Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.21.4 (trypsin)
42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Monocytes lysing a variety of tumor cells were isolated by adhesion to autologous serum-coated plastic surfaces. When the blood monocytes were co-cultured with K562 cells for 3-24 h, the supernatants contained soluble factors, termed monocyte cytotoxic factors (MCF), capable of lysing K562 and other tumor cells in a 48-h microcytotoxicity assay. The production of MCF was mediated by typical monocytes expressing a surface phenotype of CD11 (+), CD16 (-), LeuM1 (+). When target cells were pretreated with actinomycin D, they showed an increase in their susceptibility to lysis by MCF. Addition of the drug to MCF assays also resulted in an enhancement of MCF-mediated lysis. Thus, the lytic activity of MCF was detectable in an 18-h assay. The presence of interferon (IFN)-alpha or -gamma augmented the biological activity of MCF, while pretreatment of target cells with IFN did not enhance MCF activity. The absorption of MCF activity was not elevated by actinomycin D or IFN. MCF lysed target cells that were resistant to tumor necrosis factor (TNF). One result of importance is that MCF lysed autologous and allogeneic freshly isolated human tumor cells. The lysis of fresh human tumor cells by MCF was not inhibited by monoclonal antibodies directed against TNF, lymphotoxin (LT), IFN-alpha, IFN-gamma, or interleukin 1 (IL-1). Furthermore, TNF, LT, IFNs, and IL-1 did not kill fresh human tumor cells. MCF activity was stable at low temperatures but was destroyed by heating. The biological activity of MCF was reduced or abolished by serum, trypsin, chymotrypsin and proteinase K, indicating the proteinaceous nature of MCF. The lytic activity was resistant to protease inhibitors. These data indicate that MCF is a noble cytokine that acts on human fresh tumor cells.
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PMID:[Production and function of the monocyte cytotoxic factor (MCF)]. 244 Mar 86

We have investigated the effects of various interferons on the receptors for recombinant tumor necrosis factor-alpha (rTNF-alpha) and also their effects on rTNF-alpha-mediated cytotoxicity on human cervical carcinoma cell line ME-180. Preincubation of cells with interferon (IFN)-gamma causes a concentration- and time-dependent increase in rTNF-alpha receptor number without any change in the affinity constant of the receptors. The increase in receptor number is caused only by IFN-gamma and not by IFN-alpha or IFN-beta. Approximately 4-6 h of preincubation with IFN-gamma are required for maximum increase in rTNF-alpha binding to the cells, and this increase can be abolished by inhibitors of protein synthesis, suggesting de novo synthesis of rTNF-alpha receptors. The half-life of both uninduced and induced receptors of rTNF-alpha is approximately 2 h, indicating a rapid turnover. The binding of rTNF-alpha to the cells can also be eliminated by pretreatment of cells with trypsin. Following the removal of trypsin, binding of rTNF-alpha gradually increases, and this requires the synthesis of new proteins. The cytotoxic effect of rTNF-alpha on ME-180 cells is potentiated severalfold by the addition of either IFN-alpha, -beta, or -gamma. However, at similar concentrations, relatively higher potentiation of rTNF-alpha cytotoxicity is observed with IFN-gamma as compared to IFN-alpha and IFN-beta. The pre-exposure of cells to IFNs is as effective as co-exposure in enhancing cytotoxic effects of TNF-alpha. The induction of TNF-alpha receptors by IFNs is observed in different cell types regardless of their sensitivity to TNF-alpha, suggesting that increase in receptor number alone is not sufficient for the enhanced cytotoxic response. Because the enhancement of cytotoxic effects of TNF-alpha is observed by all IFNs but receptor induction in ME-180 cells occurs only with INF-gamma and because metabolic inhibitors which down-regulate TNF-alpha receptors also enhance cytotoxic response, we suggest that the induction of TNF-alpha receptor by IFNs is not a major mechanism of synergism between these cytokines.
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PMID:Induction of receptors for tumor necrosis factor-alpha by interferons is not a major mechanism for their synergistic cytotoxic response. 244 Aug 58

Glucocorticoid hormones, although able to exert profound immunosuppressive effects, do not suppress mononuclear phagocyte activation by IFN-gamma and may even enhance it. For example, expression and functional activity of the high affinity FcR for IgG on human mononuclear phagocytes (FcR gamma I) is increased by IFN-gamma and is maximal after co-treatment with IFN-gamma plus the glucocorticoid dexamethasone (DEX). To determine whether there are other mononuclear phagocyte surface Ag that are regulated in this manner, hybridomas were prepared using IFN-gamma-plus-DEX-treated human monocytes as immunogen. Five IgG1 mAb (Mac 2-8, 2-38, 2-48, 2-49, and 2-158) were developed that recognize a trypsin-sensitive mononuclear phagocyte-specific surface Ag of Mr 155,000. There was no detectable reactivity of these mAb to lymphocytes or granulocytes or to several cell lines, including U-937 and HL-60. The p155 Ag was detected on monocytes and increased significantly with time of culture or after treatment with DEX. Expression was maximal after co-treatment with rIFN-gamma plus DEX, but was inhibited or unaffected by treatment with IFN-gamma alone. For freshly isolated cells, expression of the p155 Ag was highest on peritoneal macrophages. Our results indicate that the p155 Ag is a newly identified Ag of the human mononuclear phagocyte lineage and may represent, in the least, a phenotypic marker of monocyte differentiation or maturation.
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PMID:IFN-gamma plus glucocorticoids stimulate the expression of a newly identified human mononuclear phagocyte-specific antigen. 245 Sep 16

Human immune interferon-gamma (HuIFN-gamma) labeled with 32P was used to study the structure of IFN-gamma receptor. When [32P]HuIFN-gamma was bound and crosslinked to IFN-gamma the receptor of human cells with a bifunctional crosslinker disuccinimidyl suberate (DSS), a single diffused 32P-labeled band corresponding to the IFN-gamma.receptor complex was visualized by SDS-polyacrylamide gel electrophoresis and autoradiography. The size of the [32P]-HuIFN-gamma.receptor complex was about 100-120 kD. Separation of crosslinked complex in reducing and nonreducing gels showed no size differences, suggesting the absence of interchain disulfide linkage. However, binding and formation of the crosslinked IFN-gamma. receptor complex on cells was diminished in the presence of the disulfide reducing agent dithiothreitol (DTT). The reduction was DTT-dose-dependent, suggesting that intramolecular disulfides of the receptor are important for binding. Also, [32P]HuIFN-gamma did not bind if cells were pretreated with and then washed free of DTT, suggesting an irreversible reduction of intrachain disulfide bonds, presumably of the receptor. [32P]HuIFN-gamma also specifically binds to human placental membranes. Each placenta has about 170 ng of IFN-gamma receptors. Covalent attachment of [32P]HuIFN-gamma to placental plasma membranes via DSS produced 2 crosslinked complexes with the molecular sizes of 100-120 kD and 60-70 kD. The IFN-gamma.receptor complex of placental membranes was solubilized with NP-40 after DSS treatment and partially purified with immobilized antibody to the carboxyl terminus of IFN-gamma. Treatment of the receptor complex with trypsin and papain was used to demonstrate its differential proteolytic sensitivity.
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PMID:Immune interferon receptor: chemical and enzymatic sensitivity. 246 13

Serine protease inhibitors with a specificity for trypsin inhibit interferon-gamma (INF-gamma)-induced HLA-DR expression on a hybrid human epidermal cell line (H12), dermal fibroblasts, and primary keratinocytes. Protease inhibitors with a specificity for chymotrypsin or papain fail to inhibit IFN-gamma. The inhibitory effect of the trypsin inhibitors is similar to that of glucocorticoids in that it is a transient event, fading with length of exposure to IFN-gamma, and is reversed by the addition of dibutyryl cyclic AMP (dbcAMP) and phospholipase C(PLC) from Clostridium perfringens. In H12 cells, dbcAMP and PLC enhance the IFN-gamma induction of HLA-DR, but do not induce in the absence of INF-gamma. Evidence suggests that the protease inhibitors, as well as dbcAMP and PLC, may modulate HLA-DR expression at a post-translational site as well as during IFN-gamma signal transduction. These results suggest that trypsin-like protease activity may be required for cellular HLA-DR antigen expression following exposure to IFN-gamma.
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PMID:Trypsin inhibitors inhibit induction by interferon-gamma of HLA-DR antigen expression on human skin cells. 247 85

Purified preparations of recombinant human interferon-gamma (rIFN-gamma) with Cys-Tyr-Cys at the N-terminus ([ Cys-Tyr-Cys]IFN-gamma) derived from Escherichia coli gave two closely migrating bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and two peaks on reversed-phase high-performance liquid chromatography (rpHPLC). In contrast, rIFN-gamma without Cys-Tyr-Cys and rIFN-gamma in which both Cys-1 and Cys-3 were substituted with serine behaved as a single species on both SDS-PAGE and rpHPLC. These results suggest that the N-terminal portion of rIFN-gamma is heterogeneous. To elucidate the structure of the N-terminal portion, the N-terminal peptide preparation was obtained by binding rIFN-gamma to thiopropyl-Sepharose 6B gel with disulfide linkage followed by trypsin digestion and elution with 2-mercaptoethanol. The preparation gave four peaks (NT-1, NT-2, NT-3, and NT-4, in order of elution) on rpHPLC; all four were found to be Cys-1-Lys-9 by amino acid analysis after acid hydrolysis. Various analyses indicate that NT-1 is the intact nonapeptide, that NT-3 and NT-4 are N alpha-formyl and N alpha-acetyl forms of NT-1, respectively, and that NT-2 may be S-blocked at Cys-1. It is concluded that E. coli-derived [Cys-Tyr-Cys]IFN-gamma is partially N alpha-acylated. The data also suggest that N alpha-acylation does not affect the biological activity of [Cys-Tyr-Cys]IFN-gamma.
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PMID:Escherichia coli-derived human interferon-gamma with Cys-Tyr-Cys at the N-terminus is partially N alpha-acylated. 249 19

Freshly isolated monocytes in suspension express 2000 to 4000 high affinity receptors for IFN-gamma. Because monocytes change phenotypically as they migrate out of the circulation and adhere to extracellular matrix, modulation of the expression of IFN-gamma receptors may occur. In order to determine if adherence alone modulates the receptor for IFN-gamma, we have studied receptor expression in adherent human peripheral blood monocytes. Elutriation-purified monocytes were allowed to adhere to polystyrene overnight at 37 degrees C. These cells now expressed 1 to 2 x 10(5) low affinity (Ka = 10(8) liters/M) receptors for [125I]rIFN-gamma. Binding to this receptor was specific and saturable. The expression of these receptors occurred rapidly (within 3 h) after adherence and was not inhibited by cycloheximide treatment. Binding to the receptor was abrogated by treating cells with trypsin, but was enhanced after treatment with alkaline protease or proteinase K. mAb against the high affinity receptor did not block binding to the low affinity receptor on adherent cells. The low affinity receptor transduced a signal to the cell as measured by the IFN-gamma-induced enhancement in FcR for human IgG1. The structure of the receptor on adherent cells was investigated by chemical cross-linking techniques. A receptor-[125I]rIFN-gamma complex was observed by SDS-PAGE to have a Mr of 180,000 to 200,000. Reduction of this complex with 2-ME resulted in the loss of the high Mr complex and the appearance of a doublet of lower Mr of 68,000 and 82,000. In contrast, cross-linking of monocytes in suspension yielded a complex of 110,000 to 120,000 Mr, which was unchanged upon reduction. Upon adherence, human monocytes express large numbers of a novel receptor for rIFN-gamma which is capable of stimulating the cell. This receptor appears to be composed of at least two components which are disulfide linked and structurally differs from the high affinity receptor on nonadherent monocytes.
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PMID:Characterization of a novel low affinity receptor for IFN-gamma on adherent human monocytes by radioligand binding studies and chemical cross-linking. 252 81

Plasmin reacted readily with recombinant murine interferon-gamma (rIFN-gamma) in vitro, reducing the relative molecular mass of each monomer by approximately 1,000. The amino terminus of the rIFN-gamma remained intact and no sites of internal peptide bond hydrolysis were detected, indicating that the plasmin target region is most likely near the carboxyl terminus. Cleavage of rIFN-gamma was observed with similar concentrations of trypsin or min-plasmin. By contrast, human neutrophil elastase failed to alter the structure of rIFN-gamma. The plasma proteinase inhibitor, alpha 2-antiplasmin, protected rIFN-gamma from plasmin digestion. Purified alpha 2-macroglobulin-plasmin complex cleaved rIFN-gamma; however, the activity was greatly reduced compared with the free proteinase. The antiviral activity of the rIFN-gamma was enhanced four- to fivefold by treatment with plasmin or trypsin. By contrast, naturally occurring murine IFN-gamma was inactivated by plasmin (80%), suggesting that the effect of plasmin on IFN activity can vary depending on the preparation studied. The importance of plasmin at the site of an immune reaction is well established. This investigation identifies plasmin and miniplasmin as physiologic proteinases capable of reacting with IFN-gamma in vivo.
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PMID:Cleavage of recombinant murine interferon-gamma by plasmin and miniplasmin. 252 20

We analyzed the high affinity receptor for IFN-gamma of Raji cells and human placenta by combining Scatchard analysis, cross-linking experiments, and receptor purification. Only one high affinity binding site was found, Kd 2.1 X 10(-10). The receptor is a 90-kDa glycoprotein. However, multiple cross-linked products of 110 kDa to about 250 kDa could be generated and proteins of 90, 70, and 50 kDa could be obtained upon purification. These proteins all contained the same 90-kDa receptor, or part of it. We suggest that extensive cross-linking and/or proteolysis may explain many of the conflicting results published thus far. The extracellular domain of the 90-kDa receptor protein was highly resistant to digestion with trypsin or proteinase K. Trypsin digestion neither affected the number of binding sites per cell, nor the Kd for IFN-gamma. A cluster of sites for different proteases was found in the intracellular domain. The 50-kDa fragment created by trypsin digestion had the same characteristics as the isolated 50-kDa receptor fragment. It contained the IFN-gamma binding site and the receptor's extracellular and amino-terminal domain. N-linked glycosylation contributed about 15 kDa to its molecular mass, of which 4 kDa were attributable to sialic acid residues. O-Linked glycosylation was not detected. The number of binding sites per cell and the Kd for IFN-gamma were not affected by the presence or absence of N-linked glycosylation. The receptor contained at least one critical disulfide bridge and the reduced receptor could be reactivated in vitro.
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PMID:Structure and membrane topology of the high-affinity receptor for human IFN-gamma: requirements for binding IFN-gamma. One single 90-kilodalton IFN-gamma receptor can lead to multiple cross-linked products and isolated proteins. 253 Feb 76

We have identified a late, committed stage in the differentiation of the mast cell progenitor just before granulation. Mast cell committed progenitors (MCCP) are nongranulated cells with a density of 1.060 to 1.070 g/ml which can be harvested from the mesenteric lymph node of mice infected with Nippostrongylus brasiliensis. Mast cell-committed progenitors are able to proliferate and differentiate in the absence of IL-3 or IL-4 when cultured on a monolayer of embryonic skin or 3T3 fibroblasts and can form colonies in methylcellulose supplemented with fibroblast conditioned medium. Fibroblast conditioned medium appears to contain a soluble MCCP proliferation factor that maintains biologic activity when heated to 56 degrees C for 45 min but is destroyed by incubation with either trypsin or chymotrypsin. It can be selectively precipitated with 60 to 70% saturated ammonium sulfate. The factor is not absorbed by immobilized antibodies to nerve growth factor. The MCCP proliferation activity of the factor could not be mimicked by IL-1, IL-2, IL-4, granulocyte-macrophage-CSF, granulocyte-CSF, macrophage-CSF, IFN-alpha/beta, IFN-gamma, nerve growth factor, epidermal growth factor, serum fibronectin, heparin, or a number of glycosaminoglycans. At high salt concentrations, the factor passes through a 50-kDa membrane and can be concentrated above a 5-kDa membrane. MCCP acquire a connective tissue phenotype when cultured on a fibroblast monolayer and a mucosal phenotype when cloned in the presence of conditioned medium from PWM-stimulated spleen cells. When cultured in the absence of IL-3 on a monolayer of embryonic skin or 3T3 fibroblasts, mast cell-committed progenitors produce mast cells which stain with berberine sulfate suggesting a connective tissue phenotype; however, the mast cells that develop when mast cell-committed progenitors are cultured in the presence of IL-3 or conditioned media from PWM-stimulated spleen cells do not stain with berberine sulfate. MCCP intercalate into monolayers of embryonic skin or 3T3 fibroblasts, but T cells are not able to associate with the monolayer and can be completely washed away. Attempts to enrich mast cell-committed progenitors by intercalation and elution from embryonic skin monolayers proved unsuccessful, but some enrichment of mast cell-committed progenitors could be achieved by discontinuous Percoll gradients. Thus, we have identified a way to obtain late-stage, mast cell-committed progenitors in an environment that is virtually uncontaminated with other hematopoietic progenitors.
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PMID:The mast cell-committed progenitor. I. Description of a cell capable of IL-3-independent proliferation and differentiation without contact with fibroblasts. 278 62


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