Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.2.1.31 (beta-glucuronidase)
 7,680 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

This study addresses the processing of transgenic canola seed for production of recombinant proteins by using beta-glucuronidase (rGUS) as a model protein. The major processing steps that were investigated included dry and wet grinding of the seed, solvent extraction of canola oil, and protein extraction. rGUS in canola seed was stable for at least 2 weeks of incubation at 38 degrees C and for more than 5 months at 10 degrees C. At 70 degrees C, the residual activity changed inversely to the initial moisture content of the seed. The comparison of wet and dry processing revealed no significant differences in protein recovery. rGUS was stable during the defatting of transgenic canola flakes with hexane at 66 degrees C, whereas 2-propanol extraction at the same temperature reduced the extractable enzyme activity by almost 50%. The particle size of the ground seed was important for the extraction efficiency. A faster extraction and greater protein yield was achieved by extracting particles with an average diameter equal to or smaller than 255 microm. More than 80% rGUS was extracted in one stage with sodium phosphate buffer of pH 7.5.
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PMID:Effect of processing on the recovery of recombinant beta-glucuronidase (rGUS) from transgenic canola. 1117 Apr 95

The effect of growth regulators and culture conditions on the morphogenetic response of cotyledonary leaf discs was studied in popular cucumber variety (Cucumis sativus cv. Sheetal). Organogenesis was induced directly without any intervening callus phase on Murashige and Skoog medium supplemented with different concentrations of benzyladenine and indole propionic acid. Best results (93%) were obtained in the presence of the 4 mg/L benzyladenine and 1 mg/L IPA. The elongated shoots were rooted in basal medium with 1 mg/L indole butyric acid, hardened and transferred to the field conditions. Genetic transformation system has been established for Cucumis sativus cv. Sheetal, plants by infecting cotyledonary explants with Agrobacterium tumefaciens strain LBA4404 carrying binary plasmid pBI121, which contains scorable marker, beta-glucuronidase and selectable marker nptII under the CaMV 35S promoter. Infection was most effective when explants were infected with Agrobacterium for 15 min and co-cultivated for 2 days in the co-cultivation medium. Shoots were regenerated directly from cotyledonary leaf explants in the presence of kanamycin (50 microg/ml) and analysed. Southern blot analysis confirmed that transformation had occurred. This method will allow genetic improvement of this crop by the introduction of agronomically important genes.
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PMID:In vitro organogenesis and genetic transformation in popular Cucumis sativus L. through Agrobacterium tumefaciens. 1263 5

An enantioselective HPLC method has been developed and validated for the stereospecific analysis of N-ethyl-3,4-methylenedioxyamphetamine (MDE) and its major metabolites N-ethyl-4-hydroxy-3-methoxyamphetamine (HME) and 3,4-methylenedioxyamphetamine (MDA). These compounds have been analyzed both from human plasma and urine after administration of 70 mg pure MDE-hydrochloride enantiomers to four subjects. The samples were prepared by hydrolysis of the o-glucuronate and sulfate conjugates using beta-glucuronidase/arylsulfatase and solid-phase extraction with a cation-exchange phase. A chiral stationary protein phase (chiral-CBH) was used for the stereoselective determination of MDE, HME and MDA in a single HPLC run using sodium dihydrogenphosphate, ethylendiaminetetraacetic acid disodium salt and isopropanol as the mobile phase (pH 6.44) and fluorimetric detection (lambda(ex) 286 nm, lambda(em) 322 nm). Moreover, a suitable internal standard (N-ethyl-3,4-methylenedioxybenzylamine) was synthesized and qualified for quantitation purposes. The method showed high recovery rates (>95%) and limits of quantitation for MDE and MDA of 5 ng/ml and for HME of 10 ng/ml. The RSDs for all working ranges of MDE, MDA and HME in plasma and urine, respectively, were less than 1.5%. After validation of the analytical methods in plasma and urine samples pharmacokinetic parameters were calculated. The plasma concentrations of (R)-MDE exceeded those of the S-enantiomer (ratio R:S of the area under the curve, 3.1) and the plasma half time of (R)-MDE was longer than that of (S)-MDE (7.9 vs. 4.0 h). In contrast, the stereochemical disposition of the MDE metabolites HME and MDA was reversed. Concentrations of the (S)-metabolites in plasma of volunteers were much higher than those of the (R)-enantiomers.
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PMID:Enantioselective quantitation of the ecstasy compound (R)- and (S)-N-ethyl-3,4-methylenedioxyamphetamine and its major metabolites in human plasma and urine. 1290 96

A simple, selective and reproducible high-performance liquid chromatographic (HPLC) method via enzymatic hydrolysis of glucuronide conjugates of 4-hydroxy-anethole trithione (ATX) was established for simultaneous determination of ATX. Human plasma samples were hydrolyzed by beta-glucuronidase and followed by subsequent extraction with cyclohexane-isopropanol (95:5, v/v) using mifepristone as the internal standard. Chromatography was carried out on a reverse phase C(18) column (250 mm x 4.6 mm, 5 microm) and kept at 30 degrees C, with UV detection set at 346 nm. The mobile phase consisted of a mixture of methanol and water (75:25, v/v), at a flow rate of 1 ml/min. It was validated and proved to be linear in the range of 20-1500 ng/ml, with the regression equation Y = 0.0016C-0.0069, r=0.9992. And the limit of quantification (LOQ) concentration in plasma was 20 ng/ml. The absolute recoveries of ATX at three concentrations were 32.04, 38.95 and 44.06% and the relative recoveries were 104.80, 102.53 and 107.04%, which showed that the analytical method was sensible, accurate and reproducible. The method was utilized on a double-blind, randomized, single dose, two period, and crossover bioequivalence study of ATT tablets produced by different companies in 20 healthy male Chinese subjects, with a washout between every two periods. Blood samples were collected over each period of 10h and various pharmacokinetic parameters were determined. Natural log-transformed values were compared by analysis of variance followed by classical 90% confidence interval for C(max), AUC(0-t) and AUC(0-infinity) and was found to be within the range, which indicated that the two products were bioequivalence.
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PMID:HPLC determination of 4-hydroxy-anethole trithione in plasma via enzymatic hydrolysis and its application to bioequivalence study. 1835 92

To investigate the transcriptional regulation of gene expression, chimeric fusions, between the beta-glucuronidase reporter gene (GUS) and the isolated promoter regions of the pvPDF gene (pvPDF-PRO: GUS), were constructed and introduced into Nicotiana tabacum. Analysis of transgenic pvPDF-PRO:GUS tobacco plants indicated that GUS activity was observed with all the promoter constructs with the strongest being in leaf followed by stem and roots. These results clearly demonstrate that pvPDF-PRO is a strong inducible and near-constitutive promoter and emphasize the great application potential for plant genetic engineering studies. Interestingly, a search for putative cis-acting elements in the pvPDF promoter architecture revealed the presence of some important transcription regulatory elements including: CAAT Box, TATA Box, CATA Box, and light regulatory elements (CCA1, GATA, GT-1). Taken together, these results further our understanding of the regulation of the pvPDF promoter activity.
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PMID:Cloning of a novel antifungal promoter from Phaseolus vulgaris and the determination of its activity in stably transformed Nicotiana tabacum plants. 1919 65


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