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At least 631 records · Page 35Linked to original sources
PubMed2004

High-throughput non-heme iron assay for animal tissues.

Iron has been widely studied in nearly every realm of biology. However, current methodologies, such as genetic mapping or mutation screening, have been difficult to apply due to the lack of robust high-throughput methods for quantifying iron levels from cells or tissues. The measurement of total iron levels in tissues, usually done with atomic absorption spectroscopy, is impractical for large numbers of samples and includes the contribution of heme iron from hemoglobin contained in red blood cells. The measurement of non-heme iron by reaction with a bathophenanthroline reagent, a commonly used assay reported more than 30 years ago, is also not feasible for large-scale analyses because it is cuvette-based. We therefore have modified this method to a microplate format that will facilitate large-scale analysis. The microplate assay is highly sensitive and specific, and is a simple and effective method for the measurement of non-heme iron for animal tissues that will enable the application of high-throughput of genetic methodologies.

Animals↗
PubMed2006

Micro-colony array based high throughput platform for enzyme library screening.

Enzymes are becoming increasingly important tools for synthesizing and modifying fine and bulk chemicals. The availability of biocatalysts which fulfil the requirements of industrial processes is often limited. Recruiting suited enzymes from natural (e.g. metagenomes) and artificial (e.g. directed evolution) biodiversity is based on screening libraries of microbial clones expressing enzyme variants. However, exploring the complex diversity of such libraries needs efficient screening methods. Overcoming the "screening bottleneck" requires rapid high throughput technology allowing the analysis of a large diversity of different enzymes and applying different screening conditions. Facing these facts an efficient and cost effective method for high throughput screening of large enzyme libraries at the colony level was developed. Therefore, ordered high density micro-colony arrays were combined with optical sensor technology and automated image analysis. The system generally allows the simultaneous monitoring of enzyme activities reflected by up to 7000 micro-colonies spotted on a filter in the size of a micro-titer plate. A developed replica option also allows the analysis of clones under varying external conditions. The method was verified by a model screening using esterases and was proved to provide reliable enzyme activity measurements within single micro-colonies allowing the discrimination of activity differences in the range of 10-20%.

Calibration↗
PubMed2003

Identification of metabotropic glutamate receptor antagonists using an automated high-throughput screening system.

Antagonists to the human metabotropic glutamate receptor subtype 5a(mGluR(5a)) have been implicated as potential therapeutics for the treatment of a variety of nervous system disorders, including pain, anxiety, and Parkinson's disease. To discover novel antagonists to the mGluR(5a), a functional assay measuring agonist-induced intracellular calcium release was developed. The assay was used for the high-throughput screening of a large collection of compounds in single wells using a fully automated robotic platform. Primary high-throughput screening hits were subjected to a combination of data analysis and counterscreening assays to identify several compounds with both efficacy and selectivity for the metabotropic glutamate receptor target.

Calcium↗
PubMed2004

Transcriptional-based screens for pathway-specific, high-throughput target discovery in endothelial cells.

With the sequence of the human genome at hand, target discovery strategies are needed that can rapidly identify novel gene products involved in human disease pathways. In this article, the authors describe a cell-based, high-throughput assay that can identify gene products capable of modulating the vascular endothelial growth factor (VEGF) and tumor necrosis factor alpha (TNFalpha) signaling pathways in human endothelial cells. The assay uses real-time PCR technology to measure downstream reporter mRNA transcripts induced upon cytokine stimulation in a 96-well plate format and has been adapted for use with recombinant adenoviruses. The authors specifically demonstrate modulation of cytokine-driven reporter transcripts using drug inhibitors and through adenoviral-mediated expression of known signaling intermediates of the respective pathways. In addition, they have used an arrayed library of 350 recombinant adenoviruses to screen for novel modulators of the VEGF and TNFalpha pathways. The high-throughput screening capacity and sensitivity of this system make it a useful tool for new drug target identification.

Adenoviridae↗
PubMed2003

Sensitive and high-throughput analyses of purine metabolites by dynamic pH junction multiplexed capillary electrophoresis: a new tool for metabolomic studies.

On-line sample preconcentration by a dynamic pH junction in conjunction with multiplexed capillary electrophoresis (CE) and UV detection represents a sensitive and high-throughput format for future metabolomic research, such as purine analysis. The optimization of purine focusing can be rapidly assessed by systematically altering the sample matrix properties, such as the buffer co-ion, pH and ionic strength using a 96-capillary array format. This method permits focusing of large sample injection volumes, resulting in over a 50-fold improvement in the concentration sensitivity. The limit of detection (S/N = 3) for purine metabolites was less than 8.0 x 10(-8) M under optimum conditions when using UV absorbance. Dynamic pH junction multiplexed CE demonstrated excellent linearity over a hundred-fold concentration range, as well as low inter-capillary precision in terms of normalized migration times and peak areas. The potential for clinically relevant high-throughput analyses of micromolar amounts of purine metabolites in urine was also demonstrated.

Electrophoresis, Capillary↗
PubMed2006

A high-throughput screen for compounds that inhibit aggregation of the Alzheimer's peptide.

Aggregation of the Alzheimer's peptide Abeta produces toxic multimeric species that play a key role in the development of Alzheimer's disease. Compounds that inhibit this aggregation may prove useful as therapeutic agents for the prevention or treatment of Alzheimer's disease. Although aggregation inhibitors may already exist in combinatorial libraries, finding these compounds in a cost-effective high-throughput manner poses an enormous challenge. To meet this challenge, we have developed a novel high-throughput screen capable of isolating inhibitors of Abeta aggregation from large libraries of inactive candidates. The screen uses a fusion of Abeta42 to GFP. In the absence of inhibition, the rapid misfolding and aggregation of Abeta42 causes the entire fusion protein to misfold, thereby preventing fluorescence. Compounds that inhibit Abeta42 aggregation enable GFP to fold into its native structure and be identified by the resulting fluorescent signal. By implementing the screen on a pilot library of triazine derivatives, we have identified several putative inhibitors. One of the selected compounds was studied in detail by a series of biochemical and biophysical methods. These studies confirmed that the selected compound inhibits aggregation of synthetic Abeta42 peptide. The fluorescence-based method described here is rapid and inexpensive and can be used to screen large libraries for inhibitors of Abeta42 aggregation and/or amyloidogenesis.

Alzheimer Disease↗
PubMed2000

Impact of a red-shifted dye label for high throughput fluorescence polarization assays of G protein-coupled receptors.

High throughput screening fluorescence polarization assays using G protein-coupled receptors (GPCRs) as targets have been compared using fluorescein and BODIPY TMR-labeled peptides. The red-shifted BODIPY TMR dye exhibits improved assay performance relative to fluorescein due to improvement in both ligand affinity to the GPCRs and assay precision brought about by the higher intensity probe. Furthermore, the red-shifted dye demonstrates an insensitivity to the effects of the highly colored compound tartrazine, which can produce false-negative results for assays conducted with fluorescein as a label.

Binding, Competitive↗
PubMed2000

MethyLight: a high-throughput assay to measure DNA methylation.

Cytosine-5 DNA methylation occurs in the context of CpG dinucleotides in vertebrates. Aberrant methylation of CpG islands in human tumors has been shown to cause transcriptional silencing of tumor-suppressor genes. Most methods used to analyze cytosine-5 methylation patterns require cumbersome manual techniques that employ gel electrophoresis, restriction enzyme digestion, radiolabeled dNTPs or hybridization probes. The development of high-throughput technology for the analysis of DNA methylation would significantly expand our ability to derive molecular information from clinical specimens. This study describes a high-throughput quantitative methylation assay that utilizes fluorescence-based real-time PCR (TaqMan) technology that requires no further manipulations after the PCR step. MethyLight is a highly sensitive assay, capable of detecting methylated alleles in the presence of a 10,000-fold excess of unmethylated alleles. The assay is also highly quantitative and can very accurately determine the relative prevalence of a particular pattern of DNA methylation. We show that MethyLight can distinguish between mono-allelic and bi-allelic methylation of the MLH1 mismatch repair gene in human colorectal tumor specimens. The development of this technique should considerably enhance our ability to rapidly and accurately generate epigenetic profiles of tumor samples.

Adaptor Proteins, Signal Transducing↗
PubMed2004

Beta galactosidase enzyme fragment complementation as a high-throughput screening protease technology.

The authors describe a homogeneous, high-throughput screening (HTS) assay for measuring protease activity and detection of inhibitors. The assay comprises a cyclic beta-galactosidase (beta-gal) enzyme donor peptide (ED) containing a protease-selective cleavage sequence. Alone, the cyclic peptide is inactive, but when linearized following protease cleavage, ED complements with beta-gal enzyme acceptor forming active beta-gal enzyme. This then catalyzes the formation of either fluorescent or chemiluminescent products, with beta-gal turnover providing a highly amplified signal, and thus an assay technology of high sensitivity. To demonstrate the utility of the technology, an EFC assay was developed to measure the activity of 2, caspase 3 and beta-secretase. Using a cyclic ED containing the caspase 3 substrate sequence, DEVD, the EFC assay signal was linear with respect to caspase 3 concentration. The assay was very sensitive, being able to detect activity at low picogram amounts of caspase 3. For the beta-secretase (BACE) EFC assay, a cyclic ED containing the Swedish mutant cleavage site of amyloid precursor protein (APP), SEVNLDAEFK, was used. In a similar fashion to the caspase 3 assay, the signal induced by BACE activity was linear with respect to enzyme concentration and was highly sensitive, being able to detect nanogram quantities of BACE. The assay was also more sensitive than a commercially available FRET-based assay of BACE activity. It is concluded that the EFC protease assay is a simple, flexible, and sensitive technology for HTS of proteases.

Amino Acid Sequence↗
PubMed2006

Functional characterisation of homomeric ionotropic glutamate receptors GluR1-GluR6 in a fluorescence-based high throughput screening assay.

We have constructed stable HEK293 cell lines expressing the rat ionotropic glutamate receptor subtypes GluR1(i), GluR2Q(i), GluR3(i), GluR4(i), GluR5Q and GluR6Q and characterised the pharmacological profiles of the six homomeric receptors in a fluorescence-based high throughput screening assay using Fluo-4/AM as a fluorescent Ca2+ indicator. In this assay, the pharmacological properties of nine standard GluR ligands correlated nicely with those previously observed in electrophysiology studies of GluRs expressed in Xenopus oocytes or mammalian cells. The potencies and efficacies displayed by the agonists (S)-glutamate, (S)-quisqualate, kainate, (RS)-AMPA, (RS)-ATPA, (RS)-ACPA] and (S)-4-AHCP at the six GluRs were in concordance with electrophysiological studies. Furthermore, the Ki values exhibited by the competitive antagonists NBQX and (RS)-ATPO were also in agreement with findings of previous studies. Finally, the effects of various concentrations of Ca2+ in the assay buffer and of the allosteric modulators cyclothiazide and concanavalin A on GluR signalling were examined. This study represents the most elaborate functional characterisation of multiple AMPA and KA receptor subtypes in the same assay reported to date. We propose that high throughput screening of compound libraries at the six GluR-HEK293 cell lines could be helpful in the search for structurally and pharmacologically novel ligands acting at the receptors.

Aniline Compounds↗
PubMed2005

Development and application of a high-throughput screening assay for HIV-1 integrase enzyme activities.

Integrase (IN) mediates the covalent insertion of the retroviral genome into its host chromosomal DNA. This enzymatic activity can be reconstituted in vitro with short DNA oligonucleotides, which mimic a single viral DNA end, and purified IN. Herein we report a highly efficient and sensitive high-throughput screen, HIV Integrase Target SRI Assay (HITS), for HIV-1 IN activity using 5' biotin-labeled DNA (5' BIO donor) and 3' digoxygenin-labeled DNA (3' DIG target). Following 3' processing of the 5' BIO donor, strand transfer proceeds with integration of the 5' BIO donor into the 3' DIG target. Products were captured on a streptavidin-coated microplate and the amount of DIG retained in the well was measured. The end point values, measured as absorbance, ranged from 0.9 to 1.5 for IN-mediated reactions as compared with background readings of 0.05 to 0.12. The Z factor for the assay ranged from 0.7 to 0.85. The assay was used to screen drugs in a high-throughput format, and furthermore, we adapted the assay to study mechanistic questions regarding the integration process. For example, using variations of the assay format, we showed high preference of E strand of the long terminal repeat (LTR) viral DNA as a target strand compared with its complementary A strand. The E strand is the strand processed by IN. Furthermore, we explored the reported inhibitory effect of reverse transcriptase on integration.

Biotin↗
PubMed2004

A high-throughput chemotaxis assay for pharmacological characterization of chemokine receptors: Utilization of U937 monocytic cells.

INTRODUCTION: Higher-throughput chemotaxis assays have had limited use in chemokine receptor pharmacology studies mainly because of the unavailability of optimal assay formats in addition to an incompatibility of chemotactic cell backgrounds with other pharmacological assays. Here, we developed a high-throughput 96-well chemotaxis assay for leukocytic cell lines and identified the human U937 monocytic line as an excellent cell background for both chemotaxis and the high-throughput calcium mobilization Fluorescent Imaging Plate Reader (FLIPR) assay. METHODS: Optimal chemotactic conditions were developed using the Neuroprobe MBA96 nondisposable and the Millipore MultiScreen-MIC disposable apparatuses with responses to CXC chemokine receptor (CXCR)-4 endogenously expressed on the human H9 T lymphoma line, and confirmed with Jurkat T cell and U937 monocytic cell lines. RESULTS: The U937 cell line was chosen for site-directed mutagenesis studies with CC chemokine receptor (CCR)-7 because this cell line did not endogenously express this receptor, it demonstrated a good chemotaxis index, and it showed an exceptional ability to mobilize calcium measured via FLIPR. Using the Millipore MultiScreen-MIC and FLIPR assays, alanine substitutions at K130 and Q227 caused threefold shifts in potency for the CCR7 ligand, CCL19, whereas that at K137 had no effect. DISCUSSION: Because these CCR7 mutations have previously been shown not to affect ligand binding, our results here show that these residues are specifically involved in receptor activation signals critical to chemotaxis and underscore the importance of using the U937 cell background to confirm results of chemotaxis with those of the FLIPR assay.

Amino Acid Sequence↗
PubMed2006

Protein translocation assays: key tools for accessing new biological information with high-throughput microscopy.

Redistribution technology is a cell-based assay technology that uses protein translocation as the primary readout for the activity of cellular signaling pathways and other intracellular events. Protein targets are labeled with the green fluorescent protein, and stably transfected cell lines are generated. The assays are read using a high-throughput, optical microscope-based instrument, several of which have become available commercially. Protein translocation assays can be formatted as agonist assays, in which compounds are tested for their ability to promote protein translocation, or as antagonist assays, in which compounds are tested for their ability to inhibit protein translocation caused by a known agonist. Protein translocation assays are high-content, high-throughput assays primarily used for profiling of lead series, primary screening of compound libraries, and as readouts for gene-silencing studies using siRNAs. This chapter describes two novel high-content Redistribution assay technologies: (1) The p53:hdm2 GRIP interaction assay, in which one high-content image feature is used for detection of primary hits, whereas a different feature is used to deselect compounds with unwanted mode of action, and (2) application of siRNAs to Redistribution assays, exemplified by knockdown of Akt isoforms in a FKHR translocation assay reporting on the PI3 kinase signaling pathway.

Animals↗
PubMed2004

An enzymatic assay for poly(ADP-ribose) polymerase-1 (PARP-1) via the chemical quantitation of NAD(+): application to the high-throughput screening of small molecules as potential inhibitors.

The enzyme poly(adenosine 5'-diphosphate (ADP)-ribose) polymerase (PARP-1) catalyzes the formation of (ADP)-ribose polymers on a variety of protein acceptors in a NAD+ -dependent manner. While PARP-1 is activated by DNA damage and plays a critical role in cellular survival mechanisms, its overactivation leads to a depletion of NAD+/ATP energy stores and ultimately to necrotic cell death. Due to this dual role of PARP in the cell, small-molecule inhibitors of the PARP family of enzymes have been widely investigated for use as potentiators of anticancer therapies and as inhibitors of neurodegeneration and ischemic injuries. Unfortunately, standard assays for PARP inhibition are not optimal for the high-throughput screening of compound collections or combinatorial libraries. Described herein is a highly sensitive, inexpensive, and operationally simple assay for the rapid assessment of PARP activity that relies on the conversion of NAD+ into a highly fluorescent compound. We demonstrate that this assay can readily detect PARP inhibitors in a high-throughput screen using 384-well plates. In addition, the assay can be used to determine IC50 values for PARP inhibitors that have a range of inhibitory properties. As existing PARP assays utilize specialized reagents such as radiolabeled/biotinylated NAD+ or antibodies to poly(ADP-ribose), the chemical quantitation method described herein offers a highly sensitive and convenient alternative for rapidly screening compound collections for PARP inhibition.

Acetophenones↗
PubMed2003

Horizontally transferred genes in plant-parasitic nematodes: a high-throughput genomic approach.

BACKGROUND: Published accounts of horizontally acquired genes in plant-parasitic nematodes have not been the result of a specific search for gene transfer per se, but rather have emerged from characterization of individual genes. We present a method for a high-throughput genome screen for horizontally acquired genes, illustrated using expressed sequence tag (EST) data from three species of root-knot nematode, Meloidogyne species. RESULTS: Our approach identified the previously postulated horizontally transferred genes and revealed six new candidates. Screening was partially dependent on sequence quality, with more candidates identified from clustered sequences than from raw EST data. Computational and experimental methods verified the horizontal gene transfer candidates as bona fide nematode genes. Phylogenetic analysis implicated rhizobial ancestors as donors of horizontally acquired genes in Meloidogyne. CONCLUSIONS: High-throughput genomic screening is an effective way to identify horizontal gene transfer candidates. Transferred genes that have undergone amelioration of nucleotide composition and codon bias have been identified using this approach. Analysis of these horizontally transferred gene candidates suggests a link between horizontally transferred genes in Meloidogyne and parasitism.

Amino Acid Sequence↗
PubMed2005

Establishment of an in vitro high-throughput screening assay for detecting phospholipidosis-inducing potential.

Excessive accumulation of phospholipids results in phospholipidosis (PL), which may interfere with cellular functions, leading to acute or chronic disease or even death. Electron-microscopic detection of cytoplasmic lamellar bodies is often used as a diagnostic criterion of PL, but a faster, more convenient procedure is required for high-throughput assay of the PL-inducing potential of candidate drugs. We have developed a 96-well microplate cell-culture method for detecting PL, using a phosphatidylcholine-conjugated dye (NBD-PC) and a fluoro-microplate reader. The fluorescence intensity due to NBD-PC was normalized to that of Hoechst33342, used as an indicator of cell number, to obtain the amount of NBD-PC taken up per living cell. To select a suitable cell type, we examined the PL-detection sensitivity of five cell lines, as well as human and rat primary hepatocyte cultures, with five cationic amphiphilic drugs (CAD) as PL inducers and a negative control compound. The cell lines CHO-K1 and CHL/IU gave the best results. The NBD-PC uptake per CHO-K1 cell showed a high correlation with the pathological score of PL for 24 compounds, including PL-positive and negative compounds. This high-throughput screening assay for PL-inducing potential (HTS-PL assay) offers high sensitivity and accuracy, and it allows simultaneous determination of cytotoxicity.

4-Chloro-7-nitrobenzofurazan↗
PubMed2003

High-throughput screening of kinase inhibitors by multiplex capillary electrophoresis with UV absorption detection.

Protein kinases play a major role in the transformation of cells and are often used as molecular targets for the new generation of anticancer drugs. We present a novel technique for high-throughput screening of inhibitors of protein kinases. The technique involves the use of multiplexed capillary electrophoresis (CE) for the rapid separation of the peptides, phosphopeptides, and various inhibitors. By means of UV detection, diversified peptides with native amino acid sequences and their phosphorylated counterparts can be directly analyzed without the need for radioactive or fluorescence labeling. The effects of different inhibitors and their IC(50) value were determined using three different situations involving the use of a single purified kinase, two purified kinases, and crude cell extracts, respectively. The results suggest that multiplexed CE/UV may prove to be a straightforward and general approach for high-throughput screening of compound libraries to find potent and selective inhibitors of the various protein kinases.

Calcium-Calmodulin-Dependent Protein Kinases↗
PubMed2001

High-throughput plasmid purification for capillary sequencing.

The need for expeditious and inexpensive methods for high-throughput DNA sequencing has been highlighted by the accelerated pace of genome DNA sequencing over the past year. At the Joint Genome Institute, the throughput in terms of high-quality bases per day has increased over 20-fold during the past 18 mo, reaching an average of 18.3 million bases per day. To support this unprecedented scaleup, we developed an inexpensive automated method for the isolation and purification of double-stranded plasmid DNA clones for sequencing that is tailored to meet the more stringent needs of the newer capillary electrophoresis DNA sequencing machines. The protocol is based on the magnetic bead method of solid phase reversible immobilization that has been automated by using a CRS-based robotic system. The method described here has enabled us to meet our increases in production while reducing labor and materials costs significantly.

Base Sequence↗