Cell Structure and Function
Online ISSN : 1347-3700
Print ISSN : 0386-7196
ISSN-L : 0386-7196
16 巻, 6 号
選択された号の論文の11件中1~11を表示しています
  • Minoru Horie, Hiroshi Suzuki, Seiji Hayashi, Wei-Jin Zang, Masaru Komo ...
    1991 年 16 巻 6 号 p. 433-440
    発行日: 1991年
    公開日: 2006/03/27
    ジャーナル フリー
    Using fluorescent Ca2+ indicator fura-2 and whole-cell patch-clamp techniques, we examined the effect of 2-nicotinamidoethyl nitrate (nicorandil) on the intracellular free Ca2+ concentration ([Ca2+]i) and electrical properties in single guinea pig ventricular myocytes. Nicorandil (10 nM1 mM)reduced the resting level [Ca2+]i monitored as fura-2 fluorescence ratio in a concentration-dependent manner. Dibutyryl guanosine 3':5'-cyclic monophosphate (cyclic GMP), a membranepermeable cyclic GMP analogue, mimicked the nicorandil action. Neither application of caffeine (10 mM)nor deprivation of extracellular Na+ ions could prevent the nicorandil action on [Ca2+]i. In contrast, the nicorandil effect was virtually blocked by sodium orthovanadate (40 μM), a Ca2+ pumping ATPase inhibitor. During electrophysiological experiments, nicorandil shortened action potential durations (205 ± 80 ms to 153 ± 76 ms) by increasing a glibenclamide-sensitive outward K+ conductance. However, the drug produced little hyperpolarization (2 mV) because the resting potential of ventricular myocytes was close to the K+ equilibrium potential. The involvement of voltage-dependent Ca-channel current and Na-Ca exchanger was considered to be minimal under physiological conditions. It is thus concluded that nicorandil decreases basal [Ca2+]i via cyclic GMP-mediated activation of the plasma membrane Ca2+ pump in guinea pig ventricular myocytes.
  • Toshikazu Nishimura, Norio Kawai, Ichiro Ichihara
    1991 年 16 巻 6 号 p. 441-445
    発行日: 1991年
    公開日: 2006/03/27
    ジャーナル フリー
    The transfer of endocytosed simian virus 40 (SV40) to the nuclear position was investigated ultrastructurally using cationized ferritin (CF), ferritin labelled concanavalin A (Fer-Con A) and Con A as cell membrane markers. In the cells incubated with these markers and SV40 at 4°C, and then chased for 2 h at 37°C in serum-free medium, ferritin particles representing CF and/or Fer-Con A binding sites were found in vacuoles with SV40. The membraneof some vacuoles seemedto be in contact with the outer nuclear membrane.Several ferritin particles were located in the perinuclear cisterna and within the nucleoplasm, but not within the nuclear pores. In addition, there were vacuoles with ferritin particles and SV40 near the nuclear membrane, which looked like a single diaphragm with heterochromatins inside it. The outer nuclear and vacuole membranes were often obscure in the areas where the vacuole was very close to the diaphragm. In the case of cells incubated with CF, SV40 and Con A at 4°C, chased for 2 h at 37°C, and then reacted with horseradish peroxidase (HRP), HRP activity showing Con A-binding sites was also observed along the nuclear side of the inner nuclear membrane as well as in the perinuclear cisterna along the outer membrane. These results confirm that SV40-induced endocytotic vacuoles fuse with the outer nuclear membrane, and further indicate that some endocytotic vacuoles may well interact directly with the diaphragm, suggesting another path for migration of SV40 into CV-1 cell nuclei besides the path going through the process of fusion of the vacuole membrane with the outer nuclear membrane.
  • Mitsuru Kido, Hiroshi Miwatani, Kenji Kohno, Tsuyoshi Uchida, Yoshio O ...
    1991 年 16 巻 6 号 p. 447-453
    発行日: 1991年
    公開日: 2006/03/27
    ジャーナル フリー
    We have introduced a specific point mutation into the gene for chromosomal elongation factor 2 (EF-2) in Chinese hamster ovary cells (CHO-K1) by in vivo homologous recombination. To obtain a selectable construct for gene-targeting, we modified a diphtheria toxin-resistant mutant EF-2 gene (Gly717 to Arg) by deleting its promoter and first exon so that homologous recombinants could be distinguished from randomly integrated transformants, and also by inserting a second positive selection marker, the neomycin-resistance gene, into the 3'-flanking region to increase the selective accuracy. More than 80% of the clones surviving after selection for resistance to both the toxin and neomycin were the desired homologous recombinants in which the wild type, toxin-sensitive EF-2 gene was replaced by the modified gene giving resistance to both the toxin and neomycin. This result shows that the specific point mutation was co-introduced with a second selective marker into an endogenous chromosomal gene and that the modified gene was expressed.
  • Hajime Sawada, Hiroshi Konomi
    1991 年 16 巻 6 号 p. 455-466
    発行日: 1991年
    公開日: 2006/03/27
    ジャーナル フリー
    The antigen of monoclonal antibodies which had labeled the hexagonal lattice of Descemet's membranein a specific manner was shown to be the α1 chain of type VIII collagen by immunoblotting followed by amino acid sequence analysis. With this antibody, the localization of α1 (VIII) in various tissues was studied by several immunocytochemical methods. Under light microscopy, the α1 (VIII) was found in a fine fibrillar form in various capsular tissues such as capsules of the liver, kidneys, adrenals, lungs and so on. It was also present in dense connective tissues such as the Achilles tendon, and periodontal and perivertebral ligaments. When some dense connective tissues which had been negative to the label including the intima of aorta, perimysium and Glisson's sheath of the liver, were subjected to pepsin digestion, epitopes were revealed which showed a specific immunofluorescence pattern. In many locations the pattern of localization coincided with that of elastic fiber components, and full or partial colocalization with tropoelastin or costaining with resorcin-fuchsin staining was observed. In immunoelectron microscopy, the antigen (α1 (VIII)) was localized on the surface of, but not inside, elastic fibers. However, some tissues which are rich in elastic fibers or microfibrils remained unlabeled. These included elastic fibers of the aortic media and ligamentum nuchae as well as ciliary zonules. Therefore it is suggested that α1 (VIII) is a collagen associated with microfibrils of some elastic fiber systems, but is not an intrinsic component of either elastic fibers or of microfibrils.
  • Yasuki Ogawa, Noriko Ohno, Masayo Ito, Masahiko Iizuka, Sigeyasu Kobay ...
    1991 年 16 巻 6 号 p. 467-474
    発行日: 1991年
    公開日: 2006/03/27
    ジャーナル フリー
    By using a helper-free and replication-defective recombinant retrovirus encoding the SV40 early antigens (MV40), we have established continuous macrophage (Mφ) lines. All of the lines were nonproducer M φ's with differentiated Mφ functions such as phagocytosis, cytotoxicity, and IL-1 and TNF production. To determine the effects of several cytokines on growth of mature Mφ's, the responsiveness of these established Mφ lines to various cytokines was investigated in methylcellulose culture. Their response patterns to several cytokines alone and in combination were different, implying that there might be mature Mφ subpopulations with distinct growth profiles regulated by several cytokines. On the other hand, all of the lines efficiently yielded a number of colonies in response to interleukin-4 (IL-4) alone. Moreover, IL-4 cooperated with interleukin-3 (IL-3) to enhance colony formation of all the lines. A similarly synergistic effect was observed in combination of IL-4 and macrophage-colony stimulating factor (M-CSF) in almost all the lines. Similar results were obtained with colony formation of fresh thioglycolate-induced Mφ's. These observations suggested that IL-4 was involved in growth of mature Mφ's.
    Our present results suggest that the helper-free and replication-defective MV40 is of use to obtain continuous and functional cell lines from primary Mφ's.
  • Hiroshi Yasue, Hideaki Takahashi, Takashi Awata, Paul C. Popescu
    1991 年 16 巻 6 号 p. 475-479
    発行日: 1991年
    公開日: 2006/03/27
    ジャーナル フリー
    We investigated the distribution of PRE-1 sequences (a swine major SINE) on the swine chromosomes. The investigation demonstrated that PRE-1 sequences are unevenly distributed along the chromosomes as in the case of the human and mouse SINES. The distribution pattern, however, has no simple correlation with Q-band pattern as that of human and mouse SINES. The prominent difference is as follows; PRE-1 is localized on centromeric regions, but human and mouse SINES are not [KORENBERG and RYKOWSKI (1988). Cell, 53: 391-400; BOYLE, BALLARD, and WARD(1990). Proc. Natl. Acad. Sci. U.S.A. 87: 7751-7761].
  • Shigehiko Yumura
    1991 年 16 巻 6 号 p. 481-488
    発行日: 1991年
    公開日: 2006/03/27
    ジャーナル フリー
    Cytoskeletons, or 'Triton ghosts, ' that contained mainly actin and myosin II were prepared from Dictyostelium discoideum amoebae by extraction with Triton X-100. The Triton ghosts contracted immediately upon addition of ATP. However, under high-salt conditions in the presence of ATP, they did not contract but released myosin II. Almost all of the applied myosin II became associated with ghosts when myosin-free Triton ghosts, prepared in this way, were incubated with purified actin and then with myosin II from Dictyostelium. Immunofluorescence microscopy demonstrated that the associated myosin was localized in the cortical actin layer of the ghosts. Furthermore, the ghosts reconstituted with purified myosin resumed ATP-dependent contraction. Skeletal muscle myosin could also restore contractility to ghosts from which myosin had been extracted. The amount of myosin II necessary for the contraction of the ghosts was calculated by two methods. Less than 10% of the myosin II in intact cells was necessary for the contraction. These results show that myosin II is responsible for the contraction of the Dictyostelium cytoskeleton.
  • Makoto Nishimura, Chieko Tanigaki, Shoji Okamura
    1991 年 16 巻 6 号 p. 489-494
    発行日: 1991年
    公開日: 2006/03/27
    ジャーナル フリー
    Tobacco BY-2 cells were synchronized by an aphidicolin treatment, and their β-tubulin isoforms and their mRNA were analyzed by Western, Northern and dot blottings. The relative ratio of the β-tubulin isoforms changed with the progress of cell cycle stage. By Northern blot hybridization of poly(A)+RNAs with a cloned carrot β-tubulin cDNA probe, a single band of about 1.6 kb was detected throughout the cell cycle. Dot blot hybridization showed that β-tubulin mRNA existed in all stages in the cell cycle at a relatively constant level, though it accumulated slightly more than average at Mphase and decreased during G1 phase.
  • Kazunori Ohki, Toshinori Soejima, Osamu Kohashi, Ariaki Nagayama
    1991 年 16 巻 6 号 p. 495-502
    発行日: 1991年
    公開日: 2006/03/27
    ジャーナル フリー
    We have reported the isolation and characterization of three factor-dependent macrophage cell lines from bone marrow cells of C3H/HeNmice. We have since isolated a subclone, BDM-1W3, from one of these cell lines. We found previously that BDM-1W3has a different sensitivity to bacterial lipopolysaccharide (LPS) for growth than its parental cell line, BDM-1. In this report, we show that LPS inhibits BDM-1W3 phagocytosis of antibody-coated sheep erythrocytes (Fc-mediated phagocytosis), whereas it enhanced Fc-mediated phagocytosis by BDM-1. It was observed that a loss of Fc-receptor capacity parallels a loss of phagocytic activity in LPS-treated BDM-1W3cells. LPS stimulated phagocytosis of latex beads by BDM-1and BDM-1W3, suggesting that Fc-mediated phagocytosis and phagocytosis of latex beads differ in their regulatory mechanisms. When BDM-1 cells were cultured with LPS, they underwent drastic morphological changes, whereas LPS-treated BDM-1W3 cells did not change significantly. Gamma interferon enhanced FC-mediated phagocytosis by BDM-1, while it had no significant effect on that by BDM-1W3. These cell lines should be useful for studying signal transduction mechanisms in LPS-mediated macrophage activation.
  • Shigeki Kuriyama, Masahide Yoshikawa, Shigeaki Ishizaka, Tadasu Tsujii ...
    1991 年 16 巻 6 号 p. 503-510
    発行日: 1991年
    公開日: 2006/03/27
    ジャーナル フリー
    Recent technological advances made in molecular biology and in vitro culture of human and other mammalian cells have led to broad medical and scientific acceptance of the feasibility of gene therapy for genetic diseases. Cancer might practically be one of the attractive targets for such therapy. For the treatment of cancer, it is important to manipulate the gene of interest such that it is expressed solely in cancer cells. We have developed a tissue-specific gene expression system, based on a tissue-specific promoter on a retroviral vector. A murine ecotropic retroviral vector was constructed in which the Escherichia coli β-galactosidase gene served as a reporter; it was expressed under control of the albumin enhancer element and promoter. The tissue specificity of this vector was first assessed in vitro, and β-galactosidase activity was detected exclusively in hepatoma cell lines. This recombinant retrovirus was injected directly into a subcutaneous tumor composed of transplantable murine MH-134 hepatoma cells, and expression of the gene was observed in vivo. Then this recombinant retrovirus was injected via the spleen or directly into the liver, resulting in the gene expression in dividing hepatocytes in partially hepatectomized mice, but not in nondividing hepatocytes in normal mice. Gene transfer specific to dividing hepatocytes and expression by means of retroviral vectors should possess high potential for selective elimination of hepatoma cells surrounded by nondividing normal hepatocytes.
  • Satoru Ozono, Minoru Onozuka, Kazuyoshi Sato, Yumi Ito
    1991 年 16 巻 6 号 p. 511-513
    発行日: 1991年
    公開日: 2006/03/27
    ジャーナル フリー
    Immunohistochemical analysis of progesterone receptor was carried out in rat submandibular glands. Immunoreactivity to progesterone receptors was found in cell nuclei of the intralobular duct system within male and female rat submandibular glands. The female glands contained more immunoreactive cells than the male glands. In ovariectomized rats progesterone receptor-containing cells decreased in number while testectomized glands revealed an increase. When estradiol was administered to gonadectomized rats of both sexes, the immunoreactivity in cells of the intralobular duct system markedly increased. These results suggest the possibility that progesterone may modulate various metabolic functins in the rat submandibular glands.
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