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At least 199 records · Page 11Linked to original sources
PubMed1994

Virus-vectored immunocontraception for control of wild rabbits: identification of target antigens and construction of recombinant viruses.

The need to control animal populations arises in many situations in the world from a variety of motives. Present control strategies are almost universally based on lethal procedures. Increasingly, there is dissatisfaction with such approaches from many different perspectives. In response to these concerns, the concept of controlling populations of pest species through control of their fertility has been mooted. Successful examples of this approach exist in cases of small, discrete pest populations but application of this to a widely distributed species over a broad geographical area has not yet been achieved. In this article, we report on a new approach to fertility control, virus-vectored immunocontraception, and discuss its applicability to control of wild rabbit populations. Particular emphasis is placed on the strategy for selection of a target molecule capable of inducing an immunocontraceptive response and on how the gene encoding such a molecule might be engineered into the myxoma virus for distribution into the population. The fact that the procedures for antigen identification and the viral engineering methods used are, to varying extents, generic means that the broad principles of this approach are applicable in other species.

Animals↗
PubMed2005

Phenotypic alteration and target gene identification using combinatorial libraries of zinc finger proteins in prokaryotic cells.

We have developed a method with prokaryotic organisms that uses randomized libraries of zinc finger-containing artificial transcription factors to induce phenotypic variations and to identify genes involved in the generation of a specific phenotype of interest. Combining chromatin immunoprecipitation experiments and in silico prediction of target DNA binding sequences for the artificial transcription factors, we identified ubiX, whose down-regulation correlates with the thermotolerance phenotype in Escherichia coli. Our results show that randomized libraries of artificial transcription factors are powerful tools for functional genomic studies.

Amino Acid Sequence↗
PubMed2003

Stable isotope labeling of phosphopeptides for multiparallel kinase target analysis and identification of phosphorylation sites.

An approach for multiparallel target identification and relative quantification of in vitro kinase activities in two different biological samples, using liquid chromatography/mass spectrometry (LC/MS), is described. Synthetic target peptides, containing the putative regulatory phosphorylation sites of sucrose-phosphate synthase (SPS) isoenzymes from Arabidopsis thaliana, were simultaneously in vitro phosphorylated and their phosphorylation states determined. Quantification was achieved by stable isotope labeling of the phosphoserine moiety with ethanethiol and [(2)D(5)]-ethanethiol. This revealed different kinase activities in extracts of wild-type (WT) plants and mutant plants lacking plastidic phosphoglucomutase (PGM). The multiparallel assay allowed the determination of favored substrate specificities among the putative phosphorylation sites in SPS. Additionally, we extended the method to unambiguously identify phosphorylation sites in peptides via differential labeling.

Amino Acid Sequence↗
PubMed1997

Speed of processing and stimulus complexity in low-frequency and high-frequency channels.

Studies of the microgenesis of perception led to the hypothesis that global aspects of objects are processed faster than their details. If one starts with the assumption that low-frequency information of objects corresponds to the global, and high-frequency information to the local aspects, recognising objects should rely at first on information from the low-frequency channels and afterwards from that of the high-frequency channels. The priming paradigm provides a mean of investigating experimentally the temporal availability of low-frequency and high-frequency information in object perception. In the experiments subjects had to respond to target objects preceded either by related or by unrelated priming stimuli, which consisted of low-pass-filtered and high-pass-filtered versions of the objects. With the influence of stimulus complexity controlled, pictures of objects were chosen that varied in the number of intensity changes in the high-frequency components, with those of the low-frequency components kept constant. The exposure duration of each prime was varied between 40 and 100 ms. The results indicated that target identification only profits more from low-frequency than from high-frequency primes if the high-frequency information has a high level of complexity. If the number of intensity changes in the high-frequency components of the prime is low, target identification is most strongly facilitated. The results are discussed in terms of models which focus on organising principles at different scales.

Humans↗
PubMed2004

Identification of target proteins of N-acetylglucosaminyl-transferase V and fucosyltransferase 8 in human gastric tissues by glycomic approach.

To understand better the mechanism underlying gastric cancer and search for potential markers for gastric cancer prognosis, the proteomic analysis of gastric cancer tissues was conducted using two-dimensional gel electrophoresis and lectin blot, followed by electrospray ionization-mass spectrometry. These approaches permitted identification of glyco- or putative glycosylated proteins which may be associated with tumorigenesis. The proteins identified include molecules involved in sugar metabolism, signal transduction, proteolysis, and stress, as well as several unknown proteins, which were aberrantly glycosylated as evidenced by the L-phytohemagglutinin blot.

Fucosyltransferases↗
PubMed2002

Identification of target messenger RNA substrates for the murine deleted in azoospermia-like RNA-binding protein.

The murine autosomal deleted in azoospermia-like protein (mDAZL) is a germ cell-restricted RNA-binding protein essential for sperm production. Homozygous disruption of the mDAZL gene results in the absence of germ cells beyond the spermatogonial stage. Progress into the function of DAZL in spermatogenesis has been hampered without identification of the cognate mRNA substrates that it binds to and regulates. Using the isolation of specific nucleic acids associated with proteins (SNAAP) technique recently developed in our lab, we identified mRNAs from testis that were specifically bound by mDAZL. One mRNA encoded the Tpx-1 protein, a testicular cell adhesion protein essential for the progression of spermatogenesis. A 26-nucleotide region necessary and sufficient to bind mDAZL was found within additional mRNAs isolated by the screen. These included mRNA encoding Pam, a protein associated with myc; GRSF1, an mRNA-binding protein involved in translation activation, and TRF2, a TATA box-binding protein-like protein involved in transcriptional regulation. Each mRNA containing the mDAZL binding site was specifically bound by mDAZL. A similar sequence is also present in the Cdc25A mRNA, a threonine/tyrosine phosphatase involved in cell cycle progression. The mDAZL and Cdc25A homologues are functionally linked in Drosophila and are necessary for spermatogenesis. Our demonstration that Tpx-1 and Cdc25A mRNAs are bound by mDAZL suggests that mDAZL regulates a subset of mRNAs necessary for germ cell development and cell cycle progression. Understanding how mDAZL regulates the target mRNAs will provide new insights into spermatogenesis, strategies for therapeutic intervention in azoospermic patients, and novel approaches for male contraception.

Animals↗
PubMed1995

Interaction of beta-adrenoceptor and adenosine receptor agonists on phosphorylation. Identification of target proteins in mammalian ventricles.

The influence of the adenosine derivative (--)-N6-phenylisopropyladenosine (R-PIA, 1 micron) and 5'N-ethylcarbox-amidoadenosine (NECA, 1 micron) on beta-adrenergic stimulated (isoproterenol, 10 nM) phosphorylation of sarcolemmal (15 kDa protein), sarcoplasmic reticular (phospholamban) and myofibrillar proteins (troponin I, C-protein) was studied in isolated 32P-labeled guinea-pig ventricles. The identification of the 15 kDa protein, phospholamban, troponin I and C-protein was based on their reaction with specific antibodies. Isoproterenol increased contractile parameters (developed tension, rate of tension development, rate of relaxation) and stimulated the phosphorylation state of a 15 kDa protein (now named phospholemman), of phospholamban, troponin I and C-protein (regarded as regulatory proteins). Isoproterenol concomitantly increased myocardial cyclic AMP levels. R-PIA and NECA attenuated the effects of isoproterenol on contractile parameters as well as on the phosphorylation of the regulatory proteins without affecting cyclic AMP levels. The effects of 1 microM R-PIA and 1 microM NECA on the isoproterenol-stimulated phosphorylation of regulatory proteins were blocked by the adenosine receptor antagonist 1.3-dipropyl-8-cyclopentylxanthine (DPCPX, 1 microM). Therefore, it is concluded that adenosine derivatives acting via adenosine receptors can reduce the isoproterenol-stimulated phosphorylation state of the following regulatory proteins: phospholemman, phospholamban, troponin I and C-protein.

Adenosine↗
PubMed2004

The promise of genomics to identify novel therapeutic targets.

The cataloguing of the human genome has provided an unprecedented prospectus for target identification and drug discovery. A current analysis indicates that slightly more than 3000 unique protein encoding loci are potentially amenable to pharmacological intervention (the 'druggable genome', which can be queried at http://function.gnf.org/druggable). However, the assessment of genome sequence data has not resulted in the anticipated acceleration of novel therapeutic developments. The basis for this shortfall lies in the significant attrition rates endemic to preclinical/clinical development, as well as the often underestimated complexity of gene function in higher order biological systems. To address the latter issue, a number of strategies have emerged to facilitate genomics-driven target identification and validation, including cellular profiling of gene function, in silico modelling of gene networks, and systematic analyses of protein complexes. The expectation is that the integration of these and other systems-based technologies may enable the conversion of potential genomic targets into functionally validated molecules, and result in practicable gene-based drug discovery pipelines.

Animals↗
PubMed2005

Global analysis of IL-2 target genes: identification of chromosomal clusters of expressed genes.

T lymphocytes play a central role in controlling adaptive immune responses. IL-2 critically regulates both T cell growth and death and is involved in maintaining peripheral tolerance, but the molecules involved in these and other IL-2 actions are only partially known. We now provide a comprehensive compendium of the genes expressed in T cells and of those regulated by IL-2 based on a combination of DNA microarrays and serial analysis of gene expression (SAGE). The newly identified IL-2 target genes include many genes previously linked to apoptosis in other cellular systems that may contribute to IL-2-dependent survival functions. We also studied the mRNA expression of known regulators of signaling pathways for their induction in response to IL-2 in order to identify potential novel positive and/or negative feedback regulators of IL-2 signaling. We show that IL-2 regulates only a limited number of these genes. These include suppressors of cytokine signaling (SOCS) 1, SOCS2, dual-specificity phosphatase (DUSP) 5, DUSP6 and non-receptor type phosphatase-7 (PTPN7). Additionally, we provide evidence that many genes expressed in T cells locate in chromosomal clusters, and that select IL-2-regulated genes are located in at least two clusters, one at 5q31, a known cytokine gene cluster, and the other at 6p21.3, a region that contains genes encoding the tumor necrosis factor (TNF) superfamily members TNF, LT-alpha and LT-beta.

Animals↗
PubMed2003

Identification and targeting of tumor escape mechanisms: a new hope for cancer therapy?

Conventional cancer therapy is administered in the form of surgery, radiotherapy or chemotherapy. Immunotherapy is the latest asset to the panel of anti-cancer treatments. This approach appears favorable over the other more conventional methods for various reasons: (1) it is highly specific for cancer cells and, therefore, low toxicity should be expected; (2) it recognizes and eliminates cancer cells regardless of their phase in the cell cycle; (3) tumors that developed drug resistances would still be a suitable target for immunotherapy. (4) Immunotherapy offers the possibility of preventive immunization of high-risk patients. Due to the diverse mechanisms that result in the transformation of cells and subsequent tumor development, not all cancers respond similarly to treatment. Significant effort is currently invested in the characterization of the underlying regulatory network in individual cancers responsible for tumorigenesis. Understanding tumors better allows on one hand the identification of essential pathways that can be intercepted to kill the transformed cells more specifically. On the other hand, these insights also allow us to exclude therapeutic strategies with little chance of success when dealing with tumor escape mutants thus saving valuable time and resources. Any tumor therapy puts selective pressure on tumors thus favoring the outgrowth of therapy-resistant variants. This review summarizes current knowledge on tumor escape mechanisms and some of the efforts to overcome these mechanisms.

Drug Delivery Systems↗
PubMed2006

Development of species identification tests targeting the 16S ribosomal RNA coding region in mitochondrial DNA.

This report describes the development of a species testing system based on the diversity of nucleotide sequences in mitochondrial DNA (mtDNA) among species. Five species, human, cow, pig, dog, and cat, were considered. The partial nucleotide sequences in 16S ribosomal RNA coding region were chosen as the target for discriminating the species. The sequence diversities of this approximately 400 bp long region ranged from 15.7 to 24.1% among the five species. Sequencing analysis of this target on 50 individuals of each species (53 for dogs) revealed that the nucleotide sequences were well preserved within species. Species-specific PCR for each species was also designed, and satisfactory results with regard to both sensitivity and specificity were obtained. A validation study with DNA extracted from bovine bone exposed to the environment revealed that the PCRs designed in this study worked correctly. From the results obtained, it was shown that this testing system could be a good tool for species identification. One successful case report is also demonstrated.

Animals↗
PubMed2005

Identification of target genes in breast cancer cells directly regulated by the SRC-3/AIB1 coactivator.

Steroid receptor coactivator-3 (SRC-3/AIB1) is a coactivator for nuclear receptors and other transcription factors and an oncogene that contributes to growth regulation and development of mammary and other tumor types. Because of its biological functions, it is important to identify genes regulated by SRC-3. However, because coactivators do not bind DNA directly, extensive work is required to determine whether genes identified by RNA profiling approaches are direct or indirect targets. Here, we report the use of chromatin immunoprecipitation (ChIP)-based assays that involve genomic mapping and computational analyses of immunoprecipitated DNA to identify SRC-3-binding target genes in estradiol (E2)-treated MCF-7 breast cancer cells. We identified 18 SRC-3 genomic binding sites and demonstrated estrogen receptor-alpha (ERalpha) binding to all of them. Both E2-dependent and -independent SRC-3/ERalpha-binding sites were identified. RNA polymerase II ChIP assays were used to determine the correlation between SRC-3 and ERalpha binding and recruitment of the transcriptional machinery. These assays, in conjunction with analyses of RNA obtained from E2-treated cells, lead to the identification of SRC-3/ERalpha-associated genes. The ability of SRC family coactivators to regulate the expression of one of these genes, PARD6B/Par6, was confirmed by using cells individually depleted of SRC-1, SRC-2, or SRC-3 by small interfering RNA. The method described herein can be used to identify genes regulated by non-DNA-binding factors, such as other coactivators or corepressors, as well as DNA-binding transcription factors, and provides information on their binding location that can accelerate further gene characterization.

Acetyltransferases↗
PubMed2003

Protein microarrays: new tools for pharmaceutical development.

Protein microarrays are a relatively new technology, which will dramatically impact the pharmaceutical industry. The critical need for more rapid identification of novel drug targets, and for obtaining high-quality information early in the target validation process is a major driver for the industry. High-throughput protein analytical techniques are critical for obtaining biological information beyond that which transcript analysis can provide, given that proteins are the "worker bees" in cells. The vast complexity of proteins when compared to DNA and RNA in terms of sheer number, and structural and biochemical diversity requires a higher degree of sophistication in both assay design and data analysis. High-throughput microarray technology platforms allow for simultaneous, multi-parametric analysis of complex protein mixtures. Protein microarrays have tremendous potential as a tool for the study of protein-protein, enzyme-substrate, and antibody-antigen interactions among others. They can also be used for biomarkers and drug target identification via comparative proteomic analysis of healthy and disease tissues. More recently, cellular microarrays that enable identification of cell-surface receptors and other cell-surface proteins allowing rapid screening of cell-specific, novel drug targets, are being developed. This review will focus on the technical issues and potential applications of protein microarrays in pharmaceutical discovery.

Drugs, Investigational↗
PubMed2001

Cluster analysis and promoter modelling as bioinformatics tools for the identification of target genes from expression array data.

Expression arrays yield enormous amounts of data linking genes, via their cDNA sequences, to gene expression patterns. This now allows the characterisation of gene expression in normal and diseased tissues, as well as the response of tissues to the application of therapeutic reagents. Expression array data can be analysed with respect to the underlying protein sequences, which facilitates the precise determination of when and where certain groups of genes are expressed. More recent developments of clustering algorithms take additional parameters of the experimental set-up into account, focusing more directly on co-regulated set of genes. However, the information concerning transcriptional regulatory networks responsible for the observed expression patterns is not contained within the cDNA sequences used to generate the arrays. Regulation of expression is determined to a large extent by the promoter sequences of the individual genes (and/or enhancers). The complete sequence of the human genome now provides the molecular basis for the identification of many regulatory regions. Promoter sequences for specific cDNAs can be obtained reliably from genomic sequences by exon mapping. In the many cases in which cDNAs are 5'-incomplete, high quality promoter prediction tools can be used to locate promoters directly in the genomic sequence. Once sufficient numbers of promoter sequences have been obtained, a comparative promoter analysis of the co-regulated genes and groups of genes can be applied in order to generate models describing the higher order levels of transcription factor binding site organisation within these promoter regions. Such modules represent the molecular mechanisms through which regulatory networks influence gene expression, and candidates can be determined solely by bioinformatics. This approach also provides a powerful alternative for elucidating the functional features of genes with no detectable sequence similarity, by linking them to other genes on the basis of their common promoter structures.

Algorithms↗
PubMed1996

Interference in identifying tactile patterns: response competition and temporal integration.

When two vibrotactile spatial patterns-one target and one nontarget pattern-are presented to the same location, subjects may have difficulty in identifying the target pattern. The difficulty may be in part the result of the target's and nontarget's being processed to the point of evoking competing responses. Subjects err by responding with the nontarget. Target identification may also be interfered with because the target and nontarget form a composite pattern through temporal integration, and subjects are unable to separate the features of the target and nontarget patterns. The present study addressed the issue of whether a response competition explanation of interference is consistent with the existence of temporal integration and the formation of composite patterns. A series of three experiments examined temporal integration and its relationship to response competition. The results indicate that both temporal integration and response competition can occur under similar stimulus conditions, and that both can interfere with the identification of target patterns. The results further indicate that even under conditions in which composite patterns are formed, the two patterns that constitute the composite are still identifiable. It is concluded that the interference in pattern identification attributed to response competition is not a result of temporal integration.

Adult↗
PubMedDate not supplied

Hepatitis B and C viruses: molecular identification and targeted antiviral therapies.

Four agents are in clinical development for the treatment of chronic hepatitis B infection. These nucleoside analogs are incorporated into the growing DNA chain and terminate replication. Lamivudine, a cytadine analog that inhibits the synthesis of negative strand DNA from pre-genomic RNA, predictably inhibits replication and improves liver enzymes and histology in infected individuals. Following cessation of treatment, relapse is common, and genetic causes of viral resistance have been described. Other drugs for HBV infection include famciclovir, a guanosine analog that has also shown to suppress replication in immunocompetent as well as in immunocompromised patients; lobucavir, a guanosine analog; and adevfovir, an adenine nucleotide analog. The future of drug therapy against HBV likely includes combination agents with one or more nucleoside/nucleotide analogs and immune stimulants, such as interferon, or therapeutic vaccines. Recent advances in the treatment of HCV have been less impressive. An effective vaccine is greatly needed yet development in the near future is unlikely. Recommendations for therapy of chronic HCV have been proposed following the National Institutes of Health Consensus Conference. Interferon alpha is advised in patients with elevated serum alanine aminotransferases and liver histology demonstrating active hepatitis, regardless of level of pretreatment viremia or infecting genotype. Therapy should be continued for three months, at which time response should be assessed. If a biochemical and/or virological response has been achieved, treatment should be continued for a year. Trials are underway to evaluate interferon in combination with ribavirin. Recent identification of the crystalline structure of the HCV NS3 protease promises development of effective inhibitors of this critical viral enzyme.

Animals↗
PubMed1997

In vivo cleavage of Escherichia coli BIME-2 repeats by DNA gyrase: genetic characterization of the target and identification of the cut site.

The Escherichia coli chromosome contains about 300 bacterial interspersed mosaic elements (BIMEs). These elements, located at the 3' end of genes, are composed of three types of alternating repetitive extragenic palindromes (REPs). Based on the type of REP they contain and on their ability to interact with the integration host factor (IHF), BIMEs are subdivided into two families: BIME-1 elements contain an IHF binding site flanked by converging Y and Z1 REPs, whereas BIME-2 elements contain a variable number of alternating Y and Z2 REPs without an IHF site. Although some BIMEs have been implicated in the protection of mRNA against 3' exonucleolytic degradation, the main role of elements belonging to both families remains to be elucidated. In this paper, we used oxolinic acid, a drug that reveals potential sites of DNA gyrase action, to demonstrate that DNA gyrase interacts in vivo with BIME-2 elements. The frequency of cleavage varied from one element to another, and the cleavage pattern observed in elements containing several REPs indicated that DNA gyrase cut DNA every two REPs. A single cleavage site has been identified in the Y REP in six out of seven instances, and the nucleotide sequence of a 44 bp fragment containing the scission point displayed conserved residues at six positions. The lack of one of the conserved residues accounted for the absence of cleavage in most of the Z2 REPs. Our results also showed that cleaved REPs were always associated with another REP, suggesting that a pair of diverging REPs constitutes the target of DNA gyrase. DNA gyrase cleavage at repetitive BIME-2 elements may have consequences for DNA topology and genomic rearrangements.

Base Sequence↗
PubMed2004

Proteome analysis for identification of target proteins of genistein in primary human endothelial cells stressed with oxidized LDL or homocysteine.

BACKGROUND: Epidemiological studies suggest that soy consumption contributes to the prevention of coronary heart disease. The proposed anti-atherogenic effects of soy appear to be carried by the soy isoflavones with genistein as the most abundant compound. AIM OF THE STUDY: To identify proteins or pathways by which genistein might exert its protective activities on atherosclerosis, we analyzed the proteomic response of primary human umbilical vein endothelial cells (HUVEC) that were exposed to the pro-atherosclerotic stressors homocysteine or oxidized low-density lipoprotein (ox-LDL). METHODS: HUVEC were incubated with physiological concentrations of homocysteine or ox-LDL in the absence and presence of genistein at concentrations that can be reached in human plasma by a diet rich in soy products (2.5 microM) or by pharmacological intervention (25 microM). Proteins from HUVEC were separated by two-dimensional polyacrylamide gel electrophoresis and those that showed altered expression level upon genistein treatment were identified by peptide mass fingerprints derived from tryptic digests of the protein spots. RESULTS: Several proteins were found to be differentially affected by genistein. The most interesting proteins that were potently decreased by homocysteine treatment were annexin V and lamin A. Annexin V is an antithrombotic molecule and mutations in nuclear lamin A have been found to result in perturbations of plasma lipids associated with hypertension. Genistein at low and high concentrations reversed the stressor-induced decrease of these anti-atherogenic proteins. Ox-LDL treatment of HUVEC resulted in an increase in ubiquitin conjugating enzyme 12, a protein involved in foam cell formation. Treatment with genistein at both doses reversed this effect. CONCLUSION: Proteome analysis allows the identification of potential interactions of dietary components in the molecular process of atherosclerosis and consequently provides a powerful tool to define biomarkers of response.

Annexin A5↗