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1.
Monoclonal nonspecific suppressor factor (MNSF) is a cytokine with antigen nonspecific suppressive activity. MNSFbeta (a subunit of MNSF) is a 14.5 kDa fusion protein consisting of a protein with 36% identity with ubiquitin and ribosomal protein S30. The ubiquitin-like segment (Ubi-L) may be cleaved from MNSFbeta in the cytosol. Recently, we have observed that Ubi-L covalently binds to intracellular proteins in mitogen-activated murine T-helper type 2 clone, D.10 cells. In this study, we purified a 33.5 kDa Ubi-L adduct from D.10 cell lysates by sequential chromatography on DEAE, anti-(Ubi-L) Ig-conjugated Sepharose, and hydroxylapatite. MALDI-TOF-MS fingerprinting revealed that this Ubi-L adduct consists of an 8.5 kDa Ubi-L and a Bcl2-like protein, murine orthologue of a previously cloned human BCL-G gene product with pro-apoptotic function. Murine Bcl-G mRNA was highly expressed in testis and significantly in spleen. In addition, the level of Bcl-G mRNA expression was increased in concanavalin A- and interferon gamma-activated D.10 cells. The 33.5 kDa Ubi-L adduct was expressed in spleen but not in testis, even though Bcl-G protein was highly expressed in this tissue. The antisense oligonucleotide to Bcl-G significantly decreased the level of the Ubi-L adduct formation in concanavalin A-activated D.10 cells and the proliferative response of the D.10 cells. These results suggest that the post-translational modification of Bcl-G by Ubi-L might be implicated in T-cell activation.  相似文献   

2.
Monoclonal nonspecific suppressor factor (MNSF), a lymphokine produced by murine T cell hybridoma, possesses pleiotrophic antigen-nonspecific suppressive functions. A cDNA clone encoding MNSF-beta, an isoform of the MNSF, has been isolated and characterized. MNSF-beta cDNA encodes a fusion protein consisting of a ubiquitin-like segment (Ubi-L) and ribosomal protein S30. Ubi-L appears to be cleaved from the ribosomal protein and released extracellularly in association with T cell receptor-like polypeptide. In the current study we have characterized the biochemical nature of the Ubi-L receptor on D.10 G4.1, a murine T helper clone type 2. Biotinylated Ubi-L bound preferentially to concanavalin A-stimulated but not to unstimulated D.10 cells. Detergent-extracted membrane proteins were applied to an immobilized Ubi-L column. SDS-polyacrylamide gel electrophoresis of eluted fraction revealed a band of Mr = 82,000. Biotinylated Ubi-L specifically recognized this band, confirming that the 82-kDa protein is the Ubi-L receptor. A complex of Mr = 90,000 was visualized by immunoprecipitation of 125I-Ubi-L cross-linked to the purified receptor followed by SDS-polyacrylamide gel electrophoresis and autoradiography. In addition, a 105-kDa protein was coimmunoprecipitated by anti-Ubi-L receptor (82-kDa polypeptide) antibody, indicative of the association of this protein with the Ubi-L receptor complex. Amino acid sequence analysis of the 82-kDa polypeptide revealed that the Ubi-L receptor may be a member of a cytokine receptor family.  相似文献   

3.
Monoclonal nonspecific suppressor factor (MNSF), a lymphokine produced by murine T cell hybridoma, possesses a pleiotropic antigen-nonspecific suppressive function. We have shown that 70 kDa MNSF comprises an 8 kDa ubiquitin-like polypeptide (Ubi-L) and 62 kDa T cell receptor (TCR) alpha-like molecule. Ubi-L binds specifically to its 82 kDa receptor protein on target cells. In the current study, we have further characterized the biochemical nature of the TCR(alpha)-like molecule. The 62 kDa protein was separated into two species of 46 kDa and 16 kDa on reverse-phase HPLC. Anti-TCR(alpha) monoclonal antibody recognized the 46 kDa, but not the 16 kDa protein. Anti-TCRbeta monoclonal antibody failed to recognize these proteins. Ubi-L conjugated to the 46 kDa protein, whereas Ubi-L lacking its C-terminal Gly-Gly did not. Although Ubi-L was labile both to heating at 56 degrees C and to acidification to pH 4, the Ubi-L-46 kDa protein complex was unaffected by these treatments. In addition, the 46 kDa protein elongated the Ubi-L-induced protein tyrosine phosphorylation in a concanavalin A-activated murine T helper type 2 clone, D10 cells. One of the four tryptic peptide sequences derived from the 46 kDa protein was in alignment with a related sequence found in the J(alpha) region of the TCR(alpha), including the highly conserved motif F-G-X-G-T-X-L.  相似文献   

4.
Monoclonal-nonspecific suppressor factor (MNSF) is a lymphokine derived from murine T cell hybridoma. The target tissues are both LPS-stimulated B cells and Con A-stimulated T cells. Since the action of MNSF may be mediated by its binding to specific cell surface receptors, we characterized the mode of this binding. The purified MNSF was labeled with 125I, using the Bolton-Hunter reagent. The labeled MNSF bound specifically to a single class of receptor (300 receptors per cell) on mitogen-stimulated murine B cells or T cells with an affinity of 16 pM at 24 degrees C, in the presence of sodium azide. Competitive experiments showed that MNSF bound to the specific receptor and that the binding was not shared with IL2, IFN-gamma, and TNF. Various cell types were surveyed for the capacity to specifically bind 125I-MNSF. 125I-MNSF bound to MOPC-31C (a murine plasmacytoma line) and to EL4 (a murine T lymphoma line). The presence of specific binding correlates with the capacity of the cells to respond to MNSF. These data support the view that like other polypeptide hormones, the action of MNSF is mediated by specific cell surface membrane receptor protein. Identification of these receptors will provide insight into the apparently diverse activities of MNSF.  相似文献   

5.
MNSFbeta is a ubiquitously expressed member of the ubiquitin-like family that has been implicated in various biological functions. Previous studies have demonstrated that MNSFbeta covalently binds to intracellular proapoptotic protein Bcl-G in mitogen-activated murine T cells. In this study, we further investigated the intracellular mechanism of action of MNSFbeta in macrophage cell line, Raw 264.7 cells. We present evidence that MNSFbeta.Bcl-G complex associates with ERKs in non-stimulated Raw 264.7. We found that MNSFbeta.Bcl-G directly bound to ERKs and inhibited ERK activation by MEK1. In Raw 264.7 cells treated with MNSFbeta small interfering RNA (siRNA) lipopolysaccharide (LPS)-induced ERK1/2 activation was enhanced and LPS-induced JNK and p38 activation was unaffected. SiRNA-mediated knockdown of MNSFbeta increased tumor necrosis factor alpha (TNFalpha) expression at mRNA and protein levels in LPS-stimulated Raw 264.7 cells. Finally, we found that transfection with MNSFbeta expression construct resulted in a significant inhibition of LPS-induced ERK activation and TNFalpha production. Co-transfection experiments with MNSFbeta and Bcl-G greatly enhanced this inhibition. Collectively, these findings indicate that MNSFbeta might be implicated in the macrophage response to LPS.  相似文献   

6.
Monoclonal nonspecific suppressor factor (MNSF) is a lymphokine derived from a murine T cell hybridoma. The action of MNSF is mediated by specific cell-surface receptors. Since IFN-gamma alters the cellular response to MNSF (M. Nakamura, H. Ogawa, and T. Tsunematsu, J. Immunol. 138, 1799, 1987), we investigated whether IFN-gamma has an effect on the expression of MNSF receptor on target cells. IFN-gamma enhanced the expression of MNSF receptor on both MOPC-31C cells (a murine plasmacytoma line) and EL4 (a murine T lymphoma line). Incubation with IFN-gamma increased the number of specific MNSF-binding sites by about 50 to 90%, with no significant change in binding affinity. IFN-alpha and IFN-beta also increased MNSF binding, although the effect of the saturating amounts was lower than that seen with IFN-gamma. Maximal enhancement of receptor expression was observed after about 15 hr of incubation with IFN-gamma. No demonstrable change occurred in the kinetics of internalization of 125I-MNSF bound to MOPC-31C cells preincubated without or with IFN-gamma.  相似文献   

7.
The secretion of immunoglobulin (Ig) from cultured mononuclear cells by lipopolysaccharide (LPS) stimulation is inhibited by monoclonal nonspecific suppressor factor (MNSF), a lymphokine produced by murine T cell hybridoma. In an attempt to develop a murine monoclonal antibody (MAb) with specific reactivity against MNSF, a cell fusion technique that incorporated immune murine splenocytes and HAT-sensitive murine myeloma cells was used. Cross-reactivity experiments confirmed that the MAb (MO6) does not bind to unrelated proteins such as bovine serum albumin, mouse IgG, and murine interferon-gamma (IFN-gamma). There are no effects when anti-IFN-gamma antibodies are used with MNSF. As far as biological activity is concerned, MO6 inhibits in vitro the activity of MNSF in terms of the Ig secretion from cultured lymphocytes. By using MO6, affinity chromatography and immunoblotting were performed. The MNSF on the SDS-PAGE showed a band with m.w. of approximately 70,000, indicating the formation of an aggregate in saline; but after treatment with 0.4 M pyridine-acetic acid buffer, separate bands of 24,000 and 16,000 daltons were evident. Therefore MO6 recognizes 70,000 and both 24,000 and 16,000 daltons. Thus we confirmed by using this MAb and affinity chromatography, the existence of human counterpart, human nonspecific suppressor factor (hNSF), in supernatant from concanavalin A-stimulated T cells. When hNSF was fractionated by high pressure liquid chromatography (HPLC), the activity was found in a region corresponding to 70,000 daltons. However, when fractionated in pyridine-acetic acid buffer, hNSF activity was distributed in a slightly wider range of 15,000 to 30,000 daltons. Physicochemical analysis showed that the purified hNSF is resistant to either heating at 56 degrees C or to 2-mercaptoethanol treatment; however, it is labile to acidification at pH 2.0 and is also sensitive to protease treatment, the characteristics of which were similar to those of murine MNSF. Thus MO6 was confirmed to be a pertinent tool for isolation of hNSF, as well as for murine MNSF.  相似文献   

8.
Monoclonal-nonspecific suppressor factor (MNSF), a product of murine T cell hybridoma, suppresses antibody response to lipopolysaccharide. In an attempt to clarify the functional mechanisms in vitro, we investigated the mode of action of MNSF. This factor inhibited the antibody response by B cells (depleting T cells and M?), thereby indicating that the lymphokine acts directly on B cells, without interaction between B and T cells or M?. MNSF activity was absorbed by mitogen-stimulated T or B cells, but not by resting lymphocytes. Proliferative responses to T cell and B cell mitogens were inhibited dose dependently by the addition of MNSF. Kinetic studies showed that MNSF suppressed the antibody response, in all culture periods, thereby indicating that immunoglobulin secretion and proliferation were inhibited. The effect of growth factor on MNSF-mediated suppression was investigated to search for a possible suppression of MNSF action. Interleukin 2 (IL-2) remarkably inhibited MNSF activity, and the effect of IL-1 or IL-4 was less. IL-2 was most effective when added on the fourth day of culture. MNSF also inhibits division in the plasmacytoma line MOPC-31C or in thymoma EL4, but not in L929 fibroblasts. Tumor necrosis factor (TNF) inhibits cell division of various tumor cells and suppresses the pokeweed mitogen-induced antibody response, without cytotoxic action, as does MNSF. While MNSF and TNF have similar biochemical and physiochemical properties, the cross-reaction tests showed that both are antigenically discrete lymphokines. Although MNSF lacks TNF activity, the concomitant addition of both factors to L929 increases the cytotoxic action, a finding indicative of a synergistic effect.  相似文献   

9.
Monoclonal nonspecific suppressor factor (MNSF), a product of a murine T cell hybridoma, suppresses the antibody response to lipopolysaccharide. In an attempt to clarify the N-terminal sequence, MNSF was prepared and purified by affinity chromatography with the use of an anti-MNSF monoclonal antibody (MO6), and reverse-phase high-pressure liquid chromatography. On the SDS-PAGE, the purified MNSF showed a single band with a molecular weight of 12,000. The N-terminal amino acid sequence of the protein was determined and showed no strong homology to any of the sequences of known biologically active proteins. However, the sequence revealed significant (60%) amino acid identity to transforming growth factor beta 2 (TGF beta 2).  相似文献   

10.
Post-translational modification by monoclonal nonspecific suppressor factor β (MNSFβ) has been involved in the regulation of a variety of cellular processes. Previous studies have demonstrated that MNSFβ covalently binds to the intracellular pro-apoptotic protein Bcl-G and regulates TLR-4-mediated signal transduction. Recently, we found that MNSFβ also covalently conjugates to endophilin II, a member of the endophilin A family, and inhibits the signal pathway upstream of IKK activation, but not downstream of TLR-2 signaling. In this study, we further examined the mechanism of action of MNSFβ in TLR-2-mediated signal transduction in macrophage-like cell line Raw264.7 cells. Although MNSFβ siRNA enhanced Pam(3)CDK(4) (TLR-2-specific ligand)-stimulated TNFα production, Bcl-G siRNA did not affect. MNSFβ cDNA inhibited the Pam(3)CDK(4)-stimulated TNFα production. High-molecular weight (130 kDa) MNSFβ-adduct was induced in Pam(3)CDK(4)-stimulated Raw264.7 cells. This MNSFβ-adduct was not induced by LPS, indicative of the specificity of TLR-2-mediated signal transduction. Similar observations were seen in BALB/c peritoneal macrophages. Interestingly, 40-kDa MNSFβ-adduct was tyrosine phosphorylated by Pam(3)CDK(4) stimulation. Collectively, novel MNSFβ-adducts may regulate TLR-2 signaling pathway in macrophages.  相似文献   

11.
Successful implantation requires synchronous development of and active dialogue between the maternal endometrium and the implanting blastocyst. While it is well established that appropriate maternal steroid hormones are essential for endometrial preparation for implantation, the molecular events at the actual site of implantation are still little understood. The aims of our studies were to identify genes explicitly expressed or repressed at the sites of implantation by utilising RNA differential display (DDPCR), and to establish the roles of these genes in the implantation process in a mouse model. Ten bands unique in implantation sites compared to interimplantation sites were identified by DDPCR and subsequently confirmed by Northern blotting. One of these bands contained a cDNA fragment that was highly homologous to mouse monoclonal nonspecific suppressor factor beta (MNSFbeta) or Fau. The full cDNA sequence of this gene, obtained by screening a lambdagt11 cDNA library, was essentially the same as MNSFbeta, except that it had much longer 5' untranslated region. Interestingly, both Northern and immunohistochemical analysis showed that the expression of this gene was much lower in implantation sites compared to interimplantation sites on day 4.5 of pregnancy, when embryos first attach to the uterus and initiate implantation, and on day 5.5, when implantation has advanced. These results suggest a role for MNSF during implantation and early pregnancy, possibly through regulating the proliferation and/or differentiation of uterine stromal cells. It may also be involved in the selective production of TH2-type cytokines in implantation sites to regulate the immune system at the maternal-fetal interface.  相似文献   

12.
Successful embryo implantation and pregnancy in mammals depends on the establishment of immune tolerance between the maternal immune system and fetal cells. Monoclonal nonspecific suppressor factor beta (MNSFbeta), a cytokine produced by suppressor T cells in various tissues, possesses an antigen-nonspecific immune-suppressive function, and may be involved in the regulation of the uterine immune response during embryo implantation. In this study, anti-MNSFbeta IgG administered directly into the uterine lumen, significantly inhibited mouse embryo implantation in a dose-dependent manner in vivo, and this effect was reversed by co-administration of recombinant MNSFbeta. The effects of anti-MNSFbeta IgG on the gene pattern profiles in mouse uterine tissues were examined by cDNA microarray and several changes were confirmed by real-time PCR. Anti-MNSFbeta IgG caused up-regulation (> or = 2-fold) of 71 known genes and 17 unknown genes, and decreased expression (> or = 2-fold) of 74 known genes and 43 unknown genes, including several genes previously associated with embryo implantation or fetal development. Most of the known genes are involved in immune regulation, cell cycle/proliferation, cell differentiation/apoptosis, and lipid/glucose metabolism. These results demonstrate that MNSFbeta plays critical roles during the early pregnancy via multiple pathways.  相似文献   

13.
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16.
D10.G4.1 (D10) cells, a murine conalbumin-reactive Th2 cell line, made to overexpress the beta(2) integrin LFA-1 by pharmacological manipulation or by transfection become autoreactive and are capable of inducing in vivo autoimmunity. However, whether this is specific to LFA-1 and whether overexpression of other T cell integrin molecules has the same effect are unknown. We examined the functional consequences of T cell CD49d (alpha(4) integrin) overexpression by transfecting murine CD49d cDNA into D10 cells. Similar to the LFA-1-transfected cells, the CD49d-overexpressing T cells are autoreactive and proliferate in response to APCs in an MHC class II-dependent manner in the absence of nominal Ag. Additionally, CD49d overexpression is associated with increased in vitro adhesion to endothelial cells and increased in vivo splenic homing. However, in contrast to LFA-1 overexpression, increased T cell CD49d expression is not associated with autoreactive cytotoxicity or the ability to induce in vivo autoimmunity. In addition to the novel observation that CD49d overexpression is sufficient to induce T cell autoreactivity, our results also support the hypothesis that the ability to induce in vivo autoimmunity is related to T cell cytotoxicity and not to T cell proliferation function in the D10 murine adoptive transfer model of autoimmunity.  相似文献   

17.
Nuclei from butyrate-treated murine lymphosarcoma cells were incubated with different amounts of the polyanion heparin, which is known to interact predominantly with chromatin-associated histones. Unlike isolated histone H1, histone H1 in the nuclei of butyrate-treated cells was found to display an enhanced affinity for the binding to heparin as compared to histone H1 from control cells. Dephosphorylation of histone H1 as a result of butyrate treatment of the cells is discussed as a possible factor involved in the observed higher affinity of the protein for heparin.  相似文献   

18.
T M Cao  M T Sung 《Biochemistry》1982,21(14):3419-3427
Histones have been cross-linked to DNA in chicken erythrocyte nuclei and chromatin by using ultraviolet light irradiation at 254 nm. Following irradiation, cross-linked histone-DNA adducts were isolated and purified by hydroxylapatite chromatography, and the DNA component was subjected to acid hydrolysis. Of several hydrolysis techniques investigated, trichloroacetic hydrolysis of the DNA component of the adducts was found to be most effective. Histones isolated from hydrolyzed histone-DNA adducts were characterized by gel electrophoresis and fingerprint analysis. No histone-histone protein adducts were observed. All histone fractions have been shown to cross-link DNA in nuclei or chromatin by utilizing the technique employed, but with different propensities. The order of observed cross-linking, deduced from kinetic experiments, is H1 + H5, H3 greater than H4 greater than H2A much greater than H2B. The preferential binding of the core histone H3, as compared to the other core histones, is discussed in light of recent data concerning histone-DNA interactions and nucleosome structure. The use of the ultraviolet light technique as a conformational probe to study chromatin is also discussed.  相似文献   

19.
A cDNA clone encoding the human lymphocyte differentiation Ag CD38 was isolated from a mixture of four different lymphocyte CDNA libraries expressed transiently in COS cells and screened by panning with mAb. Transfected COS cells expressed a surface protein of Mr 46,000 that was similar to the native CD38 molecule expressed on the B cell line Daudi and the T cell leukemia HPB-ALL and which was recognized by each of the CD38 specific mAb HIT-2, T16, T168, HB7, 5D2, ICO-18, and ICO-20. The CD38 cDNA sequence predicts an unusual 30-kDa polypeptide with a short N-terminal cytoplasmic tail, and a carboxyl-terminal extracellular domain carrying the four potential N-linked glycosylation sites. The absence of significant homology with other known surface Ag including members of the Ig superfamily ruled out the possibility that CD38 was the human homologue of the murine Qa2 molecule as has been suggested previously. PvuII digests of human genomic DNA revealed a polymorphism linked to the CD38 gene.  相似文献   

20.
Cloning and expression of murine lymphotoxin cDNA   总被引:10,自引:0,他引:10  
The murine lymphotoxin (LT) gene has been cloned and used to identify cDNA clones in a library prepared from activated murine T cell mRNA. A recombinant murine genomic library was screened with a human lymphotoxin cDNA probe, resulting in the isolation of the entire LT gene. The murine LT gene structure is similar to the human gene, containing three intervening sequences. An activated murine T cell cDNA library was prepared with poly(A)+ RNA isolated 7 hr after concanavalin A stimulation of an L3T4+ interleukin 2-dependent murine T cell clone. Two colonies of the cDNA library that contained inserts that hybridized with the murine LT gene probe were sequenced and were used to construct expression plasmids. The amino acid sequence deduced from the cDNA indicates that murine LT is highly homologous to human LT (74%) and is related to murine tumor necrosis factor (35% homology). The cDNA was transcribed and was translated in vitro, and was expressed in COS-1 cells. This has resulted in the production of LT biological activity.  相似文献   

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