This 18-page file contains ten figures, two tables and one text.

Ginseng (the root of Panax ginseng C. A. MEYER, family Araliaceae) is frequently used as a traditional medicine, taken orally, in China, Korea, Japan and other Asian countries.1) The major components of ginseng, the ginsenosides, are glycosides that contain an aglycone with a dammarane skeleton.2,3) These ginsenosides have been reported to show various biological activities, including immunomodulatory effect, anti-inflammatory activity and antitumor effects. 4-7)To explain these pharmacological actions, it is thought that ginseng saponins must be metabolized by human intestinal bacteria after their oral administration. [8][9][10][11][12] For example, ginsenoside Rb1, Rb2 and Rc are transformed to 20-O-b-D-glucopyranosyl-20(S)-protopanaxadiol (compound K) by human intestinal bacteria, with ginsenoside Re transformed to protopanaxatriol.Akao et al. reported that compound K was detected in the blood after the oral administration of ginsenoside Rb1 to rats. 8,10) We also reported that compound K was detected in the urine when ginsenoside Rb1 was orally administered to rats.13) Tawab et al. reported that ginsenoside F1, compound K and ginsenoside Rh1 were detected in the urine and blood when ginseng extract was orally administered to humans. 14)These results suggest that protopanaxadiol ginsenosides may be metabolized, mainly to compound K, and protopanaxatriol ginsenosides to ginsenoside Rh1 and ginsenoside F1. Thus, these findings are in contrast with the report of Hasegawa et al. who suggested that ginsenoside Re was metabolized mainly to protopanaxatriol by human intestinal microflora.15) Therefore, to clarify the metabolism of the ginsenoside Re by human intestinal microflora, the ginsenoside Re-metabolizing intestinal bacteria were isolated from human feces, their metabolic activity measured and the estrogenic effect of ginsenoside Re, as well as its main metabolites, investigated. MATERIALS AND MEHTODS Materials The, sulforhodamine B (SRB), NP40, 17b-estradiol, Dulbecco's modified Eagles medium (DMEM), fetal bovine serum (FBS) and charcoal dextran stripped FBS (CD-FBS) were purchased from Sigma Chem. Co. (U.S.A.). The general anaerobic medium (GAM) was purchased from Nissui Pharmaceutical Co., Ltd., (Tokyo, Japan). The tryptic soy broth (TS) and other media were purchased from Difco Co. (U.S.A.). The protein assay reagent was purchased from Bio-Rad Laboratories (U.S.A.). The enhanced chemiluminescence (ECL) kit was purchased from Amersham Co. (U.S.A.). The progesterone receptor (PR) was purchased from Santa Cruz Co. (U.S.A.). The protease inhibitor cocktail was purchased from Roche Co. (Germany). The poncirin, poncirenin and ponciretin were isolated as previously described.16) The ginsenosides Re and Rg2 were isolated according to the previously described method.3,15) All other chemicals were of analytical reagent grade, and all solutions used after redistillation.Isolation of Ginsenoside Re-hydrolyzing Bacteria from Human Intestinal Bacteria A suspension of the fresh feces of a healthy Korean man was diluted ...

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Max-E47 is a designed hybrid protein comprising the Max DNA-binding basic region and E47 HLH dimerization subdomain. In the yeast one-hybrid system (Y1H), Max-E47 shows strong transcriptional activation from the E-box site, 5'-CACGTG, targeted by the Myc/Max/Mad network of transcription factors; two mutants, Max-E47Y and Max-E47YF, activate more weakly from the E-box in the Y1H. Quantitative fluorescence anisotropy titrations to gain free energies of protein:DNA binding gave low nM Kd values for the native MaxbHLHZ, Max-E47, and the Y and YF mutants binding to the E-box site (14 nM, 15 nM, 9 nM, and 6 nM, respectively), with no detectable binding to a nonspecific control duplex. Because these minimalist, E-box-binding hybrids have no activation domain and no interactions with the c-MycbHLHZ, as shown by the yeast two-hybrid assay, they can potentially serve as dominant-negative inhibitors that suppress activation of E-box-responsive genes targeted by transcription factors including the c-Myc/Max complex. As proof-of-principle, we used our modified Y1H, which allows direct competition between two proteins vying for a DNA target, to show that Max-E47 effectively outcompetes the native MaxbHLHZ for the E-box; weaker competition is observed from the two mutants, consistent with Y1H results. These hybrids provide a minimalist scaffold for further exploration of the relationship between protein structure and DNA-binding function and may have applications as protein therapeutics or biochemical probes capable of targeting the E-box site.