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At least 415 records · Page 23Linked to original sources
PubMed1988

Cloning and expression in Escherichia coli of the gene for mouse tumor necrosis factor.

The mouse tumor necrosis factor (TNF) gene was isolated from a mouse genomic library. The entire sequence of the gene was determined using both an automated DNA sequencer with improved primer extension reaction conditions as well as the standard radioisotopic method. Comparison of the nucleotide sequence of the gene with that of mouse TNF cDNA showed that the mouse gene consists of four exons, like rabbit and human TNF genes. There is strong nucleotide sequence homology in the 5'-flanking region among the mouse, rabbit, and human TNF genes, suggesting that the mechanisms regulating TNF gene expression are highly conserved. Direct expression of mature mouse TNF was achieved using a plasmid constructed by site-directed mutagenesis. Purified mouse TNF produced in Escherichia coli showed cytotoxicity to mouse L cells.

Amino Acid Sequence↗
PubMed1996

A rapid method for detecting specific amplified PCR fragments in microtiter plates.

A simple method is presented to circumvent laborious and time consuming electrophoretic separations of specific PCR amplification products. Specific target DNA is amplified using nucleotides labelled with DIG-dUTP or biotin-dCTP. The labelled PCR products are separated from unincorporated nucleotides or oligonucleotides by using a positively charged DEAE cellulose matrix. Amplification products are visualized directly in the matrix using immunoenzymatic methods or streptavidin-conjugated enzymes. The detection process can be carried out within 2 h, allows the processing of large sample sizes and can potentially be automated.

DNA↗
PubMed2001

Combinatorial Heterogeneous Catalysis-A New Path in an Old Field.

Combinatorial catalysis is the systematic preparation, processing, and testing of large diversities of chemically and physically different materials libraries in a high-throughput fashion. It also embodies microfabrication, robotics, automation, instrumentation, computational chemistry, and large-scale information management (informatics), and as such carries the promise of a renaissance in catalytic reaction engineering. Significant progress has already been made in demonstrating the speed and economic advantage of combinatorial approaches by the discovery of superior catalytic materials in a matter of hours and days, as opposed to the months and years required using traditional methods. Combinatorial methods can also significantly contribute to our understanding of catalytic function by increasing our chances of discovering totally new and unexpected catalytic materials, and by expediting the recognition of trends and patterns of structure-activity relations, from which new catalytic materials can be designed more efficiently. Combinatorial catalysis undoubtedly will be the new paradigm of catalysis research as the industry faces increasing global competition and pressure for the development of environmentally friendly processes at a time when resources for research are diminishing.

Journal Article↗
PubMed1986

The formation of learning sets by the chinchilla in an auditory discrimination task.

The performance of 9 chinchillas (Chinchilla lanigera) in a completely computer-automated sound-discrimination experiment was observed using stimulus pairs from a library of 180 complex sounds. Each sound of a pair was 0.2-2.08 sec in duration, separated by an interval of 250 +/- 125 msec. A modified "go/no go" paradigm was used. Licking behavior at a water-delivery tube was occasioned by keeping Ss at about 85% of ad-lib weight by water deprivation. After habituation and blank-trials training stages, sound discrimination was required: If the sounds of a pair were identical, no response was required; if they were different, a fleeing response (crossing a mid-cage barrier) was required. Each problem set utilized a different sound pair, for a total of 90 problems. Therefore, Ss learned to perform general auditory discriminations, not sound-specific discriminations. The chinchillas quickly learned to respond, but not at a rate consistently higher than 75 %-correct. The results were compared to those of other studies of sensory discrimination in monkey and chinchilla. Suggestions were made for minor improvement of the paradigm for use in future sound-discrimination tasks.

Animals↗
PubMed2000

Molecular surveillance of European quinolone-resistant clinical isolates of Pseudomonas aeruginosa and Acinetobacter spp. using automated ribotyping.

Nosocomial isolates of Pseudomonas aeruginosa and Acinetobacter spp. exhibit high rates of resistance to antibiotics and are often multidrug resistant. In a previous study (D. Milatovic, A. Fluit, S. Brisse, J. Verhoef, and F. J. Schmitz, Antimicrob. Agents Chemother. 44:1102-1107, 2000), isolates of these species that were resistant to sitafloxacin, a new advanced-generation fluoroquinolone with a high potency and a broad spectrum of antimicrobial activity, were found in high proportion in 23 European hospitals. Here, we investigate the clonal diversity of the 155 P. aeruginosa and 145 Acinetobacter spp. sitafloxacin-resistant isolates from that study by automated ribotyping. Numerous ribogroups (sets of isolates with indistinguishable ribotypes) were found among isolates of P. aeruginosa (n = 34) and Acinetobacter spp. (n = 16), but the majority of the isolates belonged to a limited number of major ribogroups. Sitafloxacin-resistant isolates (MICs > 2 mg/liter, used as a provisional breakpoint) showed increased concomitant resistance to piperacillin, piperacillin-tazobactam, ceftriaxone, ceftazidime, amikacin, gentamicin, and imipenem. The major ribogroups were repeatedly found in isolates from several European hospitals; these isolates showed higher levels of resistance to gentamicin and imipenem, and some of them appeared to correspond to previously described multidrug-resistant international clones of P. aeruginosa (serotype O:12) and Acinetobacter baumannii (clones I and II). Automated ribotyping, when used in combination with more discriminatory typing methods, may be a convenient library typing system for monitoring future epidemiological dynamics of geographically widespread multidrug-resistant bacterial clones.

4-Quinolones↗
PubMed1999

A knowledge-based approach in designing combinatorial or medicinal chemistry libraries for drug discovery. 1. A qualitative and quantitative characterization of known drug databases.

The discovery of various protein/receptor targets from genomic research is expanding rapidly. Along with the automation of organic synthesis and biochemical screening, this is bringing a major change in the whole field of drug discovery research. In the traditional drug discovery process, the industry tests compounds in the thousands. With automated synthesis, the number of compounds to be tested could be in the millions. This two-dimensional expansion will lead to a major demand for resources, unless the chemical libraries are made wisely. The objective of this work is to provide both quantitative and qualitative characterization of known drugs which will help to generate "drug-like" libraries. In this work we analyzed the Comprehensive Medicinal Chemistry (CMC) database and seven different subsets belonging to different classes of drug molecules. These include some central nervous system active drugs and cardiovascular, cancer, inflammation, and infection disease states. A quantitative characterization based on computed physicochemical property profiles such as log P, molar refractivity, molecular weight, and number of atoms as well as a qualitative characterization based on the occurrence of functional groups and important substructures are developed here. For the CMC database, the qualifying range (covering more than 80% of the compounds) of the calculated log P is between -0.4 and 5.6, with an average value of 2.52. For molecular weight, the qualifying range is between 160 and 480, with an average value of 357. For molar refractivity, the qualifying range is between 40 and 130, with an average value of 97. For the total number of atoms, the qualifying range is between 20 and 70, with an average value of 48. Benzene is by far the most abundant substructure in this drug database, slightly more abundant than all the heterocyclic rings combined. Nonaromatic heterocyclic rings are twice as abundant as the aromatic heterocycles. Tertiary aliphatic amines, alcoholic OH and carboxamides are the most abundant functional groups in the drug database. The effective range of physicochemical properties presented here can be used in the design of drug-like combinatorial libraries as well as in developing a more efficient corporate medicinal chemistry library.

Algorithms↗
PubMed2002

Automated analysis of large sets of heteronuclear correlation spectra in NMR-based drug discovery.

Drug discovery procedures based on NMR typically require the analysis of thousands of NMR spectra. For example, in "SAR by NMR", two-dimensional NMR spectra are recorded for a target protein mixed with ligand candidates from a comprehensive library of small molecules and are compared to the corresponding spectrum for the protein alone. We present an automated procedure for the comparative analysis of large sets of heteronuclear single quantum coherence spectra, which is based on three-way decomposition and implemented as the software package MUNIN. In a single step, spectra with differences in the peak positions (indicating ligand binding) and the affected peaks are identified. By omission of peak picking, ad hoc scoring of the quality of doubtful peaks is avoided. The procedure has been tested on the bacterial ribonuclease barnase, with a protein concentration of only 50 microM, using several small molecules including the substrate analogue 3'-GMP. Sets of 51 spectra were processed simultaneously, and it is concluded that spectra with binding ligands can be unambiguously identified from much larger sets of spectra.

Bacterial Proteins↗
PubMed2006

High-content screening of functional genomic libraries.

Recent advances in functional genomics have enabled genome-wide genetic studies in mammalian cells. These include the establishment of high-throughput transfection and viral propagation methodologies, the production of large-scale cDNA and siRNA libraries, and the development of sensitive assay detection processes and instrumentation. The latter has been significantly facilitated by the implementation of automated microscopy and quantitative image analysis, collectively referred to as high-content screening (HCS), toward cell-based functional genomics application. This technology can be applied to whole genome analysis of discrete molecular and phenotypic events at the level of individual cells and promises to significantly expand the scope of functional genomic analyses in mammalian cells. This chapter provides a comprehensive guide for curating and preparing function genomics libraries and performing HCS at the level of the genome.

Animals↗
PubMed2003

Computational methods for sequence mapping of large combinatorial libraries and deduced sequence signatures.

Here we describe a computational approach for the high-throughput sequence mapping of combinatorial libraries obtained by DNA shuffling. Original algorithms and their software implementation were developed for the automated and reliable analysis of hybridization data of differentially labeled oligonucleotide probes with PCR products spotted on DNA microarrays. This novel approach allows a context-dependent sequence attribution tolerant to fluctuations in experimental conditions and is well adapted to hybridization signals of variable qualities resulting from high-throughput PCR amplification from colonies. In addition, the analysis permits the calculation of sequence signatures that are characteristic of combinatorial library structure, defects, and diversity. The approach is of interest for the characterization and the equalization (library reduction to nonredundant structures) of combinatorial libraries involved in directed evolution and could be extrapolated to high-throughput polymorphism analysis.

Biotechnology↗
PubMed2004

An automated image capture and quantitation approach to identify proteins affecting tumor cell proliferation.

To facilitate the characterization of proteins that negatively regulate tumor cell proliferation in vitro, the authors have implemented a high-throughput functional assay that measures S-phase progression of tumor cell lines. For 2 tumor cell lines-human melanoma A375 and human lung carcinoma A549-conditions were established using the cyclin-dependent kinase inhibitor, p27kip; the tumor suppressor p53, a kinase-inactive allele of the cell cycle-regulated serine/threonine kinase Aurora2; and the G1/S drug block, aphidicolin. For screening purposes, gene libraries were delivered by adenoviral infection. Cells were fixed and labeled by immunocytochemistry, and an automated image acquisition and analysis package on a Cellomics ArrayScanII was used to quantify the effects of these treatments on cell proliferation. The assay can be used to identify novel proteins involved in proliferation and serves as a more robust, reproducible, and sensitive alternative to enzyme-linked immunosorbent assay (ELISA)-based technologies.

Automation↗
PubMed2004

Transposon Express, a software application to report the identity of insertions obtained by comprehensive transposon mutagenesis of sequenced genomes: analysis of the preference for in vitro Tn5 transposition into GC-rich DNA.

Comprehensive mutant libraries can be readily constructed by transposon mutagenesis. To systematically mutagenise the genome of the Gram-positive bacterium Streptomyces coelicolor A3(2), we have employed high-throughput shuttle transposon mutagenesis of a cosmid library prepared in Escherichia coli. The location of transposon insertions is determined using automated procedures for cosmid isolation and DNA sequencing. However, a major bottleneck was the subsequent analysis of DNA sequence files. To overcome this limitation, a software application, Transposon Express, was written to allow the rapid location of transposon insertions in a sequenced genome (available at http://www.swan.ac.uk/genetics/dyson/InstallTE). Transposon Express determines the identity both of a disrupted open reading frame (ORF), and the short target site duplication created by transposition. Transposon Express also reports the orientation of the transposon and can therefore predict transcriptional coupling between an upstream promoter and a promoter-less reporter gene carried by the transposon. Analysis of a large dataset of independent insertions created using a Tn5-based transposon revealed an insertional preference for GC-rich streptomycete DNA compared to E.coli vector DNA. In addition to demonstrating the value of Transposon Express as a generic tool supporting genome-wide transposon mutagenesis programs, these data provide insight into target site selection by Tn5.

Computational Biology↗
PubMed1997

High throughput analysis and purification in support of automated parallel synthesis.

Rapid reverse-phase analytical and preparative HPLC methods have been developed for application to parallel synthesis libraries. Gradient methods, short columns, and high flow rates allow analysis of over 300 compounds per day on a single system, or purification of up to 200 compounds per day on a single preparative system. Hardware and software modifications allow continuous unattended use for maximum efficiency and throughput.

Automation↗
PubMed2005

Acquisition of Fyn-selective SH3 domain ligands via a combinatorial library strategy.

A stepwise library-based strategy has been employed to acquire a potent ligand for the SH3 domain of Fyn, a Src kinase family member that plays a key role in T cell activation. The easily automated methodology is designed to identify potential interaction sites that circumscribe the protein/peptide binding region on the SH3 domain. The library protocol creates peptide/nonpeptide chimeras that are able to bind to these interaction sites that are otherwise inaccessible to natural amino acid residues. The peptide-derived lead and the Fyn-SH3 domain form a complex that exhibits a K(D) of 25 +/- 5 nM, approximately 1000-fold more potent than that displayed by the corresponding conventional peptide ligand. Furthermore, the lead ligand exhibits selectivity against SH3 domains derived from other Src kinases, in spite of a sequence identity of approximately 80%.

Amino Acid Sequence↗
PubMed2004

Accelerated screening of phage-display output with alkaline phosphatase fusions.

When using multiple targets and libraries, selection of affinity reagents from phage-displayed libraries is a relatively time-consuming process. Herein, we describe an automation-amenable approach to accelerate the process by using alkaline phosphatase (AP) fusion proteins in place of the phage ELISA screening and subsequent confirmation steps with purified protein. After two or three rounds of affinity selection, the open reading frames that encode the affinity selected molecules (i.e., antibody fragments, engineered scaffold proteins, combinatorial peptides) are amplified from the phage or phagemid DNA molecules by PCR and cloned en masse by a Ligation Independent Cloning (LIC) method into a plasmid encoding a highly active variant of E. coli AP. This time-saving process identifies affinity reagents that work out of context of the phage and that can be used in various downstream enzyme linked binding assays. The utility of this approach was demonstrated by analyzing single-chain antibodies (scFvs), engineered fibronectin type III domains (FN3), and combinatorial peptides that were selected for binding to the Epsin N-terminal Homology (ENTH) domain of epsin 1, the c-Src SH3 domain, and the appendage domain of the gamma subunit of the clathrin adaptor complex, AP-1, respectively.

Alkaline Phosphatase↗
PubMed1983

Automated metabolic profiling analysis of urinary steroids by a gas chromatography mass spectrometry data system.

A computer system (MSSMET), using methylene unit retention indices for an off-line reverse library search analysis of selected ion chromatograms from gas-liquid chromatographic mass spectrometric data, has been applied for the qualitative and quantitative determination of daily variations in the excreted levels of urinary steroids of two individuals, using capillary column gas-liquid chromatography. Aliquots of 24 h urine collections and morning spot urine samples were examined. The daily excretion pattern of most of the major steroid metabolites was fairly consistent from day to day (i.e. 3 alpha-hydroxy-5 alpha-androstane-17-one, androsterone; 3 alpha-hydroxy-5 beta-androstane-17-one, etiocheolanolone; 3 alpha, 17 alpha, 21-trihydroxy-5 beta-pregnane-11,20-dione, THE; 3 alpha, 11 beta, 17 alpha, 21-tetrahydroxy-5 beta-pregnane-20-one, THF; 3 alpha, 11 beta, 17 alpha, 21-tetrahydroxy-5 alpha-pregnane-20-one; allo-THF; 3 alpha, 17 alpha, 20 alpha, 21-tetrahydroxy-5 beta-pregnane-11-one, cortolone; 3 alpha, 17 alpha, 20 beta, 21-tetrahydroxy-5 beta-pregnane-11-one, beta-cortolone; 5 beta-pregnane-3 alpha, 11 beta,17 alpha,20 alpha,21-pentol, cortol; 5 beta-pregnane-3 alpha, 11 beta, 17 alpha, 20 beta, 21-pentol, beta-cortol), while certain other steroid metabolites had a less consistent excretion pattern (3 beta-hydroxy-5-androstene-17-one, for example). Advantages and disadvantages of using capillary columns for the automated metabolic profile analysis of urinary steroids by reverse library search of selected mass chromatograms.

Adult↗