ABSTRACT
Subsets of CD4 T cells are defined by the cytokines that they produce; these cytokines determine the effector function of these cells. Cloned CD4 T cells fall into two subsets, producing either interferon-gamma (IFN gamma) or interleukin-4 (IL-4) in combination with other cytokines, and are called Th1 and Th2 cells, respectively. The lineage relationship between naive T cells and effector Th1- and Th2-type cells is unclear. We generated transgenic mice in which IL-4-producing cells express herpes simplex virus 1 thymidine kinase and are eliminated by ganciclovir (GANC). Activation of transgenic T cells in the presence of GANC eliminates IL-4 and IFN gamma production, showing that IL-4- and IFN gamma-producing cells express or have expressed IL-4. These results show that effector cells producing either IL-4 or IFN gamma have a common precursor, which expresses the IL-4 gene.
Subject(s)
CD4-Positive T-Lymphocytes/metabolism , Interferon-gamma/biosynthesis , Interleukin-4/biosynthesis , T-Lymphocyte Subsets/metabolism , Animals , CD4-Positive T-Lymphocytes/cytology , Cell Differentiation , Concanavalin A/pharmacology , Ganciclovir/pharmacology , Gene Expression , Interferon-gamma/pharmacology , Interleukin-12 , Interleukin-4/pharmacology , Interleukins/pharmacology , Lymphocyte Activation , Lymphocyte Depletion/methods , Mice , Mice, Transgenic , RNA, Messenger/genetics , Simplexvirus/enzymology , T-Lymphocyte Subsets/cytology , Thymidine Kinase/antagonists & inhibitors , Transforming Growth Factor beta/pharmacologyABSTRACT
Plasma membrane-stimulated vanadate-dependent NADH oxidation has been characterized in Saccharomyces cerevisiae. This activity is specific for vanadate, because molybdate, a similar metal oxide, did not substitute for vanadate in the reaction. Vanadate-dependent plasma membrane-stimulated NADH oxidation activity was dependent on the concentrations of vanadate, NADH, and NADPH and required functional plasma membranes; no stimulation occurred in the presence of boiled membranes or bovine serum albumin. The dependence of membrane-stimulated vanadate-dependent NADH oxidation was not linearly dependent on added membrane protein. The activity was abolished by the superoxide anion scavenger superoxide dismutase and was stimulated by paraquat and NADPH. These data are consistent with the previously proposed chain reaction for vanadate-dependent NADH oxidation. The role of the plasma membrane appears to be to stimulate superoxide radical formation, which is coupled to NADH oxidation by vanadate. 51V-nuclear magnetic resonance studies are consistent with the hypothesis that a phosphovanadate anhydride is the stimulatory oxyvanadium species in the phosphate buffers used at pHs 5.0 and 7.0. In phosphate buffers, compared with acetate buffers, the single vanadate resonance was shifted upfield at both pH 5.0 and pH 7.0, which is characteristic of the phosphovanadate anhydride. Since the cell contains an excess of phosphate to vanadate, the phosphovanadate anhydride may be involved in membrane-mediated vanadate-dependent NADH oxidation in vivo.
Subject(s)
NAD/metabolism , Saccharomyces cerevisiae/metabolism , Vanadates/pharmacology , Cell Fractionation , Cell Membrane/drug effects , Cell Membrane/metabolism , Cell Membrane/ultrastructure , Centrifugation, Density Gradient , Hydrogen-Ion Concentration , Kinetics , Magnetic Resonance Spectroscopy , Oxidation-Reduction , Oxygen Consumption , Succinate Dehydrogenase/metabolismABSTRACT
Interactions of oxyvanadium compounds with cellular metabolism have recently been demonstrated. Membrane-stimulated vanadate-dependent NADH oxidation has been hypothesized to involve the cellular accumulation of H2O2, which may cause the vanadate sensitivity of animals and microbes. This report shows that the vanadate-dependent NADH oxidation activity of the yeast plasma membrane requires oxygen and is present in vanadate-resistant mutants of Saccharomyces cerevisiae. In addition, the vanadate sensitivity of growth in S. cerevisiae is the same during aerobic and anaerobic growth. These results imply that neither plasma membrane-mediated vanadate-stimulated NADH oxidation, nor any other oxidative process, is the primary cause of vanadate sensitivity in yeast cells.
