Annual Editions: Environment 00/01

x,215 p.:ill.;25 c

Green Fluorescent Fusion Proteins: Powerful Tools for Monitoring Protein Expression in Live Zebrafish Embryos

AbstractThe recent development of transgenic technology in zebrafish has opened an exciting new avenue in which to explore vertebrate development. However, as in other species, the inability to easily identify live transgenic fish severely limits the potential of this promising technology. To determine whether the recently described green fluorescent protein (GFP) might provide a convenient live staining method in zebrafish, we constructed a glutathione S-transferase/GFP fusion protein (GST-GFP). GST-GFP cRNA, when injected into individual blastomeres of early zebrafish embryos, resulted in the rapid development (3 hr) of easily detectable green fluorescence which persisted for up to 4 days. GFP fluorescence was restricted to progeny of the injected cell and appeared to have no adverse effects on embryonic development despite widespread expression. Our findings demonstrate that GFP fusion proteins will provide a simple yet powerful means of monitoring production of heterologous proteins in live zebrafish

Graft smooth muscle cells specifically synthesize increased collagen

AbstractPurpose: Anastomotic intimal hyperplasia is characterized by smooth muscle cell (SMC) proliferation, but its final form is predominantly extracellular matrix. The purpose of this study was to compare collagen synthesis from graft SMC to that from adjacent native arterial SMC.Methods: Thoracoabdominal bypass grafts were excised 20 weeks after implantation into canine models. SMC harvested from six anastomotic graft segments and adjacent native aorta were passaged twice, grown to near-confluence, and then assayed for collagen synthesis and total protein synthesis. In four of these sites type I α-1 procollagen mRNA levels were measured and normalized to glyceraldehyde-3-phosphate dehydrogenase. To control for increases in collagen synthesis associated with proliferation, SMC were plated at equal densities and tritium-thymidine incorporation and DNA concentration were determined. Data (mean ± SE) were analyzed with two-factor ANOVA for repeated measures and paired Student t test and were considered significant if p < 0.05.Results: There was no difference in thymidine incorporation and total protein synthesis between groups, but collagen synthesis (graft: 52.9 ± 1.6 disintegrations per minute/ng DNA versus native: 42.6 ± 1.9 dpm/ng DNA; p = 0.03) and collagen synthesis as a percentage of total protein synthesis (graft: 7.16% ± 0.11% versus native: 5.8% ± 0.14%; p = 0.001) increased significantly in graft SMC as compared to native SMC. Type I α-1 procollagen mRNA levels were higher in graft SMC, but this difference was not significant.Conclusions: Graft SMC specifically produce more collagen than SMC from adjacent native artery. This change does not simply reflect increases in either total protein synthesis or proliferation and may, in part, be due to increased collagen gene expression. (J VASC SURG 1995;22:142-9.