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1.
Purification and biochemical characterization of an inhibitor of DNA synthesis produced by an Epstein-Barr virus-transformed B cell line 总被引:1,自引:0,他引:1
J S Beneke L F Qualtiere M C Nesheim G R Pearson 《Journal of immunology (Baltimore, Md. : 1950)》1980,124(6):2950-2955
An inhibitory factor, which has been shown to suppress the uptake of 125I-iododeoxyuridine by both lymphoid and nonlymphoid cells, was isolated from the supernatant of an Epstein-Barr virus- (EBV) transformed B cell line (1605L) established from a cotton-topped marmoset. Purification of the inhibitor, which was produced in serum-free medium by crowded cultures of the 1605L cells, was achieved by DEAE-cellulose chromatography followed by preparative polyacrylamide gel electrophoresis. The apparent m.w. of the 1605L factor was determined to be 65,000 to 70,000 by SDS-polyacrylamide gel electrophoresis. The inhibitor was sensitive to digestion by trypsin and chymotrypsin but not RNase or DNase, indicating that it was protein in nature. Exposure of the 1605L factor to 56 degrees C for 1/2 hr or pH 2 for 48 hr at 4 degrees C destroyed its inhibitory activity. The biochemical characteristics and activity of the 1605L inhibitor distinguish it from Type I interferon and several other soluble immunologic mediators known to be produced by lymphoid cell lines. 相似文献
2.
Carlo Garzelli Agostino Bazzichi Addawe Mohamed Dayah Maria Manunta Marina Incaprera Giuseppe Falcone 《FEMS microbiology letters》1992,100(1-3):449-454
A human Epstein-Barr virus (EBV)-positive lymphoblastoid B cell line, named BA-D10-4, produces a factor of a molecular mass less than 10 kDa that promotes cell proliferation of both BA-D10-4 cells and other human T or B lymphoid cell lines, either EBV-positive or -negative. The factor synergizes with higher molecular mass autocrine growth factors and makes both BA-D10-4 cells and B cell lines from Burkitt's lymphoma, but not cells from T cell leukemia, more responsive to interleukin-1 and interleukin-6. Therefore, this low molecular mass factor seems to be an autocrine growth factor per se and to have the characteristics of a competence factor. 相似文献
3.
4.
Partial purification and biochemical characterization of a T cell suppressor factor produced by human glioblastoma cells 总被引:3,自引:0,他引:3
The human glioblastoma cell line 308 constitutively secretes a soluble factor with biologic and biochemical characteristics of human monocyte-derived interleukin 1 (IL 1). The 308 cells also produce a 97,000 m.w. factor that inhibits the effects of IL 1 and interleukin 2 (IL 2) on T lymphocytes. By using sequential chromatography on Blue Affigel, hydroxyapatite, and Ultrogel AcA54, the inhibitory factor, termed glioblastoma-derived T cell suppressor factor (G-TsF), was separated from IL 1 and purified 2000-fold with respect to the protein present in the crude 308 cell supernatant. This G-TsF preparation was sensitive to tryptic proteolysis, showed a peak of pI 4.6 on isoelectric focusing, and when labeled with 125I, revealed six protein bands in the range of 30 to 100 kdaltons on SDS gel. 相似文献
5.
Identification of interleukin-6 as an autocrine growth factor for Epstein-Barr virus-immortalized B cells. 总被引:28,自引:7,他引:21
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Autocrine growth factors are believed to be important for maintenance of an immortalized state by Epstein-Barr virus (EBV), because cell-free supernatants of EBV-immortalized cell lines promote the proliferation of autologous cells and permit their growth at low cell density. In this study, we provide evidence for the existence of two autocrine growth factor activities produced by EBV-immortalized lines distinguished by size and biological activities. Much of the autocrine growth factor activity in lymphoblastoid cell line supernatants resided in a low-molecular-weight (less than 5,000) fraction. However, up to 20 to 30% of the autocrine growth factor activity resided in the high-molecular-weight (greater than 5,000) fraction. While the nature of the low-molecular-weight growth factor activity remains undefined, the high-molecular-weight growth factor activity was identified as interleukin-6 (IL-6). Culture supernatants from six EBV-induced lymphoblastoid cell lines tested contained IL-6 activity, because they promoted proliferation in the IL-6-dependent hybridoma cell line B9. In addition, a rabbit antibody to human IL-6 neutralized the capacity of the high-molecular-weight (greater than 5,000) fraction of a lymphoblastoid cell line supernatant to promote growth both in autologous EBV-immortalized cells and in B9 cells. Similarly, this high-molecular-weight autocrine growth factor activity was neutralized by a monoclonal antibody to human IL-6. Furthermore, characteristic bands, attributable to IL-6, were visualized in supernatants of each of four EBV-induced lymphoblastoid cell lines after immunoprecipitation with a rabbit antiserum to human IL-6. Thus, in addition to its previously reported properties, IL-6 is an autocrine growth factor for EBV-immortalized B cells cultured under serum-free conditions. 相似文献
6.
A growth factor has been purified to homogeneity from human pituitary glands. The pituitary growth factor (PGF) is trypsin-sensitive and acid- and heat-labile and has a molecular weight of 18,000 and an isoelectric point of 7.5. PGF was purified by heparin and copper affinity chromatography followed by carboxymethylcellulose 52. The amino-terminal amino acid sequence of PGF was established as PALPEXGGXGA and is identical with that of basic fibroblast growth factor at the identified amino acid residues. PGF was mitogenic for rabbit fetal chondrocytes and bovine corneal endothelial cells in the range of 0.015-15 ng mL-1. Heparin alone at low concentrations (0.5 microgram mL-1) was found to be weakly mitogenic for rabbit fetal chondrocytes. In combination with PGF a marked increase in cell growth was observed, which was inhibited by protamine sulfate. These data demonstrate the presence of a potent mitogen in human pituitaries that is structurally related to basic fibroblast growth factor and synergizes with heparin to promote cell growth. 相似文献
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8.
Purification and physicochemical characterization of a human cytotoxic factor produced by a human haemic cell line. 总被引:1,自引:0,他引:1
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A cytotoxic factor, produced by a human lymphoblastoid cell line [Karpas (1977) Br. J. Cancer 35, 152--160; Karpas (1977) Br. J. Cancer 36, 437--445], was purified both from the cell extracts and from the culture medium containing the cell lysate, by using ammonium sulphate precipitation, DEAE-cellulose chromatography, gel filtration and affinity chromatography on concanavalin A--Sepharose and on [3H]amino-ethanol--glass beads. Two factors, Factor I and Factor II, were separated by DEAE-cellulose chromatography. Factor I was eluted from this column at 30 mM-aminoethanol/HCl buffer, pH 8.0, whereas Factor II was bound strongly to DEAE-cellulose and was eluted only at 325 mM-aminoethanol/HCl buffer, pH 8.0. The purified Factor I migrated as a single band on polyacrylamide-gel electrophoresis. Its isoelectric point, pI, was 8.0 +/- 0.3. Its sedimentation coefficient, S20,w, was 3.5 +/- 0.1 S and its apparent molecular weight, Mr, was 65 000 +/- 1000 as determined by sedimentation-velocity and sedimentation-equilibrium measurements. A linear relationship between molecular weight and concentration was found in equilibrium runs, suggesting a non-spherical shape of the molecule. Factor I is not a glycoprotein, inasmuch as it does not bind to concanavalin A--Sepharose. It consists of two subunits (Mr 32 000 +/- 4000), migrating on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis as a single band. Factor II had pI 6.0 +/- 0.4 and Mr 75 000 +/- 3000. Factors I and II are thus different proteins. 相似文献
9.
Stratilová E Dzúrová M Breierová E Omelková J 《Zeitschrift für Naturforschung. C, Journal of biosciences》2005,60(1-2):91-96
The extracellular polygalacturonases produced by Aureobasidium pullulans isolated from waters of the Danube river were partially purified and characterized. The pH optima of polygalacturonases produced in the first phases of cultivation (48 h) and after 10 d as well as their optima of temperature, thermal stabilities, molecular masses, isoelectric points, action pattern and ability to cleave polymeric and oligomeric substrates were compared. Polygalacturonases with a random action pattern (random cleavage of pectate forming a mixture of galactosiduronides with a lower degree of polymerization) [EC 3.2.1.15] were produced only in the first phases of growth, while exopolygalacturonases [EC 3.2.1.67] with a terminal action pattern (cleavage of pectate from the nonreducing end forming D-galactopyranuronic acid as a product) were found during the whole growth. The main enzyme form with a random action pattern was glycosylated and its active site had the arrangement described previously for the active site of polygalacturonase of phytopathogenic fungi. 相似文献
10.
R Lupu A Wellstein J Sheridan B W Ennis G Zugmaier D Katz M E Lippman R B Dickson 《Biochemistry》1992,31(32):7330-7340
We have purified and characterized a novel 30-kDa glycoprotein (gp30) with TGF alpha-like properties secreted from the estrogen receptor negative breast cancer cell line MDA-MB-231. This factor was immunoprecipitated by an anti-TGF alpha polyclonal antibody and also had TGF alpha-like biological activity, as assayed by EGF radioreceptor assay and anchorage-independent assays. In addition, the novel growth factor stimulated phosphorylation of the EGF receptor and erbB-2 receptor. However, the novel growth factor, unlike EGF and TGF alpha, bound to heparin-Sepharose. Purification of gp30 was obtained to apparent homogeneity by heparin affinity chromatography and subsequent reversed-phase chromatography. Tunicamycin treatment in vivo or N-glycanase deglycosylation in vitro revealed a putative precursor of approximately 22 kDa molecular mass in contrast to the "normal" 16-kDa precursor species for TGF alpha. In vitro translation of total mRNA from MDA-MB-231 cells confirmed the size of the putative precursor. Biochemical characterization of gp30 was begun by V8 protease digestion of the deglycosylated polypeptide and the translated products. Peptide mapping of V8-digested, immunoprecipitated material suggests that the amino acid sequence of this unique protein is distinct from mature TGF alpha and not the result of a posttranslational modification of the precursor. We conclude that this TGF alpha-like (gp30) polypeptide is a novel growth factor with agonistic activity for both EGF and erbB-2 receptors. 相似文献
11.
Purification of human B cell growth factor 总被引:14,自引:0,他引:14
S R Mehta D Conrad R Sandler J Morgan R Montagna A L Maizel 《Journal of immunology (Baltimore, Md. : 1950)》1985,135(5):3298-3302
Human B cell growth factor (BCGF, 12,000 to 14,000 daltons) has been purified from lectin-stimulated, peripheral blood mononuclear cell-conditioned medium. The purification procedure involves a series of column chromatographic steps incorporating ion exchange, affinity binding, and gel filtration. This procedure is centered around a relatively high yield single chromatographic step, for the removal of co-eluting cytokines from BCGF, that is based on differential binding characteristics to the weak ion-exchange matrix, hydroxylapatite. Reverse-phase high-pressure liquid chromatographic separation on a C18-Bondapak column effectively separates the BCGF and TCGF moieties, yet is characterized by poor yields. High-pressure liquid chromatographic procedures on anion exchange and size exclusion provided the final purification step for BCGF, at an analytical level, resulting in a single band with a m.w. of 12,000 on a SDS-polyacrylamide gel. 相似文献
12.
Productive ascites growth of heterohybridomas between Epstein-Barr virus-transformed human B cells and murine P3X63Ag8.653 myeloma cells 总被引:1,自引:0,他引:1
Heterohybridomas between Epstein-Barr virus-transformed human B cells and murine myeloma P3X63Ag8.653 cells were found to produce sizable quantities of human monoclonal antibodies in nude mouse ascites. Mice injected intraperitoneally with two of such heterohybridomas yielded several milliters of ascites fluid which contained nearly 100-fold higher anti-platelet activities than those in routine culture supernatants. 相似文献
13.
Purification and biochemical characterization of recombinant hirudin produced by Saccharomyces cerevisiae 总被引:2,自引:0,他引:2
N Riehl-Bellon D Carvallo M Acker A Van Dorsselaer M Marquet G Loison Y Lemoine S W Brown M Courtney C Roitsch 《Biochemistry》1989,28(7):2941-2949
Recombinant hirudin was produced by the yeast Saccharomyces cerevisiae using the alpha-pheromone prepro sequence to direct its secretion into the culture medium. The secreted hirudin was isolated to greater than or equal to 95% purity as measured by 205-nm absorbance integration from a reverse-phase chromatogram. One major activity peak corresponding to the complete, correctly processed molecule and two minor activity peaks corresponding to C-terminally truncated forms were identified. The primary structure of the major peak, determined by N-terminal sequencing of tryptic peptides, was that predicted from the cDNA sequence, and the molecular mass analyzed by fast atom bombardment mass spectrometry (FAB-MS) was 6892.6 (calculated 6892.5). UV spectral analysis suggested that, in contrast to the natural molecule, recombinant hirudin produced by S. cerevisiae is not sulfated. 相似文献
14.
Purification and biochemical characterization of a soluble mouse interferon-gamma receptor produced in insect cells. 总被引:2,自引:0,他引:2
M Fountoulakis E J Schlaeger R Gentz J F Juranville M Manneberg L Ozmen G Garotta 《European journal of biochemistry》1991,198(2):441-450
The extracellular domain of the mouse interferon gamma receptor comprising amino acids 17-243 of the protein was produced in Spodoptera frugiperda cells infected with a recombinant baculovirus. The receptor was mainly secreted into the culture medium and was purified to homogeneity in several hundred milligram amounts. The purification procedure involved four chromatography steps and delivered a soluble and active receptor with an overall recovery of 30%. From each purification run, two pools of soluble receptor with the same interferon gamma binding capacity were isolated. Under reducing electrophoretic conditions the protein of pool I migrates as two bands of molecular masses 32 and 34 kDa and of pool II as two bands of 30 and 32 kDa. The soluble receptor of both pools carries a heterogeneous glycosylation. After deglycosylation it appears as one protein band of 27 kDa. N-linked carbohydrates contribute about 6 kDa and O-linked carbohydrates 1 kDa to its molecular mass. The nonreduced protein specifically binds interferon gamma on ligand blots and in a solid-phase binding system and competes for the binding of radiolabeled interferon gamma to the cell surface receptor. The soluble mouse interferon gamma receptor exists as a monomer in physiological buffer and binds interferon gamma in its dimeric form. It is stable at room temperature and against tryptic digestion, but is very sensitive to proteinase K digestion. The soluble mouse interferon gamma receptor produced in the insect/baculovirus expression system may prove useful to study the function of interferon gamma receptor as an antagonist of endogenous interferon gamma in the treatment of immunological and inflammatory disorders. 相似文献
15.
Reticuloendotheliosis virus strain T (REV-T)-transformed cells gave rise spontaneously to variants which secrete a factor that forms a distinct visible ring of precipitation (halo) surrounding colonies grown in soft agar. An Mr 15,000 protein was produced at higher levels by halo variants than by nonhalo-producing cells. An assay designed to detect the formation of precipitates enabled purification of an Mr 15,000 protein, p15, from serum-free medium conditioned by the growth of REV-T-transformed hematopoietic cells. Fractions enriched in p15 permitted the growth of REV-T-transformed cells under conditions where they normally failed to proliferate. 相似文献
16.
Purification of the Epstein-Barr virus-determined nuclear antigen from Epstein-Barr virus-transformed human lymphoid cell lines. 总被引:7,自引:7,他引:0
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The Epstein-Barr virus-determined nuclear antigen (EBNA) was purified from extracts of the human lymphoid cell lines Raji, Namalwa, and B95-8/MLD by two different methods. In the first approach, the apparently native antigen was purified 1,200-fold by a four-step procedure involving DNA-cellulose chromatography, blue dexptran-agarose chromatography, hydroxyapatite chromatography, and gel filtration, employing complement fixation as the assay procedure. Such EBNA preparations specifically inhibited the anticomplement immunofluorescence test for EBNA and bound to methanol/acetic acid-fixed metaphase chromosomes. The purified antigen, which has a molecular weight of 170,000 to 200,000, yielded a single protein band of molecular weight about 48,000 by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. These data indicate that native EBNA has a tetrameric structure. In the second purification method, EBNA-containing cell extracts containing radioactively labeled proteins were incubated with anti-EBNA-positive sera, and antigen-antibody complexes were adsorbed to matrix-bound staphylococcal protein A. The bound proteins were then released with an SDS-containing buffer, and denatured EBNA was separated from antibody chains by SDS-polyacrylamide gel electrophoresis and visualized by fluorography. The denatured EBNA obtained in radiochemically pure form by this procedure has a molecular weight of about 48,000, so both methods yield an EBNA monomer of the same size. 相似文献
17.
Purification and characterization of a cytotoxic factor produced by a mouse macrophage hybridoma 总被引:1,自引:0,他引:1
A mouse macrophage cytotoxic factor was purified to homogeneity from the serum-free culture supernatant of a mouse macrophage hybridoma clone, N/P-7-1, stimulated with lipopolysaccharide by gel filtration, affinity chromatography, anion-exchange chromatography, and polyacrylamide gel electrophoresis. The purified material was judged to be homogeneous as to the criteria of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and has a relative molecular mass of 17,500, as determined by SDS-PAGE, or 55,000, as determined by gel filtration on columns of both Sephacryl S-200 and TSK G3000SW. It has an isoelectric point of 5.0, and is trypsin sensitive, stable at 56 degrees C and labile at pH less than 6. The cytotoxic activity of the purified factor could not be inhibited by various sugars and lectins. The production of the factor from N/P-7-1 triggered by macrophage-activating factor for cytotoxicity, but not by mouse recombinant gamma-interferon. The factor should be synthesized after lipopolysaccharide stimulation because treatment of N/P-7-1 cells with a metabolic inhibitor, emetine or actinomycin D, prevents the production. 相似文献
18.
19.
W D Rutenberg U al-Khalidi F S Rosen E Merler 《Journal of immunology (Baltimore, Md. : 1950)》1979,122(2):723-727
Supernatants of human T lymphocytes stimulated by TT antigen release two factors that induce mitogenesis in autologous and allogeneic B lymphocytes. These factors are precipitated by 60% ammonium sulfate and 50% ethanol, and are both destroyed by heating to 70 degrees C for 5 min. By equilibrium ultracentrifugation there was a peak of mitogenic activity in the fraction with a specific gravity of 1.3147 corresponding to a partial specific volume of 0.761. After ultrafiltration through an Amicon XM50 membrane, the concentrate was chromatographed on a Sephadex G-200 column. Mitogenic activity was found only in the post-albumin fraction. When the post-albumin fraction was run on an isoelectrofocusing column, two distinct mitogenic factors were identified. The major peak of mitogenic activity (LMF) had a pI of 6.68 +/- 0.05 and the minor peak (MMF) had a pI OF 7.27 +/- 0.05. Amino acid analysis of LMF identified it as a protein and PAGE showed that LMF probably was a tetramer with a m.w. of 80,000. 相似文献
20.
Claudia Negri Antonella Cerino Roberto Chiesa e Giulia C. B. Astaldi Ricotti 《Cytotechnology》1991,5(Z1):132-133
Interleukin-6 (IL-6) was recently described to support the growth of Epstein-Barr (EBV) transformed lymphocytes. However no effect was reported on cloning of EBV transformed lymphocytes under limiting dilution conditions. In this paper we demonstrate that the Supernatant of Human Endothelial Cells (HECS) contains a factor(s) different from IL-6 which induces high cloning efficiency of EBV transformed cell lines, cultured under limiting dilution conditions. Furthermore HECS is superior to IL-6 in all the passages of EBV lymphocytes transformation. 相似文献