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PubMed1966

Fluorescence and viability of Proteus mirabilis stained directly with fluorescein isothiocyanate.

Grunberg, E. (Hoffman-La Roche, Inc., Nutley, N.J.), and R. Cleeland. Fluorescence and viability of Proteus mirabilis stained directly with fluorescein isothiocyanate. J. Bacteriol. 92:23-27. 1966.-Washed cell suspensions of Proteus mirabilis, under the proper conditions, stained well with fluorescein isothiocyanate with little or no loss of cell viability. The speed and intensity of the reaction was dependent on both the concentration of dye and pH. Within a range of pH 3.0 to 10.0, staining was most rapid at pH 5.0 to 6.0, with a slower and less intense reaction occurring at the other pH values. As the concentration of dye at either pH 5.0 or 9.0 was increased from 10 to 1,000 mug/ml, there was an increase in the rate of staining but a decrease in cell viability. After 24 hr of incubation at 4 C, pH 5.0, and a dye concentration of 10 mug/ml, all cells were stained, the majority exhibiting intense fluorescence with little or no loss of viability noted. In preliminary experiments with Staphylococcus aureus, similar results were obtained. Of various other fluorescent dyes tested, only rhodamine isothiocyanate was found to give satisfactory staining.

Fluoresceins↗
PubMed1994

Renal cortical basolateral Na+/HCO3- cotransporter: II. Detection of conformational changes with fluorescein isothiocyanate labeling.

Fluorescein isothiocyanate (FITC) fluorescently labels amino groups and has been useful in detecting conformational changes in transport proteins through quenching or enhancement of the fluorescence signal upon exposure of protein to substrates. Solubilized renal basolateral membrane proteins, enriched in Na+/HCO3- cotransporter activity, were reconstituted into liposomes and treated with FITC or its nonfluorescent analogue PITC (phenyl isothiocyanate). In the absence of Na+ and HCO3-, incubation of proteoliposomes with PITC or FITC significantly inhibited cotransporter activity. However, in the presence of Na+ and HCO3- during labeling both agents failed to inhibit cotransporter activity, indicating that these probes interact specifically with the cotransporter. In the presence of the substrates Na+ and HCO3-, PITC binds covalently to amino groups unprotected by substrates leaving the Na+/HCO3- cotransporter available for specific labeling with FITC. Addition of NaHCO3 to FITC-labeled proteoliposomes resulted in a concentration-dependent enhancement of the fluorescence signal which was inhibited by pretreatment with 4,4'-diisothiocyanostilbene 2',2-disulfonic acid (DIDS) prior to FITC labeling. SDS PAGE analysis of FITC-treated proteoliposomes showed the presence of two distinct fluorescent bands (approximate MW of 90 and 56 kD). In the presence of substrates, the fluorescence intensity of these bands was enhanced as confirmed by direct measurement of gel slice fluorescence. Thus, FITC detects conformational changes of the Na+/HCO3- cotransporter and labels proteins which may represent the cotransporter or components of this cotransporter.

Animals↗
PubMed1993

Chemical modification of the Ca(2+)-ATPase of rabbit skeletal muscle sarcoplasmic reticulum: identification of sites labeled with aryl isothiocyanates and thiol-directed conformational probes.

The Ca(2+)-ATPase protein of rabbit skeletal muscle sarcoplasmic reticulum is a single polypeptide chain of 1001 amino-acid residues. Among these residues are 24 Cys, 9 of which have previously been shown to be accessible to one or more thiol-specific reagents. Many studies on the structure and function of this Ca(2+)-ATPase have made use of sulfhydryl-directed, conformationally-sensitive probes, but the labeling sites for these probes have been directly identified in only a few cases, causing uncertainty in the interpretation of results. In the present work, we have investigated the Ca(2+)-ATPase labeling sites for three thiol-directed spectroscopic probes: fluorescein 5'-maleimide (Fmal), 4-dimethylaminophenyl-azo phenyl-4'-maleimide (DABmal), and 4-dimethylaminophenylazophenyl-4'-iodoacetamide (DABIA). Labeled Ca(2+)-ATPase was digested exhaustively with trypsin, and labeled peptides were purified and sequenced in order to identify the labeled Cys residues. Our results do not support the widely held assumptions that Cys-344 and Cys-364 are the most reactive residues with maleimide-based reagents, while Cys-670 and Cys-674 react most rapidly with iodoacetamide derivatives. We found instead that Fmal reacted most rapidly with Cys-471, followed by Cys-364, and more slowly with Cys-498, -525, -614 and -636. DABmal reacted most rapidly with Cys-364, followed by Cys-614, and more slowly with Cys-471, -498, -636 and -670. Cys-344 was not labeled by either Fmal or DABmal. DABIA reacted with the same six Cys residues, including Cys-670, as were labeled with DABmal, but in much lower yield. There was no evidence for labeling of Cys-674 with DABIA. The high reactivity of Fmal, but not the more hydrophobic DABmal, with Cys-471 is of interest because of previous studies suggesting that the accessibility of Cys-471 is influenced by ATP and that fluorescein derivatives bind to a hydrophobic pocket in the ATP binding site. Another derivative, fluorescein-5'-isothiocyanate (FITC), is thought to label the catalytic site of the Ca(2+)-ATPase and has been widely used as a conformational probe in structure-function studies on this and related proteins. We reinvestigated the chemical modification of the Ca(2+)-ATPase by FITC and 4-dimethyl-aminophenyl-4'-isothiocyanate (DABITC). Incorporation of stoichiometric amounts of FITC resulted in a nearly complete loss of ATPase activity. Labeling and inactivation of the Ca(2+)-ATPase by FITC did not occur in the presence of ATP. DABITC was less reactive than FITC, and did not inactivate the Ca(2+)-ATPase to any significant extent.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1998

Erythrosin 5'-isothiocyanate labels Cys549 as part of the low-affinity ATP binding site of Na+/K+-ATPase.

The high-affinity E1ATP site of Na+/K+-ATPase labeled with fluorescein 5'-isothiocyanate and its E2ATP site labeled with erythrosin 5'-isothiocyanate (ErITC), as was shown recently [Linnertz et al. (1998) J. Biol. Chem. 273, 28813-28821], reside on separate and adjacent catalytic alpha subunits. This paper provides evidence that specific labeling of the E2ATP binding site with ErITC resulted in a modification of the Cys549 residue in the tryptic fragment with the sequence Val545-Leu-Gly-Phe-Cys549-His550. Hence, Cys549 is part of or close to the low-affinity E2ATP binding site of Na+/K+-ATPase.

Adenosine Triphosphate↗
PubMed1984

Conformational changes in the intestinal brush border sodium-glucose cotransporter labeled with fluorescein isothiocyanate.

Fluorescein isothiocyanate (FITC) was used to label the rabbit intestinal brush border Na+-glucose carrier, identify the carrier protein on sodium dodecyl sulfate/polyacrylamide gel electrophoresis, and monitor the effect of ions and substrates on fluorescence quenching. Enriched brush border preparations were employed to study both glucose transport and FITC binding. FITC and a nonfluorescent analog (phenyl isothiocyanate, PITC) both inhibited Na+-dependent D-glucose transport irreversibly. Inhibition was blocked completely by the presence of Na+ and D-glucose during labeling. PITC was used to label nonspecific amino groups in the presence of glucose and Na+, and then the glucose carrier was labeled with FITC in the absence of substrates. Fluorescence of FITC bound to the carrier was quenched specifically with Na+ in a saturable fashion, and this indicates a Na+-dependent conformational change in the carrier. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis of FITC-labeled membranes revealed specific labeling of a 71,000-dalton peptide. We conclude that Na+ induces a conformational shift in the 71,000-dalton glucose carrier, and this is quite consistent with the kinetics of Na+-dependent glucose transport in these membranes.

Animals↗
PubMed2000

Antiplatelet and anticancer isothiocyanates in Japanese domestic horseradish, wasabi.

6-Methylsulfinylhexyl isothiocyanate (MS-ITC) was isolated from wasabi (Wasabia japonica, Japanese domestic horseradish) as a potential inhibitor of human platelet aggregation in vitro through our extensive screening of vegetables and fruits. In the course of another screening for the induction of glutathione S-transferase (GST) activity in RL34 cells. MS-ITC was inadvertently isolated from wasabi as a potential inducer of GST. MS-ITC administered to rats or mice also showed both activities in vivo. As a result from elucidation of the platelet aggregation inhibition and the GST induction mechanisms of MS-ITC, the isothiocyanate moiety of MS-ITC plays an important role for antiplatelet and anticancer activities because of its highly reactivity with sulfhydryl (-SH) groups in biomolecules (GSH, cysteine residue in a certain protein, etc.).

Antineoplastic Agents, Phytogenic↗
PubMed2006

Cell death induction by isothiocyanates and their underlying molecular mechanisms.

An important and promising group of compounds that have a chemopreventive property are organosulfur compounds, such as isothiocyanates (ITCs). In recent years, it has been shown that ITCs induce apoptosis in various cancer cell lines and experimental rodents. During the course of apoptosis induction by ITC, multiple signal-transduction pathways and apoptosis intermediates are modulated. We have also clarified the molecular mechanism underlying the relationship between cell cycle arrest and apoptosis induced by benzyl isothiocyanate (BITC), a major ITC compound isolated from papaya. The exposure of cells to BITC resulted in the inhibition of the G2/M progression that coincided with not only the up-regulated expression of the G2/M cell cycle arrest-regulating genes but also the apoptosis induction. The experiment using the phase-specific synchronized cells demonstrated that the G2/M phase-arrested cells are more sensitive to undergoing apoptotic stimulation by BITC than the cells in other phases. We identified the phosphorylated Bcl-2 as a key molecule linking the p38 MAPK-dependent cell cycle arrest with the JNK activation by BITC. We also found that BITC induced the cytotoxic effect more preferentially in the proliferating normal human colon epithelial cells than in the quiescent cells. Conversely, treatment with an excessive concentration of BITC resulted in necrotic cell death without DNA ladder formation. This review addresses the biological impact of cell death induction by BITC as well as other ITCs and the involved signal transduction pathways.

Anticarcinogenic Agents↗
PubMed2004

Benzyl isothiocyanate modifies expression of the G2/M arrest-related genes.

Naturally occurring isothiocyanates are effective chemoprotective agents against chemical carcinogenesis in experimental animals. In the present study, we clarified the molecular mechanism underlying the relationship between benzyl isothiocyanate (BITC)-induced cell cycle arrest and apoptosis. The exposure of HL-60 cells to BITC resulted in the inhibition of the G2/M progression that coincided with the apoptosis induction. We demonstrated that BITC significantly up-regulated expression of the G2/M cell cycle arrest-regulating genes including p21, GADD45, and 14-3-3sigma. Thus, these gathered data further supported that BITC has a potential to induce apoptosis selectively in the proliferating pre-cancerous cells through a cell cycle arrest-dependent mechanism.

Anticarcinogenic Agents↗
PubMed1989

Cyanine dye labeling reagents containing isothiocyanate groups.

New isothiocyanate derivatives of cyanine dyes were synthesized as fluorescent covalent labeling reagents for proteins and other biomolecules. These dyes have maximum absorbance in the red and near infrared regions of the spectrum, have high extinction coefficients and have adequate quantum yields. Incorporating two alkyl sulfonate groups in the dye structures increases their water solubility, which is beneficial for labeling biological molecules in aqueous solution. Reactivities of proteins with these new cyanines are similar to their reactivities with fluorescein isothiocyanate. These new labeling reagents are complementary to the fluorescein and rhodamine reagents, expanding the possibilities of multicolor analyses. Sheep anti-mouse-IgG antibody was labeled with a pentamethine cyanine dye (CY5.8-ITC) and used with a fluoresceinated antibody as a second reagent for detecting human T-cell subsets by flow cytometry.

Antibodies↗
PubMed1989

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

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

Animals↗
PubMed2005

A novel function of benzyl isothiocyanate in vascular smooth muscle cells: the role of ERK1/2, cell cycle regulation, and matrix metalloproteinase-9.

Dietary isothiocyanates (ITCs) have shown protective effects against certain chemically induced cancers in animal models. These inhibitory effects are associated with reduced levels of extracellular signal-regulated kinase (ERK) 1/2 activity and the arrest of the G(1) cell cycle. Benzyl isothiocyanate (BITC) treatment down-regulates cyclins and CDKs and up-regulates the expression of the CDK inhibitor p21, but up-regulation of p27 or p53 was not detected. Since antiatherogenic effects are not needed for antiproliferation, we determined whether BITC exerted inhibitory effects on matrix metalloproteinase-9 (MMP-9) activity in TNF-alpha-induced vascular smooth muscle cells (VSMCs). BITC inhibited TNF-alpha-induced MMP-9 secretion in VSMC in a dose dependent manner. This inhibition was characterized by the down-regulation of MMP-9, which is transcriptionally regulated at the NF-kappaB site, and the activation protein-1 (AP-1) site in the MMP-9 promoter. These findings indicate that BITC is an effective agent for inhibiting cell proliferation, the G1 to S phase cell cycle progress, and MMP-9 expression through the transcription factors NF-kappaB and AP-1 in TNF-alpha-induced VSMC.

Animals↗
PubMed2006

c-jun/AP-1 activation does not affect the antiproliferative activity of phenethyl isothiocyanate, a cruciferous vegetable-derived cancer chemopreventive agent.

Cruciferous vegetable-derived isothiocyanates (ITCs) display potent cancer chemopreventive activity, but also markedly stimulate oncogenic activator protein 1 (AP-1). AP-1 is well known to promote cell survival and proliferation. We examined the impact of AP-1 activation on antiproliferative activity of ITCs, using bladder cancer cells and phenethyl isothiocyanate (PEITC) as models. AP-1 transactivation induced by PEITC was almost completely suppressed by a dominant-negative c-jun (TAM67). However, suppression of AP-1 transactivation did not affect PEITC-induced apoptosis or cell-cycle arrest. Moreover, we previously showed that in response to ITC treatment c-jun was predominantly stimulated among AP-1 family members largely by c-jun N-terminal kinase (JNK) [Food Chem Toxicol 2005; 43: 1373-1380], but neither JNK inhibition nor forced expression of c-jun altered the antiproliferative activity of PEITC. In addition, cyclin D1, which is considered as an AP-1 target gene and promotes cell proliferation, was markedly elevated in PEITC-treated cells. Unexpectedly, neither TAM67 or JNK inhibition, nor forced c-jun expression had a significant impact on cyclin D1 induction by PEITC, indicating that c-jun/AP-1 does not play an important role in cyclin D1 induction by PEITC. In conclusion, despite the known role of c-jun/AP-1 as a stimulator of cell growth and proliferation, our data show that its activation does not diminish the antiproliferative activity of PEITC and is not responsible for cyclin D1 induction by PEITC.

Anticarcinogenic Agents↗
PubMed2005

beta-Phenylethyl isothiocyanate mediated apoptosis: a proteomic investigation of early apoptotic protein changes.

beta-Phenylethyl isothiocyanate (PEITC) is a promising chemopreventative agent found in abundance in watercress (Rorripa nasturtium aquaticum) as its glucosinolate precursor. In the present investigation, we sought to determine the early changes in protein expression that contribute to the mechanism(s) of PEITC-mediated apoptosis in the human hepatoma HepG2 cell line. Such data may invariably identify new molecular targets of PEITC, contributing to a greater understanding of the mechanism(s) by which isothiocyanates mediate apoptotic cascades. Using two-dimensional difference gel electrophoresis we determined the changes in global protein expression between control (0.01% dimethyl sulfoxide) and PEITC (IC50 approximately 20 microM) treated cells after 3 and 6 h, such time points being used to circumvent the effects of caspase mediate proteolysis. Comparison between PEITC treated cells with their respective controls showed that 17 protein spots were differentially expressed. Fourteen of these spots, representing 9 unique proteins, were successfully identified using matrix-assisted laser desorption / ionization-time of flight (MALDI-TOF) and MALDI tandem time of flight (TOF/TOF) mass spectrometry. We observed significant shifts in isoelectric points on two-dimensional electrophoresis gels in heat shock 27 kDa protein (HSP27), macrophage migration inhibition factor and heterogeneous nuclear ribonucleoprotein K (hnRNP K) indicating that these proteins are probably involved in protein phosphorylation. Indeed, hnRNP K was determined to be phosphorylated on key tyrosine residues as assessed by using antiphosphotyrosine antibodies. In separate experiments we also showed that c-myc is up-regulated in PEITC treated cells, and since hnRNP K is reported to induce overexpression of c-myc, we proposed that PEITC-induced apoptosis may involve a c-myc dependent apoptotic pathway in HepG2 cells.

Apoptosis↗
PubMed1999

Temporal effects of the detoxification enzyme inducer, benzyl isothiocyanate: activation of c-Jun N-terminal kinase prior to the transcription factors AP-1 and NFkappaB.

Benzyl isothiocyanate (BIT), a microconstituent found in cruciferous vegetables, is known to be a potent inducer of the detoxification enzyme, NAD(P)H: quinone reductase (QR). QR catalyzes a two-electron transfer to a wide variety of redox-cycling species, including quinones, transforming them into dihydrodiols, thereby preventing the mutation of DNA and reducing cancer risk. The upstream signaling mechanisms that lead to the induction of QR remain unclear. The 5' promoter region of the human QR gene contains the cis-acting AP-1 and NFkappaB transcription factor binding sites. When HT29 human colon cells were exposed to 25microM benzyl isothiocyanate, AP-1 binding increased, beginning at 3 hours and increasing until 16 hours. NFkappaB binding also increased, reaching a maximum at around 6 hours. We also found that c-Jun N-terminal kinase (JNK), which phosphorylates c-Jun, a component of AP-1, was activated 9-fold over controls, beginning at 60 minutes. The temporal sequence of these events supports the idea that JNK is involved in the induction of QR and that this is an initial event preceding an increase in transcription factor binding and subsequent QR activity.

Calcium-Calmodulin-Dependent Protein Kinases↗
PubMed1995

Specific reactivity of fluorescein isothiocyanate-conjugated separated IgG and IgA from celiac disease sera on human tissues.

Study of the reaction of celiac disease-related antibodies with human substrates has been hampered by the immunological cross-reactivity between fluorescein isothiocyanate-conjugated anti-human immunoglobulins and immunoglobulins normally present in human tissues. In order to overcome this problem we extracted IgG from 2 celiac disease sera (positive for specific IgG and IgA antibodies) using a genetically engineered recombinant form of streptococcal protein G covalently immobilized on 4% agarose beads. The separated IgG and IgA was conjugated with fluorescein isothiocyanate and used in direct immunofluorescence on 0 blood group human duodenum, liver, and myocardium. Staining of the lamina propria beneath the villous epithelium, the endomysium of smooth muscle layers in the duodenum, and the liver sinusoidal and pericellular myocardial matrix was observed. The reactivity of celiac disease-related antibodies with various human tissues indicates that these antibodies are the result of a systemic immune response directed against all tissues containing matrix proteins.

Animals↗
PubMed2004

Effect of organic isothiocyanates on breast cancer resistance protein (ABCG2)-mediated transport.

PURPOSE: To investigate the effect of organic isothiocyanates (ITCs), dietary compounds with chemopreventive activity, on breast cancer resistance protein (BCRP)-mediated transport. METHODS: The effect of 12 ITCs on the cellular accumulation of mitoxantrone (MX) was measured in both BCRP-overexpressing and BCRP-negative human breast cancer (MCF-7) and large cell lung carcinoma (NCI-H460) cells by flow cytometric analysis. The ITCs showing activity in MX accumulation were examined for their effect on MX cytotoxicity, and the intracellular accumulation of radiolabeled phenethyl isothiocyanate (PEITC) was measured in both BCRP-overexpressing and BCRP-negative NCI-H460 cells. RESULTS: ITCs significantly increased MX accumulation and reversed its cytotoxicity in resistant cells, but had a small or no effect in sensitive cells. The effects of ITCs on MX accumulation and cytotoxicity were ITC-concentration dependent. Significant increases in MX accumulation were observed at ITC concentrations of 10 or 30 microM, and significant reversal of MX cytotoxicity was generally observed at ITC concentrations of 10 microM. Intracellular accumulation of radiolabeled PEITC in BCRP-overexpressing cells was significantly lower than that in BCRP-negative cells and was increased significantly by the BCRP inhibitor fumitremorgin C (FTC). CONCLUSIONS: Certain ITCs are BCRP inhibitors and PEITC and/or its cellular metabolite(s) may represent BCRP substrates, suggesting the potential for diet-drug interactions.

ATP Binding Cassette Transporter, Subfamily G, Mem↗
PubMed1988

Manual gas-phase isothiocyanate degradation.

We describe a manual gas-phase isothiocyanate degradation procedure for the primary structure determination of proteins and peptides. The proteins and peptides are applied to a polybrene-coated glass fiber filter wedged into a small glass column. The phenylisothiocyanate is directly pipetted onto the filter disk. The coupling and cleavage reactions are performed in small desiccators containing trimethylamine and trifluoroacetic acid vapors, respectively. The wash and extraction steps are performed by allowing the suitable solvents to percolate through the filter disk. The extracted anilinothiazolinone is then converted to the phenylthiohydantoin and identified by any one of a number of described methods. Our results show that this method is very sensitive and that the reactions proceed faster than those of the published automated procedure. No expensive equipment is required and the manual degradation can be performed by a laboratory assistant. A large number of samples can be simultaneously subjected to the degradation under identical conditions, making this an ideal method for physicochemical investigations into the isothiocyanate degradation. We also use this method to screen HPLC fractions after enzymatic protein fragmentation. Manually sequenced glass filters can be transferred to the automated instrument for more extended degradations.

Amino Acid Sequence↗
PubMed1990

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

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

Bacteroides↗