Modification of chloroplast CF1 by fluoresceinisothiocyanate

Incubation of the chloroplast coupling factor with fluorescein isothiocyanate inhibits the Ca-ATPase activity of the enzyme and results in incorporation of fluorescein into the α and β subunits. High concentrations of ATP prevent the inhibition and reduce the incorporation of fluorescein into the α and β subunits. Ca and Mg ions increase fluorescein isothiocyanate inhibition and fluorescein incorporation into the α, β and γ subunits. It is suggested that fluorescein isothiocyanate modifies the catalytic site of the enzyme by blocking lysine residues in the α and β subunits which are involved in the binding of ATP.     

Transport of fluorescein in MDCKII-MRP1 transfected cells and mrp1-knockout mice.

The multidrug resistant-associated protein 1 (MRP1) is a membrane-bound transport protein that is involved in the efflux of organic anions and has been implicated in multidrug resistance in cancer. MRP1 has also been reported to be ubiquitously expressed in normal tissues, including the brain. The presence of functional organic anion transporters in the blood-brain and blood-CSF barriers that influence the distribution of various compounds to the brain has long been known. The purpose of this study was to examine the role of MRP1 in the brain distribution of a model organic anion, fluorescein. The substrate specificity of MRP1 for fluorescein was initially determined by examining the accumulation of fluorescein in MDCKII MRP1-transfected cells. The distribution of fluorescein in the brain was then examined in wild-type and mrp1 gene knockout mice. The results show that in MDCKII MRP1-transfected cells, the accumulation of fluorescein was significantly lower (about 40% lower) than that in wild-type MDCKII cells. MRP1 inhibitors such as probenecid, MK-571, and LY402913 enhanced fluorescein accumulation in MDCKII MRP1-transfected cells to a greater extent than in wild-type MDCKII cells. In an in vivo study, after intravenous injection of fluorescein, the fluorescein brain-to-plasma concentration ratio in mrp1 knockout mice was not significantly different than that in wild-type mice. However, when probenecid was co-administered with fluorescein in wild-type mice, the fluorescein brain-to-plasma ratio was significantly increased (1.5-fold). These findings suggest that fluorescein is a substrate for MRP1. Furthermore, the in vivo study also suggests that MRP1 has a limited role in the transport and distribution of fluorescein in the brain. Therefore, other organic anion transport proteins, including the various isoforms of the MRP family, may be responsible for the accumulation and transport of organic anions in the brain.     

Fluorogenic approach to evaluating prodrug hydrolysis and stability in live cells

Fluorescein diester which is conjugated with cell membrane permeable Arg9 peptide was proposed as probe for ester prodrug stability and drug release study in living cells. α-Amino protected d-Val and l-Ala which bear differently hindered side chains were used to afford model diesters of 5-maleimide-fluorescein. Such fluorescein diesters were further conjugated with a Cys containing cell membrane permeable Arg9 peptide via thiol-ene crosslink reaction. The resulted conjugates of fluorescein diester and Arg9 peptide were purified with HPLC and characterized with MALDI-TOF MS. Upon incubation with cultured cells, the fluorescein diesters were delivered into the cells, the following hydrolysis of fluorescein diesters and release of fluorescein inside living cells were observed by monitoring the fluorescence accumulation. Fluorescence microscopic imaging studies of HeLa cells treated with fluorescein l-Ala diester show strong fluorescence accumulation in 30?min indicating fast hydrolysis of fluorescein diester and fluorescein release; in contrast d-Val diester remains stable inside cells evidenced by margin fluorescence formation. Further flowcytometry studies on the fluorescein diester-Arg9 conjugate treated cells show that the hydrolysis t_1/2 for l-Ala diester is 15?min. The results also show that Arg9 peptide not only transports the ester probes into cell efficiently but also can retain and concentrate hydrolytic product fluorescein inside cells so that the accumulated fluorescence can be accurately quantified. This fluorogenic probe approach provides feasible applications in dynamic studies on ester prodrug hydrolysis and release, facilitating screening and optimization of prodrug structures in living cell settings.     

Photobleaching kinetics of fluorescein in quantitative fluorescence microscopy.

An investigation on the photobleaching behavior of fluorescein in microscopy was carried out through a systematic analysis of photobleaching mechanisms. The individual photochemical reactions of fluorescein were incorporated into a theoretical analysis and mathematical simulation to study the photochemical processes leading to photobleaching of fluorescein in microscopy. The photobleaching behavior of free and bound fluorescein has also been investigated by experimental means. Both the theoretical simulation and experimental data show that photobleaching of fluorescein in microscopy is, in general, not a single-exponential process. The simulation suggests that the non-single-exponential behavior is caused by the oxygen-independent, proximity-induced triplet-triplet or triplet-ground state dye reactions of bound fluorescein in microscopy. The single-exponential process is a special case of photobleaching behavior when the reactions between the triplet dye and molecular oxygen are dominant.     

Enhancer effect of fluorescein on the luminol–H2O2–horseradish peroxidase chemiluminescence: energy transfer process

The chemiluminescence of the luminol–H₂O₂–horseradish peroxidase system is increased by fluorescein. Fluorescein produces an enhancement of the luminol chemiluminescence similar to that of phenolphthalein, by an energy transfer process from luminol to fluorescein. The maximum intesity and the total chemiluminescence emission (between 380 and 580 nm) of luminol with fluorescein was more than three times greater than without fluorescein; however, the emission duration was shorter. The emission spectra in the presence of fluorescein had two maxima (425 and 535 nm) and the enhancement was dependent on pH and fluorescein concentration. A mechanism is proposed to explain these effects. © 1997 John Wiley & Sons, Ltd.     

Macrophage activation by synthetic peptides. I. Kinetic changes in the transport of fluorescein anions across the plasma membrane of macrophages

As shown by cytofluorimetric technique, fluorescein anions formed in macrophages due to hydrolysis of fluorescein diacetate (FDA) release into the extracellular medium through the probenecid-inhibitable transport system of organic acids. Technical procedures have been elaborated to record separately the process of FDA hydrolysis characterising the activity of intracellular esterases, and the fluorescein anion transport representing secretion of organic acids by macrophages. It has been established that the tetrapeptide tuftsin stimulates the cell esterase activity without affecting the rate of fluorescein efflux. The peptide KPR (Lys-Pro-Arg) decreases both the esterase activity and the fluorescein anion efflux.     

Influence of the triplet excited state on the photobleaching kinetics of fluorescein in microscopy.

The investigation in this report aimed at providing photophysical evidence that the long-lived triplet excited state plays an important role in the non-single-exponential photobleaching kinetics of fluorescein in microscopy. Experiments demonstrated that a thiol-containing reducing agent, mercaptoethylamine (MEA or cysteamine), was the most effective, among other commonly known radical quenchers or singlet oxygen scavengers, in suppressing photobleaching of fluorescein while not reducing the fluorescence quantum yield. The protective effect against photobleaching of fluorescein in the bound state was also found in microscopy. The antibleaching effect of MEA let to a series of experiments using time-delayed fluorescence spectroscopy and nanosecond laser flash photolysis. The combined results showed that MEA directly quenched the triplet excited state and the semioxidized radical form of fluorescein without affecting the singlet excited state. The triplet lifetime of fluorescein was reduced upon adding MEA. It demonstrated that photobleaching of fluorescein in microscopy is related to the accumulation of the long-lived triplet excited state of fluorescein and that by quenching the triplet excited state and the semioxidized form of fluorescein to restore the dye molecules to the singlet ground state, photobleaching can be reduced.     

Transepithelial transport of fluorescein in Caco-2 cell monolayers and use of such transport in in vitro evaluation of phenolic acid availability

Fluorescein is a marker-dye customary applied to the evaluation of tight-junctional permeability of epithelial cell monolayers. However, the true mechanism for the permeation has not been elucidated. Transepithelial transport of fluorescein in Caco-2 cell monolayers was therefore examined. Fluorescein transport was dependent on pH, and in a vectorical way in the apical-basolateral direction, but it was independent of the tight-junctional permeability of monolayers of these human intestinal cells. The permeation of fluorescein was concentration-dependent and saturable; the Michaelis constant was 7.7 mM and the maximum velocity was 40.3 nmol min(-1) (mg protein)(-1). Benzoic acid competitively inhibited fluorescein transport, suggesting that fluorescein is transported by a monocarboxylic acid transporter (MCT). Antioxidative polyphenolic compounds such as ferulic acid from dietary sources, competitively inhibited the permeation of fluorescein. These compounds probably share a transport carrier with fluorescein. Measurement of the effects of phenolic acids on fluorescein transport across Caco-2 monolayers would be a useful way to evaluate the intestinal absorption or bioavailability of dietary phenolic acids.     

Flow system fluorescence polarization measurements on fluorescein diacetate-stained EL4 cells.

We have adapted a multiparameter cell sorter to measure the distribution of fluorescence polarization in cell populations. Measurements carried out on EL4 cells show that the percent polarization of fluorescein fluorescence decreases with increasing fluorescence intensity. This inverse relationship between polarization and intensity is shown both within the cell population and by the average values of the two quantities during both the increase and decrease of fluorescence intensity. The quantitative relation between intensity and polarization is different in hypertonic than in isotonic media. These results suggest that polarization measurements carried out at a fixed time after incubation of cells with fluorescein diacetate, which is converted to fluorescein within the cells, may depend in part on the rate of fluorescein accumulation, and that agents that have been reported to change the polarization of fluorescein in living cells may do so by changing the kinetics of fluorescein accumulation.     

Intestinal uptake of nateglinide by an intestinal fluorescein transporter

Nateglinide, a novel oral hypoglycemic agent, rapidly reaches its maximum serum concentration after oral administration, suggesting that it is rapidly absorbed in the intestine. However, nateglinide itself is not transported by MCT1 or PEPT1. The aim of this study was to characterize the transporters on the apical side of the small intestine that are responsible for the rapid absorption of nateglinide. It has been reported that the uptake of fluorescein by Caco-2 cells occurs via an H+-driven transporter and that the intestinal fluorescein transporter is probably not MCT1. We examined the contribution of the fluorescein transporter to the uptake of nateglinide by Caco-2 cells. Fluorescein competitively inhibited H+-dependent nateglinide uptake. All of fluorescein transporter inhibitors examined reduced the uptake of nateglinide. Furthermore, nateglinide inhibited fluorescein uptake. We conclude that the intestinal nateglinide/H+ cotransport system is identical to the intestinal fluorescein/H+ cotransport system.     

Labeling of the cytoplasmic domain of the influenza virus hemagglutinin with fluorescein reveals sites of interaction with membrane lipid bilayers

The hemagglutinin (HA) glycoprotein of influenza virus was labeled in its cytoplasmic domain with fluorescein. Reactive amino groups in the external domain were blocked by modification of the intact virus with the membrane-impermeable reagent isethionyl acetimidate. The HA was then solubilized with the detergent octyl glucoside, and the single lysine in the cytoplasmic domain was reacted with fluorescein isothiocyanate. This protocol resulted in the incorporation of 1.3 mol of fluorescein/mol of HA. Using a virus strain lacking lysine in the cytoplasmic domain of HA, it was determined that 0.47 mol of fluorescein/mol of HA was located at an additional site(s). The fluorescein groups at both sites exist in an environment of reduced polarity as shown by a shift in excitation and emission maxima and a shift in the pKa of the fluorescein groups. The fluorescence polarization and the pKa of the fluorescein groups were greater when the HA was incorporated into liposomes than when in detergent solution. These data indicate that the fluorescein groups interact directly with the lipid bilayer, probably in the phospholipid head-group region. The fluorescence properties of the labeled HA were not responsive to the gel to liquid-crystal phase transition in the lipid bilayer. These results indicate that the boundary between the cytoplasmic domain and the hydrophobic sequence that anchors the protein to the lipid bilayer is located in the head-group region of the bilayer.     

Cadmium stimulation of Na-independent organic acid transport in the kidney tubules

The influence of Cd++ (as well as of Hg++ and Cu++) on the uptake of an organic acid (fluorescein) in superficial proximal tubules of the surviving rat kidney was studied at 20 degrees C, when the active transport of fluorescein does not depend on the external Na. In contrast to mercury and copper, cadmium stimulated the uptake of fluorescein from the beginning of incubation. The minimal effective concentration of Cd++ was 5 X 10(-6)M, the relative effect of Cd++ on the uptake being the same within the concentration range from 5 X 10(-6) to 10(-3) M. A 60 minutes pre-incubation with Cd++ at 20 degrees C resulted in a significant increase in the stimulatory effect of acetate on the fluorescein transport. The stimulation of the fluorescein transport by cadmium was prevented by ouabain or by omissing Na from the incubating medium, although neither ouabain nor the absence of Na affected the transport of fluorescein under these conditions. It is supposed that the stimulation by Cd++ of the fluorescein transport may result from the activated oxidation of NAD-linked substrates due to acceleration of the active transepithelial transport of Na ions.     

SYNTHESIS AND STUDY OF THE FLUORESCEIN CONJUGATE OF THE NUCLEOTIDE dPTP

The synthesis of a fluorescein conjugate on the non-natural base P is described. The ability of the newly synthesised fluorescein - dPTP and of a fluorescein-11-dUTP to compete with the natural nucleotide TTP was also studied. Overall the efficiency of labelling a nucleic acid with a fluorescein moiety was found to be approximately equal.     

Adsorption of fluorescein by protein crystals

Adsorption characteristics of native and cross-linked lysozyme crystals were examined using fluorescein as model adsorbate. The adsorption isotherms exhibited Langmuir or linear behavior. The affinity constant (b1) and the adsorption capacity (Qsat) for fluorescein were found to depend on the type and concentration of co-solute present in the solution. The dynamics of adsorption isotherm transition from Langmuir to linear showed that affinity of lysozyme for solutes increases in the order 2-(cyclohexylamino)ethanesulphonic acid (CHES), 4-morpholinepropanesulphonic acid (MOPS), acetate, fluorescein. Furthermore, the crystal morphology, the degree of cross-linking of the crystals, and, in particular, solution pH were identified as factors determining fluorescein adsorption by the lysozyme crystals. These factors seem to affect crystal capacity for the solute more than affinity for the solute. Adsorption of fluorescein by cross-linked tetragonal lysozyme crystals was exponentially dependent on the lysozyme net charge calculated from the final solution pH. The 3-5-fold increase in the fluorescein adsorption as a result of cross-linking is presumably due to the increasing hydrophobicity of the lysozyme crystal.     

Intestinal uptake of nateglinide by an intestinal fluorescein transporter

Nateglinide, a novel oral hypoglycemic agent, rapidly reaches its maximum serum concentration after oral administration, suggesting that it is rapidly absorbed in the intestine. However, nateglinide itself is not transported by MCT1 or PEPT1. The aim of this study was to characterize the transporters on the apical side of the small intestine that are responsible for the rapid absorption of nateglinide. It has been reported that the uptake of fluorescein by Caco-2 cells occurs via an H⁺-driven transporter and that the intestinal fluorescein transporter is probably not MCT1. We examined the contribution of the fluorescein transporter to the uptake of nateglinide by Caco-2 cells. Fluorescein competitively inhibited H⁺-dependent nateglinide uptake. All of fluorescein transporter inhibitors examined reduced the uptake of nateglinide. Furthermore, nateglinide inhibited fluorescein uptake. We conclude that the intestinal nateglinide/H⁺ cotransport system is identical to the intestinal fluorescein/H⁺ cotransport system.     

Fluorescence analysis of the size of a binding pocket of a peptide receptor at natural abundance

We have studied the topography of interaction of a family of fluorescent formyl peptides containing four (CHO-Met-Leu-Phe-Lys-fluorescein), five (CHO-Met-Leu-Phe-Phe-Lys- fluorescein), and six (CHO-Nle-Leu-Phe-Nle-Tyr-Lys-fluorescein and CHO-Met-Leu-Phe-Phe-Phe-Lys- fluorescein) amino acids with their receptor using spectroscopic methods adapted to small sample volumes. Only the fluorescent peptides containing four and five amino acids were quenched upon binding to the receptor, indicating physical contact of the chromophore with the receptor. In contrast, only the hexapeptides were accessible to antibodies to fluorescein. Taken together, these results suggest that the carboxy terminus of the tetrapeptide or the pentapeptide is protected in the receptor binding pocket while the fluorescein on the carboxy terminus of either hexapeptide is exposed and recognized by the antibody to fluorescein. These results indicate that the binding pocket accommodates at least five but no more than six amino acids.     

Estimation of the degradation of endocytosed material by flow cytofluorometry using two neoglycoproteins containing different numbers of fluorescein molecules

The fluorescence properties of fluorescein bound to protein are used to quantitate by flow cytofluorometry the degradation of fluorescein-labeled alpha-glucosylated serum albumin (fluorescein-labeled neoglycoprotein) after endocytosis by the membrane lectin of Lewis lung carcinoma cells (3LL cells). The quantum yield of fluorescein bound to a protein decreases when the number of fluorescein residues per protein molecule increases; however, after proteolytic digestion the mean fluorescence intensity of a fluorescein molecule is constant and equal to that of free fluorescein. The extent of the degradation of the endocytosed neoglycoprotein was determined with a flow cytofluorometer by using two neoglycoproteins containing either a small or a large number of fluorescein residues per neoglycoprotein molecule. At 4 degrees C, 3LL cells bind 750,000 molecules of fluorescein-labeled alpha-glucosylated serum albumin with an apparent binding constant of 2 X 10(6) 1 X mole-1. At 37 degrees C, after 4 hr incubation 2.2 X 10(6) molecules of fluorescent alpha-glucosylated serum albumin were cell-associated, and of these at least one third were degraded.     

The use of vital dyes to assess embryonic viability in the hamster, Mesocricetus auratus

Experiments were designed to assess the use of the vital dyes trypan blue and fluorescein diacetate as indicators of the viability of hamster ova and embryos. Exclusion of trypan blue and fluorescence with fluorescein diacetate showed high correlations with uptake of [3H]uridine by ova and further development of embryos in vitro. Ova killed by freezing and thawing incorporated [3H]uridine at background levels. Trypan blue exclusion and fluorescein diacetate uptake were highly correlated with each other (r = 0.99). Trypan blue and fluorescein diacetate serve as excellent indices of viability in ova and early embryos of hamsters.     

Effect of fluorochrome location in protein A on sensing efficiency of IgG

Fragment B of protein A conjugated with fluorescein at various lysine residues is prepared and separated by using a DEAE column in anion-exchange chromatography. The binding of IgG Fc to fragment B contributes to an additional positive electric potential around fragment B. The change in the local electrostatic environment and pH can then be specifically monitored by measuring the fluorescence intensity of fluorescein conjugated with fragment B before and after the introduction of IgG. The studies for the quantitative dependence of fluorescein location on the effectiveness of fluorescein for sensing the protein A-IgG reaction are presented and discussed. (c) 1993 John Wiley & Sons, Inc.