Academic literature on the topic 'Fluorescein-5-isothiocyanate (FITC)'

This paper addresses the mechanism of inhibition of the plasma membrane Ca pump by fluorescein analogues and their isothiocyanate derivatives. Eosin (i.e., tetrabromofluorescein) was found to be one of the most potent reversible inhibitors of the erythrocyte Ca pump [half-maximal inhibitory concentration (IC50) < 0.2 microM]; fluorescein itself was about four orders of magnitude less potent (IC50 approximately 1,000 microM). Eosin decreased the maximum influx and thus did not compete with ATP for the Ca pump. Irreversible inhibition produced by the isothiocyanate analogues of eosin and fluorescein [eosin 5-isothiocyanate (EITC) and fluorescein 5-isothiocyanate (FITC), respectively] was also studied. While EITC bound reversibly at the eosin site, two results suggest that EITC does not react covalently at this site: 1) eosin did not alter the time course of the EITC irreversible reaction, and 2) the concentration dependence for reversible EITC inhibition was different from the concentration dependence for irreversible EITC inhibition. ATP did slow the rate of inactivation of both EITC and FITC consistent with the idea that EITC and FITC bind to the ATP site. Our results are consistent with eosin and ATP binding to separate sites and EITC reacting covalently at the ATP site, but not the eosin site.

Fluorescein 5′-isothiocyanate (FITC) was used to modify the lysine residue in the active site of tonoplast H(+)-ATPase from etiolated mung-bean (Vigna radiata L.) seedlings. FITC caused marked inactivation of the enzyme activities of both membrane-bound and soluble ATPase and its associated H+ translocation. The SDS/PAGE pattern revealed that the FITC-binding site was in the large (A) subunit of ATPase. Inhibition could be substantially prevented by its physiological substrate ATP, pyrophosphate and nucleotides in the decreasing order: ATP greater than pyrophosphate greater than ADP greater than AMP greater than GTP greater than CTP greater than UTP. The mode of inhibition by FITC was competitive with respect to ATP. Loss of ATPase activity followed pseudo-first-order kinetics with a Ki of 0.33 mM, a minimum inactivation half-time of 110 s, and a first-order rate constant of 0.244 s-1. A double-logarithmic plot of apparent rate constant versus FITC concentration gave a slope of 0.913, indicating that inactivation results from reaction of at least one lysine residue at the catalytic site of the large subunit. Labelling studies indicated that the incorporation of approx. 1 mol of FITC/mol of ATPase is sufficient to inhibit ATPase completely. The enhancement and blue shift of emission maxima of FITC after modification of ATPase indicated that the labelled lysine residue was located in a relatively hydrophobic domain.

Fluorescein 5(6)-isothiocyanate starch maleate (FISM) nanoparticles were prepared by covalently attached fluorescein 5(6)-isothiocyanate (FITC) with starch maleate. FISM nanoparticles with a mean particle size of 87 nm were formed via self-assembly upon precipitation in ethanolic solution. FISM nanoparticles were strongly fluorescent with maximum emission wavelength of 518 nm. The fluorescence of FISM nanoparticles can be quenched by silver (Ag+) and lead (Pb2+) ions in a concentration dependent manner. We have demonstrated the first use of FISM nanoparticles as cheap and effective fluorescent sensing probes for Ag+and Pb2+ions with detection limits as low as 2.55×10−5 M and 3.64×10−5 M, respectively.

Detection of fluorescein-5-isothiocyanate (FITC)-labeled conjugates is suboptimal in two-color confocal scanning laser microscopy (CLSM). This limits the detection of small, dimly fluorescent targets. We explored the possible advantages of applying eosin-5-isothiocyanate (EITC) conjugated to avidin (Av-EITC) as an alternative for Av-FITC in CSLM. Despite the lower quantum efficiency of EITC, we found that the measured Av-EITC and Av-FITC emission intensities were similar as a result of the standard filter combinations used for simultaneous two-color detection in the Bio-Rad MRC 600 CSLM. The advantage of Av-EITC was that its fading characteristics compared very favorably to those of Av-FITC. An excitation intensity-dependent increase in Av-EITC fluorescence was observed, followed by an exponential decrease. This increase in fluorescence allows longer observation times, averaging of several scans without loss of brightness, and thus detection of dimly fluorescent targets by CSLM.

Magnetic gold nanoparticles (mGNPs) with uniform size and morphology synthesized by our sonication treatment method were covalently bound with fluorescein isothiocyanate (FITC) molecules. Driven by an external magnetic field, FITC-labelled nanoparticles were delivered into plant cells with and without cell walls, evident from sectional transmission electron microscopy images. Confocal images further indicate that the green fluorescence in canola protoplasts and walled cells indeed came from the FITC molecules, instead of the chloroplasts’ autofluorescence. FITC-labelled nanoparticles had a delivery efficiency of 95% based on confocal images. In further study, plasmids were covalently bound with mGNPs, and delivered into canola cells with and without cell walls. After culturing for 48 h followed by staining with 5-bromo-4-chloro-3-indolyl-β-d-glucuronic acid (X-Gluc), blue colour appeared in the protoplasts, while the walled canola cells showed a green colour that can be interpreted as the combination of blue and yellow from the suspension cells themselves. The presence of the blue colour indicates the expression of the GUS gene; therefore, the plasmids were successfully delivered into the canola cells. Furthermore, on examination, mGNPs were considered to be noncytotoxic by fluorescein diacetate staining.

SummaryDisintegrins are a group of snake venom peptides which inhibit human platelet aggregation by acting as glycoprotein Ilb-IIIa (GPIIb-Ilia) antagonists. They are cysteine-rich, Arg-Gly-Asp (RGD)-containing peptides, and bind to GPIIb-Ilia complex on platelet membrane with a very high affinity (Kd, 10−7 ∼ 10−8 M). In this study, we analyzed GPIIb-Ilia 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-Ilia. 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. As a result these three patients could be classified as type I thrombasthenia based on the extremely low level of GPIIb-Ilia detected by this method (<5% of normal value).

The present study aimed to demonstrate that Sideral® RM (SRM, Sucrosomial® Raw Material Iron) is transported across the excised intestine via a biological mechanism, and to investigate the effect that this transport route may produce on oral iron absorption, which is expected to reduce the gastrointestinal (GI) side effects caused by the bioavailability of non-absorbed iron. Excised rat intestine was exposed to fluorescein isothiocyanate (FITC)-labeled SRM in Ussing chambers followed by confocal laser scanning microscopy to look for the presence of fluorescein-tagged vesicles of the FITC-labeled SRM. To identify FITC-labeled SRM internalizing cells, an immunofluorescence analysis for macrophages and M cells was performed using specific antibodies. Microscopy analysis revealed the presence of fluorescein positive particulate structures in tissues treated with FITC-labeled SRM. These structures do not disintegrate during transit, and concentrate in macrophage cells. Iron bioavailability was assessed by determining the time-course of Fe3+ plasma levels. As references, iron contents in liver, spleen, and bone marrow were determined in healthy rats treated by gavage with SRM or ferric pyrophosphate salt (FP). SRM significantly increased both area under the curve (AUC) and clearance maxima (Cmax) compared to FP, thus increasing iron bioavailability (AUCrel = 1.8). This led to increased iron availability in the bone marrow at 5 h after single dose gavage.

Antidiuretic hormone (ADH) increases the osmotic water permeability (Pf) of the toad urinary bladder by insertion of water channels into the apical cell membrane. Transepithelial water flow (Jv) reduces Pf by inducing endocytosis of apical water channels despite continuous ADH stimulation. This phenomenon is termed flux inhibition. We wished to determine whether cytoplasmic dilution or transcellular Jv causes flux inhibition because both have been proposed previously as a primary regulatory mechanism for this process. Apical membrane endocytosis was quantified by monitoring the uptake of the fluid phase marker fluorescein isothiocyanate dextran (FITC-dextran). FITC-dextran fluorescence was monitored in Triton X-100 extracts of epithelial cells as the ratio of total tissue fluorescence compared with background fluorescence. The background was defined as cellular autofluorescence and nonspecific tissue staining due to the presence of small amounts of free fluorescein contaminating the FITC-dextran. FITC-dextran uptake measured under symmetric isotonic (220 mosmol/kgH2O) conditions in either the absence (1.0 +/- 0.4 SD; n = 14) or presence (1.3 +/- 0.3; n = 4) of ADH was not statistically different from that of background. In contrast, flux inhibition induced by a 180 mosmol/kgH2O apical-to-basolateral osmotic gradient increased FITC-dextran uptake to 3.4 +/- 1.3 (n = 7). FITC-dextran uptake was identical in bladders exposed to symmetric hypotonic (150 mosmol/kgH2O) solutions during ADH (3.6 +/- 0.9; n = 6) or adenosine 3',5'-cyclic monophosphate (3.1 +/- 0.4 fold; n = 3) stimulation.(ABSTRACT TRUNCATED AT 250 WORDS)

A 100-kDa protein that is a main component of the microsomal fraction from rabbit gastric mucosa is phosphorylated by cAMP-dependent protein kinase (PKA) in the presence of 0.2% Triton X-100. Microsomes from rabbit gastric mucosa possess activity of H,K-ATPase but not activity of Na,K-ATPase. Incubation of microsomes with 5 μM fluorescein 5′-isothiocyanate (FITC) results in both an inhibition of H,K-ATPase and labeling of a protein with an electrophoretic mobility corresponding to the mobility of the protein phosphorylated by PKA. The data suggest that the α-subunit of H,K-ATPase can be a potential target for PKA phosphorylation.

SummaryThe 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 Ilb/IIIa, remaining available. It was evidenced by the observation that the sum of glycoprotein Ilb/IIIa occupation by 7E3 and that of FITC-crotavirin approached the total amount of glycoprotein Ilb/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 Ilb/IIIa blockade by 7E3. At the saturation binding concentration (5 |xg/ml), FITC-crotavirin did not displace platelet bound 7E3. Gating the light-scattering profile for platelets, the binding of FITC-crotavirin to platelet glycoprotein Ilb/IIIa could be easily determined in diluted whole blood by direct stain method. The available unoccupied glycoprotein Ilb/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 fig/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 Ilb/IIIa by 7E3, a promising anti-thrombotic agent, thus providing a monitoring method for adjusting the therapeutic dose of 7E3 or its related derivatives.

La cartographie peptidique est une méthode qui permet entre autre d’identifier les modifications post-traductionnelles des protéines. Elle comprend trois étapes : 1) la protéolyse enzymatique, 2) la séparation par électrophorèse capillaire (CE) ou chromatographie en phase liquide à haute performance (HPLC) des fragments peptidiques et 3) l’identification de ces derniers. Cette dernière étape peut se faire par des méthodes photométriques ou par spectrométrie de masse (MS). Au cours de la dernière décennie, les enzymes protéolytiques immobilisées ont acquis une grande popularité parce qu’elles peuvent être réutilisées et permettent une digestion rapide des protéines due à un rapport élevé d’enzyme/substrat. Pour étudier les nouvelles techniques d’immobilisation qui ont été développées dans le laboratoire du Professeur Waldron, la cartographie peptidique par CE est souvent utilisée pour déterminer le nombre total de peptides détectés et leurs abondances. La CE nous permet d’avoir des séparations très efficaces et lorsque couplée à la fluorescence induite par laser (LIF), elle donne des limites de détection qui sont 1000 fois plus basses que celles obtenues avec l’absorbance UV-Vis. Dans la méthode typique, les peptides venant de l’étape 1) sont marqués avec un fluorophore avant l’analyse par CE-LIF. Bien que la sensibilité de détection LIF puisse approcher 10-12 M pour un fluorophore, la réaction de marquage nécessite un analyte dont la concentration est d’au moins 10-7 M, ce qui représente son principal désavantage. Donc, il n’est pas facile d’étudier les enzymes des peptides dérivés après la protéolyse en utilisant la technique CE-LIF si la concentration du substrat protéique initial est inférieure à 10-7 M. Ceci est attribué à la dilution supplémentaire lors de la protéolyse. Alors, afin d’utiliser le CE-LIF pour évaluer l’efficacité de la digestion par enzyme immobilisée à faible concentration de substrat,nous proposons d’utiliser des substrats protéiques marqués de fluorophores pouvant être purifiés et dilués. Trois méthodes de marquage fluorescent de protéine sont décrites dans ce mémoire pour étudier les enzymes solubles et immobilisées. Les fluorophores étudiés pour le marquage de protéine standard incluent le naphtalène-2,3-dicarboxaldéhyde (NDA), la fluorescéine-5-isothiocyanate (FITC) et l’ester de 6-carboxyfluorescéine N-succinimidyl (FAMSE). Le FAMSE est un excellent réactif puisqu’il se conjugue rapidement avec les amines primaires des peptides. Aussi, le substrat marqué est stable dans le temps. Les protéines étudiées étaient l’-lactalbumine (LACT), l’anhydrase carbonique (CA) et l’insuline chaîne B (INB). Les protéines sont digérées à l’aide de la trypsine (T), la chymotrypsine (CT) ou la pepsine (PEP) dans leurs formes solubles ou insolubles. La forme soluble est plus active que celle immobilisée. Cela nous a permis de vérifier que les protéines marquées sont encore reconnues par chaque enzyme. Nous avons comparé les digestions des protéines par différentes enzymes telles la chymotrypsine libre (i.e., soluble), la chymotrypsine immobilisée (i.e., insoluble) par réticulation avec le glutaraldéhyde (GACT) et la chymotrypsine immobilisée sur billes d’agarose en gel (GELCT). Cette dernière était disponible sur le marché. Selon la chymotrypsine utilisée, nos études ont démontré que les cartes peptidiques avaient des différences significatives selon le nombre de pics et leurs intensités correspondantes. De plus, ces études nous ont permis de constater que les digestions effectuées avec l’enzyme immobilisée avaient une bonne reproductibilité. Plusieurs paramètres quantitatifs ont été étudiés afin d’évaluer l’efficacité des méthodes développées. La limite de détection par CE-LIF obtenue était de 3,010-10 M (S/N = 2,7) pour la CA-FAM digérée par GACT et de 2,010-10 M (S/N = 4,3) pour la CA-FAM digérée par la chymotrypsine libre. Nos études ont aussi démontrées que la courbe d’étalonnage était linéaire dans la région de travail (1,0×10-9-1,0×10-6 M) avec un coefficient de corrélation (R2) de 0,9991.<br>Peptide mapping is a routine method for identifying post-translational modifications of proteins. It involves three steps: 1) enzymatic proteolysis, 2) separation of the peptide fragments by capillary electrophoresis (CE) or high performance liquid chromatography (HPLC), 3) identification of the peptide fragments by photometric methods or mass spectrometry (MS). During the past decade, immobilized enzymes for proteolysis have been gaining in popularity because they can be reused and they provide fast protein digestion due to the high ratio of enzyme-to-substrate. In order to study new immobilization techniques developed in the Waldron laboratory, peptide mapping by CE is frequently used, where the total number of peptides detected and their abundance are related to enzymatic activity. CE allows very high resolution separations and, when coupled to laser-induced fluorescence (LIF), provides excellent detection limits that are 1000 times lower than with UV-Vis absorbance. In the typical method, the peptides produced in step 1) above are derivatized with a fluorophore before separation by CE-LIF. Although the detection sensitivity of LIF can approach 10 12 M for a highly efficient fluorophore, a major disadvantage is that the derivatization reaction requires analyte concentrations to be approx. 10 7 M or higher. Therefore, it is not feasible to study enzymes using CE-LIF of the peptides derivatized after proteolysis if the initial protein substrate concentration is <10-7 M because additional dilution occurs during proteolysis. Instead, to take advantage of CE-LIF to evaluate the efficiency of immobilized enzyme digestion of low concentrations of substrate, we propose using fluorescently derivatized protein substrates that can be purified then diluted. Three methods for conjugating fluorophore to protein were investigated in this work as a means to study both soluble and immobilized enzymes. The fluorophores studied for derivatization of protein standards included naphthalene-2,3-dicarboxaldehyde (NDA), fluoresceine-5-isothiocyanate (FITC) and 6-carboxyfluorescein N-succinimide ester (FAMSE). The FAMSE was found to be an excellent reagent that conjugates quickly with primary amines and the derivatized substrate was stable over time. The studied substrates were -lactalbumin (LACT), carbonic anhydrase (CA) and insulin chain-B (INB). The CE-LIF peptide maps were generated from digestion of the fluorescently derivatized substrates by trypsin (T), chymotrypsin (CT) or pepsin (PEP), either in soluble or insoluble forms. The soluble form of an enzyme is more active than the immobilized form and this allowed us to verify that the conjugated proteins were still recognized as substrates by each enzyme. The digestion of the derivatized substrates with different types of chymotrypsin (CT) was compared: free (i.e., soluble) chymotrypsin, chymotrypsin cross-linked with glutaraldehyde (GACT) and chymotrypsin immobilized on agarose gel particles (GELCT), which was available commercially. The study showed that, according to the chymotrypsin used, the peptide map would vary in the number of peaks and their intensities. It also showed that the digestion by immobilized enzymes was quite reproducible. Several quantitative parameters were studied to evaluate the efficacy of the methods. The detection limit of the overall method (CE-LIF peptide mapping of FAM-derivatized protein digested by chymotrypsin) was 3.010-10 M (S/N = 2.7) carbonic anhydrase using insoluble GACT and 2.010-10 M (S/N = 4.3) CA using free chymotrypsin. Our studies also showed that the standard curve was linear in the working region (1.0×10-9-1.0×10-6 M) with a correlation coefficient (R2) of 0.9991.

Tuberculosis (TB) is an airborne infectious disease caused by the bacterium Mycobacterium Tuberculosis (MTB) and is a persistent problem in developing countries. Present methods for its detection include normal or nested Polymerase Chain Reaction (PCR) followed by electrophoresis, real-time PCR, Ziehl-Neelsen staining, and culture assay. These techniques entail various disadvantages such as high cost, long assay time and use of toxic substances. Novel loop-mediated isothermal amplification (LAMP) permits DNA to be amplified rapidly under constant temperature. The combination of LAMP and chromatographic Lateral Flow Dipstick (LAMP-LFD) by using biotinylated LAMP amplicon hybridized with Fluorescein Isothiocyanate (FITC)-labeled probes are allowed to detect MTB without electrophoresis and interpreted within 3-5 min. LAMP-LFD is as highly sensitive as PCR-electrophoresis method. Based on its sensitivity, specificity, rapidity, cost effectiveness, ease of use, and convenience, LAMP-LFD could be suitable for use in early MTB detection.

We have examined the effect of DN-9693 (piperidinyl - imidazo - quinazoline: Daiichi Seiyaku, Japan) a water soluble phosphodiesterase inhibitor on platelet aggregation, secretion and thromboxane B2 (TXB2) production. In platelet rich plasma and at concentrations of 2uM and 5uM the drug significantly inhibited aggregation induced by adenosine diphosphate, collagen and sodium arachidonate. TXB2 production and release of adenosine tri-phosphate and 14C 5-hydroxytryptamine were also significantly inhibited by the drug. Cyclic adenosine mono-phosphate accumulation was enhanced. Inhibition of ristocetin induced platelet agglutination was an unexpected finding and further experiments were undertaken to explore this. These suggested no specific effect against a plasma factor (von Willebrand factor) and reduced expression of the platelet membrane glycoprotein Ib was implicated. To investigate this further we examined the effect of DN-9693 on the binding of a monoclonal antibody (McAb) to platelet membrane glycoprotein lb (AN51). This was assessed by a FACS IV flow cytofluorimeter utilising a goat anti-mouse fluorescein isothiocyanate (FITC) labelled secondary antibody. Similar experiments were also performed with McAbs to the membrane glycoprotein complex IIb/IIIa (M148) and also to glycoprotein IIIa (C17). In platelet rich plasma, at concentrations which have been shown to inhibit aggregation, DN-9693 significantly reduced the mean fluorescence intensity of the cells coated with McAb AN51 in a dose related manner. This strongly suggested a drug effect against the glycoprotein Ib receptor site. Also, the drug appeared to enhance the binding of McAb C17 to glycoprotein IIIa. This study indicates that in addition to potent phosphodiesterase inhibitor activity, DN-9693 causes a platelet surface membrane change which is associated with reduced expression of membrane glycoprotein Ib.