems. There is a substantial correlation between the hemolytic property of a toxin and its capacity to disrupt lysosomes, lending support to the concept that erythrocytes and lysosomes are bounded by similar membranes. PMID:5874534

  9. Concentrations of MDPV in rat striatum correlate with the psychostimulant effect.

    Novellas, Judith; López-Arnau, Raúl; Carbó, Marcel Li; Pubill, David; Camarasa, Jorge; Escubedo, Elena


    3,4-methylenedioxypyrovalerone or MDPV is a synthetic cathinone with psychostimulant properties more potent than cocaine. We quantified this drug in the striatum after subcutaneous administration to rats. MDPV reached the brain around 5 min after its administration and peaked at 20-25 min later. The elimination half-life in the striatum (61 min) correlates with the decrease in the psychostimulant effect after 60 min. Around 11% of the administered dose reached the striatum and, considering a homogeneous brain distribution, we determined that around 86% of the plasma MDPV is distributed to the brain. MDPV induced a dose-dependent increase in locomotor activity, rearing behaviour and stereotypies, all prevented by haloperidol. A plot of locomotor activity or stereotypies versus MDPV striatal concentrations over time showed a direct relationship between factors. No free MDPV metabolites were detected in plasma, at any time, but hydrolysis with glucuronidase allowed us to identify mainly three metabolites, one of them for the first time in rat plasma. The present results contribute to evidence that MDPV induces hyperlocomotion mainly through a dopamine-dependent mechanism. Good correlation between behavioural effects and striatal levels of MDPV leads us to conclude that its psychostimulant effect is mainly due to a striatal distribution of the substance. The present research provides useful information on the pharmacokinetics of MDPV, and can help design new experiments with kinetics data as well as provide a better understanding of the effects of MDPV in humans and its potential interactions. © The Author(s) 2015.

  10. The Arabidopsis ATNRT2.7 nitrate transporter controls nitrate content in seeds.

    Chopin, Franck; Orsel, Mathilde; Dorbe, Marie-France; Chardon, Fabien; Truong, Hoai-Nam; Miller, Anthony J; Krapp, Anne; Daniel-Vedele, Françoise


    In higher plants, nitrate is taken up by root cells where Arabidopsis thaliana NITRATE TRANSPORTER2.1 (ATNRT2.1) chiefly acts as the high-affinity nitrate uptake system. Nitrate taken up by the roots can then be translocated from the root to the leaves and the seeds. In this work, the function of the ATNRT2.7 gene, one of the seven members of the NRT2 family in Arabidopsis, was investigated. High expression of the gene was detected in reproductive organs and peaked in dry seeds. beta-Glucuronidase or green fluorescent protein reporter gene expression driven by the ATNRT2.7 promoter confirmed this organ specificity. We assessed the capacity of ATNRT2.7 to transport nitrate in Xenopus laevis oocytes or when it is expressed ectopically in mutant plants deficient in nitrate transport. We measured the impact of an ATNRT2.7 mutation and found no difference from the wild type during vegetative development. By contrast, seed nitrate content was affected by overexpression of ATNRT2.7 or a mutation in the gene. Finally, we showed that this nitrate transporter protein was localized to the vacuolar membrane. Our results demonstrate that ATNRT2.7 plays a specific role in nitrate accumulation in the seed.

  11. The prophylactic effect of probiotic Enterococcus lactis IW5 against different human cancer cells



    Full Text Available Enterococcus lactis IW5 was obtained from human gut and the potential probiotic characteristics of this organism were then evaluated. Results showed that this strain was highly resistant to low pH and high bile salt and adhered strongly to Caco-2 human epithelial colorectal cell lines. The supernatant of E. lactis IW5 strongly inhibited the growth of several pathogenic bacteria and decreased the viability of different cancer cells, such as HeLa, AGS, HT-29, and MCF-7. Conversely, E. lactis IW5 did not inhibit the viability of normal FHs-74 cells. This strain did not generate toxic enzymes, including β-glucosidase, β-glucuronidase, and N-acetyl-β-glucosaminidase and was highly susceptible to ampicillin, gentamycin, penicillin, vancomycin, clindamycin, sulfamethoxazol, and chloramphenicol but resistant to erythromycin and tetracyclin. This study provided evidence for the effect of E. lactis IW5 on cancer cells. Therefore, E. lactis IW5, as a bioactive therapeutics, should be subjected to other relevant tests to verify the therapeutic suitability of this strain for clinical applications.

  12. The spinal muscular atrophy gene region at 5q13.1 has a paralogous chromosomal region at 6p21.3.

    Banyer, J L; Goldwurm, S; Cullen, L; van der Griend, B; Zournazi, A; Smit, D J; Powell, L W; Jazwinska, E C


    Paralogous regions are duplicated segments of chromosomal DNA that have been acquired during the evolution of the genome. Subsequent divergent evolution of the genes within paralogous regions can lead to the formation of gene families. Here, we report the identification of a region on Chromosome (Chr) 6 at 6p21.3 that is paralogous with the Spinal Muscular Atrophy (SMA) gene region on Chr 5 at 5q13.1. Partial characterization of this region identified nine sequences all of which are highly homologous to DNA sequences of the SMA gene region at 5q13.1. These sequences include four beta-glucuronidase sequences, two retrotransposon sequences, a novel cDNA, a Sequence Tagged Site (STS), and one that is homologous to exon 9 of the Neuronal Apoptosis Inhibitor Protein (NAIP) gene. The 6p21.3 paralogous SMA region may contain genes that are related to those in the SMA region at 5q13.1; however, a direct association of this region with SMA is unlikely given that no linkage of SMA with Chr 6 has been reported.

  13. Transient transformation of the obligate biotrophic rust fungus Uromyces fabae using biolistics.

    Djulic, Alma; Schmid, Annette; Lenz, Heike; Sharma, Pia; Koch, Christin; Wirsel, Stefan G R; Voegele, Ralf T


    Obligate biotrophic pathogens like the rust fungi are important plant pathogens causing enormous losses on food, forage and biomass crops. The analysis of the molecular details underlying obligate biotrophic host-parasite interactions is mainly hampered by the fact that no system for transformation is available for most obligate biotrophic organisms. Here we report the transient transformation of Uromyces fabae, an obligate biotrophic rust fungus using a biolistic approach. Biolistic bombardment of U. fabae urediospores was used to deliver different color markers (β-glucuronidase (GUS), intron green fluorescent protein (iGFP) and red fluorescent protein (DsRed) and/or a selection marker. Endogenous regulatory elements from U. fabae plasma membrane ATPase (Uf-PMA1) were used to drive expression of the transgenes. In addition to the delivery of color markers, an in planta selection procedure using the fungicide Carboxin was established allowing the propagation of transformants. In addition to mere cytoplasmic expression of the color markers, a nuclear localization signal was fused to DsRed (pRV115-NLS) targeting the fluorescent marker protein to the nuclei. A procedure for the genetic modification of U. fabae was established. The method can be easily adapted for use with other obligate biotrophic fungi. This provides the basis for a more in depth analysis of the molecular principles governing the obligate biotrophic lifestyle. Copyright © 2011 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

  14. Relationship among Shigella spp. and enteroinvasive Escherichia coli (EIEC and their differentiation

    Abu Ud-Din


    Full Text Available Shigellosis produces inflammatory reactions and ulceration on the intestinal epithelium followed by bloody or mucoid diarrhea. It is caused by enteroinvasive E. coli (EIEC as well as any species of the genus Shigella, namely, S. dysenteriae, S. flexneri, S. boydii, and S. sonnei. This current species designation of Shigella does not specify genetic similarity. Shigella spp. could be easily differentiated from E. coli, but difficulties observed for the EIEC-Shigella differentiation as both show similar biochemical traits and can cause dysentery using the same mode of invasion. Sequencing of multiple housekeeping genes indicates that Shigella has derived on several different occasions via acquisition of the transferable forms of ancestral virulence plasmids within commensal E. coli and form a Shigella-EIEC pathovar. EIEC showed lower expression of virulence genes compared to Shigella, hence EIEC produce less severe disease than Shigella spp. Conventional microbiological techniques often lead to confusing results concerning the discrimination between EIEC and Shigella spp. The lactose permease gene (lacY is present in all E. coli strains but absent in Shigella spp., whereas β-glucuronidase gene (uidA is present in both E. coli and Shigella spp. Thus uidA gene and lacY gene based duplex real-time PCR assay could be used for easy identification and differentiation of Shigella spp. from E. coli and in particular EIEC.

  15. Zinc triggers a complex transcriptional and post-transcriptional regulation of the metal homeostasis gene FRD3 in Arabidopsis relatives.

    Charlier, Jean-Benoit; Polese, Catherine; Nouet, Cécile; Carnol, Monique; Bosman, Bernard; Krämer, Ute; Motte, Patrick; Hanikenne, Marc


    In Arabidopsis thaliana, FRD3 (FERRIC CHELATE REDUCTASE DEFECTIVE 3) plays a central role in metal homeostasis. FRD3 is among a set of metal homeostasis genes that are constitutively highly expressed in roots and shoots of Arabidopsis halleri, a zinc hyperaccumulating and hypertolerant species. Here, we examined the regulation of FRD3 by zinc in both species to shed light on the evolutionary processes underlying the evolution of hyperaccumulation in A. halleri. We combined gene expression studies with the use of β-glucuronidase and green fluorescent protein reporter constructs to compare the expression profile and transcriptional and post-transcriptional regulation of FRD3 in both species. The AtFRD3 and AhFRD3 genes displayed a conserved expression profile. In A. thaliana, alternative transcription initiation sites from two promoters determined transcript variants that were differentially regulated by zinc supply in roots and shoots to favour the most highly translated variant under zinc-excess conditions. In A. halleri, a single transcript variant with higher transcript stability and enhanced translation has been maintained. The FRD3 gene thus undergoes complex transcriptional and post-transcriptional regulation in Arabidopsis relatives. Our study reveals that a diverse set of mechanisms underlie increased gene dosage in the A. halleri lineage and illustrates how an environmental challenge can alter gene regulation. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology.

  16. The 5' untranslated region of the VR-ACS1 mRNA acts as a strong translational enhancer in plants.

    Wever, Willem; McCallum, Emily J; Chakravorty, David; Cazzonelli, Christopher I; Botella, José R


    The structure and function of untranslated mRNA leader sequences and their role in controlling gene expression remains poorly understood. Previous research has suggested that the 5' untranslated region (5'UTR) of the Vigna radiata aminocyclopropane-1-carboxylate synthase synthase (VR-ACS1) gene may function as a translational enhancer in plants. To test such hypothesis we compared the translation enhancing properties of three different 5'UTRs; those from the VR-ACS1, the chlorophyll a/b binding gene from petunia (Cab22L; a known translational enhancer) and the Vigna radiata pectinacetylesterase gene (PAE; used as control). Identical constructs in which the coding region of the beta-glucuronidase (GUS) gene was fused to each of the three 5'UTRs and placed under the control of the cauliflower mosaic virus 35S promoter were prepared. Transient expression assays in tobacco cell cultures and mung bean leaves showed that the VR-ACS1 and Cab22L 5'UTRs directed higher levels of GUS activity than the PAE 5'UTR. Analysis of transgenic Arabidopsis thaliana seedlings, as well as different tissues from mature plants, confirmed that while transcript levels were equivalent for all constructs, the 5'UTRs from the VR-ACS1 and Cab22L genes can increase GUS activity twofold to fivefold compared to the PAE 5'UTR, therefore confirming the translational enhancing properties of the VR-ACS1 5'UTR.

  17. Compost Grown Agaricus bisporus Lacks the Ability to Degrade and Consume Highly Substituted Xylan Fragments.

    Jurak, Edita; Patyshakuliyeva, Aleksandrina; de Vries, Ronald P; Gruppen, Harry; Kabel, Mirjam A


    The fungus Agaricus bisporus is commercially grown for the production of edible mushrooms. This cultivation occurs on compost, but not all of this substrate is consumed by the fungus. To determine why certain fractions remain unused, carbohydrate degrading enzymes, water-extracted from mushroom-grown compost at different stages of mycelium growth and fruiting body formation, were analyzed for their ability to degrade a range of polysaccharides. Mainly endo-xylanase, endo-glucanase, β-xylosidase and β-glucanase activities were determined in the compost extracts obtained during mushroom growth. Interestingly, arabinofuranosidase activity able to remove arabinosyl residues from doubly substituted xylose residues and α-glucuronidase activity were not detected in the compost enzyme extracts. This correlates with the observed accumulation of arabinosyl and glucuronic acid substituents on the xylan backbone in the compost towards the end of the cultivation. Hence, it was concluded that compost grown A. bisporus lacks the ability to degrade and consume highly substituted xylan fragments.

  18. Definitive Drug and Metabolite Screening in Urine by UPLC-MS-MS Using a Novel Calibration Technique.

    Rosano, Thomas G; Ohouo, Patrice Y; LeQue, John J; Freeto, Scott M; Wood, Michelle


    Drug screening is an essential analytical tool for detection of therapeutic, illicit and emerging drug use. Presumptive immunoassay screening is widely used, while initial definitive testing by chromatography-coupled mass spectrometry is hampered due to complex pre-analysis steps, long chromatography time and matrix effects. The aim of this study is to develop and validate a definitive test for rapid and threshold accurate screening of 33 drugs or metabolites (analytes) in urine. Sample preparation in a 96-well plate format involves rapid glucuronidase hydrolysis followed by dilution, filtration and ultra-performance liquid chromatography-MS-MS analysis. Chromatographic separation, on an ACQUITY UPLC(®) BEH phenyl column is optimized for a 3-min MS-MS ion acquisition. Matrix effect was normalized by an innovative technique called threshold accurate calibration employing an additional analysis with an analyte spike as an internal standard undergoing the same matrix effect as an analyte in a drug-positive donor specimen. Accuracy and precision, at above and below threshold concentrations, were determined by replicate analysis of control urine pools containing 50, 75, 125 and 150% of threshold concentrations. Accuracy and selectivity were further demonstrated by concordant findings in proficiency and confirmatory testing. The study shows the applicability of definitive testing as an alternative to immunoassay screening and demonstrates a new approach to normalization of matrix effect.

  19. Functional Characterization of a Putative Nitrate Transporter Gene Promoter from Rice

    Ting-Zhang HU; Kai-Ming CAO; Mian XIA; Xi-Ping WANG


    Drought is one of the most significant abiotic stresses that influence plant growth and development. Expression analysis revealed that OsNRT1.3, a putative nitrate transporter gene in rice, was induced by drought. To confirm if the OsNRT1.3 promoter can respond to drought stress, a 2019 bp upstream sequence of OsNRT1.3 was cloned. Three OsNRT1.3 promoter fragments were generated by 5'-deletion, and fused to the β-glucuronidase (GUS) gene. The chimeric genes were introduced into rice plants. NRT2019::GUS, NRT1196: :GUS and NRT719::GUS showed similar expression patterns in seeds,roots, leaves and flowers in all transgenic rice, and GUS activity conferred by different OsNRT1.3 promoter fragments was significantly upregulated by drought stress, indicating that OsNRT1.3 promoter responds to drought stress and the 719 bp upstream sequence of OsNRT1.3 contains the drought response elements.

  20. Preventive effects of p-coumaric acid on lysosomal dysfunction and myocardial infarct size in experimentally induced myocardial infarction.

    Jyoti Roy, Abhro; Stanely Mainzen Prince, P


    The present study was designed to evaluate the preventive effects of p-coumaric acid on lysosomal dysfunction and myocardial infarct size in isoproterenol induced myocardial infarcted rats. Male albino Wistar rats were pretreated with p-coumaric acid (8 mg/kg body weight) daily for a period of 7 days after which isoproterenol (100mg/kg body weight) was injected subcutaneously into rats twice at an interval of 24h (8th and 9th day).The activity/levels of serum cardiac diagnostic markers, heart lysosomal lipid peroxidation products and the activities of lysosomal enzymes (β-glucuronidase, β-galactosidase, cathepsin-B and cathepsin-D) were significantly (Plysosomal fraction. The pretreatment with p-coumaric acid significantly (Plysosomal lipid peroxidation products and the activities of lysosomal enzymes. In addition, p-coumaric acid greatly reduced myocardial infarct size. p-Coumaric acid pretreatment (8 mg/kg body weight) to normal rats did not show any significant effect. Thus, this study showed that p-coumaric acid prevents lysosomal dysfunction against cardiac damage induced by isoproterenol and brings back the levels of lipid peroxidation products and activities of lysosomal enzymes to near normal levels. The in vitro study also revealed the free radical scavenging activity of p-coumaric acid. Thus, the observed effects are due to p-coumaric acid's free radical scavenging and membrane stabilizing properties.

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

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


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

  2. Degradation of Opioids and Opiates During Acid Hydrolysis Leads to Reduced Recovery Compared to Enzymatic Hydrolysis.

    Sitasuwan, Pongkwan; Melendez, Cathleen; Marinova, Margarita; Mastrianni, Kaylee R; Darragh, Alicia; Ryan, Emily; Lee, L Andrew


    Drug monitoring laboratories utilize a hydrolysis process to liberate the opiates from their glucuronide conjugates to facilitate their detection by tandem mass spectrometry (MS). Both acid and enzyme hydrolysis have been reported as viable methods, with the former as a more effective process for recovering codeine-6-glucuronide and morphine-6-glucuronide. Here, we report concerns with acid-catalyzed hydrolysis of opioids, including a significant loss of analytes and conversions of oxycodone to oxymorphone, hydrocodone to hydromorphone and codeine to morphine. The acid-catalyzed reaction was monitored in neat water and patient urine samples by liquid chromatography-time-of-flight and tandem MS. These side reactions with acid hydrolysis may limit accurate quantitation due to loss of analytes, possibly lead to false positives, and poorly correlate with pharmacogenetic profiles, as cytochrome P450 enzyme (CYP2D6) is often involved with oxycodone to oxymorphone, hydrocodone to hydromorphone and codeine to morphine conversions. Enzymatic hydrolysis process using the purified, genetically engineered β-glucuronidase (IMCSzyme(®)) addresses many of these concerns and demonstrates accurate quantitation and high recoveries for oxycodone, hydrocodone, oxymorphone and hydromorphone.

  3. Production and purification of two recombinant proteins from transgenic corn.

    Kusnadi, A R; Hood, E E; Witcher, D R; Howard, J A; Nikolov, Z L


    This study reports the production, purification, and characterization of recombinant Escherichia coli beta-glucuronidase (GUS) and chicken egg-white avidin from transgenic corn seed. The avidin and gus genes were stably integrated in the genome and expressed over seven generations. The accumulation levels of avidin and GUS in corn kernel were 5.7% and 0.7% of extractable protein, respectively. Within the kernel, avidin and GUS accumulation was mainly localized to the germ, indicating possible tissue preference of the ubiquitin promoter. The storage-stability studies demonstrated that processed transgenic seed containing GUS or avidin can be stored at 10 degrees C for at least 3 months and at 25 degrees C for up to 2 weeks without a significant loss of activity. The heat-stability experiments indicated that GUS and avidin in the whole kernels were stable at 50 degrees C for up to 1 week. The buffer composition also had an affect on the aqueous extraction of avidin and GUS from ground kernels. Avidin was purified in one step by using 2-iminobiotin agarose, whereas GUS was purified in four steps consisting of adsorption, ion-exchange, hydrophobic interaction, and size-exclusion chromatography. Biochemical properties of purified avidin and GUS were similar to those of the respective native proteins.

  4. Gingival crevicular fluid in the diagnosis of periodontal and systemic diseases

    Čakić Saša


    Full Text Available Gingival crevicular fluid (GCF can be found in the physiologic space (gingival sulcus, as well as in the pathological space (gingival pocket or periodontal pocket between the gums and teeth. In the first case it is a transudate, in the second an exudate. The constituents of GCF originate from serum, gingival tissues, and from both bacterial and host response cells present in the aforementioned spaces and the surrounding tissues. The collection and analysis of GCF are the noninvasive methods for the evaluation of host response in periodontal disease. These analyses mainly focus on inflammatory markers, such as prostaglandin E2, neutrophil elastase and β-glucuronidase, and on the marker of cellular necrosis - aspartat aminotransferase. Further, the analysis of inflammatory markers in the GCF may assist in defining how certain systemic diseases (e.g., diabetes mellitus can modify periodontal disease, and how peridontal disease can influence certain systemic disorders (atherosclerosis, preterm delivery, diabetes mellitus and some chronic respiratory diseases. Major factors which influence the results obtained from the analyses of GCF are not only the methods of these analyses, but the method of GCF collection as well. As saliva collection is less technique-sensitive than GCF collection, some constituents of saliva which originate from the GCF can be analyzed as more amenable to chairside utilization.

  5. Inflammatory biomarkers in saliva: assessing the strength of association of diabetes mellitus and periodontal status with the oral inflammatory burden.

    Yoon, Angela J; Cheng, Bin; Philipone, Elizabeth; Turner, Ryan; Lamster, Ira B


    To determine the strength of association of type 2 diabetes mellitus and periodontal disease with the oral inflammatory burden, as assessed by markers of inflammation in saliva. Unstimulated saliva samples were collected from 192 subjects with or without type 2 diabetes. β-glucuronidase (βG) was measured via a fluorometric array and interlukin-1β (IL-1β) via enzyme-linked immunosorbent assay. The concentration of both mediators was evaluated in relationship to clinical parameters, severity of periodontal disease and diabetes status. Regression analysis demonstrated that diabetes and periodontal disease was independently and positively correlated with increased concentration of βG in saliva (p periodontal disease with the level of βG in saliva was greater than the strength of association of the diabetic status. IL-1β concentration in saliva was primarily associated with the severity of periodontal disease (p diabetes (p = 0.50). This study examined the nature of the inflammatory response in the oral cavity as assessed by inflammatory markers in saliva. Both periodontal disease and diabetes mellitus were independently associated with the oral inflammatory burden, in which the effect of periodontal disease was more pronounced. © 2012 John Wiley & Sons A/S.

  6. Physcomitrella patens Activates Defense Responses against the Pathogen Colletotrichum gloeosporioides

    Guillermo Reboledo


    Full Text Available The moss Physcomitrella patens is a suitable model plant to analyze the activation of defense mechanisms after pathogen assault. In this study, we show that Colletotrichum gloeosporioides isolated from symptomatic citrus fruit infects P. patens and cause disease symptoms evidenced by browning and maceration of tissues. After C. gloeosporioides infection, P. patens reinforces the cell wall by the incorporation of phenolic compounds and induces the expression of a Dirigent-protein-like encoding gene that could lead to the formation of lignin-like polymers. C. gloeosporioides-inoculated protonemal cells show cytoplasmic collapse, browning of chloroplasts and modifications of the cell wall. Chloroplasts relocate in cells of infected tissues toward the initially infected C. gloeosporioides cells. P. patens also induces the expression of the defense genes PAL and CHS after fungal colonization. P. patens reporter lines harboring the auxin-inducible promoter from soybean (GmGH3 fused to β-glucuronidase revealed an auxin response in protonemal tissues, cauloids and leaves of C. gloeosporioides-infected moss tissues, indicating the activation of auxin signaling. Thus, P. patens is an interesting plant to gain insight into defense mechanisms that have evolved in primitive land plants to cope with microbial pathogens.

  7. Effect of experimental diabetes and insulin replacement on intestinal metabolism and excretion of 4-nitrophenol in rats.

    Fischer, Emil; Almási, Attila; Bojcsev, Sztojan; Fischer, Tamás; Kovács, Noémi Piroska; Perjési, Pál


    Luminal appearance of 4-nitrophenol (PNP) metabolites (4-nitrophenol-β-glucuronide (PNP-G) and 4-nitrophenol-sulfate (PNP-S)) and activity of the related metabolic enzymes have been investigated in control and experimental diabetic rats. Experimental diabetes was induced by administration of streptozotocin (65 mg/kg i.v.). PNP (500 μmol/L) was luminally perfused in the small intestine and the metabolites were determined in the perfusion solution. Effect of insulin replacement was also investigated in the diabetic rats. It was found that experimental diabetes increased the luminal appearance of PNP-G, which could be completely compensated by rapid-acting insulin administration (1 U/kg i.v.). Activities of the enzymes involved in PNP-G production (UDP-glucuronyltransferase and β-glucuronidase) were also elevated; however, these changes were only partially compensated by insulin. Luminal appearance of PNP-S was not significantly changed by administration of streptozotocin and insulin. Activities of the enzymes of PNP-S production (sulfotransferases and arylsulfatases) did not change in the diabetic rats. The results indicate that experimental diabetes can provoke changes in intestinal drug metabolism. It increased intestinal glucuronidation of PNP but did not influence sulfate conjugation. No direct correlation was found between the changes of metabolic enzyme activities and the luminal appearance of the metabolites.

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

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


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

  9. Tobacco Transcription Factor NtWRKY12 Interacts With TGA2.2 in vitro and in vivo

    Marcel evan Verk


    Full Text Available The promoter of the salicylic acid-inducible PR-1a gene of Nicotiana tabacum contains binding sites for transcription factor NtWRKY12 (WK-box at position -564 and TGA factors (as-1-like element at position -592. Transactivation experiments in Arabidopsis protoplasts derived from wild type, npr1-1, tga256 and tga2356 mutant plants revealed that NtWRKY12 alone was able to induce a PR-1a::β-glucuronidase (GUS reporter gene to high levels, independent of co-expressed tobacco NtNPR1, TGA2.1, TGA2.2 or endogenous Arabidopsis NPR1, TGA2/3/5/6. By in vitro pull-down assays with GST and Strep fusion proteins and by Fluorescence Resonance Energy Transfer assays with protein-CFP and protein-YFP fusions in transfected protoplasts, it was shown that NtWRKY12 and TGA2.2 could interact in vitro and in vivo. Interaction of NtWRKY12 with TGA1a or TGA2.1 was not detectable by these techniques. A possible mechanism for the role of NtWRKY12 and TGA2.2 in PR-1a gene expression is discussed.

  10. Expression of anti-tumor necrosis factor alpha (TNFα) single-chain variable fragment (scFv) in Spirodela punctata plants transformed with Agrobacterium tumefaciens.

    Balaji, Parthasarathy; Satheeshkumar, P K; Venkataraman, Krishnan; Vijayalakshmi, M A


    Therapeutic antibodies against tumor necrosis factor alpha (TNFα) have been considered effective for some of the autoimmune diseases such as rheumatoid arthritis, Crohn's diseases, and so on. But associated limitations of the current therapeutics in terms of cost, availability, and immunogenicity have necessitated the need for alternative candidates. Single-chain variable fragment (scFv) can negate the limitations tagged with the anti-TNFα therapeutics to a greater extent. In the present study, Spirodela punctata plants were transformed with anti-TNFα through in planta transformation using Agrobacterium tumefaciens strain, EHA105. Instead of cefotaxime, garlic extract (1 mg/mL) was used to remove the agrobacterial cells after cocultivation. To the best of our knowledge, this report shows for the first time the application of plant extracts in transgenic plant development. 95% of the plants survived screening under hygromycin. ScFv cDNA integration in the plant genomic DNA was confirmed at the molecular level by PCR. The transgenic protein expression was followed up to 10 months. Expression of scFv was confirmed by immunodot blot. Protein expression levels of up to 6.3% of total soluble protein were observed. β-Glucuronidase and green fluorescent protein expressions were also detected in the antibiotic resistant plants. The paper shows the generation of transgenic Spirodela punctuata plants through in planta transformation.

  11. A Proximal Promoter Region of Arabidopsis DREB2C Confers Tissue-specific Expression under Heat Stress

    Huan Chen; Jihyun Je; Chieun Song; Jung Eun Hwang; Chae Oh Lim


    The dehydration-responsive element-binding factor 2C (DREB2C) is a member of the CBF/DREB subfamily of proteins,which contains a single APETALA2/Ethylene responsive element-binding factor (AP2/ERF)domain.To identify the expression pattern of the DREB2C gene,which contains multiple transcription cis-regulatory elements in its promoter,an approximately 1.4 kb upstream DREB2C sequence was fused to the β-glucuronidase reporter gene (GUS) and the recombinant p1244 construct was transformed into Arabidopsis thaliana (L.) Heynh.The promoter of the gene directed prominent GUS activity in the vasculature in diverse young dividing tissues.Upon applying heat stress (HS),GUS staining was also enhanced in the vasculature of the growing tissues.Analysis of a series of 5'-deletions of the DREB2C promoter revealed that a proximal upstream sequence sufficient for the tissue-specific spatial and temporal induction of GUS expression by HS is localized in the promoter region between -204 and -34 bps relative to the transcriptional start site.Furthermore,electrophoretic mobility shift assay (EMSA) demonstrated that nuclear protein binding activities specific to a -120 to -32 bp promoter fragment increased after HS.These results indicate that the TATA-proximal region and some latent trans-acting factors may cooperate in HS-induced activation of the Arabidopsis DREB2C promoter.

  12. Hemicellulases from the ethanologenic thermophile, Thermoanaerobacter ethanolicus and related anaerobic thermophiles. Final report, September 1992--June 1996

    Wiegel, J.


    The short term goals of this application were to characterize hemicellulases from anaerobic thermophiles on the biochemical and molecular level to extend the presently limited knowledge of hemicellulases in anaerobic thermophilic bacteria. This objective includes the following tasks: (1) Traditional purification and biochemical/biophysical characterization of xylanases from the newly isolated, slightly alkalitolerant strain NDF190, and the slightly acid-tolerant strain YS485, both with high xylanolytic activities, and of the 4-O-methyl glucuronidase and arabinosidase from strain NDF190 and the acetyl (xylan) esterase from T. ethanolicus. This also includes determining the N-terminal sequences and obtaining gene probes. (2) Elucidation of the regulation of hemicellulolytic enzymes in anaerobic thermophiles. (3) To clone into E. coli and identify the multiplicity of the enzymes involved in hemicellulose degradation by T. ethanolicus and other suitable organisms. (4) To purify and characterize the recombinant enzymes with the goal of identifying the best enzymes for cloning into the ethanologenic T. ethanolicus to obtain an optimized hemicellulose utilization by this bacterium.

  13. Hemicellulases from the ethanologenic thermophile Thermoanaerobacter ethanolicus and related anaerobic thermophiles. Final report, September 1992--June 1996

    Wiegel, J.


    The SHORT TERM GOALS of this application were to characterize hemicellulases from anaerobic thermophiles on the biochemical and molecular level to extend the presently limited knowledge of hemicellulases in anaerobic thermophilic bacteria. This objective includes the following TASKS: (1) Traditional purification and biochemical/biophysical characterization of xylanases from the newly isolated, slightly alkalitolerant strain NDF190, and the slightly acid-tolerant strain YS485, both with high xylanolytic activities, and of the 4-0-methyl glucuronidase and arabinosidase from strain NDF190 and the acetyl (xylan) esterase from T. ethanolicus. This also includes determining the N-terminal sequences and obtaining gene probes. (2) Elucidation of the regulation of hemicellulolytic enzymes in anaerobic thermophiles. (3) To clone into E. coli and identify the multiplicity of the enzymes involved in hemicellulose degradation by T. ethanolicus and other suitable organisms. (4) To purify and characterize the recombinant enzymes with the goal of identifying the best enzymes for cloning into the ethanologenic T. ethanolicus to obtain an optimized hemicellulose utilization by this bacterium (one of our long term goals).

  14. Silencing and heterologous expression of ppo-2 indicate a specific function of a single polyphenol oxidase isoform in resistance of dandelion (Taraxacum officinale) against Pseudomonas syringae pv. tomato.

    Richter, Carolin; Dirks, Mareike E; Gronover, Christian Schulze; Prüfer, Dirk; Moerschbacher, Bruno M


    Dandelion (Taraxacum officinale) possesses an unusually high degree of disease resistance. As this plant exhibits high polyphenol oxidase (PPO) activity and PPO have been implicated in resistance against pests and pathogens, we analyzed the potential involvement of five PPO isoenzymes in the resistance of dandelion against Botrytis cinerea and Pseudomonas syringae pv. tomato. Only one PPO (ppo-2) was induced during infection, and ppo-2 promoter and β-glucuronidase marker gene fusions revealed strong induction of the gene surrounding lesions induced by B. cinerea. Specific RNAi silencing reduced ppo-2 expression only, and concomitantly increased plant susceptibility to P. syringae pv. tomato. At 4 days postinoculation, P. syringae pv. tomato populations were strongly increased in the ppo-2 RNAi lines compared with wild-type plants. When the dandelion ppo-2 gene was expressed in Arabidopsis thaliana, a plant having no PPO gene, active protein was formed and protein extracts of the transgenic plants exhibited substrate-dependent antimicrobial activity against P. syringae pv. tomato. These results clearly indicate a strong contribution of a specific, single PPO isoform to disease resistance. Therefore, we propose that specific PPO isoenzymes be included in a new family of pathogenesis-related (PR) proteins.

  15. Efficient gusA Transient Expression in Porphyra yezoensis Protoplasts Mediated by Endogenous Beta-tubulin Flanking Sequences

    GONG Qianhong; YU Wengong; DAI Jixun; LIU Hongquan; XU Rifu; GUAN Huashi; PAN Kehou


    Endogenous tubulin promoter has been widely used for expressing foreign genes in green algae, but the efficiency and feasibility of endogenous tubulin promoter in the economically important Porphyra yezoensis (Rhodophyta) are tmknown. In this study, the flanking sequences of beta-tubulin gene from P. yezoensis were amplified and two transient expression vectors were constructed to determine their transcription promoting feasibility for foreign gene gusA. The testing vector pATubGUS was constructed by inserting 5'- and 3'-flanking regions (Tub5'and Tub3') up- and down-stream of β-glucuronidase (GUS) gene (gusA), respectively,into pA, a derivative of pCAT(R)3-enhancer vector. The control construct, pAGUSTub3, contains only gusA and Tub3 '. These constructs were electroporated into P. yezoensis protoplasts and the GUS activities were quantitatively analyzed by spectrometry. The results demonstrated that gusA gene was efficiently expressed in P. yezoensis protoplasts under the regulation of 5'-flanking sequence of the beta-tubulin gene. More interestingly, the pATubGUS produced stronger GUS activity in P. yezoensis protoplasts when compared to the result from pBI221, in which the gusA gene was directed by a constitutive CaMV 35 S promoter. The data suggest that the integration of P. yezoensis protoplast and its endogenous beta-tubulin flanking sequences is a potential novel system for foreign gene expression.

  16. Visualized and precise design of artificial small RNAs for regulating T7 RNA polymerase and enhancing recombinant protein folding in Escherichia coli

    Yujia Zhao


    Full Text Available Small non-coding RNAs (sRNAs have received much attention in recent years due to their unique biological properties, which can efficiently and specifically tune target gene expressions in bacteria. Inspired by natural sRNAs, recent works have proposed the use of artificial sRNAs (asRNAs as genetic tools to regulate desired gene that has been applied in several fields, such as metabolic engineering and bacterial physiology studies. However, the rational design of asRNAs is still a challenge. In this study, we proposed structure and length as two criteria to implement rational visualized and precise design of asRNAs. T7 expression system was one of the most useful recombinant protein expression systems. However, it was deeply limited by the formation of inclusion body. To settle this problem, we designed a series of asRNAs to inhibit the T7 RNA polymerase (Gene1 expression to balance the rate between transcription and folding of recombinant protein. Based on the heterologous expression of Aspergillus oryzae Li-3 glucuronidase in E. coli, the asRNA-antigene1-17bp can effectively decrease the inclusion body and increase the enzyme activity by 169.9%.

  17. A Novel R2R3-MYB Transcription Factor BpMYB106 of Birch (Betula platyphylla) Confers Increased Photosynthesis and Growth Rate through Up-regulating Photosynthetic Gene Expression.

    Zhou, Chenguang; Li, Chenghao


    We isolated a R2R3-MYB transcription factor BpMYB106, which regulates photosynthesis in birch (Betula platyphylla Suk.). BpMYB106 mainly expresses in the leaf and shoot tip of birch, and its protein is localized in the nucleus. We further fused isolated a 1588 bp promoter of BpMYB106 and analyzed it by PLACE, which showed some cis-acting elements related to photosynthesis. BpMYB106 promoter β-glucuronidase (GUS) reporter fusion studies gene, the result, showed the GUS reporter gene in transgenic birch with BpMYB106 promoter showed strong activities in shoot tip, cotyledon margins, and mature leaf trichomes. The overexpression of BpMYB106 in birch resulted in significantly increased trichome density, net photosynthetic rate, and growth rate as compared with the wild-type birch. RNA-Seq profiling revealed the upregulation of several photosynthesis-related genes in the photosynthesis and oxidative phosphorylation pathways in the leaves of transgenic plants. Yeast one-hybrid analysis, coupled with transient assay in tobacco, revealed that BpMYB106 binds a MYB binding site MYB2 in differentially expressed gene promoters. Thus, BpMYB106 may directly activate the expression of a range of photosynthesis related genes through interacting with the MYB2 element in their promoters. Our study demonstrating the overexpression of BpMYB106-a R2R3-MYB transcription factor-upregulates the genes of the photosynthesis and oxidative phosphorylation pathways to improve photosynthesis.

  18. A novel soybean intrinsic protein gene, GmTIP2;3, involved in responding to osmotic stress

    Da Yong eZhang


    Full Text Available Water is essential for plant growth and development. Water deficiency leads to loss of yield and decreased crop quality. To understand water transport mechanisms in plants, we cloned and characterized a novel tonoplast intrinsic protein (TIP gene from soybean with the highest similarity to TIP2-type from other plants, thus designated GmTIP2;3. The protein sequence contains two conserved NPA motifs and six transmembrane domains. The expression analysis indicated that this gene was constitutively expressed in all detected tissues, with higher levels in the root, stem and pod, and the accumulation of GmTIP2;3 transcript showed a significant response to osmotic stresses, including 20% PEG6000 (polyethylene glycol and 100 µM ABA (abscisic acid treatments. The promoter-GUS (glucuronidase activity analysis suggested that GmTIP2;3 was also expressed in the root, stem and leaf and preferentially expressed in the stele of root and stem, and the core promoter region was 1000 bp in length, located upstream of the ATG start codon. The GUS tissue and induced expression observations were consistent with the findings in soybean. In addition, subcellular localization showed that GmTIP2;3 was a plasma membrane-localized protein. Yeast heterologous expression revealed that GmTIP2;3 could improve tolerance to osmotic stress in yeast cells. Integrating these results, GmTIP2;3 might play an important role in response to osmotic stress in plants .

  19. Encapsulated Whole Bone Marrow Cells Improve Survival in Wistar Rats after 90% Partial Hepatectomy

    Carolina Uribe-Cruz


    Full Text Available Background and Aims. The use of bone marrow cells has been suggested as an alternative treatment for acute liver failure. In this study, we investigate the effect of encapsulated whole bone marrow cells in a liver failure model. Methods. Encapsulated cells or empty capsules were implanted in rats submitted to 90% partial hepatectomy. The survival rate was assessed. Another group was euthanized at 6, 12, 24, 48, and 72 hours after hepatectomy to study expression of cytokines and growth factors. Results. Whole bone marrow group showed a higher than 10 days survival rate compared to empty capsules group. Gene expression related to early phase of liver regeneration at 6 hours after hepatectomy was decreased in encapsulated cells group, whereas genes related to regeneration were increased at 12, 24, and 48 hours. Whole bone marrow group showed lower regeneration rate at 72 hours and higher expression and activity of caspase 3. In contrast, lysosomal-β-glucuronidase activity was elevated in empty capsules group. Conclusions. The results show that encapsulated whole bone marrow cells reduce the expression of genes involved in liver regeneration and increase those responsible for ending hepatocyte division. In addition, these cells favor apoptotic cell death and decrease necrosis, thus increasing survival.

  20. The effects of co-administration of butter on the absorption, metabolism and excretion of catechins in rats after oral administration of tea polyphenols.

    Zhang, Liang; Han, Yuhui; Xu, Liwei; Liang, Yuhong; Chen, Xin; Li, Junsong; Wan, Xiaochun


    In Southwest China, tea polyphenols are usually utilized by way of butter tea. Tea polyphenols inhibit the absorption and biosynthesis of fatty acids in vivo, but the effects of butter on the pharmacokinetics of tea polyphenols have drawn less concern. A rapid UHPLC-MS/MS method was used to quantitatively determine the catechins in the plasma, feces and bile of rats after the oral administration of tea polyphenol or its combination with butter. In comparison with the single tea polyphenol treatment, the maximum plasma concentrations (Cmax) of the free EGCG, EGC, EC, GCG, GC and ECG significantly decreased after the co-administration of butter. The mean residence times (MRT) of the free EGCG, EGC, EC, GC and ECG were also significantly prolonged. When the plasma samples were treated with β-glucuronidase and arylsulfatase, the pharmacokinetic parameters of the total catechins (free and conjugated forms) were not affected by the co-administration of butter. These results indicated that the total absorption of catechins was not affected by butter, but the metabolism of catechins had been changed. Furthermore, the fecal catechins were significantly increased by butter. The total fecal amount and excretion ratio of all catechins were increased highly. The biliary excretion of EGCG, EGC, EC, GCG and GC was significantly increased by the co-administration of butter. To sum up, the butter changed the metabolism of catechins in vivo by decreasing the plasma concentration of the free catechins but increasing the conjugated catechins.

  1. Development of a UFLC-MS/MS method for the simultaneous determination of seven tea catechins in rat plasma and its application to a pharmacokinetic study after administration of green tea extract.

    Huo, Yanshuang; Zhang, Qian; Li, Qing; Geng, Bingjie; Bi, Kaishun


    A rapid, sensitive and selective ultra-fast liquid chromatography-tandem mass spectrometry (UFLC-MS/MS) method was developed and validated for simultaneous determination of seven green tea catechins including catechin (C), (-)-epicatechin (EC), gallocatechin (GC), (-)-epigallocatechin (EGC), (-)-epicatechin-3-gallate (ECG), gallocatechin-3-gallate (GCG) and (-)-epigallocatechin-3-gallate (EGCG) in rat plasma. The plasma samples were firstly hydrolysed with the mixture of β-glucuronidase and sulfatase, which were then extracted by liquid-liquid extraction with ethyl acetate-isopropanol (1:1, v/v). The analytes were separated on a Venusil MP C18 column (Venusil, China) with a gradient elution at a flow rate of 0.4mL/min. The detection was performed in negative ionization and multiple reaction monitoring (MRM) mode. All the calibration curves exhibited good linearity (r>0.9943) with intra- and inter-day precisions of less than 14.3% and the accuracy deviations ranging from -8.8% to 7.5%. The extraction recoveries of the analytes and ethyl gallate (internal standard) were all more than 72%. The validated method was successfully applied to a pharmacokinetic study of seven catechins in rat plasma after oral administration of the green tea extract at different doses of 0.4, 1.2 and 2.0g/kg.

  2. A putative colR(XC1049)-colS(XC1050) two-component signal transduction system in Xanthomonas campestris positively regulates hrpC and hrpE operons and is involved in virulence, the hypersensitive response and tolerance to various stresses.

    Zhang, Sui-Sheng; He, Yong-Qiang; Xu, Li-Ming; Chen, Bo-Wen; Jiang, Bo-Le; Liao, Jie; Cao, Jin-Rui; Liu, Dan; Huang, Yan-Qiang; Liang, Xiao-Xia; Tang, Dong-Jie; Lu, Guang-Tao; Tang, Ji-Liang


    The ColR-ColS two-component signal transduction system was originally characterized as a regulatory system involved in the capacity of root-colonizing biocontrol bacterium Pseudomonas fluorescens to colonize plant roots. There are three pairs of putative colR-colS two-component regulatory systems annotated in the phytopathogen Xanthomonas campestris pathovar campestris. Mutational studies revealed that one of them, named colR(XC1049) and colS(XC1050), is a global regulatory system involved in various cellular processes, including virulence, hypersensitive response and stress tolerance. Growth rate determination showed that, although the colR(XC1049) and colS(XC1050) mutants are not auxotrophic, colR(XC1049) and colS(XC1050) are required for the pathogen to proliferate well in standard media and host plants. Assays of beta-glucuronidase activities of plasmid-driven promoter-gusA reporters and/or semi-quantitative RT-PCR demonstrated that colR(XC1049) and colS(XC1050) positively regulate expression of hrpC and hrpE operons, and that expression of colR(XC1049) and colS(XC1050) is not controlled by key hrp regulators HrpG and HrpX.

  3. Determination of two mebeverine metabolites, mebeverine alcohol and desmethylmebeverine alcohol, in human plasma by a dual stable isotope-based gas chromatographic-mass spectrometric method.

    Tulich, L J; Randall, J L; Kelm, G R; Wehmeyer, K R


    A dual stable isotope-based GC-MS method was developed for the simultaneous determination of two metabolites of mebeverine, mebeverine alcohol and desmethylmebeverine alcohol, in human plasma. Plasma samples were treated with beta-glucuronidase to cleave the glucuronide conjugates of both compounds prior to analysis. The treated plasma was prepared for analysis by solid-phase extraction using octadecylsilane cartridges. The isolated metabolites were derivatized and analyzed by GC-MS using selected-ion monitoring. Plots of peak-area ratio were linear with metabolite concentration from 2 to 200 ng/ml and the limit of detection for both metabolites was 0.5 ng/ml. The GC-MS methodology was applied to the analysis of plasma from human subjects following peroral administration of mebeverine. Pharmacokinetic parameters for both metabolites were determined and suggest that relative systemic mebeverine exposure may potentially be assessed using metabolite kinetics, if the latter subsequently are demonstrated to be linear with mebeverine dose.

  4. POPCORN functions in the auxin pathway to regulate embryonic body plan and meristem organization in Arabidopsis.

    Xiang, Daoquan; Yang, Hui; Venglat, Prakash; Cao, Yongguo; Wen, Rui; Ren, Maozhi; Stone, Sandra; Wang, Edwin; Wang, Hong; Xiao, Wei; Weijers, Dolf; Berleth, Thomas; Laux, Thomas; Selvaraj, Gopalan; Datla, Raju


    The shoot and root apical meristems (SAM and RAM) formed during embryogenesis are crucial for postembryonic plant development. We report the identification of POPCORN (PCN), a gene required for embryo development and meristem organization in Arabidopsis thaliana. Map-based cloning revealed that PCN encodes a WD-40 protein expressed both during embryo development and postembryonically in the SAM and RAM. The two pcn alleles identified in this study are temperature sensitive, showing defective embryo development when grown at 22°C that is rescued when grown at 29°C. In pcn mutants, meristem-specific expression of WUSCHEL (WUS), CLAVATA3, and WUSCHEL-RELATED HOMEOBOX5 is not maintained; SHOOTMERISTEMLESS, BODENLOS (BDL) and MONOPTEROS (MP) are misexpressed. Several findings link PCN to auxin signaling and meristem function: ectopic expression of DR5(rev):green fluorescent protein (GFP), pBDL:BDL-GFP, and pMP:MP-β-glucuronidase in the meristem; altered polarity and expression of pPIN1:PIN1-GFP in the apical domain of the developing embryo; and resistance to auxin in the pcn mutants. The bdl mutation rescued embryo lethality of pcn, suggesting that improper auxin response is involved in pcn defects. Furthermore, WUS, PINFORMED1, PINOID, and TOPLESS are dosage sensitive in pcn, suggesting functional interaction. Together, our results suggest that PCN functions in the auxin pathway, integrating auxin signaling in the organization and maintenance of the SAM and RAM.

  5. Strong seed-specific protein expression from the Vigna radiata storage protein 8SGα promoter in transgenic Arabidopsis seeds.

    Chen, Mo-Xian; Zheng, Shu-Xiao; Yang, Yue-Ning; Xu, Chao; Liu, Jie-Sheng; Yang, Wei-Dong; Chye, Mee-Len; Li, Hong-Ye


    Vigna radiata (mung bean) is an important crop plant and is a major protein source in developing countries. Mung bean 8S globulins constitute nearly 90% of total seed storage protein and consist of three subunits designated as 8SGα, 8SGα' and 8SGβ. The 5'-flanking sequences of 8SGα' has been reported to confer high expression in transgenic Arabidopsis seeds. In this study, a 472-bp 5'-flanking sequence of 8SGα was identified by genome walking. Computational analysis subsequently revealed the presence of numerous putative seed-specific cis-elements within. The 8SGα promoter was then fused to the gene encoding β-glucuronidase (GUS) to create a reporter construct for Arabidopsis thaliana transformation. The spatial and temporal expression of 8SGα∷GUS, as investigated using GUS histochemical assays, showed GUS expression exclusively in transgenic Arabidopsis seeds. Quantitative GUS assays revealed that the 8SGα promoter showed 2- to 4-fold higher activity than the Cauliflower Mosaic Virus (CaMV) 35S promoter. This study has identified a seed-specific promoter of high promoter strength, which is potentially useful for directing foreign protein expression in seed bioreactors.

  6. Online turbulent flow extraction coupled with liquid chromatography-tandem mass spectrometry for high throughput screening of anabolic steroids in horse urine.

    Shin, Hyun Du; Suh, Joon Hyuk; Kim, Junghyun; Cho, Hyun-Deok; Lee, Su Duk; Han, Kwan Seok; Wang, Yu; Han, Sang Beom


    A high throughput method for simultaneous screening of anabolic steroids and their metabolites (4-esterendione, trenbolone, boldenone, oxandrolone, nandrolone, methandrostenolone, testosterone, 1-androstendione, ethisterone, normethandrolone, methyltestosterone, 16β-Hydroxystanozolol, epitestosterone, bolasterone, norethandrolone, danazol, stanozolol and androstadienone) in equine urine by online turbulent flow extraction coupled with liquid chromatography-tandem mass spectrometry was developed. The use of turbulent flow chromatography could simplify pretreatment of horse urine, which has complex matrices as well as high viscosity. The urine was extracted by mixed-mode cation exchange solid phase extraction, and hydrolyzed using β-glucuronidase/arylsulfatase. Then, the sample was automatically loaded on the TurboFlow Cyclone extraction column for removal of further matrix, followed by separation on a fused core C18 column before MS/MS detection. Optimization and validation of the method were discussed in detail. All analytes were rapidly detected within 10min with high sensitivity (picogram to nanogram per milliliter level), and no interference was observed. The linearity range was from 0.1-10ng/mL for nine steroids and 1.0-50ng/mL for the others, with correlation of coefficient values over 0.995. Precision and accuracy ranged from 0.1 to 14.5% and 1.7 to 12.4%, respectively. The developed method was successfully applied to the analysis of anabolic steroids in horse urine after administration of a model drug. Copyright © 2017 Elsevier B.V. All rights reserved.

  7. Long circulating enzyme replacement therapy rescues bone pathology in mucopolysaccharidosis VII murine model.

    Rowan, Daniel J; Tomatsu, Shunji; Grubb, Jeffrey H; Haupt, Bisong; Montaño, Adriana M; Oikawa, Hirotaka; Sosa, Angela C; Chen, Anping; Sly, William S


    Mucopolysaccharidosis (MPS) type VII is a lysosomal storage disease caused by deficiency of the lysosomal enzyme β-glucuronidase (GUS), leading to accumulation of glycosaminoglycans (GAGs). Enzyme replacement therapy (ERT) effectively clears GAG storage in the viscera. Recent studies showed that a chemically modified form of GUS (PerT-GUS), which escaped clearance by mannose 6-phosphate and mannose receptors and showed prolonged circulation, reduced CNS storage more effectively than native GUS. Clearance of storage in bone has been limited due to the avascularity of the growth plate. To evaluate the effectiveness of long-circulating PerT-GUS in reducing the skeletal pathology, we treated MPS VII mice for 12 weeks beginning at 5 weeks of age with PerT-GUS or native GUS and used micro-CT, radiographs, and quantitative histopathological analysis for assessment of bones. Micro-CT findings showed PerT-GUS treated mice had a significantly lower BMD. Histopathological analysis also showed reduced storage material and a more organized growth plate in PerT-GUS treated mice compared with native GUS treated mice. Long term treatment with PerT-GUS from birth up to 57 weeks also significantly improved bone lesions demonstrated by micro-CT, radiographs and quantitative histopathological assay. In conclusion, long-circulating PerT-GUS provides a significant impact to rescue of bone lesions and CNS involvement.

  8. Evaluation and Application of Two High-Iron Transgenic Rice Lines Expressing a Pea Ferritin Gene

    YE Hong-xai; LI Mei; Guo Ze-jian; Shu Qing-yao; xu Xiao-hui; BAO Jin-song; SHEN Sheng-quan


    A totaI of 105 transgenic rice lines independently transformed with a pea ferritin gene (Fer)were previously obtained.After seven generations of selfing and β-glucuronidase(GUS)assisted selection,82 transgenic lines with stable agronomic traits were got.Among the 82 transgenic lines,two high-iron transgenic rice lines Fer34 and Fer65,with the iron contents in the milled rice being 4.82 and 3.46 times of that of the wild type Xiushui 11,respectively were identified.In the two transgenic lines,the exogenous Fer gene was highly expressed,and inherited as a single locus.The transgene had no negative effect on the agronomic traits of rice plant,other mineral nutritional components,appearance quailty and eating quailty of the milled rice,indicating that these two lines were elite high-iron breeding lines.Furthermore,the practical application and further studies facilitating utilization of the two elite breeding lines were discussed.

  9. The companion cell-specific Arabidopsis disaccharide carrier AtSUC2 is expressed in nematode-induced syncytia.

    Juergensen, Katja; Scholz-Starke, Joachim; Sauer, Norbert; Hess, Paul; van Bel, Aart J E; Grundler, Florian M W


    Cyst nematodes induce a metabolically highly active syncytial cell complex in host roots. The syncytia are symplastically isolated. Because they form a strong sink, assimilates must be imported via the apoplast, thus suggesting that specific membrane-bound sugar transport proteins are expressed and activated. To identify possible candidate genes, transgenic Arabidopsis plants expressing different reporter genes under the control of different promoters from Arabidopsis sugar transporter genes were infected with the beet cyst nematode (Heterodera schachtii). With polymerase chain reaction, 13 additional sugar transporters were tested for their presence in the syncytia through the use of a syncytium-specific cDNA library. Analysis of the infected roots showed that the promoter of the sucrose (Suc) transporter AtSUC2 gene that codes for a companion cell-specific Suc transporter in noninfected plants was found to be expressed in syncytia. Its expression patterns in beta-glucuronidase and green fluorescent protein plants were monitored. Syncytium-specific gene expression was confirmed by reverse transcriptase-polymerase chain reaction. Results support the idea that AtSUC2 mediates the transmembrane transfer of Suc. AtSUC2 is the first disaccharide carrier described to be activated by pathogens.

  10. The Structural Basis of Substrate Recognition in an exo-b-d-glucosaminidase Involved in Chitosan Hydrolysis

    Van Bueren, A.; Ghinet, M; Gregg, K; Fleury, A; Brzezinski, R; Boraston, A


    Family 2 of the glycoside hydrolase classification is one of the largest families. Structurally characterized members of this family include enzymes with ?-galactosidase activity (Escherichia coli LacZ), ?-glucuronidase activity (Homo sapiens GusB), and ?-mannosidase activity (Bacteroides thetaiotaomicron BtMan2A). Here, we describe the structure of a family 2 glycoside hydrolase, CsxA, from Amycolatopsis orientalis that has exo-?-d-glucosaminidase (exo-chitosanase) activity. Analysis of a product complex (1.85 A resolution) reveals a unique negatively charged pocket that specifically accommodates the nitrogen of nonreducing end glucosamine residues, allowing this enzyme to discriminate between glucose and glucosamine. This also provides structural evidence for the role of E541 as the catalytic nucleophile and D469 as the catalytic acid/base. The structures of an E541A mutant in complex with a natural ?-1,4-d-glucosamine tetrasaccharide substrate and both E541A and D469A mutants in complex with a pNP-?-d-glucosaminide synthetic substrate provide insight into interactions in the + 1 subsite of this enzyme. Overall, a comparison with the active sites of other GH2 enzymes highlights the unique architecture of the CsxA active site, which imparts specificity for its cationic substrate.

  11. The Structural Basis of Substrate Recognition in an exo-beta-d-Glucosaminidase Involved in Chitosan Hydrolysis

    Lammerts van Bueren, A.; Ghinet, M; Gregg, K; Fleury, A; Brzezinski, R; Boraston, A


    Family 2 of the glycoside hydrolase classification is one of the largest families. Structurally characterized members of this family include enzymes with beta-galactosidase activity (Escherichia coli LacZ), beta-glucuronidase activity (Homo sapiens GusB), and beta-mannosidase activity (Bacteroides thetaiotaomicron BtMan2A). Here, we describe the structure of a family 2 glycoside hydrolase, CsxA, from Amycolatopsis orientalis that has exo-beta-D-glucosaminidase (exo-chitosanase) activity. Analysis of a product complex (1.85 A resolution) reveals a unique negatively charged pocket that specifically accommodates the nitrogen of nonreducing end glucosamine residues, allowing this enzyme to discriminate between glucose and glucosamine. This also provides structural evidence for the role of E541 as the catalytic nucleophile and D469 as the catalytic acid/base. The structures of an E541A mutant in complex with a natural beta-1,4-D-glucosamine tetrasaccharide substrate and both E541A and D469A mutants in complex with a pNP-beta-D-glucosaminide synthetic substrate provide insight into interactions in the +1 subsite of this enzyme. Overall, a comparison with the active sites of other GH2 enzymes highlights the unique architecture of the CsxA active site, which imparts specificity for its cationic substrate.

  12. Functional properties of a chitinase promoter from cabbage (Brassica oleracea var.capitata)



    The 5'-region of the chitinase gene cabch29,derived from Brassica oleracea var.capitata,has been sequenced and analyzed for cis-acting elements important in controlling gene expression in transgenic tobacco plants.Different 5'-deletion fragments were linked to reporter gene β-glucuronidase (GUS) as translational fusions,and the expression of these chimeric genes was analyzed in vegetative organs and tissues.Sequences up to-651 showed some basal GUS activity with nearly equal levels in wounded and intact tissues.The addition of further upstream sequences(-651 to-1284) enhanced expression level,and the expression driven by this fragment was inducible by a factor of two to three-fold by wounding.Histochemical analysis of different tissue from transgenic plants that contain cabch29 promoter-gus fusion gene demonstrated woundinducible and tissue-specific cabch29 promoter activity in plants containing the 1308 base pair fragment.The location of GUS activity appears to be cell-specific,being highest in vascular cells and epidermal cells of stem,leaf and roots.Meanwhile,the temporal and spatial expression of cabch29-GUS fusion gene has been investigated.Among the different vegetative organs,a high level of GUS activity was observed in stem and a moderate one in roots;whereas,wounding stress led to a high level of GUS in stem and moderate one in leaf.

  13. Stability of glufosfamide in phosphate buffers and in biological samples.

    Sun, Yuming; Chen, Xiaoyan; Xu, Haiyan; Guan, Zhongmin; Zhong, Dafang


    Glufosfamide is a new, potential chemotherapeutic agent currently under investigation. Stability of glufosfamide was investigated in sodium phosphate buffers with different pH and temperature and in biological samples. Glufosfamide and isophosphamide mustard were quantified simultaneously using a liquid chromatography-ion trap mass spectrometric method; precision and accuracy were within 15% for each analyte. Glufosfamide was stable in neutral buffers, but decomposed to form isophosphoramide mustard under acidic and basic conditions, which was pH- and temperature-dependent. The stability of glufosfamide varied in different biological samples. Results indicated that glufosfamide was unstable in some biological samples, such as the small intestine, smooth muscles, pancreas and urine, especially in the small intestine homogenate, with a half-life of 1.1 h. But the pH (<8) and beta-glucosidase of the tissue homogenate was found to have negligible contribution to the degradation of glufosfamide. The enzymatic inhibition experiment with the specific inhibitor, saccharo-1,4-lactone, demonstrated that it was glucuronidase that resulted in the degradation of glufosfamide in small intestine homogenate. Methanol was recommended to be used to homogenize the tissue in an ice water bath, and the container for urine collection should also be maintained in an ice water bath, and all the biological samples collected should be preserved in frozen condition until analysis.

  14. Germin-like protein 2 gene promoter from rice is responsive to fungal pathogens in transgenic potato plants.

    Munir, Faiza; Hayashi, Satomi; Batley, Jacqueline; Naqvi, Syed Muhammad Saqlan; Mahmood, Tariq


    Controlled transgene expression via a promoter is particularly triggered in response to pathogen infiltration. This is significant for eliciting disease-resistant features in crops through genetic engineering. The germins and germin-like proteins (GLPs) are known to be associated with plant and developmental stages. The 1107-bp Oryza sativa root GLP2 (OsRGLP2) gene promoter fused to a β-glucuronidase (GUS) reporter gene was transformed into potato plants through an Agrobacterium-mediated transformation. The OsRGLP2 promoter was activated in response to Fusarium solani (Mart.) Sacc. and Alternaria solani Sorauer. Quantitative real-time PCR results revealed 4-5-fold increase in promoter activity every 24 h following infection. There was a 15-fold increase in OsRGLP2 promoter activity after 72 h of F. solani (Mart.) Sacc. treatment and a 12-fold increase observed with A. solani Sorauer. Our results confirmed that the OsRGLP2 promoter activity was enhanced under fungal stress. Furthermore, a hyperaccumulation of H2O2 in transgenic plants is a clear signal for the involvement of OsRGLP2 promoter region in the activation of specific genes in the potato genome involved in H2O2-mediated defense response. The OsRGLP2 promoter evidently harbors copies of GT-I and Dof transcription factors (AAAG) that act in response to elicitors generated in the wake of pathogen infection.

  15. A novel PR10 promoter from Erianthus arundinaceus directs high constitutive transgene expression and is enhanced upon wounding in heterologous plant systems.

    Chakravarthi, M; Syamaladevi, Divya P; Harunipriya, P; Augustine, Sruthy Maria; Subramonian, N


    In genetic engineering, inducible promoters play an important role as the expression of genes driven by them can be turned on or off under situations like biotic or abiotic factors. There are few reports on inducible promoters that can be employed in the development of transgenic plants, particularly in sugarcane. In the present study, four wound inducible genes (Chitinase, PR1A, PR10 and HRGP) were selected and were amplified from Erianthus arundinaceus, a distant relative of sugarcane. In order to determine the gene that is highly induced upon wounding, RT-qPCR was performed, which showed that PR10 gene expression was instantaneous and higher upon wounding when compared to the other three genes. Using the random amplification of genomic ends technique, a 592 bp promoter sequence was obtained and in silico analysis of the upstream regulatory region revealed a 469 bp promoter and 123 bp of 5' untranslated region (UTR). Functional analyses of the promoter sequence (with and without 5' UTR) in tobacco, rice and sugarcane using β-glucuronidase (GUS) as the reporter gene revealed the constitutive and inducible nature of the PR10 promoter. Our studies have demonstrated that the PR10 promoter, though highly constitutive, was quickly induced upon wounding as well as on treatment with abscisic acid and methyl jasmonate hormones. This is the first report on the isolation and characterization of a PR10 promoter from a wild grass and is expected to have application for development of transgenic plants.

  16. An Arabidopsis tissue-specific RNAi method for studying genes essential to mitosis.

    Brunilís Burgos-Rivera

    Full Text Available A large fraction of the genes in plants can be considered essential in the sense that when absent the plant fails to develop past the first few cell divisions. The fact that angiosperms pass through a haploid gametophyte stage can make it challenging to propagate such mutants even in the heterozygous condition. Here we describe a tissue-specific RNAi method that allows us to visualize cell division phenotypes in petals, which are large dispensable organs. Portions of the APETALA (AP3 and PISTILLATA (PI promoters confer early petal-specific expression. We show that when either promoter is used to drive the expression of a beta-glucuronidase (GUS RNAi transgene in plants uniformly expressing GUS, GUS expression is knocked down specifically in petals. We further tested the system by targeting the essential kinetochore protein CENPC and two different components of the Spindle Assembly Checkpoint (MAD2 and BUBR1. Plant lines expressing petal-specific RNAi hairpins targeting these genes exhibited an array of petal phenotypes. Cytological analyses of the affected flower buds confirmed that CENPC knockdown causes cell cycle arrest but provided no evidence that either MAD2 or BUBR1 are required for mitosis (although both genes are required for petal growth by this assay. A key benefit of the petal-specific RNAi method is that the phenotypes are not expressed in the lineages leading to germ cells, and the phenotypes are faithfully transmitted for at least four generations despite their pronounced effects on growth.

  17. Transformation of Morinda citrifolia via simple mature seed imbibition method.

    Lee, J J; Ahmad, S; Roslan, H A


    Morinda citrifolia, is a valuable medicinal plant with a wide range of therapeutic properties and extensive transformation study on this plant has yet been known. Present study was conducted to establish a simple and reliable transformation protocol for M. citrifolia utilising Agrobacterium tumefaciens via direct seed exposure. In this study, the seeds were processed by tips clipping and dried and subsequently incubated in inoculation medium. Four different parameters during the incubation such as incubation period, bacterial density, temperature and binary vectors harbouring beta-glucuronidase (GUS) gene (pBI121 and pGSA1131), were tested to examine its effect on transformation efficiency. The leaves from the treated and germinated seedlings were analysed via Polymerase Chain Reaction (PCR), histochemical assay of the GUS gene and reverse transcription-PCR (RT-PCR). Results of the study showed that Agrobacterium strain LBA4404 with optical density of 1.0 and 2 h incubation period were optimum for M. citrifolia transformation. It was found that various co-cultivation temperatures tested and type of vector used did not affect the transformation efficiency. The highest transformation efficiency for M. citrifolia direct seed transformation harbouring pBI121 and pGSA1131 was determined to be 96.8% with 2 h co-cultivation treatment and 80.4% when using bacterial density of 1.0, respectively. The transformation method can be applied for future characterization study of M. citrifolia.

  18. Evidence for the negative regulation of phytase gene expression in Streptomyces lividans and Streptomyces coelicolor.

    Boukhris, Ines; Dulermo, Thierry; Chouayekh, Hichem; Virolle, Marie-Joëlle


    Sco7697, a gene encoding a phytase, enzyme able to degrade phytate (myo-inositol 1,2,3,4,5,6-hexakis phosphate), the most abundant phosphorus storing compound in plants is present in the genome of S. coelicolor, a soil born bacteria with a saprophytic lifestyle. The expression of this gene was previously shown to be induced in conditions of Pi limitation by the response regulator PhoP binding to an operator sequence, the PHO box, located upstream of the -35 promoter sequence. A close examination of the promoter region of sco7697 revealed the presence of another putative operator site, a Direct Repeat (DR), located downstream of the -10 promoter sequence. In order to determine whether this DR played a role in regulation of sco7697 expression, different variants of the phytase gene promoter region were transcriptionally fused to the ß-glucuronidase reporter gene (GUS). As expected, deletion of the PHO box led to abolition of sco7697 induction in conditions of Pi limitation. Interestingly, alteration of the DR correlated with a dramatic increase of GUS expression but only when PhoP was present. These results demonstrated that this DR is the site of strong negative regulation by an unknown repressor. The latter would impede the necessary activation of phytase expression by PhoP.

  19. The role of heparanase in pulmonary cell recruitment in response to an allergic but not non-allergic stimulus.

    Abigail Morris

    Full Text Available Heparanase is an endo-β-glucuronidase that specifically cleaves heparan sulfate proteoglycans in the extracellular matrix. Expression of this enzyme is increased in several pathological conditions including inflammation. We have investigated the role of heparanase in pulmonary inflammation in the context of allergic and non-allergic pulmonary cell recruitment using heparanase knockout (Hpa-/- mice as a model. Following local delivery of LPS or zymosan, no significant difference was found in the recruitment of neutrophils to the lung between Hpa-/- and wild type (WT control. Similarly neutrophil recruitment was not inhibited in WT mice treated with a heparanase inhibitor. However, in allergic inflammatory models, Hpa-/- mice displayed a significantly reduced eosinophil (but not neutrophil recruitment to the airways and this was also associated with a reduction in allergen-induced bronchial hyperresponsiveness, indicating that heparanase expression is associated with allergic reactions. This was further demonstrated by pharmacological treatment with a heparanase inhibitor in the WT allergic mice. Examination of lung specimens from patients with different severity of chronic obstructive pulmonary disease (COPD found increased heparanase expression. Thus, it is established that heparanase contributes to allergen-induced eosinophil recruitment to the lung and could provide a novel therapeutic target for the development of anti-inflammatory drugs for the treatment of asthma and other allergic diseases.

  20. The alc-GR system: a modified alc gene switch designed for use in plant tissue culture.

    Roberts, Gethin R; Garoosi, G Ali; Koroleva, Olga; Ito, Masaki; Laufs, Patrick; Leader, David J; Caddick, Mark X; Doonan, John H; Tomsett, A Brian


    The ALCR/alcA (alc) two-component, ethanol-inducible gene expression system provides stringent control of transgene expression in genetically modified plants. ALCR is an ethanol-activated transcription factor that can drive expression from the ALCR-responsive promoter (alcA). However, the alc system has been shown to have constitutive expression when used in plant callus or cell suspension cultures, possibly resulting from endogenous inducer produced in response to lowered oxygen availability. To widen the use of the alc system in plant cell culture conditions, the receptor domain of the rat glucocorticoid receptor (GR) was translationally fused to the C terminus of ALCR to produce ALCR-GR, which forms the basis of a glucocorticoid-inducible system (alc-GR). The alc-GR switch system was tested in tobacco (Nicotiana tabacum) Bright Yellow-2 suspension cells using a constitutively expressed ALCR-GR with four alternative alcA promoter-driven reporter genes: beta-glucuronidase, endoplasmic reticulum-targeted green fluorescent protein, haemagglutinin, and green fluorescent protein-tagged Arabidopsis (Arabidopsis thaliana) Arath;CDKA;1 cyclin-dependent kinase. Gene expression was shown to be stringently dependent on the synthetic glucocorticoid dexamethasone and, in cell suspensions, no longer required ethanol for induction. Thus, the alc-GR system allows tight control of alcA-driven genes in cell culture and complements the conventional ethanol switch used in whole plants.

  1. Combination of the ALCR/alcA ethanol switch and GAL4/VP16-UAS enhancer trap system enables spatial and temporal control of transgene expression in Arabidopsis.

    Jia, Hongge; Van Loock, Bram; Liao, Mingjun; Verbelen, Jean-Pierre; Vissenberg, Kris


    The experimental control of gene expression in specific tissues or cells at defined time points is a useful tool for the analysis of gene function. GAL4/VP16-UAS enhancer trap lines can be used to selectively express genes in specific tissues or cells, and an ethanol-inducible system can help to control the time of expression. In this study, the combination of the two methods allowed the successful regulation of gene expression in both time and space. For this purpose, a binary vector, 962-UAS::GUS, was constructed in which the ALCR activator and beta-glucuronidase (GUS) reporter gene were placed under the control of upstream activator sequence (UAS) elements and the alcA response element, respectively. Three different GAL4/VP16-UAS enhancer trap lines of Arabidopsis were transformed, resulting in transgenic plants in which GUS activity was detected only on ethanol induction and exclusively in the predicted tissues of the enhancer trap lines. As a library of different enhancer trap lines with distinct green fluorescent protein (GFP) patterns exist, transformation with a similar vector, in which GUS is replaced by another gene, would enable the control of the time and place of transgene expression. We have constructed two vectors for easy cloning of the gene of interest, one with a polylinker site and one that is compatible with the GATEWAY vector conversion system. The method can be extended to other species when enhancer trap lines become available.

  2. Agrobacterium-mediated transformation of Phalaenopsis by targeting protocorms at an early stage after germination.

    Mishiba, Kei-ichiro; Chin, Dong Poh; Mii, Masahiro


    A transformation procedure for phalaenopsis orchid established by using immature protocorms for Agrobacterium infection was aimed at the introduction of target genes into individuals with divergent genetic backgrounds. Protocorms obtained after 21 days of culture on liquid New Dogashima medium were inoculated with Agrobacterium strain EHA101(pIG121Hm) harboring both beta-glucuronidase (GUS) and hygromycin resistance genes. Subculture of the protocorms on acetosyringone-containing medium 2 days before Agrobacterium inoculation gave the highest transformation efficiencies (1.3-1.9%) based on the frequency of hygromycin-resistant plants produced. Surviving protocorms obtained 2 months after Agrobacterium infection on selection medium containing 20 mg l(-1) hygromycin were cut transversely into two pieces before transferring to recovery medium without hygromycin. Protocorm-like bodies (PLBs) proliferated from pieces of protocorms during a 1-month culture on recovery medium followed by transfer to selection medium. Hygromycin-resistant phalaenopsis plants that regenerated after the re-selection culture of PLBs showed histochemical blue staining due to GUS. Transgene integration of the hygromycin-resistant plants was confirmed by Southern blot analysis. A total of 88 transgenic plants, each derived from an independent protocorm, was obtained from ca. 12,500 mature seeds 6 months after infection with Agrobacterium. Due to the convenient protocol for Agrobacterium infection and rapid production of transgenic plants, the present procedure could be utilized to assess expression of transgenes under different genetic backgrounds, and for the molecular breeding of phalaenopsis.

  3. Substrate mediated enzyme prodrug therapy.

    Betina Fejerskov

    Full Text Available In this report, we detail Substrate Mediated Enzyme Prodrug Therapy (SMEPT as a novel approach in drug delivery which relies on enzyme-functionalized cell culture substrates to achieve a localized conversion of benign prodrug(s into active therapeutics with subsequent delivery to adhering cells or adjacent tissues. For proof-of-concept SMEPT, we use surface adhered micro-structured physical hydrogels based on poly(vinyl alcohol, β-glucuronidase enzyme and glucuronide prodrugs. We demonstrate enzymatic activity mediated by the assembled hydrogel samples and illustrate arms of control over rate of release of model fluorescent cargo. SMEPT was not impaired by adhering cells and afforded facile time - and dose - dependent uptake of the in situ generated fluorescent cargo by hepatic cells, HepG2. With the use of a glucuronide derivative of an anticancer drug, SN-38, SMEPT afforded a decrease in cell viability to a level similar to that achieved using parent drug. Finally, dose response was achieved using SMEPT and administration of judiciously chosen concentration of SN-38 glucuronide prodrug thus revealing external control over drug delivery using drug eluting surface. We believe that this highly adaptable concept will find use in diverse biomedical applications, specifically surface mediated drug delivery and tissue engineering.

  4. Effects of Catechol O-Methyl Transferase Inhibition on Anti-Inflammatory Activity of Luteolin Metabolites.

    Ha, Sang Keun; Lee, Jin-Ah; Cho, Eun Jung; Choi, Inwook


    Although luteolin is known to have potent anti-inflammatory activities, much less information has been provided on such activities of its hepatic metabolites. Luteolin was subjected to hepatic metabolism in HepG2 cells either without or with catechol O-methyl transferase (COMT) inhibitor. To identify hepatic metabolites of luteolin without (luteolin metabolites, LMs) or with COMT inhibitor (LMs+CI), metabolites were treated by β-glucuronidase and sulfatase, and found that they were composed of glucuronide and sulfate conjugates of diosmetin in LMs or these conjugates of luteolin in LMs+CI. LMs and LMs+CI were examined for their anti-inflammatory activities on LPS stimulated Raw 264.7 cells. Expression of iNOS and production of nitric oxide and pro-inflammatory cytokines such as TNF-α, IL-1β, and IL-6 were suppressed more effectively by the treatment with LMs+CI than LMs. Our data provide a new insight on possible improvement in functional properties of luteolin on target cells by modifying their metabolic pathway in hepatocytes.

  5. Increase of homologous recombination frequency in vascular tissue of Arabidopsis plants exposed to salt stress.

    Boyko, Alex; Hudson, Darryl; Bhomkar, Prasanna; Kathiria, Palak; Kovalchuk, Igor


    Here we analyzed the influence of salt stress on plant genome stability. Homologous recombination events were detected in transgenic Arabidopsis plants that carried in their genome a beta-glucuronidase recombination marker. Recombination events were scored as blue sectors using a stereo microscope. Exposure to 50 mM salt resulted in a 3.0-fold increase in recombination frequency. To analyze the organ and tissue specificity of recombination events, we examined cross-sections of leaves, stems and roots. We found that nearly 30% of recombination events in plants grown under normal conditions and nearly 50% of events in plants grown on salt were undetected by the conventional method. Most of the recombination events represented a cluster/group of cells (12 on average), although events with single cells were also detected. Recombination events were very frequent in leaf mesophyll cells. On average, individual recombination events located on leaves contained more cells than events located on roots or stems. Analysis of recombination events in cross-sectioned tissue of salt-treated plants revealed a shift in the distribution of recombination events towards the vascular tissue. We discuss the significance of the finding for plant stress physiology.

  6. Homeodomain leucine zipper proteins bind to the phosphate response domain of the soybean VspB tripartite promoter.

    Tang, Z; Sadka, A; Morishige, D T; Mullet, J E


    The soybean (Glycine max L. Merr. cv Williams 82) genes VspA and VspB encode vacuolar glycoprotein acid phosphatases that serve as vegetative storage proteins during seed fill and early stages of seedling growth. VspB expression is activated by jasmonates (JAs) and sugars and down-regulated by phosphate and auxin. Previous promoter studies demonstrated that VspB promoter sequences between -585 and -535 mediated responses to JA, and sequences between -535 and -401 mediated responses to sugars, phosphate, and auxin. In this study, the response domains were further delineated using transient expression of VspB promoter-beta-glucuronidase constructs in tobacco protoplasts. Sequences between -536 and -484 were identified as important for phosphate responses, whereas the region from -486 to -427 mediated sugar responses. Gel-shift and deoxyribonuclease-I footprinting assays revealed four DNA-binding sites between -611 and -451 of the soybean VspB promoter: one in the JA response domain, two in the phosphate response domain, and one binding site in the sugar response domain. The sequence CATTAATTAG present in the phosphate response domain binds soybean homeodomain leucine zipper proteins, suggesting a role for these transcription factors in phosphate-modulated gene expression.

  7. A novel cold-inducible zinc finger protein from soybean, SCOF-1, enhances cold tolerance in transgenic plants.

    Kim, J C; Lee, S H; Cheong, Y H; Yoo, C M; Lee, S I; Chun, H J; Yun, D J; Hong, J C; Lee, S Y; Lim, C O; Cho, M J


    Cold stress on plants induces changes in the transcription of cold response genes. A cDNA clone encoding C2H2-type zinc finger protein, SCOF-1, was isolated from soybean. The transcription of SCOF-1 is specifically induced by low temperature and abscisic acid (ABA) but not by dehydration or high salinity. Constitutive overexpression of SCOF-1 induced cold-regulated (COR) gene expression and enhanced cold tolerance of non-acclimated transgenic Arabidopsis and tobacco plants. SCOF-1 localized to the nucleus but did not bind directly to either C-repeat/dehydration (CRT/DRE) or ABA responsive element (ABRE), cis-acting DNA regulatory elements present in COR gene promoters. However, SCOF-1 greatly enhanced the DNA binding activity of SGBF-1, a soybean G-box binding bZIP transcription factor, to ABRE in vitro. SCOF-1 also interacted with SGBF-1 in a yeast two-hybrid system. The SGBF-1 transactivated the beta-glucuronidase reporter gene driven by the ABRE element in Arabidopsis leaf protoplasts. Furthermore, the SCOF-1 enhanced ABRE-dependent gene expression mediated by SGBF-1. These results suggest that SCOF-1 may function as a positive regulator of COR gene expression mediated by ABRE via protein-protein interaction, which in turn enhances cold tolerance of plants.

  8. Regeneration of transformed shoots from electroporated soybean (Glycine max (L.) Merr.) protoplasts.

    Dhir, S K; Dhir, S; Sturtevant, A P; Widholm, J M


    Stable transformation of soybean (Glycine max (L.) Merr.) protoplasts isolated from immature cotyledons was achieved following electroporation with plasmid DNA carrying chimeric genes encoding ß-glucuronidase (GUS) and hygromycin phosphotransferase (HPT) under the control of the cauliflower mosaic virus (CaMV) 35S promoter. Transformed colonies were stringently selected by growing 15-day-old protoplast-derived cells in the presence of 40 μg/ml of hygromycin-B for 6 weeks. Over 93% of the resistant cells and colonies exhibited GUS activity, indicating that the two marker genes borne on a single plasmid were co-introduced and co-expressed at a very high freguency. This transformation procedure reproducibly yields transformants at frequencies of 2.9-6.8 × 10(-4) (based on the number of protoplasts electroporated) or 23.0% (based on the number of control microcalli formed) counted after 6 weeks of selection. After repeated subculturing on regeneration medium, shoots were induced from 8.0% of the transformed calli. Southern hybridization confirmed the presence of both the GUS and hygromycin genes in the transformed calli and shoots.

  9. Morphine-3-D glucuronide stability in postmortem specimens exposed to bacterial enzymatic hydrolysis.

    Carroll, F T; Marraccini, J V; Lewis, S; Wright, W


    Medical examiners frequently rely on the finding of free morphine present in postmortem specimens to assist in certifying deaths associated with narcotics. In vitro hydrolysis of morphine-3-D glucuronide (M3DG) to free morphine was studied using variable specimen pH, initial degree of specimen putrefaction, storage temperature and time, and the effectiveness of sodium fluoride (NaF) preservation. Reagent M3DG was added to opiate-free fresh blood and urine and to autopsy-derived blood specimens. Reagent bovine glucuronidase was also added to certain specimens. Freshly collected and refrigerated NaF-preserved blood produced minimal free morphine, whereas four of five autopsy blood specimens produced free morphine from M3DG. Increased storage time, temperature, and initial degree of putrefaction resulted in greater free morphine generation despite the absence of viable bacteria. Hydrolysis occurring during specimen storage can generate free morphine from M3DG and may result in erroneous conclusions in certifying narcotic deaths.

  10. 豌豆核基质结合区的功能研究



    将豌豆基因组中分离的核基质结合区(Matrix attachment regions,MARs)构建在植物表达载体的T-DNA结构域中,使得报导基因β-葡糖醛酸酶(β-glucuronidase,GUS)基因位于两段MARS序列之间,将此载体与不包含MARs序列的植物表达载体经农杆菌介导转化烟草,GUS活性检测表明,MARs可以提高GUS基因的表达水平,和不包含MARs的转基因植株相比,MARs序列使GUS基因的平均表达水平提高2倍,但在瞬时表达中,MARs对GUS基因的表达没有影响。

  11. Visualization of site-specific recombination catalyzed by a recombinase from Zygosaccharomyces rouxii in Arabidopsis thaliana.

    Onouchi, H; Nishihama, R; Kudo, M; Machida, Y; Machida, C


    Excision of a DNA segment can occur in Arabidopsis thaliana by reciprocal recombination between two specific recombination sites (RSs) when the recombinase gene (R) from Zygosaccharomyces rouxii is expressed in the plant. To monitor recombination events, we generated several lines of transgenic Arabidopsis plants that carried a cryptic beta-glucuronidase (GUS) reporter gene which was designed in such a way that expression of the reporter gene could be induced by R gene-mediated recombination. We also made several transgenic lines with an R gene linked to the 35S promoter of cauliflower mosaic virus. Each transgenic line carrying the cryptic reporter gene was crossed with each line carrying the R gene. Activity of GUS in F1 and F2 progeny was examined histochemically and recombination between two RSs was analyzed by Southern blotting and the polymerase chain reaction. In seedlings and plantlets of F1 progeny and most of the F2 progeny, a variety of patterns of activity of GUS, including sectorial chimerism in leaves, was observed. A small percentage of F2 individuals exhibited GUS activity in the entire plant. This pattern of expression was ascribed to germinal recombination in the F1 generation on the basis of an analysis of DNA structure by Southern blotting. These results indicate that R gene-mediated recombination can be induced in both somatic and germ cells of A. thaliana by cross-pollination of parental transgenic lines.

  12. Arabidopsis Nitrate Transporter NRT1.9 Is Important in Phloem Nitrate Transport[W][OA

    Wang, Ya-Yun; Tsay, Yi-Fang


    This study of the Arabidopsis thaliana nitrate transporter NRT1.9 reveals an important function for a NRT1 family member in phloem nitrate transport. Functional analysis in Xenopus laevis oocytes showed that NRT1.9 is a low-affinity nitrate transporter. Green fluorescent protein and β-glucuronidase reporter analyses indicated that NRT1.9 is a plasma membrane transporter expressed in the companion cells of root phloem. In nrt1.9 mutants, nitrate content in root phloem exudates was decreased, and downward nitrate transport was reduced, suggesting that NRT1.9 may facilitate loading of nitrate into the root phloem and enhance downward nitrate transport in roots. Under high nitrate conditions, the nrt1.9 mutant showed enhanced root-to-shoot nitrate transport and plant growth. We conclude that phloem nitrate transport is facilitated by expression of NRT1.9 in root companion cells. In addition, enhanced root-to-shoot xylem transport of nitrate in nrt1.9 mutants points to a negative correlation between xylem and phloem nitrate transport. PMID:21571952

  13. Arabidopsis nitrate transporter NRT1.9 is important in phloem nitrate transport.

    Wang, Ya-Yun; Tsay, Yi-Fang


    This study of the Arabidopsis thaliana nitrate transporter NRT1.9 reveals an important function for a NRT1 family member in phloem nitrate transport. Functional analysis in Xenopus laevis oocytes showed that NRT1.9 is a low-affinity nitrate transporter. Green fluorescent protein and β-glucuronidase reporter analyses indicated that NRT1.9 is a plasma membrane transporter expressed in the companion cells of root phloem. In nrt1.9 mutants, nitrate content in root phloem exudates was decreased, and downward nitrate transport was reduced, suggesting that NRT1.9 may facilitate loading of nitrate into the root phloem and enhance downward nitrate transport in roots. Under high nitrate conditions, the nrt1.9 mutant showed enhanced root-to-shoot nitrate transport and plant growth. We conclude that phloem nitrate transport is facilitated by expression of NRT1.9 in root companion cells. In addition, enhanced root-to-shoot xylem transport of nitrate in nrt1.9 mutants points to a negative correlation between xylem and phloem nitrate transport.

  14. [Regulation pattern of the FRUITFULL (FUL) gene of Arabidopsis thaliana].

    Chu, Tingting; Xie, Hua; Xu, Yong; Ma, Rongcai


    FRUITFULL (FUL) is an MADS box gene that functions early in controlling flowering time, meristem identity and cauline leaf morphology and later in carpel and fruit development in Arabidopsis thaliana. In order to clarify the regulation of FUL expression the upstream regulatory region, -2148 bp - +96 bp and the first intron of the FUL gene were cloned, and vectors with a series of deletion of FUL promoter, and the ones fused with the first intron were constructed. Vectors harboring the fusion of cis-acting elements with the constitutive promoters of TUBULIN and ACTIN were also constructed. Beta-Glucuronidase activity assays of the transgenic Arabidopsis plants showed that two cis-elements were involved in the repression of FUL expression, with one of the two being probably the binding site of the transcriptional factor AP1. And the two CArG boxes played a important role in FUL initiation particularly. Furthermore, the first intron of FUL was shown to participate in the development of carpel and stamen as an enhancer.

  15. Update on clinical and research application of fecal biomarkers for gastrointestinal diseases

    Siddiqui, Imran; Majid, Hafsa; Abid, Shahab


    Gastrointestinal (GI) diseases comprise a large spectrum of clinical conditions ranging from indigestion to inflammatory bowel diseases (IBDs) and carcinomas. Endoscopy is the usual method employed to diagnose these condition. Another noninvasive way to assess and diagnose GI conditions are fecal biomarkers. Fecal biomarkers provide information regarding a specific disease process and are perhaps more acceptable to clinicians and patients alike because of their non-invasivity compared to endoscopy. Aim of this review was to evaluate the current status of the fecal biomarkers in clinical and research for in GI diseases. Multiple types of fecal biomarkers are discussed in this review including; markers to assess IBD, which are released as a results of an inflammatory insults to intestinal epithelia such as antimicrobial peptides (lactoferrin) or inflammation related proteins (calprotectin). While markers related to function of digestion are primarily related to partially digested food or mucosal proteins such as abnormal amount of fecal fat α1-antitrypsin, elastase and secretary IgA. The upcoming fecal biomarker like M2 pyruvate kinase and neutrophil gelatinase associated lipocalin are discussed as well. Apart from above mention, the fecal biomarkers under exploration for possible clinical use in future are also discussed. These include cathelicidins, osteoprotegerin, β-glucuronidase, Eosinophil proteins, etc. PMID:28217373

  16. [Protoplast isolation and transformation of Exserohilum turcicum].

    Zhou, Yong-Li; Tanaka, Chihiro; Matsushim, Satoshi; Tsuda, Mitsuya


    Northern corn leaf blight, caused by the fungus Exserohirum turcicum Pass. (Leonard and Suggs), is one of the major diseases in most corn-growing areas of the world. Research on gene tagging of E. turcicum has been limited due to the lack of an efficient transformation system. Since E. turcicum produces and accumulates melamin in cell walls during vegetative growth, it is difficult to efficiently isolate its protoplast. To isolate the protoplast of this pathogen with a high frequency, the effects of cell wall degradation enzymes, including beta-1,3-glucanase (Fungase, Funcelase, Novozyme and Glucanex) and beta-glucuronidase (Driselase, Uskizyme and Kitalase), enzyme concentrations, combinations, strains and medium on the isolation frequency were tested. The isolation frequencies were high enough for transformation when the combinations of (Kitalase + Glucanex + Driselase), (Kitalase + Glucanex) or (Kitalase + Uskizyme) were used. Moreover, the isolation frequencies of protoplast were significantly affected by the cultural morphologies of strain and the growth stage of mycelia. Among the plasmids tested, only plasmid pAN71 is efficient for transformation of E. turcicum. This result will provide some useful information for gene tagging of E. turcicum and other species in Exserohirum.

  17. Disposition of methyl ethyl ketoxime in the rat after oral, intravenous and dermal administration.

    Burka, L T; Black, S R; Mathews, J M


    1. The disposition of 14C-methyl ethyl ketoxime (MEKO) was determined in the male F344 rat following oral, intravenous (i.v.) and dermal administration. 2. Oral doses of 2.7, 27 and 270 mg/kg were primarily excreted as CO2 (71-49%) in decreasing percentage as the dose increased. Excretion in urine (13-26%) and as volatiles (5-18%) increased as the dose increased. Five to 6% of the dose remained in the major tissues after 72 h. 3. An i.v. dose of 2.7 mg/kg was also principally excreted as CO2 (48.8%) with excretion in urine and as expired volatiles accounting for 21.4 and 11.4%, respectively. About 7% of the administered radioactivity remained in the tissues after 72 h. 4. Following dermal administration, 13 and 26% of a 2.7 and 270 mg/kg dose, respectively, were absorbed. Volatilization from the dose site prior to placement in the metabolism cage may account for the low absorption. 5. MEKO was biotransformed to at least five polar metabolites that could only be partially resolved by anion exchange chromatography. Incubation with glucuronidase, but not sulphatase, changed the urinary metabolic profile. Methyl ethyl ketone was a major component in the volatiles.

  18. Functional characterization of a strong bi-directional constitutive plant promoter isolated from cotton leaf curl Burewala virus.

    Zainul A Khan

    Full Text Available Cotton leaf curl Burewala virus (CLCuBuV, belonging to the genus Begomovirus, possesses single-stranded monopartite DNA genome. The bidirectional promoters representing Rep and coat protein (CP genes of CLCuBuV were characterized and their efficacy was assayed. Rep and CP promoters of CLCuBuV and 35S promoter of Cauliflower mosaic virus (CaMV were fused with β-glucuronidase (GUS and green fluorescent protein (GFP reporter genes. GUS activity in individual plant cells driven by Rep, CP and 35S promoters was estimated using real-time PCR and fluorometric GUS assay. Histochemical staining of GUS in transformed tobacco (Nicotiana tabacum cv. Xanthi leaves showed highest expression driven by Rep promoter followed by 35S promoter and CP promoter. The expression level of GUS driven by Rep promoter in transformed tobacco plants was shown to be two to four-fold higher than that of 35S promoter, while the expression by CP promoter was slightly lower. Further, the expression of GFP was monitored in agroinfiltrated leaves of N. benthamiana, N. tabacum and cotton (Gossypium hirsutum plants using confocal laser scanning microscopy. Rep promoter showed strong consistent transient expression in tobacco and cotton leaves as compared to 35S promoter. The strong constitutive CLCuBuV Rep promoter developed in this study could be very useful for high level expression of transgenes in a wide variety of plant cells.

  19. Transcriptional Regulation of Expression of the Maize Aldehyde Dehydrogenase 7 Gene (ZmALDH7B6) in Response to Abiotic Stresses

    GU Ri-liang


    Aldehyde dehydrogenases (ALDHs) represent a large protein family, which includes several members that catalyze the oxidation of an aldehyde to its corresponding carboxylic acid in plants. Genes encoding members of theALDH7 subfamily have been suggested to play important roles in various stress adaptations in plants. In this study, quantitative RT-PCR analysis revealed that a maizeALDH7 subfamily member (ZmALDH7B6) was constitutively expressed in various organs, including roots, leaves, immature ears, tassels, and developing seeds. The abundance ofZmALDH7B6 mRNA transcripts in maize roots was increased by ammonium, NaCl, and mannitol treatments. To further analyze tissue-speciifc and stress-induced expression patterns, the 1.5-kb 5´-lfankingZmALDH7B6 promoter region was fused to the β-glucuronidase (GUS) reporter gene and introduced into maize plants. In roots of independent transgenic lines, there was signiifcant induction of GUS activity in response to ammonium supply, conifrming ammonium-dependent expression ofZmALDH7B6 at the transcript level. Histochemical staining showed that GUS activity driven by theZmALDH7B6 promoter was mainly localized in the vascular tissues of maize roots. These results suggested thatZmALDH7B6 is induced by multiple environmental stresses in maize roots, and may play a role in detoxifying aldehydes, particularly in vascular tissue.

  20. The effect of neonatal gene therapy on skeletal manifestations in mucopolysaccharidosis VII dogs after a decade.

    Xing, Elizabeth M; Knox, Van W; O'Donnell, Patricia A; Sikura, Tracey; Liu, Yuli; Wu, Susan; Casal, Margret L; Haskins, Mark E; Ponder, Katherine P


    Mucopolysaccharidosis (MPS) VII is a lysosomal storage disease due to deficient activity of β-glucuronidase (GUSB), and results in glycosaminoglycan accumulation. Skeletal manifestations include bone dysplasia, degenerative joint disease, and growth retardation. One gene therapy approach for MPS VII involves neonatal intravenous injection of a gamma retroviral vector expressing GUSB, which results in stable expression in liver and secretion of enzyme into blood at levels predicted to be similar or higher to enzyme replacement therapy. The goal of this study was to evaluate the long-term effect of neonatal gene therapy on skeletal manifestations in MPS VII dogs. Treated MPS VII dogs could walk throughout their lives, while untreated MPS VII dogs could not stand beyond 6 months and were dead by 2 years. Luxation of the coxofemoral joint and the patella, dysplasia of the acetabulum and supracondylar ridge, deep erosions of the distal femur, and synovial hyperplasia were reduced, and the quality of articular bone was improved in treated dogs at 6 to 11 years of age compared with untreated MPS VII dogs at 2 years or less. However, treated dogs continued to have osteophyte formation, cartilage abnormalities, and an abnormal gait. Enzyme activity was found near synovial blood vessels, and there was 2% as much GUSB activity in synovial fluid as in serum. We conclude that neonatal gene therapy reduces skeletal abnormalities in MPS VII dogs, but clinically-relevant abnormalities remain. Enzyme replacement therapy will probably have similar limitations long-term.