Exchange of mitochondrial subunits C15ORF48 and NDUFA4 occurs during macrophage activation in vitro and in inflammatory disease.

and Summary.-Transitions in the state of RNA polymerase were demonstrated during the unprimed synthesis of the r [I-C ] copolymer. No detectable change in the usual dimer-monomer pattern was noted during the lag phase (0-25 min at 370) after analysis of the reaction mixture by acrylamide gel electrophoresis. At the end of the lag phase, a major alteration in the electrophoretic pattern occurred, marked by the disappearance of the dimer-monomer bands and the concomitant appearance of a series of monomer-r [I-C] copolymer complexes. As these complexes of r[I-C] copolymer with one or more polymerase monomer were formed, an enzymatically inactive component (,y protein) of the polymerase was displaced. During the phase of rapid r[I-C] copolymer synthesis, the active form of the A. vinelandii RNA polymerase was the r[I-C] monomer lacking the o protein.RNA polymerase catalyzes two types of in vitro reactions, template-directed -5 and unprimed.6-10 Unprimed reactions are characterized by a lag phase that varies in length according to the polymer being synthesized; unprimed synthesis of the r [I-C] copolymer begins after a lag period of about 30 minutes at 37°.9 We have used acrylamide gel electrophoresis to look for transitions in the state of RNA polymerase during the course of the unprimed synthesis of the r[I-C] copolymer. At the end of the lag phase, the normal dimer-monomer11 pattern shifts to a series of monomer-r [I-C ] complexes. As the enzyme binds the newly formed polyribonucleotide, the y protein component of the polymerase is displaced. During the phase of active r[I-C] synthesis, the active form of the enzyme appears to be the r [I-C ]-monomer complex which lacks the y protein.Materials and Methods.-The T1 RNase and ethidium bromide were obtained from Calbiochem; the ITP and CTP came from P. L. Biochemicals and Boehringer, Inc., respectively.A. vinelandii RNA polymerase was prepared with the modification previously published,11 and had a specific activity of 120. Acrylamide gel electrophoresis and the in situ polymerase assay were carried out by the method of Krakow et al.11The unprimed synthesis of r[I-C] copolymer was carried out in the following reaction mixture: 0.05 M Tris buffer, pH 7.8; 0.01 M mercaptoethylamine; 2 mM MnSO4; 0.5 mM CTP; 0.5 mM ITP; and 300 ,ug RNA polymerase per ml. The reactions were incubated at 370 for the times indicated, suitable aliquots were removed, and EDTA was added (final conc. 3 mMin) to stop the reaction. The samples were placed in ice until needed for electrophoresis. 50-,.l samples were analyzed by acrylamide gel electrophoresis (5% gels) at 3 ma per gel for 60 min with the system at about 10°.The r[I-C] copolymer was prepared in an r[I-C]-directed reaction with the use of essentially the conditions listed above with the addition of 0.3 OD253 units of r[I-C] copolymer in a 20-ml reaction volume. After 120 min at 370, the reaction was terminated with the addition of 3 mM EDTA and then deproteinized with silicic acid by the method of Sueoka and Hardy.12 The r[I-...

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Glucocorticoids potently inhibit expression of many inflammatory mediators, and have been very widely used to treat both acute and chronic inflammatory diseases for more than seventy years. However, they can have several unwanted effects, amongst which immunosuppression is one of the most common. Here we investigated effects of the synthetic glucocorticoid dexamethasone on the responses of primary mouse bone marrow-derived macrophages to the pro-inflammatory agonist lipopolysaccharide (LPS). At the mRNA level, dexamethasone inhibited the LPS-induced expression of more than 100 genes that are involved in cell-intrinsic defence against viral pathogens. Expression of most of the corresponding proteins was also reduced by dexamethasone. This antiviral disarmament occurred at two distinct levels. First, dexamethasone strongly and dose-dependently inhibited the expression of the type I interferon IFN beta by LPS-activated macrophages. IFN beta mediates an autocrine positive feedback loop in LPS-treated macrophages, promoting the expression of antiviral genes and other interferon-stimulated genes. Hence reduction of IFN beta expression contributes to impaired expression of antiviral genes. Dexamethasone also acted downstream of IFN beta to inhibit expression of a subset of interferon-regulated genes. We tested a number of hypotheses based on previous publications, but found that no single mechanism could account for more than a small fraction of the broad suppressive impact of dexamethasone on macrophage type I interferon signaling, underlining the complexity of this pathway. Preliminary experiments indicated thatdexamethasone exerted similar inhibitory effects on primary human monocyte-derived or alveolar macrophages.