Beta-glucuronidase Activity Research Articles

Analysis of a new begomovirus unveils a composite element conserved in the CP gene promoters of several Geminiviridae genera: Clues to comprehend the complex regulation of late genes.

A novel bipartite begomovirus, Blechum interveinal chlorosis virus (BleICV), was characterized at the genome level. Comparative analyses revealed that BleICV coat protein (CP) gene promoter is highly divergent from the equivalent region of other begomoviruses (BGVs), with the single exception of Tomato chino La Paz virus (ToChLPV) with which it shares a 23-bp phylogenetic footprint exhibiting dyad symmetry. Systematic examination of the homologous CP promoter segment of 132 New World BGVs revealed the existence of a quasi-palindromic DNA segment displaying a strongly conserved ACTT-(N7)-AAGT core. The spacer sequence between the palindromic motifs is constant in length, but its sequence is highly variable among viral species, presenting a relaxed consensus (TT)GGKCCCY, which is similar to the Conserved Late Element or CLE (GTGGTCCC), a putative TrAP-responsive element. The homologous CP promoter region of Old World BGVs exhibited a distinct organization, with the putative TATA-box overlapping the left half of the ACTT-N7 composite element. Similar CP promoter sequences, dubbed “TATA-associated composite element” or TACE, were found in viruses belonging to different Geminiviridae genera, hence hinting unsuspected evolutionary relationships among those lineages. To get cues about the TACE function, the regulatory function of the CLE was explored in distinct experimental systems. Transgenic tobacco plants harboring a GUS reporter gene driven by a promoter composed by CLE multimers expressed high beta-glucuronidase activity in absence of viral factors, and that expression was increased by begomovirus infection. On the other hand, the TrAP-responsiveness of a truncated CP promoter of Tomato golden mosaic virus (TGMV) was abolished by site-directed mutation of the only CLE present in it, whereas the artificial addition of one CLE to the -125 truncated promoter strongly enhanced the transactivation level in tobacco protoplasts. These results indicate that the CLE is a TrAP-responsive element, hence providing valuable clues to interpret the recurrent association of the CLE with the TACE. On the basis of the aforesaid direct evidences and the insights afforded by the extensive comparative analysis of BleICV CP promoter, we propose that the TACE might be involved in the TrAP-mediated derepression of CP gene in vascular tissues.

Investigation of the AQP Family in Soybean and the Promoter Activity of TIP2;6 in Heat Stress and Hormone Responses.

Aquaporins (AQPs) are one diverse family of membrane channel proteins that play crucial regulatory roles in plant stress physiology. However, the heat stress responsiveness of AQP genes in soybean remains poorly understood. In this study, 75 non-redundant AQP encoding genes were identified in soybean. Multiple sequence alignments showed that all GmAQP proteins possessed the conserved regions, which contained 6 trans-membrane domains (TM1 to TM6). Different GmAQP members consisted of distinct Asn-Pro-Ala (NPA) motifs, aromatic/arginine (ar/R) selectivity filters and Froger’s positions (FPs). Phylogenetic analyses distinguished five sub-families within these GmAQPs: 24 GmPIPs, 24 GmTIPs, 17 GmNIPs, 8 GmSIPs, and 2 GmXIPs. Promoter cis-acting elements analyses revealed that distinct number and composition of heat stress and hormone responsive elements existed in different promoter regions of GmAQPs. QRT-PCR assays demonstrated that 12 candidate GmAQPs with relatively extensive expression in various tissues or high expression levels in root or leaf exhibited different expression changes under heat stress and hormone cues (abscisic acid (ABA), l-aminocyclopropane-l-carboxylic acid (ACC), salicylic acid (SA) and methyl jasmonate (MeJA)). Furthermore, the promoter activity of one previously functionally unknown AQP gene-GmTIP2;6 was investigated in transgenic Arabidopsis plants. The beta-glucuronidase (GUS) activity driven by the promoter of GmTIP2;6 was strongly induced in the heat- and ACC-treated transgenic plants and tended to be accumulated in the hypocotyls, vascular bundles, and leaf trichomes. These results will contribute to uncovering the potential functions and molecular mechanisms of soybean GmAQPs in mediating heat stress and hormone signal responses.

A basic helix-loop-helix 104 (bHLH104) protein functions as a transcriptional repressor for glucose and abscisic acid signaling in Arabidopsis

Transduction of glucose (Glc) signaling is critical for plant development, metabolism, and stress responses. However, identifying initial Glc sensing and response stimulating mechanisms in plants has been difficult due to dual functions of glucose as energy sources and signaling component. A basic Helix-Loop-Helix 104 (bHLH104) protein is a homolog of bHLH34 previously isolated from Arabidopsis that functions as a transcriptional activator of Glc and abscisic acid (ABA) responses. In this study, we characterized bHLH104 as a transcription factor that binds to the regulatory region of Arabidopsis Plasma membrane Glc-responsive Regulator (AtPGR) gene. The bHLH104 binds to 5′-AANA-3′ element of the promoter region of AtPGR in vitro and represses beta-glucuronidase (GUS) activity in AtPGR promoter-GUS transgenic plants. Genetic approaches show that bHLH104 positively regulates Glc and abscisic acid (ABA) response. These results suggest that bHLH104 is involved in Glc- and ABA-mediated signaling pathway. Taken together, these findings provide evidence that bHLH104 is an important transcription regulator in plant-sensitivity to Glc and ABA signaling.

Prevalence and potential virulence of Escherichia coli in ready-to-eat raw mixed vegetable salads in collective catering in Abidjan, Côte d’Ivoire

Purpose Vegetable salads, despite their recognized health benefits, are an increasingly common cause of foodborne illness worldwide. The purpose of this paper is to determine the prevalence of E. coli with virulence genes in ready-to-eat raw mixed vegetable salads sold in collective catering in Abidjan. Design/methodology/approach A total of 436 strains of E. coli were isolated from 306 ready-to-eat raw mixed vegetables salads and then identified biochemically and molecularly based on the uidA gene responsible for beta-glucuronidase activity. The virulence genes were determined by polymerase chain reaction. Findings The prevalence in vegetable salads of E. coli with virulence genes was 35.3 percent. The distribution of pathovars was 21.2 percent enterotoxigenic (ETEC), 4.9 percent enteropathogenic (EPEC), 0.7 percent Shigatoxigenic (STEC), and 7.5 percent Enteroaggregative E. coli (EAEC). It appears from the study that vegetable salads sold in collective catering in Abidjan are at risk for contamination by E. coli pathovars. Originality/value Processing conditions for these salads during preparation appear to be hygienically insufficient, so measures to control the risk of contamination are necessary.

Arabidopsis Basic Helix-Loop-Helix 34 (bHLH34) Is Involved in Glucose Signaling through Binding to a GAGA Cis-Element.

The modulation of glucose (Glc) homeostasis and signaling is crucial for plant growth and development. Nevertheless, the molecular signaling mechanism by which a plant senses a cellular Glc level and coordinates the expression of Glc-responsive genes is still incompletely understood. Previous studies have shown that Arabidopsis thaliana plasma membrane Glc-responsive regulator (AtPGR) is a component of the Glc-responsive pathway. Here, we demonstrated that a transcription factor bHLH34 binds to 5′-GAGA-3′ element of the promoter region of AtPGR in vitro, and activates beta-glucuronidase (GUS) activity upon Glc treatment in AtPGR promoter-GUS transgenic plants. Gain- and loss-of-function analyses suggested that the bHLH34 involved in the responses to not only Glc, but also abscisic acid (ABA) and salinity. These results suggest that bHLH34 functions as a transcription factor in the Glc-mediated stress responsive pathway as well as an activator of AtPGR transcription. Furthermore, genetic experiments revealed that in Glc response, the functions of bHLH34 are different from that of a bHLH104, a homolog of bHLH34. Collectively, our findings indicate that bHLH34 is a positive regulator of Glc, and may affect ABA or salinity response, whereas bHLH104 is a negative regulator and epistatic to bHLH34 in the Glc response.

Sugar industry sponsorship of germ-free rodent studies linking sucrose to hyperlipidemia and cancer: An historical analysis of internal documents.

In 1965, the Sugar Research Foundation (SRF) secretly funded a review in the New England Journal of Medicine that discounted evidence linking sucrose consumption to blood lipid levels and hence coronary heart disease (CHD). SRF subsequently funded animal research to evaluate sucrose’s CHD risks. The objective of this study was to examine the planning, funding, and internal evaluation of an SRF-funded research project titled “Project 259: Dietary Carbohydrate and Blood Lipids in Germ-Free Rats,” led by Dr. W.F.R. Pover at the University of Birmingham, Birmingham, United Kingdom, between 1967 and 1971. A narrative case study method was used to assess SRF Project 259 from 1967 to 1971 based on sugar industry internal documents. Project 259 found a statistically significant decrease in serum triglycerides in germ-free rats fed a high sugar diet compared to conventional rats fed a basic PRM diet (a pelleted diet containing cereal meals, soybean meals, whitefish meal, and dried yeast, fortified with a balanced vitamin supplement and trace element mixture). The results suggested to SRF that gut microbiota have a causal role in carbohydrate-induced hypertriglyceridemia. A study comparing conventional rats fed a high-sugar diet to those fed a high-starch diet suggested that sucrose consumption might be associated with elevated levels of beta-glucuronidase, an enzyme previously associated with bladder cancer in humans. SRF terminated Project 259 without publishing the results. The sugar industry did not disclose evidence of harm from animal studies that would have (1) strengthened the case that the CHD risk of sucrose is greater than starch and (2) caused sucrose to be scrutinized as a potential carcinogen. The influence of the gut microbiota in the differential effects of sucrose and starch on blood lipids, as well as the influence of carbohydrate quality on beta-glucuronidase and cancer activity, deserve further scrutiny.

Beta-glucuronidase activity in dried blood spots: Reduced technique with biochemical parameters determined

IntroductionMucopolysaccharidoses (MPS) occur due to deficiency in the activity of enzymes that catalyze the breakdown of glycosaminoglycans. MPS VII is caused by deficiency of the beta-glucuronidase enzyme (GUSB). ObjectivesThis study aimed to enhance the technique to measure GUSB activity by reducing the amount of reagents and the size of the DBS, as well as to determine some biochemical parameters of enzyme of healthy individuals. MethodsThe measurement of GUSB in 3 and 1.2mm DBS (with reagents reduced 2.5- and fourfold) was correlated and the precision of the technique was tested. Optimal pH, Km and Vmax, and thermostability parameters were determined and time and temperature of sample storage were established. ResultsThe correlations among the techniques were significant. Although the correlation coefficient was similar, fourfold reduction was selected. pH4.4 had the highest enzyme activity. GUSB's Km was 1.25mM, while Vmax was 594.48nmol/h/mL. After pre-incubation of the sample at 60°C, its activity dropped from 100% to 15.8% at 120min. GUSB activity significantly decreased after 45days of storage at 4, 25, and 37°C. ConclusionsThis research allowed a previously described technique for MPS VII diagnosis to be adapted for smaller amounts of sample and reagents. That will facilitate the use of smaller amounts of samples, which may be used for other techniques and to save material. Given the importance of early MPS VII diagnosis due to the severity of the disease, using reliable diagnostic techniques in DBS is essential.

Regulation of Arabidopsis thaliana plasma membrane glucose-responsive regulator (AtPGR) expression by A. thaliana storekeeper-like transcription factor, AtSTKL, modulates glucose response in Arabidopsis

Biochemical, genetic, physiological, and molecular research in plants has demonstrated a central role of glucose (Glc) in the control of plant growth, metabolism, and development, and has revealed networks that integrate light, stresses, nutrients, and hormone signaling. Previous studies have reported that AtPGR protein as potential candidates for Glc signaling protein. In the present study, we characterized transcription factors that bind to the upstream region of the AtPGR gene isolated using the yeast one-hybrid screening with an Arabidopsis cDNA library. One of the selected genes (AtSTKL) appeared to confer elevated sensitivity to Glc response. Overexpression of AtSTKLs (AtSTKL1 and AtSTKL2) increased the sensitivity to Glc during the post-germination stages. In contrast, atstkl1 and atstkl2 antisense lines displayed reduced sensitivity to high Glc concentration during the early seedling stage. Furthermore, we showed that the two AtSTKLs bind to the 5′-GCCT-3′ element of the upstream promoter region of the AtPGR gene in vitro and repress the beta-glucuronidase (GUS) activity in AtPGR promoter-GUS (P999-GUS) transgenic plants. Green fluorescent protein (GFP)-tagged AtSTKLs were localized in the nuclei of transgenic Arabidopsis cells. Collectively, these results suggest that AtSTKL1 and AtSTKL2 function both as repressors of AtPGR transcription and as novel transcription factors in the Glc signaling pathway.

Synthesis and Antitumor Properties of BQC-Glucuronide, a Camptothecin Prodrug for Selective Tumor Activation.

Major limitations of camptothecin anticancer drugs (toxicity, nonselectivity, water insolubility, inactivation by human serum albumin) may be improved by creating glucuronide prodrugs that rely on beta-glucuronidase for their activation. We found that the camptothecin derivative 5,6-dihydro-4H-benzo[de]quinoline-camptothecin (BQC) displays greater cytotoxicity against cancer cells than the clinically used camptothecin derivatives SN-38 and topotecan even in the presence of human serum albumin. We synthesized the prodrug BQC-glucuronide (BQC-G), which was 4000 times more water soluble and 20-40 times less cytotoxic than BQC. Importantly, even in the presence of human serum albumin, BQC-G was efficiently hydrolyzed by beta-glucuronidase and produced greater cytotoxicity (IC50 = 13 nM) than camptothecin, 9-aminocamptothecin, SN-38, or topotecan (IC50 > 3000, 1370, 48, and 28 nM, respectively). BQC-G treatment of mice bearing human colon cancer xenografts with naturally or artificially elevated beta-glucuronidase activity produced significant antitumor activity, showing that BQC-G is a potent prodrug suitable for selective intratumoral drug activation.

Wheat TaSP gene improves salt tolerance in transgenic Arabidopsis thaliana

A novel salt-induced gene with unknown functions was cloned through analysis of gene expression profile of a salt-tolerant wheat mutant RH8706-49 under salt stress. The gene was named Triticum aestivum salt-related protein (TaSP) and deposited in GenBank (Accession No. KF307326). Quantitative polymerase chain reaction (qPCR) results showed that TaSP expression was induced under salt, abscisic acid (ABA), and polyethylene glycol (PEG) stresses. Subcellular localization revealed that TaSP was mainly localized in cell membrane. Overexpression of TaSP in Arabidopsis could improve salt tolerance of 35S::TaSP transgenic Arabidopsis. 35S::TaSP transgenic Arabidopsis lines after salt stress presented better physiological indexes than the control group. In the non-invasive micro-test (NMT), an evident Na+ excretion was observed at the root tip of salt-stressed 35S::TaSP transgenic Arabidopsis. TaSP promoter was cloned, and its beta-glucuronidase (GUS) activities before and after ABA, salt, cold, heat, and salicylic acid (SA) stresses were determined. Full-length TaSP promoter contained ABA and salt response elements.

Effects of epidermal growth factor receptor inhibitor on proliferative cholangitis in hepatolithiasis

There is currently no effective medication to prevent stone recurrence after choledochoscopic lithotomy or to treat proliferative cholangitis (PC), which is the pathologic basis of hepatolithiasis. This study aimed to investigate whether gefitinib, an epidermal growth factor receptor (EGFR) inhibitor, inhibited cholangio hyperplasia and lithogenesis in PC. After cholangioscopic lithotomy, indwelling catheters were placed in the diseased bile duct lumens in 94 patients with hepatolithiasis. Subsequently, 49 of the 94 patients were treated with 250 mg gefitinib solution via a catheter twice a week, and they were subjected to choledochoscopic biopsy at 6 and 12 weeks. The rest 45 hepatolithiasis patients without gefitinib treatment served as controls. The expressions of EGFR, PCNA and procollagen I were significantly reduced in the patients treated with gefitinib in 12 weeks compared with those in the control group. Patients in the gefitinib group had a much lower degree of hyperplasia of the biliary epithelium, submucosal glands and collagen fibers compared with those in the control group. Gefitinib treatment significantly decreased mucin 3 expression and beta-glucuronidase activity. Postoperative gefitinib treatment could significantly inhibit PC-mediated hyperplasia and lithogenesis, which might provide a novel strategy for the prevention of biliary restenosis and stone recurrence in patients with hepatolithiasis.

Analysis of a taurine-dependent promoter in Sinorhizobium meliloti that offers tight modulation of gene expression.

BackgroundGenetic models have been developed in divergent branches of the class Alphaproteobacteria to help answer a wide spectrum of questions regarding bacterial physiology. For example, Sinorhizobium meliloti serves as a useful representative for investigating rhizobia-plant symbiosis and nitrogen fixation, Caulobacter crescentus for studying cell cycle regulation and organelle biogenesis, and Zymomonas mobilis for assessing the potentials of metabolic engineering and biofuel production. A tightly regulated promoter that enables titratable expression of a cloned gene in these different models is highly desirable, as it can facilitate observation of phenotypes that would otherwise be obfuscated by leaky expression.ResultsWe compared the functionality of four promoter regions in S. meliloti (ParaA, PtauA, PrhaR, and PmelA) by constructing strains carrying fusions to the uidA reporter in their genomes and measuring beta-glucuronidase activities when they were induced by arabinose, taurine, rhamnose, or melibiose. PtauA was chosen for further study because it, and, to a lesser extent, PmelA, exhibited characteristics suitable for efficient modulation of gene expression. The levels of expression from PtauA depended on the concentrations of taurine, in both complex and defined media, in S. meliloti as well as C. crescentus and Z. mobilis. Moreover, our analysis indicated that TauR, TauC, and TauY are each necessary for taurine catabolism and substantiated their designated roles as a transcriptional activator, the permease component of an ABC transporter, and a major subunit of the taurine dehydrogenase, respectively. Finally, we demonstrated that PtauA can be used to deplete essential cellular factors in S. meliloti, such as the PleC histidine kinase and TatB, a component of the twin-arginine transport machinery.ConclusionsThe PtauA promoter of S. meliloti can control gene expression with a relatively inexpensive and permeable inducer, taurine, in diverse alpha-proteobacteria. Regulated expression of the same gene in different hosts can be achieved by placing both tauR and PtauA on appropriate vectors, thus facilitating inspection of conservation of gene function across species.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-014-0295-2) contains supplementary material, which is available to authorized users.

The flavonoid quercetin induces acute vasodilator effects in healthy volunteers: Correlation with beta-glucuronidase activity

Quercetin exerts vasodilator, antiplatelet and antiproliferative effects and reduces blood pressure, oxidative status and end-organ damage in hypertensive humans and animal models. We hypothesized that oral quercetin might induce vasodilator effects in humans and that they might be related to the deconjugation of quercetin-3-O-glucuronide (Q3GA). Design: double blind, randomized, placebo-controlled trial. Fifteen healthy volunteers (26±5 years, 6 female) were given a capsule containing placebo, 200 or 400mg of quercetin in random order in three consecutive weeks. At 2h a dose-dependent increase in Q3GA was observed in plasma (∼0.4 and 1μM for 200 and 400mg, respectively) with minor levels of quercetin and isorhamnetin. No changes were observed in blood pressure. At 5h quercetin induced and increase in brachial arterial diameter that correlated with the product of the levels of Q3GA by the plasma glucuronidase activity. There was an increase in urinary levels of glutathione but there was no increase in nitrites plus nitrates. Quercetin and isorhamnetin also relaxed human umbilical arteries in vitro while Q3GA was without effect. In conclusions, quercetin exerts acute vasodilator effects in vivo in normotensive, normocholesterolemic human subjects. These results are consistent with the effects being due to the deconjugation of the metabolite Q3GA.

Screening of plant cell culture collection for efficient host species for Agrobacterium-mediated transient expression

Agrobacterium-mediated transient expression is an approach for short-time expression of heterologous genes in plant systems. During the last decade transient expression was regarded as a potent protocol for high scale production of foreign proteins in plants including pharmaceutically valuable proteins. In vitro grown plant cell cultures represent a suitable system for accumulation of heterologous proteins under controlled conditions. Since host characteristics may strongly influence transient expression efficiency, we performed screening of undifferentiated cell cultures for transient expression ability using GUS as a reporter. Analysis of 248 plant species belonging to 49 families from the National Germplasm Bank of the World Flora of the Institute of Cell Biology and Genetic Engineering (Kyiv, Ukraine) allowed for selection of about 50 plant species exhibiting detectable beta-glucuronidase activity.

Comparison of Salivary Beta Glucuronidase Activity in Chronic Periodontitis Patients with and without Diabetes Mellitus

The aim of the study was to estimate the salivary beta glucuronidase (β) activity in patients with chronic periodontitis with and without diabetes mellitus and to evaluate the relationship between Beta Glucuronidase activity and Periodontal clinical parameters. The study consisted of 80 patients of both sexes with age ranging from 20-60 years and they were divided into four groups. Clinical parameters such as Gingival index, Probing depth and Clinical attachment loss were measured. Salivary Beta Glucuronidase activity was measured using spectrophotometer with reagents like phenolphthalein glucuronic acid, phosphate and glycine buffer. The mean BG activity of Group IV (1.17 ± 0.27) was significantly higher than mean BGA levels of Group I, II, III. The p-value was < 0.05. The mean BGA levels of Group III (0.78 ± 0.17) was significantly higher than mean BGA levels of Group I, Group II at 5 % level. There was a significant positive linear relationship between salivary β Glucuronidase level and Probing Depth, clinical attachment level in the experimental Groups. The salivary β Glucuronidase level was higher in Diabetic patients with periodontitis than nondiabetic periodontitis patients.

The effect of leukocyte reduction filters on inflammatory mediator release during coronary artery bypass grafting

Extracorporeal circulation used during coronary artery bypass grafting triggers systemic inflammatory response with neutrophil activation which adversely affects ischaemic/reperfused myocardium. One method of myocardial protection during cardiac surgery is the use of blood cardioplegia. Its protective effect is related to cardiac cooling and metabolism reduction, oxygen supply from erythrocytes, and reactive oxygen species scavenging. However, blood cardioplegia is also associated with myocardial damage induced by undesirable morphotic blood elements. To evaluate the effect of the use of leukocyte reduction filters on the activity of polymorphonuclear neutrophils (PMN) in patients undergoing surgical myocardial revascularisation. PMN activity was evaluated based on measurements of plasma activity of granulocyte enzymes, lysozyme and beta-glucuronidase. We studied 40 patients who underwent myocardial revascularisation using extracorporeal circulation. Patients were randomly assigned to two equal groups: in Group I, blood cardioplegia was administered using leukocyte reduction filters, and in Group II, leukocyte reduction filters were not used for blood cardioplegia. Measurements were performed in plasma of arterial and coronary sinus blood samples collected before aortic clamping, immediately after unclamping, and after 25 min of reperfusion. In addition, blood cardioplegic solution samples were collected in Group I from the lines proximal and distal to the filter during first and last administration. Plasma levels of lysozyme and beta-glucuronidase were determined using previously described methods. We found a significant decrease in PMN count in filtered blood cardioplegic solution during its first administration (0.27 ± 0.07 G/L) compared to samples collected before filter passage (1.73 ± 0.049 G/L). Also during last administration, PMN count in filtered blood cardioplegic solution was decreased compared to samples collected before filter passage (0.66 ± 0.35 G/L vs. 3.64 ± 1.14 G/L, respectively). Significantly lower (p < 0.02) plasma beta-glucuronidase levels were found in arterial blood samples in Group I compared to Group II (5.59 ± 1.63 μg/mL immediately after aortic unclamping and 6.59 ± 1.98 μg/mL after 25 min of reperfusion in Group I vs. 10.19 ± 2.66 and 12.83 ± 1.88 μg/mL, respectively, in Group II). Beta-glucuronidase levels in coronary sinus blood samples collected after aortic unclamping and at the end of reperfusion were significantly higher in Group II compared to Group I (p < 0.04). In Group I, plasma lysozyme levels in arterial and venous blood samples did not show significant changes during the surgery. In contrast, plasma lysozyme level in coronary sinus blood samples at the end of reperfusion in Group II was significantly higher compared to that in pre-clamping samples (p < 0.014). With the use of leukocyte reduction filters, we found significantly lower beta-glucuronidase levels in arterial and coronary sinus blood samples. These findings seem to confirm reduced PMN activation and/or reduced myocardial infiltration by activated PMN. Plasma levels of lysozyme, a characteristic product of PMN degranulation, did not show significant differences between the study groups.

Ssp DnaE split-intein mediated split-Cre reconstitution in tobacco

The Cre/loxP system is increasingly exploited for spatial and temporal gene activation or inactivation. In this study, a novel approach for gene activation using a Cre/loxp system in tobacco is described. As the DnaE intein in Synechocystis sp. strain PCC6803 is capable of catalyzing a protein trans-splicing reaction to assemble a mature protein from two separate precursors, the N- and C-terminal ends of the Cre enzyme, split between Gly190 and Gly191, were fused to N- and C-terminals of the Ssp DnaE split intein,respectively. Subsequently, in-frame fusions of NCre/NInt and CInt/CCre are assembled into the pCAMBIA1300 cloning vector, and used for co-expression, along with the BAR selectable marker gene for BASTA herbicide resistance in tobacco. A Cre-dependent excision recombination event is monitored when tobacco leaf explants are screened for resistance to Basta, but along with absence of beta-glucuronidase activity. Based on herbicide resistance, an efficient recombination event is observed, in vivo Bar activation following co-expression of NCre/NInt and CInt/CCre fusion genes in pCAGUS/BAR transgenic lines. Moreover, the recombination efficiency is comparable to that of intact Cre gene expression. However, no Cre recombination event is observed when only the NCre and CCre genes or the NCre/NInt fusion gene and the CCre genes are co-expressed. Thus, the Ssp DnaE split intein-mediated Cre activity reconstitution observed in this study provides an alternative approach for the traditional Cre/loxP system, and this may aid in achieving dynamic regulation of gene expression in transgenic plants.

Selective Cancer Therapy by Extracellular Activation of a Highly Potent Glycosidic Duocarmycin Analogue

Conventional cancer chemotherapy is limited by systemic toxicity and poor selectivity. Tumor-selective activation of glucuronide prodrugs by beta-glucuronidase in the tumor microenvironment in a monotherapeutic approach is one promising way to increase cancer selectivity. Here we examined the cellular requirement for enzymatic activation as well as the in vivo toxicity and antitumor activity of a glucuronide prodrug of a potent duocarmycin analogue that is active at low picomolar concentrations. Prodrug activation by intracellular and extracellular beta-glucuronidase was investigated by measuring prodrug 2 cytotoxicity against human cancer cell lines that displayed different endogenous levels of beta-glucuronidase, as well as against beta-glucuronidase-deficient fibroblasts and newly established beta-glucuronidase knockdown cancer lines. In all cases, glucuronide prodrug 2 was 1000-5000 times less cytotoxic than the parent duocarmycin analogue regardless of intracellular levels of beta-glucuronidase. By contrast, cancer cells that displayed tethered beta-glucuronidase on their plasma membrane were 80-fold more sensitive to glucuronide prodrug 2, demonstrating that prodrug activation depended primarily on extracellular rather than intracellular beta-glucuronidase activity. Glucuronide prodrug 2 (2.5 mg/kg) displayed greater antitumor activity and less systemic toxicity in vivo than the clinically used drug carboplatin (50 mg/kg) to mice bearing human lung cancer xenografts. Intratumoral injection of an adenoviral vector expressing membrane-tethered beta-glucuronidase dramatically enhanced the in vivo antitumor activity of prodrug 2. Our data provide evidence that increasing extracellular beta-glucuronidase activity in the tumor microenvironment can boost the therapeutic index of a highly potent glucuronide prodrug.

Editorial: The Future of Development

We have investigated the developmental potential of mouse blastocysts cultured under a variety of conditions. A number of parameters were used as criteria for development and differentiation, namely hatching of blastocysts from the zona pellucida and their adhesion to the substratum, outgrowth and polyploidization of trophoblast cells, increase in cell number, protein content, beta-glucuronidase activity, appearance of lactate dehydrogenase A subunits, plasminogen activator production, and delta 5,3 beta hydroxysteroid dehydrogenase activity. Under optimal culture conditions, embryos grew relatively rapidly and expressed all the differentiative markers for which they were tested. Under less supportive conditions, the production of the markers was usually reduced quantitatively; the expression of some markers could also be considerably delayed or even totally prevented. In fact, embryos cultured in the least nutritive medium (one designed to support development only through pre-implantation stages) appeared to be in a state of metabolic quiescence closely resembling that of blastocysts in ovariectomy-induced delay. Overall, the results of our investigations lead us to propose that the expression of each of the aforementioned markers is probably under independent control and subject to intrinsic programming. Finally, the observation that some markers are produced by embryos in suboptimal media whereas others are not, suggests that the minimum metabolic level necessary for expression varies from one marker to another.

β-glucuronidase and β-glucosidase Activity in Stool Specimens of Children with Inflammatory Bowel Disease

The aim of the study was to analyze the differences in the activity of beta-glucuronidase and beta-glucosidase in stool specimens of children with Inflammatory Bowel Diseases (IBD) and healthy subjects. The disease activity was determined according to the PCDAI scale (Crohn disease) and Truelove-Witts scale (Ulcerative colitis). Enzyme activity was determined by spectrophotometry. There was a correlation between the level of beta - glucosidase activity in stool and patient's age in the group of healthy controls, but not in the IBD group. beta-glucosidase activity in IBD and healthy subjects stool specimens did not differ significantly. The activity of beta-glucuronidase in children with IBD was two times lower than in the healthy group and was correlated with age in children with IBD, but not in the group of healthy ones.