Embryonic E14 stem cells were differentiated to parietal yolksac-like flat cells in vitro in the absence of added feeders and LIF (Leukemia Inhibitory Factor). We cloned circular DNAs from the differentiating E14 cells. Out of 9 DNA inserts with the unique sequence, one clone showed a chromosomal rearrangement which could have occurred between a pair of short inverted repeats. Recombination mechanism is discussed in view of two other circularization events of the flanking sequences between short inverted repeats shown in differentiated P19 embryonal carcinoma cells.

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We established a retinal pigment epithelium-derived cell line from transgenic mouse harboring temperature-sensitive simian virus 40 T-antigen gene (tsSV40T) and examined its characteristics. We enucleated both eyes from a 2-month-old transgenic mouse and removed the retinal pigment epithelial (RPE) cells and neuroretinal cells. After cloning the RPE cells, we obtained a cell line (RPET). RPET cells grew well at 33°C but not at 37°C, expressing on the temperature-sensitive character of tsSV40T, and maintained characters of RPE cells such as T1-tyrosinase production, phagocytosis of rod outer segments, and presence of cytokeratin, microvilli on the cell surface and lysosome-like granules around the Golgi apparatus in the cytoplasm. Conditioned medium (CM) from a culture of neuroretinal cells harboring tsSV40T was essentially required for growth. The factor(s) in CM was heat- and acid labile, but was resistant to trypsin digestion. In the presence of 3% CM, epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) had strong growth effects on RPET cells, whereas insulin, insulin-like growth factor I (IGF-I), and IGF-II had moderate growth effects. Interestingly, none of these growth factors stimulated the RPET cells in the absence of CM. EHS-Matrix® had growth effect, whereas laminin, collagen types I and IV, and fibronectin had no marked growth effects on RPET cells. RPET cells were morphologically changed on a laminin-coated dish. They could not spread on the coated dish, and the majority of the cells floated. But when the floating cells were transferred to non-coated dishes, they immediately attached themselves. These results suggest that RPET cells are a good model for finding novel growth factor(s) and for investigating the mechanism of cell-laminin attachment.

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Analysis of the flow of light information in Paramecium, especially the participation of GTP-binding protein, was carried out. Injection of GDP-β-S into Paramecium cell abolished the light-induced inward current. The partially purified rhodopsin-like protein (RLP) that we obtained activated frog rod outer segments (ROS) GTPase. A GTP-binding protein, with Mw 57, 000, was detected by immunostaining with anti-rat Gα rabbit IgG. These results suggest that the light information flows from the light-captured RLP to the GTP-binding protein.

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We analyzed the kinase activities capable of phosphorylating the regulatory light chain of myosin-II (MRLC) from chicken gizzard in unfertilized and fertilized sea urchin egg extracts. Total kinase activity phosphorylating MRLC in vitro did not fluctuate throughout the first cell cycle. Phosphopeptide mapping analysis showed that MRLC was phosphorylated at two different sites corresponding to myosin light chain kinase purified from chicken gizzard (MLCK) and protein kinase C (PKC) phosphorylation sites, namely MLCK and PKC sites, respectively. The activity of the kinase(s) responsible for phosphorylation of MRLC at PKC sites showed a significant increase at metaphase. Phosphoamino acid analysis revealed that this increase in MRLC phosphorylation was due to phosphorylation at serine residue (Ser-1 and/or Ser-2) and at threonine residue (Thr-9). This increase in phosphorylation at PKC sites is occurred concomitantly with an increase in histone HI kinase activity. In contrast, MRLC phosphorylation at MLCK sites showed no significant changes during the first cell cycle. Butyrolactone I, a selective inhibitor of p34^cdc2 kinase, inhibited the activity of the kinase(s) responsible for phosphorylation of MRLC at PKC sites at metaphase. These results suggest that the increase in MRLC phosphorylation at PKC sites (Ser-1 and/or -2, and Thr-9) at metaphase may be induced by p34^cdc2 kinase. Thus, p34^cdc2 kinase may be involved in the regulation of MRLC phosphorylation during cell division.

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When rat 3Y1 fibroblastic cells are cultured toward confluency, the cells go through maximum cell density (overshoot) before reaching post-confluence stationary cell density. After overshooting, a number of floating cells are found in the medium. In a long-term culture, a cyclic change in cell number, an increase after each medium refreshment and subsequent cell loss within a few days has been observed so that the cell populations in the monolayer maintain post-confluence stationary cell density at a constant level. The floating cells excluded trypan blue, but they had no ability to attach to the substrate and to form colonies after being reseeded in fresh medium. They had condensed and uniformly electron-dense chromatin with sharply circumscribed edges. Their DNA contained a laddering pattern in harmony with internucleosomal cleavage. The features were those of apoptosis. When floating cells appeared, apoptotic bodies were also observed in the monolayer. Most of them were found within the cytoplasm of intact cells, suggesting that apoptotic bodies were also faded away from the culture by being rapidly engulfed by neighboring intact cells. These suggest that apoptosis and subsequent detachment from the monolayer or engulfment by neighboring intact cells, in addition to inhibition of cell division, are basic mechanisms on the process of density-dependent regulation in monolayer culture of rat 3Y1 cells.

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Actin depolymerizing factor (ADF)/cofilin is a widely distributed family of actin-binding proteins which regulate actin polymerization in a pH-dependent manner. In cultured cells, cofilin, as well as ADF, translocates from the cytoplasm into the nucleus together with actin and forms rod-like structures in response to heat shock or dimethylsulfoxide (DMSO) treatment. In order to study in vivo interaction of cofilin with actin, we examined the effects of cofilin overexpression on actin cytoskeleton in C2 myoblasts. Interestingly, no remarkable effect was observed on phalloidin-stained patterns in cells overexpressing cofilin as compared with normal cells. However, upon treatment with DMSO, cytoplasmic actin filaments were disrupted and intranuclear rod structures containing cofilin and actin were apparently larger and thicker in cells overexpressing cofilin than in normal cells. Heat shock also stimulated disruption of microfilaments and formation of both intranuclear and prominent cytoplasmic cofilin-actin rods in cofilin-transfected cells, suggesting that DMSO-treatment or heat shock triggers cofilin-actin interaction. We further found that a myosin ATPase inhibitor (BDM) induced a reduction in cytoplasmic staining with phalloidin in cofilin-transfected cells. The results suggest that myosin activity might be involved in the regulation of cofilin-actin interaction in vivo.

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In the course of investigating effective biological active substances, we detected a substance in an extract of silkworm faeces that markedly suppresses viral production. The extract, prepared with hot phosphate-buffered saline and purified with ammonium sulfate precipitation, inhibited HVJ (Sendai virus), HSV (herpes simplex virus type-1), and HIV (human immunodeficiency virus type-1), but not poliovirus, suggesting that it is effective on enveloped virus production but not on non-enveloped ones. In the case of HVJ, indirect immunofluorescent staining using anti-HVJ antibody and Northern blotting analysis showed that, while viral adsorption and entry into the host cells were not affected, the synthesis of viral specific gene was inhibited by pretreatment of the virions with the extract. The extract affected more effectively aged virion, which loses membrane function as barrier and its envelope is leaky, than young virion that maintains barrier function. The active substance was partially purified by gel filtration after treatment of the extract with 1 N NaOH solution. From analysis with SDS-PAGE (SDS-polyacrylamide gel electrophoresis), protein bands were detected with molecular masses of about 25 kDa and near 14 kDa, while sugars were also detected with lectin blotting.

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A monoclonal antibody, H-145, the antigen of which is a glycoprotein with a molecular weight of 116 kDa, inhibits the fusion of quail skeletal myoblasts transformed with the temperature-sensitive mutant of Rous sarcoma virus (ts-RSV) (QM-RSV cells). Its antigen shows a unique distribution pattern during myogenic differentiation, and is required continuously for the inhibition as reported previously. The H-145 antigen is expressed from as early as the presumptive myoblast stage, and its expression increases during differentiation. In presumptive myoblasts, H-145 antigen is mainly accumulated in the Golgi apparatus. However, on transfer to conditions for differentiation, the antigen accumulated in the Golgi apparatus begins to become dispersed in the cytoplasm, and is gradually transported to the cell surface during differentiation, suggesting that transportation of H-145 antigen to the cell surface is required for myoblast fusion. To examine this possibility, we studied the effect of bafilomycin Al, which blocks transport of intracellular proteins between trans-Golgi cisternae and the cell surface. Treatment of QM-RSV cells with bafilomycin Al inhibited myoblast fusion even at 41°C, the temperature for myogenic differentiation. Under this condition, the antigen did not diffuse to the cell surface, but remained localized in the Golgi apparatus, as on culture at 35.5°C, the non-differentiation condition. These results suggest that quantitative expression of H-145 antigen on the cell surface is a prerequisite for myoblast fusion upon differentiation of QM-RSV cells

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In adult rat cerebellum, HPC-1/syntaxin 1 is detected at high density on the plasma membrane of the non-synaptic region of parallel fibers in addition to the synaptic terminal membranes and the synaptic vesicles (Koh, S., Yamamoto, A., Inoue, A., Inoue, Y., Akagawa, Y., Kawamura, Y., Kawamoto, Y., and Tashiro, Y. (1993). J. Neurocytology 22: 995-1005). To assess the possibility that HPC-1/syntaxin 1 participates in the morphogenesis of the nervous system, we examined changes in the localization of HPC-1/syntaxin 1 during postnatal development of the molecular layer of the rat cerebellum. HPC-1/syntaxin 1 appeared in the granule cells in the outer granule cell layer in 3-days-old rat cerebellum when the formation of synapses and the appearance of a synaptic vesicle protein, synaptophysin, had not yet been observed in the molecular layer. At this stage, the granule cells began to form parallel fibers. Confocal laser microscopy and immune-electron microscopy showed that HPC-1/syntaxin 1 was localized on the extruding plasma membrane of the granule cells to form parallel fibers. In 8-days-old rats, synapses formed between the parallel fibers and the developing dendrites of Purkinje cells, and the HPC-1 immunoreactivity appeared on the axons of parallel fibers and on the synapses. In 21-days-old rats, the HPC-1/syntaxin 1 immunostaining pattern was similar to that of adult rats. These results suggest that HPC-1/syntaxin 1 is involved in the formation of the molecular layer, especially in the axonal growth of the parallel fibers.

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Protein kinase activities in myxoamoebae of a true slime mold, Physarum polycephalum, were investigated in response to heat shock. In-gel assay detected an apparent activation of a Ca²⁺-dependent, 53-kDa protein kinase that phosphorylated casein but not histone HI. This enzyme needed co-presence of Mg²⁺ ion with Ca²⁺ for its activity. Treatment with calf intestinal alkaline phosphatase did not affect the heat-inducible 53-kDa protein kinase activity at all. The effects of protein kinase inhibitors were examined, and staurosporine suppressed the activity of this enzyme completely. H-7 decreased the activity to about 20% and HA-1004 to 65%. These results suggest that this protein kinase that may phosphorylate tyrosine and serine/threonine residues of target proteins is activated by heat shock in Physarum cells, and that the activation is not regulated via phosphorylation by putative protein kinase(s) that may act at an upstream position in the signaling cascade(s).

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