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https://www.readbyqxmd.com/read/28453888/live-imaging-of-extracellular-signal-regulated-kinase-and-protein-kinase-a-activities-during-thrombus-formation-in-mice-expressing-biosensors-based-on-f%C3%A3-rster-resonance-energy-transfer
#1
Takuya Hiratsuka, Takeshi Sano, Hisashi Kato, Naoki Komatsu, Masamichi Imajo, Yuji Kamioka, Kenta Sumiyama, Fumiaki Banno, Toshiyuki Miyata, Michiyuki Matsuda
BACKGROUND: Dynamic features of thrombus formation have been visualized by conventional video wide field microscopy or confocal microscopy in live mouse. However, due to technical limitations precise spatiotemporal regulation of intracellular signaling molecule activities, which have been extensively studied in vitro, remains elusive in vivo. OBJECTIVES: By two-photon excitation microscopy of transgenic mice expressing Förster resonance energy transfer (FRET) biosensors for extracellular signal-regulated kinase (ERK) and protein kinase A (PKA), we visualized ERK and PKA activities during thrombus formation in laser-injured subcutaneous arterioles RESULTS: When a core of densely packed platelets was developed, ERK activity was increased from the basal region close to the injured arterioles...
April 28, 2017: Journal of Thrombosis and Haemostasis: JTH
https://www.readbyqxmd.com/read/28453854/the-dynamics-of-the-monomeric-restriction-endonuclease-bcni-during-its-interaction-with-dna
#2
Georgij Kostiuk, Jasmina Dikic, Friedrich W Schwarz, Giedrius Sasnauskas, Ralf Seidel, Virginijus Siksnys
Endonucleases that generate DNA double strand breaks often employ two independent subunits such that the active site from each subunit cuts either DNA strand. Restriction enzyme BcnI is a remarkable exception. It binds to the 5΄-CC/SGG-3΄ (where S = C or G, '/' designates the cleavage position) target as a monomer forming an asymmetric complex, where a single catalytic center approaches the scissile phosphodiester bond in one of DNA strands. Bulk kinetic measurements have previously shown that the same BcnI molecule cuts both DNA strands at the target site without dissociation from the DNA...
April 27, 2017: Nucleic Acids Research
https://www.readbyqxmd.com/read/28448394/in-response
#3
Sam Baete, Gerd Vercruysse, Margot Vander Laenen, Pieter De Vooght, Dimitri Dylst, Jeroen van Melkebeek, Maud Beran, Jan Van Zundert, René Heylen, Willem Boer, Sam Van Boxstael, Tom Fret, Hans Verhelst, Cathy De Deyne, Frank Jans, Pascal Vanelderen
No abstract text is available yet for this article.
April 24, 2017: Anesthesia and Analgesia
https://www.readbyqxmd.com/read/28448057/mixed-primary-cultures-of-murine-small-intestine-intended-for-the-study-of-gut-hormone-secretion-and-live-cell-imaging-of-enteroendocrine-cells
#4
Arianna Psichas, Gwen Tolhurst, Cheryl A Brighton, Fiona M Gribble, Frank Reimann
The gut is the largest endocrine organ of the body, with hormone-secreting enteroendocrine cells located along the length of the gastrointestinal epithelium. Despite their physiological importance, enteroendocrine cells represent only a small fraction of the epithelial cell population and in the past, their characterization has presented a considerable challenge resulting in a reliance on cell line models. Here, we provide a detailed protocol for the isolation and culture of mixed murine small intestinal cells...
April 20, 2017: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/28448008/a-protocol-for-using-f%C3%A3-rster-resonance-energy-transfer-fret-force-biosensors-to-measure-mechanical-forces-across-the-nuclear-linc-complex
#5
Paul T Arsenovic, Kranthidhar Bathula, Daniel E Conway
The LINC complex has been hypothesized to be the critical structure that mediates the transfer of mechanical forces from the cytoskeleton to the nucleus. Nesprin-2G is a key component of the LINC complex that connects the actin cytoskeleton to membrane proteins (SUN domain proteins) in the perinuclear space. These membrane proteins connect to lamins inside the nucleus. Recently, a Förster Resonance Energy Transfer (FRET)-force probe was cloned into mini-Nesprin-2G (Nesprin-TS (tension sensor)) and used to measure tension across Nesprin-2G in live NIH3T3 fibroblasts...
April 11, 2017: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/28447783/real-time-detection-reveals-responsive-co-transcriptional-formation-of-persistent-intramolecular-dna-and-intermolecular-dna-rna-hybrid-g-quadruplexes-stabilized-by-r-loop
#6
Yang Zhao, Jia-Yu Zhang, Zong-Yu Zhang, Tan-Jun Tong, Yu-Hua Hao, Zheng Tan
G-quadruplex (GQ) structures are implicated in important physiological and pathological processes. Millions of GQ-forming motifs are enriched near transcription start sites (TSSs) of animal genes. Transcription can induce formation of GQs which in turn regulates transcription. The kinetics of the formation and persistence of GQs in transcription is crucial for the role they play, but has not yet been explored. We established a method based on the fluorescence resonance energy transfer (FRET) technique to monitor in real-time the co-transcriptional formation and post-transcriptional persistence of GQs in DNA...
April 27, 2017: Analytical Chemistry
https://www.readbyqxmd.com/read/28446610/conformational-changes-at-cytoplasmic-inter-subunit-interactions-control-kir-channel-gating
#7
Shizhen Wang, William F Borschel, Sarah Heyman, Phillip Hsu, Colin G Nichols
The defining structural feature of inward-rectifier potassium (Kir) channels is the unique Kir cytoplasmic domain. Recently, we have shown that salt bridges located at the cytoplasmic domain subunit interfaces (CD-Is) of eukaryotic Kir channels control channel gating via stability of a novel inactivated closed state. The cytoplasmic domains of prokaryotic and eukaryotic Kir channels show similar conformational rearrangements to the common gating ligand, phosphatidylinositol bisphosphate (PIP2), although this exhibits opposite coupling to opening and closing transitions...
April 26, 2017: Journal of Biological Chemistry
https://www.readbyqxmd.com/read/28443815/molecular-mechanism-of-voltage-dependent-potentiation-of-kcnh-potassium-channels
#8
William N Zagotta, Gucan Dai
EAG-like (ELK) voltage-gated potassium channels are abundantly expressed in the brain. These channels exhibit a behavior called voltage-dependent potentiation (VDP), which appears to be a specialization to dampen the hyperexitability of neurons. VDP manifests as a potentiation of current amplitude, hyperpolarizing shift in voltage sensitivity, and slowing of deactivation in response to a depolarizing prepulse. Here we show that VDP of D. rerio ELK channels involves the structural interaction between the intracellular N-terminal eag domain and C-terminal CNBHD...
April 26, 2017: ELife
https://www.readbyqxmd.com/read/28441861/anionic-polymer-and-quantum-dot-excipients-to-facilitate-sirna-release-and-self-reporting-of-disassembly-in-stimuli-responsive-nanocarrier-formulations
#9
Chad T Greco, Jason C Andrechak, Thomas H Epps Iii, Millicent O Sullivan
The incorporation of anionic excipients into polyplexes is a promising strategy for modulating siRNA binding vs. release and integrating diagnostic capabilities; however, specific design criteria and structure-function relationships are needed to facilitate the development of nanocarrier-based theranostics. Herein, we incorporated poly(acrylic acid) (PAA) and quantum dot (QD) excipients into photolabile siRNA polyplexes to increase gene silencing efficiencies by up to 100% and enable self-reporting of nanocarrier disassembly...
April 25, 2017: Biomacromolecules
https://www.readbyqxmd.com/read/28441012/rgb-switchable-porous-electrospun-nanofiber-chemoprobe-filter-prepared-from-multifunctional-copolymers-for-versatile-sensing-of-ph-and-heavy-metals
#10
Fang-Cheng Liang, Chi Ching Kuo, Bo-Yu Chen, Chia-Jung Cho, Chih-Chien Hung, Wen-Chang Chen, Redouane Borsali
Novel red-green-blue (RGB) switchable probes based on fluorescent porous electrospun (ES) nanofibers exhibiting high sensitivity to pH and mercury ions (Hg2+) were prepared with one type of copolymer (poly(methyl methacrylatete-co-1, 8-naphthalimide derivatives-co-rhodamine derivative); poly(MMA-co-BNPTU-co-RhBAM)) by using a single-capillary spinneret. The MMA, BNPTU, and RhBAM moieties were designed to (i) permit formation of porous fibers, (ii) fluoresce for Hg2+ detection, and (iii) fluoresce for pH, respectively...
April 25, 2017: ACS Applied Materials & Interfaces
https://www.readbyqxmd.com/read/28439869/studying-protein-protein-interactions-in-planta-using-advanced-fluorescence-microscopy
#11
Marc Somssich, Rüdiger Simon
The formation of protein complexes through direct protein-protein interaction is essential for virtually every biological process, and accordingly the ability to determine the interaction properties of specific proteins is important to understand these processes. Förster resonance energy transfer (FRET) measurements are state-of-the-art confocal fluorescence microscopy- and imaging-based techniques that allow the analysis of protein interactions in vivo and in planta, in specific compartments of single cells or tissues...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28439254/distinct-conformational-dynamics-of-three-g-protein-coupled-receptors-measured-using-flash-bret-biosensors
#12
Kyla Bourque, Darlaine Pétrin, Rory Sleno, Dominic Devost, Alice Zhang, Terence E Hébert
A number of studies have profiled G protein-coupled receptor (GPCR) conformation using fluorescent biaresenical hairpin binders (FlAsH) as acceptors for BRET or FRET. These conformation-sensitive biosensors allow reporting of movements occurring on the intracellular surface of a receptor to investigate mechanisms of receptor activation and function. Here, we generated eight FlAsH-BRET-based biosensors within the sequence of the β2-adrenergic receptor (β2AR) and compared agonist-induced responses to the angiotensin II receptor type I (AT1R) and the prostaglandin F2α receptor (FP)...
2017: Frontiers in Endocrinology
https://www.readbyqxmd.com/read/28436456/synthesis-and-anti-obesity-effects-in-vivo-of-crotadihydrofuran-c-as-a-novel-ppar%C3%AE-antagonist-from-crotalaria-albida
#13
Qin-Hu Sun, Yu Zhang, Gui-Xin Chou
Crotadihydrofuran C (CC) from the herbs of Crotalaria albida is able to inhibit adipocyte differentiation and lipid accumulation. However, the effects of CC on obesity and metabolic disorders have not yet been elucidated. In our study, the first enantioselective synthesis of the 2-isopropenyl dihydrofuran isoflavone skeleton (CC) is described. The convenient and efficient synthetic protocols developed skilfully solve the problems of the ortho-para directing group and Suzuki coupling reaction using a boronic acid pinacol ester that was more stable and easy to obtain...
April 24, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28435000/context-dependent-intravital-imaging-of-therapeutic-response-using-intramolecular-fret-biosensors
#14
REVIEW
James R W Conway, Sean C Warren, Paul Timpson
Intravital microscopy represents a more physiologically relevant method for assessing therapeutic response. However, the movement into an in vivo setting brings with it several additional considerations, the primary being the context in which drug activity is assessed. Microenvironmental factors, such as hypoxia, pH, fibrosis, immune infiltration and stromal interactions have all been shown to have pronounced effects on drug activity in a more complex setting, which is often lost in simpler two- or three-dimensional assays...
April 18, 2017: Methods: a Companion to Methods in Enzymology
https://www.readbyqxmd.com/read/28433631/detection-of-camp-and-of-pka-activity-in-saccharomyces-cerevisiae-single-cells-using-fluorescence-resonance-energy-transfer-fret-probes
#15
Sonia Colombo, Serena Broggi, Maddalena Collini, Laura D'Alfonso, Giuseppe Chirico, Enzo Martegani
In Saccharomyces cerevisiae the second messenger cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA) play a central role in metabolism regulation, stress resistance and cell cycle progression. To monitor cAMP levels and PKA activity in vivo in single S. cerevisiae cells, we expressed an Epac-based FRET probe and a FRET-based A-kinase activity reporter, which were proven to be useful live-cell biosensors for cAMP levels and PKA activity in mammalian cells. Regarding detection of cAMP in single yeast cells, we show that in wild type strains the CFP/YFP fluorescence ratio increased immediately after glucose addition to derepressed cells, while no changes were observed when glucose was added to a strain that is not able to produce cAMP...
April 19, 2017: Biochemical and Biophysical Research Communications
https://www.readbyqxmd.com/read/28432259/probing-native-metal-ion-association-sites-through-quenching-of-fluorophores-in-the-nucleotide-binding-domains-of-the-abc-transporter-msba
#16
Daiki Tatsumi, Kei Nanatani, Yuto Koike, Kiyoto Kamagata, Satoshi Takahashi, Ayumu Konno, Tadaomi Furuta, Minoru Sakurai, Nobuyuki Uozumi
ATP binding cassette (ABC) transporters are ubiquitously present in prokaryotic and eukaryotic cells. Binding of ATP to the nucleotide binding domains (NBDs) elicits major conformational changes of the transporters resulting in the transport of the substrate across the membrane. The availability of a crystal structure of the NBDs enabled us to elucidate the local structure and small-scale dynamics in the NBDs. Here, we labeled the ABC transporter MsbA, a homodimeric flippase from E. coli , with a fluorescent probe, Alexa532, within the NBDs...
April 21, 2017: Biochemical Journal
https://www.readbyqxmd.com/read/28428126/interactions-of-tetracyclines-with-ovalbumin-the-main-allergen-protein-from-egg-white-spectroscopic-and-electrophoretic-studies
#17
Maria Dayanne de A Dantas, Humberto de Araújo Tenório, Thiago Inácio B Lopes, Hugo Juarez V Pereira, Anita J Marsaioli, Isis M Figueiredo, Josué Carinhanha Caldas Santos
The interactions of tetracycline (TC), oxytetracycline (OTC) and chlortetracycline (CTC) with ovalbumin (OVA), the main allergen protein of egg white, were investigated by molecular spectroscopy and electrophoresis at three pH conditions (1.5, 4.6 and 7.4). Molecular and synchronous fluorescence, UV-vis spectroscopy, electrophoresis and (1)H NMR were used to study the interaction process. Tetracyclines interact with ovalbumin fluorescence by a static quenching mechanism with non-fluorescent complex formation changing the native protein structure...
April 17, 2017: International Journal of Biological Macromolecules
https://www.readbyqxmd.com/read/28425708/revealing-multiple-pathways-in-t4-lysozyme-sub-step-conformational-motions-by-single-molecule-enzymology-and-modeling
#18
Maolin Lu, H Peter Lu
Enzyme conformational dynamics play crucial roles in catalytic functions. Obtaining molecular level insights into the conformational transition dynamics of enzyme-substrate complex from the inactive state to the active state is fundamental for understanding enzymatic function and dynamics. Here, we report our progress on the real-time conformational transition dynamics of T4 lysozyme under enzymatic reactions using single-molecule fluorescence resonance energy transfer. The time duration in forming the active enzyme-substrate complex state (ES*) show distinctive Poisson and Non-Poisson statistics, including exponential and non-exponential, convoluted Poisson distributions and Gaussian-like distributions...
April 20, 2017: Journal of Physical Chemistry. B
https://www.readbyqxmd.com/read/28425435/fret-biosensor-uncovers-camp-nano-domains-at-%C3%AE-adrenergic-targets-that-dictate-precise-tuning-of-cardiac-contractility
#19
Nicoletta C Surdo, Marco Berrera, Andreas Koschinski, Marcella Brescia, Matias R Machado, Carolyn Carr, Peter Wright, Julia Gorelik, Stefano Morotti, Eleonora Grandi, Donald M Bers, Sergio Pantano, Manuela Zaccolo
Compartmentalized cAMP/PKA signalling is now recognized as important for physiology and pathophysiology, yet a detailed understanding of the properties, regulation and function of local cAMP/PKA signals is lacking. Here we present a fluorescence resonance energy transfer (FRET)-based sensor, CUTie, which detects compartmentalized cAMP with unprecedented accuracy. CUTie, targeted to specific multiprotein complexes at discrete plasmalemmal, sarcoplasmic reticular and myofilament sites, reveals differential kinetics and amplitudes of localized cAMP signals...
April 20, 2017: Nature Communications
https://www.readbyqxmd.com/read/28424742/fret-from-single-to-multiplexed-signaling-events
#20
REVIEW
Gertrude Bunt, Fred S Wouters
Förster resonance energy transfer (FRET) is a powerful tool for the visualization of molecular signaling events such as protein activities and interactions in cells. In its different implementations, FRET microscopy has been mainly used for monitoring single events. Recently, there has been a trend of extending FRET imaging towards the simultaneous detection of multiple events and interactions. The concomitant increase in experimental complexity requires a deeper understanding of the biophysical background of FRET...
April 2017: Biophysical Reviews
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