Concanavalin A-induced cap formation in rat ascites hepatoma cells (AH 7974) and the interaction of cytoplasmic proteins with plasma membranes.

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.</p

Fluorescence and Electron Microscopic Studies of the Cytoskeletal Organi­zation of Normal, Established and Transformed Chick Embryo Cells

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. </p

Fetal calf serum increased the zona pellucida penetrability of rat oocytes matured in vitro.

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. </p

Changes in the levels of lipoperoxide and antioxidant factors in human placenta during gestation.

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.</p

Histological Observation of the Development of Follicles and Follicular Atresia in Immature Rat Ovaries

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 &#946; (ER&#946;), 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 &#946; 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 ER&#946; 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

Effects of tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate on morphology and anchorage-independent growth of normal and established chick embryo 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.</p

Quantification of PERF 15 mRNA in Tissue Sections from Rat Testes

We previously conducted basic research to quantify in situ hybridization (ISH) signals in rat testes. In this experimental model, we selected ribosomal RNA (rRNA) as the hybridizable RNA in paraffin sections, since it allowed us to easily analyze ISH signals expressed with digoxygenin (DIG)-labeled probes quantitatively through “posterization” of the images. We applied this method to analyze the quantification of transcript, PERF 15 mRNA. PERF 15 is expressed specifically in the testes and localized in the rigid cytoskeletal structure of the sperm head, and has been considered to be involved in the apoptotic process of spermatogenic cells. Quantification of the signals may help to clarify the detailed function of PERF 15. We further analyzed the signals concomitant with a confocal laser scanning microscope. The peak of PERF 15 mRNA expression was found in diplotene spermatocytes, and the amount of PERF 15 mRNA was greatest in late pachytene and diplotene spermatocytes and early spermatids, followed by early pachytene spermatocytes, and then late spermatids. PERF 15 may be involved in the events leading to meiotic division, in which apoptosis is also involved. The present study may help to determine the concentration of mRNA in tissue sections

Mechanism for drug absorption from rat-liver surface membrane: effect of dose and transport inhibitors on the pharmacokinetics of phenol red

We examined the effect of dose and transport inhibitors on the pharmacokinetics of phenol red as a model drug after application to rat liver surface in-vivo, employing a cylindrical glass cell (i.d. 9 mm, area 0.64 cm2), to elucidate the mechanism for drug absorption from liver surface membrane. Absorption ratios of phenol red in 6 h were determined to be 91.1, 91.8 and 89.9 % at a dose of 0.3, 1 and 3 mg, respectively. Also, the AUC value for plasma concentration profile of phenol red was proportional to the dose. It is thus suggested that absorption process of phenol red from rat liver surface does not approach saturability. Time course of remaining amount of phenol red in glass cell obeyed the first-order kinetics at a dose of 0.3 mg, and its rate constant Ka was calculated to be 0.0069 min-1. Moreover, no significant difference was seen in Ka value within the dose range of 0.3 - 3 mg, which was estimated by curve fitting of the plasma concentration profile of phenol red after application to rat liver surface in the two-compartment model with first-order absorption. 2,4-Dinitrophenol (0.3 mg) and probenecid (0.5 and 1 mg), inhibitors of metabolic energy and anion transport respectively, had no significant effect on the pharmacokinetics of phenol red after application to rat liver surface. These data demonstrate that specific transport mechanism such as active transport is not involved in phenol red absorption from rat liver surface membrane.without figuresグラフな

Pharmacokinetic analysis of in vivo metabolism of amino acid or dipeptide conjugates of salicylic acid in rabbit intestinal microorganisms

We analyzed the pharmacokinetics of salicylic acid (SA)-amino acid (alanine, glutamic acid, methionine, and tyrosine) or SA-dipeptide (glycylglycine) conjugates in rabbits, by using a model that takes into account the metabolism of prodrug to SA by intestinal microorganisms and, also, by model-independent analysis. The blood concentration profiles of these prodrugs and released SA following intracecal and oral administration to rabbits were obtained previously (Nakamura et al., J. Pharm. Pharmacol., 44, 295-299, 1992; Chem. Pharm. Bull., 40, 2164-2168, 1992; Int. J. Pharm., 87, 59-66, 1992; J. Pharm. Pharmacol., 44, 713-716, 1992). First, the overall in vivo behavior was evaluated by statistical moment analysis. Next, the blood concentration profiles of prodrug and SA following intracecal and oral administration were simultaneously fitted to the above model. In general, good agreement was observed between fitted lines and experimental data for every prodrug, suggesting the validity of this model. The obtained parameters characterized the difference in the rate of metabolism and absorption among the prodrugs. Lower absorbability and enhanced hydrolysis rate of the prodrug lead to prolonged blood concentration of SA.without figuresグラフな

Absorption of organic anions as model drugs following application to rat liver surface in-vivo

Absorption of organic anions (phenol red, bromphenol blue and bromosulphonphthalein) has been studied after their application to rat liver surface in-vivo, employing a cylindrical glass cell (i.d. 9 mm, area 0.64 cm2). Every drug appeared gradually in the blood with the peak level at about 1 h, after which its concentration declined slowly. Absorbed model drug was efficiently excreted into the bile. These observations appear to indicate the possibility of drug absorption from liver surface membrane. Absorption ratios of model drugs were estimated to be more than 59 % in 6 h. As to phenol red, its biliary recovery and metabolism ratio did not change as compared with that of i.v. administration.without figuresグラフな