A new class of transforming growth factor (TGF), with chemical characteristics differing from previously reported TGFs, was isolated and purified from an avian sarcoma virus-transformed rat cell line, 77N1 . Purification steps were simple and consisted of ion-exchange chromatography on diethylaminoethyl (DEAE)-Sephacel, ammonium sulfate precipitation, Chromatofocusing, and DEAE-Sephadex A-25 chromatography. The purified TGF is a heat- and acid-labile protein with a molecular mass of 12,000 daltons and isoelectric point of 5.2-5.4. Because of the acid lability of this TGF, purification was carried out at neutral pH. The TGF induced DNA synthesis in growth-arrested BALB 3T3 cells and promoted anchorage-independent growth of nontransformed BALB 3T3 cells in soft agar; the latter activity is specific for the peptide growth factors, called TGFs, but it did not compete with epidermal growth factor (EGF) for binding to the EGF membrane receptors. The TGF activity was not potentiated by EGF. The purified preparation of the TGF stimulated BALB 3T3 cells to grow progressively in soft agar at a dose of 20 ng/ml.
Transformation of rat cells by avian sarcoma viruses induced the release of growth factors into serum-free conditioned medium. An avian sarcoma virus-transformed rat cell line, 77N1, produced and released a polypeptide growth factor, classified as a transforming growth factor (TGF), which transiently promotes anchorage-dependent BALB3T3 A31 cells to form progressively growing colonies in soft agar. The TGF was isolated and partially purified from an extract of 77N1 cells by ion-exchange chromatography on a diethylaminoethyl Sephacel column followed by ammonium sulfate precipitation. The TGF was assumed to have a molecular weight of 11,000 from gel filtration on Sephadex G-50. This TGF did not compete with epidermal growth factor for binding to cell membrane receptors and was not potentiated by epidermal growth factor. The TGF was trypsin and dithiothreitol sensitive as well as heat and acid labile, indicating that it was different from previously reported TGFs of similar molecular weight and thus belonged to a new class of TGFs.
IL ‐2 plays an important role in immunological and other biological functions. This cytokine directly induces the production of several cytokines, such as IL ‐5 and IL ‐13. The mechanisms of IL ‐2‐mediated cytokine synthesis are mostly unclear; however, the involvement of IL ‐2 receptor ( IL ‐2R)β has been suggested. In this study, the signaling molecule downstream of IL ‐2Rβ was investigated, employing a proteomic approach. Full‐length IL ‐2Rβ and its mutant in which the intracellular component was truncated were introduced in an IL ‐2Rα‐ and IL ‐2Rγ‐stably transfected T cell hybridoma, S1. The differential phosphorylation profiles of protein tyrosine residues in these cells upon IL ‐2 stimulation were examined by two‐dimensional gel electrophoresis. The candidate phosphoproteins of interest were re‐covered, in‐gel digested and mass spectrometry fingerprinted. Among proteins specifically phosphorylated in full‐length IL ‐2Rβ‐expressing cells in response to IL ‐2 stimulation, protein phosphatase ( PP )1β and FK 506‐binding protein 4 were identified. Particularly, PP 1β augmented IL ‐5 and IL ‐13 expression stimulated by IL ‐2 but not by anti‐ CD 3 antibody in human peripheral CD 4 + T cells upon ectopic expression. IL ‐2‐induced cytokine expression was suppressed by overexpression of PP 1 regulatory subunit 2. A PP 1 inhibitor, tautomycin, but not a PP 2A inhibitor, okadaic acid, also inhibited the IL ‐2R‐mediated responses. It was conclusively shown that PP 1 is crucially involved in IL ‐2‐mediated IL ‐5 and IL ‐13 synthesis in human T cells.
<i>Background:</i> Mast cells (MCs) play a central role in allergic reactions through high-affinity IgE receptor (FcεRI)-mediated responses. Many attempts have been performed to investigate MC functions, though molecular bases of the intracellular signaling cascade through FcεRI, especially in human MCs, remain scant and unexplored. <i>Methods:</i> Human MCs were differentiated from CD34+ cells by culture with stem cell factor, IL-6 and IL-3. The differential phosphorylation profiles of protein tyrosine residues in the resulting MCs with or without FcεRI aggregation were examined by two-dimensional gel electrophoresis. The candidate phosphoproteins of interest were picked, in-gel digested and mass spectrometry fingerprinted. <i>Results:</i> Approximately 40 proteins in MCs were phosphorylated on their tyrosine residues in response to activation and some of them were identified. Particularly IL-31 receptor α, solute carrier family 39, syntaxin 5 and heterogeneous nuclear ribonucleoprotein are newly identified as phosphoproteins that are potentially involved in the MC signaling cascade through FcεRI. <i>Conclusion:</i> Our present phosphoproteome data may provide the clue to understand the molecular mechanisms for the activation of human MCs.
A cell growth inhibitor was isolated from an avian sarcoma virus-transformed rat cell line (77N1), from which we had previously obtained a novel transforming growth factor, TGF gamma 2. The growth inhibitor (named SGI) was found in cell extract of 77N1 cells and was separable from TGF gamma 2 by means of ion exchange chromatography. SGI inhibited the DNA synthesis of serum-starved, TGF gamma 2-stimulated BALB3T3 cells as well as the spontaneous and TGF-induced colony formation of various cells in semisolid agar. We present evidence that SGI is distinct from the ubiquitous growth modulator TGF beta, and we discuss the possible role of SGI in the neoplastic cell proliferation.
Comparing protein expression in the cerebrospinal fluid (CSF) of rheumatoid arthritis (RA) patients with that of controls, makes possible the uncovering of proteins that affect disease progression and regulate responsiveness to drugs. Two-dimensional gel electrophoresis (2-DE) and silver staining were used for identifying disease-associated CSF proteins in RA patients. First, to enhance the detection of CSF proteins and to improve the separation of their isoforms by 2-DE, CSF samples were pre-treated with an albumin and IgG removal kit, then by acetone precipitation. The 2-DE analysis revealed more than 1600 spots by the removal of albumin and immunoglobulin from CSF. The expression of the protein spots was not greatly changed in either group, but some notable changes in protein spots were observed in two RA samples. In particular, the expression of an approximately 50 kD protein increased markedly, whereas that of two sequential protein spots of 10-15 kD and with neutral pI decreased in the RA samples. These preliminary results suggest that the proteomic method is conducive to clarifying the mechanism of RA crises, and that some of the expression-changed proteins may be new candidates for disease-associated proteins of RA.
