佐々木 順造

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.