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PubMed2002

A novel mechanism of gene regulation and tumor suppression by the transcription factor FKHR.

The mammalian DAF-16-like transcription factors, FKHR, FKHRL1, and AFX, function as key regulators of insulin signaling, cell cycle progression, and apoptosis downstream of phosphoinositide 3-kinase. Gene activation through binding to insulin response sequences (IRS) has been thought to be essential for mediating these functions. However, using transcriptional profiling, chromatin immunoprecipitation, and functional experiments, we demonstrate that rather than activation of IRS regulated genes (Class I transcripts), transcriptional repression of D-type cyclins (in Class III) is required for FKHR mediated inhibition of cell cycle progression and transformation. These data suggest that a novel mechanism of FKHR-mediated gene regulation is linked to its activity as a suppressor of tumor growth.

Adenoviridae↗

PubMed1995

2-[N'-(3-arylallylidene)hydrazino]adenosines showing A2a adenosine agonist properties and vasodilation activity.

A series of 2-[N'-(3-arylallylidene)hydrazino]adenosines were prepared and studied in binding and functional assays to assess their potency for the A2a compared with the A1 adenosine receptor. These analogs possess A2a receptor affinity in the low nanomolar range associated with weak interaction with the A1 receptor. Among the compounds, in rat tissues, 2-[N'-[3-(4-nitrophenyl)allylidene] hydrazinoadenosine (5g) had the most potent affinity for the A2a receptor, the K(i) value being 23 nM. The type and position of substituents on the phenyl ring show a moderate influence on biological activity, allowing the conclusion that the latter is mostly due to the allylidenehydrazino side chain. From functional experiments 2-[N'-(2-furylmethylidene)hydrazino]adenosine (4b), 2-[N'-(3-phenylallylidene)hydrazino]adenosine (5a) and 2-[N'-[3-(2-furyl)allylidene]hydrazino]adenosine (5b) appeared to be potent in inducing vasorelaxation (an A2a-mediated response) without appreciable effects on the heart rate (an A1-mediated action). While the lack of effects on heart rate is clearly explained by the poor affinity for A1 receptors, more difficult appears the interpretation of vasorelaxant properties displayed by some compounds. Affinity for A2a has a major role, but other types of interactions, yet to be identified, may play a part.

Adenosine↗

PubMed2006

Fancy bioisosteres: novel paracyclophane derivatives as super-affinity dopamine D3 receptor antagonists.

The exploration of the chemical diversity space depends on the discovery of novel bioisosteric elements. As a continuation of our project on bilayered arene surrogates, we herein report on [2.2]paracyclophane-derived dopamine D3 receptor antagonists of type 4 and 6. For the most promising test compound 6a, bearing a 2-methoxyphenyl substituent, a stereocontrolled preparation was performed when the planar chirality of enantiomers (R)-6a (FAUC 418) and (S)-6a caused a considerable differentiation of D3 binding, which is indicated by K(i) values of 0.19 and 3.0 nM, respectively. Functional experiments showed D3 antagonist properties for the paracyclophane derivatives of type 6. To elucidate putative bioactive low-energy conformations, DFT-based studies including the calculation of diagnostic magnetic shielding properties were performed. An 89% increase in volume for the [2.2]paracyclophane moiety compared to that of the monolayered benzofurane of lead compound 3b indicates higher plasticity of GPCR binding regions than usually expected.

Amides↗

PubMed2006

2-[(4-phenylpiperazin-1-yl)methyl]imidazo(di)azines as selective D4-ligands. Induction of penile erection by 2-[4-(2-methoxyphenyl)piperazin-1-ylmethyl]imidazo[1,2-a]pyridine (PIP3EA), a potent and selective D4 partial agonist.

A series of novel 2-[(4-phenylpiperazin-1-yl)methyl]imidazoazines and aza-analogues were prepared and screened at selected dopamine, serotonin, and adrenergic receptor subtypes. 2-Substituted imidazopyridines and pyridazines presented high affinities and selectivities for D4 dopamine receptors. Whereas functional experiments indicated neutral antagonists or weak partial agonist effects for most of the target compounds, the 2-methoxyphenyl substituted 2-piperazinylmethylimidazopyridine 3c (PIP3EA) displayed substantial agonist efficacy in mitogenesis experiments and GTPgammaS binding tests, resulting in EC50 values of 3.0 (46%) and 4.5 nM (57%), respectively. Our D4 agonist 3c induced penile erection in vivo when administered to rats. This effect was inhibited by L-745,870 a D4 selective antagonist, confirming the mechanistic pathway.

Animals↗

PubMed1996

Synthesis and pharmacological characterization of all sixteen stereoisomers of 2-(2'-carboxy-3'-phenylcyclopropyl)glycine. Focus on (2S,1'S,2'S,3'R)-2-(2'-carboxy-3'-phenylcyclopropyl)glycine, a novel and selective group II metabotropic glutamate receptors antagonist.

All 16 2-(2'-carboxy-3'-phenylcyclopropyl)glycine (PCCGs) stereoisomers 32-47 have been prepared from the corresponding racemic aldehydes 12-15 following an enantiodivergent synthetic protocol. Compounds 32-47 were evaluated by a number of binding and functional experiments as potential ligands for several classes of excitatory amino acid receptors, including metabotropic glutamate receptors (mGluR1a, mGluR2, mGluR4) and ionotropic glutamate receptors (NMDA, KA, AMPA) as well as sodium-dependent and calcium/ chloride-dependent glutamate transport systems. The stereolibrary of compounds 32-47 appears to be endowed with a peculiar pharmacological profile. PCCG-2 (33) and PCCG-3 (34) displaced labeled kainate at low micromolar concentration; PCCG-9 (40) and PCCG-11 (42) weakly interacted with the NMDA site; PCCG-5 (36), PCCG-10 (41), and PCCG-12 (43) showed to be potent inhibitors of Ca2+/Cl(-)-dependent glutamate transport system. Most interestingly, PCCG-4 (35) has been shown to be able to antagonize (IC50 = 8 microM) the effects of glutamate on forskolin-stimulated cAMP formation in BHK cells expressing mGluR2. Uneffective at mGluR1, 35 is a weak mGluR4 agonist (EC50 = 156 microM) and has no effect on either ionotropic receptors or glutamate transport systems, thus demonstrating to be a novel selective mGluR2 antagonist with a 6-fold increase in potency over previously reported antagonists.

Alternative Splicing↗

PubMed1999

Structure-activity relationships in 1,4-benzodioxan-related compounds. 6. Role of the dioxane unit on selectivity for alpha(1)-adrenoreceptor subtypes.

WB 4101-related benzodioxans 3-9 were synthesized, and their biological profiles at alpha(1)-adrenoreceptor subtypes and 5-HT(1A) serotoninergic receptors were assessed by binding assays in CHO and HeLa cells membranes expressing the human cloned receptors. Furthermore, receptor selectivity of selected benzodioxan derivatives was further determined in functional experiments in isolated rat vas deferens (alpha(1A)) and aorta (alpha(1D)) and guinea pig spleen (alpha(1B)), in additional receptor binding assays in rat cortex membranes containing alpha(2)-adrenoreceptors and 5-HT(2) serotoninergic receptors, and in rat striatum membranes containing D(2) dopaminergic receptors. An analysis of the results of receptor binding experiments for benzodioxan-modified derivatives 3-9 showed high affinity and selectivity toward the alpha(1a)-adrenoreceptor subtype for compounds 3-5 and 7 and a reversed selectivity profile for 9, which was a selective alpha(1d) antagonist. Furthermore, the majority of structural modifications performed on the prototype 1 (WB 4101) led to a marked decrease in the affinity for 5-HT(1A) serotoninergic receptors, which may have relevance in the design of selective alpha(1A)-adrenoreceptor antagonists. The exception to these findings was the chromene derivative 8, which exhibited a 5-HT(1A) partial agonist profile.

Adrenergic alpha-Agonists↗

PubMed2001

Protective effect of a new hypothalamic peptide against cobra venom and trauma-induced neuronal injury.

A study of separate and combined actions of cobra venom (CV) and a new hypothalamic proline-rich polypeptide (PRP) isolated from magnocellular cells (NPV and NSO) on intoxication- and trauma-induced neuronal injury (during 3-4 weeks after hemisection with and without PRP treatment) was carried out. The registration of background and evoked impulse activity flow, changes in spinal cord (SC) inter- and motoneurons, responding to flexor, extensor, and mixed nerve stimulation in both acute and chronic experimental neurodegeneration was performed. The facilitating effect of PRP on the abovementioned neurons was revealed. High doses of CV that evoked the neurodegenerative changes demonstrated an inhibitory effect. In this case PRP treatment both before and after intoxication restored electrical neuronal activity to baseline level and higher. These results are evidence of protective action of PRP. The low doses of CV induced a facilitating effect. The combination of CV and PRP displayed an additive facilitating effect; in a number of cases the repeated administration of CV led to decrease of significant PRP effect till baseline level (for example, the inhibition after primary response prior to secondary late discharge). Greater liability of the secondary early and late long-time discharges of poststimulus responses, differently expressed in various neuron types of SC to chemical influences is of interest. PRP-induced inhibition of the paroxysmal activity related with CV action is also very interesting. Morpho-functional experiments with SC injury demonstrated the abolition of difference in the background and evoked SC neuronal activity below the section and on intact symmetric side after daily PRP administration for 3 weeks. PRP hindered the scar formation and activated neuroglia proliferation; it promoted white matter element growth, hampered the degeneration of cellular elements, and protected against tissue stress. Our results favor the combined use of PRP and CV in clinical practice for the treatment of neurodegeneration of toxic and traumatic origin, as well as specific neurodegenerative diseases such as Alzheimer's.

Animals↗

PubMed1999

Identification of cis-regulatory elements required for endosperm expression of the rice storage protein glutelin gene GluB-1.

Rice storage protein glutelin genes are coordinately regulated during seed development. A previous 5' deletion analysis using transgenic tobacco revealed that the minimum 5' region necessary for endosperm specificity was within -245 bp of the transcription start site, and included the AACA and GCN4 motifs that are highly conserved in the 5'-flanking regions of all glutelin genes. In this paper, the sequence elements essential for endosperm-specific expression are characterized in stable transgenic tobacco plants by both loss-of-function and gain-of-function experiments using this minimum promoter. Base substitution analysis shows that the proximal AACA motif between -73 and -61, and the GCN4 motif between -165 and -158 act as critical elements. An ACGT motif between -81 and -75, and Skn-I-like elements between -173 and -169 also play important roles in controlling the seed-specific expression. When the distal region between -245 and -145 containing the AACA and the GCN4 motifs or the proximal region between -113 and -46 containing the ACGT and AACA motifs is fused to a truncated promoter (-90 to +9) of the CaMV 35S gene fused to the beta-glucuronidase (GUS) reporter gene, high levels of seed-specific expression are observed in these fusions, thereby indicating that either pair of motifs is sufficient to confer seed expression in these fusions. However, when substituted for by the CaMV 35S core promoter (-46 to +1), seed expression is abolished, suggesting that the sequence between -90 and -46 of the CaMV 35S promoter containing G-box-like motif (as-1 element) is required for such specific expression in addition to AACA and GCN4 motifs. Therefore, we conclude that at least three cis-regulatory elements, the AACA motif, GCN4 motif and ACGT motif, are necessary to mediate endosperm expression of the GluB-1 glutelin gene.

Base Sequence↗

PubMed1992

Ontogeny of chick responses to maternal food calls in the Burmese red junglefowl (Gallus gallus spadiceus).

Burmese red junglefowl (Gallus gallus spadiceus) chicks were reinforced at 5 days old with a mealworm at the end of a runaway in the presence of a maternal food call. Two food calls were used, one made to a highly preferred food item (a mealworm) and one made to a less preferred food item (chick crumbs). Chicks initially approached the food call made to the mealworm faster regardless of reinforcement contingencies, but by the 3rd day of training chicks responded faster to the call that was reinforced. These results indicate that functional experience can modify preexisting response tendencies to food-calling stimuli. We discuss the implications of these results for the interpretation of published accounts that chickens use food calls deceptively.

Aging↗

PubMed2005

Casein kinase 1 gamma couples Wnt receptor activation to cytoplasmic signal transduction.

Signalling by Wnt proteins (Wingless in Drosophila) has diverse roles during embryonic development and in adults, and is implicated in human diseases, including cancer. LDL-receptor-related proteins 5 and 6 (LRP5 and LRP6; Arrow in Drosophila) are key receptors required for transmission of Wnt/beta-catenin signalling in metazoa. Although the role of these receptors in Wnt signalling is well established, their coupling with the cytoplasmic signalling apparatus remains poorly defined. Using a protein modification screen for regulators of LRP6, we describe the identification of Xenopus Casein kinase 1 gamma (CK1gamma), a membrane-bound member of the CK1 family. Gain-of-function and loss-of-function experiments show that CK1gamma is both necessary and sufficient to transduce LRP6 signalling in vertebrates and Drosophila cells. In Xenopus embryos, CK1gamma is required during anterio-posterior patterning to promote posteriorizing Wnt/beta-catenin signalling. CK1gamma is associated with LRP6, which has multiple, modular CK1 phosphorylation sites. Wnt treatment induces the rapid CK1gamma-mediated phosphorylation of these sites within LRP6, which, in turn, promotes the recruitment of the scaffold protein Axin. Our results reveal an evolutionarily conserved mechanism that couples Wnt receptor activation to the cytoplasmic signal transduction apparatus.

Amino Acid Motifs↗

PubMed2003

The transmembrane protein XFLRT3 forms a complex with FGF receptors and promotes FGF signalling.

Fibroblast growth factors (FGFs) signal through high-affinity tyrosine kinase receptors to regulate a diverse range of cellular processes, including cell growth, differentiation and migration, as well as cell death. Here we identify XFLRT3, a member of a leucine-rich-repeat transmembrane protein family, as a novel modulator of FGF signalling. XFLRT3 is co-expressed with FGFs, and its expression is both induced after activation and downregulated after inhibition of FGF signalling. In gain- and loss-of function experiments, FLRT3 and FLRT2 phenocopy FGF signalling in Xenopus laevis. XFLRT3 signalling results in phosphorylation of ERK and is blocked by MAPK phosphatase 1, but not by expression of a dominant-negative phosphatidyl inositol 3-OH kinase (PI(3)K) mutant. XFLRT3 interacts with FGF receptors (FGFRs) in co-immunoprecipitation experiments in vitro and in bioluminescence resonance energy transfer assays in vivo. The results indicate that XFLRT3 is a transmembrane modulator of FGF-MAP kinase signalling in vertebrates.

Animals↗

PubMed2005

Dishevelled mediates ephrinB1 signalling in the eye field through the planar cell polarity pathway.

An important step in retinal development is the positioning of progenitors within the eye field where they receive the local environmental signals that will direct their ultimate fate. Recent evidence indicates that ephrinB1 functions in retinal progenitor movement, but the signalling pathway is unclear. We present evidence that ephrinB1 signals through its intracellular domain to control retinal progenitor movement into the eye field by interacting with Xenopus Dishevelled (Xdsh), and by using the planar cell polarity (PCP) pathway. Blocking Xdsh translation prevents retinal progeny from entering the eye field, similarly to the morpholino-mediated loss of ephrinB1 (ref. 2). Overexpression of Xdsh can rescue the phenotype induced by loss of ephrinB1, and this rescue (as well as a physical association between Xdsh and ephrinB1) is completely dependent on the DEP (Dishevelled, Egl-10, Pleckstrin) domain of Xdsh. Similar gain- and loss-of-function experiments suggest that Xdsh associates with ephrinB1 and mediates ephrinB1 signalling through downstream members of the PCP pathway during eye field formation.

Adaptor Proteins, Signal Transducing↗

PubMed2006

ADAM10 is a principal 'sheddase' of the low-affinity immunoglobulin E receptor CD23.

CD23, the low-affinity immunoglobulin E receptor, is an important modulator of the allergic response and of diseases such as rheumatoid arthritis. The proteolytic release of CD23 from cells is considered a key event in the allergic response. Here we used loss-of-function and gain-of-function experiments with cells lacking or overexpressing candidate CD23-releasing enzymes (ADAM8, ADAM9, ADAM10, ADAM12, ADAM15, ADAM17, ADAM19 and ADAM33), ADAM-knockout mice and a selective inhibitor to identify ADAM10 as the main CD23-releasing enzyme in vivo. Our findings provide a likely target for the treatment of allergic reactions and set the stage for further studies of the involvement of ADAM10 in CD23-dependent pathologies.

ADAM Proteins↗

PubMed2004

Neural fate of ignored stimuli: dissociable effects of perceptual and working memory load.

Observers commonly experience functional blindness to unattended visual events, and this problem has fuelled an intense debate concerning the fate of unattended visual information in neural processing. Here we used functional magnetic resonance imaging (fMRI) to demonstrate that the type of task that a human subject engages in determines the way in which ignored visual background stimuli are processed in parahippocampal cortex. Increasing the perceptual difficulty of a foveal target task attenuated processing of task-irrelevant background scenes, whereas increasing the number of objects held in working memory did not have this effect. These dissociable effects of perceptual and working memory load clarify how task-irrelevant, unattended stimuli are processed in category-selective areas in human ventral visual cortex.

Adult↗

PubMed2006

Dynamic roles for G4 DNA in the biology of eukaryotic cells.

Recent advances have made a persuasive case for the existence of G4 DNA in living cells, but what--if any--are its functions? Experiments have established how G4 DNA may contribute to the biology of eukaryotic cells, and genomic analysis has identified new ways in which the potential to form G4 DNA may influence gene regulation and genomic stability. This Perspective highlights those advances and identifies some key open questions.

Animals↗

PubMed2026

Methyltransferase METTL1 regulates MSC mRNA stability via m7G modification in acute pancreatitis.

Acute pancreatitis (AP) is a serious inflammatory disease with significant morbidity, yet its underlying molecular mechanisms remain incompletely understood. This study reveals a novel epitranscriptomic pathway in AP pathogenesis centered on METTL1-mediated N7-methylguanosine (m7G) RNA modification. We found that METTL1 expression and global m7G levels were significantly elevated in serum from AP patients, pancreatic tissues of sodium taurocholate-induced AP mice, and in vitro models of LPS-polarized macrophages and STC-injured pancreatic acinar cells. Through integrated multi-omics analysis combining m7G methylome mapping and transcriptome profiling, we identified Musculin (MSC) as a key target whose mRNA stability is enhanced by METTL1-mediated m7G modification. Functional experiments demonstrated that MSC upregulation activates TNF signaling through phosphorylation of NF-κB, JNK, and MAPK proteins, thereby promoting macrophage M1 polarization and pancreatic acinar cell injury. The pathological significance of this pathway was confirmed in vivo, where pancreas-targeted knockdown of Mettl1 significantly attenuated AP severity. Furthermore, mechanistic studies using a catalytic-dead METTL1 mutant established that both the methyltransferase activity of METTL1 and subsequent TNF signaling activation are essential for driving inflammatory responses. Our findings delineate a previously unrecognized METTL1-m7G-MSC-TNF signaling axis that promotes AP progression, highlighting the therapeutic potential of targeting METTL1-mediated epitranscriptomic modification in inflammatory diseases.

Animals↗

PubMed2025

Systematic mining and quantification reveal the dominant contribution of non-HLA variations to acute graft-versus-host disease.

Human leukocyte antigen (HLA) disparity between donors and recipients is a key determinant triggering intense alloreactivity, leading to a lethal complication, namely, acute graft-versus-host disease (aGVHD), after allogeneic transplantation. Moreover, aGVHD remains a cause of mortality after HLA-matched allogeneic transplantation. Protocols for HLA-haploidentical hematopoietic cell transplantation (haploHCT) have been established successfully and widely applied, further highlighting the urgency of performing panoramic screening of non-HLA variations correlated with aGVHD. On the basis of our time-consecutive large haploHCT cohort (with a homogenous discovery set and an extended confirmatory set), we first delineated the genetic landscape of 1366 samples to quantitatively model aGVHD risk by assessing the contributions of HLA and non-HLA genes together with clinical factors. In addition to identifying multiple loss-of-function (LoF) risk variations in non-HLA coding genes, our data-driven study revealed that non-HLA genetic variations, independent of HLA disparity, contributed the most to the occurrence of aGVHD. This unexpected major effect was verified in an independent cohort that received HLA-identical sibling HCT. Subsequent functional experiments further revealed the roles of a representative non-HLA LoF gene and LoF gene pair in regulating the alloreactivity of primary human T cells. Our findings highlight the importance of non-HLA genetic risk in the new era of transplantation and propose a new direction to explore the immunogenetic mechanism of alloreactivity and to optimize donor selection strategies for allogeneic transplantation.

Humans↗

PubMed2017

Functional mapping and annotation of genetic associations with FUMA.

A main challenge in genome-wide association studies (GWAS) is to pinpoint possible causal variants. Results from GWAS typically do not directly translate into causal variants because the majority of hits are in non-coding or intergenic regions, and the presence of linkage disequilibrium leads to effects being statistically spread out across multiple variants. Post-GWAS annotation facilitates the selection of most likely causal variant(s). Multiple resources are available for post-GWAS annotation, yet these can be time consuming and do not provide integrated visual aids for data interpretation. We, therefore, develop FUMA: an integrative web-based platform using information from multiple biological resources to facilitate functional annotation of GWAS results, gene prioritization and interactive visualization. FUMA accommodates positional, expression quantitative trait loci (eQTL) and chromatin interaction mappings, and provides gene-based, pathway and tissue enrichment results. FUMA results directly aid in generating hypotheses that are testable in functional experiments aimed at proving causal relations.

Chromatin↗