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At least 163 records · Page 9Linked to original sources
PubMed2007

Identification of targets of phosphorylation in heart mitochondria.

Whereas most strategies in proteomics deal with changes in protein levels, posttranslational modifications often represent pathological consequences at the molecular level without changes in abundance and, therefore represent a critical phenomenon to study. The present report elucidates an approach to studying one such posttranslational modification, phosphorylation, which is an important event in a variety of signaling cascades initiated in the heart in response to physiological and pathological stimuli. Comparison of phosphorylation profiles in different conditions, including drug treatments, receptor stimulation or blockade, and so forth, may lead to identification of relevant targets of signal transduction cascades. Furthermore, it may be possible to devise similar schemes to detect the subset of proteins modified by ubiquitinylation, lipid acylation, and so forth, and to compare differential patterns. Phosphorylation of mitochondrial targets is increasingly being recognized. Mitochondria are critical targets of a number of signaling pathways and therefore it is important to develop suitable methods to detect phosphorylation of mitochondrial targets. One approach is to perform in vitro phosphorylation in a reconstituted system of cytosol and mitochondria.

Animals↗
PubMed2004

Characterization of antirrhinum petal development and identification of target genes of the class B MADS box gene DEFICIENS.

The class B MADS box transcription factors DEFICIENS (DEF) and GLOBOSA (GLO) of Antirrhinum majus together control the organogenesis of petals and stamens. Toward an understanding of how the downstream molecular mechanisms controlled by DEF contribute to petal organogenesis, we conducted expression profiling experiments using macroarrays comprising >11,600 annotated Antirrhinum unigenes. First, four late petal developmental stages were compared with sepals. More than 500 ESTs were identified that comprise a large number of stage-specifically regulated genes and reveal a highly dynamic transcriptional regulation. For identification of DEF target genes that might be directly controlled by DEF, we took advantage of the temperature-sensitive def-101 mutant. To enhance the sensitivity of the profiling experiments, one petal developmental stage was selected, characterized by increased transcriptome changes that reflect the onset of cell elongation processes replacing cell division processes. Upon reduction of the DEF function, 49 upregulated and 52 downregulated petal target genes were recovered. Eight target genes were further characterized in detail by RT-PCR and in situ studies. Expression of genes responding rapidly toward an altered DEF activity is confined to different petal tissues, demonstrating the complexity of the DEF function regulating diverse basic processes throughout petal morphogenesis.

Antirrhinum↗
PubMed1994

The Max transcription factor network: involvement of Mad in differentiation and an approach to identification of target genes.

The small bHLHZip protein, Max, was originally identified through its interaction with Myc family proteins and appears to be an obligate partner for Myc function. Max has now been found to interact with at least two other proteins, Mad and Mxi1. These also belong to the bHLHZip class but are otherwise unrelated to Myc. Mad has been shown to abrogate the positive transcriptional activity of Myc and to inhibit Myc in co-transformation assays. This suggests that Mad may antagonize Myc function. Mad is rapidly induced upon differentiation, a time when Myc is frequently down-regulated. We show here evidence for Mad expression upon differentiation of myeloblasts, monoblasts, and keratinocytes. Mad:Max complexes are detected during differentiation and appear to replace the Myc:Max complexes present in proliferating cell populations. Since these complexes appear to form even in the presence of Myc, there may exist mechanisms that act to inhibit Myc:Max, or to promote Mad:Max, complex formation. We speculate that Max complex switching causes a change in the transcriptional activity of groups of target genes. Mad is not induced in all differentiating cell types, suggesting that other, possibly tissue-restricted, proteins might act in similar switch mechanisms to effect changes in transcriptional programs. We have also developed an approach to identification of the gene targets for Myc:Max complexes. By employing an immunoisolation procedure, we have begun characterization of several clones whose expression levels correlate with those of c-myc. Further identification of Myc-regulated genes may allow us to determine the molecular mechanism by which Myc governs cell proliferation and differentiation.

Basic Helix-Loop-Helix Leucine Zipper Transcriptio↗
PubMed2004

Computational identification of microRNA targets.

Recent experiments have shown that the genomes of organisms such as worm, fly, human, and mouse encode hundreds of microRNA genes. Many of these microRNAs are thought to regulate the translational expression of other genes by binding to partially complementary sites in messenger RNAs. Phenotypic and expression analysis suggests an important role of microRNAs during development. Therefore, it is of fundamental importance to identify microRNA targets. However, no experimental or computational high-throughput method for target site identification in animals has been published yet. Our main result is a new computational method that is designed to identify microRNA target sites. This method recovers with high specificity known microRNA target sites that have previously been defined experimentally. Based on these results, we present a simple model for the mechanism of microRNA target site recognition. Our model incorporates both kinetic and thermodynamic components of target recognition. When we applied our method to a set of 74 Drosophila melanogaster microRNAs, searching 3'UTR sequences of a predefined set of fly mRNAs for target sites which were evolutionary conserved between D. melanogaster and Drosophila pseudoobscura, we found that many key developmental body patterning genes such as hairy and fushi-tarazu are likely to be translationally regulated by microRNAs.

Algorithms↗
PubMed2005

Competition for representation is mediated by relative attentional salience.

The biased competition model of attentional selection proposes that objects compete with one another for neural representation, with the competition rooted in stimulus and attentionally-based salience. Two experiments explore how the salience of a target item relative to flanking items impacts the speed of target identification. The results of two experiments suggest that spatially proximal items compete for shared, spatially dependent processing resources. In both experiments, subjects identified target elements embedded in multi-element displays. In Experiment 1, attentional salience was manipulated by using abrupt onsets (high-salience) and non-onsets (low-salience). Target identifications were slowest when the target was flanked by two high-salience stimuli (abrupt onsets) and fastest when the target was flanked by two low-salience items, (non-onsets). In Experiment 2, the attentional salience of display items was set through a probability manipulation involving the color of the target. The results mirrored those of Experiment 1, consistent with predictions of the biased competition model.

Adult↗
PubMed2005

A genetic approach to the de novo identification of targets of strain-specific immunity in malaria parasites.

Vaccine research in malaria has a high priority. However, identification of specific antigens as candidates for vaccines against asexual blood stages of malaria parasites has been based on largely circumstantial evidence. We describe here how genes encoding target antigens of strain-specific immunity in malaria can be directly located in the parasite's genome without prior information concerning their identity, by the method we call linkage group selection. Two genetically distinct clones of the rodent malaria parasite Plasmodium chabaudi chabaudi, each of which induces an immunity in laboratory mice that is more protective against challenge with itself than with the heterologous strain, were genetically crossed, and the uncloned cross progeny selected in mice that had been made partially immune by infection and drug cure with one or the other parental strain. Proportions of parental alleles in the selected and unselected cross progeny were compared by using quantitative genome-wide molecular markers. A small number, including groups of linked markers forming so-called selection valleys, were markedly reduced under strain-specific immune pressure. A very prominent selection valley was found to contain the gene for merozoite surface protein-1, a major candidate antigen for malaria vaccine development, at the locus at which the strongest reduction under strain-specific immune selection was detected. Closely linked to the merozoite surface protein-1 gene was a gene containing the signature motif of the ring-infected erythrocyte surface antigen family. Another affected locus, unlinked to this selection valley, contained a member of the serine repeat antigen gene family.

Animals↗
PubMed2005

Rapid identification of target genes for 3-methyl-1-butanol production in Saccharomyces cerevisiae.

Extracellular conditions determine the taste of fermented foods by affecting metabolite formation by the micro-organisms involved. To identify targets for improvement of metabolite formation in food fermentation processes, automated high-throughput screening and cDNA microarray approaches were applied. Saccharomyces cerevisiae was cultivated in 96-well microtiter plates, and the effects of salt concentration and pH on the growth and synthesis of the fusel alcohol-flavoured substance, 3-methyl-1-butanol, was evaluated. Optimal fermentation conditions for 3-methyl-1-butanol concentration were found at pH 3.0 and 0% NaCl. To identify genes encoding enzymes with major influence on product formation, a genome-wide gene expression analysis was carried out with S. cerevisiae cells grown at pH 3.0 (optimal for 3-methyl-1-butanol formation) and pH 5.0 (yeast cultivated under standard conditions). A subset of 747 genes was significantly induced or repressed when the pH was changed from pH 5.0 to 3.0. Expression of seven genes related to the 3-methyl-1-butanol pathway, i.e. LAT1, PDX1, THI3, ALD4, ILV3, ILV5 and LEU4, strongly changed in response to this switch in pH of the growth medium. In addition, genes involved in NAD metabolism, i.e. BNA2, BNA3, BNA4 and BNA6, or those involved in the TCA cycle and glutamate metabolism, i.e. MEU1, CIT1, CIT2, KDG1 and KDG2, displayed significant changes in expression. The results indicate that this is a rapid and valuable approach for identification of interesting target genes for improvement of yeast strains used in industrial processes.

Culture Media↗
PubMed1995

Comparison of MRI and ventriculographic target acquisition for posteroventral pallidotomy.

The true accuracy of MRI scanners utilized for target acquisition in functional stereotaxis remains undefined. We have performed pallidotomies at the Loma Linda University Medical Center using both MRI and MRI concomitant with positive contrast ventriculography. A retrospective study of 33 cases revealed significant differences in coordinates obtained by MRI alone and MRI concomitant with ventriculography (antero-posterior coordinates, mean error: 2.6 mm, range 0.5-6.8 mm; Z coordinates: mean error 4.6 mm, range 0-19 mm). While MRI promises excellent target identification, the generated target coordinates significantly stray from the optimal target. Ventriculography corrects the MRI coordinates and demonstrates the position of the radiofrequency probe with respect to the target and anatomic reference points.

Cerebral Ventriculography↗
PubMed2003

IL-7-dependent human leukemia T-cell line as a valuable tool for drug discovery in T-ALL.

The specific targeting of critical signaling molecules may provide efficient therapies for T-cell acute lymphoblastic leukemia (T-ALL). However, target identification and drug development are limited by insufficient numbers of primary T-ALL cells and by their high rate of spontaneous apoptosis. We established a human interleukin-7 (IL-7)-dependent T-ALL cell line, TAIL7, that maintains several biologic and signaling properties of its parental leukemia cells. TAIL7 cells are pre-T-ALL cells that proliferate in response to IL-7 and IL-4. IL-7 stimulation of TAIL7 cells prevents spontaneous in vitro apoptosis and induces cell activation and cell cycle progression. The signaling events triggered by IL-7 include down-regulation of p27(kip1) and hyperphosphorylation of retinoblastoma protein (Rb). Stimulation of TAIL7 cells by IL-7 leads to phosphorylation of Janus kinase 3 (JAK3), signal transducer and activator of transcription 5 (STAT5), Akt/PKB (protein kinase B), and extracellular-regulated kinase 1 and 2 (Erk1/2). Importantly, specific blockade of JAK3 by its inhibitor WHI-P131 abrogates the IL-7-mediated proliferation and survival of TAIL7 cells, suggesting that activation of JAK3 is critical for IL-7 responsiveness by these cells. Because TAIL7 cells seem to be a biologic surrogate for primary leukemia T cells, this cell line constitutes a valuable tool for the study of the signaling pathways implicated in T-ALL. Exploitation of this cell line should allow the identification of molecular targets and promote the rational design and validation of antileukemia signaling inhibitors.

Animals↗
PubMed2000

The intracellular target for the antiresorptive aminobisphosphonate drugs in Dictyostelium discoideum is the enzyme farnesyl diphosphate synthase.

Aminobisphosphonate (aBP) drugs inhibit osteoclast-mediated bone resorption and also growth of amoebas of Dictyostelium discoideum apparently by interaction with the same intracellular target. Identification of the target in Dictyostelium therefore could also identify the target in osteoclasts. The aBPs (100 microM alendronate and 30 microM YM-175) inhibited conversion of [14C]mevalonate into sterols by cultures of Dictyostelium amoebas. One of three enzymes (isopentenyl diphosphate [IDP] isomerase, farnesyl diphosphate [FDP] synthase, and squalene synthase) appeared to be the target for this inhibition because conversion of [14C]IDP into squalene, the immediate precursor for sterol biosynthesis, was inhibited in extracts of wild-type amoebas by alendronate (IC50 = 75 nM) or risedronate (IC50 = 30 nM) whereas, when the extract had been prepared from amoebas of strains selected for having partial resistance to the growth-inhibitory effects of alendronate (strain MR102) or risedronate (strain RB101), the values of IC50 were increased to 700 nM for alendronate (MR102 extract) or 130 nM for risedronate (RB101 extract). Neither IDP isomerase nor squalene synthase was inhibited significantly by alendronate or risedronate but both of these aBP drugs, and all others tested, inhibited FDP synthase. Determination of the nucleotide sequences of complementary DNAs (cDNAs) encoding FDP synthase in the wild-type and aBP-resistant strains of Dictyostelium indicated that there had been no changes in the amino acid sequence of the enzyme in the mutant strains. However, both mutant strains overproduce FDP synthase. It is concluded that FDP synthase is the intracellular target for the aBP drugs.

Alendronate↗
PubMed2001

Functional genomic technologies applied to the control of the human malaria parasite, Plasmodium falciparum.

Infection with any of the four species of Plasmodium single cell parasites that infects humans causes the clinical disease, malaria. Of these, it is Plasmodium falciparum that is responsible for the majority of the 1.5-2.3 million deaths due to this disease each year. Worldwide there are between 300-500 million cases of malaria annually. To date there is no licensed vaccine and resistance to most of the available drugs used to prevent and/or treat malaria is spreading. There is therefore an urgent need to develop new and effective drugs and vaccines against this devastating parasite. We have outlined a strategy using a combination of DNA-based vaccines and the data derived from the soon-to-be completed P. falciparum genome and the genomes of other species of Plasmodium to develop new vaccines against malaria. Much of the technology that we are developing for vaccine target identification is directly applicable to the identification of potential targets for drug discovery. The publicly available genome sequence data also provides a means for researchers whose focus may not be primarily malaria to leverage their research on cancer, yeast biology and other research areas to the biological problems of malaria.

Animals↗
PubMed2002

Identifying molecular targets mediating the anticancer activity of histone deacetylase inhibitors: a work in progress.

The anticancer properties of histone deacetylase inhibitors have been known for some time. However, it is only recently that the functional identities of the intracellular targets mediating the anticancer properties have started to be revealed. These targets appear to play significant roles in cell cycle control, apoptosis and differentiation. Importantly, the modulation of these activities is likely to be mediated by alterations in the acetylation status of both histone and non-histone targets. Identification of these targets, and the specific histone deacetylase enzymes that modulate them, is an important step in designing rational-based therapies for the treatment of cancer. In this review we discuss the state of progress in identifying the molecular pathways/events mediating the anticancer activity of histone deacetylase inhibitors.

Animals↗
PubMed2000

Identification of target antigen for SLA/LP autoantibodies in autoimmune hepatitis.

BACKGROUND: Autoantibodies are a hallmark of autoimmune hepatitis, but most are not disease specific. Autoantibodies to soluble liver antigen (SLA) and to liver and pancreas antigen (LP) have been described as disease specific, occurring in about 30% of all patients with autoimmune hepatitis, but no standardised assays are available. Methods We tested 2000 serum samples from patients with various liver diseases and controls for SLA autoantibodies by inhibition ELISA. Serum samples positive for SLA antibodies were used for immunoscreening of cDNA expression libraries. Identified clones were tested against a panel of serum samples positive for SLA and LP autoantibodies and control serum samples, and the epitope mapped by deletion mutants and exonuclease digestion. FINDINGS: SLA and LP autoantibodies were identical. Of 2000 serum samples screened, 35 were positive for SLA autoantibodies. These positive samples came from patients with autoimmune hepatitis; three from patients with an overlap syndrome (primary biliary cirrhosis and secondary autoimmune hepatitis). Expression cloning and absorption experiments identified a 422 aminoacid protein present in two splice variants as the sole target antigen. Aminoacids 371-409 were critical for immune recognition. INTERPRETATION: The identified cDNA encodes the primary target antigen of SLA/LP autoantibodies. The SLA/LP antigen has a previously unknown aminoacid sequence, and presumably codes for an unindentified enzyme, suggested to be UGA-suppressor tRNA-associated protein. SLA/LP autoantibodies are disease specific and recognise a dominant epitope, suggesting a specific antigen-driven immune response. Identification of the SLA/LP target antigen will allow establishment of a reliable, widely available diagnostic assay. Furthermore, its role in the pathogenesis of autoimmune hepatitis can now be studied.

Autoantibodies↗
PubMed2026

Target Antigen Identification for Antibody Drug Conjugate Therapy in Biliary Tract Cancer.

BACKGROUND: Data on antibody-drug conjugates (ADCs) target expression prevalence, intertumoral heterogeneity, genomic concordance, and its effect on clinical outcomes is limited in biliary tract cancers (BTC). METHODS: Resected primary BTC specimens, and when available, matched metastatic samples were assembled into tissue microarrays and tested for CLDN18.2, c-MET, Nectin-4, TROP2, and HER2 expression by immunohistochemistry (IHC). A subset underwent targeted next-generation sequencing using MSK-IMPACT (NCT01775072). Exploratory associations of target expression with clinicopathologic parameters, genomic alterations, recurrence-free (RFS), and overall (OS) survival were evaluated. RESULTS: 65 patients with resected BTC and 18 paired metastatic sites were identified-43% extrahepatic cholangiocarcinoma, 40% intrahepatic cholangiocarcinoma, and 17% gallbladder cancer. All evaluated target antigens were expressed; percent positivity and H-score ≥200 were: TROP2 (83%, 26%), c-MET (75%, 26%), Nectin-4 (66%, 35%), and CLDN18.2 (46%, 7.7%). HER2 overexpression occurred in 3.1% of tumors. Overall agreement among paired primary and metastatic samples on calling either positive or negative ranged from 43% to 75% with the highest observed for HER2 [75%; κ=0.29 (95%CI: -0.32 to 0.91)] and TROP2 (71%; κ not available) and lowest for c-MET, CLDN18.2, and Nectin-4. Frequently altered genes included TP53 (36%), SMAD4 (27%), ELF3 (21%). We observed no significant association between target antigen expression with genomics, RFS, or OS. CONCLUSIONS: BTC displays frequent but heterogeneous expression of multiple ADC targets. These hypothesis generating findings suggest inherent complexity of target protein quantification, target threshold determination, and target sampling discordance. Future studies will be required to refine our understanding the utlitiy of ADCs in BTC.

Journal Article↗
PubMed2007

Recommended criteria for the mass spectrometric identification of target peptides and proteins (<8 kDa) in sports drug testing.

Mass spectrometry has become an invaluable tool for the identification of prohibited peptide hormones and proteins in doping control analysis. Regulatory authorities have established criteria for identifying banned drugs in doping control specimens, but these criteria do not address the specific issues for high molecular weight protein drugs such as molecular weight determination of multiply charged molecules, analysis of chemically or enzymatically derived degradation products, identification of amino acid sequence tags, etc. Technical considerations such as sample preparation methods (e.g. immunoaffinity purification), resulting analytes (e.g. intact compounds vs. chemically or enzymatically derived peptides), ionization modes, analyzer resolution, and the information provided by respective techniques are discussed in light of sports drug testing requirements using typical application examples.

Doping in Sports↗
PubMed1999

An in vivo assay for the identification of target proteases which cleave membrane-associated substrates.

Proteases not only play a fundamental role in numerous physiological processes, but are also involved in several human diseases including Alzheimer's disease (AD). A key protease implicated in AD is the so far unidentified gamma-secretase, which cleaves the membrane-bound beta-amyloid precursor protein (betaAPP) at the C-terminus of its amyloid domain within the membrane to release the neurotoxic amyloid beta-peptide. In order to allow the isolation of proteases, which specifically cleave membrane-bound substrates within or in the vicinity of a transmembrane domain, we developed a reporter gene assay in Saccharomyces cerevisiae. This assay may allow the identification of genes encoding target proteases that specifically cleave membrane bound substrates by transforming expression libraries.

Amyloid Precursor Protein Secretases↗
PubMed2003

Identification of target areas for deep brain stimulation in human basal ganglia substructures based on median nerve sensory evoked potential criteria.

OBJECTIVE: In the interventional treatment of movement disorders, the thalamic ventral intermediate nucleus (VIM) and the subthalamic nucleus (STN) are the most relevant electrode targets for deep brain stimulation (DBS). This study tested the value of somatosensory evoked potentials (SEP) for the functional identification of VIM and STN. METHODS: Median nerve SEP were recorded from the final stimulation electrodes targeted at STN and VIM. Throughout the stereotactic procedure SEP were recorded during short electrode stops above STN/VIM and within the presumed target areas. After digital filtering, high and low frequency SEP components were analysed separately to parameterise both the 1000 Hz SEP burst and low frequency (<100 Hz) components. RESULTS: SEP recorded in the VIM target region could unequivocally be distinguished from SEP recorded in STN. The 1000 Hz burst signal was significantly larger in VIM than in STN without any overlap of amplitude values. In the low frequency band, a primary high amplitude negativity was obtained in VIM, contrasting with a low amplitude positivity in STN. SEP waveshapes in recordings above target positions resembled SEP obtained in STN. When entering VIM, a sharp amplitude increase was observed over a few millimetres only. CONCLUSIONS: Based on SEP criteria, the VIM target but not the STN region can be identified by typical SEP configuration changes, when penetrating the target zone. The approach is independent of the patient's cooperation and vigilance and therefore feasible in general anaesthesia. It provides an easy, reliable, and robust tool for the final assessment of electrode positions at the last instance during electrode implantation when eventual electrode revisions can easily be performed.

Adult↗
PubMed2006

Caenorhabditis elegans: a versatile platform for drug discovery.

Drug discovery and drug target identification are two intimately linked facets of intervention strategies aimed at effectively combating pathological conditions in humans. Simple model organisms provide attractive platforms for devising and streamlining efficient drug discovery and drug target identification methodologies. The nematode worm Caenorhabditis elegans has emerged as a particularly convenient and versatile tool that can be exploited to achieve these goals. Although C. elegans is a relatively modern addition to the arsenal of model organisms, its biology has already been investigated to an exceptional level. This, coupled with effortless handling and a notable low cost of cultivation and maintenance, allows seamless implementation of high-throughput drug screening approaches as well as in-depth genetic and biochemical studies of the molecular pathways targeted by specific drugs. In this review, we introduce C. elegans as a model organism with significant advantages toward the identification of molecular drug targets. In addition, we discuss the value of the worm in the development of drug screening and drug evaluation protocols. The unique features of C. elegans, which greatly facilitate drug studies, hold promise for both deciphering disease pathogenesis and formulating educated and effective therapeutic interventions.

Animals↗