The Depletion of Nuclear Glutathione Impairs Cell Proliferation in 3t3 Fibroblasts

BACKGROUND:Glutathione is considered essential for survival in mammalian cells and yeast but not in prokaryotic cells. The presence of a nuclear pool of glutathione has been demonstrated but its role in cellular proliferation and differentiation is still a matter of debate. PRINCIPAL FINDINGS:We have studied proliferation of 3T3 fibroblasts for a period of 5 days. Cells were treated with two well known depleting agents, diethyl maleate (DEM) and buthionine sulfoximine (BSO), and the cellular and nuclear glutathione levels were assessed by analytical and confocal microscopic techniques, respectively. Both agents decreased total cellular glutathione although depletion by BSO was more sustained. However, the nuclear glutathione pool resisted depletion by BSO but not with DEM. Interestingly, cell proliferation was impaired by DEM, but not by BSO. Treating the cells simultaneously with DEM and with glutathione ethyl ester to restore intracellular GSH levels completely prevented the effects of DEM on cell proliferation. CONCLUSIONS:Our results demonstrate the importance of nuclear glutathione in the control of cell proliferation in 3T3 fibroblasts and suggest that a reduced nuclear environment is necessary for cells to progress in the cell cycle

Biliary alpha 1-acid glycoprotein concentrations in gallstone-free controls and in patients with multiple or solitary cholesterol gallstones.

Abstract: We recently identified a promoting glycoprotein in the concanavalin A-bound fraction of gallbladder bile as a biliary form of alpha 1-acid glycoprotein (AAG), The concentration of biliary AAG appears to exert an important promoting effect on the speed of cholesterol nucleation in many patients with cholesterol gallstone disease, In the current study, we provide information about the biliary concentration of AAG as well as the amount and comparative potency of its subfractions in patients with and without cholesterol gallstone disease, The amount of total biliary AAG and the amounts of its different isoforms separated by concanavalin A affinity chromatography were measured by ELISA, Estimates of absolute concentrations of AAG for each sample were normalized to the sample total protein content to give relative AAG values. The promoting activity (potency) of immunopurified biliary AAG from gallstone patients and gallstone-free controls on cholesterol crystallization was compared by a crystal growth assay. The mean absolute concentration of AAG in gallstone-free controls was not significantly different from multiple stone patients. The relative concentration of AAG (micrograms per milligram total protein) was significantly increased in patients with multiple stones when compared to controls (P < 0.05), and both the absolute and relative concentrations of AAG (micrograms per milligram bile), were three- and to five fold higher in a number of these patients. The functional activity and distribution of AAG in different subfractions was similar in gallstone patients and gallstone-free controls, The relative concentration of biliary AAG is significantly greater in cholesterol gallstone patients with multiple stones than in gallstone-free controls, These observations suggest that raised levels of AAG may be of pathogenetic importance in a subgroup of patients with multiple gallstones

Correlation between biliary alfa1-acid glycoprotein concentration and cholesterol crystal nucleation time in gallstone disease.

Abstract: A biliary form of the alpha(1)-acid glycoprotein (AAG) promotes cholesterol crystallization in the lower-molecular-weight, concanavalin A-bound fraction of gallbladder bile, In addition, bile AAG concentration is higher in cholesterol gallstone patients with multiple stones than in control patients without gallstone disease. In this study we sought to determine whether the increased biliary concentration of AAG in cholesterol gallstone patients is accompanied by a more rapid nucleation time in patients with multiple stones. AAG concentration in native biles was measured by ELISA. Nucleation time was measured using a standard microscopy method. The concentration of biliary AAG was then related to nucleation time in biles from the same patients. Nucleation times were significantly shorter (less than or equal to 5 days) in cholesterol gallstone patients with raised AAG concentrations (P < 0.03). There was a significant (P = 0.004) negative correlation (r = -0.53) between nucleation time and the AAG concentration in cholesterol gallstone patients with multiple stones, The concentration of biliary AAG appears to exert an important influence on the speed of cholesterol nucleation in bile in many patients with cholesterol gallstone disease