Cofractionation of HMGB proteins with histone dimers.

An effective and flexible method is presented that can be used to investigate cofractionation of groups of nuclear proteins. The method was used to analyze chromatin-related proteins, of which high-mobility group B (HMGB) proteins consistently cofractionated by cation-exchange chromatography with the histone dimer (H2A-H2B). This led to the hypothesis that the two form a complex, further suggested by gel filtration, in which the HMGBs with core histones eluted as a defined high-molecular-weight peak. A necessary requirement for further studying protein interactions is that the constituents are of the highest possible purity and the pure histone dimers and tetramers used in this study were derived from pure histone octamers with their native marks. There is a growing interest in protein-protein interactions and an increasing focus on protein-interaction domains: most frequently, pull-down assays are used to examine these. The technology presented here can provide an effective system that complements pull-down assays.

The oxidised histone octamer does not form a H3 disulphide bond.

A H3 dimer band is produced when purified native histone octamers are run on an SDS-PAGE gel in a beta-mercaptoethanol-free environment. To investigate this, native histone octamer crystals, derived from chicken erythrocytes, and of structure (H2A-H2B)-(H4-H3)-(H3'-H4')-(H2B'-H2A'), were grown in 2 M KCl, 1.35 M potassium phosphates and 250-350 microM of the oxidising agent S-nitrosoglutathione, pH 6.9. X-ray diffraction data were acquired to 2.10 A resolution, yielding a structure with an Rwork value of 18.6% and an Rfree of 22.5%. The space group is P6(5), the asymmetric unit of which contains one complete octamer. Compared to the 1.90 A resolution, unoxidised native histone octamer structure, the crystals show a reduction of 2.5% in the c-axis of the unit cell, and free-energy calculations reveal that the H3-H3' dimer interface in the latter has become thermodynamically stable, in contrast to the former. Although the inter-sulphur distance of the two H3 cysteines in the oxidised native histone octamer has reduced to 6 A from the 7 A of the unoxidised form, analysis of the hydrogen bonds that constitute the (H4-H3)-(H3'-H4') tetramer indicates that the formation of a disulphide bond in the H3-H3' dimer interface is incompatible with stable tetramer formation. The biochemical and biophysical evidence, taken as a whole, is indicative of crystals that have a stable H3-H3' dimer interface, possibly extending to the interface within an isolated H3-H3' dimer, observed in SDS-PAGE gels.

Antimalarial and cytotoxic constituents of Xylaria cf. cubensis PK108.

Xylaria cf. cubensis PK108 was identified by its distinctive morphological characteristics and its internal transcribed spacers sequence analysis. The chromatographic separation and structural elucidation based on spectroscopic analysis of fungal crude extracts led to 10 compounds; tryptoquivaline L (1), fiscalin C (2), epi-fiscalin C (3), cytochalasin D (4), ergosterol (5), ergosterol peroxide (6), chevalone C (7), xylaranol B (8), helvolic acid (9) and cyclo-(L-Pro-L-Leu) (10). The bioassay screening showed that 4 displayed cytotoxicity against KB and NCI-H187 cancer cell lines with IC50 values of 3.25 and 5.95 µg mL(-1). 6 exhibited cytotoxicity against NCI-H187 with an IC50 value of 5.81 µg mL(-1). 7 and 9 showed antimalarial activity with IC50 values of 25.00 and 6.25 µg mL(-1), respectively. This result establishes Xylaria as broad spectrum bioactive compound producers.

Chemical constituents, and their cytotoxicity, of the rare wood decaying fungus Xylaria humosa.

Samples of Xylaria humosa, a rare species of Xylariaceae, were collected during an investigation into the diversity of the fungus in the Phu Khieo Wildlife Sanctuary, Thailand. Nine compounds were isolated from the species and their structures elucidated by spectroscopic methods. The compounds were ergosterol (1), ergosterol peroxide (2), two meroterpenoids, chevalone B and C (3-4), together with five indole alkaloids, tryptoquivaline L (5), tryptoquivaline M (6), fiscalin A (7), epi-fiscalin A (8) and epi-fiscalin C (9). Compounds 2-9 exhibited variable cytotoxic activity against KB, NCI-H187 and MCF-7 cell lines.

Potential antimalarial derivatives from astraodorol.

Astraodorol, a major lanostane-type triterpene isolated from the edible mushroom Astraeus odoratus, was subjected to chemical modifications. Ten derivatives have been synthesized and their biological activities were evaluated. Compounds 5, 6, 7a, 7c, 7e, 7f, and 7 g exhibited strong antimalarial activity with IC50 values of 4.85, 4.48, 4.16, 4.46, 3.45, 3.23, and 3.41 µg/mL, respectively. Compounds 7a, 7c, and 7e showed moderate cytotoxicity against NCI-H187 with IC50 values of 23.36, 34.28, and 9.84 µg/mL. Compound 7e demonstrated moderate cytotoxicity against KB, MCF-7, and Vero cell lines with IC50 values of 16.94, 49.60, and 26.48 µg/mL, respectively.

Astraodoric acids A-D: new lanostane triterpenes from edible mushroom Astraeus odoratus and their anti-Mycobacterium tuberculosis H37Ra and cytotoxic activity.

Tuberculosis (TB) is one of the chronic infectious diseases caused by Mycobacterium tuberculosis that causes about 2-3 million deaths per year. Isoniazid and rifampicin are examples of first line drugs used for TB treatment; however, they are potentially hepatotoxic. More effective and safer drugs are urgently needed, especially from natural products. Basidiomycete mushrooms are known as important sources of pharmaceutically active metabolites including an anti-TB agent. In this work, the chemical constituents of the edible mushroom Astraeus odoratus were isolated and investigated for antibacterial activity against M. tuberculosis H(37)Ra. The cytotoxic activity against cancerous cell lines was also evaluated. Four new lanostane triterpenes, astraodoric acids A-D, and new 5-hydroxyhypaphorine have been isolated together with four known compounds. The structures were elucidated by NMR spectroscopic methods, HR-ESI-MS results, and X-ray crystallographic analysis. Astraodoric acids A and B exhibited moderate antibacterial (MICs of 50 and 25 µg/mL) and cytotoxic activities (IC(50) values of 34.69 and 18.57 µg/mL against KB and 19.99 and 48.35 µg/mL against NCI-H187), respectively. The results of this study show that A. odoratus could be a significant natural source for safer antitubercular and anticancer agents.