Yoshitaka Horiuchi

Tokushima Bunri University, Tokusima, Tokushima, Japan

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Publications (9)17.63 Total impact

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    ABSTRACT: A bactericidal mechanism mediated by human serum was investigated by a field emission scanning electron microscope and a strain of drug-resistant Pseudomonas aeruginosa. When the bacteria were treated with meropenem, a carbapenem antibiotic, spheroplasts and bulges (spheroidization) appeared after 1-3 h. When 40% serum was added to the bacteria, the bacteria agglutinated within 2 min and then lysed after 5-30 min. Immunoelectron micrographic analyses showed dispositions of complement component C9 molecules on the cell surface of lysed bacteria by the serum treatment that might suggest formation of a membrane attack complex. Immunoglobulin G (IgG) depletion from the serum diminished the lytic activity and adding human intravenous immunoglobulin (IVIG) restored it, suggesting that lysis was induced by specific IgG binding to the bacteria. IVIG may help patients with less IgG against bacteria to overcome severe infection.
    Journal of Infection and Chemotherapy 12/2010; 16(6):383-7. · 1.55 Impact Factor
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    ABSTRACT: This ultrastructural study showed that nonisotopic methanolic hafnium chloride and aqueous lead solution was an excellent new electron stain for enhancing TEM contrasts of fungal and plant cell structures. The ultrastructural definition provided by the new stain was often superior to that provided by conventional staining with uranyl acetate and lead. Definition of fine ultrastructure was also supported by quantitative data on TEM contrast ratios of organelles and components in fungal and plant cells. In particular, polysaccharides, which were localized in cell walls, glycogen particles, starch grains, and plant Golgi vesicle components, were much more reactive to the new stain than to the conventional one. The new nonisotopic stain is useful for enhancing the contrast of ultrastructure in biological tissues and is a safer alternative to uranyl acetate. Microsc. Res. Tech., 2010. © 2010 Wiley-Liss, Inc.
    Microscopy Research and Technique 12/2010; · 1.59 Impact Factor
  • Masae Takahashi, Yoshitaka Horiuchi, Tadashi Tezuka
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    ABSTRACT: Hematoxylin-stainability of keratohyalin granules (KHG) using biochemical and immunohistochemical techniques is due to the presence of a fibrinogen γ-chain protein. A protein with a molecular weight of 100 kDa was stained with anti-Ted-H-1 monoclonal antibody and hematoxylin solution (hematoxylin-stainable protein). Since the amino acid sequence of the hematoxylin-stainable protein was to that of fibrinogen γ-chain protein, a peptide was synthesized and an antibody against the peptide was produced. This antibody reacted with the hematoxylin-stainable protein and fibrinogen γ-chain protein on immunoblot analysis and with KHG on immunohistochemical examination. Furthermore, a commercial anti-fibrinogen γ-chain protein antibody (Ab) also reacted with the hematoxylin-stainable protein as well as fibrinogen. In contrast, anti-fibrinogen β-chain protein Ab did not react with the hematoxylin-stainable protein. The fibrinogen γ-chain protein also stained with hematoxylin. These findings suggested that fibrinogen γ-chain protein may be a novel component protein of KHG and may induce the hematoxylin-stainability of KHG.
    Archives for Dermatological Research 11/2010; 302(9):679-84. · 2.71 Impact Factor
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    ABSTRACT: Erythropoietin supports the survival of erythroblasts. We previously demonstrated that 24 malignant human cell lines expressed erythropoietin and its receptor and that erythropoietin secretion was enhanced under anoxia. In this study, we examined the viability of 22 of these cell lines excluding two leukemia cell lines under anoxia. Twenty-two cancer cell lines of various origins were cultured under anoxia or normoxia for 4 days, and their viability was examined at 1-day intervals. The levels of lactate and ATP were measured. The expressions of hypoxia-inducible transcription factor 1alpha (HIF-1alpha) and Bcl-2 family proteins were examined by western blotting analysis. The cellular and mitochondrial features were examined by microscopy. Eleven of the 22 cancer cell lines examined showed 80% to 100% cell viability after 4 days under anoxia; 2 cell lines showed similar viability for 3 days, 3 cell lines showed similar viability for 2 days, and 6 cell lines showed similar viability for 1 day or less. These 11 death-resistant cell lines, which secrete various amounts of erythropoietin under anoxia, produced significantly more lactate during 2 days under anoxia than under normoxia, with ATP levels about 60% of those before anoxia. ATP returned to the normal level when normoxia was restored after 4 days of anoxia. However, the nonresistant cell lines responded to anoxia by yielding significantly more lactate without a reduction of the ATP level. The expression patterns of Bcl-2 family proteins revealed that apoptosis-inhibiting signals predominated over proapoptotic signals in the death-resistant cells under anoxia. The majority of the cancer cell lines examined survived under anoxia in vitro, through the Pasteur effect, in a dormant state without direct support of erythropoietin.
    International Journal of Clinical Oncology 01/2008; 12(6):455-62. · 1.73 Impact Factor
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    ABSTRACT: We obtained an antibody, anti-inner root sheath cells antibody (anti-IRSC Ab), that reacted with the inner root sheath (IRS) cells especially trichohyalin granules (THG). In order to compare the properties of anti-IRSC Ab and AE15, which is a specific monoclonal antibody against THG, histochemical and biochemical examinations were performed. In vivo localization with anti-IRSC Ab and AE15 indicated that both antibodies reacted with THG, but anti-IRSC Ab reacted with THG in the suprabulbar region of the Huxley layer, whereas AE15 reacted with THG in the suprabulbar region and upper bulbar portion of the Huxley layer, as shown by immunohistochemical and immunoelectron microscopic analyses. The results of immunoblot analysis showed that anti-IRSC Ab reacted with a protein spot at 45 kDa, pI 6.5, but AE15 reacted with high molecular weight proteins at pI 5.5. Furthermore, anti-IRSC Ab reacted with specimens of squamous cell carcinoma (SCC) but did not react with those of basal cell carcinoma (BCC). In contrast, AE15 reacted with neither SCC nor BCC. These findings suggest that anti-IRSC Ab and AE 15 recognized different component proteins in THG, and therefore indicated that THG, like as keratohyalin granules, might consist of several proteins. It is the novel finding that the anti-IRSC Ab positive substance in THG in the normal hair and SCC cells.
    Archives for Dermatological Research 05/2007; 299(1):33-9. · 2.71 Impact Factor
  • M Takahashi, Y Horiuchi, T Tezuka
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    ABSTRACT: Our previous study showed that large keratohyaline granules (KHG) in molluscum contagiosum that stained with haematoxylin also reacted with anti-Ted-H-1 monoclonal antibody (mAb), but not with antifilaggrin mAb or antiloricrin polyclonal antibody (pAb). This finding indicated that the Ted-H-1 antigenic protein is a haematoxylin-stainable protein in KHG. To clarify the identity of the major component protein of the large KHG in solar keratosis, another disorder in which large KHG are observed. An enzyme immunohistochemical study was performed using antifilaggrin mAb, anti-Ted-H-1 mAb and antiloricrin pAb. Immunofluorescent double staining and immunoelectron microscopic analyses were performed using anti-Ted-H-1 mAb and antiloricrin pAb. Antifilaggrin mAb, anti-Ted-H-1 mAb and antiloricrin pAb reacted with normal KHG in nonlesional skin of solar keratosis, while only anti-Ted-H-1 mAb reacted with the large KHG in the lesions of solar keratosis. Antifilaggrin mAb did not react with large KHG. Antiloricrin pAb reacted with the cell membrane of the stratum granulosum, but not with large KHG. These findings suggest that the haematoxylin-stainable protein in the large KHG would be a Ted-H-1 antigen protein which was neither filaggrin nor loricrin.
    British Journal of Dermatology 12/2005; 153(5):976-80. · 3.76 Impact Factor
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    ABSTRACT: Heretofore, epithelial cells have been considered to be the only source of keratin (K) polypeptides that assemble into 10-nm filaments to form an extensive cytoplasmic network in conjunction with nuclear and cytoplasmic membranes. However, K was recently found to be expressed also in cultured non-epithelial normal and tumor cells: melanocyte, fibroblast, endothelial cell, malignant melanoma, fibrosarcoma, and angiosarcoma. Nevertheless, electron microscopy was incapable of detecting the K filaments (Katagata et al., J Dermatol Sci, 30, 1-9, 2002, see ref. 11). That is, K may present as subunits in each of the cultured cells named above, not as a filament formation. We used squamous cell carcinoma observed with immunoelectron microscopy, a more precise and conclusive technique, to further confirm whether or not K filament is formed in those cultured cells. HaCaT, an immortalizd keratinocyte cell line used as a positive control, yielded elegant immunoelectron microscopic images. Considerable K filament formations existed in malignant melanoma using anti-K or anti-vimentin antibodies, as revealed by the presence of linear immune gold particles on high electron density substances. In the case of squamous cell carcinoma, the gold particles were fewer than those of malignant melanoma. By contrast, no K filaments were detected in the other non-epithelial normal and tumor cell lines: fibroblast, endothelial cell, fibrosarcoma and angiosarcoma. These results suggest that the formation of K filaments in malignant melanoma (and slight presence in squamous cell carcinoma) is a particular and cell-dependent characterization. Key words: tumor cells, keratins, filament formation, immunoelectron microscopy
    01/2005;
  • Masae Takahashi, Yoshitaka Horiuchi, Tadashi Tezuka
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    ABSTRACT: To examine the presence of bactericidal/permeability-increasing protein (BPI) in skin, which is an antibacterial protein, has cytotoxicity toward Gram-negative bacteria, and may have an important role against bacterial infection in the skin, immunohistochemical and biochemical analyses were performed. Anti-BPI/KLH Ab reacted with the cytoplasm of the inner root sheath cells of both human and rat hair follicles by immunohistochemical examination. A protein band in 10-M alkaline urea extracts of human scalp skin or 7-day-old rat skin reacted with an antibody against BPI conjugated with KLH (anti-BPI/KLH Ab). Purified skin BPI (sBPI) from rat was a single protein spot and reacted with both anti-BPI/KLH Ab and a commercially available monoclonal antibody against BPI (anti-BPI MoAb). Moreover, sBPI possessed inhibitory activity against LPS. Bactericidal/permeability-increasing protein mRNA was expressed not only in leukocytes but also in human scalp skin and cultured keratinocytes. These findings suggest that sBPI could exist in the inner root sheath cells of human and rat hair follicles, and might play a role as a barrier against anaerobic bacteria in the isthmus of hair follicles.
    Experimental Dermatology 02/2004; 13(1):55-60. · 3.58 Impact Factor
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    ABSTRACT: Erythropoietin (Epo) therapy for combatting anemia or fatigue in cancer patients has become a controversial issue. We have previously reported our study of 24 malignant human cell lines which express Epo and its receptor (EpoR) mRNAs and secrete Epo protein; blockade of Epo-signal destroyed the xenografts of female malignancies [1] and cancer cell lines [2]. We speculated that the conflicting clinical outcomes are due to the individual responsiveness to Epo of the various cancers. We measured the expression levels of Epo and EpoR mRNAs and the amount of Epo protein secreted and demonstrated the presence of EpoR protein in these 24 cell lines, some of which had anoxia-inducible Epo and/or EpoR mRNA. Additionally in seven selected cell lines with known amounts of Epo and EpoR expression, rhEpo triggered and the EpoR antagonist (EMP9) inhibited tyrosine phosphorylation of STAT5. They showed a rhEpo-induced growth that corresponded generally to the level of the constitutive activation of tyrosine phosphorylation of STAT5. Further, EMP9-suppressed growth depended inversely on the amount of Epo secretion. These data justify our speculation that the growth of very many cancers is promoted by their own Epo-signal which may or may not be accelerated by exogenous rhEpo.

Publication Stats

14 Citations
17.63 Total Impact Points

Institutions

  • 2007–2010
    • Tokushima Bunri University
      • Institute for Health Sciences
      Tokusima, Tokushima, Japan
  • 2005–2010
    • Kinki University
      • • Life Science Research Institute
      • • Department of Dermatology
      Ōsaka, Ōsaka, Japan