Fibroblast growth factors (FGFs)1 comprise a family of 23 polypeptides that induce mitogenic, angiogenic, and chemotactic responses in cells of mesodermal and neuroectodermal origin (1-3). The FGF signaling pathway also plays a significant role in normal development, and increased FGF production is associated with chronic immunologic injury as well as tumor development and metastasis (4 -6). Such a diverse array of biological effects occurs through ligand interaction with high affinity cell surface receptors (FGFR1-4) that are structurally similar and exhibit a high degree of sequence homology at the amino acid level (3, 7). The full-length FGFR exhibits three extracellular immunoglobulin-like domains, a single transmembrane domain, and a split intracellular tyrosine kinase domain (7). Ligand binding causes receptor dimerization allowing trans autophosphorylation of intracellular tyrosine residues and activation of intrinsic kinase activity (8).Activation of FGFR1 results in tyrosine phosphorylation of multiple signaling and adaptor proteins, including FRS2 (FGF receptor substrate 2/SNT-1), Shc, Grb2, Ras/Raf, Crk, phosphatidylinositol 3-kinase, SHP-2, and Src (reviewed in Ref. 9). A constitutive association of the multidocking protein FRS2 at the juxtamembrane segment of FGFR1 occurs independently of receptor activation (10
B lymphocytes that recognize soluble self-Ags are routinely found in normal individuals in a functionally inactive or anergic state. Current models indicate that this tolerant state is maintained by interactions with self-Ags that uncouple the BCR from downstream signaling pathways and increase levels of free calcium. Contrary to this expectation, B cells that harbor anti-insulin Ig transgenes (125Tg) are maintained in a tolerant state even though free calcium levels remain normal and tyrosine kinase substrate phosphorylation is preserved following BCR stimulation. Under basal conditions, intracellular levels of inositol 1,4,5-trisphosphate are increased and NFATc1 levels are reduced in 125Tg B cells. The 125Tg B cells are markedly impaired in their ability to mobilize calcium upon stimulation with ionomycin, and BCR-induced calcium mobilization from internal stores is decreased. In contrast, poisoning intracellular calcium pumps with thapsigargin increases calcium mobilization in 125Tg B cells. Changes in calcium signaling are accompanied by a failure of 125Tg B cells to translocate NFATc1 into the nucleus following stimulation with either anti-IgM or ionomycin. Thus, disassociation of BCR from multiple signaling pathways is not essential for maintaining tolerance in anti-insulin 125Tg B cells. Rather, BCRs that are occupied by autologous insulin deliver signals that induce changes in intracellular calcium mobilization and maintain tolerance by preventing activation of key transcription factors such as NFAT.
Stable cell lines expressing the human epidermal growth factor (EGF) precursor have been prepared by transfection of mouse NIH 3T3 cells with a bovine papillomavirus-based vector in which the human kidney EGF precursor cDNA has been placed under the control of the inducible mouse metallothionein I promoter. Synthesis of the EGF precursor can be induced by culturing the cells in 5 mM butyric acid or 100 ,uM ZnCl2. The EGF precursor synthesized by these cells appears to be membrane associated; none is detectable in the cytoplasm. The size of the EGF precursor expressed by these cells is 4150-180 kDa, which is larger than expected from its amino acid sequence, suggesting that it is posttranslationally modified, presumably by glycosylation. The EGF precursor was also detected in the conditioned medium from these cells, indicating that some fraction of the EGF precursor synthesized by these transfected cells may be secreted. Preliminary data suggest that this soluble form of the EGF precursor may compete with 125I-labeled EGF for binding to the EGF receptor. These cell lines should be useful for studying the processing of the EGF precursor to EGF as well as determining the properties and possible functions ofthe EGF precursor itself.
FGF-1 and FGF-2 potently induce proliferation and migration of these cells as well as angiogenesis, events that are required during normal embryogenesis and tissue repair. Although expression of FGF-1 and FGF-2 at sites of injury is therefore beneficial, some evidence suggests these growth factors may also contribute to vascular pathology by promoting excessive intimal hyperplasia (2-5). FGF-1 expression is increased at several sites of chronic immune injury, including the synovium in rheumatoid arthritis (6 -8), the myocardium in human hearts after transplantation (9 -11), and in transplanted kidneys undergoing chronic rejection (12, 13). The pathologic lesions in these sites demonstrate cellular responses typical of FGF effects on mesenchymal cells that result in vascular intimal hyperplasia, increased extracellular matrix deposition, and neoangiogenesis. In addition, these sites are characterized by chronic infiltration of T lymphocytes, suggesting there may be interactions between the immune system and fibroblast growth factors as demonstrated by the finding that T cells can produce FGF-2 (14, 15). Evidence that FGFs may have immunoregulatory effects on T cells was first provided in 1985 by Johnson and Torres (16), who showed that FGF, at physiologically relevant concentrations, could replace the requirement for IL-2 or helper cells in production of interferon-␥. Although FGF could activate intracellular signals necessary for interferon-␥ production, FGF alone could not stimulate proliferation of T cells. More recent studies show directly that some human T cells express receptors for FGF-1 and that the normally small subpopulation of FGF-responsive T cells is expanded in the peripheral blood of patients with rheumatoid arthritis and in patients after heart transplantation (8, 17). These data suggest that T cells bearing FGF receptors can be stimulated and expanded in the FGF-enriched environment at sites of immune injury and subsequently migrate to the peripheral blood. As found in the earlier studies (16), FGF alone does not stimulate proliferation of T cells but together with engagement of the T cell antigen receptor induces production of IL-2 and proliferation (17). In T cells, FGF thus functions in a manner analogous to other well described "costimulators" (18,19) to activate a second signal transduction pathway necessary for T cell proliferation and effector function.These findings suggest that FGF and FGF receptors in T cells may function quite differently than in cells in which FGF alone can stimulate proliferation, migration, and secretion of effector molecules such as plasminogen activator (20). Our efforts to investigate T cells that express FGF receptors and the mechanisms by which FGF signals in T cells have been hampered by the lack of reagents that can conveniently identify these cells and allow us to examine the fate of FGF and its receptors in T cells. To address this difficulty, the experiments reported here describe the production and characterization of a fusion protein that includes a por...
Mycobacterium ulcerans produces an exotoxin in culture which, when inoculated into guinea pig skin, causes inflammation, necrosis, edema, and other histopathological changes resembling those in infections of humans. The toxin was resistant to heat and to alkalies and was moderately acid labile. Toxic activity was destroyed by Pronase, phospholipase, lipase, amylase, and glucosidase but not by trypsin, collagenase, cellulase, lysozyme, hyaluronidase, or neuraminidase. Toxic activity was resistant to treatment with 2-mercaptoethanol, urea, guanidine hydrochloride, p-chloromercuribenzoate, ethylenediaminetetraacetate, and sodium deoxycholate but was destroyed by sodium m-periodate and sodium dodecyl sulfate. The toxin was precipitated by a wide range of ammonium sulfate concentrations. Extraction with chloroform-methanol or petroleum ether destroyed its activity. Isopycnic density gradient ultracentrifugation in KBr produced a highdensity lipoprotein layer with a 24-fold increase in specific activity. The results indicate that this toxin is a high-molecular-weight phospholipoprotein-polysaccharide complex.
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