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Publications (5)10.25 Total impact

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    ABSTRACT: Alopecia is known as a symptom of acute radiation, yet little is known concerning the mechanism of this phenomenon and the alteration of hair protein profiles. To examine this, 6-week-old male C57/BL6 mice were exposed to 6 Gy of X-ray irradiation, which caused acute alopecia. Their hair and skin were collected, and hair proteins were analyzed with liquid chromatography/electrospray-ionization mass spectrometry and immunohistochemistry. No change was observed in the composition of major hair keratins, such as Krt81, Krt83 and Krt86. However, cytokeratin Krt15 and CD34, which are known as hair follicle stem cell markers, were decreased in alopecic mice. Cytokeratin Krt5, which is known as a marker for basal and undifferentiated keratinocytes, was increased in the epidermis of alopecic mice. These findings suggest that radiation damages hair stem cells and the differentiation of keratinocytes in the epidermis. For the evaluation of radiation exposure, chromosomal aberration is considered to be the gold standard, yet our results suggest that Krt5 may be a novel biological marker for acute radiation symptoms.
    International Journal of Molecular Medicine 06/2012; 30(3):579-84. · 1.96 Impact Factor
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    ABSTRACT: Fish produce mucus substances as a defensive outer barrier against environmental xenobiotics and predators. Recently, we found a bioactive protein in the mucus layer of the flounder Platichthys stellatus, which showed antibacterial activity against Staphylococcus epidermidis, Staphylococcus aureus and methicillin-resistant S. aureus. In this study, we isolated and identified the antibacterial protein from the mucus components of P. stellatus using a series of column chromatography steps. We then performed gel electrophoresis and cDNA cloning to characterize the protein. The antibacterial protein in the mucus had a molecular mass of approximately 52 kDa with an isoelectric point of 5.3, and cDNA sequencing showed that it corresponded completely with the peptide sequence of antibacterial protein from the gill. A BLAST search suggested that the cDNA encoded an antibacterial protein sharing identity with a number of L-amino acid oxidases (LAAOs) and possessing several conserved motifs found in flavoproteins. RT-PCR using a specific primer, and immunohistochemical analysis with anti-LAAO IgG, demonstrated tissue-specific expression and localization in the gill. Moreover, the anti-LAAO IgG was able to neutralize the antibacterial activity of the protein against methicillin-resistant S. aureus. Thus, we demonstrated that this antibacterial protein, identified from P. stellatus-derived epidermal mucus, is a novel LAAO-like protein with antibacterial activity, similar to snake LAAOs.
    FEBS Journal 12/2009; 277(2):453-65. · 4.25 Impact Factor
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    ABSTRACT: OHK cells, a human lymphoma cell line, are known to produce large amounts of hyaluronan. We investigated the effect of 4-methylumbelliferone, an inhibitor of hyaluronan synthesis, on the activity of matrix metalloproteinases in OHK cells. Matrix metalloproteinase-9 was detected on gelatin zymography as the main metalloproteinase excreted into the medium of cultured OHK cells, and 4-methylumbelliferone added to the medium decreased the activity of the enzyme in a dose-dependent manner. Addition of Streptomyces hyaluronidase to the medium during cultivation did not decrease the enzyme activity. Reverse transcription-polymerase chain reaction revealed that 4-methylumbelliferone markedly decreased the level of mRNA for matrix metalloproteinase-9 in cultured OHK cells. A similar decrease of the activity of matrix metalloproteinase-9 by 4-methylumbelliferone was also observed in cultured human breast and colon carcinoma cells. These results suggest that 4-methylumbelliferone suppresses the expression of matrix metalloproteinase-9 in cultured cancer cells.
    Cell Biology International 10/2007; 31(9):1022-6. · 1.64 Impact Factor
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    ABSTRACT: Human skin fibroblasts were cultured in the presence of 4-methylumbelliferone, an inhibitor of hyaluronan synthesis. Gelatinolytic activity excreted in the medium was examined by zymography and gelatinase assay using a fluorogenic substrate. 4-Methylumbelliferone added to the medium activated the latent form of matrix metalloproteinase-2 in a dose- and time-dependent manner. Immunoblot analysis also showed the conversion of the latent form of matrix metalloproteinase-2 to its active form. This activation was observed even when the cells were cultured with both 4-methylumbelliferone and hyaluronan. Addition of Streptomyces hyaluronidase to the medium during cultivation did not activate the latent form of matrix metalloproteinase-2. Reverse transcription-polymerase chain reaction revealed that 4-methylumbelliferone markedly increased the level of mRNA for membrane type 1-matrix metalloproteinase, whereas levels of mRNA for matrix metalloproteinase-2 and tissue inhibitor of metalloproteinase-2 were little affected. These results suggest that 4-methylumbelliferone induces the expression of membrane type 1-matrix metalloproteinase, resulting in activation of matrix metalloproteinase-2, in cultured human skin fibroblasts.
    Biochemical and Biophysical Research Communications 12/2002; 298(5):646-50. · 2.41 Impact Factor
  • Toshiya Nakamura, Asami Sakamoto, Takashi Ishikawa
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    ABSTRACT: Background: Hyaluronan plays an important role in embryonal development, inflammation, wound healing, angiogenesis, and tumorigenesis. Although the activation or inhibition of matrix metalloproteinases (MMPs) has also been observed in such conditions, the relationship between hyaluronan and MMP activity is not fully investigated. Therefore, the effects of hyaluronan on MMP activity were examined in human skin fibroblasts. Methods: Quiescent subconfluent cells were cultured for 2 days in the presence of hyaluronan of differing molecular weights. The conditioned media were treated with Streptomyces hyaluronidase. The fractions precipitated with 80% saturation of ammonium sulfate were collected from the media and used as enzyme preparations. MMP activity was examined using gelatin or casein zymography. Results: Gelatin zymography showed an increase of MMP-2 (gelatinase A) activity, especially by higher-molecular weight hyaluronan preparations (800 and 1200 k). MMP activity appearing as a 47-kDa band was also increased in the same way. In contrast, casein zymography showed a decrease of MMP-3 (stromelysin-1) activity in the presence of hyaluronan, regardless of its molecular weight. Conclusions: These results suggest that hyaluronan has some effect on MMP activity and may be linked to proteolytic tissue remodeling in various physiological and pathological conditions.
    International Congress Series 01/2001; 1223:259-263.