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Background/Purpose Some health-beneficial foods, including adzuki beans (Vigna angularis), can show antihyperglycemic effects. Adzuki bean-derived polyphenols, such as (+)-catechin 7-O-β-d-glucopyranoside (C7G) and (+)-epicatechin 7-O-β-d-glucopyranoside (E7G), could be potential inhibitors of α-amylase and α-glucosidase, however, few studies of the activity of adzuki polyphenols have been performed in vivo. Thus, we evaluated the antihyperglycemic effects of crude adzuki bean extract containing polyphenols (ABP), and purified C7G or E7G using mice and Caco-2 cells.Methods Six-week-old male ICR mice were orally administered ABP (100 or 250 mg/kg), C7G or E7G (15 or 40 mg/kg), and sucrose (2 g/kg). Blood samples were taken from the lateral tail vein to measure the blood glucose levels at various times over 0–120 min. Cultured caco-2 cells, an intestinal model, were treated with ABP, C7G, or E7G. The glucose concentration in the medium was measured to examine the activity of α-glucosidase.Results Administration of adzuki polyphenols decreased blood glucose levels, especially E7G administration significantly showed antihyperglycemic effects in vivo. In Caco-2 cells, α-glucosidase activity was significantly decreased by ABP, C7G, or E7G addition in a dose-dependent manner without cytotoxicity.Conclusion These results suggest that C7G and E7G in adzuki bean extract inhibited sucrose-degrading enzymes to elicit antihyperglycemic effects in vivo, which supports the results of previous studies using adzuki beans, and may contribute to health promotion.

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.

Using recorded data of pressure and temperature, we developed a nondestructive method to estimate the precipitation rate of carbon dioxide (CO2) hydrate by considering the CO2solution density equation in association with the mass conservation equation. We applied this method to the investigation of the dynamic process of clathrate hydrate precipitation from CO2solution using a high-pressure dissolution system consisting of a high-pressure vessel and optical detecting instruments. The role of stirring was examined. The temperatures studied in the experiments were from 275 to 288 K over a pressure range of 4∼8 MPa. Experimental results showed that our method can quantitatively monitor this dynamic process. The volume ratio of precipitated hydrate to that of the pressure-vessel approached 0.09 when a steady state was reached, which took about 100 seconds; more than 60% of the total clathrate hydrate precipitated within 10 seconds at the cooling rates of this experiment. For precipitation, stirring enhanced the cooling and led to a large hydrate precipitation rate from 0.004 to 0.008 in volume ratio per second. For nucleation, however, the residual structure of the solution decreased the amount of hysteresis in the formation of hydrate nuclei during supercooling, of which induction times were reduced by 4∼9 minutes.

To study the biological impacts of CO2ocean sequestration on floating marine organisms, a full Eulerian-Eulerian scheme model has been developed in a large-eddy simulation (LES) version using one-way coupling of the equations of seawater flow to the transport equations of the bio-scalar variables. Special attention was paid to deriving the transport equation, involving non-conservative scalars to describe the degree of injury to floating organisms due to the change in the pH environment resulting from CO2dissolution. The source terms of the transport equations of bio-scalar variables are based on experimental data on zooplankton activities affected by lower pH seawater, allowing construction of empirical sub-models of three kinds of floating marine organisms: Gaidius variabilis, Paraeuchaeta Birostrata, and Multi-organisms. An example is given to show the applicability of the model to the assessment of the biological impact of CO2sequestration in the ocean. Given an initial CO2droplet diameter of 8.0 mm and an injection rate of 1.0 kg/sec, the model simulation predicts that the zooplanktons lose approximately 90% of their activity when the lowest pH inside the plume decreases from 7.57 to 5.61. These injured zooplanktons then recovered gradually to their normal state within two hours due to dilution of the plume.

Using recorded data of pressure and temperature, we developed a nondestructive method to estimate the precipitation rate of carbon dioxide (CO2) hydrate by considering the CO2solution density equation in association with the mass conservation equation. We applied this method to the investigation of the dynamic process of clathrate hydrate precipitation from CO2solution using a high-pressure dissolution system consisting of a high-pressure vessel and optical detecting instruments. The role of stirring was examined. The temperatures studied in the experiments were from 275 to 288 K over a pressure range of 4∼8 MPa. Experimental results showed that our method can quantitatively monitor this dynamic process. The volume ratio of precipitated hydrate to that of the pressure-vessel approached 0.09 when a steady state was reached, which took about 100 seconds; more than 60% of the total clathrate hydrate precipitated within 10 seconds at the cooling rates of this experiment. For precipitation, stirring enhanced the cooling and led to a large hydrate precipitation rate from 0.004 to 0.008 in volume ratio per second. For nucleation, however, the residual structure of the solution decreased the amount of hysteresis in the formation of hydrate nuclei during supercooling, of which induction times were reduced by 4∼9 minutes.

We established a precise DeLIF (Dual Emission Laser Light Fluorescence) system for the instant measurement of the pH field in laboratory experiments. This method is very important in studies of the biological impacts of CO2sequestration, i.e., effects of pH on organisms. The technique is demonstrated by applying it to the pH measurement around a liquid CO2droplet, which has a large gradient of CO2concentration near its interface, the concentration value on the boundary being known as the solubility of CO2. In the Laser Induced Fluorescence (LIF) method using quinine and an ultraviolet laser light source, the relatively unstable pulse of the laser source decreased the measurement reliability. We therefore investigated many kinds of fluorescent dye and demonstrated the DeLIF measurement, identifying a process for cancelling fluctuations of laser light intensity. Quinine and sulforhodamine were used as fluorescence dyes. The molar absorption coefficient and emission intensity of quinine depend on the pH of aqueous solutions, while those of sulforhodamine are not as much affected by pH. The ratio of these fluorescence intensities represents pH at each point of the image. As a result, the pH field was successfully measured with an uncertainty of only approximately ±0.005, for 3.0 < pH.

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 α. From these observations, important roles of both protein B23 isoforms in stimulation of DNA polymerase α activity during rat liver regeneration were suggested.

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.

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-inand peeling-outfor 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-inand peeling-outappear 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.

The density of CO2solution 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 CO2mass fraction in solution up to 0.061. It was found that the density difference between the CO2solution and pure water at the same pressure and temperature is monotonically linear with the CO2mass fraction. The slope of this linear function, calculated by experimental data fitting, is 0.275.

We have recently isolated a mammary growth factor from the conditioned medium of mouse mammary stromal fibroblasts and identified it as a mouse homologue of human HGF (hepatocyte growth factor). To elucidate the role of HGF in mouse mammary tumorigenesis, we produced recombinant mouse HGF and examined its effects on primary cultures of mouse mammary tumor cells in this study. HGF at concentrations above 20 ng/ml maximally stimulated the growth of mammary tumor cells in primary monolayer culture. HGF also stimulated the three‐dimensional growth and branching morphogenesis of mammary tumor cells cultured inside collagen gels. A comparison of the growth‐stimulating activity of HGF with that of EGF (epidermal growth factor) and KGF (keratinocyte growth factor) revealed that HGF is the most potent growth factor among the three. Immunological studies using an antibody against mouse HGF demonstrated that 74% of the growth‐stimulating activity present in the mammary fibroblast‐conditioned medium was abolished by the antibody, indicating that HGF is the major growth factor produced by the fibroblasts. These observations thus suggest a role for HGF as a mammary stromal fibroblast‐derived factor which stimulates growth and morphogenesis of adjacent mammary tumor cells in vivo.

We earlier confirmed that there are isoforms of Ca2+/calmodulin (CaM)-dependent protein kinase I (CaM kinase I) (CaM kinase Iβ1 and Iγ) beside CaM kinase Iα by cDNA cloning (Yokokura, H., Terada, O., Naito, Y., and Hidaka, H. (1997) Biochim. Biophys. Acta1338, 8–12). Here, we demonstrate the existence of an isoform-specific activation mechanism of CaM kinase I and alternative splicing specifically regulating CaM kinase I (CaM kinase Iβ2) in the central nervous system. To cast light on isoform structure-enzyme activity relationships, CaM kinase Iβ1, Iβ2, and Iα were expressed separately using a baculovirus/Sf9 cell expression system. The novel CaM kinase Iβ2 isoform demonstrated similar catalytic activity to those of CaM kinase Iβ1 and Iα. Interestingly, CaM kinase Iβ1 and Iβ2 both can activate CaM kinase Iα activity via phosphorylation at Thr177. Reverse transcribed-polymerase chain reaction analysis showed that CaM kinase Iβ2 is dominant in the cerebrum and cerebellum, whereas CaM kinase Iβ1 is present in peripheral tissues such as liver, heart, lung, kidney, spleen, and testis. CaM kinase Iβ2 was also detected with an anti-CaM kinase Iβ2 antibody in PC12 cells. The results indicate that alternative splicing is a means for tissue-specific expression of CaM kinase Iβ. Thus the Thr177residue of CaM kinase Iα is phosphorylated by not only CaM kinase kinase but also CaM kinase Iβ for activation of the enzyme.

Novel type tin enolate is easily formed by the regioselective ring cleavage of diketene with Bis(tributyltin) oxide [(n-Bu3Sn)2O]. The enolate caused carbon-carbon bond formation with aldehydes. As a result, a,ß-unsaturated methyl ketones were obtained in good yields, accompanied by decarboxylation.

Tin enolate derived from diketene and (n-Bu3Sn)2O was alkylated by alkyl halides, leading to mono-alkylated enolate via facile decarboxylation, which successively react with other electrophiles like an aldehyde and alkyl halides to furnish double-alkylated acetones.

Most of the neuronal nitric oxide synthase (nNOS) is present in the particulate fraction of tissue extracts. Here, we show that the calmodulin (CaM)-binding domain of nNOS interacts with anionic phospholipid vesicles but not with neutral ones. Identification of residues in the CaM-binding domain of nNOS as the key domain for the interaction is also documented. Recombinant wild-type nNOS was found to associate with phosphatidylserine (PS) or phosphatidic acid (PA) but not with phosphatidylethanolamine (PE) or phosphatidylcholine (PC), indicating that nNOS-phospholipid binding requires an electrostatic interaction. A synthetic peptide corresponding to residues 732–754 blocked the interaction of nNOS with PS. Furthermore, a purified fusion protein containing residues 724–755 interacted with PS in a competitive fashion with CaM. Inactive nNOS lacking CaM-binding ability, generated by mutation of (Lys732LysLeu) to (Asp732AspGlu) (Watanabe, Y., Hu, Y., and Hidaka, H.,FEBS Lett.403, 75–78, 1997) did not interact with PS. Preincubation of nNOS with PS protected subsequent limited proteolysis of the synthase byStaphylococcus aureusV8 protease, probably as a result of conformational changes in the protein. Wild-type nNOS was found almost entirely in the membrane fraction of Sf9 cells, whereas inactive nNOS was also found in cytosolic fraction in Sf9 cells expressing the mutant enzyme. These results demonstrate that the mutated hydrophobic/basic amino acid cluster in nNOS sequence, Lys732LysLeu, is essential for nNOS–PS and nNOS–CaM interactions.

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.