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PubMedDate not supplied

Direct cytotoxicity mediated by allogeneic and isogeneic pig lymphocytes modified with fluorescein isothiocyanate.

Cell mediated cytotoxicity (CMC) is restricted by the major histocompatibility complex (MHC) in some animal models but not in others. In order to determine if there was MHC restriction in a CMC experimental model, mononuclear cells from blood of two lines of pigs, (1/1) and (6/6) which are homogeneous to the MHC products (SLA-ABCD), were used. Cytotoxic (1/1) cells were induced in vitro and in vivo against (1/1) mononuclear cells modified with fluorescein isothiocyanate (FITC) and tested against (1/1)-FITC isogeneic and (6/6)-FITC allogeneic cells, without showing MHC restriction of the cytotoxicity. The cytolysis was not due to antibody and K cells because fluoresceinated ovalbumin did not inhibit the test. It was not due to the "altered self" reaction against its own MHC products that sometimes may be induced by allogeneic responses, because the (1/1) anti (6/6) cytotoxic cells did not kill haptenated cells (1/1)-FITC. According to the results there was no evidence of MHC restriction of the CMC response of haptenated cells from pigs of (1/1) haplotype.

Animals↗

PubMed1990

Improved purification of human lactate dehydrogenase isoenzyme-3 and studies with its fluorescein isothiocyanate and biotin conjugates.

Lactate dehydrogenase (L-lactate:NAD+ oxidoreductase, EC 1.1.1.27) isoenzyme-3 (LD-3) has been isolated in milligram quantities from human erythrocytes. Using an improved procedure--which involves complete hemolysis of the erythrocytes, diethylaminoethyl (DEAE)-Sephacel column chromatography, and 5'-AMP-Sepharose 4B affinity chromatography--we obtained 23,000-fold purified isoenzyme from the crude hemolysate (overall yield about 90%). The final product was homogeneous on polyacrylamide disc gel electrophoresis and had a specific activity of about 435 kU/g. Its amino acid composition is presented. With the eventual aim to make visible and isolate IgA kappa antibody-secreting B lymphocytes, we developed reproducible methods for preparing fluorescein isothiocyanate isomer-1-conjugated LD-3 with a fluorescein/LD-3 molar ratio between 1.3 and 3.3, and biotinylated LD-3 with a biotin/LD-3 molar ratio between 1.3 and 2.5. In evaluating the stability of these two conjugates, we determined that they still can react with IgA kappa to form the IgA kappa (LD-3)2 complex.

Affinity Labels↗

PubMed2006

Fluorescein isothiocyanate labelled bovine serum albumin (FITC-BSA) as a model protein drug: opportunities and drawbacks.

In the therapy of various diseases, parenterally administered protein drugs are of steadily rising importance. In order to reduce the application frequency, these proteins can be incorporated into drug delivery systems, e.g. biodegradable microparticles from poly(lactic-co-glycolic acid) (PLGA). To evaluate the characteristics of these vehicles, fluorescent labelled proteins like fluorescein isothiocyanate labelled bovine serum albumin (FITC-BSA) may be used as model drugs to allow the visualisation of the protein localisation within the microparticle and the detection of microparticles in cell cultures or tissues. However, the quantification of protein by fluorescence spectroscopy failed. In this study we focused on the mechanism of fluorescence dequenching in a multi-FITC-labelled protein and its impact on a reliable protein determination.

Dendritic Cells↗

PubMed2007

Effects of 6-(methylsulfinyl)hexyl isothiocyanate on cyclooxygenase-2 expression induced by lipopolysaccharide, interferon-gamma and 12-O-tetradecanoylphorbol-13-acetate.

6-(methylsulfinyl)hexyl isothiocyanate (6-MITC) is a bioactive compound extracted from a typical Japanese spice, wasabi (Wasabia japonica (Miq.) Matsumura). In the present study, we found that 6-MITC suppressed the expression of cyclooxygenase-2 (COX-2) induced by lipopolysaccharide (LPS), interferon-gamma (IFN-gamma), but did not suppress that induced by 12-O-tetradecanoylphorbol-13-acetate (TPA), in murine macrophage RAW264. Molecular mechanisms were investigated by targeting the transcriptional factors including activator protein-1 (AP-1), CCAAT/enhancer-binding protein delta (C/EBPdelta), CRE-binding protein (CREB) and nuclear factor kappaB (NF-kappaB), which bind to the core element of COX-2 promoter. LPS induced activation of all of these factors and 6-MITC suppressed LPS-induced activation of AP-1, C/EBPdelta, CREB, but not NF-kappaB. IFN-gamma did not induce any activation of these factors, but 6-MITC suppressed IFN-gamma-induced COX-2 expression, suggesting that the upstream region of the core element is linked for this suppression. Finally, TPA stimulated the activation of CREB and AP-1, but 6-MITC did not block TPA-induced COX-2 expression. These results suggest that LPS, IFN-gamma and TPA regulate COX-2 expression through different mechanisms, and 6-MITC acts as a potent inhibitor of COX-2 expression induced by LPS or IFN-gamma.

Animals↗

PubMed1991

[Chemical modification of steroid-hydroxylating monooxygenases with fluorescein isothiocyanate].

Chemical modifications of cytochrome P-450scc and cytochrome P-450(11) beta with fluorescein-, diiodofluorescein-, eosine- and rhodamine isothiocyanate have been carried out. At a low reagent/protein ratio and neutral pH, a selective chemical modification was known to take place which did not affect the spectral properties of cytochrome P-450scc. Covalent chromatography was found useful to discriminate between covalent modification of cytochrome P-450scc and non-specific binding of FITC with cytochrome P-450scc. Proteolytic modification of cytochrome P-450scc and structural analysis indicate that a lysine residue of the C-terminal sequence of cytochrome P-450scc is accessible to FITC. The residue was shown, by the analysis of the chymotryptic hydrolysate of the fragment F2, to be Lys338. Effect of modification with FITC on the interaction of cytochrome P-450scc with cholesterol or adrenodoxin, on the reduction kinetics and on the conversion of cholesterol to pregnenolone was also studied.

Amino Acid Sequence↗

PubMed1991

Identification of the lysine residue involved in the inactivation of lamb liver 6-phosphogluconate dehydrogenase by fluorescein 5'-isothiocyanate.

Fluorescein 5'-isothiocyanate binds almost selectively at the active site of lamb liver NADP-dependent 6-phosphogluconate dehydrogenase causing the inactivation of the enzyme. The substrate and the coenzyme protect against the loss of catalytic activity. The enzyme derivative was digested with trypsin, the labelled peptide was isolated by h.p.l.c. and its amino acid analysis allowed to establish that the inactivator binds to lysine 166 at the active site of the protein.

Animals↗

PubMed1991

Nucleotide specificity of cardiac sarcoplasmic reticulum. Inhibition of GTPase activity by ATP analogue in fluorescein isothiocyanate-modified calcium ATPase.

Unlike skeletal muscle sarcoplasmic reticulum, canine cardiac sarcoplasmic reticulum hydrolyzes GTP in ways that are similar and different from ATP hydrolysis. Also, ATP and ATP analogues inhibit GTPase activity noncompetitively with a Ki compatible with the high affinity ATP-binding site (c.f. Tate, C.A., Bick, R.J., Blaylock, S., Youker, K., Scherer, N.M., and Entman, M.L. (1989) J. Biol. Chem. 264, 7809-7813). This suggested that ATP and GTP may enter the reaction pathway at separate nucleotide-binding sites on the CaATPase. To test this hypothesis, cardiac sarcoplasmic reticulum was incorporated with fluorescein isothiocyanate (FITC), which apparently binds at or near the ATP-binding site of the enzyme, preventing ATP binding. After FITC incorporation, calcium-dependent ATPase activity, but not GTPase activity, was completely inhibited. Adenyl-5'-yl imidodiphosphate (AMP-P(NH)P), but not guanyl-5'-yl imidodiphosphate, protected against FITC incorporation and the inhibition of calcium-dependent ATPase activity; at least 100 microM AMP-P(NH)P was required for some protection. Despite FITC incorporation, AMP-P(NH)P still inhibited the GTPase activity with a Ki of 3-7 microM. Direct photo-affinity labeling with either 0.2 microM [alpha-32P]ATP or 0.2 microM [alpha-32P]GTP demonstrated that FITC incorporation did not prevent ATP or GTP binding. The mechanism of FITC inhibition of calcium-dependent ATPase activity was related to the prevention of all calcium-dependent, but not calcium-independent, reactions with both nucleotides.

Adenosine Triphosphate↗

PubMed1986

o-Isothiocyanate dihydropyridine (oNCS-DHP), a long-acting, reversible inhibitor of the Ca++ channel.

The binding characteristics and pharmacological properties of o-isothiocyanate dihydropyridine [oNCS-DHP; 2,6-dimethyl-3,5-dicarbomethoxy-4-(2-isothiocyanatophenyl)-1, 4-dihydropyridine] were investigated in guinea pig heart and ileum. [3H]oNCS-DHP bound to a single population of high-affinity sites (Bmax = 107 fmol/mg of protein and Kd = 0.99 nM) in cardiac membranes, with a specificity characteristic of dihydropyridine receptors. After incubation of membranes with the tracer (0.5 nM), addition of excess nifedipine (1 microM) caused a dissociation of [3H]oNCS-DHP from its binding site. The reversibility of [3H]oNCS-DHP binding was confirmed by the lack of affinity labeling of cardiac membranes as determined by sodium dodecylsulfate-polyacrylamide gel electrophoresis. oNCS-DHP inhibited the inward Ca++ current of isolated guinea pig cardiac myocytes as determined in voltage-clamp experiments. In isolated perfused guinea pig hearts, oNCS-DHP caused a concentration-dependent increase in coronary artery flow and a decrease in left ventricular pressure. The effects of the highest concentration (0.3 microM) were still near maximal after a 1-h washout. Suppression of K+ depolarization-induced contractures of isolated ileal longitudinal muscle strips by oNCS-DHP remained maximal even after 5 h of washout. In all of the three biological test systems investigated, the Ca++ channel activator Bay K 8644 caused a complete and rapid reversal of the inhibitory effects of oNCS-DHP. Thus, it can be concluded that oNCS-DHP does not bind irreversibly to Ca++ channel dihydropyridine receptors in guinea pig heart and ileum. However, the o-isothiocyanatophenyl substituent on the dihydropyridine molecule confers upon the compound a very long duration of Ca++ channel blocking activity.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗

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↗

PubMedDate not supplied

Age-related changes in the metabolism and excretion of allyl isothiocyanate. A model compound for glutathione conjugation.

The objective of this study was to evaluate age-related changes in the in vivo metabolism and excretion of allyl isothiocyanate (AITC), which is used as a model compound to assess glutathione conjugation. AITC is a constituent of oil of mustard and a food additive that has been shown to induce transitional cell papillomas in the urinary bladder of male Fischer rats. Male Fischer rats, ages 3, 16, and 27 months, were dosed orally with 14C-AITC (25 mg/kg). Urine, feces, volatiles, and expired air were collected for 72 hr. Biliary excretion was also examined after iv administration of 10 mg/kg of 14C-AITC. There was no significant difference between these age groups in the percentage of the total dose of AITC eliminated in urine, which is the major route of excretion. The percentage of the dose excreted as volatiles increased in the 27-month animals. These animals also showed a decrease in the production of 14CO2. An age-related decrease in fecal excretion of AITC-derived radioactivity was observed. However, the percentage of the dose excreted in bile increased at 16 months and then decreased in the senescent rats as compared to the 3-month age group. The age-related decrease in 14CO2 production suggests a change in the oxidative metabolism of AITC in senescent animals. However, there was no change in the relative amounts of mercapturic acid excreted in urine, suggesting that glutathione conjugation was unchanged with age.(ABSTRACT TRUNCATED AT 250 WORDS)

Age Factors↗

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↗

PubMed1989

Inhibition of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-induced DNA adduct formation and tumorigenicity in the lung of F344 rats by dietary phenethyl isothiocyanate.

F344 rats fed diets containing phenethyl isothiocyanate (PEITC, 3 mumol/g diet), a cruciferous vegetable component, before and during treatment with the tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), developed about 50% fewer lung tumors than NNK-treated rats fed control diets. NNK-induced liver and nasal cavity tumors in rats were, however, not affected by this dietary treatment. The effects of PEITC diets on the formation of DNA adducts by NNK were also investigated in these target tissues. DNA methylation and pyridyloxobutylation by NNK were both decreased by 50% in lung of rats fed PEITC diets compared to that of rats fed control diets, but the levels of DNA methylation were not affected in liver and nasal mucosa. These results correlated with those from the carcinogenicity bioassay, suggesting that DNA alkylations could be used as indicators for screening inhibitors of NNK tumorigenesis. A slight increase in the number of tumors of the exocrine pancreas was observed in PEITC-fed rats with or without NNK treatments. However, these incidences were not statistically significant when compared to the control groups. The potential toxicity of PEITC at concentrations ranging from 0.75 mumol to 6 mumol/g diet was evaluated in a 13-week study. The only toxicity caused by this treatment was minimal fatty metamorphosis in the liver. Considering the widespread human exposure to NNK through tobacco use, it is of practical importance to demonstrate inhibition of lung tumors induced by this carcinogen. These results provide a basis for studies designed to discover agents of better efficacy for the prevention of NNK-induced tumorigenesis.

Alkylation↗

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↗

PubMedDate not supplied

Characterization of benzyl isothiocyanate and phenyl acetonitrile from papayas by mass spectrometry.

Two unidentified analytical responses in a papaya extract were structurally determined by mass spectrometry to be benzyl isothiocyanate and phenyl acetonitrile. Both these compounds have previously been shown to result from degradation of benzylglucosinolate that occurs naturally in the seeds of the fruit. Characterization by mass spectrometry has now provided a convenient mechanism to detect both these degradation compounds in extracts resulting from routine pesticide residue analysis.

Acetonitriles↗

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↗

PubMedDate not supplied

[Safety of animal husbandry products produced by using allyl isothiocyanate-preserved feeds].

To examine the safety of foods (meat and milk) obtained from animals whose feeds were preserved with allyl isothiocyanate (AITC), the authors investigated the status and development of animals, some aspects of protein, lipid and carbohydrate metabolism, some enzymes, hemopoiesis and reproduction function of Wistar rats fed diets containing the above products (55 g dry milk or 50 g boiled meat per 100 g diet). To measure the residual quantity of allylamine (AA), the main metabolite of AITC, a chromato-fluorometric method with the use of dansyl chloride was devised, permitting the detection of 2 ng AA in the spot. The relative standard deviation was 0.14-17. The degree of AA extraction amounted to 96%. The method made it possible to detect the residual quantity of AA in the kidneys (up to 12.2 +/- 2.0 micrograms/kg) and in the liver (up to 83.1 +/- 12.4 micrograms/kg). The data obtained evidence that AA cannot be used as conservant for manufacturing feeds for agricultural animals.

Allylamine↗