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https://www.readbyqxmd.com/read/29320704/extracellular-vesicles-provide-a-means-for-tissue-crosstalk-during-exercise
#1
Martin Whitham, Benjamin L Parker, Martin Friedrichsen, Janne R Hingst, Marit Hjorth, William E Hughes, Casey L Egan, Lena Cron, Kevin I Watt, Rhiannon P Kuchel, Navind Jayasooriah, Emma Estevez, Tim Petzold, Catherine M Suter, Paul Gregorevic, Bente Kiens, Erik A Richter, David E James, Jørgen F P Wojtaszewski, Mark A Febbraio
Exercise stimulates the release of molecules into the circulation, supporting the concept that inter-tissue signaling proteins are important mediators of adaptations to exercise. Recognizing that many circulating proteins are packaged in extracellular vesicles (EVs), we employed quantitative proteomic techniques to characterize the exercise-induced secretion of EV-contained proteins. Following a 1-hr bout of cycling exercise in healthy humans, we observed an increase in the circulation of over 300 proteins, with a notable enrichment of several classes of proteins that compose exosomes and small vesicles...
January 9, 2018: Cell Metabolism
https://www.readbyqxmd.com/read/29301854/autophagy-is-required-for-sortilin-mediated-degradation-of-apolipoprotein-b100
#2
Jaume Amengual, Liang Guo, Alanna Strong, Julio Madrigal-Matute, Haizhen Wang, Susmita Kaushik, Jeffrey L Brodsky, Daniel J Rader, Ana Maria Cuervo, Edward A Fisher
Rationale: Genome-Wide Association Studies identified single nucleotide polymorphisms (SNPs) near the SORT1 locus strongly associated with decreased plasma low-density lipoprotein cholesterol (LDL-C) levels and protection from atherosclerotic cardiovascular disease and myocardial infarction. The minor allele of the causal SORT1 SNP locus creates a putative C/EBPα binding site in the SORT1 promoter, thereby increasing sortilin expression by 12-fold in liver, which is rich in this transcription factor. Our previous studies in mice have showed reductions in plasma LDL-C and its principal protein component, apolipoprotein B (apoB) with increased SORT1 expression, and in vitro studies suggested that sortilin promoted the presecretory lysosomal degradation of apoB associated with the LDL precursor, very-low density lipoprotein (VLDL)...
January 4, 2018: Circulation Research
https://www.readbyqxmd.com/read/29290400/evaluation-of-reversed-phase-nano-liquid-chromatography-conditions-by-using-reversed-phase-thin-layer-chromatography-based-on-hansen-solubility-parameters-for-the-analysis-of-amphiphilic-glycosylsphingolipid-transformations
#3
Yoshimi Kanie, Mizuki Taniuchi, Osamu Kanie
Pulse chase analysis is often used in investigating dynamics of cellular substances. Fluorescently labeled lactosyl sphingosine molecule is useful in chasing its transformation, however the analysis of such metabolites in attomole level is of extreme difficult due to the presence of large amount of endogenous amphiphilic molecules such as glycosphingolipids, sphingomyerin, and glycerophospholipids. Nano LC suites for analyzing the attomole scale metabolites, therefore removal of endogenous substances prior to nano LC and finding appropriate nano LC conditions are necessary...
December 23, 2017: Journal of Chromatography. A
https://www.readbyqxmd.com/read/29279083/discovery-of-a-recombinant-babesia-canis-supernatant-antigen-that-protects-dogs-against-virulent-challenge-infection
#4
K Moubri, J Kleuskens, J Van de Crommert, N Scholtes, T Van Kasteren, S Delbecq, B Carcy, E Précigout, A Gorenflot, Th Schetters
Soluble parasite antigens (SPA) in supernatants of in vitro cultures of Babesia canis can be used to vaccinate dogs against virulent B. canis infection. The moment that immunity becomes apparent coincides with the appearance of antibodies against SPA in the serum of the vaccinated animals. This so-called vaccination-challenge serum (VC-serum) was used to precipitate antigens from B. canis culture supernatants in agarose gels. This antigen preparation was then used to analyse the reactivity of sera from vaccinated dogs on western blots...
January 15, 2018: Veterinary Parasitology
https://www.readbyqxmd.com/read/29243248/uptake-kinetics-and-storage-capacity-of-dissolved-inorganic-phosphorus-and-corresponding-n-p-dynamics-in-ulva-lactuca-chlorophyta
#5
Alexander Lubsch, Klaas Timmermans
Dissolved inorganic phosphorus (DIP) is an essential macronutrient for maintaining metabolism and growth in autotrophs. Little is known about DIP-uptake kinetics and internal P-storage capacity in seaweeds, such as Ulva lactuca (Chlorophyta). Ulva lactuca is a promising candidate for biofiltration purposes and mass commercial cultivation. We exposed U. lactuca to a wide range of DIP concentrations (1 - 50 μmol · L-1 ) and a non-limiting concentration of dissolved inorganic nitrogen (DIN; 5000 μmol · L-1 ) under fully controlled laboratory conditions in a 'pulse-and-chase' assay over 10 days...
December 14, 2017: Journal of Phycology
https://www.readbyqxmd.com/read/29236258/using-tet-off-cells-and-rnai-knockdown-to-assay-mrna-decay
#6
Thomas D Baird, J Robert Hogg
Cellular mRNA levels are determined by the competing forces of transcription and decay. A wide array of cellular mRNA decay pathways carry out RNA turnover either on a constitutive basis or in response to changing cellular conditions. Here, we outline a method to investigate mRNA decay that employs RNAi knockdown of known or putative decay factors in commercially available Tet-off cell systems. Reporter mRNAs of interest are expressed under the control of a tetracycline-regulated promoter, allowing pulse-chase mRNA decay assays to be conducted...
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/29236247/5-bromouridine-ip-chase-bric-seq-to-determine-rna-half-lives
#7
Toshimichi Yamada, Naoto Imamachi, Rena Onoguchi-Mizutani, Katsutoshi Imamura, Yutaka Suzuki, Nobuyoshi Akimitsu
Analysis of RNA stability at genome-wide level is an advanced method in RNA biology that examines the half-life of each transcript. In particular, a pulse-labeling method using uridine analogs enables the determination of half-life of each transcript under physiologically undisturbed conditions. The technique involves pulse labeling of endogenous RNAs in mammalian cells with 5'-bromouridine (BrU), followed by measuring the chronological decrease of BrU-labeled RNAs using deep sequencing (BRIC-seq). Here, we describe a detailed protocol and technical tips for BRIC-seq...
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/29232161/characterization-of-novel-missense-variants-of-serpina1-gene-causing-alpha-1-antitrypsin-deficiency
#8
Nerea Matamala, Beatriz Lara, Gema Gomez-Mariano, Selene Martínez, Diana Retana, Taiomara Fernandez, Ramona Angeles Silvestre, Irene Belmonte, Francisco Rodriguez-Frias, Marçal Vilar, Raquel Sáez, Igor Iturbe, Silvia Castillo, María Molina-Molina, Anna Texido, Gema Tirado-Conde, Jose Luis Lopez-Campos, Manuel Posada, Ignacio Blanco, Sabina Janciauskiene, Beatriz Martinez-Delgado
SERPINA1 gene is highly polymorphic, with more than one hundred variants described in databases. The SERPINA1 encodes alpha-1 antitrypsin (AAT) protein, and the severe deficiency of AAT is a major contributor to pulmonary emphysema and liver diseases. We report seven new variants in Spanish patients with AAT deficiency. All variants involved amino acid substitutions in different exons: PiSDonosti (S+Ser14Phe), PiTijarafe (Ile50Asn), PiSevilla (Ala58Asp), PiCadiz (Glu151Lys), PiTarragona (Phe227Cys), PiPuerto Real (Thr249Ala) and PiValencia (Lys328Glu)...
December 12, 2017: American Journal of Respiratory Cell and Molecular Biology
https://www.readbyqxmd.com/read/29216926/pro-inflammatory-adjuvant-properties-of-pigment-grade-titanium-dioxide-particles-are-augmented-by-a-genotype-that-potentiates-interleukin-1%C3%AE-processing
#9
Sebastian Riedle, Laetitia C Pele, Don E Otter, Rachel E Hewitt, Harjinder Singh, Nicole C Roy, Jonathan J Powell
BACKGROUND: Pigment-grade titanium dioxide (TiO2) particles are an additive to some foods (E171 on ingredients lists), toothpastes, and pharma-/nutraceuticals and are absorbed, to some extent, in the human intestinal tract. TiO2 can act as a modest adjuvant in the secretion of the pro-inflammatory cytokine interleukin 1β (IL-1β) when triggered by common intestinal bacterial fragments, such as lipopolysaccharide (LPS) and/or peptidoglycan. Given the variance in human genotypes, which includes variance in genes related to IL-1β secretion, we investigated whether TiO2 particles might, in fact, be more potent pro-inflammatory adjuvants in cells that are genetically susceptible to IL-1β-related inflammation...
December 8, 2017: Particle and Fibre Toxicology
https://www.readbyqxmd.com/read/29202457/recapitulation-of-developmental-mechanisms-to-revascularize-the-ischemic-heart
#10
Karina N Dubé, Tonia M Thomas, Sonali Munshaw, Mala Rohling, Paul R Riley, Nicola Smart
Restoring blood flow after myocardial infarction (MI) is essential for survival of existing and newly regenerated tissue. Endogenous vascular repair processes are deployed following injury but are poorly understood. We sought to determine whether developmental mechanisms of coronary vessel formation are intrinsically reactivated in the adult mouse after MI. Using pulse-chase genetic lineage tracing, we establish that de novo vessel formation constitutes a substantial component of the neovascular response, with apparent cellular contributions from the endocardium and coronary sinus...
November 16, 2017: JCI Insight
https://www.readbyqxmd.com/read/29190188/biomeasures-and-mechanistic-modeling-highlight-pk-pd-risks-for-a-monoclonal-antibody-targeting-fn14-in-kidney-disease
#11
Xiaoying Chen, Vahid Farrokhi, Pratap Singh, Mireia Fernandez Ocana, Jenil Patel, Lih-Ling Lin, Hendrik Neubert, Joanne Brodfuehrer
Discovery of the upregulation of fibroblast growth factor-inducible-14 (Fn14) receptor following tissue injury has prompted investigation into biotherapeutic targeting of the Fn14 receptor for the treatment of conditions such as chronic kidney diseases. In the development of monoclonal antibody (mAb) therapeutics, there is an increasing trend to use biomeasures combined with mechanistic pharmacokinetic/pharmacodynamic (PK/PD) modeling to enable decision making in early discovery. With the aim of guiding preclinical efforts on designing an antibody with optimized properties, we developed a mechanistic site-of-action (SoA) PK/PD model for human application...
November 30, 2017: MAbs
https://www.readbyqxmd.com/read/29189775/visualizing-endocytic-recycling-and-trafficking-in-live-neurons-by-subdiffractional-tracking-of-internalized-molecules
#12
Merja Joensuu, Ramon Martínez-Mármol, Pranesh Padmanabhan, Nick R Glass, Nela Durisic, Matthew Pelekanos, Mahdie Mollazade, Giuseppe Balistreri, Rumelo Amor, Justin J Cooper-White, Geoffrey J Goodhill, Frédéric A Meunier
Our understanding of endocytic pathway dynamics is restricted by the diffraction limit of light microscopy. Although super-resolution techniques can overcome this issue, highly crowded cellular environments, such as nerve terminals, can also dramatically limit the tracking of multiple endocytic vesicles such as synaptic vesicles (SVs), which in turn restricts the analytical dissection of their discrete diffusional and transport states. We recently introduced a pulse-chase technique for subdiffractional tracking of internalized molecules (sdTIM) that allows the visualization of fluorescently tagged molecules trapped in individual signaling endosomes and SVs in presynapses or axons with 30- to 50-nm localization precision...
December 2017: Nature Protocols
https://www.readbyqxmd.com/read/29167274/distinct-cellular-mechanisms-underlie-smooth-muscle-turnover-in-vascular-development-and-repair
#13
Urmas Roostalu, Bashar Aldeiri, Alessandra Albertini, Neil E Humphreys, Maj Simonsen-Jackson, Jason K Wong, Giulio Cossu
Rationale: Vascular smooth muscle turnover has important implications for blood vessel repair and for the development of cardiovascular diseases, yet lack of specific transgenic animal models has prevented it's in vivo analysis. Objective: To characterize the dynamics and mechanisms of vascular smooth muscle turnover from the earliest stages of embryonic development to arterial repair in the adult. Methods and Results: We show that CD146 is transiently expressed in vascular smooth muscle development. By using CRISPR-Cas9 genome editing and in vitro smooth muscle differentiation assay we demonstrate that CD146 regulates the balance between proliferation and differentiation...
November 22, 2017: Circulation Research
https://www.readbyqxmd.com/read/29164873/plasma-protein-turnover-rates-in-rats-using-stable-isotope-labeling-global-proteomics-and-activity-based-protein-profiling
#14
Jordan Ned Smith, Kimberly J Tyrrell, Joshua R Hansen, Dennis George Thomas, Taylor A Murphree, Anil K Shukla, Teresa Luders, James M Madden, YunYing Li, Aaron T Wright, Paul D Piehowski
Protein turnover is important for general health on cellular and organism scales providing a strategy to replace old, damaged, or dysfunctional proteins. Protein turnover also informs of biomarker kinetics, as a better understanding of synthesis and degradation of proteins increases the clinical utility of biomarkers. Here, turnover rates of plasma proteins in rats were measured in vivo using a pulse-chase stable isotope labeling experiment. During the pulse, rats (n=5) were fed 13C6-labeled lysine ("heavy") feed for 23 days to label proteins...
November 22, 2017: Analytical Chemistry
https://www.readbyqxmd.com/read/29163579/arabidopsis-phosphatidic-acid-phosphohydrolases-are-essential-for-growth-under-nitrogen-depleted-conditions
#15
Yushi Yoshitake, Ryoichi Sato, Yuka Madoka, Keiko Ikeda, Masato Murakawa, Ko Suruga, Daisuke Sugiura, Ko Noguchi, Hiroyuki Ohta, Mie Shimojima
The Arabidopsis homologs of mammalian lipin, PAH1 and PAH2, are cytosolic phosphatidic acid phosphohydrolases that are involved in phospholipid biosynthesis and are essential for growth under phosphate starvation. Here, pah1 pah2 double-knockout mutants were found to be hypersensitive to nitrogen (N) starvation, whereas transgenic plants overexpressing PAH1 or PAH2 in the pah1 pah2 mutant background showed a similar growth phenotype as compared with wild type (WT) under N starvation. The chlorophyll content of pah1 pah2 was significantly lower than that of WT, whereas the chlorophyll content and photosynthetic activity of the transgenic plants were significantly higher than those of WT under N-depleted conditions...
2017: Frontiers in Plant Science
https://www.readbyqxmd.com/read/29158258/a-photo-cross-linking-approach-to-monitor-folding-and-assembly-of-newly-synthesized-proteins-in-a-living-cell
#16
Ryoji Miyazaki, Naomi Myougo, Hiroyuki Mori, Yoshinori Akiyama
Many proteins form multimeric complexes that plays crucial roles in various cellular processes. Studying how proteins are correctly folded and assembled into such complexes in a living cell is important for understanding the physiological roles and the qualitative and quantitative regulation of the complex. However, few methods are suitable for analyzing these rapidly occurring processes. Site-directed in vivo photo-cross-linking is an elegant technique that enables analysis of protein-protein interactions in living cells with high spatial resolution...
November 20, 2017: Journal of Biological Chemistry
https://www.readbyqxmd.com/read/29109155/temporal-fluxomics-reveals-oscillations-in-tca-cycle-flux-throughout-the-mammalian-cell-cycle
#17
Eunyong Ahn, Praveen Kumar, Dzmitry Mukha, Amit Tzur, Tomer Shlomi
Cellular metabolic demands change throughout the cell cycle. Nevertheless, a characterization of how metabolic fluxes adapt to the changing demands throughout the cell cycle is lacking. Here, we developed a temporal-fluxomics approach to derive a comprehensive and quantitative view of alterations in metabolic fluxes throughout the mammalian cell cycle. This is achieved by combining pulse-chase LC-MS-based isotope tracing in synchronized cell populations with computational deconvolution and metabolic flux modeling...
November 6, 2017: Molecular Systems Biology
https://www.readbyqxmd.com/read/29101475/insight-into-the-pathology-of-a-col1a1-signal-peptide-heterozygous-mutation-leading-to-severe-osteogenesis-imperfecta
#18
U Lindert, M Gnoli, M Maioli, M F Bedeschi, L Sangiorgi, M Rohrbach, C Giunta
Osteogenesis imperfecta or "brittle bone disease" is a congenital disorder of connective tissue causing the bone to break easily. Around 85-90% of cases are due to autosomal dominant mutations in the genes encoding type I collagen, the major organic component of bone. Genotype-phenotype correlations have shown that quantitative defects of collagen type I lead to mild OI, whereas structural defects show a wide clinical range from mild to perinatal lethal. This may partially be explained by the type of amino acid substitution and the relative location in the domain structure...
November 3, 2017: Calcified Tissue International
https://www.readbyqxmd.com/read/29100014/tgf-%C3%AE-induced-quiescence-mediates-chemoresistance-of-tumor-propagating-cells-in-squamous-cell-carcinoma
#19
Jessie A Brown, Yoshiya Yonekubo, Nicole Hanson, Ana Sastre-Perona, Alice Basin, Julie A Rytlewski, Igor Dolgalev, Shane Meehan, Aristotelis Tsirigos, Slobodan Beronja, Markus Schober
Squamous cell carcinomas (SCCs) are heterogeneous tumors sustained by tumor-propagating cancer cells (TPCs). SCCs frequently resist chemotherapy through still unknown mechanisms. Here, we combine H2B-GFP-based pulse-chasing with cell-surface markers to distinguish quiescent from proliferative TPCs within SCCs. We find that quiescent TPCs resist DNA damage and exhibit increased tumorigenic potential in response to chemotherapy, whereas proliferative TPCs undergo apoptosis. Quiescence is regulated by TGF-β/SMAD signaling, which directly regulates cell-cycle gene transcription to control a reversible G1 cell-cycle arrest, independent of p21CIP function...
November 2, 2017: Cell Stem Cell
https://www.readbyqxmd.com/read/29091273/analysis-of-disulfide-bond-formation
#20
Ineke Braakman, Lydia Lamriben, Guus van Zadelhoff, Daniel N Hebert
In this unit, protocols are provided for detection of disulfide bond formation in cultures of intact cells and in an in vitro translation system containing isolated microsomes or semi-permeabilized cells. First, the newly synthesized protein of interest is biosynthetically labeled with radioactive amino acids in a short pulse. The labeled protein then is chased with unlabeled amino acids. At different times during the chase, a sample is collected, membranes are lysed with detergent, and the protein is isolated by immunoprecipitation, as described...
November 1, 2017: Current Protocols in Protein Science
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