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https://www.readbyqxmd.com/read/28092053/sh2-domain-based-fret-biosensor-for-measuring-bcr-abl-activity-in-living-cml-cells
#1
Mari Fujioka, Yumi Asano, Shigeyuki Nakada, Yusuke Ohba
Fluorescent proteins (FPs) displaying distinct spectra have shed their light on a wide range of biological functions. Moreover, sophisticated biosensors engineered to contain single or multiple FPs, including Förster resonance energy transfer (FRET)-based biosensors, spatiotemporally reveal the molecular mechanisms underlying a variety of pathophysiological processes. However, their usefulness for applied life sciences has yet to be fully explored. Recently, our research group has begun to expand the potential of FPs from basic biological research to the clinic...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28091627/development-of-a-single-step-immunoassay-microdevice-based-on-a-graphene-oxide-containing-hydrogel-possessing-fluorescence-quenching-and-size-separation-functions
#2
Akihiro Shirai, Kaho Nakashima, Kenji Sueyoshi, Tatsuro Endo, Hideaki Hisamoto
An immunoassay, which is an indispensable analytical method both in biological research and in medical fields was successfully integrated into a "single-step" by developing a microdevice composed of a graphene oxide (GO)-containing hydrogel and a poly (dimethylsiloxane) (PDMS) microchannel array with a polyethylene glycol (PEG) coating containing a fluorescently-labelled antibody. Here we used 2-hydroxyethylmethacrylate (HEMA) as a monomer that is easily, and homogeneously, mixed with GO to synthesize the hydrogel...
January 16, 2017: Analyst
https://www.readbyqxmd.com/read/28090611/performance-of-nano-assembly-logic-gates-with-a-dna-multi-hairpin-motif
#3
Lulu Zhang, Annie M Bluhm, Kuan-Jen Chen, Nicholas E Larkey, Sean M Burrows
DNA nano-assemblies have far-reaching implications for molecular computers. Boolean logic gates made from DNA respond to specific combinations of chemical or molecular inputs. In complex samples an assortment of other chemicals and molecules may interfere with the gate's recognition and response mechanisms. For logic gates to accept an increasing number of inputs, while maintaining selectivity, their design must only respond when specific input combinations are available simultaneously. Here we present proof-of-principle for a fluorescent-based nano-assembly logic gate for three inputs...
January 16, 2017: Nanoscale
https://www.readbyqxmd.com/read/28087633/the-abl-pathway-bifurcates-to-balance-enabled-and-rac-signaling-in-axon-patterning-in-drosophila
#4
Ramakrishnan Kannan, Jeong-Kuen Song, Tatiana Karpova, Akanni Clarke, Madhuri Shivalkar, Benjamin Wang, Lyudmila Kotlyanskaya, Irina Kuzina, Qun Gu, Edward Giniger
The Abl tyrosine kinase signaling network controls cell migration, epithelial organization, axon patterning and other aspects of development. While individual components are known, the relationships among them remain mysterious. We now use FRET measurements of pathway activity, analysis of protein localization and genetic epistasis to dissect the structure of this network in Drosophila We find that the adaptor protein Disabled stimulates Abl kinase activity. Abl suppresses the actin regulatory factor Enabled, and we find that Abl also acts through the GEF Trio to stimulate the signaling activity of Rac GTPase: Abl gates the activity of the spectrin repeats of Trio, allowing them to relieve intramolecular repression of Trio GEF activity by the Trio N-terminal domain...
January 13, 2017: Development
https://www.readbyqxmd.com/read/28081500/a-highly-sensitive-quantum-dots-dna-nanobiosensor-based-on-fluorescence-resonance-energy-transfer-for-rapid-detection-of-nanomolar-amounts-of-human-papillomavirus-18
#5
Mojtaba Shamsipur, Vahid Nasirian, Kamran Mansouri, Ali Barati, Asad Veisi-Raygani, Soheila Kashanian
A very sensitive and convenient nanobiosensor based on fluorescence resonance energy transfer (FRET) was developed for the detection of a 22-mer oligonucleotides sequence in Human Papillomavirus 18 virus (HPV18) gene. For this purpose, water-soluble CdTe quantum dots (QDs) were synthesized and, subsequently, amino-modified 11-mer oligonucleotide as one of the two necessary probes was attached to QDs surface to form functional QDs-DNA conjugates. Right after addition of the QDs-DNA and a second Cyanine5 (Cy5)-labeled 11-mer oligonucleotide probe to the DNA target solution, the sandwiched hybrids were formed...
January 3, 2017: Journal of Pharmaceutical and Biomedical Analysis
https://www.readbyqxmd.com/read/28081290/membrane-microdomains-and-the-cytoskeleton-constrain-athir1-dynamics-and-facilitate-the-formation-of-an-athir1-associated-immune-complex
#6
Xueqin Lv, Yanping Jing, Jianwei Xiao, Yongdeng Zhang, Yingfang Zhu, Russell Julian, Jinxing Lin
Arabidopsis hypersensitive induced reaction (AtHIR) proteins function in plant innate immunity. However, the underlying mechanisms by which AtHIRs participate in plant immunity remain elusive. Here, using VA-TIRFM and FLIM-FRET, we revealed that AtHIR1 is present in membrane microdomains and co-localizes with the membrane microdomain marker REM1.3. Single-particle tracking analysis revealed that membrane microdomains and the cytoskeleton, especially microtubules, restrict the lateral mobility of AtHIR1 at the plasma membrane and facilitate its oligomerization...
January 12, 2017: Plant Journal: for Cell and Molecular Biology
https://www.readbyqxmd.com/read/28071848/gxxxg-mediated-parallel-and-antiparallel-dimerization-of-transmembrane-helices-and-its-inhibition-by-cholesterol-single-pair-fret-and-2d-ir-studies
#7
Yoshiaki Yano, Kotaro Kondo, Yuta Watanabe, Tianqi O Zhang, Jia-Jung Ho, Shinya Oishi, Nobutaka Fujii, Martin T Zanni, Katsumi Matsuzaki
Small-residue-mediated interhelical packings are ubiquitously found in helical membrane proteins, although their interaction dynamics and lipid dependence remain mostly uncharacterized. We used a single-pair FRET technique to examine the effect of a GXXXG motif on the association of de novo designed (AALALAA)3 helices in liposomes. Dimerization occurred with sub-second lifetimes, which was abolished by cholesterol. Utilizing the nearly instantaneous time-resolution of 2D IR spectroscopy, parallel and antiparallel helix associations were identified by vibrational couplings across helices at their interface...
January 10, 2017: Angewandte Chemie
https://www.readbyqxmd.com/read/28068385/the-arginine-pairs-and-c-termini-of-the-sso7c4-from-sulfolobus-solfataricus-participate-in-binding-and-bending-dna
#8
Bo-Lin Lin, Chin-Yu Chen, Chun-Hsiang Huang, Tzu-Ping Ko, Cheng-Hung Chiang, Kuan-Fu Lin, Yuan-Chih Chang, Po-Yen Lin, Hui-Hsu Gavin Tsai, Andrew H-J Wang
The Sso7c4 from Sulfolobus solfataricus forms a dimer, which is believed to function as a chromosomal protein involved in genomic DNA compaction and gene regulation. Here, we present the crystal structure of wild-type Sso7c4 at a high resolution of 1.63 Å, showing that the two basic C-termini are disordered. Based on the fluorescence polarization (FP) binding assay, two arginine pairs, R11/R22' and R11'/R22, on the top surface participate in binding DNA. As shown in electron microscopy (EM) images, wild-type Sso7c4 compacts DNA through bridging and bending interactions, whereas the binding of C-terminally truncated proteins rigidifies and opens DNA molecules, and no compaction of the DNA occurs...
2017: PloS One
https://www.readbyqxmd.com/read/28067372/selective-turn-on-and-modulation-of-resonant-energy-transfer-in-single-plasmonic-hybrid-nanostructures
#9
Łukasz Bujak, Tatsuya Ishii, Dharmendar Kumar Sharma, Shuzo Hirata, Martin Vacha
Förster resonant energy transfer (FRET) is a nonradiative process by which the energy of light absorbed by a donor molecule is transferred to an acceptor molecule over a distance of several nanometers. FRET plays a crucial role in photosynthesis and nature-inspired artificial light-harvesting systems that are being explored for use in energy conversion applications. Localized plasmons of metal nanoparticles can potentially lead to a significant increase of FRET efficiency and effective donor-acceptor distance...
January 9, 2017: Nanoscale
https://www.readbyqxmd.com/read/28065272/activity-assays-for-rhomboid-proteases
#10
E Arutyunova, K Strisovsky, M J Lemieux
Rhomboids are ubiquitous intramembrane serine proteases that are involved in various signaling pathways. This fascinating class of proteases harbors an active site buried within the lipid milieu. High-resolution structures of the Escherichia coli rhomboid GlpG with various inhibitors revealed the catalytic mechanism for rhomboid-mediated proteolysis; however, a quantitative characterization was lacking. Assessing an enzyme's catalytic parameters is important for understanding the details of its proteolytic reaction and regulatory mechanisms...
2017: Methods in Enzymology
https://www.readbyqxmd.com/read/28064103/why-do-some-titanium-alloy-total-hip-arthroplasty-modular-necks-fail
#11
Samo K Fokter, Andrej Moličnik, Rajko Kavalar, Primož Pelicon, Rebeka Rudolf, Nenad Gubeljak
BACKGROUND: Increased modularity in total hip arthroplasty (THA) through extra junction between the neck and the femoral stem is gaining popularity among orthopaedic community. However, the advantage of the additional junction is shadowed by an increased risk of mechanical failure. The aim of this study was to describe the exact mechanism of fracture of the modular femoral neck in an uncemented stem. METHODS: Clinical, metallurgical, and mechanical analysis including finite-element modelling and elemental-sensitive tissue analysis with the micro-PIXE method was performed on two patients treated with fully modular primary THA made from Titanium alloy of the same oval taper-cone design...
December 27, 2016: Journal of the Mechanical Behavior of Biomedical Materials
https://www.readbyqxmd.com/read/28063999/relationship-between-intracellular-ph-metabolic-co-factors-and-caspase-3-activation-in-cancer-cells-during-apoptosis
#12
Tatiana F Sergeeva, Marina V Shirmanova, Olga A Zlobovskaya, Alena I Gavrina, Varvara V Dudenkova, Maria M Lukina, Konstantin A Lukyanov, Elena V Zagaynova
A complex cascade of molecular events occurs in apoptotic cells but cell-to-cell variability significantly complicates determination of the order and interconnections between different processes. For better understanding of the mechanisms of programmed cell death, dynamic simultaneous registration of several parameters is required. In this paper we used multiparameter fluorescence microscopy to analyze energy metabolism, intracellular pH and caspase-3 activation in living cancer cells in vitro during staurosporine-induced apoptosis...
January 4, 2017: Biochimica et Biophysica Acta
https://www.readbyqxmd.com/read/28063492/interfacial-enzymes-membrane-binding-orientation-membrane-insertion-and-activity
#13
S A Tatulian
Most interfacial enzymes undergo activation upon membrane binding. Interfacial activation is determined not only by the binding strength but also by the specific mode of protein-membrane interactions, including the angular orientation and membrane insertion of the enzymes. This chapter describes biophysical techniques to quantitatively evaluate membrane binding, orientation, membrane insertion, and activity of secreted phospholipase A2 (PLA2) and lipoxygenase (LO) enzymes. Procedures for recombinant production and purification of human pancreatic PLA2 and human 5-lipoxygenase (5-LO) are also presented...
2017: Methods in Enzymology
https://www.readbyqxmd.com/read/28063388/nanostructured-biosensor-for-detecting-glucose-in-tear-by-applying-fluorescence-resonance-energy-transfer-quenching-mechanism
#14
Longyi Chen, Wai Hei Tse, Yi Chen, Matthew W McDonald, James Melling, Jin Zhang
In this paper, a nanostructured biosensor is developed to detect glucose in tear by using fluorescence resonance energy transfer (FRET) quenching mechanism. The designed FRET pair, including the donor, CdSe/ZnS quantum dots (QDs), and the acceptor, dextran-binding malachite green (MG-dextran), was conjugated to concanavalin A (Con A), an enzyme with specific affinity to glucose. In the presence of glucose, the quenched emission of QDs through the FRET mechanism is restored by displacing the dextran from Con A...
December 23, 2016: Biosensors & Bioelectronics
https://www.readbyqxmd.com/read/28062602/methods-used-to-study-the-oligomeric-structure-of-g-protein-coupled-receptors
#15
Hui Guo, Su An, Richard J Ward, Yang Yang, Ying Liu, Xiao-Xi Guo, Qian Hao, Tian-Rui Xu
G-protein coupled receptors (GPCRs), which constitute the largest family of cell surface receptors, were originally thought to function as monomers, but are now recognized as being able to act in a wide range of oligomeric states and indeed, it is known that the oligomerization state of a GPCR can modulate its pharmacology and function. A number of experimental techniques have been devised to study GPCR oligomerization including those based upon traditional biochemistry such as blue-native polyacrylamide gel-electrophoresis (BN-PAGE), co-immunoprecipitation and protein-fragment complementation assays, those based upon resonance energy transfer, fluorescence resonance energy transfer (FRET), time-resolved FRET, FRET spectrometry and bioluminescence resonance energy transfer (BRET)...
January 6, 2017: Bioscience Reports
https://www.readbyqxmd.com/read/28060890/developing-and-testing-a-bayesian-analysis-of-fluorescence-lifetime-measurements
#16
Bryan Kaye, Peter J Foster, Tae Yeon Yoo, Daniel J Needleman
FRET measurements can provide dynamic spatial information on length scales smaller than the diffraction limit of light. Several methods exist to measure FRET between fluorophores, including Fluorescence Lifetime Imaging Microscopy (FLIM), which relies on the reduction of fluorescence lifetime when a fluorophore is undergoing FRET. FLIM measurements take the form of histograms of photon arrival times, containing contributions from a mixed population of fluorophores both undergoing and not undergoing FRET, with the measured distribution being a mixture of exponentials of different lifetimes...
2017: PloS One
https://www.readbyqxmd.com/read/28060336/screening-for-endocrine-activity-in-water-using-commercially-available-in-vitro-transactivation-bioassays
#17
Alvine C Mehinto, B Sumith Jayasinghe, Darcy R Vandervort, Nancy D Denslow, Keith A Maruya
In vitro transactivation bioassays have shown promise as water quality monitoring tools, however their adoption and widespread application has been hindered partly due to a lack of standardized methods and availability of robust, user-friendly technology. In this study, commercially available, division-arrested cell lines were employed to quantitatively screen for endocrine activity of chemicals present in water samples of interest to environmental quality professionals. A single, standardized protocol that included comprehensive quality assurance/quality control (QA/QC) checks was developed for Estrogen and Glucocorticoid Receptor activity (ER and GR, respectively) using a cell-based Fluorescence Resonance Energy Transfer (FRET) assay...
December 4, 2016: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/28058420/sensitized-zno-nanorod-assemblies-to-detect-heavy-metal-contaminated-phytomedicines-spectroscopic-and-simulation-studies
#18
Damayanti Bagchi, Tuhin Kumar Maji, Samim Sardar, Peter Lemmens, Chinmoy Bhattacharya, Debjani Karmakar, Samir Kumar Pal
The immense pharmacological relevance of the herbal medicine curcumin including anti-cancer and anti-Alzheimer effects, suggests it to be a superior alternative to synthesised drugs. The diverse functionalities with minimal side effects intensify the use of curcumin not only as a dietary supplement but also as a therapeutic agent. Besides all this effectiveness, some recent literature reported the presence of deleterious heavy metal contaminants from various sources in curcumin leading to potential health hazards...
January 6, 2017: Physical Chemistry Chemical Physics: PCCP
https://www.readbyqxmd.com/read/28058293/quantification-of-small-molecule-protein-interactions-using-fret-between-tryptophan-and-the-pacific-blue-fluorophore
#19
Molly M Lee, Blake R Peterson
We report a new method to quantify the affinity of small molecules for proteins. This method is based on Förster resonance energy transfer (FRET) between endogenous tryptophan (Trp) residues and the coumarin-derived fluorophore Pacific Blue (PB). Tryptophan residues are frequently found in proteins near ligand-binding sites, making this approach potentially applicable to a wide range of systems. To improve access to PB, we developed a scalable multigram synthesis of this fluorophore, starting with inexpensive 2,3,4,5-tetrafluorobenzoic acid...
December 31, 2016: ACS Omega
https://www.readbyqxmd.com/read/28055118/fret-in-a-synthetic-flavin-and-bilin-binding-protein
#20
Julian Simon, Aba Losi, Kai-Hong Zhao, Wolfgang Gärtner
The last decade has seen development and application of a large number of novel fluorescence-based techniques that have revolutionized fluorescence microscopy in life sciences. Preferred tags for such applications are genetically encoded fluorescent proteins (FP), mostly derivatives of the green fluorescent protein (GFP). Combinations of FPs with wavelength-separated absorption/fluorescence properties serve as excellent tools for molecular interaction studies, e.g., protein-protein complexes or enzyme-substrate complexes, based on the FRET phenomenon (Förster resonance energy transfer)...
January 5, 2017: Photochemistry and Photobiology
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