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PubMed1982

The active site structure of Na+- and K+-stimulated ATPase. Location of a specific fluorescein isothiocyanate reactive site.

Fluorescein 5'-isothiocyanate (FITC) has been shown to specifically inactivate the Na+- and K+-stimulated adenosine triphosphatase ((Na,K)-ATPase) at low concentrations (Karlish, S. J. D. (1979) Na+,K+ATPase Structure and Kinetics 115-128). The site of modification of purified dog kidney (Na,K)-ATPase by FITC has been investigated by enzymatic cleavage and fluorescence resonance energy transfer. The binding of FITC, which occurs at a stoichiometry of approximately one site per ATP binding site, causes an ATP-protectable inactivation of ATPase activity suggesting that it is reacting at the ATP hydrolysis site. The FITC reaction site apparently is located near the center of the COOH-terminal 77,000-dalton peptide fragment obtained by chymotryptic cleavage of the alpha subunit. Addition of ouabain to the native enzyme in the presence of chymotrypsin enhances cleavage at this site and releases the fluorescein moiety from the membrane. It is further shown that the distance from the FITC reaction site to the ouabain binding site, as judged by fluorescence resonance energy transfer from anthroyl ouabain to FITC, is approximately 74 A. These results demonstrate that ouabain inhibits the (Na,K)-ATPase by causing a protein conformational change which extends an unusually large distance across the membrane.

Animals↗

PubMed1983

Sugar transport by the bacterial phosphotransferase system. Preparation of a fluorescein derivative of the glucose-specific phosphocarrier protein IIIGlc and its binding to the phosphocarrier protein HPr.

In diauxic growth, the bacterial phosphoenolpyruvate: glycose phosphotransferase system (PTS) regulates the utilization of certain compounds which are not PTS substrates. It has recently been shown that this PTS regulation is mediated via one of the PTS phosphocarrier proteins, IIIGlc. In the present studies, IIIGlc was derivatized with the fluorescent reagent fluorescein-5-isothiocyanate. One mol of label was incorporated per mol of protein and the label was located at the NH2-terminal amine, as shown by tryptic peptide mapping and one-step Edman-type degradation. The fluorescent moiety was found to be stable and resistant to photodecomposition. The fluorescent IIIGlc was purified and shown to be fully active in its ability to accept phosphate from phospho-HPr (the histidine-containing phosphocarrier protein of the phosphotransferase system), but only 20% active in catalyzing the transfer of the phosphate to methyl alpha-glucoside via the membrane-bound II-BGlc protein. The decay of the fluorescence intensity was dominated by a single component (90%) with a lifetime of 4 ns. The decay of the fluorescence emission anisotropy was determined for excitation in both a negative and positive transition of fluorescein and was best described in terms of a biexponential function, indicating internal motion of the fluorophore and possible anisotropic rotation of the protein as a whole. The formation of a complex between IIIGlc and HPr was demonstrated by using the techniques of time-resolved and steady state fluorescence emission measurements, resonance energy transfer, and equilibrium gel filtration.

Amino Acid Sequence↗

PubMed1984

Protein-enhanced fluorescein chemiluminescence used in an immunoassay for rubella antibody in serum.

The chemiluminescence of fluorescein derivatives is greatly enhanced when they are conjugated to proteins. We exploited this property in devising a chemiluminescence immunoassay for rubella antibodies. In this sequential "sandwich"-type assay, tubes coated with rubella antigen are used for antibody separation and goat antibody to human IgG conjugated with fluorescein isothiocyanate is used for detection of bound rubella antibodies.

Antibodies, Viral↗

PubMed1984

Evaluation of injured intestine with the aid of fluorescein.

In an animal model, a new diagnostic use for fluorescein as an aid in determining intestinal viability after blunt trauma, has been evaluated. Areas of traumatized intestine that do not fluoresce are invariably necrotic; fluorescing tissue is always viable. Fluorescein is a simple and rapid means of evaluating traumatized intestine, and it may prove to be a useful adjunct to the clinical examination.

Animals↗

PubMed1981

In vivo labelling of the spleen and mesenteric lymph nodes with fluorescein isothiocyanate for lymphocyte migration studies.

Lymphocytes in normal young pigs were labelled in vivo with fluorescein isothiocyanate in the spleen using an extracorporeal perfusion system and in mesenteric lymph nodes by direct injection into the nodes. Labelled lymphocytes leave the spleen at a high rate via the splenic vein and migrate to different lymphoid organs. Emigrants from mesenteric lymph nodes left the nodes more slowly and revealed a different homing pattern. Evidence is presented that a considerable number of lymphocytes from the parenchyma leave the nodes via the vein and not by the classical route of recirculating lymphocytes via the efferent lymphatics. Fluorescein labelling of lymphocytes in their normal micro-environment is a suitable method for lymphocyte migration studies.

Animals↗

PubMed1982

The binding of fluorescein-labeled protein synthesis initiation factor 2 to Escherichia coli 30 S ribosomal subunits determined by fluorescence polarization.

The binding of initiation factor IF2 to 30 S ribosomal subunits was studied quantitatively by steady state fluorescence polarization techniques. Covalent fluorescent derivatives of IF2 were prepared by treating the pure factor with fluorescein isothiocyanate. Derivatives (F-IF2) containing about 1 fluorescein molecule/protein bound to 30 S ribosomal subunits as measured by sucrose gradient centrifugation; however, their activity is slightly impaired in assays for stimulation of formylmethionyl-tRNA binding to 70 S ribosomes. Upon addition of 30 S ribosomal subunits to F-IF2 solutions, a significant increase in fluorescence polarization results, but no change in quantum yield or fluorescence lifetime is detected. Fluorescence polarization values were measured at different F-IF2 and 30 S ribosomal subunit concentrations and the equilibrium association constant and number of ribosomal binding sites were calculated. The association constant for F-IF2 in buffer containing 100 mM NH4Cl and 10 mM Mg acetate is 1.5 +/- 0.2 X 10(7) M-1; the number of binding sites is 0.9 +/- 0.2. Competition of F-IF2 with IF2 showed that the nonderivatized factor binds somewhat more tightly than F-IF2, with an association constant of 2.7 X 10(7) M-1. The binding of F-IF2 is strongly inhibited by raising the NH4Cl concentration from 20 mM to 150 mM, but is rather insensitive to Mg acetate concentrations between 2.5 and 10 mM. Addition of initiation factors IF1 and IF3 causes a 3-fold increase in the association constant for F-If2 binding to 30 S subunits.

Ammonium Chloride↗

PubMed1983

Examination of the intracellular ionic environment and of ionophore action by null point measurements employing the fluorescein chromophore.

Fluorescein and carboxyfluorescein have found recent application as probes of intracellular pH. The present study examines several parameters required for interpretation of the spectral information derived from fluorescein and carboxyfluorescein generated intracellularly from their permeant diacetate derivatives. Coefficients were determined for the pH dependence of the difference absorbance, of the absorbance ratios, and of the fluorescence emission intensity ratios at selected wavelength pairs for carboxyfluorescein in aqueous buffers. The effect of light scattering on the apparent pH reported by carboxyfluorescein in dilute cell suspensions was assessed. An apparent intracellular acidification associated with increasing internal dye concentration was found to result probably from interactions of the intracellular probe with itself. Working within the experimental limitations imposed by these considerations, protocols utilizing either direct measurement of absorbance or fluorescence or determination of the null spectral response observed upon release of internal carboxyfluorescein all indicate that the cytosolic space of bovine epididymal sperm is maintained at pH 6.5-6.6. The monovalent-cation-specific, carboxylic acid ionophores, nigericin and monensin, were utilized to produce transmembrane proton gradients in cells that were allowed to generate intracellular carboxyfluorescein in a preliminary incubation, then resuspended in media buffered at the same pH as the sperm cytosol but of varying cation composition. By interpolation to the null response, the initial internal Na+ and K+ concentrations in bovine sperm were estimated as 14 +/- 2 and 120 +/- 5 mM, respectively. The ability of initial transmembrane gradients of either protons or of monovalent cations to promote equivalent changes in internal pH following ionophore addition to sperm suspensions supports application of a simple model that predicts steady state cation distributions. With the assumption that the cytosolic proton buffer has a pKa near the determined internal pH, these experiments allow the additional calculation that this buffer is present at a concentration of 190 +/- 20 meq/liter in the bovine sperm.

Animals↗

PubMedDate not supplied

[The fluorescein infiltration test in the differential diagnosis of precancer and cancer of the lip and oral mucosa].

Infiltration fluorescein test based on examination of the strength of tissue structures subject to dystrophy in cancer is suggested to improve the results of differential diagnosis of cancer and precancer. The diagnosis is made depending on the appearance or absence of fluorescein drops on a tumor surface in response to injection of fluorochrome dose into the tumor. A fluorescent stomatoscope is used to assess the test results. Clinical trials in 85 patients have demonstrated that the test parameters and methods of evaluation are essential to obtain correct results. A high diagnostic value of the test (9.6 +/- 3.3%) recommend it as a malignancy test during fluorescent cheilostomatoscopy.

Adult↗

PubMedDate not supplied

[Role of sodium fluorescein dye binding during its accumulation in cells].

The accumulation of sodium fluorescein inside K562 cells in culture was investigated. It was found that the great part of the dye was bound to the enzymes inside the cell. The efficiency of the binding was found to depend upon the pH value inside the cell. This pH value was measured in the experiment. The fraction of the fluorescein bound in the cell, the dissociation constant, the concentration of binding cites in the cell and the binding energy are estimated.

Binding Sites↗

PubMed1993

[Technical concepts of retrograde venous perfusion with fluorescein].

Regional intravenous drug application has been suggested to increase peripheral tissue concentrations in severe arterial insufficiency. Within this study basic operational characteristics of the retrograde venous perfusion technique (RVP) were evaluated in 10 patients with intermittent claudication. Fluorescein was used to visualize the intracutaneous distribution of the injected fluid. Subjects were divided into 2 groups. The "cocktail-group" received 100 ml of a homogenous fluorescein dilution. To compare the effect of injecting a small sample, 45 ml of unstained solution were given before and after 10 ml of concentrated dye ("fraction-group"). Photographic recording of skin fluorescence distal to the arterial tourniquet revealed a rapid and complete staining of the lower leg including the toes in the cocktail-group. In the fraction-group, however, skin fluorescence appeared markedly delayed and, more over, did not spread down to the toes in 2 cases. In conclusion, drugs can be applied by RVP provided that they are dissolved in a sufficient amount of fluid of 100 ml at least.

Chemotherapy, Cancer, Regional Perfusion↗

PubMed1997

Real-time monitoring of hairpin ribozyme kinetics through base-specific quenching of fluorescein-labeled substrates.

Current methods for evaluating the kinetics of ribozyme-catalyzed reactions rely primarily on the use of radiolabeled RNA substrates, and so require tedious electrophoretic separation and quantitation of reaction products for each data point in any experiment. Here, we report the use of fluorescent substrates for real-time analysis of the time course of reactions of the hairpin ribozyme. Fluorescence of 3' fluorescein-labeled substrates was quenched upon binding to the hairpin ribozyme or its isolated substrate-binding strand (SBS), under conditions of ribozyme or SBS excess. This decrease was accompanied by an increase in anisotropy, and resulted from a base-specific quenching by a guanosine residue added to the 5' end of the SBS, close to fluorescein in the complex. Fluorescence quenching was used to determine rate constants for substrate binding (1.4 x 10(8) M(-1) min(-1)), cleavage (0.15 min(-1)), and substrate dissociation (0.010 min(-1)) by a structurally well-defined ribozyme at 25 degrees C in 50 mM Tris-HCI, pH 7.5, 12 mM MgCl2. These rates are in excellent agreement with those obtained using traditional radioisotopic methods. Measurements of dissociation rates provided physical support for interdomain interactions within the substrate-ribozyme complex. We estimate that 2.1 kcal/mol of additional substrate binding energy is provided by the B domain of the ribozyme. Part of this free energy apparently stems from coaxial stacking of helices in the hinge region between domains, and it is plausible that the remainder might be contributed by direct interactions with loop B. The fluorescence quenching and dequenching methods described here should be readily adaptable to studying a wide variety of RNA interactions and reactions using ribozymes and other model systems.

Fluorescein↗

PubMed1997

Detection of melanoma by monitoring the intracellular fluorescein fluorescence polarization changes in lymphocytes.

The effectiveness of detecting melanoma by measuring the intracellular fluorescein fluorescent polarization (IFFP) of patients' SCM (structuredness of the cytoplasmic matrix)-responding lymphocytes was examined. SCM-responding lymphocytes from 46 melanoma patients and 32 healthy volunteers were labeled with fluorescein diacetate and challenged with different stimuli, and the resulting polarization was determined. The polarizations (P) obtained upon stimulation with nothing (P-0), encephalitogenic factor (P-EF), phytohaemagglutinin (P-PHA), or melanoma antigen (P-MEL), and the ratios RR(ef) (P-EF/P-PHA) and RR(mel) (P-MEL/P-PHA) were lower for SCM-responding lymphocytes from the patients as a group than for those of the controls. The specificity and sensitivity of the IFFP tests (using cutoff values) to detect melanoma were 90.6 and 73.9%, respectively. The IFFP tests may facilitate the discrimination between melanoma patients and healthy subjects, and may be used in follow-up of patients with melanoma.

Algorithms↗

PubMed2003

Effects of aldose reductase inhibitors and galactose withdrawal on fluorescein angiographic lesions in galactose-fed dogs.

OBJECTIVE: To assess the effect of aldose reductase inhibitor (ARI) M79175 (2-methyl-6-fluoro-spiro-chroman-4-5'-imidazolidine-2',4'-dione) administration and galactose withdrawal on the progression of retinal changes using fluorescein angiography in galactose-fed dogs. METHODS: Thirty male beagles were randomized into 4 groups. Three dogs were fed a normal control diet containing 30% nonnutritive fiber for 74 months (control group), 11 dogs a 30% galactose diet for 74 months (continuous galactose group), 8 dogs a 30% galactose diet for 36 months followed by replacement with a normal diet for 36 months (galactose withdrawal group), and 8 dogs a 30% galactose diet supplemented with M79175 for 34 months followed by replacement with a normal diet and removal of M79175 treatment for 38 months (ARI-treated galactose withdrawal group). Stereoscopic color fundus photography and fluorescein angiography, performed at baseline and follow-up, were assessed for the clinical development of retinopathy, including the first appearance of hyperfluorescence, varying severity of retinal nonperfusion, and retinal neovascularization. Histopathologic features were examined in selected dogs. RESULTS: All dogs in the 3 groups fed the 30% galactose diet developed areas of hyperfluorescence and nonperfusion. Of these dogs, only those supplemented with the ARI did not develop areas of nonperfusion greater than or equal to half the field and retinal neovascularization. Parametric survival analysis showed significant differences (galactose withdrawal group vs ARI-treated galactose withdrawal group) in the median times to the development of nonperfusion greater than or equal to half the field (P =.003) and retinal neovascularization (P =.03). CONCLUSION: Normalization of glycemic control with galactose withdrawal and ARI treatment may delay the onset and progression of retinal lesions in galactose-fed dogs. Clinical Relevance Perfect glycemic control after a period of poor control does not completely prevent the progression of retinal lesions. Therapy with ARIs may potentially be important in the prevention of retinal lesions associated with diabetic eye disease.

Aldehyde Reductase↗

PubMed2004

Fluorescein filling defects and quantitative morphologic analysis of the optic nerve head in glaucoma.

OBJECTIVES: To evaluate absolute filling defects of the optic nerve head in normal tension glaucoma (NTG) and primary open-angle glaucoma (POAG) and to compare the filling defects with topographic analysis of the optic disc. METHODS: Twenty-five patients with NTG, 25 patients with POAG, and 25 age-matched controls were included. Fluorescein angiograms were performed by means of a scanning laser ophthalmoscope. The extent of absolute filling defects of the optic nerve head was assessed using digital image analysis of early-phase angiograms. Topographic measurements of the optic disc were acquired using the Heidelberg Retina Tomograph II. RESULTS: Absolute filling defects were significantly larger (P =.001) and were seen more often (P<.001) in patients with NTG (n = 18) and POAG (n = 19) compared with controls (n = 3). Rim area (P =.006), rim volume (P =.007), cup-disc area ratio (P =.008), linear cup-disc ratio (P =.005), maximum cup depth (P =.002), cup shape measure (P =.03), and nerve fiber layer thickness (P =.008) and cross-sectional area (P =.006) were significantly different between patients with glaucoma and controls. Absolute filling defects were significantly correlated with cup area (r = 0.31; P =.007), rim area (r = -0.38; P<.001), rim volume (r = -0.35; P =.002), cup-disc area ratio (r = 0.49; P<.001), linear cup-disc ratio (r = 0.48; P<.001), cup shape measure (r = 0.27; P =.02), and nerve fiber layer thickness (r = -0.33; P =.004) and cross-sectional area (r = -0.30; P =.009). CONCLUSIONS: Fluorescein filling defects of the optic disc are present in NTG and POAG. The extent of these filling defects is correlated with the morphologic disc damage.

Blood Pressure↗

PubMed1975

Ceroid-lipofuscinosis (Batten disease). Fluorescein angiography, electrophysiology, histopathology, ultrastructure, and a review of amaurotic familial idiocy.

Three children with ceroid-lipofuscinosis and their mother wer investigated fluorescein angiographically and electrophysiologically after definitive diagnosis of the oldest child had been made from a brain biopsy specimen studied biochemically, histopathologically, and ultrastructurally. The diagnostic features of the two classes of familial amaurotic idiocy (the gangliosidoses and the ceroidlipofuscinoses) are reviewed with emphasis on the importance of the fundus picture and fluorescein angiographic study in differentiating the two classes of disease and in identifying affected siblings.

Adult↗

PubMed1976

Cilioretinal arteries. A study based on review of stereo fundus photographs and fluorescein angiographic findings.

The incidence of cilioretinal arteries in 2,000 eyes of 1,000 consecutive patients was determined by review of stereo color fundus photographs and fluorescein angiographs. One or more cilioretinal arteries were present in 49.5% of all patients or in 32.1% of the eyes. The arteries occurred bilaterally in 14.6% and contributed to some portion of the macular circulation in 18.7% of the patients. A great deal of variability of size, number, and distribution of cilioretinal vessels was observed. Prior studies that were based on direct visualization of the fundus have reported cilioretinal arteries in 7% to 29.6% of patients examined. Careful review of stereo fundus photographs and early phase fluorescein angiographs aided in the observation of these vessels that might otherwise have been unrecognized. We believe the method of review accounts for the high incidence of cilioretinal arteries in our series.

Ciliary Body↗

PubMed1976

Synchronous bilateral fluorescein angiography. A new technique.

Simultaneous angiography of both eyes allows the synchronous measurement of the arm-retina-time and the retinal circulation time in each eye. The dye-filling pattern of retinal arteries, choroid, disc, and cilioretinal vessels can be compared. A new method of simultaneous fluorescein angiography of both eyes, using two fundus cameras, is demonstrated. Each camera is operated by a separate power unit, thereby providing sufficient light energy for one picture every 0.6 second. The fluorescein is injected by means of an automatic injector in order to create a compact dye bolus. Simultaneous angiograms of a healthy patient and of a patient with unilateral carotid occlusion are shown.

Choroid↗

PubMed1977

Fluorescein angiographic defects of the optic disc in ocular hypertension.

Fluorescein angiograms of the optic disc were studied in 23 normal and 29 ocular hypertensive eyes. Significant differences in the frequency distributions of areas of defects were obtained between normal and ocular hypertensive eyes. Increased areas of filling defects were noted in ocular hypertensive eyes as compared to normals. There were also significant correlations of the areas of filling defects with age and systolic blood pressure in the ocular hypertensive eyes that were not present in the normals. These observations support the concept that fluorescein angiography of the optic disc demonstrates localized areas of impaired circulation that increase with ocular pressure, age, and systolic blood pressure. This technique may be useful in separating normal from the ocular hypertensive patient who shows changes in the circulation of the optic disc with increased ocular pressure.

Adolescent↗