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.

scRepli-Seq: A Powerful Tool to Study Replication Timing and Genome Instability.

Advances in "omics" technology have made it possible to study a wide range of cellular phenomena at the single-cell level. Recently, we developed single-cell DNA replication sequencing (scRepli-seq) that measures replication timing (RT) by copy number differences between replicated and unreplicated genomic DNA in replicating single mammalian cells. This method has been used to reveal previously unrecognized static and dynamic natures of several hundred kilobases to a few megabases-scale chromosomal units called RT domains. Because RT domains are highly correlated to A/B compartments detected by Hi-C, scRepli-seq data can be used to predict the 3D organization of the genome in the nuclear space. scRepli-seq, which essentially measures the copy number, can also be applied to study genome instability.

The Temporal Order of DNA Replication Shaped by Mammalian DNA Methyltransferases.

Multiple epigenetic pathways underlie the temporal order of DNA replication (replication timing) in the contexts of development and disease. DNA methylation by DNA methyltransferases (Dnmts) and downstream chromatin reorganization and transcriptional changes are thought to impact DNA replication, yet this remains to be comprehensively tested. Using cell-based and genome-wide approaches to measure replication timing, we identified a number of genomic regions undergoing subtle but reproducible replication timing changes in various Dnmt-mutant mouse embryonic stem (ES) cell lines that included a cell line with a drug-inducible Dnmt3a2 expression system. Replication timing within pericentromeric heterochromatin (PH) was shown to be correlated with redistribution of H3K27me3 induced by DNA hypomethylation: Later replicating PH coincided with H3K27me3-enriched regions. In contrast, this relationship with H3K27me3 was not evident within chromosomal arm regions undergoing either early-to-late (EtoL) or late-to-early (LtoE) switching of replication timing upon loss of the Dnmts. Interestingly, Dnmt-sensitive transcriptional up- and downregulation frequently coincided with earlier and later shifts in replication timing of the chromosomal arm regions, respectively. Our study revealed the previously unrecognized complex and diverse effects of the Dnmts loss on the mammalian DNA replication landscape.

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.

The CDK-PLK1 axis targets the DNA damage checkpoint sensor protein RAD9 to promote cell proliferation and tolerance to genotoxic stress.

Genotoxic stress causes proliferating cells to activate the DNA damage checkpoint, to assist DNA damage recovery by slowing cell cycle progression. Thus, to drive proliferation, cells must tolerate DNA damage and suppress the checkpoint response. However, the mechanism underlying this negative regulation of checkpoint activation is still elusive. We show that human Cyclin-Dependent-Kinases (CDKs) target the RAD9 subunit of the 9-1-1 checkpoint clamp on Thr292, to modulate DNA damage checkpoint activation. Thr292 phosphorylation on RAD9 creates a binding site for Polo-Like-Kinase1 (PLK1), which phosphorylates RAD9 on Thr313. These CDK-PLK1-dependent phosphorylations of RAD9 suppress checkpoint activation, therefore maintaining high DNA synthesis rates during DNA replication stress. Our results suggest that CDK locally initiates a PLK1-dependent signaling response that antagonizes the ability of the DNA damage checkpoint to detect DNA damage. These findings provide a mechanism for the suppression of DNA damage checkpoint signaling, to promote cell proliferation under genotoxic stress conditions.

Direct visualization of replication dynamics in early zebrafish embryos.

We analyzed DNA replication in early zebrafish embryos. The replicating DNA of whole embryos was labeled with the thymidine analog 5-ethynyl-2'-deoxyuridine (EdU), and spatial regulation of replication sites was visualized in single embryo-derived cells. The results unveiled uncharacterized replication dynamics during zebrafish early embryogenesis.

Direct Visualization of DNA Replication Dynamics in Zebrafish Cells.

Spatiotemporal regulation of DNA replication in the S-phase nucleus has been extensively studied in mammalian cells because it is tightly coupled with the regulation of other nuclear processes such as transcription. However, little is known about the replication dynamics in nonmammalian cells. Here, we analyzed the DNA replication processes of zebrafish (Danio rerio) cells through the direct visualization of replicating DNA in the nucleus and on DNA fiber molecules isolated from the nucleus. We found that zebrafish chromosomal DNA at the nuclear interior was replicated first, followed by replication of DNA at the nuclear periphery, which is reminiscent of the spatiotemporal regulation of mammalian DNA replication. However, the relative duration of interior DNA replication in zebrafish cells was longer compared to mammalian cells, possibly reflecting zebrafish-specific genomic organization. The rate of replication fork progression and ori-to-ori distance measured by the DNA combing technique were ∼ 1.4 kb/min and 100 kb, respectively, which are comparable to those in mammalian cells. To our knowledge, this is a first report that measures replication dynamics in zebrafish cells.