Effects of a negative supercoil on the local denaturation of the DNA double helix were studied at the single-molecule level. The local denaturation in λDNA and λDNA containing the SV40 origin of DNA replication (SV40ori-λDNA) was directly observed by staining single-stranded DNA regions with a fusion protein comprising the ssDNA binding domain of a 70-kDa subunit of replication protein A and an enhanced yellow fluorescent protein (RPA-YFP) followed by staining the double-stranded DNA regions with YOYO-1. The local denaturation of λDNA and SV40ori-λDNA under a negative supercoil state was observed as single bright spots at the single-stranded regions. When negative supercoil densities were gradually increased to 0, -0.045, and -0.095 for λDNA and 0, -0.047, and -0.1 for SV40ori-λDNA, single bright spots at the single-stranded regions were frequently induced under higher negative supercoil densities of -0.095 for λDNA and -0.1 for SV40ori-λDNA. However, single bright spots of the single-stranded regions were rarely observed below a negative supercoil density of -0.045 and -0.047 for λDNA and SV40ori-λDNA, respectively. The probability of occurrence of the local denaturation increased with negative superhelicity for both λDNA and SV40ori-λDNA.
ABSTRACT.Purpose: The transforming growth factor-b (TGF-b) family includes three multifunctional proteins, TGF-b1, TGF-b2 and TGF-b3, expressed in ocular tissue, which are involved in regulating cell differentiation, cell proliferation and other cell functions. TGF-b is present in aqueous humour and regulates corneal endothelial cells. This study explores the mechanism by which TGF-b regulates the cell cycle in cultured corneal endothelial cells. Methods: The expression of specific receptors for the TGF-b family was investigated at the protein level by affinity cross-linking with radio-iodinated TGF-b1 and immunoprecipitation with specific antibodies to TGF-b receptors. Regulation of entry into the S-phase of the cell cycle was determined by 5-bromo-2 0 deoxyuridine (BrdU) incorporation into the cells. The signal transduction pathways were investigated using various blocking agents for protein kinase transducers involved in intracytoplasmic signal transduction. Results: Cultured bovine corneal endothelial cells were confirmed to express TGF-b type 1 and type 2 receptors and endoglin. In the confluent state, TGFb1 and TGF-b2 stimulated the cells to progress to the S-phase of the cell cycle through platelet-derived growth factor-B (PDGF-B) chain production and protein kinase C. Conclusions: TGF-b accelerated cell cycle progression from the G0/G1 phase to the S-phase in cultured corneal endothelial cells, under our experimental conditions, through pathways involving protein kinase C. These pathways are related to the cross-talk between TGF-b and other cytokines. The conditions employed in the present experiments may be useful for investigating the complex cross-talk between various cytokines and growth factors.
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