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At least 307 records · Page 17Linked to original sources
PubMed2004

RNA interference: from model organisms towards therapy for neural and neuromuscular disorders.

Experimental RNA interference (RNAi) leading to the selective knockdown of gene function is induced by introducing into cells either double stranded RNA (dsRNA), or short interfering RNA (siRNA) fragments into which dsRNA is cut. The siRNA triggers degradation of homologous messenger RNA (mRNA). Widely used as a research tool in the genetic model organisms Caenorhabditis elegans, Drosophila melanogaster and mouse to investigate the function of individual genes, RNAi has also been deployed in genome-wide, specific gene-knockdown screens. Recent rapid progress in the application of RNAi to mammalian cells, including neurons and muscle cells, offers new approaches to drug target identification and validation. Advances in targeted delivery of RNAi-inducing molecules has raised the possibility of using RNAi directly as a therapy for a variety of human genetic and other neural and neuromuscular disorders. Here, we review examples of the application of RNAi to worm, fly and mouse models of such diseases aimed at understanding their pathophysiology and we address problems to be solved in developing RNAi-based therapies.

Animals↗
PubMed2006

Variations on a theme: Hox and Wnt combinatorial regulation during animal development.

Shockingly few transcription factors and cell signaling pathways are utilized to pattern organs and to specify the fate of a seemingly endless variety of unique cell types during animal development. This dichotomy led to the hypothesis that each factor is used in multiple tissues and that a combinatorial code of factors determines cell fate or tissue identity in a unique fashion. Two recent papers describe temporal changes in the interplay between Hox transcription factors, which specify positional identity, and Wnt signaling, which provides spatial information and promotes asymmetric cell division. These changes guide cells through a series of discrete steps, leading to unique fates. Variations between these two studies highlight the diversifying potential of combinatorial regulation, in short, that the pathways through which these molecules interact can vary even between adjacent cells. Shared features include cross-regulatory interactions to redeploy patterning genes in a tissue-specific manner for organogenesis and coregulation of common downstream targets. Identification of additional combinatorial gene targets and elucidation of their underlying molecular mechanisms are important future tasks in developmental biology and the study of evolution.

Animals↗
PubMed1999

Regulation of the cytoskeleton and cell adhesion by the Rho family GTPases in mammalian cells.

Members of the Rho family of small Ras-like GTPases--including RhoA, -B, and -C, Rac1 and -2, and Cdc42--exhibit guanine nucleotide-binding activity and function as molecular switches, cycling between an inactive GDP-bound state and an active GTP-bound state. The Rho family GTPases participate in regulation of the actin cytoskeleton and cell adhesion through specific targets. Identification and characterization of these targets have begun to clarify how the Rho family GTPases act to regulate cytoskeletal structure and cell-cell and cell-substratum contacts in mammalian cells. The Rho family GTPases are also involved in regulation of smooth muscle contraction, cell morphology, cell motility, neurite retraction, and cytokinesis. However, the molecular mechanisms by which the Rho family GTPases participate in the regulation of such processes are not well established.

Cell Adhesion↗
PubMed2005

[The application of RNAi to the medical genetics].

Experimental RNA interference (RNAi) leading to the selective knockdown of gene function is induced by introducing into cells either double stranded RNA (dsRNA), or short interfering RNA (siRNA) fragments into which dsRNA is cut. The siRNA triggers degradation of homologous messenger RNA (mRNA). Widely used as a research tool in the genetic model organisms Caenorhabditis elegans, Drosophila melanogaster and mouse to investigate the function of individual genes, RNAi has also been deployed in genome-wide, specific gene-knockdown screens. Recent rapid progress in the application of RNAi to mammalian cells, including neurons and muscle cells, offers new approaches to drug target identification and validation. Advances in targeted delivery of RNAi-inducing molecules have raised the possibility of using RNAi directly as a therapy for a variety of human genetic and other neural and neuromuscular disorders. Here, we review examples of the application of RNAi to worm, fly and mouse models of such diseases aimed at understanding their pathophysiology.

Gene Silencing↗
PubMed2005

Current efforts in the analysis of RNAi and RNAi target genes.

RNAi is RNA interference by short RNAs. It influences gene-expression by down-regulation of mRNAs, typically by complementarity to the 3' UTR (untranslated region) of the mRNA. microRNAs (miRNAs) are short RNAs acting as natural RNAi. miRNAs mediate down-regulation of many mRNAs from developmental genes and transcription factor genes. Natural examples for this additional level of post-transcriptional control are increasing. Suitable computer-based search strategies for new miRNA candidates include precursor folding as well as different compositional search strategies. Example programs for this are presented. New own and other data are provided for an overview on such strategies. A strategy feasible in plants for miRNA target identification is direct base pairing of miRNAs to potential mRNA target 3' UTRs. Correct identification in animals usually requires comparative genomics and conserved UTR regions pairing to conserved miRNA substructures. A number of example programs and target examples for these tasks are examined. Finally, strategies and programs for artificial gene silencing by designed RNAi are explained.

Animals↗
PubMed1991

The effects of target discriminability and retinal eccentricity on saccade latencies: an analysis in terms of variable-criterion theory.

Two experiments are reported that studied changes in saccade latencies (SLs) in a target-identification task as a function of target discriminability and eccentricity. SLs were found to be longer when target discriminability was low and eccentricity was large, both factors showing a significant interaction. Variable-criterion theory (Grice, 1968) was used to derive a working model of the perceptual and decisional processes influencing SLs in the present task. Application of the measurement operations of the theory provided a good quantitative description of individual SL distributions. The analyses in terms of variable-criterion theory suggest that the perceptual process influencing SLs starts earlier and operates at a faster rate when the saccade target can be discriminated on the basis of global-visual features (e.g., size) than when local features (e.g., gaps) have to be extracted.

Adult↗
PubMed2006

4-Aminopyrimidines as novel HIV-1 inhibitors.

A series of 4-aminopyrimidines (1) was identified as novel HIV inhibitors of unknown molecular target. Structural modifications were carried out to establish its SAR and identify the linking site for target identification. A number of analogs were found to possess single digit inhibitory activity for HIV replication. Several analogs with various potential linkers, including a biotinated analog, also exhibited excellent potency, and could serve as tools for the identification of novel anti-HIV targets.

Anti-HIV Agents↗
PubMed2007

Protein i: interference at protein level by intrabodies.

Immunoglobulin molecules have long been used in biomedical research as in vitro tools for identification, purification and functional manipulation of target proteins. The specificity and diversity of immunoglobulins can be exploited to target a wide range of intracellular proteins by expressing them in vivo. Such antibody molecules, which are expressed intracellularly and directed to defined sub-cellular compartments, are termed 'intrabodies'. They represent a new and versatile tool that has the potential to manipulate diverse biological processes. Gene knockout, antisense and small interfering RNA knockdown techniques, are employed to characterize the function of many gene products and to validate these gene products as potential drug targets for novel therapeutics, but they have their limitations. The experimental results to date suggest that intrabodies represent a powerful alternative to modulate protein function and analyze its effects. Moreover, they can also be utilized to target specific domains of a particular target protein. Even though their clinical application might take a long time, they can play an important role for target identification and validation in functional genomics and/or proteomics. Here we review the latest advances in the concept, construction and applications of Intrabodies.

HIV Infections↗
PubMed2001

In vitro protein display in drug discovery.

Nucleic acid-encoded libraries have been used at different stages of the drug discovery process for the identification of polypeptide ligands and for target identification. Traditionally, phage display screening systems have been used to explore large libraries of peptides and proteins. Lately, novel protein selection technologies have been developed that work entirely in vitro and use the polymerase chain reaction (PCR) rather than cells to amplify genetic material. The simplicity of the linkage between the protein and its encoding nucleic acid leads to several advantages, including the use of larger libraries without the biases of cell-based amplification, greater control over binding conditions and the ease with which PCR-based mutagenesis and recombination can be incorporated. This review focuses on the latest improvements in this new generation of in vitro protein display techniques and discusses their applications to the drug discovery process.

Animals↗
PubMed2003

Accelerating comparative genomics using parallel computing.

In the past decade there has been an increase in the number of completely sequenced genomes due to the race of multibillion-dollar genome-sequencing projects. The enormous biological sequence data thus flooding into the sequence databases necessitates the development of efficient tools for comparative genome sequence analysis. The information deduced by such analysis has various applications viz. structural and functional annotation of novel genes and proteins, finding gene order in the genome, gene fusion studies, constructing metabolic pathways etc. Such study also proves invaluable for pharmaceutical industries, such as in silico drug target identification and new drug discovery. There are various sequence analysis tools available for mining such useful information of which FASTA and Smith-Waterman algorithms are widely used. However, analyzing large datasets of genome sequences using the above codes seems to be impractical on uniprocessor machines. Hence there is a need for improving the performance of the above popular sequence analysis tools on parallel cluster computers. Performance of the Smith-Waterman (SSEARCH) and FASTA programs were studied on PARAM 10000, a parallel cluster of workstations designed and developed in-house. FASTA and SSEARCH programs, which are available from the University of Virginia, were ported on PARAM and were optimized. In this era of high performance computing, where the paradigm is shifting from conventional supercomputers to the cost-effective general-purpose cluster of workstations and PCs, this study finds extreme relevance. Good performance of sequence analysis tools on a cluster of workstations was demonstrated, which is important for accelerating identification of novel genes and drug targets by screening large databases.

Algorithms↗
PubMed2006

Discovery of new small molecules and targets towards angiogenesis via chemical genomics approach.

Chemical genetics/genomics is an inter- and multi-disciplinary research engine, which utilizes small molecules to explore the function of genes and accelerate the drug discovery. Bioactive small molecules that are permeable to cellular membrane and bind to its cognate target protein can exert the phenotype changes of the cells or organisms. Functional target proteins of these small molecules have been successfully identified by affinity, genetics and genomics based target identification. The specific molecular recognition of small molecule with target protein has facilitated to decipher the mode of actions of small molecules and developed better drug based on their structure activity relationship. Based on this idea, we have applied chemical genomics to angiogenesis, a new blood vessel formation, resulting in the identification of new small molecules as well as targets. In this review, our application of chemical genomics towards a cellular phenotype, angiogenesis, will be demonstrated.

Amino Acid Sequence↗
PubMedDate not supplied

New horizons in the development of antiepileptic drugs: the search for new targets.

The past decades have brought many advances to the treatment of epilepsy. However, despite the continued development and release of new antiepileptic drugs, many patients have seizures that do not respond to drug therapy or have related side effects that preclude continued use. Even in patients in whom pharmacotherapy is efficacious, current antiepileptic drugs do not seem to affect the progression or underlying natural history of epilepsy. Furthermore, there is currently no drug available which prevents the development of epilepsy, e.g., after head trauma or stroke. Thus, there are at least three important goals for the future: (1) better understanding of processes leading to epilepsy, thus allowing to create therapies aimed at the prevention of epilepsy in patients at risk; (2) development of disease-modifying therapies, interfering with progression of epilepsy, and (3) improved understanding of neurobiological mechanisms of pharmacoresistance, allowing to develop drugs for reversal or prevention of drug resistance. The Second Workshop on New Horizons in the Development of Antiepileptic Drugs explored these three goals for improved epilepsy therapy, with a focus on the search for new drug targets for prevention of epilepsy, for interfering with progression of epilepsy, and for interfering with drug resistance in epilepsy. A special topic dealt with gene expression analysis for target identification. Furthermore, pharmacological and non-pharmacological targets for curing epilepsy were explored. In this conference review, the current status of antiepileptic therapies is critically assessed, and innovative approaches for future therapies are highlighted.

Animals↗
PubMed2006

Phosphoinositide 3-kinase: from viral oncoprotein to drug target.

The catalytic subunit p110alpha of the phosphoinositide 3-kinase (PI3K) and the serine-threonine protein kinase Akt have been extensively studied as retroviral oncoproteins. The experimental tools developed with the retroviral vectors are now being applied to PI3K mutations in human cancer. The most frequently occurring mutants of p110alpha are oncogenic in vitro and in vivo, show gain of enzymatic function, activate Akt, and their oncogenic activity is sensitive to rapamycin. The related isoforms p110beta, gamma and delta induce oncogenic transformation as wild-type proteins. Mutated p110alpha proteins are ideal drug targets. Identification of small molecule inhibitors that specifically target these mutant proteins is a realistic and urgent goal.

Animals↗
PubMed2005

Finding new drug targets in the 21st century.

The past 30 years have witnessed a steady decline in the number of new drug targets. This review concentrates on the initial process of target identification and argues that current problems have resulted from a decrease in clinical research, an overemphasis on the discovery of new targets through an understanding of the molecular causes of disease and the adoption of cell and animal models that are poor predictors of human disease. To resolve this situation, we argue for increased clinical research and show that an intervention at the physiological level, using drugs to target at the extracellular signalling pathways, will facilitate identification of novel drug targets in the 21st century.

Animals↗
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↗
PubMed2002

Latency effects in orientation popout.

A target that differs in orientation from neighboring lines and "pops out" has been found to evoke larger responses in cortical V1 cells than lines in the uniform texture surround which do not popout (e.g., Journal of Neurophysiology 67 (1992) 961). If this is more than a coincidence of observations, physiological properties of contextual modulation should be reflected in the perception of salience. In particular, as the differential suppression from texture surround has been reported to be delayed, target salience may be affected by the history of surrounding lines, i.e. by their orientation before the target was presented. This was tested using a feature flicker paradigm in which target and background lines changed their orientations (Experiment 2). All subjects (N = 4) indicated a benefit in target detection when target orientation was not previously present in the surround. A control experiment showed that this effect was not caused by the purely temporal aspects of asynchronous stimulus presentation (Experiment 3). To distinguish this effect from other sources of delayed processing, Experiment 1 compared the performance in target detection and target identification tasks, for single-lines and popout targets. All subjects required longer stimulus presentation time to identify the orientation of a single line than to detect the line itself, indicating that orientation coding needs longer processing than encoding stimulus onset. However, most subjects needed even longer presentations to detect popout, suggesting that the processing of orientation contrast adds to this delay. In an appendix, putative response variations of V1 cells to asynchronous flicker are computed.

Adult↗
PubMed1984

Does rate of processing determine ease of target detection?

In a complex target identification task, Krumhansl (1982) reported that ease of target detection was inversely related to the exposure duration of contextual elements in the display. This relationship held at brief exposures of the contextual elements. However, in Krumhansl's design, brightness and duration covaried. Hence, the results cannot be ascribed unambiguously to the effect of duration alone. In the present two experiments, brightness and duration were varied independently. The outcome showed that brightness differences were both a necessary and a sufficient condition for producing the relationships observed by Krumhansl. In fact, when brightness was equalized across all durations the relationship was reversed. Although unable to account for the effects of duration, Krumhansl's formal model remains viable as a predictor of the effects of brightness.

Attention↗
PubMed2005

Serum response factor regulates a muscle-specific microRNA that targets Hand2 during cardiogenesis.

Gradients of signalling and transcription factors govern many aspects of embryogenesis, highlighting the need for spatiotemporal control of regulatory protein levels. MicroRNAs are phylogenetically conserved small RNAs that regulate the translation of target messenger RNAs, providing a mechanism for protein dose regulation. Here we show that microRNA-1-1 (miR-1-1) and miR-1-2 are specifically expressed in cardiac and skeletal muscle precursor cells. We found that the miR-1 genes are direct transcriptional targets of muscle differentiation regulators including serum response factor, MyoD and Mef2. Correspondingly, excess miR-1 in the developing heart leads to a decreased pool of proliferating ventricular cardiomyocytes. Using a new algorithm for microRNA target identification that incorporates features of RNA structure and target accessibility, we show that Hand2, a transcription factor that promotes ventricular cardiomyocyte expansion, is a target of miR-1. This work suggests that miR-1 genes titrate the effects of critical cardiac regulatory proteins to control the balance between differentiation and proliferation during cardiogenesis.

Algorithms↗