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PubMed2003

Comparison of isothiocyanate yield from wasabi rhizome tissues grown in soil or water.

The isothiocyanate (ITC) yield of wasabi, the Japanese horseradish (Wasabi japonica), was measured on its release from glucosinolates in the rhizomes of plants grown in two traditional ways. Mature plants of 18 months old were harvested from two different commercial farms located in the South Island of New Zealand. At one farm, the plants were grown in raised soil beds, while the plants at the other farm were grown in gravel irrigated by river water. Following harvest, the rhizomes from each growth medium were divided into five size groups based on the weight and length of the rhizomes. The different sized rhizomes were also subdivided into proximal, medial, and distal portions of the rhizomes and each portion was further subdivided into epidermis plus cortex, and vascular plus pith. The individual and total ITC contents of each portion (proximal, medial, and distal) of the rhizomes were measured using dichloromethane extraction followed by the GC-FPD. The total ITC content of the rhizomes grown in soil increased (13 times) linearly from 6 to 114 g of rhizome weight, while the mean ITC content of the water-grown wasabi increased (10 times) nonlinearly for similar sized rhizomes. Water-grown rhizomes in the weight range from 18 to 45 g gave significantly (P = 0.030) higher total ITC (1-2 times) than similarly sized soil-grown rhizomes. Analysis of the tissues showed that the total and the individual ITCs were found in significantly higher levels (73 and 64%, respectively) in the skin and cortex tissue compared to the vascular and pith tissues. Analysis of the ITC content of the different locations of the wasabi rhizome showed that the distal portion of the rhizome contained significantly higher levels of both total and individual ITCs compared to the medial and proximal portions of the rhizome.

Isothiocyanates↗
PubMed2006

Improvement in isolation and identification of food-derived peptides in human plasma based on precolumn derivatization of peptides with phenyl isothiocyanate.

For the isolation and detection of food-derived peptides in blood, an approach based on the derivatization of peptides with phenyl isothiocyanate (PITC) was developed. This approach allows hydrophilic peptides to be resolved and specifically detected by reversed-phase (RP) HPLC. For the rapid capturing and clarification of peptides in human plasma, solid-phase extraction by using a mini spin column (5 mmx5 mm) packed with a strong cation exchanger was used. The clarified peptide fraction was further fractionated by size-exclusion chromatography (SEC). The peptides in the SEC fractions were derivatized with PITC, and the derivatives were resolved by RP-HPLC by using an ammonium acetate buffer or a trifluoroacetic acid system. An automatic peptide sequencer based on Edman degradation with a modified program can directly analyze the resolved derivatives. Some synthetic peptides and food-derived peptides in human plasma were successfully isolated and identified by this approach.

Chromatography, Gel↗
PubMed2000

Effect of hydrolysis time on the determination of amino acids in samples of soybean products with ion-exchange chromatography or precolumn derivatization with phenyl isothiocyanate.

Accurate determination of amino acid levels in soy products facilitates optimum diet formulation and amino acid supplementation. A study was carried out to investigate the effect of hydrolysis time and method of amino acid measurement on amino acid levels. Correction factors to standardize amino acid levels to 24 h of hydrolysis were also determined. Six different soybean products were evaluated. Hydrolysis was carried out for 10 different periods of time. Amino acids were analyzed by both ion-exchange chromatography and precolumn derivatization with phenyl isothiocyanate. Both hydrolysis time and measurement method affected (P < 0.05) amino acid levels. Standard hydrolysis conditions (hydrolysis in 6 M HCl at 110 degrees C for 24 h) rarely provide the maximal amino acid values. Therefore, sequential hydrolyses curves were very useful and should be used.

Amino Acids↗
PubMed2000

Factors affecting the dissolution and degradation of oriental mustard-derived sinigrin and allyl isothiocyanate in aqueous media.

Sinigrin, the predominant glucosinolate in the oriental mustard Brassica juncea, is mainly degraded upon the enzymatic action of myrosinase under normal conditions to give allyl isothiocyanate (AITC) in an aqueous media. Because AITC is considered to be the principal nematicidal ingredient in B. juncea, its stability in aqueous media is an important issue in achieving efficient nematode control. Pure sinigrin and AITC were found to be relatively stable in buffered water in the pH range of 5.00-7.00 but less stable at pH 9.00. Both sinigrin and AITC were more stable in soil water (supernatant of a 1:1 water/air-dried soil mixture) than in buffered water at the same pH range of 5.00-9.00. Sinigrin dissolved from the mustard bran or ground seed into water very quickly and was degraded by codissolved myrosinase to AITC. The AITC that formed from the degradation of sinigrin was found to be more stable in the soil water than in the buffered water. Buffer capacity was considered to be one of the factors that contributed to the stabilization of AITC in the soil water, but other unknown factors from both bran or seed and soil may also have contributed to the stabilization.

Glucosinolates↗
PubMed2000

Reactions of allyl isothiocyanate with alanine, glycine, and several peptides in model systems.

The nucleophilic addition reactions of allyl isothiocyanate (AITC) with alanine, glycine, and five alanine and/or glycine containing di- and tripeptides were investigated in model aqueous solutions of pH 6, 8, and 10 at 25 degrees C for 2-4 weeks. The formation of primary adducts, i.e., N-allylthiocarbamoyl amino acids (ATC-amino acids) or ATC-peptides, their transformation products, i.e., 3-allyl-2-thiohydantoins originating by cyclization of ATC-amino acids or by cleavage of ATC-peptides, and several other minor components were observed. The results revealed that both addition and cleavage rates rise proportionally to pH, whereas the formation of 2-thiohydantoins from ATC-amino acids is controlled by H(3)O(+) concentration. Depending on pH, differences in reaction rates of the additions are determined by either pK(a)(NH(2)) of amino compounds or electrical effects and steric hindrance of the molecules. The latter factors are crucial also for differences in cleavage rates of ATC-peptides. With regard to the pK(a) values and simultaneous AITC decomposition by aqueous nucleophiles, the reactions with amino acids and oligopeptides are predominant reaction pathways of AITC in solutions of pH 10 and 8, respectively. Reaction mechanism of the cleavage of 2-thiohydantoins from ATC-peptides in alkaline and mild acidic solutions is different from the conventional Edman scheme used for anhydrous acid medium.

Alanine↗
PubMed2000

Synthesis and evaluation of isothiocyanate-containing derivatives of the delta-opioid receptor antagonist Tyr-Tic-Phe-Phe (TIPP) as potential affinity labels for delta-opioid receptors.

Derivatives of the delta-opioid receptor-selective peptide antagonist H-Tyr-Tic-Phe-Phe-OH (TIPP) containing an isothiocyanate moiety at the para position of either Phe(3) or Phe(4) were prepared as potential affinity labels for delta-opioid receptors. The synthesis was accomplished using a general solution-phase synthetic procedure which allows for introduction of affinity labeling groups late in the synthesis of a variety of small peptide substrates. The target peptides and their corresponding amines were then evaluated in radioligand binding experiments using Chinese hamster ovary (CHO) cells expressing delta- and mu-opioid receptors. The peptides [Phe(p-NCS)(3)]TIPP (2) and [Phe(p-NCS)(4)]TIPP (4) showed affinity for delta-receptors comparable to the parent compound TIPP (IC(50) = 12 and 5 nM, respectively, vs 6 nM for TIPP). Both peptides 2 and 4 were able to inhibit radioligand binding to delta-receptors in a wash-resistant manner at a concentration of 10 nM. Therefore, the peptides [Phe(p-NCS)(3)]TIPP (2) and [Phe(p-NCS)(4)]TIPP (4) represent two affinity labels that may prove useful in the study of delta-opioid receptors.

Affinity Labels↗
PubMed1997

Isothiocyanate derivatives of 9-[3-(cis-3,5-dimethyl-1-piperazinyl)propyl]carbazole (rimcazole): irreversible ligands for the dopamine transporter.

Cocaine has been reported to bind to the dopamine transporter in a biphasic fashion, and it has been hypothesized that the low-affinity component may play a modulatory role in cocaine's psychomotor stimulant effects. In an effort to gain further insight into the roles of the two sites, we have prepared a series of irreversible ligands based on rimcazole (9-[3-(cis-3,5-dimethyl-1-piperazinyl)propyl]carbazole, 2), a compound that has been postulated to bind only to the low-affinity site. The alkylating moiety (isothiocyanate) is attached to the distal nitrogen of the piperazine ring via alkyl chains of varying lengths or directly attached to one of the aromatic groups. It was found that substitution on the piperazine nitrogen caused an initial decrease in affinity that was recovered as the alkyl chain length increased. Importantly, the analogue 16, with the highest affinity for the dopamine transporter (DAT), binds in a monophasic and irreversible manner, as evidenced by the greatly diminished binding of [3H]WIN 35,428 in tissue that had been preincubated with the ligand and then thoroughly washed using centrifugation. The dose-dependent reduction in Bmax was accompanied by a concentration-related decrease in KD values. This shift in KD to a lower value suggests that the preincubation with 16 produced a preferential irreversible binding to the low-affinity [3H]WIN 35,428 site on the dopamine transporter. These ligands may prove to be important tools with which to study the significance of the low-affinity site on the DAT. Since rimcazole does not share the behavioral profile of cocaine, and in fact appears to play a modulatory role, these compounds may provide leads for a novel cocaine-abuse treatment.

Animals↗
PubMed1996

In-vivo buccal delivery of fluorescein isothiocyanate-dextran 4400 with glycodeoxycholate as an absorption enhancer in pigs.

Buccal delivery of fluorescein isothiocyanate labeled dextran 4400 (FD4) was investigated in-vivo in pigs. The delivery device consisted of an application chamber with a solution of FD4 and was adhered to the buccal mucosa for 4 h using an adhesive patch. A randomized crossover study including intravenous administration and buccal delivery without and with 10 mM sodium glycodeoxycholate (GDC) as absorption enhancer was performed in five pigs. After buccal administration, steady state plasma levels were rapidly reached. Coadministration of 10 mM GDC increased the absolute bioavailability of FD4 from 1.8 +/- 0.5% to 12.7 +/- 2.0%. Since FD4 is a macromolecular and hydrophilic compound such as peptide and protein drugs, buccal delivery would provide an adequate alternative to the parenteral administration of these drugs.

Absorption↗
PubMed1996

Effects of protease inhibitors on the absorption of phenol red and fluorescein isothiocyanate dextrans from the rat intestine.

The absorption enhancement effects of three types of protease inhibitors, aprotinin, bacitracin, and soybean trypsin inhibitor, on the small and large intestinal absorption of phenol red (PR) and fluorescein isothiocyanate dextrans (FDs) were examined in rats. Of these protease inhibitors, only bacitracin enhanced the absorption of PR and FDs from the rat small and large intestine. Thus, we suggest that bacitracin has not only a protease-inhibitory but also an absorption-enhancing capability. We examined the effects of various protease inhibitors on intestinal mucosal toxicity by measuring the leakage of Evans blue (EB) from the systemic circulation. Although there was a significant increase in the leakage of EB in the presence of bacitracin, it was considerably less than that in the case of polyoxyethylene 9-lauryl ether (BL-9), which was used as a positive control. Therefore, bacitracin may be a good model adjuvant for improving the intestinal absorption of poorly absorbable drugs because it did not cause serious intestinal mucosal damage, as seen in the case of BL-9.

Animals↗
PubMed1999

Inactivation of cytochrome P450 2E1 by benzyl isothiocyanate.

The cytochrome P450 enzymes constitute a family of phase I enzymes that play a prominent role in the metabolism of a great variety of endogenous and xenobiotic compounds. In this study, the kinetics for the inactivation of cytochrome P450 2E1 by benzyl isothiocyanate (BITC) were elucidated. BITC is a naturally occurring compound found in cruciferous vegetables such as broccoli. BITC inhibited the 7-ethoxy-4-(trifluoromethyl)coumarin (7-EFC) O-deethylation activity of purified and reconstituted P450 2E1 in a time- and concentration-dependent manner. The concentration of inactivator needed for half-maximal inactivation (K(I)) was 13 microM, and the maximum rate of inactivation at saturation (k(inact)) was 0.09 min-1. The partition ratio for the inactivation of P450 2E1 by BITC was found to have an approximate value of 27. Inactivation of P450 2E1 by BITC was dependent on the presence of NADPH. Following incubation for 5 min with BITC, a 65% loss in enzymatic activity was observed, while approximately 74% of the spectrally detectable enzyme remained. 7-Ethoxycoumarin (7-EC), a substrate of P450 2E1, protected P450 2E1 from BITC inactivation, reducing the loss in 7-EFC O-deethylation activity from 50 to 18% when a 1:20 molar ratio of BITC:7-EC was used. Inactivation of P450 2E1 by BITC was irreversible, and no activity was regained after extensive washes to remove BITC. Addition of cytochrome b(5) to the reconstituted system did not affect the rate of inactivation. Reductase activity was unaffected by BITC. The results reported here indicate that BITC is a mechanism-based inactivator of cytochrome P450 2E1 and that the inactivation was primarily due to a modification of the apoprotein by BITC.

Cytochrome P-450 CYP2E1↗
PubMed2003

Involvement of Nrf2 and JNK1 in the activation of antioxidant responsive element (ARE) by chemopreventive agent phenethyl isothiocyanate (PEITC).

PURPOSE: Phenethyl isothiocyanate (PEITC) has been of great interest as a promising cancer chemopreventive agent. To better understand its chemopreventive activity, we examined the effect of PEITC on the antioxidant responsive element (ARE), which is an important gene regulatory element of many phase II drug-metabolizing/detoxification enzymes as well as cellular defensive enzymes. METHODS: HeLa cells were transiently transfected with different cDNA plasmids using calcium phosphate precipitation. Subsequently, the cells were maintained in fresh media, and various concentrations of PEITC were added to the transfected cells. After harvesting and lysing of the cells, ARE-luciferase reporter gene activity was measured and normalized against beta-galactosidase activity. RESULTS: Treatments of HeLa cells with PEITC transiently stimulated ARE-reporter gene expressions in a dose-dependent manner. Overexpression of wild-type NF-E2 related factor-2 (Nrf2) dramatically increased ARE-reporter gene expression in a dose-dependent manner. Similar effects were seen when wild-type c-Jun N-terminal kinase 1 (JNK1) was transfected, although the transactivating potential of JNK1 was much less than that of Nrf2. Cotransfection of Nrf2 and JNK1 showed additional enhancement of ARE reporter gene expression, implying that JNK1 might be an upstream activator of Nrf2. To support this, overexpression of dominant-negative JNK1 suppressed Nrf2-induced ARE reporter gene expression in a dose-dependent manner. When PEITC was added, slight enhancement of ARE reporter gene expression was observed in either Nrf2- or JNK1-transfected cells. Finally, ARE reporter activity induced by PEITC was substantially attenuated by transfection of either dominant-negative mutant of Nrf2 or dominant-negative mutant of JNK1. CONCLUSION: Taken together, these data suggest that JNK1 acts as an upstream activator of Nrf2 and that PEITC activates ARE-mediated phase II drug metabolism gene expressions via the JNK1- and Nrf2-dependent pathways.

Anticarcinogenic Agents↗
PubMed2004

The new isothiocyanate 4-(methylthio)butylisothiocyanate selectively affects cell-cycle progression and apoptosis induction of human leukemia cells.

We investigated proliferation and apoptosis induction in Jurkat T-leukemia cells by the new isothiocyanate 4-(methylthio)butylisothiocyanate (MTBITC). To help elucidate whether the effects of MTBITC are specific for cancer cells, we tested MTBITC on freshly isolated, non-transformed human peripheral T lymphocytes. The effects of MTBITC are leukemic-cell-specific and consist of derangements in a critical point of cell-cycle control (G2/M transition). In fact, an increase in the proportion of G2 cells (from about 18% to 50%) was apparent following 24 h of treatment, associated with a decrease in the protein expression of cyclin B1. The expression of cyclin-dependent kinase (CDK) 1 was more mildly attenuated by MTBITC. Our results demonstrated that high concentrations of MTBITC can also induce apoptosis, through an increase of p53 and bax, but not bcl-2, protein expression. No effects of MTBITC were demonstrated on non-transformed T lymphocytes. Taking into account its in vitro antineoplastic activity and selectivity toward leukemia cells, MTBITC can be viewed as a conceptually promising agent in cancer therapy.

Apoptosis↗
PubMed2004

Binding of fluorescein isothiocyanate to insulin: a fluorimetric labeling study.

The determination of the fluorophore to the protein molar ratio has been studied using fluorescence spectroscopy. The tyrosine fluorescence is measured from insulin (Ins) solutions at wavelengths lambda(ex)/lambda(em) = 276/300 nm and from fluorescein isothiocyanate (FITC) solutions at lambda(em)/lambda(em) = 494/518 nm. Series of solutions prepared from insulin and FITC are tested for conjugation, recording their fluorimetric intensities. Fluorimetric titrations with different formal concentrations are followed either by intrinsic and extrinsic emission intensities at lambda(ex)/lambda(em) = 276 or 494/518 nm and by their typical emission spectra at pH 9.0. All results denoted a binding ratio of 3 moles of FITC/mole of Ins.

Animals↗
PubMed2006

Sensitisation for cisplatin-induced apoptosis by isothiocyanate E-4IB leads to signalling pathways alterations.

A new synthetic isothiocyanate (ITC) derivative, ethyl 4-isothiocyanatobutanoate (E-4IB), appeared to be an effective modulator of cellular proliferation and potent inducer of apoptosis. In cooperation with cisplatin, this compound exerted synergistic effects in human ovarian carcinoma A2780 cells. In the present study we investigated in more detail E4IB-sensitisation for cisplatin-induced apoptosis. Sequential administration of both cytostatic agents led to increased intracellular platinum accumulation, glutathione level depletion and mitochondrial membrane potential dissipation. These events were accompanied with poly (ADP-ribosyl) polymerase cleavage, stimulation of caspase-3 activity, upregulation of p53, FasL and Gadd45alpha, cyclin B1 downregulation and an increase in mitogen-activated protein kinases JNK, ERK and p38 phosphorylation as well as PI3K level alterations. The presented results might have implications for developing new strategies aimed at therapeutic benefit of natural or synthetic ITCs in cooperation with various anticancer drugs.

Antineoplastic Agents↗
PubMed1981

Isolation and characterization of polypeptide at the picomole level. Pre-column formation of peptide derivatives with dimethylaminoazobenzene isothiocyanate.

Polypeptides coupled with dimethylaminoazobenzene isothiocyanate through their amino groups to form dimethylaminoazobenzenethiocarbamoyl- (DABTC-)peptides can be separated by reversed-phase high-pressure liquid chromatography and detected in the visible region (436 nm). As little as 1 ng (2 pmol) of a DABTC-pentapeptide can be identified against a stable base-line with the signal-to-noise ratio of 10. The DABTC-peptides can also be recovered from the column, and their N-terminal amino acids (obtained by direct treatment with aqueous acid) and amino acid compositions and sequences can be all analysed at the picomole level. The power of this method is demonstrated by the complete separation and characterization of model peptides, peptide hormones and peptides derived from enzymic fragmentation of proteins. This new technique should provide a sensitive and efficient tool for peptide analysis at the nanogram level.

Amino Acids↗
PubMed1983

A new isotype sequence (V kappa 27) of the variable region of kappa-light chains from a mouse hybridoma-derived anti-(streptococcal group A polysaccharide) antibody containing an additional cysteine residue. Application of the dimethylaminoazobenzene isothiocyanate technique for the isolation of peptides.

The first complete sequence of the variable region of a kappa-light chain (V kappa) from a mouse anti-(streptococcal group A polysaccharide) antibody (immunoglobulin 7S34.1) is reported. Immunoglobulin 7S34.1 was isolated from the ascitic fluid of hybridoma 7S34.1 previously cloned in vitro. A newly developed technique for the isolation of peptides by using pre-column formation of peptide derivatives with dimethylaminoazobenzene isothiocyanate also served to complete the sequence. The sequence of the variable region of the kappa-light chain of immunoglobulin 7S34.1 defines a new mouse V kappa isotype (V kappa 27) and is the first mouse immunoglobulin light-chain variable region to be shown to have an extra cysteine residue at position 48.

Amino Acid Sequence↗
PubMed1992

Inhibition of tonoplast ATPase from etiolated mung bean seedlings by fluorescein 5'-isothiocyanate.

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.

Adenosine Triphosphatases↗