Reporter Gene Assay System Research Articles

Detection of Aryl Hydrocarbon Receptor Activation by Some Chemicals in Food Using a Reporter Gene Assay.

The purpose of this study was to examine whether a simple bioassay used for the detection of dioxins (DXNs) could be applied to detect trace amounts of harmful DXN-like substances in food products. To identify substances with possible DXN-like activity, we assessed the ability of various compounds in the environment to bind the aryl hydrocarbon receptor (AhR) that binds specifically to DXNs. The compounds tested included 19 polycyclic aromatic hydrocarbons (PAHs), 20 PAH derivatives (nitrated, halogenated, and aminated derivatives), 23 pesticides, six amino acids, and eight amino acid metabolites. The AhR binding activities (AhR activity) of these compounds were measured using the chemical activated luciferase gene expression (CALUX) reporter gene assay system. The majority of the PAHs exhibited marked AhR activity that increased in a concentration-dependent manner. Furthermore, there was a positive link between AhR activity and the number of aromatic rings in the PAH derivatives. Conversely, there appeared to be a negative correlation between AhR activity and the number of chlorine residues present on halogenated PAH derivatives. However, there was no correlation between AhR activity and the number and position of substituents among nitrated and aminated derivatives. Among the pesticides tested, the indole-type compounds carbendazim and thiabendazole showed high levels of activity. Similarly, the indole compound tryptamine was the only amino acid metabolite to induce AhR activity. The results are useful in understanding the identification and characterization of AhR ligands in the CALUX assay.

MiRNA-100 promotes proliferation of human leukemia cells HL-60 by targeting carboxy-terminal domain small phosphatase-like protein

Objective To investigate the effect of miRNA-100 on the proliferation of human leukemia cells HL-60, and to explore the mechanism of this action. Methods The bioinformatics software and database were applied to predict and analyze target genes of miRNA-100. The vector contained the target gene 3'UTR portion cloned into a luciferase reporter construct. A luciferase reporter assay was performed following co-transfection of small molecular miRNA-100 mimics and target gene wild- type or mutant plasmid into HEK-293T cells. HL-60 cells were transfected with miRNA-100 mimics or anti-miRNA-100. After transfection, Western blot was applied to validate the expression of carboxy-terminal domain small phosphatase-like protein (CTDSPL), and the viability of HL-60 was measured by using cell counting kit (CCK-8) assay at 24 h, 48 h, 72 h, 96 h. Results Online software predicted that CTDSPL was likely to be the target gene of miRNA-100.Dual luciferase reporter gene assay system showed that miRNA-100 could significantly suppress the activity of reporter gene containing CTDSPL 3'-UTR which decreased by about 57.1%(P=0.000 7). Western blot showed that the expression of CTDSPL was increased after being transfected with miRNA-100 antisense oligonucleotides and decreased after being transfected with miRNA-100 mimics.At the same time, the growth rate of cells treated with miRNA-100 mimics or CTDSPL miRNA-100 was increased compared with that in control by CCK-8 test(P<0.05). Conclusions CTDSPL is a downstream target gene of miRNA-100.miRNA-100 can promote leukemia cell proliferation by inhibiting the expression of CTDSPL. Key words: miRNA-100; Carboxy-terminal domain small phosphatase-like protein; Proliferation

Effects of multi-component mixtures of polyaromatic hydrocarbons and heavy metal/loid(s) on Nrf2-antioxidant response element (ARE) pathway in ARE reporter-HepG2 cells.

Exposure to polyaromatic hydrocarbons (PAHs) and heavy metal/loid(s) has been demonstrated to induce an oxidative stress response in mammalian cells. The combined effect of PAHs and heavy metal/loid(s) on the oxidative stress response has not been reported extensively. The Nrf2 antioxidant response pathway plays an important role in cellular antioxidant defense against oxidative stress-induced cell damage. In this study, we have determined the combined effect of four PAHs (benzo[a]pyrene (B[a]P), naphthalene (Nap), phenanthrene (Phe) and pyrene (Pyr)) and three heavy metal/loid(s) (arsenic (As), cadmium (Cd) and lead (Pb)) on the Nrf2 antioxidant pathway using the ARE reporter-HepG2 cell line. The mixture study was carried out for binary, ternary, quaternary and seven-component combinations of PAHs and heavy metal/loid(s). Initially, individual dose responses for the PAHs (B[a]P, Nap, Phe and Pyr) and heavy metal/loid(s) (As, Cd and Pb), as well as their respective concentrations that induced an induction ratio of 1.5 (ECIR1.5), were determined. The luciferase assay system was used to quantify the induction of the Nrf2 antioxidant pathway. The individual dose response study showed that both PAHs and heavy metal/loid(s) activated the Nrf2 antioxidant pathway in ARE reporter-HepG2 cells. Among these chemicals, Cd was the most potent inducer, followed by B[a]P and As. Based on the individual dose response findings, PAHs and heavy metal/loid(s) were mixed at equipotent ratios using a fixed concentration ratio, and the effects of the mixtures of PAHs and heavy metal/loid(s) (binary to seven-component) on the Nrf2 antioxidant pathway were determined. The mixture effects were predicted by using the concentration addition (CA) model. Overall, the results showed that the multi-component mixtures of PAHs and heavy metal/loid(s) induced an oxidative stress response in ARE reporter-HepG2 cells, and that the CA model is an appropriate model to predict the interaction effect of these selected mixtures. A human cell line-based reporter gene assay system was successfully used to determine the mixture effects of two groups of common contaminants on oxidative stress response pathway.

On-chip enzymatic assay for chloramphenicol acetyltransferase using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Herein, we report a chloramphenicol (CAP) acetyltransferase (CAT) activity assay based on self-assembled monolayers on gold as an alternative to conventional CAT reporter gene assay systems, which sometimes require toxic materials and complicated steps that limit their use. A CAP derivative presented on a monolayer was converted to the acetylated CAP by CAT in the presence of acetyl-CoA. The conversion was directly monitored by observing the molecular weight changes in CAP using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. CAT activity was determined under various reaction conditions by changing reaction times, CAT and acetyl-CoA concentrations. As a practical application, we identified gene expression in bacteria that were transformed with pCAT plasmid DNA. Our strategy can provide a simple and rapid assay that eliminates some commonly used but potentially detrimental steps in enzymatic assays, such as radioactive labeling and complicated separation and purification of analytes prior to detection.

Comparative evaluation of different cultivars of Flos Chrysanthemi by an anti-inflammatory-based NF-κB reporter gene assay coupled to UPLC-Q/TOF MS with PCA and ANN

Ethnopharmacological relevanceFlos Chrysanthemi (FC), a commonly used traditional Chinese medicine, has five major cultivars (“Boju”, “Chuju”, “Gongju”, “Hangbaiju” and “Huaiju”) from different sources. However, the active constituents of these cultivars have not been studied or characterized with respect to their bioactivity, which is a serious problem when considering quality and safety. Aim of the studyTo evaluate the differences among the five cultivars of FC, and to establish a method for the standardization and quality control of FC related to its bioactivity. Materials and methodsIn this study, the different ingredients in five cultivars of FC were identified by UPLC-Q/TOF and PCA, and the anti-inflammatory ingredients of FC were predicted and screened by artificial neural network (ANN) and an NF-κB luciferase reporter gene assay system. Using this comprehensive method, we successfully screened the anti-inflammatory markers of different cultivars of FC. ResultsNineteen marker ingredients were confirmed to contribute strongly to the cluster, and eleven compounds in the five cultivars of FC were found to exert potential anti-inflammatory effects. Among these compounds, the NF-κB inhibitor activity of apigenin-7-O-6″-malonyl-glucoside, luteolin-7-O-rutinoside, quercetin-7-O-galactoside, quercetin-3-O-glucoside, apigenin-7-O-rutinoside and apigenin-7-O-glucoside were first reported here. Chlorogenic acid, luteolin-7-O-glucoside, 3,5-dicaffeoylquinic acid and luteolin were confirmed to be the most important anti-inflammatory marker ingredients useful for the quality control of FC. ConclusionsThe proposed efficient and systematic method is helpful for the standardization and quality control of FC. Moreover, this comprehensive strategy may prove to be a powerful technique for the rapid establishment of quality control procedures related to bioactivity for other herbal samples and foods.

Brain-derived neurotrophic factor is a novel target gene of the has-miR-183/96/182 cluster in retinal pigment epithelial cells following visible light exposure.

Light-induced retinal injury is clinically and experimentally well-documented. It may be categorized into three types: Photothermal, photomechanical and photochemical injuries. To date, the variation in the hsa-miR-183/96/182 cluster and its potential target genes in human primary retinal pigment epithelial (RPE) cells, following visible light exposure, has not been reported. In the present study, RPE cells were exposed to 4 h of constant visible light. The expression of the hsa-miR-183/96/182 cluster was determined using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and its potential target genes were investigated. Additionally, hsa-miR-183, hsa-miR-96, hsa-miR-182 and has-miR-183/96/182 mimics were designed and synthesized in vitro, and transfected into the RPE cells. Subsequently, the expression of brain-derived neurotrophic factor (BDNF) mRNA and protein was measured, using RT-qPCR and western blotting, respectively. The regulation of miRNAs to the BDNF gene were then validated using a dual luciferase reporter gene assay system. The expression of hsa-miR-183, hsa-miR-96 and hsa-miR-182 significantly increased in RPE cells following 4 h of visible light exposure, compared with RPE cells that had been exposed to dark conditions (P<0.01). Following RPE cell transfection with mimics, BDNF mRNA and protein expression in the RPE cells was significantly downregulated compared with control RPE cells (P<0.05, P<0.01, respectively). Similarly, the ratio of Renilla luciferase/firefly luciferase significantly decreased in the RPE cells of the mimic + wild type (WT) group compared with cells of the psiCHECK(TM)-2 (a vector lacking the sequence of the BDNF gene), wild type and mimic + mutation groups (P<0.05, P<0.01). The present study suggests that BDNF is a target gene of the has-miR-183-96-182 cluster in RPE cells. The present study suggests an underlying protective mechanism against retinal light injury and may provide a novel target for the prevention and treatment of light-induced retinal injury.

MiR-125b promotes proliferation of human acute myeloid leukemia cells by targeting Bak1

To investigate miR- 125b regulation mechanism by identifying miR-125b target genes and its function in acute myeloid leukemia (AML). The bioinformatics software and database were applied to predict and analyze target genes of miR-125b. The vector contained the target gene 3'-UTR portion cloned into a luciferase reporter construct. A luciferase reporter assay was performed following co-transfection of small molecular miR-125b mimics and target gene wild-type or mutant plasmid into HEK-293T cells. Further in leukemia cell lines NB4 and HL-60, the protein level of target gene was measured by Western blot after overexpression miR-125b. Finally, the viabilities of NB4 and HL-60 cells were measured by CCK-8 assay at 24 h, 48 h, 72 h, 96 h after electroporation. Bcl-2-antagonist/killer 1 (Bak1), a pro-apoptotic gene, was a target gene of miR-125b by software predicts. Reporter vector containing the 3'-UTR Bak1 wild and mutation sites were co-transfected with small molecule analogues of miR-125b in HEK-293T cells. Dual luciferase reporter gene assay system showed that miR-125b significantly suppressesthe reporter gene activity containing Bak1 3'-UTR by about 53.8% (P<0.05), but it didn't suppresses the reporter gene activity containing 3'-UTR Bak1 mutation. Western blot showed that miR-125b mimics significantly down-regulated the expression of Bak1 in human leukemia cell lines NB4 and HL-60. Meanwhile, the growth rate of cells treated with miR-125b obviously increased compared with that in control by CCK-8 test (P<0.05). Our findings strongly indicated that BAK1 was a downstream target gene of miR-125b, and miR-125b promoted proliferation in human AML cells at least partially by targeting Bak1, so we speculated that miR-125b as an oncogene could be a potential therapeutic target for treating AML.

Potencies of Red Seabream AHR1- and AHR2-Mediated Transactivation by Dioxins: Implication of Both AHRs in Dioxin Toxicity

To evaluate species- and isoform-specific responses to dioxins and related compounds (DRCs) via aryl hydrocarbon receptor (AHR) in the red seabream ( Pagrus major ), we constructed a reporter gene assay system. Each expression plasmid of red seabream AHR1 (rsAHR1) and AHR2 (rsAHR2) together with a reporter plasmid containing red seabream CYP1A 5'-flanking region were transfected into COS-7 cells. The cells were treated with graded concentrations of seven DRC congeners including 2,3,7,8-TCDD, 1,2,3,7,8-PeCDD, 1,2,3,4,7,8-HxCDD, 2,3,7,8-TCDF, 2,3,4,7,8-PeCDF, 1,2,3,4,7,8-HxCDF, and PCB126. Both rsAHR1 and rsAHR2 exhibited dose-dependent responses for all the tested congeners. The rsAHR isoform-specific TCDD induction equivalency factors (rsAHR1- and rsAHR2-IEFs) were calculated on the basis of 2,3,7,8-TCDD relative potency derived from the dose-response of each congener. The rsAHR1-IEFs of PeCDD, HxCDD, TCDF, PeCDF, and HxCDF were estimated as 0.17, 0.29, 2.5, 1.5, and 0.27, respectively. For PCB126, no rsAHR1-IEF was given because of less than 10% 2,3,7,8-TCDD maximum response. The rsAHR2-IEFs of PeCDD, HxCDD, TCDF, PeCDF, HxCDF, and PCB126 were estimated as 0.38, 0.13, 1.5, 0.93, 0.20, and 0.0085, respectively. The rsAHR1/2-IEF profiles were different from WHO toxic equivalency factors for fish. In silico docking simulations supported that both rsAHRs have potentials to bind to these congeners. These results suggest that dioxin toxicities may be mediated by both rsAHRs in red seabreams.

Extract of Kuding Tea Prevents High-Fat Diet-Induced Metabolic Disorders in C57BL/6 Mice via Liver X Receptor (LXR) β Antagonism

ObjectiveTo investigate the effects of ilex kudingcha C. J. Tseng (kuding tea), a traditional beverage in China, on the metabolic disorders in C57BL/6 mice induced by high-fat diets.DesignFor the preventive experiment, the female C57BL/6 mice were fed with a standard diet (Chow), high-fat diet (HF), and high-fat diet mixed with 0.05% ethanol extract of kuding tea (EK) for 5 weeks. For the therapeutic experiment, the C57BL/6 mice were fed high-fat diet for 3 months, and then mice were split and EK was given with oral gavages for 2 weeks at 50 mg/day/kg. Body weight and daily food intake amounts were measured. At the end of treatment, the adipocyte images were assayed with a scanning electron microscope, and the fasting blood glucose, glucose tolerance test, serum lipid profile and lipids in the livers were analyzed. A reporter gene assay system was used to test the whether EK could act on nuclear receptor transcription factors, and the gene expression analysis was performed with a quantitative PCR assay.ResultsIn the preventive treatment, EK blocked the body weight gain, reduced the size of the adipocytes, lowered serum triglyceride, cholesterol, LDL-cholesterol, fasting blood glucose levels and glucose tolerance in high-fat diet-fed C57BL/6 mice. In the therapeutic treatment, EK reduced the size of the white adipocytes, serum TG and fasting blood glucose levels in obese mice. With the reporter assay, EK inhibited LXRβ transactivity and mRNA expression of LXRβ target genes.ConclusionWe observed that EK has both preventive and therapeutic roles in metabolic disorders in mice induced with high-fat diets. The effects appear to be mediated through the antagonism of LXRβ transactivity. Our data indicate that kuding tea is a useful dietary therapy and a potential source for the development of novel anti-obesity and lipid lowering drugs.

Quantitative evaluation of the molting hormone activity in coleopteran cells established from the Colorado potato beetle, Leptinotarsa decemlineata

A novel reporter gene assay system using BCIRL-Lepd-SL1, a cell line from the coleopteran Colorado potato beetle (Leptinotarsa decemlineata), was established. Cells were transiently transfected with the reporter plasmid that was composed of a firefly luciferase gene with upstream ecdysone response elements, and an internal control plasmid that constitutively produces Renilla reniformis luciferase. Transfected cells were incubated with various molting hormone agonists, and the activity of these agonists was quantitatively determined by measuring luminescence emission. Transcription-inducing activity for ecdysone, 20-hydroxyecdysone and ponasterone A in terms of EC50 (50% effective concentration) were determined to be 1μM (pEC50=5.99), 68nM (pEC50=7.17) and 1.3nM (pEC50=8.88), respectively. Among tested diacylhydrazine (DAH)-type compounds, 11 compounds were active (pEC50=3.56∼6.41), but two compounds were inactive. The EC50 values were linearly correlated to their binding affinity except for one compound. While several ecdysone reporter systems were developed before that employ dipteran and lepidopteron cell lines, the assay system described here is only the second one that employs a coleopteran cell line. This reporter system will allow screening in high-throughput format for new and more potent molting accelerating compounds that specifically target coleopteran pests.

A Secreted Placental Alkaline Phosphatase-Based Reporter Assay System for Screening of Compounds Acting at an Octopamine Receptor Stably Expressed in a Mammalian Cell Line

Octopamine receptors are attractive insecticide targets. To screen compounds acting at octopamine receptors simply and rapidly, we constructed a chemiluminescent reporter gene assay system that detects secreted placental alkaline phosphatase transcriptionally regulated by the cAMP response element for a silkworm octopamine receptor. This system proved useful in high-throughput screening to develop octopamine receptor-specific insecticides.

Production of bioactive extracellular domain of pig and chicken activin type IIB receptors in Pichia pastoris

Myostatin (MSTN) is a potent negative regulator for skeletal muscle growth, and binds to activin type IIB receptor (ActRIIB) for its cellular signal transduction. Administration of the extracellular domain of ActRIIB (ActRIIB-ECD) improved skeletal muscle growth in laboratory animals, suggesting that ActRIIB-ECD can be a useful pharmacological agent to improve skeletal muscle growth of meat-producing animals. In the current study, pig and chicken ActRIIB-ECDs were produced in the Pichia pastoris GS115, and the recombinant proteins were purified from induced culture media by Ni-NTA affinity chromatography. The digestion of pig and chicken ActRIIB-ECDs with PNGase F and glycoprotein staining demonstrated an N-linked glycosylation of these recombinant proteins. Glycoprotein staining also indicated an additional presence of glycosylation in chicken ActRIIB-ECD. Both the pig and chicken ActRIIB-ECDs were shown to inhibit MSTN activity in a reporter gene assay system in vitro. When MSTN-inhibitory potencies were compared by analyzing EC 50 values, no difference in MSTN-inhibitory potency was observed between the glycosylated and N-deglycosylated forms of pig or chicken ActRIIB-ECD, suggesting that glycosylation does not affect the bioactivity of ActRIIB-ECD. MSTN-inhibitory potency of chicken ActRIIB-ECD was greater ( P < 0.01) than that of pig ActRIIB-ECD. Results of this study demonstrate that bioactive pig and chicken ActRIIB-ECDs can be produced from P. pastoris. In addition, the study indicates that the N-glycosylation status of ActRIIB-ECD does not affect its bioactivity in vitro.

Forced Expression of Heat Shock Protein 27 (Hsp27) Reverses P-Glycoprotein (ABCB1)-mediated Drug Efflux and MDR1 Gene Expression in Adriamycin-resistant Human Breast Cancer Cells

Mutant p53 accumulation has been shown to induce the multidrug resistance gene (MDR1) and ATP binding cassette (ABC)-based drug efflux in human breast cancer cells. In the present work, we have found that transcriptional activation of the oxidative stress-responsive heat shock factor 1 (HSF-1) and expression of heat shock proteins, including Hsp27, which is normally known to augment proteasomal p53 degradation, are inhibited in Adriamycin (doxorubicin)-resistant MCF-7 cells (MCF-7/adr). Such an endogenous inhibition of HSF-1 and Hsp27 in turn results in p53 mutation with gain of function in its transcriptional activity and accumulation in MCF-7/adr. Also, lack of HSF-1 enhances nuclear factor κB (NF-κB) DNA binding activity together with mutant p53 and induces MDR1 gene and P-glycoprotein (P-gp, ABCB1), resulting in a multidrug-resistant phenotype. Ectopic expression of Hsp27, however, significantly depleted both mutant p53 and NF-κB (p65), reversed the drug resistance by inhibiting MDR1/P-gp expression in MCF-7/adr cells, and induced cell death by increased G(2)/M population and apoptosis. We conclude from these results that HSF-1 inhibition and depletion of Hsp27 is a trigger, at least in part, for the accumulation of transcriptionally active mutant p53, which can either directly or NF-κB-dependently induce an MDR1/P-gp phenotype in MCF-7 cells. Upon Hsp27 overexpression, this pathway is abrogated, and the acquired multidrug resistance is significantly abolished so that MCF-7/adr cells are sensitized to Dox. Thus, clinical alteration in Hsp27 or NF-κB level will be a potential approach to circumvent drug resistance in breast cancer.

Evaluation of Relative Potencies for in Vitro Transactivation of the Baikal Seal Aryl Hydrocarbon Receptor by Dioxin-Like Compounds

To evaluate the sensitivity and responses to dioxins and related compounds (DRCs) via aryl hydrocarbon receptor (AHR) in Baikal seals (Pusa sibirica), we constructed an in vitro reporter gene assay system. Baikal seal AHR (BS AHR) expression plasmid and a reporter plasmid containing CYP1A1 promoter were transfected in COS-7 cells. The cells were treated with six representative congeners, and dose-dependent responses were obtained for all the congeners. EC50 values of 2,3,7,8-TCDD, 1,2,3,7,8-PeCDD, 2,3,7,8-TCDF, 2,3,4,7,8-PeCDF, and PCB126 were found to be 0.021, 1.8, 0.16, 2.4, and 2.5 nM, respectively. As the response did not reach the maximal plateau, EC50 value for PCB118 could not be obtained. The TCDD-EC50 for BS AHR was as high as that for dioxin sensitive C57BL/6 mouse AHR. The in vitro dose responses were further analyzed following an established systematic framework and multiple (20, 50, and 80%) relative potencies (REPs) to the maximum TCDD response. The estimates revealed lower REP ranges (20-80%) of PeCDD and PeCDF for BS AHR than for mouse AHR. Average of the 20, 50, and 80% REPs was designated as Baikal seal specific TCDD induction equivalency factor (BS IEF). The BS IEFs of PeCDD, TCDF, PeCDF, PCB126, and PCB118 were estimated as 0.010, 0.018, 0.0078, 0.0059, and 0.00010, respectively. Total TCDD induction equivalents (IEQs) that were calculated using BS IEFs and hepatic concentrations in wild Baikal seals corresponded to only 12-31% of 2005 WHO TEF-derived TEQs. Nevertheless, about 50% of Baikal seals accumulated IEQs over the TCDD-EC50 obtained in this study. This assessment was supported by the enhanced CYP1A1 mRNA expression found in 50% of the specimens contaminated over the TCDD-EC50. These findings suggest that the IEFs proposed from this in vitro assay could be used to predict AHR-mediated responses in wild seals.

Butyrate-rich Colonic Microenvironment Is a Relevant Selection Factor for Metabolically Adapted Tumor Cells

The short chain fatty acid (SCFA) butyrate is a product of colonic fermentation of dietary fibers. It is the main source of energy for normal colonocytes, but cannot be metabolized by most tumor cells. Butyrate also functions as a histone deacetylase (HDAC) inhibitor to control cell proliferation and apoptosis. In consequence, butyrate and its derived drugs are used in cancer therapy. Here we show that aggressive tumor cells that retain the capacity of metabolizing butyrate are positively selected in their microenvironment. In the mouse xenograft model, butyrate-preselected human colon cancer cells gave rise to subcutaneous tumors that grew faster and were more angiogenic than those derived from untreated cells. Similarly, butyrate-preselected cells demonstrated a significant increase in rates of homing to the lung after intravenous injection. Our data showed that butyrate regulates the expression of VEGF and its receptor KDR at the transcriptional level potentially through FoxM1, resulting in the generation of a functional VEGF:KDR autocrine growth loop. Cells selected by chronic exposure to butyrate express higher levels of MMP2, MMP9, α2 and α3 integrins, and lower levels of E-cadherin, a marker for epithelial to mesenchymal transition. The orthotopic model of colon cancer showed that cells preselected by butyrate are able to colonize the animals locally and at distant organs, whereas control cells can only generate a local tumor in the cecum. Together our data shows that a butyrate-rich microenvironment may select for tumor cells that are able to metabolize butyrate, which are also phenotypically more aggressive.

Improving rainbow trout ( Oncorhynchus mykiss) growth by treatment with a fish ( Paralichthys olivaceus) myostatin prodomain expressed in soluble forms in E. coli

Strategies for enhancing skeletal muscle growth of aquaculture species are needed to meet the challenges of the increasing worldwide consumption of fish. One approach appears to be the inhibition of the activity of myostatin (MSTN), a member of the transforming growth factor-β superfamily that acts as a potent negative regulator of skeletal muscle growth. In mammalian vertebrates, MSTN activity is negatively regulated by MSTN prodomain, the N-terminal part of proMSTN cleaved during post-translational MSTN processing, but the role of MSTN prodomain on MSTN activity in fish is less clear. This study was designed to express a fish ( Paralichthys olivaceus) MSTN-1 prodomain in soluble forms in E. coli, to examine its capacity to suppress MSTN activity in vitro, and to investigate the effect of treating fish with the MSTN-1 prodomain on fish growth. It was found that Rosetta-gami 2(DE3)pLysS strains transformed with pET32a or pMALc2x expression constructs carrying the P. olivaceus MSTN-1 prodomain (poMSTNpro) sequence could express soluble, cytoplasmic poMSTNpros. Affinity-purified poMSTNpros inhibited MSTN bioactivity in an in vitro reporter gene assay system. For the first time, it was observed that treating rainbow trout ( Oncorhynchus mykiss) with either of the recombinant poMSTNpros improved the body mass growth of the fish up to 42% without affecting length growth. The results indicate that MSTN-1 prodomain suppresses MSTN activity in fish like it does in mammalian vertebrates. At the same time, the results demonstrate the potential of MSTN-based biotechnology to improve fish growth in aquaculture.

Design and synthesis of 1,4-dihydropyridine derivatives as BACE-1 inhibitors

BACE-1 has been shown to be an attractive therapeutic target in Alzheimer's disease (AD). Using a 1,4-dihydropyridine (DHP) scaffold, we synthesized new inhibitors of BACE-1 by modifying the known BACE inhibitor 2 containing a hydroxyethylamine (HEA) motif. Using structure-based drug design based on computer-aided molecular docking, the isophthalamide ring of 2 was replaced with a 1,4-dihydropyridine ring as a brain-targeting strategy. Several of the new dihydropyridine derivatives were synthesized and their BACE-1-inhibitory activities were evaluated using a cell-based, reporter gene assay system that measures the cleavage of alkaline phosphatase (AP)-APP fusion protein by BACE-1. Most of the 1,4-DHP analogs showed BACE-1-inhibitory activities with IC 50 values in the range 8–30 μM, suggesting that the 1,4-DHP skeleton may be utilized to develop brain-targeting BACE-1 inhibitors.

Abstract B91: Induction of RXR transcriptional activity and consequent upregulation of p21 by 3-amino-6-(3-aminopropyl)-5,6-dihydro-5, 11-dioxo-11H-indeno[1,2-c]isoquinoline dihydrochloride in MCF-7 breast cancer cells

Abstract Retinoid X receptor (RXR) is known to play various roles in embryogenesis, metabolic processes, differentiation, and apoptosis. Recently, RXR has been studied as a target for cancer chemoprevention and treatment. To discover potential cancer chemopreventive agents acting through RXRs, we developed a cell line-based RXR response element (RXRE)-luciferase reporter gene assay system. Following extensive screening, 3-amino-6-(3aminopropyl)-5,6-dihydro-5,11-dioxo-11H-indeno[1,2-c]isoquinoline dihydrochloride (AM6-36) was found to induce RXRE-luciferase activities in a dose-dependent manner. Subsequently, the substance was found to inhibit the proliferation of MCF-7 breast cancer cells in a concentration-dependent manner. Prompted by these results, we examined the underlying mechanism of AM6-36 anti-proliferative activity using MCF-cells as a model. In brief, at lower concentrations, G2/M arrest was observed, and S phase arrest was enhanced at higher concentrations. Based on DNA microarray analysis, AM6-36 was found to up-regulate the expression of CDKN1A, a target gene of RXR, by ∼40-fold. In accordance with this response, the protein level of CDKN1A (p21WAF1/CIP1) was increased in the presence of AM6-36. Induction of p21 by AM6-36 was abrogated following transient transfection of RXR siRNA, demonstrating the effect of AM6-36 on the expression of p21 is closely related to modulation of RXR transcriptional activity. These data suggest AM6-36 is a promising lead compound for the treatment or prevention of breast cancer. A synthetic procedure has been devised for large-scale production, moderate absorption and slow metabolism has been demonstrated with rats (p.o. administration), and more advanced anti-tumor evaluation is underway. (Supported by program project P01 CA48112 awarded by the National Cancer Institute.) Citation Information: Cancer Prev Res 2010;3(1 Suppl):B91.

Polymorphisms of sepiapterin reductase gene alter promoter activity and may influence risk of bipolar disorder

In a previous investigation, we observed altered expression of sepiapterin reductase (SPR) in cultured neural cells chronically exposed to paroxetine. SPR is an enzyme, which catalyzes the final step in the synthesis of tetrahydrobiopterin (BH4). BH4 is an essential cofactor for synthesis of many neurotransmitters including serotonin. Given the pivotal role of SPR in neurotransmitter production, we sought to test the hypothesis that SPR would influence susceptibility to mood disorders and patient response to antidepressants. We tested for association of SPR promoter polymorphisms with antidepressant response in a well-characterized triad cohort of mood disorders. We evaluated the functional effect of these variants using the Dual-Luciferase Reporter Gene Assay System in two independent cell lines. Two promoter single nucleotide polymorphisms (rs1876487 and rs2421095) in SPR were identified that occurred in three distinct haplotypes. We found a statistically significant association of haplotype pair 2,3 with bipolar I disorder [odds ratio: 5.47; 95% confidence interval: (1.68-17.88); P<0.005] and the personality measure self-transcendence (P = 0.020). Moreover, we found preliminary evidence that individuals with haplotype pair 2,3 responded better to the treatment with selective serotonin reuptake inhibitors. Reporter gene assays revealed a 1.4-fold to 1.6-fold decrease in the transcription rate of the two less common haplotypes (2 and 3) compared with haplotype 1, in the two cell lines investigated. This reduced transcription rate for SPR promoter haplotypes 2 and 3 may impact on BH4-mediated neurotransmitter production, thus suggesting a biological process through which SPR gene variants might influence antidepressant response and susceptibility to bipolar disorder.

Current advanced bioluminescence technology in drug discovery

Background:Bioluminescence technology is based on the luciferin–luciferase reaction and is generally well known as a reporter gene assay system that uses firefly luciferase. It has revolutionized the field of transcriptional analysis owing to its usability and quantitative capability. Several methods for transcription analysis have emerged in the past two decades. Recently, novel bioluminescence techniques that differ from typical approaches were developed for the detection of transcriptional regulation or direct protein–protein interactions. Objective: As each method has its own characteristics, this review summarizes the latest bioluminescence methods that are applicable to the field of drug discovery research. Methods: Considering the diversity of related techniques, this review covers several aspects that have been divided into the following classes: variation of reporter gene assays, secretion properties, protein–protein interaction assays in living cells and bioluminescence imaging of living cells. Results/conclusions: The practical application of several luciferins and/or luciferases and the generation of novel applications by incorporating fluorescent molecules into bioluminescence techniques will become increasingly important because bioluminescence technology has a significant potential depending on how we use it.