The mechanism by which nonsteroidal antiestrogen inhibits Ca²⁺- and phospholipid-dependent protein kinase (PKC) activity was investigated. Antiestrogenic agents, clomiphene and tamoxifen, inhibited the PKC-dependent phosphorylation of histone and r-annexin I in a dose-dependent manner. Ki values for the agents were different for two substrate proteins. The inhibitory action of the agents depended on the membrane-substrate protein interaction. Phosphorylation of cytoplasmic proteins obtained from rat uterus and mammary gland, including annexin I, by endogenous PKC was also inhibited by low concentrations of these agents. These results suggest that the inhibitory action of nonsteroidal antiestrogens occurs through their inhibitory effect on the membrane-substrate protein interaction.

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To optimize conditions for retroviral-mediated transfer of a recombinant gene to hepatocytes, the pMNSM-Tk-lacZ vector, which we had constructed to express the bacterial β-galactosidase gene, was transduced to rat hepatocytes under various conditions, and the expression of β-galactosidase activity was examined by cytochemical staining. Compared to the hepatocytes of adult rats, those of newborns were about 50-100 times more sensitive to transduction with the β-galactosidase gene in vitro. The sensitivity was high in the newborn hepatocytes when the virus was infected on days 1 and 2 after initiation of culture. However, the sensitivity to infection did not correlate with the DNA synthetic activity. The gene transfer was feasible not only to hepatocytes in monolayer culture but also to those in spheroid culture. The spheroidal aggregates containing hepatocytes transduced with the β-galactosidase gene could be transplanted into the spleen of syngenic adult rat, although the expression was very low.

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The behavior of individual Dictyostelium amebae was quantitatively analyzed with the computer-assisted "Dynamic Morphology System" (Soil, Voss, Varnum-Finney and Wessels, (1988) J. Cell. Biochem., 37: 177-192.). The same amoebae were then fixed and analyzed for filamentous (F-) actin and myosin (myosin-II, or "coventional" myosin) by fluorescence microscopy using the "agar-overlay method" (Yumura, and Fukui (1985) Nature, 314: 194-196.). This procedure provides a novel description of the behavior and morphometric changes preceding the static analysis of cytoskeletal organization in the same cell. It is demonstrated that when translocating cells make contact with an etched-smooth glass interface, 14 % cross the interface, 20 %
either reverse direction or migrate along the interface, and the remaining 45 % stay at the site. Cells contacting the interface from the smooth or etched side show equivalent behavioral responses. Upon contact with the interface, they project numerous lamellipodia and pseudopodia. While the lamellipodial projections exhibit cycles of spreading and retraction, the pseudopodia show lateral scanning motion, analogous to "substrate exploration" in fibroblasts (Albrecht-Buehler (1976) J. Cell Biol., 69: 275-286.). F-actin is localized in the lamellipodia and pseudopodia of amoebae contacting the interface. There is also discernable cortical F-actin, while conventional myosin appears to be excluded from the cortex and dispersed throughout the cytoplasm. The myosin displays a transient filamentous lattice at the base of newly forming lamellipodia. The ultrastructural study suggests that the new lamellipodia are formed on the dorsal surface and subsequently make contact with the substrate, indicating the dorsoventral sequence of polarity of the motile/sensory cellular organs. The present study demonstrates substrate exploration in Dictyostelium amoebae, and suggests its coupling to dynamic reorganization of the actomyosin cytoskeleton. The possible role of single-headed small myosin(s) (myosin-I, or minimyosin) is discussed.

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The localization of surface antigens and the binding activity of two monoclonal antibodies, HAM2 and HAM4, which recognize the rat major histocompatibility complex (MHC) antigen class I and the rat hepato-renal antigen respectively, on dissociated (free) hepatocytes was examined by light (LM) and electron
microscopy (EM), and by radioimmunoassay (RIA). Fixed hepatocytes, fixed before dissociation, and fresh hepatocytes, dissociated by collagenase, were treated by direct staining with HAM2- or HAM4-immunogold complexes (HAM2-gold and HAM4-gold). Some of the directly stained hepatocytes were further mixed with anti-mouse IgG-gold complex (IgG-gold) to supplement the direct staining.
The polarity of the sinusoidal and contiguous faces and the bile canaliculus, i.e. the in situ morphology, was well preserved in the fixed hepatocytes, while the fresh cells had lost the polarity and were round. On the fixed hepatocytes HAM2-gold particles were distributed predominantly on the sinusoidal face, while HAM4-gold particles were localized on both the bile canalicular and sinusoidal faces. No different antigen distribution on the fresh cells was detected with the two antibodies. Supplementation by IgG-gold was noticeable in most cases. The extent of binding activity in both the immunogold and RIA experiments was lower in the fixed cells than in fresh cells.
These results suggest that HAM2 and HAM4 are useful monoclonal antibodies for detecting the localization of the MHC class I antigen and the hepato-renal antigen on the hepatocytes, respectively.

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tsJT16 is a G₀/G₁ ts mutant from the Fischer rat fibroblast line. It has a ts defect in a function operating early after growth stimulation with fetal bovine serum (FBS). A primarily induced gene product, p70, was not synthesized at 40°C after stimulation with serum, while c-fos and c-myc mRNAs accumulated under the same condition. This paper reports that p70 was synthesized following stimulation of G₀-arrested cells with platelet-derived growth factor, epidermal growth factor (EGF), and 12-0-tetradecanoylphorbol-13-acetate (TPA) at 34°C, but not at 40°C. However, it was synthesized at both temperatures after addition of A23187. In protein kinase C-deprived cells, peptide growth factors and A23187 induced p70 at 34°C, whereas TPA did not. Fibroblast growth factor and insulin did not induce p70. Induction of c-fos and c-myc occurred at both temperatures after the stimulation with FBS, TPA or A23187. These results indicated that the defect in tsJT16 to induce p70 is likely to be located at the common downstream of protein kinase C-dependent and -independent pathways, but is independent from the pathway of calcium mobilization.

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A monoclonal type M-immunoglobulin (IgM) was generated in mice against a nuclease-urea extract of HeLa metaphase chromosomes. This antibody stains metaphase chromosomes from a variety of mammalian cultured cell types by indirect immunofluorescence. Antibody 12C7 reacts by western transfer technique with histone H1 in all the cell lines tested. The antibody cross-reacts with H1, and H1⁰ in human cells. Proteolytic digestions of H1 suggest that the epitope is localized in the carboxy-terminal domain of the histone H1 molecule. Digestion with trypsin demonstrates that the antibody 12C7 does not react with the globular domain of histone H1. The C-terminal domain of H1 subtypes therefore seems to have a conserved determinant which does exist in H1, H1⁰, and probably in H5. This antibody has applications in studying the role of that domain of H1 in processes like chromosome condensation and variations in chromatin structure which influence gene expression.

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To elucidate the cytotoxic mechanism of tumor necrosis factor (TNF), we isolated TNF-resistant sublines of L929 cells. As compared with L929 cells, TNF-resistant cells retained similar number and affinity of TNF-binding sites, and showed a similar growth rate. TNF stimulated arachidonate release from L929 cells, while no stimulation was observed at all in TNF-resistant cells tested. The cytotoxic action of TNF on L929 cells was inhibited by indomethacin, suggesting that prostaglandin may be involved in the action. Therefore, TNF-stimulated prostaglandin production was examined in L929 and TNF-resistant sublines. The amount of PGE₂ produced by L929 cells was increased more than 5-fold after the addition of TNF, whereas the amount of PGE₂ did not change in the resistant sublines following addition of the factor. TNF-stimulated arachidonate release and PGE₂ production were reversed by islet-activating protein (IAP)-treatment of L929 cells. These results suggest that arachidonate release and subsequent prostaglanding production are important for the cytotoxic action of TNF and that these processes are mediated by GTP-binding protein (G protein) that is coupled to the TNF-receptor.

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We describe here the use of a confocal laser scanning microscope for imaging fast dynamic changes of the intracellular calcium ion concentration ([Ca²⁺]_i) in isolated ventricular cell pairs. The scanning apparatus of our system, paired galvanometer mirrors, can perform narrow band scanning of an area of interest at a high temporal resolution of less than 70 msec per image. The actual [Ca²⁺]_i is obtained directly through the fluorescence intensity of injected fluo-3, which responds to changes of [Ca²⁺]_i in optically sectioned unit volumes of the cell. Images of the calcium wave obtained during propagation between paired cells revealed that the wave front is contant in shape and propagates at constant velocity without any delay at the cell-to-cell junction. The confocal laser scanning microscope with depth-discriminating ability is a valuable tool for taking pictures of the sequence of biological events in living cells.

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The lipoprotein secreted by cultured eel hepatocytes was fractionated by density gradient ultracentrifugation and compared with eel serum lipoproteins. Eel hepatocytes were cultured for 7 to 10 days as a monolayer in Williams' medium E containing 5 % fetal bovine serum and 0.16 μM insulin on a dish precoated with fibronectin of horse serum. The only lipoprotein secreted by eel hepatocytes was a very-low-density lipoprotein like one which consisted of 69 % triglyceride, 15 % phospholipid, 4 % cholesterol, and 12 % protein. On the other hand, very-low-density lipoprotein and high density lipoprotein were found in eel serum, in which high density lipoprotein was a main lipoprotein. The secreted lipoprotein contained apo B and apo A as the main protein components. Furthermore, the lipoprotein contained proapo A-I in addition to apo A-I, which was proved by comparing the amino acid composition of both proteins. In our discussion, we noted that the lipoprotein secreted by eel hepatocytes was a good material for the study of high-density lipoprotein formation.

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