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Volume 14 (1989)
- Issue 6 Pages 649-
- Issue 5 Pages 501-
- Issue 4 Pages 375-
- Issue 3 Pages 279-
- Issue 2 Pages 141-
- Issue 1 Pages 1-
Volume 14, Issue 2
Displaying 1-14 of 14 articles from this issue
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Hiroshi Komada, Hiroshi Nakabayashi, Toshimichi Yoshida, Hideki Takana ...1989 Volume 14 Issue 2 Pages 141-150
Published: 1989
Released on J-STAGE: April 18, 2008
JOURNAL FREE ACCESSThe cytosolic free calcium ion concentration ([Ca2+]i) of individual lymphocytes was measured by microfluorometry with dual excitation wavelengths using quin 2 or fura-2. Fura-2 was a more suitable fluorescent Ca2+ indicator than quin 2 for measurements of single cells because the standard curve calibrated for fura-2 had a good linearity, and the standard deviation (SD) of the value of the intensity ratio of fura-2-loaded cells was much smaller than that of quin 2-loaded cells. The [Ca2+]i in quiescent lymphocytes was about 1 x 10-7 M, and an increase in the [Ca2+]i was observed within a few minutes of ionomycin, protein A, phorbol myristate acetate (PMA) or concanavalin A (Con A) stimulation. Ionomycin-induced proliferation occurred when the initial [Ca2+]i was approximately 3 x 10-7 M or greater. The increase in the [Ca2+]i induced by Con A occurred transiently, and another rise in the [Ca2+]i was observed in the stage prior to the S-phase. These results indicate that Ca2+ is necessary for stimulated lymphocytes to enter the cell cycle and S-phase.View full abstractDownload PDF (808K) -
Yasunori Mivamoto, Masako Izumi, Shozo Ishizaka, Masao Hayashi1989 Volume 14 Issue 2 Pages 151-162
Published: 1989
Released on J-STAGE: April 18, 2008
JOURNAL FREE ACCESSWe have investigated the adsorption of cell-spreading activity in human serum onto polystyrene plates after treatment of the serum with sodium dodecyl sulfate (SDS). Vitronectin in human serum was remarkably adsorbed onto the plate after boiling the serum with 0.1% SDS for 5 min. SDS was effective over the concentration range from 0.05 to 0.25%. Increase of the vitronectin adsorption was accompanied by an increase of cell spreading on the plates. The cell-spreading activity in SDS-treated serum was impeded by anti-vitronectin antibody but not by anti-fibronectin antibody. After treatment with SDS, fibronectin-depleted serum could induce cell spreading but vitronectin-depleted serum could not. These results indicate that vitronectin alone was the cell-spreading factor in SDS-treated human serum. However, SDS-treated pure vitronectin itself did not retain the cell-spreading activity. The activity was recovered when bovine serum albumin was added to pure vitronectin before or after boiling with 0.1% SDS. Therefore, vitronectin adsorbed from SDS-treated serum might retain the cell-spreading activity with the aid of serum protein. Treatment of serum with SDS provides an easy, specific, and efficient method of coating polystyrene plates with vitronectin.View full abstractDownload PDF (1302K) -
Sadaki Yokota, Masaru Himeno, Keitaro Kato1989 Volume 14 Issue 2 Pages 163-171
Published: 1989
Released on J-STAGE: April 18, 2008
JOURNAL FREE ACCESSLocalization of acid phosphatase (ACPase) in rat liver was investigated by immunocytochemical techniques. Rat liver was fixed by perfusion and cut into thick tissue slices, which were embedded in Epon or Lowicryl K4M. For light microscopy (LM), semithin Epon sections were stained for the enzyme ACPase by an indirect immunoenzyme technique. For electron microscopy (EM), ultra-thin Lowicryl K4M sections were stained by a protein A-gold technique. By means of LM, granular reaction deposits were observed in hepatocytes and sinus-lining cells. Stained granules were present in the juxtanuclear cytoplasm, but they did not correspond to a typical staining pattern for the Golgi complex. EM revealed that gold particles indicating ACPase antigens were present on lysosomes and on some vesicles locating in the trans Golgi region. Endosomelike vesicles were strongly positive for the labeling. Golgi cisterna were mostly negative, but weak signals were noted in dilated saccules. The plasma membranes on the sinusoidal and bile canalicular sides were labeled by a few gold particles. The results indicate that ACP ase is present in endosomes and in a restricted area of plasma membrane, as well as in the lysosomal system.View full abstractDownload PDF (1498K) -
Kanji Ishizaki, Hae Ryong Chang, Tadashi Eguchi, Mitou Ikenaga1989 Volume 14 Issue 2 Pages 173-181
Published: 1989
Released on J-STAGE: April 18, 2008
JOURNAL FREE ACCESSHigh voltage electric pulses, 1000 V/cm, were found to induce cell fusion efficiently when delivered to cells growing in a monolayer culture. The maximum yield of fused cells was about 30% of cells remaining after treatment. Colonies of interspecies hybrid cells between mouse and others also appeared with a frequency of 3 x 10-4-2 x 10-5 after culturing fused cells in a selection medium that permits the growth of only hybrid cells. This method of electrofusion was applied for complementation analysis of DNA repair-deficient xeroderma pigmentosum cells. Efficient cell fusion was also observed with these human diploid fibroblasts and the resulting heterodikaryons showed a recovery of ultraviolet light-induced unscheduled DNA synthesis to the same extent as in normal cells.View full abstractDownload PDF (754K) -
Hiroshi Miwatani, Yoshihiro Yoneda, Hideo Maki, Yasufumi Kan-eda, Tsuy ...1989 Volume 14 Issue 2 Pages 183-192
Published: 1989
Released on J-STAGE: April 18, 2008
JOURNAL FREE ACCESSPreviously an Mr 46, 000 protein (named p46) was shown to be induced in the culture medium of human hepatoblastoma cells, Huh-6 C1-5, treated with 12-0-tetradecanoyl phorbol-13-acetate (TPA). For further characterization of p46, Huh-7 C1-4, another line of well differentiated liver cells, was incubated in the presence and absence of TPA, and proteins were labeled with [35S]methionine, and the proteins secreted into the medium were analyzed by one-and two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis. A protein of Mr 46, 000 was observed in the culture medium of Huh-7 C1-4 cells treated by TPA or plated at low density, but only slightly in the culture medium of Huh-7 Cl-4 cells plated at high density. As these results suggested that the expression of p46 was regulated by a factor that was ac-tivated in the growth state and by TPA treatment, the effect of introduction of the competence gene c-myc into Huh-7 C1-4 cells was examined. All 37 indepen-dent transfected colonies obtained showed enhanced expression of p46. As a control, the c-Ha-ras gene was introduced into Huh-7 C1-4 cells and shown not to enhance expression of p46. These results strongly suggested that the expres-sion of p46 was regulated by the competence gene c-myc.View full abstractDownload PDF (1010K) -
Hiroshi Komada, Hiroshi Nakabayashi, Mari Hara, Masayuki Hara, Takao T ...1989 Volume 14 Issue 2 Pages 193-197
Published: 1989
Released on J-STAGE: April 18, 2008
JOURNAL FREE ACCESSInterleukin 2 (IL-2) initiated proliferation of the cells of T-enriched population stimulated with non-mitogenic dose of concanavalin A (Con A), whereas it could not induce proliferation of the cells treated with non-mitogenic lectin wheat germ agglutinin (WGA). Use of monoclonal antibody (MAb) directed against interleukin 2 receptor (IL-2R) showed that IL-2 significantly increased expression of IL-2R on the cells of T-enriched population stimulated with Con A, whereas it could not cause any significant enhancement of expression of IL-2R on the cells treated with WGA. We concluded that IL-2 activated T cells in combination with non-mitogenic does of Con A, and induc-ed both expression of IL-2R and proliferation.View full abstractDownload PDF (464K) -
Yoshiki Shiba, Yoshinobu Kanno1989 Volume 14 Issue 2 Pages 199-208
Published: 1989
Released on J-STAGE: April 18, 2008
JOURNAL FREE ACCESSThe effects of type I and IV collagens on the survival and proliferation of cells were investigated to clarify a possible involvement of the substratum in the regulation of cell function. BSC-1 cells attached, spread and sustained their viability in the absence of calf serum on culture dishes coated with type IV collagen, but were unable to spread and survive on untreated culture dishes. The effects of adding type IV collagen in solution were similar to those of type IV coating. The fraction of the solution of type IV collagen with molecular mass of more than 100 kDa enhanced spreading and survival of cells, but the fraction of less than 100 kDa did not. Type I collagen did not support cell viability in the absence of calf serum. Moreover, coating of culture dishes with type I collagen, but not with type IV collagen, inhibited DNA synthesis and cell proliferation in the presence of calf serum. The cells grown on type I collagen were long, thin and spindle-shaped, and their stress fibers were not well developed, but the cells grown on type IV collagen, as well as those grown on untreated culture dishes, were polygonal in shape with well-developed stress fibers. These results indicate that the interactions of BSC-1 cells with the substratum, when it is derived from type I and IV collagens, differentially modulate the survival and proliferation of BSC-1 cells.View full abstractDownload PDF (2589K) -
Takashi Oka, Yasuo Nakaoka1989 Volume 14 Issue 2 Pages 209-216
Published: 1989
Released on J-STAGE: April 18, 2008
JOURNAL FREE ACCESSParamecium cells adapted to 2 mM K+ were voltage clamped and transient inward currents induced by step-depolarizations were studied. When the external K+ concentration was increased to 8 mM, the inward current was largely suppressed. Similar suppression of the inward current was induced by positive shifts of the holding potential above the resting level. The suppression of inward current is, therefore, primarily caused by depolarizing changes in the membrane potential. During adaptation to 8 mM K+, the inward current recovered to about 70% of that in the cells adapted to 2 mM K+, the resting membrane repolarized, and the steady state inactivation of the inward current was shifted to a more positive level than the cells adapted to 2 mM K+. When mutant cells which lack depolarization-sensitive Ca-current were adapted to 8 mM K+, a step-depolarization induced a fast activation of outward current, which would subtract from the inward current in the wild-type cells, suggesting that the transient inward current of cells adapted to 8 mM K+ is partially reduced by the fast activation of outward current.View full abstractDownload PDF (701K) -
Noboru Yamamoto, Kimiko Imazato, Aki Masumoto1989 Volume 14 Issue 2 Pages 217-229
Published: 1989
Released on J-STAGE: April 18, 2008
JOURNAL FREE ACCESSABSTRACTS. Cocultures of adult rat hepatocytes with nonparenchymal liver cells (NPC) resulted in stimulation of DNA synthesis of hepatocytes and their survival for more than one month. These cells were found capable of syn-thesizing albumin and 3-methylcholanthrene-inducing cytochrome P-450. A conditioned medium obtained by culturing NPC was effective to the same degree as the coculture with NPC for stimulating DNA synthesis in hepatocytes. The factor(s) responsible for this was inactivated by heating or trypsin treatment, but it was not capable of passing through a cellulose tubing. It is thus evident that soluble proteinaceous substance (s) secreted from NPC stimulates the growth of hepatocytes in a primary culture.View full abstractDownload PDF (6563K) -
Hiroaki Fukumoto, Yasuhisa Matsui, Masuo Obinata1989 Volume 14 Issue 2 Pages 231-239
Published: 1989
Released on J-STAGE: April 18, 2008
JOURNAL FREE ACCESSThe murine erythroleukemia (MEL) cell line, TSA8, becomes responsive to erythropoietin after induction with dimethyl sulfoxide (DMSO). We examined the signalling pathways involved in the commitment of TSA8 cells to become the erythroid progenitor cells responsive to erythropoietin, com-paring them with the pathway used in an erythropoietin-induced change of the progenitor cells.
Amiloride, an inhibitor of the Na+/H+ antiporter, completely blocked the commitment of TSA8 cells to become responsive to erythropoietin at a concen-tration that did not affect cell proliferation, while it showed no effect on the differentiation or proliferation of the erythroid progenitor cells derived from TSA8 cells by erythropoietin. Ethyleneglycol-bis (β-aminoethyl ether) N, N, N', N'-tetra acetic acid (EGTA) inhibited the commitment of TSA8 cells to CFU-E-like cells without affecting colony formation. In contrast, EGTA did not inhibit erythropoietin-induced differentiation of the progenitor cells, but did inhibit their proliferation. These results indicate that erythropoietin uses different signalling pathways from those used in the induction of the commit-ment of TSA8 cells.View full abstractDownload PDF (812K) -
Yukihisa Hamaguchi, Fuyuki Iwasa, Masaru Toriyama, Hikoichi Sakai1989 Volume 14 Issue 2 Pages 241-248
Published: 1989
Released on J-STAGE: April 18, 2008
JOURNAL FREE ACCESSThe localizations of tubulin and calmodulin were investigated in the mouse oocyte during the second meiosis by fluorescently labeling and microinjecting these proteins prepared from porcine brain tissue. When in-jected, both tubulin and calmodulin were quickly incorporated into the pre-formed meiotic apparatus of the oocyte at metaphase. The localization of label-ed tubulin was coincident with that of birefringence. However, the localization of labeled calmodulin was somewhat different: the fluorescence of calmodulin was intense in the polar regions of the spindle. After the chromosomes began to move, followed by parthenogenetic activation upon microinjection of a calcium buffer, these two fluorescent proteins, localized in the meiotic apparatus, mov-ed to the interzonal region of the spindle during anaphase. At late anaphase and throughout telophase, calmodulin was excluded from the mid-bodylike struc-tures in the interzonal region, whereas tubulin did accumulate in these struc-tures.View full abstractDownload PDF (6639K) -
Shohei Maekawa, Sachiko Endo, Hikoichi Sakai1989 Volume 14 Issue 2 Pages 249-259
Published: 1989
Released on J-STAGE: April 18, 2008
JOURNAL FREE ACCESSMonoclonal antibodies reactive to a high molecular weight pro-tein in MDBK cells were prepared and used to localize this protein in cultured cells. An insoluble fraction of MDBK cells after extraction with Triton X-100 was used as an immunogen and an immunofluorescent cell staining method was used to screen hybridoma cells. Three mAbs recognized a protein having nearly the same mobility as that of the microtubule-associated protein 2 in SDS-PAGE but no immunological reactivity of bovine microtubule proteins to these mAbs distinguished these proteins. Immunoblotting and cell staining experiments showed its presence in the intermediate filament-nuclear matrix fraction. Dou-ble staining experiments of cells using these mAbs and antibody against vimen-tin cleared the association of this protein with vimentin filaments. Results of im-munoelectron microscopy confirmed its localization on the intermediate filaments.View full abstractDownload PDF (15156K) -
Koichiro Shiokawa, Tsunevuki Yamazaki, Yuchang Fu, Kosuke Tashiro, Kun ...1989 Volume 14 Issue 2 Pages 261-269
Published: 1989
Released on J-STAGE: April 18, 2008
JOURNAL FREE ACCESSWe injected circular forms of several different DNAs into fertilized eggs of Xenopus laevis, and studied the persistence and expression of the injected DNAs during early embryonic development. When we injected plasmids which contained Drosophila amylase genes, the copy number of the injected DNA increased only slightly during cleavage, started to decrease at the blastula stage, then became very small at the tadpole stage. In such embryos, Drosophila amylase activity was detected at and after the gastrula stage. When we injected other kinds of circular DNAs (pX1r101A, cDm2055, pII25.1, pBR322, and pSP-64-L14), their copy number did not increase throughout the early stages. When circular plasmids that contained bacterial chloramphenicol acetyltransferase (CAT) genes were injected, their copy number usually did not increase, but sometimes, for unknown reasons, it increased extensively throughout the blastula to gastrula stages. In both cases, CAT enzyme activity started to be expressed during the blastula to gastrula stages and disappeared at the 2 day-old tadpole stage. The level of CAT enzyme activity was roughly pro-portional to the amount of CAT mRNA formed, and also to the copy number of injected genes. From these results, we concluded that in Xenopus embryos, exogenously-injected circular DNAs are preserved for the most part as circular DNAs, and that the increase in their copy number within the embryos is not prerequisite for the expression of their genetic information.View full abstractDownload PDF (6826K) -
Frances M. Davis, David A. Wright, Joseph E. Penkala, Potu N. Rao1989 Volume 14 Issue 2 Pages 271-277
Published: 1989
Released on J-STAGE: April 18, 2008
JOURNAL FREE ACCESSBy microinjecting monoclonal antibodies that bind specifically to mitotic and meiotic cells of a variety of species, we studied the biological ac-tivity of antigens recognized by these antibodies. The antibodies recognize a family of phosphoprotein antigens that are found throughout the cytoplasm of mitotic cells and particularly at microtubule organizing centers, including centrosomes and kinetochores. Their binding is dependent on phosphorylation of the polypeptides. Immunoglobulins were introduced into Xenopus laevis and Rana pipiens oocytes or cleaving embryos using glass micropipettes. The ability of the antibody-injected oocytes to undergo mitosis or meiosis was compared with those injected with control mouse immunoglobulins. The antibodies failed to block chromosome condensation and germinal vesicle breakdown in pro-gesterone-treated oocytes. However, functional mitotic spindles were not assembled in cleavage stage frog embryos injected with antibodies. In vitro, the binding of the antibodies to the antigens inhibited the dephosphorylation of the antigens by alkaline phosphatase. The antibody binding to the activated microtubule organizing centers (MTOC) seems to block not only the nucleation of microtubules and the organization of the mitotic spindle, but also the dephosphorylation of proteins associated with the MTOC that normally occurs at the mitosis-G1 transition.View full abstractDownload PDF (2823K)
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