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PubMedDate not supplied

The brainstem origin of monoaminergic projections to the spinal cord of the North American opossum: a study using fluorescent tracers and fluorescence histochemistry.

The retrograde transport of fluorescent markers has been combined with the glyoxylic acid and Falck-Hillarp techniques to identify the origin of monoamine axons within the spinal cord of the North American opossum. Catecholamine axons arise from neurons located within the ventrolateral medulla, dorsal to the superior olivary complex, within the dorsolateral and rostrolateral pons and within the periventricular nuclei of the hypothalamus. Such neurons are most numerous within the dorsolateral pons where they are found dorsal and lateral to the motor trigeminal nucleus, within the nucleus locus coeruleus pars alpha and adjacent reticular formation as well as within the ventral part of the nucleus locus coeruleus. Neurons containing the fluorescent marker and catecholamines were interspersed with others containing only the injected marker with the possible exception of the nucleus locus coeruleus. Spinal axons of the indoleamine type arise from neurons within the nuclei pallidus, obscurus and magnus raphe, the nucleus reticularis gigantocellularis, the nucleus reticularis gigantocellularis pars ventralis, the nucleus reticularis pontis pars ventralis and the nucleus dorsalis raphe. The latter nucleus only innervates rostral cervical levels. Most of the above areas also contain many non-indoleamine neurons which were labelled by the injected marker. This was particularly true of the nucleus magnus raphe and the adjacent nucleus reticularis points pars ventralis after injections of fluorescent markers into the superficial dorsal horn.

Animals↗
PubMed1991

Analysis of plasma catecholamines by high-performance liquid chromatography with fluorescence detection: simple sample preparation for pre-column fluorescence derivatization.

Analysis of plasma catecholamines (norepinephrine, epinephrine and dopamine) by high-performance liquid chromatography using 1,2-diphenylethylenediamine as a fluorescent reagent is described. We have developed an automatic catecholamine analyser, based on pre-column fluorescence derivatization and column switching. The analysis time for one assay was 15 min. The correlation coefficients of the linear regression equations were greater than 0.9996 in the range 10-10,000 pg/ml. The detection limit, at a signal-to-noise ratio of 3, was 2 pg/ml for dopamine. A new method of sample preparation for the pre-column fluorescence derivatization of plasma catecholamines was used. In order to protect the catecholamines from decomposition, an ion-pair complex between boric acid and the diol group in the catecholamine was formed at a weakly alkaline pH. The stabilities of plasma catecholamines were evaluated at several temperatures. After complex formation, the catecholamines were very stable at 17 degrees C for 8 h, and the coefficients of variation for norepinephrine, epinephrine and dopamine were 1.2, 4.2 and 9.3%, respectively.

Autoanalysis↗
PubMed2004

Multiplexed fluorescence detection in microfabricated devices with both time-resolved and spectral-discrimination capabilities using near-infrared fluorescence.

We examined the feasibility of using a two-color time-resolved detection scheme with microdevices for DNA sequencing applications. A home-built dual-color optical-fiber-based time-resolved near-infrared (IR) fluorescence microscope successfully coupled lifetime discrimination with color discrimination, increasing fluorescence multiplexing capabilities. The instrument was constructed by using two pulsed-diode lasers (680/780-nm excitation) and two avalanche photodiodes as the basic building blocks. The data were processed using electronics configured in a time-correlated single-photon counting format. The use of near-IR fluorescence detection greatly simplified the hardware and allowed low detection limits (< 0.1nM). We examined the separation of a single-base tract on a microchip and compared the performance with that of conventional capillary gel electrophoresis. The microchip was fabricated in glass and contained an effective separation length of 7.0 cm. It was found that, without incorporating a solid-phase reversible immobilization cleanup procedure, the calculated lifetime of the dye label on the microchip was longer and the standard deviation was larger than those of the same sample analyzed using capillary electrophoresis. Using cleanup steps, the accuracy and precision of the measurements improved. Lifetimes of four near-IR dyes (AlexaFluor680, IRD700, IRD800, and IRD40) used in this study were determined to be 986 ps (RSD=2.1%), 1551 ps (RSD=1.8%), 520 ps (RSD=3.3%), and 788 ps (RSD=4.9%), respectively, in a microchannel filled with poly(dimethylacrylamide) (POP-6) gel. The lifetimes calculated using maximum likelihood estimators provided favorable precision on the microchip, where small numbers of photocounts were collected. An M13mp18 template was sequenced on the microchip using a two-color two-lifetime format with POP-6 as the sieving polymer. Read lengths of 294 bp with calling accuracies of 90.8 and 83.7% were achieved in each color channel. The relatively low calling accuracy and the short read length resulted primarily from the short separation channel, which yielded low electrophoretic resolution.

DNA↗
PubMed2006

A substrate for deubiquitinating enzymes based on time-resolved fluorescence resonance energy transfer between terbium and yellow fluorescent protein.

Deubiquitinating enzymes (DUBs) proteolytically cleave ubiquitin from ubiquitinated proteins, and inhibition of DUBs that rescue oncogenic proteins from proteasomal degradation is of emerging therapeutic interest. Recently, USP2 and UCH37 have been shown to deubiquitinate tumor-growth-promoting proteins, and other DUBs have been shown to be overexpressed in cancer cells. Therefore inhibition of DUBs is of interest as a potential therapeutic strategy for treating cancer. DUBs require the presence of properly folded ubiquitin protein in the substrate for efficient proteolysis, which precludes the use of synthetic peptide substrates in DUB activity assays. Because of the requirement for full-length ubiquitin, substrates suitable for use in fluorescent assays to identify or study DUB inhibitors have been difficult to prepare. We describe the development of a time-resolved fluorescence resonance energy transfer (FRET)-based DUB substrate that incorporates full-length ubiquitin that is site-specifically labeled using genetically encoded yellow fluorescent protein (YFP) and a chemically attached terbium donor. The intact substrate shows a high degree of FRET between terbium and YFP, whereas DUB-dependent cleavage leads to a decrease in FRET.

Amino Acid Sequence↗
PubMed2004

Native fluorescence detection of flavin derivatives by microchip capillary electrophoresis with laser-induced fluorescence intensified charge-coupled device detection.

To widen the scope of laser-induced fluorescence (LIF) for detection in microchip capillary electrophoresis (CE), a microchip CE LIF-ICCD (intensified charge-coupled device) system based on a tunable wavelength dye laser pumped by a pico-second pulse nitrogen laser for excitation and a spectrograph with ICCD for detection had developed to demonstrate the enhancement in detection sensitivity by the following three approaches: direct detection of native fluorescence, improvement of signal-to-noise ratio by pulse laser excitation and time delay detection, and selective spectral acquisition by multi-channel detection. Riboflavin, flavin mononucleotide (FMN) and flavin-adenine dinucleotide (FAD) have been selected as they are dietetically important and microchip CE provides a promising onsite detection method. The results indicate a strong effect of wavelength on detection sensitivity and the need to tune wavelength for direct detection. Under optimized conditions (excitation 450 nm, emission 520 nm, gate delay time 45 ns, 20 mM phosphate buffer at pH 7.1), the following results were obtained under static condition: Working ranges (0.6-350 microg/l, r > 0.99), detection limits (0.15-1.0 microg/l) and peak height repeatability (1.8-2.2% R.S.D.), all within the applicability range for body fluids or beverages such as human urine and cow milk. Baseline separation of three flavins was obtained under dynamic condition and the fluorescence spectra acquired assist the identification of alkaline-degraded products of riboflavin. Thus, the capability to check peak purity and identify unknown peaks has been demonstrated.

Electrophoresis, Capillary↗
PubMed1997

Fluorescent recovery after photobleaching (FRAP) of a fluorescent transferrin internalized in the late transferrin endocytic compartment of living A431 cells: theory.

In previous works, other authors characterized a compartment (LCT) of A431 carcinoma cells in which markers of transferrin endocytose had accumulated during a long chase period. This compartment, was essentially formed by large stationary vacuoles. A few small vesicles budded from these vacuoles, rapidly saltated along microtubules and eventually fused with other vacuoles, causing an intracellular transport of the marker bound to the limiting membrane (M. De Brabander, R. Nuygens, H. Geerts, C.R. Hopkins, Cell. Mot. Cytoskel. 9 (1988) 30). In the present paper, we derived the fluorescence recovery after photobleaching (FRAP) of a fluorescent marker of LCT. We assumed that the rate of the intracellular transport of the marker was controlled by the fission-fusion process between vesicles and vacuoles. We showed that the concentration of a bleached fluorescent marker was a decreasing exponential function of the time elapsed from the beginning of the recovery phase. The rate constant of this exponential was equal to the product of the vesicle surface by the number of vesicles which fused with a unit of vacuole surface during one second. If a fraction of the marker spontaneously reactivated itself with a much higher rate constant of reaction than the rate constant of the transport process, the fractional FRAP of the marker was the sum of the fractional FRAP of both processes occurring separately. In a companion paper (F. Azizi, P. Wahl, Biochim. Biophys. Acta 1327 (1997) 75-88), our FRAP experiments will be described and analysed with the mathematical expressions derived in the present paper.

Biological Transport, Active↗
PubMed1985

Picosecond fluorescence of cryptomonad biliproteins. Effects of excitation intensity and the fluorescence decay times of phycocyanin 612, phycocyanin 645, and phycoerythrin 545.

The fluorescence of purified biliproteins (phycocyanin 645, phycocyanin 612, and phycoerythrin 545) from three cryptomonads, Chroomonas species, Hemiselmis virescens, and Rhodomonas lens, and C-phycocyanin from Anacystis nidulans has been time resolved in the picosecond region with a streak camera system having less than or equal to 2-ps jitter. The fluorescence lifetimes of phycocyanins from Chroomonas species and Hemiselmis virescens are 1.5 +/- 0.2 ns and 2.3 +/- 0.2 ns, respectively, regardless of the fluence of the 30 ps, 532-nm excitation pulse. (Fluence [or photons/cm2] = f intensity [photons/cm2s]dt.). In contrast, that of C-phycocyanin is 2.3 +/- 0.2 ns when the excitation fluence is 8.2 X 10(11) photons/cm2 and decreases to a decay approximated by an exponential decay time of 0.65 +/- 0.1 ns at 7.2 X 10(16) photons/cm2. The cryptomonad phycoerythrin fluorescence decay lifetime is also dependent on intensity, having a decay time of 1.5 +/- 0.1 ns at low fluences and becoming clearly biphasic at higher fluences (greater than 10(15) photons/cm2). We interpret the shortening of decay times for C-phycocyanin and phycoerythrin 545 in terms of exciton annihilation, and have discussed the applicability of exciton annihilation theories to the high fluence effects.

Cyanobacteria↗
PubMed1998

Cholesterol distribution in living cells: fluorescence imaging using dehydroergosterol as a fluorescent cholesterol analog.

Cholesterol is an important constituent of most mammalian cell membranes and its concentration in various cellular membranes is tightly regulated. Although there is much information about cholesterol distribution and trafficking in cells, it is primarily derived from indirect measurements, and the results obtained using different approaches are often conflicting. A cholesterol analog that faithfully mimics the properties of cholesterol and can be followed in living cells would thus be very useful. In this study, we report the fluorescence imaging of such an analog, dehydroergosterol (DHE), in living cells. DHE differs from cholesterol in having three additional double bonds and an extra methyl group. In model systems, DHE closely mimics the behavior of native cholesterol. Using triple-labeling studies, we show that DHE colocalizes extensively with endocytosed transferrin, an endocytic recycling compartment marker, and with a marker for the trans-Golgi network, Tac-TGN38. This distribution of DHE is qualitatively similar to that observed when cells are labeled with the fluorescent cholesterol-binding polyene antibiotic, filipin, although there are differences in apparent proportions of DHE and filipin that are localized at the plasma membrane. Another cholesterol derivative, 25-NBD-cholesterol, has a structure that is compromised by the presence of a bulky NBD group and does not distribute to the same organelles as DHE or filipin. In addition, we show in this manuscript that kinetic processes can be followed in living cells by monitoring recovery of DHE fluorescence in a photobleached region over time. Our observations provide evidence for the presence of a large intracellular cholesterol pool in the endocytic recycling compartment and the trans-Golgi network that might play important roles in the trafficking of lipids, lipid-anchored proteins, and transmembrane proteins that preferentially partition into cholesterol-enriched membrane domains. In addition, this intracellular cholesterol pool might be involved in the maintenance of cellular cholesterol homeostasis.

4-Chloro-7-nitrobenzofurazan↗
PubMed2002

Direct monitoring of the expression of the green fluorescent protein-extracellular signal-regulated kinase 2 fusion protein in transfected cells using capillary electrophoresis with laser-induced fluorescence detection.

Proper subcellular localization of the extracellular signal-regulated kinases (ERKs) is important in regulating physiological functions such as proliferation and differentiation in the pheochromocytoma cell line (PC12 cells). Thus, a direct visualization method is necessary to observe ERK localization within the cell or in crude cellular extracts. In this paper, a determination method was established for the detection of ERK2 localization in PC12 cells using green fluorescent protein (GFP) and capillary electrophoresis with laser-induced fluorescence (LIF). GFP as a reporter or labeling tag for gene expression in biochemistry and cell biology was used for the detection of ERK2 localization in PC12 cells. PC12 cells were transfected with GFP-ERK2 plasmid construct that was inserted into a variant GFP gene (enhanced green fluorescent protein), and successfully expressed GFP-ERK2 fusion proteins. GFP-ERK2 fusion proteins were detected within 5 min by CE analysis using an uncoated fused-silica capillary with LIF. Optimum conditions for GFP-ERK2 fusion proteins detection were 100 mM 3-(cyclohexylamino)-1-propanesulfonic acid buffer containing 100 mM sodium dodecylsulfate, pH 11, running at 20 degrees C. This result offers new opportunity in screening for the determination of localization of intracellular components, protein-protein interactions and kinase activity within the cells.

Animals↗
PubMed2001

Microminiaturized immunoassays using quantum dots as fluorescent label by laser confocal scanning fluorescence detection.

An immunoassay readout method based on fluorescent imaging analysis with laser confocal scanning is described. The ZnS-coated CdSe quantum dots (ZnS/CdSe QDs) were linked to a detection antibody. Immunoassay was carried out on a glass chip using a sandwich assay approach, where antibody covalently bound to a glass chip was allowed to capture antigen specially. Afterwards, the detection antibody labeled with QD was allowed to bind selectively to the captured antigen. The fluorescent signals of the sandwich conjugate were detected by a laser confocal scanner. A diode laser was used to excite efficiently the fluorescent signals while bovine serum albumin was used to eliminate nonspecific binding sites. The detection limit of this approach was up to 10(-9) M under current experimental conditions. The specificity of the QDs-labeled immunoglobulin (IgG) was tested by an experiment using goat IgG and human IgG samples. The result was consistent with the binding specificity in a sandwich-type assay. The potential of this method to function as a simple and efficient readout strategy for immunoassay in biochip is discussed.

Animals↗
PubMed1997

A novel use for a carbodiimide compound for the fixation of fluorescent and non-fluorescent calcium indicators in situ following physiological experiments.

The inability to determine the precise intracellular location of non-fluorescent organic calcium chelators such as BAPTA is a persistent problem which has precluded much detailed analysis of the chelators' spatial or temporal dynamics in live cells. Similarly, following physiological experiments with fluorescent indicators like Fura-2, it has often been desirable to maintain the dye within the cell for later analysis by additional histological techniques. Based on chemical considerations, and its prior use in tissue fixation, we examined the water soluble reagent 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) as a potential fixative for diverse calcium chelators. The utility of EDC, but not other common fixatives, was confirmed through electrophysiological means, through a novel ELISA, which exploits anti-BAPTA antibodies to assess the extent and kinetics of fixation; by autoradiography of neurons loaded with [14C]-BAPTA, and by immunocytochemistry and imaging of intracellular BAPTA or Calcium Green in neurons. At concentrations > 0.1 mg/ml, EDC caused virtually instantaneous, irreversible, fixation of > 95% of BAPTA free acid. Fixation of intracellular BAPTA was confirmed in hippocampal brain slices loaded with BAPTA/AM ester, and showed biphasic kinetics consistent with rapid loading and subsequent extrusion of the chelator. Immunocytochemistry on neurons microinjected with BAPTA free acid and the dye Lucifer Yellow showed BAPTA-specific staining which was distributed in the cell similarly to that of the accompanying marker dye. Application of EDC also efficiently fixed in situ analogs of BAPTA such as Calcium Green (a fluorescent Ca2+ indicator) as shown by confocal imaging of EDC-fixed brain slices loaded with this indicator. Taken together, these data show that EDC is an effective, inexpensive and versatile fixative for calcium chelators in diverse cells. The availability of a suitable fixative now makes it possible to determine the distributions of such chelators at both the light and, possibly, the electron microscope level. Two important features of EDC, arise from its specificity for free carboxyl groups. First, the ability to fix, selectively, the chelators but not their AM esters; and, second, its enormous potential as a fixative for the numerous other carboxyl-containing chelators, dyes and pH indicators currently available.

Animals↗
PubMed2003

Purification of adult hepatic progenitor cells using green fluorescent protein (GFP)-transgenic mice and fluorescence-activated cell sorting.

BACKGROUND/AIMS: Recent advances in stem cell research have revealed that hepatic stem/progenitor cells may play an important role in liver development and regeneration. However, a lack of detectable definitive markers in viable cells has hindered their primary culture from adult livers. METHODS: Enzymatically dissociated liver cells from green fluorescent protein (GFP)-transgenic mice, which express GFP highly in liver endodermal cells, were sorted by GFP expression using a fluorescence-activated cell sorter. Sorted cells were characterized, and also low-density cultured for extended periods to determine their proliferation and clonal differentiation capacities. RESULTS: When CD45(-)TER119(-) side-scatter(low) GFP(high) cells were sorted, alpha-fetoprotein-positive immature endoderm-characterized cells, having high growth potential, were present in this population. Clonal analysis and electron microscopic evaluation revealed that each single cell of this population could differentiate not only into hepatocytes, but also into biliary epithelial cells, showing their bilineage differentiation activity. When surface markers were analyzed, they were positive for Integrin-alpha6 and -beta1, but negative for c-Kit and Thy1.1. CONCLUSIONS: Combination of GFP-transgenic mice and fluorescence-activated cell sorting enabled purification of hepatic progenitor cells from adult mouse liver. Further analysis of this population may lead to purification of their human correspondence that would be an ideal cell-source candidate for regenerative medicine.

Age Factors↗
PubMed2004

Investigation into the applicability of the centrifugal microfluidics platform for the development of protein-ligand binding assays incorporating enhanced green fluorescent protein as a fluorescent reporter.

The incorporation of a protein-ligand binding assay into a centrifugal microfluidics platform is described. The platform itself is a disc-shaped polymer substrate, upon which a series of microfluidic channels and reservoirs have been machined. Centrifugal microfluidics platforms require no internal moving parts, and fluid propulsion is achieved solely through rotation of the disc. Fluid flow is controlled by passive valves, the opening of which is dependent on the angular frequency of the rotating platform, the channel dimensions, and the physical properties of the fluid. To evaluate the effectiveness of incorporating a protein-based assay onto the centrifugal microfluidics analytical platform, a class-selective, homogeneous assay for the detection of phenothiazine antidepressants was employed. This class of drugs is known to bind to calmodulin, a calcium binding protein. Specifically, a fusion protein between calmodulin and enhanced green fluorescent protein was utilized. Calmodulin undergoes a conformational change upon binding to phenothiazines that alters the fluorescence properties of the attached fluorescent protein, which can be correlated to the concentration of the drug present. Another important aspect of this work was to study the efficacy of the platform to perform reconstitution assays. To do this, the biological reagent was dried on the platform and rehydrated to carry out the assay. The ability to prealiquot reagents on the platform should enhance its versatility and portability. The integration of protein-based assays in this platform should be useful in the design of analytical systems for high-throughput screening of pharmaceuticals and clinical diagnostics.

Antidepressive Agents↗
PubMed1989

Characterization of the tryptophan fluorescence from sarcoplasmic reticulum adenosinetriphosphatase by frequency-domain fluorescence spectroscopy.

We examined the tryptophan decay kinetics of sarcoplasmic reticulum Ca2+-ATPase using frequency-domain fluorescence. Consistent with earlier reports on steady-state fluorescence intensity, our intensity decays reveal a reproducible and statistically significant 2% increase in the mean decay time due to calcium binding to specific sites involved in enzyme activation. This Ca2+ effect could not be eliminated with acrylamide quenching, which suggests a global effect of calcium on the Ca2+-ATPase, as opposed to a specific effect on a single water-accessible tryptophan residue. The tryptophan anisotropy decays indicate substantial rapid loss of anisotropy, which can be the result of either intramolecular energy transfer or a change in segmental flexibility of the ATPase protein. Energy transfer from tryptophan to TNP-ATP in the nucleotide binding domain, or to IEADANS on Cys-670 and -674, indicates that most tryptophan residues are 30 A or further away from these sites and that this distance is not decreased by Ca2+. In light of known structural features of the Ca2+-ATPase, the tryptophan fluorescence changes are attributed to stabilization of clustered transmembrane helices resulting from calcium binding.

Adenosine Triphosphate↗
PubMed2004

EosFP, a fluorescent marker protein with UV-inducible green-to-red fluorescence conversion.

A gene encoding a fluorescent protein from the stony coral Lobophyllia hemprichii has been cloned in Escherichia coli and characterized by biochemical and biophysical methods. The protein, which we named EosFP, emits strong green fluorescence (516 nm) that changes to red (581 nm) upon near-UV irradiation at approximately 390 nm because of a photo-induced modification involving a break in the peptide backbone next to the chromophore. Single-molecule fluorescence spectroscopy shows that the wild type of EosFP is tetrameric, with strong Forster resonance coupling among the individual fluorophores. We succeeded in breaking up the tetramer into AB and AC subunit dimers by introducing the single point mutations V123T and T158H, respectively, and the combination of both mutations yielded functional monomers. Fusion constructs with a variety of proteins were prepared and expressed in human cells, showing that normal biological functions were retained. The possibility to locally change the emission wavelength by focused UV light makes EosFP a superb marker for experiments aimed at tracking the movements of biomolecules within the living cell.

Animals↗
PubMed1978

Fluorescent labeling of hormone receptors in viable cells: preparation and properties of highly fluorescent derivatives of epidermal growth factor and insulin.

Highly fluorescent analogs of insulin and epidermal growth factor were prepared by the covalent attachment of these peptides to alpha-lactalbumin molecules that were highly substituted (i.e., seven to one) with rhodamine molecules. The alpha-lactalbumin was specifically linked to the lysine residue of insulin or to the alpha-amino group of epidermal growth factor. The insulin derivative retained 1.15% of its potency in stimulating glucose oxidation in fat cells but retained about 8.3% of its binding affinity toward receptors. The epidermal growth factor derivative was completely active in binding to fibroblast receptors and 40% as potent as the native hormone in stimulating DNA synthesis. These highly fluorescent derivatives were suitable for the specific visual labeling of receptor sites in viable cells and for measuring the lateral mobilities of the receptor-hormone complexes by fluorescent photobleaching recovery techniques. By these methods it was shown that the hormone-receptor complexes can move laterally in the plane of the plasma membrane with a diffusion coefficient of (3-5) X 10(-10) cm2/sec.

Adipose Tissue↗
PubMed1999

Shedding light on the dark and weakly fluorescent states of green fluorescent proteins.

Recent experiments on various similar green fluorescent protein (GFP) mutants at the single-molecule level and in solution provide evidence of previously unknown short- and long-lived "dark" states and of related excited-state decay channels. Here, we present quantum chemical calculations on cis-trans photoisomerization paths of neutral, anionic, and zwitterionic GFP chromophores in their ground and first singlet excited states that explain the observed behaviors from a common perspective. The results suggest that favorable radiationless decay channels can exist for the different protonation states along these isomerizations, which apparently proceed via conical intersections. These channels are suggested to rationalize the observed dramatic reduction of fluorescence in solution. The observed single-molecule fast blinking is attributed to conversions between the fluorescent anionic and the dark zwitterionic forms whereas slow switching is attributed to conversions between the anionic and the neutral forms. The predicted nonadiabatic crossings are seen to rationalize the origins of a variety of experimental observations on a common basis and may have broad implications for photobiophysical mechanisms in GFP.

Amino Acid Sequence↗