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PubMed1999

Rapid detection of the factor V Leiden (1691 G > A) and haemochromatosis (845 G > A) mutation by fluorescence resonance energy transfer (FRET) and real time PCR.

A rapid method based on fluorescence resonance energy transfer (FRET) and real time polymerase chain reaction (PCR) was used to identify the haemochromatosis genotype in 112 individuals and the factor V genotype in 134 individuals. The results were compared with conventional methods based on restriction enzyme digestion of PCR products. The two methods agreed in 244 of the 246 individuals; for the other two individuals, sequencing showed that they had been incorrectly genotyped by the standard method but correctly genotyped by FRET. The simplicity, speed, and accuracy of real time PCR analysis using FRET probes make it the method of choice in the clinical laboratory for genotyping the haemochromatosis and factor V genes.

Computer Systems↗
PubMed2003

The detection of Simian virus 40 in mesotheliomas from New Zealand and England using real time FRET probe PCR protocols.

AIMS: To detect Simian virus 40 (SV40) DNA in mesotheliomas from New Zealand and from England using novel real time FRET probe polymerase chain reaction (PCR) protocols. METHODS: Twenty four mesotheliomas from New Zealand (Central North Island) and 32 mesotheliomas from England (Greater Manchester region) were examined. Two real time FRET probe PCR protocols were optimised and their analytical sensitivity compared using dilutions of SV40 DNA. A conventional SV40 large tumour antigen protocol with detection by probe hybridisation and chemiluminescent Southern blotting was also optimised. RESULTS: Both real time PCR protocols had the same analytical sensitivity, detecting down to 10(-6) pg of SV40 DNA for each reaction, approximately one SV40 copy. All of the 56 mesothelioma samples contained amplifiable beta globin DNA, but none contained amplifiable SV40 DNA with the conventional large T antigen PCR-Southern blotting protocol, or the two real time FRET probe PCR protocols. The positive and negative controls gave the expected results. There was no evidence of inhibition. CONCLUSIONS: There is abundant evidence in the literature for the presence of SV40 in mesotheliomas. However, this study found no evidence of SV40 in mesotheliomas from England and New Zealand. The extensive use of SV40 contaminated polio vaccine in New Zealand does not seem to have resulted in SV40 associated mesotheliomas.

DNA, Viral↗
PubMed2005

FRET-based voltage probes for confocal imaging: membrane potential oscillations throughout pancreatic islets.

Insulin secretion is dependent on coordinated pancreatic islet physiology. In the present study, we found a way to overcome the limitations of cellular electrophysiology to optically determine cell membrane potential (V(m)) throughout an islet by using a fast voltage optical dye pair. Using laser scanning confocal microscopy (LSCM), we observed fluorescence (Förster) resonance energy transfer (FRET) with the fluorescent donor N-(6-chloro-7-hydroxycoumarin-3-carbonyl)-dimyristoylphosphatidyl-ethanolamine and the acceptor bis-(1,3-diethylthiobarbiturate) trimethine oxonol in the plasma membrane of essentially every cell within an islet. The FRET signal was approximately linear from V(m) -70 to +50 mV with a 2.5-fold change in amplitude. We evaluated the responses of islet cells to glucose and tetraethylammonium. Essentially, every responding cell in a mouse islet displayed similar time-dependent changes in V(m). When V(m) was measured simultaneously with intracellular Ca2+, all active cells showed tight coupling of V(m) to islet cell Ca2+ changes. Our findings indicate that FRET-based, voltage-sensitive dyes used in conjunction with LSCM imaging could be extremely useful in studies of excitation-secretion coupling in intact islets of Langerhans.

Animals↗
PubMed2002

A comparison of ALPHAScreen, TR-FRET, and TRF as assay methods for FXR nuclear receptors.

New developments in detection technologies are providing a variety of biomolecular screening strategies from which to choose. Consequently, we performed a detailed analysis of both separation-based and non-separation-based formats for screening nuclear receptor ligands. In this study, time-resolved fluorescence resonance energy transfer (TR-FRET), ALPHAScreen, and time-resolved fluorescence (TRF) assays were optimized and compared with respect to sensitivity, reproducibility, and miniaturization capability. The results showed that the ALPHAScreen system had the best sensitivity and dynamic range. The TRF assay was more time consuming because of the number of wash steps necessary. The TR-FRET assay had less interwell variation, most likely because of ratiometric measurement. Both the ALPHAScreen and the TR-FRET assays were miniaturized to 8-microl volumes. Of the photomultiplier tube-based readers, the ALPHAScreen reader (ALPHAQuest) presented the advantage of faster reading times through simultaneous reading with four photomultiplier tubes.

Animals↗
PubMed2003

A widely applicable, high-throughput TR-FRET assay for the measurement of kinase autophosphorylation: VEGFR-2 as a prototype.

Homogeneous time-resolved fluorescence resonance energy transfer (TR-FRET) assays represent a highly sensitive and robust high-throughput screening (HTS) method for the quantification of kinase activity. Traditional TR-FRET kinase assays detect the phosphorylation of an exogenous substrate. The authors describe the development and optimization of a TR-FRET technique that measures the autophosphorylation of vascular endothelial growth factor receptor 2 (VEGFR-2) kinase and extend its applicability to a variety of other kinases. The VEGFR-2 assay demonstrated dose-dependent inhibition by compounds known to modulate the catalytic activity of this receptor. In addition, kinetic analysis of a previously characterized VEGFR-2 inhibitor was performed using the method, and results were consistent with those obtained using a different assay format. Because of the known involvement of VEGFR-2 in angiogenesis, this assay should facilitate HTS for antiangiogenic agents. In addition, this general technique should have utility for the screening for inhibitors of kinases as potential therapeutic agents for many other disease indications.

Antibodies↗
PubMed2002

Specificity of DNA triple helix formation analyzed by a FRET assay.

BACKGROUND: A third DNA strand can bind into the major groove of a homopurine duplex DNA to form a DNA triple helix. Sequence specific triplex formation can be applied for gene targeting, gene silencing and mutagenesis. RESULTS: We have analyzed triplex formation of two polypurine triplex forming oligodeoxynucleotides (TFOs) using fluorescence resonance energy transfer (FRET). Under our conditions, the TFOs bind to their cognate double strand DNAs with binding constants of 2.6 x 10(5) and 2.3 x 10(6) M(-1). Our data confirm that the polypurine TFO binds in an antiparallel orientation with respect to the polypurine DNA strand and that triplex formation requires Mg2+ ions whereas it is inhibited by K+ ions. The rate of formation of triple helices is slow with bimolecular rate constants of 5.6 x 10(4) and 8.1 x 10(4) min(-1) M(-1). Triplex dissociation was not detectable over at least 30 hours. Triplex formation is sequence specific; alteration of a single base pair within the 13 base pairs long TFOs prevents detectable triplex formation. CONCLUSION: We have applied a FRET assay to investigate the specificity of DNA triple helix formation. This assay is homogeneous, continuous and specific, because the appearance of the FRET signal is directly correlated to triplex formation. We show that polypurine TFOs bind highly specifically to polypurine stretches in double stranded DNA. This is a prerequisite for biotechnical applications of triple helices to mediate sequence specific recognition of DNA.

DNA↗
PubMed2005

Development of ERK Activity Sensor, an in vitro, FRET-based sensor of Extracellular Regulated Kinase activity.

BACKGROUND: Study of ERK activation has thus far relied on biochemical assays that are limited to the use of phospho-specific antibodies and radioactivity in vitro, and analysis of whole cell populations in vivo. As with many systems, fluorescence resonance energy transfer (FRET) can be utilized to make highly sensitive detectors of molecular activity. Here we introduce FRET-based ERK Activity Sensors, which utilize variants of Enhanced Green Fluorescent Protein fused by an ERK-specific peptide linker to detect ERK2 activity. RESULTS: ERK Activity Sensors display varying changes in FRET upon phosphorylation by active ERK2 in vitro depending on the composition of ERK-specific peptide linker sequences derived from known in vivo ERK targets, Ets1 and Elk1. Analysis of point mutations reveals specific residues involved in ERK binding and phosphorylation of ERK Activity Sensor 3. ERK2 also shows high in vitro specificity for these sensors over two other major MAP Kinases, p38 and pSAPK/JNK. CONCLUSION: EAS's are a convenient, non-radioactive alternative to study ERK dynamics in vitro. They can be utilized to study ERK activity in real-time. This new technology can be applied to studying ERK kinetics in vitro, analysis of ERK activity in whole cell extracts, and high-throughput screening technologies.

Journal Article↗
PubMed2003

Visualization of Rab5 activity in living cells by FRET microscopy and influence of plasma-membrane-targeted Rab5 on clathrin-dependent endocytosis.

Rab5 is a small GTPase that controls endocytosis and early endosome dynamics. To visualize active, GTP-loaded Rab5 in living cells, we developed molecular sensors consisting of the Rab5-binding fragments of Rabaptin5 or EEA.1 fused to yellow fluorescent protein (YFP). Interaction of these sensors with GTP-bound Rab5 fused to cyan fluorescent protein (CFP) resulted in fluorescence resonance energy transfer (FRET) between CFP and YFP. Activated Rab5 was detected by FRET microscopy in endosomal compartments and often concentrated in microdomains in the endosomal membrane. Although the plasma membrane-localized activity of Rab5 was not detected by light microscopy, overexpression of a GDP-bound mutant of CFP-Rab5(S34N) inhibited internalization of the epidermal growth factor receptor by retaining receptors in clathrin-coated pits. To test whether the Rab5(S34N) mutant affects endocytosis directly at the plasma membrane, CFP-Rab5 was fused to the plasma membrane targeting sequence of K-Ras containing a CAAX motif. The resulting chimeric CFP-Rab5-CAAX was located mainly in the plasma membrane and was capable of binding GTP as judged by FRET microscopy with the Rabaptin5-based sensor. Interestingly, EEA.1 sensor did not follow activated Rab5-CAAX to the plasma membrane, suggesting that the interaction of EEA.1 with Rab5 plays a secondary role in EEA.1 targeting. Overexpression of CFP-Rab5(S34N)CAAX prevented endocytosis of receptors by retaining them in coated pits. These data suggest that the dominant-negative effect of the Rab5(S34N) mutant on the late stages of endocytosis can be mediated through the inhibition of cytosol-associated or plasma-membrane-associated rather than endosome-associated regulators of Rab proteins.

Animals↗
PubMed2004

Microscopic analysis of fluorescence resonance energy transfer (FRET).

We describe here detailed practical procedures for implementing various approaches of fluorescence resonance energy transfer (FRET) in microscope-based measurements. A comprehensive theoretical formalism is developed and the different experimental procedures are outlined. A step-by-step protocol is provided for preparing the specimens for FRET measurements, data acquisition procedures, analysis, and quantification. Particular emphasis is given to exemplify the FRET applications in the study of protein-protein interactions.

Animals↗
PubMed1993

[Fretting in modular design implant systems].

Fretting is a problem of load-bearing implants (hip, knee) especially when modular in design and employing different materials. In contrast, fretting has not yet been observed in dental implants. In the case of ceramic femoral heads and cups (e.g. BIOLOX) no problems with fretting have so far been reported. The state-of-the-art is discussed and assessed.

Biomechanical Phenomena↗
PubMed2004

Adenovirus type-5 entry and disassembly followed in living cells by FRET, fluorescence anisotropy, and FLIM.

We have used fluorescence resonance energy transfer (FRET) to follow the process of capsid disassembly for adenovirus (Ad) serotype 5 (Ad5) in living CHO-CAR cells. Ad5 were weakly labeled on their capsid proteins with FRET donor and acceptor fluorophores. A progressive decrease in FRET efficiency recorded during Ad5 uptake revealed that the time course of Ad5 capsid disassembly has two sequential protein dissociation rates with half-times of 3 and 60 min. Fluorescence anisotropy measurements of the segmental motions of fluorophores on Ad5 indicate that the first rate is linked to the detachment from the capsid of the protruding, flexible fiber proteins. The second rate was shown to report on the combined dissociation of protein IX, penton base, and hexons, which form the rigid icosahedral capsid shell. Fluorescence lifetime imaging microscopy measurements using a pH-sensitive probe provided information on the pH of the microenvironment of Ad5 particles during intracellular trafficking, and confirmed that the fast fiber dissociation step occurred at the onset of endocytosis. The slower dissociation phase was shown to coincide with the escape of Ad5 from endocytic compartments into the cytosol, and its arrival at the nuclear membrane. These results demonstrate a rapid, quantitative live-cell assay for the investigation of virus-cell interactions and capsid disassembly.

Adenoviruses, Human↗
PubMed2005

Identification of plasma membrane macro- and microdomains from wavelet analysis of FRET microscopy.

In this study, we sought to characterize functional signaling domains by applying the multiresolution properties of the continuous wavelet transform to fluorescence resonance energy transfer (FRET) microscopic images of plasma membranes. A genetically encoded FRET reporter of protein kinase C (PKC)-dependent phosphorylation was expressed in COS1 cells. Differences between wavelet coefficient matrices revealed several heterogeneous domains (typically ranging from 1 to 5 microm), reflecting the dynamic balance between PKC and phosphatase activity during stimulation with phorbol-12,13-dibutyrate or acetylcholine. The balance in these domains was not necessarily reflected in the overall plasma membrane changes, and observed heterogeneity was absent when cells were exposed to a phosphatase or PKC inhibitor. Prolonged exposure to phorbol-12,13-dibutyrate and acetylcholine yielded more homogeneous FRET distribution in plasma membranes. The proposed wavelet-based image analysis provides, for the first time, a basis and a means of detecting and quantifying dynamic changes in functional signaling domains, and may find broader application in studying fine aspects of cellular signaling by various imaging reporters.

Adaptor Proteins, Signal Transducing↗
PubMed2004

Using live FRET imaging to reveal early protein-protein interactions during T cell activation.

The emerging challenge for proteomics in general and lymphocyte biology in particular is to understand protein-protein interactions in the dynamic context of the living cell. Particularly interesting are the molecular dynamics of the T cell receptor-CD3 complex and other immunoreceptors in immune synapses. Fluorescence (or Förster) resonance energy transfer (FRET) is one of the few techniques that are capable of giving dynamic information about the nanometer-range proximity between molecules, as opposed to simply the subcellular co-localization that is provided by fluorescence microscopy. Spectral changes in fluorescence intensity and down modulation of donor lifetime are the basis for rapidly developing approaches to real-time FRET imaging. With two-photon excitation, FRET can now be extended to in vivo imaging.

Animals↗
PubMed2006

[Investigation of calculating the FRET efficiency of donor-acceptor pair].

The research on the method of calculating FRET efficiency is important to organic white LED research. In the present article, a new method of calculating the FRET efficiency of donor-acceptor pair by means of emission and excitation spectra was investigated. This method is easy to use to process data, needs only normal experiment equipments and doesn't need the parameters such as fluorescence quantum yield. However it can only be used to calculate comparative result. With this method, the FRET efficiency of fluorescein sodium, alpha-naphthol, bromocresol purple and riboflavin with different concentrations was studied, and the results match the experimental results completely, so this method has proved to be reasonable.

English Abstract↗
PubMed1998

Ultramicrotomy on fretting wear debris.

A TEM-sample preparation method for small amounts of fretting wear debris is presented. After embedding in a resin, the debris are ultramicrotomed to ultra-thin sections. In this way, valuable observation of nanocrystalline fretting wear debris originating from TiN-coatings could be rapidly obtained.

Alloys↗
PubMed2004

Application of spectral imaging microscopy in cytomics and fluorescence resonance energy transfer (FRET) analysis.

BACKGROUND: Specific signal detection has been a fundamental issue in fluorescence microscopy. In the context of tissue samples, this problem has been even more pronounced, with respect to spectral overlap and autofluorescence. METHODS: Recent improvements in confocal laser scanning microscopy combine sophisticated hardware to obtain fluorescence emission spectra on a single-pixel basis and a mathematical procedure called "linear unmixing" of fluorescence signals. By improving both the specificity of fluorescence acquisition and the number of simultaneously detectable fluorochromes, this technique of spectral imaging (SI) allows complex interrelations in cells and tissues to be addressed. RESULTS: In a comparative approach, SI microscopy on a quantitative basis was compared to conventional bandpass (BP) filter detection, demonstrating substantial superiority of SI with respect to detection accuracy and dye combination. An eight-color immunofluorescence protocol for tissue sections was successfully established. Moreover, advanced use of SI in fluorescence resonance energy transfer (FRET) applications using enhanced green fluorescence protein (EGFP) and enhanced yellow fluorescence protein (EYFP) in a confocal set up could be demonstrated. CONCLUSIONS: This novel technology will help to perform complex multiparameter investigations at the cellular level by increasing the detection specificity and permitting simultaneous use of more fluorochromes than with classical techniques based on emission filters. Moreover, SI significantly extends the possibilities for specialized microscopy applications, such as the visualization of macromolecular interactions or conformational changes, by detecting FRET.

Flow Cytometry↗
PubMed2005

Computer program for analyzing donor photobleaching FRET image series.

BACKGROUND: The photobleaching fluorescence resonance energy transfer (pbFRET) technique is a spectroscopic method to measure proximity relations between fluorescently labeled macromolecules using digital imaging microscopy. To calculate the energy transfer values one has to determine the bleaching time constants in pixel-by-pixel fashion from the image series recorded on the donor-only and donor and acceptor double-labeled samples. Because of the large number of pixels and the time-consuming calculations, this procedure should be assisted by powerful image data processing software. There is no commercially available software that is able to fulfill these requirements. METHODS: New evaluation software was developed to analyze pbFRET data for Windows platform in National Instrument LabVIEW 6.1. This development environment contains a mathematical virtual instrument package, in which the Levenberg-Marquardt routine is also included. As a reference experiment, FRET efficiency between the two chains (beta2-microglobulin and heavy chain) of major histocompatibility complex (MHC) class I glycoproteins and FRET between MHC I and MHC II molecules were determined in the plasma membrane of JY, human B lymphoma cells. RESULTS: The bleaching time constants calculated on pixel-by-pixel basis can be displayed as a color-coded map or as a histogram from raw image format. CONCLUSION: In this report we introduce a new version of pbFRET analysis and data processing software that is able to generate a full analysis pattern of donor photobleaching image series under various conditions. .

Algorithms↗
PubMed1994

Cellular uptake of titanium and vanadium from addition of salts or fretting corrosion in vitro.

The use of titanium and titanium-6% aluminum-4% vanadium alloy for dental and orthopedic implants has increased in the last decade. The implants are presumed to be compatible because osseointegration, bony apposition, and cell attachment are known. However, the cellular association of titanium and vanadium have remained unknown. This study examined the uptake of salts or fretting corrosion products. Titanium was not observed to be toxic to the cells. Vanadium was toxic at levels greater than 10 micrograms/mL. The percentage of cellular association of titanium was shown to be about 10 times that of vanadium. The percentage of cellular association of either element was greater from fretting corrosion than from the addition of salts. The presence of vanadium did not affect the cellular uptake of titanium. The presence of titanium decreased the cell association of vanadium.

Alloys↗