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Two highly potent cytotoxic 26-membered macrolides, isocaribenolide-I (1) and a chlorohydrin 2, together with known amphidinolide N (3), have been isolated from a free-swimming dinoflagellate Amphidinium species (KCA09053 and KCA09056 strains) collected off Iriomote Island, Japan. The structures of 1 and 2 were determined to be a congener of 3 with an isobutyl terminus and the chlorohydrin form of 3, respectively, by detailed analyses of spectroscopic data. The relative stereochemistries of 1 and 2 were elucidated by the conformational analyses based on NMR data.

Molecular cytogenetic investigation was carried out on the scleractinian coral, Acropora pruinosa (A. pruinosa). Conventional Giemsa staining techniques for karyotyping, such as G- and C-banding, were conducted. Karyotype analysis showed mosaicism of cells with two different chromosome numbers, 28 and 29, in the same embryo. The C-band positive portions appeared on the centromeres of most chromosomes and along the entire length of the unpaired chromosome 15 in cells with 29 chromosomes. Fluorescence in situ hybridization (FISH) revealed that the loci of rRNA genes (rDNA), 5S and 18S/28S, were located on chromosomes 4 and 2, respectively. Whole genome hybridization (WGH) with sperm DNA showed distinct signals not only on all centromeric regions but also on a whole unpaired chromosome 15. Comparative genomic hybridization (CGH) using DNAs from sperm and unfertilized eggs revealed that the unpaired chromosome 15 has sperm-specific DNA sequences. We therefore hypothesized the existence of a sex-related chromosome in A. pruinosa. In this study, we have presented a tentative karyotype of this coral, based on banding results, and described FISH, WGH, and CGH results. Moreover, we successfully cloned and sequenced three clones; AP-5S for 5S rDNA, AP-18S for 18S/28S rDNA, and AP-unpaired chromosome 15q for the unpaired chromosome 15-specific DNA. These molecular cytogenetic approaches will help establish a more exact karyotype of corals and promote understanding of coral genetics, including chromosome evolution. These findings will help to verify the method of classification of complex scleractinian corals.

We performed conventional and molecular cytogenetic studies on the

Two highly potent cytotoxic 26-membered macrolides, isocaribenolide-I (1) and a chlorohydrin 2, together with known amphidinolide N (3), have been isolated from a free-swimming dinoflagellate Amphidinium species (KCA09053 and KCA09056 strains) collected off Iriomote Island, Japan. The structures of 1 and 2 were determined to be a congener of 3 with an isobutyl terminus and the chlorohydrin form of 3, respectively, by detailed analyses of spectroscopic data. The relative stereochemistries of 1 and 2 were elucidated by the conformational analyses based on NMR data.

Th17 cells and the cytokine they produce, interleukin (IL)-17, play an important role in tumor progression in humans and in mice. IL-6 and IL-23 are critical cytokines for the differentiation and propagation of Th17 cells, respectively. Bacterial lipopolysaccharides (LPS) are known to stimulate immune cells to produce such inflammatory cytokines. Contrary to Escherichia coli (E. coli) LPS, LPS from Spirulina has low toxicity and barely induces in vivo production of IL-6 and IL-23 in mice. We examined the antitumor effects of Spirulina LPS compared to E. coli LPS in an MH134 hepatoma model. Administration of Spirulina LPS suppressed tumor growth in C3H/HeN mice, but not in Toll-like receptor 4 (TLR4)-mutant C3H/HeJ mice, by reducing serum levels of IL-17 and IL-23, while increasing interferon (IFN)-γ levels. The antitumor activity and IFN-γ production were mediated by T cells. Moreover, in vitro experiments showed that Spirulina LPS impaired the antigen-presenting function that supports the generation of IL-17-producing cells in a toll-like receptor (TLR)4-dependent manner. Of note, injection of anti-IL-17 antibody in tumor-bearing C3H/HeN mice in the absence of Spirulina LPS markedly suppressed tumor growth and augmented IFN-γ responses. Thus, our results support the notion that IFN-γ and IL-17/IL-23 mutually regulate Th17 and Th1 responses in tumor-bearing hosts, and Spirulina LPS modulates the balance of the IFN-γ-IL-17/IL-23 axis towards IFN-γ production, which leads to tumor inhibition. Furthermore, Spirulina LPS effectively inhibited the spontaneous development of mammary tumors. This study has important implications for the exploitation of TLR-based immunomodulators for cancer immunotherapy.

Dimagnesium silicide is an eco-friendly thermoelectric compound whose constituent elements of both Mg and Si are non-toxic and exist in abundance on the earth. In this study, we attempted to control the thermal conductivity of Al-doped Mg 2 Si by grain refinement. Three types of Si powders, i.e., commercial coarse powder, its pulverized powder and commercial fine powder were prepared for synthesizing the Al-doped Mg 2 Si. Mg powder and one of the Si powders were weighed with Mg/Si mole ratio of 67/33, and mixed with Al powder with amount of 0.1 at.%. The Al-doped Mg 2 Si compounds were synthesized using three different Si powders via a liquid-solid phase reaction process under unified synthesizing conditions. A part of the synthesized Mg 2 Si powder using the coarse Si powder was pulverized. Four kinds of Mg 2 Si powders were sintered by pulse discharge sintering method under unified sintering conditions. The sintered samples of the synthesized Mg 2 Si powders made from the fine and the milled Si powders and of the milled Mg 2 Si powder had the grain-refined microstructure. Especially, the sintered sample of the milled Mg 2 Si powder was effective for grain refinement and for reduction of thermal conductivity, and had the best thermoelectric performance of ZT = 1.15 at 873 K.

Two new macrolides, iriomoteolides-10a (1) and -12a (2), have been isolated from a marine dinoflagellate Amphidinium sp. (KCA09053 strain), and their structures were elucidated on the basis of a detailed two dimensional (2D)-NMR analysis. Compound 1 is a novel 21-membered Amphidinium macrolide, which contains one tetrahydrofuran ring, two ketone carbonyls, two hydroxyl groups, and six one-carbon branches. Compound 2 is a new 12-membered macrolide related to amphidinolide Q. Compound 1 exhibited cytotoxic activity against human cervix adenocarcinoma HeLa and murine hepatocellular carcinoma MH134 cells.

The phototropic bacterium Synechocystis sp. strain PCC 6803 is able to adapt its morphology in order to survive in a wide range of harsh environments. Under conditions of high salinity, planktonic cells formed cell aggregates in culture. Further observations using crystal violet staining, confocal laser scanning microscopy, and field emission-scanning electron microscopy confirmed that these aggregates were Synechocystis biofilms. Polyamines have been implicated in playing a role in biofilm formation, and during salt stress the content of spermidine, the major polyamine in Synechocystis , was reduced. Two putative arginine decarboxylases, Adc1 and Adc2, in Synechocystis were heterologously expressed in Escherichia coli and purified. Adc2 had high arginine decarboxylase activity, whereas Adc1 was much less active. Disruption of the adc genes in Synechocystis resulted in decreased spermidine content and formation of biofilms even under nonstress conditions. Based on the characterization of the adc mutants, Adc2 was the major arginine decarboxylase whose activity led to inhibition of biofilm formation, and Adc1 contributed only minimally to the process of polyamine synthesis. Taken together, in Synechocystis the shift from planktonic lifestyle to biofilm formation was correlated with a decrease in intracellular polyamine content, which is the inverse relationship of what was previously reported in heterotroph bacteria. IMPORTANCE There are many reports concerning biofilm formation in heterotrophic bacteria. In contrast, studies on biofilm formation in cyanobacteria are scarce. Here, we report on the induction of biofilm formation by salt stress in the model phototrophic bacterium Synechocystis sp. strain PCC 6803. Two arginine decarboxylases (Adc1 and Adc2) possess function in the polyamine synthesis pathway. Inactivation of the adc1 and adc2 genes leads to biofilm formation even in the absence of salt. The shift from planktonic culture to biofilm formation is regulated by a decrease in spermidine content in Synechocystis . This negative correlation between biofilm formation and polyamine content, which is the opposite of the relationship reported in other bacteria, is important not only in autotrophic but also in heterotrophic bacteria.

Photoautotrophic bacteria have developed mechanisms to maintain K + homeostasis under conditions of changing ionic concentrations in the environment. Synechocystis sp. strain PCC 6803 contains genes encoding a well-characterized Ktr-type K + uptake transporter (Ktr) and a putative ATP-dependent transporter specific for K + (Kdp). The contributions of each of these K + transport systems to cellular K + homeostasis have not yet been defined conclusively. To verify the functionality of Kdp, kdp genes were expressed in Escherichia coli , where Kdp conferred K + uptake, albeit with lower rates than were conferred by Ktr. An on-chip microfluidic device enabled monitoring of the biphasic initial volume recovery of single Synechocystis cells after hyperosmotic shock. Here, Ktr functioned as the primary K + uptake system during the first recovery phase, whereas Kdp did not contribute significantly. The expression of the kdp operon in Synechocystis was induced by extracellular K + depletion. Correspondingly, Kdp-mediated K + uptake supported Synechocystis cell growth with trace amounts of external potassium. This induction of kdp expression depended on two adjacent genes, hik20 and rre19 , encoding a putative two-component system. The circadian expression of kdp and ktr peaked at subjective dawn, which may support the acquisition of K + required for the regular diurnal photosynthetic metabolism. These results indicate that Kdp contributes to the maintenance of a basal intracellular K + concentration under conditions of limited K + in natural environments, whereas Ktr mediates fast potassium movements in the presence of greater K + availability. Through their distinct activities, both Ktr and Kdp coordinate the responses of Synechocystis to changes in K + levels under fluctuating environmental conditions.

A novel linear polyketide, amphirionin-2 ( 1 ), with two unique hexahydrofuro[3,2- b ]furan moieties has been isolated from the cultivated algal cells of a benthic dinoflagellate Amphidinium sp. (strain KCA09051). The structure was elucidated on the basis of detailed analyses of 2D NMR data, and the absolute configuration of C-5 was determined by using modified Mosher’s method. Amphirionin-2 ( 1 ) exhibited potent cytotoxic activity against human colon carcinoma Caco-2 cells and human lung adenocarcinoma A549 cells.

A linear polyketide, amphirionin-4 (1), has been isolated from cultivated algal cells of the marine dinoflagellate Amphidinium species. The structure was elucidated on the basis of detailed analyses of 1D and 2D NMR data, and the absolute configurations of C-4 and C-8 were determined using the modified Mosher's method. Amphirionin-4 (1) exhibited extremely potent proliferation-promoting activity on murine bone marrow stromal ST-2 cells (950% promotion) at a concentration of 0.1 ng/mL.

A novel linear polyketide, amphirionin-5 (1), has been isolated from the cultivated algal cells of the benthic dinoflagellate Amphidinium sp. (strain KCA09053), and the structure was elucidated on the basis of detailed analyses of 1D and 2D NMR data. Amphirionin-5 (1) resulted in a 282% increase in the proliferation of murine bone-marrow derived stromal ST-2 cells and 320% increase in the proliferation of murine osteoblastic MC3T3-E1 cells.

Iriomoteolide-13a (1) has been isolated from a benthic dinoflagellate Amphidinium sp. (strain KCA09053) as a new 22-membered macrolide containing one hexahydrofuro[3,2-b]furan ring, one tetrahydropyran ring, two tetrahydrofuran rings, three one-carbon branches, and three hydroxyl groups including two hemiketals. The structure of 1 was assigned on the basis of a detailed 2D NMR analysis. Compound 1 exhibited cytotoxic activity against human cervix adenocarcinoma HeLa cells (IC50: 0.5 μg/mL).

Peridinin and fucoxanthin, which are natural carotenoids isolated from a symbiotic dinoflagellate, Symbiodinium sp., and a brown alga, Petalonia fascia, respectively, were compared for inhibitory effects on delayed-type hypersensitivity in mice. The number of eosinophils at the site of inflammation and in peripheral blood was compared for the administration of peridinin and fucoxanthin applied by painting and intraperitoneally. Peridinin, but not the structurally-related fucoxanthin, significantly suppressed the number of eosinophils in both the ear lobe and peripheral blood. Furthermore, peridinin applied topically, but not administered intraperitoneally, suppressed the level of eotaxin in the ears of sensitized mice. Fucoxanthin weakly suppressed the concentration of eotaxin in ears only by intraperitoneal administration. Although both carotenoids inhibited the migration of eosinophils toward eotaxin, the inhibitory effect of peridinin was higher than that of fucoxanthin. Peridinin may be a potential agent for suppressing allergic inflammatory responses, such as atopic dermatitis, in which eosinophils play a major role in the increase of inflammation.

We performed a molecular cytogenetic investigation of the scleractinian coral Acropora solitaryensis, which is dominant in the temperate region of Japan (30-35°N). Molecular cytogenetic analysis, using fluorescence in situ hybridization (FISH), was carried out for karyotyping and gene mapping. We propose the karyotype of this coral (2n = 30) based on C-banding and FISH analyses. FISH mapping of the rRNA gene was carried out with a probe generated by PCR amplification using rRNA gene primers. Furthermore, the telomeres and centromeres of all chromosomes were visualized using FISH. By comparative genomic hybridization using DNA from sperm and unfertilized eggs of this coral, we offer evidence suggesting the existence of sex chromosomes in this species. Collectively, these data advance our understanding of coral genetics.

In order to analyze the damage of human epithelial cells, we used human quasi-normal FPCK-1-1 cells derived from a colonic polyp in a patient with familial adenomatous polyposis as a monolayer, which is co-cultured with peptidoglycan (PGN)-stimulated THP-1 cells. Co-cultured FPCK-1-1 cells showed a decreased transepithelial electrical resistance (TER) and the lower level of claudin-2. When Spirulina complex polysaccharides were added one day before the start of the co-culture, there was no decrease of TER and claudin-2 (early phase damage). In contrast, when Spirulina complex polysaccharides were added to FPCK-1-1 cells after the level of TER had decreased, there was no recovery at the level of claudin-2, though the TER level recovered (late phase damage). The mucosa reconstitution is suggested to be involved in the recovery from the damaged status. Interestingly, autonomous recovery of FPCK-1-1 cells from both the early and late phase damage requires the production of IL-22, because anti-IL-22 antibodies inhibited recovery in these cases. Antibodies against either TLR2 or TLR4 inhibited the production of IL-22 from FPCK-1-1 colon epithelial cells, suggesting that signals through TLR2 and TLR4 are necessary for autonomous recovery of FPCK-1-1 colon epithelial cells by producing IL-22. In conclusion, we have established a useful model for the study of intestinal damage and recovery using human colon epithelial cells and our data suggest that damage to human colon epithelial cells can, at least in part, be recovered by the autonomous production of IL-22 in response to Spirulina complex polysaccharides.

This study is the first to report that Spirulina complex polysaccharides (CPS) suppress glioma growth by down-regulating angiogenesis via a Toll-like receptor 4 signal. Murine RSV-M glioma cells were implanted s.c. into C3H/HeN mice and TLR4 mutant C3H/HeJ mice. Treatment with either Spirulina CPS or Escherichia coli (E. coli) lipopolysaccharides (LPS) strongly suppressed RSV-M glioma cell growth in C3H/HeN, but not C3H/HeJ, mice. Glioma cells stimulated production of interleukin (IL)-17 in both C3H/HeN and C3H/HeJ tumor-bearing mice. Treatment with E. coli LPS induced much greater IL-17 production in tumor-bearing C3H/HeN mice than in tumor-bearing C3H/HeJ mice. In C3H/HeN mice, treatment with Spirulina CPS suppressed growth of re-transplanted glioma; however, treatment with E. coli LPS did not, suggesting that Spirulina CPS enhance the immune response. Administration of anti-cluster of differentiation (CD)8, anti-CD4, anti-CD8 antibodies, and anti-asialo GM1 antibodies enhanced tumor growth, suggesting that T cells and natural killer cells or macrophages are involved in suppression of tumor growth by Spirulina CPS. Although anti-interferon-γ antibodies had no effect on glioma cell growth, anti-IL-17 antibodies administered four days after tumor transplantation suppressed growth similarly to treatment with Spirulina CPS. Less angiogenesis was observed in gliomas from Spirulina CPS-treated mice than in those from saline- or E. coli LPS-treated mice. These findings suggest that, in C3H/HeN mice, Spirulina CPS antagonize glioma cell growth by down-regulating angiogenesis, and that this down-regulation is mediated in part by regulating IL-17 production.

Two new macrolides, iriomoteolides-10a (1) and -12a (2), have been isolated from a marine dinoflagellate Amphidinium sp. (KCA09053 strain), and their structures were elucidated on the basis of a detailed two dimensional (2D)-NMR analysis. Compound 1 is a novel 21-membered Amphidinium macrolide, which contains one tetrahydrofuran ring, two ketone carbonyls, two hydroxyl groups, and six one-carbon branches. Compound 2 is a new 12-membered macrolide related to amphidinolide Q. Compound 1 exhibited cytotoxic activity against human cervix adenocarcinoma HeLa and murine hepatocellular carcinoma MH134 cells.

Flagellar operons are divided into three classes with respect to their transcriptional hierarchy in Salmonella enterica serovar Typhimurium. The class 1 gene products FlhD and FlhC act together in an FlhD 4 C 2 heterohexamer, which binds upstream of the class 2 promoters to facilitate binding of RNA polymerase. In this study, we showed that flagellar expression was much reduced in the cells grown in poor medium compared to those grown in rich medium. This nutritional control was shown to be executed at a step after class 1 transcription. We isolated five Tn 5 insertion mutants in which the class 2 expression was derepressed in poor medium. These insertions were located in the ydiV ( cdgR ) gene or a gene just upstream of ydiV . The ydiV gene is known to encode an EAL domain protein and to act as a negative regulator of flagellar expression. Gene disruption and complementation analyses revealed that the ydiV gene is responsible for nutritional control. Expression analysis of the ydiV gene showed that its translation, but not transcription, was enhanced by growth in poor medium. The ydiV mutation did not have a significant effect on either the steady-state level of flhDC mRNA or that of FlhC protein. Purified YdiV protein was shown in vitro to bind to FlhD 4 C 2 through interaction with FlhD subunit and to inhibit its binding to the class 2 promoter, resulting in inhibition of FlhD 4 C 2 -dependent transcription. Taking these data together, we conclude that YdiV is a novel anti-FlhD 4 C 2 factor responsible for nutritional control of the flagellar regulon.

In 2009, the three participating countries – Japan, Taiwan, and the Philippines – completed a round each of holding the Annual Symposium; thus, from this year, Kochi University has been exploring means of further pursuing research development among the collaborating partners. As an outcome, placing emphasis on the Kuroshio Triangle, we decided to reveal our concept of linkage among mountains, rivers, and seas. At the same time, we also decided to take up the topic of how recent societal changes and modifications to the natural environment have affected coastal and land-based environments. This Symposium will especially focus on islands/archipelagos of the Kuroshio region that have engaged in cultural exchange from a very early time in history. Here, universities and institutions representing the three countries will form a nucleus for a network to which will be added Malaysia’s Sarawak State, which possesses vast tropical forests, and West Kalimantan of Indonesia. With this development, we expect to achieve even more enhanced information exchange concerning the effectsof climate change on the ecology of coastal and land-based areas of the Kuroshio region. The addition of these countries to our network will expand the Kuroshio Triangle to form a new domain,named here as “The Kuroshio-Related S-Shaped Zone”. Representative universities from Malaysiaand Indonesia will be concerned with addressing ecological and environmental problems that extend beyond national boundaries.

Algae are eaten as health food in many Asian countries. We summarize our recent results regarding fine tuning reagents of the immune responses. the immunoregulatory role of polysaccharides from edible algae on delayed-type hypersensitivity (DTH) and antitumor immune responses using mice. These algae are divided into two types; those which manipulate immune responses through toll-like receptor 4 (TLR 4), and those which use different receptors to modify the immune response. Petalonia binghamiae polysaccharides attenuate DTH and tumor growth by reducing the production of inflammatory cytokine, IL-17 through TLR4.The serum level of IL-17 was decreased significantly, only when P. binghamiae polysaccharides wereadministered intraperitoneally in the DTH response. Intraperitoneal administration of other brown algae polysaccharides, such as alginic acid did not suppress the DTH response nor reduced the serum level of IL-17. Although orally administered P. binghamiae polysaccharides did not reduce the serum level of IL-17, they inhibited the migration of eosinophils. These results suggest that the inhibition of either the generation of IL-17 or the migration of eosinophils is effective in regulating inflammation,and that these two events were separately regulated. Here, we present data suggesting that either the peritoneal or oral administration of P. binghamiae polysaccharides efficiently inhibits the migration of eosinophils to the site of inflammation in a DTH model using IL-5 transgenic mice. Alginic acid suppressedtumor growth in both C3H/HeN and C3H/HeJ mice and did not suppress the DTH response as strongly as P. binghamiae polysaccharides. We propose the potential usefulness of edible algae as fine tuning reagents of the immune responses.

Algae are eaten as healthy foods in Asian countries. We summarize our recent results on the immunoregulatory role of polysaccharide fractions from edible algae on immediate hypersensitivity, delayed-type hypersensitivity, and antitumor immune responses. They are divided into two types. One is to manipulate immune response through toll-like receptor 4 (TLR 4). The other uses different receptors to modify the immune response. Petalonia binghamiae polysaccharide fraction and Spirulina pacifica complex polysaccharide attenuate the delayed-type hypersensitivity and tumor growth by reducing the production of inflammatory cytokine, IL-17 through TLR4. This is suggested from the results that these polysaccharide fractions could suppress the delayed-type hypersensitivity and tumor growth in C3H/HeN but not in tolllike receptor 4 mutant, C3H/HeJ mice. Alginic acid, the polysaccharide from brown algae suppressed tumor growth in both C3H/HeN and C3H/HeJ mice and did not suppress delayed-type hypersensitivity response significantly, when administered intraperitoneally. We propose the potential usefulness of edible algae as the fine tuning reagents of the immune response. We also summarize the recent advancement in the area of regulation of immune responses in relation to these polysaccharides.

Candida spp. are an opportunistic pathogen causing serious local and systemic infections, especially in immuno-compromised hosts such as the elderly and HIV-positive patients. Hinokitiol C 10 H 12 O 2 (β-thujaplicin) is a component of the essential oils isolated from Cupressaceae and shows antibacterial activities for various bacteria. The aim of this study was to demonstrate the preventive effects of hinokitiol on the adherence of seven species of Candida to oral epithelial cells and to establish a safe and useful method for oral hygiene. A short-time treatment (30 min) with 0.25 mM hinokitiol showed 30–70% inhibition of adherence of Candida spp. to oral epithelial cells, inhibited about 11% biofilm formation, and did not inhibit the cell growth of Candida spp. Furthermore, short treatment with 0.25 mM hinokitiol was a safe method for oral hygiene against Candida infection because it did not inhibit the cell growth of commensal bacteria, such as oral streptococci existing in normal flora, or damage the epithelial cells. However, long-time treatment and a high concentration of hinokitiol demonstrated both the adherence inhibition of Candida and damage to commensal bacteria and epithelial cells. Our data suggest an appropriate procedure to apply hinokitiol that may be beneficial for the prevention of opportunistic pathogens such as Candida spp. in the oral cavity. The clinical and daily use of hinokitiol under an appropriate procedure may be a preventive and realistic therapy for Candida infection in immune-compromised hosts.

Aims/Background: To investigate how systemic overexpression of IL-4, IL-5 and IFN-γ affects the severity of experimental conjunctivitis (EC) in mice. Methods: We electroporated the tibialis anterior muscle of naive BALB/c mice with IL-4, IL-5, IFN-γ or a control gene and then harvested the mice blood and conjunctivas to measure eosinophil content in these tissues. To evaluate the effects of cytokine gene electroporation on the early phase reaction (EPR), cytokine gene-electroporated ragweed (RW)-immunized mice were intravenously injected with Evans blue (EB) and then challenged them with RW in eye drops. Thirty minutes later, we harvested their conjunctivas to extract EB and evaluate the EPR. Additionally, 24 hours after RW challenge, conjunctivas were harvested from cytokine gene-electroporated RW-immunized mice, which had not received intravenous injection of EB, to measure conjunctival eosinophilia. Results: Significantly more eosinophils were detected in the blood and conjunctivas of IL-5-electroporated mice in which EC was not induced. The intensity of the EPR was significantly greater in IFN-γ-electroporated mice. Significantly greater eosinophil infiltration was seen in the conjunctivas of IL-5-electroporated EC-developing mice. Conclusions: It appears that systemic IL-5 and IFN-γ play different roles in the development of EC.

Inhibitory effects on degranulation of rat basophilic leukemia (RBL-2H3) cells and mouse eosinophils by marine algae extracts were examined. More than 50% of the degranulation of RBL-2H3 cells was inhibited by water extracts of Ecklonia cava and Chrysymenia wrightii at a concentration of 100 μg/mL. More than 50% of the degranulation of RBL-2H3 cells was inhibited by methanol extracts of Petalonia binghamiae, Scytosiphon lomentaria, Undaria pinnatifida, Porphyra dentata, Codium fragile and Ulva japonica at a concentration of 200 μg/mL. Most inhibitory substances in the methanol extracts were partitioned into ethyl acetate and hexane layers. The ethyl acetate-partitioned layer of methanol extract of Petalonia binghamiae had higher inhibitory effects than the hexane-partitioned layer on the degranulation of RBL-2H3 cells. By contrast, the hexanepartitioned layer of the same extract had a higher inhibitory effect than the ethyl acetate-partitioned layer on the degranulation of mouse eosinophils. The ethyl acetate-partitioned layer of methanol extract of Petalonia binghamiae was further separated into eight fractions by silica gel column chromatography. Most inhibited the degranulation of RBL-2H3 cells, but not that of mouse eosinophils significantly. These results suggest methanol extract of Petalonia binghamiae contains materials that inhibit the degranulation of basophils and eosinophils differentially.

We previously described an inverse correlation between galectin-9 (Gal-9) expression and metastasis in patients with malignant melanoma and breast cancer. This study verified the ability of Gal-9 to inhibit lung metastasis in experimental mouse models using highly metastatic B16F10 melanoma and Colon26 colon cancer cells. B16F10 cells transfected with a secreted form of Gal-9 lost their metastatic potential. Intravenous Gal-9 administration reduced the number of metastases of both B16F10 and Colon26 cells in the lung, indicating that secreted Gal-9 suppresses metastasis. Analysis of adhesive molecule expression revealed that B16F10 cells highly express CD44, integrin alpha1, alpha 4, alpha V, and beta1, and that Colon26 cells express CD44, integrin alpha2, alpha 5, alpha V, and beta1, suggesting that Gal-9 may inhibit the adhesion of tumor cells to vascular endothelium and the extracellular matrix (ECM) by binding to such adhesive molecules. Indeed, Gal-9 suppressed the binding of hyaluronic acid to CD44 on both B16F10 and Colon26 cells, and also suppressed the binding of vascular cell adhesion molecule-1 to very late antigen-4 on B16F10 cells. Furthermore, Gal-9 inhibited the binding of tumor cells to ECM components, resulting in the suppression of tumor cell migration. The present results suggest that Gal-9 suppresses both attachment and invasion of tumor cells by inhibiting the binding of adhesive molecules on tumor cells to ligands on vascular endothelium and ECM.

Galectin-9 (Gal-9) belongs to the galectin family, which exhibits affinity for beta-galactosides. Gal-9 has a variety of biological activities; however, its role in allergic inflammation is unknown.We evaluated the effect of a stable form of the human protein on allergic airway inflammation in a mite allergen-induced asthma model.Human stable Gal-9 was given by intravenous injection to mice during antigen challenge. The effect of Gal-9 on airway inflammation and airway hyperresponsiveness (AHR) was then evaluated.Gal-9 reduced AHR as well as Th2-associated airway inflammation. Furthermore, administration of Gal-9 as well as anti-CD44 monoclonal antibody inhibited the infiltration of peripheral blood Th2 cells into the airway. Interestingly, Gal-9 directly bound the CD44 adhesion molecule and inhibited interactions with hyaluronan (HA). Consistent with the concept that CD44-HA interactions mediate the migration of T cells into the lung, Gal-9 blocked CD44-dependent adhesion of BW5147 mouse T cells to HA.We conclude that Gal-9 inhibits allergic inflammation of the airway and AHR by modulating CD44-dependent leukocyte recognition of the extracellular matrix.

Bacterial H antigens are specified by flagellin molecules, which constitute the flagellar filament. Escherichia coli 781-55 and E2987-73 are the type strains for H44 and H55 antigens, respectively. Unlike E. coli K-12, they possess two flagellin genes, fliC and fllA, on their chromosomes. However, they are monophasic, expressing exclusively the fllA genes, which specify the type antigens. In this study, the flagellin genes were cloned from these strains and their structure and expression were analyzed. It was found that the fliC genes encode apparently intact flagellin subunits but possess inefficient sigma28-dependent promoters, which may result in these genes being silent. The chromosomal locations of the fllA genes are approximately, but not exactly, identical with that of the phase-2 flagellin gene, fljB, of diphasic Salmonella strains. However, unlike the Salmonella fljB gene, the invertible H segment and the fljA gene responsible for the control of flagellar phase variation are both absent from the fllA loci. The fllA genes are highly homologous to the E. coli fliC gene but distantly related to the Salmonella fljB gene. These results suggest a hypothesis that the fllA genes may have emerged by an intra-species lateral transfer of the fliC gene. This hypothesis is further supported by the observation that the fllA genes are flanked by several IS elements and located within cryptic prophage elements.

Karyotype analysis was performed on the scleractinian coral Coelastrea aspera Verrill, 1866, commonly found along temperate coasts in Japan (30–35°N) and in coastal waters in the Indian and Pacific oceans. G-banding of C. aspera was successfully performed, although the banding pattern was not as clear as that in mammals. The karyogram clearly revealed that this coral had a homogeneously staining region (hsr) in chromosome 11. This hsr consisted of ribosomal RNA (rRNA) related genes, which was demonstrated by fluorescence in situ hybridization (FISH) with probes generated using 28S ribosomal DNA (rDNA) primers and those generated through chromosome microdissection. In addition, we conducted silver-stained nucleolus organizer region (Ag-NOR) analysis and found Ag depositions in the interphase nuclei but not on rRNA gene loci and hsr(s) in the mitotic stage. The hsr of this coral was observed in approximately 50% of the metaphase spreads analyzed. This may explain the diversity of coral rDNA based on the molecular study of sequence analysis. Furthermore, it was discovered that human telomere and Alu repeated sequences were present in this C. aspera. Probes derived from human Alu sequences are expected to play an important role in the classification of corals. Overall, our data can be of great value in discriminating among scleractinian coral species and understanding their genetics, including chromosomal evolution.

The transcription factor PU.1 regulates erythrocyte differentiation. We previously reported that F5-5 erythroblasts differentiate into erythrocytes in response to activin by degrading PU.1, and that inhibiting PU.1- degradation suppresses F5-5 cell differentiation into erythrocytes. These findings suggest that regulating PU.1 degradation is critical for terminal differentiation of erythrocytes. Here, we investigate the mechanism underlying PU.1 degradation during successive differentiation of erythrocytes. Using 2D-MS proteomic analysis, we show that proteasome subunits and proteins required for degradation by proteasomes immunoprecipitate with PU.1 in response to activin. Furthermore, a proteasome inhibitor, lactacystin, partially suppresses differentiation of F5-5 cells into erythrocytes in response to activin, and partially inhibits PU.1 degradation. Our results indicate that degradation of PU.1 necessary for erythrocyte differentiation occurs, in part, through the proteasome pathway.

Shigella strains are nonmotile. The master operon of flagellar synthesis, flhDC , was analyzed for genetic damage in 46 Shigella strains representing all known serotypes. In 11 strains (B1, B3, B6, B8, B10, B18, D5, F1B, D10, F3A, and F3C) the flhDC operon was completely deleted. PCR and sequence analysis of the flhDC region of the remaining 35 strains revealed many insertions or deletions associated with insertion sequences, and the majority of the strains were found to be defective in their flhDC genes. As these genes also play a role in regulation of nonflagellar genes, the loss may have other consequences or be driven by selection pressures other than those against flagellar motility. It has been suggested that Shigella strains fall mostly into three clusters within Escherichia coli , with five outlier strains, four of which are also within E. coli (G. M. Pupo, R. Lan, and P. R. Reeves, Proc. Natl. Acad. Sci. USA 97:10567-10572, 2000). The distribution of genetic changes in the flhDC region correlated very well with the three clusters and outlier strains found using housekeeping gene DNA sequences, enabling us to follow the sequence of mutational change in the flhDC locus. Two cluster 2 strains were found to have unique flhDC sequences, which are most probably due to recombination during the exchange of the adjacent O-antigen gene clusters.

Simple heat-conduction and thermoacoustic self-sustained oscillation branches are discussed employing current thermodynamics and thermoacoustic theory. Supposing a short regenerator where the local entropy production rate is homogeneous, thermodynamic stability is discussed analytically. Minimizing the entropy production rate of the short regenerator gives both simple heat conduction and the thermoacoustic self-sustained branches for a given oscillation mode. Necessary conditions for maintaining the thermoacoustic self-sustained branch restrict the oscillation mode. The conditions required for attaining stable oscillation in the short regenerator are also discussed.

In IL-5 transgenic mice (C3H/HeN-TgN(IL-5)-Imeg), in which 50% of peripheral blood leukocytes are eosinophils, the development of infection by Leishmania amazonensis was clearly suppressed. To determine mechanistically how this protozoan parasite is killed, we performed in vitro killing experiments. Either IL-4 or IFN-gamma effectively stimulated eosinophils to kill Leishmania amazonensis promastigotes, and most of the killing was inhibited by catalase but not by the NO inhibitor L-N5-(1-iminoethyl)-ornithine, suggesting that hydrogen peroxide is responsible for the killing of L. amazonensis by eosinophils. There was no significant degranulation of eosinophils in the culture, because eosinophil peroxidase was not detected in culture supernatants when L. amazonensis promastigotes were killed by activated eosinophils. Such resistance was also observed in BALB/c mice, which are highly susceptible to L. amazonensis. Expression plasmids for IL-4, IL-5, and IFN-gamma were transferred into muscle by electroporation in vivo starting 1 week before infection. Expression plasmid for IL-5 was most effective in slowing the development of infection among three expression plasmids. Expression plasmid for IL-4 was slightly effective and that for IFN-gamma had no effect on the progress of disease. These results suggest that IL-5 gene transfer into muscle by electroporation is useful as a supplementary protection method against L. amazonensis infection.

We have established a culture system for the development of eosinophils from murine embryonic stem (ES) cells. After transferring ES cells from embryonic fibroblast cells onto macrophage colony-stimulating factor-deficient stromal cells, OP9, ES cells were cultured in the presence of interleukin (IL)-5 with either IL-3 or granulocyte-macrophage colony stimulating factor (GM-CSF) for 20 d to obtain approximately 50% eosinophils. Electron microscopy confirmed the presence of crystallized major basic protein (MBP) in the granules of some of these cells. Neither IL-5, IL-3, GM-CSF nor eotaxin alone could induce eosinophils as efficiently as the conditions described above. Eotaxin induced eosinophil development in combination with either IL-3 or IL-5. Levels of GATA-1, Friend of GATA (FOG)-1, PU.1, CCAAT/enhancer binding protein (C/EBP)alpha, C/EBPbeta, IL-3 receptor alpha (IL-3Ralpha), GM-CSF receptor alpha (GM-CSFRalpha), and MBP mRNAs were increased in ES cells 10 d after transfer onto OP9 cells. In contrast, C/EBPepsilon, IL-5Ralpha, and eosinophil peroxidase mRNAs were induced in response to IL-3 and IL-5 after transfer onto OP9 cells. Eosinophils that developed in this system expressed Gr-1, F4/80, B220, CCR3, IL-3Ralpha, IL-5Ralpha, and DX5. Finally, eosinophils developed from ES cells produced reactive oxygen species in response to Leishmania as do peripheral blood eosinophils.

Thermoacoustic pheomena are classified into heat-pumping, prome-mover and dream-pipe including simple heat-conduction. Necessary conditions for thermoacoustic heat-engines are discussed using the first and second laws of thermodynamics and thermoacoustic theory. These conditions restirict possible regions of the temperature gradient and the phase difference between oscillations of pressure and displacement.

The entropy flow is rearranged in accordance with the new thermoacoustic theory. The local entropy production rate for thermoacoustic phenomena is formulated. The entropy production rate due to the effective heat conduction and that due to the dissipation of the work flow are positive. The results of formulation are rewritten in terms of normalized amplitude and impedance. Some empirical facts related to the normalized impedance are qualitatively discussed.