Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “functional experiments”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 361 records · Page 20Linked to original sources
PubMed2001

Zebrafish admp is required to restrict the size of the organizer and to promote posterior and ventral development.

Bone morphogenetic proteins (Bmps) and their roles during early dorsoventral patterning of the vertebrate embryo are well understood. The role and regulation of a more distant member of this family, the anti-dorsalizing morphogenetic protein (Admp), however, are less clear. Here, we report the isolation and characterization of zebrafish admp. Unlike other bmps, admp is exclusively expressed on the dorsal side. Expression starts at blastula stages in the region of the organizer, giving rise to anterior neuroectoderm and axial mesoderm. During the course of gastrulation, both the neuroectodermal and the mesodermal admp transcripts vanish in an anterior-posterior wave. The maintenance of admp expression is positively influenced by Nodal signaling and by Bozozok (Boz), an organizer-promoting homeodomain protein acting as a repressor of early bmp2b expression. Despite the positive effect of boz on admp expression, Boz and Admp have rather opposite effects on zebrafish patterning, as revealed in gain- and loss-of-function experiments. Upon overexpression, admp has Bmp-like activities causing a smaller organizer and enhanced ventral specification, very similar to the phenotype caused by the loss of boz function in mutant embryos. Antisense-based admp knockdown, on the other side, leads to an enlarged organizer and impaired ventral and posterior development, as observed in embryos after boz overexpression. This finding indicates that admp is required for the development of embryonic structures normally suppressed by organizer activities. The seeming discrepancy between the regulative and functional relationship of boz and admp is discussed, and models are proposed according to which Admp might be part of a negative feedback loop to pattern and confine the organizer region.

Animals↗
PubMed2006

Defects in eye development in transgenic mice overexpressing the heparan sulfate proteoglycan agrin.

The importance of heparan sulfate proteoglycans (HSPGs) in neurodevelopment is becoming increasingly clear. However, studies on HSPGs are hampered by pleiotropic effects when synthesis or modification of heparan sulfate itself is targeted, and by redundancy when the core proteins are altered. Gain-of-function experiments can sometimes circumvent these issues. Here we establish that transgenic mice overexpressing the HSPG agrin have severe ocular dysgenesis. The defects occur through a gain-of-function mechanism and penetrance is dependent on agrin dosage. The agrin-induced developmental defects are highly variable, and include anophthalmia, persistence of vitreous vessels, and fusion of anterior chamber structures. A frequently observed defect is an optic stalk coloboma leading to the misdifferentiation of the optic stalk as retina, which becomes continuous with the forebrain. The defects in optic-stalk differentiation correlate with reduced sonic hedgehog immunoreactivity and overexpansion of the PAX6 domain from the retina into the optic stalk. The ocular phenotypes associated with agrin overexpression are dependent on genetic background, occurring with high penetrance in inbred C57BL/6J mice. Distinct loci sensitizing C57BL/6J mice to agrin-induced dysgenesis were identified. These results indicate that agrin overexpression will provide a tool to explore the molecular interactions of the extracellular matrix and cell surface in eye development, and provide a means for identifying modifier loci that sensitize mice to developmental eye defects.

Agrin↗
PubMed2003

Control of HIV-1 env RNA splicing and transport: investigating the role of hnRNP A1 in exon splicing silencer (ESS3a) function.

The control of HIV-1 viral RNA splicing and transport plays an important role in the successful replication of the virus. Previous studies have identified both an exon splicing enhancer (ESE) and a bipartite exon splicing silencer (ESS3a and ESS3b) within the terminal exon of HIV-1 that are involved in modulating both splicing and Rev-mediated export of viral RNA. To define the mechanism of ESS3a function, experiments were carried out to better define the cis and trans components required for ESS3a activity. Mutations throughout the 30-nt element resulted in partial loss of ESS function. Combining mutations was found to have an additive effect, suggesting the presence of multiple binding sites. Analysis of interacting factors identified hnRNP A1 as one component of the complex that modulates ESS3a activity. However, subsequent binding analyses determined that hnRNP A1 interacts with only one portion of ESS3a, suggesting the involvement of another host factor. Parallel analysis of the effect of the mutations on Rev-mediated export determined that there is not a direct correlation between the effect of the mutations on splicing and RNA transport. Consistent with this hypothesis, replacement of ESS3a with consensus hnRNP A1 binding sites was found to be insufficient to block Rev-mediated RNA export.

Base Sequence↗
PubMed2005

Inhibited cell growth and protein functional changes from an editing-defective tRNA synthetase.

The genetic code is established in aminoacylation reactions catalyzed by aminoacyl-tRNA synthetases. Many aminoacyl-tRNA synthetases require an additional domain for editing, to correct errors made by the catalytic domain. A nonfunctional editing domain results in an ambiguous genetic code, where a single codon is not translated as a specific amino acid but rather as a statistical distribution of amino acids. Here, wide-ranging consequences of genetic code ambiguity in Escherichia coli were investigated with an editing-defective isoleucyl-tRNA synthetase. Ambiguity retarded cell growth at most temperatures in rich and minimal media. These growth rate differences were seen regardless of the carbon source. Inclusion of an amino acid analogue that is misactivated (and not cleared) diminished growth rate by up to 100-fold relative to an isogenic strain with normal editing function. Experiments with target-specific antibiotics for ribosomes, DNA replication, and cell wall biosynthesis, in conjunction with measurements of mutation frequencies, were consistent with global changes in protein function caused by errors of translation and not editing-induced mutational errors. Thus, a single defective editing domain caused translationally generated global effects on protein functions that, in turn, provide powerful selective pressures for maintenance of editing by aminoacyl-tRNA synthetases.

Amino Acyl-tRNA Synthetases↗
PubMed2005

The apoptotic response to pneumolysin is Toll-like receptor 4 dependent and protects against pneumococcal disease.

Pneumolysin, the cholesterol-dependent cytolysin of Streptococcus pneumoniae, induces inflammatory and apoptotic events in mammalian cells. Toll-like receptor 4 (TLR4) confers resistance to pneumococcal infection via its interaction with pneumolysin, but the underlying mechanisms remain to be identified. In the present study, we found that pneumolysin-induced apoptosis is also mediated by TLR4 and confers protection against invasive disease. The interaction between TLR4 and pneumolysin is direct and specific; ligand-binding studies demonstrated that pneumolysin binds to TLR4 but not to TLR2. Involvement of TLR4 in pneumolysin-induced apoptosis was demonstrated in several complementary experiments. First, macrophages from wild-type mice were significantly more prone to pneumolysin-induced apoptosis than cells from TLR4-defective mice. In gain-of-function experiments, we found that epithelial cells expressing TLR4 and stimulated with pneumolysin were more likely to undergo apoptosis than cells expressing TLR2. A specific TLR4 antagonist, B1287, reduced pneumolysin-mediated apoptosis in wild-type cells. This apoptotic response was also partially caspase dependent as preincubation of cells with the pan-caspase inhibitor zVAD-fmk reduced pneumolysin-induced apoptosis. Finally, in a mouse model of pneumococcal infection, pneumolysin-producing pneumococci elicited significantly more upper respiratory tract cell apoptosis in wild-type mice than in TLR4-defective mice, and blocking apoptosis by administration of zVAD-fmk to wild-type mice resulted in a significant increase in mortality following nasopharyngeal pneumococcal exposure. Overall, our results strongly suggest that protection against pneumococcal disease is dependent on the TLR4-mediated enhancement of pneumolysin-induced apoptosis.

Amino Acid Chloromethyl Ketones↗
PubMed2002

Autocrine-paracrine VEGF loops potentiate the maturation of megakaryocytic precursors through Flt1 receptor.

The expression/function of vascular endothelial growth factor (VEGF) receptors (VEGFR1/Flt1 and VEGFR2/KDR/Flk1) in hematopoiesis is under scrutiny. We have investigated the expression of Flt1 and kinase domain receptor (KDR) on hematopoietic precursors, as evaluated in liquid culture of CD34(+) hematopoietic progenitor cells (HPCs) induced to unilineage differentiation/maturation through the erythroid (E), megakaryocytic (Mk), granulocytic (G), or monocytic (Mo) lineage. KDR, expressed on 0.5% to 1.5% CD34(+) cells, is rapidly downmodulated on induction of differentiation. Similarly, Flt1 is present at very low levels in HPCs and is downmodulated in E and G lineages; however, Flt1 is induced in the precursors of both Mo and Mk series; ie, its level progressively increases during Mo maturation, and it peaks at the initial-intermediate culture stages in the Mk lineage. Functional experiments indicate that Mk and E, but not G and Mo, precursors release significant amounts of VEGF in the culture medium, particularly at low O(2) levels. The functional role of VEGF release on Mk maturation is indicated by 2 series of observations. (1) Molecules preventing the VEGF-Flt1 interaction on the precursor membrane (eg, soluble Flt1 receptors) significantly inhibit Mk polyploidization. (2) Addition of exogenous VEGF or placenta growth factor (PlGF) markedly potentiates Mk maturation. Conversely, VEGF does not modify Mo differentiation/maturation. Altogether, our results suggest that in the hematopoietic microenvironment an autocrine VEGF loop contributes to optimal Mk maturation through Flt1. A paracrine loop involving VEGF release by E precursors may also operate. Similarly, recent studies indicate that an autocrine loop involving VEGF and Flt1/Flk1 receptors mediates hematopoietic stem cell survival and differentiation.

Antibodies, Monoclonal↗
PubMed1998

Control of lymphocyte function through CD27-CD70 interactions.

In contrast to the expression of other TNFR/TNF family members, expression of CD27 and its ligand CD70 is predominantly confined to lymphocytes. High expression levels of CD27 appear to be dependent on proper ligation of antigen receptors, whereas for the induction of CD70 expression additional (co-stimulatory and/or pro-inflammatory) signals are required. Next to membrane-bound CD27 also a soluble form of CD27 is produced in the course of the immune response. Soluble CD27 (sCD27) is found in body fluids and can be used to monitor local and systemic immune activation. In addition, elevated serum concentrations of sCD27 are found in patients with B cell malignancies and levels of sCD27 strongly correlate with tumor load. Based on functional experiments and in vitro expression regulation data, we propose that interactions between activated lymphocytes in vivo can result in CD27-CD70 interactions that may regulate the size and function of antigen-primed lymphocyte populations.

Animals↗
PubMed1987

Knowledge about hand shaping and knowledge about objects.

Our two experiments investigated associations between cognitive representations of objects and hand-shape categories. Hand configurations were partitioned according to prehensility and the size of the contacting surface, resulting in the classes: pinch, poke, palm, and clench. Experiment 1 elicited object names in response to configuration-name cues, provided ratings of the relevance of each configuration to a set of objects, and probed for the functions determining such relevance. Cueing with a configuration class elicited an associated object category with substantial intersubject agreement, and vice versa. Both the object categories and the functions associated with the four hand-configuration classes differed substantially, although the same object could be associated to some extent with multiple configurations, given variations in function. Experiment 2 elicited the names of hand-configuration classes in response to unfamiliar forms, which varied systematically in depth and the size of the projecting picture-plane surface. The modal response, response time, and degree of intersubject agreement were directly related to these variables. These structural variables, however, did not adequately predict shaping responses to real objects, as ascertained from Experiment 1. The results have implications for cognitive representation of motor categories and hand shaping in response to objects.

Journal Article↗
PubMed2006

The tailless nuclear receptor acts as a dedicated repressor in the early Drosophila embryo.

Tailless is an orphan nuclear receptor that controls terminal body patterning in Drosophila. Genetic analyses have revealed both positive and negative regulatory interactions of Tailless with various target genes, leading to the idea that, like many other nuclear receptors, Tailless mediates both activation and repression of transcription. In this paper, we have examined the consequences of converting Tailless into an obligate repressor and compared the activities of the resulting protein with those of wild-type Tailless. We find that this repressor form of Tailless behaves like the intact protein in gain- and loss-of-function experiments, being sufficient to support normal embryonic development and establish accurate patterns of gene expression even for positive Tailless targets such as hunchback and brachyenteron. This suggests that Tailless functions exclusively as a transcriptional repressor in the embryo and that the observed positive interactions of Tailless with specific targets are secondary effects involving repression of repressors. We provide evidence that knirps is one such repressor gene acting between Tailless and its indirect positive targets. Finally, our results indicate that Tailless exerts an active mechanism of repression via its ligand-binding domain and that this activity is largely independent of the activation function 2 (AF2) motif characteristic of most nuclear receptors.

Amino Acid Sequence↗
PubMedDate not supplied

Interleukin-2-induced proliferation of CD4-CD8- human thymocytes. In vitro expression of CD3 and CD8 antigens and cytolytic activity.

Human thymocytes lacking both CD4 and CD8 differentiation antigens were prepared by treating total thymocyte suspensions with a mixture of anti-CD4 and anti-CD8 monoclonal antibodies and complement. The resulting populations contained less than 2% CD4+, CD8+ or WT31+ cells and variable percentages (less than 20%) of CD3+ cells. These cell populations were cultured in recombinant IL-2 in the presence of peripheral blood mononuclear cells as feeder cells. Cells underwent extensive proliferation accompanied by a progressive increase of CD3+ and CD8+ cells. On the other hand, appearance of neither WT31+, alpha/beta-positive T cell receptor (TCR), nor CD4+ cells could be observed in several independent experiments. Functional analyses revealed the appearance and the progressive increase of cytolytic activity against the natural killer (NK)-sensitive K562 cells as well as the NK-resistant fresh melanoma cells. Experiments of T cell cloning indicated that both the expression of CD8 and CD3 antigens and the appearance of cytolytic activity were consequent to cell maturation occurring at the level of CD4-CD8- non-cytolytic cell precursors. In these experiments, more than 30% of cells underwent clonal expansion and all the clonal progenies obtained displayed cytolytic activity and expressed the CD3+WT31- surface phenotype. The expression of CD8 was variable, whereas no CD4+ clones could be obtained. Cells expressing such surface phenotype are known to belong to the TCR gamma-positive T lymphocyte subset lacking the typical alpha/beta TCR and thus appear to be the only T cell type capable of in vitro proliferation and maturation under easily reproducible culture conditions.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1994

CD27: marker and mediator of T-cell activation?

CD27 is a lymphocyte-specific member of the tumour necrosis factor receptor (TNF-R) family, expression of which is tightly regulated during T-cell ontogeny. Recently, the ligand for CD27 was identified and was shown to be identical to CD70, a novel member of the TNF family. Functional experiments show that the interaction between CD27 and its ligand generates a co-stimulatory signal for T-cell activation. Here, Rogier Hintzen and colleagues integrate the phenotypic and functional data available on CD27 and its ligand, and propose a role for CD27 in the amplification of T-cell responses.

Animals↗
PubMed1990

Functional impairment in Mexican Americans and non-Hispanic whites with diabetes.

There are virtually no data available describing the functional status of diabetic individuals. We therefore measured functional status using the Sickness Impact Profile (SIP) in 393 diabetic subjects and 486 nondiabetic control subjects identified from the San Antonio Heart Study, a population-based study of diabetes among Mexican Americans and non-Hispanic whites. The SIP is a validated instrument that assesses the presence of health-related behavior changes and activity restrictions in 12 different categories. Functional impairment, defined as a SIP score of 2.0% or greater, was present among 36.6% of diabetic subjects. Following adjustments for age, Mexican Americans were 1.63 times more likely to experience functional impairment that non-Hispanic whites, although this difference was not statistically significant (95% confidence interval: 0.92-2.89). The categories in which subjects experienced impairment varied widely, but the category with the highest prevalence of impairment was "eating" (greater than 40%). The prevalence of functional impairment was 45.9% among diabetic subjects with vascular complications, 31.8% among diabetic subjects without complications, and 16.7% among nondiabetic control subjects. Among all diabetic subjects impairment increased with age, duration of diabetes, fasting glucose, and BMI, and with insulin use and the presence of hypertension. In a multiple logistic regression model these factors (with the exception of insulin use) remained associated with the presence of functional impairment even after adjustment for the presence of vascular complications. If the factors responsible for this excess of functional impairment can be identified, an intervention might be designed which can lead to improvement in the quality of life for diabetic individuals.

Adult↗
PubMed1995

Pharmacological implications of inward rectifier K+ channels regulation by cytoplasmic polyamines.

The powerful combination of molecular biology and electrophysiology has allowed extraordinary progress in the field of ion channel structure-function. In fact, only 10 years have passed since the first amino acid sequence of a voltage-dependent ion channel, the Na+ channel, was deduced [1], and already the structural domains involved in ion channel permeation, block and gating have been identified in many channel types. Despite this progress, in most cases the correlation between specific domains and ion channel function is still speculative at present, due to the absence of direct structural information [2]. In this review we will describe recent progress in the field of structure-function of one class of K+ channels, the inward rectifiers (IRKs). In particular, we will review the sequences of structure-function experiments which have led to the discovery of a novel regulation of IRKs by cytoplasmic organic polycationic substances like polyamines (PAs). This discovery represents a paradigm for how structure-function information has preceded and made possible the identification of physiological mechanisms of ion channel regulation. Owing to the important role played by IRKs in the regulation of resting membrane potential, a major determinant of cellular transport and volume [3], and to the established link between PAs and cell growth and division, the direct regulation of IRKs by PAs assumes a critical importance for the pharmacological control of cell growth and neoplastic transformation.

Animals↗
PubMed2002

Stability of time trade-off utilities for health states associated with the treatment of prostate cancer.

BACKGROUND: Patients diagnosed with localized prostate cancer face several treatment options. Patient preferences for treatment side effects often dominate the decision making process. We proposed to learn more about the nature of patient preferences, or utilities, for these side effects. METHODS: Two hundred and fifteen men were consecutively enrolled from three institutions for assessment after prostate needle biopsy. Baseline and 6 month follow-up assessments were done using the University of California, Los Angeles Prostate Cancer Index (UCLA PCI), and a laptop utility assessment application, U-Titer II. Patient utility was assessed for current pelvic functions as well as hypothetical pelvic dysfunctions. We calculated stability of utility scores and correlations between utility scores and UCLA PCI scores. RESULTS: Utility scores for current pelvic functions exhibited a significant 'ceiling effect.' Utility scores for current pelvic functions and hypothetical impaired states were stable after 6 months in patients with negative biopsies. In patients who underwent treatment, utility for current sexual function decreased by 0.13 units (p < 0.00) and utility for current urinary function decreased by 0.09 units (p < 0.01). Utility for hypothetical stress urinary incontinence rose in men with a >25-point drop in UCLA PCI score. CONCLUSION: Utilities for some 'current' pelvic functions decreased in tandem with UCLA PCI scores in men who experienced >25-point changes in these scores. Utilities for some 'hypothetical' pelvic dysfunctions rose as men began to actually experience functional changes in those areas.

Adult↗
PubMed2002

Apical heterodimeric cystine and cationic amino acid transporter expressed in MDCK cells.

The luminal uptake of L-cystine and cationic amino acids by (re)absorptive epithelia, as found in the small intestine and the proximal kidney tubule, is mediated by the transport system b(0,+), which is defective in cystinuria. Expression studies in Xenopus laevis oocytes and other nonepithelial cells as well as genetic studies on cystinuria patients have demonstrated that two gene products, the glycoprotein rBAT and the multitransmembrane-domain protein b(0,+)AT, are required for system b(0,+) function. To study the biosynthesis, surface expression, polarity, and function of this heterodimer in an epithelial context, we established stable Madin-Darby canine kidney (MDCK) cell lines expressing rBAT and/or b(0,+)AT. Confocal immunofluorescence microscopy shows that both subunits depend on each other for apical surface expression. Immunoprecipitation of biosynthetically labeled proteins indicates that b(0,+)AT is stable in the absence of rBAT, whereas rBAT is rapidly degraded in the absence of b(0,+)AT. When both are coexpressed, they associate covalently and rBAT becomes fully glycosylated and more stable. Functional experiments show that the expressed transport is of the high-affinity b(0,+)-type and is restricted to the apical side of the epithelia. In conclusion, coexpression experiments in MDCK cell epithelia strongly suggest that the intracellular association of rBAT and b(0,+)AT is required for the surface expression of either subunit, which together form a functional heterocomplex at the apical cell membrane.

ATP-Binding Cassette Transporters↗
PubMed2006

Optic cup and facial patterning defects in ocular ectoderm beta-catenin gain-of-function mice.

BACKGROUND: The canonical Wnt signaling pathway has a number of critical functions during embryonic development and, when activated aberrantly, in the genesis of cancer. Current evidence suggests that during eye development, regulation of Wnt signaling is critical for patterning the surface ectoderm that will contribute to multiple components of the eye. Wnt signaling loss-of-function experiments show that a region of periocular ectoderm will form ectopic lentoid bodies unless the Wnt pathway modifies its fate towards other structures. Consistent with this, Wnt signaling gain of function in the ocular region ectoderm results in a suppression of lens fate. RESULTS: Here we demonstrate that ectoderm-specific Wnt signaling gain-of-function embryos exhibit additional defects besides those noted in the lens. There are profound facial defects including a foreshortened snout, malformation of the nasal region, and clefting of the epidermis along the ocular-nasal axis. Furthermore, despite the restriction of Wnt pathway gain-of-function to the surface ectoderm, the optic cup is inappropriately patterned and ultimately forms a highly convoluted, disorganized array of epithelium with the characteristics of retina and retinal pigmented epithelium. CONCLUSION: We suggest that activation of the Wnt pathway in surface ectoderm may disrupt the normal exchange of signals between the presumptive lens and retina that coordinate development of a functional eye.

Animals↗
PubMed2005

The roles of FGF signaling in germ cell migration in the mouse.

Fibroblast growth factor (FGF) signaling is thought to play a role in germ cell behavior. FGF2 has been reported to be a mitogen for primordial germ cells in vitro, whilst combinations of FGF2, steel factor and LIF cause cultured germ cells to transform into permanent lines of pluripotent cells resembling ES cells. However, the actual function of FGF signaling on the migrating germ cells in vivo is unknown. We show, by RT-PCR analysis of cDNA from purified E10.5 germ cells, that germ cells express two FGF receptors: Fgfr1-IIIc and Fgfr2-IIIb. Second, we show that FGF-mediated activation of the MAP kinase pathway occurs in germ cells during their migration, and thus they are potentially direct targets of FGF signaling. Third, we use cultured embryo slices in simple gain-of-function experiments, using FGF ligands, to show that FGF2, a ligand for FGFR1-IIIc, affects motility, whereas FGF7, a ligand for FGFR2-IIIb, affects germ cell numbers. Loss of function, using a specific inhibitor of FGF signaling, causes increased apoptosis and inhibition of cell shape change in the migrating germ cells. Lastly, we confirm in vivo the effects seen in slice cultures in vitro, by examining germ cell positions and numbers in embryos carrying a loss-of-function allele of FGFR2-IIIb. In FGFR2-IIIb(-/-) embryos, germ cell migration is unaffected, but the numbers of germ cells are significantly reduced. These data show that a major role of FGF signaling through FGFR2-IIIb is to control germ cell numbers. The data do not discriminate between direct and indirect effects of FGF signaling on germ cells, and both may be involved.

Alternative Splicing↗
PubMed2005

Quantitative trait nucleotide analysis using Bayesian model selection.

Although much attention has been given to statistical genetic methods for the initial localization and fine mapping of quantitative trait loci (QTLs), little methodological work has been done to date on the problem of statistically identifying the most likely functional polymorphisms using sequence data. In this paper we provide a general statistical genetic framework, called Bayesian quantitative trait nucleotide (BQTN) analysis, for assessing the likely functional status of genetic variants. The approach requires the initial enumeration of all genetic variants in a set of resequenced individuals. These polymorphisms are then typed in a large number of individuals (potentially in families), and marker variation is related to quantitative phenotypic variation using Bayesian model selection and averaging. For each sequence variant a posterior probability of effect is obtained and can be used to prioritize additional molecular functional experiments. An example of this quantitative nucleotide analysis is provided using the GAW12 simulated data. The results show that the BQTN method may be useful for choosing the most likely functional variants within a gene (or set of genes). We also include instructions on how to use our computer program, SOLAR, for association analysis and BQTN analysis.

Bayes Theorem↗