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Pulse and chase

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
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
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
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
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
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 p21(CIP) function...
November 2, 2017: Cell Stem Cell
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
Yi-Ru Hong, Chak Hin Lam, Kui-Thong Tan
Although many protein labeling probes have been developed to elucidate the trafficking and turnover processes of cell surface proteins, real-time tracking of intracellular proteins remains a challenging task. Herein, we describe a new design to construct a cell-permeable, photostable, and far-red fluorescent turn-on probe to enable no-wash, organelle-specific, and long-term visualization of intracellular SNAP-tagged proteins in living cells. When the probe was used in dual-color pulse chase labeling experiments to differentiate between preLamin and mature Lamin, our results reveal that the shape of mature Lamin can be altered by the newly synthesized preLamin and that this alteration is progressive, cumulative, and due to a concentration-dependent dominant-negative effect of preLamin...
November 3, 2017: Bioconjugate Chemistry
Terry Brissac, Anukul T Shenoy, LaDonna A Patterson, Carlos J Orihuela
Streptococcus pneumoniae (Spn, the pneumococcus) is the leading cause of community-acquired pneumonia and is now recognized to be a direct contributor to adverse acute cardiac events. During invasive pneumococcal disease, Spn can gain access to the myocardium, kill cardiomyocytes, and form bacteria-filled "microlesions" causing considerable acute and long-lasting cardiac damage. While the molecular mechanisms responsible for bacterial translocation into the heart have been elucidated, the initial interactions of heart-invaded Spn with cardiomyocytes remain unclear...
October 23, 2017: Infection and Immunity
Vahid Farrokhi, Xiaoying Chen, Hendrik Neubert
BACKGROUND: The half-life of target proteins is frequently an important parameter in mechanistic pharmacokinetic and pharmacodynamic (PK/PD) modeling of biotherapeutics. Clinical studies for accurate measurement of physiologically relevant protein turnover can reduce the uncertainty in PK/PD model-based predictions, for example, of the therapeutic dose and dosing regimen in first-in-human clinical trials. METHODS: We used a targeted mass spectrometry work flow based on serial immunoaffinity enrichment of multiple human serum proteins from a [5,5,5-(2)H3]-L-leucine tracer pulse-chase study in healthy volunteers...
October 20, 2017: Clinical Chemistry
Assaf Gal, Sanja Sviben, Richard Wirth, Anja Schreiber, Benedikt Lassalle-Kaiser, Damien Faivre, André Scheffel
Many organisms form minerals from precursor phases that crystallize under strict biological control. The dynamic intracellular processes of formation, transport, and deposition of these precursor phases are challenging to identify. An unusual situation is recently revealed for the calcifying alga Emiliania huxleyi, as the cells contain a compartment filled with a concentrated Ca and P phase but the final calcite crystals, which are nucleated in a different compartment, are P-free. Thus, the connection of the Ca-P-rich pool to the mineralization process remains unclear...
October 2017: Advanced Science (Weinheim, Baden-Wurttemberg, Germany)
Christine N Rodriguez, Hoang Nguyen
Hair follicle stem cells (HFSCs) are noted for their relative quiescence and therefore can be distinguished from other cells by their differential history of cell division. Replicating cells can be labeled by pulsing the animals repeatedly with 5-bromo-2'-deoxyuridine (BrdU) or tritiated thymidine ([(3)H]TdR), thymidine analogs that get incorporated into DNA during DNA synthesis. Because dividing cells dilute the label after each cell division, frequently dividing cells will lose the label over time while slow cycling cells will retain the label and thus are termed label retaining cells (LRCs)...
2018: Methods in Molecular Biology
Alexey Uvarovskii, Christoph Dieterich
Motivation: Metabolic labelling of RNA is a well-established and powerful method to estimate RNA synthesis and decay rates. The pulseR R package simplifies the analysis of RNA-seq count data that emerge from corresponding pulse-chase experiments. Results: The pulseR package provides a flexible interface and readily accommodates numerous different experimental designs. To our knowledge, it is the first publicly available software solution that models count data with the more appropriate negative-binomial model...
October 15, 2017: Bioinformatics
Biao Li, Amanda Sierra, Juan Jose Deudero, Fatih Semerci, Andrew Laitman, Marek Kimmel, Mirjana Maletic-Savatic
BACKGROUND: Adult hippocampal neurogenesis, the process of formation of new neurons, occurs throughout life in the hippocampus. New neurons have been associated with learning and memory as well as mood control, and impaired neurogenesis has been linked to depression, schizophrenia, autism and cognitive decline during aging. Thus, understanding the biological properties of adult neurogenesis has important implications for human health. Computational models of neurogenesis have attempted to derive biologically relevant knowledge, hard to achieve using experimentation...
October 3, 2017: BMC Systems Biology
Eab Gilbert, M K Vickaryous
As for many lizards, the leopard gecko (Eublepharis macularius) can self-detach its tail to avoid predation and then regenerate a replacement. The replacement tail includes a regenerated spinal cord with a simple morphology: an ependymal layer surrounded by nerve tracts. We hypothesized that cells within the ependymal layer of the original spinal cord include populations of neural stem/progenitor cells (NSPCs) that contribute to the regenerated spinal cord. Prior to tail loss, we performed a bromodeoxyuridine pulse-chase experiment and found that a subset of ependymal layer cells (ELCs) were label-retaining after a 140-day chase period...
October 4, 2017: Journal of Comparative Neurology
Vivek Chowdhary, Kun-Yu Teng, Sharda Thakral, Bo Zhang, Cho-Hao Lin, Nissar Wani, Lei Bruschweiler-Li, Xiaoli Zhang, Laura James, Dakai Yang, Norman Junge, Rafael Brüschweiler, William M Lee, Kalpana Ghoshal
Acetaminophen toxicity is a leading cause of acute liver failure (ALF). We found that miRNA-122 (miR-122) is down-regulated in liver biopsy specimens of patients with ALF and in acetaminophen-treated mice. A marked decrease in the primary miR-122 expression occurs in mice on acetaminophen overdose because of suppression of its key transactivators, hepatocyte nuclear factor (HNF)-4α and HNF6. More importantly, the mortality rates of male and female liver-specific miR-122 knockout (LKO) mice were significantly higher than control mice when injected i...
September 28, 2017: American Journal of Pathology
Yong Wang, Shoujun Wang, Alex Rockwood, Bradley M Luther, Reed Hollinger, Alden Curtis, Chase Calvi, Carmen S Menoni, Jorge J Rocca
We demonstrate the generation of 0.85 PW, 30 fs laser pulses at a repetition rate of 3.3 Hz with a record average power of 85 W from a Ti:sapphire laser. The system is pumped by high-energy Nd:glass slab amplifiers frequency doubled in LiB3O5 (LBO). Ultrahigh-contrast λ=400  nm femtosecond pulses were generated in KH2PO4 (KDP) with >40% efficiency. An intensity of 6.5×10(21)  W/cm(2) was obtained by frequency doubling 80% of the available Ti:sapphire energy and focusing the doubled light with an f/2 parabola...
October 1, 2017: Optics Letters
James D P Rhodes, Judith H I Haarhuis, Jonathan B Grimm, Benjamin D Rowland, Luke D Lavis, Kim A Nasmyth
To ensure disjunction to opposite poles during anaphase, sister chromatids must be held together following DNA replication. This is mediated by cohesin, which is thought to entrap sister DNAs inside a tripartite ring composed of its Smc and kleisin (Scc1) subunits. How such structures are created during S phase is poorly understood, in particular whether they are derived from complexes that had entrapped DNAs prior to replication. To address this, we used selective photobleaching to determine whether cohesin associated with chromatin in G1 persists in situ after replication...
September 19, 2017: Cell Reports
Virginia P Ronchi, Elizabeth D Kim, Christopher M Summa, Jennifer M Klein, Arthur L Haas
To understand the mechanism for assembly of Lys(48)-linked polyubiquitin degradation signals, we previously demonstrated that the E6AP/UBE3A ligase harbors two functionally distinct E2∼ubiquitin-binding sites: a high-affinity Site 1 required for E6AP Cys(820)∼ubiquitin thioester formation and a canonical Site 2 responsible for subsequent chain elongation. Ordered binding to Sites 1 and 2 is here revealed by observation of UbcH7∼ubiquitin-dependent substrate inhibition of chain formation at micromolar concentrations...
November 3, 2017: Journal of Biological Chemistry
Kavita Srivastava, Anke Jentsch, Juergen Kreyling, Bruno Glaser, Guido L B Wiesenberg
Temperate ecosystems are susceptible to drought events. The effect of a severe drought (104 days) followed by irrigation on the plant C uptake, its assimilation and input of C in soil were examined using a triple (13)CO2 pulse-chase labelling experiment in model grassland and heathland ecosystems. First (13)CO2 pulse at day 0 of the experiment revealed much higher (13)C tracer uptake for shoots, roots and soil compared to the second pulse (day 44), where all plants showed significantly lower (13)C tracer uptake...
September 15, 2017: Isotopes in Environmental and Health Studies
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