Heterokaryon Identification Through Simultaneous Fluorescence of Tetramethylrhodamine Isothiocyanate and Fluorescein Isothiocyanate Labelled Protoplasts

Protoplasts were separately stained with the fluorescent dyes fluorescein iso-thiocyanate (FITC) and tetramethylrhodamine isothiocyanate (TRITC). Following fusion, doubly stained heterokaryons were identified under fluorescence microscopy by using the Zeiss filter set 48 77 05 (excitation filter 450-490 nm, dichroic reflector 510 nm, and barrier filter 520 nm) which allowed simultaneous fluorochrome emissions. Previously, either emisson spectrum, but not both, was possible for any single filter set.     

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.     

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.     

Fluorescent energy transfer measurements on fluorescein isothiocyanate modified cytochrome P-450 LM2

The distance between the heme iron and the N-terminus of cytochrome P-450 LM2 was determined by fluorescence energy transfer measurements. Fluorescein isothiocyanate which was covalently bound to the N-terminal methionine was used as donor chromophor. The Ro value between fluorescein isothiocyanate and the heme was calculated to be 3.98 nm. The distance between the nitrogen of the N-terminal methionine and the heme was estimated with 2.84 +/- 0.23 nm excluding most likely the N-terminal amino acid of cytochrome P-450 LM2 to participate in the electron transfer to the heme iron. A cytochrome P-450 LM2 membrane model is proposed.     

Optimization of an Acridine Orange–Bisbenzimide Procedure for the Detection of Apoptosis-Associated Fluorescence Colour Changes in Etoposide-Treated Cell Cultures

This study was initiated in order to investigate the possibility of improving fluorescence microscopy as a method for evaluating apoptosis in cells by combining two fluorescent dyes with different staining characteristics. Cells were vitally stained with bisbenzimide (1.3 microM) and Acridine Orange (6.6 microM) and observed using the following filter configuration: excitation 380 nm, beamsplitter 395 nm and longpass filter 397 nm. Control cells exhibited clear blue fluorescent nuclei and red fluorescing lysosomes. In cells treated with etoposide to induce apoptosis, two distinct occurrences were observed: a change in the spectrum of emitted light from bisbenzimide bound to the nuclear region and an increase in lysosomal Acridine Orange fluorescence. The two occurrences together permit a more unbiased detection of apoptosis than most assays. Only one filter set is required for evaluation and the resulting images can be easily evaluated visually or processed further by image analysis.     

The metabolism of benzyl isothiocyanate and its cysteine conjugate.

1. The corresponding cysteine conjugate was formed when the GSH (reduced glutathione) or cysteinylglycine conjugates of benzyl isothiocyanate were incubated with rat liver or kidney homogenates. When the cysteine conjugate of benzyl isothiocyanate was similarly incubated in the presence of acetyl-CoA, the corresponding N-acetylcysteine conjugate (mercapturic acid) was formed. 2. The non-enzymic reaction of GSH with benzyl isothiocyanate was rapid and was catalysed by rat liver cytosol. 3. The mercapturic acid was excreted in the urine of rats dosed with benzyl isothiocyanate or its GSH, cysteinyl-glycine or cysteine conjugate, and was isolated as the dicyclohexylamine salt. 4. An oral dose of the cysteine conjugate of [14C]benzyl isothiocyanate was rapidly absorbed and excreted by rats and dogs. After 3 days, rats had excreted a mean of 92.4 and 5.6% of the dose in the urine and faeces respectively, and dogs had excreted a mean of 86.3 and 13.2% respectively. 5. After an oral dose of the cystein conjugate of [C]benzyl isothiocyanate, the major 14C-labelled metabolite in rat urine was the corresponding mercapturic acid (62% of the dose), whereas in dog urine it was hippuric acid (40% of the dose). 5. Mercapturic acid biosynthesis may be an important route of metabolism of certain isothiocyanates in some mammalian species.     

Mechanism of oxidative DNA damage induced by carcinogenic allyl isothiocyanate

Several isothiocyanates have been proposed as promising chemopreventive agents for human cancers. However, it has been reported that allyl isothiocyanate exhibit carcinogenic potential, and benzyl isothiocyanate and phenethyl isothiocyanate have tumor-promoting activities. We investigated whether these isothiocyanates could cause DNA damage, using (32)P-labeled DNA fragments obtained from the human p53 tumor suppressor gene and the c-Ha-ras-1 protooncogene. Allyl isothiocyanate caused Cu(II)-mediated DNA damage and formation of 8-oxo-7, 8-dihydro-2'-deoxyguanosine (8-oxodG) more strongly than benzyl and phenethyl isothiocyanates. Catalase and bathocuproine, a Cu(I)-specific chelator, inhibited Cu(II)-mediated DNA damage by these isothiocyanates, suggesting involvement of H(2)O(2) and Cu(I). Isothiocyanates induced DNA damage frequently at thymine and cytosine residues in the presence of Cu(II). A UV-visible spectroscopic study revealed an association between the generation of superoxide and the yield of SH group from isothiocyanates. Furthermore, the yield of 8-oxodG formation was correlated with their superoxide-generating ability. Allyl isothiocyanate significantly induced 8-oxodG formation in HL-60 cells, but not in H(2)O(2)-resistant HP100 cells, suggesting the involvement of H(2)O(2) in cellular DNA damage. We conclude that oxidative DNA damage may play important roles in carcinogenic processes induced by allyl isothiocyanate.     

Analgesic activity and selectivity of isothiocyanate derivatives of fentanyl analogs for opioid receptors

The analgesic activity and opioid receptor binding characteristics were studied for the isothiocyanate ohmefentanyl (OMFIT), and isothiocyanate carfentanil (CarFIT), isothiocyanate 4-methoxymethylfentanyl (MethoFIT), isothiocyanate 3-methylfentanyl (superFIT) and their amide analogs. Antinociceptive activity was evaluated using the mouse hot plate test; selectivity for opioid receptor was determined in bioassay and binding assay. SuperFIT, CarFIT, OMFIT and MethoFIT exhibited an analgesic ED50 lower than those of their parent compounds without isothiocyanate (SCN) group. Furthermore these compounds exhibited potent inhibitory actions on the electrically evoked contractions of mouse vas deferens, which could be antagonized by naloxone, but their actions were weaker than those of their parent compounds without SC N-group. The inhibitory actions of these compounds on binding of [3H]OMF to mouse brain membrane was weaker than those of their parent compounds without SCN-group. CarFIT and MethoFIT showed weaker inhibitory actions on the binding of [3H] DADLE than their parent compounds without SCN-group, but SuperFIT and OMFIT stronger than their parent compounds, 3-methylfentanyl and ohmefentanyl. The selectivity of these isothiocyanate derivatives for delta opioid receptors increased. In conclusion, introducing isothiocyanato-group into 1-position of phenyl ring of ohmefentanyl and other fentanyl analogs would enhance the selectivity of these compounds for delta-opioid receptors, but decrease their analgesic activity.     

Allyl isothiocyanate is mutagenic in Salmonella typhimurium

Allyl isothiocyanate, a naturally occurring compound, component of oil of mustard and human food plants such as cabbage, cauliflower and horseradish, has up to now been regarded as nonmutagenic in bacterial mutagenicity testing systems. Recently, however, it was found to cause transitional-cell papillomas in the urinary bladder of male F344 rats. Contrary to earlier reports, in this study allyl isothiocyanate showed clear mutagenicity for Salmonella typhimurium TA100 in the preincubation assay after longer, non-standard preincubation times (greater than 20 min). The mutagenicity is expressed only in the presence of a rat-liver homogenate metabolising system, i.e. it is indirect. However, high concentrations of rat-liver homogenate suppress the mutagenicity of allyl isothiocyanate. SKF525, inhibitor of microsomal oxygenase, reduces the mutagenic potential which on the other hand is increased in the presence of 1,1,1-trichloropropene-2-oxide, inhibitor of epoxide hydrolase. This indicates the occurrence of an epoxide intermediate in allyl isothiocyanate metabolism. Another metabolic pathway, namely hydrolysis to allyl alcohol and oxidation to acrolein, a known mutagen, also seems possible as cyanamide, inhibitor of aldehyde dehydrogenase, can slightly increase the mutagenic potential. The reason(s) for allyl isothiocyanate's requirement for long preincubation times to express mutagenicity still requires elucidation, and the question arises: is allyl isothiocyanate a single, exceptional case or not?     

Interaction of oxidized glutathione with allyl isothiocyanate

Isothiocyanates formed from glucosinolates in Brassica species have a strong affinity for amino acids and proteins, especially for their thiol, sulphide and terminal amino groups. To investigate the action of isothiocyanate on cystine residues in proteins and peptides, the present study on the interaction between allyl isothiocyanate and oxidized glutathione under physiological conditions was undertaken. Oxidized glutathione was oxidatively cleaved to some modified glutathiones by the attack of allyl isothiocyanate on its disulphide bond. Two new modified products were isolated from the reaction mixture by gel chromatography and HPLC, and their structures were determined by NMR and mass spectral analyses as glutathionyl N-allyldithiocarbaramate and its allyl thiohydantoin derivative. The formation of these products indicated oxidative cleavage of the disulphide bond in the cystine residue; the electrophilic attack of the isothiocyanate on the sulphur atom must cleave the disulphide bond oxidatively to dithiocarbamate and sulphenate, as in the case of cystine.     

C-terminal sequence analysis of peptides using triphenylgermanyl isothiocyanate

The Schlack-Kumpf degradation, also called the isothiocyanate method, is thought to be a promising approach to chemical C-terminal sequencing of peptides and proteins. The derivatizing reagent is most crucial to this method. A new derivatizing reagent, triphenylgermanyl isothiocyanate (TPG-ITC), has been synthesized and applied to C-terminal peptide sequencing. The chemistry involves activation with acetic anhydride, derivatization with TPG-ITC, and cleavage of the derivatized C-terminal amino acid thiohydantoin with sodium hydroxide. A series of reaction conditions, including activation reagent volume, activation time, and derivatization temperature and time, have been investigated using a model peptide covalently attached to 1,4-phenylene diisothiocyanate (DITC)-glass beads. This procedure has been successfully used to sequence eight C-terminal residues of a model peptide at low nanomole levels. TPG-ITC is a white solid with relatively long shelf-life. According to our previous article (B. Mo, J. Li, and S. P. Liang, 1997, Anal. Biochem. 252, 169-176), TPG-ITC is a type II derivatizing reagent. Compared with acetyl isothiocyanate and trimethylsilyl isothiocyanate, TPG-ITC is much more stable and efficient for use in peptide C-terminal sequencing.     

Benzyl isothiocyanate is the chief or sole anthelmintic in papaya seed extracts

Papaya (Carica papaya) seeds were extracted in an aqueous buffer or in organic solvents, fractionated by chromatography on silica and aliquots tested for anthelmintic activity by viability assays using Caenorhabditis elegans. For all preparations and fractions tested, anthelmintic activity and benzyl isothiocyanate content correlated positively. Aqueous extracts prepared from heat-treated seeds had no anthelmintic activity or benzyl isothiocyanate content although both appeared when these extracts were incubated with a myrosinase-containing fraction prepared from papaya seeds. A 10 h incubation of crude seed extracts at room temperature led to a decrease in anthelmintic activity and fractionated samples showed a lower benzyl isothiocyanate content relative to non-incubated controls. Benzyl thiocyanate, benzyl cyanide, and benzonitrile were not detected in any preparations and cyanogenic glucosides. which were present, could not account for the anthelmintic activity detected. Thus, our results are best explained if benzyl isothiocyanate is the predominant or sole anthelmintic agent in papaya seed extracts regardless of how seeds are extracted.     

Binding of fluorescein isothiocyanate conjugated lectins to rat spermatogenic cells in tissue sections

Summary The testes from three months old Spague-Dawley rats were fixed in Bouin's fluid or neutral buffered 10% formalin, embedded in paraffin, sectioned and after deparaffination stained with the following fluorescein isothiocyanate coupled lectins: PNA, WGA, Con A, RCA, SBA, DBA and UEA. The results show that there are considerable differences in the staining pattern of various spermatogenic cells between different lectins. The fixation in Bouin's fluid enhanced the staining of all the lectins compared to formalin fixation in which only a weak staining could be seen in the acrosomes of spermatids after WGA or PNA staining. PNA and WGA stained specifically the acrosome of the developing spermatids, which was seen from the beginning of the acrosome formation and lasted up to late spermiogenesis. However, the staining with PNA decreased in the late spermatids whereas the intensity of the staining remained unchanged with WGA. Con A did not stain the acrosome but stained unspecifically the cytoplasm of all spermatogenic cells. RCA stained faintly the acrosome throughout the spermatid differentiation. DBA and UEA stained specifically the chromosomes of B spermatogonia. DBA also faintly stained the cell membranes of early spermatids. SBA did not show any specific staining of the spermatogenic cells. Based on this it is suggested that the carbohydrates and glycoproteins which are known to be present in the acrosome are formed already in the beginning of the acrosome formation. The decrease in the PNA staining in late spermatids possibly reflects the fact that the receptor molecules are not synthesized in late spermatids but are formed in earlier developmental stages and are thereafter preserved in the acrosome. The enhancement of lectin binding caused by Bouin's fixative might also be applied to other tissues where previous experiments with formalin fixed tissue have failed to show any staining.     

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.     

Antibacterial and mutagenic activities of new isothiocyanate derivatives

Nine newly synthetized isothiocyanate derivatives were demonstrated to posses antibacterial and genotoxic activitiesin vitro. 4-Hydroxybutyl isothiocyanate exhibited a broad antibacterial effect, with MIC values of 762 μmol/L forStaphylococcus aureus andEscherichia coli. Ethyl 4-methylsulfoxidobutanoate had the highest antibacterial activity in Gram-positive bacteria, the MIC value being 425 μmol/L forS. aureus. The highest tested concentrations of ethyl 4-isothiocyanatobutanoate and 4-hydroxybutyl isothiocyanate produced a bacteriocidal effect in Gram-positive bacteria. The compounds showed no mutagenic effects onSalmonella typhimurium tester strains TA 98 and TA 100, either in the absence or in the presence of a metabolically active microsomal S9 fraction from rat liver using standard Ames test.     

Mortality and behavior in Heterodera glycines juveniles following exposure to isothiocyanate compounds

For this report, we examined the toxic effects of three plant-derived isothiocyanate compounds on second-stage juveniles (J2) of Heterodera glycines. We found significant differences among compounds in the concentration required to affect nematodes, according to mortality and behavioral measurements. The concentrations required to affect behavior were significantly lower than those required for mortality. Both mortality and behavioral measurements were used to investigate whether nematodes in a quiescent state display decreased sensitivity to isothiocyanates compared with actively moving nematodes. Mortality measurements revealed that quiescent nematodes were significantly less sensitive to isothiocyanates than active nematodes. All behavioral measurements following exposure to benzyl- and phenyl isothiocyanate showed significant differences in sensitivity between quiescent and active nematodes. However, significant differences between quiescent and active nematodes were observed in only one of the five behavioral measurements following exposure to allyl isothiocyanate. These results expand the list of plant-derived compounds toxic to H. glycines and illustrate the impact of behavioral quiescence on nematode sensitivity to exogenous toxins.     

Single-nucleotide polymorphism analysis using fluorescence resonance energy transfer between DNA-labeling fluorophore,fluorescein isothiocyanate,and DNA intercalator,POPO-3, on bacterial magnetic particles

To develop an analytical system for single-nucleotide polymorphisms (SNPs), the fluorescence resonance energy transfer (FRET) technique was employed on a bacterial magnetic particle (BMP) surface. A combination of fluorescein isothiocyanate (FITC; excitation 490 nm/emission 520 nm) labeled at the 5' end of DNA and an intercalating compound (POPO-3, excitation 534 nm/emission 570 nm) was used to avoid the interference from light scattering caused by nanoparticles. After hybridization between target DNA immobilized onto BMPs and FITC-labeled probes, fluorescence from POPO-3, which was excited by the energy from the FITC, was detected. The major homozygous (ALDH2*1), heterozygous (ALDH2*1/*2), and minor homozygous (ALDH2*2) genotypes in the blood samples were discriminated by this method. The assay described herein allows for a simple and rapid SNP analysis using a fully automated system.     

Determination of fungal glucosamine using HPLC with 1-napthyl isothiocyanate derivatization and microwave heating

A rapid method for the determination of fungal glucosamine (GlcN) from Aspergillus sp BCRC 31742 was developed. The hydrochlorination process using microwave effectively reduced reaction time needed for GlcN analysis. The analytical method consisted of two steps: (1) hydrochlorination of fungal cells and (2) derivatization process. Fungal GlcN hydrochloride was reacted with 1-napthyl isothiocyanate (1-NITC) as the derivatizing agent to enhance the sensitivity of GlcN and so to achieve high resolution. This method was specific for quantification of GlcN hydrochloride at the wavelength of 230 nm. The standard deviation and relative error of the analytical results were less than 5%. By using microwave heating, the reaction time of hydrochlorination process was shortened from 24 h to 3 min. Thus, the overall time needed for analyzing GlcN from fungal sources was reduced from 5 h (thermal method) to 2 h (microwave method).     

Genotoxic effects of allyl isothiocyanate (AITC) and phenethyl isothiocyanate (PEITC)

Two isothiocyanates (ITCs) commonly found in human diet, allyl isothiocyanate (AITC) and phenethyl isothiocyanate (PEITC), were tested for genotoxic effects in a battery of assays: Salmonella/microsome assay with TA 98 and TA 100, differential DNA repair assay with E. coli and micronucleus (MN) induction assay with human derived Hep G2 cells. Albeit to a different degree, both ITCs induced genotoxic effects in all test systems. AITC was more genotoxic in bacterial test systems than in Hep G2 cells; in contrast, the effect of PEITC was stronger in Hep G2 cells. In in vivo assays with E. coli indicators in which mice were exposed to relatively high doses of the compounds (90 and 270 mg/kg), AITC induced moderate but significant effects; PEITC failed to induce significant effects in any of the organs. To find out the reason for the weak genotoxicity of AITC and PEITC under in vivo test conditions, we exposed E. coli indicator cells to the test substances in the absence or presence of rat liver homogenate (with and without cofactors), bovine serum albumin (BSA) and human saliva. All of them markedly attenuated the genotoxicity of AITC and PEITC, implying that the test substances are detoxified by direct non-enzymatic binding to proteins. Additional experiments carried out on the mechanistic aspects of AITC and PEITC-induced genotoxicity showed that the compounds induce the formation of thiobarbituric acid reactive substances (TBARS) in Hep G2 cells. Furthermore, in in vitro assays with E. coli, radical scavengers reduced the differential DNA damage induced by AITC and PEITC. The latter two findings give a clue that reactive oxygen species might be involved in the genotoxic effect of the ITCs. Although ITCs have been repeatedly advocated as very promising anticancer agents, the data presented here indicate that the compounds are genotoxic, and probably carcinogenic, in their own right.     

Antitumour activity of sphingoid base adducts of phenethyl isothiocyanate

N-(N'-Phenethylthiocarbamoyl) derivatives of sphingosine and sphinganine were prepared. They had antitumour activity: GC50 values of 0.64+/-0.02 microM (N = 18) and 1.6+/-0.01 microM (N = 18). respectively, with human leukaemia 60 cells in vitro. This antitumour effect may contribute to the suppression of carcinogenesis associated with dietary phenethyl isothiocyanate and sphingolipid bases.