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Proximity ligation assay

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https://www.readbyqxmd.com/read/27906063/characterization-of-a-multiprotein-complex-involved-in-excitation-transcription-coupling-of-skeletal-muscle
#1
Manuel Arias-Calderón, Gonzalo Almarza, Alexis Díaz-Vegas, Ariel Contreras-Ferrat, Denisse Valladares, Mariana Casas, Héctor Toledo, Enrique Jaimovich, Sonja Buvinic
BACKGROUND: Electrical activity regulates the expression of skeletal muscle genes by a process known as "excitation-transcription" (E-T) coupling. We have demonstrated that release of adenosine 5'-triphosphate (ATP) during depolarization activates membrane P2X/P2Y receptors, being the fundamental mediators between electrical stimulation, slow intracellular calcium transients, and gene expression. We propose that this signaling pathway would require the proper coordination between the voltage sensor (dihydropyridine receptor, DHPR), pannexin 1 channels (Panx1, ATP release conduit), nucleotide receptors, and other signaling molecules...
April 11, 2016: Skeletal Muscle
https://www.readbyqxmd.com/read/27903903/novel-roles-of-hp1a-and-mcm10-in-dna-replication-genome-maintenance-and-photoreceptor-cell-differentiation
#2
Nicole Vo, Dang Ngoc Anh Suong, Natsuki Yoshino, Hideki Yoshida, Sue Cotterill, Masamitsu Yamaguchi
Both Mcm10 and HP1a are known to be required for DNA replication. However, underlying mechanism is not clarified yet especially for HP1. Knockdown of both HP1a and Mcm10 genes inhibited the progression of S phase in Drosophila eye imaginal discs. Proximity Ligation Assay (PLA) demonstrated that HP1a is in close proximity to DNA replication proteins including Mcm10, RFC140 and DNA polymerase ϵ 255 kDa subunit in S-phase. This was further confirmed by co-immunoprecipitation assay. The PLA signals between Mcm10 and HP1a are specifically observed in the mitotic cycling cells, but not in the endocycling cells...
November 29, 2016: Nucleic Acids Research
https://www.readbyqxmd.com/read/27892504/soluble-siglec-5-associates-to-psgl-1-and-displays-anti-inflammatory-activity
#3
Marion Pepin, Soraya Mezouar, Julie Pegon, Vincent Muczynski, Frédéric Adam, Elsa P Bianchini, Amine Bazaa, Valerie Proulle, Alain Rupin, Jerome Paysant, Laurence Panicot-Dubois, Olivier D Christophe, Christophe Dubois, Peter J Lenting, Cécile V Denis
Interactions between endothelial selectins and the leukocyte counter-receptor PSGL1 mediates leukocyte recruitment to inflammation sites. PSGL1 is highly sialylated, making it a potential ligand for Siglec-5, a leukocyte-receptor that recognizes sialic acid structures. Binding assays using soluble Siglec-5 variants (sSiglec-5/C4BP and sSiglec-5/Fc) revealed a dose- and calcium-dependent binding to PSGL1. Pre-treatment of PSGL1 with sialidase reduced Siglec-5 binding by 79 ± 4%. In confocal immune-fluorescence assays, we observed that 50% of Peripheral Blood Mononuclear Cells (PBMCs) simultaneously express PSGL1 and Siglec-5...
November 28, 2016: Scientific Reports
https://www.readbyqxmd.com/read/27890932/il-10-production-by-cll-cells-is-enhanced-in-the-anergic-ighv-mutated-subset-and-associates-with-reduced-dna-methylation-of-the-il10-locus
#4
S Drennan, A D'Avola, Y Gao, C Weigel, E Chrysostomou, A J Steele, T Zenz, C Plass, P W Johnson, A P Williams, G Packham, F K Stevenson, C C Oakes, F Forconi
Chronic lymphocytic leukemias (CLL) with unmutated (U-CLL) or mutated (M-CLL) IGHV have variable features of immunosuppression, possibly influenced by those CLL cells activated to produce IL-10. The two subsets differ in their levels of anergy, defined by low surface IgM levels/signaling capacity, and in their DNA methylation profile, particularly variable in M-CLL. We have now found that levels of IL-10 produced by activated CLL cells were highly variable. Levels were higher in M-CLL than in U-CLL and correlated with anergy...
November 28, 2016: Leukemia: Official Journal of the Leukemia Society of America, Leukemia Research Fund, U.K
https://www.readbyqxmd.com/read/27872311/methyltransferase-like-protein-16-binds-the-3-terminal-triple-helix-of-malat1-long-noncoding-rna
#5
Jessica A Brown, Charles G Kinzig, Suzanne J DeGregorio, Joan A Steitz
Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), a cancer-promoting long noncoding RNA, accumulates in cells by using a 3'-triple-helical RNA stability element for nuclear expression (ENE). The ENE, a stem-loop structure containing a U-rich internal loop, interacts with a downstream A-rich tract (ENE+A) to form a blunt-ended triple helix composed of nine U•A-U triples interrupted by a C•G-C triple and C-G doublet. This unique structure prompted us to explore the possibility of protein binding...
November 21, 2016: Proceedings of the National Academy of Sciences of the United States of America
https://www.readbyqxmd.com/read/27861175/transient-receptor-potential-vanilloid-4-and-serum-glucocorticoid-regulated-kinase-1-are-critical-mediators-of-lung-injury-in-overventilated-mice-in-vivo
#6
Laura Michalick, Lasti Erfinanda, Ulrike Weichelt, Markus van der Giet, Wolfgang Liedtke, Wolfgang M Kuebler
BACKGROUND: Mechanical ventilation can cause lung endothelial barrier failure and inflammation cumulating in ventilator-induced lung injury. Yet, underlying mechanotransduction mechanisms remain unclear. Here, the authors tested the hypothesis that activation of the mechanosensitive Ca channel transient receptor potential vanilloid (TRPV4) by serum glucocorticoid-regulated kinase (SGK) 1 may drive the development of ventilator-induced lung injury. METHODS: Mice (total n = 54) were ventilated for 2 h with low (7 ml/kg) or high (20 ml/kg) tidal volumes and assessed for signs of ventilator-induced lung injury...
November 18, 2016: Anesthesiology
https://www.readbyqxmd.com/read/27856957/nuclear-localized-lsr-a-novel-regulator-of-breast-cancer-behavior-and-tumorigenesis
#7
Denise K Reaves, Katherine A Hoadley, Katerina Fagan-Solis, Dereje D Jima, Michael Bereman, Lynnelle Thorpe, Jyla Hicks, David McDonald, Melissa A Troester, Charles M Perou, Jodie M Fleming
: Lipolysis Stimulated Lipoprotein Receptor (LSR) has been found in the plasma membrane and is believed to function in lipoprotein endocytosis and tight junctions. Given the impact of cellular metabolism and junction signaling pathways on tumor phenotypes and patient outcome, it is important to understand how LSR cellular localization mediates its functions. We conducted localization studies, evaluated DNA binding, and examined the effects of nuclear LSR in cells, xenografts, and clinical specimens...
November 17, 2016: Molecular Cancer Research: MCR
https://www.readbyqxmd.com/read/27846223/combgap-promotes-ovarian-niche-development-and-chromatin-association-of-ecr-binding-regions-in-br-c
#8
Anna Hitrik, Malka Popliker, Dana Gancz, Zohar Mukamel, Aviezer Lifshitz, Omer Schwartzman, Amos Tanay, Lilach Gilboa
The development of niches for tissue-specific stem cells is an important aspect of stem cell biology. Determination of niche size and niche numbers during organogenesis involves precise control of gene expression. How this is achieved in the context of a complex chromatin landscape is largely unknown. Here we show that the nuclear protein Combgap (Cg) supports correct ovarian niche formation in Drosophila by controlling ecdysone-Receptor (EcR)- mediated transcription and long-range chromatin contacts in the broad locus (BR-C)...
November 2016: PLoS Genetics
https://www.readbyqxmd.com/read/27846146/identification-and-validation-of-novel-subtype-specific-protein-biomarkers-in-pancreatic-ductal-adenocarcinoma
#9
Laura Kuhlmann, Wiebke M Nadler, Alexander Kerner, Sabrina A Hanke, Elisa M Noll, Christian Eisen, Elisa Espinet, Vanessa Vogel, Andreas Trumpp, Martin R Sprick, Christoph P Roesli
OBJECTIVES: Pancreatic ductal adenocarcinoma (PDAC) has been subclassified into 3 molecular subtypes: classical, quasi-mesenchymal, and exocrine-like. These subtypes exhibit differences in patient survival and drug resistance to conventional therapies. The aim of the current study is to identify novel subtype-specific protein biomarkers facilitating subtype stratification of patients with PDAC and novel therapy development. METHODS: A set of 12 human patient-derived primary cell lines was used as a starting material for an advanced label-free proteomics approach leading to the identification of novel cell surface and secreted biomarkers...
November 11, 2016: Pancreas
https://www.readbyqxmd.com/read/27829249/dcr3-tff3-and-midkine-are-novel-serum-biomarkers-in-small-intestinal-neuroendocrine-tumors
#10
Katarina Edfeldt, Kosmas Daskalakis, Christofer Bäcklin, Olov Norlén, Eva Tiensuu Janson, Gunnar Westin, Per Hellman, Peter Stålberg
Small intestinal neuroendocrine tumors (SI-NETs) are amine- and peptide producing neoplasms. Most patients display metastases at the time of diagnosis, they have an unpredictable individual disease course and the tumors are often therapy resistant. Chromogranin A (CgA) and 5-hydroxyindoleacetic acid (5-HIAA) are the clinically most used biomarkers today, but there is a great need for novel diagnostic and prognostic biomarkers and new therapeutic targets. Sixty-nine biomarkers were screened in serum from 23 SI-NET patients and 23 healthy controls using multiplex PLA (proximity ligation assay)...
November 9, 2016: Neuroendocrinology
https://www.readbyqxmd.com/read/27826057/adenylyl-cyclase-5-links-changes-in-calcium-homeostasis-to-camp-dependent-cyst-growth-in-polycystic-liver-disease
#11
Carlo Spirli, Valeria Mariotti, Ambra Villani, Luca Fabris, Romina Fiorotto, Mario Strazzabosco
BACKGROUND/AIMS: Genetic defects in Polycystins -1 or -2 (PC1 or PC2) cause polycystic liver disease associated with ADPKD (PLD-ADPKD). Progressive cyst growth is sustained by a cAMP-dependent Ras/ERK/HIFα pathway leading to increased autocrine/paracrine VEGF-A signalling. In PC2-defective cholangiocytes, store-operated Ca(2+) entry (SOCE), intracellular and endoplasmic reticulum [Ca(2+)]ER levels are reduced, while cAMP production in response to [Ca(2+)]ER depletion is increased. We hypothesized that in PC2-defective cells, in response to [Ca(2+)]ER depletion, the Ca(2+)-inhibitable adenylyl-cyclases AC5 or AC6 are activated by the ER chaperon STIM1 resulting in cAMP/PKA-dependent Ras/ERK/HIFα pathway activation...
November 5, 2016: Journal of Hepatology
https://www.readbyqxmd.com/read/27825739/use-of-a-multiplex-ligation-dependent-probe-amplification-method-for-the-detection-of-deletions-duplications-in-the-gba1-gene-in-gaucher-disease-patients
#12
Suelen P Basgalupp, Marina Siebert, Filippo Pinto E Vairo, Anisse Marques Chami, Louise Lapagesse de Camargo Pinto, Gerson da S Carvalho, Ida Vanessa D Schwartz
Gaucher disease (GD) is caused by the deficient activity of β-glucocerebrosidase due to pathogenic mutations in the GBA1. This gene has a pseudogene (GBAP) with 96% of sequence homology. Recombination (Rec) events in the GBA1 seem to be facilitated by an increased degree of homology and proximity to the GBAP. The objectives of this study were to validate the P338-X1 GBA kit (MRC-Holland) for Multiplex Ligation-dependent Probe Amplification (MLPA) and to detect larger deletions/duplications present in GBA1 in GD patients from Brazil...
October 20, 2016: Blood Cells, Molecules & Diseases
https://www.readbyqxmd.com/read/27815219/existence-of-muscarinic-acetylcholine-receptor-machr-and-fibroblast-growth-factor-receptor-fgfr-heteroreceptor-complexes-and-their-enhancement-of-neurite-outgrowth-in-neural-hippocampal-cultures
#13
V Di Liberto, D O Borroto-Escuela, M Frinchi, V Verdi, K Fuxe, N Belluardo, G Mudò
BACKGROUND: Recently, it was demonstrated that G-protein-coupled receptors (GPCRs) can transactivate tyrosine kinase receptors in absence of their ligands. In this work, driven by the observation that mAChRs and fibroblast growth factor receptors (FGFRs) share signalling pathways and regulation of brain functions, it was decided to explore whether mAChRs activation may transactivate FGFRs and, if so, to characterize the related trophic effects in cultured hippocampal neurons. METHODS: Oxotremorine-M transactivation of FGFRs and related trophic effects were tested in primary hippocampal neurons...
November 1, 2016: Biochimica et Biophysica Acta
https://www.readbyqxmd.com/read/27807477/chromatin-proteins-and-rna-are-associated-with-dna-during-all-phases-of-mitosis
#14
Kathryn L Black, Svetlana Petruk, Tyler K Fenstermaker, Jacob W Hodgson, Jeffrey L Caplan, Hugh W Brock, Alexander Mazo
Mitosis brings about major changes to chromosome and nuclear structure. We used recently developed proximity ligation assay-based techniques to investigate the association with DNA of chromatin-associated proteins and RNAs in Drosophila embryos during mitosis. All groups of tested proteins, histone-modifying and chromatin-remodeling proteins and methylated histones remained in close proximity to DNA during all phases of mitosis. We also found that RNA transcripts are associated with DNA during all stages of mitosis...
2016: Cell Discovery
https://www.readbyqxmd.com/read/27806331/xpo1-inhibitor-combination-therapy-with-bortezomib-or-carfilzomib-induces-nuclear-localization-of-i%C3%AE%C2%BAb%C3%AE-and-overcomes-acquired-proteasome-inhibitor-resistance-in-human-multiple-myeloma
#15
Joel G Turner, Trinayan Kashyap, Jana L Dawson, Juan Gomez, Alexis A Bauer, Steven Grant, Yun Dai, Kenneth H Shain, Mark Meads, Yosef Landesman, Daniel M Sullivan
Acquired proteasome-inhibitor (PI) resistance is a major obstacle in the treatment of multiple myeloma (MM). We investigated whether the clinical XPO1-inhibitor selinexor, when combined with bortezomib or carfilzomib, could overcome acquired resistance in MM. PI-resistant myeloma cell lines both in vitro and in vivo and refractory myeloma patient biopsies were treated with selinexor/bortezomib or carfilzomib and assayed for apoptosis. Mechanistic studies included NFκB pathway protein expression assays, immunofluorescence microscopy, ImageStream flow-cytometry, and proximity-ligation assays...
October 28, 2016: Oncotarget
https://www.readbyqxmd.com/read/27798680/loukoumasomes-are-distinct-subcellular-structures-from-rods-and-rings-and-are-structurally-associated-with-map2-and-the-nuclear-envelope-in-retinal-cells
#16
Jake W Noble, Diana V Hunter, Calvin D Roskelley, Edward K L Chan, Julia Mills
"Rods and rings" (RR) and loukoumasomes are similarly shaped, subcellular macromolecular structures with as yet unknown function. RR, so named because of their shape, are formed in response to inhibition in the GTP or CTP synthetic pathways and are highly enriched in the two key enzymes of the nucleotide synthetic pathway. Loukoumasomes also occur as linear and toroidal bodies and were initially inferred to be the same as RR, largely due to their shared shape and size and the fact that it was unclear if they shared the same subcomponents...
2016: PloS One
https://www.readbyqxmd.com/read/27789473/kv7-channel-activation-underpins-epac-dependent-relaxations-of-rat-arteries
#17
Jennifer B Stott, Vincenzo Barrese, Iain A Greenwood
OBJECTIVE: To establish the role of Kv7 channels in EPAC (exchange protein directly activated by cAMP)-dependent relaxations of the rat vasculature and to investigate whether this contributes to β-adrenoceptor-mediated vasorelaxations. APPROACH AND RESULTS: Isolated rat renal and mesenteric arteries (RA and MA, respectively) were used for isometric tension recording to study the relaxant effects of a specific EPAC activator and the β-adrenoceptor agonist isoproterenol in the presence of potassium channel inhibitors and cell signaling modulators...
October 27, 2016: Arteriosclerosis, Thrombosis, and Vascular Biology
https://www.readbyqxmd.com/read/27784654/calcitonin-gene-related-peptide-rescues-proximity-associations-of-its-receptor-components-calcitonin-receptor-like-receptor-and-receptor-activity-modifying-protein1-in-rat-uterine-artery-smooth-muscle-cells-exposed-to-tumor-necrosis-factor-alpha
#18
Yuanlin Dong, Madhu Chauhan, Michael Belfort, Chandra Yallampalli
Calcitonin gene-related peptide (CALCB), adrenomedullin (ADM) and adrenomedullin 2 (ADM2)/intermedin (IMD) play critical roles in vascular adaptation during pregnancy through calcitonin-receptor-like-receptor (CALCRL) and receptor activity-modifying proteins (RAMPs). This study was designed to assess the predominant RAMP that associates with CALCRL to form a functional receptor in the rat uterine artery smooth muscle (RUASM). We also determined if these receptor component associations are decreased by TNF-α and if CALCB, ADM or ADM2 can rescue CRLR/RAMP associations...
October 26, 2016: Biology of Reproduction
https://www.readbyqxmd.com/read/27771510/methionine-synthase-and-methionine-synthase-reductase-interact-with-mmachc-and-with-mmadhc
#19
Christine Bassila, Rose Ghemrawi, Justine Flayac, D Sean Froese, Matthias R Baumgartner, Jean-Louis Guéant, David Coelho
An increasing number of studies indicate that each step of the intracellular processing of vitamin B12 or cobalamin (Cbl) involves protein-protein interactions. We have previously described a novel interaction between methionine synthase (MS) and MMACHC and its effect on the regulation of MMACHC activity. Our goal is to further characterize the interactions of MS with other potential partners in a so-called MS interactome. We dissected the interactions and their alterations by co-immunoprecipitation and DuoLink proximity ligation assays in fibroblasts with cblG, cblE, and cblC genetic defects affecting respectively the expression of MS, methionine synthase reductase (MSR) and MMACHC and in HepG2 cells transfected with corresponding siRNAs...
October 19, 2016: Biochimica et Biophysica Acta
https://www.readbyqxmd.com/read/27761829/proximity-ligation-assay-to-quantify-foxp3-acetylation-in-regulatory-t-cells
#20
Jing Jiao, Rongxiang Han, Wayne W Hancock, Ulf H Beier
Determining protein acetylation by immunoprecipitation and immunoblotting can be challenging, especially if the tissue of interest is low in quantity, and when good quality acetylation site-specific antibodies are not available. Proximity ligation assays allow a sensitive and quantitative method to assess Foxp3 acetylation in regulatory T cells, with as little as 1.5 × 10(5) cells within two days turnaround time. This method is of potential use in other similar scenarios, when post-translational modifications of a protein of interest need to be determined with only a small amount of sample and in the absence of specific antibodies that can assess the post-translational modification in the protein of interest...
2017: Methods in Molecular Biology
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