論文 - 阪口　政清

Manumycin A, inhibitor of ras farnesyltransferase, inhibits proliferation and migration of rat vascular smooth muscle cells 査読

H Kouchi, K Nakamura, K Fushimi, M Sakaguchi, M Miyazaki, T Ohe, M Namba

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS   264 ( 3 )   915 - 920   1999年11月

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ACADEMIC PRESS INC  

Restenosis after angioplasty is thought to be caused by proliferation and migration of vascular smooth muscle cells (VSMCs), and it is a most serious problem in medical treatment. A low dose (50 ng/ml) of manumycin A, an inhibitor of p21(ras) (ras) farnesylation, significantly inhibited proliferation of rat VSMCs stimulated by the platelet-derived growth factor (PDGF). The mitoinhibitory effect of manumycin A was dose- and time-dependent but was independent of cell density. Western blot analysis showed that manumycin A reduced the amount of functional ras localized at the cytoplasmic membrane and inhibited the phosphorylation of p42/44 mitogen-activated protein kinase (MAPK). Manumycin A also inhibited VSMC migration and disorganized alpha actin fibers, as shown by immnofluorecence staining. These results indicate that the interruption of the ras/MAPK signal transduction pathway and the disorganization of alpha actin fibers are the main cause of manumycin A inhibition of VSMC proliferation and migration induced by PDGF. (C) 1999 Academic Press.

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Transferrin synthesized in cultured human fibroblasts is associated with tubulins and has iron binding capacity 査読

Masakiyo Sakaguchi, Tadashi Kondo, Hong Pu, Masayoshi Namba

Cell Structure and Function   24 ( 1 )   5 - 9   1999年

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

In a previous report, using immunocytochemical and fluorescence-labeling techniques, we demonstrated that transferrin is synthesized in cultured human fibroblasts and that it is associated with tubulins in the cells. These morphological findings led us to attempt to elaborate those issues in more detail by biochemical methods. In this report, we were able to prove the association of transferrin produced in cells with tubulins. In addition, the transferrin associated with tubulins was found to bind to iron. These results suggest that endogenous transferrin plays a role in preventing damage caused by free radicals which can be induced by the interaction of iron with the hydrogen peroxide produced in cells.

DOI： 10.1247/csf.24.5

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Characteristics of intracellular transferrin produced by human fibroblasts: Its posttranscriptional regulation and association with tubulin 査読

Tadashi Kondo, Masakiyo Sakaguchi, Masayoshi Namba

Experimental Cell Research   242 ( 1 )   38 - 44   1998年7月

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Academic Press Inc.  

Transferrin (Tf), an iron-binding protein, was investigated in the cultured human fibroblast which is a major cell type in connective tissues. Tf is a major iron-transporting protein and has an important role in iron metabolism. Using two-dimensional gel electrophoresis, we demonstrated the eight subtypes of Tf produced by cultured normal human fibroblasts and their down-regulation after the immortalization of human cells, an essential early step of in vitro transformation. However, the amount of Tf mRNA in the immortalized cells was equal to that in the normal human fibroblasts, suggesting that the down-regulation occurred at the posttranscriptional level. The amount of Tf receptor increased in the immortalized cells in spite of a decrease in the amount of intracellular Tf. Interestingly the produced Tf associated with microtubules. These findings suggest a novel aspect of Tf characteristics in human fibroblasts.

DOI： 10.1006/excr.1998.4080

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Two-dimensional gel electrophoretic analysis of the changes after immortalization of human cells: Decrease of intracellular alpha-2-macroglobulin fragment 査読

T Kondo, M Sakaguchi, H Yamada, M Namba

ELECTROPHORESIS   19 ( 10 )   1836 - 1840   1998年7月

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：WILEY-V C H VERLAG GMBH  

To study the mechanisms of immortalization of human cells, an early step in cancer development, we compared the cellular proteins of normal and immortalized human fibroblasts. Two-dimensional gel electrophoresis showed that one spot with a molecular mass 20 kDa and an isoelectric point of 6.0, became significantly smaller after immortalization of human cells. Further, the spot was rarely observed in four human liver cancer cell lines. Investigation of the N-terminal amino acids revealed that the spot was a fragment of alpha-2-macroglobulin. Although the 20 kDa fragment contains methionine, the spot was not labeled with [S-35]methionine. Thus we concluded that the spot might be derived from the culture medium. These results indicated that intracellular metabolism of alpha-2-macroglobulin, which is a multifunctional protease inhibitor, changed after the cells were transformed.

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Differential localization of two types of transferrin: Produced by human fibroblasts or incorporated from culture medium 査読

Masakiyo Sakaguchi, Tadashi Kondo, Hong Pu, Masayoshi Namba

Cell Structure and Function   23 ( 2 )   69 - 72   1998年

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Japan Society for Cell Biology  

In a previous paper, we demonstrated that cultured human fibroblasts synthesize transferrin (Tf). Two types of Tf are present
one is produced by the cells and the other is internalized from the culture medium. To study the metabolism of intracellular Tf, we investigated the subcellular localization of the two types of Tf in human fibroblasts by immunocytochemical and fluorescence-labeling techniques. The internalized Tf was found to be localized in the perinuclear area, and the synthesized Tf was associated with microtubules, forming a fibrous structure in the cytoplasm. When the cells were treated with colchicine which depolymerizes microtubules irreversibly, the synthesized Tf lost its fibrous structure and spread out in cytoplasm, but the internalized Tf remained around the nucleus. These results suggest that the two types of Tf are regulated differently in the cells.

DOI： 10.1247/csf.23.69

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