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Exploring molecular networks directly in the cell.

The hierarchy of cell function comprises at least four distinct functional levels: genome, transcriptome, proteome, and toponome. The toponome is the entirety of all protein networks traced out directly as patterns on the single cell level in the natural environment of cells in situ (e.g. tissues). In this work a photonic microscopic robot technology (MELK) capable of tagging and imaging hundreds (and possibly thousands) of different molecular components (e.g. proteins) of morphologically-intact fixed cells and tissue have been developed. MELK data sets represent multidimensional vectors of the topologically determined arrangements of proteins within the cell. The data, assembled in a toponome dictionary of the cell, give rise to a new concept for target and drug lead discovery.

Eukaryotic Cells↗

Long identical repeats in the mouse gamma 2b switch region and their implications for the mechanism of class switching.

A computer sorting method was used to construct a dictionary by which long identical repeats of nucleotides could be identified and all available sequences of immunoglobulin switch regions were examined. The genomic mouse gamma 2b switch region contains two sets of four long identical repeats comprising 102, 72, 98 and 109 nucleotides respectively. The first is separated from the second by 347 nucleotides which contain the first 46 nucleotides of the 98 nucleotide set as a third partial repeat. These sets overlap the 49-bp separation between identical five nucleotide repeats GAGCT, GGGGT, ACCAG and CGAGC. Switches from S mu to S gamma 2b in and between these sets involve deletions of all or part of a set. Frequencies and locations of short repeats show differences between S mu, S epsilon, and S alpha and the other switch regions; these could determine specificity and locations of switches.

Animals↗

Automated support for pharmacovigilance: a proposed system.

Governments, manufacturers, and other entities are interested in adverse event surveillance of marketed medical products. FDA's Center for Drug Evaluation and Research redesigned the post-marketing adverse reaction surveillance process to use the advantages of new technology. As part of this effort, a 'Pharmacovigilance Working Group' designed a new strategy for the review and analyses of adverse event reports received by FDA. It created requirements which divided signal detection into five tiers: (1) Single 'urgent' reports would be sent to reviewers' workstations nightly for immediate attention. Reviewers would be able to customize definitions of 'urgent' (events that should not wait for aggregate review). (2) Single urgent reports would be placed in a context matrix containing historical counts of similar events to aid in initial interpretation. (3) In this first level of aggregate review, graphical displays would highlight patterns within all the reports, both urgent and non-urgent, and (4) periodic drug-specific tabled-based reports would display the newly received reports across a pre-defined variety of displays. These four tiers would produce passive and criteria-based results which would be presented to safety reviewers' electronic workstations. (5) Active query capabilities (routine, such as age, sex, and year distributions, as well as ad hoc) would be available for exploring alerted issues. The historical database would be migrated into the new format. All historical and new reaction data would be coded with the new MedDRA (Medical Dictionary for Regulatory Activities) scheme. The strategy was to design a full data capture system which effectively exploits current computing advances and technical performance to automate many aspects of initial adverse event review, supporting more efficient and effective clinical assessment of safety signals.

Adverse Drug Reaction Reporting Systems↗

Reports of withdrawal syndrome with the use of SSRIs: a case/non-case study in the French Pharmacovigilance database.

The SSRIs can be associated with withdrawal reactions and the objective of this study is to test the existence of an association between reports of withdrawal syndromes with the selective serotonin re-uptake inhibitors in the French spontaneous reports database. All reactions are coded according to the WHO ART dictionary. Cases are reports of reactions of interest (withdrawal syndrome). Non-cases are all reports of reactions other than those being studied. We calculated the odds ratio (OR) as the ratio of the odds of the association of reports of withdrawal syndrome with SSRIs in cases and non-cases. SSRIs are clearly associated with a higher risk of withdrawal syndrome (OR: 5.05, 95% CI: 3.81-6.68) and in particular with venlafaxine and paroxetine (OR: 12.16, 95% CI: 6.17-23.35 and OR: 8.47, 95% CI: 5.63-12.65, respectively). The risk of withdrawal reactions appears to be greater with short half-life drugs such as paroxetine and venlafaxine. The precise mechanisms have not been identified.

Adult↗

Structural classification of thioredoxin-like fold proteins.

Protein structure classification is necessary to comprehend the rapidly growing structural data for better understanding of protein evolution and sequence-structure-function relationships. Thioredoxins are important proteins that ubiquitously regulate cellular redox status and various other crucial functions. We define the thioredoxin-like fold using the structure consensus of thioredoxin homologs and consider all circular permutations of the fold. The search for thioredoxin-like fold proteins in the PDB database identified 723 protein domains. These domains are grouped into eleven evolutionary families based on combined sequence, structural, and functional evidence. Analysis of the protein-ligand structure complexes reveals two major active site locations for the thioredoxin-like proteins. Comparison to existing structure classifications reveals that our thioredoxin-like fold group is broader and more inclusive, unifying proteins from five SCOP folds, five CATH topologies and seven DALI domain dictionary globular folding topologies. Considering these structurally similar domains together sheds new light on the relationships between sequence, structure, function and evolution of thioredoxins.

Amino Acid Motifs↗

SPINS: a laboratory information management system for organizing and archiving intermediate and final results from NMR protein structure determinations.

Recent technological advances and experimental techniques have contributed to an increasing number and size of NMR datasets. In order to scale up productivity, laboratory information management systems for handling these extensive data need to be designed and implemented. The SPINS (Standardized ProteIn Nmr Storage) Laboratory Information Management System (LIMS) addresses these needs by providing an interface for archival of complete protein NMR structure determinations, together with functionality for depositing these data to the public BioMagResBank (BMRB). The software tracks intermediate files during each step of an NMR structure-determination process, including: data collection, data processing, resonance assignments, resonance assignment validation, structure calculation, and structure validation. The underlying SPINS data dictionary allows for the integration of various third party NMR data processing and analysis software, enabling users to launch programs they are accustomed to using for each step of the structure determination process directly out of the SPINS user interface.

Computer Graphics↗

Rebuilding flavodoxin from C alpha coordinates: a test study.

The tertiary structure of flavodoxin has been model built from only the X-ray crystallographic alpha-carbon coordinates. Main-chain atoms were generated from a dictionary of backbone structures. Side-chain conformations were initially set according to observed statistical distributions, clashes were resolved with reference to other knowledge-based parameters, and finally, energy minimization was applied. The RMSD of the model was 1.7 A across all atoms to the native structure. Regular secondary structural elements were modeled more accurately than other regions. About 40% of the chi 1 torsional angles were modeled correctly. Packing of side chains in the core was energetically stable but diverged significantly from the native structure in some regions. The modeling of protein structures is increasing in popularity but relatively few checks have been applied to determine the accuracy of the approach. In this work a variety of parameters have been examined. It was found that close contacts, and hydrogen-bonding patterns could identify poorly packed residues. These tests, however, did not indicate which residues had a conformation different from the native structure or how to move such residues to bring them into agreement. To assist in the modeling of interacting side chains a database of known interactions has been prepared.

Flavodoxin↗

The vitamin D3 hydroxylase-associated protein is a propionamide-metabolizing amidase enzyme.

Previously we isolated a novel protein that coimmunoprecipitates with the 1,25-dihydroxyvitamin D3-24R-hydroxylase and 25-hydroxyvitamin D3-1 alpha-hydroxylase. This kidney-specific protein found in the inner membrane of mitochondria is named the vitamin D3 hydroxylase-associated protein (VDHAP). To determine a putative function for this protein, an extensive computer search of the deduced amino acid sequence of VDHAP was performed. A BLAST homology search identified amino acid residues 133 through 321 in acetamidase from Aspergillus nidulans that exhibit 38% amino acid identify and 65% amino acid similarity to VDHAP. A protein consensus sequence dictionary, MOTIFS, identified an amidase consensus sequence in VDHAP. This sequence, G-G-S-S-G-G-E-G-A-L-I-A-G-G-G-S-L-L-G-I-G-S-D-V-A-G-S-I-R-L-P-S, in VDHAP is located between amino acids 223 and 254. Propionamide, acetamide, and acrylamide were identified as substrates for an amidase activity in soluble chicken kidney mitochondria. Propionamide is the best substrate with a Vmax of 16.7 nmol NH4+/min/mg protein and an apparent Km of 7.9 mM in soluble chicken kidney mitochondria. A VDHAP monoclonal antibody, IVC2G8, immunoprecipitates 78% of the total propionamidase activity in soluble chicken kidney mitochondria. These results suggest that VDHAP is a propionamidase enzyme in soluble chicken kidney mitochondria and a member of the amidase signature gene family.

Amides↗

Design and implementation of a relational database used in the management of patients with retinoblastoma.

Clinical data are most useful in the management of patients with complex medical problems if they are accurate, reliable, and easily accessible by physicians and the medical community at large. Furthermore, the data are most valuable when they can be shared among cooperating institutions. We describe a computer system which exhibits a uniform taxonomy, an integrated on-line dictionary of clinical terms, a coherent temporal layout, and persistent spatial integrity with regard to the values of the variables. The system is user friendly and provides real time data access which can be retrieved by structured query language or may be programmed to be used as part of an international network in the management of patients with retinoblastoma, a malignant and potentially fatal tumor of childhood. Furthermore, because of its design flexibility, this system provides for potential application to other ophthalmic disorders, such as malignant uveal melanoma, and other areas of medicine as well.

Databases, Factual↗

Hidden Markov models in computational biology. Applications to protein modeling.

Hidden Markov Models (HMMs) are applied to the problems of statistical modeling, database searching and multiple sequence alignment of protein families and protein domains. These methods are demonstrated on the globin family, the protein kinase catalytic domain, and the EF-hand calcium binding motif. In each case the parameters of an HMM are estimated from a training set of unaligned sequences. After the HMM is built, it is used to obtain a multiple alignment of all the training sequences. It is also used to search the SWISS-PROT 22 database for other sequences that are members of the given protein family, or contain the given domain. The HMM produces multiple alignments of good quality that agree closely with the alignments produced by programs that incorporate three-dimensional structural information. When employed in discrimination tests (by examining how closely the sequences in a database fit the globin, kinase and EF-hand HMMs), the HMM is able to distinguish members of these families from non-members with a high degree of accuracy. Both the HMM and PROFILESEARCH (a technique used to search for relationships between a protein sequence and multiply aligned sequences) perform better in these tests than PROSITE (a dictionary of sites and patterns in proteins). The HMM appears to have a slight advantage over PROFILESEARCH in terms of lower rates of false negatives and false positives, even though the HMM is trained using only unaligned sequences, whereas PROFILESEARCH requires aligned training sequences. Our results suggest the presence of an EF-hand calcium binding motif in a highly conserved and evolutionary preserved putative intracellular region of 155 residues in the alpha-1 subunit of L-type calcium channels which play an important role in excitation-contraction coupling. This region has been suggested to contain the functional domains that are typical or essential for all L-type calcium channels regardless of whether they couple to ryanodine receptors, conduct ions or both.

Algorithms↗

Protein folds, functions and evolution.

The evolution of proteins and their functions is reviewed from a structural perspective in the light of the current database. Protein domain families segregate unequally between the three major classes, the 32 different architectures and almost 700 folds observed to date. We find that the number of new topologies is still increasing, although 25 new structures are now determined for each new topology. The corresponding analysis and classification of function is only just beginning, fuelled by the genome data. The structural data revealed unexpected conservations and divergence of function both within and between families. The next five years will see the compilation of a definitive dictionary of protein families and their related functions, based on structural data which reveals relationships hidden at the sequence level. Such information will provide the foundation to build a better understanding of the molecular basis of biological complexity and hopefully to facilitate rational molecular design.

Animals↗

The elements for a classification of units of genetic information with a combinatorial component.

An integrative approach to the study of the regulation of gene expression has been undertaken here. The main goal of this approach is to make explicit the common rules that govern the relative location of regulatory sites within operons and other units of genetic information (UGIs). A classification that emphasizes the regulatory properties of UGIs can be achieved by partitioning UGIs into short sequences with defined properties. Such a classification scheme can be precisely defined as a Grammar with a component of combinatorial (rewriting) rules, and a dictionary component. Sequences have then to be grouped into classes such that any sequence of the same class can mutually substitute and produce novel regulatable UGIs. It is shown here that individual nucleotides cannot define such classes--they are far from equivalent to phonemes. Neither pairs, triplets or any short sequence with a defined number of nucleotides can define productive substitutions. Defined sequences like promoter, operator and activator binding sites are the smallest elements of combinatorial rules within the defined range of transcription initiation of sigma 70 Escherichia coli promoters.

Animals↗

Is DNA a language?

DNA sequences usually involve local construction rules that affect different scales. As such their "dictionary" may not follow Zipf's law (a power law) which is followed in every natural language. Indeed, analysis of many DNA sequences suggests that no linguistics connections to DNA exist and that even though it has structure DNA is not a language. Computer simulations and a biological approach to this problem further support these results.

Animals↗

Deciphering the language of the genome.

The non-coding DNA in eukaryotic genomes encodes a language which programs organismal growth and development. We show that a linguistic and cryptographic approach can be used to deduce the syntax of this programming language for gene regulation and to compile a dictionary of enhancers which form its words.

Animals↗

Nuclear transfer in farm animal species.

CLONE 'a group of two or more individuals with identical genetic makeup derived, by asexual reproduction, from a single common parent or ancestor' (The Chambers Dictionary 1993, Chambers Harrap). The term clone was originally applied to plants but has subsequently been used in a much broader context to include a person or thing closely similar to another, a copy or replica. In animals, true clones, as defined above, may be produced by embryo splitting or blastomere separation either artificially, or as occurs naturally in the production of identical twins. In these individuals all of the components making up the individual, including nuclear genetic material (the genome) and other maternally derived factors are derived from a single unique embryo which is the result of sexual reproduction. The term clone has been applied to animals produced by the technique of nuclear transfer. In this asexual process, nuclear genetic material is transferred from a donor cell (karyoplast) into a recipient cell (cytoplast) from which the genetic material has been removed. In farm animals the cytoplast of choice is the matured oocyte (or unfertilised egg) thus the animals developing from this technique are not true clones as each cytoplast is often derived from a different animal. The resultant animals may therefore be more aptly described as 'genomic copies'. In mammals, successful development of embryos reconstructed by nuclear transfer was originally restricted to using early embryos as nuclear donors, however, recent progress has demonstrated successful development using nuclei from embryonic, foetal and adult derived cell populations. Numerous factors affect the development of embryos reconstructed by nuclear transfer including; the cell cycle stage of the recipient cell, the cell cycle stage of the donor nucleus, the differentiated state of the donor nucleus, activation of the recipient cell, the culture method. In addition, there are variations in success between species, these may be related to differences in organisation of the cytoskeleton and/or the meiotic spindle in the recipient cell,differences in cell cycle control during early development, the onset of zygotic transcription or differences in the metabolic requirements of early embryos in vitro. The aim of this article is to describe and discuss some of these factors in relation to the successful development of nuclear transfer reconstructed embryos and in particular to the 'reprogramming' or 'remodeling' of the donor genetic material to attain successful development.

Animals↗

A machine learning approach to computer-aided molecular design.

Preliminary results of a machine learning application concerning computer-aided molecular design applied to drug discovery are presented. The artificial intelligence techniques of machine learning use a sample of active and inactive compounds, which is viewed as a set of positive and negative examples, to allow the induction of a molecular model characterizing the interaction between the compounds and a target molecule. The algorithm is based on a twofold phase. In the first one--the specialization step--the program identifies a number of active/inactive pairs of compounds which appear to be the most useful in order to make the learning process as effective as possible and generates a dictionary of molecular fragments, deemed to be responsible for the activity of the compounds. In the second phase--the generalization step--the fragments thus generated are combined and generalized in order to select the most plausible hypothesis with respect to the sample of compounds. A knowledge base concerning physical and chemical properties is utilized during the inductive process.

Amino Acid Sequence↗

A nonlinear systems approach to Fechner's paradox.

It is possible to predict the topology of isointensity plots under conditions of extreme imbalance of the stimulus inputs, without making any assumptions specific to the circumstances in which Fechner's Paradox is sometimes observed. This is done by extending a nonlinear model for a sensory channel, by postulating a form of cross-coupling or interference between two channels which represents other phenomena in psychophysics. It is noted that the form in which data are usually reported is not an adequate basis for testing all the predictions of a nonlinear model in sensory psychophysics. The physiologist Panum (1958), and later Fechner (1860) reported that the apparent brightness of an object viewed binocularly could, under conditions where the input to one eye was diminished by filtering, be less than its brightness viewed monocularly by the unfiltered eye. To a first approximation, binocular brightness is more like an averaging of two monocular inputs than a summation of those same inputs. For over 120 years this phenomenon, which came to be called "Fechner's Paradox", though Panum should perhaps have had some credit, has been the subject of experimental investigation and associated mathematical modelling. If one consults a dictionary of psychological terms, for example Evans (1978), one may read something like Fechner's Paradox: The name give to the observation that something [which is] viewed binocularly seems to increase in brightness when one eye is closed. And yet we now know that this definition is misleading, because the same phenomenon in pooling two sensory inputs has its analogues in audition (Lehky 1983) and in olfaction (Gregson 1986). Gilchrist and McIver (1985) have now shown an analogue of the paradox exists in ocular contrast sensitivity. The definition also goes awry when the input luminance to one eye is zero, or when the luminance and ocular adaptation are closely matched for the two eyes. It is wiser, in the light of results reporting individual differences in the existence and extent of the paradox, and its sensitivity to stimulus conditions, to side with Blake and Fox (1973) when they observed that it is not unreasonable to suppose that various stimulus conditions might yield varying amounts of summation or even inhibition. Empirical reviews of relevant data in vision have been given by Roelofs and Zeeman (1914), Blake and Fox (1973), and Blake et al. (1981), but a theoretical model of interest as a starting point is that of Lehky (1983).

Computer Simulation↗

Language patterns of opponents to a child protection program.

A community effort was made to help sexually abused girls and a "pro-incest lobby" was uncovered. This report focuses on the language used to deny or obscure the occurrence of father-daughter incest. As children's rights advocates, the authors attempted to provide a lexicon, a dictionary for the clinician, so that efforts to condone incest will not be overlooked but refuted. Both in the past and the present, instances of incest have been rationalized away by health care professionals, members of the legal profession and the community at large. The incidence of incest is higher than the increasing number of actual cases reported. The vocabulary that systematically drives certain resistances and defenses to the surface, in opposition to child protection in such cases, is analyzed by the authors. The "dynamics" involved in the realization of incest as a psychologically damaging situation for the child are complex. The social influences on the individual and the married couple, that serve to make incest a closely kept secret, are examined. The authors express hope that their linguistic analysis will help to alert all persons who work in the broad field of child abuse and child protection.

Adolescent↗