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PubMed1996

[Traveling on the data highway--online information for urologists].

Global exchange of information is one of the major sources of scientific progress in medicine. Electronic media have recently supplemented the standard methods of scientific communication such as congresses, books and journals. Currently, 30 million computers are part of the world-wide, exponentially growing network, the ""information superhighway''. Numerous resources offered by the network are becoming increasingly useful in clinical medicine. In Germany, three major networks are currently available: T-Online/Datex-J, Compuserve and Internet. The majority of information relevant to urologists can be found in the Internet. This information includes medical journals that can either partly or completely be read online, online library catalogs, scientific images (pathology and radiology), and databases on urologic oncology and basic research in urology. Even outside academic institutions access to the "information superhighway" is easily available. Currently, the "information superhighway" constitutes a new source of database-type information; in the near future, medical education, clinical consultation and worldwide cooperation in clinical studies will be new fields of scientific communication employing worldwide computer networks.

Forecasting↗
PubMed1992

Multi-priming sequencing: a DNA sequencing method involving restriction enzyme-digested DNA fragments as primers.

An improved strategy for fluorescence-labeled dideoxy chain termination sequencing involving restriction enzyme-digested DNA fragments as primers, which are prepared from the DNA to be sequenced, is described. By using modified nucleoside triphosphates for strand protection in chain termination reactions, newly synthesized chains were detached from a primer at the regenerated recognition site by means of suitable restriction enzyme digestion. The digests could be analyzed with commercial automated DNA sequencers. Thus, by using restriction DNA fragments (double-stranded) as primers, sequence information was obtained from both "minus" and "plus" single-stranded DNA templates without subcloning. Nor is the synthesis of oligonucleotide primers needed. This method, named "Multi-Priming Sequencing," was proven to be time-saving, economical, and effective compared to conventional methods.

Base Sequence↗
PubMed2005

ESPSearch: a program for finding exact sequences and patterns in DNA, RNA, or protein.

ESPSearch is a computer program for rapidly identifying nucleic acid or amino acid sequences of any length within any source sequence from promoters to entire genomes to protein libraries. ESPSearch utilizes a user-constructed database to identify many sequences simultaneously, including target sequences with wildcards and mismatches and user-specified patterns of those recognized sequences. Here we use ESPSearch to identify a variety of possible binding sites for dimeric artificial transcription factors within several p53 recognition sites and the promoter of the BAX gene. Heterodimeric and homodimeric proteins are designed using human zinc fingers by identifying groups of zinc finger binding sites meeting particular pattern constraints. ESPSearch is also used to estimate the specificity of each artificial transcription factor by searching the entire genome. Next, the specificity of several possible small interfering RNA (siRNA) sequences is determined by searching both the whole genome and the library of known human mRNAs. Finally, ESPSearch identifies proteins containing different forms of the LXXLL motif used in nuclear receptor-coactivator interactions from the human proteome, making use of user-defined groups of amino acids. ESPSearch could also be applied to other tasks involving sequence and pattern recognition on small and large scales. ESPSearch is freely available at http://web.chemistry.gatech.edu/-doyle/espsearch/.

Database Management Systems↗
PubMed1994

Rat kidney carboxylesterase. Cloning, sequencing, cellular localization, and relationship to rat liver hydrolase.

We recently purified from rat liver microsomes a carboxylesterase, designated hydrolase B, that catalyzes the hydrolysis of para-nitrophenylacetate with low affinity (Km approximately 400 microM) and is relatively insensitive to the inhibitory effects of phenylmethylsulfonyl fluoride. A carboxylesterase with identical properties is also present in rat kidney microsomes, at levels comparable to those in liver microsomes. The kidney enzyme is immunochemically indistinguishable from hydrolase B by Western immunoblotting and Ouchterlony double diffusion analysis. This study describes the cloning and sequencing of hydrolase B. A 1809-base pair (bp) cDNA was isolated from a rat kidney cDNA library screened with antibody against hydrolase B. Screening the same cDNA library by two-step polymerase chain reaction with external and internal primers based on the sequence of the 1809-bp cDNA and a primer based on the sequence of the adjoining lambda gt11 arm yielded a 279-bp cDNA that overlapped by 179 bp with the 1809-bp-sequence. Together these two cDNAs spanned a 1909-bp sequence with an opening reading frame encoding 561 amino acids, which includes all 543 amino acid residues in the mature protein plus an 18-amino acid signal peptide at the N terminus. The mature protein encoded by this kidney cDNA matches perfectly the N-terminal amino acid sequence of purified hydrolase B for 30 amino acid residues, as determined by automated Edman degradation. The mature protein contains 5 cysteine residues, two potential N-linked glycosylation sites, and a C-terminal tetrapeptide (His-Asn-Glu-Leu) that matches the HXEL consensus sequence for retaining proteins in the lumen of the endoplasmic reticulum. Based on alignment of conserved amino acid sequences in several mammalian carboxylesterases, and based on the mechanism of catalysis of serine proteases, the catalytic triad in hydrolase B is apparently composed of the nucleophile Ser203, the basic amino acid His448, and the acidic amino acid Asp97 or Glu228. Northern blots probed with the 1809-bp cDNA identified high levels of a approximately 2-kilobase mRNA for hydrolase B in liver and kidney. Little or no mRNA for hydrolase B was detected in testis, lung, prostate, brain, and heart, which confirms the tissue distribution of hydrolase B based on catalytic activity and Western immunoblotting. Immunocytochemical studies established that hydrolase B is localized in the centrilobular region of the liver and in the proximal tubules of the kidney, where it presumably plays a role in the metabolism of xenobiotics and possibly endogenous lipids, although a precise physiological role for hydrolase B remains to be determined.

Amino Acid Sequence↗
PubMed2005

Chemical complementation: a definitive phenotypic strategy for identifying small molecule inhibitors of elusive cellular targets.

Forward Pharmacology seeks to identify small or large molecules that modulate a normal or abnormal biological process in living cells or whole organisms and historically has been responsible for the discovery of many clinically used drugs. Forward Pharmacology approaches have become particularly attractive because advances in combinatorial chemistry and laboratory automation have made it possible to generate and interrogate large compound collections in a short period of time. Because many drug discovery efforts are now directed against specific biochemical targets, however, the utility of Forward Pharmacology is limited by the fact that assays to investigate compounds in biological systems are often phenotypic rather than target specific. We discuss here a novel strategy to discover target-based small molecules in intact cells using contemporary Forward Pharmacology in cells with specific genetic manipulations. The method, which we have termed "chemical complementation", is defined as the ability of small molecules to reverse a genetically induced phenotypic change in intact cells. Chemical complementation represents an extension of the commonly used genetic complementation approach, where cDNA libraries are used to investigate the function of genes based on their ability to rescue a specific genetic defect. We present examples of how chemical complementation has been used to identify and credential cell-active, small molecule inhibitors of 2 dual-specificity phosphatases, Cdc25A and MKP-3, which heretofore have eluded small molecule drug discovery efforts.

Animals↗
PubMed1986

Amino acid sequence of the b subunit of human factor XIII, a protein composed of ten repetitive segments.

Factor XIII is a plasma protein that participates in the final stages of blood coagulation. The complete amino acid sequence of the b subunit of human factor XIII was determined by a combination of cDNA cloning and amino acid sequence analysis. A lambda gt11 cDNA library prepared from human liver mRNA was screened with an affinity-purified antibody against the b subunit of human factor XIII. Nine positive clones were isolated from 2 X 10(6) phage and plaque-purified. The largest cDNA insert was sequenced and shown to contain 2180 base pairs coding for a portion of the leader sequence (19 amino acids), the mature protein (641 amino acids), a stop codon (TGA), a 3' noncoding region (187 nucleotides), and a poly(A) tail. When the b subunit of human factor XIII was digested with cyanogen bromide, nine peptides were isolated by gel filtration and reverse-phase high-performance liquid chromatography. Amino acid sequence analyses of these peptides were performed with an automated sequenator, and 299 amino acid residues were identified. These amino acid sequences were in complete agreement with the amino acid sequence predicted from the cDNA. The b subunit of factor XIII contained 10 repetitive homologous segments, each composed of about 60 amino acids and 4 half-cystine residues. Each of these repeated segments is a member of a family of repeats present in human beta 2-glycoprotein I, complement factor B, and haptoglobin alpha 1 chain. Three potential Asn-linked carbohydrate attachment sites were also identified in the b subunit of factor XIII.

Amino Acid Sequence↗
PubMed2005

A graphical framework for specification of clinical guidelines at multiple representation levels.

Formalization of a clinical guideline for purposes of automated application and quality assessment mainly involves conversion of its free-text representation into a machine comprehensible representation, i.e., a formal language, thus enabling auto-mated support. The main issues involved in this process are related to the collaboration between the expert physician and the knowledge engineer. We introduce GESHER - a graphical framework for specification of clinical guidelines at multiple representation levels. The GESHER architecture facilitates incremental specification through a set of views adapted to each representation level, enabling this process to proceed smoothly and in a transparent fashion, fostering extensive collaboration among the various types of users. The GESHER framework supports specification of guidelines at multiple representation levels, in more than one specification language, and uses the DeGeL digital guideline library architecture as its knowledge base. The GESHER architecture also uses a temporal abstraction knowledge base to store its declarative knowledge, and a standard medical-vocabularies server for generic specification of key terms, thus enabling reuse of the specification at multiple sites.

Computer Graphics↗
PubMed1999

Application of multilayer feed-forward neural networks to automated compound identification in low-resolution open-path FT-IR spectrometry.

A drawback of current open-path Fourier transform infrared (OP/FT-IR) systems is that they need a human expert to determine those compounds that may be quantified from a given spectrum. In this work, multilayer feed-forward neural networks with one hidden layer were used to automatically recognize compounds in an OP/FT-IR spectrum without compensation of absorption lines due to atmospheric H2O and CO2. The networks were trained by fast-back-propagation. The training set comprised spectra that were synthesized by digitally adding randomly scaled reference spectra to actual open-path background spectra measured over a variety of path lengths and temperatures. The reference spectra of 109 compounds were used to synthesize the training spectra. Each neural network was trained to recognize only one compound in the presence of up to 10 other interferences in an OP/FT-IR spectrum. Every compound in a database of vaporphase reference spectra can be encoded in an independent neural network so that a neural network library can be established. When these networks are used for the identification of compounds, the process is analogous to spectral library searching. The effect of learning rate and band intensities on the convergence of network training was examined. The networks were successfully used to recognize five alcohols and two chlorinated compounds in field-measured controlled-release OP/FT-IR spectra of mixtures of these compounds.

Neural Networks, Computer↗
PubMed1991

The amino acid sequence of the aspartate aminotransferase from baker's yeast (Saccharomyces cerevisiae).

1. The single (cytosolic) aspartate aminotransferase was purified in high yield from baker's yeast (Saccharomyces cerevisiae). 2. Amino-acid-sequence analysis was carried out by digestion of the protein with trypsin and with CNBr; some of the peptides produced were further subdigested with Staphylococcus aureus V8 proteinase or with pepsin. Peptides were sequenced by the dansyl-Edman method and/or by automated gas-phase methods. The amino acid sequence obtained was complete except for a probable gap of two residues as indicated by comparison with the structures of counterpart proteins in other species. 3. The N-terminus of the enzyme is blocked. Fast-atom-bombardment m.s. was used to identify the blocking group as an acetyl one. 4. Alignment of the sequence of the enzyme with those of vertebrate cytosolic and mitochondrial aspartate aminotransferases and with the enzyme from Escherichia coli showed that about 25% of residues are conserved between these distantly related forms. 5. Experimental details and confirmatory data for the results presented here are given in a Supplementary Publication (SUP 50164, 25 pages) that has been deposited at the British Library Document Supply Centre, Boston Spa. Wetherby, West Yorkshire LS23 7 BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1991) 273, 5.

Amino Acid Sequence↗
PubMed2006

Automated SNP detection from a large collection of white spruce expressed sequences: contributing factors and approaches for the categorization of SNPs.

BACKGROUND: High-throughput genotyping technologies represent a highly efficient way to accelerate genetic mapping and enable association studies. As a first step toward this goal, we aimed to develop a resource of candidate Single Nucleotide Polymorphisms (SNP) in white spruce (Picea glauca [Moench] Voss), a softwood tree of major economic importance. RESULTS: A white spruce SNP resource encompassing 12,264 SNPs was constructed from a set of 6,459 contigs derived from Expressed Sequence Tags (EST) and by using the bayesian-based statistical software PolyBayes. Several parameters influencing the SNP prediction were analysed including the a priori expected polymorphism, the probability score (PSNP), and the contig depth and length. SNP detection in 3' and 5' reads from the same clones revealed a level of inconsistency between overlapping sequences as low as 1%. A subset of 245 predicted SNPs were verified through the independent resequencing of genomic DNA of a genotype also used to prepare cDNA libraries. The validation rate reached a maximum of 85% for SNPs predicted with either PSNP > or = 0.95 or > or = 0.99. A total of 9,310 SNPs were detected by using PSNP > or = 0.95 as a criterion. The SNPs were distributed among 3,590 contigs encompassing an array of broad functional categories, with an overall frequency of 1 SNP per 700 nucleotide sites. Experimental and statistical approaches were used to evaluate the proportion of paralogous SNPs, with estimates in the range of 8 to 12%. The 3,789 coding SNPs identified through coding region annotation and ORF prediction, were distributed into 39% nonsynonymous and 61% synonymous substitutions. Overall, there were 0.9 SNP per 1,000 nonsynonymous sites and 5.2 SNPs per 1,000 synonymous sites, for a genome-wide nonsynonymous to synonymous substitution rate ratio (Ka/Ks) of 0.17. CONCLUSION: We integrated the SNP data in the ForestTreeDB database along with functional annotations to provide a tool facilitating the choice of candidate genes for mapping purposes or association studies.

Algorithms↗
PubMed2004

A single-vial analytical and quantitative gas chromatography-mass spectrometry assay for terpene synthases.

A quantitative assay for the analysis of sesquiterpene synthases, wherein each reaction mixture is formulated in glass gas chromatography vials, overlaid with organic solvent such as ethyl acetate, and subsequently vortexed to extract hydrocarbon reaction products into the organic phase after a suitable incubation period, was developed. The product-enriched organic phase is then sampled in an automated fashion and injected directly into a gas chromatograph-mass spectrometer without further workup for analysis and quantification of hydrocarbon products. Application of the vial assay to the analysis of amorpha-4,11-diene synthase (ADS), a sesquiterpene synthase, demonstrated the sensitivity of the assay for detection of major and minor reaction products and most notably for the identification of several sesquiterpene products that had escaped previous detection. A steady-state kinetic analysis of tobacco 5-epi-aristolochene synthase (TEAS), another sesquiterpene synthase, validated the quantitative nature of the assay, providing an alternative means to the established method of using radiolabeled substrate, extraction, and scintillation counting. This simplified assay provides a standardized method to facilitate analysis of terpene synthases and diverse mutant enzyme libraries by supplanting the common practice of using larger scale reactions, multiple extractions, and evaporative concentration of the organic phase prior to gas chromatography-mass spectrometry (GC-MS) analysis.

Alkyl and Aryl Transferases↗
PubMed2002

Science's signal transduction knowledge environment: the connections maps database.

Knowledge environment (KE) describes the collection of electronic networking tools that have been and continue to be developed by AAAS and Stanford University libraries. Knowledge environments use practical, production-quality tools to systematize the consensus knowledge within a scientific domain and facilitate users' access to that knowledge. Science's Signal Transduction Knowledge Environment (STKE) is the first in this new concept in electronic publishing that combines the traditional, albeit electronic, publishing of articles, such as reviews, perspectives, and protocols, with tools for organizing and collating information in the cross-disciplinary field of signal transduction. One of the major tools developed for the STKE is the Connections Map database and the software (called CMADES [Connections Maps Authority Data Entry Software]) created to facilitate data entry by Pathway Authorities. The Connections Maps are a graphical representation of a database of information about the molecules involved in cellular signaling cascades. CMADES automates many of the functions involved in adding data into the Connections Maps database, such as references and descriptors, as well as allowing the Authorities to indicate the relationships between the components in the pathway through the use of a graphing tool. CMADES and the Connections Maps represent evolving tools that assist the Authorities in systemizing information regarding a particular system at the organism- and cell-specific level and the canonical level, as well as provide the STKE user with organized and expert-supplied information about signal transduction pathways.

Biology↗
PubMed1988

Profile scanning for three-dimensional structural patterns in protein sequences.

Profile analysis measures the similarity between a target sequence and a group of aligned sequences (the probe). The probe sequences are used to produce a position-specific scoring table (the profile) that can be aligned with any sequence (the target) using standard dynamic programming methods. We are developing a library of profiles, each describing a different structural motif. This allows any target sequence to be rapidly scanned for the presence of structural motifs. Levels of significance for the comparison of target sequences with the profile are determined in advance, permitting an objective decision to be made as to whether a protein is likely to possess a structural motif.

Amino Acid Sequence↗
PubMed2001

[Use of phage display for detecting single-nucleotide differences in genes].

Experimental strategy has been developed for selection of mismatched DNA binding phages from library of E. coli f1 filamentous phages carrying random peptide inserts on the surface of bacteriophage particles. The strategy is based on the use of phage display technique, DNA heteroduplexes (with single nucleotide variations), and paramagnetic beads. DNA heteroduplexes have been obtained from biotin-labeled PCR product. During the first stage the phage particles were incubated with DNA heteroduplexes possessing mismatched nucleotides. The next step after elimination of free phages and separation of bound phages from DNA heteroduplexes was subtraction of phages binding with DNA heteroduplexes (without mismatched nucleotides). Phages selected by this method were capable of discriminating DNA heteroduplexes with single nucleotide variations from DNA homoduplexes. Phages immobilized on solid base retain their activity and specificity, and therefore can be used for developing a new screening automated method for detecting point mutations and gene polymorphism.

Base Sequence↗
PubMed2000

Automated search of natively folded protein fragments for high-throughput structure determination in structural genomics.

Structural genomic projects envision almost routine protein structure determinations, which are currently imaginable only for small proteins with molecular weights below 25,000 Da. For larger proteins, structural insight can be obtained by breaking them into small segments of amino acid sequences that can fold into native structures, even when isolated from the rest of the protein. Such segments are autonomously folding units (AFU) and have sizes suitable for fast structural analyses. Here, we propose to expand an intuitive procedure often employed for identifying biologically important domains to an automatic method for detecting putative folded protein fragments. The procedure is based on the recognition that large proteins can be regarded as a combination of independent domains conserved among diverse organisms. We thus have developed a program that reorganizes the output of BLAST searches and detects regions with a large number of similar sequences. To automate the detection process, it is reduced to a simple geometrical problem of recognizing rectangular shaped elevations in a graph that plots the number of similar sequences at each residue of a query sequence. We used our program to quantitatively corroborate the premise that segments with conserved sequences correspond to domains that fold into native structures. We applied our program to a test data set composed of 99 amino acid sequences containing 150 segments with structures listed in the Protein Data Bank, and thus known to fold into native structures. Overall, the fragments identified by our program have an almost 50% probability of forming a native structure, and comparable results are observed with sequences containing domain linkers classified in SCOP. Furthermore, we verified that our program identifies AFU in libraries from various organisms, and we found a significant number of AFU candidates for structural analysis, covering an estimated 5 to 20% of the genomic databases. Altogether, these results argue that methods based on sequence similarity can be useful for dissecting large proteins into small autonomously folding domains, and such methods may provide an efficient support to structural genomics projects.

Algorithms↗
PubMed2005

Robotic sequential analysis of a library of metalloporphyrins as electrocatalysts for voltammetric nitric oxide sensors.

A library of 83 metalloporphyrins with varying substitution pattern at the meso-position of the porphyrins and different central metal ions in the core region has been synthesized in small quantities using a parallel synthesis strategy. By means of a specially designed electrochemical robotic device integrating a 96-well microtitre plate and an easily movable assembly of working, counter and reference electrodes, the different porphyrins were automatically applied in sequence to an in-well electrochemical preparation and testing of NO sensors. Screening the entire compound collection suggested initial considerations concerning the influence of varied functionalities of the metalloporphyrins on their electrocatalytic properties for the oxidation of NO and helped to identify the quality of the investigated catalyst candidates. As compared to manually performed quality tests, the proposed strategy of automation has advantages in convenience, rapidity and especially reproducibility avoiding any inaccuracies introduced by manually performing all steps of the complex sensor formation and testing sequence.

Animals↗
PubMed2005

A robust homogeneous binding assay for alpha4beta2 nicotinic acetylcholine receptor.

AIM: To develop a homogeneous high-throughput screening (HTS) assay based on scintillation proximity assay (SPA) technology for identification of novel alpha4beta2 nicotinic acetylcholine receptor (nAChR) modulators. METHODS: Membrane preparation of HEK293 cells expressing alpha4beta2 nAChR, [(3)H]cytisine and wheat germ agglutinin (WGA)-coupled microbeads were used to develop an HTS assay based on SPA technology. This method was validated against a conventional filter binding approach and applied to large-scale screening of a library containing 32 000 synthetic compounds. Intracellular calcium measurement was carried out to verify the bioactivities of the hits found by the SPA assay. RESULTS: IC(50) values of 2 reference compounds (epibatidine and RJR 2403) determined by SPA and filter binding methods were comparable and consistent with those reported elsewhere. A total of 54 compounds, showing more than 60% competitive inhibition on [(3)H]cytisine binding to alpha4beta2 nAChR, were identified initially following an HTS campaign. Secondary screening confirmed that 17 compounds with novel chemical structures possessed relatively high binding affinity to alpha4beta2 nAChR (K(i)<2 micromol/L). Eight compounds displayed antagonistic effects with >50% inhibition on ABT-594-induced calcium mobilization while none showed any agonist activity. CONCLUSIONS: This homogeneous binding assay is a highly efficient, amenable to automation and robust tool to screen potential alpha4beta2 nAChR modulators in an HTS setting. Its application may be expanded to other membrane receptors and ion channels.

Alkaloids↗
PubMed2004

High-throughput siRNA-based functional target validation.

The drug discovery process pursued by major pharmaceutical companies for many years starts with target identification followed by high-throughput screening (HTS) with the goal of identifying lead compounds. To accomplish this goal, significant resources are invested into automation of the screening process or HTS. Robotic systems capable of handling thousands of data points per day are implemented across the pharmaceutical sector. Many of these systems are amenable to handling cell-based screening protocols as well. On the other hand, as companies strive to develop innovative products based on novel mechanisms of action(s), one of the current bottlenecks of the industry is the target validation process. Traditionally, bioinformatics and HTS groups operate separately at different stages of the drug discovery process. The authors describe the convergence and integration of HTS and bioinformatics to perform high-throughput target functional identification and validation. As an example of this approach, they initiated a project with a functional cell-based screen for a biological process of interest using libraries of small interfering RNA (siRNA) molecules. In this protocol, siRNAs function as potent gene-specific inhibitors. siRNA-mediated knockdown of the target genes is confirmed by TaqMan analysis, and genes with impacts on biological functions of interest are selected for further analysis. Once the genes are confirmed and further validated, they may be used for HTS to yield lead compounds.

Base Sequence↗