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531.
Live Imaging of Glucose Homeostasis in Nuclei of COS-7 Cells 总被引:3,自引:0,他引:3
Measuring subcellular glucose levels deep in tissues can provide new insights into compartmentalization and specialization of glucose metabolism among different cells. As shown previously, a FRET-based glucose-sensor consisting of two GFP-variants and the Escherichia coli periplasmic glucose/galactose binding protein was successfully expressed in the cytosol of COS7-cells and used to determine cytosolic glucose levels. Recording cytosolic fluorescence intensities in cells located in deeper layers of tissues is often difficult due to loss of signal intensity caused by effects of other cell layers on excitation and emission light. These interfering effects may be reduced by restricting fluorophores to occupy only a fraction of the assayed tissue volume. This can be accomplished by confining fluorophores to a sub-compartment of each cell in the tissue, such as the nucleus. The glucose-sensor was targeted to nuclei of COS7-cells. To determine, whether nuclear glucose levels can be used to track cytosolic changes, nuclear glucose concentrations were quantified as the cells were challenged with external glucose over a range of 0.5 to 10 mM and compared to cytosolic levels. Internal glucose concentrations in both compartments were similar, corresponding to approximately 50% of the external concentration. Taken together, these results indicate that nuclear glucose levels can be used to determine cytosolic levels indirectly, permitting more reliable quantification of fluorescence intensities and providing a tool for measurements not only in cell cultures but also in tissues. 相似文献
532.
Fluorescence Resonance Energy Transfer (FRET) is a powerful tool to determine distances between chromophores bound to macromolecules, since the efficiency of the energy transfer from an initially excited donor to an acceptor strongly depends on the distance between the two dye molecules. The structure of the noncovalent complex of double-strand DNA (dsDNA) with thiazol orange dimers (TOTO) allows FRET analysis of two intercalated chromophores. By intercalation of two different TOTO dyes we observe an energy transfer from TOTO-1 as donor and TOTO-3 as acceptor. In this manner we are able to determine the mean distance between two proximate TOTO molecules bound to dsDNA. Thus the maximum number of binding positions for this type of intercalation dyes in the dsDNA can be obtained. Furthermore the dependency of the acceptor emission on the donor concentration is analysed. The emission of TOTO-3 reaches a maximum when the acceptor-to-donor ratio is 1:10. 相似文献
533.
A practical high-throughput protein detection system is described, based on synthetic peptide arrays consisting of designed alpha-helical peptides, detected by fluorescence resonance energy transfer (FRET). Initially a model alpha-helical peptide known to interact with a structured protein, calmodulin, was selected to establish the strategy for high-throughput detection. In comparison to peptides with a single probe, a much higher FRET response has been observed with two fluorescent probes (7-diethylaminocoumarin-3-carboxylic acid and 5(6)-carboxy-fluorescein) at both termini of the synthetic peptides. To establish a reproducible high-throughput detection system, peptides were also immobilized onto a solid surface for detection of the target proteins. A small library of 112 different peptides was constructed, based on a model of the alpha-helical peptide with systematic replacement of residues carrying specific charges and/or hydrophobicities. The library was used to effectively characterize various proteins, giving their own 'protein fingerprint' patterns. The resulting 'protein fingerprints' correlate with the recognition properties of the proteins. The present microarray with designed synthetic peptides as the capturing agents is promising for the development of protein detection chips. 相似文献
534.
Hong Hai Tomoo Miura Tomoyoshi Kobayashi Yuichiro Maéda Masao Miki 《Journal of fluorescence》2000,10(2):193
Contraction of vertebrate striated muscle is regulated by the strong Ca2+-dependent interaction among troponin (Tn), tropomyosin (Tm), and actin on the thin filament. Using fluorescence resonance energy transfer (FRET), the interactions between Tm and the Tn complex or between Tm and the Tn subunit, TnI or TnC, with or without other troponin subunits, were characterized in the presence or absence of F-actin and Ca2+ ions. Cys-190 of Tm was selectively labeled with the acceptor probe, 4-dimethylaminophenylazophenyl 4-maleimide. Troponin was selectively labeled at position 9 or 133 of TnI and position 98 of TnC with a donor probe, 5-(2-iodoacetylaminoethyl)aminonaphtha lene 1-sulfonic acid. FRET measurements indicate that the interaction between TnI and Tm alone is very weak, but that in the presence of F-actin, TnI binds to the proper binding site on Tm even in the absence of TnT. The distances between Cys-190 of Tm on F-actin and Cys-9 or Cys-133 of Tnl or Cys-98 of TnC in the reconstituted Tn were determined to be 52.8, 53.7, Å and 56.5 Å, respectively, in the absence of Ca2+, indicating that the Tnl—TnC complex, the globular portion of Tn, is located near Cys-190 of Tm on the reconstituted thin filaments. Upon binding of Ca2+ to TnC, these distances increased by 5.6 and 1.4 Å or decreased by 5.4 Å, respectively. These Ca2+-induced changes in Tn—Tm seem to occur only when F-actin is present, suggesting that the stable complex formation of TnI with the outer domain of F-actin upon removal of Ca2+ is a very important event during inhibition. 相似文献
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537.
By using a facile, wet-chemical approach, luminescent LaF3:Ce3+/Tb3+ single-crystal nanoparticles were prepared from nitrate and sodium fluoride precursors in a mixture of ethanol and ethylene glycol. These nanoparticles were functionalized with glucose. A novel fluorescence resonance energy transfer method for nonenzymatic glucose determination has been developed by using these glucose-modified nanocrystals. Under the chosen conditions, concentrations of glucose between 0.5 and 25.0 mmol L-1 in aqueous solutions were successfully determined. Owing to their high luminescence and good dispersibility in water, these nanocrystals are also potential fluorescent biolabels for other biological and clinical applications, such as in fluorescence imaging and for immunoassays. 相似文献
538.
Roberto J. Brea María Jesús Pérez‐Alvite Michele Panciera Prof. Dr. Manuel Mosquera Prof. Dr. Luis Castedo Prof. Dr. Juan R. Granja 《化学:亚洲杂志》2011,6(1):110-121
Cyclic octapeptides composed of α‐amino acids alternated with cis‐3‐aminocycloalkanecarboxylic acids, self‐assemble as drumlike dimers through β‐sheet‐like, backbone‐to‐backbone hydrogen bonding. Heterodimerization appears to be significantly more favored than homodimerization, and this represents a novel approach for the design and fabrication of highly stable heterodimeric assemblies. A multicomponent equilibrium network based on fluorescently derivatized self‐assembling α,γ‐cyclic octapeptides has been successfully used to form light‐harvesting/light‐converting ensembles with a distinctive organization of donor and acceptor units able to act as efficient artificial photosystems. 相似文献
539.
A new dimethylaminocinnamaldehyde linked rhodamine based fluorescence receptor 3 is synthesized which shows fluorescence resonance energy transfer in the presence of Fe2+ ions, thus enhancing rational partition in between donor and acceptor emissions and permitting separated measurement of emissions of both fluorophores. 相似文献
540.
Adrian Alvarez José M. Costa-Fernández Rosario Pereiro Alfredo Sanz-Medel Alfonso Salinas-Castillo 《Trends in analytical chemistry : TRAC》2011,30(9):1513-1525
This review deals with the emerging field of fluorescent conjugated polymers for the development of chemical and/or biochemical sensors. As a result of their amplified physical properties due to a “molecular wire effect”, these materials offer excellent characteristics to develop different sensing schemes (e.g., employing direct superquenching or relying on development of fluorescence-resonance-energy-transfer formats). The versatility of their synthesis procedures allows us to introduce the desired functional groups to achieve analytically useful interactions with analytes [e.g., from transition-metal ions to explosives, or even, in recent years, relevant biomolecules (e.g., proteins or DNA, where conformational changes play a decisive role in detection)]. 相似文献