Expression of cre recombinase as a reporter of signal transduction in mammalian cells

BACKGROUND: Cell-based reporter assays, which rely on a reporter gene under the control of a regulated promoter, are widely used to screen chemical libraries for novel receptor ligands. Here, we describe a reporter system that is based on ligand-induced DNA recombination to express the reporter gene. This system converts a transient activation of a signal transduction pathway into an amplified, constitutive and heritable expression of the reporter gene. RESULTS: We constructed gene fusions of Cre recombinase and mammalian promoters regulated by calcium, nuclear receptors or cyclic AMP. Reporter systems, comprising a Cre gene fusion and a loxP/reporter gene, were used to study the kinetics and dose responses to compounds that activate or inhibit the corresponding signal transduction pathway. We compared these reporters with conventional reporter systems in which the reporter gene is under the direct control of the responsive promoter. Reporter gene expression of the Cre reporters was greater than that of conventional reporters and could be measured more than a week after adding the stimulus. For all pathways studied here, the dose responses of the Cre reporters are nearly identical to those of conventional reporter systems. CONCLUSIONS: We have shown that Cre recombinase can be regulated by a variety of signal transduction pathways. It should therefore be possible to use receptor ligands to induce phenotypic conversion of mammalian cells for use in a variety of applications. One such application is high-throughput screening, and we developed loxP/luciferase reporter genes that provide an amplified and sustained luminescent response.     

Dual-bioactivity-based liquid chromatography-coupled quadrupole time-of-flight mass spectrometry for NF-κB inhibitors and β2AR agonists identification in Chinese Medicinal Preparation Qingfei Xiaoyan Wan

Traditional Chinese medicine (TCM) preparations have been used as an effective multitarget strategy for the treatment of complex diseases; however, their bioactive constituents are undefined and difficult to identify. In this study, a simple and dual-target method based on ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry combined with dual-bioactive (NF-κB and β₂-adrenergic receptor) luciferase reporter assay systems was developed for the rapid determination of various bioactive compounds of TCM preparations. Qingfei Xiaoyan Wan, a TCM preparation used for the clinical therapy of asthma, was analyzed with this method. Potential anti-inflammatory and spasmolytic constituents were screened using NF-κB and β₂-adrenergic receptor activity luciferase reporter assay systems and simultaneously identified according to the time-of-flight mass spectrometry data. One β₂-adrenergic receptor agonist (ephedrine) and four structural types of NF-κB inhibitors (arctigenin derivatives, cholic acid derivatives, chlorogenic acid, and sinapic acid) were characterized. Tracheloside was considered a new NF-κB inhibitor. Further cytokine and chemokine detection confirmed the anti-inflammatory effects of the potential NF-κB inhibitors. The integration of ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry and dual-bioactive human cell functional evaluation systems proved to be a simple and effective strategy for the rapid screening of various bioactive compounds in TCM preparations used to treat complex diseases.     

Analytical approach for monitoring endocrine-disrupting compounds in urban waste water treatment plants

The presence of endocrine-disrupting compounds in influent and effluent water samples from four waste water treatment plants located in Italy was studied. The estrogen-like activity of the water samples was measured using a chemiluminescent recombinant yeast assay which is based on genetically engineered yeast cells that express the human estrogen receptor. This receptor, once activated, elicits the expression of the reporter gene lac-Z and, consequently, the production of β-galactosidase, which is then measured by chemiluminescence. To control and minimize sample matrix effects, an external control based on a modified yeast strain stably expressing β-galactosidase was developed and also used in the assay. Rapid and sensitive chemiluminescent enzyme immunoassays were also developed and validated for the quantification of 17β-estradiol, estrone, and estriol in waste water samples. Results from both methods were compared with a reference high-performance liquid chromatography and electrospray ionization tandem mass spectrometry (HPLC ESI-MS-MS) method developed for the quantification of natural estrogens. The recombinant yeast assay revealed a significant estrogenic activity in the influent samples, ranging from 80 to 400 pmol/L 17β-estradiol equivalents (EEQ), which was reduced by 70–95 % in the effluent samples. The yeast assay also showed a systematic 20–30 % overestimation of estrogenic activity relative to the HPLC ESI-MS-MS method, suggesting the presence of other compounds in the samples with estrogenic activity. The chemiluminescent enzyme immunoassays showed the presence of estrogens in the influent samples (mean concentrations: 350–450 pmol/L for estrone, 5–100 pmol/L for 17β-estradiol, 25–300 pmol/L for estriol), with significantly lower concentrations detected in the respective effluent samples. The waste water treatment was able to reduce natural estrogen concentrations by 40–95 %, although a high variability was observed. The enzyme immunoassay data correlated well with data obtained by the HPLC ESI-MS-MS method. Although the recombinant yeast assay represents a useful tool for a first-level screening of estrogenic activity due to its simplicity and high analytical throughput, sample matrix effects observed in waste water of industrial origin were found to strongly affect the yeast cells response, even when properly corrected for using the external control, thereby limiting its use to urban waste water. Its integration with chemiluminescent enzyme immunoassays would improve its performance by reducing false negative results, thereby enabling its use in extensive studies monitoring for the presence of endocrine-disrupting compounds in urban treatment plant effluents.     

褶合光谱法测定β-半乳糖苷酶的活性

运用褶合光谱法建立了 β 半乳糖苷酶的最佳褶合光谱以及该酶在最佳测试条件下的线性方程 :Qj=1.90 6× 10 -2 +4 .946× 10 -3 C +1.90 9× 10 -4 C2 ,r =1.0 0 0 0 ;对已瞬时转染 β 半乳糖苷酶表达载体的COS 1细胞提取液样品中的 β 半乳糖苷酶的活性进行了测定 ,并与吸光度值分析法相对照。结果表明 :本法快速、简便、准确 ,在生化分析中有广泛的应用价值     

Luminescent enzyme-linked receptor assay for estrogenic compounds

The analytics of endocrine-disrupting compounds has become a major issue during recent years. Several test systems have been developed for endocrine-disrupting chemicals. Yeast reporter gene assays and MCF-7 cell-based proliferation assays (E-screen) are particularly popular. A correlation of an enzyme-linked receptor assay (ELRA) with a yeast reporter gene assay is shown. In addition, the development of an ultra-sensitive luminescent ELRA with a detection limit of 20 ng/L for 17-estradiol in the sample is reported. Data for real sample analysis are shown in this paper. ELRA characteristics are compared with cell-based assays, and the issue of detection limits is addressed. In this context, the detection limits of the cell-based assays have been claimed to be below the ELRA detection limits. However, it is clarified that the given detection limits for the yeast estrogen screen and the E-screen are usually based on concentrations of 17-estradiol in the well, not in the sample, whereas ELRA detection limits are concentrations in the sample.     

Potent estrogen agonists based on carborane as a hydrophobic skeletal structure. A new medicinal application of boron clusters

BACKGROUND: Carboranes (dicarba-closo-dodecaboranes) are a class of carbon-containing polyhedral boron-cluster compounds having remarkable thermal stability and exceptional hydrophobicity. Applications of the unique structural and chemical properties offered by icosahedral carboranes in boron neutron capture therapy have received increasing attention over the past 30 years. However, these features of carboranes may allow another application as a hydrophobic pharmacophore in biologically active molecules that interact hydrophobically with receptors. RESULTS: We have designed candidate estrogen-receptor-binding compounds having carborane as a hydrophobic skeletal structure and synthesized them. The most potent compound bearing a carborane cage exhibited activity at least 10-fold greater than that of 17beta-estradiol in the luciferase reporter gene assay. Estrogen receptor-alpha-binding data for the compound were consistent with the results of the luciferase reporter gene assay. The compound also showed potent in vivo effects on the recovery of uterine weight and bone loss in ovariectomized mice. CONCLUSION: Further development of the potent carborane-containing estrogenic agonists described here, having a new skeletal structure and unique characteristics, should yield novel therapeutic agents, especially selective estrogen receptor modulators. Furthermore, the suitability of the spherical carborane cage for binding to the cavity of the estrogen receptor-alpha ligand-binding domain should provide a basis for a similar approach to developing novel ligands for other steroid receptors.     

Miniaturization of a panel of high-throughput yeast-cell-based nuclear receptor assays in 384- and 1536-well microplates

A panel of luminescent Saccharomyces cerevisiae cell-based nuclear receptor assays, consisting of human estrogen receptors α and β, androgen receptor, and aryl hydrocarbon receptor, was miniaturized from the standard 96-well microplate format to high-throughput 384- and 1536-well microplate formats. In these assays, firefly luciferase lacking the peroxisome targeting sequence was used as a reporter and D-luciferin substrate was pre-mixed with the yeast cells before the incubation step, eliminating cell lysis and substrate addition steps, and allowing multiple readings at any desired time point. All of the assays were highly functional in the 384-well format, and most functioned well in the 1536-well format. The detection limit of the estrogen receptor α assay was even lower in the miniaturized microplate formats than in the original 96-well format. The panel of yeast-cell-based nuclear receptor assays can be used for high-throughput chemical testing and environmental monitoring of potential endocrine-disrupting activity of compounds and samples.     

Screening for metal ligands by liquid chromatography--ligand-exchange--electrospray mass spectrometry

Electrospray ionization mass spectrometry is applied for the selective detection of metal ligands after a post-column continuous-flow ligand-exchange reaction. The detection is based on the specific release of a reporter ligand from a metal-reporter ligand complex by a high affinity ligand. Constant infusion and direct-injection experiments are performed to optimize the method. The on-line coupling of a liquid chromatographic separation prior to the continuous flow ligand-exchange reaction enables the screening for high affinity ligands in complex samples. The feasibility of the method is demonstrated by using several ligands with a different affinity for either Cu(II) or Zn(II) ions. The selectivity of the ligand-exchange detection method can be tuned by the choice of the reporter ligand. This is demonstrated by using either 2,2'-bipyridyl or 5-methyl-1,10-phenanthroline as reporter ligands.     

Self-Immolative Activation of β-Galactosidase-Responsive Probes for In Vivo MR Imaging in Mouse Models

Our lab has developed a new series of self-immolative MR agents for the rapid detection of enzyme activity in mouse models expressing β-galactosidase (β-gal). We investigated two molecular architectures to create agents that detect β-gal activity by modulating the coordination of water to Gd^III. The first is an intermolecular approach, wherein we designed several structural isomers to maximize coordination of endogenous carbonate ions. The second involves an intramolecular mechanism for q modulation. We incorporated a pendant coordinating carboxylate ligand with a 2, 4, 6, or 8 carbon linker to saturate ligand coordination to the Gd^III ion. This renders the agent ineffective. We show that one agent in particular (6-C pendant carboxylate) is an extremely effective MR reporter for the detection of enzyme activity in a mouse model expressing β-gal.     

Towards rapid on-site phage-mediated detection of generic Escherichia coli in water using luminescent and visual readout

Wild-type T4 bacteriophage and recombinant reporter lac Z T4 bacteriophage carrying the β-galactosidase gene were used for detection of generic Escherichia coli by monitoring the release of β-galactosidase upon phage-mediated cell lysis. The reaction was performed on a paper-based portable culture device to limit the diffusion of reagents and, hence, increase the sensitivity of the assay, and to avoid handling large sample volumes, making the assay suitable for on-site analysis. Chromogenic (chlorophenol red-β-d-galactopyranoside, CPRG) and bioluminescent (6-O-β-galactopyranosyl-luciferin, Beta-Glo^®) β-galactosidase substrates were tested in the assay. Water samples were first filtered through 0.45-μm pore size filters to concentrate bacteria. The filters were then placed into the paper-based device containing nutrient medium and incubated at 37 °C for 4 h. Bacteriophage with the respective indicator substrate was added to the device, and signal (color, luminescence) development was recorded with a digital camera, luminometer, or luminescence imaging device. It was demonstrated that as low as 40 or <10 colony-forming units (cfu)?ml^?1 of E. coli can be detected visually within 8 h when wild-type T4 bacteriophage or recombinant lacZ T4 bacteriophage were used in the assay, respectively. Application of the bioluminescent β-galactosidase substrate allowed reliable detection of <10 cfu ml^?1 within 5.5 h. The specificity of the assay was demonstrated using a panel of microorganisms including Aeromonas hydrophila, Enterobacter cloacae, E. coli, and Salmonella Typhimurium. Scheme for rapid E. coli assay including filtration of water sample, short incubation on the filter in a paper-based culture device, addition of bacteriophage and [beta]-galactosidase substrate, and recording/processing of the accumulated color or luminescence signal.      

Determination of glucose and lactose in food products with the use of biosensors based on Berlin blue

The immobilization of enzymes into polyelectrolyte membranes with the use of organic solvents was applied to the development of the biosensing elements of biosensors. The following domestically produced preparations were used: the enzymes glucose oxidase and β-galactosidase and a perfluorosulfonated polymer. The compositions of mono-and bienzymic polyelectrolyte membranes were optimized. The glucose and lactose biosensors based on Berlin blue (as a signal transducer) and polyelectrolyte membranes exhibited high sensitivity, low detection limits, and fast response. The results of the analysis of milk whey in a flow-injection system that included biosensors completely correlated with measurement data obtained by a standard chromatographic technique.     

Utilization of Cheese Whey Using Synergistic Immobilization of β-Galactosidase and <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> Cells in Dual Matrices

Whey is a byproduct of the dairy industry, which has prospects of using as a source for production of various valuable compounds. The lactose present in whey is considered as an environmental pollutant and its utilization for enzyme and fuel production, may be effective for whey bioremediation. The dairy yeast Kluyveromyces marxianus have the ability to utilize lactose sharply as the major carbon source for the production of the enzyme. Five strains were tested for the production of the β-galactosidase using whey. The maximum β-galactosidase activity of 1.74 IU/mg dry weight was achieved in whey using K. marxianus MTCC 1389. The biocatalyst was further immobilized on chitosan macroparticles and exhibited excellent functional activity at 35 °C. Almost 89 % lactose hydrolysis was attained for concentrated whey (100 g/L) and retained 89 % catalytic activity after 15 cycles of reuse. Finally, β-galactosidase was immobilized on chitosan and Saccharomyces cerevisiae on calcium alginate, and both were used together for the production of ethanol from concentrated whey. Maximal ethanol titer of 28.9 g/L was achieved during fermentation at 35 °C. The conclusions generated by employing two different matrices will be beneficial for the future modeling using engineered S. cerevisiae in scale-up studies.     

Tuning glycosidase inhibition through aglycone interactions: pharmacological chaperones for Fabry disease and GM1 gangliosidosis

Competitive inhibitors of either α-galactosidase (α-Gal) or β-galactosidase (β-Gal) with high affinity and selectivity have been accessed by exploiting aglycone interactions with conformationally locked sp(2)-iminosugars. Selected compounds were profiled as potent pharmacological chaperones for mutant lysosomal α- and β-Gal associated with Fabry disease and GM(1) gangliosidosis.