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Pulse and chase

Paul C Guest
Pulse-chase radiolabeling of cells with radioactive amino acids is a common method for tracking the biosynthesis of proteins. Radiolabeled newly synthesized proteins can be analyzed by a number of techniques such as two dimensional gel electrophoresis (2DE). This chapter presents a protocol for the biosynthetic labeling of pancreatic islets with (35)S-methionine in the presence of basal and stimulatory concentrations of glucose, followed by subcellular fractionation to produce a secretory granule fraction and analysis of the granule protein contents by 2DE...
2017: Methods in Molecular Biology
Guo Zhu, Sherri L Rankin, Jon D Larson, Xiaoyan Zhu, Lionel Ml Chow, Chunxu Qu, Jinghui Zhang, David W Ellison, Suzanne J Baker
Loss of the tumor suppressor gene PTEN exerts diverse outcomes on cancer in different developmental contexts. To gain insight into the effect of its loss on outcomes in the brain, we conditionally inactivated the murine Pten gene in neonatal neural stem/progenitor cells. Pten inactivation created an abnormal perivascular proliferative niche in the cerebellum that persisted in adult animals but did not progress to malignancy. Proliferating cells showed undifferentiated morphology and expressed the progenitor marker Nestin but not Math1, a marker of committed granule neuron progenitors...
November 4, 2016: Cancer Research
Anna E Beaudin, E Camilla Forsberg
Hematopoietic stem cells (HSCs) have long been considered the continuous source of all hematopoietic cells for the life of an individual. Recent findings have questioned multiple aspects of this view, including the ability of life-long HSCs to contribute to tissue-resident immune cells. Here we discuss the most recent findings on the source of B1a cells, innate-like lymphocytes that primarily reside in serous cavities. Powerful experimental approaches including barcoding, single cell transplantation, in vivo lineage tracing, and HSC-specific pulse-chase labeling have provided novel insights on B1a cell generation during ontogeny...
October 31, 2016: Blood
Annabel Y Minard, Martin K L Wong, Rima Chaudhuri, Shi-Xiong Tan, Sean J Humphrey, Benjamin L Parker, Jean Y Yang, D Ross Laybutt, Gregory J Cooney, Adelle C F Coster, Jacqueline Stöckli, David E James
Hyperinsulinemia, which is associated with aging and metabolic disease, may lead to defective protein homeostasis (proteostasis) due to hyperactivation of insulin-sensitive pathways such as protein synthesis. We investigated the effect of chronic hyperinsulinemia on proteostasis by generating a time-resolved map of insulin-regulated protein turnover in adipocytes using metabolic pulse-chase labeling and high resolution mass spectrometry. Hyperinsulinemia increased the synthesis of nearly half of all detected proteins and did not affect protein degradation despite suppressing autophagy...
December 2, 2016: Journal of Biological Chemistry
David B Guiliano, Helen North, Eleni Panayoitou, Elaine C Campbell, Kirsty McHugh, Fiona G M Cooke, Marine Silvestre, Paul Bowness, Simon J Powis, Antony N Antoniou
Objective - HLA-B27 is associated with the inflammatory spondyloarthropathies (SpAs). Of significance, subtypes HLA-B*27:06 and HLA-B*27:09 are not associated with the SpAs. These subtypes primarily differ from the HLA-B*27:05 disease associated allele at residues 114 and 116 of the heavy chain, part of the F pocket of the antigen-binding groove. Dimerisation of HLA-B27 during assembly has been implicated in disease onset. This study investigated the factors influencing differences in dimerisation between disease associated and non-associated HLA-B27 alleles...
October 9, 2016: Arthritis & Rheumatology
Erik McShane, Celine Sin, Henrik Zauber, Jonathan N Wells, Neysan Donnelly, Xi Wang, Jingyi Hou, Wei Chen, Zuzana Storchova, Joseph A Marsh, Angelo Valleriani, Matthias Selbach
Do young and old protein molecules have the same probability to be degraded? We addressed this question using metabolic pulse-chase labeling and quantitative mass spectrometry to obtain degradation profiles for thousands of proteins. We find that >10% of proteins are degraded non-exponentially. Specifically, proteins are less stable in the first few hours of their life and stabilize with age. Degradation profiles are conserved and similar in two cell types. Many non-exponentially degraded (NED) proteins are subunits of complexes that are produced in super-stoichiometric amounts relative to their exponentially degraded (ED) counterparts...
October 20, 2016: Cell
Simon Pouil, Michel Warnau, François Oberhänsli, Jean-Louis Teyssié, Paco Bustamante, Marc Metian
Diet is an important pathway for metal uptake in marine organisms, and assimilation efficiency (AE) is one of the most relevant parameter to quantify trophic transfer of metals along aquatic food webs. The most commonly used method to estimate this parameter is pulse-chase feeding using radiolabelled food. This approach is, however, based on several assumptions that are not always tested in experimental context. The present work aimed at validating the approach by assessing single-feeding and multiple-feeding approaches, using a model species (the turbot Scophthalmus maximus)...
October 5, 2016: Environmental Toxicology and Chemistry
Meriam Boubakri, Taro Chaya, Hiromi Hirata, Naoko Kajimura, Ryusuke Kuwahara, Akiko Ueno, Jarema Malicki, Takahisa Furukawa, Yoshihiro Omori
In the retina, aberrant opsin transport from cell bodies to outer segments leads to retinal degenerative diseases such as retinitis pigmentosa. Opsin transport is facilitated by the intraflagellar transport (IFT) system that mediates the bidirectional movement of proteins within cilia. In contrast to functions of the anterograde transport executed by IFT complex B (IFT-B), the precise functions of the retrograde transport mediated by IFT complex A (IFT-A) have not been well studied in photoreceptor cilia. Here, we analyzed developing zebrafish larvae carrying a null mutation in ift122 encoding a component of IFT-A...
November 18, 2016: Journal of Biological Chemistry
Francesca De Marchis, Andrea Pompa, Michele Bellucci
The classical Golgi pathway is not the only mechanism for vacuolar protein transport in plants because alternative transport mechanisms have been described. The existence of these alternative pathways can be demonstrated using several chemicals and here we describe the use of brefeldin A (BFA), endo-β-N-acetylglucosaminidase H (Endo-H), and tunicamycin, on isolated tobacco leaf protoplasts. Two main methods are illustrated in this chapter, protoplast pulse-chase followed by protein immunoprecipitation, and protoplast immunofluorescence...
2016: Methods in Molecular Biology
Daniela Justa-Schuch, Maria Silva-Garcia, Esther Pilla, Michael Engelke, Markus Kilisch, Christof Lenz, Ulrike Möller, Fumihiko Nakamura, Henning Urlaub, Ruth Geiss-Friedlander
The aminopeptidase DPP9 removes dipeptides from N-termini of substrates having a proline or alanine in second position. Although linked to several pathways including cell survival and metabolism, the molecular mechanisms underlying these outcomes are poorly understood. We identified a novel interaction of DPP9 with Filamin A, which recruits DPP9 to Syk, a central kinase in B-cell signalling. Syk signalling can be terminated by degradation, requiring the ubiquitin E3 ligase Cbl. We show that DPP9 cleaves Syk to produce a neo N-terminus with serine in position 1...
September 10, 2016: ELife
Lucia Fuchslueger, Michael Bahn, Roland Hasibeder, Sandra Kienzl, Karina Fritz, Michael Schmitt, Margarete Watzka, Andreas Richter
Drought periods are projected to become more severe and more frequent in many European regions. While effects of single strong droughts on plant and microbial carbon (C) dynamics have been studied in some detail, impacts of recurrent drought events are still little understood.We tested whether the legacy of extreme experimental drought affects responses of plant and microbial C and nitrogen (N) turnover to further drought and rewetting. In a mountain grassland, we conducted a (13)C pulse-chase experiment during a naturally occurring drought and rewetting event in plots previously exposed to experimental droughts and in ambient controls (AC)...
September 2016: Journal of Ecology
Ryan E Schaub, Yolande A Chan, Mijoon Lee, Dusan Hesek, Shahriar Mobashery, Joseph P Dillard
Neisseria gonorrhoeae releases peptidoglycan (PG) fragments during infection that provoke a large inflammatory response and, in pelvic inflammatory disease, this response leads to the death and sloughing of ciliated cells of the Fallopian tube. We characterized the biochemical functions and localization of two enzymes responsible for the release of proinflammatory PG fragments. The putative lytic transglycosylases LtgA and LtgD were shown to create the 1,6-anhydromuramyl moieties, and both enzymes were able to digest a small, synthetic tetrasaccharide dipeptide PG fragment into the cognate 1,6-anhydromuramyl-containing reaction products...
September 8, 2016: Molecular Microbiology
Karin Nienhaus, G Ulrich Nienhaus
Proteins of the green fluorescent protein (GFP) family are indispensable for fluorescence imaging experiments in the life sciences, particularly of living specimens. Their essential role as genetically encoded fluorescence markers has motivated many researchers over the last 20 years to further advance and optimize these proteins by using protein engineering. Amino acids can be exchanged by site-specific mutagenesis, starting with naturally occurring proteins as templates. Optical properties of the fluorescent chromophore are strongly tuned by the surrounding protein environment, and a targeted modification of chromophore-protein interactions requires a profound knowledge of the underlying photophysics and photochemistry, which has by now been well established from a large number of structural and spectroscopic experiments and molecular-mechanical and quantum-mechanical computations on many variants of fluorescent proteins...
November 9, 2016: Journal of Physics. Condensed Matter: An Institute of Physics Journal
Hongliang Wang, Andrew W Tai
BACKGROUND & AIMS: Like all positive-sense RNA viruses, hepatitis C virus (HCV) induces host membrane alterations for its replication. In chronically infected cells, it is not known whether these viral replication organelles are being continually resupplied by newly synthesized viral proteins in situ, or whether they are generated de novo. Here we aimed to study temporal events in replication organelles formation and maturation. METHODS: Here we use pulse-chase labeling in combination with confocal microscopy, correlative light electron microscopy and biochemical methods to identify temporally distinct populations of replication organelles in living cells and study the formation, morphogenesis as well as compositional and functional changes of replication organelles over time...
September 4, 2016: Journal of Hepatology
Xiaomo Jiang, Feng Cong
Wnt pathways are critical for embryonic development and adult tissue homeostasis in all multicellular animals. Many regulatory mechanisms exist to control proper signaling output. Recent studies suggest that cell surface Wnt receptor level is controlled by ubiquitination, and serve as a critical regulatory point of Wnt pathway activity as it determines the responsiveness of cells to Wnt signal. Here, we describe flow cytometry, cell surface protein biotinylation, and immunofluorescence pulse-chase methods to probe the surface expression, ubiquitination, and internalization of the Wnt receptors FZD and LRP6...
2016: Methods in Molecular Biology
Xiang Deng, Oxana A Zhironkina, Varvara D Cherepanynets, Olga S Strelkova, Igor I Kireev, Andrew S Belmont
In higher eukaryotic interphase nuclei, the 100- to >1,000-fold linear compaction of chromatin is difficult to reconcile with its function as a template for transcription, replication, and repair. It is challenging to imagine how DNA and RNA polymerases with their associated molecular machinery would move along the DNA template without transient decondensation of observed large-scale chromatin "chromonema" fibers [1]. Transcription or "replication factory" models [2], in which polymerases remain fixed while DNA is reeled through, are similarly difficult to conceptualize without transient decondensation of these chromonema fibers...
September 26, 2016: Current Biology: CB
Annalena La Porte, Jennifer Cano, Xuhong Wu, Doyel Mitra, Ganjam V Kalpana
: INI1/hSNF5/SMARCB1/BAF47 is an HIV-specific integrase (IN)-binding protein that influences HIV-1 transcription and particle production. INI1 binds to SAP18 (Sin3a-associated protein, 18 kDa), and both INI1 and SAP18 are incorporated into HIV-1 virions. To determine the significance of INI1 and the INI1-SAP18 interaction during HIV-1 replication, we isolated a panel of SAP18-interaction-defective (SID)-INI1 mutants using a yeast reverse two-hybrid screen. The SID-INI1 mutants, which retained the ability to bind to IN, cMYC, and INI1 but were impaired for binding to SAP18, were tested for their effects on HIV-1 particle production...
November 1, 2016: Journal of Virology
Qian Chai, Stacy R Webb, Zhaoshuai Wang, Rebecca E Dutch, Yinan Wei
Proteins are constantly synthesized and degraded in living cells during their growth and division, often in response to metabolic and environmental conditions. The synthesis and breakdown of proteins under different conditions reveal information about their mechanism of function. The metabolic incorporation of non-natural amino acid azidohomoalanine (AHA) and subsequent labeling via click chemistry emerged as a non-radioactive strategy useful in the determination of protein kinetics and turnover. We used the method to monitor the degradation of two proteins involved in the multidrug efflux in Escherichia coli, the inner membrane transporter AcrB and its functional partner membrane fusion protein AcrA...
November 2016: Analytical and Bioanalytical Chemistry
Renata Slavíková, David Püschel, Martina Janoušková, Martina Hujslová, Tereza Konvalinková, Hana Gryndlerová, Milan Gryndler, Martin Weiser, Jan Jansa
Quantification of carbon (C) fluxes in mycorrhizal plants is one of the important yet little explored tasks of mycorrhizal physiology and ecology. (13)CO2 pulse-chase labelling experiments are increasingly being used to track the fate of C in these plant-microbial symbioses. Nevertheless, continuous monitoring of both the below- and aboveground CO2 emissions remains a challenge, although it is necessary to establish the full C budget of mycorrhizal plants. Here, a novel CO2 collection system is presented which allows assessment of gaseous CO2 emissions (including isotopic composition of their C) from both belowground and shoot compartments...
August 22, 2016: Mycorrhiza
David A Gold, Nagayasu Nakanishi, Nicholai M Hensley, Volker Hartenstein, David K Jacobs
The moon jellyfish Aurelia exhibits a dramatic reorganization of tissue during its metamorphosis from planula larva to polyp. There are currently two competing hypotheses regarding the fate of embryonic germ layers during this metamorphosis. In one scenario, the original endoderm undergoes apoptosis and is replaced by a secondary endoderm derived from ectodermal cells. In the second scenario, both ectoderm and endoderm remain intact through development. In this study, we performed a pulse-chase experiment to trace the fate of larval ectodermal cells...
August 18, 2016: Development Genes and Evolution
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