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PubMed2003

Use of a fluorescence labelled, carbohydrate-binding module from Phanerochaete chrysosporium Cel7D for studying wood cell wall ultrastructure.

UNLABELLED: The alpha-amino group of the carbohydrate-binding module (CBM) from Phanerochaete chrysosporium cellulase Cel7D was covalently labelled with fluorescein isothiocyanate. The fluorescein-labelled CBM was characterised regarding substrate binding, showing specificity only to cellulose and not to mannan and xylan. Conjugation of fluorescein isothiocyanate to CBM did not affect its binding to cellulose. The labelled CBM was successfully used as a probe for detecting cellulose in lignocellulose material such as never dried spruce and birch wood as well as pulp fibres. ABBREVIATIONS: Bmax, maximum binding capacity; BMCC, bacterial microcrystalline cellulose; CBH, cellobiohydrolase; CBM, carbohydrate-binding module; CBM-FITC, carbohydrate-binding module labelled with fluorescein isothiocyanate; CBM1, carbohydrate-binding module of family 1; FITC, fluorescein isothiocyanate; KD, dissociation constant; RP-HPLC, reversed phase HPLC; S1-S3, Secondary cell wall layers 1-3.

Betula↗
PubMed2005

Synthesis and spectroscopic characterisation of new ESIPT fluorescent protein probes.

Three new benzazole isothiocyanate fluorescent dyes, 2-(4'-isothiocyanate-2'-hydroxyphenyl)benzoxazole, 2-(4'-isothiocyanate-2'-hydroxyphenyl)benzothiazole and 2-(4'-isothiocyanate-2'-hydroxyphenyl)benzimidazole were synthesised, purified until optical purity grade and characterised by spectroscopic techniques. UV/VIS and steady-state fluorescence were also applied to characterise the photophysical behaviour of the dyes. These dyes exhibit an intense fluorescence emission with a large Stokes shift, inherent to the class of benzazoles which relax by the excited state intramolecular proton transfer (ESIPT) mechanism. The dyes were studied for labeling bovine serum albumin (BSA), resulting conjugates BSA-dye with a remarkable photostability under UV/VIS radiation in relation to classical protein labels. The resulting conjugates presented fluorescence in the blue-green region. Direct fluorescence detection of protein-labeled with those dyes after polyacrylamide gel electrophoresis indicates their potential use as fluorescent probes for proteins.

Azoles↗
PubMed1998

Evaluation of the capacity of dendritic cells derived from cord blood CD34+ precursors to present haptens to unsensitized autologous T cells in vitro.

Langerhans cells play a key role in contact hypersensitivity reactions. The application of haptens on the skin leads to many modifications of these cells, including the increase of major histocompatibility complex II expression, allogeneic stimulation potency, and migration towards lymph nodes to activate T cells. Moreover, it has been shown that Langerhans cells cultured in vitro are able to prime naive T cells in response to hapten contact. From CD34+ progenitors present in cord blood, we generated dendritic cells of which some presented the phenotypic markers of Langerhans cells. We show that these cells are able to sensitize syngeneic naive (CD45RA+) T cells to haptens such as trinitrophenyl conjugate of trinitrobenzene sulfonic acid (TNP) and fluoroscein isothiocyanate. The response to TNP is higher than to fluoroscein isothiocyanate, whereas sodium dodecyl sulfate, an irritant molecule used as a control, never caused this effect. Phenotypic analysis of cellular suspensions and experiments of cell sorting lead to the conclusion that only CD1a+ cells are able to induce a primary response of syngeneic T cells to TNP or fluoroscein isothiocyanate. Furthermore, we have shown a close relationship between the differentiation state of dendritic cells and their ability to prime T lymphocytes. Dendritic cells are able to present haptens in an efficient manner between day 10 and 14 of culturing CD34+ progenitors, whereas they were efficient in presenting alloantigens from day 6 until after day 20. This dissociation suggests the need of an active metabolic process for hapten presentation in the direct treatment of dendritic cells with haptens. This model of hapten presentation was used for a panel of fragrance molecules and other molecules considered as weaker haptens than TNP and fluoroscein isothiocyanate.

Antigens, CD1↗
PubMed1961

Studies on fluorescent antibody staining. I. Non-specific fluorescence with fluorescein-coupled sheep anti-rabbit globulins.

1. A study has been made of the non-specific fluorescent staining of splenic imprints treated with fluorescent sheep antibody globulins. 2. In tissue imprints made with the spleens of antigen-stimulated animals, no morphological distinction was evident between areas showing non-specific fluorescence and specific fluorescence. 3. Elimination of non-specific fluorescence was not achieved by any one, or any combination of the following: (a) conjugating only gamma globulins with fluorescein isothiocyanate; (b) removal of dialyzable fluorescent products on sephadex, followed by concentration through the use of pressure dialysis; (c) use of crystalline preparations of fluorescein isothiocyanate. 4. Individual preparations of fluorescent antibodies were separated by gradient elution chromatography on diethylaminoethyl (DEAE) cellulose into fractions possessing different numbers of fluorescein radicals per molecule of globulin. 5. The coupling ratio of 50 mg fluorescein isothiocyanate (FITC) per gm of protein, as commonly advocated, can not be recommended for the precise localization of antibody globulin in tissues owing to the capacity of the coupled products to give non-specific fluorescent staining. When crystalline preparations of FITC are used instead of the amorphous product at 50 mg/gm protein, far too high non-specific fluorescence results. 6. A fraction with bright specific fluorescence and no or negligible nonspecific fluorescence was obtained from each fluorescent antibody that was prepared by using 6 to 8 mg of crystalline fluorescein isothiocyanate per gm of globulin and was then subjected to DEAE-cellulose chromatography and gradient elution to eliminate the most highly coupled molecules.

Animals↗
PubMed2002

Inhibition of lung tumorigenesis in A/J mice by N-acetyl-S-(N-2-phenethylthiocarbamoyl)-L-cysteine and myo-inositol, individually and in combination.

Isothiocyanates, their N-acetylcysteine conjugates, and myo-inositol (MI) are inhibitors of lung tumorigenesis in A/J mice. However, chemoprevention by combinations of these compounds in different temporal sequences has not been examined. This is important for developing practical approaches to lung cancer chemoprevention in smokers and ex-smokers. We used a tumor model in which A/J mice are treated with 8 weekly doses of benzo[a]pyrene (B[a]P) plus 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and killed 19 weeks after the final treatment. In Experiment 1, isothiocyanates or their N-acetylcysteine conjugates were added to the diet (1 or 3 micro mol/g) from 1 week before until 1 week after carcinogen treatment. The compounds were 2-phenethyl isothiocyanate (PEITC), 3-phenylpropyl isothiocyanate (PPITC), N-acetyl-S-(N-benzyl-thiocarbamoyl)-L-cysteine (BITC-NAC), N-acetyl-S-(N-2-phenethylthiocarbamoyl)-L-cysteine (PEITC-NAC), and N-acetyl-S-(N-3-phenylpropylthiocarbamoyl)-L-cysteine (PPITC-NAC). Significant reductions in lung tumor multiplicity were observed in mice treated with PEITC, PEITC-NAC, PPITC and PPITC-NAC. PEITC-NAC was chosen for combination studies with MI (Experiment 2). Mice were treated with B[a]P plus NNK without or with PEITC-NAC (3 micro mol/g diet), MI (55.5 micro mol/g diet), or PEITC-NAC plus MI (3 micro mol plus 55.5 micro mol/g diet). Different temporal sequences of dietary additions were investigated: carcinogen treatment phase; post-carcinogen treatment phase; entire experiment; 50% of carcinogen treatment phase until termination; and 75% of carcinogen treatment phase until termination. All treatments reduced lung tumor multiplicity except PEITC-NAC post-carcinogen or from 75% of the carcinogen treatment phase. Reduction of lung tumor multiplicity by PEITC-NAC plus MI was greater than that in the mice treated with the agents alone in all temporal sequences. When all results were combined, PEITC-NAC plus MI was significantly more effective than the agents alone. There was a significant trend for reduction in lung tumor multiplicity with increased duration of treatment by the chemopreventive agents. These results provide a basis for further development of mixtures of PEITC-NAC and MI for chemoprevention of lung cancer.

Animals↗
PubMed1994

ADP-induced changes in ordering of spin-labelled myosin heads in muscle fibres.

Rotational dynamics and ordering of myosin heads in glycerinated skeletal muscle fibres were studied using an isothiocyanate-based spin label attached to the fast-reacting thiol sites of myosin and were compared with data obtained for maleimide and iodoacetamide spin labels attached to the same sites. The ordering of probe molecules on the millisecond time scale in the rigor state, at sarcomere length 2.2-2.3 +/- 0.1 microns, was static. Isothiocyanate probe molecules showed greater mobility; the segment holding the label rotated in the microsecond time range. In the saturation transfer EPR time domain, MgADP did not produce a significant change in the mobility of spin labels. The spectra of isothiocyanate spin-labelled fibres were analyzed in terms of two narrow distributions with mean angles of 75 degrees and 56 degrees. In the rigor state, the fractions represented approximately 76% and 24% of the total EPR absorbance. In the presence of MgADP, the conventional EPR spectra showed large changes in the ordering of isothiocyanate probe molecules towards a new distribution, the population with a theta value of 56% increased from 24% to 71% at the expense of the 75% population with no change in the mean angles of the distributions. In the case of maleimide and iodoacetamide spin-labelled fibres, however, the effect of MgADP on the probe angular distribution was small.

Adenosine Diphosphate↗
PubMed1995

Na+/K(+)-ATPase with a blocked E1ATP site still allows backdoor phosphorylation of the E2ATP site.

The role of simultaneously existing ATP-binding sites in the catalytic process of Na+/K(+)-ATPase is unclear. In order to learn whether blocking the E1ATP site affects the properties of the E2ATP site, the E1ATP site was inactivated by either fluorescein 5'-isothiocyanate, the non-phosphorylating Cr(H2O)4AdoPP[CH2]P or the phosphorylating Cr(H2O)4ATP. The properties of the remaining E2ATP site were studied by measuring 'backdoor phosphorylation' in the presence of ouabain, or K(+)-activated hydrolysis of p-nitrophenyl phosphate. The involvement of the E2ATP site was further tested by the effects of Co(NH3)4ATP, a specific inactivator of this site. When the E1ATP site was inactivated by fluorescein 5'-isothiocyanate or the non-phosphorylating Cr(H2O)4AdoPP[CH2]P, backdoor phosphorylation and the activity of K(+)-activated p-nitrophenylphosphatase remained unchanged. Both processes were lost, however, when the E2ATP site was additionally inactivated by Co(NH3)4ATP. Inactivation of the E1ATP site by fluorescein 5'-isothiocyanate or Cr(H2O)4AdoPP[CH2]P decreased the affinity of the p-nitrophenylphosphatase activity of the E2ATP site for the substrate p-nitrophenyl phosphate by four times. This is consistent with a former report showing that dephosphorylation in a fluorescein 5'-isothiocyanate-inactivated Na+/K(+)-ATPase has a lowered sensitivity for ATP [Scheiner-Bobis, G., Antonipillai, J. & Farley, R. A. (1993) Biochemistry 32, 9592-9599]. Inactivation of the E1ATP site by the phosphorylating Cr(H2O)4ATP, however, led to a loss of the property of the E2ATP site to hydrolyse K(+)-dependent p-nitrophenyl phosphate and to achieve backdoor phosphorylation. Evidently, ATP sites coexist in Na+/K(+)-ATPase, and binding of ATP to one site affects the property of the other site [Scheiner-Bobis, G., Esmann, M. & Schoner, W. (1989) Eur. J. Biochem. 183, 173-178]. Although the enzyme can be phosphorylated from both ATP sites, phosphorylation of the E1ATP site excludes the phosphorylation of the E2ATP site.

Adenosine Triphosphate↗
PubMed1999

Changes in intestinal absorption of 5-fluorouracil-treated rats.

5-Fluorouracil chemotherapy is often accompanied by gastrointestinal toxicity. In this study, we investigated the effect of 5-fluorouracil on the epithelial barrier function of rat small intestine by examining the absorption of a poorly absorbable marker, fluorescein isothiocyanate-labelled dextran (molecular weight 4,400). We further evaluated the intestinal absorption of 5-fluorouracil in rats treated orally with 5-fluorouracil once daily for 4 days. The small intestinal absorption of fluorescein isothiocyanate-labelled dextran and 5-fluorouracil was tested using in situ closed loop intestine technique and in vitro everted intestine technique, respectively. After administration of 5-fluorouracil to rats for 4 days, the body weight of rats decreased significantly and the fluorescein isothiocyanate-labelled dextran concentration in plasma increased significantly, compared with that of control rats to which the saline solution alone was administered. Moreover, the intestinal absorption of 5-fluorouracil in the 5-fluorouracil-treated rats was enhanced significantly, compared with that of control rats. The administration of 5-fluorouracil to rats caused body weight loss and epithelial barrier dysfunction of the small intestine in rats as shown by the increased permeation of the high molecular weight compound, fluorescein isothiocyanate-labelled dextran. The increased absorption of 5-fluorouracil after this treatment suggest that the 5-fluorouracil toxicity might be amplified by its treatment.

Animals↗
PubMedDate not supplied

Radioprotective activity of naturally occurring organosulfur compounds.

The radioprotective effects of naturally occurring sulfur compounds and isothiocyanates such as diallyl sulfide (DAS), diallyl disulfide (DADS), allyl methyl sulfide (AMS), allyl isothiocyanate (AITC) and phenyl isothiocyanate (PITC) have been investigated in whole body irradiated Swiss albino mice. Administration of these sulfur compounds could reduce the serum content of alkaline phosphatase (ALP), which was elevated after irradiation (23.9 +/- 1.82 KA units). The elevated liver content of glutamate pyruvate transaminase (GPT) in control animals (76.2 +/- 2.2 U/mL) after irradiation was significantly reduced in DAS (58.93 +/- 4 U/mL) and AMS (55.7 +/- 2.2 U/mL) treated animals. Elevated levels of lipid peroxides in serum and liver of irradiated control animals were also significantly reduced by treatment with these sulfur compounds. The glutathione (GSH) content in liver and intestinal mucosa was drastically reduced after irradiation. All the sulfur compounds and isothiocyanates could effectively enhance the GSH content of intestinal mucosa and liver. Findings at histopathological analysis of the intestine proved to be correlated with the above results.

Allyl Compounds↗
PubMed2005

Trigeminal sensitization and desensitization in the nasal cavity: a study of cross interactions.

Chemical irritation in the human nasal cavity is poorly documented. In this field, an important issue concerns the differential responses produced by successive stimulation. Repeated identical chemical irritant stimuli can produce increases or decreases in responses (two phenomena known as self-sensitization or self-desensitization). In the same way, different molecules can interact and produce cross-sensitization or cross-desensitization. The aim of this study was to contribute to this question using two specific molecules, acetic acid (AA) and allyl isothiocyanate (AIC). As the self-sensitization and -desensitization for AIC is known, a first experiment in the present work investigated the response, acute effects and time course of sensitization or desensitization to acetic acid. A second experiment tested the responses of acetic acid after a previous stimulation with allyl isothiocyanate (mustard oil) and inversely with a short inter-stimulus interval (ISI of 45 s). A third experiment similar to the second used a long inter-stimulus interval (ISI of 3 min 30). Twelve healthy subjects participated in the study using psychophysical (intensity ratings) and psychophysiological (skin conductance response) measurements. Firstly, the results showed that repeated nasal stimulation with acetic acid produced a self-desensitization whatever the ISI. Secondly, the results showed a cross-desensitization of allyl isothiocyanate by previous acetic acid stimulation. In contrast, previous stimulation with allyl isothiocyanate had no effect on the following acetic acid response. These findings confirm that trigeminal sensitization and desensitization in the nasal cavity do not follow the same processes in relation to molecules used.

Acetic Acid↗
PubMed1998

Glutathione transferase null genotype, broccoli, and lower prevalence of colorectal adenomas.

Cruciferous vegetables, especially broccoli, may prevent cancer through anticarcinogenic compounds. For example, broccoli contains isothiocyanates that induce carcinogen-detoxifying enzymes. Glutathione transferase enzymes conjugate isothiocyanates, leading to excretion. We hypothesized that broccoli consumption in combination with the glutathione transferase M1 (GSTM1) null genotype would be associated with a lower prevalence of colorectal adenomas because of higher isothiocyanate levels. We used a case-control study of mainly asymptomatic subjects aged 50-74 years who underwent a screening sigmoidoscopy at either of two Southern California Kaiser Permanente Medical Centers during 1991-1993. Cases (n = 459) had a first-time diagnosis of histologically confirmed adenomas detected by flexible sigmoidoscopy. Controls (n = 507) had no polyp detected. Subjects had a 45-min in-person interview for information on various risk factors and basic demographic data and completed a 126-item, semiquantitative food frequency questionnaire. Blood samples were used for GSTM1 genotyping. Subjects with the highest quartile of broccoli intake (an average of 3.7 servings per week) had an odds ratio of 0.47 (95% confidence interval, 0.30-0.73) for colorectal adenomas, compared with subjects who reportedly never ate broccoli. When stratified by GSTM1 genotype, a protective effect of broccoli was observed only among subjects with the GSTM1 null genotype (P for trend, 0.001; P for interaction, 0.01). The observed broccoli-GSTM1 interaction is compatible with an isothiocyanate mechanism.

Adenoma↗
PubMed1988

Reversed-phase liquid chromatographic separation of enantiomeric and diastereomeric bases related to chloramphenicol and thiamphenicol.

The important antimicrobial agents chloramphenicol and thiamphenicol are N-acylated amines whose chemical structures include two chiral centers. Each drug is the single enantiomer of R,R configuration. The N-deacylated bases of the drugs are important intermediates in their synthesis and optical resolution. In this report, reversed-phase HPLC methods are described for the separation of enantiomeric and diastereomeric bases of the two drugs and of two closely related bases used in some syntheses of the drugs. The stereoisomeric bases were derivatized with a homochiral isothiocyanate and the resulting diastereomeric thioureas were separated on C18 columns with methanol:water mixtures as mobile phases and detection at 254 nm. The four stereoisomeric bases of chloramphenicol and those of its unnitrated analogue were thus separable after derivatization with 2,3,4,6-tetra-O-acetyl-beta-D-glucopyranosyl isothiocyanate. This reagent also allowed the separation of the D-threo isomer of the p-mercaptomethyl analogue of thiamphenicol base from its stereoisomers. The stereoisomers of thiamphenicol base were similarly separated with (R)-alpha-methylbenzyl isothiocyanate as the derivatizing agent. The diastereomers of chloramphenicol base and of thiamphenicol base were chromatographically separable after derivatization with the nonchiral reagent benzyl isothiocyanate. The procedures developed may be useful in the determination of the stereoisomeric composition of the drugs in research and in quality control, and may be applicable to other similar agents whose chemistry and pharmacology are receiving considerable attention.

Capsules↗
PubMed1978

The effect of a disulfonic acid stilbene on proximal cell membrane potential in Necturus kidney.

The effects of 0.5 mM 4-acetamido-4'-isothiocyano-stilbene-2,2' disulfonic acid on the electrical properties of the peritubular membrane were studied in the proximal tubule of the perfused Necturus kidney. The addition of stilbene isothiocyanate disulfonic acid in peritubular perfusate resulted in an average 4.5 mV hyperpolarization with no detectable changes of peritubular membrane input conductance. The depolarization elicited by high-K media was enhanced by 18% in the presence of stilbene isothiocyanate disulfonic acid, an observation indicating that the inhibitor increased the contribution of potassium to membrane potential, presumably by decreasing anionic permeabilities. The hyperpolarizing effect of stilbene isothiocyanate disulfonic acid was abolished when peritubular bicarbonate was removed from the medium and isoosmotically replaced by chloride. These data suggest that (a) intracellular bicarbonate activity is higher than that predicted from passive distribution, (b) stilbene isothiocyanate disulfonic acid decreases P HCO3, thus hyperpolarizing the membrane, (c) chloride distribution appears to be passive when bicarbonate is removed from the peritubular perfusate. The state of Cl distribution when extracellular bicarbonate is at physiologic concentration cannot be assessed from the present data.

Animals↗
PubMed1995

Effects of sperm preparation and bull fertility on in vitro penetration of zona-free bovine oocytes.

The objectives of this study were to develop and validate a zona-free bovine oocyte penetration assay for detecting relative differences in bovine sperm fertility and to determine the effect of different sperm preparation methods on oocyte penetration. Oocytes were incubated with heparin-capacitated spermatozoa which either were or were not induced to acrosome-react with lysophosphatidylcholine. Heparin-capacitated spermatozoa treated with lysophosphatidyl-choline penetrated more oocytes and had more penetrations per oocyte than spermatozoa capacitated in heparin but not induced to acrosome-react with lysophosphatidylcholine. Spermatozoa stained with Hoechst 33342, fluorescein isothiocyanate or tetramethyl rhodamine isothiocyanate, alone or in combination, penetrated similar numbers and percentages of zona-free bovine oocytes as the similar to non-stained spermatozoa. When spermatozoa from the same ejaculate were stained with either fluorescein isothiocyanate or tetramethyl rhodamine isothiocyanate and competed in penetrating the same oocytes, the number of penetrations generated by the 2 differently stained spermatozoa was similar. Spermatozoa from bulls of differing in vivo fertilities were labeled with different fluorescent dyes, and their relative abilities to penetrate the same oocytes were assessed. Comparisons between spermatozoa from high and low fertility bulls demonstrated that high fertility spermatozoa had a significant oocyte penetrating advantage over low fertility spermatozoa in 13 of 16 paired competitions. We concluded that the results of the competitive penetration of zona-free bovine oocytes by fluorochrome-labeled spermatozoa from bulls of different fertilities were indicative of their relative in vivo fertility.

Journal Article↗
PubMed2001

Inhibition of cytochrome P450 isozymes by curcumins in vitro and in vivo.

To study the mechanism(s) of turmeric-mediated chemoprevention and to compare the chemopreventive efficacy of turmeric/curcumin(s) against benzo[a]pyrene (B(a)P) and 4-methylnitrosamino-1-(3-pyridyl)-1-butanone (NNK, a tobacco-specific carcinogen), the effects of turmeric/curcumin (C), demethoxycurcumin (dmC), bis-demethoxycurcumin (bdmC) and phenyl and phenethyl-isothiocyanates (PITC and PEITC) on the dealkylation of ethoxyresorufin (ER), methoxyresorufin (MR) and pentoxyresorufin (PR) by rat liver microsomes (in vitro) were studied. These reactions are predominantly mediated by cytochrome P450 (CYP450) isozymes 1A1, 1A2 and 2B1, respectively. In vitro incubation of rat liver microsomes with each of the compounds--C, dmC, bdmC, PITC and PEITC--showed a dose-dependent decrease in carbon monoxide binding to microsomes and also showed a dose-dependent inhibition of CYP 1A1, 1A2 and 2B1 activity, as judged by a decrease in formation of resorufin from respective biochemical probes used. Both the isothiocyanates inhibited activity of CYP 2B1 more readily than that of CYP 1A1/1A2. Significantly lower concentrations of curcumin(s) than isothiocyanates achieved 50% inhibition of activity of CYP 1A1 and 1A2, while concentrations of C (4 microM), bdmC (2.5 microM) required to inhibit CYP 2B1 were slightly higher than that of PEITC (1.3 microM), suggesting curcumin(s) to be effective inhibitors of CYP 2B1 as well. Pretreatment of rats with 1% turmeric through the diet resulted in a significant decrease in induction of B(a)P-induced CYP 1A1 and 1A2 and phenobarbitone (PB)-induced CYP 2B1 in liver, lung and stomach, although the extent of the decrease was different. These results suggest that turmeric/curcumin(s) as in the case of isothiocyanate, PEITC, are likely to inhibit activation of carcinogens metabolized by CYP450 isozymes, namely, CYP 1A1, 1A2 and 2B1.

Animals↗
PubMed2004

Mustard oils and cannabinoids excite sensory nerve fibres through the TRP channel ANKTM1.

Wasabi, horseradish and mustard owe their pungency to isothiocyanate compounds. Topical application of mustard oil (allyl isothiocyanate) to the skin activates underlying sensory nerve endings, thereby producing pain, inflammation and robust hypersensitivity to thermal and mechanical stimuli. Despite their widespread use in both the kitchen and the laboratory, the molecular mechanism through which isothiocyanates mediate their effects remains unknown. Here we show that mustard oil depolarizes a subpopulation of primary sensory neurons that are also activated by capsaicin, the pungent ingredient in chilli peppers, and by Delta(9)-tetrahydrocannabinol (THC), the psychoactive component of marijuana. Both allyl isothiocyanate and THC mediate their excitatory effects by activating ANKTM1, a member of the TRP ion channel family recently implicated in the detection of noxious cold. These findings identify a cellular and molecular target for the pungent action of mustard oils and support an emerging role for TRP channels as ionotropic cannabinoid receptors.

Animals↗
PubMed2004

Differential carbohydrate epitope recognition of globotriaosyl ceramide by verotoxins and a monoclonal antibody.

The role of renal expression of the glycosphingolipid verotoxin receptor, globotriaosylceramide, in susceptibility to verotoxin-induced hemolytic uremic syndrome is unclear. We show that a single glycosphingolipid can discriminate multiple specific ligands. Antibody detection of globotriaosylceramide in renal sections does not necessarily predict verotoxin binding. The deoxyglobotriaosylceramide binding profile for verotoxin 1, verotoxin 2 and monoclonal anti-globotriaosylceramide are distinct. Anti-globotriaosylceramide had greater dependency on the intact alpha-galactose and reducing glucose of globotriaosylceramide than verotoxin 1, while verotoxin 2 was intermediate. These ligands differentially stained human kidney sections. Glomerulopathy is the primary verotoxin-associated pathology in hemolytic uremic syndrome. For most samples, verotoxin 1 immunostaining within adult glomeruli was observed (type A). Some samples, however, lacked glomerular binding (type B). Anti-globotriaosylceramide (and less effectively, verotoxin 2) stained all glomeruli. Verotoxin 1/anti-globotriaosylceramide tubular staining was comparable. Type B glomerular/tubular globotriaosylceramide showed minor, but significant, fatty acid compositional differences. Verotoxin 1 type B glomerular binding became evident following pretreatment with cold acetone, or methyl-beta-cyclodextrin, used to deplete cholesterol. Direct visualization, using fluorescein isothiocyanate-verotoxin 1B, showed paediatric, but no adult glomerular staining; this was confirmed by anti-fluorescein isothiocyanate immunostaining. Acetone induced fluorescein isothiocyanate-verotoxin 1B glomerular staining in type A, but poorly in type B samples. Comparison of fluorescein isothiocyanate-verotoxin 1B and native verotoxin 1B deoxyglobotriaosylceramide analogue binding showed an alteration in subspecificity. These studies indicate a marked heterogeneity of globotriaosylceramide expression within renal glomeruli and differential binding of verotoxin 1/verotoxin 2/anti-globotriaosylceramide to the same glycosphingolipid. Verotoxin 1 derivatization can induce subtle changes in globotriaosylceramide binding to significantly affect tissue binding. Heterogeneity in glomerular globotriaosylceramide expression may play a significant (cholesterol-dependent?) role in determining renal pathology following verotoxemia.

Adult↗
PubMed1979

Segregation of pre-stalk and pre-spore cells of Dictyostelium discoideum: observations consistent with selective cell cohesion.

Aggregating Dictyostelium discoideum cells were dissociated and separated into two populations, pre-stalk and pre-spore cells, by centrifugation in a Percoll gradient. After harvesting, one population of cells was labeled with fluorescein isothiocyanate and the other with tetramethylrhodamine isothiocyanate. Equal numbers of the two cell populations were then mixed and observed while they aggregated and differentiated to form fruiting bodies. Within minutes after mixing, the two populations showed evidence of segregation and tended to form separate streams of cells as they migrated toward aggregation centers. Many separate clusters of tetramethylrhodamine isothiocyanate-labeled or fluorescein isothiocyanate-labeled cells were found within an aggregation center. Thereafter there was further segregation. The denser cells from the gradient tended to assume the position of stalk cells whereas the lighter cells migrated to the position characteristic of spore cells. The patterns of segregation suggest that selective cohesiveness of one or both cell types plays a role in morphogenesis.

Cell Aggregation↗