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PubMed1999

Fundus photographic, fluorescein angiographic, and indocyanine green angiographic signs in successful laser chorioretinal venous anastomosis for central retinal vein occlusion.

OBJECTIVE: To describe the fundus signs and angiographic signs that accompany development of a laser-induced chorioretinal venous anastomosis in central retinal vein occlusion and to describe the chronology of the signs. DESIGN: Noncomparative, consecutive case series. PARTICIPANTS: Fifteen eyes of 15 patients were treated. INTERVENTION: The argon laser was used in the original method of McAllister and Constable to form an anastomosis in five eyes, and the modified method of McAllister involving the argon laser followed by the YAG laser was used for ten eyes. MAIN OUTCOME MEASURES: Changes in vessel diameters, retinal blood flow, and morphology of anastomosis over time as documented photographically and angiographically. RESULTS: The earliest fluorescein angiographic sign of success is a hyperfluorescent spindle at 1 week. The earliest indocyanine green angiographic sign is direct connection of retinal venous and choroidal venous circulations at 2 weeks. The earliest fundus photographic and, hence, ophthalmoscopic sign is asymmetry in venous diameter at the disc at 3 weeks. No sign is present in all successful cases. The most commonly observed sign is fluorescein flow around a corner in a retrograde direction toward the anastomosis in 80% of cases. Drainage of only a fraction of the retina occurred in 93% of cases. Fifteen eyes with successful anastomoses had mean improvement of 2.3 +/- 2.4 (standard deviation [SD]) Snellen lines of best-corrected visual acuity compared to 0.2 +/- 2.3 (SD) lines for 9 eyes with unsuccessful anastomoses (P = 0.0439). CONCLUSION: Recognition of the variety and typical chronology of postoperative fundus and angiographic signs in laser-induced chorioretinal anastomosis will help prevent premature retreatment and guide appropriately timed additional treatment for failed initial attempts. Fluorescein angiography and indocyanine green angiography are necessary components of intensive postoperative follow-up of these patients. The follow-up care is more difficult than the technical aspects of the surgery itself. Successful anastomoses help by taking part of the flow away from the compromised central vein, not by providing global venous bypass. This technique remains controversial, unproven, and in need of a randomized clinical trial to determine its role in the management of nonischemic central retinal vein occlusion.

Anastomosis, Surgical↗
PubMed2001

Potential problems with fluorescein diacetate assays of cell viability when testing natural products for antimicrobial activity.

There are two potential problems in the use of fluorescein diacetate (FDA) as a measure of cell viability. The first is the hydrolysis of FDA to fluorescein in the absence of live cells and the second is the quenching of fluorescence by assay solutions. We show that common media components such as tryptone, peptone and yeast extract all promote hydrolysis of FDA in the absence of live cells, as do Tris-HCl and sodium phosphate buffers. As a consequence, various microbiological media promote hydrolysis of FDA in the absence of live cells. Different media were also shown to reduce the amount of visible fluorescence of fluorescein. Diluting the medium decreases the background hydrolysis of FDA as well as increases the amount of visible fluorescence. Both problems should be considered when using FDA as an indicator of cell viability when testing natural products for antimicrobial activity.

Anti-Infective Agents↗
PubMed2003

Influence of phloretin and 6-ketocholestanol on the skin permeation of sodium-fluorescein.

In the present study, we investigated the feasibility of enhancing the transport of the model drug sodium-fluorescein across rat, porcine and human skin by treating it with phloretin and 6-ketocholestanol. Both 6-ketocholestanol and phloretin were incorporated into unilamellar liposomes and used as a skin pre-treatment. Afterwards standard diffusion experiments with Franz-type diffusion cells were performed. The results indicate a positive effect of 6-ketocholestanol on the sodium-fluorescein diffusion in all skin types, whereas phloretin had no significant influence compared to the control in human skin. In contrast to this, phloretin had a significant positive effect in rat as well as in porcine skin after 30 h of diffusion. In addition to the permeation studies, the human skin samples were analysed by confocal laser scanning microscopy for direct visualisation of the sodium-fluorescein. As expected the results of the diffusion experiments were confirmed by this microscopic method.

Animals↗
PubMed1979

The intravenous fluorescein test: use in timing of groin flap division.

Successful division or delay of the arterial groin flap requires an accurate prediction of viability. The fluorescein dye test is an objective test to determine the earliest possible time a pedicle can be divided. It is an easy, quick, and comfortable test that can be repeated many times without injuring or altering the flap. It consists of an intravenous injection of fluorescein while the pedicle is temporarily and reversibly occluded. All areas with adequate revascularization fluoresce under ultraviolet light. In three groin flaps intended for hand resurfacing, interval fluorescence testing was the sole criterion for successful pedicle division. In an experimental neurovascular island skin flap in the rat, the validity of the fluorescein test was confirmed. In the clinical situation and in the experimental model, fluorescence is an accurate indicator of groin flap revascularization and a predictor for the timing of safe and early groin flap division.

Animals↗
PubMed1998

Periorbital urticaria and topical fluorescein.

Topical fluorescein application is a routine component of the ophthalmic examination. Although complications of fluorescein angiography have been well documented, adverse reactions to topical application of this agent have not been previously reported. This report describes a case of topical fluorescein-induced urticaria.

Administration, Cutaneous↗
PubMed1998

Fluoresceinated phosphoethanolamine for flow-cytometric measurement of lipid peroxidation.

A new lipophilic fluorescein probe (fluor-DHPE) has been identified that can assay lipid peroxidation in mammalian cells on a cell-by-cell or selected-cell-subpopulation basis by flow cytometry. Application of this approach requires that the fluorescent probe be nonexchangeable among cells. Fluorescein is an appropriate fluorophore, since its fluorescence matches the specifications of common flow cytometers and the compound loses its fluorescence upon reaction with peroxyl radicals. Upon examination of four lipophilic derivatives of fluorescein, fluor-DHPE was found to be the only probe that was nonexchangeable among labeled and unlabeled rat RBC for at least 24 h. The exposure of fluor-DHPE-labeled RBC to benzoyl peroxide followed by mixing the sample with RBC unexposed to peroxide led to a decrease in fluorescence. Furthermore, the flow cytometer could clearly select the subpopulation of cells undergoing lipid peroxidation from those cells that were not. Fluor-DHPE-labeled-RBC obtained from rats and exposed to cumene hydroperoxide also displayed a gradual decrease in fluorescence. This decrease was preventable by either regulation of the vitamin E content in the animal diet or in vitro supplementation of cells with vitamin E. We conclude that fluor-DHPE is a stable and nonexchangeable probe for monitoring lipid peroxidation in cell subpopulations by flow cytometry.

Animals↗
PubMed1997

Synthesis and characterization of insulin-fluorescein derivatives for bioanalytical applications.

Human insulin was labeled with fluorescein isothiocyanate (FITC) and fully characterized to yield four distinct insulin-FITC species. High-performance liquid chromatography and electrospray mass spectrometry were used to determine the extent and location of fluorescein conjugation. By changing the reaction conditions (i.e., pH, time, and FITC/insulin ratio) the selectivity of the fluorescein conjugation was altered, and all conjugates could be separated. The isolated species of insulin-FITC were labeled at the following residues: A1(Gly), B1(Phe), A1(Gly)B1(Phe), and A1(Gly)B1(Phe)B29(Lys). All four insulin-FITC conjugates were then used to develop fluorescence polarization binding assays with monoclonal and polyclonal anti-insulin antibodies. The assay sensitivity differed between the conjugates depending on the site of modification (B1 > A1 > A1B1 > A1B1B29). Also, the type of antibody used had an important role in the binding of insulin-FITC conjugates. Finally, for the first time the biological activity of the four conjugates was demonstrated by an autophosphorylation assay. The positional substitution dramatically affected the biological activity, confirming insights into the residues responsible for the insulin binding region. The B1 conjugate was found to retain almost all biological activity while the A1 and A1B1 conjugates had approximately 10 times lower activity. The trisubstituted species (labeled at A1, B1, and B29) was determined to be least active.

Amino Acid Sequence↗
PubMedDate not supplied

Preparation of 5- and 6-(aminomethyl)fluorescein.

5(6)-Carboxyfluorescein is protected as the diacetate then reduced to 5(6)-(hydroxymethyl)fluorescein diacetate. The separated isomers are subjected to a Mitsunobu reaction with dibenzyl imidodicarbonate, yielding diprotected 5- and 6-(aminomethyl)fluorescein diacetate. Methanolysis of the acetates followed by deprotection with HBr/acetic acid gives 5- and 6-(aminomethyl)fluorescein hydrobromide.

Acetic Anhydrides↗
PubMedDate not supplied

Chemoenzymatic synthesis of 3'-O-(carboxyalkyl)fluorescein labels.

A general and versatile method is described for the synthesis of fluorescent labels. Coupling of the 3'-phenol of fluorescein methyl ester with hydroxyalkyl benzyl esters, followed by benzyl ester hydrolysis, provided a series of fluorescein carboxyalkyl ethers. Use of the Mitsunobu reaction allowed for the introduction of linkers of different lengths onto the 3'-phenol of fluorescein. Chemoenzymatic benzyl ester hydrolysis was achieved with LPL-80 lipase, providing pH-independent labels useful for the preparation of fluorescent conjugates.

Fluoresceins↗
PubMed1991

Kinetic assay of fluorescein mono-beta-D-galactoside hydrolysis by beta-galactosidase: a front-face measurement for strongly absorbing fluorogenic substrates.

A novel enzymatic assay method was developed for fluorogenic substrates that have significant intrinsic absorbance and fluorescence under the assay conditions. Fluorescein mono-beta-D-galactoside (FMG) was chosen as the substrate for the fluorescence enzymatic assay because of the high fluorescence of its hydrolytic product (fluorescein) and suitability of being hydrolyzed by beta-galactosidase. The fluorescence-concentration relationships for fluorescein and for FMG in both the right-angle detection mode of a fluorometer and the front-face detection mode of a fluorescence plate reader were exactly established and used to determine the kinetics of the enzyme assay. The results show that only front-face detection in the fluorescence plate reader can overcome the fluorescence concentration quenching that inevitably results from high absorbance by the intrinsically absorbing substrate in the conventional fluorometer, which utilizes right-angle detection. Only with front-face detection was the fluorescent assay of FMG hydrolysis under conditions of high optical density possible. The enzymatic measurements on the fluorescence plate reader were particularly efficient for determination of the enzyme kinetics because of the high rate of data collection. In this assay system, Michaelis-Menten constant Km and enzymatic catalysis rate k2 of FMG were determined as 117.6 microM and 22.7 mumol-(min.mg)-1, respectively. The results and methods described in this paper can be generalized for any assay using a fluorogenic substrate whether or not it has a high background absorbance.

Fluoresceins↗
PubMed1991

Kinetic fluorescence measurement of fluorescein di-beta-D-galactoside hydrolysis by beta-galactosidase: intermediate channeling in stepwise catalysis by a free single enzyme.

Kinetic fluorescence measurements were employed to quantitative to stepwise hydrolysis of fluorescein di-beta-D-galactoside (FDG) by beta-galactosidase and the intermediate fluorescein mono-beta-D-galactoside (FMG) channeling. The kinetic parameters, Michaelis-Menten constant Km and enzymatic catalysis rate k2, for FDG hydrolysis to FMG by beta-galactosidase were obtained as 18.0 microM and 1.9 mumol.(min-mg)-1, respectively. The FMG intermediate is hydrolyzed via two modes: (1) FMG that is in free solution binding to the enzyme substrate binding site in competition with FDG and then being hydrolyzed (binding mode); (2) FMG being directly hydrolyzed into the final products of fluorescein and galactose before the FMG can diffuse away from the enzyme active site (channeling mode). The extent of the FMG channeling mode was found to depend on the FDG hydrolysis rate but to be independent of the free enzyme concentration. A channeling factor, defined as the ratio of the real FMG hydrolysis rate with both binding and channeling modes over that which would be observed with an exclusive binding mode, was used to quantitate the effect of the intermediate channeling. The FMG channeling factor was determined to be close to 1 at low FDG concentration (about 5.1 microM), where the slow FDG hydrolysis rate gives an ineffective channeling and where the FMG is then hydrolyzed mainly via the binding mode. However, the channeling factor dramatically increases at higher FDG concentrations (greater than Km), strongly indicating that the effective FMG channeling mode, resulting from the considerable FDG hydrolysis rate at high FDG concentrations, becomes a primary pathway to channel a steady system hydrolysis with a high rate.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding, Competitive↗
PubMed1990

Activation mechanism of retinal rod cyclic GMP phosphodiesterase probed by fluorescein-labeled inhibitory subunit.

The cyclic GMP phosphodiesterase (PDE) of vertebrate retinal rod outer segments (ROS) is kept inactive in the dark by its gamma subunits and is activated following illumination by the GTP form of the alpha subunit of transducin (T alpha-GTP). Recent studies have shown that the stoichiometry of the inhibited holoenzyme is alpha beta gamma 2. T alpha-GTP and gamma act reciprocally. We have investigated the activation mechanism using fluorescein-labeled gamma subunit (gamma F) as a probe. gamma F containing a single covalently attached fluorescein was prepared by reaction of PDE with 5-(iodoacetamido)fluorescein and purification by reversed-phase high-pressure liquid chromatography (HPLC). gamma F, like native gamma, inhibits the catalytic activity of trypsin-activated PDE and transducin-activated PDE. Inhibition by gamma F was overcome by further addition of T alpha-GTP. gamma F binds very weakly to ROS membranes stripped of PDE and other peripheral membrane proteins. gamma F added to ROS membranes became incorporated into a component that could be extracted with a low ionic strength buffer. HPLC gel filtration showed that gamma F became part of the PDE holoenzyme. Incorporation occurred in less than 1 min in the presence of light and GTP, but much more slowly (t1/2 approximately 500 s) in the absence of GTP. This result indicates that transducin activates PDE by binding to the holoenzyme and accelerating the dissociation of gamma from the inhibitory sites. The binding of gamma F to trypsin-activated PDE alpha beta was monitored by steady-state emission anisotropy measurements and compared with PDE activity.(ABSTRACT TRUNCATED AT 250 WORDS)

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed2002

Ultrafast electron transfer in the complex between fluorescein and a cognate engineered lipocalin protein, a so-called anticalin.

Anticalins are a novel class of engineered ligand-binding proteins with tailored specificities derived from the lipocalin scaffold. The anticalin FluA complexes fluorescein as ligand with high affinity, and it effects almost complete quenching of its steady-state fluorescence. To study the underlying mechanism, we have applied femtosecond absorption spectroscopy, which revealed excited-state electron transfer within the FluA*Fl complex to be responsible for the strong fluorescence quenching. On the basis of a comparison of redox potentials, either tryptophan or tyrosine may serve as electron donor to the bound fluorescein group in its excited singlet state, thus forming the fluorescein trianion radical within 400 fs. The almost monoexponential rate points to a single, well-defined binding site, and its temperature independence suggests an (almost) activationless process. Applying conventional electron transfer theory to the ultrafast forward and slower back-rates, the resulting electronic interaction is rather large, with approximately 140 cm(-1) for tyrosine, which would be consistent with a coplanar arrangement of both aromatic moieties within van der Waals distance. The weak residual steady-state fluorescence originates from a small (approximately 10%) component with a time constant in the 40-60 ps range. These results demonstrate the power of time-resolved absorption spectroscopy as a diagnostic tool for the elucidation of a fluorescence quenching mechanism and the temporal profiles of the processes involved. The high structural and dynamic definition of the complexation site suggests the anticalin FluA to be a promising model in order to tailor and probe electronic interactions and energetics in proteins.

Carrier Proteins↗
PubMed2004

Synthesis and characterization of zinc sensors based on a monosubstituted fluorescein platform.

The synthesis of a new fluorescein carboxaldehyde asymmetrically substituted on the xanthene (top) ring is reported. This molecule is a key precursor for two of three monofunctionally derivatized fluorescein-based Zn(II) sensors presented in this work. Detailed preparative routes to, and photophysical characterization of, these sensors are described. The sensors are based on the previously reported ZP4 motif (Burdette, S. C.; Frederickson, C. J.; Bu, W.; Lippard, S. J. J. Am. Chem. Soc. 2003, 125, 1778-1787) and incorporate a di(2-picolyl)amine-containing aniline-derivatized ligand framework. By varying the nature of the substituent (X) para to the aniline nitrogen atom, which is responsible for PET quenching of the unbound ZP dye, we investigated the extent to which such electronic tuning might improve the fluorescent properties of asymmetrical ZP sensors. Although a comparison of probes with X = H, F, Cl, OMe reveals that the photophysical behavior of these dyes is not readily predictable, our methodology illustrates the ease with which aniline-based ligands may be linked to fluorescein dyes.

Biosensing Techniques↗
PubMed2001

Fluorescent sensors for Zn(2+) based on a fluorescein platform: synthesis, properties and intracellular distribution.

Two new fluorescent sensors for Zn(2+) that utilize fluorescein as a reporting group, Zinpyr-1 and Zinpyr-2, have been synthesized and characterized. Zinpyr-1 is prepared in one step via a Mannich reaction, and Zinpyr-2 is obtained in a multistep synthesis that utilizes 4',5'-fluorescein dicarboxaldehyde as a key intermediate. Both Zinpyr sensors have excitation and emission wavelengths in the visible range ( approximately 500 nm), dissociation constants (K(d1)) for Zn(2+) of <1 nM, quantum yields approaching unity (Phi = approximately 0.9), and cell permeability, making them well-suited for intracellular applications. A 3- to 5-fold fluorescent enhancement is observed under simulated physiological conditions corresponding to the binding of the Zn(2+) cation to the sensor, which inhibits a photoinduced electron transfer (PET) quenching pathway. The X-ray crystal structure of a 2:1 Zn(2+):Zinpyr-1 complex has also been solved, and is the first structurally characterized example of a complex of fluorescein substituted with metal binding ligands.

Animals↗
PubMed2005

Evolution of fluorescein as a platform for finely tunable fluorescence probes.

Fluorescence imaging is the most powerful technique currently available for continuous observation of dynamic intracellular processes in living cells. Suitable fluorescence probes are naturally of critical importance for fluorescence imaging, but only a very limited range of biomolecules can currently be visualized because of the lack of flexible design strategies for fluorescence probes. At present, design is largely empirical. Here we show that the carboxylic group of traditional fluorescein dyes, formerly considered indispensable, has been replaced with other substituents, affording various kinds of new fluoresceins. Further, by breaking out of the traditional structure of fluorescein, we developed the first and totally rational design strategy for novel fluorescence probes based on a strict photochemical basis. The value of this approach is exemplified by its application to develop a novel, highly sensitive, and membrane-permeable fluorescence probe for beta-galactosidase, which is the most widely used reporter enzyme.

Biosensing Techniques↗
PubMed2004

Rational principles for modulating fluorescence properties of fluorescein.

Rational design strategies based on practical fluorescence modulation mechanisms would enable us to rapidly develop novel fluorescence probes for target molecules. Here, we present a practical and general principle for modulating the fluorescence properties of fluorescein. We hypothesized that (a) the fluorescein molecule can be divided into two moieties, i.e., the xanthene moiety as a fluorophore and the benzene moiety as a fluorescence-controlling moiety, even though there is no obvious linker structure between them, and (b) the fluorescence properties can be modulated via a photoinduced electron transfer (PeT) process from the excited fluorophore to a reducible benzene moiety (donor-excited PeT; d-PeT). To evaluate the relationship between the reduction potential of the benzene moiety and the fluorescence properties, we designed and synthesized various derivatives in which the reduction potential of the benzene moiety was fine tuned by introducing electron-withdrawing groups onto the benzene moiety. Our results clearly show that the fluorescence properties of fluorescein derivatives were indeed finely modulated depending upon the reduction potential of the benzene moiety. This information provides a basis for a practical strategy for rational design of novel functional fluorescence probes.

Drug Design↗
PubMed2001

Fluorescein-assisted viscodissection for easier phacoemulsification.

TITLE: Fluorescein-assisted viscodissection for easier phacoemulsification. SETTINGS: Department of Ophthalmology, Cairo University. PURPOSE: The description of a new surgical technique to help overcome difficulties in the learning curve of phacoemulsification. METHODS: A mixture of Fluorescein 0.25% and methyl cellulose 0.25% is used for hydrodissection. Standard phacoemulsification is then continued until the initial groove reaches the plane between the lens cortex and the capsule when the fluorescein flows towards the anterior chamber in a Green Smoke. This sign indicates a proper depth for attempting to crack the nucleus into two halves.

Contrast Media↗