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At least 1,045 records · Page 58Linked to original sources
PubMed2006

A high throughput fluorescent assay for measuring the activity of fatty acid amide hydrolase.

Fatty acid amide hydrolase (FAAH) is the enzyme responsible for the rapid degradation of fatty acid amides such as the endocannabinoid anandamide. Inhibition of FAAH activity has been suggested as a therapeutic approach for the treatment of chronic pain, depression and anxiety, through local activation of the cannabinoid receptor CB1. We have developed a high throughput screening assay for identification of FAAH inhibitors using a novel substrate, decanoyl 7-amino-4-methyl coumarin (D-AMC) that is cleaved by FAAH to release decanoic acid and the highly fluorescent molecule 7-amino-4-methyl coumarin (AMC). This assay gives an excellent signal window for measuring FAAH activity and, as a continuous assay, inherently offers improved sensitivity and accuracy over previously reported endpoint assays. The assay was validated using a panel of known FAAH inhibitors and purified recombinant human FAAH, then converted to a 384 well format and used to screen a large library of compounds (>600,000 compounds) to identify FAAH inhibitors. This screen identified numerous novel FAAH inhibitors of diverse chemotypes. These hits confirmed using a native FAAH substrate, anandamide, and had very similar rank order potency to that obtained using the D-AMC substrate. Collectively these data demonstrate that D-AMC can be successfully used to rapidly and effectively identify novel FAAH inhibitors for potential therapeutic use.

Amidohydrolases↗
PubMed1992

Development of a biological dosimeter for translocation scoring based on two-color fluorescence in situ hybridization of chromosome subsets.

Recently fluorescence in situ hybridization protocols have been developed which allow the painting of individual chromosomes using DNA-libraries from sorted human chromosomes. This approach has the particular advantage that radiation induced chromosome translocations can be easily detected, if chromosomes of distinctly different colors take part in the translocation event. To enhance the sensitivity of this approach two metaphase chromosome subsets A and B (A: chromosomes 1, 2, 4, 8, 16; B: 3, 5, 9, 10, 13) were simultaneously painted in green and red color. Counterstaining of the chromosomes with DAPI resulted in a third subset which exhibited blue fluorescence only. Green-red, green-blue and red-blue translocation chromosomes could be easily detected after irradiation of lymphocyte cultures with 137Cs-gamma-rays. Analyses of painted chromosomes can be combined with conventional GTG-banding analyses. This new biological dosimeter should become useful to monitor both long term effects of single irradiation events and the cumulative effects of multiple or chronic irradiation exposures. In contrast to translocation scoring based on the analysis of banded chromosomes, this new approach has the particular advantage that a rapid, automated scoring of translocations can now be envisaged.

Chromosomes↗
PubMed1998

Analysis of EST-driven gene annotation in human genomic sequence.

We have performed a systematic analysis of gene identification in genomic sequence by similarity search against expressed sequence tags (ESTs) to assess the suitability of this method for automated annotation of the human genome. A BLAST-based strategy was constructed to examine the potential of this approach, and was applied to test sets containing all human genomic sequences longer than 5 kb in public databases, plus 300 kb of exhaustively characterized benchmark sequence. At high stringency, 70%-90% of all annotated genes are detected by near-identity to EST sequence; >95% of ESTs aligning with well-annotated sequences overlap a gene. These ESTs provide immediate access to the corresponding cDNA clones for follow-up laboratory verification and subsequent biologic analysis. At lower stringency, up to 97% of annotated genes were identified by similarity to ESTs. The apparent false-positive rate rose to 55% of ESTs among all sequences and 20% among benchmark sequences at the lowest stringency, indicating that many genes in public database entries are unannotated. Approximately half of the alignments span multiple exons, and thus aid in the construction of gene predictions and elucidation of alternative splicing. In addition, ESTs from multiple cDNA libraries frequently cluster over genes, providing a starting point for crude expression profiles. Clone IDs may be used to form EST pairs, and particularly to extend models by associating alignments of lower stringency with high-quality alignments. These results demonstrate that EST similarity search is a practical general-purpose annotation technique that complements pattern recognition methods as a tool for gene characterization.

Base Sequence↗
PubMed2001

Transfected cell lines as tools for high throughput screening: a call for standards.

During 1999, Journal of Biomolecular Screening presented a series of Point-Counterpoint articles that addressed a question posed by editor Bill Janzen: "What is the future of HTS?" These articles discussed many of the global issues involved in HTS, such as target identification and library size, as well as the scientific and technical challenges facing the field. In this perspective we address a related, but very focused, issue that is increasingly important for many of us in the HTS community: the use of stably transfected cell lines as an integral part of screening strategies. Transfected cell lines provide powerful tools for assay design, but at the same time they introduce complex variables into the screening system. Although it is difficult to develop precise definitions and standards for biologicals such as cell lines, we propose that the development of guidelines for the nomenclature and use of transfected cell lines is essential for their use in HTS.

Animals↗
PubMed1999

A genetic strategy to eliminate self-activator baits prior to high-throughput yeast two-hybrid screens.

Large-scale sequencing projects have predicted high numbers of gene products for which no functional information is yet available. Hence, large-scale projects, such as gene knockouts, gene expression profiles, and protein-interaction mapping, are currently under way to initiate the understanding of the function of these gene products. The high-throughput strategies that are currently being developed to generate protein-interaction maps include automated versions of the yeast two-hybrid system. These strategies rely on the large-scale construction of DNA-binding domain/protein-of-interest hybrid constructs (DB-X baits). An inherent problem of large-scale two-hybrid systems is that a high percentage of cloned sequences encode polypeptides that, when fused to DB, can activate transcription in the absence of any two-hybrid-interacting partner protein. Here, we describe and validate a genetic strategy that efficiently eliminates such self-activator baits prior to screening procedures. The strategy is based on a negative-growth selection and is compatible with high-throughput settings.

DNA, Fungal↗
PubMed2007

Development of a homogeneous calcium mobilization assay for high throughput screening of mas-related gene receptor agonists.

AIM: To develop homogeneous calcium mobilization assay for high-throughput screening (HTS) of mas-related gene (Mrg) receptor agonists. METHODS: CHO-K1 cells stably expressing the full-length MrgD receptor and a calcium-sensitive dye were used to develop an HTS assay based on intracellular calcium influx. This method was applied to large-scale screening of a library containing 8000 synthetic compounds and natural product extracts. cAMP measurements were carried out to verify the bioactivities of the hits found by the calcium mobilization assay. Similar approaches were also employed in the identification of the MrgA1 receptor agonists following HTS of 16,000 samples. RESULTS: EC(50) values of the positive control compounds (beta-alanine for MrgD receptor and dynorphin A for MrgA1 receptor) determined by the calcium mobilization assay were consistent with those reported in the literature, and the Z' factors were 0.65 and 0.50 for MrgD and MrgA1 receptor assay, respectively. About 31 compounds for the MrgD receptor and 48 compounds for the MrgA1 receptor showing > or =20% of the maximal agonist activities found in the controls were initially identified as hits. Secondary screening confirmed that 2 compounds for each receptor possessed specific agonist activities. Intracellular cAMP level measurements indicated that the 2 confirmed hits displayed the functionality of the MrgD receptor agonists. CONCLUSION: A series of validation studies demonstrated that the homogeneous calcium mobilization assay developed was highly efficient, amenable to automation and a robust tool to screen potential MrgD and MrgA1 receptor agonists. Its application may be expanded to other G-protein coupled receptors that mobilize calcium influx upon activation.

Animals↗
PubMed2005

Homogeneous, bioluminescent protease assays: caspase-3 as a model.

Using caspase-3 as a model, the authors have developed a strategy for highly sensitive, homogeneous protease assays suitable for high-throughput, automated applications. The assay uses peptide-conjugated aminoluciferin as the protease substrate and a firefly luciferase that has been molecularly evolved for increased stability. By combining the proluminescent caspase-3 substrate, Z-DEVD-aminoluciferin, with a stabilized luciferase in a homogeneous format, the authors developed an assay that is significantly faster and more sensitive than fluorescent caspase-3 assays. The assay has a single-step format, in which protease cleavage of the substrate and luciferase oxidation of the aminoluciferin occurs simultaneously. Because these processes are coupled, they rapidly achieve steady state to maintain stable luminescence for several hours. Maximum sensitivity is attained when this steady state occurs; consequently, this coupled-enzyme system results in a very rapid assay. The homogeneous format inherently removes trace contamination by free aminoluciferin, resulting in extremely low background and yielding exceptionally high signal-to-noise ratios and excellent Z' factors. Another advantage of a luminescent format is that it avoids problems of cell autofluorescence or fluorescence interference that can be associated with synthetic chemical and natural product libraries. This bioluminescent, homogeneous format should be widely applicable to other protease assays.

Caspase 3↗
PubMed2003

BioQuery: an object framework for building queries to biomedical databases.

SUMMARY: BioQuery is an application that helps scientists automate database searches. Users can build and store queries to public biomedical databases, and receive periodic updates on the results of those queries when new data is available. The application is implemented on a portable object framework that can provide database-searching capability to other applications. This framework is easily extensible, allowing users to develop plug-ins that provide access to new databases. BioQuery thus provides end-users with a complete database searching interface and updating service, and gives developers a toolkit to provide database-searching capability to their applications. AVAILABILITY: Free to all users: http://www.bioquery.org.

Biomedical Research↗
PubMed1990

SPLICE, a computer program for automated extraction of information from GenBank sequence entries.

SPLICE, a software tool for the extraction of sequences from files in GenBank tape format, has been developed. The program can analyze the features table in this format and use any of the information provided to write the corresponding sequences into a standard sequence file format suitable for use with sequence analysis programs. Sequences that are present as several subsequent fragments in a single GenBank file, such as those encoding a peptide, can be spliced together by the program. Further, sequences that are present in more than one Genbank file, such as an exon which spans several different files, can also be spliced into one sequence. SPLICE runs under the MS/DOS and Unix operating systems, can be called as a sub-process by other programs and can process batches of files.

Animals↗
PubMed1999

DENS (differential extension with nucleotide subsets): application to the sequencing of human genomic DNA and cDNA.

Here we describe further development of our method of DNA sequencing by Differential Extension with Nucleotide Subsets (DENS) and its application to the sequencing of human genomic DNA and full-insert cDNA. Essentially, DENS is primer walking without custom primer synthesis; instead, DENS uses a presynthesized library of octamer primers degenerate in two positions (4,096 tubes/sequences for a complete library). DENS converts an octamer selected from this library into a long primer on the template, at the intended site only. This is done using a two-step procedure which starts with a limited extension of the octamer (at 20 degrees C) in the presence of only two of the four possible dNTPs. The primer is extended by five bases or more at the intended priming site, which is deliberately selected to maximize the extension length (as are the two-dNTP set and the primer itself). The subsequent termination reaction at 60 degrees C then accepts the primer extended at the intended site, but not at alternative sites, where the initial extension (if any) is generally much shorter. This paper presents a set of rules for selection of DENS priming sites. We also compare different ways of template preparation for DENS sequencing. The data were obtained from primer walking on three human genomic DNA subclones of 3 to 4 kbp and four cDNA clones containing inserts of 1.9, 2.3, 3.8, and 4.9 kbp. Full-length sequences were obtained from both strands of each subclone by automated dye-terminator fluorescent DNA sequencing using DENS with degenerate octamer primers. We compared the following types of DNA templates: single-stranded and double-stranded phagemid DNA, double-stranded PCR products, asymmetric PCR products, and single-stranded DNA produced by digestion with Lambda Exonuclease of double-stranded PCR product phosphorylated at one end (Exo-PCR). While all of the preps were found to work, the best results were obtained with Exo-PCR and phagemid single-stranded DNA. Exo-PCR directly from overnight bacterial culture with no plasmid prep of any kind yielded templates for DENS as good as Exo-PCR from purified DNA. We found that the Tm of the differentially extended octamers is an important factor in the success of DENS. Clustering of successful reactions was clearly distinguished in the Tm range of 50-66 degrees C, with success rates of 70% for Exo-PCR and 65% for ss phagemid templates.

Base Sequence↗
PubMed2006

SVM Classifier - a comprehensive java interface for support vector machine classification of microarray data.

MOTIVATION: Graphical user interface (GUI) software promotes novelty by allowing users to extend the functionality. SVM Classifier is a cross-platform graphical application that handles very large datasets well. The purpose of this study is to create a GUI application that allows SVM users to perform SVM training, classification and prediction. RESULTS: The GUI provides user-friendly access to state-of-the-art SVM methods embodied in the LIBSVM implementation of Support Vector Machine. We implemented the java interface using standard swing libraries. We used a sample data from a breast cancer study for testing classification accuracy. We achieved 100% accuracy in classification among the BRCA1-BRCA2 samples with RBF kernel of SVM. CONCLUSION: We have developed a java GUI application that allows SVM users to perform SVM training, classification and prediction. We have demonstrated that support vector machines can accurately classify genes into functional categories based upon expression data from DNA microarray hybridization experiments. Among the different kernel functions that we examined, the SVM that uses a radial basis kernel function provides the best performance. The SVM Classifier is available at http://mfgn.usm.edu/ebl/svm/.

Cluster Analysis↗
PubMed1998

A software system for data analysis in automated DNA sequencing.

Software for gel image analysis and base-calling in fluorescence-based sequencing consisting of two primary programs, BaseFinder and GelImager, is described. BaseFinder is a framework for trace processing, analysis, and base-calling. BaseFinder is highly extensible, allowing the addition of trace analysis and processing modules without recompilation. Powerful scripting capabilities combined with modularity and multilane handling allow the user to customize BaseFinder to virtually any type of trace processing. We have developed an extensive set of data processing and analysis modules for use with the program in fluorescence-based sequencing. GelImager is a framework for gel image manipulation. It can be used for gel visualization, lane retracking, and as a front end to the Washington University Getlanes program. The programs were designed using a cross-platform development environment, currently allowing them to run in Windows NT, Windows 95, Openstep/Mach, and Rhapsody. Work is ongoing to deploy the software on additional platforms, including Solaris, Linux, and MacOS. This software has been thoroughly tested and debugged in the analysis of >2 million bp of raw sequence data from human chromosome 19 region q13. Overall sequencing accuracy was measured using a significant subset of these data, consisting of approximately 600 sequences, by comparing the individual shotgun sequences against the final assembled contigs. Also, results are reported from experiments that analyzed the accuracy of the software and two other well-known base-calling programs for sequencing the M13mp18 vector sequence. [The sequence data described in this paper have been submitted to the GenBank data library under accession no. AF025422]

Algorithms↗
PubMed2005

Object detection via feature synthesis using MDL-based genetic programming.

In this paper, we use genetic programming (GP) to synthesize composite operators and composite features from combinations of primitive operations and primitive features for object detection. The motivation for using GP is to overcome the human experts' limitations of focusing only on conventional combinations of primitive image processing operations in the feature synthesis. GP attempts many unconventional combinations that in some cases yield exceptionally good results. To improve the efficiency of GP and prevent its well-known code bloat problem without imposing severe restriction on the GP search, we design a new fitness function based on minimum description length principle to incorporate both the pixel labeling error and the size of a composite operator into the fitness evaluation process. To further improve the efficiency of GP, smart crossover, smart mutation and a public library ideas are incorporated to identify and keep the effective components of composite operators. Our experiments, which are performed on selected training regions of a training image to reduce the training time, show that compared to normal GP, our GP algorithm finds effective composite operators more quickly and the learned composite operators can be applied to the whole training image and other similar testing images. Also, compared to a traditional region-of-interest extraction algorithm, the composite operators learned by GP are more effective and efficient for object detection.

Algorithms↗
PubMed2000

Octamer-primed sequencing technology: development of primer identification software.

Octamer sequencing technology (OST) is a primer-directed sequencing strategy in which an individual octamer primer is selected from a pre-synthesized octamer primer library and used to sequence a DNA fragment. However, selecting candidate primers from such a library is time consuming and can be a bottleneck in the sequencing process. To accelerate the sequencing process and to obtain high quality sequencing data, a computer program, electronic OST or eOST, was developed to automatically identify candidate primers from an octamer primer library. eOST integrates the base calling software PHRED to provide a quality assessment for target sequences and identifies potential primer binding sites located within a high quality target region. To increase the sequencing success rate, eOST includes a simple dynamic folding algorithm to automatically calculate the free energy and predict the secondary structure within the template in the vicinity of the octamer-binding site. Several parameters were found to be important, including base quality threshold, the window size of the template sequence segment, and the threshold [Delta] G value. OST, coupled with the eOST software, can be used to sequence short DNA fragments or in the finishing assembly stage of large-scale sequencing of genomic DNA.

Algorithms↗
PubMed2000

Genome-wide protein interaction maps using two-hybrid systems.

Automated sequence technology has rendered functional biology amenable to genomic scale analysis. Among genome-wide exploratory approaches, the two-hybrid system in yeast (Y2H) has outranked other techniques because it is the system of choice to detect protein-protein interactions. Deciphering the cascade of binding events in a whole cell helps define signal transduction and metabolic pathways or enzymatic complexes. The function of proteins is eventually attributed through whole cell protein interaction maps where totally unknown proteins are partnered with fully annotated proteins belonging to the same functional category. Since its first description in the late 1980's, several versions of the Y2H have been developed in order to overcome the major limitations of the system, namely false positives and false negatives. Optimized versions have been recently applied at multi-molecular and genomic scale. These genome-wide surveys can be methodologically divided into two types of approaches: one either tests combinations of predefined polypeptides (the so-called matrix approach) using various short-cuts to speed up the process, or one screens with a given polypeptide (bait) for potential partners (preys) present in complex libraries of genomic or complementary DNA (library screening). In the former strategy, one tests what one knows, for example pair-wise interactions between full-length open reading frames from recently sequenced and annotated genomes. Although based on a one-by-one scheme, this method is reported to be amenable to large-scale genomics thanks to multicloning strategies and to the use of small robotics workstations. In the latter, highly complex cDNA or genomic libraries of protein domains can be screened to saturation with high-throughput screening systems allowing the discovery of yet unidentified proteins. Both approaches have strengths and drawbacks that will be discussed here. None yields a full proteome-wide screening since certain proteins (e.g. some transcription factors) are not usable in Y2H. Novel two-hybrid assays have been recently described in bacteria. Applications of these time- and cost-effective assays to genomic screening will be discussed and compared to the Y2H technology.

Animals↗
PubMedDate not supplied

Fast, easy, and efficient method for the purification of phenolic isomers using a selective solid-phase scavenging process.

The need for fast and efficient purification methods that can be easily handled and moreover automated is considerably increasing with the new techniques of high-throughput chemical synthesis. Following our previous work on the use of simple polymeric scavengers in fast reactions and purifications of organic substances, this article presents the results found during the development of a new method for the purification of phenolic substances. The purification method was found to be regulated by the interaction of acidic phenol groups with a basic polystyrene resin. Furthermore, the scavenging of phenolic isomers proved to be very selective for a given isomer. But the most interesting aspect of this method is that it is based on a simple contact in situ with the resin and that the adsorption/desorption process of the phenol was found to be solvent-dependent. The phenols can thus be freed from impurities, or other isomers, by a simple and fast contact with the resin in the first solvent, filtration, and washings, followed by liberation of the purified phenol by a last soaking in another solvent for desorption. The method was successfully applied to the purification of a crude reaction mixture issued from the Fries rearrangement of phenyl acetate, as well as to small libraries of phenolic derivatives.

Journal Article↗
PubMed2006

Three-dimensional model-based object recognition and segmentation in cluttered scenes.

Viewpoint independent recognition of free-form objects and their segmentation in the presence of clutter and occlusions is a challenging task. We present a novel 3D model-based algorithm which performs this task automatically and efficiently. A 3D model of an object is automatically constructed offline from its multiple unordered range images (views). These views are converted into multidimensional table representations (which we refer to as tensors). Correspondences are automatically established between these views by simultaneously matching the tensors of a view with those of the remaining views using a hash table-based voting scheme. This results in a graph of relative transformations used to register the views before they are integrated into a seamless 3D model. These models and their tensor representations constitute the model library. During online recognition, a tensor from the scene is simultaneously matched with those in the library by casting votes. Similarity measures are calculated for the model tensors which receive the most votes. The model with the highest similarity is transformed to the scene and, if it aligns accurately with an object in the scene, that object is declared as recognized and is segmented. This process is repeated until the scene is completely segmented. Experiments were performed on real and synthetic data comprised of 55 models and 610 scenes and an overall recognition rate of 95 percent was achieved. Comparison with the spin images revealed that our algorithm is superior in terms of recognition rate and efficiency.

Algorithms↗
PubMed1996

Molecular characterization, over-expression and purification of a novel dipeptidase from Lactobacillus helveticus.

A dipeptidase gene (pepD) from an industrial Lactobacillus helveticus strain was isolated by colony hybridization. An open reading frame (ORF) of 1422 base pairs (bp) with a coding capacity for a 53.5-kDa protein (PepD) was identified. The ORF was preceded by a typical prokaryotic promoter region, and an inverted repeat structure with delta G of -51.0 kJ mol-1 was found downstream of the coding region. The deduced amino acid sequence of the 53.5-kDa protein revealed no marked homologies when compared to the data bases of EMBL and SWISS-PROT. The 5'end of the 1.6-kb pepD transcript was determined both by a conventional primer extension method and using an automated sequencer. pepD was found to be maximally expressed at late exponential growth. The pepD gene was cloned into an expression vector to over-produce PepD in Escherichia coli JM105. Purification of PepD to homogeneity was achieved using three chromatographic steps. PepD was able to hydrolyze a number of dipeptides with the exception of those containing a proline residue. Optimal PepD activity was observed at pH 6.0 and 55 degrees C. The enzyme was inhibited by p-hydroxymercuribenzoate and reactivated by dithiothreitol whereas ethylenediaminetetraacetate had no inhibitory effect on PepD. The enzymatic properties of PepD suggest that it represents a novel dipeptidase type among lactic acid bacteria.

Amino Acid Sequence↗