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At least 847 records · Page 47Linked to original sources
PubMed1984

Pathophysiology of the blood-retinal barrier in experimental diabetes. Vitreous fluorophotometry using carboxyfluorescein and fluorescein.

Carboxyfluorescein resembles fluorescein in size and spectral characteristics but is much less lipid soluble. Both dyes were used to differentiate between two groups of factors that influence penetration across the blood-retinal barrier: (1) factors that depend on lipid solubility, such as the area of the barrier, and (2) factors independent of lipid solubility, such as opened intercellular junctions or necrotic cells. Vitreous fluorophotometry was performed on normal and diabetic rats after injection of either dye. After the results were adjusted for sources of error, midvitreous-plasma dye ratios for carboxyfluorescein and fluorescein were of the same order of magnitude in normal rats. Ratios for both dyes increased in diabetic rats, and the increases were similar in magnitude. Our results suggest that lipid solubility contributes little to inward transport of these dyes in both the normal and diabetic states.

Animals↗
PubMed1986

Vitreous fluorophotometry in juvenile diabetics after oral fluorescein.

Vitreous fluorophotometry was performed in young patients (8 to 19 years old) affected by insulin-dependent diabetes mellitus, with no signs of diabetic retinopathy. Oral fluorescein was administered, and measures were used to prevent hypoglycemia during the examination. The results showed a breakdown of the blood-retinal barrier in nine (43%) of the 21 patients. The oral fluorescein method allows a satisfactory evaluation of ocular fluorescence profiles, with minimal risk of systemic side effects.

Administration, Oral↗
PubMed1985

Flow cytometry of leukocytes after intravenous fluorescein angiography.

Blood specimens were obtained for T lymphocyte analysis from patients undergoing intravenous fluorescein angiography (IVFA). Mononuclear cells were prepared by differential density centrifugation and analyzed by flow cytometry (FCM). Prior to IVFA, there was very little autofluorescence in the mononuclear cell population. After IVFA, there was an increase in the fluorescence of the mononuclear cells. Though barely visible by fluorescence microscopy, the fluorescence was uniformly distributed throughout the cytoplasm. By FCM, fluorescein was detected in 27% of the lymphocytes, 61% of the monocytes, and 75% of the granulocytes. Care must be taken when interpreting flow-cytometric lymphocyte subset data because of the increased nonspecific fluorescence of mononuclear cells in patients who have undergone IVFA.

Flow Cytometry↗
PubMed1989

A quantitative study of fluorescein isothiocyanate-dextran transport in the microcirculation of the isolated perfused rat liver.

Hepatic extraction of solutes depends on microvascular angioarchitecture, hemodynamics and solute concentrations. These factors may contribute to the heterogeneity observed in solute transport and uptake in the hepatic lobules. However, predictions of liver extraction based on black-box models require assumptions about these factors and the microvascular transport mechanisms involved. Consequently, the purpose of this study was to investigate solute transport and uptake by hepatocytes. Livers from male Sprague-Dawley rats were perfused at physiological flowrates and portal pressures on the stage of an in vivo microscope using a low-hematocrit Ringer solution. A bolus of fluorescein isothiocyanate-dextrans (17,900, 39,000, 65,600 or 156,900 MW), which are considered inert fluid-phase markers, was injected into the portal vein. Fluorescein isothiocyanate fluorescence, as a measure of solute concentration, was video recorded in periportal or centrivenular regions of the lobules. Spatial and temporal fluorescence data, measured in sinusoids and hepatocytes, were fit to one-dimensional transport models to determine estimates for an intracellular effective diffusion coefficient and for hepatocyte permeability. The calculated effective diffusion coefficients were 2.5 times larger for dextrans less than 66,000 MW, but were not different between the periportal and centrivenular regions. Also, the values did not show the inverse log-log molecular weight dependency for dextrans seen in other microvascular tissues.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1985

Drug absorption from inhalation aerosols administered by positive-pressure ventilation. I: Administration of a characterized, solid disodium fluorescein aerosol under a controlled respiratory regime to the beagle dog.

An apparatus and novel method is described for administration of well-characterized inhalation aerosols, under strictly controlled respiratory regimes, direct to the respiratory tract (RT) of the beagle dog by positive-pressure ventilation. The method enables the study of systemic absorption kinetics of compounds delivered as inhalation aerosols as a function of the aerosol particle size and respiratory variables provided their intrinsic pharmacokinetics are linear. Aerosol characteristics are determined by sampling the aerosol at a point close to its entry to the endotracheally intubated animal. The chosen positive-pressure ventilatory regime, which is monitored as airway pressure and exhaled volume versus time, can be held constant for the aerosol administration period. The methodology is illustrated by administration of a solid polydispersed aerosol of disodium fluorescein. Resultant plasma concentrations (C) were determined as a function of time by sampling from an indwelling venous cannula. The pharmacokinetic analysis of resultant C versus time data, together with that from an intravenous control experiment, is described to determine the amount absorbed as a function of time. Following aerosol administration according to the chosen respiratory regime, fluorescein was rapidly absorbed from the RT. The methodology will enable systematic variation of the particle size and positive-pressure respiratory regime in order to determine effects on drug absorption kinetics.

Absorption↗
PubMed1988

Preparation and characterization of disodium fluorescein powders in association with lauric and capric acids.

Hygroscopic growth of pharmaceutical aerosol powders is a factor governing their deposition, absorption, and pharmacologic effect in the respiratory tract. Surface association of hydrophobic molecules may reduce this effect. Disodium fluorescein was coated with the hydrophobic materials lauric and capric acids. The nature and extent of the association was investigated by a variety of techniques, with the notable use of X-ray photoelectron spectroscopy to examine the powder surface directly. It was concluded that the fatty acids associate at the phenol--sodium bond of the disodium fluorescein, and that the extent of the bulk interaction was governed by a fatty acid-, solute-, and concentration-dependent expanded surface area effect which resulted in much higher surface concentrations than might be expected by simple adsorption.

Caproates↗
PubMed1994

Antibody-antigen binding constants determined in solution-phase with the threshold membrane-capture system: binding constants for anti-fluorescein, anti-saxitoxin, and anti-ricin antibodies.

Affinities of various monoclonal and polyclonal antibodies for fluorescein-containing antigens, saxitoxin and ricin, were determined by using a light addressable potentiometric sensor-based system (Threshold). The dissociation constants, determined from Scatchard plots, ranged from 2 x 10(-7) to approximately 3 x 10(-12) M. Dissociation constants for fluorescein and saxitoxin were compared with values determined by independent means. This technique was found to be quick, simple, reproducible, and accurate.

Antibodies↗
PubMed1986

Fluorescein and tetramethyl rhodamine as haptens in enzyme immunohistochemistry.

Fluorescein (Fl) and tetramethyl rhodamine (Rh) were evaluated as possible candidates for a double hapten sandwich system in enzyme immunohistology. Monoclonal antibodies were raised against Fl and Rh. Their fine-specificity was tested with a competition-like assay. A pair of Mab's was selected for immunohistology in which they functioned as a bridge between Fl/Rh conjugated antibodies and Fl/Rh labeled peroxidase and alkaline phosphatase, respectively. The binding of fluorescein labeled antibodies could be successfully demonstrated in histological slides. A large variability in the efficacy of staining was observed in the case of rhodamine labeled antibodies. The phenomenon is explained by assuming that tetramethyl rhodamine isothiocyanate reacts preferentially with lysine residues near to, or embedded in, hydrophobic regions in a protein. This condition may reduce the accessibility of the Rh moiety for anti-Rh antibodies.

Alkaline Phosphatase↗
PubMed1994

Influence of electrical stimulation of locus coeruleus on the rat blood-brain barrier permeability to sodium fluorescein.

The role of central adrenergic innervation of the brain capillaries is still a matter of discussion. The hypothesis that these nerves control the blood-brain barrier permeability was tested by electrically stimulating the locus coeruleus, the major central adrenergic nucleus, in the anaesthetized rat. Frequencies of 5, 15, and 30 Hz were used. A frequency dependent increase in blood-brain barrier permeability to sodium fluorescein was verified. Prior administration of the alpha-adrenoceptor antagonist phenoxybenzamine (10 mg/kg i.p., 24 h before electrical stimulation) totally blocked the effect of 15 Hz stimulation. The same dose of pindolol (a beta-adrenoceptor antagonist) given 1 h before electrical stimulation potentiated the effect of 5 Hz stimulation. Thus, blood-brain barrier permeability is increased, in a frequency dependent manner, by electrical stimulation of the locus coeruleus. The results obtained with phenoxybenzamine and pindolol suggest an opposite effect of alpha and beta-adrenoceptors on the control of sodium fluorescein transport through the blood-brain barrier.

Adrenergic Fibers↗
PubMed1985

Outward transport of fluorescein from the vitreous in normal human subjects.

Vitreous fluorophotometry was performed on eight normal subjects to compare the outward and inward permeabilities of fluorescein into the vitreous. The results indicate that diffusion alone does not account for the outward flow of fluorescein in the vitreous. The possible underlying factors and significance of these findings are discussed.

Biological Transport, Active↗
PubMed1986

Blood-retinal barrier permeability to carboxyfluorescein and fluorescein in monkeys.

Lipid solubility is a major determinant of permeability across the blood-brain barrier, to which the blood-retinal barrier (BRB) has many similarities. Carboxyfluorescein is a dye with about 1/1000 the lipid solubility of fluorescein, but their molecular sizes and spectral characteristics are similar. We studied the importance of lipid solubility in BRB permeability by comparing the BRB permeabilities to these two dyes. Dye in the vitreous and plasma of four monkeys was measured by fluorophotometry. The estimated inward permeability coefficients (Pin) were 11 +/- 7.4 X 10(-6) cm/min (mean and SD) for carboxyfluorescein and 21 +/- 5.9 X 10(-6) cm/min for fluorescein. The ratio of the means was 1/1.9, far from the expected 1/1000. This finding suggests that the BRB does not function as a continuous lipid membrane and that other factors are more important determinants of permeability for these dyes than lipid solubility.

Animals↗
PubMed1989

Fundamental and clinical studies on fluorescence laparoscopy after intravenous injection of fluorescein-sodium.

In rats with experimental liver injuries and humans with various liver diseases, fluorescence from the liver surface after intravenous injection of 10% fluorescein-sodium was investigated. Our improved fluorescence laparoscopy allowed clear fluorescent images. In normal rats and in cases of nonspecific reactive hepatitis, fluorescence was first found in the portal area of the liver surface; it was then observed spreading into the lobules towards the central veins. In damaged livers of both rats and human, bright fluorescence was observed in arterioles, necrotic foci, and fibrotic areas on the liver surface. The patterns of fluorescence which appeared in the early phase after intravenous injection of fluorescein-sodium seemed to reflect the shapes and distribution patterns of the portal areas. These results suggest that observations with improved fluorescence laparoscopy are useful for diagnoses of liver diseases.

Adult↗
PubMed1984

Fluorescein isothiocyanate-labeled casein assay for proteolytic enzymes.

A simple inexpensive sensitive protease assay was developed using soluble fluorescein isothiocyanate (FITC)-labeled casein. Casein was reacted with FITC to form the fluorescein thiocarbamoyl derivative. This substrate is cleaved by trypsin, chymotrypsin, elastase, subtilisin, and thermolysin in a linear time-dependent manner. These enzymes can be measured in the nanogram and subnanogram range using this assay. The assay is reproducible, has a low blank, and uses casein, which resembles natural substrates of most proteases.

Animals↗
PubMed1988

Fluorescein labeling of Fab' while preserving single thiol.

We determined the conditions for labeling rabbit Fab' with fluorescein isothiocyanate to provide maximal fluorescence intensity compatible with the preservation of integrity of its single thiol residue. We found that we could reproducibly substitute Fab' with two fluorescein moieties in 30 min at room temperature at pH 8.9. We achieved a high level of fluorescence intensity while we preserved the integrity of more than half of the existing thiols.

Animals↗
PubMed1990

Fluorometric determination of bacterial protease activity using fluorescein isothiocyanate-labeled proteins as substrates.

Intact fluorescein isothiocyanate-labeled proteins have relatively low background fluorescence at excitation and emission wavelengths of 495 and 525 nm, respectively. Degradation of these substrates leads to exposure of covalently linked fluorescein isothiocyanate molecules and to a concomitant increase in relative fluorescence at these wavelengths. The increase in relative fluorescence is proportional to the degree of protein degradation. This phenomenon provides the basis for a sensitive assay for bacterial protease activity. There is no requirement for the removal of undegraded substrate from the assay mixture prior to the measurement of fluorescence. Assays can be performed in 96-well microtiter trays, enabling a large number of samples and their respective controls to be processed simultaneously and repeated determinations of fluorescence values may be made on the same assay.

Bacteroides↗
PubMed1985

High-affinity and low-affinity vanadate binding to sarcoplasmic reticulum Ca2+-ATPase labeled with fluorescein isothiocyanate.

Conditions were found that allowed both the fluorescence detection of vanadate binding to the Ca2+-ATPase of skeletal muscle sarcoplasmic reticulum and the vanadate-induced formation of two-dimensional arrays of the enzyme. The fluorescence intensity of fluorescein isothiocyanate-labeled Ca2+-ATPase increased with high-affinity vanadate binding (Ka = 10(6) M-1) as reported by Pick and Karlish (Pick, U. and Karlish, S.D. (1982) J. Biol. Chem. 257, 6120-6126). The Ca2+ and Mg2+ dependencies for high-affinity vanadate binding were similar but not identical to those for orthophosphate. In addition, it was found that there is low-affinity (Ka = 380 M-1) vanadate binding, which causes a 25% decrease in fluorescence. The Ca2+ and Mg2+ dependencies of the low-affinity vanadate binding were different from those of orthophosphate or high-affinity vanadate binding. The covalent attachment of fluorescein isothiocyanate (FITC) in the ATP site of the Ca2+-ATPase did not affect the formation of two-dimensional arrays, as detected by negatively stained electron micrographs. Vanadate concentrations high enough to saturate the low-affinity binding caused two-dimensional arrays as reported by Dux and Martonosi (Dux, L. and Martonosi, A. (1983) J. Biol. Chem. 258, 2599-2603). In addition, freeze-fracture replicas of quick-frozen specimens showed rows of indentations in the inner leaflet of the bilayer that corresponds to the arrays seen on the outer leaflet. This appearance of indentations suggests that low-affinity vanadate binding causes a transmembrane movement of the Ca2+-ATPase. By contrast, high-affinity vanadate binding was shown to cause neither array formation nor the appearance of indentations.

Animals↗
PubMed1983

Fluorescent energy transfer measurements on fluorescein isothiocyanate modified cytochrome P-450 LM2.

The distance between the heme iron and the N-terminus of cytochrome P-450 LM2 was determined by fluorescence energy transfer measurements. Fluorescein isothiocyanate which was covalently bound to the N-terminal methionine was used as donor chromophor. The Ro value between fluorescein isothiocyanate and the heme was calculated to be 3.98 nm. The distance between the nitrogen of the N-terminal methionine and the heme was estimated with 2.84 +/- 0.23 nm excluding most likely the N-terminal amino acid of cytochrome P-450 LM2 to participate in the electron transfer to the heme iron. A cytochrome P-450 LM2 membrane model is proposed.

Animals↗
PubMed1984

The amino acid sequence of the fluorescein isothiocyanate reactive site of lamb and rat kidney Na+- and K+-dependent ATPase.

Fluorescein 5'-isothiocyanate has been used to label ouabain sensitive and insensitive (Na,K)-ATPases from lamb and rat kidney, respectively. The labeled enzymes were digested with trypsin to generate soluble peptides, which were purified by high performance liquid chromatography and sequenced on a gas phase sequenator. The sequence of the labeled peptide from both species is His-Leu-Leu-Val-Met-Lys-Gly-Ala-Pro-Glu-Arg. Thus, it appears that the primary structure of the fluorescein 5'-isothiocyanate reactive site, and therefore presumably the ATP binding site, is completely conserved in ouabain sensitive and ouabain insensitive (Na,K)-ATPases.

Amino Acid Sequence↗