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At least 1,549 records · Page 86Linked to original sources
PubMed2001

A cGMP-dependent protein kinase assay for high throughput screening based on time-resolved fluorescence resonance energy transfer.

Activation of cyclic GMP-dependent protein kinase (cGK) is an important event in the regulation of blood pressure and platelet function. Upstream signals are the generation of nitric oxide (NO) by NO synthases and the subsequent rise in cyclic GMP levels mediated by NO-dependent guanylyl cyclases (GCs). The identification of new cGK activators by high throughput screening (HTS) may lead to the development of a novel class of therapeutics for the treatment of cardiovascular diseases. Therefore, a homogeneous, nonradioactive assay for cGK activity was developed using a biotinylated peptide derived from vasodilator-stimulated phosphoprotein (VASP), a well-characterized natural cGK substrate. The phosphorylated peptide could be detected by a VASP-specific monoclonal phosphoserine antibody and a fluorescent detection system consisting of a europium-labeled secondary antibody and allophycocyanin (APC)-labeled streptavidin. Fluorescence resonance energy transfer (FRET) from europium to APC was detected in a time-resolved fashion (TR-FRET). Activation and inhibition constants for known substances determined by this new fluorescence-based assay correlated well with published results obtained by conventional radioactive cGK activity assays. The assay proved to be sensitive, robust, highly specific for cGK, and suitable for HTS in 96- and 384-well formats. This assay is applicable to purified enzymes as well as to complex samples such as human platelet extracts.

Alkaloids↗
PubMed2006

Converting a breast cancer microarray signature into a high-throughput diagnostic test.

BACKGROUND: A 70-gene tumor expression profile was established as a powerful predictor of disease outcome in young breast cancer patients. This profile, however, was generated on microarrays containing 25,000 60-mer oligonucleotides that are not designed for processing of many samples on a routine basis. RESULTS: To facilitate its use in a diagnostic setting, the 70-gene prognosis profile was translated into a customized microarray (MammaPrint) containing a reduced set of 1,900 probes suitable for high throughput processing. RNA of 162 patient samples from two previous studies was subjected to hybridization to this custom array to validate the prognostic value. Classification results obtained from the original analysis were then compared to those generated using the algorithms based on the custom microarray and showed an extremely high correlation of prognosis prediction between the original data and those generated using the custom mini-array (p < 0.0001). CONCLUSION: In this report we demonstrate for the first time that microarray technology can be used as a reliable diagnostic tool. The data clearly demonstrate the reproducibility and robustness of the small custom-made microarray. The array is therefore an excellent tool to predict outcome of disease in breast cancer patients.

Biomarkers, Tumor↗
PubMed2004

Microarray TRAP--a high-throughput assay to quantitate telomerase activity.

Telomeric repeat amplification protocol (TRAP)--a sensitive, PCR-based assay to detect telomerase activity was quintessential to the evaluation of telomerase role in telomere maintenance, cell proliferation, tumour development, and cell immortalization. The assay, however, suffers from many limitations. The most significant are: lack of telomerase activity quantification, changes of the enzyme activity product size and/or ratio, and complex post-amplification procedures which limit the assay throughput. Here we report the development of the microarray TRAP (MTRAP) assay which combines advantages of microarray technology with a modified TRAP assay. The MTRAP was designed and optimized on rice cell suspension telomerase extract to enable telomerase specific, reliable, and linear quantification in high throughput mode, with sensitivity comparable to those of radioisotope-based TRAP assays. The MTRAP has a built-in system guaranteeing the amplification of telomerase activity products unchanged in length and/or ratio and built-in control for false negatives. Thus, our MTRAP assay provides new reliable tool for experiments requiring massive quantitation of telomerase activity.

Enzyme Activation↗
PubMed2004

A high-throughput proteo-genomics method to identify antibody targets associated with malignant disease.

Identification of antibody targets associated with malignant disease is indispensable to developing passive and active antibody-based therapeutics or diagnostic agents. We have developed a novel technique combining Western blotting, genetic profiling, and mass spectroscopy that allows for the rapid and unambiguous identification of such antigens in a high-throughput manner. Herein, we demonstrate this technique, designated Ab SCAN, by deducing the known target of a monoclonal antibody and by identifying a new antigen that was observed to be the frequent target of humoral immune responses in prostate cancer patients. In both instances, a specific antigen emerged as the sole protein candidate. The newly identified antigen, mannose-6-phosphate/IGF II receptor, may be an important naturally immunogenic antigen involved in prostate cancer. The Ab SCAN technique is uniquely suited to the analysis of longitudinal serum samples from clinical studies and could be a powerful tool to correlate humoral immune responses directed against discreet antigens with clinical events.

Antibodies, Monoclonal↗
PubMed2005

High-throughput screening of protein surface activity via flow injection analysis-pH gradient-dynamic surface tension detection.

Using flow injection analysis (FIA), a pH gradient is blended in real time with a protein sample as the pH-dependent protein surface activity is measured by a dynamic surface tension detector (FIA-pH-DSTD). This instrumental system was developed as a high-throughput method for the screening of protein surface activity at the air/liquid interface as a function of pH. This method utilizes the continuous flow, drop-based dynamic surface tension detector in combination with flow injection sample introduction and blending of a steady-state concentration of protein sample with a pH gradient ranging from pH 2.0 to pH 11.5. Dynamic surface tension is measured through the differential pressure across the air/liquid interface of repeatedly growing and detaching drops. Continuous surface tension measurement is achieved for each eluting drop of 2-s length (2 muL), providing insight into both the kinetic and thermodynamic behaviors of molecular orientation processes at the liquid/air interface. Three-dimensional data are obtained, with surface tension first converted to surface pressure, which is collected as a function of elution time versus drop time. In FIA-pH-DSTD, a commercial pH probe is used to measure pH during elution time, enabling surface pressure throughout drop time to be subsequently plotted as a function of eluting pH. An automated DSTD calibration procedure and data analysis method is applied, which allows simultaneous use of two different solvents, permitting real-time dynamic surface tension data to be obtained. The method was applied to the analysis of 14 commercial purified proteins, yielding characteristic features of surface activity as a function of pH. The reproducibility of the measurement and selectivity advantage of the DSTD was shown for the analysis of serum albumins from various mammalian sources. Several applications were also suggested and discussed in order to show the potential of the method for protein and food chemistry studies and in the study of protein-polymer interactions.

Journal Article↗
PubMed2004

Gene expression-based high-throughput screening(GE-HTS) and application to leukemia differentiation.

Chemical genomics involves generating large collections of small molecules and using them to modulate cellular states. Despite recent progress in the systematic synthesis of structurally diverse compounds, their use in screens of cellular circuitry is still an ad hoc process. Here, we outline a general, efficient approach called gene expression-based high-throughput screening (GE-HTS) in which a gene expression signature is used as a surrogate for cellular states, and we describe its application in a particular setting: the identification of compounds that induce the differentiation of acute myeloid leukemia cells. In screening 1,739 compounds, we identified 8 that reliably induced the differentiation signature and, furthermore, yielded functional evidence of bona fide differentiation. The results indicate that GE-HTS may be a powerful, general approach for chemical screening.

Acute Disease↗
PubMed2002

Assessing pregnancy risks of azole antifungals using a high throughput aromatase inhibition assay.

UNLABELLED: Human aromatase (CYP19) converts C19 androgens to aromatic C18 estrogenic steroids. Its activity is critical for early and mid pregnancy maintenance and in regulating parturition in late pregnancy. Past studies have utilized placental microsome tritiated water release assay to assess drug-hormone interactions with estrogen synthesis. We compared data from human placental assays with BD Gentest's high throughput recombinant CYP19 enzyme assay using the fluorometric substrate dibenzylfluorescein. We tested a panel of azole antifungal agents that are commonly administered to women of childbearing potential, for their potential to inhibit aromatase. Potency varied by several orders of magnitude. Plasma and tissue levels of some azole drugs following oral or topical administration are at or above these IC50 values. These include the oral agents fluconazole and ketoconazole, and the topical agents econazole, bifonazole, clotrimazole, miconazole, and sulconazole. CONCLUSIONS: 1. Recombinant enzyme assay data are comparable to the human placental assay data in both SAR rank order and potency. 2. Plasma and tissue levels of some azole drugs following oral or topical administration are at or above these IC50 values. Therefore, some azole drugs may disrupt estrogen production in pregnancy, affecting pregnancy outcome. 3. Recombinant CYP19 assay using the fluorometric substrate dibenzylfluorescein, demonstrates rapid screening potential for chemicals that may affect pregnancy outcome as a result of CYP19 inhibition.

Animals↗
PubMed2006

A mutation in Arabidopsis cytochrome b5 reductase identified by high-throughput screening differentially affects hydroxylation and desaturation.

As a model for analyzing the production of novel fatty acids in oilseeds, we used the genetic and molecular techniques available for Arabidopsis to characterize modifying mutations affecting the accumulation of hydroxy fatty acids in the seeds of Arabidopsis plants that express a transgene for the castor bean fatty acid hydroxylase, FAH12. We developed a high-throughput analytical system and used it to identify three complementation classes of mutations with reduced hydroxy fatty acid accumulation from among Arabidopsis M3 seed samples derived from chemical mutagenesis. We identified one of the mutations by positional cloning as a single base pair change in a gene encoding NADH:cytochrome b5 reductase (CBR1, At5g17770). When expressed in yeast, the mutant form of the enzyme was less active and less stable than the wild-type enzyme. Characterization of homozygous mutant lines with and without the FAH12 transgene (FAH12 cbr1-1 and cbr1-1, respectively) indicated that the only detectable consequence of the cbr1-1 mutation was on desaturase and hydroxylase reactions in the developing seed. The leaf and root fatty compositions, as well as the growth, development and seed production of mutant plants were indistinguishable from wild type. Interestingly, while the cbr1-1 mutation reduced the accumulation of hydroxy fatty acids in seeds by 85%, the effects on 18:1 and 18:2 desaturation reactions were much less (<25% and <60%, respectively). These results suggest that there is competition in developing seeds among the several reactions that utilize reduced cytochrome b5.

Amino Acid Sequence↗
PubMed2006

New developmental insights from high-throughput biological analysis in Caenorhabditis elegans.

The use of Caenorhabditis elegans as a model system for understanding animal development and human disease has long been recognized as an efficient tool of discovery. Recent developments, particularly in our understanding of RNA-mediated interference and its ability to modify gene activity, have facilitated the use of C. elegans in determining gene function via high-throughput analysis. These new strategies have provided a framework that allows investigators to analyse gene function globally at the genomic level and will likely become a prototypic model for biological analysis in the post-genome era.

Animals↗
PubMed2005

High-throughput screening of interactions between G protein-coupled receptors and ligands using confocal optics microscopy.

Interactions of extracellular ligands with proteins in the cellular plasma membrane are the starting point for various intracellular signaling cascades. In the pharmaceutical industry, particular attention has been paid to G protein- coupled receptors (GPCRs), which are involved in various disease processes. In so-called high-throughput screening (HTS) campaigns, large medicinal chemistry compound libraries were searched for bioactive molecules that would either induce or inhibit the activity of a specific disease-relevant GPCR. In the respective drug discovery assays, the test compound typically competes with the physiological ligand for a binding site on the receptor. The transmembrane receptor is prepared in the form of membrane fragments or, as described here, in so-called virus-like particles (VLiPs). As hundreds of thousands of test compounds must be analyzed, there is a strict need for low volume binding assays to save the expensive bioreagents, and to reduce the consumption of the test compounds. In this chapter, we describe the application of confocal optics microscopy to measure GPCR ligand interactions in low microliter assay volumes.

Animals↗
PubMed2006

A nonradioactive high-throughput/high-content assay for measurement of the human serotonin reuptake transporter function in vitro.

Both the tricyclic and specific serotonin reuptake inhibitor classes of antidepressants act primarily by inhibiting the reuptake of released serotonin by the human serotonin reuptake transporter (hSERT). In this article, the authors describe the use of a fluorescent substrate of the transporter (4-(4-(dimethylamino)-styrl)-N-methylpyridinium, ASP) to develop a microplate-based high-throughput screen for hSERT function. The assay is sensitive to known inhibitors of serotonin uptake, including fluoxetine (Prozac), with the correct rank order of potency and IC(50) values close to those reported in the literature for tritiated serotonin uptake. The authors also describe the validation of the assay for natural product screening using a test set of 2400 pure phyto-chemicals and 80 plant extracts. The mean Z of the screened plates was 0.53. Hit rates, confirmation rates, and validation of the hits in a "classical" assay for serotonin uptake are all reported. The assay can also be read in "high-content" mode using a subcellular imaging device, which allows direct detection of possible assay interference by acutely cytotoxic compounds. Among the compounds identified were several previously reported inhibitors of the hSERT, as well as compounds having structural similarity to the tricyclic antidepressant drugs.

Cell Line↗
PubMedDate not supplied

Automated high-throughput synthesis of artificial glycopeptides. Small-molecule probes for chemical glycobiology.

A fully automated method for the synthesis of artificial glycopeptides having two (similar or different) carbon-linked glycosyl moieties on a dipeptide scaffold has been developed. By use of this approach that combines the diversity of peptide/pseudopeptide and glycosides, different glycoside moieties can be incorporated onto the peptide/pseudopeptide backbone in a highly controlled manner. The approach utilizes a stepwise reductive amination with glycoside aldehyde derivatives (model 1) or (ii) glycoside reductive amination followed by glycoside amide bond formation (model 2). Further, an automated method has been utilized in the high-throughput library synthesis of 4 x 96 artificial glycopeptides. These libraries were tested as chemical probes/inhibitors of enzyme systems that convert a glucose moiety into rhamnose prior to incorporation of the rhamnose unit and the conversion of UDP-galactopyranose to UDP-galactofuranose via UDP-galactopyranose mutase enzyme during the biosynthesis of the mycobacterium cell wall.

Combinatorial Chemistry Techniques↗
PubMed2005

Identification of inhibitors of the kinase activity of oncogenic V600E BRAF in an enzyme cascade high-throughput screen.

The Cancer Genome Project has identified several oncogenic mutations in BRAF that represent important opportunities for cancer drug discovery. The V600E BRAF mutation accounts for approximately 90% of the mutations identified. A strong case has emerged from molecular, cellular, and structural studies for the identification and development of inhibitors of this mutated BRAF protein. The authors have developed and run a high-throughput screen to find inhibitors of V600E BRAF using an enzyme cascade assay in which oncogenic BRAF activates MEK1, which in turn activates ERK2, which then phosphorylates the transcription factor ELK1. A phosphospecific antibody, Europium-labeled secondary antibody, and a time-resolved fluorescent readout were used to measure phosphorylation of ELK1. Overall assay variation was 12.4%. The assay was used to screen 64,000 compounds with an overall Z' factor of 0.58 +/- 0.12. A series of 3,5, di-substituted pyridines were identified as inhibitors of the cascade assay. These compounds did not inhibit a shortened activated MEK1 to ELK1 cascade but were active (0.5-27.9 microM) in a V600E BRAF assay and represent a potential starting point for future drug discovery and development.

Drug Evaluation, Preclinical↗
PubMed2003

High-throughput TAIL-PCR as a tool to identify DNA flanking insertions.

Thermal asymmetric interlaced polymerase chain reaction (TAIL-PCR) is a fast and efficient method to amplify unknown sequences adjacent to known insertion sites in Arabidopsis. Nested, insertion-specific primers are used together with arbitrary degenerate primers (AD primers), which are designed to differ in their annealing temperatures. Alternating cycles of high and low annealing temperature yield specific products bordered by an insertion-specific primer on one side and an AD primer on the other. Further specificity is obtained through subsequent rounds of TAIL-PCR, using nested insertion-specific primers. The increasing availability of whole genome sequences renders TAIL-PCR an attractive tool to easily identify insertion sites in large genome tagging populations through the direct sequencing of TAIL-PCR products. For large-scale functional genomics approaches, it is desirable to obtain flanking sequences for each individual in the population in a fast and cost-effective manner. In this chapter, we describe a TAIL-PCR method amenable for high-throughput production (HT-TAIL-PCR) in Arabidopsis. Based on this protocol, HT-TAIL-PCR may be easily adapted for other organisms.

Arabidopsis↗
PubMed2006

A high-throughput cell-based assay for inhibitors of ABCG2 activity.

ABCG2 is a member of the adenosine triphosphate (ATP)-binding cassette family of multidrug transporters associated with resistance of tumor cells to many cytotoxic agents. Evaluation of modulators of ABCG2 activity has relied on methods such as drug sensitization, biochemical characterization, and transport studies. To search for novel inhibitors of ABCG2, a fluorescent cell-based assay was developed for application in high-throughput screening. Accumulation of pheophorbide a (PhA), an ABCG2-specific substrate, forms the basis for the assay in NCI-H460/MX20 cells overexpressing wild-type ABCG2. Treatment of these cells with 10 microM fumitremorgin C (FTC), a specific ABCG2 inhibitor, increased cell accumulation of PhA to 5.6 times control (Z' 0.5). Validation included confirmation with known ABCG2 inhibitors: FTC, novobiocin, tariquidar, and quercetin. Verapamil, reported to inhibit P-glycoprotein but not ABCG2, had insignificant activity. Screening of a library of 3523 natural products identified 11 compounds with high activity (> or = 50% of FTC, confirmed by reassay), including 3 flavonoids, members of a family of compounds that include ABCG2 inhibitors. One of the inhibitors detected, eupatin, was moderately potent (IC50 of 2.2 microM) and, like FTC, restored sensitivity of resistant cells to mitoxantrone. Application of this assay to other libraries of synthetic compounds and natural products is expected to identify novel inhibitors of ABCG2 activity.

ATP Binding Cassette Transporter, Subfamily G, Mem↗
PubMed2006

Precise physical models of protein-DNA interaction from high-throughput data.

A cell's ability to regulate gene transcription depends in large part on the energy with which transcription factors (TFs) bind their DNA regulatory sites. Obtaining accurate models of this binding energy is therefore an important goal for quantitative biology. In this article, we present a principled likelihood-based approach for inferring physical models of TF-DNA binding energy from the data produced by modern high-throughput binding assays. Central to our analysis is the ability to assess the relative likelihood of different model parameters given experimental observations. We take a unique approach to this problem and show how to compute likelihood without any explicit assumptions about the noise that inevitably corrupts such measurements. Sampling possible choices for model parameters according to this likelihood function, we can then make probabilistic predictions for the identities of binding sites and their physical binding energies. Applying this procedure to previously published data on the Saccharomyces cerevisiae TF Abf1p, we find models of TF binding whose parameters are determined with remarkable precision. Evidence for the accuracy of these models is provided by an astonishing level of phylogenetic conservation in the predicted energies of putative binding sites. Results from in vivo and in vitro experiments also provide highly consistent characterizations of Abf1p, a result that contrasts with a previous analysis of the same data.

Binding Sites↗
PubMed2000

High-throughput profiling of the mitochondrial proteome using affinity fractionation and automation.

Recent studies have demonstrated the need for complementing cellular genomic information with specific information on expressed proteins, or proteomics, since the correlation between the two is poor. Typically, proteomic information is gathered by analyzing samples on two-dimensional gels with the subsequent identification of specific proteins of interest by using trypsin digestion and mass spectrometry in a process termed peptide mass fingerprinting. These procedures have, as a rule, been labor-intensive and manual, and therefore of low throughput. The development of automated proteomic technology for processing large numbers of samples simultaneously has made the concept of profiling entire proteomes feasible at last. In this study, we report the initiation of the (eventual) complete profile of the rat mitochondrial proteome by using high-throughput automated equipment in combination with a novel fractionation technique using minispin affinity columns. Using these technologies, approximately one hundred proteins could be identified in several days. In addition, separate profiles of calcium binding proteins, glycoproteins, and hydrophobic or membrane proteins could be generated. Because mitochondrial dysfunction has been implicated in numerous diseases, such as cancer, Alzheimer's disease and diabetes, it is probable that the identification of the majority of mitochondrial proteins will be a beneficial tool for developing drug and diagnostic targets for associated diseases.

Animals↗
PubMed2004

Development of a quantitative, high-throughput cell-based enzyme-linked immunosorbent assay for detection of colony-stimulating factor-1 receptor tyrosine kinase inhibitors.

Inhibitors of receptor tyrosine kinases are implicated as therapeutic agents for the treatment of many human diseases including cancer, inflammation and diabetes. Cell-based assays to examine inhibition of receptor tyrosine kinase mediated intracellular signaling are often laborious and not amenable to high-throughput cell-based screening of compound libraries. Here we describe the development of a nonradioactive, sandwich enzyme-linked immunosorbent assay (ELISA) to quantify the activation and inhibition of ligand-induced phosphorylation of the colony-stimulating factor-1 receptor (CSF-1R) in 96-well microtiter plate format. The assay involves the capture of the Triton X-100 solubilized human CSF-1R, from HEK293E cells overexpressing histidine epitope-tagged CSF-1R (CSF-1R/HEK293E), with immobilized CSF-1R antibody and detection of phosphosphorylation of the activated receptor with a phosphotyrosine specific antibody. The assay exhibited a 5-fold increase in phosphorylated CSF-1R signal from CSF-1R/HEK293E cells treated with colony-stimulating factor (CSF-1) relative to treated vector control cells. Additionally, using a histidine epitope-specific capture antibody, this method can also be adapted to quantify the phosphorylation state of any recombinantly expressed, histidine-tagged receptor tyrosine kinase. This method is a substantial improvement in throughput and quantitation of CSF-1R phosphorylation over conventional immunoblotting techniques.

Automation↗