Time-resolved
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We present a method for the determination of the lignan enterolactone in plasma (serum). This compound, produced by intestinal bacteria from matairesinol and secoisolariciresinol in fiber-rich food, is a biomarker related to the intake of a healthy diet. The method is based on time-resolved fluoroimmunoassay using a europium chelate as a label. After synthesis of 5'-O-carboxymethoxyenterolactone the compound is coupled to bovine serum albumin and then used as antigen in immunization of rabbits. The tracer with the europium chelate is synthesized using the same 5'-derivative of enterolactone. After enzymatic hydrolysis and ether extraction the immunoassay is carried out using the VICTOR 1420 multilabel counter (Wallac Oy, Turku, Finland). No antiserum cross-reactivity with available lignans, isoflavonoids, or flavonoids could be detected. The intraassay and interassay coefficients of variation at different concentrations vary 4.6-6.0 and 5.5-9.9, respectively. The working range of the assay is 1.5-540 nmol/liter. We measured enterolactone in serum/plasma of 224 Finnish subjects: 98.8% of the subjects had values <100 nmol/liter, 38.0% had 20-39.9 nmol/liter, and 34.4% had <20 nmol/liter. Copyright 1998 Academic Press.
- MeSH
- fluoroimunoanalýza * metody MeSH
- gama-butyrolakton * analogy a deriváty krev MeSH
- lidé MeSH
- lignany * krev MeSH
- plynová chromatografie s hmotnostně spektrometrickou detekcí MeSH
- senzitivita a specificita MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- práce podpořená grantem MeSH
- Research Support, U.S. Gov't, P.H.S. MeSH
Fluorescence methods are versatile tools for obtaining dynamic and topological information about biomembranes because the molecular interactions taking place in lipid membranes frequently occur on the same timescale as fluorescence emission. The fluorescence intensity decay, in particular, is a powerful reporter of the molecular environment of a fluorophore. The fluorescence lifetime can be sensitive to the local polarity, hydration, viscosity, and/or presence of fluorescence quenchers/energy acceptors within several nanometers of the vicinity of a fluorophore. Illustrative examples of how time-resolved fluorescence measurements can provide more valuable and detailed information about a system than the time-integrated (steady-state) approach will be presented in this review: 1), determination of membrane polarity and mobility using time-dependent spectral shifts; 2), identification of submicroscopic domains by fluorescence lifetime imaging microscopy; 3), elucidation of membrane leakage mechanisms from dye self-quenching assays; and 4), evaluation of nanodomain sizes by time-resolved Förster resonance energy transfer measurements.
During translation, a conserved GTPase elongation factor-EF-G in bacteria or eEF2 in eukaryotes-translocates tRNA and mRNA through the ribosome. EF-G has been proposed to act as a flexible motor that propels tRNA and mRNA movement, as a rigid pawl that biases unidirectional translocation resulting from ribosome rearrangements, or by various combinations of motor- and pawl-like mechanisms. Using time-resolved cryo-EM, we visualized GTP-catalyzed translocation without inhibitors, capturing elusive structures of ribosome•EF-G intermediates at near-atomic resolution. Prior to translocation, EF-G binds near peptidyl-tRNA, while the rotated 30S subunit stabilizes the EF-G GTPase center. Reverse 30S rotation releases Pi and translocates peptidyl-tRNA and EF-G by ~20 Å. An additional 4-Å translocation initiates EF-G dissociation from a transient ribosome state with highly swiveled 30S head. The structures visualize how nearly rigid EF-G rectifies inherent and spontaneous ribosomal dynamics into tRNA-mRNA translocation, whereas GTP hydrolysis and Pi release drive EF-G dissociation.
- MeSH
- aminoacyl-tRNA metabolismus MeSH
- elektronová kryomikroskopie * MeSH
- elongační faktor G chemie metabolismus MeSH
- Escherichia coli chemie metabolismus MeSH
- fosfáty metabolismus MeSH
- guanosintrifosfát chemie metabolismus MeSH
- malé podjednotky ribozomu bakteriální chemie metabolismus MeSH
- messenger RNA metabolismus MeSH
- proteosyntéza MeSH
- ribozomy chemie metabolismus MeSH
- RNA transferová metabolismus MeSH
- vazba proteinů MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
Time-resolved confocal microspectrofluorometry and fluorescence microscopy imaging were applied to monitor the cellular uptake of fluorescent-labeled oligonucleotides (ONs) delivered by a porphyrin molecule. The fate of porphyrin-ON complexes inside living cells has also been monitored. Due to intrinsic fluorescence of the porphyrin and sensitivity of its characteristics to microenvironment, multicomponent analysis of time-resolved fluorescence provides unique information about stability of the porphyrin-ON complexes, ON interactions with their target sequences, and ON and porphyrin distributions after delivery inside the cells. Time-resolved confocal microspectrofluorometry indeed delivers additional information compared with fluorescence confocal microscopy imaging widely employed to study ON uptake.
- MeSH
- antisense oligonukleotidy chemie MeSH
- buněčné jádro metabolismus MeSH
- buňky 3T3 MeSH
- časové faktory MeSH
- financování organizované MeSH
- fluorescence MeSH
- fluorescenční mikroskopie metody přístrojové vybavení MeSH
- kationty MeSH
- konfokální mikroskopie metody přístrojové vybavení MeSH
- lékové transportní systémy MeSH
- melanom experimentální MeSH
- myši MeSH
- oligonukleotidy chemie MeSH
- porfyriny chemie MeSH
- senzitivita a specificita MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- přehledy MeSH
Experimental and theoretical foundations for femtosecond time-resolved circular dichroism (TRCD) spectroscopy of excitonic systems are presented. In this method, the system is pumped with linearly polarized light and the signal is defined as the difference between the transient absorption spectrum probed with left and with right circularly polarized light. We present a new experimental setup with a polarization grating as key element to generate circularly polarized pulses. Herein the positive (negative) first order of the diffracted light is left-(right-)circularly polarized and serves as a probe pulse in a TRCD experiment. The grating is capable of transferring ultrashort broadband pulses ranging from 470 nm to 720 nm into two separate beams with opposite ellipticity. By applying a specific chopping scheme we can switch between left and right circular polarizations and detect transient absorption (TA) and TRCD spectra on a shot-to-shot basis simultaneously. We perform experiments on a squaraine polymer, investigating excitonic dynamics, and we develop a general theory for TRCD experiments of excitonically coupled systems that we then apply to describe the experimental data in this particular example. At a magic angle of 54.7° between the pump-pulse polarization and the propagation direction of the probe pulse, the TRCD and TA signals become particularly simple to analyze, since the orientational average over random orientations of complexes factorizes into that of the interaction with the pump and the probe pulse, and the intrinsic electric quadrupole contributions to the TRCD signal average to zero for isotropic samples. Application of exciton theory to linear absorption and to linear circular dichroism spectra of squaraine polymers reveals the presence of two fractions of polymer conformations, a dominant helical conformation with close interpigment distances that are suggested to lead to short-range contributions to site energy shifts and excitonic couplings of the squaraine molecules, and a fraction of unfolded random coils. Theory demonstrates that TRCD spectra of selectively excited helices can resolve state populations that are practically invisible in TA spectroscopy due to the small dipole strength of these states. A qualitative interpretation of TRCD and TA spectra in the spectral window investigated experimentally is offered. The 1 ps time component found in these spectra is related to the slow part of exciton relaxation obtained between states of the helix in the low-energy half of the exciton manifold. The dominant 140 ps time constant reflects the decay of excited states to the electronic ground state.
- Publikační typ
- časopisecké články MeSH
