Effect of Royal Sun Medicinal Mushroom Agaricus brasiliensis KA21 (Agaricomycetes) Cultivated Outdoors on Wound Healing and Maintaining Skin Elasticity.

Royal sun medicinal mushroom Agaricus brasiliensis KA21 has various beneficial pharmacological effects; however, these effects are influenced by its cultivation conditions. A. brasiliensis KA21 is rich in ß-glucan, which promotes wound healing. This study evaluated the wound healing-promoting effects of A. brasiliensis KA21 and the influence of cultivation conditions on these effects. Upon evaluation of its effect on wound healing, the outdoor-cultivated A. brasiliensis KA21 (KAOD) promoted wound healing in mice, and the effect of KAOD was stronger than that of the indoor-cultivated one (KAID). In addition, A. brasiliensis KA21 promoted the synthesis of collagen I and III, which are involved in promoting wound healing; KAOD strongly induced collagen III production. Furthermore, KAOD suppressed the decrease in skin elasticity after acetone application in our mouse model, which was not observed for KAID. These results show that KAOD is useful as a supplement in surgery and injury healing for humans and animals, improving immunity against pathogens in wound areas and promoting wound healing. In addition, KAOD may be useful in the field of skin aesthetics for repairing skin damage and maintaining skin elasticity.

Effects of Outdoor-Grown Royal Sun Medicinal Mushroom Agaricus brasiliensis KA21 (Agaricomycetes) Fruiting Body on Canine Malassezia Dermatitis.

Brazil-grown outdoor-cultivated Agaricus brasiliensis KA21 fruiting body (KA21) significantly increases the production of serum anti-beta-glucan antibody. Therefore, KA21 ingestion may be useful for the prevention and alleviation of fungal infections. This study aimed to determine the effects of KA21 in fungal infections in animals. KA21 was administered to nine dogs infected with Malassezia. Notably, the anti-beta-glucan antibody titer remained unchanged or tended to decrease in the oral steroid arm, whereas in the non-steroid arm, antibody titer increased in almost all animals after KA21 ingestion. Dogs showing improved clinical symptoms exhibited increased anti-beta-glucan antibody titers. The results of this study suggest that KA21 ingestion may alleviate the symptoms of Malassezia and other fungal infections and that continuous ingestion may help prolong recurrence-free intervals. Additionally, the ingestion of KA21 during oral steroid dosage reduction or discontinuation may enable smoother steroid withdrawal.

Dectin-1 Reactivity to Paramylon Derived from Euglena gracilis EOD-1.

Euglena gracilis is a microalga that has recently attracted attention because of its bioactivities. Paramylon (PM), a major ß-1,3-glucan, constitutes 70-80% of the cells of the E. gracilis EOD-1 strain. Dectin-1 is a pattern recognition receptor that recognizes ß-glucan. However, it is unclear whether PM binds to dectin-1. In this study, we investigated the reactivity of EOD1PM with dectin-1 by analyzing the binding of soluble murine and human dectin-1-Fc fusion protein (m dectin-1 Fc, h dectin-1 Fc) to EOD1PM using flow cytometry and enzyme-linked immunosorbent assay (ELISA). m Dectin-1 Fc bound to EOD1PM particles when m dectin-1-Fc is added. Furthermore, the binding specificity was examined in a competitive reaction following addition of a soluble antigen. It was found that the binding of m dectin-1-Fc to EOD1PM was not inhibited by the addition of dextran or ovalbumin but by the addition of solubilized EOD1PM or Candida cell wall- solubilized ß-glucan. In addition, the h dectin-1-Fc fusion protein was found to specifically bind to EOD1PM. These results suggest that dectin-1 recognizes and binds to the ß-glucan structure of EOD1PM. Dectin-1 is expressed in leukocytes as a ß-glucan receptor and is involved in the expression of various biological activities; therefore, the dectin-1 pathway may be involved in the biological activity of EOD1PM.

Development of a Highly Sensitive ß-Glucan Detection System Using Scanning Single-Molecule Counting Method.

To overcome the limitations of the Limulus amebocyte lysate (LAL) assay method for the diagnosis of invasive fungal infection, we applied a reaction system combining recombinant ß-glucan binding proteins and a scanning single-molecule counting (SSMC) method. A novel (1→3)-ß-D-glucan recognition protein (S-BGRP) and a (1→6)-ß-glucanase mutant protein were prepared and tested for the binding of (1→6)-branched (1→3)-ß-D-glucan from fungi. S-BGRP and (1→6)-ß-glucanase mutant proteins reacted with ß-glucan from Candida and Aspergillus spp. Although LAL cross-reacted with plant-derived ß-glucans, the new detection system using the SSMC method showed low sensitivity to plant (1→3)-ß-D-glucan, which significantly improved the appearance of false positives, a recognized problem with the LAL method. Measurement of ß-glucan levels by the SSMC method using recombinant ß-glucan-binding proteins may be useful for the diagnosis of fungal infections. This study shows that this detection system could be a new alternative diagnostic method to the LAL method.

Binding Specificity of a New Artificial ß-Glucan Recognition Protein and Its Application to ß-Glucan Detection in Mushroom Extracts.

ß-1,3-D-glucan (BG) activates innate immunity and enhances immune responses. Fungi, such as mushrooms, produce a relatively large amount of BG, the structure and molecular weight of which varies depending on the species of fungi. This study was conducted to develop a detection probe for quantifying or detecting BG from fungi using BG-binding proteins. The binding properties of a new ß-glucan recognition protein (BGRP) against various BGs were compared. With reference to the amino acid sequences of BGRP in insects, an artificial BGRP (supBGRP) was designed with higher production efficiency using gene recombination technology. SupBGRP was produced in Escherichia coli with high efficiency, and its reactivity with BG from fungi was the highest among the BG-binding proteins examined. SupBGRP exhibited high reactivity with 1,6-branched BG and will be useful for the quantification and detection of fungal BG.

Possibility of Japanese Cedar Pollen Causing False Positives in the Deep Mycosis Test.

Because Japanese cedar pollen (JCP) contains beta-1,3-d-glucan (BG), there is concern that its lingering presence in the atmosphere, especially during its scattering period, may cause false positives in the factor-G-based Limulus amebocyte lysate (LAL) assay used to test for deep mycosis (i.e., G-test). Hence, we examined whether the LAL assay would react positively with substances contained in JCP by using the G-test to measure JCP particles and extracts. BG was purified from the JCP extract on a BG-specific affinity column, and the percentage extractability was measured using three different BG-specific quantitative methods. The G-test detected 0.4 pg BG in a single JCP particle and 10 fg from a single particle in the extract. The percentage extractability of JCP-derived BG was not significantly different among the three quantitative methods. As the JCP particles should technically have been removed during serum separation, they should be less likely to be a direct false-positive factor. However, given that the LAL-assay-positive substances in the JCP extract were not distinguishable by the three BG-specific quantitative methods, we conclude that they may cause the background to rise. Therefore, in Japan false positives arising from JCP contamination should be considered when testing patients for deep mycosis.

Open-Label Study of the Influence of Food Containing the Royal Sun Mushroom, Agaricus brasiliensis KA21 (Agaricomycetes), on ß-Glucan-Specific Antibody Production in Healthy Human Volunteers.

The edible mushroom Agaricus brasiliensis contains a large amount ß-glucan, which is mainly composed of a ß-1,6-glucan structure. In this study, we investigated the effect of A. brasiliensis strain KA21 on the anti-ß-glucan antibody titer in healthy humans and the role of antibodies as an immunomodulator. Twenty-two healthy volunteers were fed the dried fruiting body of A. brasiliensis (900 or 1500 mg/day) for 12 weeks. The anti-ß-glucan antibody titer in the serum was determined by enzyme-linked immunosorbent assay. Immunoglobulin G (IgG) against ß-glucan was significantly upregulated after intake of A. brasiliensis. Murine experiments demonstrated improvement of anti-ß-glucan antibody production after intraperitoneal injection of Agaricus-derived ß-glucan. To understand the role of antibody against ß-glucan in exclusion of pathogenic fungi, we examined the interaction between HL-60 cells and antibody-treated heat-killed Candida albicans. Flow cytometry analysis indicated the upregulation of Candida-positive HL-60 cells after treatment with human IgG, whereas the competitive assay demonstrated that the main epitope of Candida-reacted IgG was the ß-1,6-glucan structure. Binding between HL-60 and IgG-opsonized C. albicans was suppressed by anti-Fcγ receptor 1 (FcγRI) neutralizing antibody. Finally, using FcγRI-expressed cells with the nuclear factor of activated T-cell reporter assay, we demonstrated that higher titers of anti-ß-glucan IgG can induce stronger Fc receptor-mediated cell activation through the formation of an antibody-ß-glucan complex. In conclusion, oral ingestion of A. brasiliensis KA21 promotes anti-ß-glucan antibody production and may contribute to preventing fungal infection through the activation of immune cells by forming antibody-ß-glucan complexes via an FcγR-dependent pathway.

Latent 1,3-ß-D-glucan acts as an adjuvant for allergen-specific IgE production induced by Japanese cedar pollen exposure.

BACKGROUND: The pollen grains of several plant species contain 1,3-ß-D-glucan (BG). BG activates dendritic cells (DCs) and subsequently regulates the innate immune responses. Within Japan, the most common disease associated with type-I hypersensitivity is Japanese cedar pollinosis. However, the role of BG in Japanese cedar pollen (JCP) remains unclear. This study examined the localization and immunological effects of BG in JCP. METHODS: The localization of BG in JCP grain was determined by immunohistochemical staining using a soluble dectin-1 protein probe and a BG recognition protein (BGRP). The content of BG extracted from JCP was measured by a BGRP-based ELISA-like assay. The cytokine production by bone marrow-derived DCs (BMDCs) obtained from wild-type and BG receptor (dectin-1) knock-out mice was examined in vitro. The mice were intranasally administered JCP grains and the specific serum Ig levels were then quantified. RESULTS: BG was detected in the exine and cell wall of the generative cell and tube cell of the JCP grain. Moreover, BG in the exine stimulated production of TNF-α and IL-6 in the BMDCs via a dectin-1-dependent mechanism. Meanwhile, JCP-specific IgE and IgG were detected in the serum of wild-type mice that had been intranasally administered with JCP grains. These mice also exhibited significantly enhanced sneezing behavior. However, dectin-1 knock-out mice exhibited significantly lower JCP-specific IgE and IgG levels compared to wild-type mice. CONCLUSIONS: Latent BG in JCP can act as an adjuvant to induce JCP-specific antibody production via dectin-1.

Coronary Vasculitis Induced in Mice by Cell Wall Mannoprotein Fractions of Clinically Isolated Candida Species.

Kawasaki disease (KD) is an inflammatory disease that was identified by Professor Tomisaku Kawasaki in 1961. Candida albicans-derived substances (CADS) such as the hot water extract of C. albicans and Candida water-soluble fractions (CAWS) induce coronary vasculitis similar to KD in mice. An increasing proportion of deep-seated candidiasis cases are caused by non-albicans Candida and are often resistant to antifungal drugs. We herein investigated whether the mannoprotein fractions (MN fractions) of clinically isolated Candida species induce vasculitis in mice. We prepared MN fractions from 26 strains of Candida species by conventional hot water extraction and compared vasculitis in DBA/2 mice. The results obtained revealed that the induction of vasculitis and resulting heart failure were significantly dependent on the species; namely, death rates on day 200 were as follows: Candida krusei (100%), Candida albicans (84%), Candida dubliniensis (47%), Candida parapsilosis (44%), Candida glabrata (32%), Candida guilliermondii (20%), and Candida tropicalis (20%). Even for C. albicans, some strains did not induce vasculitis. The present results suggest that MN-induced vasculitis is strongly dependent on the species and strains of Candida, and also that the MN fractions of some non-albicans Candida induce similar toxicity to those of C. albicans.

Quantification of ß-Glucan from Culinary-Medicinal Mushrooms Using Novel Artificial ß-Glucan Recognition Protein.

(1->3)-ß-D-glucans (BGs), found in culinary-medicinal mushrooms, exhibit an immunostimulatory effect; hence, it is important to measure the content of BGs contained in mushrooms. BGs content in a mushroom extract was measured using a recombinant BG-binding protein, supBGRP, and compared with the existing BG assay using BGs antibody. The specificity of supBGRP enzyme immunoassay (EIA) was evaluated using a commercially available polysaccharide reagent. The supBGRP did not react to barley glucan, dextran, mannan, pustulan, and xylan, but reacted to sonifilan, and only slightly to curdlan. Among the BGs tested, supBGRP was most reactive to lentinan. The glucans were extracted using hot water and alkaline solution from the fruit body of the following edible mushrooms: Pleurotus ostreatus, Grifola frondosa, Lentinus edodes, Hypsizygus marmoreus, Flammulina velutipes, and Auricularia polytricha. All BGs extracted from edible mushrooms were detectable; in particular, the reactivity of supBGRP toward the alkaline-extracted fraction from Lentinus edodes was higher than that toward polyclonal antibody for BGs. The results suggest that supBGRP had a specific reaction to BG. The supBGRP seems to be superior to antibodies due to easy availability as a reagent and stability as a protein molecule for measurement of BGs.

Outdoor-Cultivated Royal Sun Medicinal Mushroom Agaricus brasiliensis KA21 (Agaricomycetes) Reduces Anticancer Medicine Side Effects.

We investigated whether outdoor-cultivated Agaricus brasiliensis (KA21) could reduce the side effects caused by the anticancer medicine 5-fluorouracil (5-FU). The adverse effects of 5-FU were analyzed in mice by orally administering the drug every day for 5 days. Leukopenia, diarrhea, body weight loss, anorexia, kidney injury, gastrointestinal tract injury, and hair loss were evaluated as side effects. We determined whether these side effects were reduced by the intake of outdoor-cultivated A. brasiliensis. The side effects were reduced in mice receiving the outdoor-cultivated A. brasiliensis but not in those receiving the indoor-cultivated A. brasiliensis. These results suggest that outdoor-cultivated A. brasiliensis is beneficial in reducing the side effects of the anticancer medicine and might, therefore, be useful in improving the quality of life of patients with cancer. Furthermore, because antioxidants have been reported to contribute to the suppression of the side effects of anticancer medicines, the antioxidant activities of different mushrooms were determined. The antioxidant activity of the outdoor-cultivated A. brasiliensis was the highest among all the tested mushrooms. These findings make it clear that the cultivation conditions of A. brasiliensis are important in suppressing the side effects of anticancer medicines. Strong antioxidant activity might be one of the mechanisms through which this pharmacological activity is mediated.

Coronary Vasculitis Induced in Mice by the Cell Wall Mannoprotein of Candida krusei.

Kawasaki disease (KD) is an inflammatory disease that was identified by Professor Tomisaku Kawasaki in 1961. Candida albicans-derived substances, such as the hot water extract of C. albicans (CADS) and Candida water-soluble fraction (CAWS), induced coronary vasculitis similar to KD in mice. An increasing proportion of deep-seated candidiasis cases are caused by non-albicans Candida and are often resistant to antifungal drugs. We herein investigated whether the hot water extract of C. krusei, inherently resistant to fluconazole, induces vasculitis in mice. Three strains of C. krusei, NBRC1395, NBRC1162, and NBRC10737, were cultured in natural (Y) and chemically defined (C) media and cell wall mannoprotein (MN) fractions were prepared by autoclaving cells (CKY1395MN, CKC1395MN, CKY1162MN, CKC1162MN, CKY10737MN, and CKC10737MN). All MN fractions reacted strongly with Concanavalin A (Con A) and dectin-2 and induced anaphylactoid shock in ICR mice. MNs induced severe coronary vasculitis in DBA/2 mice, resulting in cardiac hypertrophy. MNs also induced coronary vasculitis in C57Bl/6 mice. These results suggest that the MNs of non-albicans Candida, such as C. krusei, induce similar toxicity to those of C. albicans.

Quantification of Mushroom-Derived Soluble ß-1,6-Glucan Using the Function-Modified Recombinant ß-1,6-Glucanase.

Mushroom is one of the major sources of ß-glucan used in medical applications and traditional therapies. Thus, structure analysis and quantification of ß-glucan content is crucial to evaluate medicinal mushrooms. Most studies concerning mushroom-derived ß-glucan have been focused on ß-1,3-glucans. However, recent investigations suggest that ß-1,6 glucans have important roles for immunomodulating activity. Therefore, to elucidate the fine structure of various mushroom-derived ß-glucans, we recently developed a novel ß-1,6 glucan detection system using the function-modified recombinant ß-1,6-glucanase. In this study, we performed an ELISA-like assay using modified ß-1,6-glucanase and soluble dectin-1-Fc as the probes for ß-1,6-glucan and ß-1,3-glucan, respectively. Reactivity of ELISA to crude hot water extracts of edible mushrooms (Grifola frondosa, Agaricus bisporus, Pleurotus tuoliensis, P. eryngii, P. ostreatus, Hypsizygus marmoreus, and Lentinus edodes) was compared and L. edodes showed the strongest reactivity among them. An additional 19 different products of fresh L. edodes (shiitake mushroom) commercially available in Japan were also analyzed. This revealed limited differences in amounts of ß-1,6-glucan and ß-1,3-glucan in each shiitake mushroom. Furthermore, structural analysis of some purified ß-glucans derived from medicinal mushrooms was performed, and their action for inducing tumor necrosis factor-α production from the murine bone marrow-derived dendritic cells was investigated. We found relation between reactivity to modified ß-1,6-glucanase and its cytokine inducing activity. This assay could be useful for evaluating the strains of edible or medicinal mushrooms, which may be used as alternative medicines.

N-Terminal (1→3)-ß-d-Glucan Recognition Proteins from Insects Recognize the Difference in Ultra-Structures of (1→3)-ß-d-Glucan.

Recognition of (1→3)-ß-d-glucans (BGs) by invertebrate ß-1,3-d-glucan recognition protein (BGRP) plays a significant role in the activation of Toll pathway and prophenoloxidase systems in insect host defense against fungal invasion. To examine the structure diversity of BGRPs for the recognition of physiochemically different BGs, the binding specificity of BGRPs cloned from four different insects to structure different BGs was characterized using ELISA. Recombinant BGRPs expressed as Fc-fusion proteins of human IgG1 bound to the solid phase of BGs. Based on the binding specificities, the BGRPs were categorized into two groups with different ultrastructures and binding characters; one group specifically binds BGs with triple-helical conformation, while the other group recognizes BGs with disordered conformations like single-helical or partially opened triple helix. The BGRPs from the silkworm and the Indian meal moth bound to the BGs with a triple-helical structure, whereas BGRPs from the red flour beetle and yellow mealworm beetle showed no binding to triple-helical BGs, but bound to alkaline-treated BGs that have a partially opened triple-helical conformation. This evidence suggests that the insect BGRPs can distinguish between different conformations of BGs and are equipped for determining the diversity of BG structures.

Effects of Euglena gracilis EOD-1 Ingestion on Salivary IgA Reactivity and Health-Related Quality of Life in Humans.

Euglena gracilis EOD-1, a microalgal strain known for high yields of the ß-1, 3-glucan paramylon, is suggested to function as a dietary fiber and enhance immunity. Here, we aimed to investigate the effects of E. gracilis EOD-1 biomass (EOD1BM) ingestion on immunoglobulin A (IgA) antibody titers in saliva, its reactivity, and the health-related quality of life (QOL) in humans. Reacting human immunoglobulin preparations and saliva with paramylon granules revealed the presence of anti-paramylon antibodies in the blood and saliva. We conducted a placebo-controlled, double-blind, crossover study involving 13 healthy subjects who ingested the placebo or EOD1BM for 4 weeks. Saliva was collected from each subject before and after ingestion, and IgA titers and E. gracilis EOD-1 paramylon (EOD1PM) reactivity were compared. In the EOD1BM Ingestion group, the anti-EOD1PM IgA content and titer increased after EOD1BM ingestion. No such change was observed in the Placebo group. Furthermore, the health-related QOL, especially mental health, increased in the EOD1BM Ingestion group. Thus, EOD1BM ingestion led to the production of paramylon (PM)-specific IgA antibody and increased salivary IgA antibody titers. We demonstrate that EOD1BM ingestion enhanced the immunity in the mucosal surface, evoked an antigen-specific response, and increased the health-related QOL, thereby contributing to health improvement.

Solubilized melanin suppresses macrophage function.

Melanin-producing Cryptococcus and Aspergillus are highly invasive and can suppress or escape the immune system of the host. Since non-melanin-producing strains do not affect the immune system, melanin may play a role in immune system suppression. Artificial melanin synthesized using conventional methods is insoluble, making structural and functional analysis of this chemical difficult. In this study, we describe a melanin solubilization method based on polymerization of homogentisic acid (solubilizing component) and an equivalent amount of L-DOPA in the presence of laccase. In addition, we investigated the effect of melanin on the immune system. Homogentisic acid and L-DOPA mixed melanin (HALD), the synthetic solubilized melanin, did not exert a cytotoxic effect on mouse macrophages. HALD suppressed cytokine and reactive oxygen species production by macrophages when they were stimulated by fungal components. HALD also suppressed the phagocytosis of fungal components by macrophages. These results suggest that HALD can suppress the function of macrophages without causing cytotoxicity.

Protection against Gut Inflammation and Sepsis in Mice by the Autodigested Product of the Lingzhi Medicinal Mushroom, Ganoderma lingzhi (Agaricomycetes).

Ganoderma lingzhi (reishi) (GL) is a widely used medicinal mushroom in the treatment of several diseases, including metabolic syndrome and cancer. We recently performed autodigestion of GL and found enhanced release of hypotensive peptides and immunomodulating beta-1,3-glucan. In the present study, we examined the protective effects of G. lingzhi and its autodigested product (AD-GL) against gut inflammation and endogenous sepsis induced in mice by the oral administration of indomethacin (IND). Gut inflammation was assessed by measuring the lengths of the intestines and colon, and sepsis was evaluated by the survival period. G. lingzhi and AD-GL were mixed with animal feed (2.5%) that was available ad libitum during the experimental period. The murine model was established by the repeated oral administration of IND (once a day, 5 mg/kg from day 0). On day 3, the lengths of the small intestine and colon were measured, and the average lengths of the intestines were significantly shorter in the control and G. lingzhi-administered groups than in the AD-GL-administered group. This finding suggests that AD-GL protected against gut inflammation due to IND-induced ulceration and subsequent microbial translocation. Furthermore, the median numbers of survival days in the control group, the G. lingzhi group, and the AD-GL group were 5, 6, and 11, respectively. The concentrations of the inflammatory cytokines, tumor necrosis factor (TNF)-α and interleukin-6, in the blood were significantly reduced in the mice administered AD-GL. In the in vitro cell culture, G. lingzhi and AD-GL fractions released a significantly higher concentration of TNF-α from the spleen, and the splenocytes of mice administered AD-GL hot water extract showed a greater potential to produce cytokines in response to pathogen-associated molecular patterns. These results strongly suggest the protection of the gut mucosa from inflammation, and therefore the prevention of sepsis, by the administration of AD-GL. Autodigestion appears to be a promising protocol that enhances the usefulness of G. lingzhi as a functional food.

The ATP Transporter VNUT Mediates Induction of Dectin-1-Triggered Candida Nociception.

Candida albicans infection can cause skin, vulvar, or oral pain. Despite the obvious algesic activity of C. albicans, the molecular mechanisms of fungal nociception remain largely unknown. Here we show that the C. albicans-specific signaling pathway led to severe mechanical allodynia. We discovered that C. albicans-derived ß-glucan stimulated nociceptors depending on Dectin-1, and two pathways in inflammatory pain. The major pathway operates via the Dectin-1-mediated ATP-P2X3/P2X2/3 axis through intercellular relationships between keratinocytes and primary sensory neurons, which depends on the ATP transporter vesicular nucleotide transporter (VNUT). The other pathway operates via the Dectin-1-mediated PLC-TRPV1/TRPA1 axis in primary sensory neurons. Intriguingly, C. albicans-derived ß-glucan has the ability to enhance histamine-independent pruritus, and VNUT inhibitor clodronate can be used to treat unpleasant feelings induced by ß-glucan. Collectively, this is the first report to indicate that Dectin-1 and VNUT mediated innate sensory mechanisms that detect fungal infection.

Amphiphobic Septa Enhance the Mechanical Stability of Free-Standing Bilayer Lipid Membranes.

Artificial bilayer lipid membranes (BLMs) provide well-defined systems for investigating the fundamental properties of membrane proteins, including ion channels, and for screening the effect of drugs that act on them. However, the application of this technique is limited due to the low stability and low reconstitution efficiency of the process. We previously reported on improving the stability of BLM based on the fabrication of microapertures having a tapered edge in SiO2/Si3N4 septa and efficient ion channel incorporation based on vesicle fusion accelerated by a centrifugal force. Although the BLM stability and incorporation probability were dramatically improved when these approaches were used, some BLMs were ruptured when subjected to a centrifugal force. To further improve the BLM stability, we investigated the effect of modifying the surface of the SiO2/Si3N4 septa on the stability of BLM suspended in the septa. The modified surfaces were characterized in terms of hydrophobicity, lipophobicity, and surface roughness. Diffusion coefficients of the lipid monolayers formed on the modified surfaces were also determined. Highly fluidic lipid monolayers were formed on the amphiphobic substrates that had been modified with long-chain perfluorocarbons. Free-standing BLMs formed in amphiphobic septa showed a much higher mechanical stability, including tolerance to water movement and applied centrifugal forces with and without proteoliposomes, than those formed in the septa that had been modified with a short alkyl chain. These results demonstrate that highly stable BLMs are formed when the surface of the septa has amphiphobic properties. Because highly fluidic lipid monolayers that are formed on the septa seamlessly connect with BLMs in a free-standing region, the high fluidity of the lipids contributes to decreasing potential damage to BLMs when mechanical stresses are applied. This approach to improve the BLM stability increases the experimental efficiency of the BLM systems and will contribute to the development of high-throughput platforms for functional assays of ion channel proteins.

Activation of macrophages by a laccase-polymerized polyphenol is dependent on phosphorylation of Rac1.

Various physiologically active effects of polymerized polyphenols have been reported. In this study, we synthesized a polymerized polyphenol (mL2a-pCA) by polymerizing caffeic acid using mutant Agaricus brasiliensis laccase and analyzed its physiological activity and mechanism of action. We found that mL2a-pCA induced morphological changes and the production of cytokines and chemokines in C3H/HeN mouse-derived resident peritoneal macrophages in vitro. The mechanisms of action of polymerized polyphenols on in vitro mouse resident peritoneal cells have not been characterized in detail previously. Herein, we report that the mL2a-pCA-induced production of interleukin-6 (IL-6) and monocyte chemotactic protein-1 (MCP-1) in C3H/HeN mouse-derived resident peritoneal cells was inhibited by treatment with the Rac1 inhibitor NSC23766 trihydrochloride. In addition, we found that mL2a-pCA activated the phosphorylation Rac1. Taken together, the results show that mL2a-pCA induced macrophage activation via Rac1 phosphorylation-dependent pathways.