Times of the induction and of commitment to sporulation and variations in intracellular nucleotide (ATP, ADP, AMP, CTP, CDP, UTP, UDP) and cyclic nucleotide (cAMP, cGMP) concentrations were determined from the time at which starved plasmodia were exposed to continuous light until sporulation about 10 h after illumination. Induction took place after about 1 h, a burst of cAMP and cGMP concentrations occurring at about the same time. Plasmodia acquired the commitment after about 4.5 h, which corresponded to the second-rising phase of the oscillating nucleotide concen-trations. All the nucleotides studied oscillated virtually in phase, evidence that oscillation is related to conversion between monomeric and polymeric nucleotides. Anaerobic conditions suppressed both sporulation and oscillation of the intracellular nucleotide concentrations. The rate of O₂ uptake decreased on illumination, but did not oscillate. Sporulation is discussed in terms of nonlinear dynamics as a bifurcation from stable to oscillatory branches in an open nonequilibrium system.

View full abstract

Mouse FM3A cells propagated well in serum-free medium containing 0.5 % serum albumin and 1 μg of insulin/ml. The vitamin B₁₂ (B12) requirement of the cells depended on the population density. This requirement disappeared when a sufficiently large cell population was present. A combination of 1-100 ng of B12/ml and 4 μg of hypoxanthine/ml resulted in a synergistic increase in cell growth at low cell densities. A similar growth response was obtained when the B12 plus hypoxanthine was replaced by 4 μg of hypoxanthine/ml in combination with 100 ng of thymidine/ml, 1 μg of folic acid/ml or 1 μg of folic acid/ml, even though 1 μg of folic acid/ml already was present in the medium. Experiments on single cell inoculation showed that colony size and the yield of cells grown in B12-supplemented medium were much larger than those for cells grown in B12-free medium. A more critical population-dependent B12 requirement was demonstrated in mouse Ehrlich and L cells and their hybrids. At less than 100 cells there was no propagation in serum-free medium lacking B12, folic acid and thymidine; whereas, a satisfactory growth response was obtained in medium supplemented with these substances.

View full abstract

We previously established a serum-free hormone-supplemented medium for the induction of adipocyte differentiation of 3T3-L1 cells (Gamou, S. and N. Shimizu. in "Growth and Differentiation of Cells in Defined Environment", H. Murakami et al., ed., Kodansha/Springer-Verlag, pp. 173-178, 1985). Under those conditions the stage of the cell's commitment to adipocyte differentiation was separated from the stage of expression of the adipocyte phenotype. In the current study, the relationship between cell division of the growth-arrested 3T3-L1 cells and their entry into the differen-tiation program was examined by autoradiography at the individual cell level. It was found that cells treated with the inducers dexamethasone and 1-methyl-3-isobutylxanthine went through DNA synthesis (S phase) prior to lipid accumulation and that insulin enhanced this differentiation process. Under these serum-free hormone-supplemented conditions, the tumor promoter dihydroteleocidin B was found to be a strong inhibitor of adipocyte differ-entiation.

View full abstract

To study the mechanism of lipoprotein transport through arterial endothelial cells, porcine endothelial cells were cultured on gelated type I collagen supported by a dacron sheet, and the transport of low density lipoprotein (LDL) labeled with rhodamine B isothiocyanate (RB-LDL) through the cells was measured. Light and scanning electron microscopy showed that the cells on the gel were confluent. There was little RB-LDL transport through the endothelial monolayer at 0°C. RB-LDL transport through the monolayer at 37°C was dose-dependent saturable at 0.4 mg protein/ml. The transport was energy-dependent, since its rate was affected by temperature and was inhibited by a combination of 2-deoxyglucose (50 mM) and NaN₃ (10 mM). RB-LDL was shown not to be degraded during transport.

View full abstract

RNAs on the cell surfaces of two nonleukemic and two leukemic strains of mouse ascites tumor cells were studied by fractionating the RNAs released from the cell surface by gentle pronase treatment after sucrose density gradient centrifugation, by indirect membrane immunofluorescence that used anti-RNA antibody and by cell electrophoresis.
RNA was extracted from the cell supernatants of Ehrlich ascites tumor and sarcoma 180 cells (nonleukemic) that had been treated or not treated with pronase (1 μg/ml, 37°C, 20 min) followed by sucrose density gradient centri-fugation. It was clearly demonstrated that the amounts of ribosomal RNA (18S and 28S) released after pronase treatment were approximately 80 % greater than that of nonpronase-treated cells. Ehrlich ascites tumor cells that had been treated with actinomycin D (100 μg/kg body weight of mouse, 16 h) in vivo released an amount of ribosomal RNA after pronase treatment only 20 % greater than the value for untreated cells. Actinomycin D treatment greatly reduced both the cell surface negative charge and the cell surface immunofluorescence when rabbit anti-RNA antibody was used. Under the same experimental conditions with actinomycin D, only ribosomal RNA synthesis showed preferential inhibition, not the syntheses of poly A-containing messenger RNA, 4S or other small-size RNAs.
In contrast, L1210 and C1498 cells (leukemic) showed no change in the amounts of ribosomal RNA released after pronase treatment. L1210 cells also showed no change in the surface negative charge after being treated with actinomycin D. These results suggest that the RNA on the surfaces of nonleukemic ascites tumor cells is composed predominantly of 18S and 28S ribosomal RNA and that these RNAs might be responsible, in part if not completely, for the surface negative charges on nonleukemic tumor cells.

View full abstract

Light-mediated hydrolysis of phosphatidylinositol-4, 5-bis-ph osphate (TPI) to 1, 2-diacylglycerol and D-myo-inositol 1, 4, 5-triphosphate (IP3) has been reported in the visual photoreceptor cells. We have investigated the localization of the TPI antigenic sites in dark-and light-adapted rat retinas using rabbit anti TPI antibodies (Ab). Sprague-Dawley rats were dark-, or light-adapted, or were exposed to a light flash. The eyes were fixed immedi-ately and the tissue sections stained with the rabbit anti TPI Ab. The per-oxidase-antiperoxidase method was used to find the localization of the TPI antigenic site. Image analysis of the sections was performed to obtain optical density profiles of the stain. Dark-adapted retinas showed intense staining of the rod outer segment (OS) layer but much less staining of the rod inner segment layer. Compared with the OS of dark-adapted retinas, those of the flash-bleached retinas were stained much less. The OS of fully bleached retinas showed little or no staining. The anti TPI Ab-protein A-gold conjugate intense-ly stained disks from dark-adapted retina but those from bleached retina much less. Our results suggest that rapid hydrolysis of TPI in rat rod outer segments occurs in vivo in response to light.

View full abstract

A heat shock-resistant variant of Chinese hamster cell line (CHO) was isolated from ethane methane sulfonate-treated CHO cells through selection by repeated exposures to elevated temperature. The variant, desig-nated HR-01, was one to two order of magnitudes more resistant to lethal heat shock (46.0°C) than the parental CHO strain (p-CHO). The heat shock resistance characteristic of this variant was stable. In addition, the HR-01 variant showed more elongated cell morphology, and was more adherent to substrate than p-CHO. When total proteins of p-CHO and HR-01 cells were compared in two-dimensional polyacrylamide gel electrophoresis, HSP90, a heat shock protein of Mr 90, 000, was found to be the only protein that was expressed at a significantly higher level in HR-01 cells than in p-CHO cells. Because of the known intriguing molecular properties of HSP90, the HR-01 variant would be useful for further investigation of functions of HSP90 as well as the mechanism of acquiring heat shock resistance in mammalian cells.

View full abstract

Existing replica methods designed for adherent somatic cells are not applicable to non-adherent cells of tumor origin. We have developed a new, efficient replica method for the negative selection of mutants of non-adherent mouse FM3A cells. Mutagenized cells are grown clonally on soft-agar plates then allowedto proliferate upwards through layered polyester disks, thus producing replicas with the same colony patterns. Isolation of mutants with increased requirements for myo-inositol by this method is described.

View full abstract

Vacuolar sap was separated by intracellular perfusion and the distribution of proteases was studied in the giant alga Chara australis. Caseinolytic and hemoglobin-digesting activities were found to be higher at low pH, and about 85 % of total activity was localized in the central vacuole. The optimal pH for carboxypeptidase activity measured using N-carbo-benzoxy-L-phenylalanyl-L-alanineas a substrate was around 5, and about 95 % of total activity was localized in the vacuole. Moreover a substantial portion (40 %) of aminopeptidase activity, measured at pH 5.5 using L-leucine-β-naphthylamide as a substrate, was found in the vacuole.

View full abstract