The interference of various SH-blocking chemicals with the insulin-controlled regulation of the hepatic carbohydrate metabolism was studied in rat hepatocyte primary cultures. The organic mercurials PCMB, PCMBS, mersalyl the disulphide agents DTP, CPDS, disulfiram and the SH-alkylating reagent NEM were used as experimental SH-blocking model compounds. All studied compounds, except for NEM, induced an increased glycogen deposition comparable with the physiological insulin-induced glycogen-deposition. PCMBS appeared to be the most effective insulin-mimicking anabolic trigger. The action of the insulin molecule itself was potentiated by PCMBS as well as demonstrated by increased glycogen deposition, induced pyruvate kinase (PK) and decreased phosphoenol-pyrnvate carboxykinase (PEP-CK) activity. However, cell-exposure to insulin and PCMBS in relatively high doses was destructive, as demonstrated by decreased glycogen levels, most probably as a result of insulin-receptor overstimulation and metabolic stress. Thus, SH-blocking compounds are able to trigger insulin-dependent metabolic processes. The relatively non-permeant organic mercurial PCMBS proved to be the most effective insulin-mimicking SH-blocking compound.

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Both slowly and rapidly fluctuating currents were observed after incorporating a heavy sarcoplasmic reticulum (HSR) vesicle or a purified ryanodine receptor (RyR) of rabbit skeletal muscle into a lipid bilayer. The responses to the cis side Ca²⁺, ruthenium red and ryanodine confirmed that both were the currents through one Ca²⁺ release channel. From the analysis of conductance states, RyR is supposed to have two different configurations. The current through one configuration has a high reversal potential (HRP) (-18 mV) and that through another one has a low reversal potential (LRP) (- 11 mV). The current with HRP fluctuates rapidly but the current with LRP always fluctuates slowly. From the analysis of both rapidly and slowly fluctuating currents, it was shown that there are four pores in RyR. Among the conductance states of the slowly fluctuating current with LRP, the conductance of the 4OP (four opened pores) state was smaller than four times that of 1OP (one opened pore). To explain this fact, we proposed a model of the channel, a large central pore in series with four small parallel pores. The calculation according to the model has a good fit with the bilayer measurement.

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To investigate the mechanism of myogenic differentiation, we are using quail myoblast cells (QM cells) transformed with a temperature-sensitive mutant of Rous sarcoma virus (ts-RSV), termed QM-RSV cells. At 35.5°C, a permissive temperature for RSV, QM-RSV cells repeatedly proliferate without differentiation, but, at 41°C, a nonpermissive temperature, myogenic differentiation proceeds. This temperature dependency of the differentiation is derived from protein kinase activity of pp60^v-src, as tyrosine dephosphorylation is necessary for myogenic differentiation of QM-RSV cells. In this study, it was demonstrated that among four fusion inhibitors, aspirin, doxorubicin, HMBA and TPA, three of the inhibitors, except for TPA, inhibited inyogenin gene expression. Moreover, HMBA inhibited myoblast fusion accompanying inhibition of tyrosine dephosphorylation of certain proteins, and recovered the tyrosine kinase activity of pp60^v-src to a certain extent. To study the effect of HMBA on the intracellular localization of pp60^v-src, detergent-soluble and detergent-resistant fractions were prepared with Triton X-100. As a result, it was shown that pp60^v-src mainly exists in detergent-resistant fraction at 35.5°C. While almost all of the pp60^v-src at 41°C exists in detergent-soluble fraction. HMBA treatment retained pp60^v-src in detergent-resistant fraction even at 41°C. These results suggest that HMBA inhibits myogenic differentiation of QM-RSV cells by affecting the regulation of pp60^v-src.

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The ends of stress fibers in the basal portion of cells (basal stress fibers) are anchored to focal adhesions, and stress fibers in the apical part of cells (apical stress fibers) are attached to the apical membrane, forming a structure (the apical plaque; KATOH, K. et al. (1995). Cell Motil. Cytoskel., 31:177-195) resembling the focal adhesion. In addition to these two sites, stress fibers also make lateral contact with the plasma membrane but little is known about the molecular composition of this type of stress fiber-membrane interaction sites. Several actin-membrane association types are known, each employing a different set of proteins, and the focal adhesion and the erythrocyte membrane skeleton are the best characterized systems. We investigated by immunofluorescence microscopy if there is any morphological basis for the involvement of the erythrocyte membrane proteins in the stress fiber-plasma membrane association sites in cultured human fibroblasts. Our results indicated that fodrin (nonerythrocyte type spectrin) and ankyrin were generally associated with the plasma membrane, but that they were clearly excluded from the focal adhesion, the apical plaque and the stress fiber. Thus, it appears that the spectrin and the integrin based actin-membrane association systems are mutually excluded in the fibroblast membrane undercoat. Protein localization at the lateral stress fiber-membrane association site was also studied. Our data indicated that, while talin, vinculin, paxillin, fibronectin receptor and integrin β1 were present at the three stress fiber-membrane association sites, vitronectin receptor and integrin α_vwere absent from the apical plaque and the lateral association site. While the plasma membrane at the focal adhesion adheres tightly to a solid substrate, the cell surface of the apical plaque is free. Although the lateral association site faces the substrate, the molecular composition of this site is similar to that of the apical plaque.

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The sexual cycle of the cellular slime mold Dictyostelium discoideum offers a suitable system to analyze the mechanism of cell recognition during mating. Sexual cell fusion in D. discoideum typically occurs between complementary heterothallic strains. In addition, several bisexual strains are known which undergo sexual cell fusion with heterothallic strains of either mating type, but cannot do so by themselves. In the present study, trypsin digestion of cell surface molecules was found to induce self-fusion in a bisexual strain WS2162, suggesting the presence on the cell surface of a self-recognition molecule whose homophilic interaction interferes with the cell fusion mechanism.

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Fluorescence microscopy, with dyes analog of ceramide, and transmission electron microscopy, were used to analyze lipid traffic during interaction of Toxoplasma gondii with host cells. It is C6-NBD-Ceramide (C6-NBD-Cer), a fluorescent analog of ceramide, stained the Golgi complex where was metabolized into fluorescent shingolipid and glucosylceramide, and translocated via the Golgi complex to the plasma membrane of living cells. In uninfected cells, C6-NBD-Cer initially concentrated at the perinuclear region, and after its fluorescent products were present in the cytoplasm and the plasma membrane. In infected cells, the probe initially is stained the Golgi complex. After 4 hours incubation with C6-NBD-Cer, the parasites within the parasitophorous vacuole began to be stained, and at 5 hours incubation, the parasites are completely fiuorecent. The Golgi complex, as revealed by fluorescent probe and electron microscopy, maintained its perinuclear position throughout the evolution of intracellular parasitism. These results suggested that the intracellular parasite used the lipid pathway of the host cell.

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An in vitro model of apoptosis and differentiation in human embryonal carcinoma (EC) cells was developed to study the mode of cell death and mechanisms of cell death in early development. Death of these cells was induced by treatment with retinoic acid (RA) under the same conditions as those for induction of differentiation. The manner of this cell death was apoptosis, not necrosis, with the morphological criterion for apoptosis. Serum deprivation likewise caused apoptosis in both undifferentiated and differentiated EC cells. In differentiated EC cells, DNA fragmentation was observed in a smear pattern lacking the ladder pattern typically associated with apoptosis. However, in differentiated EC cells, DNA fragmentation occurred in various sizes.
The expression of a carbohydrate antigen, Le^Y, a reported marker of apoptotic cancer cells, was increased by the treatment with RA. However, two-color analysis by flow cytometry with nick end labelling method revealed that Le^Y expression was closely correlated with cellular differentiation but not apoptosis after RA treatment in the human EC cell system. Collection of Le^Y positive cells by the magnetic bead method demonstrated that this expression was not due to apoptosis but rather to differentiation. On the other hand, Le^Y expression associated with apoptosis was induced by serum deprivation in both undifferentiated and differentiated EC cells.
These results show that a subpopulation of undifferentiated EC cells takes the apoptotic pathway by induction of differentiation. The results also suggest that the population of cells taking an apoptotic pathway differs from a population of cells taking a differentiation pathway. This in vitro system for apoptosis in human EC cells will be useful for studies concerning apoptosis or programmed cell death in human developmental biology.

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A serum from a patient with HBV hepatitis was found to contain autoantibodies reacting with various mammalian cells. Immunofluorescence staining of cultured cells with the autoantibodies revealed that the antigen was localized at perinuclear regions, where the Golgi markers α-mannosidase II and β-COP were colocalized. The autoantigen disappeared from the perinuclear regions upon incubation with the fungal metabolite brefeldin A, and the immunostainable structures were fragmented into vesicles by treatment with nocodazole. These results strongly indicate that the antigen is localized at the Golgi complex. Immunoblots of cell lysates showed that the autoantibodies recognized a single protein with a molecular mass of 230 kDa in a variety of cell lines, indicating that the 230-kDa antigen is a conserved protein among mammalian species. We designated this protein GCP230 (Golgi complex-associated protein with a molecular mass of 230 kDa). When a postnuclear fraction was prepared and centrifuged, GCP230 was recovered in both cytosol and membrane fractions. Peripheral interaction of GCP230 with membranes was confirmed by phase separation in Triton X-114 solution and by extraction with sodium carbonate. Taken together, these results indicate that GCP230 is a peripheral membrane protein of the Golgi derived from the cytosol, although its function is not known at present.

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Ciliated protozoa, a group of unicellular eukaryotes, have two kinds of nuclei, a macronucleus (somatic nucleus) and a micronucleus (germinal nucleus) in a single cell. We previously reported that amicronucleate cells of Tetrahymena thermophila induced by nocodazole gradually lost their oral apparatus (OA) and ciliary rows but that amacronucleate cells and empty cells did not. Since the macronucleus is responsible for the gene expression in the vegetative phase, the effects of actinomycin D and cycloheximide on the disorganization of the OA in amicronucleate cells induced by nocodazole were investigated. These inhibitors prevented the disorganization of the OA in amicronucleate cells. Amicronucleate cells did not grow even in the medium supplemented with high concentration of Fe, Cu and folinic acid which allow cells to grow without formation of food vacuoles. The results suggest that the macronucleus in the amicronucleate cells plays an active role in the induction of disorganization of the OA and malfunctions of nutrient uptake from the cell surface and/or in the fundamental cell division mechanisms, resulting in the death of amicronucleate cells.

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We cultured isolated alveolar type II epithelial cells on a collagen gel matrix. At confluence, cultured type II cells were exposed to air. Under these conditions, the cellular density of the type II cells increased and they nodularly aggregated. The cultured cells consisted mainly of flattened epithelia intermingled with cuboidal cells. In the cytoplasm and at the apical surface of cuboidal cells, a surfactant protein was detected by immunohistochemistry and electron microscopy. Electron microscopic examination revealed that the surfactants in the cytoplasm increased when the cells were exposed to air. On the other hand, the flattened cells morphologically resembled type I cells in vivo. Air exposure treatment on the collagen gel matrix promoted the maintenance of the characteristic differentiation of alveolar epithelial cells. This culture system seemed to provide an appropriate physiological environment in which to study differentiation and disorders of pulmonary alveoli.

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7H6 antigen is a novel tight junction-associated protein first identified in our laboratory with a monoclonal antibody generated by immunizing mice with a bile canaliculus-rich membrane fraction obtained from adult rat liver. Although the antigen is found preferentially at the tight junction of epithelial cells in various organs in adult mammals, the relationship between 7H6 antigen and other tight junction proteins remains obscure. In the present study we examined the relationship between the immimohistochemical distribution of 7H6 antigen and occludin in chick intestinal epithelial cells during development in vivo, since occludin is the only known membrane protein composing tight junctions which is found thus far exclusively in avian tissues.
7H6 antigen was found as coarse dots along the basolateral cell membrane of intestinal epithelium in the chick embryo, whereas it was found as a fine dashed line along the cell border in the chicken. In contrast, occludin was found linearly together with ZO-1 at the cell border of intestinal epithelia both in the chick embryo and chicken. The possible significance of such different localization is briefly discussed.

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