Kenichi Yamaguchi

Nagasaki University, Nagasaki, Nagasaki, Japan

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Publications (61)124.4 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: Alginate is an acidic linear polysaccharide with immune-modulating activities. In this study, we found that enzymatically digested alginate oligomer (AO) with various degrees of polymerization (DP; 2-5) induced a higher level of nitric oxide (NO) production in RAW264.7 cells than undigested alginate polymer (AP). Reverse transcription-polymerase chain reaction and western blot analyses revealed that the expression level of inducible NO synthase in AO-treated RAW264.7 cells was higher than that in AP-treated cells. AO induced nuclear translocation of nuclear factor (NF)-κB p65 subunit in RAW264.7 cells to a greater extent than AP. Although AO and AP induced similar extents of phosphorylation in three mitogen-activated protein (MAP) kinases, c-Jun N-terminal kinase inhibitor exhibited the most potent inhibitory effect on NO induction in AO- and AP-treated RAW264.7 cells, among three MAP kinase inhibitors that were tested.
    Bioscience Biotechnology and Biochemistry 06/2015; DOI:10.1080/09168451.2015.1052768
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    ABSTRACT: We previously found that ascophyllan, a sulfated polysaccharide isolated from brown seaweed Ascophyllum nodosum, exhibited antitumor activity in sarcoma-180 tumor-bearing mice. In this study, we found that ascophyllan inhibited the migration and adhesion of B16 melanoma cells by reducing the expression of N-cadherin and enhancing the expression of E-cadherin in a concentration-dependent manner. Transwell invasion assay revealed that ascophyllan suppressed the invasion ability of B16 cells. It also inhibited the expression of matrix metalloprotease-9 (MMP-9) mRNA and the secretion of MMP-9 protein in B16 cells, a process that may involve the extracellular signal-regulated kinase (ERK) signaling pathway. Furthermore, ascophyllan administered intraperitoneally at 25 mg/kg showed anti-metastatic activity in a mouse model of metastasis induced by intravenous injection of B16 cells, and the number of lung surface metastatic nodules in ascophyllan-treated mice was significantly reduced compared to that in the untreated control mice. Since splenic natural killer cell activity enhanced in the mice injected with ascophyllan intraperitoneally, we suggest that ascophyllan may exhibit in vivo anti-metastatic activity on B16 melanoma cells through activation of the host immune system in addition to a direct action on cancer cells. Copyright © 2015. Published by Elsevier Inc.
    Biochemical and Biophysical Research Communications 01/2015; 458(4). DOI:10.1016/j.bbrc.2015.01.061
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    ABSTRACT: In the present study, the chemical structure and chemoprotective activity of Pyropia yezoensis protein (PYP) were investigated using sodium dodecyl sulfate (SDS)‑polyacrylamide gel electrophoresis, automated protein sequencing, matrix‑assisted laser desorption/ionization‑quadrupole ion trap‑time‑of‑flight mass spectrometry and a chemoprotective assay using a synthetic peptide. The PYP fraction was demonstrated to contain two proteins: PYP1 (10 kDa, SDS‑resistant dimer) and PYP2 (10 kDa). PYP1 is a novel protein showing sequence homology with the hypothetical function‑unknown proteins of Chondrus crispus (Rhodophyta) and Emiliania huxleyi (Haptophyceae). PYP2 is a paralog of an extrinsic protein of photosystem II found in other Rhodophyta. The synthetic peptide PYP1 (1‑20), corresponding to the N‑terminal 20 residues of PYP1 (ALEGGKSSGGGEATRDPEPT), exhibits chemoprotective activity against acetaminophen‑induced cell death in Chang liver cells, indicating that PYP1 is a chemoprotectant of the PYP fraction. A possible association between the structure of PYP and its chemoprotective activity is discussed.
    International Journal of Molecular Medicine 11/2014; 35(1). DOI:10.3892/ijmm.2014.1992
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    ABSTRACT: Polysaccharides prepared from marine algae sometimes contain contaminants such as polyphenols and endotoxins that may mislead their bona fide biological activities. In this study, we examined bioactive contaminants in commercially available fucoindan from Fucus vesiculosus, along with ascophyllan and fucoidan from Ascophyllum nodosum. E vesiculosus fucoidan inhibited the growth of Vibrio alginolyticus in a concentration-dependent manner (0-1,000 mu g mL(-1)). However, the antibacterial activity of the fucoidan significantly reduced after methanol-extraction, and the methanol-extract showed a potent antibacterial activity The extract also showed cytotoxicity to RAW264.7 and 15937 cells, and induced apoptotic nuclear morphological changes in U937 cells. These results suggest that the antibacterial activity of the fucoidan is partly due to the methanol-extractable contaminants that can also contribute to the cytotoxicity on RAW264.7 and U937 cells. On the other hand, the activities to induce secretion of nitric oxide and tumor necrosis factor-a from RAW264.7 cells were observed in the fucoidan even after methanol extraction, and the extract had no such activities. Our observations suggest that commercially available fucoidan should be purified prior to biochemical use.
    09/2014; 29(3):237-247. DOI:10.4490/algae.2014.29.3.237
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    ABSTRACT: We evaluated the antitumor activity of crude extract and ascophyllan prepared from Ascophyllum nodosum in sarcoma-180 solid tumor-bearing mice with continuous intraperitoneal (i.p.) administration at a dose of 50 mg/kg body weight/day or oral administration at a dose of 500 mg/kg body weight/day. Ascophyllan and crude extract administered via the oral route showed greater antitumor effects than via i.p. route, and the tumor sizes in mice treated with ascopyllan and crude extract were reduced by a mean of 68.7±6.8% and 42.4±24.8% by the oral route, and 41.4±16.1% and 13.6±20.6% by i.p. route compared to control mice. Splenic natural killer cell activity in the mice treated with ascophyllan and crude extract by i.p. route was significantly enhanced, while only a slight increase of this activity was observed in orally-treated mice. Furthermore, increase in spleen weight of tumor-bearing mice was slightly suppressed by oral administration of ascophyllan, whereas i.p. administration resulted in further enlargement. Analysis of serum cytokines revealed that oral treatment with ascophyllan resulted in significant increase of tumor necrosis factor-α and interleukin-12 levels. Since ascophyllan showed no direct cytotoxic effect on sarcoma-180 cells, orally-administered ascophyllan is suggested to exhibit its antitumor activity through the activation of the host immune system.
    Anticancer research 04/2014; 34(4):1663-71.
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    Tsukasa Kuwabara, Kenichi Yamaguchi
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    ABSTRACT: http://archive.org/details/interactionism20130517
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    ABSTRACT: Hyoscyamus albus is a well-known source of the tropane alkaloids, hyoscyamine and scopolamine, which are biosynthesized in the roots. To assess the major biochemical adaptations that occur in the roots of this plant in response to iron deficiency, we used a small-scale proteomic approach in which 100 mg of root tips were treated with and without Fe, respectively, for 5 days. Two-dimensional mini gels showed that 48 spots were differentially accumulated between the two conditions of Fe availability and a further 36 proteins were identified from these spots using MALDI-QIT-TOF mass spectrometry. The proteins that showed elevated levels in the roots lacking Fe were found to be associated variously with carbohydrate metabolism, cell differentiation, secondary metabolism, and oxidative defense. Most of the proteins involved in carbohydrate metabolism were increased in abundance, but mitochondrial NAD-dependent malate dehydrogenase was decreased, possibly resulting in malate secretion. Otherwise, all the proteins showing diminished levels in the roots were identified as either Fe-containing or ATP-requiring. For example, a significant decrease was observed in the levels of hyoscyamine 6β-hydroxylase (H6H), which requires Fe and is involved in the conversion of hyoscyamine to scopolamine. To investigate the effects of Fe deficiency on alkaloid biosynthesis, gene expression studies were undertaken both for H6H and for another Fe-dependent protein, Cyp80F1, which is involved in the final stage of hyoscyamine biosynthesis. In addition, tropane alkaloid contents were determined. Reduced gene expression was observed in the case of both of these proteins and was accompanied by a decrease in the content of both hyoscyamine and scopolamine. Finally, we have discussed energetic and Fe-conservation strategies that might be adopted by the roots of H. albus to maintain iron homeostasis under Fe-limiting conditions.
    Frontiers in Plant Science 08/2013; 4:331. DOI:10.3389/fpls.2013.00331
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    ABSTRACT: To investigate the role of sulfate groups on the macrophage-stimulating activities of ascophyllan, we prepared desulfated ascophyllan, and its effects on RAW264.7 cells were compared with native ascophyllan. The chemical structural analysis revealed that nearly 21% of sulfate groups of ascophyllan were removed by desulfation reaction, while no significant changes in the molecular mass and monosaccharide composition occurred after desulfation. NO- and cytokine- (TNF-α and G-CSF) inducing activities of the desulfated ascophyllan on RAW264.7 cells were significantly decreased as compared to native ascophyllan. Furthermore, the activity of desulfated ascophyllan to induce reactive oxygen species (ROS) generation from RAW264.7 cells decreased to almost negligible level. Our results suggest that the level of sulfate groups of ascophyllan is an important structural element responsible for the macrophage-stimulating activities. Probably, even the limited removal of sulfate residues sensitive to desulfation reaction may result in significant decrease in the bioactivities of ascophyllan.
    Carbohydrate research 06/2013; 380C:124-129. DOI:10.1016/j.carres.2013.05.018
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    ABSTRACT: Antioxidant activities of sulfated polysaccharide ascophyllan from Ascophyllum nodosum was investigated in vitro by various assays, and compared with those of fucoidan. A chemiluminescence (CL) analysis using a luminol analog, L-012, showed that ascophyllan scavenges superoxide, and the activity is greater than fucoidan. However, in the presence of 10 μg/ml of ascophyllan or 10 μg/ml and 100 μg/ml of fucoidan, slightly enhanced CL-responses were observed. Since EDTA-treatment resulted in disappearance of the enhancement effects, it was suggested that metal ions especially iron ions in the polysaccharides might be involved in this phenomenon. In fact, metal element analysis revealed that ascophyllan and fucoidan inherently contain iron and other metal elements. EDTA-treatment resulted in significant increase in Fe2+-chelating activities of these polysaccharides. In an electron spin resonance (ESR)-spin trapping analysis in which direct UV-radiation to hydrogen peroxide was used as a source of hydroxyl radical, ascophyllan and fucoidan showed potent hydroxyl radical scavenging activity with similar extent. Reducing power of ascophyllan was stronger than that of fucoidan. Our results indicate that ascophyllan can exhibit direct and potent antioxidant activity.
    International journal of biological macromolecules 05/2013; 59. DOI:10.1016/j.ijbiomac.2013.04.035
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    ABSTRACT: Lactate dehydrogenase (LDH)-release assay was applied to estimate the toxic potential of harmful algal species at the cellular level. African green monkey kidney (Vero), yellowtail fin epithelia (MJF), and rainbow trout gill (RTgill-W1) cells were used as target cells. A live cell suspension of Karenia mikimotoi (SUO-1) induced the release of LDH from these cell lines, while the activity of another strain, FUK, was much lower. The cell-free culture supernatants and ruptured cell suspensions of both strains of K. mikimotoi were less effective on LDH-release assay. Exposure experiments against abalone and shrimp revealed that SUO-1 showed much stronger lethal effects on these organisms than FUK. Among six phytoplankton species, three species known to be harmful algal species induced the release of LDH to different extents depending on the cell line, whereas the other three species, known to be non-toxic, showed no effects on any cell lines. These results suggest that LDH-release assay is a useful micro-plate assay for estimation of the toxic potential of harmful phytoplankton.
    Bioscience Biotechnology and Biochemistry 02/2013;
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    ABSTRACT: Four genes of Takifugu rubripes, tentatively designated Tr1-Tr4, encoding homologs of pufferfish saxitoxin- and tetrodotoxin-binding protein, were identified by BLAST search and 3'-RACE. RT-PCR and MALDI-TOF mass spectrometry allowed the identification and discrimination of Tr isoforms from the non-toxically cultured specimens. The expression of Tr1 and Tr3 mRNAs exclusively in the liver and the presence of their products as 120-kDa plasma proteins were confirmed.
    Bioscience Biotechnology and Biochemistry 01/2013; 77(1):208-12. DOI:10.1271/bbb.120701
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    ABSTRACT: In this study, we investigated the bioactivity of ascophyllan in terms of reactive oxygen species (ROS) generation. In RAW264.7 cells, we found that ascophyllan induced ROS generation in a concentration-dependent manner, but with bell-shaped profile. Immunoblot analysis demonstrated that ascophyllan promoted the translocation of cytosolic subunits (p67phox and p47phox) of NADPH oxidase to the plasma membrane. Among mitogen activated protein (MAP) kinase inhibitors tested, JNK inhibitor showed the most potent inhibitory effect on ascophyllan-induced ROS production. Consistently, significant level of phosphorylated JNK MAP kinase was detected in ascophyllan-treated RAW264.7 cells. Our findings suggest for the first time that ascophyllan can stimulate macrophages to produce ROS through the activations of NADPH oxidase and JNK MAP kinase.
    International journal of biological macromolecules 09/2012; 52:164–169. DOI:10.1016/j.ijbiomac.2012.09.008
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    ABSTRACT: Splenic natural killer (NK) cell activity against YAC-1 cells increased in mice intraperitoneally injected with ascophyllan. Ascophyllan enhanced the cytotoxicity of RAW264.7 cells toward YAC-1 cells in a concentration-dependent manner. The cytotoxicity of ascophyllan-stimulated RAW264.7 cells as to YAC-1 cells was suppressed with N(G)-nitro-L-arginine methyl ester hydrochloride (L-NAME), an inhibitor of nitric oxide (NO) synthase, suggesting the involvement of NO in the cytotoxicity of ascophyllan-stimulated RAW264.7 cells.
    Bioscience Biotechnology and Biochemistry 08/2012; 76(8):1573-6. DOI:10.1271/bbb.120232
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    ABSTRACT: A time-course analysis of reactive oxygen species (ROS) generation in fertilized eggs of the devil stinger (Inimicus japonicus) from 0 h post-fertilization (hpf) to the early larval stage indicated that the ROS level was highest in the 22 hpf embryo, and declined thereafter. Phorbol myristate acetate (PMA) had no effect on ROS generation by the 22 hpf embryo, whereas PMA significantly increased larval ROS generation, suggesting that the ROS generation mechanisms of the 22 hpf embryo and larva are different at least in terms of PMA-responsiveness. Our results suggest the presence of a specific ROS generation system in devil stinger embryo which can be transitionally activated during embryogenesis.
    Bioscience Biotechnology and Biochemistry 08/2012; 76(8):1561-4. DOI:10.1271/bbb.120184
  • Toxicon 08/2012; 60(2):153. DOI:10.1016/j.toxicon.2012.04.116
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    ABSTRACT: The antioxidant and macrophage-stimulating activities of polyguluronic acid (PG) and polymannuronic acid (PM) prepared from alginate were examined. A chemiluminescence (CL) method using a luminol analog, L-012, showed that both PM and PG scavenge superoxide produced by hypoxanthine-xanthine oxidase system in a concentration-dependent manner. At 100 μg/ml, PG showed slightly stronger superoxide scavenging activity than PM. In an electron spin resonance (ESR)-spin trapping method in which the Fenton reaction was used as hydroxyl radical generation system, we found that both PM and PG showed potent hydroxyl radical scavenging activity to a similar extent. Because PM and PG showed no chelating activity on Fe(2+), it was confirmed that PM and PG can directly scavenge hydroxyl radical. No significant scavenging activity of PM and PG toward hydrogen peroxide was observed. Interestingly, the macrophage-stimulation activity of PG as measured by nitric oxide (NO)-production from mouse macrophage cell line RAW264.7 cells was evidently stronger than that of PM. Our results suggest that RAW264.7 cells might be able to distinguish the conformational differences between PM and PG, and respond differently to them, whereas the effects of such structural differences between PM and PG on the radical scavenging activities may not be so significant.
    Carbohydrate research 02/2012; 352:88-93. DOI:10.1016/j.carres.2012.02.005
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    ABSTRACT: Alginate is a natural acidic linear polysaccharide that is produced by brown seaweeds. It is currently used in a broad range of commercial enterprises, such as the food and medical products industries. Recent evidence has demonstrated that alginate oligosaccharides may function as growth promoting agents for certain plant cells, including those of some green algae. Chlamydomonas reinhardtii is a green alga that is used as a model organism in fundamental molecular biology studies; it is also a producer of biohydrogen. In the present study, we examined effects of two types of alginate oligosaccharide mixtures (AOMs), which were prepared by either enzymatic degradation (ED) or acid hydrolysis (AH), on the growth of C. reinhardtii. Growth was significantly promoted by AOM (ED) in a concentration-dependent manner. The maximum effect was observed on day 4 of treatment. The fatty acid composition of C. reinhardtii was also influenced by AOM (ED); the levels of C16:0, C18:2 cis and C18:3 n-3 increased in treated cells. AOM (AH) and the other saccharides that we tested did not affect the growth of C. reinhardtii. The effects that we identified could promote efficient biomass production by reducing culture times and by changing cellular fatty acid levels.
    Journal of Bioscience and Bioengineering 01/2012; 113(1):112-6. DOI:10.1016/j.jbiosc.2011.09.009
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    Zedong Jiang, Yoichiro Hama, Kenichi Yamaguchi, Tatsuya Oda
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    ABSTRACT: Porphyran, extracted from an edible red alga (Porphyra yezoensis), is a sulphated polysaccharide with a wide variety of biological activities including anti-tumour, antioxidant and immuno-modulating activities. In this study, we examined the effect of porphyran on nitric oxide (NO) production in mouse macrophage cell line RAW264.7 cells. Although no significant activity of porphyran to induce NO or tumour necrosis factor-α (TNF-α) production in RAW264.7 cells was observed at the concentration range tested (10-500 µg/ml), it was found for the first time that porphyran inhibited NO production and expression of inducible nitric oxide synthase (iNOS) in RAW264.7 cells stimulated with lipopolysaccharide (LPS). In the presence of 500 µg/ml porphyran, NO production and expression of iNOS in LPS-treated RAW264.7 cells were completely suppressed. On the other hand, porphyran showed only a marginal effect on the secretion of TNF-α from LPS-stimulated RAW264.7 cells. Electrophoretic mobility shift assay (EMSA) using infrared dye labelled oligonucleotide with nuclear factor-κB (NF-κB) consensus sequence suggested that porphyran inhibited the LPS-induced NF-κB activation. The LPS-inducible nuclear translocation of p65, and the phosphorylation and degradation of IκB-α were also inhibited by the pre-treatment with porphyran. Our results obtained in in vitro analysis suggest that porphyran suppresses NO production in LPS-stimulated macrophages by the blocking of NF-κB activation.
    Journal of Biochemistry 01/2012; 151(1):65-74. DOI:10.1093/jb/mvr115
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    Zedong Jiang, Takasi Okimura, Kenichi Yamaguchi, Tatsuya Oda
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    ABSTRACT: Ascophyllan isolated from the brown alga Ascophyllum nodosum is a fucose-containing sulfated polysaccharide, which has similar but distinct characteristic monosaccharide composition and entire chemical structure to fucoidan. In this study, we examined the effects of ascophyllan, fucoidan isolated from A. nodosum (A-fucoidan), and fucoidan from Sigma (S-fucoidan) as a representative fucoidan derived from other source (Fucus vesiculosus) on mouse macrophage cell line RAW264.7 cells. No significant cytotoxic effects of ascophyllan and A-fucoidan on RAW264.7 cells were observed up to 1000μg/ml, while S-fucoidan showed cytotoxic effect in a concentration-dependent manner. Ascophyllan induced extremely higher level of nitric oxide (NO) production from RAW264.7 cells than those induced by fucoidans over the concentration range tested (0-200μg/ml). Reverse transcription polymerase chain reaction (RT-PCR) and western blot analysis revealed that expression level of inducible NO synthase (iNOS) in ascophyllan-treated RAW264.7 cells was much higher than the levels detected in the cells treated with fucoidans. Furthermore, the activities of ascophyllan to induce the secretion of tumor necrosis factor-α (TNF-α) and granulocyte colony-stimulating factor (G-CSF) from RAW264.7 cells were also greater than those induced by fucoidans especially at lower concentration range (3.1-50μg/ml). The activities of ascophyllan to induce NO and cytokine production in mouse peritoneal macrophages were also stronger than those of fucoidans. Electrophoretic mobility shift assay (EMSA) using infrared dye labeled nuclear factor-kappa B (NF-κB) and AP-1 consensus sequences suggested that ascophyllan can strongly activate these transcription factors. Marked increase in the nuclear translocation of p65, and the phosphorylation and degradation of IκB-α were also observed in ascophyllan-treated RAW264.7 cells. Analysis using mitogen-activated protein (MAP) kinase inhibitors and western blot analysis suggested that c-Jun N-terminal kinase (JNK) and p38 MAP kinase are mainly involved in ascophyllan-induced NO production.
    Nitric Oxide 11/2011; 25(4):407-15. DOI:10.1016/j.niox.2011.10.001
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    ABSTRACT: Heterocapsa circularisquama showed much higher toxic effects on short-necked clams than Chattonella marina. Clams exposed to H. circularisquama exhibited morphological changes concomitant with an accumulation of mucus-like substances in the gills, a profound reduction in filtration activity, and lysosomal destabilization in hemocytes. Chattonella marina was less effective than H. circularisquama, and Heterocapsa triquetra was almost harmless in all these criteria. These results suggest that H. circularisquama exerted its lethal effect on short-necked clams through gill tissue damage and subsequent induction of physiological stress.
    Bioscience Biotechnology and Biochemistry 10/2011; 75(10):2052-5. DOI:10.1271/bbb.110382

Publication Stats

613 Citations
124.40 Total Impact Points

Institutions

  • 2003–2015
    • Nagasaki University
      • • Graduate School of Fisheries Science and Environmental Studies
      • • Department of Science and Technology
      • • Faculty of Fisheries
      • • Department of Biochemistry
      Nagasaki, Nagasaki, Japan
  • 2011
    • Korea Basic Science Institute KBSI
      • Jeju Center
      Sŏul, Seoul, South Korea
  • 2007
    • National Research Institute of Fisheries and Environment of Inland Sea
      Hirosima, Hiroshima, Japan
  • 2004
    • Kyushu University
      Hukuoka, Fukuoka, Japan