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Hirohisa Kimura, Takao Taki, Shyoji Hoshi, Shyoji Yamamoto, Makoto Mat ...
1986 Volume 11 Issue 2 Pages
89-97
Published: 1986
Released on J-STAGE: April 18, 2008
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Flow cytometric analysis using anti-glycolipid antiserum was used on rat bone marrow cells to determine the relation between the glycolipid species expressed on cell surfaces and cell differentiation. Four kinds of antibodies against gangliotriaosylceramide (Gg
3Cer), gangliotetraosylceramide (Gg
4Cer), fucogangliotetraosylceramide (IV
2aFuc-Gg
4Cer) and IV3aGal-fucogangliotetraosylceramide (IV
3aGalIV
2aFuc-Gg
4Cer, blood group B lipid) were used. The cells sorted out by each anti-glycolipid antiserum were stained with May-Grünwald-Giemsa reagent and identified by microscopy. In the erythropoietic group, only polychromatic erythroblasts had these four glyco-lipids on their cell surfaces ; none appeared on differentiated erythrocytes. These glycolipids were expressed during the early stages of immature granulo-cytes, especially in the promyelocyte and myelocyte stages of eosinophilic and neutrophilic granulocytes. Very limited populations of lymphocytes were sorted out as asialoganglioside-expressing cells. We concluded that asialo-gangliosides are useful differentiation markers for the erythropoietic and granulopoietic cells of rat bone marrow, and that anti-asialoganglioside antibody-flow cytometry is a very useful technique with which to isolate immature granulocytes and erythropoietic cells from rat bone marrow cells.
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Akiko Iwata, Mineo Iwata, Eizo Nakano
1986 Volume 11 Issue 2 Pages
99-107
Published: 1986
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Distributions of fibronectin, microtubules and microfilaments in the melanophore of the medaka,
Oryzias latipes, were studied using im-munofluorescence. We found fluorescent fibers of fibronectin running from the central region to the periphery of the melanophore, but patterns differed depending on whether melanophores had been treated with methanol. Fi-brous structures were present in methanol-treated melanophores, whereas only diffuse fluorescence was seen in untreated ones. These fibrous structures, thought to be microtubules, were arranged radially. Actin fibers also showed a radial arrangement, but were not as well organized. In addition, granules with actin immunofluorescence were present.
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Kosuke Tashiro, Masami Inoue, Yoshiyuki Sakaki, Koichiro Shiokawa
1986 Volume 11 Issue 2 Pages
109-114
Published: 1986
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A circular rDNA-containing recombinant plasmid, pXlr101A, and its vector pBR322 were microinjected into the cytoplasm of fertilized
Xenopus laevis eggs. The DNAs extracted from injected embryos at various stages of development were analyzed by hybridization with 32P-labeled pBR322 as the probe. Neither pXlr101A nor pBR322 were replicated, but they were maintained until the tailbud stage, disappearing during the muscular response stage. When pX1r101A-injected embryos were raised until the 2-week-old tadpole stage, sequences homologous to pBR322 were detectable in two Eco RI fragments of the pX1r101A-injected tadpole DNA. The sizes of the Eco RI fragments suggested that the plasmid sequences were preserved most probably in the chromosome-integrated form.
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Kohzaburo Fujikawa-Yamamoto, Nariko Miyashita, Shizuo Odashima
1986 Volume 11 Issue 2 Pages
115-124
Published: 1986
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A simple method with which to determine the cell cycle parameters, T
G1, T
S and T
G2M (the durations of the G
1, S and G
2+M phases) is described. V79 Chinese hamster lung cells were used to evaluate the method. After continuous labeling with bromodeoxyuridine (BrdU), V79 cells were stained with anti BrdU-monoclonal antibody with FITC (fluorescein isothio-cyanate) and with PI (propidium iodide). The individual cells were checked by flow cytometry for green and red fluorescences whose signal intensities cor-responded to the BrdU and cellular DNA contents. The durations of G
1, S and G
2+M phases of V79 cells were determined by measuring the cell fractions containing the nonlabeled G
1, labeled S and nonlabeled G
2+M phases. The reliability of this method is discussed.
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Eisuke Sato, Yoshiki Takehara, Junzo Sasaki, Irsuyoshi Matsuno, Kozo U ...
1986 Volume 11 Issue 2 Pages
125-134
Published: 1986
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Polymorphonuclear leukocytes undergo a series of morpho-logical and biochemical changes in response to various chemical stimuli. Transmembrane potential change is an early event that follows stimulation and membrane depolarization may act as a trigger for superoxide generation. To determine if there is a correlation between membrane depolarization and superoxide generation, we investigated the effects of different membrane modulators on stimulus-dependent depolarization. The membrane modulators mepacrine, chlorpromazine and cepharanthine inhibited the superoxide generation produced by chemotactic peptide, FMLP, and/or digitonin in neutrophils. Inhibitory profiles of the activation parameters, however, demon-strate that membrane depolarization is not associated with superoxide gener-ation : FMLP-induced depolarization was inhibited by the modulators tested and was accompanied by the suppression of superoxide generation, but the depolarization produced by digitonin was stimulated somewhat by these drugs. Our results indicate that receptor-mediated membrane depolarization is not a necessary event for the activation of superoxide generation by digitonin.
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Toshikazu Nishimura, Norio Kawai, Makoto Kawai, Kunihiro Notake, Ichir ...
1986 Volume 11 Issue 2 Pages
135-141
Published: 1986
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The interaction between simian virus 40(SV40)-induced endo-cytotic vacuoles and the nuclear membrane was investigated using cationized ferritin (CF) and concanavalin A (Con A) as cell membrane markers. These markers bound to the cell surfaces of CV-1 cells together with SV40 at 4°C. Following incubation of these modified cells at 37°C in serum-free medium, the cell membranes showed many invaginations. After incubation for 60 min at 37°C in the same medium, many various-sized vacuoles were present that contained membrane-bound CF, Con A and SV40. After 2 h of incubation at 37°C, Con A was present in some areas of the perinuclear cisterna along the nuclear membrane. The control experiment, however, showed no localization of Con A-binding on the nuclear membrane. These results provide evidence that SV40-induced endocytotic vacuoles migrate toward the nucleus and fuse with its membrane.
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Yasuko M. Morimoto, Eisuke Sato, Koichi Nobori, Ryutaro Takahashi, Koz ...
1986 Volume 11 Issue 2 Pages
143-155
Published: 1986
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When phospholipases of plasma membranes are activated by certain stimuli, unsaturated fatty acids are liberated. Because unsaturated fatty acids enhance the transmembrane movement of calcium ions, the fatty acids released may modulate intracellular calcium homeostasis in various cells, including neutrophils. To determine the physiological function of these unsaturated fatty acids, we studied the effects of various fatty acids on superoxide generation and on changes in intracellular calcium contents of guinea pig neutrophils. Some unsaturated fatty acids, arachidonate and linoleate, stimulated the rate of superoxide generation concomitant with the increase in the amount of intracellular calcium.
In contrast, the saturated fatty acid, myristate, stimulated the generation of superoxide without affecting the content of intracellular calcium. The stimulating actions of arachidonate and myristate were increased dramatically by the presence of a low concentration (1 μM) of extracellular calcium ion. The rate of superoxide generation in fatty acid-treated neutrophils was inhib-ited by chlorpromazine, an inhibitor of such calcium-binding proteins as C-kinase. These and other observations suggest that liberated unsaturated fatty acids increase the amount of intracellular calcium and enhance C-kinase activity also that the increased activity of the enzyme is involved in the chain of events leading to the stimulation of superoxide generation in fatty acid-treated neutrophils.
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Isao Adachi, Masako Takahashi, Hirokazu Adachi, Isamu Horikoshi
1986 Volume 11 Issue 2 Pages
157-163
Published: 1986
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EL-4 tumor cells were assayed
in vitro for their ability to aggre-gate two kinds of platelets. An inhibition study showed that the EL-4 tumor cell can induce platelet aggregation by at least two different mechanisms. One, mediated by thrombin, was dominant with rabbit platelets because hirudin, which specifically inhibits thrombin, considerably suppressed the rabbit platelet aggregation induced by EL-4 tumor cells. In contrast, EL-4 cells induced the aggregation of human platelets even in citrated PRP. It is the apyrase-sensitive pathway that is believed to work in human platelets.
The human platelet responses to EL-4 tumor cells clearly differed from those of rabbit platelets in terms of inhibition by hirudin and apyrase and of reactivity in citrated PRP. Both phospholipase A
2 and dibutyryl cAMP strongly inhibited EL-4 tumor cell-induced platelet aggregation in both rabbit and human platelets. These two compounds may block a vital step in platelet aggregation that is elicited by the EL-4 tumor cells. Our results show that hu-man platelet response to tumor cells is not necessarily deducable from ex-perimental data obtained with animal platelets.
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Masakuni Furusato, Yasushi Kikuchi, Shin Wakui, Tetsuro Wada, Izuru Ma ...
1986 Volume 11 Issue 2 Pages
165-174
Published: 1986
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The Nuclear Body Appearance Rate in seventeen human prostatic cases was statistically analyzed according to three lesion areas-the hyperplastic nodule, non-nodule and atrophic-in the secretory epithelium and basal cells. It was meaningfuly high in the secretory epithelium of the hyperplastic nodule, but not in the other two lesion areas. There was no meaningful result in basal cells.
Though there is a wide variety of reported data on the human prostate, particular care must be taken in analysis at the electron microscopic level, keeping in mind that even in a single specimen this organ has a notable variety of tissue changes. The Nuclear Body Appearance Rate reflects cellular hyperactivity although it does not have a specific known function at present.
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Hiroaki Asou, Noriko Mutou, Shusuke Hirano, Tamaki Takagi, Kagemasa Ka ...
1986 Volume 11 Issue 2 Pages
175-180
Published: 1986
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Localization of ganglioside GM
1 in cholinergic neurons from the septal area of a primary culture newborn rat brain was studied with a double avidin-biotin complex system. Cholinergic neurons were identified by double immunolabeling techniques that use choline acetyltransferase (ChAT) and neurofilament (NF) protein-antibodies. ChAT-positive neurons also were stained for ganglioside GM
1 by using an avidin-biotin complex technique.
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Hirao Kohno, Shigeru Taketani, Rikio Tokunaga
1986 Volume 11 Issue 2 Pages
181-190
Published: 1986
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The treatment of human leukemia K562 cells with 12-0-tetradecanoyl phorbol-13-acetate (TPA) caused a decrease of transferrin receptors. The mechanism of the decrease of the receptors with TPA has been investigated. In cells incubated with TPA, the rate of biosynthesis of transferrin receptors was reduced to 10-20 % of that in untreated cells. Pulse-chase experiments showed that turnover of the receptors in TPA-treated cells was accelerated over that in untreated cells. These results indicated that the decrease of transferrin receptors in TPA-treated cells was caused by reduced biosynthesis and accelerated degradation of the receptors.
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Hiroko Hama-Inaba, Tadahiro Shiomi, Koki Sato, Akemi Ito, Michiki Kasa ...
1986 Volume 11 Issue 2 Pages
191-197
Published: 1986
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A high-voltage generating machine which could generate semi-rectangular pulses in PBS solution was constructed, and the effects of field strength and duration of the pulse on electric pulse-mediated transfor-mation of mouse mammary carcinoma FM3A cells by a linear form of plasmid pSV2neo DNAs were examined. In parallel, cell survival and growth after pulsing were analyzed. When the field strength and duration of the pulse were increased, the transformation frequency increased, although the cell survival rate decreased. Under the best conditions, the transformation frequen-cy was 2×10
-4, which was 80 times higher than that obtained by the calcium phosphate coprecipitation method.
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Mitsuyuki Kuroki, Naoki Kamo, Yonosuke Kobatake, Yukichi Abe, Shin-ich ...
1986 Volume 11 Issue 2 Pages
199-204
Published: 1986
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Stimulation of polymorphonuclear leukocytes (PMN) by tetravalent concanavalin A (α-ConA) induces membrane depolarization preceding the onset of superoxide anion (O
2-) production. Both divalent and monovalent ConA analogues were studied to evaluate the role of valence. Monovalent ConA (m-ConA) was inactive in stimulating O
2-production and divalent derivatives were less active than native a-ConA. Similarly, membrane depolarization was dependent on the valency of ConA. m-ConA did not induce a marked change in membrane potential, whereas sustained depolarization occurred with multivalent ConA. The formation of multiple linked interactions between surface receptors may be an important early event in the activation of PMN by ConA.
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A Study of the Injection Volume
Shunichi Kurata, Yoji Ikawa
1986 Volume 11 Issue 2 Pages
205-207
Published: 1986
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A DNA transfection method by laser microbeam pricking has been recently reported (Kurata, S.
et al. Exp. Cell Res. 162, 372 (1986)). The volume of external fluid transferred into the cell by the method was determined through the injection of diphtheria toxin fragment A (Yamaizumi, M.
et al. Cell 15, 245 (1978)). Using these results and the results on laser DNA transfection efficiency (Kurata, S.
et al. Exp. Cell Res. 162, 372 (1986)), the approximate number of DNA molecules necessary to transform the recipient cell was estimated.
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