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.

Intensity-modulated radiation therapy with the central shielding technique for patients with uterine cervical cancer†.

We aimed to examine outcomes and toxicities of intensity-modulated radiation therapy (IMRT) with the central shielding (CS) technique for patients with uterine cervical cancer. This retrospective study included 54 patients with International Federation of Gynecology and Obstetrics IB-IVA cancer. Whole pelvic radiotherapy or extended-field radiotherapy were performed at the dose of 50.4 Gy in 28 fractions with helical tomotherapy (HT). Six patients had para-aortic lymph node metastases. The CS technique with HT was utilized after a total dose of 28.8-41.4 Gy to reduce doses to the rectum and bladder. The prescribed dose of intracavitary brachytherapy was mainly 18-24 Gy in three or four fractions at point A. Concurrent chemotherapy was used for 47 patients (87%). Median follow-up time was 56 months. Seventeen patients (31%) developed recurrence. The recurrence of the cervix was observed in two patients (4%). The 5-year rates of the locoregional control, progression-free survival (PFS) and overall survival were 79, 66 and 82%, respectively. Among several factors evaluated, histological type of adenocarcinoma was only a significantly worse prognostic factor for PFS by multivariate analysis (hazard ratio, 4.9 [95% confidence interval, 1.3-18], P = 0.018). Grade 2 or higher late toxicities were observed in nine patients (17%). Two patients (4%) each had grade 3 proctitis and grade 3 ileus, respectively. No grade 4 toxicity or treatment-related death was observed. The results suggest that IMRT with the CS technique allows a high local control without increasing the risk of complications for cervical cancer patients.

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.

cAMP-CRP acts as a key regulator for the viable but non-culturable state in Escherichia coli.

A variety of bacteria, including Escherichia coli, are known to enter the viable but non-culturable (VBNC) state under various stress conditions. During this state, cells lose colony-forming activities on conventional agar plates while retaining signs of viability. Diverse environmental stresses including starvation induce the VBNC state. However, little is known about the genetic mechanism inducing this state. Here, we aimed to reveal the genetic determinants of the VBNC state of E. coli. We hypothesized that the VBNC state is a process wherein specific gene products important for colony formation are depleted during the extended period of stress conditions. If so, higher expression of these genes would maintain colony-forming activities, thereby restraining cells from entering the VBNC state. From an E. coli plasmid-encoded ORF library, we identified genes that were responsible for maintaining high colony-forming activities after exposure to starvation condition. Among these, cpdA encoding cAMP phosphodiesterase exhibited higher performance in the maintenance of colony-forming activities. As cpdA overexpression decreases intracellular cAMP, cAMP or its complex with cAMP-receptor protein (CRP) may negatively regulate colony-forming activities under stress conditions. We confirmed this using deletion mutants lacking adenylate cyclase or CRP. These mutants fully maintained colony-forming activities even after a long period of starvation, while wild-type cells lost most of this activity. Thus, we concluded that the lack of cAMP-CRP effectively retains high colony-forming activities, indicating that cAMP-CRP acts as a positive regulator necessary for the induction of the VBNC state in E. coli.

IOP-lowering effect of isoquinoline-5-sulfonamide compounds in ocular normotensive monkeys.

Rho-associated coiled coil-formed protein kinase (ROCK) inhibitors are under development as a new class of antiglaucoma agents. Based on the potent ROCK inhibitor H-1152, previously developed by us, we explored the possibility of related compounds as antiglaucoma agents and synthesized seven types of H-1152-inspired isoquinoline-5-sulfonamide compounds (H-0103-H-0107, H-1001, H-1005). Although all of these compounds potently inhibited ROCK (IC50=18-48 nM), only H-0104 and H-0106 exerted strong intraocular pressure (IOP)-lowering effects into the eyes of monkeys. These results suggested the possibility that there is no direct relationship between ROCK inhibition and IOP-lowering effects, indicating that the initial screening of compounds based on ROCK inhibitory activity may be an unsuitable strategy for developing antiglaucoma agents with potent IOP-lowering effects.

Xanthine oxidase inhibitory activity and hypouricemic effect of aspalathin from unfermented rooibos.

Rooibos is rich in flavonoids such as aspalathin, which is a unique C-glycosyl dihydrochalcone, that is used as a traditional herbal tea. This study was designed to evaluate the in vitro xanthine oxidase (XOD) inhibitory activity of the aspalathin-rich fraction (ARF) and purified aspalathin from rooibos. The hypouricemic effects of the ARF and aspalathin on hyperuricemic mice were also assessed. The ARF was prepared from aqueous extract of unfermented rooibos leaves and stems, and it was collected by column chromatography; the aspalathin content in this fraction was 21.4%. The ARF and aspalathin inhibited XOD in a dose-dependent manner. The concentrations of the ARF and aspalathin required to inhibit XOD at 50% (IC50 ) were 20.4 µg/mL (4.4 µg/mL aspalathin equivalents) and 4.5 µg/mL, respectively. Lineweaver-Burk plot analysis indicated that aspalathin was a competitive inhibitor of XOD, and the inhibition constant (Ki) was 3.1 µM. In hyperuricemic mice induced by inosine-5'-monophosphate, treatment with the ARF and aspalathin significantly suppressed the increased plasma uric acid level in a dose-dependent manner. The suppressed plasma uric acid level in mice could be attributed to the XOD inhibitory activity of the ARF and aspalathin. Further study is required to determine the effect of aspalathin or its metabolites on XOD activity in vivo.

Hippocampal leptin suppresses methamphetamine-induced hyperlocomotion.

Leptin is an anorexigenic peptide which is synthesized in white adipose tissue. The actions of leptin are mediated by the leptin receptor which is abundantly localized in the hypothalamus and is involved in energy regulation and balance. Recently, there has been evidence suggesting that the leptin receptor is also present in the hippocampus and may be involved with hippocampal excitability and long-term depression. To investigate the physiological function of leptin signalling in the hippocampus, we studied the effects of leptin on methamphetamine-induced ambulatory hyperactivity by utilizing intra-hippocampal infusion (i.h.) in mice. Our results show that the infusion of leptin (5 ng each bilaterally i.h.) does not affect the basal ambulatory activity but significantly suppresses methamphetamine-induced ambulatory hyperactivity as compared to saline-infused controls. Interestingly, higher dose of leptin increases the suppression of the methamphetamine-induced ambulatory hyperactivity. The i.h. infusion of leptin did not activate the JAK-STAT pathway, which is the cellular signalling pathway through which leptin acts in the hypothalamus. The infusion of leptin also did not affect activation of p42/44 MAPK which is known to be another leptin-induced signalling pathway in the brain. These results demonstrate that leptin has a novel potential suppressive effect on methamphetamine-induced hyperlocomotion and also suggest that there must be an alternative pathway in the hippocampus through which leptin signalling is being mediated.

BDNF-mediated cerebellar granule cell development is impaired in mice null for CaMKK2 or CaMKIV.

The Ca(2+)/calmodulin-activated kinases CaMKK2 and CaMKIV are highly expressed in the brain where they play important roles in activating intracellular responses to elevated Ca(2+). To address the biological functions of Ca(2+) signaling via these kinases during brain development, we have examined cerebellar development in mice null for CaMKK2 or CaMKIV. Here, we demonstrate that CaMKK2/CaMKIV-dependent phosphorylation of cAMP response element-binding protein (CREB) correlates with Bdnf transcription, which is required for normal development of cerebellar granule cell neurons. We show in vivo and in vitro that the absence of either CaMKK2 or CaMKIV disrupts the ability of developing cerebellar granule cells in the external granule cell layer to cease proliferation and begin migration to the internal granule cell layer. Furthermore, loss of CaMKK2 or CaMKIV results in decreased CREB phosphorylation (pCREB), Bdnf exon I and IV-containing mRNAs, and brain-derived neurotrophic factor (BDNF) protein in cerebellar granule cell neurons. Reexpression of CaMKK2 or CaMKIV in granule cells that lack CaMKK2 or CaMKIV, respectively, restores pCREB and BDNF to wild-type levels and addition of BDNF rescues granule cell migration in vitro. These results reveal a previously undefined role for a CaMKK2/CaMKIV cascade involved in cerebellar granule cell development and show specifically that Ca(2+)-dependent regulation of BDNF through CaMKK2/CaMKIV is required for this process.

The effect of the H-1152P, a potent Rho-associated coiled coil-formed protein kinase inhibitor, in rabbit normal and ocular hypertensive eyes.

PURPOSE: We wanted to determine the intraocular pressure (IOP)-lowering effect of H-1152P by utilizing a rabbit ocular hypertension-glaucoma model and normal eyes. H-1152P is a potent, Rho-associated, coiled, coil-forming protein kinase (ROCK) inhibitor. METHODS: IOPs were monitored by a pneumatonometer in New Zealand White rabbits that were given topically administered H-1152P or vehicle alone. Animals were divided into four groups followed by topical administration of 0.1, 1.0, 10, and 28 mM H-1152P. To study the IOP-lowering effects on an elevated IOP model, a rabbit ocular hypertension model was created by water loading. All studies were carried out by monitoring of IOPs on H-1152P-administered right eyes and phosphate-buffered saline (PBS)-administered left eyes. RESULTS: In normotensive IOP rabbits, topical administration of H-1152P significantly decreased IOPs by 46.1 +/- 5.0% at 1% (28 mM) solution. This effect was dose dependent, as the maximum reduction of IOPs were observed between 60 and 90 min after topical administration (3.6 +/- 0.9 mmHg, 5.4 +/- 0.7 mmHg, 6.8 +/- 0.7 mmHg, and 7.2 +/- 1.9 mmHg at 0.1, 1.0, 10, and 28 mM H-1152P). In addition, in the rabbit ocular hypertension model, the topical administration of H-1152P (28 mM) significantly lowered IOPs starting at 30 minutes and lasting up to 300 minutes after water loading. The maximum IOP reduction, however, was observed at 90 minutes after water loading (10.6 +/- 2.3 mmHg). No serious side effects were observedin ocular tissues except for some conjunctival congestion that shortly disappeared within 3 hours. CONCLUSION: Topical administration of H-1152P potently decreased rabbit normotensive IOPs in a dose-dependent manner, and the duration of the IOP lowering was also elongated in a dose-dependent manner. In addition, H-1152P has a potent IOP-lowering effect on an ocular hypertension model. These result suggested that H-1152P could be a candidate for the next generation of glaucoma therapy.

The effect of the Rho-associated protein kinase inhibitor, HA-1077, in the rabbit ocular hypertension model induced by water loading.

PURPOSE: The aim of this study was to investigate the intraocular pressure (IOP)-lowering effect of a new anti-glaucoma drug, the Rho-associated protein kinase (ROCK) inhibitor, HA-1077, in a rabbit ocular hypertension model. METHODS: Experiments were carried out in 18 male New Zealand white rabbits, with ocular hypertension induced by water loading. Animals were divided into three groups followed by topical administration of 1 mM, 2 mM, and 3 mM HA-1077 in the left eye. As a control, phosphate buffered saline was administered in the opposite eye. RESULTS: After administration of HA-1077 eye drops, there was a significant time- and dose-dependent decrease of the IOP. While minor conjunctival injection was seen in a few cases, no abnormalities of the anterior chamber or fundus were observed. CONCLUSIONS: This is the first report of the effect of the ROCK inhibitor, HA-1077, on the IOP in an ocular hypertension model. Study results indicated that HA-1077 has a strong IOP-lowering effect.

Investigation of binding proteins for anti-platelet agent K-134 by Drug-Western method.

K-134 ((-)-6-[3-[3-cyclopropyl-3-[(1R, 2R)-2-hydroxycyclohexyl]ureido]-propoxy]-2(1H)-quinolinone) is a novel anti-platelet agent with anti-hyperplastic activities. We found previously that K-134 is a potent phosphodiesterase-3 (PDE3) inhibitor. In the present study, we found other K-134-binding proteins by Drug-Western method. We isolated two clones that can bind directly to K-134, cofilin-2, and CD36 in vitro. Comparison of their amino acid sequences showed similarity over a short stretch [KxxxxVxIxWxxE] in part in the collagen-binding region of CD36. K-134 inhibited binding between CD36 and collagen type-I; however, other PDE3 inhibitors, cilostazol, amrinone, and an inactive derivative of K-134, 4S-OH-K-134, showed little or no effect on binding. It was strongly suggested that the direct binding between K-134 and CD36 is a characteristic effect of K-134, and the homologous stretch may be necessary for binding to K-134. These results also suggested that these interactions are involved in the mechanisms of the anti-platelet and anti-hyperplastic effects of K-134.

Subcellular localization of estrogen receptor beta in mouse hippocampus.

While estrogen receptors have been known to represent estrogen-dependent transcription factors as part of the nuclear receptor family, a putative membrane-bound form of estrogen receptors has been suggested. Since estrogen receptor beta (ERbeta) is reportedly abundant in the hippocampus and other regions of the central nervous system, subcellular localization of ERbeta in mouse hippocampus was investigated. ERbeta was predominantly found in nuclear, synaptosomal and synaptic membrane fractions, particularly this last fraction. Immunocytochemical investigation using the NG108-15 neuroblastoma-glioma hybridoma cell line indicated that ERbeta is predominantly localized in cell membranes and nuclei. These results suggest that ERbeta localizes on synaptic membranes and may represent an important regulator of intracellular signal transduction from membrane to cytosol in hippocampal neurons.

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.

Role of hippocampal alpha(2A)-adrenergic receptor in methamphetamine-induced hyperlocomotion in the mouse.

Psychostimulants, such as methamphetamine (METH), induce psychological dependence and we recently suggested that hippocampal alpha(2A)-adrenergic receptor (alpha(2A)-AR) is involved in METH-induced modulation of central nervous systems. The present study shows that pretreatment with yohimbine dose-dependently decreased the ambulatory hyperactivities induced by METH (2 mg/kg) in mice. Moreover, specific knock-down of the hippocampal alpha(2A)-AR with infusion of anti-sense oligo DNA of alpha(2A)-AR significantly suppressed ambulatory activity induced by METH administration. Infusion of sense oligo DNA of alpha(2A)-AR into mouse hippocampus exerted no effects on the ambulatory activity. These observations strongly suggest the involvement of hippocampal alpha(2A)-AR in the regulation of ambulatory activity induced by METH administration.

Measurement of the density of CO(2) solution by Mach-Zehnder interferometry.

The density of CO(2) solution was measured by using Mach-Zehnder interferometry in the pressure range from 5.0 to 12.5 MPa, at temperatures from 273.25 to 284.15 K, and CO(2) mass fraction in solution up to 0.061. It was found that the density difference between the CO(2) solution and pure water at the same pressure and temperature is monotonically linear with the CO(2) mass fraction. The slope of this linear function, calculated by experimental data fitting, is 0.275.

Numerical visualization of two-phase plume formation in a stratification flow environment.

Evolution of two-phase plumes driven by air bubble buoyancy in a stratification ambient in a rectangular tank is visualized numerically by means of two-phase flow theory and large-eddy simulation technology. With a focus on the discrete nature of the buoyant dispersed phase and the role of momentum exchange between two phases in plume formation, we investigated the phenomena of mass entraining-in and peeling-out for continuous phase plume, which may result from a complicated and intricate interplay with phase interaction and dynamic stability of the stratification ambient, respectively. Numerical simulations show that although mass entraining-in and peeling-out appear to be distinguished entirely in the vertical direction, they interact or couple locally within inner of the plume and present a discontinuity in nature. The numerically visualized three-dimensional density field also indicates the same plume characteristics.

Flow characteristics of two immiscible liquid layers subjected to a horizontal temperature gradient.

Marangoni convection, driven by an interfacial instability due to a surface tension gradient, presents a significant problem in crystal growth in normal microgravity environments. It is important to suppress and control the convection phenomenon for better material processing, especially in crystal growth by the liquid encapsulated Czochralski or liquid encapsulated floating zone techniques, in which the melt is encapsulated in an immiscible medium. Marangoni convection can occur on the liquid-liquid interface and on the gas-liquid free surface. Buoyancy driven convection can also affect and complicate the flow. In the study we report here, experiments were carried out with two liquid layers, silicone oil and fluorinert, in an open and enclosed rectangular cavity. The flow in the cavity was subjected to a horizontal temperature gradient. The interactive flow near the liquid-liquid interface was measured by the particle image velocimetry technique. The measured flow field is in agreement with numerical predictions. Free surface fluctuations with several dominant frequencies were also measured.