Properties of pyranose dehydrogenase purified from the litter-degrading fungus Agaricus xanthoderma.

We purified an extracellular pyranose dehydrogenase (PDH) from the basidiomycete fungus Agaricus xanthoderma using ammonium sulfate fractionation and ion-exchange and hydrophobic interaction chromatography. The native enzyme is a monomeric glycoprotein (5% carbohydrate) containing a covalently bound FAD as its prosthetic group. The PDH polypeptide consists of 575 amino acids and has a molecular mass of 65 400 Da as determined by MALDI MS. On the basis of the primary structure of the mature protein, PDH is a member of the glucose-methanol-choline oxidoreductase family. We constructed a homology model of PDH using the 3D structure of glucose oxidase from Aspergillus niger as a template. This model suggests a novel type of bi-covalent flavinylation in PDH, 9-S-cysteinyl, 8-alpha-N3-histidyl FAD. The enzyme exhibits a broad sugar substrate tolerance, oxidizing structurally different aldopyranoses including monosaccharides and oligosaccharides as well as glycosides. Its preferred electron donor substrates are D-glucose, D-galactose, L-arabinose, and D-xylose. As shown by in situ NMR analysis, D-glucose and D-galactose are both oxidized at positions C2 and C3, yielding the corresponding didehydroaldoses (diketoaldoses) as the final reaction products. PDH shows no detectable activity with oxygen, and its reactivity towards electron acceptors is rather limited, reducing various substituted benzoquinones and complexed metal ions. The azino-bis-(3-ethylbenzthiazolin-6-sulfonic acid) cation radical and the ferricenium ion are the best electron acceptors, as judged by the catalytic efficiencies (k(cat)/K(m)). The enzyme may play a role in lignocellulose degradation.

New derivatives of silybin and 2,3-dehydrosilybin and their cytotoxic and P-glycoprotein modulatory activity.

Large series of O-alkyl derivatives (methyl and benzyl) of silybin and 2,3-dehydrosilybin was prepared. Selective alkylation of the silybin molecule was systematically investigated. For the first time we present here, for example, preparation of 19-nor-2,3-dehydrosilybin. All prepared silybin/2,3-dehydrosilybin derivatives were tested for cytotoxicity on a panel of drugs sensitive against multidrug resistant cell lines and the ability to inhibit P-glycoprotein mediated efflux activity. We have identified effective and relatively non-cytotoxic inhibitors of P-gp derived from 2,3-dehydrosilybin. Some of them were more effective inhibitors at concentrations lower than a standard P-gp efflux inhibitor cyclosporin A. Another group of 2,3-dehydrosilybin derivatives also had better inhibitory effects on P-gp efflux but a cytotoxicity comparable with that of parent 2,3-dehydrosilybin. Structural requirements for improving inhibitory activity and reducing toxicity of 2,3-dehydrosilybin were established. Effect of E-ring substitution as well as an influence of the substituent size at the C-7-OH position of A-ring on P-gp-inhibitory activity was evaluated for the first time in this study.

Effects of silymarin flavonolignans and synthetic silybin derivatives on estrogen and aryl hydrocarbon receptor activation.

Silymarin, a standardized mixture of flavonolignans, or its major constituents could be effective for prevention and treatment of hepatic damage or skin cancer. However, their potential side effects, such as modulation of endocrine functions via the disruption of estrogen receptor (ER) and/or aryl hydrocarbon receptor (AhR) activation, are largely unknown. In the present study, we investigated impact of silymarin, its constituents and a series of their synthetic derivatives on ER- and AhR-mediated activities using in vitro reporter gene assays. We found that none of the compounds under study affected the AhR-mediated activity in rat hepatoma cells. Contrary to that, several compounds behaved as either partial or full ER agonists. Silymarin elicited partial ER activation, with silybin B being probably responsible for a majority of the weak ER-mediated activity of silymarin; silybin A and other flavonolignans were found to be inactive and potent ER agonist taxifolin is only a minor constituent of silymarin. To our knowledge, this is probably the first time, when receptor-specific in vitro effects of separated diastereomers have been demonstrated. In contrast to silymarin constituents, the synthetic silybin derivatives, potentially useful as chemoprotective agents, did not modulate the ER-mediated activity, with exception of 23-O-pivaloylsilybin. Interestingly, 7-O-benzylsilybin potentiated ER-mediated activity of 17beta-estradiol despite possessing no estrogenic activity. In conclusion, our data suggest that estrogenicity of some silymarin constituents should be taken in account as their potential side effect when considered as chemopreventive compounds. These results also stress the need to study biological activities of purified or synthesized diastereomers of silybin derivatives.

Clustered ergot alkaloids modulate cell-mediated cytotoxicity.

Dimers of agroclavine (1) and terguride (2), as well as a series of terguride oligomers, for example trimers (5, 6), tetramer (7), hexamer (8) and functionalized tergurides for further complex clustering were synthesized. Terguride oligomers were screened for their direct cellular toxicity on lymphoma cell lines in vitro and for their immunomodulating activities, represented by the natural killer (NK) cell-mediated cytotoxicity, as the most sensitive screening marker during immune responses. Dimers linked via aromatic spacer showed a high toxicity (1 microM) to lymphoma cells, which was not detected in other derivatives. In vitro and ex vivo experiments performed on mouse spleen lymphocytes in the presence of terguride oligomers demonstrated an immunosuppressive effect of dimers with aromatic spacer (4c-d) and NK cell stimulatory effect of terguride hexamer (8) and trimer with aliphatic spacer (5c). There is a considerable evidence that indolic part of molecule contributes to immunosuppressive action of terguride, which is potentiated in dimers carrying aromatic linker. This effect can be reversed by higher oligomerization of the respective alkaloids.