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

The promoter of the nit1 gene, encoding the predominantly expressed isoform of the Arabidopsis thaliana (L.) Heynh. nitrilase isoenzyme family, fused to the beta-glucuronidase gene (uidA) drives beta-glucuronidase expression in the root system of transgenic A. thaliana and tobacco plants. This expression pattern was shown to be controlled developmentally, suggesting that the early differentiation zone of root tips and the tissue surrounding the zone of lateral root primordia formation may constitute sites of auxin biosynthesis in plants. The root system of A. thaliana was shown to express functional nitrilase enzyme. When sterile roots were fed [2H]5-L-tryptophan, they converted this precursor to [2H]5-indole-3-acetonitrile and [2H]5-indole-3-acetic acid. This latter metabolite was further metabolized into base-labile conjugates which were the predominant form of [2H]5-indole-3-acetic acid extracted from roots. When [1-13C]-indole-3-acetonitrile was fed to sterile roots, it was converted to [1-13C]-indole-3-acetic acid which was further converted to conjugates. The results prove that the A. thaliana root system is an autonomous site of indole-3-acetic acid biosynthesis from L-tryptophan.
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PMID:Indole-3-acetic acid is synthesized from L-tryptophan in roots of Arabidopsis thaliana. 976 5

We developed a high-performance liquid chromatography (HPLC) method for quantitating p-hydroxy-N-benzylamphetamine glucuronide (pHBAG) and p-hydroxy-benzphetamine glucuronide (pHBZG), which are urinary metabolites of benzphetamine, in humans. Urine samples were hydrolysed with beta-glucuronidase (EC 3.2.1.31) at 37 degrees C overnight and the treated urine was applied to a solid phase extraction column. After washing the column with water, 0.01 mol/L acetic acid and methanol, pHBA and pHBZ were eluted with dichloromethane:isopropanol:28% ammonium hydroxide (78.4:19.6:2.0 v/v). The eluate was evaporated and the residue was dissolved in acetonitrile: 5 mmol/L 1-pentane sulphonic acid (5:95 v/v) and analysed by HPLC with gradient elution. The amounts of urinary pHBAG and pHBZG excreted by two human subjects after oral administration of 10 mg benzphetamine hydrochloride were determined. About 10-15% of benzphetamine was found to be excreted as pHBAG and pHBZG, and almost all of these metabolites were excreted within 24 h. Urine samples should be collected as early as possible after ingestion of benzphetamine to detect pHBAG and pHBZG.
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PMID:Development of a method for the quantitation of benzphetamine metabolites in human urine by high-performance liquid chromatography. 983 92

We developed a method for simultaneous analysis of benzphetamine (BZ) and its metabolites, p-hydroxy-N-benzylamphetamine (pHBA), p-hydroxybenzphetamine (pHBZ), amphetamine (AP), methamphetamine and p-hydroxymethamphetamine by micellar electrokinetic chromatography (MEKC). Urine samples from 0-15 h (3-h intervals) after oral administration of BZ (10 mg) were hydrolyzed with beta-glucuronidase (EC 3.2.1.31) at 37 degrees C overnight. The treated urine was applied to a solid phase extraction column Bond Elut Certify. After sequentially washing the column with water, 0.1 mol/l acetic acid and methanol, the samples were eluted with dichloromethane:isopropanol:28% ammonium hydroxide=78.4:19.6:2.0 (v/v %). The eluate was evaporated and the residue dissolved in running buffer was analyzed by MEKC. In urine from 0-3 h, AP, pHBZ and pHBA were detected. After that, only pHBA, which is one of the major metabolites of BZ in human urine, could be detected in the urine by the present method. A method for quantitation of pHBA by MEKC is described here. The effects of acetonitrile and sodium dodecyl sulfate in the running buffer of MEKC on the separation of BZ and its metabolites are also reported.
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PMID:Simultaneous analysis of benzphetamine and its metabolites, and quantitation of urinary p-hydroxy-N-benzylamphetamine by micellar electrokinetic chromatography. 985 14

The ethanol extracts of Opuntia ficus-indica fructus (EEOF) and Opuntia ficus-indica stem (EEOS) were prepared and used to evaluate the pharmacological effects of cactus. Both the extracts inhibited the writhing syndrome induced by acetic acid, indicating that they contains analgesic effect. The oral administrations of EEOF and EEOS suppressed carrageenan-induced rat paw edema and also showed potent inhibition in the leukocyte migration of CMC-pouch model in rats. Moreover, the extracts suppressed the release of beta-glucuronidase, a lysosomal enzyme in rat neutrophils. It was also noted that the extracts showed the protective effect on gastric mucosal layers. From the results it is suggested that the cactus extracts contain anti-inflammatory action having protective effect against gastric lesions.
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PMID:Studies on the pharmacological action of cactus: identification of its anti-inflammatory effect. 987 11

The expression of nitrilase in Arabidopsis during the development of the clubroot disease caused by the obligate biotroph Plasmodiophora brassicae was investigated. A time course study showed that only during the exponential growth phase of the clubs was nitrilase prominently enhanced in infected roots compared with controls. NIT1 and NIT2 are the nitrilase isoforms predominantly expressed in clubroot tissue, as shown by investigating promoter-beta-glucuronidase fusions of each. Two peaks of beta-glucuronidase activity were visible: an earlier peak (21 d post inoculation) consisting only of the expression of NIT1, and a second peak at about 32 d post inoculation, which predominantly consisted of NIT2 expression. Using a polyclonal antibody against nitrilase, it was shown that the protein was mainly found in infected cells containing sporulating plasmodia, whereas in cells of healthy roots and in uninfected cells of inoculated roots only a few immunosignals were detected. To determine which effect a missing nitrilase isoform might have on symptom development, the P. brassicae infection in a nitrilase mutant (nit1-3) of Arabidopsis was investigated. As a comparison, transgenic plants overexpressing NIT2 under the control of the cauliflower mosaic virus 35S promoter were studied. Root galls were smaller in nit1-3 plants compared with the wild type. The phenotype of smaller clubs in the mutant was correlated with a lower free indole-3-acetic acid content in the clubs compared with the wild type. Overexpression of nitrilase did not result in larger clubs compared with the wild type. The putative role of nitrilase and auxins during symptom development is discussed.
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PMID:Expression and localization of nitrilase during symptom development of the clubroot disease in Arabidopsis. 1067 30

SYPRO Tangerine stain is an environmentally benign alternative to conventional protein stains that does not require solvents such as methanol or acetic acid for effective protein visualization. Instead, proteins can be stained in a wide range of buffers, including phosphate-buffered saline or simply 150 mM NaCl using an easy, one-step procedure that does not require destaining. Stained proteins can be excited by ultraviolet light of about 300 nm or with visible light of about 490 nm. The fluorescence emission maximum of the dye is approximately 640 nm. Noncovalent binding of SYPRO Tangerine dye is mediated by sodium dodecyl sulfate (SDS) and to a lesser extent by hydrophobic amino acid residues in proteins. This is in stark contrast to acidic silver nitrate staining, which interacts predominantly with lysine residues or Coomassie Blue R, which in turn interacts primarily with arginine and lysine residues. The sensitivity of SYPRO Tangerine stain is similar to that of the SYPRO Red and SYPRO Orange stains - about 4-10 ng per protein band. This detection sensitivity is comparable to colloidal Coomassie blue staining and rapid silver staining procedures. Since proteins stained with SYPRO Tangerine dye are not fixed, they can easily be eluted from gels or utilized in zymographic assays, provided that SDS does not inactivate the protein of interest. This is demonstrated with in-gel detection of rabbit liver esterase activity using alpha-naphthyl acetate and Fast Blue BB dye as well as Escherichia coli beta-glucuronidase activity using ELF-97 beta-D-glucuronide. The dye is also suitable for staining proteins in gels prior to their transfer to membranes by electroblotting. Gentle staining conditions are expected to improve protein recovery after electroelution and to reduce the potential for artifactual protein modifications such as the alkylation of lysine and esterification of glutamate residues, which complicate interpretation of peptide fragment profiles generated by mass spectrometry.
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PMID:Fluorescence detection of proteins in sodium dodecyl sulfate-polyacrylamide gels using environmentally benign, nonfixative, saline solution. 1072 49

The level of indole-3-acetic acid (IAA) was locally modified in cambial tissues of transgenic aspen (Populus tremula L. x Populus tremuloides Michx.). We also demonstrate the use of a linked reporter gene to visualize the expression of the iaa genes. The rate-limiting bacterial IAA-biosynthetic gene iaaM and the reporter gene for beta-glucuronidase (GUS), uidA, were each fused to the cambial-region-specific Agrobacterium rhizogenes rolC promoter and linked on the same T-DNA. In situ hybridization of the iaaM gene confirmed that histochemical analysis of GUS activity could be used to predict iaaM gene expression. Moreover, quantitative fluorometric analysis of GUS activity allowed estimation of the level of de novo production of IAA in transgenic lines carrying a single-copy insert of the iaaM, uidA T-DNA. Microscale analysis of the IAA concentration across the cambial region tissues showed an increase in IAA concentration of about 35% to 40% in the two transgenic lines, but no changes in the radial distribution pattern of IAA compared with wild-type plants. This increase did not result in any changes in the developmental pattern of cambial derivatives or the cambial growth rate, which emphasizes the importance of the radial distribution pattern of IAA in controlling the development of secondary xylem, and suggests that a moderate increase in IAA concentration does not necessarily stimulate growth.
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PMID:Cambial-region-specific expression of the Agrobacterium iaa genes in transgenic aspen visualized by a linked uidA reporter gene. 1085 83

The tomato (Lycopersicon esculentum cv Ailsa Craig) polygalacturonase genes TAPG1 (LYCes;Pga1;2) and TAPG4 (LYCes;Pga1;5) are abundantly expressed in both abscission zones and the pistils of mature flowers. To further investigate the spatial and temporal expression patterns for these genes, the TAPG gene promoters were ligated to beta-glucuronidase (GUS) reporter genes and transformed into tomato. GUS expression with both constructs was similar and entirely consistent with the expression patterns of the native gene transcripts. GUS activity was observed in the weakening abscission zones of the leaf petiole, flower and fruit pedicel, flower corolla, and fruit calyx. In leaf petiole and flower pedicel zones this activity was enhanced by ethylene and inhibited by indole-3-acetic acid. On induction of abscission with ethylene, GUS accumulation was much earlier in TAPG4:GUS than in TAPG1:GUS transformants. Moreover, TAPG4:GUS staining appeared to predominate in the vascular bundles relative to surrounding cortex cells whereas TAPG1:GUS was more evenly distributed across the separation layer. Like the native genes, GUS was also expressed in the stigma. Activity was not apparent in pistils until the flowers had opened and was confined to the stigma and style immediately proximal to it. A minimal promoter construct consisting of a 247-bp 5'-upstream element from TAPG1 was found to be sufficient to direct GUS expression in both abscission zones and the stigma.
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PMID:Analysis of gene promoters for two tomato polygalacturonases expressed in abscission zones and the stigma. 1088 36

An efficient protocol for the establishment of transgenic opium poppy (Papaver somniferum L.) and California poppy (Eschscholzia californica Cham.) root cultures using A. grobacterium rhizogenes is reported. Five strains of A. rhizogenes were tested for their ability to produce hairy roots on wounded opium poppy seedlings and California poppy embryogenic calli. Three of the strains induced hairy root formation on both species, whereas two others either caused the growth of tumorigenic calli or produced no response. To characterize the putative transgenic roots further, explant tissues were co-cultivated with the most effective A: rhizogenes strain (R1000) carrying the pBI121 binary vector. Except for the co-cultivation medium, all formulations included 50 mg l(-1) paromomycin to select for transformants and 200 mg l(-1) timentin to eliminate the Agrobacterium. Four weeks after infection, paromomycin-resistant roots appeared on 92-98% of explants maintained on hormone-free medium. Isolated hairy roots were propagated in liquid medium containing 1.0 mg l(-1) indole-3-acetic acid to promote rapid growth. Detection of the neomycin phosphotransferase gene, high levels of beta-glucuronidase (GUS) transcripts and enzyme activity, and GUS histochemical localization confirmed the integrative transformation of root cultures. Transgenic roots grew faster than wild-type roots, and California poppy roots grew more rapidly than those of opium poppy. With the exception of a less compact arrangement of epidermal cells and more root hairs, transformed roots of both species displayed anatomical features and benzylisoquinoline alkaloid profiles that were virtually identical to those of wild-type roots. Transgenic root cultures of opium poppy and California poppy are a simple, reliable and well-defined model system to investigate the molecular and metabolic regulation of benzylisoquinoline alkaloid biosynthesis, and to evaluate the genetic engineering potential of these important medicinal plants.
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PMID:Agrobacterium rhizogenes-mediated transformation of opium poppy, Papaver somniferum l., and California poppy, Eschscholzia californica cham., root cultures. 1094 28

It is believed that geminiviral DNA replication is coupled to the cell-cycle regulatory complex of the plant cell and that the virus-early (complementary or C sense) gene products REP and REPA may be able to manipulate the regulation of the cycle. In this study, we examined expression from the promoters of Maize streak virus (MSV) in transgenic maize plants and cells to determine whether they showed cell-cycle specificity. Histochemical staining of plant roots containing "long and short" C-sense promoter sequences upstream of the GUS (beta-glucuronidase) reporter gene showed that promoter activity was restricted to the meristematic region of the roots and was enhanced by 2,4-dichlorophenoxy acetic acid (2,4-D) treatment. Analysis of reporter gene and cell-cycle-specific gene transcript levels coupled with flow cytometric data in synchronized transgenic maize cells revealed that all of the MSV promoters showed cell-cycle specificity. The coat protein gene promoter showed highest activity in early G2, whereas the C-sense promoter sequences produced two peaks of activity in the S and G2 cell-cycle phases.
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PMID:Cell-cycle, phase-specific activation of Maize streak virus promoters. 1133 25


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