Green tea polyphenol EGCg induces cell fusion via reactive oxygen species.

Background: Osteoclasts are multinucleated cells formed by macrophage cell fusion that are responsible for bone resorption. Previously, we found that treating osteoclastic progenitor cells with (-)-epigallocatechin gallate (EGCg) increased cell fusion. In this study, we aimed to identify factors involved in the cell fusion induced by EGCg. Methods: We hypothesized that EGCg-induced oxidative stress might be involved in cell fusion, and used macrophage cell line RAW264.7 cells. We evaluated cell fusion activity after adding the antioxidants N-acetyl-l-cysteine (NAC) or catalase in addition to EGCg. The mRNA expressions of genes related to cell fusion and bone resorption were quantified by real-time PCR. Finally, we added hydrogen peroxide and examined its effects on cell fusion and TRAP activity. Results: EGCg-induced cell fusion was strongly inhibited by the addition of NAC in a dose-dependent manner (EGCg with 5 mM NAC; decreased to 1.5%; p < 0.05), while the inhibitory effect of catalase was limited (EGCg with 500 U/mL catalase; decreased to 27.7%; p < 0.05). DC-STAMP expression was significantly upregulated by EGCg compared with the untreated group, and the upregulation was significantly suppressed by 5 mM NAC. Conversely, Nfatc1 and TRAP expression were not upregulated by EGCg. These results suggest that EGCg induces DC-STAMP expression via reactive oxygen species production, which regulates cell fusion but does not affect the osteoclastic pathway. Although treatment with hydrogen peroxide promoted the formation of multinucleated cells, no increase in TRAP activity was observed, which was similar to EGCg treatment. Conclusions: This study suggests that the increased cell fusion by EGCg may be induced by oxidative stress due to reactive oxygen species production.

Cholesterol-lowering activity of adzuki bean (Vigna angularis) polyphenols.

BACKGROUND: Adzuki beans (ABs; Vigna angularis) were reported to show potential for prevention of cholesterol absorption and lowering of the blood cholesterol level. However, the main active compounds and some cellular effects remain unknown. In this study, we evaluated the potential cholesterol-lowering effects of (+)-catechin 7-O-ß-D-glucopyranoside (C7G) and (+)-epicatechin 7-O-ß-D-glucopyranoside (E7G), identified as abundant polyphenols in ABs. METHODS AND RESULTS: To investigate the cholesterol-lowering activity in vitro, cholesterol micelles, bile acids, and Caco-2 cells as an intestinal model were used in the study. C7G and E7G each inhibited micellar solubility in a dose-dependent manner, and their inhibitory activity was as strong as that of (+)-catechin (IC50 values: C7G, 0.23 ± 0.03 mg/ml; E7G, 0.22 ± 0.02 mg/ml; (+)-catechin, 0.26 ± 0.11 mg/ml). The AB polyphenols showed binding activity toward bile acids and changed them into an insoluble form. When Caco-2 cells were treated with C7G or E7G, the amount of incorporated cholesterol was significantly decreased compared with vehicle-treated control cells, and no cytotoxicity was observed under the experimental conditions used. Meanwhile, quantitative real-time PCR revealed that the mRNA level of the cholesterol transporter NPC1L1 remained unchanged in the treated cells. CONCLUSIONS: Taken together, the present findings suggest that C7G and E7G are the main active compounds in ABs, and have the ability to inhibit micellar solubility, bind to bile acids, and suppress cholesterol absorption. The present study supports the health benefits of ABs as a medicinal food and the application of AB polyphenols as medicinal supplements to suppress cholesterol elevation.

Tea extract increases cell fusion via regulation of cell surface DC-STAMP.

Mononuclear osteoclast precursor cells fuse with each other to become mature multinucleated osteoclasts, which is regulated by dendritic cell-specific transmembrane protein (DC-STAMP). We evaluated the effects of tea extract and catechins on cell-cell fusion and DC-STAMP expression to elucidate their relationship with osteoclast development. When tea extract or epigallocatechin gallate (EGCg) was applied to RAW264.7 cells, multinucleated cells were increased significantly, while tartrate-resistant acid phosphatase (TRAP) activity was hardly upregulated. Flow cytometric analysis revealed that EGCg suppressed DC-STAMP expression on the cell surface, which is similar to osteoclast development. These observations suggest that TRAP activity is not activated even when suppression of both surface DC-STAMP expression and multinucleation occurs, which might be mediated by another pathway.

Isolation and Characterization of Antihyperglycemic Compounds from Vigna angularis Extracts.

Functional foods that inhibit α-amylase and α-glucosidase activity are effective for regulating the blood glucose level and preventing hyperglycemia. Extracts of adzuki beans (ABs, Vigna angularis), widely eaten in East Asia, can inhibit α-amylase and α-glucosidase activity. In this study, we identified and evaluated the components in an AB water extract (ABWE) after boiling, which is an essential process for cooking ABs. The ABWE before boiling inhibited α-amylase and α-glucosidase activity and the boiled ABWE showed slightly stronger inhibitory effects. High-performance liquid chromatography, liquid chromatography-mass spectrometry, and nuclear magnetic resonance analyses identified (+)-catechin 7-O-ß-d-glucopyranoside (C7G), (+)-epicatechin 7-O-ß-d-glucopyranoside (E7G), and (+)-catechin as the bioactive components in boiled ABWE. Interestingly, the quantity of E7G significantly increased after boiling (from 0% to 17.1 ± 1.3%). E7G showed stronger inhibition of α-amylase and α-glucosidase than C7G; the IC50 values for α-amylase were 0.74 ± 0.04 mg/mL (C7G) and 0.40 ± 0.09 mg/mL (E7G), and for α-glucosidase the IC50 values were 0.085 ± 0.032 mg/mL (C7G) and 0.051 ± 0.007 mg/mL (E7G). Our findings suggest that C7G and E7G are the main active components in ABWE as they inhibit α-amylase and α-glucosidase and their inhibitory effect is not lost after boiling. These results support the effectiveness of boiled ABs in the promotion of health. PRACTICAL APPLICATION: We identified (+)-catechin 7-O-ß-d-glucopyranoside (C7G), (+)-epicatechin 7-O-ß-d-glucopyranoside (E7G), and (+)-catechin in adzuki bean extracts and commercially available boiled adzuki bean products. Interestingly, the E7G content was increased by boiling, and this compound showed strong inhibitory activity toward α-amylase and α-glucosidase. These results support the consumption of boiled adzuki beans to prevent acute rises in blood glucose level.

Antiosteoporotic Effects of Acer palmatum Extract on Osteoclastogenesis and Osteoblastogenesis.

Osteoporosis is a systemic skeletal disease that causes bone weakness and fragility. Consuming bone-beneficial nutrients through diet can prevent and treat osteoporosis. Acer palmatum (Japanese maple) leaves are used to make tea, but there have been few reports of their health benefits, especially regarding bone homeostasis. In this study, we evaluated the effects of A. palmatum hot water extract (APE) on osteoclastogenesis and osteoblastogenesis in cultured cells. APE suppressed the number of tartrate-resistant acid phosphatase-positive multinucleated osteoclasts in RANKL induced RAW264.7 cells. Furthermore, APE facilitated Alkaline phosphatase activity and calcium deposition during osteoblast differentiation in MC3T3-E1 cells. High-performance liquid chromatography analysis was performed to investigate the effective components of APE, and four flavonoids orientin, isoorientin, vitexin, and isovitexin were identified with the LC-MS analysis. Treatment with fractionated APE suppressed osteoclastogenesis and facilitated osteoblastogenesis in cultured cells. These findings suggest that APE contains antiosteoporotic compounds; thus, APE might have health promoting effects that help prevent osteoporosis by inhibiting osteoclastogenesis and facilitating osteoblastogenesis.

Identification of a catalytic residue of Clostridium paraputrificum N-acetyl-beta-D-glucosaminidase Nag3A by site-directed mutagenesis.

Clostridium paraputrificum M-21 beta-N-acetylglucosaminidase 3A (Nag3A) is an enzyme classified in family 3 of the glycoside hydrolases. To identify catalytic residues of this enzyme, mutations were introduced into highly conserved Glu and Asp residues. Replacement of Asp175 with Ala abolished the catalytic activity without change in the circular dichroism spectrum, strongly suggesting that this residue is a catalytic residue, a nucleophile/base or a proton donor. Since the K(m) values of mutant enzymes D119N, D229N, D229A and D274N increased 17 to 41 times as compared with that of wild-type enzyme, Asp119, Asp229, and Asp274 appear to be involved in substrate recognition and binding. Taking previous studies into consideration, we presume that Asp303 is the catalytic nucleophile and Asp175 is the proton donor of C. paraputrificum Nag3A.

Potential ability of hot water adzuki (Vigna angularis) extracts to inhibit the adhesion, invasion, and metastasis of murine B16 melanoma cells.

The 40% ethanol eluent of the fraction of hot-water extract from adzuki beans (EtEx.40) adsorbed onto DIAION HP-20 resin has many biological activities, for example, antioxidant, antitumorigenesis, and intestinal alpha-glucosidase suppressing activities. This study examined the inhibitory effect of EtEx.40 on experimental lung metastasis and the invasion of B16-BL6 melanoma cells. EtEx.40 was found significantly to reduce the number of tumor colonies. It also inhibited the adhesion and migration of B16-BL6 melanoma cells into extracellular matrix components and their invasion into reconstituted basement membrane (matrigel) without affecting cell proliferation in vitro. These in vivo data suggest that EtEx.40 possesses a strong antimetastatic ability, which might be a lead compound in functional food development.

Expression of two isoform mRNAs of nucleolar protein B23 during rat liver regeneration.

Nucleolar protein B23 exists in two isoforms designated as B23.1 and B23.2, differing only in their carboxy-terminal short sequences. To clarify the levels of protein B23 isoform mRNAs in the cell cycle, we have investigated the expressions of the two mRNAs during rat liver regeneration. Both of B23 isoforms transcripts increased after a partial hepatectomy. Peaks of the mRNAs were observed after 9 h (fivefold) with B23.1 and 12 h (twofold) with B23.2, respectively. These two peaks were slightly preceding the onset of DNA synthesis, which was revealed by the activity of DNA polymerase alpha. From these observations, important roles of both protein B23 isoforms in stimulation of DNA polymerase a activity during rat liver regeneration were suggested.

Tryptophans 286 and 288 in the C-terminal region of protein B23.1 are important for its nucleolar localization.

Nucleolar protein B23 can shuttle between the nucleolus and cytoplasm. However, the mechanism involved in the protein moving and staying in the nucleolus is not fully understood. To identify the nucleolar localization signal sequence of protein B23, we examined the subnuclear location of B23.1 mutant proteins fused with green fluorescent protein in HeLa cells. The results suggested that the two C-terminal tryptophan residues (Trp-286 and Trp-288) of protein B23.1 were important in this phenomenon.

Specific interactions between cryogel components: role of extra domain A containing fibronectin in cryogelation.

Cryogel is a physical gel formed by heterophilic aggregation of extra domain A containing fibronectin [EDA(+)FN], plasma fibronectin (pFN), fibrinogen (Fbg) and heparin (Hep), which are found in high concentrations in the blood of patients suffering from rheumatoid arthritis. In this study, we clarify the specific interactions between cryogel components in terms of the affinity constant (K(A)), obtained by surface plasmon resonance (SPR). It is found that Fbg self-interactions occur at lower temperatures, and that K(A) of Fbg-Hep changes with temperature. Specifically, K(A) (2.0 x 10(8) [M(-1)]) of Fbg-Hep at 5 degrees C increases significantly from that (1.0x10(7) [M(-1)]) at 40 degrees C. K(A) of EDA(+)FN-Hep increases with temperature, by approximately 100-fold between 40 degrees C (K(A)=10(12) [M(-1)]) and 20 degrees C (K(A)=10(10) [M(-1)]). Although K(A) of the FN fragments of Hep-binding domain containing an EDA region [EDA(+)HBD(+)] and Hep increases with temperatures above 30 degrees C, K(A)s of HBD(+)-Hep and EDA(+)-Hep are not temperature-dependent. Therefore, EDA(+)HBD(+), formed as a special structure for high Hep affinity, exhibits temperature-dependent interaction with Hep. These results suggest that the main role of EDA(+)FN in cryogelation is to support the interaction with Hep.

Phosphorylation-dependent migration of retinoblastoma protein into the nucleolus triggered by binding to nucleophosmin/B23.

Underphosphorylated retinoblastoma (Rb) protein inhibits progression around the cell cycle by binding to transcription factors like E2F; subsequent hyperphosphorylation of Rb protein releases E2F from the complex so that it can then drive the cell into S phase. We immunolocalized Rb protein in human cells during the cell cycle. Rb protein translocated into nucleoli after DNA replication completed, and the nucleolar Rb was shown to be in the hyperphosphorylated form by immunoblotting. This form, but not its underphosphorylated counterpart, interacted with the nucleolar protein nucleophosmin/B23. The two formed a salt-resistant complex in vitro, and the two could be immunoprecipitated together from nucleolar extracts. These results suggest that hyperphosphorylated Rb protein is imported into nucleoli late in S or G2 phase with nucleophosmin/B23. Analysis of the nucleolar location of Rb protein using various deletion mutants tagged with the green fluorescent protein implicated pocket A of Rb protein as the region responsible for nucleolar targeting; this region also interacted with nucleophosmin/B23. Nucleolar translocation of Rb mutant was inhibited by introducing nucleophosmin/B23 antisense oligomer. These results suggest that nucleolar translocation of Rb protein is promoted by the binding with nucleophosmin/B23 via the pocket A region.