Aconitase is a member of a family of iron-sulfur-containing (de)hydratases whose activities are modulated in bacteria by superoxide radical (O2-.)-mediated inactivation and iron-dependent reactivation. The inactivation-reactivation of aconitase(s) in cultured mammalian cells was explored since these reactions may impact important and diverse aconitase functions in the cytoplasm and mitochondria. Conditions which increase O2-. production including exposure to the redox-cycling agent phenazine methosulfate (PMS), inhibitors of mitochondrial ubiquinol-cytochrome c oxidoreductase, or hyperoxia inactivated aconitase in mammalian cells. Overproduction of mitochondrial Mn-superoxide dismutase protected aconitase from inactivation by PMS or inhibitors of ubiquinol-cytochrome c oxidoreductase, but not from normobaric hyperoxia. Aconitase activity was reactivated (t1/2 of 12 +/- 3 min) upon removal of PMS. The iron chelator deferoxamine impaired reactivation and increased net inactivation of aconitase by O2-.. The ability of ubiquinol-cytochrome c oxidoreductase-generated O2-. to inactivate aconitase in several cell types correlated with the fraction of the aconitase activity localized in mitochondria. Extracellular O2-. generated with xanthine oxidase did not affect aconitase activity nor did exogenous superoxide dismutase decrease aconitase inactivation by PMS. The results demonstrate a dynamic and cyclical O2-.-mediated inactivation and iron-dependent reactivation of the mammalian [4Fe-4S] aconitases under normal and stress conditions and provide further evidence for the membrane compartmentalization of O2-..
Activated monocytes or macrophages can release soluble cytotoxic molecules capable of lysing tumour cells in vitro and thus represent an important component of the host defence mechanisms against malignancy. The recent availability of pure recombinant or natural human lymphokines and monokines and their respective polyclonal or monoclonal antibodies now makes it possible to dissect the interactions of these factors in the induction and performance of the cytotoxic event by the monocytes. Our studies indicate that pretreatment of monocytes with alpha-IFN or gamma-IFN, and also interleukin (IL)-1 or tumour necrosis factor (TNF) results in enhanced monocyte cytotoxicity. Although all these substances induce the production of IL-1 by monocytes, TNF mediates the enhanced cytotoxicity induced in monocytes by gamma-IFN, IL-1 and, in an autocrine manner, by TNF itself. Neither TNF, IL-1, gamma-IFN nor alpha-IFN mediate spontaneous monocyte cytotoxicity or that induced by alpha-IFN. Our studies thus reveal new interactions between the two monokines IL-1 and TNF and provide a dual role for TNF, as immunomodulator and mediator of monocyte cytotoxicity induced by certain specific lymphokine and monokine molecules.
We report a general mass spectrometric approach for the rapid identification and characterization of proteins isolated by preparative two-dimensional polyacrylamide gel electrophoresis. This method possesses the inherent power to detect and structurally characterize covalent modifications. Absolute sensitivities of matrix-assisted laser desorption ionization and high-energy collision-induced dissociation tandem mass spectrometry are exploited to determine the mass and sequence of subpicomole sample quantities of tryptic peptides. These data permit mass matching and sequence homology searching of computerized peptide mass and protein sequence data bases for known proteins and design of oligonucleotide probes for cloning unknown proteins. We have identified 11 proteins in lysates of human A375 melanoma cells, including: a-enolase, cytokeratin, stathmin, protein disulfide isomerase, tropomyosin, Cu/Zn superoxide dismutase, nucleoside diphosphate kinase A, galaptin, and triosephosphate isomerase. We have characterized several posttranslational modifications and chemical modifications that may result from electrophoresis or subsequent sample processing steps. Detection of comigrating and covalently modified proteins illustrates the necessity of peptide sequencing and the advantages of tandem mass spectrometry to reliably and unambiguously establish the identity of each protein. This technology paves the way for studies of cell-type dependent gene expression and studies of large suites of cellular proteins with unprecedented speed and rigor to provide information complementary to the ongoing Human Genome Project.
Human immune interferon (IFN-gamma) differs from leukocyte interferon (IFN-alpha) and fibroblast interferon (IFN-beta) in cell origin, inducing agents, physical and biological properties and amino acid sequence. These differences have led to interest in possible differences in the biological properties of IFN-gamma compared with IFN-alpha and IFN-beta. IFN-gamma has the same broad range of biochemical and biological actions as do IFN-alpha and IFN-beta, although relative potencies vary depending on the cell type and function investigated. There has so far been no direct evidence that IFN-gamma alters normal cell functions differently from other interferons. We report here striking qualitative and quantitative differences in the intracellular response of human fibroblasts to IFN-gamma compared with IFN-alpha and IFN-beta. Two-dimensional gel electrophoresis demonstrates, in addition to the induction of a common group of polypeptides, the existence of a set of polypeptides whose synthesis is uniquely induced by IFN-gamma.
The lymphotoxin b receptor (LTbR), and its ligand, LTa1b2, have been proposed to play a key role in the development and organization of lymphoid tissues. The LTbR is expressed on a variety of human primary and transformed cells, but strikingly absent on T or B lymphocytes and primary monocytes or peripheral dendritic cells, although LTbR is detected on some myeloid leukemic lines. In the developing thymus LTbR is prominent along the trabeculae and into the medulla upto corticomedullary junction. In the spleen, LTbR is prominently expressed by cells in the red pulp and along the borders of red and white pulp which colocalizes with reticular stromal cells. The LTbR is expressed on a human follicular dendritic cell line, FDC-1, and signals expression of CD54 when ligated with the LTa1b2 complex. These results support the concept that directional interactions between LTa1b2 bearing lymphocytes and LTbR bearing stromal cells are involved in the organization of lymphoid tissue.
T cells of patients with rheumatoid arthritis (RA) do not control the rate of B lymphoblast transformation induced by Epstein-Barr virus (EBV) as efficiently as T cells from healthy individuals; thus, lymphoblast cell lines are established more readily in RA lymphocytes in vitro after EBV infection. In the present experiments, we have asked whether this T cell regulation can be reproduced by lymphocytes. We found that normal T cells, activated in allogeneic or autologous mixed leukocyte reactions (MLR), produce lymphokines that inhibit in vitro EBV-induced B cell proliferation. Allogeneic MLR supernatants inhibited EBV-induced DNA synthesis 62 +/- 4% (mean +/- SE) at 10 d post-infection, whereas autologous MLR supernatants suppressed it 50 +/- 3%. RA T cell supernatants produced in an allogeneic MLR suppressed as well as normal T cell supernatants (64 +/- 5% inhibition). In contrast, supernatants from RA autologous MLR had little inhibitory activity. EBV-induced DNA synthesis at 10 d was reduced only 8 +/- 3%, compared with the 50 +/- 3% suppressive activity of normal autologous MLR supernatants. The magnitude of the proliferative responses in the autologous MLR regenerating the lymphokines was similar in the normal and RA populations. After depletion of adherent cells from the RA auto-MLR stimulators, supernatant inhibitory activities increased to normal levels (from 11 +/- 6 [SE] to 52 +/- 6% [SE]). The inhibitory factor involved in the regulation of in vitro EBV infection is a protein with a molecular weight of approximately 50,000. Its activity is eliminated by hearing at 56 degrees C and by exposure to acid at pH 2. The inhibitory activity is blocked by mixing the MLR supernatants with a polyvalent antisera or monoclonal antibodies specific for human gamma interferon. Gamma interferon produced by activating T cells in allo- or auto-MLR can reproduce T cell-mediated regulation of EBV-induced B cell proliferation, and the failure of RA auto-MLR to generate that lymphokine parallels the defective T cell regulation of EBV-induced B cell proliferation characteristic of RA lymphoid cells.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.