Resident macrophages which were harvested from the mouse peritoneal cavity showed the attachment activity to opsonized erythrocytes (OE) without the treatment of chondroitin sulfates (CSA) or phorbol esters. Phorbol ester (phorbol 12-myristate 13-acetate or phorbol 12, 13-diacetate) rapidly activated an opsonin-dependent erythrophagocytosis in resident macrophages, whereas CSA slowly activated it in vivo and in vitro. An additive effect of phorbol esters was observed in macrophages which were cultured with CSA in vitro or stimulated by the intraperitoneal injection of CSA for 1 or 2 day(s). In the case of macrophages stimulated by the intraperitoneal injection of CSA for 3 or 4 days the erythrophagocytic activity was at very high level and the additive effect of phorbol esters vanished. These results indicate that CSA plays a role in the induction of opsonin-dependent ingestion activity of resident macrophages.

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Estrogen has been suggested to influence the cell cycle of haploid yeast cells in the early G₁ phase of mitosis, its effect possibly being mediated by control of the level of cAMP (TANAKA, S. et al. (1989). Cell, 57: 675-681). Therefore, we were interested in whether estrogen also affects the meiotic phase of diploid yeast cells. Accordingly, we measured the amounts of adenylate cyclase mRNA and intracellular cAMP, the proportions of dividing cells and 4n cells and the doubling times of diploid yeast cells during the presporulation stage in the presence and absence of estrogen. The amount of adenylate cyclase mRNA was found to decrease rapidly within 24 hours after inoculation of cells onto sporulation-promoting plates (YPA plates). The cAMP level of these cells also decreased rapidly. Mitotic cell division continued for 18 hours after cell inoculation, but about 24 hours after inoculation, the amount of cAMP per cell had decreased to a minimum and the cells began to enter meiosis. By contrast, when the cells were inoculated onto YPA plates in the presence of estrogen, their intracellular cAMP and adenylate cyclase mRNA levels became higher than those in control cultures without estrogen and cell division continued for 24 hours. But after 30 hours their intracellular cAMP level decreased to a minimum and they began to enter meiosis. These results show that estrogen delayed the entry of diploid yeast cells into meiosis on sporulation-promoting plates and suggest that its effect may be mediated by control of the level of cAMP.

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A method was developed for determining the number of nuclei of hepatocytes cultured on collagen gel using a nonionic detergent, Nikkol BO-10TX. The cells were recovered in a test tube after solubilizing the gel by incubating it with the detergent in 0.1 M citric acid and then centrifuging the mixture. Nuclei were isolated from the cells with the same detergent solution and collected by centrifugation. The numbers of nuclei in cultures, scored with a hemocytometer or an electronic particle counter, were proportional to the lactate dehydrogenase activities of the cells. This method was also applicable for scoring the number of nuclei of hepatocytes cultured on collagen-coated plastic.

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Human thyroid epithelial cells were isolated from surgically resected human thyroid gland with collagenase and cultured for one week under EGF-supplemented conditions to allow them to proliferate. Then the cells were transferred to the following three-dimensional culture systems. One was a culture of isolated cells between floating double layers of collagen gel, designated the "floating sandwich method." The other was a culture of isolated cells mixed with collagen gel, designated the "dispersed embedding method." Many folliclelike structures with lumina of appreciable size were obtained by the former method. The cells cultured by the floating sandwich method exhibited a distinct polarity shown by the presence of numerous microvilli at the apical surface and close contact with collagen gels at the basal surface. On the other hand, only a few folliclelike structures were obtained by the dispersed embedding method, in which the folliclelike structures were small in size and the cells showed less distinct polarity than those observed in the floating sandwich method. Thus, the floating sandwich method appears to be suitable for studying the process and mechanism of in vitro organization of follicular structures by human thyroid epithelial cells.

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Intact A431 cells were labeled with [γ-³²P]ATP. The major phosphorylation product of the ecto-kinase activity of A431 cells had the molecular mass of 170 kd and was identified as EGF receptor by specific immunoprecipitation. This phosphorylation was not stimulated by EGF added to the reaction buffer, but replacement of MgCl₂ by MnCl₂ in the buffer remarkably stimulated phosphorylation. An exogenous protein substrate, α-casein, was also phosphorylated by intact A431 cells. The analyses for phospho-amino acids of both EGF receptor and α-casein revealed that phosphorylation occurred mainly at phosphotyrosine residues. Tryptic phospho-peptides of the EGF receptor of intact A431 cells labeled with [γ-³²P]ATP were fractionated by HPLC. The elution patterns were essentially the same as that of the autophosphorylated EGF receptor, indicating that the phosphorylation sites of EGF receptor labeled in vivo with [γ-³²P]ATP are located in three tyrosine residues in the carboxyl terminus. These results indicate that the carboxyl-terminal tyrosine kinase domain of a small fraction of the EGF receptor molecules of an A431 cell is exposed on the outer surface of the cells.

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Modulation of vascular endothelial cell growth by basement membrane heparan sulfate was investigated using four lines of normal and transformed cells. The growth of transformed endothelial cells, but not normal cells, on reconstituted basement membrane was severely suppressed when heparan sulfate, one of the components of the membrane, was specifically degraded by an enzyme, heparitinase. Similarly, when cells were grown on surfaces coated with heparan sulfate, as little as 60 pg/cm² of heparan sulfate caused growth enhancement of transformed cells, but suppression of normal cells. These results together with our previous observations (IMAMURA, T and MITSUI, Y. (1987) Exp. Cell Res., 172: 92-100) argue that transformed cells have reversed a mechanism by which basement membrane heparan sulfate functions as a physiological suppressor for the growth of normal endothelial cells.

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Actin bundles in proximal tubules of the rat kidney were examined by immunofluorescence and confocal laser microscopy with special reference to their three-dimensional distribution and identification as stress fibers. Renal tubular segments were prepared from the fresh renal cortex by simple homogenization and centrifugation, and fixed in formaldehyde for staining with fluorescent dye-labeled phalloidin. Segments of the proximal tubules could be identified easily on the bases of their diameter, the height of epithelial cells and prominent brush borders. Confocal laser microscopy clearly demonstrated the overall distribution of actin bundles in the whole-mount proximal tubular segments. Actin bundles in the basal cytoplasm of epithelial cells were observed to run parallel to each other and at a right angle to the tubular axis. In the stereo views reconstructed from serial optical sections, the basal actin bundles appeared as straight rods with both ends tapered. They varied in length and width and extended rather short distances of not more than 10 μm. Often, two or more actin bundles were longitudinally aligned in tandem. Some bundles showed irregular bandings along their length. Each bundle was composed of tightly packed actin filaments which could be decorated with heavy meromyosin subfragment-1 to display a bi-directional arrangement within the bundle. Immunostaining of cryostat sections showed that actin bundles contained myosin and vinculin. Enzymatically isolated proximal tubules contracted upon addition of Mg-ATP. These observations collectively suggest that the actin bundles at the base of renal promixal tubule epithelial cells can be listed among the examples of stress fibers in situ.

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A quail cell line (QT6-c) was co-transfected with pTEX vector expressing RNA complementary to chicken integrin β₁ subunit mRNA (Anti-Int) and pRSV neo vector by a calcium phosphate method. Transfectants showing reduced expression of quail integrin β₁ subunit were selected with an immunoblot assay, and a few positive clones were examined in detail. Northern blot and immunoblot analyses revealed that the Anti-Int caused a clear reduction of the transcript encoding integrin β₁ subunit depending on culture conditions. The number of cell surface integrins also decreased in proportion to the decrement of the total amount of integrin β₁ subunits. When one transfectant (QA23)was cultured in a serum-free medium, cell shape changed from fibroblast-like to neuron-like morphology accompanied by a low growth rate, and the cells did not form focal contact on fibronectin. A similar morphological change occurred in QT6-c cells when the cells were infected with Rous Sarcoma virus, which could produce the Anti-Int. The QA23 cells did not attach to fibronectin as efficiently as did the original QT6-c cells. These data suggest that reduced expression of integrin β₁ subunit affects cell growth as well as cell morphology by disordering the interaction between integrins and matrix proteins and/or cytoplasmic proteins.

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The distribution and arrangement of microtubules (MTs) in skeletal muscle fibers of the rat and mouse diaphragm were examined by thin-section electron microscopy. In the central portion of muscle fibers, most MTs ran longitudinally between myofibrils and beneath the sarcolemma, and some MTs ran transversely predominantly at the level of the I band, especially of the A-I junction, thus forming a lattice-like arrangement. At the fiber periphery, MTs were aggregated in the perinuclear region, from which they radiated to take a longitudinal course beneath the sarcolemma and to run in a transverse direction at the I-band level. In the end portion of muscle fibers, MTs were abundant and ran longitudinally into sarcoplasmic processes. MTs were often found to be spatially associated with membranous organelles. Quantitative analyses indicated that the longitudinally running MTs were remarkably more numerous in the peripheral zone of muscle fibers than in the deeper zones. The density of MTs in the central portion was almost the same in both red and white muscle fibers. The density was significantly higher at the fiber ends, though it varied considerably among different fibers. These results are discussed with special reference to the possible involvement of MTs in intracellular transport as well as structural support.

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In higher eucaryotes, heat shock factor (HSF) exists in a cryptic form in unstressed cells. We investigated molecular forms of human HSF before and after activation by sucrose density gradient centrifugation and by gel mobility shift assay using a ³²P-labeled heat shock element (HSE). We found that the in vivo or in vitro activated HSF, which is capable of binding to HSE, and its inactive form present in unstressed cells have different sedimentation coefficient; the former is 8 S whereas the latter is 4-5 S. Both the 8 S and 4-5 S forms contain the HSF polypeptide which has the ability to bind to HSE upon activation. The inactive 4-5 S form acquires HSE-binding ability when activated by heat shock or other stimuli. This HSF activity was greatly reduced, however, during recentrifugation in sucrose density gradient and, in addition, the residual activity was not recovered in 8 S fractions. Transformation of the inactive 4-5 S form of HSF to the stable, active 8 S form was achieved when the inactive form was activated and mixed with cytosols of unstressed cells.

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