We designed and constructed an antibiotic screening system by using antibiotic responsive genes as reporters. Plasmid pCS26 carrying a promoterless luminescence reporter, luxCDABE, was used as the vector and the promoter regions of antibiotic responsive genes/operons from Escherichia coli were cloned upstream of the lux reporter to form the first part of the screening reporter array. Random promoter library of Salmonella enterica and Pseudomonas aeruginosa were screened for antibiotic responsive clones which consist of the second part of the screening array. The selected final reporter array responded to different antibiotics in distinct patterns and enabled in vivo high-throughput screening for antibiotics. Unknown antibiotics could, in general, be classified by analyzing the response patterns. This screening system is both sensitive and efficient and should prove to be a useful tool for screening new antibiotic compounds.
Anti-Bacterial Agents ; pharmacology ; Drug Evaluation, Preclinical ; methods ; Gene Expression Regulation, Bacterial ; drug effects ; Promoter Regions, Genetic ; genetics ; Pseudomonas aeruginosa ; drug effects ; genetics ; Salmonella enterica ; drug effects ; genetics

OBJECTIVE: To investigate the effects of DNA hypomethylation on mRNA and protein expression of perforin promotor in T cells. METHODS: T cells were isolated from the peripheral venous blood of healthy donors by density gradient centrifugation. CD4(+) and CD8(+) subsets were isolated using Miltenyi beads and protocols provided by the manufacturer. Where indicated the T cells were stimulated with PHA for 24 h, then treated with 5-azaC for an additional 72 h. Genomic DNA, mRNA, and protein were isolated from untreated and 5-azaC-treated T cells. Purified DNA was treated with sodium bisulfite, the desired sequences were amplified in sequential fragments using nested PCR. The amplified fragments were cloned into bacteria DH5 alpha and 5 independent clones for each of the amplified fragments were sequenced. The expression of perforin was determined using real time RT-PCR and Western blot. RESULTS: The perforin mRNA and protein in the CD4(+) and CD8(+) subsets treated with 5-azaC were significantly higher than those in the untreated subsets (P<0.05). The results of bisulfite genomic sequencing showed that the methylation of perforin promotor was significantly reduced in the treated cells compared with the untreated cells (P<0.05). CONCLUSION The mRNA and protein expression of perforin significantly increases in the CD4(+) and CD8(+) T cells treated with 5-azaC,which is associated with DNA hypomethylation of perforin promoter in T cells.
Adult ; Azacitidine ; pharmacology ; Cells, Cultured ; DNA Methylation ; drug effects ; Humans ; Perforin ; genetics ; Promoter Regions, Genetic ; genetics ; T-Lymphocyte Subsets ; metabolism

OBJECTIVETo study the inhibition effect of HCV NS5A on p53 protein transactivity and its possible mechanism.

METHODSLuciferase reporter gene system was used for the study of p53 transactivity on p21 promoter and electrophorectic mobility-shift assay (EMSA) was applied to observe whether HCV NS5A could suppress the binding ability of p53 protein to its specific DNA sequence.

RESULTSEndogenous p53 protein could stimulate p21 promoter activity, and the relative luciferase activity increased significantly (3.49 x 10(5) vs 0.60 x 10(5), t = 5.92, P<0.01). Exogenous p53 protein also up-regulated p21 promoter driving luciferase expression, comparing to the control group (0.47 x 10(5)), the relative luciferase activity increased (5.63 x 10(5)) obviously (t = 10.12, P<0.01). HCV NS5A protein inhibited both endogenous and exogenous p53 transactivity on p21 promoter in a dose-dependent manner (F > or = 20.71, P<0.01). In the experiment of EMSA, p53 could bind to its specific DNA sequence, but when co-transfected with HCV NS5A expressing vector, the p53 binding affinity to its DNA decreased.

CONCLUSIONHCV NS5A can inhibit p53 protein transactivity on p21 promoter through its inhibiting of p53 binding ability to the specific DNA sequence.

Hepacivirus ; genetics ; Humans ; Promoter Regions, Genetic ; Transcriptional Activation ; drug effects ; Tumor Suppressor Protein p53 ; drug effects ; genetics ; metabolism ; physiology ; Viral Core Proteins ; genetics ; Viral Nonstructural Proteins ; genetics ; pharmacology

OBJECTIVE: To construct a luciferase reporter gene vector carrying human nuclear factor of activated T cells 2 (NFATc2) gene promoter and examine the effects of metformin and lipopolysaccharide (LPS) on the transcriptional activity of NFATc2 gene. METHODS: The promoter sequence of human NFATc2 gene was acquired from UCSC website for PCR amplification. NFATc2 promoter fragment was inserted into pGL3-basic plasmid double cleaved with Kpn Ⅰ and Hind Ⅲ. The resultant recombinant plasmid pGL3-NFATC2-promoter was co-transfected with the internal reference plasmid pRL-TK in 293F cells, and luciferase activity in the cells was detected. Reporter gene vectors of human NFATc2 gene promoter with different fragment lengths were also constructed and assayed for luciferase activity. The changes in transcription activity of NFATc2 gene were assessed after treatment with different concentrations of metformin and LPS for 24 h. We also examined the effect of mutation in RUNX2-binding site in NFATC2 gene promoter on the regulatory effects of metformin and LPS on NFATc2 transcription. RESULTS: We successfully constructed pGL3-NFATc2-promoter plasmids carrying different lengths (2170 bp, 2077 bp, 1802 bp, 1651 bp, 1083 bp, 323 bp) of NFATc2 promoter sequences as verified by enzymatic digestion and sequencing. Transfection of 293F cells with the plasmid carrying a 1651 bp NFATc2 promoter (pGL3-1651 bp) resulted in the highest transcriptional activity of NFATc2 gene, and the luciferase activity was approximately 3.3 times that of pGL3-2170 bp (1.843 ± 0.146 vs 0.547 ± 0.085). Moderate (5 mmol/L) and high (10 mmol/L) concentrations of metformin significantly upregulated the transcriptional activity of pGL3-1651 bp by up to 2.5 and 3 folds, respectively. LPS at different doses also upregulated the transcriptional activity of pGL3-1651 bp by at least 1.6 folds. The mutation in the RUNX2 binding site on pGL3-1651 bp obviously reduced metformin- and LPS-induced enhancement of pGL3-1651bp transcription by 1.7 and 2 folds, respectively. CONCLUSION pGL3-NFATc2-promoter can be transcribed and activated in 293F cells, and LPS and metformin can activate the transcription of pGL3- NFATc2-promoter in a RUNX2-dependent manner.
Core Binding Factor Alpha 1 Subunit/genetics* ; Humans ; Lipopolysaccharides/pharmacology* ; Luciferases/genetics* ; Metformin/pharmacology* ; NFATC Transcription Factors/genetics* ; Promoter Regions, Genetic ; T-Lymphocytes ; Transcription, Genetic/drug effects* ; Transfection

AIMTo investigate the molecular mechanism underlying the effect of linoleic acid on plasminogen activator inhibitor type-1 (PAI-1) expression in HepG2 cells.

METHODSHepG2 cells were exposed to different concentrations of linoleic acid and PAI-1 expression was determined by RT-PCR and colorimetric assay. Luciferase reporter gene plasmids containing four sequentially truncated fragments of the PAI-1 promoter region (-804 to +17) were constructed, and plasmids carrying constructs of Smad binding element (SBE)-site directed deletions in PAI-1 promoter were also generated using overlap extention PCR and transiently transfected into HepG2 cells, the transcriptional activity of PAI-1 was demonstrated by the luciferase activity.The effect of linoleic acid on Smad3 and Smad4 protein levels in cultured HepG2 cells was measured by Western blot analysis.

RESULTS(1) Linoleic acid remarkably increased PAI-1 mRNA expression and transcription in varying concentrations. (2) The level of PAI-1 transcription was gradually decreased induced by linoleic acid when transfected the SBE- site directed-deletions plasmids in PAI-1 promoter at -734/-731. (3) Protein levels of both Smad3 and 4 in HepG2 cells were increased by linoleic acid.

CONCLUSIONLinoleic acid regulated the expression of PAI-1 from transcriptional level in HepG2 cells and SBE involved in the regulation, and both Smads protein and Smad signaling pathway acted main role in this procession.

Gene Expression Regulation ; drug effects ; Hep G2 Cells ; Humans ; Linoleic Acid ; pharmacology ; Plasminogen Activator Inhibitor 1 ; genetics ; Promoter Regions, Genetic ; Signal Transduction ; drug effects ; Smad Proteins ; metabolism

OBJECTIVETo screen the active component of Wuzhuyutang (WZYT, Evodiae prescription) and investigate the regulatory effects of the components in WZYT on the TPH2 promoter, and to explore the possible molecular mechanism of WZYT on migraine.

METHODBy transfecting a TPH2 promoter regulating Red Fluorescent Protein expressing plasmid into PC12 cell, the global fluorescence intensities and calculations of fluorescent cells after components treatment were statistically evaluated.

RESULTDifferent regulatory effects of different components in WZYT with different concentrations on TPH2 promoter were observed.

CONCLUSIONTPH2 promoter drove Red Fluorescent Protein expressing cell line can be used as system screening components targeting TPH2 promoter activity. The possible mechanism of WZYT on migraine may due to its stimulating effects on TPH2 promoter, and promote the synthesis and release of 5-HT in cerebral.

Animals ; Drugs, Chinese Herbal ; chemistry ; pharmacology ; Evodia ; chemistry ; Humans ; Migraine Disorders ; drug therapy ; enzymology ; genetics ; PC12 Cells ; Promoter Regions, Genetic ; drug effects ; Rats ; Tryptophan Hydroxylase ; genetics ; metabolism

BACKGROUNDBoth in vitro and in vivo data have demonstrated the TGFBI gene functions as a putative tumor suppressor and is frequently downregulated in human tumors of different histological types. The hypermethylation of the TGFBI promoter, as one of the main regulatory mechanisms, is associated with TGFBI silencing. In this study, we used a methylation-specific PCR (MSP) method to evaluate the methylation status of the TGFBI promoter in human leukemias.

METHODSReal-time RT-PCR and methylation-specific PCR approaches were performed to define the TGFBI expression and promoter methylation in human leukemia cell lines and clinical samples. Genomic DNA was isolated from peripheral blood mononuclear cells from leukemia patients, bisulfite-converted, and analyzed by the MSP method.

RESULTSHypermethylation of the TGFBI promoter occurred in leukemia cell lines and demethylation treatment reexpressed TGFBI at a substantially increased level in most of leukemia cell lines tested. Furthermore, a much higher level of CpG island methylation and a significantly lower TGFBI expression were also identified in clinical leukemia samples.

CONCLUSIONThe results suggest an important role of promoter methylation in regulating TGFBI expression in leukemia, which provides a useful diagnostic marker for clinical management of human leukemias.

Cell Line, Tumor ; CpG Islands ; genetics ; DNA Methylation ; drug effects ; genetics ; Epigenesis, Genetic ; drug effects ; genetics ; Extracellular Matrix Proteins ; genetics ; Humans ; Leukemia ; epidemiology ; Promoter Regions, Genetic ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Sulfites ; pharmacology ; Transforming Growth Factor beta ; genetics

OBJECTIVETo study the molecular mechanism of cyclooxygenase-2 (COX-2), one of effective ingredient of brucine, in inducing non-small cell lung cancer cell apoptosis.

METHODCOX-2 promoter, transcription factor deletion mutants and COX-2 mRNA 3'-UTR-containing report plasmids were transfected with Renillia to non-small cell lung cancer A549 cell, in order to detect the activity of report gene luciferase and minimum cis-acting element of COX-2 promoter inhibited by brucine. The influence of brucine on IkappaB phosphorylation and the nuclear translocation of p65 were detected by immunoblotting assay.

RESULTBrucine significantly suppressed LPS-induced COX-2 promoter activation, but revealed minor impact on COX-2 mRNA stability. NF-kappaB in the vicinity of COX-2 promoter-262 was an important cis-acting element of brucine for inhibiting the activity of COX-2 promoter. Brucine was found to inhibit the phosphorylation of IkappaBalpha as well as the nuclear translocation of p65.

CONCLUSIONBrucine can improve A549 cells apoptosis by inhibiting the activity of NF-kappaB and the subsequent COX-2 gene expression.

Biological Transport ; drug effects ; Carcinoma, Non-Small-Cell Lung ; genetics ; metabolism ; Cell Line, Tumor ; Cyclooxygenase 2 ; genetics ; Humans ; NF-kappa B ; metabolism ; Phosphorylation ; drug effects ; Promoter Regions, Genetic ; drug effects ; genetics ; RNA Stability ; drug effects ; Strychnine ; analogs & derivatives ; pharmacology

OBJECTIVETo investigate the effect of Lang-chuang-ding Decoction (, LCD) on the expression of DNA methylation of CD70 gene promoter in peripheral blood mononuclear cells (PBMCs) of females with systemic lupus erythematosus (SLE).

METHODSPBMCs isolated from female patients with SLE or healthy donors were cultured and treated with LCD medicated serum or normal serum for 24 or 48 h. The mRNA expressions of CD70 gene in PBMCs were detected by reverse transcription polymerase chain reaction (PCR); the DNA methylation of the CD70 gene promoter region was detected by methylation-specific PCR.

RESULTSAfter treated with medicated serum for 48 h, the mRNA expression levels of CD70 in PBMCs of SLE patients were signifificantly higher than those of healthy donors (P<0.05); the DNA methylation levels of CD70 promoter region in PBMCs of SLE patients treated with medicated serum for 48 h were signifificantly higher than those treated with fetal bovine serum (P<0.01).

CONCLUSIONLCD could inhibit CD70 gene expression in PBMCs of SLE patients by promoting the DNA methylation of CD70 gene promoter.

Adult ; CD27 Ligand ; genetics ; metabolism ; DNA Methylation ; drug effects ; genetics ; Drugs, Chinese Herbal ; pharmacology ; Female ; Gene Expression Regulation ; drug effects ; Humans ; Leukocytes, Mononuclear ; drug effects ; metabolism ; Lupus Erythematosus, Systemic ; drug therapy ; genetics ; Promoter Regions, Genetic ; RNA, Messenger ; genetics ; metabolism

OBJECTIVETo detect aberrant methylation in the promoter region of fetal endometriosis susceptibility gene homeobox-10 (HOXA10) in women with and without folic acid supplementation and explore the effect of folic acid in optimizing intrauterine environment.

METHODSThirty-six cord blood specimens were collected between January, 2010 and December, 2012 from pregnant women with endometriosis, including 22 with folic acid treatment and 15 without. Methylation-specific polymerase chain reaction (MSP) and bisulfite salt modified sequencing (BSP) were employed to detect aberrant methylation of HOXA10 gene in these specimens.

RESULTSThe methylation rate of HOXA10 gene differed significantly between pregnant women with endometriosis taking folic acid and those who did (P<0.05).

CONCLUSIONFolic acid treatment can significantly reduce the methylation rate of fetal endometriosis susceptibility gene HOXA10.

DNA Methylation ; drug effects ; Endometriosis ; genetics ; metabolism ; Female ; Fetus ; metabolism ; Folic Acid ; pharmacology ; therapeutic use ; Homeodomain Proteins ; genetics ; metabolism ; Humans ; Pregnancy ; Promoter Regions, Genetic