Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.31665/JFB.2024.18373

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.33555/jffn.v5i1.119

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1177/1934578X231183603

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/j.focha.2023.100253

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/j.phytol.2022.10.002

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：MDPI AG  

Cuminum cyminum L. (cumin) seeds are widely used as a spice. Although we previously reported that the aqueous extract of cumin seeds suppresses the degranulation of rat basophilic RBL-2H3 cells, it has not been clarified whether the extract alleviates actual allergy symptoms in vivo. Therefore, in this study, we investigated the effect of oral administration of cumin seed aqueous extract (CAE) in ovalbumin (OVA)-induced allergic rhinitis. BALB/c mice were randomly divided into the following three groups: control group (five mice), OVA group (five mice), and OVA + CAE group (five mice). Allergic rhinitis was induced by sensitization (intraperitoneal, 25 μg OVA and 1.98 mg aluminum hydroxide gel) followed by challenge (intranasal, 400 μg OVA). The oral administration of CAE (25 mg/kg) reduced the sneezing frequency of OVA-induced allergic rhinitis model mice. In addition to reducing the serum immunoglobulin E and IL-4 levels, the oral administration of CAE reduced the production of T-helper type-2 (Th2) cytokines (IL-4, IL-5, IL-10, and IL-13) in the splenocytes of the model mice. Furthermore, a significant increase in the ratio of Th1 to Th2 cells was observed in the CAE-administered group. Our findings suggest that the ingestion of CAE improves T cell balance, the dominant state of Th2, and alleviates allergic rhinitis symptoms.

DOI： 10.3390/foods11203224

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Language：English   Publishing type：Research paper (scientific journal)  

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：MDPI AG  

Coriander (Coriandrum sativum L.) is classified in the Apiaceae family and used as an herb. Coriander leaf has been reported to possess various health functions. Here, we report the anti-allergic effect of aqueous coriander leaf extract (ACLE). ACLE with 1.0 mg/mL or higher concentration significantly inhibited degranulation of RBL-2H3 cells in a concentration-dependent manner with no cytotoxicity. ACLE suppressed the increase in the intracellular Ca2+ concentration in response to antigen-specific stimulation. Immunoblot analysis demonstrated that ACLE significantly downregulates phosphorylation of phosphatidylinositol 3-kinase and tends to downregulate phosphorylation of Syk kinase in the signaling pathways activated by antigen-mediated stimulation. Oral administration of ACLE did not alter the sneezing frequency of pollinosis model mice stimulated with cedar pollen, but significantly reduced the serum IgE level. Our data show anti-allergic effects of coriander leaf in both cultured cells and pollinosis mice. These results suggest that coriander leaf has the potential to be a functional foodstuff with anti-allergy effects.

DOI： 10.3390/nutraceuticals2030013

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：MDPI AG  

Cuminum cyminum L. (cumin) is an annual plant of the Umbelliferae family native to Egypt. We previously showed that the aqueous extract of cumin seeds suppresses degranulation by downregulating the activation of antigen-induced intracellular signaling molecules in rat basophilic leukemia RBL-2H3 cells. However, the active substances in the extract have not yet been identified. Accordingly, herein, we aimed to ascertain the water-soluble substances present in cumin seeds that inhibit degranulation, which led to the identification of umbelliferose, a characteristic trisaccharide present in plants of the Umbelliferae family. Our study is the first to reveal the degranulation-suppressing activity of umbelliferose, and quantification studies suggest that cumin seed powder contains 1.6% umbelliferose. Raffinose, an isomer of umbelliferose, was also found to significantly suppress antigen-induced degranulation, but less so than umbelliferose. Both umbelliferose and raffinose contain sucrose subunits in their structures, with galactose moieties bound at different sites. These differences in structure suggest that the binding of galactose to the sucrose subunit at the α1-2 bond contributes to its strong degranulation-inhibiting properties.

DOI： 10.3390/molecules27134101

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Royal Society of Chemistry (RSC)  

We found that p-synephrine inhibits the production of proinflammatory cytokines in LPS-stimulated RAW264.7 cells. This inhibitory effect could be attributed to the downregulation of p38 MAPK and NF-κB cascades mediated by β-adrenergic receptors.

DOI： 10.1039/d2fo00299j

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Hasanuddin University, Faculty of Law  

Obesity is one of the major health problems which could trigger the development of many other non-communicable diseases. Tamarind (Tamarindus indica) is a multi-purpose plant that provides many health benefits, including anti-obesity potentials. In Indonesia, it is often used as a traditional food component in many dishes. A recent in vivo study proved that tamarind might reduce the bodyweight of obese rats. To further investigate the anti-obesity effect of tamarind, an in vitro study using 3T3-L1 pre-adipocytes was conducted. During differentiation, 3T3-L1 cells synthesize and accumulate lipid in the form of triglycerides in the cytoplasm. Tamarind water and ethanol extracts were incorporated into the medium during the differentiation stage of the cells. Oil red O staining assay was used to measure the total lipid accumulated. Results showed that both tamarind water and ethanol extracts exhibited non-cytotoxicity effects on 3T3-L1 cells. In addition, tamarind water extract at 10 mg/mL decreased the total lipid accumulated significantly to 80.5% compared with that of control, while tamarind ethanol extract showed less lipid accumulation inhibitory effect at any doses tested. This study revealed that tamarind water extract has the potential to reduce lipid accumulation, thus showing an anti-obesity effect.

DOI： 10.20956/canrea.v5i1.630

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Authorship：Last author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/j.legalmed.2021.101988

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：MDPI AG  

Inflammation is related to various life-threatening diseases including cancer, neurodegenerative diseases, and metabolic syndrome. Because macrophages are prominent inflammatory cells, regulation of macrophage activation is a key issue to control the onset of inflammation-associated diseases. In this study, we aimed to evaluate the potential anti-inflammatory activity of Citrus unshiu leaf extract (CLE) and to elucidate the mechanism underlying its anti-inflammatory effect. We found the inhibitory activity of CLE on the secretion of proinflammatory cytokines and a chemokine from mouse macrophage-like RAW 264.7 cells and mouse peritoneal macrophages. The inhibitory activity of CLE was attributed to downregulated JNK, p38 MAPK, and NF-κB signaling pathways, leading to suppressed gene expression of inflammation-associated proteins. Oral administration of CLE significantly decreased the serum level of proinflammatory cytokines IL-6 and TNFα and increased that of anti-inflammatory cytokine IL-10 in lipopolysaccharide-induced systemic inflammation mice. In addition, oral administration of CLE decreased secretion and gene expression of several proinflammatory proteins in the liver and spleen of the model mice. Overall results revealed that C. unshiu leaf is effective to attenuate inflammatory responses in vitro and in vivo.

DOI： 10.3390/plants10081708

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Authorship：Last author   Language：English  

DOI： 10.33555/jffn.v3i1.68

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Authorship：Lead author,　Corresponding author   Language：Japanese   Publishing type：Research paper (scientific journal)  

DOI： 10.3136/nskkk.68.356

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Authorship：Last author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Media Peternakan  

DOI： 10.5398/tasj.2021.44.4.511

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Authorship：Last author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.17509/ijost.v6i2.34544

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Academic Research and Community Service Swiss German University  

Allergy rhinitis (AR), as reported by the World Allergy Organization (WAO), is one of the highest prevalence allergies affecting 10-30% of all adults and up to 40% of children. In Indonesia, current evidence showed that the prevalence of AR is increasing. Averrhoa bilimbi Linn. fruit (AF), or locally known as belimbing wuluh, has been scientifically proven to treat many diseases due to the abundant of polyphenol content which was shown to have the potential to treat allergies. Therefore, this study was aimed to investigate the anti-allergy potential of AF in vitro. The anti-allergy effect of Averrhoa bilimbi Linn. fruit water extract (AFWE) was examined using RBL-2H3 cells. At first, the cytotoxicity effect of AFWE was determined by WST-8 assay. The release of β-hexosaminidase by RBL-2H3 cells was also measured to evaluate degranulation suppression activity of AFWE. Lastly, calcium assay was employed to investigate the intracellular calcium concentration ([Ca­2+]i). Results demonstrated that AFWE does not show any cytotoxicity at any given concentration. In addition, AFWE at 1.25 mg/mL showed sufficient inhibitory effect towards degranulation by RBL-2H3 cells. Moreover, the degranulation-suppressing activity of AFWE was resulted from the inhibition of calcium-dependent signaling pathways. Unfortunately, the properties of active substances from AFWE have not been investigated. To conclude, this study indicated that AFWE has potential as an alternative treatment for allergic diseases.

DOI： 10.33555/jffn.v2i2.62

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Authorship：Last author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.bmcl.2020.127191

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Language：English   Publishing type：Research paper (international conference proceedings)   Publisher：IOP Publishing  

DOI： 10.1088/1757-899x/821/1/012028

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Other Link： https://iopscience.iop.org/article/10.1088/1757-899X/821/1/012028

Authorship：Last author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1186/s41702-020-0061-z

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.molimm.2019.12.020

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1021/acs.jmedchem.9b00994

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Language：English   Publishing type：Research paper (scientific journal)  

Ficifolidione, a natural insecticidal compound isolated from the essential oils of Myetaceae species, is a spiro phloroglucinol with an isobutyl group at the C-4 position. We found that ficifolidione showed cytotoxicity against cancer cells via apoptosis. Replacement of the isobutyl group by n-propyl group did not influence the potency, but the effect of the replacement of this group by a shorter or longer alkyl group on the biological activity remains unknown. In this study, ficifolidione derivatives with alkyl groups such as methyl, n-pentyl, and n-heptyl group-instead of the isobutyl group at the C-4 position-were synthesized to evaluate their cytotoxicity against the human promyelocytic leukaemia cell line HL60 and their insecticidal activity against mosquito larvae. The biological activities of their corresponding 4-epimers were also evaluated. As a result, the conversion of the isobutyl group to another alkyl group did not significantly influence the cytotoxicity or insecticidal activity. In HL60 cells treated with the n-heptyl-ficifolidione derivative, the activation of caspase 3/7 and the early stages of apoptosis were detected by using immunofluorescence and flow cytometric techniques, respectively, suggesting that the cytotoxicity should be induced by apoptosis even though the alkyl group was changed.

DOI： 10.3390/molecules24224081

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Authorship：Last author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1038/s41598-019-52317-z

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1080/09168451.2019.1618697

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier {BV}  

DOI： 10.1016/j.jff.2019.103422

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1007/s10616-019-00323-4

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Authorship：Last author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1021/acs.jafc.9b03171

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1007/s10616-019-00309-2

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1007/s10616-019-00296-4

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Authorship：Last author   Language：English   Publishing type：Research paper (scientific journal)  

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer Netherlands  

Lysozyme from hen egg has been reported to possess an anti-inflammatory effect. However, little is known about its detailed mechanism. The mechanism of anti-inflammatory effect of lysozyme was examined in this study. When mouse macrophage-like cell line RAW264.7 cells and mouse peritoneal macrophages were activated with lipopolysaccharide (LPS) and then treated with lysozyme, the production of tumor necrosis factor-α and interleukin-6 was significantly suppressed. The effect was induced by suppressing the gene expression levels of both cytokines. Phagocytosis activity of peritoneal macrophages was not altered by the treatment with lysozyme, suggesting that lysozyme shows the anti-inflammatory effect without inhibiting the phagocytotic response of macrophages. In addition, lysozyme inhibited phosphorylation of c-jun N-terminal kinase (JNK) and was taken up by macrophages within 1 h after treatment of the cells with lysozyme. Overall results suggest that lysozyme is taken up intracellularly and suppresses LPS-induced inflammatory responses by inhibiting JNK phosphorylation.

DOI： 10.1007/s10616-017-0184-2

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer Netherlands  

Japanese black vinegar (JBV) is a traditional vinegar manufactured with steamed unpolished rice. After screening, beneficial effects of JBV on IgE-mediated allergic responses were found. In this study, acetic acid-free JBV was used to evaluate its antiallergic effects. JBV suppressed degranulation of rat basophilic leukemia RBL-2H3 cells in a dose-dependent manner without cytotoxicity. The inhibitory effect of JBV on the degranulation seemed to be caused by the bioactive ingredients other than proteins, because the activity was not affected by heat treatment or protease digestion. JBV inhibited the elevation in the intracellular Ca2+ concentration induced by antigen. Immunoblot analysis revealed that JBV suppresses degranulation of RBL-2H3 cells by downregulated phosphorylation of PI3K, Akt, and PLCγ1. In addition, oral administration of JBV significantly suppressed passive cutaneous anaphylaxis reaction in mice and an allergic symptom in Cry j1-induced pollinosis model mice. Thus, JBV has a potential as a health-promoting food with the antiallergy effect.

DOI： 10.1007/s10616-018-0208-6

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Society for Indonesian Biodiversity  

Asam kandis (dried Garcinia xanthocymus Hook.f.ex.T.Anderson) is popularly used as a spice in many Indonesian cuisines. Although Garcinia xanthocymus has been reported to possess various pharmacological properties, its immunologic functions have been poorly studied. Therefore, in this study, we aimed to determine immunoglobulin E (IgE) production-suppressing effect of Asam Kandis extract (AKE) using mouse hybridoma aDNP-Hy cells. AKE was prepared through 24 hours maceration in distilled water. IgE production-suppressing activity of AKE by aDNP-Hy cells was examined in time-wise manner. Cell viability and IgE mRNA expression were assessed using trypan blue assay and qRT-PCR, respectively. IgE production-suppressing activity of heat-treated (100°C) AKE was also examined. As a result, AKE significantly reduced IgE production by aDNP-Hy cells in a concentrationdependent manner both in 24 and 36 h incubation periods. Furthermore, AKE exhibited its IgE production-suppressing activity without any cytotoxicity and also significantly decreased IgE mRNA expression. Heat-treated AKE showed no IgE production-suppressing activity, indicating that the bioactive substances are heat labile. More interestingly, heat-treated AKE showed cytotoxic effect by aDNPHy cells. We showed that AKE exhibits IgE production-suppressing effect by aDNP-Hy cells. Further studies are necessary to discover underlying bioactive compounds of asam kandis and substance that responsible for the cytotoxic effect of heat-treated AKE.

DOI： 10.13057/biodiv/d190145

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Authorship：Last author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Society for Indonesian Biodiversity  

Indonesia is known to be home to various fermented foods with many-reported usage as potential sources of probiotics. Tempoyak (fermented durian) is among one of the Indonesian fermented foods that are rarely studied for its bioactivities. This study was conducted to evaluate the potential bioactivities of tempoyak, particularly the immunostimulatory aspects. Water extract of tempoyak was prepared by suspending the freeze-dried tempoyak sample in distilled water. Immunostimulatory activity of tempoyak water extract was evaluated using mouse macrophage cell line RAW 264.7. ELISA was used to screen cytokine productions (IL-6 and TNF-α) by RAW 264.7 cells following treatment with tempoyak water extract. In addition, real-time RT-PCR was also used to determine IL-6 and TNF-α mRNA expression. We showed that water extract of tempoyak exerts immunostimulatory effects towards RAW 264.7 cells. This was observed from the increased production of IL-6 and TNF-α in a dose-dependent manner. This was also supported by increased IL-6 and TNF-α mRNA expression. Our finding suggests that tempoyak has immunostimulatory effects towards murine macrophage cell line RAW 264.7. However, further studies are needed to identify the specific compounds responsible for inducing immunostimulatory effects.

DOI： 10.13057/biodiv/d190143

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Publishing type：Research paper (scientific journal)  

DOI： 10.20956/canrea.v1i2.99

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Japan Society for Bioscience Biotechnology and Agrochemistry  

Immunoglobulin E (IgE) is involved in the onset of allergic reaction, and the suppression of IgE production leads to alleviation of allergic symptoms. We found that mango peel ethanol extract (MPE) significantly suppresses IgE production by human myeloma cell line U266 cells, suggesting that MPE has an anti-allergic effect by inhibiting the production of IgE. Although mangiferin is contained in mango, which suppresses IgE production by U266 cells, it was not contained in MPE. We investigated the suppressive effect of MPE in 2,4-dinitrofluorobenzene (DNFB)-induced allergic contact dermatitis model mice. The elevation of serum IgE level was significantly suppressed by oral administration of MPE. Intake of MPE also suppressed the expression level of IL-4 in the DNFB-challenged ears, suggesting that MPE suppresses the IL-4mediated maturation into IgE-producing cells. Our findings indicate that MPE has a potential to alleviate the increase in serum IgE level that is feature of type I allergy.

DOI： 10.1080/09168451.2017.1412250

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY  

BACKGROUNDCoriandrum sativum L. seed is generally used as a spice and crude drug. Although many functions of the various components in C. sativum L. seed have been reported, the immunostimulatory effect of water-soluble components in C. sativum L. seed has not been studied. In the present study, we focused on the immunostimulatory effect of C. sativum L. seed aqueous extract (CAE) on macrophages as a novel health function of C. sativum L. seed components.
RESULTSCAE significantly enhanced the production of tumor necrosis factor (TNF)- and interleukin (IL)-6 in both RAW264.7 cells and peritoneal macrophages by enhancing the expression levels of these cytokine genes. CAE also stimulated nitric oxide (NO) production and the phagocytosis activity in RAW264.7 cells. We suggest that the activity of CAE is a result of the upregulation of mitogen-activated protein kinase and nuclear factor-B cascades via TLR4. In addition, IL-6 production by peritoneal macrophages collected from CAE-administered mice was significantly enhanced, suggesting that CAE could stimulate macrophage activity in vivo.
CONCLUSIONThe findings of the present study suggest that CAE contains a novel water-soluble component with an immunostimulatory effect on macrophages. CAE would contribute to activating host defense against pathogens by stimulating the innate immunity. (c) 2017 Society of Chemical Industry

DOI： 10.1002/jsfa.8341

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

The new lignano-9,9'-lactones (alpha,beta-dibenzyl-y-butyrolactone lignans), which showed the higher cytotoxicity than arctigenin, were synthesized. The well-known cytotoxic arctigenin showed activity against HL-60 cells (EC50 =12 IA/I), however, it was inactive against HeLa cells (EC50 > 100 mu M). The synthesized (3,4-dichloro, 2'-butoxy)-derivative 55 and (3,4-dichloro, 4'-butyl)-derivative 66 bearing the lignano9,9'-lactone structures showed the EC50 values of 10 mu M and 9.4 mu M against HL-60 cells, respectively. Against HeLa cells, the EC50 value of the derivative 66 was 27 mu M. By comparing the activities with the corresponding 9,9'-epoxy structure (tetrahydrofuran compounds), the importance of the lactone structure of 55 and 66 for the higher activities was shown. The substituents on the aromatic ring of the lignano-9,9'-lactones affected the cytotoxicity level, observing more than 10-fold difference. (C) 2017 Published by Elsevier Ltd.

DOI： 10.1016/j.bmcl.2017.06.070

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：MDPI AG  

Porang is a local plant of Indonesia, which has a high content of glucomannan. In this study, porang glucomannan (PG) was esterified with octenyl succinic anhydride (OSA) to enhance emulsion properties to be widely used in food industry. OSA-modified PG (OSA-PG) enhanced the phagocytosis activity of macrophage-like J774.1 cells and mouse peritoneal macrophages. In addition, OSA-PG increased the production of IL-6 and TNF-alpha by enhancing their gene expression. Immunoblot analysis displayed that OSA-PG tended to activate both nuclear factor-kappa B and mitogen-activated protein kinase cascades. Treatment of OSA-PG with polymyxin B revealed that cytokine production induced by OSA-PG was not caused by endotoxin contamination. Our findings also indicated that OSA-PG activates macrophages through not only Toll-like receptor (TLR) 4, but another receptor. Overall findings suggested that OSA-PG has a potential as an immunomodulatory food factor by stimulating macrophages.

DOI： 10.3390/molecules22071187

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Authorship：Last author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ASSOC RESEARCH VISION OPHTHALMOLOGY INC  

PURPOSE. To evaluate the effects of mandarin orange yogurt containing nobiletin and beta-lactoglobulin on the allergic conjunctivitis induced by a conjunctival allergen challenge (CAC).
METHODS. Experiment 1 was performed on 26 asymptomatic patients (age, 25.3 +/- 5.3 years) with proven seasonal allergic conjunctivitis due to cedar pollen. We compared the degree of conjunctivitis induced by CAC before and after ingesting mandarin orange yogurt for 2 weeks. Experiment 2 was a double-blind, placebo-controlled trial performed on 31 patients (age, 32.5 +/- 12.2 years). A diet containing mandarin orange yogurt was compared to a diet containing yogurt lacking the mandarin orange on the conjunctivitis induced by CAC. The temperature of the inferior bulbar conjunctiva was measured before and 20 minutes after the CAC with an ocular surface thermographer (OST). The degree of conjunctival injection and chemosis was graded by slit-lamp biomicroscopy. The changes in the symptoms were evaluated by a questionnaire.
RESULTS. In experiment 1, the scores of redness (3.07 +/- 3.03 vs. 1.05 +/- 1.70), chemosis (2.84 +/- 2.27 vs. 0.81 +/- 1.11), itching (4.34 +/- 3.05 vs. 1.39 +/- 2.12), and temperature (0.73 +/- 0.42 degrees C vs. 0.45 +/- 0.43 degrees C) were significantly lower (P < 0.001) after a diet of mandarin orange yogurt for 2 weeks. In experiment 2, the scores of redness (1.03 +/- 0.18 vs. 1.28 +/- 0.52; P = 0.0156), itching (1.93 +/- 1.92 vs. 2.82 +/- 2.21; P = 0.0133), and surface temperature (0.54 +/- 0.21 degrees C vs. 0.31 +/- 0.25 degrees C; P < 0.001) were significantly lower in the mandarin orange yogurt group than in the control yogurt group.
CONCLUSIONS. Mandarin orange yogurt can be an effective nutritional intervention for allergic conjunctivitis.

DOI： 10.1167/iovs.16-21206

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

An array of foodstuffs was screened for the suppressive effect on production of IgE, a key molecule in the type I allergic reaction, using U266 cells. An ethanol extract from passion fruit seeds (PFS) has emerged as a food material with the IgE production-suppressive effect as the result of screening. Real-time RT-PCR analysis revealed that the IgE production-suppressive effect was caused by downregulated IgE gene expression. In addition, PFS selectively suppressed IgE production and did not affect the production of IgG, IgA, or IgM by mouse splenocytes. Resveratrol and piceatannol were identified as bioactive ingredients contained in passion fruit seeds. The effect of oral administration of PFS, resveratrol, and piceatannol was evaluated using a mouse model of allergic contact dermatitis. Both stilbenoids significantly suppressed IgE production in orally administered mice. Overall results indicated that passion fruit seeds are a promising candidate with the antiallergy effect for development of functional foods. (C) 2017 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.jff.2017.02.030

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER  

Dried bonito is a preserved food used in Japan, which contains abundant flavor ingredients and functional substances. We focused on the immunostimulatory effect of dried bonito extract (DBE) on mouse macrophage-like J774.1 cells, RAW264.7 cells, and mouse primary peritoneal macrophages. DBE significantly stimulated the production of tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) by both J774.1 cells and peritoneal macrophages by enhancing the cytokine gene expression levels. In addition, DBE stimulated nitric oxide production by enhancing the expression of inducible nitric oxide synthase in RAW264.7 cells. DBE also increased the phagocytosis activity of J774.1 cells. Immunoblot analysis revealed that DBE has an immunostimulatory effect on macrophages through activation of mitogenactivated protein kinase and nuclear factor-jB cascades. TNF-alpha production enhanced by DBE was partially inhibited by treatment with TLR4 inhibitor TAK-242, whereas IL-6 production enhanced by DBE was almost inhibited. These results suggested that DBE is thought to strongly stimulate the TLR4 signaling pathway for macrophage activation, and its activation is also involved in other signaling. Finally, the phagocytosis activity of peritoneal macrophages from DBE-administered BALB/ c mice increased significantly, suggesting that DBE has the potential to stimulate macrophage activity in vivo. In conclusion, these data indicated that DBE contributes to activating host defense against pathogens by activating innate immunity.

DOI： 10.1007/s10616-016-0053-4

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (international conference proceedings)   Publisher：AMER INST PHYSICS  

Snake fruit (Salacca edulis Reinw) cultivar Pondoh Hitam is a tropical fruit produced in Indonesia. It is consumed freshly or processed and believed as the most delicious snake fruit cultivar. Snake fruit flesh contains high polisaccharides such as pectin and dietary fiber. Therefore, snake fruit is a potential immunostimulator candidates but the immunological effect of snake fruit flesh has not been reported. In the present study, immunostimulatory activity of snake fruit flesh extract (SFFE) on macrophages activation was evaluated.
SFFE was prepared by extracting from snake fruit flesh with water, methanol 70%, and ethanol 70% for 15 h at 4 degrees C. Then obtained SFFE was used to stimulated cytokine production in vitro using J774.1 cell line. The extract giving strongest stimulation was sellected for in vivo assay to stimulate cytokines production and gene expression using peritoneal macrophage (P-mac) of BALB/c mice.
The results showed that SFFE exhibited immunostimulatory activities. Immunostimulatory activity could be indicated by macrophages activation characteristics such as cytokines production. Water extract of SFFE gave strongest stimulation on cytokines production in vitro and sellected for in vivo assay. In vivo assay showed that SFFE stimulated cytokines production as well as their gene expression levels. The optimum stimulation was demonstrated by SFFE 16.7 mg/g. Overall findings suggest that SFFE has a potent beneficial effects to promote the body health through activating macrophages.

DOI： 10.1063/1.4982287

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER  

Chlorophyll c2 extracted from Sargassum horneri improved allergic symptoms in an animal model of allergic rhinitis. In the present study, we explored the efficacy of chlorophyll c2 in patients with seasonal allergic rhinitis. This was a single-center, randomized, double-blind placebo-controlled trial. Sixty-six patients aged 20-43 years, each with a 2-year history of seasonal allergic rhinitis, were randomly assigned to receive either a single daily dose (0.7 mg) of chlorophyll c2 or placebo for 12 weeks. The use of medications including H1-antihistamines and topical nasal steroids was recorded by rescue medication scores (RMSs) noted after 4, 8, and 12 weeks of treatment. Disease-specific quality of life was measured using the Japan Rhinitis Quality of Life Questionnaire (JRQLQ) both before and after 4, 8, and 12 weeks of treatment. The RMS at 8 weeks was significantly better in the chlorophyll c2 than the placebo group (mean RMS difference = -3.09; 95 % confidence interval = -5.96 to -0.22); the mean RMS at 4 weeks was only slightly better in the chlorophyll c2 group. The JRQLQ scores did not differ significantly between the two groups. Chlorophyll c2 would have a potential to be an alternative treatment for allergic rhinitis.

DOI： 10.1007/s00405-016-4133-z

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Nobiletin is a major citrus flavonoid possessing several biological activities. Nobiletin has been reported to display anti-inflammatory activity and to reduce monocyte chemoattractant protein (MCP)-1 secretion by 3T3-L1 adipocytes. However, its mode of action remains unclear. In the present study, we investigated the suppressive effect of nobiletin on MCP-1 expression in 3T3-L1 cells and its underlying molecular mechanism. Nobiletin notably down regulated the MCP-1 mRNA level during adipocyte differentiation. Nobiletin also reduced MCP-1 secretion by preadipocytes (undifferentiated cells), differentiated adipocytes, and hypertrophied adipocytes. Immunoblot analysis showed that nobiletin inhibits the phosphorylation level of extracellular signal-regulated kinase (ERK) in both preadipocytes and differentiated adipocytes. Moreover, nobiletin suppressed MCP-1 secretion by adipocytes induced by tumor necrosis factor (TNF)-alpha. (C) 2016 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.jff.2016.09.025

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER  

Snake fruit (Salacca edulis Reinw.) is a tropical fruit produced in Indonesia. Snake fruit peel is normally discarded as waste. In the present study, it was revealed that snake fruit peel has high bioactivities on stimulation of the immune system. Snake fruit peel extract (SFPE) was prepared by extracting snake fruit peel powder in water for 15 h at 4 A degrees C. SFPE enhanced phagocytotic activity of murine macrophage-like J774.1 cells. Production of cytokine such as tumour necrosis factor (TNF)-alpha and interleukin (IL)-6 was also stimulated by SFPE. The gene expression levels for these cytokines were elevated. Immunoblot analysis revealed that SFPE enhanced not only nuclear factor (NF)-kappa B but also mitogen-activated protein kinases signalling cascades such as JNK and p38 in macrophage. Overall findings suggested that SFPE has a potential beneficial effect to promote our body health through the stimulation of macrophage.

DOI： 10.1007/s10616-015-9925-2

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

To estimate the effect of methyl group of dihydroguaiaretic acid, which shows many kinds of biological activities, on biological activity, both enantiomers of 9'-dehydroxyimperanene (5, 6) and 7,8-dihydro-9'-dehydroxyimperanene (7, 8) lacking one of the methyl groups of dihydroguaiaretic acid were synthesized. (S)-7,8-Dihydro-9'-dehydroxyimperanene (7) showed 4-6-fold higher cytotoxic activity than all stereoisomers of dihydroguaiaretic acid (2-4). The IC50 values of (S)-7,8-dihydro-9'-dehydroxyimperanene (7) against HL-60 and HeLa cells were 6.1 mu M and 5.6 mu M, respectively. Though only one of three stereoisomers of dihydroguaiaretic acid showed antibacterial activity against a gram negative bacterium, both enantiomers of 5-8 showed antibacterial activity against a gram negative bacterium. This is a Letter on biological activity of 9-norlignan, in which one of methyl groups of lignan is absent. (C) 2016 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.bmcl.2016.05.020

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：TAYLOR & FRANCIS LTD  

We herein report the immunostimulatory effect of spinach aqueous extract (SAE) on mouse macrophage-like J774.1 cells and mouse primary peritoneal macrophages. SAE significantly enhanced the production of interleukin (IL)-6 and tumor necrosis factor- by both J774.1 cells and peritoneal macrophages by enhancing the expression levels of these cytokine genes. In addition, the phagocytosis activity of J774.1 cells was facilitated by SAE. Immunoblot analysis revealed that SAE activates mitogen-activated protein kinase and nuclear factor-B cascades. It was found that SAE activates macrophages through not only TLR4, but also other receptors. The production of IL-6 was significantly enhanced by peritoneal macrophages from SAE-administered BALB/c mice, suggesting that SAE has a potential to stimulate macrophage activity in vivo. Taken together, these data indicate that SAE would be a beneficial functional food with immunostimulatory effects on macrophages.

DOI： 10.1080/09168451.2016.1146068

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Authorship：Last author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

BACKGROUND: Equol is a metabolite of daidzein that is produced by intestinal microbiota. The oestrogenic activity of equol is stronger than daidzein. Equol-producing bacteria are believed to play an important role in the gut. The rod-shaped and Gram-positive anaerobic equol-producing intestinal bacterium Slackia TM-30 was isolated from healthy human faeces and its effects on urinary phyto-oestrogen, plasma and faecal lipids were assessed in adultmice.
RESULTS: The urinary amounts of equol in urine were significantly higher in mice receiving the equol-producing bacterium TM-30 (BAC) group than in the control (CO) group (P<0.05). However, no significant differences were observed between the urinary amounts of daidzein, dihydrodaidzein, enterodiol, and enterolactone between the BAC and CO groups. No significant differences in the plasma lipids were observed between the two groups. The lipid content (% dry weight) in the faeces sampled on the final day of the experiment tended to be higher in the BAC group than in the CO group (P= 0.07).
CONCLUSION: Administration of equol-producing bacterium TM-30 affected the urinary amounts of phyto-oestrogens and the faecal lipid contents of mice. The equol-producing bacterium TM-30 likely influences the metabolism of phyto-oestrogen via changes in the gastrointestinal environment. (C) 2015 Society of Chemical Industry

DOI： 10.1002/jsfa.7490

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Nobiletin and beta-lactoglobulin were previously demonstrated to suppress mast cell de granulation in vitro and in vivo. In this study, the multiple anti-allergic effects of both substances by simultaneous treatment were examined in order to determine if they could further mitigate allergic symptoms. Concurrent treatment with nobiletin and beta-lactoglobulin effectively suppressed de-granulation of RBL-2H3 cells by inhibiting increases in intracellular Ca2+ concentration. Immunoblot analysis showed that phosphorylation of phosphoinositide 3-kinase, Akt, and Syk in RBL-2H3 cells was all inhibited by the concurrent presence of nobiletin and beta-lactoglobulin. Moreover, concurrent administration of nobiletin and beta-lactoglobulin reduced passive cutaneous anaphylaxis (PCA) reaction in mice compared with nobiletin or beta-lactoglobulin administered alone. In addition, the concurrent administration of freeze-dried Citrus unshiu peel powder and yogurt whey containing these bioactive substances also reduced PCA reaction and allergic symptoms of a murine model of Japanese cedar pollinosis. (C) 2016 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.jff.2016.01.045

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：TAYLOR & FRANCIS LTD  

DOI： 10.1080/09168451.2015.1123603

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ROYAL SOC CHEMISTRY  

The effects of small dried sardine on IgE-mediated allergic responses are herein reported. Small dried sardine extract digested with an acidic protease from Aspergillus niger suppressed degranulation of rat basophilic leukemia RBL-2H3 cells in a dose-dependent manner without cytotoxicity. Size-exclusion chromatography analysis and a dialysis experiment indicated that the molecular weight of bioactive components contained in enzyme-treated dried sardine extract (EDS extract) ranges from 4800 to 14 000. Immunoblot analysis revealed that EDS extract does not affect the signaling pathway via the spleen tyrosine kinase Syk. Although degranulation of RBL-2H3 cells induced by either antigen or a calcium ionophore A23187 was suppressed by EDS extract, the elevation in intracellular Ca2+ concentration was not affected. Immunofluorescence staining revealed that EDS extract significantly suppresses microtubule formation in RBL-2H3 cells during degranulation induced by antigen. In addition, oral administration of EDS extract significantly suppressed passive cutaneous anaphylaxis reaction in mice and an allergic symptom in Cry j1-and Cry j2-induced pollinosis model mice. In conclusion, small dried sardine treated with a protease has potential as a health-promoting foodstuff with an anti-allergic effect.

DOI： 10.1039/c6ra14967g

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：INFORMA HEALTHCARE  

Methoxychlor, an organochlorine insecticide developed to replace DDT (dichlorodiphenyltrichloroethane), has been reported to induce mast cell degranulation and to enhance IgE-mediated allergic responses. However, the mechanisms underlying these effects are not clear. To clarify potential mechanisms, the effects of methoxychlor on degranulation of mast cells were examined. Degranulation responses were evaluated using RBL-2H3 cells and mouse bone marrow-derived mast cells with either the antigen-induced or calcium ionophore-induced stimulation. Phosphorylation of enzymes related to signaling events associated with mast cell degranulation was analyzed by immunoblotting. Effects on vascular permeability in the passive cutaneous anaphylaxis reaction were evaluated following oral administration of methoxychlor to BALB/c mice. The results indicated that methoxychlor caused increased mast cell degranulation in the presence of antigen, whereas it had no effect on calcium ionophore-induced degranulation of RBL-2H3 cells. Immunoblot analyses demonstrated that the phosphorylation level of phosphoinositide 3-kinase (which plays a central role in mast cell signaling) was increased by methoxychlor during antigen-induced degranulation. In addition, methoxychlor activated the signaling pathway via the high-affinity IgE receptor by inducing phosphorylation of Syk and PLC gamma 1/2, which transfer the signal for degranulation downstream. Lastly, oral administration of methoxychlor exhibited a tendency to promote vascular permeability in passive cutaneous anaphylaxis model mice. Taken together, the results here suggested that methoxychlor enhanced degranulation through Fc epsilon RI-mediated signaling and promoted allergenic symptoms involved in mast cell degranulation.

DOI： 10.3109/1547691X.2014.962122

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Jellyfish has recently attracted much attention for various applications, including functional foods due to its abundance of collagen. Jellyfish collagen, extracted from Nemopilema nomurai, was found to stimulate murine macrophage-like J774.1 cells. However, few reports have determined the immunostimulatory effects of jellyfish collagen on the innate immune response. We herein demonstrate the effect of jellyfish collagen on mouse bone marrow-derived dendritic cells (BMDCs). Jellyfish collagen stimulated TNF-alpha, IL-6, IL-1 beta and IL-12 production by BMDCs as the result of the elevation of gene expression level of these cytokines. In addition, jellyfish collagen-treated BMDCs have more wrinkles and longer pseudopodia on the cell surface compared with the control cells. Jellyfish collagen also stimulated cell-surface MHC-II expression level. Furthermore, jellyfish collagen downregulated phagocytosis capacity of BMDCs. Thus, our findings suggest that JC has the potential to activate DCs and thereby contribute to health promotion. (C) 2015 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.jff.2015.02.008

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Ishizuchi dark tea is a microorganism-fermented tea produced in Ehime, Japan. We herein describe the anti-allergic effect of Ishizuchi dark tea. The water extract from Ishizuchi dark tea suppressed degranulation of RBL-2H3 cells in a dose-dependent manner with no apparent cytotoxicity. Fractionation and beta-hexosaminidase release assay of the tea components revealed theabrownins as the major bioactive substance in Ishizuchi dark tea. Phosphorylation of Syk, a tyrosine kinase, tended to be suppressed by treating RBL-2H3 cells with theabrownins. In addition, Ishizuchi dark tea suppressed an allergic symptom in the Cry j1-induced pollinosis model mice. Taken together, these data indicated that Ishizuchi dark tea would be a beneficial functional food with an anti-allergic effect. (C) 2015 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.jff.2015.02.045

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

(-)-Dihydroguaiaretic acid (DGA) and its derivatives having 3-hydroxyphenyl (3-OH-DGA) and variously substituted phenyl groups instead of 3-hydroxy-4-methoxyphenyl groups were synthesized to measure their larvicidal activity against the mosquito Culex pipiens Linnaeus, 1758 (Diptera: Culicidae). Compared with DGA and 3-OH-DGA (LC50 (M), 3.52 x 10(-5) and 4.57 x 10(-5), respectively), (8R,8'R)-lignan-3-ol (3) and its 3-Me (10), 2-OH (12), 3-OH (13), and 2-OMe (15) derivatives showed low potency (ca. 6-8 x 10(-5) M). The 4-Me derivative (11) showed the lowest potency (12.1 x 10(-5) M), and the 2-F derivative (4) showed the highest (2.01 x 10(-5) M). All of the synthesized compounds induced an acute toxic symptom against mosquito larvae, with potency varying with the type and position of the substituents. The 4-F derivative (6), which killed larvae almost completely within 45 min, suppressed the O-2 consumption of the mitochondrial fraction, demonstrating that this compound inhibited mitochondrial O-2 consumption contributing to a respiratory inhibitory activity.

DOI： 10.1021/jf504816a

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Ficifolidione (1), a moderately active insecticidal compound from two species of Myrtaceae, and its derivatives were synthesized to evaluate their insecticidal activity. X-ray crystallographic analyses and specific rotation values of ficifolidione and its C-4 (2) demonstrated that the structure of ficifolidione differs from the reported absolute structure; that is, the C-4 configuration of ficifolidione should have an S configuration. The reported insecticidal activity of ficifolidione (1) and its C-4 epimer (2) against adult houseflies (Musca domestica), mosquito larvae (Culex pipiens), and cutworms (Spodoptera litura) was not observed. The cytotoxicities of ficifolidione and its derivatives (14) against four cell lines, Sf9, Colon26, HL60, and Vero, were also measured because ficifolidione has a phloroglucinol-derived moiety, a motif that is often present in the structure of cytotoxic chemicals. Compound 1 exhibited IC50 values of ca. 32, 9, 3, and 12 mu M for Sf9, Colon26, HL60, and Vero cells, respectively, indicating that ficifolidione possesses selective cytotoxicity against the four cell lines. In HL60 cells treated with 1, DNA fragmentation and the activation of procaspase 3 were observed, suggesting that the cytotoxicity is induced by apoptosis.

DOI： 10.1021/np5006746

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Lipid vesicles are potentially useful as microcapsules for drug and/or gene delivery. We developed cationic lipid vesicles consisting mainly of sorbitan monooleate (Span 80) and cationic peptide lipid (CPL), and evaluated the CPL vesicles as gene transfection vectors. The optimum CPL concentration for gene transfection into HeLa cells was found to be 20 wt % of total lipid, and such CPL vesicles did not exhibit significant cytotoxicity. Co-culture Of poly-L-lysine and plasmids prior to making CPL vesicle-plasmid complexes was effective. Lipofection using LipofectAMINE was suppressed in 10% serum-supplemented medium. The transfection efficiency of 20 wt % CPL vesicles, however, was not affected by serum in the medium when plasmids were treated with poly-L-lysine.

DOI： 10.1271/bbb.69.1453

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We have already reported that lactate dehydrogenase (LDH) activates lymphocytes in vitro and in vivo. In this paper, we report the activating effects of LDH on the macrophage-like cell line J774.1. LDH was found to enhance production of IL-6 and TNF-alpha by J774.1 cells in a dose-dependent manner. Transcription levels of IL-6 and TNF-alpha in J774.1 cells were also enhanced by supplementation with LDH. From immunoblot analysis, it was revealed that LDH enhances the phosphorylation level of JNK in J774.1 cells. Moreover, the JNK inhibitor SP600125 decreased production of IL-6 and TNF-alpha induced by LDH. NF-kappa B translocation to the nucleus was also facilitated by LDH. These results was revealed that LDH enhances production of IL-6 and TNF-alpha by J774.1 cells via the increase of JNK phosphorylation and NF-kappa B translocation to the nucleus. Our data indicated that macrophages may be activated by LDH released from damaged tissues and cells in our body.

DOI： 10.1007/s10616-013-9646-3

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Various foodstuffs were screened for the ability to prevent mast cell degranulation using a rat basophilic leukaemia cell line (RBL-2H3 cells); whey emerged as a candidate to alleviate allergic symptoms. Further exploration of the suppressive effect of whey on mast cell degranulation led to identification of beta-lactoglobulin (BLG) as one of the active substances. The potential of BLG to modulate allergic responses of mast cells using the RBL-2H3 cells in vitro with the passive cutaneous anaphylaxis (PCA) reaction model mice in vivo is described. BLG was shown to suppress degranulation of RBL-2H3 cells. The molecular mechanism underlying the inhibitory effect of BLG on degranulation of RBL-2H3 cells included reduced phosphorylation of the tyrosine kinase Syk, phosphoinositide 3-kinase, and phospholipase C-gamma 1/2. Furthermore, oral administration of BLG tended to suppress the PCA reaction in the anti-dinitrophenol (DNP) IgE-sensitised, DNP-human serum albumin-challenged BALB/c mice, presenting a new biological functionality of BLG. (C) 2014 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.idairyj.2014.05.006

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Chlorophyll c2 in a Sargassum horneri extract suppressed the degranulation of rat basophilic leukaemia RBL-2H3 cells. In this contribution, the mode of action of S. horneri extract was revealed. The extract inhibited the elevation of intracellular Ca2+ concentration induced by the antigen. Immunoblot analysis showed that S. horneri extract down-regulated the phosphorylation of both Syk and PI3K in the signalling pathways involved in degranulation triggered by antigen-antibody interaction. In addition, oral administration of S. horneri extract inhibited mice nasal rubbing behaviour sensitized with anti-DNP IgE. These findings suggest that S. horneri extract has anti-allergic activity via the suppression of degranulation of mast cells and basophils. (C) 2014 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.jff.2014.06.002

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All stereoisomers of methoxybutane and fluorobutane type of 1,7-seco-2,7'-cyclolignane were synthesized and cytotoxic activities of these compounds were compared with those of all stereoisomers of butane and butanol type compounds. Both enantiomers of butane type secocyclolignane showed higher cytotoxic activity (IC50 = 16-20 mu M) than methoxy type compounds, whereas none was observed for all the stereoisomers of butanol type secocyclolignane, however, (-)-Kadangustin J showed stereospecific cytotoxic activity (IC50 = 47-67 mu M). Since (R)-9'-fluoro derivative 23 was most potent (IC50 = 19 mu M) among the corresponding fluoro stereoisomers, (R)-9'-alkyl derivatives were synthesized, hydrophobic 9'-heptyl derivative 27 showing highest activity (IC50 = 3.7 mu M against HL-60, IC50 = 3.1 mu M against HeLa) in this experiment. Apoptosis induction caused by Caspase 3 and 9 for (R)-9'-heptyl derivative 27 was observed in the research on the mechanism. A degradation of DNA into small fragments was also shown by DNA ladder assay. (C) 2014 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.bmcl.2014.07.033

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ROYAL SOC CHEMISTRY  

Bengkoang (Pachyrhizus erosus (L.) Urban) is an edible root tuber containing fairly large amounts of carbohydrates and crude fibers. Our previous studies showed that the bengkoang fiber extract (BFE) stimulates activation of macrophages, leading to induction of phagocytotic activity and cytokine production. In the present study we investigated the mechanism underlying activation of murine macrophages by BFE. BFE increased production of TNF-alpha, IL-6, and nitric oxide by J774.1 cells. In addition BFE also facilitated the gene expression levels of inducible nitric oxide synthase. We examined the effect of a TLR4 inhibitor on cytokine production to investigate the membrane receptor of macrophage activation by BFE. Treatment of J774.1 cells with the TLR4 inhibitor significantly inhibited production of IL-6 and TNF-alpha, suggesting that TLR4 is the target membrane receptor for BFE. The main signal molecules located downstream of TLR4 such as JNK, p38, ERK, and NF-kappa B were activated by BFE treatment. The immunostimulatory effect of BFE was cancelled by the pectinase treatment, suggesting that the active ingredient in BFE is pectin-like molecules. Overall results suggested that BFE activates J774.1 cells via the MAPK and NF-kappa B signaling pathways.

DOI： 10.1039/c3fo60360a

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Cytotoxic activities of synthesized lignan derivatives were estimated by WST-8 reduction assay against HL-60 and HeLa cells to show the structure-activity relationship. The activities of some effective compounds were examined against Colon 26 and Vero cells. Dietary secoisolariciresinol (SECO, 1) and its metabolite, 9,9'-anhydrosecoisolariciresinol (2), did not show the cytotoxic activity. On the other hand, all stereoisomers of dihydroguaiaretic acid (DGA, 9,9'-dehydroxysecoisolariciresinol, 3-5) exhibited the activity (IC50: around 30 mu M). The IC50 value of (8R,8'R)-9-butyl DGA derivative 13 was around 6 mu M. This fact means that the hydrophobic group was advantageous for higher activity at 9- and 9'-positions. By the evaluation of the effect of 7and 7'-aryl group on the activity, we discovered the highest activity of (8R,8'R)-7-(3-hydroxy-4-methoxyphenyl)-7'-(2-ethoxyphenyl) DGA derivative 47 showing around 1 mu M of IC50 value, which is about 24-fold higher activity than that of natural (8R,8'R)-DGA. The derivative of dietary lignan showed the high cytotoxic activity.

DOI： 10.1021/jf5010572

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Tributyltin (TBT) has long been recognized as a major environmental pollutant that can cause significant damage to the cellular functions as well as disruption of endocrine homeostasis. TBT induces apoptosis accompanied by production of reactive oxygen species (ROS) in mammalian and yeast cells. We observed that the budding yeast cells exposed to this compound at low concentrations exhibited cell growth arrest, but not cell death. Flow cytometric analysis of yeast cells without synchronization and morphological assessment of cells synchronized at M phase by nocodazole treatment indicated that TBT-exposed Saccharomyces cerevisiae cells were arrested at G1 phase of the cell cycle. This arrest was recovered by the addition of N-acetylcysteine, suggesting the involvement of ROS production by TBT. This is the first study to evaluate the action of TBT on cell cycle events.

DOI： 10.2131/jts.39.311

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPANESE SOC TOXICOLOGICAL SCIENCES  

Tributyltin (TBT) has long been recognized as a major environmental pollutant that can cause significant damage to the cellular functions as well as disruption of endocrine homeostasis. TBT induces apoptosis accompanied by production of reactive oxygen species (ROS) in mammalian and yeast cells. We observed that the budding yeast cells exposed to this compound at low concentrations exhibited cell growth arrest, but not cell death. Flow cytometric analysis of yeast cells without synchronization and morphological assessment of cells synchronized at M phase by nocodazole treatment indicated that TBT-exposed Saccharomyces cerevisiae cells were arrested at G1 phase of the cell cycle. This arrest was recovered by the addition of N-acetylcysteine, suggesting the involvement of ROS production by TBT. This is the first study to evaluate the action of TBT on cell cycle events.

DOI： 10.2131/jts.39.311

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

We previously reported that jellyfish collagen stimulates both the acquired and innate immune responses. In the acquired immune response, jellyfish collagen enhanced immunoglobulin production by lymphocytes in vitro and in vivo. Meanwhile, in the innate immune response jellyfish collagen promoted cytokine production and phagocytotic activity of macrophages. The facts that jellyfish collagen plays several potential roles in stimulating cytokine production by macrophages have further attracted us to uncover its mechanisms. We herein describe that the cytokine production-stimulating activity of jellyfish collagen was canceled by a Toll-like receptor 4 (TLR4) inhibitor. Moreover, jellyfish collagen stimulated phosphorylation of inhibitor of kappa B alpha (I kappa B alpha), promoted the translocation of nucleus factor-kappa B (NF-kappa B), and activated c-Jun N-terminal kinase (JNK). A JNK inhibitor also abrogated the cytokine production-stimulating activity of jellyfish collagen. These results suggest that jellyfish collagen may facilitate cytokine production by macrophages through activation of NF-kappa B and JNK via the TLR4 signaling pathways. (C) 2013 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.molimm.2013.11.003

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Nobiletin is a polymethoxy flavonoid present in fruit tissues of citrus species and has been reported to suppress degranulation of RBL-2H3 cells. However, the detailed mechanism is still unknown. In this study the mechanism of suppressive effect of nobiletin on degranulation of RBL-2H3 cells was examined. Nobiletin suppressed degranulation of both RBL-2H3 cells and bone marrow-derived mast cells induced by antigen stimulation. Nobiletin also suppressed degranulation stimulated by the calcium ionophore A23187 and thapsigargin, a specific inhibitor of the Ca2+-dependent ATPase. Elevation of intracellular free Ca2+ concentration was also suppressed by nobiletin when RBL-2H3 cells were stimulated by either antigen or A23187. Immunoblot analysis revealed that the inhibitory effect results from down-regulated phosphorylation of both phosphatidylinositol 3-kinase (PI3K) and phospholipase C gamma 1 in the signaling pathways related to degranulation stimulated by antigen. Furthermore, oral administration of nobiletin suppressed vascular permeability in passive cutaneous anaphylaxis model mice. The tendency in all experiments obtained by treating with nobiletin was highly similar to that obtained by wortmannin, a specific inhibitor of PI3K. These findings suggest that nobiletin suppresses Ca2+ influx and consequently exerts the anti-allergy activity in a similar way to wortmannin. (C) 2013 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.jff.2013.12.005

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Bengkoang (Pachyrhizus erosus (L.) Urban) is one of the most popular edible root vegetables in Indonesia. Bengkoang contains fairly large amounts of carbohydrates and crude fiber. The purpose of this research is to evaluate the immunomodulatory effect of the bengkoang fiber extract (BFE) in vitro and in vivo. BFE was prepared by heating the powder of bengkoang fiber suspended in distilled water at 121 degrees C for 20 min. BFE facilitated IgM production by the human hybridoma cell line HB4C5 cells. In addition, production of IgM, IgG, and IgA by mouse primary splenocytes was facilitated by BFE in a dose-dependent manner. BFE also significantly facilitated production of both interleukin-5 and interleukin-10 by splenocytes. Immunoglobulin production by lymphocytes from the spleen, Peyer's patch, and mesenteric lymph node were significantly activated by oral administration of BFE to mice for 14 days. The serum immunoglobulin levels of IgG, IgM, and IgA were also significantly enhanced. Furthermore, cytokine production by lymphocytes from the spleen, Peyer's patch, and mesenteric lymph node were also facilitated by oral administration of BFE. These results suggest that BFE has positive effects on the immune system in vitro and in vivo.

DOI： 10.1007/s10616-013-9539-5

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Academic Journals Inc.  

The research objective is to obtain the cellular mechanism(s) of Syzygium polyanthum leaves for its development into commercial herbal product for certain degenerative diseases. The approach was to conduct activity-guided fractionation of metabolite extracts based on the antioxidant and the apoptotic modulation activities using cultured cells. Antioxidant activity of the active fractions were screened using l,l-diphenyl-2-picrylhydrazil (DPPH) free radical assay and thiobarbituric acid (TBA) assay. The cellular effects were investigated using cultures of HB4C5 human hybridoma and mouse colon 26 adenocarcinoma cells. The flavonoids extract which has the lowest IC50 value of 14.84 jig mL_1 for DPPH assay among other metabolites, was found to have significant cytotoxic effect on both HB4C5 and colon 26 cells. Specific assays for apoptosis using caspase 3-gene expression and annexin-V flow cytometry analyses, supported the effect of cell proliferation inhibition which was not due to apoptosis. Subsequent cell cycle analysis indicated that crude extract may induce cell cycle arrest of HB4C5 cells at Gl to S phase, whereas active compound fractions of flavonoids stacked the cell cycle at G2/M phase. These observations suggest that flavonoid extracts of Syzygium polyanthum posses active compound(s) with anti-proliferative effect. This is a new finding for therapeutic potential of Syzygium polyanthum leaves in Indonesian traditional medicines. © 2014 Academic Journals Inc.

DOI： 10.3923/ajdd.2014.90.101

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The cytotoxic activities of sesquilignans, (7S,8S,7'R,8'R)- and (7R,8R,7'S,8'S)-morinol A and (7S,8S,7'S,8'S)- and (7R,8R,7'R,8'R)-morinol B were compared, showing no significant difference between stereoisomers (IC50=24-35 mu M). As a next stage, the effect of substituents at 7, 7', and 7 ''-aromatic ring on the activity was evaluated to find out the higher activity of (7S,8S,7'R,8'R)-7,7',7 ''-phenyl derivative 18 (IC50 = 6-7 mu M). In the research on the structure-activity relationship of 7 ''-position of (7S,8S,7'R,8'R)-7,7',7 ''-phenyl derivative 18, the most potent compounds were 7,7',7 ''-phenyl derivative 18 (IC50 = 6 mu M) against HeLa cells. Against HL-60 cells, 7 ''-(4-nitrophenyl)-7,7'-phenyl derivative 33 and 7 ''-hexyl-7,7'-phenyl derivative 37 (IC50=5 mu M) showed highest activity. We discovered the compounds showed four to sevenfold potent activity than that of natural (7S,8S,7'R,8'R)-morinol A. It was also confirmed that the 7'-benzylic hydroxy group have an important role for exhibiting activity, on the other hand, the resonance system of cinnamyl structure is not crucial for the potent activity. (C) 2013 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.bmcl.2013.06.067

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

The relationship between antifungal activity against Alternaria alternata and structure on the 7-phenyl group of (+)-dihydroguaiaretic acid ((+)-DGA) was clarified by employing 38 synthesized (+)-DGA derivatives. The results were identified by quantitative structure activity relationship (QSAR) analysis employing the Hansch-Fujita method. Some compounds showed higher activity than (+)-DGA. The compound showing highest activity was 3,5-difluorophenyl derivative 37. It was suggested that the small electron-withdrawing group at the meta-position of the 7-phenyl group is important for the higher activity by antifungal test and Hansch-Fujita analysis. The whitening activity of 3-hydroxy-4-methoxyphenyl derivative 28, 3-hydroxy-4-methoxyphenyl derivative 29, and 3-hydroxy-4-isopropoxyphenyl derivative 30 against A. alternata Japanese pear pathotype was also discovered.

DOI： 10.1021/jf4015526

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPANESE SOC TOXICOLOGICAL SCIENCES  

ortho-Phenylphenol has been employed in post-harvest treatment of citrus fruits. Although o-phenylphenol has been reported to cause carcinomas in the urinary tract in rats, toxicity to the immune organs is still unknown. Herein, we report that administration of o-phenylphenol induces thymic atrophy and loss of thymocytes in female BALB/c mice. The influence seems to result from inhibition of the thymocyte development, because increased and decreased populations of the CD4(-) CD8(-) double-negative and CD4(+) CD8(+) double-positive thymocytes were observed in the o-phenylphenol-administered mice, respectively. ortho-Phenylphenol is metabolized to phenylhydroquinone by cytochrome P450 monooxygenases. Phenylhydroquinone made cell cycle of thymocytes to be arrested through reduced expression of the genes associated with G(2)/M phase and through phosphorylation of p53 at Ser15. Phosphorylation of p53 at Ser15 was upregulated by activation of not only ATR but also Erk1/2 and p38, leading to increase of apoptosis. Gene expression of cytochrome P450 1A1 (CYP1A1) was promoted in thymocytes from the o-phenylphenol-administered mice. Overall, our results suggest that o-phenylphenol induces CYP1A1 expression and is metabolized into phenylhydroquinone by the expressed GYP 1A1 in thymocytes. The produced phenylhydroquinone in turn induces inhibition of thymocyte development through cell cycle arrest and apoptosis in the p53-dependent pathway.

DOI： 10.2131/jts.38.325

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Bengkoang (Pachyrhizus erosus (L) Urban) is an edible root vegetable containing fairly large amounts of carbohydrates and crude fibers. The purpose of this study is to evaluate the effects of the bengkoang fiber extract (BFE) on macrophages in vitro and in vivo. BEF was prepared by heat-extraction from the bengkoang fiber in distilled water at 121. °C for 20. min. BFE stimulated the phagocytotic activity of J774.1 cells. In addition, BFE significantly facilitated production of tumor necrosis factor (TNF)-α and interleukin (IL)-6 by J774.1 cells and mouse peritoneal macrophages. BFE also facilitated the gene expression levels of inducible nitric oxide synthase and cyclooxygenase-2. The phagocytotic activity of mouse peritoneal macrophages was significantly enhanced by oral administration of BFE to BALB/c mice for 7. days. Ex vivo analysis revealed that production and gene expression of TNF-α and IL-6 in peritoneal macrophages from the BFE-administered mice were significantly enhanced. These results demonstrated that BFE activates macrophages in vitro and in vivo. © 2012 Elsevier Ltd.

DOI： 10.1016/j.jff.2012.12.005

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

The anti-allergic effect of alcohol extracts from edible seaweeds was evaluated by the degranulation of a rat basophilic leukemia cell line (RBL-2H3). Extracts prepared from the brown algae Sargassum horneri, Ecklonia cava and Ecklonia kurome, inhibited the degranulation of RBL-2H3 cells, whereas those from green algae and red algae did not. The active substance in S. horneri was purified and identified as chlorophyll c2, a major pigment in brown algae. The purified chlorophyll c2 preparation also suppressed the degranulation of RBL-2H3 cells. In contrast, chlorophyll a and b did not affect cell degranulation. Therefore, chlorophyll c2 from brown algae might be useful for the prevention or mitigation of allergic symptoms. (c) 2012 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.jff.2012.10.006

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The inhibitory effect of an aqueous extract from spinach on degranulation of RBL-2H3 cells is herein reported. The extract significantly suppressed antigen-induced degranulation in a dose-dependent manner without affecting cell viability. Active substances in the extract were heat-stable and trypsin-resistant with molecular weights ranging from 500 Da to 14 kDa. The extract inhibited elevation of the intracellular Ca2+ concentration caused by stimulation by antigen, while not suppressing degranulation induced by a calcium ionophore A23187. Immunoblot analysis revealed that the inhibitory effect results from down-regulation of phosphorylation of both Syk kinase and phosphatidylinositol 3-kinase in the signalling pathways involved in degranulation caused by the antigen-antibody interaction. Taken together, these findings suggest that aqueous spinach extract has an anti-allergic activity that controls degranulation. (C) 2012 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.foodchem.2012.08.079

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Five strains indigenous lactic acid bacteria i.e. Lactobacillus plantarum-pentosus T14, Lactobacillus plantarum-pentosus T20, L. plantarum T32, L. plantarum T33 and L. plantarum-pentosus T35 were tested for their capabilities to transform isoflavone glucosides to aglycones in the kerandang crude extract. Changes in growth, pH, titratable acidity (TA), β-glucosidase activity was investigated during fermentation at 37°C for 24 h. Isoflavone transformation was analyzed using UPLC (Ultra Performance Liquid Chromatography). The antioxidant properties were evaluated using 1,1-diphenyl-2-picrylhydrazyl (DPPH) and ferrous ion-chelating ability method. The result showed that initial cell population of 106 - 107 CFU/ml rapidly increased and reached 109 CFU/ml in MRS-kerandang extract after 9 to 12 h of fermentation and constant to 24 h fermentation. All five strains produced lactic acid followed by the decreasing of pH. Lactobacillus plantarum-pentosus T14 showed the highest β-glucosidase activity is 558 ± 9.8 mU/ml culture at 12 h fermentation. All five strains were able to transform isoflavone glucoside to aglycone. L. plantarum-pentosus T14 have a better ability to transform, followed by L. plantarum-pentosus T20, L. plantarum T32, L. plantarum T33 and L. plantarum-pentosus T35.

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Catecholic drugs had been reported to be metabolized through conjugation reactions, particularly methylation and sulfation. Whether and how these two Phase II conjugation reactions may occur in a concerted manner, however, remained unclear. The current study was designed to investigate the methylation and/or sulfation of five catecholic drugs. Analysis of the spent media of HepG2 cells metabolically labeled with [S-35]sulfate in the presence of individual catecholic drugs revealed the presence of two [S-35]sulfated metabolites for dopamine, epinephrine, isoproterenol, and isoetharine, but only one [S-35]sulfated metabolite for apomorphine. Further analyses using tropolone, a catechol O-methyltransferase (COMT) inhibitor, indicated that one of the two [S-35]sulfated metabolites of dopamine, epinephrine, isoproterenol, and isoetharine was a doubly conjugated (methylated and sulfated) product, since its level decreased proportionately with increasing concentrations of tropolone added to the labeling media. Moreover, while the inhibition of methylation resulted in a decrease of the total amount of [S-35]sulfated metabolites, sulfation appeared to be capable of compensating the suppressed methylation in the metabolism of these four catecholic drugs. A two-stage enzymatic assay showed the sequential methylation and sulfation of dopamine, epinephrine, isoproterenol, and isoetharine mediated by, respectively, the COMT and the cytosolic sulfotransferase SULT1A3. Collectively, the results from the present study implied the concerted actions of the COMT and SULT1A3 in the metabolism of catecholic drugs. (c) 2012 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bcp.2012.08.009

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Using 21 newly synthesized 7,7'-dioxo-9,9'-epoxylignane derivatives having a modified 7-phenyl group, we examined the relationship between their structure and antifungal activity against plant pathogens such as Bipolaris oryzae to determine the effects of various substituents on the antifungal activity. Compared with the lead compound having a 4-OH-3-CH3O-phenyl moiety, several analogs showed higher antifungal activity against B. oryzae, including the compound having an unsubstituted phenyl group and those having either of the following phenyl substituents: 2-OH, 4-CH3O, 4-C2H5O, 4-n-C3H7O, 4-n-C4H9O, 4-CF3O, 4-C2H5, or 4-Cl. On the other hand, the activity of compounds having a branched substituent, such as 4-i-C3H7O or 4-i-C3H7, on the 7-phenyl group or a multi-substituted phenyl group was equipotent or inferior to that of the lead compound. These results as well as correlations between the antifungal activity of the test compounds and the physicochemical parameters of the varied substituents suggest that the position of substitution on the 7-phenyl group and the incorporation of substituents with optimal physicochemical properties are important for exerting the antifungal activity. (C) 2012 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.bmcl.2012.08.078

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Effects of auraptene on immune cells in vitro and in vivo were examined. IgM production by human hybridoma HB4C5 cells was enhanced by auraptene. Auraptene also stimulated IgA and IgG production by mouse primary splenocytes, and IgA and IgM production by lymphocytes from mesenteric lymph nodes in vitro. To examine the effect of auraptene on immune system in vivo, auraptene was orally administered to mice for 14 days. Immunoglobulin production by splenocytes and lymphocytes from mesenteric lymph nodes were significantly activated by intake of auraptene. Moreover, IL-4, IFN-gamma, and TNF-alpha production by splenocytes was accelerated under concanavalin A-activated condition. B cell population in spleen was increased by auraptene intake. (C) 2012 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.jff.2012.06.005

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Lactate dehydrogenase (LDH) has been demonstrated to have an immunoglobulin production-stimulating effect on an IgM-producing human hybridoma cell line and on human peripheral blood lymphocytes in vitro. Here, we examined the immunostimulatory activity of LDH on mouse immune system in vitro and in vivo. LDH stimulated IgA, IgG, and IgM production by lymphocytes from spleen, mesenteric lymph node, and Peyer's patch in vitro. BALB/c mice were administered with LDH at 1.0 or 5.0 mg/kg/day for 2 weeks to examine the immunostimulatory activity of LDH in vivo. The IgA level in mouse serum was increased by administration at 5.0 mg/kg/day. Moreover, IgA and IgG production by Peyer's patch lymphocytes from mice administered at 1.0 mg/kg/day of LDH was accelerated. IgA, IL-4, IL-5, IL-10, IFN-gamma, and TNF-alpha production by spleen lymphocytes were also facilitated by administration of LDH. It was revealed that oral administration of LDH stimulates immunoglobulin and cytokine production by lymphocytes in vivo. (C) 2012 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.jff.2012.07.005

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Oysters are one of foodstuffs containing a relatively high amount of cadmium. Here we report on establishment of an immunochromatographic assay (ICA) method of cadmium levels in oysters. Cadmium was extracted with 0.1 mol L-1 HCl from oysters and cleaned up from other metals by the use of an anion-exchange column. The behavior of five metals Mn, Fe, Cu, Zn, and Cd was monitored at each step of extraction and clean-up procedure for the ICA method in an inductively coupled plasma-mass spectrometry (ICP-MS) analysis. The results revealed that a simple extraction method with the HCl solution was efficient enough to extract almost all of cadmium from oysters. Clean-up with an anion-exchange column presented almost no loss of cadmium adsorbed on the column and an efficient removal of metals other than cadmium. When a spiked recovery test was performed in the ICA method, the recovery ranged from 98% to 112% with relative standard deviations between 5.9% and 9.2%. The measured values of cadmium in various oyster samples in the ICA method were favorably correlated with those in ICP-MS analysis (r(2)=0.97). Overall results indicate that the ICA method established in the present study is an adequate and reliable detection method for cadmium levels in oysters. (C) 2012 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.talanta.2012.04.028

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Beta-cryptoxanthin (beta-Crp), a mono-hydroxylated beta-carotene, is a major dietary provitamin A xanthophyll. The immunostimulatory effect of beta-Crp was examined using human hybridoma HB4C5 cells and mouse primary lymphocytes in vitro and using mice in vivo. IgM production by the HB4C5 cells and both IgA and IgG productions by mouse primary lymphocytes from mesenteric lymph node and spleen were accelerated in vitro by the treatment of beta-Crp in dose-dependent manners. Afterwards 6-week-old female BALB/c mice were administered with a low or high dose of beta-Crp for 14 days, mesenteric lymph node and spleen were excised and the immunoglobulin production levels were evaluated using lymphocytes from mesenteric lymph node and spleen in vivo. Results exhibited that all of IgA, IgG, and IgM productions by lymphocytes from mesenteric lymph node were increased. Especially, the IgG production increased with statistically significant differences in both of the low and high dose groups against the control group. The immunostimulatory effect on splenocytes was also observed although not as clear as on lymphocytes from mesenteric lymph node. Overall results suggest that beta-Crp may stimulate the humoral immunity in mammals. Hence, beta-Crp might have a potentially significant impact on human health and on prevention of diseases. (c) 2012 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.jff.2012.04.001

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

As we previously reported, jellyfish collagen stimulates immunoglobulin and cytokine productions by lymphocytes in vitro and in viva Herein, the effects of collagens from jellyfish and bovine Achilles tendon on the innate immune response, especially on the activity of macrophages were evaluated. Macrophage functions were examined by the phagocytotic activity, cytokine production activity, and gene expression. The phagocytosis of the mouse J774.1 cells was enhanced by both collagens. Collagens from jellyfish and bovine Achilles tendon also stimulated the productions of tumor necrosis factor-alpha and interleukin-6 by J774.1 cells and mouse primary peritoneal macrophages as the result of elevated gene expression levels of these cytokines. Oral administration of collagens elevated cytokine production by peritoneal macrophages in mice. On the contrary, collagens suppressed gene expression of PPAR gamma 1 in macrophages. It is supposed from this result that collagens may stimulate the cytokine production by suppressing the PPAR gamma 1 expression. Collagens from jellyfish and bovine Achilles tendon enhanced not only the acquired immune response but also the innate immune response through the activation of macrophages. (C) 2012 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.jff.2012.02.011

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Background: Nobiletin is a citrus flavonoid which possesses the flavone structure with six methoxy groups. Although nobiletin has been reported to display anti-inflammatory, anti-tumor, and anti-diabetes activities, its effect on adipocyte differentiation remained unclear. In the present study, we investigated the effect of nobiletin on the differentiation of 3T3-L1 preadipocytes into adipocytes.
Methods: 3T3-L1 preadipocytes were treated with nobiletin under various differentiation conditions. The effect of nobiletin on adipocyte differentiation was evaluated by oil red 0 staining, real-time RT-PCR, and Western blotting.
Results: Nobiletin significantly suppressed the differentiation of 3T3-L1 preadipocytes into adipocytes, upon induction with insulin together with a cAMP elevator such as 3-isobutyl-1-methylxanthine (IBMX), by downregulating the expression of the gene encoding peroxisome proliferator-activated receptor (PPAR) gamma 2. In addition, nobiletin decreased the phosphotylation of CAMP-response element-binding protein (CREB) and strongly enhanced the phophorylation of signal transducer and activator of transcription (STAT) 5.
General significance: Nobiletin has a suppressive effect on the differentiation of preadipocytes into adipocytes when cells were induced with a general differentiation cocktail such as insulin, IBMX, and dexamethasone. (C) 2011 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.bbagen.2011.11.015

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Arrowroot (Maranta arundinacea. L) is an underutilized local crop potentially to be developed as carbohydrate source and functional food in Indonesia. The objectives of this research are to evaluate the immunostimulatory effects of arrowroot extracts in vitro by using animal cell culture techniques, and in vivo by using BALB/c mice. The arrowroot tuber extracts were prepared by heat-treatment at 121 degrees C for 20 min in distilled water. The IgM production stimulatory activity of arrowroot tuber extracts against human hybridoma HB4C5 cells and mouse splenocytes was assessed. The result indicated that the arrowroot tuber extract stimulated IgM production by HB4C5 cells and immunoglobulin (IgG, IgA and IgM) production by splenocytes in vitro. In addition, the arrowroot tuber extracts strongly enhanced interferon c production by splenocytes. In vivo study indicated that the diet containing arrowroot extracts increased the serum IgG, IgA and IgM levels in mice. These results revealed that the arrowroot tuber extracts have immunostimulatory effects in vivo as well as in vitro.

DOI： 10.1007/s10616-011-9403-4

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Immunostimulating effects of water extract from bulbus arteriosus in tuna were examined and found to enhance IgM production by human hybridoma HB4C5 cells. When tuna bulbus arteriosus extract was heated, the IgM production-stimulating activity was decreased or lost. To identify the active substance, the extract was partially purified by anion-exchange chromatography, and proteins contained in positive fractions were analyzed by LC-MS/MS. The positive fractions contained lactate dehydrogenase, triosephosphate isomerase, enolase, and haemoglobin. Each of these four proteins was verified to enhance IgM production by HB4C5 cells with the commercially available purified proteins, suggesting that all of these four proteins are active substances in the extract. The immunostimulatory effect of these proteins was also examined on mouse primary spleen lymphocytes in vitro. Lactate dehydrogenase enhanced IgA, IgG, and IgM production and triosephosphate isomerase enhanced IgA and IgG production. In particular, lactate dehydrogenase had the strongest Ig production-stimulating effect on HB4C5 cells and on mouse primary lymphocytes. Thus, fish viscera may serve as an important raw material for the enhancement of the acquired immune system, once processed. (C) 2011 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.jff.2011.12.002

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ROYAL SOC CHEMISTRY  

Flaxseed lignan, secoisolariciresinol has been reported to possess health benefits. We previously synthesized each stereoisomer of secoisolariciresinol and found that (-)-secoisolariciresinol reduces lipid accumulation and induces adiponectin production in 3T3-L1 adipocytes. Here we show the effects of (-)-secoisolariciresinol on high-fat diet-induced obesity in C57BL/6 male mice. Oral administration of (-)-secoisolariciresinol for 28 consecutive days significantly suppressed the gain of body weight. Increased serum adiponectin level and decreased gene expression of fatty acid synthase and sterol regulatory element-binding protein-1c in liver, which are related to fatty acid synthesis, were observed in the mice orally administered with (-)-secoisolariciresinol. In addition, subcutaneous injection of (-)-secoisolariciresinol also significantly suppressed the gain of body weight. Serum leptin levels were significantly increased by treating with (-)-secoisolariciresinol or (-)-enterolactone. Subcutaneous injection of (-)-secoisolariciresinol, (-)-enterolactone, or (-)-enterodiol promoted gene expression of acyl-CoA oxidase, carnitine palmitoyl transferase-1, and peroxisome proliferator-activated receptor alpha, which are related to beta-oxidation. Overall results suggest that (-)-secoisolariciresinol exerts a suppressive effect on the gain of body weight of mice fed a high-fat diet by inducing gene expression of adiponectin, resulting in the altered expression of various genes related to the synthesis and beta-oxidation of fatty acids.

DOI： 10.1039/c1fo10166h

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Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：JAPAN SOC FOOD SCIENCE TECHNOLOGY  

In this study, we aimed to search for peptides derived from casein with new anti-allergic action, and to examine the degranulation inhibitory effects associated with this anti-allergic action. Furthermore, amino acids making up the peptide with anti-allergic action were investigated for degranulation inhibitory effects, cytokine production and electron density. The peptides tested were low molecular weight HAQ, DMES, EQPI and KIKE, which are present in CE90GMM, a peptide mixture derived from casein. Using rat basophilic leukemia RBL-2H3 cells, we measured beta-hexosaminidase levels released during degranulation in the antigen-antibody reaction in the presence of these peptides, and evaluated the anti-allergic effects. Cytokine production (TNF-alpha, IL-4) was evaluated by ELISA. Optimized structure and electron density of amino acids were analyzed using a three-dimensional of electrostatic potential. Significant inhibition of,beta-hexosaminidase release was seen with CE90GMM stimulation at 0.25 mg/mL. With the four types of peptide at levels ranging from 0.01 to 0.5 mg/mL, significant inhibition of beta-hexosaminidase release was observed, but the effect varied with the dose. Our analysis also suggested that the peptides with anti-allergic action possess aromatic rings and neutral/hydrophobic amino acids in the side chain. (Received May 22, 2012: Accepted Jul. 19, 2012)

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In this study, we aimed to search for peptides derived from casein with new anti-allergic action, and to examine the degranulation inhibitory effects associated with this anti-allergic action. Furthermore, amino acids making up the peptide with anti-allergic action were investigated for degranulation inhibitory effects, cytokine production and electron density. The peptides tested were low molecular weight HAQ, DMES, EQPI and KIKE, which are present in CE90GMM, a peptide mixture derived from casein. Using rat basophilic leukemia RBL-2H3 cells, we measuredβ- hexosaminidase levels released during degranulation in the antigen-antibody reaction in the presence of these peptides, and evaluated the anti-allergic effects. Cytokine production (TNF-a, IL-4) was evaluated by ELISA. Optimized structure and electron density of amino acids were analyzed using a three-dimensional of electrostatic potential. Significant inhibition of β-hexosaminidase release was seen with CE90GMM stimulation at 0.25 mg/mL. With the four types of peptide at levels ranging from 0.01 to 0.5 mg/mL, significant inhibition of β-hexosaminidase release was observed, but the effect varied with the dose. Our analysis also suggested that the peptides with antiallergic action possess aromatic rings and neutral/hydrophobic amino acids in the side chain.

DOI： 10.3136/nskkk.59.556

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We focused on the biological activity of the collagen extracts obtained from the giant edible jellyfish, Nemopilema nomurai. Jellyfish collagen extracts stimulates the production of immunoglobulins (Igs) and cytokines by human hybridoma cells and human peripheral blood lymphocytes. Therefore, we examined the immunoregulatory function of jellyfish collagen extracts in mice. Intake of jellyfish collagen extracts facilitated the Ig production activity of lymphocytes from spleen and Peyer's patch. Furthermore, the levels of Igs in the serum clearly increased after the administration of jellyfish collagen extracts. Intake of bovine collagen from Achilles' tendon also activated lymphocytes activity in mice. The activity of total and antigen-specific Ig production in splenocytes from OVA-challenged mice was also enhanced by collagen intake. However, the total and OVA-specific IgE levels in the serum were not affected by the collagen intake. These results suggested that jellyfish collagen extracts stimulates an immune response in vivo, without inducing allergic complications.

DOI： 10.1007/s10616-011-9371-8

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPANESE SOC TOXICOLOGICAL SCIENCES  

Heavy oil is one of the most serious pollutants in marine ecosystem. The poisonous influences of the chemical substances contained in heavy oil on many kinds of marine organisms are widely studied. However, the influence of the chemical compounds in heavy oil on our health has not been cleared yet. In order to reveal the poisonous influences of these chemical compounds on mammalian reproductive system, water-soluble fraction (WSF) extracted from heavy oil was administrated to mice for 2 weeks. WSF-administrated mice were crossed with either WSF- or distilled water-administrated group for mating experiment. When WSF-administrated male mice were used as a father, it reduced not only mating ratio, but also neonatal male ratio. The numbers of sperms of WSF-administrated male mice were decreased. In addition, abnormality of sperms such as bent or twisted tail was increased approximately 6-fold by WSF intake. The level of testosterone in serum from WSF-administrated mice was lower than that from control mice. Testosterone is the most important for the spermatogenesis in vertebrate. It is supposed from these findings, the decrease in the number of sperms may relate with the reduction of sex hormone level in serum. It is suggested from these results that the chemical substances in WSF affected the sperm function in reproductive system of male mice.

DOI： 10.2131/jts.36.487

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：TAYLOR & FRANCIS LTD  

Matairesinol is one of the lignan compounds found in a variety of plant foodstuffs. We investigated the immunomodulatory effects of (-)-matairesinol in vivo and ex vivo by using mice. Although we found no significant differences in the IgG, IgA and IgM levels in the serum, the IgE level was strongly suppressed by the uptake of (-)-matairesinol in both intact and ovalbumin-immunized mice. The immunoglobulin produced by lymphocytes from the spleen was not activated by the intake of (-)-matairesinol. However, lymphocytes in such gut-associated lymphatic tissues as Peyer's patches and mesenteric lymph nodes were activated by the administration of (-)-matairesinol.

DOI： 10.1271/bbb.100781

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We have found that (-)-virgatusin and related compounds have antimicrobial and antifungal activity. To identify further biological activities of these compounds, we tested the activity of acridine orange efflux, which shows ionophore-like disruption of cellular ion homeostasis activity. After testing 31 compounds, we found that verrucosin and a related compound had disruption activity.

DOI： 10.1271/bbb.110004

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Paraquat (PQ) is one of the most frequently used pesticides in worldwide. In most countries, PQ is used without restrictions. To investigate the effect of PQ on myogenesis, cultures of C2C12, a useful model to study differentiation of myoblasts into myotubes, were exposed to various concentrations of PQ. Myotube formation did not occur in the presence of 50 mu M PQ. Although cell death was not observed at this concentration, growth inhibition was evident in the growth medium. Production of myosin heavy chain, a myogenesis marker protein, decreased dose dependently with the concentration of PQ, which was added to the C2C12 cell culture during differentiation. Inhibition of myogenesis by PQ was not reversed by the addition of ascorbic acid. These results show that PQ is a strong inhibitor of muscle differentiation in vitro.

DOI： 10.2131/jts.36.243

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The immunostimulation effects of yellowtail heart extracts were examined. Screening various parts of the yellowtail viscera, we found that extracts from the yellowtail heart enhanced IgM production by human hybridoma HB4C5 cells. Yellowtail heart extracts heated at 121 degrees C for 20 min and dialyzed showed the highest IgM production-stimulating activity toward HB4C5 cells. Also, immunoglobulin production by mouse spleen lymphocytes was stimulated by yellowtail heart extracts in vitro, and lymphocytes derived from mice administered the extract for 20d were activated in vivo. Yellowtail heart extracts were partially purified by anion-exchange chromatography, and fractions containing a 33 kDa-protein exhibited immunostimulating activity. LC-MS/MS analysis revealed that the 33 kDa-protein was most similar to tropomyosin-4 from various fishes. Purified tropomyosin from porcine muscle enhanced IgM production by HB4C5 cells. This means that tropomyosin-4 is one of the immunostimulating substances in the yellowtail heart.

DOI： 10.1271/bbb.100713

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPANESE SOC TOXICOLOGICAL SCIENCES  

ATP-binding cassette (ABC) transporter plays an important role for resistance against xenobiotics. There are eleven ABC transporter genes in the genome of fission yeast Schizosaccharomyces pombe. We examined the role of ABC transporter against the toxicity of tributyltin chloride (TBT), a widespread environmental pollutant, in cell growth. Among individual ABC transporter mutants, the growth of a mutant deficient in Bfr1p, a plasma membrane-embedded transporter, was extremely sensitive to TBT. The lethal TBT concentration inducing 50% of cell death (LC50) was 25 mu M for the parent strain and 10.2 mu m for the bfr1 Delta mutant. Thus, Bfr1p was responsible for TBT resistance in S. pombe.

DOI： 10.2131/jts.36.117

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The water-soluble fraction of kale (Brassica oleracea L. var. acephala DC.) had immunoglobulin (Ig) production stimulating activity in human hybridoma HB4C5 cells and human peripheral blood lymphocytes. The biochemical and physical properties of the main active substance in kale were found to be a heat-stable protein with a molecular weight higher than 50 kDa. The Ig production-stimulating factors were assumed to act on the translational and/or secreting processes of Igs. This Ig production-stimulating effect was also observed in lymphocytes from the mesenteric lymph node and Peyer's patches of mice that had been administered with the kale extract for 14 d. The partially purified kale extract was analyzed by LC-ESI-MS/MS, the result indicating ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) as an active substance. Rubisco from spinach indeed exhibited Ig production-stimulating activity in HB4C5 cells. These findings provide another beneficial aspect of kale as a health-promoting foodstuff.

DOI： 10.1271/bbb.100490

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The relationship between the structure of naturally occurring (7R,7'R,8R,8'R)-morinol B and its antifungal activity was examined. 3-Demethoxy morinol B showed much higher activity than the natural compound. The activity of the 4-butoxy-3-demethoxy derivative was higher than that of 3-demethoxy morinol B.

DOI： 10.1271/bbb.100422

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The IgE-suppressive activity of (-)-matairesinol is demonstrated, and the structure-activity relationship of (-)-matairesinol clarified. 3',4-Dihydroxy-3,4'-dimethoxylignano-9,9'-lactone showed higher IgE-suppressive activity than (-)-matairesinol without any cytotoxic activity. Some derivatives bearing a longer and more bulky alkoxy group at the 3 or 4 position showed IgE-accelerative activity.

DOI： 10.1271/bbb.100275

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Improved syntheses of optically pure (-)- and (+)-morinol C, and (-)- and (+)-morinol D were achieved by employing the Mizoroki-Heck reaction to construct the cinnamyl moiety. The protective group of the alkene substrate affected the yield of this key reaction. The reaction with a combination of the acetate-protected olefin and 4-methoxyphenylboronic acid gave the best result producing morinol C and D. All stereoisomers of morinol C and D showed cytotoxic activity, with (R,R)morinol C showing the highest antibacterial activity.

DOI： 10.1271/bbb.100255

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BENTHAM SCIENCE PUBL LTD  

Cytosolic sulfotransferases (SULTs) are traditionally known as the Phase II drug-metabolizing or detoxifying enzymes that serve for the detoxification of drugs and other xenobiotics. These enzymes in general catalyze the transfer of a sulfonate group from the active sulfate, 3'-phophoadenosine 5'-phosphosulfate (PAPS), to low-molecular weight substrate compounds containing hydroxyl or amino group(s). Despite considerable efforts made in recent years, some fundamental aspects of the SULTs, particularly their ontogeny, cell type/tissue/organ-specific distribution, and physiological relevance, particularly their involvement in drug metabolism and detoxification, still remain poorly understood. To better understand these fundamental issues, we have embarked on developing the zebrafish as a model for studies concerning the SULTs. To date, fifteen zebrafish SULTs have been cloned, expressed, purified, and characterized. These zebrafish SULTs, which fall into four major SULT gene families, exhibited differential substrate specificities and distinct patterns of expression at different stages during embryogenesis, through larval development, and on to maturity. The information obtained, as summarized in this review, provides a foundation for further investigation into the physiological and pharmacological involvement of the SULTs using the zebrafish as a model.

DOI： 10.2174/138920010791636158

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Proteose peptone (PP) is a heat-stable and acid-soluble protein in milk whey. We reveal in this study the IgE production-suppressing activity of the PP fraction in bovine milk. The PP fraction suppressed IgE production by human myeloma cell line U266 cells by depressing the IgE mRNA expression. The suppressive activity of the PP fraction was facilitated by trypsin digestion. An oral administration of the PP fraction significantly decreased the levels of total and ovalbumin (OVA)-specific IgE in the serum collected from OVA-sensitized mice. However, the serum levels of other Ig classes in OVA-sensitized mice were not affected by the intake of the PP fraction. The PP fraction suppressed the mRNA expression level of IgE in mice splenocytes collected from OVA-sensitized mice. Moreover, the B cell population in the spleen was decreased, while the T cell population was increased by administering the PP fraction. These results suggest that the PP fraction modified the B/T cell balance.

DOI： 10.1271/bbb.90809

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The lectin Eucheuma serra agglutinin (ESA) is known from previous studies to specifically bind to high-mannose type N-glycans and to induce apoptotic cancer cell death in vitro. In this study, Span 80 vesicles, with an average diameter between about 200 and 400 nm, containing immobilized ESA were prepared from the nonionic surfactant Span 80, also known as sorbitan monooleate. The vesicles were investigated in vitro and in vivo to evaluate the vesicles's potential applicability as novel drug delivery system. The results obtained arepromising since the following was observed: (i)vesicular ESAhad the same hemagglutinating activity as free ESA, demonstrating its biological activity when bound to the vesicles; (ii) vesicles containing immobilized ESA decreased the viability of Colo201 cancer cells in vitro while the growth of normal cells was not affected; (iii) the vesicles showed binding to Colo201 cells in vitro and caused inhibition of cancer cell growth in nude mice to which the vesicle-treated cells were added; (iv) the vesicles diminished tumor growth after intravenous administration to nude mice which contained an implanted Colo201 tumor; (v) the vesicles showed a tendency to

DOI： 10.1016/j.ijpharm.2010.01.033

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPANESE SOC TOXICOLOGICAL SCIENCES  

Paraquat is one of the most widely used herbicides in the world and has been known to injure lungs, liver and skin in animals and human. Hence, it is important to understand the manner of paraquat in mammals. We studied the effect of paraquat on the immune function of mouse in vitro and in vivo. When splenocytes were cultured in vitro with various concentrations of paraquat, IgA productivity was not affected while IgG and IgM productivity decreased. On the other hand, Oral administration of paraquat for 1,2 or 3 weeks increased IgA level but decreased IgM levels in serum of mice. Similarly IgA productivity increased while IgM productivity decreased. These results suggest that paraquat perturbs the lymphocytes immunoglobulin productivity in an immunoglobulin class-dependent manner.

DOI： 10.2131/jts.35.257

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

The lectin Eucheuma serra agglutinin (ESA) is known from previous studies to specifically bind to high-mannose type N-glycans and to induce apoptotic cancer cell death in vitro. In this study, Span 80 vesicles, with an average diameter between about 200 and 400 nm, containing immobilized ESA were prepared from the nonionic surfactant Span 80, also known as sorbitan monooleate. The vesicles were investigated in vitro and in vivo to evaluate the vesicles's potential applicability as novel drug delivery system. The results obtained are promising since the following was observed: (i) vesicular ESA had the same hemagglutinating activity as free ESA, demonstrating its biological activity when bound to the vesicles; (ii) vesicles containing immobilized ESA decreased the viability of Colo201 cancer cells in vitro while the growth of normal cells was not affected; (iii) the vesicles showed binding to Colo201 cells in vitro and caused inhibition of cancer cell growth in nude mice to which the vesicle-treated cells were added; (iv) the vesicles diminished tumor growth after intravenous administration to nude mice which contained an implanted Colo201 tumor; (v) the vesicles showed a tendency to accumulate at the site of the tumor 6 h after iv. administration to nude mice. Thus, all measurements carried out indicate that this type of Span 80 vesicle can be considered as promising alternatives to conventional phospholipid-based vesicles. (C) 2010 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.ijpharm.2010.01.033

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DOI： 10.1271/bbb.2010E1

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Language：English   Publishing type：Research paper (international conference proceedings)   Publisher：SPRINGER  

DOI： 10.1007/978-90-481-3892-0_62

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Language：English   Publishing type：Research paper (international conference proceedings)   Publisher：SPRINGER  

DOI： 10.1007/978-90-481-3892-0_57

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Language：English   Publishing type：Research paper (international conference proceedings)   Publisher：SPRINGER  

DOI： 10.1007/978-90-481-38920_35

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPANESE SOC TOXICOLOGICAL SCIENCES  

It is well known that heavy oil such as pollutant caused serious influences on the marine ecosystem. We may suffer from various disorders in our body via intake of marine foods polluted with heavy oil. However the influences of heavy oil on our immune system have not yet been clarified. Here we show the effects of heavy oil extracts, water-soluble fraction (WSF), methanol-soluble fraction (MSF) and ethanol-soluble fraction (ESF), on immunoglobulin production of mouse splenocytes. All extracts increased IgA productivity of splenocytes. In oral administration, shrinkage of the immune organs such as spleen or thymus was observed in only WSF-administrated mice at least during 7 days. The amount of IgG production level in splenocytes cultured medium and sera were reduced by each extract administration. A flowcytometry method, to monitor splenocytes of WSF-administrated mice, has been set up using double staining with B and T cell-specific surface antibody. The results from cell population analysis indicated that B cells, including plasma cells producing antibody were reduced. The decrease in IgG level in sera was caused by reduction of plasma cells in spleen. Hence, it is suggested that reduction of Ig production was affected by the chemical compounds contained in WSF possibly such as polycyclic aromatic hydrocarbons (PAHs) through the estrogen receptor expressed in lymphocytes.

DOI： 10.2131/jts.34.459

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPANESE SOC TOXICOLOGICAL SCIENCES  

Tributyltin chloride (TBT), an environmental Pollutant, is toxic to a variety of eukaryotic and prokaryotic organisms. Although it has been reported that TBT induces apoptotic cell death in mammalian, the action of TBT on eukaryotic microorganisms has not yet been fully investigated. In this study we examined the mechanism involved in cell death caused by TBT exposure in Saccharomyces cerevisiae. The median lethal concentration of TBT was 10 mu M for the parent strain BY4741 and 3 mu M for the pdr5A mutant defective in a major multidrug transporter, respectively. Fluorescence microscopic observations revealed nuclear condensation and chromatin fragmentation in cells treated with TBT indicating that cells underwent an apoptosis-like cell dearth. TBT-induced cell death was suppressed by deletion of the ycal gene encoding a homologue of the mammalian caspase. In parallel, reactive oxygen species (ROS) were produced by TBT. These results suggest that TBT induces apoptosis-like cell death in yeast via an Yca1 p-dependent pathway possibly downstream of the ROS production. This is the first report on TBT-induced apoptotic cell death in yeast.

DOI： 10.2131/jts.34.541

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：TAYLOR & FRANCIS LTD  

Sorghum, Sorghum bicolor (L.) Moench, is the fifth most important cereal crop in the world. In this study, we identified the IgE production-suppressing activity of white sorghum bran extracts. White sorghum is one of the genotypes of sorghum. White sorghum bran extracts in 10 mm sodium phosphate buffer (pH 7.4) suppressed IgE production in human myeloma cell line U266. The extracts suppressed IgE production by decreasing mRNA transcription level of IgE, but they did not affect IgA or IgG production of mice splenocytes in vitro. Heat treatment and trypsin digestion did not affect IgE production-suppressing activity. The white sorghum bran extracts were fractionated by ultrafiltration, and the molecular weight of the active substance was estimated to be less than 1,000.

DOI： 10.1271/bbb.90245

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：TAYLOR & FRANCIS LTD  

The inhibitory effect of (-)-, (+)-matairesinol and (-)-, (+)-secoisolariciresinol on the discoloration of dark muscle (chiai in Japanese) of two-year-old yellow-tail (hamachi in Japanese) was evaluated by measuring the X and a* values. (-)-Matairesinol was most effective for retaining the red color of dark muscle in this experiment.

DOI： 10.1271/bbb.80830

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：TAYLOR & FRANCIS LTD  

The relationship between the stereochemistry and antimicrobial activity of butane-type lignans was clarified. All stereoisomers; of dihydroguaiaretic acid (DGA) showed both antibacterial and antifungal activity. The (+)- and (-)-7,7'-dioxodihydroguaiaretic acid (ODGA) also showed both antibacterial and antifungal activity, while meso-ODGA did not show antibacterial activity, but showed antifungal activity. No activity of any stereoisomer of secoisolariciresinol (SECO) was apparent.

DOI： 10.1271/bbb.90167

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The syntheses of all stereoisomers of tetrasubstituted tetrahydrofuran lignan were accomplished, and the antimicrobial activity was examined. The 9,9'-diol compound bearing (7R,7'R,8R,8'R) and (7R,7'S,8R,8'R) stereochemistry showed the strongest antibacterial activity against Listeria denitrificans and Bacillus subtilis, respectively. It was also found that (-)-virgatusin bearing (7S,7'R,8S,8'S) stereochemistry had strongest antifungal activity.

DOI： 10.1271/bbb.90107

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPANESE SOC TOXICOLOGICAL SCIENCES  

It is well known that heavy oil pollution results ill various negative impacts on the marine environment. Although there is a low possibility of direct exposure to heavy oil, the chemical substances contained in heavy oil may be released into the environment and accumulated by marine organisms which in turn can be taken by humans via the food chain. In this study, we examined the biological risk of heavy oil extract using the common mouse, whose genetic backgrounds and immune system are well known and relatively homologous to humans. Water-soluble fraction (WSF) was extracted from heavy oil with water and the extract orally administrated to female or male mice for 7 days. In the WSF administrated group, cystoma-like formation was observed in the ovary in approximately 80% of female mice. On the other hand, we found that the prostate gland size in male mice was markedly reduced in comparison with male control mice. Continuous administration of WSF for 28 days resulted in continued hypertrophy of the cystoma around the ovary and atrophy ill the prostate gland. In addition, it was revealed that chemical substances within WSF have estrogenic activity. A major component of heavy oil, polycyclic aromatic hydrocarbons (PAHs), is known to present estrogenic activity. It is likely that the cystoma-like formation in female mice and atrophy of prostate gland in male resulted of estrogenic substances present in the WSF which might be the PAHs.

DOI： 10.2131/jts.34.239

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As we have reported, flaxseed lignan, (+)-secoisolariciresinol (SECO), (-)-SECO, and meso-SECO were stereoselectively synthesized and their biological functions were evaluated. In the present study, we focused on the effects of SECOs on the regulation of 3T3-L1 adipocytes, and identified the structure-activity relationships. Optically active SECO and meso-SECO were tested for their effects on lipid metabolism in 3T3-L1 adipocytes. (-)-SECO accelerated adiponectin production of 3T3-L1 adipocytes. On the other hand, (+)- and meso-SECO suppressed the production of adiponectin. In addition, triglyceride (TG) accumulation in 3T3-L1 adipocytes was significantly suppressed by all three SECOs tested here, as was 17 beta-estradiol, when the SECOs were added to the medium during induction of 3T3-L1 preadipocytes to adipocytes. Especially, (-)-SECO strongly reduced TG accumulation. It is well-known that SECO has estrogen-like activity. Hence the estrogen-like activity of each SECO compound was assessed. Only (-)-SECO had estrogen-like activity.

DOI： 10.1271/bbb.80393

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The antimicrobial activity of all stereoisomers of morinols A and B was tested. All stereoisomers of morinols A and B showed antifungal activity against Alternaria alternata, especially (-)-morinol B which showed the strongest activity. The natural component, (+)-morinol A, and unnatural stereoisomer, (7S,7'S,8R,8'R)-morinol B, showed antibacterial activity against the gram-positive bacteria, Bacillus subtilis and Listeria denitrificans.

DOI： 10.1271/bbb.80536

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Language：English   Publishing type：Research paper (international conference proceedings)   Publisher：SPRINGER  

The jellyfish extract enhanced IgM production of human hybridoma HB4C5 cells 34-fold. IgM and IgG production of human peripheral blood lymphocytes (hPBL) were also accelerated 2.8- and 1.4-fold, respectively. Moreover, IFN-gamma and TNF-alpha production by hPBL Were stimulated 100- and 17-fold, respectively. Collagenase treatment inactivated the immunostimulation activity of the jellyfish extract. In addition, purified collagen from bovine Achilles' tendon accelerated IgM production of hybridoma cells. These facts mean that collagen has immunostimulation effect, and the active substance in jellyfish extract is collagen. As the result of the investigation about the mode of action, colla-en stimulates both transcription and translation activities to enhance Ig production.

DOI： 10.1007/978-1-4020-9646-4_45

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：TAYLOR & FRANCIS LTD  

The antioxidant activity of butane-type lignans was evaluated. Secoisolariciresinol (SECO) and dihydroguaiaretic acid (DGA) showed higher radical scavenging activity than that of 7,7'-dioxodihydroguaiaretic acid (ODGA). SECO and DGA inhibited the oxidation of unsaturated fatty acid. Both enantiomers of DGA were also lipoxygenase inhibitors, but neither enantiomer of SECO inhibited the lipoxygenase activity.

DOI： 10.1271/bbb.80461

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We have previously demonstrated that collagen from jellyfish simulated immunoglobulin and cytokine production by human-human hybridoma line HB4C5 cells and by human peripheral blood lymphocytes (hPBL). The mode of action of the collagen as an immunostimulatory factor was investigated. The expression levels of immunoglobulin mRNAs in HB4C5 cells, and those of tumor necrosis factor (TNF)-alpha, interferon (IFN)-gamma, and transforming growth factor (TGF)-beta in hPBL were up-regulated by jellyfish collagen. In addition, this collagen activated IgM production by transcription-suppressed HB4C5 cells that had been treated with actinomycin D. This collagen also enhanced IgM production by translation-suppressed HB4C5 cells that had been treated with sodium fluoride, but was ineffective in accelerating IgM production by HB4C5 cells treated with cycloheximide. Moreover, the intracellular IgM level in HB4C5 cells treated with the post-translation inhibitor, monensin, was increased by this collagen. These results suggest that collagen from jellyfish stimulated not only the transcription activity, but also the translation activity for enhanced immunoglobulin and cytokine production.

DOI： 10.1271/bbb.80154

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All stereoisomers of goniodiol were synthesized from yeast-reduction products. The C-6 chiral centers were converted from the chiral centers of the yeast-reduction products. Stereoselective conversion of the alkene, which had been prepared from the yeast-reduction product, to glycol constructed the C-7 and C-8 stereochemistry. (+)-Goniodiol and 7-epi-(+)-goniodiol showed the highest antibacterial activity (MIC, 3.1 mM) against Yersinia intermedia.

DOI： 10.1271/bbb.80262

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER  

Immunoglobulin production stimulating activity of polyamines was investigated. Spermidine, thermine and triethylenetetraamine (TETA) stimulated IgM production of human-human hybridoma, HB4C5 cells under serum-free condition. IgM production of HB4C5 cells was accelerated 5.9-, 5.3-, and 3.7-fold by spermidine at 4.5 mM, thermine at 2 mM and TETA at 2.5 mM, respectively. However, putrescine did not enhance IgM production. Spermidine enhanced IgM productivity of the hybridoma cells in spite of its growth suppression activity. TETA also inhibited cell proliferation and the effect on the acceleration of IgM productivity disappeared during 5 days because of its cytotoxicity. On the other hand, thermine facilitated IgM productivity of the hybridoma cells without growth suppression. The laser confocal microscopic analysis revealed that IgM content inside HB4C5 cells was increased by thermine. This result suggests that thermine facilitates IgM synthesis in hybridoma cells.

DOI： 10.1007/s10616-007-9115-y

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The relationship between the antibacterial activity and structure of 9-O,9'-O-demethyl (+)-virgatusin (Virg 3) was examined. The conversion of hydroxy groups on the 9 and 9' positions to amino groups increased the activity. It was found that the 3'-methoxy group was more important for higher activity than the X-methoxy group on the 7'-phenyl group, and that the 3,4-methylenedioxy group on the 7-phenyl group was necessary for activity.

DOI： 10.1271/bbb.70783

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Tetra-substituted tetrahydrofuran compounds were stereoselectively prepared from benzylic hemiacetal in the neutral condition by employing the simple reagent, H-2, and a Pd catalyst. The stereoselective conversion of benzylic hemiacetal to two different stereoisomers of the tetrasubstituted tetrahydrofuran compound was observed. One of these tetrahydrofuran compounds was converted to the virgatusin stereoisomer to estimate its antimicrobiological activity.

DOI： 10.1271/bbb.70554

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Language：Japanese   Publisher：公益社団法人 化学工学会  

DOI： 10.11491/scej.2008.0.629.0

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：TAYLOR & FRANCIS LTD  

The first stereoselective synthesis of meso-secoisolariciresinol is reported. A comparison of the cytotoxic and immunosuppressive activity between meso-secoisolariciresinol and optically active secoisolariciresinols was similarly performed for the first time. Both enantiomers of secoisolariciresinol accelerated IgM production, although meso-secoisolariciresinol did not affect IgM production. Only meso-secoisolariciresinol showed cytotoxic activity.

DOI： 10.1271/bbb.70358

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The benzyl mesylate was employed to construct the tetrasubstituted tetrahydrofuran lignan with avoiding Friedel-Crafts type of reaction. The optically pure 7,8-trans, 7 ',8 '-trans, 7,7 '-cis, and 8,8 '-cis-virgatusin stereoisomers were synthesized. The enantiomeric excess was >> 99%.

DOI： 10.1271/bbb.70236

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The effect of the benzylic structure of lignan on antioxidant activity was evaluated. Secoisolariciresinol (1) and 3,4-bis(4-hydroxy-3-methoxybenzyl)tetrahydrofuran (2), which have two secondary benzylic positions without oxygen, showed the highest antioxidant activity. Optically active verrucosin (4) was synthesized for the first time in this experiment.

DOI： 10.1271/bbb.70275

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The effect of oxidation degree at the benzylic position of 2,3-dibenzyl-4-butanolide and 3,4-dibenzyltetrahydrofuran lignans on the antimicrobiological activity was examined. The highest oxidation degree at the benzylic position of 2,3-dibenzyl-4-butanolide gave the greatest activity, and 3,4-dibenzoyltetrahydrofuran showed the highest antifungal activity. The relationship between stereochemistry and activity was also examined. Both enantiomers of cis-matairesinol were synthesized for the first time, one of the cis-matairesinols showing antibacterial activity.

DOI： 10.1271/bbb.70168

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

The 7',8'-stereochemistry of the tetrahydropyran sesquineolignans morinols A and B was determined as threo via synthetic studies and by comparison of NMR data of 7',8'-threo-morinol and 7',8'-erythro-morinol. This study also confirmed that the biosynthetic process produces enantiomeric mixtures of morinols A and B. This was ascertained by comparing the specific rotations of synthesized morinols A and B with those of naturally occurring morinols A and B.

DOI： 10.1021/np060461j

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The relationship between antibacterial activity of tetra-substituted tetrahydrofuran lignans (1-4) and their absolute configurations was tested. Only compound 4 among two virgatusins and two related compounds exhibited growth inhibitory activity against the Gram-positive bacteria Bacillus subtilis, Staphylococcus aureas and Listeria denitrificans. Compound 4 affected the growth of B. subtilis in a bactericidic manner, and its ability to dissipate the cytoplasmic membrane potential was investigated using the fluorescence probe 3,3'-dipropylthiadicarbocyanine iodide. These results suggested that compound 4 damages cells by causing the loss of the proton motive force and disruption of the cellular integrity of the membrane, leading to cell death. In addition, it was shown that the antibacterial activity of a lignan was closely related to its absolute configuration and functional groups.

DOI： 10.1271/bbb.60429

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Language：Japanese   Publisher：公益社団法人 化学工学会  

DOI： 10.11491/scej.2007f.0.993.0

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Japan Society for Bioscience, Biotechnology, and Agrochemistry  

Antifungal activities of the optically pure (>99%ee) (−)- and (+)-virgatusin, a tetra-substituted tetrahydrofuran lignan, were tested. (−)-Virgatusin, which is a natural product, showed highest antifungal activity against <I>Colletotrichum lagenarium</I>. Research on its structure-activity relationship was also performed. It was shown that two methoxy groups on 9 and 9′ positions and a 3,4-methylenedioxyphenyl group on the 7 position of virgatusin were essential for high fungal growth inhibition. The part on 7′-phenyl group was not essential for activity. The 7′-(4-methoxyphenyl) derivative showed higher activity than that of (−)-virgatusin.

DOI： 10.1271/bbb.60696

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The cytotoxic activity for colon 26 cell line of matairesinol, oxidized matairesinol, 9,9'-epoxylignan and oxidized 9,9'-epoxylignan were examined. (-)Matairesinol (Mat 1) showed greatest cytotoxic activity (LC50 = 9 mu g/ml) of the lactone-type lignans. 7,7'-Oxomatairesinol having same steric configuration as that of (-)-matairesinol showed greater activity (LC50 = 25 mu g/ml) than hydroxy or mono-oxomatairesinol. The activities of 9,9'-epoxylignan and 7,7-oxo-9,9'-epoxylignan having same steric configurations as (-)-matairesinol were weaker than that of corresponding matairesinols. Different activity levels were observed between enantiomers.

DOI： 10.1271/bbb.60353

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Certain edible large jellyfishes belonging to the order Rhizostomeae are consumed in large quantities in China and Japan. The exumbrella part of the edible jellyfish Stomolophus nomurai was cut and soaked in dilute hydrochloric acid solution (pH 3.0) for 12 h, and heated at 121 degrees C for 20 min. The immunostimulation effects of the jellyfish extract were examined. The jellyfish extract enhanced IgM production of human hybridoma HB4C5 cells 34-fold. IgM and IgG production of human peripheral blood lymphocytes (PBL) were also accelerated, 2.8- and 1.4-fold respectively. Moreover, production of interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha by human PBL was stimulated 100- and 17-fold respectively. Collagenase treatment inactivated the immunostimulation activity of the jellyfish extract. In addition, purified collagen from bovine Achilles' tendon accelerated IgM production of hybridoma cells. These facts mean that collagen has an immunostimulation effect, and that the active substance in jellyfish extract is collagen.

DOI： 10.1271/bbb.60076

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：LIPPINCOTT WILLIAMS & WILKINS  

Eucheuma serra agglutinin (ESA) is a lectin derived from a marine red alga E. serra and binds specifically to mannose-rich sugar chains. Previous reports have indicated that ESA associates with several cancer cells via sugar chains on cell surfaces and induces apoptotic cell death. In this study, we investigated the effect of ESA on Colon26 mouse colon adenocarcinoma cells both in vitro and in vivo. ESA induced cell death against Colon26 cells in vitro, and the expression of caspase-3 and the translocation of phosphatidylserine in ESA-treated Colon26 cells suggested that this cell death was induced through apoptosis. An intravenous injection of ESA significantly inhibited the growth of Colon26 tumors in BALB/c mice; moreover, DNA fragmentation was detected in tumor cells following ESA treatment. These results indicated that ESA is effective as an anti-cancer drug not only in vitro but also in vivo. The side-effects of ESA were not considered to be serious because the decrease in body weight of the mice injected with it was negligible. These observations suggest that ESA has the potential to be an effective anti-tumor drug.

DOI： 10.1097/01.cad.0000224458.13651.b4

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The radical and superoxide scavenging activities of oxidized matairesinols were examined. It could be assumed that the free benzylic position was important for higher radical scavenging activity. The different level of activity was observed between 7'-oxomatairesinol (Mat 2) and 7-oxomatairesinol (Mat 3). The activity of 8-hydroxymatairesinol was lower than that of matairesinol (Mat 1). The superoxide scavenging activity of the oxidized matairesinols was also demonstrated for the first time. It is assumed that the pKa value of phenol in the oxidized matairesinols affected this activity.

DOI： 10.1271/bbb.60096

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Language：English   Publishing type：Research paper (international conference proceedings)   Publisher：SPRINGER  

Fat-free bovine milk fermented by more than 10 kinds of various lactic acid bacteria and yeast enhanced monoclonal antibody production of human hybridoma HB4C5 cells 2.8-fold in serum-free medium. Immunoglobulin production of human peripheral blood lymphocytes (PBL) was also stimulated in vitro. IgM and IgG production of human PBL was accelerated up to 2.8-fold and 5.4-fold, respectively. Interferon-gamma production of human PBL was also accelerated 6.0-fold by 50 mu g/ml of the fermented milk. However, interleukin-4 production of PBL was not affected. The activity was enhanced 2.5-fold by the thermal treatment for 30 min at 65 degrees C, and was completely lost by trypsin digestion. The findings suggested that the active substance in the fermented milk was heat stable protein. Gel-filtration and the SDS-PAGE analysis revealed that the molecular weight of the active protein, which was not detected in fat-free bovine milk before fermentation, was 19.0 kDa.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：TAYLOR & FRANCIS INC  

The molecular compositions of the commercial nonionic surfactants Span 80 and Span 85 were analyzed by reversed phase high performance liquid chromatography (HPLC). Both surfactants are mixtures of fatty acid esters, containing monoesters, diesters, triesters, and tetraesters. While diesters dominate in the case of Span 80, Span 85 contains mainly tetraesters. Vesicles were prepared from Span 80 (or Span 85) by a two-step emulsification method that involved homogenization and separation steps in which a portion of the surfactants was removed. The composition of the vesicles was analyzed by HPLC with respect to the different esters present. Although commercial Span 80 and Span 85 differ considerably in their molecular compositions, the ester profiles of the vesicles formed were in both cases rather similar and dominated by diesters. Therefore, the particular vesicle preparation method leads to a molecular selection of mainly those components that are prone to form bilayers.

DOI： 10.1080/01932690600859903

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Fat-free bovine milk fermented by 12 kinds of lactic acid bacteria and yeast enhanced monoclonal antibody production of human hybridoma HB4C5 cells 2.8-fold in serum-free medium. Immunoglobulin production of human peripheral blood lymphocytes (PBL) was also stimulated in vitro. IgM and IgG production of human PBL was accelerated up to 2.8-fold and 5.4-fold, respectively. Interferon-gamma production of human PBL was also accelerated 6.0-fold by 50 microg/ml of the fermented milk. However, interleukin-4 production of PBL was not affected, and tumor necrosis factor-alpha production was suppressed. The activity was enhanced 2.5-fold by the thermal treatment for 30 min at 65 degrees C and was completely lost by trypsin digestion. The findings suggested that the active substance in the fermented milk was heat stable protein. Gel-filtration and the SDS-PAGE analysis revealed that the molecular weight of the active substance was estimated as 19.0 kDa, which was not detected in fat-free bovine milk before fermentation. N-terminal amino acid sequence of the 19.0 kDa protein was highly homologous to proteose-peptone component 3 (PP3). Since molecular weight of PP3 is 28 kDa, it is suggested that the 19.0 kDa protein is derived from degradation of PP3 during fermentation of fat-free milk. Moreover, PP3 purified from fat-free milk also enhanced IgM production of HB4C5 cells.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Fat-free bovine milk fermented by 12 kinds of lactic acid bacteria and yeast enhanced monoclonal antibody production of human hybridoma HB4C5 cells 2.8-fold in serum-free medium. Immunoglobulin production of human peripheral blood lymphocytes (PBL) was also stimulated in vitro. IgM and IgG production of human PBL was accelerated up to 2.8-fold and 5.4-fold, respectively. Interferon-gamma production of human PBL was also accelerated 6.0-fold by 50 mu g/ml of the fermented milk. However, interleukin-4 production of PBL was not affected, and tumor necrosis factor-alpha production was suppressed. The activity was enhanced 2.5-fold by the thermal treatment for 30 min at 65 degrees C and was completely lost by trypsin digestion. The findings suggested that the active substance in the fermented milk was heat stable protein. Gel-filtration and the SDS-PAGE analysis revealed that the molecular weight of the active substance was estimated as 19.0 kDa, which was not detected in fat-free bovine milk before fermentation. N-terminal amino acid sequence of the 19.0 kDa protein was highly homologous to proteose-peptone component 3 (PP3). Since molecular weight of PP3 is 28 kDa, it is suggested that the 19.0 kDa protein is derived from degradation of PP3 during fermentation of fat-free milk. Moreover, PP3 purified from fat-free milk also enhanced IgM production of HB4C5 cells. (c) 2005 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.bbagen.2005.05.008

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The first highly enantioselective syntheses of tetra-substituted tetrahydrofuran lignan, (-)- and (+)-virgatusin, were achieved. Hemiacetal 15 was stereoselectively obtained from Evans's syn-aldol product 8 as a single isomer. This hemiacetal 15 was converted to (-)-virgatusin via hydrogenolysis. (+)-Virgatusin was also synthesized through the same process. The enantiomeric excess of the both enantiomers was determined as more than 99% ee. © The Royal Society of Chemistry 2005.

DOI： 10.1039/b501151e

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE INC  

By searching the zebrafish expressed sequence tag database, we had identified two partial cDNA clones encoding the 5 '- and 3 ' regions of a putative cytosolic sulfotransferase (SULT). Using the reverse transcription-polymerase chain reaction (RT-PCR) technique, a full-length cDNA encoding this zebrafish SULT was amplified, cloned, and sequenced. Analysis of the sequence data revealed that this novel zebrafish SULT displays 49, 46, and 45% amino acid sequence identity to human SULT1A1, mouse SULT1D1, and rat SULT1C1 This zebrafish SULT therefore appears to belong to the SULT1 cytosolic SULT gene family. Recombinant zebrafish SULT (designated SULT1 isoform 4), expressed using the pGEX-2TK prokaryotic expression vector and purified from transformed Escherichia coli cells, migrated as a 35 kDa protein upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Among the endogenous compounds tested as substrates, the purified SULT1 isoform 4 displayed significant sulfating activities toward thyroid hormones, estrone.. and dehydroepiandrosterone. The enzyme also showed activities toward a number of xenobiotics including some flavonoids, isoflavonoids, and other phenolic compounds, with a pH optimum at 7.0. A thermostability experiment revealed the enzyme to be relatively stable over a temperature range between 28 and 37 degrees C. Among 10 divalent metal cations tested, Fe2+ Hg2+, Co2+ Zn2+ Cu2+, and Cd2+ exhibited dramatic inhibitory effects on the activity of the enzyme. Developmental expression study using RT-PCR revealed that the zebrafish SULT1 isoform 4 showed a low level of expression in the segmentation period during the embryonic development, which gradually increased to a high level of expression throughout the larval stage onto maturity. (c) 2005 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.abb.2005.02.029

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The cationic vesicle composed of Span80 and cationic peptide lipid (CPL) was prepared. The cytotoxicity of the Span80-CPL cationic vesicle was very low compared with Span80 vesicle. Antibody was able to be immobilized on vesicle surface by mediation of protein A. The antigen targeting ability of the antibody-immobilized vesicle (immunovesicle) derived from antibody was evaluated. Our results suggested that the Span80-CPL immunovesicles specifically associate with target cells by the antibody mediation, and the substance capsulated in immunovesicle was transferred into the target cells. This means that the Span80-CPL immunovesicle is expected to achieve a high local concentration of an encapsulated drug at the target.

DOI： 10.1007/s10616-005-3764-5

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Language：English   Publishing type：Research paper (international conference proceedings)   Publisher：The Society of Chemical Engineers, Japan  

We will report a targeting and an anti-cancer effect of a lipid vesicle, to which <I>Euchuma Serra Agglutinin (ESA)</I> immobilized, to human colon cancer cell (Colo201) in mouse xenopathic model. The lipid vesicles were mainly composed of Span80 (sorbitan monooleate). First, the specific binding between the lipid vesicle and the cancer cell was studied for the application of the vesicle to DDS. It was preliminary confirmed that the vesicle flew smoothly in superior artery of a rabbit. As a targeting ligand (combined with the vesicle) at a Colo201, <I>Euchuma Serra Agglutinin (ESA)</I> was chosen. The ESA is a new lectin derived from a marine red alga and specifically combines with mannose-rich carbohydrate-chain, which often exists on the surface of cancer cells abundantly. <I>ESA</I> was found to specifically combine with many cancer cells, especially to colon adenocarcinoma (Colo201) and induce an apoptosis to the cancer cell. Secondly, the <I>in vivo</I> experiments toward to colon cancer therapy were carried out administrating the <I>ESA-immobilized</I> vesicles into nude mice in which human colon cancer-cell Colo201 had been implanted. Preliminary autoradiogram experiments showed the free <I>ESA</I> selectively accumulated in the tumor of the mice. Then, we prepared either <I>ESA-immobilized</I> vesicle or the vesicle entrapping anti-tumor drug (Taxotere) for using colon-cancer xenopathic model in nude mouse. Autoradiograms also showed the <I>ESA-immobilized</I> vesicles were specifically accumulated into the tumor rather than the other organ. The most effective anti-tumor effect was observed in using the <I>ESA-immobilized</I> vesicle entrapping Taxotere.

DOI： 10.11491/apcche.2004.0.500.0

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Language：English   Publishing type：Research paper (international conference proceedings)   Publisher：The Society of Chemical Engineers, Japan  

The purpose of this work is a construction of the basic manner of a human brain-tumor therapy by using an immunovesicle (antibody-immobilized vesicle) in DDS. The ERM5-1 cell was applied as a model cell of human brain-tumor, which was a mouse cell made by transgenic technology. The human brain-tumor cell was found to have a specific antigen of EGFR (Epidermal Growth Factor Receptor). The ERM5-1 cell has the antigen EGFR and the cell can be applied as a model cell of human brain-tumor cell. Anti-EGFR antibody was immobilized on the surface of the vesicle as follows. The human anti-EGFR antibody (IgG) was preliminary immobilized to a protein A, which has a binding site to Fc fragment of IgG. The EGFR-protein A complex was immobilized to a vesicle by means of the two-step emulsification. Thus, the human anti-EGFR antibody was successfully immobilized orientated to the outer direction of the vesicle surface. It was fluorometrically confirmed that the specific antigen-antibody affinity between the antibody of the vesicle and EGFR on the ERM5-1 cell well functioned <I>in vitro</I> experiments. In the vesicle, Taxsotere of anti-tumor drug was entrapped for increasing the anti-tumor effect of the vesicle. It was also confirmed from <I>in vitro</I> experiments that anti-tumor effect of the immunovesicle to the ERM5-1 cell was superior to a normal vesicle (non-immunovesicle). Moreover, the <I>in vivo</I> experiments of anti-tumor effect to the ERM5-1 cell were carried out administrating the Taxotere-entrapped immunovesicle into nude mice to which ERM5-1 cell was implanted. Superior anti-tumor effect to the ERM5-1 cell in the mice could be observed to that of the normal vesicle.

DOI： 10.11491/apcche.2004.0.501.0

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PORTLAND PRESS  

By searching the zebrafish EST (expressed-sequence tag) database, we have identified two partial cDNA clones encoding the 5' and 3' regions of a putative zebrafish sulphotransferase (ST). Using the reverse transcription-PCR technique, a full-length cDNA encoding this zebrafish ST was successfully cloned. Sequence analysis revealed that this novel zebrafish ST displays 44 %, 43 % and 40 % amino acid identity with mouse SULT2B1, human SULT2B1b and human SULT2A1 ST respectively. This zebrafish ST therefore appears to belong to the SULT2 cytosolic ST gene family. Recombinant zebrafish ST, expressed using the pGEX-2TK prokaryotic expression system and purified from transformed Escherichia coli cells, migrated as a 34 kDa protein upon SDS/PAGE. Purified zebrafish ST displayed a strong sulphonating activity toward DHEA (dehydroepiandrosterone), with a optimum pH of 9.5. The enzyme also exhibited activities toward several neurosteroids with differential K-m and V-max values. A thermostability experiment revealed the enzyme to be relatively stable over a temperature range between 20 degreesC and 43 degreesC. Among ten different divalent metal cations tested, Fe2+ and Cd2+ exhibited small, but significant, stimulatory effects, whereas Hg2+ and Cu2+ displayed considerably stronger inhibitory effects on the DHEA-sulphonating activity of the enzyme. These results constitute the first study on the molecular cloning, expression, and characterization of a zebrafish cytosolic SULT2 ST.

DOI： 10.1042/BJ20031050

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

Environmental estrogen-like chemicals are increasingly recognized as a potential hazardous factor for wildlife as well as humans. We have recently embarked on developing a zebrafish model for investigating the role of sulfonation in the metabolism and adverse functioning of environmental estrogens. Here, we report on a systematic investigation of the sulfonation of representative environmental estrogens (bisphenol A, 4-n-octylphenol, 4-n-nolylphenol, diethylstilbestrol, and 17alpha-ethynylestradiol) by zebrafish cytosolic sulfotransferases (STs). Of the seven enzymes tested, four zebrafish STs (designated ZF ST #2, ZF ST #3, ZF ST #4, and ZF DHEA ST) exhibited differential sulfonating activities toward the five environmental estrogens tested, with ZF ST #3 being more highly active than the other three. It was further demonstrated that bisphenol A, 4-n-octylphenol, and 4-n-nonylphenol exerted concentration-dependent inhibition of the sulfonation of 17beta-estradiol, implying a potential role of these environmental estrogens in interfering with the sulfonation, and possibly homeostasis, of endogenous estrogens. Kinetic studies revealed that the mechanism underlying the inhibition by bisphenol A or 4-n-nonylphenol to be of the competitive type. (D 2003 Elsevier Inc. All rights reserved.

DOI： 10.1016/S0006-291X(03)01524-9

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BLACKWELL PUBLISHING LTD  

As a first step toward developing a zebrafish model for investigating the role of sulfation in counteracting environmental estrogenic chemicals, we have embarked on the identification and characterization of cytosolic sulfotransferases (STs) in zebrafish. By searching the zebrafish expressed sequence tag database, we have identified two cDNA clones encoding putative cytosolic STs. These two zebrafish ST cDNAs were isolated and subjected to nucleotide sequencing. Sequence data revealed that the two zebrafish STs are highly homologous, being approximate to 82% identical in their amino acid sequences. Both of them display approximate to 50% amino acid sequence identity to human SULT1A1, rat SULT1A1, and mouse SULT1C1 ST. These two zebrafish STs therefore appear to belong to the SULT1 cytosolic ST gene family. Recombinant zebrafish STs (designated SULT1 STs 1 and 2), expressed using the pGEX-2TK prokaryotic expression system and purified from transformed Escherichia coli cells, migrated as approximate to 35 kDa proteins on SDS/PAGE. Purified zebrafish SULT1 STs 1 and 2 displayed differential sulfating activities toward a number of endogenous compounds and xenobiotics including hydroxychlorobiphenyls. Kinetic constants of the two enzymes toward two representative hydroxychlorobiphenyls, 3-chloro-4-biphenylol and 3,3',5,5'-tetrachloro-4,4'-biphenyldiol, and 3,3',5-triiodo-l-thyronine were determined. A thermostability experiment revealed the two enzymes to be relatively stable over the range 20-43 degreesC. Among 10 different divalent metal cations tested, Co2+, Zn2+, Cd2+, and Pb2+ exhibited considerable inhibitory effects, while Hg2+ and Cu2+ rendered both enzymes virtually inactive.

DOI： 10.1046/j.1432-1033.2003.03608.x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

Using the reverse transcriptase-polymerase chain reaction technique, a full-length cDNA encoding a novel zebrafish sulfotransferase was cloned and sequenced. Sequence analysis indicated that this zebrafish sulfotransferase belongs to the SULT1 cytosolic sulfotransferase gene family. The recombinant form of the zebrafish sulfotransferase, purified from Escherichia coli cells, displayed sulfating activities toward a number of endogenous compounds, in particular dopamine and thyroid hormones, in addition to xenobiotics including some flavonoids, isoflavonoids, and other phenolic compounds. The zebrafish sulfotransferase exhibited substrate dependence in pH optimum. In comparison with those determined with dopamine as substrate, the zebrafish sulfotransferase displayed much lower K-m and higher V-max with n-propyl gallate as substrate. A thermostability experiment revealed the enzyme to be relatively stable over a temperature range between 20 and 43 degreesC. Among 10 divalent metal cations tested, Hg2+, Co2+, Zn2+, Cd2+, Cu2+, and Pb2+ exhibited dramatic inhibitory effects on the activity of the zebrafish sulfotransferase. (C) 2003 Elsevier Science (USA). All rights reserved.

DOI： 10.1016/S003-9861(03)00172-3

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：TAYLOR & FRANCIS LTD  

HB4C5 cells, a human-human hybridoma, were cultured in microtubes. After 24 h of cultivation, growth of the cells cultured in microtubes was 1.2- to 1.5-fold that in culture dishes or 96-well culture plates. The production of IgM was 2.6- to 3.3-fold that in the 96-well culture plates.

DOI： 10.1271/bbb.67.393

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPANESE BIOCHEMICAL SOC  

Cytosolic sulfotransferases (STs) are traditionally known as Phase II drug-metabolizing or detoxifying enzymes that facilitate the removal of drugs and other xenobiotic compounds. In this study, we carried out a systematic investigation on the sulfation of drug compounds by two major human phenol STs (PSTs), the monoamine (M)-form and simple phenol (P)-form PSTs. Activity data obtained showed the differential substrate specificity of the two enzymes for the thirteen drug compounds tested. Kinetic studies revealed that the M-form PST displayed stereoselectivity for the chiral drug, isoproterenol. The effects of divalent metal cations on the activity of the Worm and P-form PSTs toward representative drug compounds were quantitatively evaluated. Results obtained indicated that the drug-sulfating activities of the two human PSTs were partially or completely inhibited or stimulated by the ten divalent metal cations tested at a 5 mM concentration. The two enzymes appeared to be less sensitive to the effects of physiologically more abundant metal cations such as Mg2+ and Ca2+, but more sensitive to the detrimental effects of other metal cations that may enter the body as environmental contaminants.

DOI： 10.1093/jb/mvg033

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

By searching the zebrafish expressed sequence tag (EST) database, we have identified a cDNA clone encoding a putative zebrafish cytosolic sulfotransferase (ST). This cDNA was isolated and subjected to nucleotide sequencing. Analysis of the sequence data revealed that this novel zebrafish ST displays 32-35% amino acid sequence identity to members of all major cytosolic ST gene families. Therefore, this zebrafish ST, while belonging to the cytosolic ST gene superfamily, appears to be independent from all known constituent ST gene families. Recombinant zebrafish ST, expressed using the pET23c prokaryotic expression vector and purified from transformed Escherichia coli cells, migrated as a 34-kDa protein upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Purified zebrafish ST displayed sulfating activities toward dopamine and thyroid hormones (T, and T,), with a pH optimum spanning 7-9. The enzyme also exhibited activities toward a number of xenobiotics including some flavonoids, isoflavonoids, and other phenolic compounds. A thermostability experiment revealed the enzyme to be relatively stable over a temperature range between 20 and 48 degreesC. Among 10 divalent metal cations tested. Fe-+, Hg++, Co++, Zn+-, Cu++, and Cd++ exhibited dramatic inhibitory, effects on the activity of the enzyme. These results constitute a first study on the cloning, expression, and characterization of a zebrafish cytosolic ST. (C) 2002 Elsevier Science (USA). All rights reserved.

DOI： 10.1016/S0006-291X(02)02915-7

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

The human monoamine-form phenol sulfotransferase (PST), SULT1A3, has a unique 3,4-dihydroxyphenylalanine (Dopa)/tyrosine-sulfating activity that is stereospecific for their D-form enantiomers and can be stimulated dramatically by Mn2+. This activity is not present in the simple phenol-form PST, SULT1A1, which is otherwise &gt;93% identical to SULT1A3 in amino acid sequence. The majority of the differences between these two proteins reside in two variable regions of their sequences. Through the characterization of chimeric PSTs where these two regions were exchanged between them, it was demonstrated that variable Region II of SULT1A3 is required for the stereospecificity of its Dopa/tyrosine-sulfating activity, whereas variable Region I of SULT1A3 is required for the stimulation by Mn2+ of this activity. Further studies using point-mutated SULT1A3s mutated at amino acid residues in these two regions and deletional mutants missing residues 84-86 and 84-90 implicate residue Glu-146 (in variable Region II of SULT1A3), as well as the presence of residues 84-90 of variable Region I, in the stereospecificity in the absence of Mn2+. Residue Asp-86 (in variable Region I of SULT1A3), on the other hand, is critical in the Mn2+ stimulation of the Dopa/tyrosine-sulfating activity of SULT1A3. A model is proposed, with reference to the reported x-ray crystal structure of SULT1A3, to explain how the normal role of SULT1A3 in dopamine regulation may be subverted in the presence of Mn2+. These studies could be relevant in understanding the stereoselective action of SULT1A3 on chiral drugs.

DOI： 10.1074/jbc.M203108200

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：KLUWER ACADEMIC PUBL  

Lactate dehydrogenase (LDH) derived from rabbit muscle enhanced IgM production by human-human hybridoma HB4C5 cells 12.4-fold at 320 mug/ml under serum-free conditions. LDHs from pig muscle and pig heart also accelerated IgM production 8.4- and 6.4-fold, respectively. The immunoglobulin production stimulating activity of LDH was not accompanied by activation of cell proliferation. LDH from rabbit muscle facilitated IgM and IgG production by human peripheral blood lymphocytes. This means LDH stimulates immunoglobulin production not only by the specified hybridoma cell line, but also by unspecified immunoglobulin producers. LDH from rabbit muscle enhanced IgM production of transcription-suppressed HB4C5 cells treated with actinomycin D. The immunoglobulin production-stimulating factors (IPSFs) effect of LDH was slightly weakened by sodium fluoride ( translation inhibitor) treatment of HB4C5. Moreover, the amount of intracellular IgM of monensin-treated HB4C5 cells was obviously enhanced by LDH. This result means that the IPSF effect of LDH is irrelevant to the post-translation activity of target cells. It is expected from these findings that LDH from rabbit muscle accelerates the translation step to enhance immunoglobulin productivity. The immunoglobulin production-stimulating activity of LDH was inhibited by colchicine, endocytosis inhibitor. This fact suggests that it is necessary for LDH to be taken by target cells to act as an IPSF.

DOI： 10.1023/B:CYTO.0000015838.06536.de

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Kinetic studies were performed to dissect the mechanism underlying the remarkable Mn2+ stimulation of the Dopa/tyrosine-sulfating activity of the human monoamine (M)-form phenol sulfotransferase (PST). The activities and the stimulation by Mn2+ are highly stereospecific for the D-form enantiomers of tyrosine and Dopa. Analysis of the kinetic results strongly suggests that tyrosine-Mn2+ and tyrosine-Mn2+-tyrosine complexes are obligatory substrates, whereas Dopa-Mn2+ complexes may be better substrates than Dopa alone. This activation of the Dopa/tyrosine-sulfating activity of M-form PST by Mn2+ via complex formation between Mn2+ and the substrate is the first reported case of a regulatory mechanism in this important class of enzymes. Our previous studies using point-mutated M-form PSTs established that the Mn2+ (in the substrate-Mn2+ complex) exerts its stimulatory effect by binding predominantly to the Asp-86 residue at the active site. We present here further studies using dopamine as substrate to bolster this conclusion. The possible physiological implications of this rather unusual specificity for the D-amino acid and its derivatives and the stimulation by Mn2+ are discussed in the context of protective and detoxification mechanisms that may operate in neurodegenerative processes in the brain. The, Mn2+ stimulation of the activity of M-form PST toward D-enantiomers of Dopa/tyrosine may have implications for other substrates (including chiral drugs) and for the other cytosolic sulfotransferases that are involved in the regulation of endogenous metabolites as well as in detoxification.

DOI： 10.1074/jbc.M200785200

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Environmental xenoestrogens have been implicated in human reproductive disorders and an increased incidence of breast cancer. Sulfation, a Phase II detoxification mechanism involving the cytosolic sulfotransferases (STs), may be an important mechanism in vivo for fending off these compounds. In this study, we report on the molecular cloning, expression, and purification of two human cytosolic STs, SULT2B1a and SULT2b1b. The activities of these two enzymes, as well as the other eight known human cytosolic STs previously prepared, toward representative environmental xenoestrogens were examined. Activity data showed that P-form (SULT1A1) PST displayed the highest activity toward these compounds, while SULT1C ST #2 also showed considerable activity, indicating that these enzymes may play a more important role in detoxification of environmental xenoestrogens. SULT1C ST #1, SULT2B1a ST, SULT2B1b ST and NST showed negligible or undetectable activity toward these compounds. The other four enzymes, M-form (SULT1A3) PST, SULT1B2 ST, SULTA1 ST and SULT1E ST showed intermediate levels of activity toward some of these compounds. Kinetic studies on the sulfation of xenoestrogens by P-form (SULT1A1) PST were performed. The results are interpreted in the context of the endocrine-disrupting nature of these xenoestrogens. (C) 2002 Elsevier Science B.V All rights reserved.

DOI： 10.1016/S0304-4165(02)00416-6

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Lysozyme from hen egg white was identified as an immunoglobulin production stimulating factor (IPSF) that enhances immunoglobulin production by hybridomas and lymphocytes. The IPSF activity of lysozyme was facilitated by heat treatment. The heat treatment of lysozyme at 83degreesC for 30 min activated its specific IPSF effect 30.0-fold compared with that of native lysozyme. The IPSF activity of lysozyme heat-treated at 83degreesC in 4 M urea solution was enhanced 8.4-fold than that of native lysozyme. However, lysozyme that was not heated in 4 M urea solution completely lost its IPSF activity. This means that the IPSF activity of this enzyme in 4 M urea was reactivated by thermal treatment. Moreover, coexistence of 0.5 mM 2-mercaptoethanol (2-ME) during heating in 4 M urea solution extremely enhanced the IPSF activity up to 77.8-fold. The uptake of lysozyme by hybridoma cells was enhanced by heat denaturation in 4 M urea. The hydrophobicity of lysozyme was extremely increased by heat-treatment in 2-ME containing urea solution. It is expected from these findings that the increase in the hydrophobicity caused the enhancement of incorporation of lysozyme into target cells, and resulted in the acceleration of IgM production. (C) 2002 Elsevier Science B.V. All rights reserved.

DOI： 10.1016/S0304-4165(02)00272-6

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：TAYLOR & FRANCIS LTD  

A histone mixture (H1, H2A, H2B, H3, and H4) derived from calf thymus stimulated IgM production by human-human hybridoma HB4C5 cells. On the contrary, the histone mixture did not increase IgM production by the human Burkitt's lymphoma cell line NAT-30, IgG production by the human B lymphoblastoid cell line HMy-2, and IgE production by the human myeloma cell line U266. The immunoglobulin pro duction-stimulating activity of the histone mixture was inactivated by trypsin or chymotrypsin digestion. In addition, confocal laser microscopic analysis had shown that HB4C5 cells incorporated a lot of histone but other cell lines did not incorporate it as much.
These facts strongly suggest that histone acts as an immunoglobulin pro duction-stimulating factor (IPSF) after internalization into the human B cell lines and the native structure of histone is required for the IPSF activity.

DOI： 10.1271/bbb.66.1241

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Language：English   Publishing type：Research paper (international conference proceedings)   Publisher：SPRINGER  

Lysozyme from hen egg white was identified as an immunoglobulin production stimulating factor (IPSF) which enhances immunoglobulin production by hybridomas and lymphocytes. The IPSF activity of lysozyme was facilitated by heat treatment. The heat treatment of lysozyme at 83degreesC for 30 min activated its stimulating effect 15.9-fold compared with that of native lysozyme. The IPSF activity of lysozyme heat-treated in 4 M urea solution was enhanced 8.8-fold than that of native lysozyme. However, the IPSF activity of lysozyme in 4 M urea solution without heat treatment was completely lost. The uptake of lysozyme by hybridoma cells was enhanced by heat denaturation in 4 M urea. This indicates that heat-treated lysozyme in 4 M urea is easily taken by HB4C5 cells and expresses high IPSF activity in the cells. Moreover, coexistence of 2-mercaptoethanol during heat denaturation in 4 M urea solution extremely enhanced the IPSF activity of lysozyme up to 77.8-fold.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE INC  

Alcohol dehydrogenase-I (ADH-I; EC 1.1.1.1) derived from horse liver stimulated IgM production by human-human hybridoma, HB4C5 cells and human peripheral blood lymphocytes. The immunoglobulin production stimulating activity of ADH-I was inactivated by chymotrypsin digestion, even though the enzymatic activity was completely preserved. This fact suggests that the immunoglobulin production stimulating effect of ADH-I is irrelevant to its enzymatic function. This also means that the effect is a novel function of this enzyme. ADH-I enhanced IgM production by transcription-suppressed HB4C5 cells treated with actinomycin D. Moreover, the enzyme was effective to accelerate IgM production by translation-suppressed HB4C5 cells treated with sodium fluoride and cycloheximide. Although the secretion of IgM by HB4C5 cells treated with monensin for suppression of the post-transcription process was inhibited, the intracellular IgM content of the cells was obviously increased by the addition of ADH-I. It is supposed from these results that ADH-I accelerates the translation activity to enhance immunoglobulin productivity. In addition, a laser confocal microscopic analysis revealed that ADH-I from horse liver was subsequently incorporated by HB4C5 cells. (C) 2001 Elsevier Science Inc. All rights reserved.

DOI： 10.1016/S0141-0229(01)00371-4

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：KLUWER ACADEMIC PUBL  

Eucheuma serra agglutinin (ESA) derived from a marine red alga, Eucheuma serra, is a lectin that specifically binds to mannose-rich carbohydrate chains. ESA is a monomeric molecule, with a molecular weight of 29,000. ESA induced cell death against several cancer cell lines, such as colon cancer Colo201 cells and cervix cancer HeLa cells. DNA ladder detection and the induction of caspase-3 activity suggested that the cell death induced by ESA against cancer cells was apoptosis. ESA bound to the cell surface of Colo201 cells in the sugar chain dependent manner. This means that the binding of ESA to the cell surface is specific for mannose-rich sugar chains recognized by ESA. The binding of ESA to the cell surface of Colo201 cells was slightly suppressed by the high concentrations of serum because of the competition with serum components possessing the mannose-rich sugar chain motifs. On the other hand, a lipid vesicle is a very useful microcapsule constructed by multilamellar structure, and adopted as drug or gene carrier. ESA was immobilized on the surface of the lipid vesicles to apply the lipid vesicles to cancer specific drug delivery system. ESA-immobilized lipid vesicles were effectively bound to cancer cell lines compared with plane vesicles.

DOI： 10.1023/A:1014057407251

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

As we demonstrated before, hell egg white lysozyme stimulates immunoglobulin production by a human-human hybridoma line, HB4C5 cells and human peripheral blood lymphocytes. Then, the mode of actions of lysozyme as an immunoglobulin production stimulating factor was investigated. The immunoglobulin production stimulating activity of lysozyme was inactivated by trypsin digestion, even though the enzymatic activity was completely preserved. This fact suggests that the immunoglobulin production stimulating effect of lysozyme is irrelevant to its enzymatic function. Furthermore, this means that the effect is a novel function of this enzyme. Lysozyme enhanced IgM production by transcription-suppressed HB4C5 cells treated with actinomycin D. However, the enzyme was ineffective to accelerate IgM production by translation-suppressed HB4C5 cells treated with cycloheximide or sodium fluoride. In addition, the intracellular IgM content of HB4C5 cells treated with monensin for suppression of the post-transcription activity was obviously increased by lysozyme, although the secretion of IgM was inhibited. These findings suggest that lysozyme accelerates the translation process to enhance immunoglobulin productivity. (C) 2000 Elsevier Science B.V. All rights reserved.

DOI： 10.1016/S0304-4165(00)00041-6

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Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：Japan Society for Bioscience Biotechnology and Agrochemistry  

DOI： 10.1271/nogeikagaku1924.74.1107

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DOI： 10.1271/nogeikagaku1924.74.986

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE INC  

D-fructose-1,6-bisphosphate aldolase (EC 4.1.2.13) derived from rabbit muscle facilitated immunoglobulin production by human-human hybridoma HB4C5 cells under serum-free condition. IgM production by HB4C5 cells was stimulated by the addition of aldolase from rabbit muscle at the concentrations of more than 100 mu g ml(-1). The enzyme also enhanced IgM and IgG production by human peripheral blood lymphocytes about 5.3-fold and 1.4-fold, respectively. This fact suggests that aldolase from rabbit muscle stimulates immunoglobulin production not only by the specified hybridoma cell line, but also by unspecified immunoglobulin producers; however, yeast aldolase was ineffective in accelerating immunoglobulin production by HB4C5 cells. The IPSF activity was lost by trypsin digestion at 100 mu g ml(-1); however, limited fragmentation by the lower concentrations of trypsin facilitated the IPSF activity of rabbit muscle aldolase about twofold even though the enzymatic activity was inactivated. These results imply that the immunoglobulin production-stimulating effect of aldolase is irrelevant to its enzymatic function, and the activity was defined as a novel role of rabbit muscle aldolase. (C) 1998 Elsevier Science Inc.

DOI： 10.1016/S0141-0229(98)00087-8

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The lipid vesicles prepared were composed of such as artificial lipids of Span80 and cationic lipids (DDAB and N(+)C(5)Ala2C(16)). Firstly, the cationic vesicles were prepared by the addition of the cationic lipids to Span80. The addition enhanced the affinity between the vesicles and the cells, such as MCF7 (human breast adenocarcinoma), COLO201 (colon adenocarcinoma) and the human-human hybridoma (HB4C5 cell), which was a fusion product of a human lymphocyte from lung cancer patient and a human lymphoma line, NAT-30. Nextly, the anti-IgM antibody was immobilized to vesicle (immunovesicle). The targeting antigen was the monoclonal IgM produced at the surface of the HB4C5 cells. The binding and the fusion between the vesicles and HB4C5 cells were confirmed and the addition of the cationic lipids to Span80 enhanced the fusion between the immunovesicles and HB4C5 cells. Moreover, the membrane fluidity and the HLB value of the lipids was also found to strongly effect on the vesicle fusion.

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Lysozyme derived from hen egg white stimulated immunoglobulin (Ig) production by human-human hybridoma, HB4C5 cells producing monoclonal IgM. IgM production by HB4C5 cells was enhanced more than 13-fold by lysozyme at 380 mu g/ml in serum-free medium. The mode of actions of lysozyme as an Ig production stimulating factor (IPSF) was investigated. Lysozyme enhanced Ig production by transcription-suppressed HB4C5 cells. However, the enzyme was ineffective to accelerate IgM production by translation-suppressed HB4C5 cells. In addition, the intracellular IgM content of HB4C5 cells, which were suppressed post-transcription process, was obviously increased by the addition of lysozyme. These findings suggest that lysozyme accelerates translation process to enhance Ig productivity.

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The investigations on a specific adsorption between a lipid vesicle and a cancer cell. were carried out. The vesicles were prepared by means of a two-step emulsification technique. The main component of the lipid membrane of the vesicle was Span80 (sorbitan monooleate). The attention was given to Eucheuma serra (algal lectin: abbreviated as ESA), which showed the specific affinity to a high-mannose. The ESA-immobilized vesicle was found to be specifically adsorbed to the cancer cells, such as human breast adenocarcinoma (MCF7), colon adenocarcinoma (COLO201), and human-human hybridoma (HB4C5). The vesicle flew smoothly in the superior artery of rabbit. Then, the immobilized vesicle is expected to be useful for a cancer therapy in a DDS.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：KLUWER ACADEMIC PUBL  

Alcohol dehydrogenase-I (ADH-I) derived from horse liver stimulated IgM production by human-human hybridoma, HB4C5 cells and lymphocytes. The IPSF activity of ADH-I was suppressed by coexistence of short DNA whose chain length is less than 200 base pairs (bp) and fibrous DNA in a dose-dependent manner. These DNA preparations completely inhibited the IPSF activity at the concentration of 250 mu g/ml and 1.0 mg/ml, respectively. DNA sample termed long DNA whose average chain length is 400-7000 bp slightly stimulated IPSF activity at 0.06 mu g/ml. However, long DNA suppressed IPSF activity by half at 1.0 mg/ml. The laser confocal microscopic analysis had revealed that ADH-I was incorporated by HB4C5 cells. The uptake of ADH-I was strongly inhibited by short DNA and fibrous DNA. However, long DNA did not suppress the internalization of ADH-I into HB4C5 cells. These findings indicate that short DNA and fibrous DNA depress IPSF activity of ADH-I by inhibiting the internalization of this enzyme. According to the gel-filtration analysis using HPLC, ADH-I did not directly interact with short DNA. It is expected from these findings that short DNA influences HB4C5 cells to suppress the internalization of ADH-I. Moreover, these facts also strongly suggest that ADH-I acts as IPSF after internalization into the cell.

DOI： 10.1023/A:1008024322602

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：TAYLOR & FRANCIS LTD  

We have previously identified glyceraldehyde-3-phosphate dehydrogenase as an immunoglobulin production stimulating factor (IPSF) which facilitated immunoglobulin production by hybridomas and lymphocytes. The IPSF activity of this enzyme was suppressed by the coexistence of some sorts of nucleotides. We now report that the IPSF effect of GAPDH was suppressed by the coexistence of DNA, the inhibiting effect of degraded DNA being inferior to that of long-chain DNA. Both single-stranded and double-stranded synthetic polyribonucleotides also inhibited the IPSF activity of GAPDH. Moreover, nicotinamide adenine dinucleotide (NAD(+)) repressed the IPSF effect.

DOI： 10.1271/bbb.62.1237

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Rabbit muscle enolase stimulates immunoglobulin production by a human hybridoma line, HB4C5 cells under serum-free condition. IgM productivity of HB4C5 cells was enhanced more than 20-fold by this enzyme at 220 mu g/ml. Human peripheral blood lymphocytes were also facilitated their IgM and IgG productivity in the serum-free medium. However, baker's yeast enolase was ineffective to accelerate immunoglobulin production by HB4C5 cells, in spite of the same specific enzymatic activity as rabbit muscle enolase. There were differences in sensitivities against heat treatment and trypsin digestion between IPSF and enzymatic activities of enolase. These results imply that the immunoglobulin production stimulating effect of rabbit muscle enolase is irrelevant to its enzymatic function and reaction products. This fact also means that this enzyme has another function other than enzymatic one in glycolysis. Rabbit muscle enolase enhanced IgM production of transcription-suppressed HB4C5 cells treated with actinomycin D. Cycloheximide treatment of HB4C5 cells was useless to inhibit the expression of immunoglobulin production stimulating activity. However, inhibition of post-transcriptional process by monensin invalidated the activity of enolase. These findings suggest that enolase from rabbit muscle accelerates the steps between translation and post-translational processes to enhance immunoglobulin productivity. In addition, laser confocal microscopic analysis revealed that enolase from rabbit muscle was subsequently incorporated by HB4C5 cells. (C) 1998 Elsevier Science B.V.

DOI： 10.1016/S0304-4165(97)00137-2

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The polyamine spermine was assessed for enhancement of IgM production by human-human hybridoma, HB4C5 cells, under serum-free conditions. IgM production of HB4C5 cells was stimulated approximately 6-fold by the addition of 7.3 mM of spermine. The facilitating effect was observed soon after inoculation. In spite of suppression of cell growth, the IgM production rate was accelerated for at least 5 days without medium change. Moreover, laser confocal microscopic analysis revealed that the IgM content inside HB4C5 cells was increased by spermine treatment. These findings suggest that spermine enhances specific IgM productivity of the hybridoma line. Spermine also facilitated IgM production by human peripheral blood lymphocytes under serum-free conditions. This result implies that spermine enhances immunoglobulin production of not only specific hybridoma lines, but also nonspecific immunoglobulin producers. Immunoglobulin production stimulating activity of spermine was accelerated 2-fold by the addition of DNA whith a chain length of about 400-7000 base pairs (bp). However, degraded short-chain DNA fragments (less than 200 bp) did not facilitate the immunoglobulin production stimulating activity of spermine.

DOI： 10.1023/A:1007939213564

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Immunoglobulin production stimulating activity of alcohol dehydrogenase [EC 1.1.1.1] was assessed. Alcohol dehydrogenase-I (ADH-I) derived from horse liver stimulated IgM production by human-human hybridoma, HB4C5 cells producing human lung cancer specific monoclonal IgM. IgM production of HB4C5 cells was enhanced more than 6 ford by the addition of ADH-I at 400 mu g/ml under serum-free condition. However, yeast derived ADHs, such as ADH-II and -III were ineffective to accelerate immunoglobulin production of the hybridoma line. These results imply that the immunoglobulin production stimulating effect of ADH-I is irrelevant to its enzymatic function, and defined as a novel feature of ADH-I. This enzyme also stimulated IgM and IgG production by human peripheral blood lymphocytes 2.9 fold and 1.4 fold, respectively. This fact suggests that ADH-I stimulates immunoglobulin production not only by specific hybridoma cell line, but also by non-specific immunoglobulin producers.

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Lysozyme [EC 3.2.1.17] derived from hen egg white stimulated immunoglobulin production by human-human hybridoma, HB4C5 cells producing human lung cancer specific monoclonal IgM. IgM production by HB4C5 cells was enhanced more than 13-fold by the addition of lysozyme at 380 mu g/ml in a serum-free medium. The immunoglobulin production stimulating effect of lysozyme was observed immediately after inoculation and maintained for 5 days. Lysozyme enhanced immunoglobulin production by the hybridoma line without growth promotion. This enzyme also accelerated IgM and IgG production of human peripheral blood lymphocytes 5.3-fold and 2.3-fold, respectively. These results suggest that lysozyme stimulates immunoglobuling production of not only specific hybridoma line, but also non-specific immunoglobulin producers. However, although the enzymatic activity of lysozyme was almost lost by heat-treatment at 100 degrees C for 30 min, the IPSF activity was retained. This fact suggests that IPSF activity of lysozyme does not come from its enzymatic activity or reaction products. All these findings clearly indicate that lysozyme has a novel function as an immunoglobulin production stimulating factor.

DOI： 10.1023/A:1007936629501

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Language：English   Publishing type：Research paper (international conference proceedings)   Publisher：KLUWER ACADEMIC PUBL  

Basic proteins, such as lysozyme, alcohol dehydrogenase, aldolase, and enolase, enhanced immunoglobulin production of human-human hybridoma HB4C5 cells and human peripheral blood lymphocytes. These basic proteins stimulated immunoglobulin production of hybridomas and lymphocytes under serum-fi-ee conditions. IgM production of HB4C5 cells was accelerated by these basic proteins more than 6-fold. However, the growth of this hybridoma was not enhanced by basic proteins. The immunoglobulin production stimulating effects of these basic proteins were observed immediately after inoculation and the enhanced productivities were maintained for at least 5 days. IgM and IgG production of lymphocytes were also stimulated by these proteins without promoting growth. These results suggest that the four kinds of basic proteins examined here accelerate the immunoglobulin productivity of each cell.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC FERMENTATION BIOENGINEERING, JAPAN  

Rice fermented seasoning (RFS) enhanced IgM production by human peripheral blood lymphocytes (PBL) in vitro. The IgM production by the PBL cultured in serum-free medium supplemented with 3% RFS was enhanced about 3-fold. However, rice fermented extracts did not enhance IgG production by human PBL cultured under similar conditions. RFS showed no mitogenic activity toward the PBL in this study. These results indicate that RFS stimulates immunoglobulin productivity of each IgM-producing PBL.

DOI： 10.1016/0922-338X(96)89459-1

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Immunoglobulin production stimulating factor-II alpha, which enhances immunoglobulin production of human and mouse hybridomas was purified from cell lysate of human Burkitt's lymphoma, Namalwa cells, and identified as glyceraldehyde-3-phosphate dehydrogenase, The enhancement of immunoglobulin production with this enzyme was not linked with its enzymatic activity, The enzyme enhanced immunoglobulin productivity of transcription-suppressed hybridomas, but did not enhance that of translation-suppress hybridomas. From these results, it is suggested that this enzyme takes part in the post-translational control or the enhancement of translation activity to stimulate immunoglobulin production of hybridomas.

DOI： 10.1016/0014-5793(95)00607-B

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC POLYMER SCIENCE JAPAN  

The effects of sodium poly(styrene sulfonate) on an antibody production and cell growth of a hybridoma cell were studied. Although sodium poly(styrene sulfonate) inhibited the antibody production and cell growth at lower concentration with increasing molecular weight in serum-free cultures, sodium poly(styrene sulfonate)s with medium weight-average molecular weights (M(w)) such as 35000 and 18000 suppressed antibody production without inhibiting cell growth at appropriate concentrations. Sodium poly(styrene sulfonate) (M(w)=35000) decreased the extracellular concentration of a mu heavy chain forming an antibody, while it had no effect on an intracellular antibody or mu heavy chain concentration. This suggests that the sodium poly(styrene sulfonate) suppresses secreting the antibody assembled by mu heavy chains and lambda light chains out of the cell without inhibiting the synthesis and association of both chains in the cell.

DOI： 10.1295/polymj.27.719

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Human-human hybridoma cells secreting a human monoclonal antibody were cultured in a serum-free medium containing various organic pH buffers in order to clarify their effects on cell growth and antibody production. Organic pH buffers having either one sulfonic acid and several acyclic amine moieties, or several cyclic amine moieties containing two amino nitrogen did not inhibit cell growth; while other organic buffers sulfonic acid moiety plus several cyclic amine moieties containing one amino nitrogen slightly decreased cell growth, but enhanced antibody production. Using Fujita's organic conceptual diagram, a relationship between the organicity and inorganicity of a pH buffer to cell growth and antibody production was found. pH buffers with large inorganicity and small organicity values were favorable for cell growth, and buffers with small inorganicity and large organicity values were preferred to enhance antibody production. Although the pH buffering range affects cell growth, its effect on antibody production is not clear. In conclusion, 2-morpholinoethanesulfonic acid (MES), 3-morpholinopropanesulfonic acid (MOPS) and 1, 2-N, N'-bis[N '', N'''-di(2-sulfonoethyl)piperazinyl]ethane (Bis-PIPES) are shown to be the most optimal of the buffers tested, because they enhanced antibody production without decreasing the cell growth among the pH buffers tested here.

DOI： 10.1007/BF00749399

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Immunoglobulin M productivity of a human-human hybridoma line, HB4C5, was enhanced by the addition of histones H1, H2A, and H2B in serum-free medium, which are lysine-rich histones. On the contrary arginine-rich histones, histone H3 and H4, did not stimulate immunoglobulin production at all. Another arginine-rich protein, protamine also did not significantly stimulate immunoglobulin production. These results suggested that there is a correlation between amino acid residues of basic proteins and stimulation of immunoglobulin production. In this connection, immunoglobulin production stimulating activities of poly-lysine and poly-arginine were investigated. Both poly-L-lysine and poly-D-lysine stimulated IgM productivity of the human-human hybridomas. These results show that the enhancing effect of lysine residues was derived from its physiochemical features. On the other hand, poly-L-arginine did not enhance the productivity.

DOI： 10.1271/bbb.58.2212

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New bis-piperazine-type pH buffer agents were synthesized and their buffering properties were evaluated. The compounds proved to have two-fold larger pH buffering ability than 2-[4-(2-hydroxyethyl)-1 -piperazinyl]ethanesulfonic acid (HEPES), a Good's buffer traditionally used to control the pH value of culture media.
Human-human hybridoma HB4C5 cells were cultured in a serum-free medium containing these buffer agents. The cell growth and antibody production, using 1,2-N,N'-bis [N'',N'''-di(2-sulfonoethyl)piperazinyl]ethane, were greater than when HEPES.

DOI： 10.1271/bbb.58.1370

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Two immunoglobulin production stimulating factors (IPSF) have been found in human Burkitt's lymphoma Namalwa cells. One IPSF named IPSF-IIalpha was purified and identified as glyceraldehyde-3-phosphate dehydrogenase as previously reported. We report here purification, identification and characterization of IPSF-IIbeta. IPSF-IIBeta was purified by the serial use of ammonium sulfate fractionation, hydrophobic interaction column chromatography, anion-exchange column chromatography and gel filtration. The IPSF-IIbeta was estimated as a 46 KD monomeric polypeptide by gel filtration and SDS-PAGE. Partial amino acid sequence of the 46 KD protein was analyzed for 26 amino acid residues. The sequence very closely coincided with enolase (EC 4.2. 1.11) derived from various origins and, it was completely homologous with that of human enolase alpha-chain. Rabbit muscle enolase stimulated IgM production of hybridoma lines, and IPSF-IIbeta had the enzymic activity. These results suggested that IPSF-IIbeta was alpha-enolase or its isozyme. IPSF activities of IPSF-IIbeta was stable in alkaline conditions whereas the enzymic activity was rapidly lost in alkaline conditions. Though IPSF-IIbeta stimulated IgM production of both human-human and mouse-mouse hybridoma lines in serum-free condition, it partially suppressed IgE production of mouse-mouse hybridoma lines.

DOI： 10.1007/BF00570890

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Language：English   Publishing type：Research paper (international conference proceedings)   Publisher：KLUWER ACADEMIC PUBL  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JOHN WILEY & SONS INC  

Large-scale, high-density freezing of hybridomas was studied to apply frozen cells to start high-density culture. We showed here that hybridomas can be frozen at 1.5 x 10(8) cells/mL, without decrement in viability and proliferating activity. Blood transporting bags were used for large-scale freezing to store 25 mL of cell suspension with a cell density, 1.5 X 10(8) cells/mL. The number of cells stored in a bag (3.0 X 10(9) cells) was enough to start a high-density culture at a 10 times higher cell density (6.0 X 10(6) cells/mL) than normal inoculation, and the cells proliferated to 10(7) cells/mL within 2 days. These results indicate that the large-scale freezing method is useful for large-scale culture of mammalian cells.

DOI： 10.1002/bit.260380920

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：KLUWER ACADEMIC PUBL  

Amino acid sequence of the 36 KD protein which is the active subunit of immunoglobulin production stimulating factor-II-alpha (IPSF-II-alpha) derived from Burkitt's lymphoma Namalwa cells was analyzed for the 20 amino acids from N-terminus. The N-terminal amino acid sequence of this protein coincided very closely with glyceraldehyde-3-phosphate dehydrogenase (GPD; EC 1.2.1.12) derived from various origins. Especially, it was completely homologous with that of human liver GPD. Several GPD's derived from human erythrocyte, rabbit muscle and Bacillus stearothermophilus also stimulated IgM production of hybridomas, as well as IPSF-II-alpha. Conversely, IPSF-II-alpha had GPD enzymic activity as strong as rabbit muscle and B. stearothermophilus, and stronger than human erythrocyte GPD. These results suggested that 36 KD subunit of IPSF-II-alpha was a GPD, or GPD like protein. The level of mRNA for IgM was not enhanced by IPSF-II-alpha in hybridoma cells, though the IgM productivity of the cell was remarkably stimulated by the protein, indicating that IPSF-II-alpha does not stimulate immunoglobulin production by enhancement of transcription.

DOI： 10.1007/BF00373028

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An immunoglobulin production stimulating factor (IPSF) in human lymphoblastoid Namalwa cells was purified by the serial use of ammonium sulfate fractionation, hydrophobic interaction chromatography and gel filtration, and named IPSF-II alpha. IPSF-II alpha was estimated as a 112 KD protein composed of a 40 KD polypeptide and two 36 KD polypeptides. The 36 KD protein extracted from SDS-polyacrylamide gel showed IPSF activity, but not the 40 KD protein. The IPSF activity was reasonably stable in alkaline but unstable in acidic solution and heat-unstable. In a serum-free medium, IPSF-II alpha stimulated IgM production of human-human and mouse-mouse hybridomas 4-15 and 2-fold, respectively, although its growth stimulatory effect on hybridomas was negligible. The factor did not stimulate IgG production in either human or mouse hybridomas in the same serum-free medium. These results suggested that IPSF-II alpha was a new cellular factor for stimulating IgM productivity of hybridomas.

DOI： 10.1007/BF00556295

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Language：English   Publishing type：Research paper (international conference proceedings)   Publisher：BUTTERWORTH-HEINEMANN  

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DOI： 10.1002/bit.260360715

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPAN SOC BIOSCI BIOTECHN AGROCHEM  

DOI： 10.1271/bbb1961.53.2987

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC IN VITRO BIOLOGY  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Kluwer Academic Publishers  

Potassium or sodium phosphate was found to stimulate the production of human monoclonal antibody by human-human hybridoma HB4C5. The addition of 15 mM Na-phosphate (pH 7.4) into serum-free culture medium increased the antibody production up to 4-fold, when seeded at cell density of 1×105 cells/ml in dishes. At the higher cell density of 5×105 cells/ml, K-phosphate was more effective than Na-phosphate, at the same concentration. In large-scale continuous culture, the addition of 10 mM Na-phosphate into serum-free culture medium stimulated antibody production by HB4C5 cells 6-fold. © 1989 Kluwer Academic Publishers.

DOI： 10.1007/BF00365416

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DOI： 10.1007/BF02628461

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Authorship：Lead author   Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (trade magazine, newspaper, online media)  

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Authorship：Lead author   Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (trade magazine, newspaper, online media)  

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Authorship：Lead author   Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (trade magazine, newspaper, online media)  

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Authorship：Lead author   Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (trade magazine, newspaper, online media)  

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Authorship：Lead author   Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (trade magazine, newspaper, online media)  

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Authorship：Corresponding author   Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

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Authorship：Lead author,　Corresponding author   Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (trade magazine, newspaper, online media)  

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Authorship：Lead author   Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (trade magazine, newspaper, online media)  

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Authorship：Lead author   Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (trade magazine, newspaper, online media)  

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Authorship：Lead author   Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (trade magazine, newspaper, online media)  

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Authorship：Lead author   Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (other)  

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Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (other)  

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Authorship：Corresponding author   Language：English   Publishing type：Article, review, commentary, editorial, etc. (international conference proceedings)  

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Authorship：Corresponding author   Language：English   Publishing type：Article, review, commentary, editorial, etc. (international conference proceedings)  

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Authorship：Corresponding author   Language：English   Publishing type：Article, review, commentary, editorial, etc. (international conference proceedings)  

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Authorship：Corresponding author   Language：English   Publishing type：Article, review, commentary, editorial, etc. (international conference proceedings)  

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Authorship：Corresponding author   Language：English   Publishing type：Article, review, commentary, editorial, etc. (international conference proceedings)  

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Authorship：Corresponding author   Language：English   Publishing type：Article, review, commentary, editorial, etc. (international conference proceedings)  

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Authorship：Lead author   Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (trade magazine, newspaper, online media)  

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Authorship：Lead author   Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (trade magazine, newspaper, online media)  

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Authorship：Corresponding author   Language：English   Publishing type：Article, review, commentary, editorial, etc. (international conference proceedings)  

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