Effects of UV on normal human epidermal keratinocytes were studied by measuring the intracellular free Ca²⁺ concentration ([Ca2⁺]i) using fluorescence ratio imaging (fura-2-AM). Upon UV irradiation the [Ca²⁺]i increased sharply after a certain lag time, and the UV sensitivity was higher at lower temperatures. Statistically the distribution of [Ca²⁺]i became broader as the mean values became larger, and the number of affected cells increased sharply above a certain fluenee (light intensity x time [photons/cm²]) at all wavelengths studied (200-400 nm). The action spectrum showed a single peak at about 230 nm and decreased gradually toward longer-wavelength UVregions.

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Separate treatment of PC12h cells with basic fibroblast growth factor (bFGF) and with epidermal growth factor (EGF) induced a selective decrease in the incorporation of radioactive phosphate into a 100, 000-dalton soluble protein during phosphorylation with (γ-³²P)ATP of soluble extracts from the cells, as was seen previously with nerve growth factor (NGF). This 100, 000-dalton soluble protein was designated in earlier studies as nerve growth factor-sensitive protein 100 (Nsp 100). The inhibitory effects of bFGF and EGF on Nsp 100 phosphorylation were prevented by pretreatment of PC12h cells with the calcium chelator, EGTA. Treatment of PC12h cells with the plant lectin wheat germ agglutinin (WGA), which binds to N-acetylglucosamine and sialic acid residues on glycoconjugates, blocked the inhibitory effects of bFGF, EGF, and NGF on Nsp 100 phosphorylation. The blockage by WGA was reversed by the addition of the lectin-specific sugar N-acetylglucosamine to the PC12h cultures. Although pretreatment of PC12h cells with succinylated WGA, which has the ability to bind to N-acetylglucosamine but not to sialic acid residues, failed to block the inhibitory effect of NGF on Nsp 100 phosphorylation as described previously, it did prevent the inhibitory effect of bFGF on this phosphorylation. These data suggest that in PC12h cells bFGF and EGF induce a decrease in the phosphorylation of Nsp 100 mediated through a Ca²⁺- dependent mechanism, as in the case of NGF. Furthermore, the blockage of the bFGF-induced inhibition of Nsp 100 phosphorylation by WGAand its succinylated form indicates that N-acetylglucosamine residues of bFGF receptor molecules might be involved in the mechanism by which bFGF inhibits the phosphorylation. On the other hand, staurosporine, a protein kinase inhibitor, blocked the NGF-induced decreGase in the phosphorylation of Nspl00, but failed to block the bFGF-induced one.

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Humansquamous cell carcinoma cells (NA cells) possess a large number of epidermal growth factor (EGF) receptors and their growth is inhibited by EGF. Recently, we isolated a series of variants which escaped EGF-mediated growth inhibition. The variant ER11 cells expressed a decreased level of EGFreceptors and grew in an EGF-dependent fashion. Treatment of ERll cells with EGFresulted in the activation of protein kinase C, which was followed by the enhancement of 80-kDa protein phosphorylation as observed in NA cells. Thus, EGF can activate not only tyrosine kinase but also protein kinase C in both NA and ER11 cells. The EGF dependent growth stimulation in ER11 cells was inhibited by 12-0-tetradecanoylphorbol 13-acetate (TPA). Exposure of NA and ER11 cells to TPA for 30 h resulted in the down-regulation of protein kinase C. In these protein kinase C-deficient cells, EGF was able to activate autophosphorylation of the EGF receptor. The EGF-activated EGF receptor kinase phosphorylated numerous cellular proteins even in the protein kinase C-deficient cells. However, there were less tyrosine-phosphorylated proteins in ER11 cells than in NA cells. These results suggested that protein kinase C is necessary for the EGF-dependent growth stimulation of ER11 cells and that several tyrosine-phosphorylated proteins commonlyobserved in both NA and ER11 cells seem essential for cell proliferation.

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The distribution of fibronectin (FN) and laminin (LM) at developing capillaries during various developmental stages, from capillary sprouts to relatively developed capillaries, was studied by light- and electron- microscopy immunocytochemistry. By light-microscope, FN immunoreactivity was diffusely distributed throughout the stroma of the granulation tissues, while for LMit was preferentially distributed at the perivascular region with the various developmental stages of the immature capillaries. Ultrastructural study revealed that capillary sprouts were closely surrounded by plentiful deposits of immunoreaction with the FN, but only faintly for LM. Relatively developed capillaries with large and tall endothelium were surrounded by plentiful immunoreactiye products with both FNand LM, and immunoreactivities in the cisternae of rER of the endothelium and/or the pericytes were also shown. Cytoplasmic interdigitations between the endothelium and the pericyte of developing capillaries were recognized without an immunoreaction for FNand LM. These results mean that the capillary sprouts are associated with a prepatterned FN-rich and LM-poorperivascular matrix, whereas relatively developed capillaries are associated with a FN- and LM-rich perivascular matrix which would have been produced by the capillary endothelium and/or pericytes.

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Three temperature-sensitive cell-cycle mutants of rat 3Y1 fibroblasts (3Y1tsD123, 3Y1tsG125, and 3Y1tsH203, each belonging to distinct complementation groups) were transformed with plasmid DNA carrying Harvey murine sarcoma virus cDNA. The criteria for transformation were increase in saturation cell density, capability to clone in soft agar, and alteration in the cellular morphology. At 39.8°C (restrictive temperature of the parental cell lines), all the transformed sublines of each mutant ceased to proliferate and were arrested reversibly in the G1 phase of the cell cycle like the parental lines. At both 39.8°C and 33.8°C (permissive temperature for the parental lines), all the untransformed parental lines synthesized p21^ras at low rate. At 33.8°C, all the transformed sublines synthesized p21^ras at much higher rate and expressed the morphological phenotype characteristic to v-H-ras-induced transformation. At 39.8°G, the rate of p21^ras synthesis was not changed in the transformed sublines of 3Y1tsD123 and 3Y1tsG125, and the morphology of transformed phenotype also remained intact. In the transformed subline of 3YltsH203, the rate of p21^ras synthesis was lowered at 39.8°C to that seen in the untransformed parental line, and the transformed phenotype in morphology disappeared. In all of the transformed sublines, the amount of v-H-ras mRNA markedly expressed at both 33.8°C and 39.8°C. We conclude that in both v-H-ras-tranformed sublines of 3YltsD123 and 3YltsG125 the temperature arrest is not unlocked by the process specifically causing the transformed phenotype, and that in the v-H-ras-transformed subline of 3YltsH203, the process specifically causing the transformed phenotype is blocked by the mutation after transcription of the v-H-ras oncogene.

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Spindle pole bodies (SPBs) attached to nuclei were isolated from Saccharomyces cerevisiae; they nucleated microtubules to form asters in vitro. This aster-forming activity was stable in 30%dimethylsulfoxide or 10% 2-propanol and could be solubilized with KC1. The KC1 extract contained many protein components, which aggregated upon dialysis against a low concentration salt solution. When incubated with tubulin, the aggregates formed asters. Measurements of the elongation rates of the astral microtubules indicated that the microtubules were nucleated from the SPBsor from the aggregates reconstructed from the KC1 extract by dialysis.
The plus end was distal to the astral center, as in the case of the microtubule organizing center (MTOC) of mammalian cells. We suggest that the proteins extracted with KC1 are responsible for microtubule nucleation in SPBs and that this SPBseems to have the same mechanism for microtubule nucleation as the MTOC in higher eukaryotes.

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Human mammary carcinoma cell line MCF-7 cells grown on type I collagen gels floating in a medium occasionally invaginated into the gels as a cell mass and formed cylindrical or domed structures within it. The 0.05% Triton-insoluble cytoskeleton of such cellular structures sedimented as a white flocculent layer at the boundary between 60 and 70% sucrose layers by ultracentrifugation, and consisted of 4 basal components: 54-kD (β-tubulin), 45-kD, 42-kD (actin), and 39-kD polypeptides. By contrast, the isolated cytoskeleton of MCF-7 cells grown as monolayers on plastic substratum formed a finer cytoskeletal network with a smaller buoyant density and consisted of two distinct polypeptides with apparent molecular sizes of 80-kD and 65-kD in addition to the 4 basal components found in the morphologically developing cells. The present results indicate that the cytoskeleton of MCF-7cells forming the three-dimensional cellular structures within collagen gels is lacking in these two polypeptides, and that it has a coarser cytoskeletal network with a greater buoyant density than that of the monolayeredcells on plastic.

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