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)

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

Different molecular masses have been assigned to the human interferon-gamma receptor (HuIFN-gamma-R) by several authors. After extensive purification from Raji cells, this receptor was shown in a previous work to consist of two major protein species with molecular masses of 92 kD and 50 kD, as revealed by SDS-PAGE. We show here that the 50-kD band is most probably a degradation product of the 92-kD band due to a trypsin-like protease active during the purification process. The native protein of Raji cells seems, therefore, to have a molecular mass of 92 kD. The same molecular mass was found with Colo 205 cells (derived from a colon carcinoma). However, in conditions where degradation does not occur, the HuIFN-gamma-R shows a certain polymorphism: in IM-9 cells, another B-cell line, two bands exist with molecular masses of 95 kD and 85 kD, and in Wish cells, an amnion-derived cell line, one (or two) band(s) can be detected around 87 kD. This polymorphism is due at least in part to a variable extent of N-glycosylation from line to line and also within the same line, since after tunicamycin treatment of the Raji, IM-9, and Wish cells, very similar bands are obtained with a molecular mass of 72 kD.
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PMID:Molecular characterization of the human interferon-gamma receptor: analysis of polymorphism and glycosylation. 253 62

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

In the present study we have evaluated the effect of recombinant human fibroblast, IFN-beta ser, and immune, IFN-gamma, interferon, alone and in combination, on the proliferation of fifteen early passage human glioblastoma cell cultures. Explant cultures were established from glioblastoma tumor tissue obtained at the time of surgery. After sufficient outgrowth, cultures were dispersed with trypsin/versene and maintained as independent cell lines. IFN-beta ser induced a greater than or equal to 50% reduction in the 7 day growth of 6 of the 15 cultures. The majority of cultures, 9 of 15, displayed less than or equal to 50% growth suppression in comparison with control cultures after 7 days exposure to 2000 Units/ml of IFN-beta ser. When treated with 2000 Units/ml of IFN-gamma, only 1 of the 15 glioblastoma cultures exhibited a greater than or equal to 50% reduction in growth. In contrast, when treated with the combination of IFN-beta ser plus IFN-gamma, 1000 Units/ml of each interferon preparation, 12 of 15 cultures were inhibited by greater than or equal to 50% after 7 days growth. The combination of interferons was effective in suppressing glioblastoma growth both in cultures displaying relative sensitivity and those exhibiting innate resistance to either or both types of interferon when employed alone. One glioblastoma culture, G-7, was studied through 45 passages and displayed the same sensitivity at different passages to growth inhibition when exposed to IFN-beta ser, IFN-gamma or both interferons. Based on previous clinical studies indicating that IFN-beta or IFN-gamma when administered alone to patients do not generally alter the clinical progression of malignant gliomas, the present results suggest that the combination of IFN-beta plus IFN-gamma may prove more effective than either agent alone in the clinical treatment of patients with glioblastoma multiforme.
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PMID:Enhanced in vitro growth suppression of human glioblastoma cultures treated with the combination of recombinant fibroblast and immune interferons. 283 97

Purified mouse interferon gamma (MuIFN-gamma), a lymphokine having potent antiviral, immunomodulatory, and growth inhibitory activities, is internalized (t1/2 less than 1.0 min) by mouse L929 fibroblasts via receptor-mediated endocytosis. Individual MuIFN-gamma molecules, identified by a postembedding immuno-gold technique, are then transported to the cell nucleus, perhaps through nuclear pores, into areas of dense chromatin. Purified, isolated nuclei of L929 cells bind radiolabeled MuIFN-gamma specifically and with high affinity (Kd = 2 X 10(-10) M). These nuclear membrane receptors, distinct from those for MuIFN-beta, number about 24,000/nucleus. Treatment of nuclei with trypsin prevents binding of MuIFN-gamma. The demonstration of rapid cellular uptake and transport of MuIFN-gamma into the dense chromatin, perhaps facilitated by nuclear receptors, suggests that IFN-gamma molecules, alone or bound to receptor, may directly affect genome regulation.
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PMID:Transport of gamma-interferon into the cell nucleus may be mediated by nuclear membrane receptors. 294 74

Monocytes isolated from human blood by centrifugal elutriation exhibited little ability to ingest rabbit erythrocytes (ER), zymosan particles, or desialated sheep erythrocytes. In contrast, 85 to 95% of these cells rosetted with C3b- or C3bi-bearing sheep erythrocytes (ES) or ingested IgG-coated ES. Preincubation of the monocytes with human lymphocytes increased their ability to ingest ER. the ER phagocytosis-inducing activity was contained in the 105,000 X G supernatant of lymphocyte lysates. These supernatants increased the percentage of ingesting monocytes from 5 to 15% to 80% within 60 min. The soluble factor was found to be relatively heat stable, inactivated by trypsin, and distinct from IFN-gamma. Its m.w. is less than 13,000. It was present in B and T lymphocytes and also in U937 cells. These results suggest that the ability of human monocytes to ingest nonopsonized particulate activators of the alternative complement pathway is a cytokine-inducible property and that the effect of the cytokine on complement receptor- or Fc receptor-dependent adherence or ingestion of opsonized particles is minor.
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PMID:Phagocytosis by human monocytes of unopsonized particulate activators of the human alternative complement pathway: induction by cytokine. 308 15

The complete amino acid sequence of recombinant human gamma-interferon (HuIFN-gamma) produced in Escherichia coli was determined using a gas-phase protein sequencer. The sequence was established by automated Edman degradation on the intact protein and its peptides obtained after Staphylococcus aureus V8 protease or trypsin digestion. The result was identical to the amino acid sequence predicted from the nucleotide sequence of the cloned HuIFN-gamma cDNA except that it was missing the four carboxy-terminal residues, Arg-Ala-Ser-Gln.
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PMID:Determination of the complete amino acid sequence of recombinant human gamma-interferon produced in Escherichia coli. 309 40

Natural human interferon gamma(IFN-gamma) was purified from the conditioned medium of peripheral blood leukocytes using selective silica gel adsorption and antibody-affinity chromatography. SDS-PAGE and Western blot analysis demonstrated three major species with molecular masses of 25 kDa, 20 kDa and 17 kDa. Structural analysis of this natural IFN-gamma preparation demonstrated a pyroglutamate residue at the amino terminus and a heterogeneous carboxyl terminus. The longest and most predominant polypeptide was 138 amino acids in length, which is five residues shorter than the sequence predicted from the cDNA. The presence of multiple-carboxyl-terminal forms indicated possible proteolytic processing during induction or protein purification. Limited proteolytic digestion of full-length recombinant IFN-gamma with endoproteinase Lys-C and trypsin revealed that the carboxyl-terminal 15 residues could be released under conditions in which the core portion of the polypeptide chain remained intact. Thus, the heterogeneity of natural IFN-gamma may be explained by partial proteolytic degradation of the molecule and differences in the degree of glycosylation as previously reported [Rinderknecht, E., O'Conner, B. H. & Rodriguez, H. (1984) J. Biol. Chem. 259, 6790-6797].
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PMID:Structural characterization of human interferon gamma. Heterogeneity of the carboxyl terminus. 310 13


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