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PubMed1989

[Detection of retinal vascular endothelial damage by fluorescein isothiocyanate-labelled platelet].

Fluorescein isothiocyanate (FITC)-labelled platelets were prepared in Sprague Dawley rats, and brought back into the rat through their femoral veins. Their ability to detect the photocoagulation-induced damage in the occluded retinal vessels was examined by epifluorescence microscopy in whole mount retinal specimens and cryostat specimens. The photocoagulation effects on the major retinal vessels by yellow dye (577 nm) and orange red (600 nm) laser, with parameters of 100 mu, 0.05 sec, 70 m W and 3 exposures were compared. The occluded vessels showed extensive accumulation of labelled platelets in the specimens. The portions photocoagulated by the yellow laser also showed accumulation, but not those by the orange red laser. This difference was considered to be due to the difference of hemoglobin absorption at each wavelength.

Animals↗
PubMed1989

Fluorescein isothiocyanate specifically modifies lysine 338 of cytochrome P-450scc and inhibits adrenodoxin binding.

Treatment of cytochrome P-450scc with fluorescein isothiocyanate (FITC) resulted in covalent labeling with 1.0 +/- 0.1 eq of FITC. Reverse-phase high performance liquid chromatography of tryptic and chymotryptic digests of the labeled protein revealed that a single FITC-labeled peptide accounted for 75% of the label. This peptide was found to be specifically labeled at lysine 338 by amino acid sequencing. The modification of lysine 338 with FITC resulted in 85 +/- 15% inhibition of adrenodoxin binding to cytochrome P-450scc. In a complementary experiment it was found that if a complex between adrenodoxin and native cytochrome P-450scc was formed in the presence of cholesterol and then treated with FITC, there was almost no labeling of lysine 338. The modification of lysine 338 by FITC was not inhibited by 22(R)-hydroxycholesterol, the first intermediate in the side chain cleavage reaction which binds to the active site 300 times more tightly than cholesterol itself. These experiments suggest that lysine 338 is located at the binding site for adrenodoxin and electrostatically interacts with one of the carboxylate groups on adrenodoxin that has been implicated in binding. The fluorescence emission of the FITC label on cytochrome P-450scc was only 14% as large as that of an equivalent concentration of FITC-labeled bovine serum albumin, suggesting that it was quenched by Forster energy transfer to the heme group.

Adrenodoxin↗
PubMed1985

Morphologic studies of uveoscleral outflow in normotensive and glaucomatous beagles with fluorescein-labeled dextran.

Aqueous humor leaves the anterior chamber through 2 pathways: the trabecular meshwork of the iridocorneal angle and the unconventional uveoscleral route. In the latter route, aqueous humor leaves the anterior chamber, passes caudally through the trabecular meshwork and sclerociliary cleft to enter the supraciliary and suprachoroidal spaces. The fluid is absorbed by the choroidal and scleral circulation. Fluorescein-labeled dextran was slowly infused into the posterior chamber of healthy and glaucomatous Beagles for 30 minutes. The eyes were fixed in a mixture of 70% alcohol and formalin, placed in epoxy resin for histologic evaluation, and examined by fluorescent microscopy. Fluorescence was detected in the healthy dogs throughout the uveoscleral pathway. In the glaucomatous dogs, the uveoscleral outflow was decreased or completely absent.

Animals↗
PubMed1989

[Anaphylactic shock caused by fluorescein].

Authors report one case of death caused by anaphylactic collapsus after a fluorescein angiography. They make a review of all the complications described and particularly insist about the anaphylactic reactions. They conclude on the modalities of treatment.

Aged↗
PubMedDate not supplied

Fluorescein angiography. Part 1: Technique and normal study.

1. Fluorescein angiography is a relatively noninvasive diagnostic test which provides the ophthalmic practitioner with anatomic and physiologic information about the ocular structures. 2. Once the dye solution is injected, the photographer observes the fundus through the camera until it appears in the retinal vessels. Photographs are then taken in a rapid sequence. 3. The retinal arteries fill rapidly and evenly within two seconds following the initial choroidal flush. The dye circulates through the whole arterial tree in the body, and in the late stage angiogram recirculation can be seen in the retinal blood vessels.

Eye↗
PubMed1988

The fluorescein isothiocyanate-binding site of the plasma-membrane H+-ATPase of Neurospora crassa.

The mammalian (Na+,K+), Ca2+-, and (H+,K+)-ATPases contain a well-characterized lysine residue that reacts with fluorescein 5'-isothiocyanate (FITC); enzymatic activity is protected by ATP, suggesting that the residue is located in or near the nucleotide-binding domain. In this study, the plasma-membrane H+-ATPase of Neurospora crassa is also shown to be sensitive to FITC. The reaction occurs with pseudo first-order kinetics, has a pKa of 8.0, and is stimulated by Mg2+. Enzymatic activity is protected by MgADP with a Kd of 0.2-0.3 mM, close to the Ki with which MgADP serves as a competitive inhibitor of ATP hydrolysis. A tryptic peptide labeled with FITC in the absence, but not in the presence, of MgADP has been isolated and sequenced. The FITC-sensitive residue is Lys474, located in a region that exhibits significant homology with the mammalian cation-transporting ATPases.

Amino Acid Sequence↗
PubMed1986

Prolonged monitoring of the blood-aqueous barrier with fluorescein-labeled albumin.

Aqueous fluorophotometry has proved to be a useful indicator of changes in the blood-aqueous barrier after surgical, immunologic, or laser manipulations. Previously used fluorescent tracers have been unable to follow rapid changes continuously in the blood-aqueous barrier. Fluorescein-labeled homologous serum albumin, however, provides extremely stable and high plasma levels of fluorescence due to active renal reabsorption with very low levels in the normal aqueous because of its high molecular weight. This feature allows prolonged, continuous, and highly sensitive monitoring of the blood-aqueous barrier before, during, and after a manipulation. The usefulness of this technique is demonstrated in a model that has been well studied with other methods: the response to argon laser iris photocoagulation.

Animals↗
PubMed1986

Numbers and phenotype of lymphocytes emigrating from sheep bone marrow after in situ labelling with fluorescein isothiocyanate.

In normal young lambs the bone marrow was selectively labelled with fluorescein isothiocyanate by a temporary perfusion of one hind-leg. One day later, the incidence of bone marrow emigrants in different lymph nodes, spleen, Peyer's patches, thymus, non-perfused bone marrow and blood was determined. The emigrants were also phenotyped by the use of monoclonal antibodies and classified into monocytes or lymphocyte subsets. Large numbers of lymphocytes left the bone marrow of the perfused leg during 1 day. Considerable numbers of cells migrated to other bone marrow compartments. Varying numbers of mononuclear emigrants were found in peripheral lymphoid organs, with labelling indices ranging from 1.06% in the blood to 0.004% in the thymus. In the spleen, comparable numbers of B- and T-lymphocyte emigrants from the bone marrow were found, whereas in the blood, lymph nodes and jejunal Peyer's patches many more emigrants were T lymphocytes than B lymphocytes. In the prescapular lymph nodes, for instance, 90.4% of emigrants were T cells but only 9.6% were B cells. Based on the large numbers of lymphocytes emigrating from the bone marrow, their phenotypes and their entry into other bone marrow compartments, it it can be concluded that the bone marrow of young lambs is an integral part of the migratory route of lymphocytes.

Animals↗
PubMed1986

Dendritic cells and the initiation of contact sensitivity to fluorescein isothiocyanate.

Lymph node cells taken 24 hr after skin-painting mice with the contact sensitizer fluorescein isothiocyanate (FITC) induce delayed-type hypersensitivity in recipient mice. Skin-painting increased the number of dendritic cells (DC) in the draining lymph nodes without significantly changing the number of lymphocytes at 24 hr. The antigen was preferentially located on the DC. Raising the dose of FITC increased both the number of DC and the amount per cell. The addition of these DC to syngeneic lymph node cells at a ratio as low as 1:300 initiated proliferative responses in vitro. The level of proliferation was related to the amount of antigen on the DC. Mice given 50,000 of these fluorescent DC developed specific contact sensitivity reactions. DC exposed in vitro to FITC also acquired antigen and were able to initiate proliferative responses in vitro and to sensitize recipient mice. The DC may therefore be the prime cell involved in the induction of delayed-type hypersensitivity.

Animals↗
PubMed1988

Effect of papain digestion on the specificity of fluorescein-labeled immunoglobulins.

During initial studies, we found that many fluorescein isothiocyanate-labeled anti-immunoglobulin conjugates were unstable and tended to aggregate and precipitate when used for indirect immunofluorescence microscopy. In some instances, the precipitate was extensive enough to interfere with interpretation of the test results. Attempts to resolve this problem resulted in a procedure by which such conjugates were digested with papain to Fab and Fc fragments before use. Aggregation and precipitation were prevented, while desired antibody activity was retained. Digestion with papain also reduced the diffuse background fluorescence (commonly referred to as nonspecific fluorescence or staining) that is often associated with conjugates before they are sorbed with tissue powders or chromatographed to remove highly labeled immunoglobulin molecules.

Animals↗
PubMed1988

Fluorescein conjugated lectins identify different carbohydrate residues on Glossina peritrophic membranes.

Fluorescein-lectin conjugates were used as markers to determine the presence of surface carbohydrates on the peritrophic membranes of 6 Glossina species. Inter- and intra-specific variation in exposed carbohydrate residues was observed. Peritrophic membranes from non-teneral flies appeared to have less exposed surface carbohydrates than those of tenerals. Teneral G. m. morsitans susceptible to trypanosome infection had exposed carbohydrate residues recognised by APA lectin, but these residues were absent in a line of this species refractory to trypanosome infection. It is suggested that at least some of the surface carbohydrates of the peritrophic membrane bind endogenous gut lectin and parasite-surface carbohydrates may influence trypanosome development in Glossina spp.

Acetylgalactosamine↗
PubMed1988

[Intra-operative imaging of the ureter with sodium fluorescein].

Surgical interventions on the abdominal, pelvic and retroperitoneal organs are frequently complicated by damage or alteration of the ureter. To prevent intraoperative damage of the ureter the authors suggest a method of visual identification of the ureter by luminescence analysis. A 10% solution of natrium fluorescein (14-20 mg/kg) was used as the fluorochrome. After experimental validation the method was used successfully in 12 patients.

Animals↗
PubMed1987

Microcytotoxicity assay for platelet antibodies using fresh and frozen fluorescein-labeled target platelets.

We describe our results with a microtoxicity assay that uses fluorescein-labeled platelets for the detection of auto-, iso-, and allo-immune platelet antibodies. The system has a number of advantages in that it is not only rapid but can accommodate multiple test specimens, requires minimal reagents, and can utilize both fresh or frozen donor platelets as the target cells. The latter attribute may allow for the development of a platelet blood banking system with more efficient platelet cross-matching capabilities.

Antibodies↗
PubMedDate not supplied

[Measurement of capillary permeability in chronic venous insufficiency using sodium fluorescein video-densitometry].

The measurement of capillary permeability in chronic venous insufficiency (CVI) requires techniques able to modify local microcirculatory hemodynamics and therefore the results. As for instance, venous occlusion in the Landis test or occlusive plethysmography. We are presenting a selective method which visualizes and quantifies capillary permeability without modifying the hemodynamics. A skin area on the lower extremity is observed on a microscope with blue filters. This optical device is linked with a camera transmitting information to a TV monitor and a video cassette recording system. When focusing is done, the patient's brachial vein is injected with an IV bolus of 1 ml if sodium fluorescein (Na flu). This dye diffuses rapidly from the intra toward the extra-capillary compartment. A densitometer connected to a graph measures the capillary permeability selected dynamics. The capillary permeability to Na flu is evaluated qualitatively on the TV screen (pericapillary halo), and quantitatively by the densitometric curve. In practice, this technique is advocated in the physiopathological study of any edema, especially in early CVI.

Capillary Permeability↗
PubMed1985

Characterization of monoclonal anti-fluorescein antibodies from autoimmune New Zealand mice.

Previous studies of murine IgM hybridoma protein 18-2-3, derived from an (NZB/NZW)F1 secondary response to fluorescein (FL) presented on T-dependent carrier, demonstrated a high binding affinity for FL (KA = 2.9 X 10(10) M-1) and cryoprecipitation, which could be abrogated upon FL binding. Based on these unusual properties and their possible association with defective immune regulation in lupus-prone mice, further studies were carried out to evaluate the basis of 18-2-3 cryoprecipitation, expression of characteristics related to the 18-2-3 clonotype, and structure/function aspects of additional homogeneous IgM and IgG antibodies of similar origin and specificity. Solubility experiments in which the effect of ionic strength on macroscopic aggregation was measured indicated that 18-2-3 intrinsically possessed both cryoglobulin and euglobulin properties in the absence of auxiliary gamma-globulin components. Rates of hapten fluorescence quenching by 18-2-3 were limited by factors other than diffusion and were dependent on solution temperature and ionic strength. Thirty-seven additional IgM and IgG monoclonal antibodies were shown to possess normal low-temperature solubility and hapten fluorescence-quenching properties, suggesting that 18-2-3 was derived from a relatively rare B cell progenitor. Collective results from FL binding and spectrotype analyses indicated that the majority of proteins were diverse with respect to variable region structure and binding mechanisms but unusually restricted in binding affinities (KA less than 5 X 10(6) M-1). Relative subclass frequencies for 30 monoclonal IgG proteins (IgG1 greater than IgG2b greater than IgG2a greater than IgG3) were consistent with polyclonal IgG subclass expression in normal mice in response to T-dependent immunogen.

Animals↗
PubMed1985

Interaction of gelatin with a fluorescein-labeled 42-kDa chymotryptic fragment of fibronectin.

A 42-kDa gelatin binding fragment of human plasma fibronectin was labeled with fluorescein and its fluid-phase interaction with gelatin was investigated. At 25 degrees C in 0.1 M Tris, 0.15 M NaCl, pH 7.3, a dissociation constant, Kd = 0.6 microM, was obtained from the dependence of fluorescence polarization on gelatin concentration. An identical value was obtained for the unlabeled fragment by competition. Binding was unaffected by higher concentrations of NaCl up to 1.0 M, but increased as much as 20-fold at low ionic strength. The dependence of Kd on temperature revealed that dissociation of the complex is accompanied by an increase in entropy. Thus, the interaction is not dominated by either hydrophobic or electrostatic forces; an important role for hydrogen binding is proposed.

Chymotrypsin↗
PubMedDate not supplied

Influence of organic solvents and pH on some characteristics of influenza virus A(H1N1). Note 1. Influence of organic solvents on the spectral characteristics of fluoresceine isothiocyanate-conjugated influenza virus A(H1N1).

The fluorescence of fluoresceine isothiocyanate (FITC)-conjugated influenza virus A(H1N1) was enhanced following addition of ethanol, acetone or dimethylsulfoxide (DMSO) and decreased after addition of dioxane; light scattering values increased in the presence of any of these organic solvents. The effect of the organic solvents could be correlated with the excess values delta epsilon of the dielectric constant. Out of the four organic solvents it was DMSO that caused the slightest changes in the fluorescence and light scattering of the FITC/virus system.

Dose-Response Relationship, Drug↗
PubMed1985

Streamlined fluorescein photography.

A simple, accurate inexpensive method for obtaining Polaroid pictures after the intravenous injection of fluorescein is presented. The method relies on an interference filter system and modifications permitting the use of high speed film. The resultant photographic images are equivalent to the ultraviolet light fluorescence. The photographs may be used as either a diagnostic tool or for documentation purposes.

Fluorescein↗