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PubMed1983

Catalytic and regulatory ATP-binding sites of the red cell Ca2+ pump studied by irreversible modification with fluorescein isothiocyanate.

Catalytic and regulatory binding sites for ATP on the red cell Ca2+ pump have been investigated using fluorescein isothiocyanate (FITC). Both (Ca2+ + Mg2+)-ATPase activity and ATP-dependent Ca2+ flux are selectively and irreversibly inactivated by FITC and the pump is protected from FITC by the presence of ATP. The time course of inactivation by FITC is characteristically biphasic. Analysis of the kinetics of inactivation by FITC and protection by ATP reveals the participation of both high and low affinity binding sites for ATP and FITC. The sites binding ATP or reacting with FITC do not, however, appear to co-exist on the same enzyme molecules. Thus, "flip-flop" mechanisms for (Ca2+ + Mg2+)-ATPase, involving negative interactions between high and low affinity ATP sites, are considered unlikely. The two affinities for ATP are most simply explained by assuming that the Ca2+ pump protein exists in alternative conformational forms, E1 having a high affinity for ATP and E2 having a low affinity for ATP. Ca2+ pumping and (Ca2+ + Mg2+)-ATPase involve interconversion between these forms. It is suggested that regulation of Ca2+ pump activity by Mg-ATP reflects acceleration of the conformational transition between the E1 and E2 forms, as well as a previously described acceleration of phosphoenzyme hydrolysis (Muallem, S., and Karlish, S. J. D. (1981) Biochim. Biophys. Acta 647, 73-86; Garrahan, P. J., and Rega, A. F. (1978) Biochim. Biophys. Acta 513, 59-65).

Adenosine Triphosphate↗

PubMed1983

Granules of blood eosinophils are stained directly by anti-immunoglobulin fluorescein isothiocyanate conjugates.

Direct staining of the granules of blood eosinophils by anti-immunoglobulin fluorescein isothiocyanate (FITC) conjugates was observed when feline blood smears were tested for presence of feline leukemia virus (FeLV) antigen by immunofluorescent antibody. When blood smears of other species including swine, horses, cattle, dogs, sheep, birds, and human beings were examined, direct staining of eosinophils by FITC conjugates was also detected. This FITC staining was restricted to eosinophils and was not observed in neutrophils, lymphocytes, and platelets. Direct FITC staining of eosinophils does not represent a problem in immunofluorescent test for the detection of FeLV infection in cats, as long as the eosinophils, which can easily be recognized as such, are excluded from the spectrum of interpreted cells.

Animals↗

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

[Double labeling of antibodies with fluorescein isothiocyanate (FITC) and horseradish peroxidase (HPOD)].

This paper presents a method for labeling antibodies with fluorescein isothiocyanate (FITC) and additional with horseradish peroxidase (HPOD). With this double labelled antisera the fluorescence-serologic antibody technique (FAT) as well as the enzyme-serologic antibody technique (EAT) was done bit by bit for the same object. How the presented pictures demonstrate the result of both techniques, FAT and EAT, are the same with the described method. So, it is possible after fluorescence-microscopical exploitation through a following histochemical proof of the HPOD to get permanent preparations with the same biological result.

Animals↗

PubMed1981

Inhibition of carcinogen-induced neoplasia by sodium cyanate, tert-butyl isocyanate, and benzyl isothiocyanate administered subsequent to carcinogen exposure.

The effects of sodium cyanate, tert-butyl isocyanate, and benzyl isothiocyanate on carcinogen-induced neoplasia were studied in experiments in which the test compound was fed starting 1 week following completion of carcinogen administrations. Under these conditions, all three test compounds exerted an inhibitory effect on the occurrence of 7,12-dimethylbenz(a)anthracene-induced neoplasia of the breast of Sprague-Dawley rats. In a second experiment model, sodium cyanate inhibited the occurrence of 1,2-dimethylhydrazine-induced neoplasia of the large bowel of female CF-1 mice. Thus, a new group of compounds has been identified which has inhibitory capacities against neoplasia when given subsequent to carcinogen exposure.

9,10-Dimethyl-1,2-benzanthracene↗

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↗

PubMed1995

Assessment of hepatobiliary function and placental barrier integrity in pregnant rats by combination of bromosulphophthalein and fluorescein isothiocyanate plasma disappearance tests.

The permeability of the placental barrier to bromosulphophthalein (BSP) is believed to be very low. Whether this property is shared by other cholephilic organic anions, such as fluorescein isothiocyanate (FITC), is not known. When BSP was injected (140 mumol/kg body wt) into the left jugular vein of non-pregnant or pregnant female rats (at the 21st day of pregnancy), a similar and rapid plasma disappearance was observed during the first few minutes; afterwards, a slower disappearance phase was found. This phase was different in these groups, that is, a longer retention of BSP in the maternal bloodstream of pregnant rats was accompanied by a slower BSP output into bile. It was impossible to demonstrate the presence of BSP in fetal blood or the placenta by colorimetric methods. These results are consistent with the modifications occurring in the hepatic handling of BSP during pregnancy together with a marked impermeability of the placenta to the dye, at least in the mother-to-fetus direction. After intravenous FITC (10 mumol/kg body wt) administration to the mother, the compound was rapidly transferred into both the maternal bile and the fetal blood. Thereafter, FITC refluxed back from the fetal-placental compartment to the maternal blood as soon as the maternal liver reduced its plasma concentrations, which were first higher (approximately threefold) and then similar to those found in fetal blood. The reversible retention of FITC by the fetal-placental compartment accounts for a significant delay in both FITC bile output and plasma disappearance as compared with those observed in non-pregnant rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1995

Effects of phenethyl isothiocyanate on the tissue distribution of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and metabolites in F344 rats.

Phenethyl isothiocyanate (PEITC), a naturally occurring chemopreventive agent, inhibits lung tumor induction in rats by the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). In this study, we examined the mechanism of tumor inhibition by determining the effects of dietary PEITC on levels of NNK and its metabolites in various tissues of NNK-treated F344 rats. F344 rats were fed control or PEITC-containing diets (3 mumol/g diet) before and throughout NNK treatment. To study NNK metabolism and distribution under both short-term and chronic NNK/NNK+PEITC treatments, control and PEITC-treated groups were divided into four subgroups. Subgroups were treated with either a single injection of [5-3H]NNK (1.76 mg/kg) or a total of 12, 24, and 36 doses of NNK administered three times/week. After a final injection of [5-3H]NNK in each subgroup, the rats were sacrificed at various time intervals, and NNK and its metabolites in lung, liver, nasal mucosa, pancreas, kidney, stomach, and serum were measured. Time-course curves for the tissue metabolites were generated, and the areas-under-the-curves were compared. We observed that lung, liver, and nasal mucosa, target tissues of NNK carcinogenesis in F344 rats, were also the tissues that had the highest levels of alpha-hydroxylation metabolites relative to NNK and its carbonyl reduction metabolite, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL). The most pronounced effect of PEITC was a reduction in levels of alpha-hydroxylation metabolites in most tissues examined (except nasal mucosa). The ratio of alpha-hydroxylation products to NNK + NNAL in most tissues was decreased by PEITC, indicating that alpha-hydroxylation of NNK/NNAL was inhibited. PEITC did not significantly affect the total levels of NNK and its metabolites in the lung and most tissues examined, indicating that PEITC does not alter the amount of NNK reaching the lung. These results support the hypothesis that inhibition of NNK-induced lung tumorigenesis by PEITC is a result of decreased metabolic activation of NNK.

Animals↗

PubMed1994

Analysis of human platelet glycoprotein IIb-IIIa by fluorescein isothiocyanate-conjugated disintegrins with flow cytometry.

Disintegrins are a group of snake venom peptides which inhibit human platelet aggregation by acting as glycoprotein IIb-IIIa (GPIIb-IIIa) antagonists. They are cysteine-rich, Arg-Gly-Asp (RGD)-containing peptides, and bind to GPIIb-IIIa complex on platelet membrane with a very high affinity (Kd, 10(-7)-10(-8) M). In this study, we analyzed GPIIb-IIIa complex on platelet membrane by flow cytometry using fluorescein isothiocyanate (FITC)-conjugated disintegrins as probes. Of these FITC-conjugated disintegrins, FITC-Rhodostomin is the most sensitive probe because Rhodostomin was conjugated with more FITC molecules than Trigramin and Halysin were. The binding fluorescence intensity of FITC-Trigramin (FITC-Tg), FITC-Halysin (FITC-Hy) and FITC-Rhodostomin (FITC-Rn) was measured in both resting and ADP-activated platelets of diluted human platelet-rich plasma. The binding fluorescence of FITC-disintegrins was abolished by EDTA and 7E3, a monoclonal antibody against GPIIb-IIIa. ADP markedly increased the fluorescence intensity of FITC-Tg and FITC-Hy bound on platelets especially when lower doses of these probes were used, whereas it had little effect on that of FITC-Rn. Therefore, FITC-Tg and FITC-Hy can be used for the detection of the activated platelets as noted by a higher ratio of fluorescence intensity (approx. 2-4) between ADP-activated and resting platelets as compared with that (approx. 1-1.3) in the case of FITC-Rn as the probe. The platelets from three patients with Glanzmann's thrombasthenia were probed with FITC-disintegrins.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMedDate not supplied

Analysis of methyl isothiocyanate in wine by gas chromatography with dual detection.

Methyl isothiocyanate (MITC) was extracted from a 5 mL wine sample with 2 mL dichloromethane, and the extract was washed with 5% sodium chloride solution. MITC was determined by gas chromatography with a dual nitrogen-phosphorus detector (NPD) and a flame photometric detector (FPD, S mode) (DB-210 column, 30 m x 0.53 mm id, column temperature, 70 degrees C). The limit of detection and quantitation of MITC with NPD-GC were 0.003 and 0.01 ppm, respectively. Average recoveries of MITC added to wine at 1 and 0.1 ppm were over 95%. This method is simple and rapid for routine analysis of MITC in wine.

Chromatography, Gas↗

PubMed1994

Identification and quantification of the N-acetylcysteine conjugate of allyl isothiocyanate in human urine after ingestion of mustard.

Allyl isothiocyanate (AITC) is a constituent of cruciferous vegetables. It occurs widely in the human diet as a natural ingredient or food additive. AITC possesses numerous biochemical and physiological activities. It is cytotoxic and tumorigenic at high doses and also is a modulator of enzymes involved in metabolism of xenobiotics, including carcinogens. It is plausible that the wide consumption of dietary AITC may have profound effects on human health. To facilitate investigations of the effects of dietary AITC in humans, a method of measuring its uptake is needed. In this study, a urinary marker was developed for quantifying AITC uptake in humans. Four adult volunteers were asked to eat a meal containing brown mustard as the source of AITC. The 48-h urine samples were collected from these individuals and analyzed by reverse phase high performance liquid chromatography. A major urinary metabolite was found, which was identified as N-acetyl-S-(N-allylthiocarbamoyl)-L-cysteine, the N-acetylcysteine conjugate of AITC, by comparing its retention time and UV, nuclear magnetic resonance, and mass spectra with those of the synthetic standard. After ingestion of mustard, the AITC conjugate was detected in urine collected from 0 to 12 h. No conjugate was found in urine samples collected after 12 h. The major portion of this metabolite was excreted within 8 h. The average total excretion of AITC conjugate was 5.4 +/- 1.7 (SD) mg after consumption of 10 g of mustard and 12.8 +/- 2.0 mg when 20 g of mustard was consumed. Thus, a dose-dependent excretion of this metabolite was demonstrated.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylcysteine↗

PubMed1993

[A method for measurement of circulating blood volume with fluorescein isothiocyanate labeled red blood cells].

We developed a new method to measure the total circulating blood volume using fluorescein isothiocyanate (FITC) labeled autologous red blood cell as an indicator. Red blood cells were labeled by incubation with 0.1 mg.ml-1 FITC in PBS (pH 7.2) for 30 min at 37 degrees C. Eighteen dogs were given an intravenous injection of FITC labeled red blood cells, which were approximately 0.1% of the total circulating red blood cells estimated from the body weight. The ratio of labeled cells to the normal circulating red blood cells was determined by a flow cytometry. The total blood volume was calculated from its dilution rate. The average circulating blood volume determined by this method was 76.8 +/- 9.2 ml.kg-1 (mean +/- SD). This result coincides well with the values reported previously. This method using non radioactive tracers enables a reproducible measurement of circulating blood volume and its change with time.

Animals↗

PubMed1993

Mechanisms of inhibition of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone bioactivation in mouse by dietary phenethyl isothiocyanate.

The tobacco-specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1- butanone (NNK), induces lung tumors in mice, rats, and hamsters. Phenethyl isothiocyanate (PEITC), which occurs as gluconasturtiin in cruciferous vegetables, is a potent inhibitor of NNK-induced carcinogenesis. The present study investigated the enzymatic basis for the bioactivation of NNK and the mechanisms of the inhibition of this process by dietary PEITC in mice. The apparent Km for the formation of keto aldehyde, keto alcohol, and NNK-N-oxide in lung microsomes was 4.9, 2.6, and 1.8 microM and, in liver microsomes, 5.5, 5.1, and 8.8 microM, respectively. Immunoinhibition studies suggested that cytochrome P450s (P450s) 2A1 and 2B1 or related forms are the major enzymes involved in the oxidative metabolism of NNK in mouse lung microsomes. When female A/J mice were fed diets containing 0, 1, or 3 mumol of PEITC/g of diet for 4 wk, the dietary PEITC had no significant effects on the food consumption and body weight of the mice. NNK oxidation in the lung microsomes of mice consuming the 1 or 3 mumol of PEITC/g of diet was decreased by 13 to 27% or 30 to 50%, respectively. In liver microsomes, whose NNK oxidative metabolism rates were about twice those of lung microsomes on a per mg of protein basis, the activities were decreased by 14 to 31% by the 3 mumol of PEITC/g of diet. The apparent Km remained unchanged, and the apparent Vmax decreased in the lung and liver microsomes of PEITC-fed mice, suggesting a noncompetitive nature of the inhibition. When added to the incubation mixture, PEITC decreased NNK metabolism in a concentration-dependent manner and exhibited a competitive inhibition with apparent Ki values of 51 to 93 nM. Dietary PEITC decreased the hepatic P450 content by 25%, but increased (2-fold) the O-dealkylase activities of 7-pentoxyresorufin (indicative of P450 2B1) and 7-ethoxyresorufin (indicative of P450 1A) in the liver microsomes of mice consuming the 3 mumol of PEITC/g of diet. The P450 2B level was increased in liver microsomes but slightly decreased in the lung microsomes. The p450 2E1 level was increased by dietary PEITC by 1.2- and 1.6-fold in the liver and lung microsomes, respectively. The activities of glutathione S-transferase and NAD(P)H-quinone oxidoreductase in liver and lung microsomes were not affected appreciably by the dietary PEITC treatment. The results suggest that chronic consumption of PEITC decreases the rate of metabolic activation of NNK by chemical inactivation and competitive inhibition of the enzyme(s) responsible for NNK oxidation.

Animals↗

PubMed1993

The interaction of fluorescein isothiocyanate with the ryanodine receptor/Ca2+ release channel of sarcoplasmic reticulum.

Fluorescein 5'-isothiocyanate (FITC) markedly inhibited ryanodine binding to rabbit skeletal muscle junctional sarcoplasmic reticulum. Half-maximal inhibition was obtained with about 20 microM and complete inhibition by 80 microM FITC. Inhibition was enhanced in the presence of high salt and at alkaline pH. The pH dependence of the inactivation of ryanodine binding suggested that FITC bound to a very reactive lysine epsilon-amino group with a pK alpha of about 7.5 or above. Kinetic analysis of the time course of inactivation of ryanodine binding by various concentrations of FITC suggested that the inactivation resulted from the modification of 2 or more amino acid residues. The inhibition of ryanodine binding by FITC was partially prevented by ATP, ADP, adenyl imidodiphosphate, and 3-O-(benzoyl)-benzoyl-ATP (Bz-ATP) but not by AMP. FITC modification of sarcoplasmic reticulum membranes inhibited the photoaffinity labeling by [alpha-32P]Bz-ATP of the 450-kDa protein and the ryanodine receptor with half-maximal inhibition at about 100 microM. The results suggest that the inhibition of ryanodine binding is mainly due to FITC modification of sites which are not involved in ATP binding. The FITC moiety is bound to the 160-, 96-, 76-, and 60-kDa ryanodine receptor tryptic fragments, and the FITC site is apparently on the 21.5-, 18-, and 17-kDa fragments which are formed by the V8 protease. Covalent modification by FITC dramatically affected the activity of single Ca2+ release channels incorporated into planar lipid bilayers. FITC caused a marked increased in channel open probability mainly to a noisy approximately 60% subconductance state. FITC-modified channels were no longer affected by ryanodine but were still abolished by Mg2+ and ruthenium red. We suggest that FITC modifies reactive lysine residues involved in channel activation by transmembrane charge movement in the t-tubular system.

Adenine Nucleotides↗

PubMed1993

Mechanism of K+ interaction with fluorescein 5'-isothiocyanate-modified Na+,K(+)-ATPase.

The rate of K+ binding to fluorescein 5'-isothiocyanate-modified Na+,K(+)-ATPase has been measured by stopped-flow fluorometry and shown to depend sigmoidally on potassium concentration. By assuming two K+ ions must bind to cause a rate-limiting conformational change, analytical expressions for the rate and the amplitude were derived that fit the experimental data and correctly calculate the half-maximum K+ concentration in amplitude titrations from kinetically estimated rate and dissociation constants. This is the first time that sigmoidal dependence of the time constant for K+ reaction with Na+,K(+)-ATPase on potassium concentration has been observed. The conclusion that two K+ ions are required to cause the E1-->E2 conformational change explains discrepancies between equilibrium and kinetic estimates of the half-maximum K+ concentration in published studies of the reaction and reconciles the mechanism of the conformational change in Na+,K(+)-ATPase with the transport stoichiometry of sodium pump. It is strong, additional evidence that fluorescein reports a conformational change in dephosphoenzyme that is involved in monovalent cation transport. The ratio of the K+ dissociation constants is consistent either with ordered binding to two sites with intrinsic constants differing by a factor of 4 or with binding to two independent and identical sites.

Fluorescein-5-isothiocyanate↗

PubMed1993

Labeling of lysine 492 with pyridoxal 5'-phosphate in the sarcoplasmic reticulum Ca(2+)-ATPase. Lysine 492 residue is located outside the fluorescein 5-isothiocyanate-binding region in or near the ATP binding site.

Sarcoplasmic reticulum vesicles were treated with 2 mM pyridoxal 5'-phosphate (PLP) at 25 degrees C and pH 7.0 for 6 min and reduced by NaBH4. Both the activity of the Ca(2+)-ATPase and the capacity for high affinity Mg-ATP binding were greatly reduced. Acetyl phosphate hydrolysis or phosphoenzyme formation from Pi was not inhibited. The enzyme was protected by high affinity Mg-ATP binding against the PLP-induced inhibition. A similar protective effect was obtained by Mg-AMP as well as by Mg-ADP. Acetyl phosphate or Pi gave no protection. The PLP-treated vesicles were solubilized in SDS, and the Ca(2+)-ATPase was purified by size exclusion high performance liquid chromatography (HPLC). Mapping the fluorescently labeled peptides in the tryptic digest by reversed phase HPLC revealed a single fluorescent peak, which was protected by Mg-ATP against labeling. Sequencing showed that Lys-492 is the residue labeled with PLP. These results demonstrate that Lys-492 is located in or near the ATP binding site but not in the phosphorylation site or the Pi binding site. When Lys-515 was entirely prelabeled with fluorescein 5-isothiocyanate (FITC), the subsequent labeling of Lys-492 with PLP was not prevented. This finding demonstrates that Lys-492 is located outside the FITC-binding region. It has been widely accepted that FITC occupies the adenosine-binding region within the ATP binding site. In contrast to FITC, Mg-AMP strongly inhibited the labeling of Lys-492 with PLP. These findings lead to the conclusion that Lys-492 is located outside the adenosine-binding region, most probably in or near the region occupied by the alpha-phosphoryl group of Mg-ATP bound to the ATP binding site.

Adenosine Diphosphate↗

PubMed1996

Complete inhibition of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-induced rat lung tumorigenesis and favorable modification of biomarkers by phenethyl isothiocyanate.

Phenethyl isothiocyanate (PEITC), which occurs in certain cruciferous vegetables, was tested for its ability to inhibit lung tumorigenesis in rats induced by the tobacco-specific nitrosamine 4-(methylnitrosamino-1-(3-pyridyl)-1-butanone (NNK) in a study involving virtually lifelong administration of both compounds. In addition, two biomarkers of NNK metabolism [4-hydroxy-1-(3-pyridyl)-1-butanone-releasing hemoglobin adducts and 4-(methylnitrosamino-1-3-pyridyl-1-butanol and its glucuronide in urine] were quantified in randomly selected rats during the course of the study. The rats were assigned to groups as follows: NNK, 2 ppm in drinking water, 60 rats; NNK, 2 ppm in drinking water and PEITC, 3 micromol/g NIH-07 diet, 60 rats; PEITC, 3 micromol/g NIH-07 diet, 20 rats; and untreated controls, 20 rats. NNK was added to the drinking water for 111 weeks and PEITC to the diet for 1 prior to NNK administration and then throughout the 111-week course of treatment. There were no significant differences in body weights or survival among the groups. There were no significant effects of PEITC on blood chemistry or hematology. NNK induced lung tumors (adenoma and/or adenocarcinoma) in 70% of the rats. In the group treated with NNK plus PEITC, 5% of the rats had lung tumors, which was not different from that of control rats. PEITC also appeared to inhibit progression of benign to malignant pancreatic tumors. At intervals during the study, blood was withdrawn from selected rats, and 4-hydroxy-1-(3-pyridyl)-1-butanone-releasing hemoglobin adducts, which are formed upon metabolic activation of NNK, were quantified. The hemoglobin adducts were significantly repressed throughout the study in the rats treated with NNK plus PEITC compared to those treated with NNK. The 24-h urine sample of several rats was analyzed for 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol glucuronide. A 4-6-fold increase in the sum of these metabolites was observed in the rats treated with NNK plus PEITC compared to those treated with NNK. This is also consistent with inhibition of metabolic activation of NNK by PEITC. Collectively, the results of this study provide strong evidence for the efficacy of PEITC as a chemopreventive agent against NNK-induced pulmonary carcinogenesis in rats and indicate that two biomarkers of NNK metabolism, measurable in tobacco consumers, can be modulated in a predictable way by PEITC administration.

Animals↗

PubMed1996

Measurement of glycoprotein IIb/IIIa blockade by flow cytometry with fluorescein isothiocyanate-conjugated crotavirin, a member of disintegrins.

The blockade of platelet membrane glycoprotein IIb/IIIa by a monoclonal antibody, 7E3, was measured by flow cytometry using a fluorescein isothiocyanate-conjugated disintegrin, FITC-crotavirin, as the probe. After treatment of platelets with 7E3 or 7E3 F(ab')2, there is a good correlation between the inhibition of platelet aggregation and the blockade of FITC-crotavirin's binding to platelets. The content of glycoprotein IIb/IIIa for the subsequent binding of FITC-crotavirin to the 7E3-pretreated platelets highly correlated to the extent of glycoprotein IIb/IIIa, remaining available. It was evidenced by the observation that the sum of glycoprotein IIb/IIIa occupation by 7E3 and that of FITC-crotavirin approached the total amount of glycoprotein IIb/IIIa expressed on the platelet membrane. This indicates that the percentage inhibition of FITC-crotavirin's binding at the saturation dose reflects the extent of glycoprotein IIb/IIIa blockade by 7E3. At the saturation binding concentration (5 micrograms/ml), FITC-crotavirin did not displace platelet bound 7E3. Gating the light-scattering profile for platelets, the binding of FITC-crotavirin to platelet glycoprotein IIb/IIIa could be easily determined in diluted whole blood by direct stain method. The available unoccupied glycoprotein IIb/IIIa of platelets in the 7E3 or 7E3 F(ab')2-pretreated whole blood were measured by flow cytometry at the saturation binding dose of FITC-crotavirin (4 micrograms/ml) and the data showed that the higher deconcentration of antibody added into whole blood, the lower debinding of FITC-crotavirin to platelets. This technique may provide an alternative rapid method for measuring the blockade of glycoprotein IIb/IIIa by 7E3, a promising anti-thrombotic agent, thus providing a monitoring method for adjusting the therapeutic dose of 7E3 or its related derivatives.

Antibodies, Monoclonal↗