Endogenous Β-glucuronidase Activity Research Articles

Correlation of endogenous β-glucuronidase activity with differentiation of in vitro cultures of Scutellaria baicalensis

The study examined the relationship between the contents of principal flavones of Scutellaria baicalensis Georgi and the activity of endogenous b-glucuronidase (sGUS) in differentiated and undifferentiated in vitro cultures grown on solid and liquid medium. Using RP-HPLC, it was shown that sGUS activity did not correlate with the increase in biomass of cultures, but depended on tissue differentiation. In hairy roots cultured both on liquid and solid medium, it was ten times higher than in undifferentiated cultures. At the same time, the share of aglycone forms in the total content of flavones during the culture cycle depended on the growing conditions. It was significantly higher on agar medium than on liquid medium. A correlation between sGUS activity and total aglycone content, as well as wogonin content, was observed only in hairy roots grown in the liquid medium.

The Relationship Between Endogenous β-Glucuronidase Activity and Biologically Active Flavones-Aglycone Contents in Hairy Roots of Baikal Skullcap.

Here, we examine the relationship between contents of principal flavones in hairy roots of Scutellaria baicalensis with the activity of the β-glucuronidase (sGUS) enzyme during a culturing cycle. Using RP-HPLC, we show that the highest contents of aglycones, baicalin and wogonin is observed at the growth days 8, 14, and 71 and reach 45, 41, and 62% (based on the total weight of hairy roots of the Baikal skullcap), correspondingly. Their accumulation is accompanied by increase of the sGUS activity, which we determined fluorometrically. Moreover, the enzyme activity is characterized by significant and reasonable correlation only with the wogonin contents. Our results confirm a significant role of sGUS at the final steps of the metabolism in root-specific flavones of Baikal skullcap and suggest how one can optimize the conditions of culturing the hairy roots for biotechnological production of individual flavonoids. For example, at the culturing day 71 wogonin constituted over 80% of all flavones extracted from cells.

Factor affecting the endogenous β-glucuronidase activity in rapeseed haploid cells: How to avoid interference with the Gus transgene in transformation studies

The gus gene is one of the most frequently used reporter genes in transgenic plants. However, this gene can only be used if the selected plant species does not show endogenous GUS activity. Rapeseed ( Brassica napus) microspores and microspore-derived embryos (MDEs) were found to exhibit high activity of endogenous β-glucuronidase which interferes with the expression of bacterial β-glucuronidase that was transferred into these tissues by biolistic transformation. In order to eliminate this background activity from rapeseed MDEs, different pHs of the assay buffer (5.8, 7 and 8) with or without methanol in the reaction buffer and incubation of these tissues at different temperatures (24 °C, 38 °C and 55 °C) were investigated. To avoid this problem in microspores, two incubation temperatures (38 °C and 55 °C) at different periods after GUS assay (4, 24 and 48 h) and in the presence of 1 mM potassium ferricyanide and 1 mM potassium ferrocyanide were tested. The endogenous GUS activity was significantly decreased in transformed and untransformed MDEs, when the phosphate buffer was adjusted to pH 8 and 28% methanol in the reaction solution was used. In rapeseed microspores, use of 1 mM potassium ferricyanide and 1 mM potassium ferrocyanide in the reaction buffer enhanced the expression rate of gus transgene rather than endogenous GUS activity where the high levels of gus transgene expression was observed 4 h after histochemical GUS assay. Incubation of rapeseed microspores and MDEs at 55 °C completely eliminated the endogenous GUS activity. In this study, we also examined changes in endogenous GUS activity in rapeseed MDEs at several stages including the globular, heart, torpedo and cotyledonary stages. The level of endogenous GUS activity was increased 4.33 folds in heart embryos, 6.54 folds in torpedo embryos and 8.5 folds in cotyledonary embryos. Furthermore, the level of GUS activity increased 1.72 folds in MDEs of B. napus in 12-h treatment with 2 μM gibberellic acid.

Construction and Characterization of a Lactose-Inducible Promoter System for Controlled Gene Expression in Clostridium perfringens

Clostridium perfringens is a Gram-positive anaerobic pathogen which causes many diseases in humans and animals. While some genetic tools exist for working with C. perfringens, a tightly regulated, inducible promoter system is currently lacking. Therefore, we constructed a plasmid-based promoter system that provided regulated expression when lactose was added. This plasmid (pKRAH1) is an Escherichia coli-C. perfringens shuttle vector containing the gene encoding a transcriptional regulator, BgaR, and a divergent promoter upstream of gene bgaL (bgaR-P(bgaL)). To measure transcription at the bgaL promoter in pKRAH1, the E. coli reporter gene gusA, encoding β-glucuronidase, was placed downstream of the P(bgaL) promoter to make plasmid pAH2. When transformed into three strains of C. perfringens, pAH2 exhibited lactose-inducible expression. C. perfringens strain 13, a commonly studied strain, has endogenous β-glucuronidase activity. We mutated gene bglR, encoding a putative β-glucuronidase, and observed an 89% decrease in endogenous activity with no lactose. This combination of a system for regulated gene expression and a mutant of strain 13 with low β-glucuronidase activity are useful tools for studying gene regulation and protein expression in an important pathogenic bacterium. We used this system to express the yfp-pilB gene, comprised of a yellow fluorescent protein (YFP)-encoding gene fused to an assembly ATPase gene involved in type IV pilus-dependent gliding motility in C. perfringens. Expression in the wild-type strain showed that YFP-PilB localized mostly to the poles of cells, but in a pilC mutant it localized throughout the cell, demonstrating that the membrane protein PilC is required for polar localization of PilB.

Agrobacterium tumefaciens-transient genetic transformation of Habanero pepper (Capsicum chinense Jacq.) leaf explants

Most of the pepper species of the genus Capsicum have been recalcitrant to efficient Agrobacterium tumefaciens -mediated stable or transient, genetic transformation. In the present work, we optimized a protocol for transient transformation of the Habanero pepper ( Capsicum chinense Jacq.) through the standardization of several experimental factors. These included the age of the plants, the temperature, the length of co-cultivation, the application of a negative (vacuum) and/or a positive (infiltration) pressure, along with micro injection, the use of acetosyringone during the bacterial culturing, and modification of the pH during the GUS assay to eliminate the endogenous β-glucuronidase activity. The standardized protocol, which yielded nearly 55% fully transformed leaf explants, was used to successfully mobilize two empty binary vectors (pCAMBIA2301 and pCAMex), as well as the C. chinense cDNAs encoding the pathogenesis-related protein 10 and esterase, respectively.

Metabolism of quercetin-7- and quercetin-3-glucuronides by an in vitro hepatic model: the role of human β-glucuronidase, sulfotransferase, catechol- O-methyltransferase and multi-resistant protein 2 (MRP2) in flavonoid metabolism

Quercetin-3- and quercetin-7-glucuronides are major products of small intestine epithelial cell metabolism (J. Nutr. 130 (2000) 2765) but it is not known if quercetin glucuronides can be further processed in the liver or if they are excreted directly. Using the HepG2 hepatic cell model, we show that highly purified quercetin-7- and quercetin-3-glucuronides can follow two pathways of metabolism: (i) methylation of the catechol functional group of both quercetin glucuronides (44% of quercetin-7-glucuronide at a rate of 2.6 nmol/hr/10 6 cells, and 32% of quercetin-3-glucuronide at a rate of 1.9 nmol/hr/10 6 cells, over 48 hr) or (ii) hydrolysis of the glucuronide by endogenous β-glucuronidase followed by sulfation to quercetin-3′-sulfate (7% of quercetin-7-glucuronide at a rate of 0.42 nmol/hr/10 6 cells and 10% of quercetin-3-glucuronide at a rate of 0.61 nmol/hr/10 6 cells, over 48 hr). In contrast, quercetin-4′-glucuronide was not metabolised, and interestingly this is not a major product of the small intestine absorption process. The conversion of the quercetin-7- and quercetin-3-glucuronide to the mono-sulfate conjugate shows intracellular deglucuronidation by β-glucuronidase activity, allowing transient contact of the free aglycone with the cellular environment. Inhibition of methylation using a catechol- O-methyltransferase inhibitor shifted metabolism towards sulfation, as indicated by an increase in quercetin-3′-sulfate formation (increase in rate to 1.13 and 1.43 nmol/hr/10 6 cells for quercetin-7-glucuronide and quercetin-3-glucuronide, respectively). Efflux of quercetin metabolites from HepG2 cells (methylated glucuronide and sulfate conjugates) was not altered by verapamil, a p-glycoprotein inhibitor, but efflux was competitively inhibited by MK-571, a multidrug resistant protein inhibitor, indicating a role for multidrug resistant protein in the efflux of quercetin conjugates from HepG2 cells. These results show that HepG2 cells can absorb and turnover quercetin glucuronides and that human endogenous β-glucuronidase activity could modulate the intracellular biological activities of dietary antioxidant flavonoids.

Immunohistological localization of a basic class IV chitinase in Beta vulgaris leaves after infection with Cercospora beticola

The subcellular distribution of a basic class IV chitinase (Ch4) in Cercospora beticola-infected sugar beets has been studied by immunohistological analysis. Ch4 was primarily deposited in extracellular globuli proximal to the necrosis. High levels of Ch4 were observed in the vicinity of the necrotic lesions, low levels were found at distant sites from the necrosis. This plant-pathogen interaction thus gives rise to a local induction of Ch4, whereas no apparent systemic induction of Ch4 was found. After infection with Cercospora, a tolerant sugar beet cultivar accumulated Ch4 faster than a susceptible one, and the protein was spatially distributed over a comparatively larger area in the tolerant variety. In the tolerant beet cultivar symptoms appear a few days later, and the number of necroses are reduced and smaler than in the sensitive cultivar. In uninfected sugar beet leaves, Ch4 is present in low amounts in epidermis, stomata and xylem cells. A specific autofluorescence and a high endogenous β-glucuronidase activity in Cercospora were used to demonstrate that this pathogen did not penetrate the tissue beyond the necrosis.

Detection of endogenous beta-glucuronidase activity in Aspergillus niger.

An endogenous beta-glucuronidase that hydrolyses the chromogenic substrate 5-bromo-4-chloro-3-indolyl-beta-D-glucuronide (X-gluc) in Aspergillus niger is reported. The activity was induced when the fungus was grown in media containing xylan, but was either very low, or absent, when grown on glucose. Endogenous beta-glucuronidase was primarily located in newly formed hyphae, and was apparent at pH values between 3 and 6. Hydrolysis of X-gluc was sensitive to the inhibitor D-saccharic acid 1,4-lactone and was irreversibly inactivated by heating. The bacterial uid A beta-glucuronidase reporter gene was strongly expressed in the hyphae of transformed A. niger but, in contrast to the endogenous activity, the enzyme was also active at pH 7-8.5. Histochemical localization of uidA expression in A. niger, without interference from the endogenous beta-glucuronidase activity, was achieved by staining at this pH.

Detection, expression and specific elimination of endogenous β-glucuronidase activity in transgenic and non-transgenic plants

With the aim of making a system using the GUS gene as a selection gene, the effects of two toxin glucoronides were tested on transgenic tobacco cells ( Nicotiana tabacum L.) containing a β-glucuronidase (GUS) gene from Escherichia coli and on non-transgenic cells. No significant difference in toxicity was observed between transgenic and non-transgenic cells. We found that this most probably was due to the activity of an endogeneous GUS enzyme, which could be detected in all plant species tested, e.g. tobacco, sugar beet ( Beta vulgaris L.), oilseed rape ( Brassica napus L.), pea ( Pisum sativum L.), wheat ( Triticum sativum L.) and rhubarb ( Rheum rhaponticum L.) This indicates that GUS may be ubiquitous to plants contrary to earlier assmptions. The endogenous enzyme is active at pH 4–5 and the activity is eliminated without reducing the introduced GUS activity when pH is elevated. In addition the endogenous GuS can be selectively inhibited at high temperatures. Modifications according to these findings can be employed in standard GUS assays to avoid misinterpretations when the expression of tissue specific promoters is tested.

An improved assay for β-glucuronidase in transformed cells: Methanol almost completely suppresses a putative endogenous β-glucuronidase activity

The bacterial β-glucuronidase (GUS) gene is often introduced into plants as a reporter gene fused to a promoter because of advantages over other reporter genes. However, many plants have non-neglegible levels of endogenous GUS or GUS-like activity which can interfere with the activity originating from the introduced GUS gene, especially if this level is a low one. To eliminate the endogenous GUS activity, we studied the effect of some water-soluble organic solvents on decreasing it and found that addition of methanol at 20% volume to a GUS reaction mixture was very effective, lowering the endogenous activity to 0–15% of the origin in all plant extracts tested. Moreover, methanol at this concentration enhanced by 1.4-fold the activity originating from the introduced GUS gene or GUS purified from E. coli. This method enabled us to determine GUS gene expression more reliably, after introduction of GUS gene fusion into rice protoplasts. In histochemical GUS analysis of transgenic tobacco, this method was also effective for suppressing the endogenous GUS activity.

D-glucaro-1,4-lactone: its excretion in the bile and urine and effect on the biliary secretion of beta-glucuronidase after oral administration in rats.

This experiment was designed to test the hypothesis that orally administered D-glucaro-1,4-lactone might be excreted in the bile and thus suppress the activity of biliary beta-glucuronidase, which is believed to play a key role in the development of pigment gallstones. D-Glucaro-1,4-lactone, 50 to 2,600 mu moles, was fed to adult Sprague-Dawley rats which had a bile fistula and were kept in metabolic cages for bile and urine collection. A total of 21 feeding experiments were carried out. Quantitation of D-glucaro-1,4-lactone and total D-glucaric acid as the sum of D-glucaric acid and its lactones in the bile and urine involved extraction of bile with tetrahexylammonium chloride, adjustment of pH, boiling and determination of percentage inhibition of beta-glucuronidase activity. The maximal velocity of beta-glucuronidase in the bile was also determined by the enzyme kinetic method. The results showed that 11% of administered D-glucaro-1,4-lactone was excreted in the urine and only 0.2% in the bile, with D-glucaro-1,4-lactone accounting for 20% of the total excreted D-glucaric acid. The concentration and excretory rate of total D-glucaric acid and D-glucaro-1,4-lactone in the urine, but not in the bile, were proportional to the amount of D-glucaro-1,4-lactone fed. The mean concentration of D-glucaro-1,4-lactone in the bile after feeding was 0.06 mM, which was capable of suppression of 75% of beta-glucuronidase activity. Oral administration of D-glucaro-1,4-lactone decreased biliary beta-glucuronidase concentration, slowed bile flow rate and hence decreased biliary beta-glucuronidase secretion.(ABSTRACT TRUNCATED AT 250 WORDS)