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PubMed2005

Fluorescent leukotriene B4: potential applications.

Leukotriene B4 (LTB4) is a potent lipid mediator of inflammation that acts primarily via a seven-transmembrane-spanning, G-protein-coupled receptor denoted BLT1. Here, we describe the synthesis and characterization of fluorescent analogs of LTB4 that are easy to produce, inexpensive, and without the disadvantages of a radioligand. Fluorescent LTB4 is useful for labeling LTB4 receptors for which no antibodies are available and for performing one-step fluorescence polarization assays conducive to high-throughput screening. We found that orange and green fluorescent LTB4 were full agonists that activated the LTB4 receptor BLT1 with EC50 values of 68 and 40 nM, respectively (4.5 nM for unmodified LTB4). Flow cytometric measurements and confocal imaging showed that fluorescent LTB4 colocalized with BLT1. Fluorescence polarization measurements showed that orange fluorescent LTB4 bound to BLT1 with a Kd of 66 nM and that this binding could be displaced by unlabeled LTB4 and other BLT1-specific ligands. Fluorescent LTB4 analogs were also able to displace tritiated LTB4. Orange fluorescent LTB4 binding to enhanced green fluorescent protein-tagged BLT1 could be observed using fluorescence resonance energy transfer. In addition to being a useful alternative to radiolabeled LTB4, the unique properties of fluorescently labeled LTB4 allow a variety of detection technologies to be used.

Cell Membrane↗

PubMed1993

Evaluation of porfimer sodium fluorescence for measuring tissue transformation.

BACKGROUND: Neoplastic tissue can be detected by its increased fluorescence compared with surrounding normal tissue after the injection of the tumor-localizing compound porfimer sodium (Photofrin; Quadra Logic Technologies, Vancouver, BC, Canada). In vivo fluorescence photometry is a nonimaging photodetector technique that detects specific 690 nm fluorescence of the porphyrin by subtracting nonspecific 612 nm excitation from 630 nm excitation. The technique was applied in the developmental stages of the 9,10 dimethyl-1,2-benzanthracene (DMBA)-induced hamster buccal cheek pouch carcinoma model to (1) quantitate and characterize porfimer sodium fluorescence and uptake as it relates to lesion progression and biochemical changes and (2) determine whether porfimer sodium-induced fluorescence will vary with promotional and inhibitory stimuli. METHODS: Groups of Syrian Golden hamsters had their cheek pouch buccal mucosa exposed to a 0.5% DMBA in acetone three times per week for 6 weeks (premalignant lesions), 12 weeks (squamous cell carcinomas), or other specified durations. The rate of malignant transformation was either promoted (by either carbon dioxide laser incision or continued DMBA application) or inhibited (by the administration of either somatostatin analogue RC-160 [D-Phe-Cys-Tyr-D-Trp-Lys-Val-Cys-Trp-NH2] or bombesin antagonist RC-3095 [D-Tpi-Gln-Trp-Ala-Val-Gly-His-Leu psi (CH2NH)Leu-NH2]). Groups of DMBA-exposed hamsters were subsequently injected with 1.0 mg/kg of porfimer sodium during the various stages of tumor development. Twenty-four hours after injection, fluorescence levels were measured by in vivo fluorescence photometry. Samples of tumors, dysplastic mucosal tissue, and normal-appearing oral mucosa were biopsied and used for either tissue extraction assays, histopathologic examination, or tyrosine kinase activity assay as an index of rate of transformation. RESULTS: Results demonstrated that porfimer sodium is retained in DMBA-treated tissue. Fluorescence is completely accounted for by porfimer sodium uptake. The duration of exposure to carcinogen is proportional to porfimer sodium fluorescence. This relationship establishes that premalignant lesions can be distinguished from normal tissue by porfimer sodium uptake and fluorescence. The changes in increased tyrosine kinase activity paralleled the increase in porfimer sodium fluorescence. Alterations in the rate of tissue transformation produced equivalent alterations in porfimer sodium-induced fluorescence. CONCLUSIONS: These results suggest that porfimer sodium uptake and fluorescence can be used in a prognostic manner to diagnose and determine the course of transformation of individual lesions.

9,10-Dimethyl-1,2-benzanthracene↗

PubMed2006

Regions of alpha-amino-5-methyl-3-hydroxy-4-isoxazole propionic acid receptor subunits that are permissive for the insertion of green fluorescent protein.

The green fluorescent protein can be fused to the ends of a mature glutamate receptor subunit to produce functional, fluorescent receptors. However, there are good reasons to search for internal regions of receptor subunits that can tolerate green fluorescent protein insertion. First, internal insertions of green fluorescent protein may produce functional, fluorescent subunits that traffic more correctly. Second, fluorescent proteins inserted near interacting surfaces of subunits could potentially create reagents suitable for fluorescence resonance energy transfer measurements. Finally, internal green fluorescent protein insertions could potentially produce subunits capable of signaling conformational changes through intrinsic changes in fluorescence intensity. To identify regions of receptor subunits that are permissive for green fluorescent protein insertion, we used a series of recombinant transposons to create fluorescent protein insertions in three alpha-amino-5-methyl-3-hydroxy-4-isoxazole propionic acid receptor subunits. A combined analysis of the relative fluorescence intensity and glutamate-gated ion channel function of 69 different green fluorescent protein fusion proteins identified permissive zones for the creation of bright and fully functional receptor subunits in the C-terminal portion of the amino terminal domain, the intracellular tail of the carboxy terminal domain, and within the pore-forming regions of the channel.

Amino Acid Sequence↗

PubMed1988

Dynamic fluorescence properties of bacterial luciferase intermediates.

Three fluorescent species produced by the reaction of bacterial luciferase from Vibrio harveyi with its substrates have the same dynamic fluorescence properties, namely, a dominant fluorescence decay of lifetime of 10 ns and a rotational correlation time of 100 ns at 2 degrees C. These three species are the metastable intermediate formed with the two substrates FMNH2 and O2, both in its low-fluorescence form and in its high-fluorescence form following light irradiation, and the fluorescent transient formed on including the final substrate tetradecanal. For native luciferase, the rotational correlation time is 62 or 74 ns (2 degrees C) derived from the decay of the anisotropy of the intrinsic fluorescence at 340 nm or the fluorescence of bound 8-anilino-1-naphthalenesulfonic acid (470 nm), respectively. The steady-state anisotropy of the fluorescent intermediates is 0.34, and the fundamental anisotropy from a Perrin plot is 0.385. The high-fluorescence intermediate has a fluorescence maximum at 500 nm, and its emission spectrum is distinct from the bioluminescence spectrum. The fluorescence quantum yield is 0.3 but decreases on dilution with a quadratic dependence on protein concentration. This, and the large value of the rotational correlation time, would be explained by protein complex formation in the fluorescent intermediate states, but no increase in protein molecular weight is observed by gel filtration or ultracentrifugation. The results instead favor a proposal that, in these intermediate states, the luciferase undergoes a conformational change in which its axial ratio increases by 50%.

Flavin Mononucleotide↗

PubMed2006

A modular strategy for tailoring fluorescent biosensors from ribonucleopeptide complexes.

Fluorescent biosensors that facilitate reagentless sensitive detection of small molecules are crucial tools in the areas of therapeutics and diagnostics. However, construction of fluorescent biosensors with desired characteristics, that is, detection wavelengths and concentration ranges for ligand detection, from macromolecular receptors is not a straightforward task. An ATP-binding ribonucleopeptide (RNP) receptor was converted to a fluorescent ATP sensor without chemically modifying the nucleotide in the ATP-binding RNA. The RNA subunit of the ATP-binding RNP and a peptide modified with a pyrenyl group formed a stable fluorescent RNP complex that showed an increase in the fluorescence intensity upon binding to ATP. The strategy to convert the ATP-binding RNP receptor to a fluorescent ATP sensor was applied to generate fluorescent ATP-binding RNP libraries by using a pool of RNA subunits obtained from the in vitro selection of ATP-binding RNPs and a series of fluorophore-modified peptide subunits. Simple screening of the fluorescent RNP library based on the fluorescence emission intensity changes in the absence and presence of the ligand afforded fluorescent ATP or GTP sensors with emission wavelengths varying from 390 to 670 nm. Screening of the fluorescence emission intensity changes in the presence of increasing concentrations of ATP allowed titration analysis of the fluorescent RNP library, which provided ATP sensors responding at wide concentration ranges of ATP. The combinatorial strategy using the modular RNP receptor reported here enables tailoring of a fluorescent sensor for a specific ligand without knowledge of detailed structural information for the macromolecular receptor.

Adenosine Triphosphate↗

PubMed1966

Fluorescence studies on some hydroxypyridines including compounds of the vitamin B6 group.

1. The variations with pH (from 36n-sulphuric acid to 10n-sodium hydroxide) of the excitation and fluorescence wavelengths and fluorescence intensity of 2-, 3- and 4-hydroxypyridine and their O- and N-methyl derivatives were investigated. 2. 4-Hydroxy- and 4-methoxy-pyridine were non-fluorescent at all pH values. 3. The cations and dipolar ions of the 3-hydroxypyridine derivatives and the anion of 3-hydroxypyridine were fluorescent, but the neutral forms were not. 4. All the forms of the 2-hydroxypyridine derivatives were fluorescent. 5. Pyridoxol, pyridoxal and its 5-phosphate, pyridoxamine and pyridoxic acid and its lactone were studied similarly. All these compounds, except pyridoxal 5-phosphate, were more fluorescent than 3-hydroxypyridine. 6. The most fluorescent forms of these compounds are the anions, except for pyridoxol, where the dipolar ion was the most fluorescent form. The least fluorescent forms are the neutral molecules. The dipolar ions were appreciably fluorescent in all cases. 7. The most fluorescent form examined was the dianion of pyridoxic acid lactone. 8. The cations were all fluorescent except the cations of 2- and 3-methoxypyridine. All the cations showed excited-state ionization. The excited pK(a) values of these cations were determined and the results are discussed with reference to Weller's (1952) equation relating ground- and excited-state dissociation constants. 9. The pK(a) values for all ionizations undergone by the compounds examined were determined from fluorescence data. 10. Stokes shifts for the various ionic and neutral species of the compounds examined were calculated and are discussed.

Chemical Phenomena↗

PubMed2001

Aging and effects of ultraviolet A exposure may be quantified by fluorescence excitation spectroscopy in vivo.

The fluorescence properties of skin chromophores such as tryptophan and collagen cross-links might be useful markers of aging and photoaging. As the fluorescence of pepsin-digestible collagen cross-links was found to increase with aging and decrease with photoaging we investigated the characteristics of this dependence. In vivo fluorescence excitation spectra (emission at 380 nm) of SKH hairless mouse model skin are characterized by two bands centered near 295 nm and 335 nm due, respectively, to epidermal tryptophan moieties and pepsin-digestible collagen cross-links. Several groups of hairless mice were followed over a period of 18 mo to document changes in skin fluorescence with aging. Other groups of animals were exposed to either broad band or narrowband ultraviolet A radiation to determine the effects of ultraviolet A exposure on the fluorescence of the dermal collagen cross-links and to determine an action spectrum for the induced changes. We also found that the intensity of pepsin-digestible collagen cross-links in vivo increases linearly with age and that the fluorescence of epidermal tryptophan decreases linearly with age. We found that the fluorescence of pepsin-digestible collagen cross-links decreases immediately following exposure to ultraviolet A whereas epidermal tryptophan fluorescence increases. Both changes were dose dependent but the increase in tryptophan fluorescence occurred exclusively in young animals (2--6 mo old). We found that the ultraviolet-induced fluorescence decrease of pepsin-digestible collagen cross-links is wavelength specific. The action spectrum for the ultraviolet A effect on the in vivo fluorescence of pepsin-digestible collagen cross-links shows a distinct maximum at 335 nm that corresponds to the maximum in the fluorescence excitation spectrum due to pepsin-digestible collagen cross-links. Our results seem to indicate that in vivo fluorescence of epidermal tryptophan moieties and collagen cross-links in the dermal matrix may serve as markers for skin aging, for photoaging, and for immediate assessment of exposure to ultraviolet A radiation.

Aging↗

PubMed1993

Flavin dynamics in oxidized Clostridium beijerinckii flavodoxin as assessed by time-resolved polarized fluorescence.

The time-resolved fluorescence characteristics of flavin in oxidized flavodoxin isolated from the anaerobic bacterium Clostridium beijerinckii have been examined. The fluorescence intensity decays were analyzed using the maximum-entropy method. It is demonstrated that there exist large differences in fluorescence behaviour between free and protein-bound FMN. Three fluorescence lifetime components are found in oxidized flavodoxin, two of which are not present in the fluorescence-intensity decay of free FMN. The main component is distributed at 30 ps, with relative contribution of 90%. Another minor component (4% contribution) is distributed at 0.5 ns. The third component is distributed at 4.8 ns (6%), coinciding with the main distribution present in the fluorescence decay of free FMN. The results allowed us to determine the dissociation constant, Kd = 2.61 x 10(-10) M (at 20 degrees C). Collisional fluorescence-quenching experiments revealed that the flavin moiety responsible for the longest fluorescence lifetime is, at least partially, exposed to the solvent. The shortest lifetime is not affected significantly, indicating that it possibly originates from an active-site conformation in which the flavin is more or less buried in the protein and not accessible to iodide. The fluorescence anisotropy behaviour of free and protein-bound FMN was examined and analyzed with the maximum-entropy method. It was found that an excess of apoflavodoxin is required to detect differences between free and protein-bound FMN. In free FMN one single distribution of rotational correlation times is detected, whereas in flavodoxin the anisotropy decay is composed of more than one distribution. Associative analysis of fluorescence anisotropy decays shows that part of the 4.8 ns fluorescence lifetime present in the flavodoxin fluorescence decay, is coupled to a rotational correlation time similar to that of free FMN in solution, while another part of this lifetime is coupled to a longer correlation time of about 1 ns. This finding is in accordance with earlier studies [Barman, B. G. & Tollin, G. (1972) Biochemistry 11, 4746-4754] in which it was proposed that the first binding step of the flavin to the protein involves the phosphate group rather than another part of the FMN. The two shortest fluorescence lifetimes, which do not carry information on the long-term rotational behaviour of the protein, seem nonetheless to be associated with a longer rotational correlation time which is comparable to overall protein tumbling. These lifetime components probably originate from a complex in which the flavin-ring system is more or less immobilized within the protein matrix.

Clostridium↗

PubMed2006

Fundus near infrared fluorescence correlates with fundus near infrared reflectance.

PURPOSE: To analyze the occurrence of near infrared (NIR) fluorescence in relation to NIR reflectance, blue-light-excited autofluorescence, angiograms, and funduscopy. METHODS: Observational consecutive case series in patients with macular diseases. Imaging was performed with a confocal scanning laser ophthalmoscope for NIR reflectance, blue-light-excited autofluorescence, NIR fluorescence, and fluorescein and indocyanine green (ICG) angiograms. In cases in which NIR fluorescence was observed, five to nine images were averaged. The leakage of the scanning laser ophthalmoscope was analyzed. RESULTS: In the 291 eyes analyzed, NIR fluorescence was observed in 51 and was graded weak in 27 with wet age-related macular degeneration (AMD, 10 cases), dry AMD with pigment clumping (n=7), chronic central serous choroidopathy (CSC; n=5), choroidal nevi (n=2), subretinal hemorrhages (n=2), and chloroquine maculopathy (n=1). Strong NIR fluorescence was found in 24 eyes, with wet AMD (n=14), subretinal hemorrhages (n=8), and choroidal nevi (n=2). Except for four eyes, we observed a strong correlation of NIR fluorescence and increased NIR reflectance at identical fundus location (92.2%). NIR fluorescence corresponded with increased blue-light-excited autofluorescence in 21 of 31 patients with AMD and in 4 of 5 patients with chronic CSC, but in none of the 4 patients with nevi. Funduscopy showed that structures with NIR fluorescence were pigmented or consisted of degraded blood. Barrier filter leakage of the imaging system was 6.2x10(-6). CONCLUSIONS: The high correlation of NIR fluorescence and reflectance indicated that part of the observed NIR fluorescence is pseudofluorescence, whereas gray-scale analysis indicated that both NIR autofluorescence and pseudofluorescence contribute to the NIR fluorescence images. Quantification of leakage of the imaging system indicated a significant part of the observed NIR fluorescence is NIR autofluorescence. As NIR fluorescence derives from pigmented lesions, melanin is a possible source if NIR reflectance is also increased. Comparison with blue-light-excited autofluorescence showed differences between AMD and patients with nevi. NIR autofluorescence was also detected in single cases of maculopathy without corresponding NIR reflectance.

Choroid Diseases↗

PubMed2006

[Fluorescence characteristics of dissolved organic matter during algal bloom in Jiaozhou Bay].

Fluorescence excitation-emission matrix spectroscopy (EEMS) was employed to characterize the dissolved organic matter (DOM) in algal bloom seawater of Jiaozhou Bay in February, 2004. The relationships of DOM fluorescence characteristics with phytoplankton multiplication, dissolved organic carbon (DOC), salinity, dissolved oxygen and pH were discussed. In addition, the protein-like and humic-like fluorescence changes fore and aft cross-flow ultrafiltration (CFF) were primarily evaluated. The results show that the intensities of high and low-excitation protein-like fluorescence, which have the same sources, are stronger than that of humic-like during algal bloom. Both correlations between protein-like fluorescence intensities and chlorophyll-a and between humic-like fluorescence intensities and chlorophyll-a are relevant to the phytoplankton multiplication. As a whole, the fluorescence intensities of protein-like and humic-like increases with chlorophyll-a increasing. Furthermore the ratios of new to old DOM increase with phytoplankton biomass increasing. Good positive correlations between protein-like fluorescence intensities and DOC and between the ratios of protein-like to humic-like fluorescence intensities and DOC suggest that the DOC is mainly composed of newly produced matter during algal bloom. Some inorganic factors, such as salinity, dissolved oxygen and pH, have little effects on protein-like and humic-like fluorescence. Fore and aft the CFF experiments, the positions of protein-like and humic-like fluorescence peaks have little alteration, the balance of fluorescence intensity is not so good as organic carbon mass balance as a whole. Fluorescence balance should be used only as a supplementary technique for evaluating contaminations or loss by organic carbon mass balance.

Carbon↗

PubMed1995

[In vivo fundus fluorescence measurements in patients with age related macular degeneration].

UNLABELLED: This study was performed to measure and characterize the intrinsic fluorescence of the ocular fundus in patients with age-related macular degeneration (AMD). METHOD: Fluorescence spectral measurements from discrete retinal locations were made using the fundus spectrophotometer with excitations at 470 and 510 nm. Two normal subjects and seven patients with different stages of AMD were investigated. RESULTS: The spectral characteristics of fundus fluorescence are consistent with those of lipofuscin in the retinal pigment epithelium (RPE). The fluorescence spectrum is broad, with a maximum at about 620 nm. The shape and intensity of the fluorescence spectra are affected by age, site of measurement, pathology, ocular media absorption, and excitation wavelength. Spectra from areas with drusen reveal an additional fluorophore, with maximum around 560 nm, probably emanating from drusen and Bruch's membrane. Measurements in atrophic reveal a decrease of lipofuscin fluorescence and/or a contribution likely due to choroidal and sclera collagen fluorescence. Fluorescence from lipofuscin is more efficiently excited at 510 nm, whereas that of drusen and subretinal structures is relatively more efficient with 470 nm excitation, allowing for discrimination of various fluorophores. CONCLUSION: The spectral characteristics of RPE lipofuscin could be identified and quantified in AMD patients. In addition, the spectra are affected by other fluorophores such as drusen and choroid contributions in atrophy. Fluorescence spectra measurements in AMD patients allow for discrimination of lipofuscin fluorescence, drusen fluorescence, and choroidal or scleral fluorescence. The non-invasive measurement of lipofuscin and drusen fluorescence in AMD may be helpful in monitoring the disease, understanding its evolution, and testing therapeutic concepts.

Aged↗

PubMed2004

Chromatic shifts in the fluorescence emitted by murine thymocytes stained with Hoechst 33342.

BACKGROUND: Many methods in flow cytometry rely on staining DNA with a fluorescent dye to gauge DNA content. From the relative intensity of the fluorescence signature, one can then infer position in cell cycle, amount of DNA (i.e., for sperm selection), or, as in the case of flow karyotyping, to distinguish individual chromosomes. This work examines the staining of murine thymocytes with a common DNA dye, Hoechst 33342, to investigate nonlinearities in the florescence intensity as well as chromatic shifts. METHODS: Murine thymocytes were stained with Hoechst 33342 and measured in a flow cytometer at two fluorescence emission bands. In other measurements, cells were stained at different dye concentrations, and then centrifuged. The supernatant was then used for a second round of staining to test the amount of dye uptake. Finally, to test for resonant energy transfer, we measured fluorescence anisotropy at two different wavelengths. RESULTS: The fluorescence of cells stained with Hoechst 33342 is a nonlinear process that shows an overall decrease in intensity with increased dye uptake, and spectral shift to the red. Along with the spectral shift of the fluorescence to the longer wavelengths, we document decreases in the fluorescence anisotropy that may indicate resonant energy transfer. CONCLUSIONS: At low concentrations, Hoechst 33342 binds to the minor groove of DNA and shows an increase in fluorescence and a blue shift upon binding. At higher concentrations, at which the dye molecules can no longer bind without overlapping, the blue fluorescence decreases and the red fluorescence increases until there is approximately one dye molecule per DNA base pair. The ratio of the blue fluorescence to the red fluorescence is an accurate indicator of the cellular dye concentration.

Animals↗

PubMed2005

The use of a fluorescent dye, Nile red, to evaluate the lipid content of single mammalian oocytes.

This study aimed to investigate the use of Nile red, a fluorescent dye specific for intracellular lipid droplets, to quantify the lipid content of single mammalian oocytes. It was hypothesized that a higher amount of lipid present in lipid droplets in an oocyte would result in a higher amount of emitted fluorescent light. Following fixation and subsequent staining of denuded oocytes, the fluorescence of the whole oocyte was visualized by fluorescence microscopy and quantified with a photometer and photomultiplier connected to the microscope. The peak of fluorescence was observed in the yellow spectrum (590 nm) and the fluorescence was restricted to the lipid droplets corresponding to apolar lipids. Nile red concentrations ranging from 0.1 to 10 microg/ml yielded similar results. After fixation, a minimum of 2 h staining was necessary to reach maximal fluorescence which remained stable for several hours. The position of the microscopic focus within the oocyte had no influence on the amount of measured fluorescence. Successive measurements of the same oocyte yielded very similar results indicating the repeatability of the method. Finally, the technique was validated by comparing the lipid content of bovine, porcine and murine immature oocytes, which are known to contain different amounts of lipids. After staining, the fluorescence of murine oocytes was 2.8-fold lower than the fluorescence of bovine oocytes which in turn were 2.4 times less fluorescent than porcine oocytes. Based on this study, it can be said that this rather fast and easy technique allows for the relative quantification of the lipid content (present in the lipid droplets) of one single oocyte. The different amounts of emitted fluorescent light in bovine, porcine and murine oocytes correlated with the known lipid contents in these three species. This technique could be used to compare the lipid content of oocytes originating from different donors, from different sized follicles or cultured in various conditions.

Animals↗

PubMedDate not supplied

Validation of in vivo fluorochrome concentrations measured using fluorescence molecular tomography.

Fluorescence molecular tomography (FMT) has emerged as a means of quantitatively imaging fluorescent molecular probes in three dimensions in living systems. To assess the accuracy of FMT in vivo, translucent plastic tubes containing a turbid solution with a known concentration of Cy 5.5 fluorescent dye are constructed and implanted subcutaneously in nude mice, simulating the presence of a tumor accumulating a fluorescent molecular reporter. Comparisons between measurements of fluorescent tubes made before and after implantation demonstrate that the accuracy of FMT reported for homogeneous phantoms extends to the in vivo situation. The sensitivity of FMT to background fluorescence is tested by imaging fluorescent tubes in mice injected with Cy 5.5-labeled Annexin V. For small tube fluorochrome concentrations, the presence of background fluorescence results in increases in the reconstructed concentration. This phenomenon is counteracted by applying a simple subtraction correction to the measured fluorescence data. The effects of varying tumor photon absorption are simulated by imaging fluorescent tubes with varying ink concentrations, and are found to be minor. These findings demonstrate the in vivo quantitative accuracy of fluorescence tomography, and encourage further development of this imaging modality as well as application of FMT in molecular imaging studies using fluorescent reporters.

Algorithms↗

PubMed1983

New approach for visualizing estrogen receptors in target cells using inherently fluorescent ligands and image intensification.

Four fluorescent estrogen ligands were investigated as agents for visualization of estrogen receptors in cells: 2-(2,4-dihydroxyphenyl)-6-hydroxy-3-benzofurancarboxylic acid delta-lactone (coumestrol) and 9(11)-dehydro-12-oxoestradiol [12-oxo-1,3,5-(10),9(11)-estratetraene-3, 17 beta-diol] (12-oxoestradiol), which are inherently fluorescent compounds; and tamoxifen [Z)-1-[4-(2-dimethylaminoethoxy)phenyl]-1,2-diphenyl-1-butene) and 4-hydroxytamoxifen [Z)-1-[4-(2-dimethylaminoethoxy) phenyl]-1-(4-hydroxyphenyl)-2-phenyl-1-butene), which become maximally fluorescent only after ultraviolet irradiation. By conventional fluorescence techniques, these agents can be detected down to 10(-8) M in water, but only to 10(-6) to 10(-7) M in protein solutions; however, by photon-counting spectrofluorimetry, coumestrol and 12-oxoestradiol can be detected in protein solutions down to 5 X 10(-10) M. Three of these compounds have good affinity for the estrogen receptor: coumestrol (20%); 12-oxoestradiol (12%); and 4-hydroxytamoxifen (37%), relative to estradiol (100%). Under conditions where autoradiographic controls indicate that most of the estrogen receptor of MCF-7 human breast cancer cells is in the nucleus, we could demonstrate nuclear fluorescence using 10(-9) M concentrations of coumestrol, 12-oxoestradiol, and 4-hydroxytamoxifen. This nuclear fluorescence was abolished by a 200-fold excess of diethystilbestrol and could only be observed through a fluorescence microscope equipped with a microchannel image intensifier and a video camera detector that together provide a sensitivity enhancement of approximately 10(4). These studies indicate that the estrogen receptor in breast cancer cells can be visualized by fluorescence techniques, provided that the visualizing ligands have adequate affinity and specificity for the receptor and appropriate fluorescence characteristics, and provided that the fluorescence instrument has adequate sensitivity to observe fluorescence emission from cells treated with nM concentrations of the fluorescent agents.

Breast Neoplasms↗

PubMed1997

Photobleaching during and re-appearance after photodynamic therapy of topical ALA-induced fluorescence in UVB-treated mouse skin.

Photodynamic therapy (PDT) using protoporphyrin IX (PpIX) induced by topically applied 5-aminolevulinic acid (ALA) seems a promising alternative for the treatment of superficial non-melanoma skin cancer and actinic keratosis. In this study, the kinetics of new PpIX fluorescence arising after a PDT treatment that had photobleached the original fluorescence were determined. Our purpose was to examine the feasibility of multiple irradiations, following a single topical ALA application, to increase PDT efficacy. In addition, photobleaching during PDT and the fluorescence spectra during and after PDT were studied. As a model we used hairless mice with and without UVB-induced skin lesions. ALA was applied to the skin for 4 hr. An illumination was delivered either immediately after application or 6 hr after the end of the application (at interval of maximum fluorescence). During PDT, the fluorescence of normal skin decreased at a faster rate than the fluorescence of the skin lesions. In the fluorescence study after PDT, the areas treated immediately post-application showed a fluorescence increase over time similar to that in non-treated areas on the same mice. A remarkable result was that the fluorescence of areas treated at maximum fluorescence increased, whereas the fluorescence of non-treated areas did not increase over time. With both treatment intervals the new fluorescence showed a characteristic PpIX spectrum. Our results demonstrate that a second illumination, when new PpIX fluorescence has been formed, may increase PDT efficacy after topical ALA application. This finding has been demonstrated previously for systemic ALA administration.

Administration, Topical↗

PubMed2006

Quenchers induce wavelength dependence on protein fluorescence lifetimes.

We have analysed the picosecond resolved fluorescence emission decay of horseradish peroxidase A2 and of HEW lysozyme acquired with a streak camera. Analyses of the fluorescence decay data of both proteins revealed that the dynamics of the decay is dependent on the emission wavelength. Our data strongly indicates that resonance energy transfer occurring between aromatic residues and different protein fluorescence quencher groups, and the nature of the quencher groups, are the causes of the observed wavelength dependent mean lifetime distribution. Using the global analysis data to calculate the fluorescence mean lifetime at each wavelength revealed that for lysozyme, the mean fluorescence lifetime increased with observation wavelength, whereas the opposite was the case for peroxidase. Both proteins contain strong fluorescence quencher groups located in close spatial proximity to the protein's aromatic residues. Lysozyme contains disulfide bridges as the main fluorescence quencher whereas peroxidase contains a heme group. Both for lysozyme and horseradish peroxidase there is a clear correlation between the observed fluorescence mean lifetime of the protein at a particular emission wavelength and the respective quencher's extinction coefficient at the respective wavelength. Furthermore, our study also reports a comparison of the analyses of the fluorescence data done with three different methods. Analyses of the fluorescence decay at 10 different fluorescence emission wavelengths revealed significant differences in both fluorescence lifetimes and the pre-exponential factor distributions. Such values differed from the values recovered from the integrated decay curves and from global analyse.

Acetates↗

PubMed2000

Formycin A and its N-methyl analogues, specific inhibitors of E. coli purine nucleoside phosphorylase (PNP): induced tautomeric shifts on binding to enzyme, and enzyme-->ligand fluorescence resonance energy transfer.

Steady-state and time-resolved emission spectroscopy were used to study the interaction of Escherichia coli purine nucleoside phosphorylase (PNP) with its specific inhibitors, viz. formycin B (FB), and formycin A (FA) and its N-methylated analogues, N(1)-methylformycin A (m(1)FA), N(2)-methylformycin A (m(2)FA) and N(6)-methylformycin A (m(6)FA), in the absence and presence of phosphate (P(i)). Complex formation led to marked quenching of enzyme tyrosine intrinsic fluorescence, with concomitant increases in fluorescence of FA and m(6)FA, independently of the presence of P(i). Fluorescence of m(1)FA in the complex increased only in the presence of P(i), while the weak fluorescence of FB appeared unaffected, independently of P(i). Analysis of the emission, excitation and absorption spectra of enzyme-ligand mixtures pointed to fluorescence resonance energy transfer (FRET) from protein tyrosine residue(s) to FA and m(6)FA base moieties, as a major mechanism of protein fluorescence quenching. With the non-inhibitor m(2)FA, fluorescence emission and excitation spectra were purely additive. Effects of enzyme-FA, or enzyme-m(6)FA, interactions on nucleoside excitation and emission spectra revealed shifts in tautomeric equilibria of the bound ligands. With FA, which exists predominantly as the N(1)-H tautomer in solution, the proton N(1)-H is shifted to N(2), independently of the presence of P(i). Complex formation with m(6)FA in the absence of P(i) led to a shift of the amino-imino equilibrium in favor of the imino species, and increased fluorescence at 350 nm; by contrast, in the presence of P(i), the equilibrium was shifted in favor of the amino species, accompanied by higher fluorescence at 430 nm, and a higher affinity for the enzyme, with a dissociation constant K(d)=0.5+/-0.1 microM, two orders of magnitude lower than that for m(6)FA in the absence of P(i) (K(d)=46+/-5 microM). The latter was confirmed by analysis of quenching of enzyme fluorescence according to a modified Stern-Volmer model. Fractional accessibility values (f(a)) varied from 0.31 for m(1)FA to 0.70 for FA, with negative cooperative binding of m(1)FA and FB, and non-cooperative binding of FA and m(6)FA. For all nucleoside ligands, the best model describing binding stoichiometry was one ligand per native enzyme hexamer. Fluorescence decays of PNP, FA and their mixtures were best fitted to a sum of two exponential terms, with average lifetimes ( ) affected by their interactions. Complex formation resulted in a 2-fold increase in of FA, and a 2-fold decrease in of enzyme fluorescence. The amplitude of the long-lifetime component also increased, confirming the shift of the tautomeric equilibrium in favor of the N(2)-H species. The findings have been examined in relation to enzyme-nucleoside binding deduced from structural studies.

Binding Sites↗