Acta Medica Okayama 1206 2008 alpha-lipoic acid suppresses 6-hydroxydoparnine-induced ROS generation and apoptosis through the stimulation of glutathione synthesis but not by the expression of heme oxygenase-1 1 12 EN Hirofumi Fujita Masahiko Shiosaka Tetsuya Ogino Yuya Okimura Toshihiko Utsumi Eisuke F. Sato Reiko Akagi Masayasu Inoue Kozo Utsumi Junzo Sasaki We previously reported that the generation of reactive oxygen species (ROS) is the initial event in cell death induced by 6-hydroxydopamine (6-OHDA), an experimental model of Parkinsonism. Since recent studies suggested the important role of antioxidant activity of alpha-lipoic acid (LA) in the suppression of apoptosis of various types, we studied the effect on 6-OHDA-induced apoptosis of PC12 cells. Biochemical analysis revealed that LA suppressed the 6-OHDA-induced ROS generation, increase of caspase-like activity and chromatin condensation. The suppression of 6-OHDA-induced apoptosis by LA required pre-incubation of PC12 cells with LA for 12-24 h. LA increased the intracellular levels of heme oxygenase-1 (HO-1) and glutathione (GSH) and stimulated the expression of GSH synthesis-related genes such as cystine/glutamate antiporter and gamma-glutamylcysteine synthetase (gamma-GCS). However, Sn-mesoporphyrin IX, an inhibitor of HO-1, did not attenuate the LA-induced suppression of apoptosis. In contrast, buthionine sulfoximine, an inhibitor of gamma-GCS, attenuated the LA-induced suppression of ROS generation and chromatin condensation. in addition, a transcription factor Nrf2, which regulates the expression of antioxidant enzymes such as gamma-GCS, translocated to the nucleus by LA. These results suggested that LA suppressed the 6-OHDA induced-apoptosis by the increase in cellular glutathione through stimulation of the GSH synthesis system but not by the expression of HO-1. No potential conflict of interest relevant to this article was reported.

Okayama University Medical School Acta Medica Okayama 0386-300X 61 5 2007 Histological Observation of the Development of Follicles and Follicular Atresia in Immature Rat Ovaries 283 298 EN Koji Takagi Teruo Yamada Yukari Miki Teruo Umegaki Makoto Nishimura Junzo Sasaki Original Article 10.18926/AMO/32892 To clarify the development of follicular growth and atresia in the immature ovary, rats. ovaries and blood were removed at fixed points during the period from 0 to 35 days after birth (Day 0 to Day 35). The ovaries were immunohistochemically examined, and blood concentrations of serum follicle-stimulating hormone (FSH) and estrogen (E) were measured. We investigated how time-course changes in follicular cell proliferation, estrogen receptor β (ERβ), apoptosis, and FSH and E concentrations are connected with follicular growth and atresia. Apoptosis was found in the ova from Day 0 to Day 3. On Day 15, apoptosis occurred in some granulosa cell nuclei in some follicles, but BrdU uptake and the presence of cyclin D2 and ER β could be observed in other granulosa cells. From Day 17, apoptosis increased in the follicular granulosa cells, and BrdU uptake and the presence of cyclin D2 and ERβ were decreased. Follicular atresia continued, reaching a peak on Day 30. Serum FSH and E concentrations increased until Day 15, then markedly decreased after Day 17. The mechanism of apoptosis in the ova from Day 0 to 3 has not been clarified. However, the onset of follicular atresia was caused by apoptotic degeneration from Days 15 to 17. These results showed that the oocytes were selected by apoptosis at 2 points in the time-course of the maturation of the ovary. No potential conflict of interest relevant to this article was reported.

Okayama University Medical School Acta Medica Okayama 0386-300X 37 5 1983 Inhibition of Phospholipase A2 and Platelet Aggregation by Grycyrrhizin, an Anti-inframmation Drug 385 391 EN Eiji Okimasu Yasunori Moromizato Sadahiro Watanabe Junzo Sasaki Noriyuki Shiraishi Yasuko M Morimoto Masanobu Miyahara Kozo Utsumi Article 10.18926/AMO/32426 We studied the effect of glycyrrhizin, a compound known as an anti-inflammatory and antiallergic drug, on the membrane permeability change induced by phospholipase A2 (PLA2) and on platelet aggregation. Glycyrrhizin was found to inhibit the PLA2-induced carboxyfluorescein (CF) release from D,L-dipalmitoyl phosphatidylcholine (DPPC) liposomes. Part of this inhibitory effect of glycyrrhizin on PLA2 is accounted for by the physical state of the substrate, the DPPC liposome membrane. Glycyrrhizin also inhibited collagen-induced platelet aggregation in a concentration dependent manner, which may in part account for its inhibitory effect on PLA2. No potential conflict of interest relevant to this article was reported.

Okayama University Medical School Acta Medica Okayama 0386-300X 40 6 1986 Fluorescence and electron microscopic study of intracellular F-actin in concanavalin A-treated and cytochalasin B-treated Ehrlich ascites tumor cells. 301 311 EN Sadahiro Watanabe Yoshiki Takehara Yoshitaka Fujii Eiji Okimasu Yasunori Moromizato Junzo Sasaki Article 10.18926/AMO/31915 To investigate the involvement of actin filaments in concanavalin A (Con A)-induced cap formation and cytochalasin B (CB)-induced zeiotic knob migration, the distribution of F-actin was studied in Con A-treated and CB-treated Ehrlich ascites tumor cells (EATC) by fluorescence microscopy using heavy meromyosin conjugated with a fluorescent dye, N-(7-dimethylamino-4-methylcoumarinyl) maleimide, (DACM-HMM). In non-treated cells, the diffuse fluorescence of DACM-HMM was observed in the cytoplasm, particularly intensely under the plasma membrane and around the nucleus. In Con A- and CB-treated cells, the fluorescence was seen at Con A-induced-capped and CB-induced-knob-accumulated regions. This fluorescence was more intense in CB-treated cells. To study the actin filaments in these fluorescent regions more clearly, the soluble components of the cells were eliminated by treatment with Triton X-100 or saponin solution containing a low concentration of glutaraldehyde, and the detergent-treated and saponin-treated cells were observed under a transmission electron microscope. Concentrated actin filaments were observed directly beneath the Con A-induced capping area and CB-induced zeiotic knob-accumulation area. The area of concentrated actin filaments appeared to correspond to the electron dense area observed in the identical region in the cells fixed without detergent treatment. More actin filaments were observed in CB-treated cells than in Con A-treated ones. No potential conflict of interest relevant to this article was reported.

Okayama University Medical School Acta Medica Okayama 0386-300X 41 4 1987 Concanavalin A-induced cap formation in rat ascites hepatoma cells (AH 7974) and the interaction of cytoplasmic proteins with plasma membranes. 145 154 EN Yasunori Moromizato Sadahiro Watanabe Junzo Sasaki Article 10.18926/AMO/31734 Concanavalin A (Con A) induced cap formation in rat ascites hepatoma cells (AH7974). In these Con A-treated cells, the association of cytoplasmic proteins with cell membranes was suggested by observing their Triton shells. The transition from G-actin to F-actin occurred in these cells. The association of membrane lipid with cytoplasmic proteins extracted from AH cells was studied by the isolation of protein-bound liposomes and phase transition release. The analysis of isolated liposomes revealed that many cytoplasmic proteins which specifically associated with liposomes were cytoskeletal elements including F-actins. The association of proteins with liposomes was affected by the lipid composition of the liposomal membrane and by the Ca2+ concentration of the incubation medium. The strong interaction of liposomal membrane with cytoplasmic proteins or isolated cytoskeletal proteins was demonstrated also by phase transition release using carboxy fluorescein-containing liposomes. These experiments showed that there was a strong affinity between lipid membrane and cytoskeletal elements including F-actins and that the amount of F-actin increased due to Con A treatment. The association of the submembranous microfilaments with the cell membrane may contribute to capping of the cells caused by Con A. No potential conflict of interest relevant to this article was reported.

Okayama University Medical School Acta Medica Okayama 0386-300X 35 3 1981 A scanning electron microscopic study of the two-step effect of cytochalasin B on Ehrlich ascites tumor cells. 197 204 EN Junzo Sasaki Masaaki Imanaka Sadahiro Watanabe Nagayasu Otsuka Shu Nakamoto Masaharu Mori Article 10.18926/AMO/31288 The effect of cytochalasin B (CB) on the surface structure of Ehrlich ascites tumor cells was investigated using the scanning electron microscope. The effect occurs in two steps: formation of zeiotic knobs on the cell surface and subsequent grouping of the knobs at one pole of the cell. The early step of zeiotic knob formation occurs at low concentrations of CB (0.5-1 microgram/ml) at 37 degrees C and at high concentrations of the drug (5-10 microgram/ml) at low temperature but within 1 min at 37 degrees C. This step is only partially inhibited by 5 x 10(-3) M sodium azide. The subsequent grouping of zeiotic knobs lasts for more than 2 min at 37 degrees C and occurs only in the case of high concentrations of CB. It is inhibited by sodium azide and is often associated with grouping of the microvilli, which are then lost from all of the cell surface except the area of knob-grouping. No potential conflict of interest relevant to this article was reported.

Okayama University Medical School Acta Medica Okayama 0386-300X 42 6 1988 Light and electron microscopic observation of specific atrial granules using water-miscible resin as an embedding medium. 311 316 EN Takako Nomura Nagayasu Otsuka Yukiko Tanaka Junzo Sasaki Article 10.18926/AMO/31003 A mixture of glycol methacrylate (GMA) and Quetol 523 was examined as an embedding medium for atrial tissue to be selectively stained for specific atrial granules. Semi-thin sections of rat atrial tissue embedded in this resin were stained with lead hematoxylin and observed under a light microscope. Atrial granules were found to be specifically stained blue black with lead hematoxylin. The same semithin sections stained with OsO4 vapor were examined electron microscopically and the atrial granules could be distinguised clearly from other cytoplasmic components. The GMA-Quetol 523 mixture is a useful embedding medium for studying the distribution of specific atrial granules by light and electron microscopy. No potential conflict of interest relevant to this article was reported.

Okayama University Medical School Acta Medica Okayama 0386-300X 42 4 1988 Exocytotic features of rat specific atrial granules. 201 206 EN Takako Nomura Hideo Kurokawa Shigeto Kanda Kiminao Mizukawa Junzo Sasaki Nagayasu Otsuka Article 10.18926/AMO/30996 To clarify the mode of secretion of specific atrial granules, rat atrial muscle cells were examined by transmission electron microscopy. Atrial granule formation and exocytotic features of granules were clearly seen. Abrupt breaks in the unit membrane structure of mature granules were observed in thin sections, but these breaks were not detected in freeze-fracture replicas. These findings support the concept that the granule contents are released to the extracellular space by exocytosis. No potential conflict of interest relevant to this article was reported.

Okayama University Medical School Acta Medica Okayama 0386-300X 42 4 1988 Fluorescence and Electron Microscopic Studies of the Cytoskeletal Organization of Normal, Established and Transformed Chick Embryo Cells 193 200 EN Junzo Sasaki Sadahiro Watanabe Takako Nomura Tazuko Fujiwara Hajime Ogura Article 10.18926/AMO/30995 The cytoskeletons of two established chick embryo cell (CEC) lines were examined by fluorescence and electron microscopy and compared with those of control cells and cells transformed by Rous sarcoma virus (RSV). In normal CEC, many stress fibers were observed. On the other hand, stress fibers were disorganized in nontransformed spontaneously established CEC, non-tumorigenic CEC partially transformed with a chemical carcinogen, and tumorigenic RSV-transformed CEC. In the normal CEC, actin filaments formed several bundles along the processes of the cell. Stereo-images of the peripheral region revealed bundles of filaments which were located along the attached side to the substrate. A fine well preserved network of filaments was also observed. On the other hand, in spontaneously established, partially transformed and RSV-transformed CEC, a fine network of filaments, but no actin cables, was found. These results support previous evidence that the cytoskeletal changes themselves are not directly related to the transformation or tumorigenicity of cells. No potential conflict of interest relevant to this article was reported.

Okayama University Medical School Acta Medica Okayama 0386-300X 43 4 1989 Effects of tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate on morphology and anchorage-independent growth of normal and established chick embryo cells. 249 252 EN Hajime Ogura Tazuko Fujiwara Sadahiro Watanabe Junzo Sasaki Article 10.18926/AMO/30858 The effects of 12-O-tetradecanoyl-phorbol-13-acetate (TPA) on the organization of cytoskeleton and growth of normal and established chick embryo cells (CEC) were studied. The cytoskeleton of normal CEC formed stress fibers, while that of the CEC lines established in our laboratory formed no stress fibers. TPA treatment of normal CEC resulted in disorganization of the stress fibers into amorphous structure, while that of the established CEC lines induced no reorganization of the cytoskeleton. TPA had no promotional effect in vitro or in vivo on tumor growth in normal or the established CEC. No potential conflict of interest relevant to this article was reported.

Okayama University Medical School Acta Medica Okayama 0386-300X 36 6 1982 "Pseudo-cap" formation in Ehrlich ascites tumor cells induced by cytochalasin B. 483 486 EN Masaharu Mori Shu Nakamoto Keizi Kirizuka Yoshito Sadahira Michiyasu Awai Satimaru Seno Junzo Sasaki Kozo Utsumi Brief Note 10.18926/AMO/30673 Cytochalasin B (CB) treatment induces or accelerates the capping phenomenon in some cells. In Ehrlich ascites tumor cells (EATC) CB treatment apparently induced the capping of Con A binding sites as observed under a fluorescent microscope. However, electron microscopic examinations revealed that the CB treatment did not induce a rearrangement of Con A binding sites, but rather it only induced a change in cell shape. On the contrary, CB treatment inhibited the capping phenomenon induced by treatment with Con A. Electron microscopic observations may give exact information on the distribution of lectin binding sites. No potential conflict of interest relevant to this article was reported.

Okayama University Medical School Acta Medica Okayama 0386-300X 36 4 1982 Cationized ferritin-induced cap formation and the effect of cytochalasin B. 307 312 EN Junzo Sasaki Masaaki Imanaka Sadahiro Watanabe Masaharu Mori Shu Nakamoto Keizi Kirizuka Nagayasu Otsuka Brief Note 10.18926/AMO/30662 No potential conflict of interest relevant to this article was reported.

Okayama University Medical School Acta Medica Okayama 0386-300X 44 2 1990 Changes in the levels of lipoperoxide and antioxidant factors in human placenta during gestation. 103 111 EN Yoshiki Takehara Tamotsu Yoshioka Junzo Sasaki Article 10.18926/AMO/30438 The concentration of lipoperoxides in maternal blood increases as gestation progresses. The concentration in pregnant women at 40 weeks gestation is 1.6 times higher than in nonpregnant women. The concentration in the cord blood, however, is 70% lower than that in maternal blood. To study the role of placental tissue in the difference in the lipoperoxide concentration between the cord blood and maternal blood, we investigated the lipoperoxide concentration, antioxidant activities and in vitro lipoperoxide formation in placental tissue during pregnancy. The lipoperoxide concentration was 50% lower in placental tissue of 40 weeks gestation than in tissue of 5-11 weeks gestation. Catalase and superoxide dismutase activities in placental tissues increased as gestation progressed, while glutathione peroxidase activity and alpha-tocopherol concentration did not change significantly during the gestational period. The in vitro formation of lipoperoxides in placental tissue decreased as gestation progressed. These results show that placental tissue suppresses lipoperoxide formation in the late gestational age, lowers the concentration of lipoperoxides in the blood and protects the fetus against oxygen toxicity. No potential conflict of interest relevant to this article was reported.

Okayama University Medical School Acta Medica Okayama 0386-300X 44 4 1990 Fetal calf serum increased the zona pellucida penetrability of rat oocytes matured in vitro. 203 208 EN Yoshitaka Fujii Tamotsu Yoshioka Junzo Sasaki Article 10.18926/AMO/30425 We examined the effect of fetal calf serum (FCS) on meiotic division, subsequent fertilization, and first cleavage to the 2-cell stage of rat oocytes during in vitro maturation. FCS had no effect on the nuclear progression from dictiate to metaphase of the second maturation in vitro and, FCS had no effect on the first cleavage to the 2-cell stage of fertilized oocytes. However, FCS efficiently increased penetration rate of oocytes and shortened the time required for dissolution of the zona pellucida by alpha-chymotrypsin. These results showed that FCS did not affect cytoplasmic maturation necessary for oocytes to develop to the 2-cell stages. We found that FCS only affects the zona pellucida and does not affect the nucleus or cytoplasm of rat oocytes. FCS may prevent hardening of the zona pellucida. No potential conflict of interest relevant to this article was reported.

岡山大学医療技術短期大学部 Acta Medica Okayama 0917-4494 5 1995 アドリアマイシン耐性細胞の酸素呼吸 95 98 EN Shoji Kawasaki Takako Nomura Junko Matsuura Junzo Sasaki Xian Shu Gao Jun-ichi Asaumi kouji Nishikawa Yoshio Hiraki Kozo Utsumi 10.18926/11928 エールリッヒ腹水癌細胞を用いアドリアマイシンに対する耐性細胞（ADR耐性細胞）を樹立した。電子顕微鏡を用い撮影写真から細胞質当たりのミトコンドリア（MT）の割合を面積比で求めた。親株に比較して1μg/ml ADR耐性細胞では1.32倍、10μg/ml ADR耐性細胞では1.47倍であった。これらの細胞の呼吸を測定した。耐性細胞の内発呼吸は親株に比較して増加していた。1μg/ml ADR耐性細胞では1.45倍、10μg/ml ADR耐性細胞では1.49倍であり、MTの増加量とほぼ同じ割合であった。これらのことから、細胞が耐性になるとエネルギー消費が高まるために細胞内MTが増加し、その結果呼吸（酸素消費）が増加することが推察された。 No potential conflict of interest relevant to this article was reported.

Acta Medica Okayama 1975 Mechanism of histamine release by alpha-cymotrypsin from isolated rat mast cells EN No potential conflict of interest relevant to this article was reported.