Tuning of donor-acceptor linker in rhodamine-coumarin conjugates leads remarkable solvent dependent FRET efficiency for Al3+ imaging in HeLa cells

2016 ◽  
Vol 234 ◽  
pp. 222-230 ◽  
Author(s):  
Susanta Adhikari ◽  
Sandip Mandal ◽  
Avijit Ghosh ◽  
Subhajit Guria ◽  
Debasis Das
Keyword(s):  
Hela Cells ◽  
Donor Acceptor ◽  
Fret Efficiency

scholarly journals MnO2nanosheet mediated “DD–A” FRET binary probes for sensitive detection of intracellular mRNA

2017 ◽  
Vol 8 (1) ◽  
pp. 668-673 ◽  
Author(s):  
Min Ou ◽  
Jin Huang ◽  
Xiaohai Yang ◽  
Ke Quan ◽  
Yanjing Yang ◽  
...  
Keyword(s):  
Sensitive Detection ◽  
Donor Acceptor ◽  
Fret Efficiency

The donor donor–acceptor (DD–A) FRET model has proven to have a higher FRET efficiency than donor–acceptor acceptor (D–AA), donor–acceptor (D–A), and donor donor–acceptor acceptor (DD–AA) FRET models.

scholarly journals Protein interaction quantified in vivo by spectrally resolved fluorescence resonance energy transfer

2004 ◽  
Vol 385 (1) ◽  
pp. 265-277 ◽  
Author(s):  
Valerică RAICU ◽  
David B. JANSMA ◽  
R. J. Dwayne MILLER ◽  
James D. FRIESEN
Keyword(s):  
Energy Transfer ◽  
Fluorescence Resonance Energy Transfer ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Receptor Protein ◽  
Acceptor Pair ◽  
Donor Acceptor ◽  
Fret Efficiency ◽  
Fluorescence Resonance

We describe a fluorescence resonance energy transfer (FRET)-based method for finding in living cells the fraction of a protein population (αT) forming complexes, and the average number (n) of those protein molecules in each complex. The method relies both on sensitized acceptor emission and on donor de-quenching (by photobleaching of the acceptor molecules), coupled with full spectral analysis of the differential fluorescence signature, in order to quantify the donor/acceptor energy transfer. The approach and sensitivity limits are well suited for in vivo microscopic investigations. This is demonstrated using a scanning laser confocal microscope to study complex formation of the sterile 2 α-factor receptor protein (Ste2p), labelled with green, cyan, and yellow fluorescent proteins (GFP, CFP, and YFP respectively), in budding yeast Saccharomyces cerevisiae. A theoretical model is presented that relates the efficiency of energy transfer in protein populations (the apparent FRET efficiency, Eapp) to the energy transferred in a single donor/acceptor pair (E, the true FRET efficiency). We determined E by using a new method that relies on Eapp measurements for two donor/acceptor pairs, Ste2p–CFP/Ste2p–YFP and Ste2p–GFP/Ste2p–YFP. From Eapp and E we determined αT≈1 and n≈2 for Ste2 proteins. Since the Ste2p complexes are formed in the absence of the ligand in our experiments, we conclude that the α-factor pheromone is not necessary for dimerization.

scholarly journals Energy Transfer in Dendritic Systems Having Pyrene Peripheral Groups as Donors and Different Acceptor Groups

Polymers ◽  
2018 ◽  
Vol 10 (10) ◽  
pp. 1062 ◽  
Author(s):  
Pasquale Porcu ◽  
Mireille Vonlanthen ◽  
Andrea Ruiu ◽  
Israel González-Méndez ◽  
Ernesto Rivera
Keyword(s):  
Energy Transfer ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Fullerene C60 ◽  
Energy Transfer Efficiency ◽  
Transfer Efficiency ◽  
Feature Article ◽  
Donor Acceptor ◽  
Fret Efficiency

In this feature article, a specific overview of resonance energy transfer (FRET) in dendritic molecules was performed. We focused mainly on constructs bearing peripheral pyrene groups as donor moieties using different acceptor groups, such as porphyrin, fullerene C60, ruthenium-bipyridine complexes, and cyclen-core. We have studied the effect of all the different donor-acceptor pairs in the energy transfer efficiency (FRET). In all cases, high FRET efficiency values were observed.

Water-Soluble Conjugated Polyelectrolytes with Molecular Bumper for Efficient FRET Biosensor

2006 ◽  
Vol 965 ◽  
Author(s):  
Han Young Woo ◽  
Doojin Vak ◽  
Guillermo C Bazan
Keyword(s):  
Photophysical Properties ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Structural Difference ◽  
Water Soluble ◽  
Conjugated Polyelectrolytes ◽  
Design Synthesis ◽  
Donor Acceptor ◽  
Thermodynamic Driving Force ◽  
Fret Efficiency

ABSTRACTWe report the design, synthesis and photophysical properties of two new water-soluble conjugated polyelectrolytes, poly(9,9'-bis(6-N,N,N-trimethylammoniumhexyl)fluorene-alt-1,4-(2,5-bis(6-N,N,N-trimethylammoniumhexyloxy))phenylene) tetrabromide (P1i) and poly((10,10'-bis(6-N,N,N-trimethylammoniumhexyl)-10H-spiro(anthracene-9,9'-fluorene))-alt-1,4-(2,5-bis(6-N,N,N-trimethylammoniumhexyloxy))phenylene) tetrabromide (P2i). They have same electronic conjugation but the main structural difference is the presence of the anthracenyl substituent orthogonal to the polymer main backbone in P2i. Fluorescence resonance energy transfer (FRET) from the two polymers as FRET donors to fluorescein-labeled single stranded DNA (ssDNA-Fl) was studied. It is observed the emission from ssDNA-Fl via FRET by excitation of P2i with a FRET efficiency of ∼60%. Fluorescein is not emissive within the ssDNA-Fl/P1i complex. We also observed clear PL quenching (Φ = 0.8 → Φ = 0.27) of fluorescein for P2i after electrostatic complexation with ssDNA-Fl. It suggests a quenching pathway of Fl emission through photo-induced charge transfer (PCT) in the electrostatic complex. Both P1i and P2i have same HOMO-LUMO electronic structures and a similar thermodynamic driving force for either FRET or PCT. It appears that PCT operates to a larger extent with P1i, proposing that the presence of the “molecular bumper” in P2i increases Fl emission by increasing the donor-acceptor distance, which decreases more acutely PCT quenching, relative to FRET.

scholarly journals Myristoylated rhinovirus VP4 protein activates TLR2-dependent proinflammatory gene expression

2019 ◽  
Vol 317 (1) ◽  
pp. L57-L70 ◽  
Author(s):  
J. Kelley Bentley ◽  
Mingyuan Han ◽  
Suraj Jaipalli ◽  
Joanna L. Hinde ◽  
Jing Lei ◽  
...  
Keyword(s):  
Affinity Chromatography ◽  
Mrna Expression ◽  
Hela Cells ◽  
Membrane Surface ◽  
Airway Epithelial Cells ◽  
Mouse Bone Marrow ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells ◽  
Fret Efficiency

Asthma exacerbations are often caused by rhinovirus (RV). We and others have shown that Toll-like receptor 2 (TLR2), a membrane surface receptor that recognizes bacterial lipopeptides and lipoteichoic acid, is required and sufficient for RV-induced proinflammatory responses in vitro and in vivo. We hypothesized that viral protein-4 (VP4), an internal capsid protein that is myristoylated upon viral replication and externalized upon viral binding, is a ligand for TLR2. Recombinant VP4 and myristoylated VP4 (MyrVP4) were purified by Ni-affinity chromatography. MyrVP4 was also purified from RV-A1B-infected HeLa cells by urea solubilization and anti-VP4 affinity chromatography. Finally, synthetic MyrVP4 was produced by chemical peptide synthesis. MyrVP4-TLR2 interactions were assessed by confocal fluorescence microscopy, fluorescence resonance energy transfer (FRET), and monitoring VP4-induced cytokine mRNA expression in the presence of anti-TLR2 and anti-VP4. MyrVP4 and TLR2 colocalized in TLR2-expressing HEK-293 cells, mouse bone marrow-derived macrophages, human bronchoalveolar macrophages, and human airway epithelial cells. Colocalization was absent in TLR2-null HEK-293 cells and blocked by anti-TLR2 and anti-VP4. Cy3-labeled MyrVP4 and Cy5-labeled anti-TLR2 showed an average fractional FRET efficiency of 0.24 ± 0.05, and Cy5-labeled anti-TLR2 increased and unlabeled MyrVP4 decreased FRET efficiency. MyrVP4-induced chemokine mRNA expression was higher than that elicited by VP4 alone and was attenuated by anti-TLR2 and anti-VP4. Cytokine expression was similarly increased by MyrVP4 purified from RV-infected HeLa cells and synthetic MyrVP4. We conclude that, during RV infection, MyrVP4 and TLR2 interact to generate a proinflammatory response.

Platinum Compounds as Stains for Electron Microscopy

1974 ◽  
Vol 32 ◽  
pp. 230-231
Author(s):  
S. K. Aggarwal ◽  
P. McAllister ◽  
R. W. Wagner ◽  
B. Rosenberg
Keyword(s):  
Electron Microscopy ◽  
Hela Cells ◽  
Uranyl Acetate ◽  
Sarcoma 180 ◽  
Platinum Compounds ◽  
Kb Cells ◽  
En Bloc ◽  
Human Lymphoma ◽  
Ultrathin Sections ◽  
Blue Coloration

Uranyl acetate has been used as an electron stain for en bloc staining as well as for staining ultrathin sections in conjunction with various lead stains (Fig. 1). Present studies reveal that various platinum compounds also show promise as electron stains. Certain platinum compounds have been shown to be effective anti-tumor agents. Of particular interest are the compounds with either uracil or thymine as one of the ligands (cis-Pt(II)-uracil; cis-Pt(II)-thymine). These compounds are amorphous, highly soluble in water and often exhibit an intense blue coloration. These compounds show enough electron density to be used as stains for electron microscopy. Most of the studies are based on various cell lines (human AV, cells, human lymphoma cells, KB cells, Sarcoma-180 ascites cells, chick fibroblasts and HeLa cells) while studies on tissue blocks are in progress.

Electron Microscopy of Mycoplasma Pneumoniae

1971 ◽  
Vol 29 ◽  
pp. 258-259 ◽  
Author(s):  
E. S. Boatman ◽  
G. E. Kenny
Keyword(s):  
Electron Microscopy ◽  
Light Microscopy ◽  
Growth Phase ◽  
Hela Cells ◽  
Sulfonic Acid ◽  
Gamma Globulin ◽  
Horse Serum ◽  
Morphological Aspects ◽  
The Family

Information concerning the morphology and replication of organism of the family Mycoplasmataceae remains, despite over 70 years of study, highly controversial. Due to their small size observations by light microscopy have not been rewarding. Furthermore, not only are these organisms extremely pleomorphic but their morphology also changes according to growth phase. This study deals with the morphological aspects of M. pneumoniae strain 3546 in relation to growth, interaction with HeLa cells and possible mechanisms of replication.The organisms were grown aerobically at 37°C in a soy peptone yeast dialysate medium supplemented with 12% gamma-globulin free horse serum. The medium was buffered at pH 7.3 with TES [N-tris (hyroxymethyl) methyl-2-aminoethane sulfonic acid] at 10mM concentration. The inoculum, an actively growing culture, was filtered through a 0.5 μm polycarbonate “nuclepore” filter to prevent transfer of all but the smallest aggregates. Growth was assessed at specific periods by colony counts and 800 ml samples of organisms were fixed in situ with 2.5% glutaraldehyde for 3 hrs. at 4°C. Washed cells for sectioning were post-fixed in 0.8% OSO4 in veronal-acetate buffer pH 6.1 for 1 hr. at 21°C. HeLa cells were infected with a filtered inoculum of M. pneumoniae and incubated for 9 days in Leighton tubes with coverslips. The cells were then removed and processed for electron microscopy.

Use of Uranium-Labeled Antibodies for Electron Microscopic Localization of Various Antigens in Mammalian Cells

1967 ◽  
Vol 25 ◽  
pp. 136-137
Author(s):  
J. P. Petrali ◽  
E. J. Donati ◽  
L. A. Sternberger
Keyword(s):  
Endoplasmic Reticulum ◽  
Hela Cells ◽  
Mammalian Cells ◽  
Specific Antiserum ◽  
Electron Microscopic ◽  
E Coli ◽  
Cytoplasmic Membranes ◽  
Viral Progeny ◽  
Ultrathin Sections ◽  
Electron Microscopic Localization

Specific contrast is conferred to subcellular antigen by applying purified antibodies, exhaustively labeled with uranium under immunospecific protection, to ultrathin sections. Use of Seligman’s principle of bridging osmium to metal via thiocarbohydrazide (TCH) intensifies specific contrast. Ultrathin sections of osmium-fixed materials were stained on the grid by application of 1) thiosemicarbazide (TSC), 2) unlabeled specific antiserum, 3) uranium-labeled anti-antibody and 4) TCH followed by reosmication. Antigens to be localized consisted of vaccinia antigen in infected HeLa cells, lysozyme in monocytes of patients with monocytic or monomyelocytic leukemia, and fibrinogen in the platelets of these leukemic patients. Control sections were stained with non-specific antiserum (E. coli).In the vaccinia-HeLa system, antigen was localized from 1 to 3 hours following infection, and was confined to degrading virus, the inner walls of numerous organelles, and other structures in cytoplasmic foci. Surrounding architecture and cellular mitochondria were unstained. 8 to 14 hours after infection, antigen was localized on the outer walls of the viral progeny, on cytoplasmic membranes, and free in the cytoplasm. Staining of endoplasmic reticulum was intense and focal early, and weak and diffuse late in infection.

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