We previously investigated the biochemical characteristics of microtubule-associated proteins (MAPs) of the adrenal medulla and adrenal cortex and found that they contain a new kind of MAP with a molecular weight of 190, 000 (190 kD MAP) as a major species (Kotani, S., H. Murofushi, S. Maekawa, C. Sato, and H. Sakai. Eur. J. Biochem. 156, 23-29, 1986). We now have used an affinity purified anti-(190 kD MAP) antibody and show by indirect immunofluorescent microscopy the association of this MAP with microtubules in situ in TIG-3 cells (human embryonic lung fibroblasts). The 190 kD MAP was present along the interphase and mitotic microtubules, and there was no marked difference between the staining pattern with anti-tubulin and that with anti-(190 kD MAP) antibodies, evidence that the localization of 190 kD MAP is not restricted to the subset of microtubules. We also isolated MAPs from TIG-3 cells and identified their 190 kD MAP as a major heat-stable component. Several other unidentified polypeptides were recovered in the MAP fraction specifically.

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Immunoblots of proteins extracted from the skin of a small viviparous fish (Xiphophorus) showed that a monoclonal antibody against human urokinase recognizes multiple molecular weight species of antigens. The immunoaffinity-purified antigens had serine-protease activity for the hydrolysis of a chromogenic substrate and could convert human plasminogen to plasmin in a manner similar to that for human urokinase in vitro. Two antigens with apparent molecular weights of 55 and 50 kilodaltons that had been purified on a fibrin-Celite column were separable on SDS-polyacrylamide gels and were characterized as major plasminogen activators on fibrin-agar indicator plates. The ¹²⁵I-tryptic peptide maps of both antigens were similar to that of human urokinase; therefore, the fish activators and human urokinase are structurally and functionally related.

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Epidermal growth factor (EGF) induced the formation of thin sheetlike extensions (lamellipodia) and filamentous extensions at the edges of colonies of A431 cells. To determine the necessary processes for the induction of the morphological changes mediated by EGF, the effects of a variety of ions on these changes were examined. In a NaCl solution supplemented with CaCl₂, MgCl₂ and glucose, no EGF-induced morphological changes were observed. However, when the NaCl was replaced with LiCl, fingerlike extensions were formed, but sheetlike extensions were not. Addition of vanadate to the NaCl solution also induced fingerlike extensions in cells treated with EGF. In contrast, sheetlike lamellipodia were formed in EGF-treated cells by the addition of K⁺ or PO₄^3-to the NaCl solution or by the addition of PO₄^3- to the LiCl solution. These findings indicate that Li⁺, K⁺, PO₄^3- and vanadate are involved in the processes of EGF-induced morphological changes. Since vanadate and Li⁺ have been shown to inhibit phosphatases, an EGF-dependent phosphorylation step may play an important role in the induction of the morphological changes observed.

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When stimulated with lipopolysaccharide (LPS), primary cultures of Kupffer cells from the rat secreted a hepatocyte-stimulating factor (Kupffer factor) which induced the synthesis of α₂-macroglobulin (α₂M) by hepatocytes. The effect of this Kupffer factor was found only when dexame-thasone was present in the hepatocyte culture medium. Dexamethasone alone stimulated the α₂M synthesis, to some extent, in a dose-dependent manner. When both Dex and the Kupffer factor were added to the medium, cultured hepatocytes synthesized 100-fold α₂M, quantitative evidence that explains the in vivo phenomenon in which the serum α₂M concentration increases more than 100-fold in the acute phase. A co-culture study with hepatocytes and Kupffer cells indicated that the latter cells have a major function in the induction of α₂M synthesis in vivo. A hybridization study with rat α₂M cDNA showed that the concentration of mRNA increased in cultured hepatocytes in the presence of the Kupffer factor, but only when dexamethasone was present.
From these results we concluded that Kupffer cells secrete a hepatocyte-stimulating factor when stimulated by LPS and that glucocorticoid is essential for the induction of α₂M in rat hepatocytes. The effect of the Kupffer factor and glucocorticoid appears to be regulated in the pretranslational, probably the transcriptional, phase.

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Fluorescently labeled tubulin was quickly incorporated into the mitotic apparatus when injected into a live sand dollar egg. After a rectangular area (1.6 × 16 μm) of the mitotic spindle was photobleached at metaphase or anaphase by the irradiation of a laser microbeam, redistribution of fluorescence was almost complete within 30 sec. The photobleached area did not change in shape during the redistribution. During the period of redistribution, the bleached area moved slightly toward the near pole at metaphase and anaphase (means: 1.6 and 1.8 μm/min, respectively). These results indicate that redistribution was not due to the exchange of tubulin subunits only at the ends of microtubules but to their rapid exchange at sites along the microtubules in the bleached region. Furthermore, treadmilling of tubulin molecules along with the spindle microtubules possibly occurred at the rate of 1.6μm/min at metaphase.
Birefringence of the mitotic apparatus increased with a large increase in both the number and length of astral rays shortly after taxol was injected. However, the microtubules did not all seem to elongate at the same rate but appeared to become equalized in length. Chromosome movement stopped within 60 sec after the injection. Centrospheres became large and the labeled tubulin already incorporated into the centrospheres was excluded from the enlarged centrospheres. Shortly after the labeled tubulin was injected following the injection of taxol, it accumulated in the peripheral region of the centro-spheres, suggesting that microtubules first assembled at this region. Fluorescently labeled tubulin in the mitotic apparatus in the egg after injection of taxol was redistributed much more slowly after photobleaching than in uninjected eggs.

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We previously reported that proteins from serum-free con-ditioned medium (PCM) of rat embryo fibroblasts promote the adhesion of nonmalignant rat liver epithelial cells (RL34) seeded on a collagen substratum (Yamada, M. and Okigaki, T., Cell Biol. Int. Rep. 7, 1115(1983)). We now have compared the adhesion and growth of RL34 cells seeded on an extracellu-lar matrix (RLECM), a product of RL34 cells, or on a collagen (type I) sub-stratum with or without PCM in the medium.
The adhesion rate for RL34 cells on RLECM was higher than on collagen, and once RL34 cells adhered to the collagen substratum they showed no growth, whether or not PCM was present. Cells that adhered to RLECM grew, and the growth rate was markedly higher in medium containing PCM than in medium without it.
Immunological, enzymatic and chemical analyses revealed that the adhesion-and growth-promoting activities of RLECM involved trypsin-sensitive proteins other than collagens, fibronectin (FN) or laminin (LN), and that heparan sulfate proteoglycan may be a major component of RLECM.

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Secondary hyphae of Lyophyllum ulmarium were shown to tolerate slow freezing, which allowed extracellular freezing, to -196°C. A freeze-fracture study showed that under this non-lethal freezing condition, the plasma membrane of the secondary hyphae did not show any ultrastructural changes as compared with the control, except gross cellular shrinkage. Tertiary hyphae of Lyophyllum ulmarium, on the other hand, were completely injured by slow freezing to -196°C, and the plasma membrane showed distinct intramembrane particle aggregation as a result of direct membrane contact caused by severe cellular deformation. It is suggested that the absence of freezing injury in the secondary hyphae was due to ultrastructural preservation of the plasma membrane, which resulted from avoidance of severe cellular deformation, while occurrence of freezing injury in the tertiary hyphae is considered to be due to ultrastructural changes in the plasma membrane caused by severe cellular deformation.

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Large numbers of growth cones were present in 6-day-old primary cultures of cerebral hemispheres from fetal rats. The average size of the growth cones was 24 by 28 μm. Many of these growth cones had both veil-like lamellipodia and filopodia. A few cones remained in 21-day-old cultures. These also had lamellipodia and filopodia. Ganglioside GM₁. was present in both 6-day-old and 21-day-old cultured growth cones.

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Phagocytosis of microspheres in V79 Chinese hamster lung cells was investigated by flow cytometry. Fluorescent microspheres (1.8 μm diameter) were used as the ingesta. Change in the number of V79 cells containing fluorescent microspheres was measured as an index of phagocytic activity. With time there was a sigmoidal increase in cells containing microspheres. The phagocytosis of microspheres is partially explained by two parameters introduced to describe the sigmoidal curves.

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A monoclonal antibody, M2590, that recognizes hematoside (GM₃) was used to analyze the immunostaining localization of GM₃ of the surface of transformed and non-transformed hamster embryo fibroblasts and B16 melanoma cells. The reactivity of GM₃ with the antibody changed markedly depending on the cell density. At the sparse density cells were clearly made visible by the antibody, but at the confluency the accessibility of the antibody to GM₃ was greatly decreased. This density dependent change in the reactivity of GM₃ was found for both normal and transformed cells. The staining pattern of GM₃ was examined in relation to the actin fibers made visible with NBD-Phallacidin during cell spreading. When the cells were still round, the GM₃ on microspikes or blebs was highly reactive with the antibody, and by the time cells showed circumferencial staining of their actin fibers, GM₃ had been distributed over the entire cell surface as punctuated spots. GM₃ also was visible in substrate attachment materials (SAM). Trypsin treatment of SAM diminished the reactivity of GM₃ with the antibody. The antibody did not inhibit cell attachment or spreading on a substratum coated with fibronectin or laminin.

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Adenovirus type 12 (Ad12) Ela-transformed rat cells (HY1) grew in methocel medium containing 9 % fetal calf serum (FCS), but the frequency of colony formation was very low (the order of 10^-4). The addition of conditioned medium or a high concentration of serum (20 % FCS) to the methocel medium accelerated colony formation, and plating efficiency increased 10-to 100-fold. In contrast, in stationary culture, HY1 cells grew well even in 1 %-FCS medium. These results indicate that HYI cells require high concentrations of growth factors for anchorage-independent growth. The effects of conditioned medium or FCS also were demonstrated in several transformed cell lines induced by transfection of combined sets of Ad12-transforming genes (Ela, Elb and E4). These growth behaviors suggest that the first step in cell transformation with adenovirus 12 is the acquisition of responsiveness to growth factors in methocel culture, which must be the function of the Ad12-Ela gene products. The function of the other two Ad12-transforming genes was discussed.

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Phorbol-12, 13-dibutyrate, epidermal growth factor, and insulin raised the intracellular pH ( [pH]i), presumably through the activation of a Na⁺/H⁺ antiporter. Addition of amiloride or replacement of extracellular Na⁺ by choline which abolishes the cytoplasmic alkalinization prevented the stimulation of hexose transport by these agents. Furthermore, monensin, a Na⁺/H⁺ ionophore which increases the [pH]i, stimulated hexose transport. This stimulation was also prevented by the replacement of extracellular Na+ by choline. These observations suggest that stimulation of the Na+/11+ antiporter may have stimulated the increase in hexose transport.

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c-myc gene was amplified in the human ovarian tumor line OS-4 5-6-foldand in the OvCa-1 line 3-4-fold. The relative amount of the transcript from the c-myc gene to the total RNA in OS-4 was 6-fold that for normal cells.

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Two-dimensional gel electrophoresis revealed two protein spots, a (molecular weight 70, 000, pI 4.6) and b (molecular weight 69, 000, pI 4.4) in PC-12 cells (rat pheochromocytoma cells). When the cells were induced to differentiate with nerve growth factor, the amount of protein in spot a, and later spot b increased with time, then the amount in both spots gradually decrease to undetectable level. These spots were not detected in adult rat brain nor in other cell lines of rat and mouse. Thus, these proteins can be used as markers to follow the differentiation of PC-12 cells.

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Surface antigens of Saccharomyces cerevisiae protoplast were visualized immunocytochemically with protein A-gold or ferritin conjugated antibodies. Ferritin particles were densely distributed at the surface of yeast protoplast after treatment with rabbit anti-protoplast serum and ferritin conjugated antibody against rabbit IgM. Control experiments demonstrated that the binding of these ferritin markers was specific for surface antigens. The composition of these surface antigens, however, remain to be investigated.

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