Repository hosted by TU Delft Library

In view of controversies about assessment of the β-carotene cleavage activity, methodological aspects and problems of the dioxygenase assay are described. Using rat and hamster intestinal preparations the method was optimized on retinal formation, the only cleavage product we could demonstrate. It appeared that the cell fraction with the highest cleavage activity was the 9,000g supernatant (S-9). Maximal retinal formation was obtained with SDS, taurocholate and egg lecithin in the buffer and 3 μg β-carotene dissolved in acetone. Ethanol, THF/DMSO (1:1) or propylene glycol as solvent for β-carotene reduced retinal formation to 55, 24, and 19%, respectively. Retinal formation increased proportionally with the amount of protein S-9 used and was linear up to 40-60 minutes of incubation. Incubation with α-carotene or β-cryptoxanthin resulted in a retinal formation of 29 and 55% of the amount formed from β-carotene. Addition of 9 μg of lutein to an incubation with 3 μg β-carotene reduced retinal formation, while lycopene had no effect. In conclusion, the β-carotene cleavage assay with S-9 as enzyme source described in this report, seems a useful tool to study (dietary) determinants of β-carotene cleavage activity, but for other purposes adaptation of the method is required. Chemicals/CAS: alpha-carotene, 432-70-2; Bcdo protein, rat, EC 1.14.99.36; beta Carotene, 7235-40-7; beta-Carotene 15,15'-Monooxygenase, EC 1.14.99.36; Carotenoids, 36-88-4; cryptoxanthin, 472-70-8; lycopene, 502-65-8; Oxygenases, EC 1.13.-; Retinaldehyde, 116-31-4; Tritium, 10028-17-8; Xanthophylls

In this study a polymorphism in the conjugating activity of human erythrocyte cytosol towards the dihaloethane, ethylene dibromide (EDB; 1,2-dibromoethane) was found. Two out of 12 human erythrocyte cytosols did not catalyze the formation of glutathione (GSH) conjugates of [1,2-14C]EDB. Ten cytosols formed the S,S'-ethylenebis(GSH) conjugate at a rate ranging from 0.5 to 3.2 (mean 1.76 ± 0.95) pmol min-1 (mg protein)-1. The activity of the cytosols towards EDB was compared with the activity towards 1,2-epoxy-3-(p-nitrophenoxy)-propane (EPNP) and 1-chloro-2,4-dinitrobenzene (CDNB). The GSH conjugates formed from EDB, EPNP and CDNB were all quantified by HPLC. Every cytosol was active with the classical GST substrate CDNB (2.04 ± 0.74 nmol min-1 (mg protein)-1). The two samples not showing any detectable activity towards EDB were also inactive towards EPNP: The activity towards EDB correlated significantly with EPNP (r(s) = 0.90, P < 0.005; Spearman's rank correlation), but not with CDNB (r(s) = 0.36, P > 0.10). In the incubations with EPNP, the alpha-, mu-, and pi- class glutathione S-transferase (GST) inhibitor S-hexyl(GSH) was included, indicating that the class-theta GST is the principal GST class conjugating EDB in erythrocyte cytosol. The apparent polymorphism of GST-theta which has recently been recognized to be crucial for several mono- and dihalomethanes, will thus also have considerable implications for the risk assessment of EDB.

Cytosolic calcium concentrations (Ca(i)) of barley aleurone protoplasts after stimulation with the plant hormone abscisic acid (ABA) were measured by using the calcium-sensitive fluorescent dye Indo-1. The measuredd basal Ca(i) is about 200 nM. Stimulation with ABA induces a strong dose-dependent decrease in Ca(i) to a minimal value of about 50 nM. This decrease occurs within 5 s. The Ca2+ antagonists La3+ and Cd2+ inhibit the ABA-induced Ca(i) decrease in a dose-dependent manner, while the Ca2+ channel blockers verapamil and nifedipine give no inhibition. The induction of Ca(i) decrease by ABA is consistent with activation of the plasma membrane Ca2+-ATPase by ABA. The possible role of this ABA-induced Ca(i) decrease in ABA signal transduction and in counteracting the effects of gibberellic acid are discussed. Chemicals/CAS: Histocompatibility Antigens Class IIChemicals/CAS: Abscisic Acid, 21293-29-8; Ca(2+)-Transporting ATPase, EC 3.6.1.38; Calcium, 7440-70-2; Fluorescent Dyes; indo-1, 96314-96-4; Indoles; Nifedipine, 21829-25-4; Verapamil, 52-53-9

Curcumin, an α,β-unsaturated carbonyl compound, reacts with glutathione, leading to the formation of two monoglutathionyl curcumin conjugates. In the present study, the structures of both glutathione conjugates of curcumin were identified by LC-MS and one- and two-dimensional 1H NMR analysis, and their formation in incubations with human intestinal and liver cytosol and purified human glutathione S-transferases and also in human Caco-2 cells was characterized. The results obtained demonstrate the site for glutathione conjugation to be the C1 atom, leading to two diastereoisomeric monoglutathionyl curcumin conjugates (CURSG-1 and CURSG-2). The formation of both glutathionyl conjugates appeared to be reversible. The monoglutathionyl curcumin conjugates decompose with a t1/2 of about 4 h to curcumin and other unidentified degradation products. Both human intestinal and liver cytosol catalyzed curcumin glutathione conjugation. At saturating substrate concentrations, human GSTM1a-1a and GSTA1-1 are shown to be especially active in the formation of CURSG-1, whereas GSTP1-1 and GSTA2-2 have no preference for the formation of CURSG-1 or CURSG-2. GSTT1-1 hardly catalyzes the glutathione conjugation of curcumin. In the Caco-2 human intestinal monolayer transwell model, CURSG-1 and CURSG-2 were formed at a ratio of about 2:1 followed by their excretion, which appeared to be three times higher to the apical (lumen) side than to the basolateral (blood) side. Given that GSTM1a-1a and GSTP1-1 are present in the intestinal epithelial cells, it can be concluded that efficient glutathione conjugation of curcumin may already occur in the enterocytes, followed by an efficient excretion of these glutathione conjugates to the lumen, thereby reducing the bioavailability of (unconjugated) curcumin. In conclusion, the present study identifies the nature of the diastereoisomeric monoglutathionyl curcumin conjugates, CURSG-1 and CURSG-2 formed in biological systems, and reveals that conjugate formation is catalyzed by GSTM1a-1a, GSTA1-1, and/or GSTP1-1 with different stereoselective preference. The formation of glutathione conjugates can already occur during intestinal transport, after which the monoglutathionyl conjugates are efficiently excreted to the intestinal lumen, thereby influencing the bioavailability of curcumin and, as a result, its beneficial biological effects. © 2007 American Chemical Society.

The effect of cimetidine on the metabolism of zaleplon (ZAL) in human liver subcellular fractions and precision-cut liver slices was investigated. 2. ZAL was metabolized to a number of products including 5-oxo-ZAL (M2), which is known to be formed by aldehyde oxidase, N-desethyl-ZAL (DZAL), which is known to be formed by CYP3A forms, and N-desethyl-5-oxo-ZAL (M1). 3. Human liver microsomes catalysed the NADPH-dependent metabolism of ZAL to DZAL. Kinetic analysis of three microsomal preparations revealed mean (±SEM) S₅₀ and V_max of 310±24 μM and 920±274 pmol/min/mg protein, respectively. 4. Human liver cytosol preparations catalysed the metabolism of ZAL to M2. Kinetic analysis of three cytosol preparations revealed mean (±SEM), K_m and V_max of 124±14 μM and 564±143 pmol/min/mg protein, respectively. 5. Cimetidine inhibited ZAL metabolism to DZAL in liver microsomes and to M2 in the liver cytosol. With a ZAL substrate concentration of 62 μM, the calculated mean (±SEM, n=3) IC₅₀ were 596±103 and 231±23 μM for DZAL and M2 formation, respectively. Kinetic analysis revealed that cimetidine was a competitive inhibitor of M2 formation in liver cytosol with a mean (±SEM, n=3) K_i of 155±16 μM. 6. Freshly cut human liver slices metabolized ZAL to a number of products including M1, M2 and DZAL. 7. Cimetidine inhibited ZAL metabolism in liver slices to M1 and M2, but not to DZAL. Kinetic analysis revealed that cimetidine was a competitive inhibitor of M2 formation in liver slices with an average (n = 2 preparations) K_i of 506 μM. 8. The results demonstrate that cimetidine can inhibit both the CYP3A and aldehyde oxidase pathways of ZAL metabolism in the human liver. Cimetidine appears to be a more potent inhibitor of aldehyde oxidase than of CYP3A forms and hence in vivo is likely to have a more marked effect on ZAL metabolism to M2 than on DZAL formation. 9. The results also demonstrate that precision-cut liver slices may be a useful model system for in vitro drug-interaction studies. Chemicals/CAS: Acetamides; Cimetidine, 51481-61-9; Enzyme Inhibitors; Hypnotics and Sedatives; Pyrimidines; zaleplon, 151319-34-5

Caspases play a very important role in initiating and executing apoptotic processes in animal cells. In this study we show that plant mitochondria were able to initiate the activation of caspase 3 in a Xenopus cell free system. Caspase 3-like activity was found to be present in plant cells and could only be inhibited by the specific caspase 3 inhibitor N-acetyl-Asp-Glu-Val-Asp-fluoromethylketone (Ac-DEVD-fmk) and not by cysteine protease inhibitors. By micro-injection of the caspase 3 substrate in living Chara cells we showed that caspase 3-like activity was mainly present in the cytosol rather than in the vacuole. This is the first time that in vivo caspase 3-like activity has been demonstrated in plants. Copyright (C) 2000 Federation of European Biochemical Societies. Chemicals/CAS: Caspase 3, EC 3.4.22.-; Caspases, EC 3.4.22.-; Detergents; Octoxynol, 9002-93-1; Plant Proteins; Protease Inhibitors; Sodium Chloride, 7647-14-5

Expression and post-translational modification of barley 14-3-3 isoforms, 14-3-3A, 14-3-3B and 14-3-3C, were investigated using isoform-specific antibodies. Although all three isoforms were shown to be present in the cytosolic, the nuclear and the microsomal cell fractions, differences in post-translational modification were identified for the different cell fractions. Germination-related modifications of 14-3-3 proteins were observed in the cytosol and the microsomal fraction, but not in the nucleus. In vitro proteolytic cleavage of 14-3-3 proteins using trypsin suggests that for 14-3-3A this change was caused by proteolytic cleavage of the unconserved C-terminal region. Copyright (C) 2000 Federation of European Biochemical Societies. Chemicals/CAS: 14-3-3 Proteins; Antibodies; Plant Proteins; Protein Isoforms; Proteins; Trypsin, EC 3.4.21.4; Tyrosine 3-Monooxygenase, EC 1.14.16.2