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)

Oleic acid binds in a saturable fashion to human plasma fibronectin (FN). Analysis of the binding indicated the presence of a high affinity binding site with nKa approximately equal to 10 uM-1. Furthermore, it was found that binding of sodium oleate to FN modulated its susceptibility to degradation by various proteinases. FN saturated with sodium oleate was hydrolysed at a higher rate by trypsin, cathepsin D, thermolysin and pancreatic elastase than native FN. In contrast, sodium oleate inhibits the activity of two human granulocyte proteinases, human leucocyte elastase (HLE) and cathepsin G on either FN or on their respective specific synthetic substrates (at concentrations ranging from 10(-6) mM to 10 mM). Cathepsin G inhibition was non-competitive and gave a Ki in the 10 uM range similar to the previously reported inhibitory constant of oleic acid toward HLE.
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PMID:Effect of sodium oleate on the hydrolysis of human plasma fibronectin by proteinases. 329 75

We have identified a neutrophil chemotactic factor (NCF) in supernatants from human blood mononuclear cells (MNC) cultured in the presence of phytohaemagglutinin (PHA). Maximal activity was observed 48 hr after culture. Following gel filtration, NCF eluted as a single major peak, together with proteins, having a molecular size of approximately 10,000 MW. The material gave a single band on SDS-PAGE but was heterogeneous following chromatofocusing (pIs approximately 6.8-7.0, 5.5-6.0 and 5.0). The biological activity of the partially purified material was abolished by trypsin and chymotrypsin treatment. NCF was heat stable (70 degrees, 60 min) and promoted both directional migration (chemotaxis) of neutrophils and, to a lesser extent, stimulated random locomotion (chemokinesis). The factor was not associated with detectable amounts of IL-1, IL-2 or interferon-gamma (IFN-gamma). MNC-derived NCF had a molecular size lower than recombinant granulocyte-monocyte colony-stimulating factor (rGM-CSF) and recombinant tumour necrosis factor (rTNF), and was considerably more active in chemotaxis. Optimal chemotactic concentrations of partially purified MNC-derived NCF were of comparable potency to FMLP and LTB4 and had about 60% of the activity of optimal concentrations of C5a, C5a-des-Arg and platelet-activating factor (PAF). These experiments indicate that the human MNC-derived NCF is a potent chemo-attractant distinct from other cytokines previously reported to promote neutrophil locomotion.
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PMID:The identification and partial characterization of a human mononuclear cell-derived neutrophil chemotactic factor apparently distinct from IL-1, IL-2, GM-CSF, TNF and IFN-gamma. 329 7

In 1984, we reported that while immunoreactive levels of serum alpha-1 proteinase inhibitor (API) increased significantly in nine patients with advanced solid tumors, the functional activity of the inhibitor, as measured by the serum trypsin inhibitory capacity, did not increase proportionately. This suggested that a portion of the circulating API was functionally inert. We have now assayed immunoreactive titers and trypsin inhibitory capacity of serum API of 49 patients with advanced carcinomas and 27 healthy controls. Immunoreactive levels of API (expressed as percentage of normal pooled serum which was taken as 100%) in cancer subjects were significantly elevated as compared to normals (mean +/- SE: 233 +/- 9.0% versus 102 +/- 2.0%, P less than 0.05). Although the trypsin inhibitory capacity of the cancer group (16.0 +/- 0.9 units/ml) was significantly elevated (P less than 0.05) as compared to normals (9.9 +/- 0.1 units/ml), this increase was less than that in the immunoreactive titer of API, suggesting the existence of functionally inert API in serum. The fraction of API which was functionally active in this group of cancer patients was 71.0 +/- 3.0% which was significantly less than the normal 98.0 +/- 2.0% (P less than 0.05). In 12 patients followed serially, both immunoreactive levels of API and the trypsin inhibitory capacity increased significantly at the time of clinical progression of disease. There was a significant correlation between increasing absolute granulocyte count and increasing trypsin inhibitory capacity (correlation coefficient 0.66; P less than 0.001). Neither disease progression nor increasing granulocyte count, however, was associated with increasing proportion of functionally inactive API. The inactive form of API had the same molecular weight as the native molecule as shown by gel permeation chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis/Western blot analysis of cancer sera. Therefore, the inactive form was not due to a complex between API and a tumor-derived protease or to proteolytic fragmentation of the native API. Elastase inhibitory capacity of cancer sera with subactive API was essentially identical with trypsin inhibitory capacity indicating that the active site methionine was not oxidized in the inert API. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis/Western blot analysis showed that both normal and cancer serum API existed as two mass variants, at Mr 58,000 and 56,000. Both variants formed complexes with elastase and were functionally active.
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PMID:Serum alpha-1 proteinase inhibitor in advanced cancer: mass variants and functionally inert forms. 349 70

Mouse splenocytes are induced by pokeweed mitogen to secrete a factor that stimulates mouse hemopoetic (spelling per Nomina Histologica in the Nomina Anatomica, 5th edition, 1983, Williams and Wilkins, Baltimore) progenitor cells to undergo proliferation and differentiation into granulocytes and macrophages in a semi-solid culture system. The granulocyte and macrophage colony-stimulating factor (GM-CSF) was purified with a four-step procedure that includes ultrafiltration, chromatography on DEAE-agarose, Sephacryl S-200, and chromatofocusing gel. The isoelectric point (pI) of 4.2 of the GM-CSF was determined by analytical isoelectrofocusing gel electrophoresis. The sensitivity of the biological activity of GM-CSF to digestion by trypsin and neuraminidase suggests that GM-CSF is a glycoprotein with its sugar moieties at the active site. The GM-CSF is also sensitive to heat denaturation at 60 degrees C or higher suggesting that a three-dimensional conformation is required for its biological activity. The molecular weight of GM-CSF is approximately 57,000 Daltons as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate.
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PMID:Properties and purification of a colony-stimulating factor of granulocytes and macrophages produced by mouse spleen cells. 349 11

We undertook a study to determine whether cytokines exist which are responsible for the activation of polymorphonuclear neutrophilic granulocytes (PMN) besides the already well-known stimuli. Lucigenin-dependent chemiluminescence was used to measure human PMN activation. Addition of supernatants from mononuclear cells stimulated with bacterial lipopolysaccharide produced a long-lasting activation of granulocytes. Induction of chemiluminescence was dose-dependent and inhibitable by superoxide dismutase. Fractionation of mononuclear cells by adherence to plastic dishes or counterflow elutriation proved that monocytes were able to generate granulocyte-activating mediators (GRAM). Production of GRAM was dependent on the dose of the stimulus and appeared to be maximal after 24 h of incubation. Addition of cycloheximide resulted in significantly decreased release of GRAM. Partial characterization of the activity showed GRAM to be heat-labile and sensitive to trypsin, indicating a protein nature of GRAM. The activity fractionated into 2 distinct peaks, one corresponding to 60 kD and another below 10 kD. The interleukin 1 activity did not appear to co-fractionate with GRAM. Evidence presented suggests that the activity corresponds to factors unlikely to have been described previously.
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PMID:Granulocyte-activating mediators (GRAM): I. Generation by lipopolysaccharide-stimulated mononuclear cells. 352 11

Lysostaphin, a staphylococcus-derived staphylocidal substance, has widely been used in assays of granulocyte phagocytic and bactericidal capability. It rapidly kills extracellular bacteria. Thus, a separate determination of intracellular surviving bacteria can be performed. One prerequisite for this approach is the safe inactivation of lysostaphin (usually brought about by trypsin) before the intracellular bacteria are externalized for plating. This inactivation has been found by others to be incomplete. Data are presented demonstrating a safe inactivation of lysostaphin by trypsin, if the pH value is maintained within the alkaline range. A low variation of results is obtained by plotting the total number of bacteria killed per incubate vs the logarithm of initial bacterial inoculum or of the intracellular surviving bacteria, leading to linear regression lines. The variation of the results increases greatly for initial bacteria/granulocyte proportions of greater than 5/1. The results obtained for two different St. aureus strains are significantly different. Dexamethasone pretreatment (12 mg p.o. within 8 h) had no detectable influence, when fresh blood was assayed, while blood storage at room temperature for 12 h (without dexamethasone pretreatment) led to a significant functional impairment, mainly of bactericidal capability when analyzed in a pairwise fashion. A major limitation of this kind of assays is that killed bacteria cannot be determined directly.
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PMID:Lysostaphin-based assay of human granulocyte functions: a reevaluation. 353 97

We have carried out a comparative study of mature murine granulocytes with two immature haemopoietic cell lines (multipotential cells, FDCP-Mix, and granulocyte progenitor cells, FDCP-2) with respect to the structure and composition of their surface membrane glycopeptides. The glycopeptides were labelled biosynthetically by incubation of the cells for 1-3 days with [3H]glucosamine. Cell-associated glycopeptides were released by treatment with trypsin and the trypsin extract was exhaustively digested with Pronase to remove most residual peptide. Radiolabelled materials were fractionated by chromatography on lectin affinity columns connected in the series: lentil lectin (LCA), concanavalin A (Con A) and wheat germ agglutinin (WGA). Lectin-binding glycopeptides were eluted with appropriate competing sugars and further analysed by gel filtration, base/borohydride elimination and susceptibility to degradation by glycosidases including endo-beta-galactosidase. Abundant quantities of N-linked polylactosamine-type glycopeptides, which bound only to the WGA columns, were identified on mature granulocytes but the molecules were highly-branched (i.e. resistant to endo-beta-galactosidase). In contrast, there seemed to be very little branching in the polylactosamine chains from FDCP-2 cells, whilst corresponding carbohydrates from multipotential FDCP-Mix cells gave evidence for both linear and branched domains in the same, large complex glycans. O-Linked tetrasaccharides of general structure: NeuAc-Gal-(NeuAc)-GalNAc were found in clusters on WGA-binding glycopeptides from all cell types, these components being especially prominent on mature granulocytes. FDCP-2 cells were distinguished by the presence of monosialylated and non-sialylated counterparts of the foregoing tetrasaccharides. The relative amount of LCA-binding glycopeptides was low on FDCP-Mix cells by comparison with FDCP-2 cells and mature granulocytes. Our findings therefore demonstrate that notable differences in gross composition and molecular fine structure of surface membrane glycopeptides are detectable in haemopoietic cells at different stages of development. The relationship of these differences to the biological properties of cell surfaces remains to be established.
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PMID:Developmentally-related changes in surface membrane glycopeptides of murine haemopoietic cells. 359 80

To determine whether steroidal or non-steroidal anti-inflammatory agents inhibit granulocyte migration, we measured granulocyte adherence to and migration across the intact endothelial layer of bovine pulmonary artery intimal explants. Explants were placed endothelium uppermost in chemotaxis chamber with either fetal calf serum (FCS) or zymosan activated plasma (ZAP) in medium 199 in the lower well and 5 X 10(6) separated 51Cr labelled granulocytes/ml in medium 199 + FCS in the upper well. Methylprednisolone (0.3 and 3.0 mg/ml), indomethacin (5 and 50 microM) and ibuprofen (10 and 100 microM) were also added to the upper well of some chambers. After 30, 60, 120 or 180 minutes of incubation the chambers were dismantled. Granulocyte adherence was assessed by rinsing the explant in 0.1% trypsin; the number of radioactive counts in the trypsin wash represented the number of adherent cells. Those remaining in the explant represented the number of granulocytes that migrated into the explant. At each time studied, chemotaxin-induced granulocyte migration was 2-3 times that of unstimulated or random migration (180 min incubation with FCS = 30.5% +/- S.E. 2.1; with ZAP in lower well = 61.6 +/- 3.4). Both unstimulated and chemotaxin-induced migration was significantly decreased from 30 minutes by 3.0 mg/ml of methylprednisolone (180 min incubation with ZAP in lower well = 22.3 +/- 5.8), but not by 0.3 mg/ml. However, one hour pretreatment of either the granulocytes or the explant with 3.0 mg/ml methylprednisolone had no significant effect on granulocyte migration. In contrast, granulocyte migration was unaltered by treatment with either indomethacin or ibuprofen. Methylprednisolone, indomethacin and ibuprofen had little effect on granulocyte adherence. We conclude that granulocyte migration across an intact endothelial layer is inhibited by high dose corticosteroids but not by cyclooxygenase inhibitors. This suggests a plausible rationale for use of high doses of corticosteroids in clinical states where granulocytes may mediate tissue injury.
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PMID:The effect of steroidal and nonsteroidal anti-inflammatory agents on granulocyte migration into bovine pulmonary artery intimal explants. 369 31

We have previously shown that human megakaryocyte colony-stimulating activity (Meg-CSA) is present in sera from patients with bone marrow megakaryocyte aplasia. In this report, we demonstrate that Meg-CSA is also present in sera from dogs rendered aplastic by 1000 rad total body irradiation. Canine serum Meg-CSA has activity comparable to human when assayed in plasma clot cultures containing human bone marrow mononuclear cells. Because of the uniform high potency and ready availability of aplastic canine sera, it was utilized initially for Meg-CSA purification. Sequential ammonium sulfate precipitation to approximately 80% saturation resulted in recovery of 59%-69% of the serum protein and of 75%-103% of the original serum Meg-CSA. The fraction precipitable between ammonium sulfate saturations of 0% and 44%-50% (fraction I) contained 53%-76% of the initial serum Meg-CSA and 25%-32% of the initial serum protein. This represents an enrichment of Meg-CSA specific activity by over 100%. The Meg-CSA eluted from Sephacryl S-300 in a single peak corresponding to a molecular weight of 175,000. This Meg-CSA peak also contained IgG, but the Meg-CSA did not bind to protein A-agarose. Meg-CSA was 90% inactivated by trypsin digestion for 4 h at 37 degrees C and by exposure to 5mM dithiothreitol for 2 h at room temperature. Exposure to either 6 M guanidine for 1 h at room temperature or 8 M urea for 1 h at 4 degrees C resulted in a 70% loss of Meg-CSA. At culture concentrations capable of stimulating maximal megakaryocyte colony formation, fraction I supported no colony growth by myeloid (CFU-GM) or late erythroid (CFU-E) human hematopoietic progenitor cells. Erythroid burst-promoting activity (BPA) was not detected in fraction I from two of three different aplastic canine sera tested. Therefore, Meg-CSA is functionally distinct from granulocyte-monocyte colony-stimulating factor (GM-CSF), erythropoietin, and BPA. The data indicate that serum Meg-CSA is a 175,000-dalton protein (megakaryocyte colony-stimulating factor, Meg-CSF) in which higher order structure and disulfide bonding are necessary for biologic activity. Partially purified Meg-CSF manifests functional specificity for the CFU-Meg hematopoietic progenitor cell.
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PMID:Human megakaryocyte colony-stimulating factor in sera from aplastic dogs: partial purification, characterization, and determination of hematopoietic cell lineage specificity. 387 46

Soluble factors that enhance maturation of murine B lineage precursor cells in vitro were partially purified from the serum of very young NZB mice and characterized biochemically and biologically. Activity was initially detected by induction of colony-forming activity and surface immunoglobulin (sIg) on normal sIg- marrow cells as well as responsiveness of a pre-B cell line. Pooled sera from 4- to 5-wk-old NZB mice were initially fractionated on Sephacryl S-300 and Sephadex G-100 superfine columns. Fractions with activity (corresponding to m.w. of 15,000 to 45,000) were pooled and further separated. The activity was eluted as a single peak by hydrophobic (phenyl-Sepharose, with 0.8 M (NH4)2SO4) and lentil lectin affinity chromatography but resolved into three distinct peaks in preparative isoelectric focusing (IEF), with pI values of 3.5, 7.8, and 8.4. The latter two merged into a single peak with a pI value of 8.8 when the sample was further treated with neuraminidase before IEF. These three IEF fractions, each of which were enriched at least 1000-fold in specific activity relative to starting serum, were then characterized. Each was stable at pH 2 but sensitive to trypsin, 10 M urea, and heat treatment (56 degrees C for 1 hr). In nonreduced SDS-poly-acrylamide gel electrophoresis, their mobilities corresponded to m.w. of 17,000 for peak I (pI 3.5), 15,000 for peak II (pI 7.8), and 15,000 for peak III (pI 8.4). Interleukin 1, interleukin 2, interleukin 3, colony-stimulating factor for granulocyte and macrophage progenitors, antiviral, or B cell growth factor type I-like activities were not demonstrable. Peaks II and III, but not peak I, induced Ig secretion of anti-stimulated B cells. Peak I was also less effective than peaks II and III in induction of sIg on an established pre-B cell line. However, all fractions were equally effective in enhancing maturation of normal sIg- B lineage cells. Thus, serum from 4- to 5-wk-old NZB mice contains at least two distinct soluble factors that can enhance the maturation of sIg- B lineage cells in vitro. The biologic and biochemical characteristics of these factors appear to differ from those of previously well-defined cytokines.
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PMID:Humoral factors in very young NZB mice that enhance the maturation of normal B cell precursors. Partial purification and characterization. 392 91


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