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At least 1,243 records · Page 69Linked to original sources
PubMed2003

High-throughput inhibition screening of major human cytochrome P450 enzymes using an in vitro cocktail and liquid chromatography-tandem mass spectrometry.

A method has been developed for the high-throughput inhibition screening of the major human cytochrome P450 (CYP) enzymes (CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4) using an in vitro substrate cocktail and liquid chromatography-tandem mass spectrometry (LC-MS-MS). A cocktail consisting of the selective substrates phenacetin (CYP1A2), tolbutamide (CYP2C9), omeprazole (CYP2C19), bufuralol (CYP2D6), and midazolam (CYP3A4) was incubated with human liver microsomes. The metabolic reactions were terminated with methanol containing dextrorphan as an internal standard. Following centrifugation, the supernatant was analyzed by LC-MS-MS employing a fast gradient. The concentrations of the substrate metabolites-paracetamol, 4-hydroxytolbutamide, 5-hydroxyomeprazole, 1'-hydroxybufuralol, and 1'-hydroxymidazolam-in each sample were determined by LC-MS-MS in a single assay. The method was validated by incubating known CYP inhibitors (furafylline, CYP1A2; sulfaphenazole, CYP2C9; s-mephenytoin, CYP2C19; quinidine, CYP2D6; and troleandomycin, CYP3A4) with the individual substrates they were known to inhibit and with the substrate cocktail. IC50s (microM) determined using the substrate cocktail were in good agreement with those obtained with individual substrates (furafylline, 2.9 vs. 2.0; sulfaphenazole, 0.75 vs. 0.72; s-mephenytoin, 170 vs. 180; quinidine, 0.17 vs. 0.24; troleandomycin, 2.6 vs. 3.2) and with previously reported values in the literature.

Cytochrome P-450 Enzyme System↗
PubMedDate not supplied

Simultaneous in-situ monitoring of parallel polymerization reactions using light scattering; a new tool for high-throughput screening.

A recently introduced technique, simultaneous multiple sample light scattering (SMSLS), was used to monitor parallel polymerization reactions in situ. SMSLS is designed for real-time, high-throughput screening and provides a time-dependent light scattering signature for each reaction, which contains both qualitative and semiquantitative information. Qualitatively, the signature immediately indicates whether the reaction occurs or not, whether there is an initial lag period, and how long the reaction takes until it stops. The signature also provides estimates of the reaction rate and weight average molecular mass M(w), and its shape can help identify mechanistic aspects, for example, controlled versus free radical polymerization, presence of impurities, etc. The method is inherently adapted to small sample volumes and requires no special sample preparation or postpolymerization characterization. The demonstration here involved the free radical polymerization of acrylamide under varying conditions and should be readily applicable to a wide variety of other reactions. Results were cross-checked with multi-detector gel permeation chromatography.

Journal Article↗
PubMed2004

Characterization of CHO cells stably expressing a G alpha 16/z chimera for high throughput screening of GPCRs.

G protein-coupled receptors (GPCRs) are important therapeutic targets for drug discovery. The identification and characterization of new ligands ideally requires the use of high throughput assays that are applicable to all GPCR subtypes. To circumvent the problem of different GPCRs coupling to distinct intracellular second messenger pathways, we describe a new method that uses the chimeric Galpha protein 16z25 to facilitate this process. Stably expressed in Chinese hamster ovary cells, 16z25 allows G(i/o)- and G(s)-coupled receptors to mobilize intracellular Ca(2+) upon agonist stimulation. We have generated nine cell lines each stably expressing 16z25 and a GPCR. All cell lines respond to appropriate agonist stimulation in fluorometric imaging plate reader (FLIPR) assays with robust and potent Ca(2+) mobilization. Several of these lines have been pharmacologically characterized using agonists and antagonists. We also demonstrate that the coexpression of GPCR and 16z25 does not interfere with the receptors' ability to activate endogenous signaling pathways. The ability of 16z25 to functionally mediate the agonist stimulation of a broad spectrum of GPCRs indicates that the use of cell lines stably coexpressing this chimera and GPCRs will simplify the drug screening process and aid in the deorphanization of new receptors.

Amino Acid Sequence↗
PubMed2005

High throughput purification of amino-modified oligonucleotides and its application to novel detection system of gene expression.

We report here a new amino-modifier reagent, which enable high-throughput purification of amino-modified oligonucleotides. Either monomethoxytrityl (MMT) or trifluoroacetyl (TFA) group has been used as the protecting group for the primary amine when amino-terminal oligonucleotides are prepared. Generally, the removal of MMT requires the stringent acidic treatment after the oligonucleotide synthesis. We chemically synthesized a novel amino-modifier with the MMT protection. It was found that the new amino-modifier released MMT group under mild acidic condition, and then rapid purification of diverse amino-modified oligonucleotides could be achieved with cartridge column of reverse phase. Furthermore, we tried to construct the new detection system to study gene expression using the amino-modified oligonucleotides. The new amino-modifier will be useful for molecular biology by facilitating the construction of oligonucleotide library and improving the chemical reactivity.

Amines↗
PubMed2002

A high-throughput Arabidopsis reverse genetics system.

A collection of Arabidopsis lines with T-DNA insertions in known sites was generated to increase the efficiency of functional genomics. A high-throughput modified thermal asymmetric interlaced (TAIL)-PCR protocol was developed and used to amplify DNA fragments flanking the T-DNA left borders from approximately 100000 transformed lines. A total of 85108 TAIL-PCR products from 52964 T-DNA lines were sequenced and compared with the Arabidopsis genome to determine the positions of T-DNAs in each line. Predicted T-DNA insertion sites, when mapped, showed a bias against predicted coding sequences. Predicted insertion mutations in genes of interest can be identified using Arabidopsis Gene Index name searches or by BLAST (Basic Local Alignment Search Tool) search. Insertions can be confirmed by simple PCR assays on individual lines. Predicted insertions were confirmed in 257 of 340 lines tested (76%). This resource has been named SAIL (Syngenta Arabidopsis Insertion Library) and is available to the scientific community at www.tmri.org.

Agrobacterium tumefaciens↗
PubMed2006

Application of the use of high-throughput technologies to the determination of protein structures of bacterial and viral pathogens.

The Structural Proteomics In Europe (SPINE) programme is aimed at the development and implementation of high-throughput technologies for the efficient structure determination of proteins of biomedical importance, such as those of bacterial and viral pathogens linked to human health. Despite the challenging nature of some of these targets, 175 novel pathogen protein structures (approximately 220 including complexes) have been determined to date. Here the impact of several technologies on the structural determination of proteins from human pathogens is illustrated with selected examples, including the parallel expression of multiple constructs, the use of standardized refolding protocols and optimized crystallization screens.

Animals↗
PubMed2006

High-throughput affinity mass spectrometry.

Affinity mass spectrometry (AMS) is a proteomics approach for selectively isolating target protein(s) from complex biological fluids for mass spectrometric analysis. The resulting high-content mass spectrometry (MS) data show the unique MS protein signatures (wild-type, posttranslationally modified, as well as genetically modified forms of the protein target) that are present within a biological sample. Information regarding such protein diversity is normally lost in classical proteomic or immunoassay analyses. This chapter presents a step-by-step description of high-throughput AMS in the population proteomic screening of the human plasma protein cystatin C.

Amino Acid Sequence↗
PubMed2001

High-throughput organic synthesis of peptide mimetics.

This article is a subjective review of the literature from 1999 to March 2001. It seeks to highlight current progress in the high-throughput synthesis of peptide mimetics. The review is loosely classified into sections based on the type of secondary structural feature which is being mimicked (reverse turn, beta-strand, peptide surrogates) or a common motif. The examples chosen have demonstrated the ability to produce a large number of compounds rapidly or, in our opinion, the method could produce large numbers of compounds if desired.

Animals↗
PubMedDate not supplied

Development of combinatorial chemistry methods for coatings: high-throughput weathering evaluation and scale-up of combinatorial leads.

Combinatorial screening of materials formulations followed by the scale-up of combinatorial leads has been applied for the development of high-performance coating materials for automotive applications. We replaced labor-intensive coating formulation, testing, and measurement with a "combinatorial factory" that includes robotic formulation of coatings, their deposition as 48 coatings on a 9x12-cm plastic substrate, accelerated performance testing, and automated spectroscopic and image analysis of resulting performance. This high-throughput (HT) performance testing and measurement of the resulting properties provided a powerful set of tools for the 10-fold accelerated discovery of these coating materials. Performance of coatings is evaluated with respect to their weathering, because this parameter is one of the primary considerations in end-use automotive applications. Our HT screening strategy provides previously unavailable capabilities of (1) high speed and reproducibility of testing by using robotic automation and (2) improved quantification by using optical spectroscopic analysis of discoloration of coating-substrate structure and automatic imaging of the integrity loss of coatings. Upon testing, the coatings undergo changes that are impossible to quantitatively predict using existing knowledge. Using our HT methodology, we have developed several cost-competitive coatings leads that match the performance of more costly coatings. These HT screening results for the best coating compositions have been validated on the traditional scales of coating formulation and weathering testing. These validation results have confirmed the improved weathering performance of combinatorially developed coatings over conventional coatings on the traditional scale.

Coated Materials, Biocompatible↗
PubMed2004

Denaturing high-performance liquid chromatography (DHPLC) as a reliable high-throughput prescreening method for aberrant promoter methylation in cancer.

Aberrant promoter hypermethylation of CpG dinucleotides is a frequent and significant mechanism of tumor suppressor gene (TSG) silencing in cancer. As increasing numbers of downregulated putative TSGs are emerging from large-scale expression profiling studies, high-throughput techniques are needed to screen for hypermethylation. DHPLC has been established as a reliable, highly sensitive technique for mutation analysis. In this study, the use of DHPLC as a prescreening method for the identification of CpG methylation was developed by analyzing DNA samples with different, well-characterized methylation patterns of the CDKN2A/p16 promoter. Bisulfite treatment of genomic DNA was followed by PCR-amplification of unmethylated as well as methylated CDKN2A/p16 promoter sequences. PCR products were denatured and renatured, permitting the formation of heteroduplex DNA detectable by DHPLC. Methylation of all CpG-sites results in a single peak (homoduplex) with a shift in retention time, whereas partial methylation can be recognized by additional signals representing diverse heteroduplex structures. After method development, 35 DNA samples from primary bladder and breast carcinomas were analyzed in a blinded fashion, revealing complete or partial methylation of the p16 promoter in eight cases and a heterozygous mutation in one case. In conclusion, DHPLC is a highly sensitive and convenient method for methylation screening.

Breast Neoplasms↗
PubMedDate not supplied

Dual expression system suitable for high-throughput fluorescence-based screening and production of soluble proteins.

Many studies that aim to characterize the proteome structurally or functionally require the production of pure protein in a high-throughput format. We have developed a fast and flexible integrated system for cloning, protein expression in Escherichia coli, solubility screening and purification that can be completely automated in a 96-well microplate format. We used recombination cloning in custom-designed vectors including (i) a (His)(6) tag-encoding sequence, (ii) a variable solubilizing partner gene, (iii) the DNA sequence corresponding to the TEV protease cleavage site, (iv) the gene (or DNA fragment) of interest, (v) a suppressible amber stop codon, and (vi) an S.tag peptide-encoding sequence. First, conditions of bacterial culture in microplates (250 microL) were optimized to obtain expression and solubility patterns identical to those obtained in a 1-L flask (100-mL culture). Such conditions enabled the screening of various parameters in addition to the fusion partners (E. coli strains, temperature, inducer...). Second, expression of fusion proteins in amber suppressor strains allowed quantification of soluble and insoluble proteins by fluorescence through the detection of the S.tag. This technique is faster and more sensitive than other commonly used methods (dot blots, Western blots, SDS-PAGE). The presence of the amber suppressor tRNA was shown to affect neither the expression pattern nor the solubility of the target proteins. Third, production of the most interesting soluble fusion proteins, as detected by our screening method, could be performed in nonsuppressor strains. After cleavage with the TEV protease, the target proteins were obtained in a native form with a unique additional N-terminal glycine.

Blotting, Western↗
PubMed2003

A beta-lactamase-dependent Gal4-estrogen receptor beta transactivation assay for the ultra-high throughput screening of estrogen receptor beta agonists in a 3456-well format.

Estrogen action is mediated via two estrogen receptor (ER) subtypes, ERalpha and ERbeta. Selective ER modulators with balanced high affinity for ERalpha and ERbeta have been developed as therapeutics for the treatment of a variety of diseases, including hormone-responsive breast cancer and osteoporosis. Recent data based primarily on the evaluation of ER-knockout mice have revealed that ERalpha and ERbeta may regulate separate and distinct biological processes. The identification of ERbeta specific ligands could further enhance our understanding of ERbeta biology. In addition, compounds targeting ERbeta may prove useful as therapeutic agents with activity profiles distinguishable from that of estradiol. To discover novel selective ligands for ERbeta, we developed and characterized a cell-based Gal4-ERbeta beta-lactamase reporter gene assay (GERTA) in CHO cells for the ligand-induced activation of the human ERbeta. The sensitivity and selectivity of this assay were found to be comparable to those of an ER ligand-binding assay. The assay was optimized for screening in an ultra high throughput 3456-well nanoplate format and was successfully used to screen a large compound collection for ERbeta agonists. Compounds identified in a primary screen were tested in an in vitro ligand-binding assay to characterize further the selectivity and potency for ERbeta.

Animals↗
PubMed2002

Hierarchical high-throughput SNP genotyping of the human Y chromosome using MALDI-TOF mass spectrometry.

We have established the use of a primer extension/mass spectrometry method (the PinPoint assay) for high-throughput SNP genotyping of the human Y chromosome. 118 markers were used to define 116 haplogroups and typing was organised in a hierarchical fashion. Twenty multiplex PCR/primer extension reactions were set up and each sample could be assigned to a haplogroup with only two to five of these multiplex analyses. A single aliquot of one enzyme was found to be sufficient for both PCR and primer extension. We observed 100% accuracy in blind validation tests. The technique thus provides a reliable, cost-effective and automated method for Y genotyping, and the advantages of using a hierarchical strategy can be applied to any DNA segment lacking recombination.

DNA Primers↗
PubMed2004

Inhibitors of protein synthesis identified by a high throughput multiplexed translation screen.

The use of small molecule inhibitors of cellular processes is a powerful approach to understanding gene function that complements the genetic approach. We have designed a high throughput screen to identify new inhibitors of eukaryotic protein synthesis. We used a bicistronic mRNA reporter to multiplex our assay and simultaneously screen for inhibitors of cap-dependent initiation, internal initiation and translation elongation/termination. Functional screening of >90 000 compounds in an in vitro translation reaction identified 36 inhibitors, 14 of which are known inhibitors of translation and 18 of which are nucleic acid-binding ligands. Our results indicate that intercalators constitute a large class of protein synthesis inhibitors. Four non-intercalating compounds were identified, three of which block elongation and one of which inhibits initiation. The novel inhibitor of initiation affects 5' end-mediated initiation, as well as translation initiated from picornaviral IRESs, but does not significantly affect internal initiation from the hepatitis C virus 5'-untranslated region. This compound should be useful for delineating differences in mechanism of initiation among IRESs.

Animals↗
PubMed2003

mRNA accessible site tagging (MAST): a novel high throughput method for selecting effective antisense oligonucleotides.

A solution-based method, mRNA accessible site tagging (MAST), has been developed to map the accessible sites of any given mRNA in high throughput fashion. mRNA molecules were immobilized and hybridized to randomized oligonucleotide libraries. Oligonucleotides specifically hybridized to the mRNA were sequenced and found to be able to precisely define the accessible sites of the mRNA. A number of ways were used to validate the accessible sites defined by the MAST process. Mapping of rabbit beta-globin mRNA demonstrates the efficacy and advantage of MAST over other technologies in identifying accessible sites. Antisense oligonucleotides designed according to the accessible site map of human RhoA and Renilla luciferase mRNA result in knockdown effects that are in good correlation with the degrees of accessibility. The MAST methodology can be applied to mRNA of any length using a universal protocol.

Animals↗
PubMed2003

Large-scale discovery of induced point mutations with high-throughput TILLING.

TILLING (Targeting Induced Local Lesions in Genomes) is a general reverse-genetic strategy that provides an allelic series of induced point mutations in genes of interest. High-throughput TILLING allows the rapid and low-cost discovery of induced point mutations in populations of chemically mutagenized individuals. As chemical mutagenesis is widely applicable and mutation detection for TILLING is dependent only on sufficient yield of PCR products, TILLING can be applied to most organisms. We have developed TILLING as a service to the Arabidopsis community known as the Arabidopsis TILLING Project (ATP). Our goal is to rapidly deliver allelic series of ethylmethanesulfonate-induced mutations in target 1-kb loci requested by the international research community. In the first year of public operation, ATP has discovered, sequenced, and delivered >1000 mutations in >100 genes ordered by Arabidopsis researchers. The tools and methodologies described here can be adapted to create similar facilities for other organisms.

Alkylating Agents↗
PubMed2006

Application of high-throughput technologies to a structural proteomics-type analysis of Bacillus anthracis.

A collaborative project between two Structural Proteomics In Europe (SPINE) partner laboratories, York and Oxford, aimed at high-throughput (HTP) structure determination of proteins from Bacillus anthracis, the aetiological agent of anthrax and a biomedically important target, is described. Based upon a target-selection strategy combining ;low-hanging fruit' and more challenging targets, this work has contributed to the body of knowledge of B. anthracis, established and developed HTP cloning and expression technologies and tested HTP pipelines. Both centres developed ligation-independent cloning (LIC) and expression systems, employing custom LIC-PCR, Gateway and In-Fusion technologies, used in combination with parallel protein purification and robotic nanolitre crystallization screening. Overall, 42 structures have been solved by X-ray crystallography, plus two by NMR through collaboration between York and the SPINE partner in Utrecht. Three biologically important protein structures, BA4899, BA1655 and BA3998, involved in tRNA modification, sporulation control and carbohydrate metabolism, respectively, are highlighted. Target analysis by biophysical clustering based on pI and hydropathy has provided useful information for future target-selection strategies. The technological developments and lessons learned from this project are discussed. The success rate of protein expression and structure solution is at least in keeping with that achieved in structural genomics programs.

Bacillus anthracis↗
PubMed2003

A widely applicable, high-throughput TR-FRET assay for the measurement of kinase autophosphorylation: VEGFR-2 as a prototype.

Homogeneous time-resolved fluorescence resonance energy transfer (TR-FRET) assays represent a highly sensitive and robust high-throughput screening (HTS) method for the quantification of kinase activity. Traditional TR-FRET kinase assays detect the phosphorylation of an exogenous substrate. The authors describe the development and optimization of a TR-FRET technique that measures the autophosphorylation of vascular endothelial growth factor receptor 2 (VEGFR-2) kinase and extend its applicability to a variety of other kinases. The VEGFR-2 assay demonstrated dose-dependent inhibition by compounds known to modulate the catalytic activity of this receptor. In addition, kinetic analysis of a previously characterized VEGFR-2 inhibitor was performed using the method, and results were consistent with those obtained using a different assay format. Because of the known involvement of VEGFR-2 in angiogenesis, this assay should facilitate HTS for antiangiogenic agents. In addition, this general technique should have utility for the screening for inhibitors of kinases as potential therapeutic agents for many other disease indications.

Antibodies↗