ABSTRACT
Aberrant activity of enhancer of zeste homolog 2 (EZH2) is associated with a wide range of human cancers. The interaction of EZH2 with embryonic ectoderm development (EED) is required for EZH2's catalytic activity. Inhibition of the EZH2-EED complex thus represents a novel strategy for interfering with the oncogenic potentials of EZH2 by targeting both its catalytic and non-catalytic functions. To date, there have been no reported high-throughput screening (HTS) assays for inhibitors acting at the EZH2-EED interface. In this study, we developed a fluorescence polarization (FP)-based HTS system for the discovery of EZH2-EED interaction inhibitors. The tracer peptide sequences, positions of fluorescein labeling, and a variety of physicochemical conditions were optimized. The high Z' factors (>0.9) at a variety of DMSO concentrations suggested that this system is robust and suitable for HTS. The minimal sequence requirement for the EZH2-EED interaction was determined by using this system. A pilot screening of an in-house compound library containing 1600 FDA-approved drugs identified four compounds (apomorphine hydrochloride, oxyphenbutazone, nifedipine and ergonovine maleate) as potential EZH2-EED interaction inhibitors.
Subject(s)
Enhancer of Zeste Homolog 2 Protein/antagonists & inhibitors , Enhancer of Zeste Homolog 2 Protein/metabolism , High-Throughput Screening Assays/methods , Polycomb Repressive Complex 2/antagonists & inhibitors , Polycomb Repressive Complex 2/metabolism , Protein Multimerization/drug effects , Apomorphine/pharmacology , Enhancer of Zeste Homolog 2 Protein/chemical synthesis , Ergonovine/pharmacology , Fluorescence Polarization , Humans , Hydrogen-Ion Concentration , Limit of Detection , Nifedipine/pharmacology , Oxyphenbutazone/pharmacology , Peptide Fragments/antagonists & inhibitors , Peptide Fragments/chemical synthesis , Peptide Fragments/metabolism , Protein Binding/drug effects , TemperatureABSTRACT
Emerging evidence has demonstrated that genomes are organized into higher-order structures in vivo and long range interactions between genomic regions largely contribute to the regulation of gene expression. Hematopoiesis, orchestrated by the precise spatial regulation and organization of hematopoietic transcription factors, serves as a good model for exploring these issues. The chromosome conformation capture (3C) methodology and its high throughput based technology provide an innovative solution for analyzing the regulation of functional elements through inter-chromosomal and intra-chromosomal interactions, and contacts of functional components in nuclei, thus leading to a more comprehensive understanding of human genome and gene expression. This review focuses on the recent progress of 3C and its derivatives, and their applications in unraveling the mechanisms of transcriptional regulation in hematopoiesis.
Subject(s)
Chromosomes , Genetic Techniques , Hematopoiesis/genetics , Nucleic Acid Conformation , Gene Expression Regulation , Genomics , HumansABSTRACT
Abrin is a plant toxin, which can be easily isolated from the seeds of Abrus precatorius. It may be used as a biological warfare agent. In order to detect abrin in food samples, a two-layer sandwich format enzyme-linked immunosorbent assay based on the monoclonal antibody (mAb) (as capture antibody) and rabbit polyclonal serum (as detecting antibody) was developed and applied for the determination of abrin in some food matrices. The linear range of the mAb was 1-100 µg L(-1) with a detection limit of 0.5 µg L(-1) for abrin in phosphate buffered saline (PBS). The recoveries of abrin from sausage, beer and milk samples ranged 97.5-98.6%, 95.8-98.4% and 94.8-9.6%, respectively, with a coefficient of variation (CV) of 3.7% or less. The newly developed sandwich ELISA using the mAb appears to be a reliable and useful method for detection of abrin in sausage, beer and milk.
Subject(s)
Abrin/analysis , Beer/analysis , Enzyme-Linked Immunosorbent Assay/methods , Food Contamination/analysis , Meat Products/analysis , Milk/chemistry , Toxins, Biological/analysis , Animals , Antibodies, Monoclonal/analysis , Cattle , RabbitsABSTRACT
Mercury ions (Hg(II)) are considered highly toxic and hazardous element even at low levels. The contamination of Hg(II) is a global problem. To develop selective and sensitive technique for the detection of Hg(II) has attracted considerable attention. In this study, a multi-component chemically reactive detection conjugate for determination of Hg(II) has been synthesized and a competitive format assay was proposed. In the technique, the chemically reactive capture conjugate was coated on the plate. The reactive detection conjugate was then captured by the capture conjugate. TMB solution was added and catalyzed by HRP molecules immobilized on AuNPs. Finally, the developed enzymatic signal was measured at 450 nm. The linear range of the assay was 0.35-350 ppb with a detection limit of 0.1 ppb. The average recoveries of Hg(II) from mineral water, tap water and lake water were 100.03%, 103.13% and 102.03%, respectively. All coefficients of variation (CVs) were less than 10%. The results are closely correlated with those from inductively coupled plasma mass spectrometry (ICP-MS), which indicated that the developed technique is a reliable method for and sensitive detection of Hg(II) in water samples.
Subject(s)
Drinking Water/chemistry , Fresh Water/chemistry , Mercury/analysis , Mineral Waters/analysis , Water Pollutants, Chemical/analysis , Animals , Benzidines/chemistry , Binding, Competitive , Calibration , Cattle , Horseradish Peroxidase/chemistry , Humans , Hydrogen-Ion Concentration , Lakes , Limit of Detection , Metal Nanoparticles/chemistry , Serum Albumin, Bovine/chemistry , Silver/chemistry , Spectrophotometry, AtomicABSTRACT
A novel probe based on colloidal gold nanoparticles (AuNPs) modified with goat anti-mouse IgG and horseradish peroxidase (HRP) was synthesized and an enhanced enzyme-linked immunosorbent assay (ELISA) based on the probe was developed. In the assay, the synthesized probe is bound with a monoclonal antibody (McAb) which is competitively bound by coated BSA-ITCBE-Pb(II) on plate and Pb(II) in samples. The HRP, used here for signal amplification catalytically oxidize the substrate and generate optical signals that is related to the concentration of Pb(II) and can be measured spectrophotometrically. For the monodisperse AuNPs having high surface areas, it can be conjugated with more amount of HRP than that of IgG. Therefore, compared with traditional ELISA, the signal amplification of catalytically oxidized substrate was enhanced. The detection limit for this novel modified AuNPs probe-based assay was 9 pg mL(-1). The recoveries obtained by standard Pb(II) addition to real samples, including a commercial mineral water, tap water, and lake water were all from 94.9% to 102.9%. And the coefficient of variation (CV) value of all samples was less than 10%. The results indicated that the enhanced assay gave higher sensitivity and reliable reproducibility. It could provide a general detection format for low-molecular weight contaminants.
