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PubMed2006

Benzyl isothiocyanate-induced DNA damage causes G2/M cell cycle arrest and apoptosis in human pancreatic cancer cells.

Benzyl isothiocyanate (BITC) has been shown to inhibit chemically induced pancreatic cancer in experimental animals. However, the mechanism responsible for the anticancer effects of BITC is not clearly understood. In this study, we tested whether BITC treatment would affect the growth of Capan-2 human pancreatic cancer cells. BITC (10 micromol/L) treatment caused marked phosphorylation of H2A.x (2.6-fold) and permanent damage to Capan-2 cells. BITC-mediated G2/M arrest was associated with up-regulation of cyclin dependent kinase inhibitor p21(Waf1/Cip1) and the activation of checkpoint kinase 2, whereas the expressions of other G2/M regulatory proteins, including CyclinB1, Cdc2, and cell division cycle 25C (Cdc25C), were down-regulated by 19, 51, and 70%, respectively, compared with control. These changes resulted in a 55% inhibition of Cdc2 kinase activity. In addition, the decline in the expression of Cdc25C was completely blocked when the cells were treated with lactacystin (proteasome inhibitor) prior to BITC treatment. However, G2/M arrest and apoptosis induced by BITC were partially blocked by pretreatment of cells with lactacystin. Taken together, the results of this study suggest the involvement of multiple signaling pathways targeted by BITC in mediating G2/M cell cycle arrest and apoptosis in Capan-2 cells and warrant further investigation.

Acetylcysteine↗
PubMed1999

Genotoxic effects of benzyl isothiocyanate, a natural chemopreventive agent.

Benzyl isothiocyanate (BITC) is contained in cruciferous plants which are part of the human diet. Numerous reports indicate that BITC prevents chemically induced cancer in laboratory animals and it has been postulated that BITC might also be chemoprotective in humans. On the other hand, evidence is accumulating that this compound is a potent genotoxin in mammalian cells by itself. To further elucidate the potential hazards of BITC, we investigated its genotoxic effects in different in vitro genotoxicity tests and in animal models. In in vitro experiments [differential DNA repair assay with Escherichia coli, micronucleus assay with human HepG2 cells and single cell gel electrophoresis (SCGE) assay with hepatocytes and gastrointestinal tract cells] pronounced dose-dependent genotoxic effects were found at low dose levels (</=5 microg/ml). In contrast, substantially weaker effects were obtained in in vivo experiments with laboratory rodents: in the differential DNA repair assay with E.coli cells, only moderate genotoxic effects were seen in indicator cells recovered from various organs of mice after treatment with high doses (between 90 and 270 mg/kg), while in SCGE assay with rats a change in the DNA migration pattern was seen at a dose level of 220 mg/kg body wt. These findings indicate that BITC is detoxified under in vivo test conditions. This assumption was supported by the results of in vitro experiments which showed that the genotoxic effects of BITC are markedly reduced by bovine serum albumin and human body fluids such as saliva and gastric juice. Additional experiments carried out on the mechanistic aspects of the genotoxicity of BITC showed that this compound causes formation of thiobarbituric acid-reactive substances in HepG2 cells and that its DNA damaging properties are diminished by alpha-tocopherol, vitamin C, sodium benzoate and beta-carotene, indicating the possible involvement of free radicals in the genotoxicity of BITC. The doses of BITC required to cause measurable DNA damage in laboratory rodents exceeded by far the dietary exposure levels of humans, but are similar to those which were required to inhibit chemically induced cancer in earlier animal experiments.

Adult↗
PubMed2004

Effects of alpha-naphthyl isothiocyanate and a heterocyclic amine, PhIP, on cytochrome P-450, mutagenic activation of various carcinogens and glucuronidation in rat liver.

To elucidate the mechanism underlying suppression by alpha-naphthyl isothiocyanate (ANIT) of mammary carcinogenesis induced by 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), we evaluated hepatic levels of cytochrome P-450 (CYP) enzymes, mutagenic activation of environmental carcinogens and UDP-glucuronyltransferase (UDPGT) activities in female Sprague-Dawley rats fed a high fat diet. Immunoblot analyses revealed induction of CYP1A1, newly found 51 and 53 kDa proteins and constitutive CYP1A2 and 2B2 by intragastric treatment with 85 mg/kg PhIP eight times for 11 days. Although the extents of induction were not as high as in the case of PhIP, 3 weeks feeding of 400 p.p.m. ANIT induced CYP1A1 and the 51 and 53 kDa proteins. CP1A2 level was decreased by the feeding of ANIT. The mutagenicity in strain TA98 of PhIP, four other heterocyclic amines (HCAs) and benzo[a]pyrene was greatly enhanced in the presence of liver S9 mix prepared from rats pretreated with PhIP but not with ANIT. The mutagenicities of these five HCAs were significantly decreased in the presence of liver S9 from rats pretreated with a combination of PhIP and ANIT as compared with that pretreated with PhIP alone. The level of hepatic CYP1A2, which is known to be involved in the metabolic activation of PhIP, was consistently decreased in liver microsomes from rats administered PhIP plus ANIT as compared with that from rats administered PhIP alone. On the other hand, UDPGT activity towards 4-nitrophenol (4-NP) was enhanced using liver microsomes prepared from rats pretreated with a combination of PhIP and ANIT as compared with those pretreated with PhIP or ANIT alone. These results show that chemoprevention by ANIT against PhIP-induced rat mammary carcinogenesis can be explained by a dual action mechanism, i.e. a reduction in metabolic activation by hepatic CYP1A2 and an enhancement of detoxification by 4-NP UDPGT. The role of the newly found 51 and 53 kDa proteins in activation of HCAs is also discussed.

Animals↗
PubMed1998

Fluorescein 5'-isothiocyanate-modified Na+, K+ -ATPase, at Lys-501 of the alpha-chain, accepts ATP independent of pyridoxal 5'-diphospho-5'-adenosine modification at Lys-480.

The modification of Na+,K+-ATPase with increasing pyridoxal 5'-diphospho-5'-adenosine (AP2PL) concentrations resulted in saturation of the approximately 0.5 mol AP2PL probe incorporation into the Lys-480/mol catalytic alpha-chain and reduced the Na+,K+-ATPase activity to around half without affecting the phosphorylation by acetyl phosphate (AcP), and led to increases in the AP2PL fluorescence caused by ATP and AcP. Further modification with fluorescein 5'-isothiocyanate (FITC) resulted in approximately 0.9 mol FITC probe incorporation into the Lys-501/mol alpha-chain and reduced the activity to below 5% without affecting the phosphorylation by AcP and these fluorescence increases. The ATP binding capacity of the AP2PL-FITC enzyme was shown to be at least 50% of that of the control enzyme (approximately 0.8 mol/mol alpha-chain). This is the first direct demonstration that Na+-bound FITC-modified enzymes accept ATP with an affinity for ATP (K(1/2) > 150 microM) reduced by two orders of magnitude. The data also suggest half site reactivity of Lys-480 as to AP2PL and all site reactivity of Lys-501 as to FITC in the catalytic subunits.

Adenosine Diphosphate↗
PubMed2006

The principal urinary metabolites of dietary isothiocyanates, N-acetylcysteine conjugates, elicit the same anti-proliferative response as their parent compounds in human bladder cancer cells.

Isothiocyanates (ITCs) are a class of well-known cancerpreventive phytochemicals, but are primarily disposed of and concentrated in the urine as N-acetylcysteine conjugates (NAC-ITCs) in vivo. Because human urinary bladder cancers occur almost exclusively in the bladder epithelium, which is directly exposed to the urine stored in the bladder, we undertook to examine the anti-cancer activity of NAC-ITCs in cultured human bladder cancer cells. In this paper, we report that the NAC conjugates of four naturally occurring ITCs, including allyl ITC, benzyl ITC (BITC), phenethyl ITC and sulforaphane, potently inhibited the growth of cells derived from both low-grade superficial and high-grade invasive human bladder cancers and drug-resistant bladder cancer cells. Moreover, the growth-inhibitory potencies were similar between the conjugates and their parent compounds. Further study of NAC-BITC and BITC as model compounds showed that both compounds accumulated in cells predominantly as the glutathione conjugate of BITC, but the accumulation of the former was slower. Moreover, both compounds also demonstrated the same anti-proliferative mechanisms: causing the cleavage of the same set of caspases (caspase-3, -8 and -9) in apoptosis induction, arresting cells in the same phases (S and G2/M) and targeting the same cell cycle regulator (Cdc25C), although a longer treatment time or slightly higher doses were needed for NAC-BITC to achieve the same effect as BITC, presumably due to slower cellular uptake of NAC-BITC. These data show that the NAC-ITCs are biologically similar to their parent compounds and are highly effective against human bladder cancer cells.

Acetylcysteine↗
PubMed2003

Superparamagnetic iron oxide particles transactivator protein-fluorescein isothiocyanate particle labeling for in vivo magnetic resonance imaging detection of cell migration: uptake and durability.

Conjugation of dextran-coated superparamagnetic iron oxide (SPIO) particles with transactivator protein (Tat)-peptide and fluorescein isothiocyanate (FITC) allows cells to readily uptake SPIO particles. This makes possible high-resolution, real-time imaging of these cells by magnetic resonance imaging (MRI). First, we need to understand how various subpopulations take up and maintain SPIO particles. In this report, we have focused on differences in T cells, B cells, and macrophages with respect to cross-linked (CL)-SPIO Tat-FITC particle uptake over 72 hours. We have found that cells quickly take up the particles and that the bead loss that does occur is not related to cell death or apoptosis. In contrast with reports in the literature, we have observed migration of the Tat-peptide conjugates primarily to the cytoplasm rather than the nucleus.

Animals↗
PubMed2001

Study of the role of antimicrobial glucosinolate-derived isothiocyanates in resistance of Arabidopsis to microbial pathogens.

Crude aqueous extracts from Arabidopsis leaves were subjected to chromatographic separations, after which the different fractions were monitored for antimicrobial activity using the fungus Neurospora crassa as a test organism. Two major fractions were obtained that appeared to have the same abundance in leaves from untreated plants versus leaves from plants challenge inoculated with the fungus Alternaria brassicicola. One of both major antimicrobial fractions was purified to homogeneity and identified by 1H nuclear magnetic resonance, gas chromatography/electron impact mass spectrometry, and gas chromatography/chemical ionization mass spectrometry as 4-methylsulphinylbutyl isothiocyanate (ITC). This compound has previously been described as a product of myrosinase-mediated breakdown of glucoraphanin, the predominant glucosinolate in Arabidopsis leaves. 4-Methylsulphinylbutyl ITC was found to be inhibitory to a wide range of fungi and bacteria, producing 50% growth inhibition in vitro at concentrations of 28 microM for the most sensitive organism tested (Pseudomonas syringae). A previously identified glucosinolate biosynthesis mutant, gsm1-1, was found to be largely deficient in either of the two major antimicrobial compounds, including 4-methylsulphinylbutyl ITC. The resistance of gsm1-1 was compared with that of wild-type plants after challenge with the fungi A. brassicicola, Plectosphaerella cucumerina, Botrytis cinerea, Fusarium oxysporum, or Peronospora parasitica, or the bacteria Erwinia carotovora or P. syringae. Of the tested pathogens, only F. oxysporum was found to be significantly more aggressive on gsm1-1 than on wild-type plants. Taken together, our data suggest that glucosinolate-derived antimicrobial ITCs can play a role in the protection of Arabidopsis against particular pathogens.

Arabidopsis↗
PubMed1985

Induction and suppression of contact sensitivity to fluorescein isothiocyanate (FITC).

Although both fluorescein isothiocyanate (FITC) and trinitrophenyl (TNP) covalently couple primarily to the epsilon-amino group of lysine residues of surface glycoprotein, FITC is structurally different from TNP. In this paper, we investigated the immune regulatory mechanisms in contact sensitivity (CS) to FITC by the administration of FITC and FITC-conjugated epidermal cells (FITC-EC) via various routes. Mice injected with FITC via i.p. or i.v. route did not induce CS but induced hyporesponsiveness to the following sensitization with FITC painting. Mice injected with FITC via s.c. route induced neither CS nor hyporesponsiveness to the following FITC painting. Administration of FITC via i.v. route was demonstrated to induce hapten-specific suppressor T cells. Inoculation of FITC-EC via s.c. or i.p. route induced CS, whereas injection of FITC-EC via i.v. route did not induce CS but induced hyporesponsiveness to the following FITC painting. The results are compared with the previous data obtained by trinitrobenzene sulfonate and trinitrophenyl-conjugated epidermal cells.

Animals↗
PubMed1988

Increased intratumor concentration of fluorescein-isothiocyanate-labeled neocarzinostatin in rats under angiotensin-induced hypertension.

On the basis of the observation that the tumor tissue blood flow selectively increases under angiotensin (AT)-induced hypertension, the change of the drug concentration in the tumor and normal tissues was examined in male Donryu rats. The intratumor concentration of fluorescein isothiocyanate-labeled neocarzinostatin was about 2-fold higher in the AT-induced hypertension group than in the control up to 20 min after the drug injection. In the normal organs or the uninvolved organs of the tumor-bearing rats, however, no clear increase was seen in the experimental group compared with the control, as anticipated from the observation of the tissue blood flow. The present study supports the hypothesis that the enhanced anticancer effect in chemotherapy under AT-induced hypertension formerly reported is due to the tumor-selective enhancement of the drug delivery.

Angiotensin II↗
PubMed1993

Inhibitory effects of benzyl isothiocyanate and benzyl thiocyanate on diethylnitrosamine-induced hepatocarcinogenesis in rats.

The effects of two aromatic thiocyanates, benzyl isothiocyanate (BITC) and benzyl thiocyanate (BTC), on diethylnitrosamine (DEN)-induced hepatocarcinogenesis were examined in rats. A total of 108 male ACI/N rats, 5 weeks old, were divided into 6 groups (18 rats in each). Group 1 was given a single i.p. injection of DEN (200 mg/kg body weight) one week after the start of the experiment and then kept on the basal diet until the end of the experiment (1 year). Groups 2 and 3 were treated with DEN and received dietary BITC (100 ppm) or BTC (100 ppm), respectively, throughout the experimental duration. Groups 4 and 5 were not given the carcinogen and were fed the diet containing BITC or BTC, respectively. Group 6 was kept on the basal diet alone and served as a control. Liver neoplasms were seen in Groups 1, 2 and 3. Incidence and average number of liver neoplasms in Group 2 were significantly smaller than in Group 1 (P < 0.0005 and P < 0.001, respectively). The incidence of liver neoplasms in Group 3 was slightly lower than in Group 1, although the difference was not statistically significant. The numbers of glutathione S-transferase placental form (GST-P)-positive foci in Group 2 and gamma-glutamyltranspeptidase (GGT)-positive foci in Groups 2 and 3 were significantly smaller than those in Group 1 (P < 0.001). The average and unit areas of GST-P- or GGT-positive foci in Group 2 or 3 were also significantly smaller than those in Group 1 (P < 0.05). These results suggest that BITC and BTC are chemopreventive agents for DEN-induced liver tumorigenesis.

Animals↗
PubMed1996

Chemopreventive effects of 3-phenylpropyl isothiocyanate on hamster lung tumorigenesis initiated with N-nitrosobis(2-oxopropyl)amine.

The chemopreventive effects of 3-phenylpropyl isothiocyanate (PPITC) were investigated in N-nitrosobis(2-oxopropyl)amine (BOP)-initiated hamsters. A total of 120 female 5-week-old hamsters were divided into 6 groups. Animals in groups 1-3, each consisting of 30 hamsters, were twice sc injected 7 days apart as an initiation treatment. Hamsters in groups 1 and 2 were respectively given 100 microM and 10 microM of PPITC by gavage 2 h prior to each BOP treatment. Animals in group 3 were treated with BOP alone, serving as an initiation-positive control. Animals in groups 4-6, each consisting of 10 hamsters, were given 100 microM or 10microM of PPITC alone, or non-treated, thus being available as matched negative controls to groups 1-3. At termination (experimental week 51 after the first BOP injection), the incidences of lung adenomas and/or adenocarcinomas were significantly decreased in groups 1 and 2 as compared to the group 3 value (p<0.01). The combined lung tumor incidences were inhibited by 94% and 59% at 100 and 10 microM doses, respectively. The inhibitory effects of PPITC were thus dose-dependent. The data for multiplicity of lung tumors dramatically illustrated the inhibitory effects of PPITC, and there were also statistically significant differences in the chemopreventive effect between 100 microM and 10 microM PPITC treatments. On the other hand, the PPITC treatments did not significantly modulate the development of neoplastic lesions in the pancreas,liver and kidney, although the treatments did show inhibitory tendencies, except on the liver lesions. Under present experimental conditions, PPITC itself did not exhibit tumorigenicity or apparent toxicity. The results in the present study thus clearly indicate that PPITC has an effective chemopreventive action on BOP-induced lung tumorigenesis in hamsters.

Animals↗
PubMed1997

Mechanistic insights into chemopreventive effects of phenethyl isothiocyanate in N-nitrosobis(2-oxopropyl)amine-treated hamsters.

The influence of phenethyl isothiocyanate (PEITC) on cell kinetics in the target organs of N-nitrosobis(2-oxopropyl)amine (BOP) tumorigenicity and on xenobiotic-metabolizing enzymes was investigated in hamsters. Female 5-week-old Syrian hamsters were given a single s.c. dose of 0, 20 or 50 mg/kg of BOP 2 h after receiving PEITC by gavage at a dose of 0, 100 or 250 mumol/animal (0, 16.3 or 40.8 mg/animal). Six and 22 h after the BOP administration, hamsters were killed and tissues were sampled. Proliferating cell nuclear antigen immunohistochemistry demonstrated significant reduction (P < 0.05-0.001) by PEITC of the labeling indices in the pancreatic acini and ducts, bronchioles, and renal tubules of the BOP-treated animals in a dose-dependent manner. In the lungs, the PEITC pretreatment significantly (P < 0.001) reduced the O6-methyldeoxyguanosine levels as compared to the BOP-alone value. Immunoblot analysis of liver cytochrome P450 isoenzymes showed CYP 2B1 to be mainly involved in the metabolic activation of BOP. PEITC significantly (P < 0.05) inhibited the induction of several isoenzymes, including CYP 2B1, while lowering the hepatic glutathione S-transferase activity as well as glutathione levels, regardless of BOP administration. Our results thus suggest that PEITC exerts its chemopreventive activity against BOP initiation of carcinogenesis in hamsters by decreasing cell turnover and DNA methylation in the target organs, and by influencing hepatic xenobiotic-metabolizing phase I enzymes, although the relationship, if any, of the latter with the former events remains to be investigated.

Animals↗
PubMed2003

Enhancement of urinary bladder carcinogenesis by combined treatment with benzyl isothiocyanate and N-butyl-N-(4-hydroxybutyl)nitrosamine in rats after initiation.

Previously we reported that benzyl isothiocyanate (BITC) strongly enhanced rat urinary bladder carcinogenesis after initiation with N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN), while potently inhibiting BBN-induction of lesions when given simultaneously with the carcinogen. In the present experiment, the effects of simultaneous treatment with BITC and low-dose BBN on the post-initiation period of rat urinary bladder carcinogenesis were examined. After treatment with 500 ppm BBN for 4 weeks for initiation, groups of 20, 6-week-old, F344 male rats were given 25 ppm BBN alone, basal diet alone, or 100 or 1000 ppm BITC in the diet together with or without 25 ppm BBN in their drinking water for 36 weeks and then killed for autopsy. Further groups consisting of 10 rats each were similarly given BITC or the basal diet together with or without 25 ppm BBN, without initiation treatment. In the initiated groups receiving subsequent BBN exposure, papillary and nodular hyperplasia, dysplasia and carcinoma incidences were significantly increased, and they were further increased by the combined treatment with 100 and 1000 ppm BITC in a dose-dependent manner. In the non-initiation groups, carcinomas were only observed in a single rat in each of the BBN-treated control and BBN/BITC 100 ppm treatment groups. The results indicate that simultaneous treatment with BITC and a low dose of BBN does not inhibit, but rather enhances rat urinary bladder carcinogenesis after appropriate initiation, and further suggest that BITC may be a human risk factor, at least in high-risk populations.

Animals↗
PubMed2006

Effects of phthalate esters on the sensitization phase of contact hypersensitivity induced by fluorescein isothiocyanate.

BACKGROUND: Many different types of phthalate ester are used as plasticizers and are thus found in the air. There have been several studies that suggest an association between allergies and phthalate esters. We previously found that di-butyl phthalate (DBP) has an adjuvant effect in a mouse contact hypersensitivity model, in which fluorescein isothiocyanate (FITC) is involved as an immunogenic hapten. OBJECTIVE: We examined whether other phthalate esters enhance the process of sensitization to FITC by facilitating the trafficking of FITC-presenting dendritic cells or macrophages from skin sites to draining lymph nodes. METHODS: Mice were epicutaneously sensitized with FITC dissolved in acetone containing a phthalate ester. Sensitization was evaluated as ear swelling after a challenge with FITC. Draining lymph node cells obtained 24 h after skin sensitization were examined for FITC fluorescence by means of flow cytometry. FITC-positive cells were characterized with anti-CD11c and anti-CD11b by three-colour flow cytometry. RESULTS: When mice were sensitized with FITC in acetone containing DBP or di-n-propyl phthalate (DPP), strong enhancement of the ear-swelling response was observed. Di-methyl phthalate (DMP) and di-ethyl phthalate (DEP) were less effective but produced some enhancement. Consistent enhancement was not observed with di-(2-ethylhexyl) phthalate or di-isononyl phthalate. Upon sensitization in the presence of DBP or DPP, the number of FITC-positive dendritic cells (total CD11c+ as well as CD11c+/CD11b+) was increased in draining lymph nodes. As to the other four phthalate esters, there was no significant increase in the FITC-positive cell number in the draining lymph nodes. CONCLUSION: During the process of sensitization to FITC, DBP, and DPP exert strong adjuvant effects that are associated with enhancement of trafficking of antigen-presenting dendritic cells from the skin to draining lymph nodes.

Air Pollutants↗
PubMed1988

Polymerization of actin modified with fluorescein isothiocyanate.

Solution properties of skeletal muscle actin, modified at lysine-61 with fluorescein isothiocyanate (FITC) [Burtnick, L.D. (1984) Biochim. Biophys. Acta 791, 57-62], were re-examined in this work by light scattering, analytical ultracentrifugation, fluorescence, electron microscopy and myosin ATPase activity measurements. Fluorescence measurements using trace amounts of actin labeled with N-(1-pyrenyl)iodoacetamide showed that the FITC modification inhibited but did not block completely the polymerization of actin by KCl and MgCl2. Sedimentation velocity runs of FITC-actin, incubated with 100 mM KCl and 2 mM MgCl2, revealed the presence in these solutions of polymeric, oligomeric and monomeric species. The critical concentration for FITC-actin polymerization under these conditions was 12 microM. As judged by electron microscopy, FITC-actin polymers were similar to but generally shorter than standard F-actin filaments. Light scattering measurements indicated that FITC modification inhibited also the polymerization of actin by myosin subfragment 1 (S1) but the resulting complexes were indistinguishable from standard, decorated actin filaments. MgATPase measurements showed that FITC-actin, polymerized by preincubation with S1, activated the MgATPase activity of S1 while the monomeric labeled protein did not. Thus, in analogy to native actin, the activating function of FITC-actin depended on the formation of actin filaments. Results presented in this study suggest that the region around lysine-61 of actin plays an important role in actin-actin contact and is less crucial to actomyosin interaction.

Actins↗
PubMed1989

Reaction of fluorescein isothiocyanate with an ATP-binding site on the phosphorylase kinase alpha subunit.

Phosphorylase kinase can be labeled specifically on the alpha subunit with fluorescein 5'-isothiocyanate (FITC) which concomitantly inactivates the enzyme (T. G. Sotiroudis and S. Nikolaropoulus (1984) FEBS Lett. 176, 421-425). Labeled peptides have been purified and their primary structure has been determined. The amino acid sequence of the fluorescein-labeled tryptic peptide is Lys-Met-Gln-Asp-Gly-Tyr-Phe-Gly-Gly-Ala-Arg. The environment of this fluorescein-labeled lysine has been determined by sequencing peptides isolated from a Staphylococcus aureus V8 digest and two further cyanogen bromide fragments of the purified [14C]carboxymethylated alpha subunit. The partial sequences obtained have then been localized in the primary structure of the alpha subunit [Zander et al. (1988) Proc. Natl Acad. Sci. USA 85, 2929-2933]. Both the incorporation of the fluorescent label and enzymatic inactivation are inhibited by ATP only at pH 7.0; ADP and AMP do not protect. Kinetic analysis reveals a competition between ATP and FITC; a Ki for ATP of 728 +/- 100 microM has been determined.

Adenosine Triphosphate↗
PubMed1993

Horse plasma gelsolin labelled with fluorescein isothiocyanate responds to calcium and actin.

Reaction between horse plasma gelsolin and fluorescein-5-isothiocyanate (FITC) resulted in incorporation of 4.8 +/- 0.6 fluorescein groups/gelsolin molecule. The sites of modification were not clustered in any one portion of the gelsolin polypeptide chain; all major peptides produced by proteolytic digestion with alpha-chymotrypsin exhibited a fluorescence characteristic of fluorescein. FITC-gelsolin has a peptide-backbone circular dichroism spectrum at 20 degrees C that is indistinguishable from that of native gelsolin, but FITC-gelsolin is considerably more resistant than native gelsolin to thermally induced precipitation. FITC-gelsolin is fully able to carry out severing of F-actin filaments, the prime function of gelsolin in plasma. An opening up of the structure of gelsolin on binding Ca2+ is evident from an increased susceptibility of FITC-gelsolin to quenching by I-. Ca2+ dependence of the interaction between gelsolin and actin is evident in titrations both of intensity and polarization of the fluorescence of FITC-gelsolin solutions. A Ca(2+)-sensitive interaction between gelsolin and tropomyosin also is observed.

Actins↗
PubMed2005

The time-dependent effect of gluconasturtiin and phenethyl isothiocyanate on metabolic and antioxidative parameters in rats.

The effect of gluconasturtiin (GNST) and phenethyl isothiocyanate (PEITC) on some metabolic changes and antioxidative parameters in the rat was tested using different doses of PEITC and duration of GNST or PEITC ingestion. Their effect on antioxidative processes was previously observed, however, their influence on metabolic changes is still poorly characterized. In the performed experiment, the effect of GNST (0.5 mg/kg BW) and PEITC (0.1 mg/kg BW or 0.3 mg/kg BW) administered intragastrically after 4 h or 14 days to growing male rats was studied. PEITC at both doses after 4 h of its administration caused a considerable increase in liver cholesterol and triglyceride content with a concomitant drop in the amount of glycogen. Blood glucose, free fatty acids, phospholipids and total, free, esterified cholesterol as well as cholesterol in high-density lipoprotein were not altered. GNST, at its short-time ingestion, augmented significantly the concentration of triglycerides in blood serum. The compounds tested had no influence on metabolic changes after a longer period of action with the exception of glycogen values in liver, which were substantially augmented by PEITC at both doses. Our trial revealed a lack of GNST and PEITC influence on the content of liver sulphhydryl groups and on glutathione peroxidase and glutathione-S-transferase activities. The only distinct change in the content of malonodialdehyde was observed after short-time action of lower dose of PEITC. Our research showed that the short-term PEITC action constituted a significant factor interfering with liver metabolism. Although PEITC has been repeatedly advocated as very promising anticancer agent, in our experiment, the lower dose of PEITC was revealed as a pro-oxidative substance. These inconsistent properties seem to depend on its dose and time of action.

Animals↗