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Various spatiotemporal patterns of dark and light stripes are formed in the Ag and Sb co-electrodeposition system. In this research, we investigate the time-evolution of the stripe width of a spatiotemporal stripe pattern with a size of ca. 10 μm. The results show that the stripe width increases with time and is eventually saturated. Such a phenomenon is predicted by some theories of spinodal decomposition of materials with chemical reactions in a nonequilibrium system. This suggests that the pattern is formed by the spinodal decomposition of metal alloys, Ag and Sb, with chemical reactions in a nonequilibrium system, which has not yet been reported in a metallic system.

Fibroblast growth factors (FGFs) play a role in biological processes such as cell growth and development, angiogenesis, and wound healing. Several genes have been shown to be induced by FGFs, but the underlying mechanisms have not been elucidated. We investigated the effect of FGF-2 (basic FGF) on the urokinase-type plasminogen activator (uPA) gene in NIH 3T3 fibroblasts. We found that the uPA gene is transcriptionally induced by FGF-2 as well as by 12-O-tetradecanoylphorbol-13 -acetate involving a PEA3/AP1 element located 2.4 kb upstream of the transcription initiation site; neither induction requires ongoing protein synthesis. Unlike 12-O-tetradecanoylphorbol-13-acetate induction, FGF-2 induction was not impaired by protein kinase C down-regulation. Analyses of various signaling molecules by Western blotting, extracellular signal-regulated kinase (ERK) activity assays, and transient transfection assays (cotransfection of a uPA-reporter gene construct with expression vectors for wild-type or dominant negative type of these molecules or for ERK-specific protein phosphatase MKP-1) showed that a Ras/Raf-1/MEK/ERK-2/JunD pathway is induced by FGF-2 and 12-O-tetradecanoylphorbol-13-acetate, leading to the activation of the uPA gene.

Expression of the urokinase-type plasminogen activator (uPA) gene in LLC-PK1 cells can be induced by signals mediated by both cAMP-dependent protein kinase (PKA) and Ca(2+)- and phospholipid-dependent protein kinase (PKC). We have utilized the tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA) to down-regulate PKC, in order to test for an effect on the PKA-mediated induction of the uPA gene expression. Incubation of cells for 24 h with 100 ng/ml TPA caused a marked decrease of PKC protein, both in cytosolic and particulate fractions, and an 85% reduction of total PKC activity. After down-regulation of PKC, uPA mRNA accumulation induced by 8-Br-cAMP was 5-10-fold higher than in control cells. Both uPA mRNA stability and uPA gene transcription rates induced by 8-Br-cAMP were increased by PKC down-regulation (6- and 1.8-fold, respectively). Although total PKA activity was reduced by 20% in extracts from PKC-depleted cells, activation of PKA by 8-Br-cAMP was 2.5-fold higher than in control cells. This enhanced activation of PKA in PKC-depleted cells also occurred in response to other cAMP derivatives and to cAMP induced endogenously by the activation of adenylate cyclase with forskolin, but was not due to down-regulation-associated changes in the rate of cAMP synthesis. Our results demonstrate that in LLC-PK1 cells, down-regulation of PKC results in an enhanced induction of uPA gene expression by cAMP-mediated signals without alterations in adenylate cyclase activity, suggesting a mechanism distal to adenylate cyclase.

An antibody that specifically recognized phosphothreonine 72 in ets-2 was used to determine the phosphorylation status of endogenous ets-2 in response to colony-stimulating factor 1 (CSF-1)/c-fms signaling. Phosphorylation of ets-2 was detected in primary macrophages, cells that normally express c-fms, and in fibroblasts engineered to express human c-fms. In the former cells, ets-2 was a CSF-1 immediate-early response gene, and phosphorylated ets-2 was detected after 2 to 4 h, coincident with expression of ets-2 protein. In fibroblasts, ets-2 was constitutively expressed and rapidly became phosphorylated in response to CSF-1. In both cell systems, ets-2 phosphorylation was persistent, with maximal phosphorylation detected 8 to 24 h after CSF-1 stimulation, and was correlated with activation of the CSF-1 target urokinase plasminogen activator (uPA) gene. Kinase assays that used recombinant ets-2 protein as a substrate demonstrated that mitogen-activated protein (MAP) kinases p42 and p44 were constitutively activated in both cell types in response to CSF-1. Immune depletion experiments and the use of the MAP kinase kinase inhibitor PD98059 indicate that these two MAP kinases are the major ets-2 kinases activated in response to CSF-1/c-fms signaling. In the macrophage cell line RAW264, conditional expression of raf kinase induced ets-2 expression and phosphorylation, as well as uPA mRNA expression. Transient assays mapped ets/AP-1 response elements as critical for basal and CSF-1-stimulated uPA reporter gene activity. These results indicate that persistent activation of the raf/MAP kinase pathway by CSF-1 is necessary for both ets-2 expression and posttranslational activation in macrophages.

Expression of polyomavirus middle-T antigen (middle-T) is involved in the formation of various tumors in vivo, e.g. hemangiomas and mammary gland tumors. Several genes have been shown to be activated in middle-T-expressing cells, but the underlying mechanisms have only been partially elucidated. Among the genes regulated by middle-T, the urokinase-type plasminogen activator (uPA) gene seems to be of primary importance for the development of the transformed phenotype. We have found that the uPA gene is highly expressed in eEnd2 cells derived from a hemangioma expressing middle-T. NIH3T3 cells show negligible levels of uPA mRNA but its expression was highly induced by infecting with a middle-T-expressing retrovirus. Middle-T did not affect uPA mRNA stability. Transient cotransfection experiments using a uPA-receptor gene construct and a middle-T expression vector showed that high uPA mRNA levels are due to increased uPA promoter activity. Analyses of various signaling molecules by transient cotransfection assays and in vitro kinase assays established that a signaling pathway involving c-Src, SOS, Ras, Raf-1 and ERK is activated by middle-T in NIH3T3 cells, resulting in the activation of the uPA gene promoter via PEA3/AP1 elements. In contrast, in eEND2 cells uPA gene induction is only partially dependent on this pathway, suggesting the involvement of additional signaling molecules in endothelial cells.