The induction of JE/MCP-1 gene by TPA was transcriptionally suppressed by antioxidants such as pyrrolidine dithiocarbamate (PDTC) or trimethylthiourea (TMTU) in Balb3T3 cells, whereas that of other early response genes, c-fos or egr-1, was not affected by these agents. Induction of the JE gene by TNFα or serum was not completely inhibited by these antioxidants. The antioxidants inhibited an increase in intracellular oxidized state of cells treated with TPA. Next we examined the transcriptional regulatory region of the rat JE gene to determine the genomic target of active oxygen species. The chloramphenicol acetyltransferase (CAT) reporter gene, containing the 5 -upstream region approximately 2.6 kb DNA from the cap site, was transfected into Balb 3T3 cells. The CAT activity induced by TPA increased in parallel with the endogenous JE mRNA level, and the increase was inhibited by the antioxidants. The essential region for this response in the upstream region was within the -2.6 to -2.0 kb region, and further denned to -2, 224 to -2, 069 bp which contained an NFκB-binding element. Gel shift analysis indicated that the nuclear factors that bound to this essential element contained NFκB, and that NFκB activity was stimulated by TPA and inhibited by PDTC. These results suggest that active oxygen species are involved in induction of the JE gene caused by TPA in Balb 3T3 cells, through NFκB activation.

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SI proteins A-D are hnRNP proteins which were originally isolated from cell nuclei of various tissues, by selective extraction at pH 4.9 from the supernatants of nuclei mildly treated with DNase I or RNase A. In the present study, a hybridoma was isolated which produced a monoclonal antibody that reacted specifically with SI proteins C2 and D2. When the antibody was used in indirect immunofluorescence staining of cultured cells, it stained, in addition to the nuclei, the cytoskeleton-like fibrous structures in the cytoplasm. We demonstrate that the cytoskeletal filaments are vimentin intermediate filaments. This is the first report on the hnRNP protein-association with cytoskeleton, and will help to clarify cytoplasmic mRNA localization as well as cytoplasmic distribution of hnRNP proteins.

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α-1, 3-Fucosyltransferase (Fuc TIV) cDNA was placed under the control of β-actin, cytomegalovirus enhancer/promoter and transfected into embryonic stem cells. The transfected cell clones with integrated cDNA (positive clones) differentiated more efficiently into myocardial cells than the clones without integrated cDNA (negative clones) or parental cells. Furthermore, myocardial cells differentiated from the positive clones survived longer than those differentiated from the negative clones or parental cells. These results indicate that Lewis X structure, the product of Fuc T IV, enhances myocardial differentiation. The mechanism of the phenomenon is discussed in relation to integrin action.

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We established an in vitro cell-free system with which to evaluate the effects of basic substances and acidic ionophores on the internal pH and integrity of FITC-dextran (FD)-loaded lysosomes isolated from the rat liver. In this system, basic substances and acidic ionophores not only increased the internal pH dose-dependently, but also disrupted the lysosomes in the presence of Mg-ATP, which was detected as the release of FD from lysosomes. All of the vacuoligenic bases and acidic ionophores, but none of the non-vacuoligenic bases or neutral ionophores disrupted the lysosomes, suggesting that this phenomenon is an in vitro manifestation of vacuole formation induced in vivo by basic substances and acidic ionophores. Lysosome disruption required a functional proton pump as well as permeant anions. It was inhibited by inhibitors of the lysosomal proton pump, including bafilomycin A₁, N-ethylmaleimide (NEM), and N, N'-dicyclohexylcarbodiimide (DCCD), or when permeant anions were replaced with impermeant anions. It was also suppressed by increasing the osmotic pressure of the surrounding medium, suggesting that it was caused by osmotic swelling of lysosomes induced by protonated bases or cations characteristic of particular ionophores that accumulated within lysosomes driven by the proton pump. Furthermore, this lysosomal disruption was inhibited by cytosolic factors. This phenomenon will provide an in vitro system for studies on osmoregulation and the intracellular dynamics of the lysosomal system, including membrane fusion.

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Actin filament organization may play an important role in the maintenance of differentiated functions in epithelial cells. We previously reported our success in inducing and maintaining gap junctions, which are two kinds of differentiated function, in primary rat hepatocytes cultured with 2% DMSO and 10^-7 M glucagon. In the present study, we demonstrated the formation of actin filament networks in the hepatocytes cultured with 2% DMSO and 10^-7 M glucagon. Actin filaments in hepatocytes cultured in medium with only 2% DMSO added from 96 h after plating were concentrated under the plasma membrane and were observed to be circumferential. In hepatocytes cultured in the medium with both 2% DMSO and 10^-7 M glucagon added from 96 h, not only the circumferential actin filaments but also the formation of actin filament networks were observed and the networks developed well with time in culture. The networks were observed as a dome-like structure under the cell face and terminated at the circumferential actin filaments. They were composed of electrondense star-like vertices connected by microfilament bundles of varying length and were also very sensitive to the actin disrupter cytochalasin B. However, during the network formation, there were no significant increases in the amounts of actin protein and mRNA. The actin filament networks of the hepatocytes in this culture system might be closely related to the maintenance of differentiated functions.

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Slime, the extracellular matrix of Physarum plasmodium, is secreted by the exocytosis of a vesicles that contain a slime precursor. Using an antibody raised against biochemically purified slime, we detected the intracellular localization of the slime vesicle. Slime vesicles are abundant in the advancing front of the plasmodium, as confirmed by electron microscopic observation in two different cross-sectional angles. Screening various reagents, we found that rhodamine-phosphatidylethanolamine (Rh-PE) binds specifically to slime in both its intravesicular and extracellular forms, as confirmed by immunoelectron microscopy using an antibody against fluorochrome rhodamine. The plasmodia vitally stained with Rh-PE exhibited dynamic fluorescent patterns during the course of locomotion. The fluorescence was conspicuous at the periphery of the leading pseudopods and oscillated according to the shuttle streaming that accompanied the relaxation and contraction of the periphery; it was intense in the relaxation phase when pseudopods extended, and became weak in the contraction phase when pseudopods contracted. The results collectively mean that the slime vesicles carried by the cytoplasmic streaming accumulated prior to secretion at the advancing margin of the plasmodium.

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Four different β-tubulin clones were isolated from carrot genomic and cDNA libraries. Their nucleotide sequences were determined1 and their predicted amino acids were compared with each other. The predicted amino acid composition of the C-terminal region of three of them (β-1, 3, 4) resembled one another, but that of one isotype (β-2) was divergent. The β-2 tubulin included two hydroxyl amino acids, serine and threonine, and consisted of a lower number of negatively charged amino acids than the others in the C-terminal region.
The predicted hydrophobicity profile of the β-2 tubulin around the residue 200 is less hydrophobic than β-1, but it is still more hydrophobic than those of animal and fungal β-tubulins. The β-2 gene was transcribed in cultured cells and flowers, while the β-1 gene was ubiquitously transcribed in cultured cells, roots, shoots and flowers.
When the predicted amino acids of plant tubulin were compared with those of other organisms, substitutions from non-polar amino acids to those with hydroxyl group were conspicuous in the region corresponding to the third exon in the plant genes.

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