Selective Homologous Expression of Recombinant Manganese Peroxidase Isozyme of Salt-Tolerant White-Rot Fungus Phlebia sp. MG-60, and Its Salt-Tolerance and Thermostability.

Phlebia sp. MG-60 is the salt-tolerant, white-rot fungus which was isolated from a mangrove forest. This fungus expresses three kinds of manganese peroxidase (MGMnP) isozymes, MGMnP1, MGMnP2 and MGMnP3 in low nitrogen medium (LNM) or LNM containing NaCl. To date, there have been no reports on the biochemical salt-tolerance of these MnP isozymes due to the difficulty of purification. In present study, we established forced expression transformants of these three types of MnP isozymes. In addition, the fact that this fungus hardly produces native MnP in a high-nitrogen medium (HNM) was used to perform isozyme-selective expression and simple purification in HNM. The resulting MGMnPs showed high tolerance for NaCl compared with the MnP of Phanerochaete chrysosporium. It was worth noting that high concentration of NaCl (over 200 mM to 1200 mM) can enhance the activity of MGMnP1. Additionally, MGMnP1 showed relatively high thermo tolerance compared with other isozymes. MGMnPs may have evolved to adapt to chloride-rich environments, mangrove forest.

Vaccinium virgatum Aiton Leaves Extract Suppressed Lipid Accumulation and Uric Acid Production in 3T3-L1 Adipocytes.

Blueberry (Vaccinium virgatum Aiton; Kinisato 35 Gou) leaves have recently attracted increasing attention as a useful material for the prevention of lifestyle diseases. Here, we examined the effects of the hot water extract of blueberry leaves (BLEx) on lipogenesis and uric acid production in 3T3-L1 adipocytes. The results showed that BLEx suppressed lipid accumulation and the mRNA expression of differentiation markers in 3T3-L1 adipocytes. A fractionation study showed that the highly polymerized proanthocyanidin-rich fraction was responsible for this effect. Upon maturation to adipocytes, 3T3-L1 cells produced uric acid and tumor necrosis factor-α, and hypoxia stimulated the production of uric acid and xanthine oxidoreductase activity. BLEx suppressed the production of uric acid under these conditions. Although BLEx inhibited the enzymatic activity of xanthine oxidase, this activity was observed in several fractions containing catechin, epicatechin, chlorogenic acid, rutin, and low molecular weight proanthocyanidins. Taken together, these results indicate that BLEx contains various compounds with the ability to suppress lipid accumulation and uric acid production in adipocytes.

Onion (Allium cepa L.)-Derived Nanoparticles Inhibited LPS-Induced Nitrate Production, However, Their Intracellular Incorporation by Endocytosis Was Not Involved in This Effect on RAW264 Cells.

The aim of this study was to evaluate the involvement of nanoparticles prepared from Allium cepa L. as anti-inflammatory agents. In the present study, we identified nanoparticles from Allium cepa L. using the ultracentrifugation exosome purification method. The nanoparticles were referred to as 17,000× g and 200,000× g precipitates, and they contained quercetins, proteins, lipids, and small-sized RNA. The nanoparticles inhibited nitric oxide production from lipopolysaccharide (LPS)-stimulated RAW264 cells without cytotoxic properties. Cellular incorporation was confirmed by laser microscopic observation after PKH26 staining. The inhibition of caveolae-dependent endocytosis and macropinocytosis significantly prevented the incorporation of the nanoparticles but had no effect on the inhibition of nitric oxide in RAW264 cells. Collectively, the identified nanoparticles were capable of inhibiting the LPS response via extracellular mechanisms. Taken together, the way of consuming Allium cepa L. without collapsing the nanoparticles is expected to provide an efficient anti-inflammatory effect.

Enhanced water dispersibility and permeability through a Caco-2 cell monolayer of ß-cryptoxanthin extracted from kumquats by complexation with casein.

ß-Cryptoxanthin (BCX) possesses potential therapeutic and health benefits. However, BCX absorption is low because of its poor aqueous solubility. In this study, a complex between BCX and casein (Cas) was prepared to improve the water dispersibility and bioavailability of BCX. BCX was recovered quantitatively from freeze-dried kumquat powder through solid-liquid extraction and saponification. The complexation significantly improved the apparent solubility of BCX under acidic and neutral conditions. A cell membrane permeation test using a Caco-2 cell monolayer was performed to evaluate the bioavailability of the BCX-Cas complex. This complex and a blank sample were digested in vitro and added to the apical side of the Caco-2 cell membrane. The quantity of BCX that permeated using the BCX-Cas complex after 24 h was 22.7 times greater than that of the blank. Thus, complexation of BCX with Cas improved dramatically the bioavailability of BCX from a kumquat extract.

Blueberry Leaf Polyphenols Prevent Body Fat Accumulation in Mice Fed High-fat, High-sucrose Diet.

Blueberry leaf is currently a popular dietary supplement. Effects of dietary blueberry leaf and its active components on body fat accumulation were examined. C57BL/6J mice were fed high-fat, high-sucrose diet with or without 3% blueberry leaf extract (BLEx) or 3% concentrated-polyphenolic BLEx (CP BLEx) for 8 weeks. Compared to mice fed a high-fat, high-sucrose diet without blueberry leaf, BLEx and CP BLEx significantly reduced body weight and adipose tissue weight gain. Adipocytes were also smaller and and liver lipid accumulatioin was significantly inhibited in mice fed either BLEx or CP BLEx. These effects tended to be more pronounced in mice fed CP BLEx compared to in mice fed BLEx. Together, results suggest that blueberry leaf inhibits body fat accumulation typically observed in mice fed a high-fat, high-sucrose diet, and that inhibition is attributable to polyphenolic components in leaf extracts.

Vaccinium ashei leaves extract alleviates insulin resistance via AMPK independent pathway in C2C12 myotube model.

Blueberry (Vaccinium ashei) leaf has recently attracted attention as an anti-obesity food component. In this study, we examined the effects of blueberry leaf extract (BLEx) on insulin signaling in C2C12 differentiated myoblasts. The results showed that BLEx promotes the intracellular uptake of 2-[N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-D-glucose (2-NBDG) and phosphorylation of Akt under insulin stimulation. In addition, pretreatment with BLEx ameliorated TNF-α-nduced insulin resistance with regard to 2-NBDG uptake and Akt phosphorylation. Moreover, BLEx prevented the TNF-α-induced activation of JNK and NF-kB pathways and phosphorylation of IRS-1 at serine residue. BLEx failed to induce phosphorylation of AMPK as well as did not prevent the restoration of 2-NBDG uptake under TNF-α-induced insulin resistance. Overall, skeletal muscle is a putative target for the anti-diabetic effect of BLEx by amelioration of insulin resistance.

Preparation of Conjugated Linoleic Acid Microemulsions and their Biodistribution.

Conjugated linoleic acid (CLA) has several beneficial biological properties. Specifically, trans10, cis12-CLA, one of the CLA isomers, has strong physiologic activity against cancer and obesity. However, compared with cis9, trans11-CLA, a naturally occurring CLA isomer, trans10, cis12-CLA tends to be easily metabolized. Therefore, to make efficient use of its biological properties, it is necessary to overcome the rapid clearance of trans10, cis12-CLA from the blood. Here, we employed premix membrane emulsification to prepare two oil-in-water CLA microemulsions (CLA-ME), 100 nm CLA-ME and 200 nm CLA-ME, and investigated their pharmacokinetics in a mouse model. We report that 100 nm CLA-ME contributed to the concentration of blood CLA for longer than 200 nm CLA-ME, indicating that small CLA microparticles were more suitable for maintaining blood trans10, cis12-CLA levels in vivo. However, both CLA-ME could be hardly detected in blood and other tissues 24 h after administration, suggesting that additional strategies for prolonging CLA-ME half-life are required.

Suppressing effect of cordycepin on the lipopolysaccharide-induced nitric oxide production in RAW 264.7 cells.

In this study, we focused on the anti-inflammatory effect of cordycepin, 3'-deoxyadenosine. Cordycepin potently suppressed nitric oxide production in lipopolysaccharide-stimulated RAW 264.7 cells in an adenosine receptor-independent manner. In addition, inhibitors for adenosine kinase and nucleoside transporter abrogated the action of cordycepin. Thus, we considered that intracellular metabolism cordycepin is important for the anti-inflammatory effect of cordycepin.

Expression of a manganese peroxidase isozyme 2 transgene in the ethanologenic white rot fungus Phlebia sp. strain MG-60.

BACKGROUND: The white-rot fungus Phlebia sp. strain MG-60 was proposed as a candidate for integrated fungal fermentation process (IFFP), which unifies aerobic delignification and semi-aerobic consolidated biological processing by a single microorganism based on its ability to efficiently degrade lignin and ferment the sugars from cellulose. To improve IFFP, the development of a molecular breeding method for strain MG-60 is necessary. The purpose of this study is to establish the transformation method for the strain MG-60 and to obtain the over-expressing transformants of lignin-degrading enzyme, manganese peroxidase. FINDINGS: In the present study, the expression vector regulated by Phlebia brevispora glyceraldehyde-3-phosphate dehydrogenase promoter and terminator was constructed. A polyethylene glycol transformation method for the ethanol-fermenting white-rot fungus Phlebia sp. MG-60 was established with high transformation efficiency, and the manganese peroxidase isozyme 2 gene (MGmnp2) transformants were obtained, showing higher MnP activity than control transformants. MGmnp2 transformants showed higher selective lignin degradation on Quercus wood powder. CONCLUSIONS: This first report of MG-60 transformation provides a useful methodology for widely accessible to interested researches. These results indicate the possibility of metabolic engineering of strain MG-60 for improving IFFP.

Important role of ß1-integrin in fucoidan-induced apoptosis via caspase-8 activation.

Fucoidan induces apoptosis by activating caspase-8 in human MCF-7 breast cancer cells, but the detailed mechanism for this is not understood. We demonstrate here that fucoidan interacted with the cell surface, and silencing the ß1-integrin gene expression inhibited fucoidan-induced apoptosis accompanied by caspase-8 activation. Fucoidan induced formation of the ß1-integrin-caspase-8 complex. These data indicate that ß1-integrin is an important factor for the cell-surface binding of fucoidan and plays an important role in fucoidan-induced apoptosis. Fucoidan also induced recruitment of caspase-8 to the ß1-integrin intracellular domain, cleaved it into the activated protein by direct combination with ß1-integrin, and induced apoptosis via the caspase cascade in MCF-7 cells.

Induction of endoreduplication by a JNK inhibitor SP600125 in human lung carcinoma A 549 cells.

The effect of the pan c-Jun N-terminal kinase (JNK) inhibitor SP600125 on the proliferation of human lung carcinoma A549 cells has been evaluated. We have shown that SP600125 completely inhibited the proliferation of A549 cells, the cycle arrest being in G2/M phase. When cells were treated with SP600125 for >12h, a cell population with DNA content of 4n to 8n was detected. Moreover, the effect of SP600125 on the expression of cell cycle related proteins was an upregulation of p53 protein accompanied by an increase in its molecular mass. Prolonged SP600125 treatment downregulated p21, Bax and Mdm2 expression, but increased the level of the cellular p53-Mdm2 complex. Taken together, we show that SP600125 could induce G2/M cell cycle arrest and endoreduplication in a p21 independent manner, and that SP600125 could also post-translationally modify p53 to modify its function. Our data show that basic JNK activity plays an important role in the progression of the cell cycle at G2/M cell phase.