Interaction of polyphenols with proteins: binding of (-)-epigallocatechin gallate to serum albumin, estimated by induced circular dichroism.

The binding of (-)-epigallocatechin gallate (EGCG), a representative natural polyphenol, to human serum albumin (HSA) and bovine serum albumin (BSA) was investigated using induced circular dichroism (CD). The site of the binding EGCG-HSA was analyzed based on the competition with drugs with known binding sites on HSA, such as phenylbutazone (PB) and diazepam (DP). Double-reciprocal plot analyses showed the competitive relations with the site-I- (PB and tolbutamide, TB) and site-II-binding drugs (DP and ibuprofen, IP) indicating the binding of EGCG to sites I and II on HSA, while digitoxin (DG), a site-III-binding drug, did not affect the binding of EGCG. In an analogous way, the competitive relations were observed between EGCG and the site-I- (PB and TB) and site-II-binding (ethacrynic acid, EA) drugs for the binding of EGCG and BSA. The site-III drug DG also showed competitive binding with EGCG to BSA. The binding of EGCG to the albumins indicated its affinity to sites I and II on HSA, while competitive binding for all three sites was observed on BSA.

Correlation among geometric, densitometric, and mechanical properties in mandible and femur of osteoporotic rats.

We have previously demonstrated bone loss of the mandible and femur in experimental osteoporotic rats and its prevention by medication, using peripheral quantitative computed tomography (pQCT). In the present study, the mechanical properties of the mandible and femur and the correlation to their geometric and densitometric properties were studied in ovariectomized rats with or without etidronate treatment. Fifty-four Wistar strain SPF female rats, 26 weeks old, were randomly assigned to four groups: (1) Basal group (12 rats, 1.0% Ca diet); (2) Sham group (Sham-operated, 12 rats, 0.1% Ca diet); (3) OVX group (ovariectomized, 15 rats, 0.1% Ca diet); (4) Treated group (OVX + etidronate, 15 rats, 0.1% Ca diet). Total bone mineral density (BMD), cortical BMD, cross-sectional cortical bone area, cross-sectional cortical bone thickness, crosssectional moment of inertia (CSMI), and polar strength index (SSI) of the mandible and femur were measured by pQCT. The failure load of mandible and femur was evaluated by three-point bending. The failure load of both bones was significantly lower in the Sham group compared with the Basal group. The OVX group further had a 8% and 7% decrease in the failure load for mandible and femur, respectively, compared to the Sham group. Treatment with etidronate led to an increase in the failure load compared with the OVX group. The failure load was related to the pQCT-assessed variables, especially with cortical bone area and total BMD. Moreover, the geometric and densitometric properties and failure load in the mandible showed a correlation to those in the femur.

Differences of cell growth and cell cycle regulators induced by basic fibroblast growth factor between nifedipine responders and non-responders.

Differences of cell proliferation, cell cycle, and G(1)/S transition regulatory proteins of gingival fibroblasts derived from nifedipine-reactive patient (NIFr) and nifedipine-non-reactive patient (NIFn) in the presence of basic fibroblast growth factor (bFGF) were investigated to elucidate the mechanism of gingival overgrowth associated with nifedipine, one of the Ca(2+)-channel blockers. The proliferation rate of NIFr cells in the presence of bFGF significantly increased than NIFn cells. The proportion of NIFr cells that had undergone progression to the S and G(2)/M phases from the G(0)/G(1) phase significantly increased compared to that in NIFn cells. Increases of pRB (Ser807/811), pCDK2 (Thr160), CDK2, and cyclin E protein levels in NIFr cells were greater than those in NIFn cells. The elevations of pRB (Ser780), RB, and cyclin A protein levels in NIFr cells did not differ from those of NIFn cells. The growth of NIFr cells was greater than that of NIFn cells as a result of the active G(1)/S transition of NIFr cells, as assessed by the increments of cyclin E, pCDK2, and pRB (ser807/811) protein in NIFr cells.

Size exclusion chromatographic analysis of polyphenol-serum albumin complexes.

Formation of water-soluble polyphenol-protein complexes was investigated by size-exclusion chromatography (SEC). The combination of (-)-epigallocatechin gallate (EGCG) and bovine serum albumin (BSA), which did not form a precipitate after the solutions were mixed, showed an SEC peak due to complex formation 2-24 h after mixing. Peak size of the complex varied with time, suggesting slow change of the conformation of the protein accompanied by complexation. Formation of the complex was substantiated by ultrafiltration of the mixture; the complex did not pass through a membrane with a 100,000 nominal molecular weight limit (NMWL). The SEC profile varied with the combination of compounds. The peaks due to the complexes showed that the apparent value of the number average molecular weight (M(n)) of the EGCG-BSA complex was 2.8x10(5), while that of a pentagalloylglucose (PGG)-BSA complex was 9.5x10(5) under the conditions used. Dimeric hydrolyzable tannins, oenothein B and cornusiin A, also caused changes in the SEC profile of BSA, although the combinations did not show peaks attributable to formation of such large complexes observed for EGCG and PGG. Procyanidin B3 and (+)-catechin did not cause changes in the SEC profile of BSA. With cytochrome c, EGCG did not show any chromatographic changes.

Theasinensin A, a tea polyphenol formed from (-)-epigallocatechin gallate, suppresses antibiotic resistance of methicillin-resistant Staphylococcus aureus.

When (-)-epigallocatechin gallate (EGCG), the main constituent of tea polyphenols, was kept in a neutral buffer, it decomposed rapidly to give theasinensin A as the major product. Theasinensin A suppressed the oxacillin resistance of methicillin-resistant Staphylococcus aureus (MRSA). In the presence of theasinensin A (3.5 x 10(-5) M), the minimum inhibitory concentrations (MICs) of oxacillin decreased from 256 or 64 microg/mL to 4 microg/mL for the MRSA strains used. The presence of this compound (3.5 x 10(-5) M) also decreased the MIC of other beta-lactam (penicillin G, from 32 microg/mL to 0.125-0.5 microg/mL; ampicillin, from 16-32 microg/mL to 0.5-1 microg/mL) and aminoglycoside (streptomycin, from 4 - 16 microg/mL to 0.125-4 microg/mL) antibiotics for the MRSA strains.