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The scope of the ESR technique has been expanded to enable direct measurement of the individual propagating radical concentrations in a binary free radical copolymerization. The ESR spectra observed during the copolymerization of the deuterated monomers styrene-d5 and cyclohexyl methacrylate-d5 in benzene at 60 °C could be accounted for as the superimposed spectra of the homopolymerization systems, the outermost regions of the spectrum unambiguously assigned to macroradicals with styrene-d5 as the terminal unit. The penultimate model with an implicit penultimate unit effect (PUE) was fitted to the ratio of the propagating radical concentrations (A12) and the copolymer composition, resulting in the radical reactivity ratios s1 = k211/k111 = 0.45 and s2 = k122/k222 = 0.50. Simultaneous fitting of A12 and the copolymerization propagation rate coefficient (&kmacr;p) resulted in lower s values (s1 = 0.08, s2 = 0.10). Optimization procedures employing the penultimate model with an explicit PUE and the entire data set (copolymer composition, A12 and &kmacr;p) indicated that the total sum of residuals could be further decreased compared with when the restriction of an implicit PUE was imposed on the model, but this was at the expense of the quality of the prediction of copolymer composition, which then became unacceptable.

Cbfa1 is an essential transcription factor for osteoblast differentiation and bone formation. We investigated functional differences among three isoforms of Cbfa1: Type I (originally reported as Pebp2alphaA by Ogawa et al. (Ogawa, E., Maruyama, M., Kagoshima, H., Inuzuka, M., Lu, J., Satake, M., Shigesada, K., and Ito, Y. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 6859-6863), Type II (originally reported as til-1 by Stewart et al. (Stewart, M., Terry, A., Hu, M., O'Hara, M., Blyth, K., Baxter, E., Cameron, E., Onions, D. E., and Neil, J. C. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 8646-8651), and Type III (originally reported as Osf2/Cbfa1 by Ducy et al. (Ducy, P., Zhang, R., Geoffroy, V., Ridall, A. L., and Karsenty, G. (1997) Cell 89, 747-754). A reverse transcriptase-polymerase chain reaction analysis demonstrated that these isoforms were expressed in adult mouse bones. The transient transfection of Type I or Type II Cbfa1 in a mouse fibroblastic cell line, C3H10T1/2, induced the expression of alkaline phosphatase (ALP) activity. This induction was synergistically enhanced by the co-introduction of Xenopus BMP-4 cDNA. In contrast, the transient transfection of Type III cDNA induced no ALP activity. In C3H10T1/2 cells stably transfected with each isoform of Cbfa1, the gene expression of ALP was also strongly induced in cells transfected with Type I and Type II Cbfa1 but not in cells with Type III Cbfa1. Osteocalcin, osteopontin,and type I collagen gene expressions were induced or up-regulated in all of the cells stably transfected with each isoform of Cbfa1, and Type II transfected cells exhibited the highest expression level of osteocalcin gene. A luciferase reporter gene assay using a 6XOSE2-SV40 promoter (6 tandem binding elements for Cbfa1 ligated in front of the SV40 promoter sequence), a mouse osteocalcin promoter, and a mouse osteopontin promoter revealed the differences in the transcriptional induction of target genes by each Cbfa1 isoform with or without its beta-subunit. These results suggest that all three of the Cbfa1 isoforms used in the present study are involved in the stimulatory action of osteoblast differentiation, but they exert different functions in the process of osteoblast differentiation.

We isolated the cDNA encoding a novel member of the human fibroblast growth factor (FGF) family from the lung. The cDNA encodes a protein of 208 amino acids with high sequence homology (95.6%) to rat FGF-10, indicating that the protein is human FGF-10. Human FGF-10 as well as rat FGF-10 has a hydrophobic amino terminus ( approximately 40 amino acids), which may serve as a signal sequence. The apparent evolutionary relationships of human FGFs indicate that FGF-10 is closest to FGF-7. Chromosomal localization of the human FGF-10 gene was examined by in situ hybridization. The gene was found to map to the 5p12-p13 region. Human FGF-10 (amino acids 40 to 208 with a methionine residue at the amino terminus) was produced in Escherichia coli and purified from the cell lysate. Recombinant human FGF-10 (approximately 19 kDa) showed mitogenic activity for fetal rat keratinizing epidermal cells, but essentially no activity for NIH/3T3 cells, fibroblasts. The specificity of mitogenic activity of FGF-10 is similar to that of FGF-7 but distinct from that of bFGF. In structure and biological activity, FGF-10 is similar to FGF-7.

We isolated the cDNA encoding a novel member of the fibroblast growth factor (FGF) family from rat embryos by homology-based polymerase chain reaction. The FGF-related cDNA encodes a protein of 215 amino acids (approximately 24 kDa), which has a conserved approximately 120-amino acid core with approximately 30-60% amino acid sequence identity with the FGF family. This protein with a hydrophobic amino terminus appears to be a secreted protein. The cDNA was translated in a coupled in vitro transcription-translation system. The molecular mass of the translation product was observed to be approximately 26 kDa. The expression of the FGF-related mRNA in the rat embryo and adult tissues was determined by Northern analysis and in situ hybridization. The mRNA was expressed in several discrete regions of the embryo. In adult tissues, the mRNA was preferentially expressed in the lung. The expression profile of the FGF-related mRNA was different from those of other FGF family mRNAs. As this protein is the 10th documented protein related to FGFs, we tentatively term this protein FGF-10.