Elution profile of RJ protein using cholic acid-conjugated EAH Sepharose 4B column chromatography and 10% SDS-PAGE patterns of bile acid-binding proteins derived from RJ.

<p>(A) Elution profile of RJ protein using cholic acid-conjugated EAH Sepharose 4B column chromatography. Twenty-five milliliters of RJ protein (10-kDa cut-off RJ) solution (118 mg protein) in 0.02% NaN₃ containing 10 mM Tris-HCl (pH 8.0) were applied to the column and washed with (a) 0.5 M NaCl containing 10 mM Tris-HCl buffer (pH 8.0), (b) 0.5% sodium deoxycholate containing 10 mM Tris-HCl buffer (pH 8.0), and (c) 8 M urea containing 10 mM Tris-HCl buffer (pH 8.0). (B) 10% SDS-PAGE patterns of bile acid-binding proteins derived from RJ by cholic acid-conjugated column chromatography. Lane 1, protein standard; lane 2, bile acid-binding proteins eluted with 0.5% sodium deoxycholate from the column. The amount of applied protein in lane 2 was 4.2 µg. The bile acid-binding proteins consist of MRJP1, MRJP2, and MRJP3.</p

Taurocholate binding ability and micellar solubility of cholesterol <i>in vitro</i>.

<p>(A) Binding of taurocholate to casein, MRJP1, MRJP2, or FDRJ <i>in vitro</i>. Values are means, with their standard errors represented by vertical bars (<i>n</i> 3 per group). Protein concentration was 100 mg/ml respectively. Within a row, means with different superscript letters are significantly different (P<0.05) by Tukey's test. (B) Micellar solubility of cholesterol in the presence of casein, MRJP1, MRJP2, or FDRJ <i>in vitro</i>. Protein concentration was 10 mg/ml in each case. Values are means, with their standard errors represented by vertical bars (<i>n</i> 3 per group). Within a row, means with different superscript letters are significantly different (P<0.05) by Tukey's test.</p

Effects of MTH or CTH on CYP7A1 protein level in HepG2 cells by Western blot analysis.

<p>HepG2 cells were treated with MTH (1 mg/ml) or CTH (1 mg/ml) for 48 h. Protein was prepared from the treated cells and used for Western blot analyses. Values are means, with their standard errors represented by vertical bars (<i>n</i> 4 per group). Asterisks indicate different from CTH (*P<0.05) by Student's <i>t</i>-test.</p

Effects of dietary casein or MRJP1 on body and relative liver weights, food intake, serum and liver lipids, fecal steroid excretion in rats¹.

1<p>Values are means ± SEM, n = 10. Asterisks indicate different from casein (*P<0.05, **P<0.01, ***P<0.001).</p>2<p>Values were calculated as follows: LDL + VLDL cholesterol  =  Total cholesterol - HDL cholesterol.</p>3<p>Atherogenic index calculated by Serum Total cholesterol/Serum HDL cholesterol.</p>4<p>Total steroids  =  neutral steroids + acidic steroids.</p

Effects of MRJP1 or Casein on rat liver CYP7A1 protein level by Western blot analysis.

<p>Total protein extracts from rat liver by MRJP1 or casein treatment for 7 days and used for Western blot analysis. Values are means, with their standard errors represented by vertical bars (<i>n</i> 4 per group).</p

Effects of dietary casein, MRJP¹ or β-sitosterol on body and relative liver weights, food intake, serum choleseterol levels in rats.

1<p>Values are means ± SEM, n = 8. Within a row, means with different superscript letters are significantly different (P<0.05) by Tukey's test.</p

Typical elution profiles of RJ proteins by HPLC and 15% SDS-PAGE patterns of the isolated RJ proteins.

<p>(A) Typical elution profile of RJ protein by HPLC. Elution profile of RJ protein (10-kDa cut-off RJ) by size-exclusion chromatography using a HiLoad 26/60 Superdex 200 p.g. column. Thirteen milliliters of 10-kDa cut-off RJ solution (182 mg protein) in 150 mM NaCl containing 20 mM phosphate (Na₂HPO₄/NaH₂PO₄) buffer (pH 7.5) were applied to the column. The molecular weights of the eluted proteins were calibrated using standard proteins, as follows. Peak A, 515 kDa; peak B, 290 kDa; peak C, 157 kDa; peak D, 79 kDa; peak E, 55 kDa; peak F, 5 kDa. (B) 15% SDS-PAGE patterns of peak B and peak E. Lane 1, molecular weight standards; lane 2, protein containing peak B from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105073#pone-0105073-g002" target="_blank">Fig. 2(A)</a>; lane 3, protein containing peak E from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105073#pone-0105073-g002" target="_blank">Fig. 2(A)</a>. The amount of applied protein in lanes 2 and 3 was 5 µg each. The protein contained in peak B from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105073#pone-0105073-g002" target="_blank">Fig. 2(A)</a> was detected as a 55-kDa protein. The protein contained in peak E of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105073#pone-0105073-g002" target="_blank">Fig. 2(A)</a> was detected as 2 major protein bands (55 kDa and 49 kDa)respectively. (C) Elution profile of peak E by anion exchange chromatography using a HiPrep QFF 16/10 column. Five millilters of peak E protein solution (125 mg protein) in 20 mM Tris-HCl (pH 8.0) was applied to the column. (D) 15% SDS-PAGE pattern of proteins derived from anion exchange chromatography. Lane 1, molecular weight standards; lane 2, protein containing the peak E1 and E2 of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105073#pone-0105073-g002" target="_blank">Fig. 2(C)</a>. The amount of applied protein in lane 2 was 8 µg.</p

Chemical compositions of casein, FDRJ and MRJP1.

1<p>Casein (Meiji Dairy Corporation).</p>2<p>10-HDA; 10-hydroxy-2-decenoic acid.</p><p>This is included in lipids.</p>3<p>69.3 g lipids/kg contains 0.53 g polyphenols/kg.</p>4<p>0.2 g lipids/kg contains 0.06 mgpolyphenols/kg.</p

Effects of MRJP1 or casein on cholesterol absorption in Caco-2 cells.

<p>Values are means, with their standard errors represented by vertical bars (<i>n</i> 6 per group). Asterisks indicate different from Casein (*P<0.05) by Student's <i>t</i>-test.</p

Effects of MRJP1, casein or their tryptic hydrolysates on hepatic mRNA levels related to cholesterol metabolism in rats or HepG2 cells.

<p>(A) Effects of oral administration of MRJP1 or casein on hepatic mRNA levels related to cholesterol metabolism in rats. Values are means, with their standard errors represented by vertical bars (<i>n</i> 10 per group). Asterisks indicate different from CTH (*P<0.05) by Student's <i>t</i>-test. (B) Effects of MTH or CTH on the mRNA levels mRNA levels related to cholesterol metabolism in HepG2 cells. HepG2 cells were treated with MTH (1 mg/ml) or CTH (1 mg/ml) for 24 h. Total RNA was prepared from the treated cells and used for quantitative real-time PCR analyses. Values are means, with their standard errors represented by vertical bars (<i>n</i> 3 per group). Asterisks indicate different from CTH (*P<0.05, **P<0.01) by Student's <i>t</i>-test.</p