Characean cells have contributed significantly to various areas of plant cell biology such as cell motility and membrane transport. Since characean cells are very large, various kinds of operations can easily be applied to them. Development of techniques of intracellular perfusion and permeabilization of plasma membrane has facilitated studies on functions of the plasma membrane and the vacuolar membrane(or tonoplast) which is specific to plant cells. The present article is aimed at reviewing the contribution of characean cells to the study of electrophysiological characteristics of plant membranes. Our attention was mainly focused on experiments using plasma membrane-permeabilized cells and intracellularly perfused cells.

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The role of microtubule associated proteins (MAPs) on the dynamic instability of microtubules was examined under a dark-field microscope using bovine brain tubulin purified by DEAE-Sepharose column chromatography. In the absence of MAPs, the transition from the shortening phase to the growing phase (the rescue) occurred rarely both in self-assembled microtubules and seeded ones, especially at the plus end. Even under the conditions unfavorable to stabilize microtubule, the addition of a small amount of crude MAPs or purified microtubule associated protein 2 (MAPs) to the microtubules allowed them to undergo the rescue. At increased concentrations of MAPs or MAP2, both the length change required for a rescue during shortening phase ("shortening length") and for a catastrophe (transition from the growing to the shortening phase) ("growth length") decreased. Under these conditions, the rescue often occurred at the same site where previous rescues occurred. Distribution of immunofluorescent MAP2 antibodies along individual microtubules showed that MAP2 molecules bound onto microtubules by forming discrete clusters. The number of MAP2 molecules per cluster was estimated to be between 25 and 60. Because both the "shortening length" and the distance between MAP2 clusters in a microtubule decreased with increased MAPs concentration, we suggest that the MAP2 clusters may form the specific site at which the shortening of the microtubule readily stops. MAP2 possibly regulates the dynamic instability by stopping the shortening, which is a prerequisite for the rescue.

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We previously found that type V collagen repressed the attachment and spread of aortic smooth muscle cells. The present study was carried out to investigate the effects of type V collagen on the formation of fibronectin and F-actin filaments of human dermal fibroblasts in relation to cell attachment and spread, using an immunofluorescent technique and morphometry. The number and area of the cells attached to type V collagen at 1 and 3 hours after seeding were significantly lower than those of cells on other substrates, including collagen types I, III and IV, and bovine serum albumin. However, there was no significant difference in the attachment and spread among the cells on these substrates after 24 hours. Cultured fibroblasts exhibited two patterns of fibronectin ; one was a clear, linear fibronectin localized mainly in the cellular margins, and the other was a granular or flocculent fibronectin found in the perinuclear areas. The former was stained in non-permeabilized cells, but not in trypsin-treated cells (cell surface fibronectin). In contrast, the latter was not detected in nonpermeabilized cells, but was found in trypsin-treated cells (perinuclear fibronectin). Most of the cells cultured on type V collagen did not form either linear cell surface fibronectin or F-actin filaments at 3 hours. In contrast, many cells on collagen types I, III, and IV developed both cell surface fibronectin and F-actin filaments, the distributions of which were partially coincident. Colocalization of linear cell surface fibronectin and F-actin filaments was found in cells on all of the substrates after 24 hours. Perinuclear fibronectin showed similar patterns, and was not colocalized with F-actin filaments on different substrates at 3 and 24 hours of culture. Solid-phase substrates induced a better cellular attachment at 3 hours than serum adhesive factors. The administration of monensin, which inhibits the secretion of protein products, decreased the intensity of the fluorescence of cell surface fibronectin in fibroblasts, which was observed in a clear line. These results suggest that the retardation of the initial attachment and spread of fibroblasts on type V collagen is related to an inhibition in the formation of the fibronexus, a close transmembranous association of individual fibronectin fibers and F-actin filaments.

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Human hepatoma cell lines (Hep 3B-TS, PLC/PRF/5, and Hep G2), sensitive to growth inhibition by transforming growth factor β1 (TGF-β1), express TGF-β receptors type I, type II, and type III. We report that TGF-β1 protein selectively increased steady-state levels of the mRNA for the serine/threonine kinase receptor 1 (SKR1), a member of the TGF-β superfamily receptor genes in these cells, whereas TGF-βl had little effect on expression of the TGF-β receptor type II gene. This increase of SKR1 mRNA in Hep 3B-TS cells could be detected by Northern blot analysis within 3h of addition of TGF-β1 to the cells, and enhanced message levels peaked at 12h as long as TGF-β1 was present in the culture medium. Hep 3B-TR cells which were resistant to TGF-β1 due to lack of both TGF-β receptors type I and type II, expressed SKR1 mRNA, but it was not induced by TGF-β1 protein. The increased expression of SKR1 mRNA in the cells was actinomycin D-sensitive and was not dependent on new protein synthesis. The results indicate that TGF-β1 selectively induces SKR1 message at a transcriptional level by a positive regulator.

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Production of HVJ(Sendai virus) virion is suppressed in the infected LLC-MK₂ cells in the low Ca²⁺ condition. To clarify this phenomenon, the effect of Ca²⁺ on HVJ morphogenesis was examined under normal and low Ca²⁺ conditions. The HVJ production was observed in standard medium (containing 1.8 mM Ca²⁺) within 24 hr after infection and increased with the culture time. Indirect immunofluorescent staining with an anti-HVJ envelope antibody showed that the viral glycoproteins, HANA (HN) and F proteins, were synthesized within 10 hr after infection, diffused to the cell surface, and then many patches strongly stained with this antibody were formed on the cell surface. In contrast, when the infected cells were cultured in low Ca²⁺ medium, viral glycoproteins were synthesized as in the case of standard medium. However, the glycoproteins were almost all accumulated in the Golgi apparatus, suggesting that expression of viral glycoproteins at the cell surface was suppressed, and the patches were hardly seen on the cell surface. When the low Ca²⁺ medium was replaced by standard medium, the accumulated viral glycoproteins were rapidly diffused and expressed on the cell surface, HVJ production was restored and patches formed on the cell surface. By confocal laser scanning microscopy, the results were seen more clearly ; Ca²⁺ is necessary for the expression of viral glycoproteins at the cell surface. By electron microscopy under normal conditions, groups of viral budding sites were seen collected in restricted areas, corresponding to the patches observed on immunofluorescent staining, but the budding sites were not seen on the cells cultured in low Ca²⁺ medium.These results suggest that Ca²⁺ specifically affects the expression of viral glycoproteins on the cell surface, and that Ca²⁺ is possibly associated with the assembly of HVJ-specific components necessary for formation of viral budding sites.

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Previously we have reported that Ca²⁺ concentration in culture medium affects the morphogenesis of HVJ from infected cells. We further investigated the effects of Ca²⁺ on HVJ morphogenesis in LLC-MK₂ cells, focusing on M (matrix) protein which plays an important role in the assembly of viral components. On culture in standard medium (containing 1.8 mM Ca²⁺), most of the M protein accumulated on the apical surface in patches. On double immunofluorescent staining with anti-M protein monoclonal antibody and polyclonal antibody against HVJ glycoproteins, M protein and viral glycoproteins were observed in the same patches at the cell surface. By contrast, in low Ca²⁺ medium, M protein was evenly dispersed over the cell surface, especially in the regions of cell-cell contact, and was not seen in patches. Immunoblot analysis for M protein in the infected cells showed that the total amount of M protein was almost the same irrespective of Ca²⁺ concentration in the culture medium. But, there was a distinct difference in the forms of M protein found in the cells : most of the M protein in the cells cultured in standard medium was of the non-phosphorylated form, whereas the phosphorylated form of M protein was distinctly increased in the cells cultured in low Ca²⁺ medium. These results suggest that Ca²⁺-deficiency in the medium blocks dephosphorylation of phosphorylated M protein necessary for patch formation, resulting in disturbance of viral morphogenesis.

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The localization of estrogen receptors (EsR) in the tumor tissues of submandibular glands was examined in female rats, using the indirect immunoperoxidase method in combination with the in situ hybridization technique. Tumors were experimentally produced by 9, 10-dimethyl-1, 2-benzanthracene (DMBA), and the tumor tissues were fixed with formalin or paraformaldehyde and then embedded in paraffin. In the DMBA-induced submandibular gland tumors, immunoreactivity to EsR-peroxidase conjugate was found in nuclei of the tumor cells which occupied the peripheral rim of the tumor cell nests. In contrast, the reactivity in the normal submandibular glands without tumor was mostly confined to nuclei of the duct cells. When EsR mRNA expression was analyzed in the tumor tissue by in situ hybridization with a cDNA probe, its distribution was identical with that of immunoreactivity to EsR. These data suggest that the ductal cells of the submandibular gland are responsive to ovarial steroids, and that estrogens may play an important role in the maintenance of growth of the submandibular gland tumors.

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tsJT16 is a cell-cycle temperature-sensitive (ts) mutant derived from rat fibroblasts whose functional defect appears soon after the growth stimulation from G0 phase. In addition to c-fos, c-myc and ornithine decarboxylase gene, 7 primarily inducible genes, c-jun, KC, JE, 2F1, 2A9, egr-1, and egr-2, were further shown to be expressed after serum stimulation at both permissive and nonpermissive temperatures. However, expression of secondarily inducible genes, cyclin D1 and D3 and cdk2, was ts and was cycloheximide sensitive. Expression of cyclin C was not inhibited by cycloheximide but it was ts. Failure in expression of G1 cyclins and Cdk2 is suggested to be a causal event for inability of growth induction of tsJT16 at the nonpermissive temperature.

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