Specific binding of eel serum high-density lipoprotein (HDL) to eel hepatocytes was demonstrated by using a synthesized fluorescent lipophilic dye. HDL binding was inhibited by the addition of unlabeled HDL. The binding of HDL to the hepatocytes was saturated at concentrations over 100 μg HDL protein /ml and Kd value was 20 μg HDL protein/ml. A fluorescent photomicrograph of the cultured eel hepatocytes which were incubated with the dye showed the bright, circumferential plasma membranes stained with the dye. ¹²⁵I-HDL was incorporated into the acid insoluble- and soluble-fractions of the cultured hepatocytes during incubation at 28°C for 1 h. There are three remarkable characteristics of the effect of HDL on the cultured hepatocytes. One is that the addition of HDL to the hepatocytes induced the efflux of cholesterol, triacylglycerol, and phospholipid from the hepatocytes. The second characteristic is that the efflux of the intracellular lipids was carried out with very-low-density-like or chylomicron-like lipoprotein secreted by the hepatocytes. The third characteristic is that HDL specifically stimulated the synthesis of the lipoprotein and had no effect on the synthesis of intracellular proteins and the secreted proteins except for the lipoprotein.

抄録全体を表示

We developed a cell-spreading model of permeabilized BHK cells to analyze molecular interactions in the cell-spreading machinery. BHK cells in suspension were allowed to attach onto the fibronectincoated cover slips at 37°C for 20 min. The cells were then permeabilized with 50 μg/ml saponin for 10 min at 4°C. Approximately 50 percent of the cell models spread in response to exogenous 1 Mm ATP for 90 min at 37°C. Anti-actin antibody penetrated into almost all the cell models which spread ATP-dependently. These indicate that half number of the cell models permeabilized with saponin is spread by the exogenous ATP and has pores larger than an IgG molecule.

抄録全体を表示

Mineralization was induced by glucocorticoid treatment in a human osteoblastic cell line derived from normal bone in vitro, designated SV-HFO, immortalized with simian virus 40 (CHIBA, H. et al. (1993). Jpn. J. Cancer Res., 84 : 290-297). Mineralization was revealed by electron microscopy, von Kossa staining and electron spectroscopic analysis, which indicated that the Ca/P ratio was approximately 1.70, corresponding to the value of hydroxyapatite. The effect was dose- (10^-8-10^-6 M) and time-dependent (days 7-28), was greatest at day 28, and was preceded by expression of alkaline phosphatase (ALP) and osteopontin (OPN). The ALP activity induced was highest at day 7, whereas OPN reached its highest level at day 28. When the induction of ALP activity was inhibited by 10^-4M levamisole, mineralization of SV-HFO cells by glucocorticoid treatment was markedly reduced, suggesting that elevated ALP activity in the early phase is important in the mineralization of human osteoblastic cells. Glucocorticoid treatment did not alter cell proliferation. These results indicated that glucocorticoids play crucial roles in the formation of mineralized matrix in human osteoblasts by inducing differentiation of SV-HFO cells without modulating their proliferative activity.

抄録全体を表示

Effects of the protein phosphatase inhibitors, tautomycin and calyculin A on protein phosphorylation and cytoskeleton of human platelets. It has been discovered recently that many cytotoxic compounds isolated from a variety of sources are potent phosphatase inhibitors. Two of these, tautomycin (TM) and calyculin-A (CL-A) were applied to human platelets to investigate the role of protein phosphorylation on cytoskeletal structure and function. Exposure to 10 μM TM or 0.1μM CL-A induced marked morphological changes. The granules were centralized and surrounded by actin filaments, but there was no evidence of granule release. Myosin became more centralized, was occluded from the granulomere, but was not confined to the micro filament ring. These changes occurred without an increase in cytosolic Ca²⁺ concentrations, as determined by measurements with fura-2. TM and CL-A induced an overall increase in protein phosphorylation. Phosphorylation of the 20, 000 dalton light chain of myosin increased markedly and multiple phosphorylation sites were indicated. Cytoskeletons were prepared from control, thrombin- and TM-treated platelets, the latter prepared in the absence of external calcium. The major difference in protein composition was the increased content of myosin associated with the cytoskeleton from TM-treated platelets where the dominant phosphoprotein was the 20, 000 dalton light chain. These results suggest that myosin phosphorylation drives the initial shape changes, and via a contractile process results in the formation of the micro filament ring and centralization of granules.

抄録全体を表示

Thyroid follicles in vivo are embedded in extracellular matrix (ECM). The composing epithelial cells are in close contact with ECM at the basal side. To examine cell-to-ECM interactions, we studied adhesion, proliferation and differentiation of porcine follicle cells monolayer-cultured on type I and IV collagen, fibronectin or laminin. At 3 h in culture, laminin had the lowest rate of cell adhesion. In proliferation, type IV collagen induced the highest level of nuclear bromodeoxyuridine intake. In a functional differentiation, laminin had about 3 times as much triiodothyronine production as the other ECM molecules. In confluent, culture cells, we also examined an expression of c-fos protein, a transcription factor that plays crucial roles in signal transduction. Immunocytochemistry detected the protein mainly in the nuclei. Western blot showed that laminin induced the highest level of its expression. Thyrotropin (TSH, 10 mU/ml) did not affect adhesion of the cells on any of the substrata or proliferation of the cells on fibronectin ; nor did TSH affect c-fos protein expression of the cells on the substrata except for fibronectin. Our results suggest that type IV collagen and laminin, major components of basement membrane, play positive roles in proliferation and differentiation of follicle cells, respectively, while laminin has no positive effect on adhesion of the cells at early culture ; that the cells express c-fos protein even in contact inhibition of growth and its expression is regulated in part by ECM ; and that ECM controls some behaviors of the cells in a TSH-dependent or TSH-independent manner.

抄録全体を表示

In the present work the haemocytes of mussels Mytilus galloprovincialis (Mollusca, Bivalvia) have been studied by light and electron microscopy in order to describe their main morphological features and to relate these to their roles in immune defence. The haemocytes belong to two definitive differentiated types, hyalinocytes and granulocytes. The former shows the presence of several fine pseudopodial protrusions, large nucleus with clumps of dense chromatin, scant cytoplasm, a well developed Golgi apparatus, lysosomes, several mitochondria (some with characteristic inclusions), coated pits and peripherally placed membrane-bound endocytic vesicles, considerable amounts of endoplasmic reticulum and ribosomes. The granulocytes generally possess an organelle-free ectoplasmic zone, numerous membrane-delimited dense granules of various types, coated pits and vesicles, endocytic and phagocytic vesicles, multivesicular bodies, several peroxisome-like organelles, mitochondria with inclusions, scant endoplasmic reticulum and small Golgi apparatus. These cells show the presence of few lipid droplets and variable amounts of glycogen particles. Some of the substructural features of the granules are documented here to indicate their probable biogenesis, growth and relationship with the endolysosomal compartment. In addition, in vitro phagocytosis experiments demonstrate that both hyalinocytes and granulocytes uptake latex and zymosan particles, granulocytes being much more active in phagocytosis than hyalinocytes.

抄録全体を表示

The relationship existing between cell morphology and cell metabolism, and the role of mechanical load in bone remodelling are well-known. In osteoblasts, PGE₂ mediates part of the response to mechanical stress and induce cell shape changes. We studied the influence of gravity variations on osteoblast morphology and its relationship with PGE₂ synthesis during a parabolic flight. ROS 17/2.8 osteosarcoma cells flew 15 or 30 parabolae. We measured cell area and shape factor after fluorescein staining with a semi-automatic image analyser and PGE₂ levels by RIA. Significant flight-induced shape changes consisted in a decrease in cell area and an increase in shape factor (cell irregularity), in some cells, as compared to ground controls. This heterogeneity in cell response might be explained by a cell-cycle sensitivity to mechanical stress. A 45 min pretreatment with indomethacin inhibited the flight-induced increase in cell irregularity whereas cell area remained decreased. PGE₂ levels were higher in flight than in ground controls. Linear regression analysis showed a significant negative relationship between cell area and PGE₂ synthesis. We concluded that ROS 17/2.8 are highly sensitive to gravitational variations and that PGE₂ is partly implicated in cell shape changes observed during parabolic flight. However, other mechanisms than PGE₂ synthesis condition ROS 17/2.8 morphology in response to mechanical changes.

抄録全体を表示

The differentiation of quail myoblasts transformed with a temperature-sensitive mutant of Rous sarcoma virus (QM-RSV cells) depends on culture temperature. At 35.5°C, the permissive temperature for RSV, QM-RSV cells repeatedly proliferate without differentiation. However, myogenic differentiation proceeds at 41°C, the nonpermissive temperature for RSV. To obtain useful inhibitors for the analysis of myogenic differentiation in QM-RSV cells, various drugs of myoblast fusion reported previously were examined in the QM-RSV system. Thirteen of twenty-seven drugs tested blocked myoblast fusion of QM-RSV cells. Among inhibitors that blocked the fusion of QM-RSV cells, four of them, acetylsalicylic acid (aspirin), 12-o-tetradecanoyl phorbol-13-acetate (TPA), doxorubicin, and N, N'-hexamethylenebisacetamide (HMBA), were chosen for further analysis. Two parameters of myogenic differentiation, myotube formation by myoblast fusion and creatine kinase activity, were examined. Aspirin, doxorubicin, and HMBA, inhibited myoblast fusion by acting on both steps before and after commitment for myoblast fusion. TPA affected the step before commitment for fusion. The effects of these inhibitors on creatine kinase activity were not always in parallel with myoblast fusion, suggesting that the process of myoblast fusion and the expression of creatine kinase activity are separate phenomena. Aspirin and doxorubicin did not affect creatine kinase activity. On the contrary, doxorubicin accelerated creatine kinase activity about twofold when the cells were treated with this drug after commitment in spite of strong inhibition of myoblast fusion. The expression of Mb-N3 and H145 antigens, which are closely related to the differentiation of QM-RSV cells, was affected variously by these inhibitors and sodium orthovanadate, an inhibitor of protein-phosphotyrosine phosphatase, suggesting that these inhibitors act on different steps during differentiation.

抄録全体を表示

We analyzed the direct effects of tyrosine phosphorylation on the structure and functions of tight junctions in temperature-sensitive v-src-transfected MDCK cells. When ts-v-src MDCK cells were plated at a low density at the nonpermissive temperature, they formed small, tight colonies with a typical epithelial appearance. When these colonies were cultured at the permissive temperature, cadherin-based cell adhesion was suppressed, so that individual cells scattered and assumed a fibroblastic appearance. This resulted in the destruction of tight junctions, making it difficult to analyze the direct effects of tyrosine phosphorylation on tight junctions. To suppress cell scattering, we cultured ts-v-src MDCK cells at confluence. Under these conditions, even at the permissive temperature, the cells assumed an epithelial appearance, and the structure of tight junctions were mostly maintained both at the immunofluorescence and electron microscopy levels. The transepithelial electrical resistance (TER) dropped to about 70% of the initial value after the temperature shift from the nonpermissive to the permissive. This temperature shift facilitated the tyrosine phosphorylation of the two tight junction proteins, ZO-1 and ZO-2. We concluded that the direct effects of tyrosine phosphorylation on tight junctions are not so remarkable as those on adherens junctions.

抄録全体を表示

Conditioned medium obtained from rat liver (M cells) induced a greater than 3 fold increase in the proliferation of rat bone marrow macrophages. Non-specific esterase staining of the proliferated cells gave a positive result. The conditioned media obtained from 8 other established cell lines and 2 primary human cell cultures were also examined for their effects on cultured macrophages. All of the conditioned media tested increased macrophage proliferation. In addition, the conditioned media were examined to determine whether they induced TNF-α production in macrophages. TNF-α was not detected in the culture medium of the macrophages in the absence of conditioned medium derived from other cell cultures, but TNF-α production could be induced by the addition of these conditioned media. Conditioned media obtained from the cells of different species or tumor cell lines were more effective in inducing TNF-α production than those obtained from rat cell lines or normal tissue cell lines.

抄録全体を表示

We investigated the effects of protein tyrosine phosphatase inhibitors, pervanadate and phenyl-arsine oxide (PAO), on the β1 and β3 integrin-mediated cell-substrate interaction using normal diploid human fibroblast. Pervanadate treatment of the cells in suspension state resulted in highly elevated levels of cellular protein tyrosine phosphorylation accompanied by loss of β1 integrin-mediated adhesion to substrata (i.e., collagen and laminin). In contrast, β3 integrin-mediated adhesion to substrata (i.e., fibronectin and vitronectin) of these cells was less affected. Moreover, pervanadate could reverseβ1 integrin-dependent adhesion, and cells already adhered on collagen or laminin, but not on fibronectin or vitronectin, came off within 30 min upon pervanadate treatment. These effects are likely to be directly mediated by increased cellular protein tyrosine phosphorylation, because another chemical compound, PAO, which also inhibits protein tyrosine phosphatase through a quite different mechanism, also exhibited the specific deterioration ofβ1 integrin-mediated cell-substrate interaction. Upon treatment with these protein tyrosine phosphatase inhibitors, the well developed actin stress fibers were disrupted resulting in the rounding up of cells on fibronectin and vitronectin substrate though they were still attached to theβ3 integrin-dependent substrates. Using immunoprecipitation and anti-phosphotyrosine immunoblotting, β1 integrin itself was shown not to be tyrosine phosphorylated. These results indicate that affinity regulation ofβ1 and β3 integrin is differentially controlled and that the specific regulation ofβ1 integrin is due to certain cellular component(s) whose activity is modulated by tyrosine phosphorylation/dephosphorylation.

抄録全体を表示