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)

In the murine system, a number of cytokines (including interleukin-3 [IL-3], IL-4, and stem cell factor [SCF]) promote the growth of mast cells (MCs). However, so far little is known about factors controlling differentiation of human MCs. Recent data suggest that human MCs express receptors (R) for SCF. The aim of the present study was to investigate whether recombinant human (rh) SCF induces differentiation of human MCs from their precursor cells. For this purpose, bone marrow (BM; normal donors, n = 6) and peripheral blood (PB; normal donors, n = 11) mononuclear cells (MNC) were cultured in the presence of rhSCF, rhIL-3, rhIL-4, rhIL-9, recombinant human macrophage colony-stimulating factor (rhM-CSF), or control medium in long-term (8 weeks) suspension cultures. After 4 weeks, up to 5% of the MNC (BM and PB) cultured in the presence of rhSCF, but not in the presence of other cytokines, were found to exhibit the characteristics of MCs. These MCs expressed the YB5.B8-reactive domain of the SCF R as well as IgE R, as determined by combined toluidine blue/immunofluorescence staining. Myeloid antigens, likewise expressed on human basophils (ie, CD11b, CDw65, and Bsp-1), could not be detected on these cells. Furthermore, rhSCF, but not rhIL-3, rhIL-4, rhIL-9, or rhM-CSF, induced dose- and time-dependent increases in the formation of cellular tryptase (an MC-specific enzyme) (rhSCF [100 ng/mL], 1,308 +/- 679 ng/mL v control medium, 18 +/- 6 ng/mL tryptase on day 35 of PB cell cultures), as well as an increase in cellular histamine. After 6 to 8 weeks, when other mature hematopoietic cells decreased, MCs still could be detected in culture, with up to 40% of all cells being MCs. To test whether rhSCF also activates tissue MCs, we performed histamine release experiments (dispersed tissue; lung, n = 3; uterus, n = 3). SCF was found to enhance (by up to 3.4-fold) the capacity of the MCs to release histamine upon cross-linkage of IgE R with anti-IgE. Together, these observations suggest that rhSCF induces in vitro differentiation of human MCs from their BM and PB precursor cells in long-term culture and upregulates MC releasability.
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PMID:Induction of differentiation of human mast cells from bone marrow and peripheral blood mononuclear cells by recombinant human stem cell factor/kit-ligand in long-term culture. 138 99

The MET proto-oncogene encodes a transmembrane tyrosine kinase receptor for HGF (p190MET). In this work, p190MET was immunoprecipitated, allowed to phosphorylate in the presence of [gamma-32P]ATP, and digested with trypsin. A major phosphopeptide was purified by reverse phase chromatography. The phosphorylated tyrosine was identified as residue 1235 (Tyr1235) by Edman covalent radiosequencing. A synthetic peptide derived from the corresponding MET sequence was phosphorylated by p190MET in an in vitro assay and coeluted in reverse phase chromatography. Tyr1235 lies within the tyrosine kinase domain of p190MET, within a canonical tyrosine autophosphorylation site that shares homology with the corresponding region of the insulin, CSF-1 and platelet-derived growth factor receptors, and of p60src and p130gag-fps. The p190MET kinase is constitutively phosphorylated on tryosine in a gastric carcinoma cell line (GTL16), due to the amplification and overexpression of the MET gene. Metabolic labeling of GTL-16 cells with [32P]orthophosphate followed by immunoprecipitation and tryptic phosphopeptide mapping of p190MET showed that Tyr1235 is a major site of tyrosine phosphorylation in vivo as well. Since phosphorylation activates p190MET kinase, we propose a regulatory role for Tyr1235.
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PMID:Identification of the major autophosphorylation site of the Met/hepatocyte growth factor receptor tyrosine kinase. 165 90

The effects of lamina propria mononuclear cell culture supernatant on epithelial cell DNA synthesis were studied using cells isolated from patients with inflammatory bowel disease and normal controls. Supernatants from resting and phytohaemagglutinin stimulated cells were studied and supernatants that strongly promoted DNA synthesis were pooled, and growth factor activity partially characterised. The effects of recombinant interleukins-1 beta,2,3,interferon-gamma, and granulocyte macrophage colony stimulating factor were tested in the same system. Resting lamina propria mononuclear cells produce factors that increase DNA synthesis. Production of these factors is increased by phytohaemagglutinin stimulation. No significant differences were found in production of these factors between patients with inflammatory bowel disease and normal controls. The molecular weight of the active factor(s) lies in the region 31-48 kD. Chromatofocusing produced two peaks of activity, one in the region pk 5.5 and one around pk 6.4. The activity was heat and acid pH labile. Activity was not destroyed, however, by 0.05% trypsin. Recombinant granulocyte macrophage colony stimulating factor was a weak stimulus to epithelial DNA synthesis, interleukin-1 beta was weakly inhibitory but other cytokines tested did not have any effect. Granulocyte macrophage colony stimulating factor is probably important in controlling epithelial cell growth.
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PMID:Production of epithelial cell growth factors by lamina propria mononuclear cells. 174 Feb 74

We have shown that certain murine tumors grow more slowly and spread less readily in immune deficient animals. We have also demonstrated that immunologic factors explain certain aspects of this difference. In the work presented we demonstrate that a subpopulation of splenocytes produce a factor(s) that enhances tumor cell proliferation in vitro. We also describe an in vitro model to determine the level of tumor stimulatory activity. We found that the tumor cell growth-enhancing activity (TEA) is heat stable but sensitive to trypsin digestion, low pH and beta-mercaptoethanol. TEA production is found to be insensitive to mitogen stimulation such as concanavalin A, lipopolysaccharide, and phytohemagglutinin. Among the known growth factors and interleukins we have tested (interleukin 1-7, basic FGF, EGF, TGF-beta PDGF, GM-CSF, and MCSF), none appear to account for TEA activity.
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PMID:Initial description of a tumor enhancing activity produced by murine splenocytes. 188 89

An adherent cell line, termed TC-1, has been isolated from long-term liquid culture of murine marrow cells by repeated exposure of the adherent cells to 0.1% trypsin. This is an alkaline phosphatase-positive cell line showing variable staining with acid phosphatase and alpha-naphthyl acetate esterase. On electron microscopy, the cells have moderate amounts of rough endoplasmic reticulum and variable numbers of polyribosomes. Some cells contain large clusters of laked glycogen particles. Intermediate junctions are present between some cells. Conditioned medium from this cell line produced from 384 to 638 units of CSF-1 per milliliter by radioimmunoassay and a CSF-1-dependent synergistic activity, which stimulates giant macrophage colony formation of marrow cells in soft agar. The conditioned medium also stimulates 3H-TdR incorporation by marrow cells in liquid culture and induces secondary adherent cell lines. The growth factor(s) produced by the TC-1 stromal cell line may be important in the regulation of early stages of hematopoietic differentiation. Two subclones, TC-1-C-11 and TC-1-C-3, have been isolated from passage 25 of the TC-1 cells by a penicylinder separation technique. The TC-1-C-11 is phenotypically like the parent TC-1 line and produces macrophage growth factors. The TC-1-C-3 grows as an epithelioid monolayer with visible junctions among adjacent cells under phase contrast microscopy. This subclone produces retrovirus and is capable of providing anchorage support for hematopoietic stem cells. The TC-1 cell line and its subclones may provide models for the control of early stem cell proliferation and differentiation.
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PMID:Hematopoietic factor production by a cell line (TC-1) derived from adherent murine marrow cells. 241 62

1. Colony-stimulating factor (CSF-1) was isolated from a large volume of fresh normal human urine by 5 steps of purification and enrichment. 2. The purification factor is 100,000 fold and the purified compound exhibits a 2.16 x 10(7) U/mg of protein sp. act. 3. The isolated CSF-1 is a sialoglycoprotein with 41.5% of carbohydrate. The almost complete removal of this carbohydrate moiety (up to 91%) was achieved by incubation with trifluoromethane sulfonic acid. 4. The deglycosylated CSF-1 (DG-CSF-1) possesses an apparent Mr 38,000 compared to native CSF-1 with an initial Mr 57,000 (Goa et al., 1988). 5. The features of the interaction of radio-iodinated [125I]CSF-1 with single cell suspensions from various human tissues (bone marrow, spleen, blood, peritoneal cavity, alveolar lavage, lymph node and thymus), were studied. 6. The binding activity of peritoneal macrophages was the highest among the cells examined and erythrocytes, thymus and blood granulocytes showed no CSF-1 binding. 7. On incubation with [125I]CSF-1 at 0 degrees C, cellular binding of [125I]CSF-1 reached a stable maximum within 16 hr. This is in contrast to the association behaviour at higher temperature. 8. At 37 degrees C, cellular associated [125I]CSF-1 levels reached, within 90 min, an unstable maximum which was up to 10 times less than that occurring under the same conditions at 0 degree C. From the Scatchard plot analysis, we obtained the affinity constant and the number of receptor(s). 9. The binding site is sensitive to trypsin. 10. The receptor alone, (labelled by cross-linking to [125I]CSF-1 with di-succinylimidyl-suberate), is a polypeptide with an approx. Mr 110,000. 11. Our results showed that the receptor of CSF-1 is a tyrosin-kinase.
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PMID:The specific binding activities of human urinary radioiodinated colony-stimulating factor-1 to various human tissue cells. 252 35

In amphibians, zygotes microinjected with cytosol of unactivated eggs are arrested at metaphase of mitosis. The factor responsible for this effect has been designated 'cytostatic factor, (CSF)'. CSF is inactivated by Ca2+ addition to cytosols. During storage of the Ca(2+)-containing cytosols, a stable CSF activity develops. Therefore, the first Ca(2+)-sensitive CSF and the second Ca(2+)-insensitive CSF have been referred to as primary CSF (CSF-1) and secondary CSF (CSF-2), respectively. We have partially purified CSF-1, which had been stabilized with NaF and ATP, and CSF-2 from cytosols of Rana pipiens eggs by ammonium sulphate (AmS) precipitation and sucrose density gradient centrifugation or gel filtration, and investigated their molecular characteristics. CSF-1 was sensitive to protease, but resistant to RNAse, and inactivated within 2 h at 25 degrees C. CSF-1 could be sedimented in a sucrose density gradient from a fresh cytosol or its crude fraction precipitated at 20-30% saturation of AmS, showing the sedimentation coefficient 3S. When analyzed by SDS-polyacrylamide gel electrophoresis (PAGE), all the proteins in partially purified CSF-1 samples entered the gel and were separated into numerous peptide bands. In contrast, CSF-2 was an extremely large molecule, being eluted from Sepharose columns as molecules larger than 2 x 10(6), and failed to enter the gel when analyzed by SDS-PAGE. It could be purified 40 times from cytosols. CSF-2 was a highly stable molecule, being neither inactivated nor dissociated at pH 11.5 or by 4M-NaCl and LiCl and 8 M-urea. It was also resistant to RNAse treatment. However, CSF-2 could be broken down into small peptides of variable sizes by trypsin, alpha-chymotrypsin, and papain, but not by S. aureus V8 protease, although it was less sensitive to proteases than CSF-1. The dose-dependency test showed that the activity of CSF-2 is independent of its concentration and that an amount of CSF-2 could cause cleavage arrest earlier when injected into a blastomere in a larger volume.
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PMID:Molecular characteristics of cytostatic factors in amphibian egg cytosols. 256 70

Recent work from our laboratory has shown that NK cells rapidly release preformed factor(s) that stimulate monocyte oxidative metabolism and microbicidal activity. We have hypothesized that such factors could also activate macrophage (M phi) tumor lysis and might be stored in the cytoplasmic granules. Granules were isolated from the RNK large granular lymphocyte leukemias by nitrogen cavitation and Percoll fractionation of the cell homogenate. Utilizing CSF-1 differentiated murine bone marrow-derived M phi and P815 tumor target cells, a M phi-activating factor (MAF) was found. The MAF activity was identified in two peaks, the first was coincident with dense granule enzymes and was 60 times more concentrated per mg protein than a second peak in the cytosol fractions. Solubilization in 2 M NaCl was necessary to recover activity from both peaks. Granule NK-MAF required the simultaneous presence of LPS in order to induce tumoricidal activity. Kinetics of NK-MAF activation peaked after 12 h of exposure. The NK-MAF was short lived in the solubilized granules; however, its heat resistance allowed us to prepare enriched and stable preparations. Treatment of NK-MAF with pepsin but not trypsin completely abrogated its activity. The NK-MAF passed through an ultrafiltration membrane with a nominal cut-off of 10 kDa. This work indicates that NK cell granules contain a small heat-stable peptide capable of activating M phi tumoricidal activity.
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PMID:Identification of a macrophage-activating factor in granules of the RNK large granular lymphocyte leukemia. 264 45

Incubation of murine bone marrow-derived macrophages (BMM) in medium containing recombinant macrophage colony-stimulating factor (rM-CSF) stimulated influx, efflux, and the net accumulation of the fluid-phase pinocytic marker, lucifer yellow (LY). Stimulation was dose dependent, occurred within 5 min of addition of the growth factor, and was sustained. Previous experiments had shown that BMM treated with PMA were stimulated to accumulate LY, but compared with rM-CSF-treated cells, the onset of stimulation in PMA-treated macrophages was slower. In further comparisons of rM-CSF- and PMA-stimulated LY accumulation, it was found that rM-CSF-stimulated pinocytosis could be abolished by pretreatment with 0.5 mg/ml trypsin, whereas neither unstimulated nor PMA-stimulated LY accumulation was affected by trypsin pretreatment. These findings indicate that the rM-CSF response was initiated at the cell surface, while the PMA response occurred via intracellular (or trypsin-resistant) receptors. However, once initiated, the pinocytic responses elicited by either agent were very similar. First, rM-CSF-treated cells, like PMA-treated cells, showed extensive ruffling and formation of large phase-bright pinosomes. Second, both rM-CSF- and PMA-stimulated LY accumulation could be inhibited by treatment of cells with the cytoskeleton destabilizing drugs nocodazole, colchicine, or cytochalasin D. Finally, rM-CSF, like PMA, was found to stimulate efflux of LY from cells preloaded with the dye. Thus, both rM-CSF and PMA stimulate the net rate of solute flow through the macrophage endocytic compartment.
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PMID:Macrophage colony-stimulating factor (rM-CSF) stimulates pinocytosis in bone marrow-derived macrophages. 268 16

In primary cultures of rat preadipocytes (PA) isolated from epididymal or perirenal depots, rat serum is more effective than other animal sera (fetal calf, newborn calf, human, horse, rabbit, cat, sheep, goat, dog, pig) in promoting adipogenic conversion, biochemical differentiation, and mitogenesis. Only mouse serum is comparable to rat serum. This activity is attributable to a specific growth factor (preadipocyte stimulating factor, PSF). An assay for PSF in rat serum was devised using PA from perirenal fat of 3-month-old Fischer 344 rats grown first to confluence in FCS for 8 days and then for the next 3 days in test serum, followed by measurement of triglyceride (TG) and glycerol-3-phosphate dehydrogenase (GPDH). Rat serum induces dose-dependent rapid cell division, which coincides with accumulation of TG and increase of GPDH; for routine quantitation, TG is assayed. The biochemical characteristics of PSF in serum are as follows: stable at 4 degrees C for up to 1 year; inactivated at 100 degrees C (80% loss, 30 min) but stable at 56 degrees C for 1 hr; stable at pH 2-12; non-dialyzable; completely resistant to pepsin, trypsin, and chymotrypsin but destroyed by pronase and subtilisn; stable to DTT and periodate; and m.w. between 68 kDa (Sephacryl-300) and 58 kDa (Sephacryl-300 in 5 M urea). PSF activity is greater in serum from Wistar than from Fischer 344 rats, while activity of serum from Zucker obese (fa/fa) rats is at least as great as that from Wistar rats and, like serum of rats made obese by feeding a high-fat, high-carbohydrate diet, is not suppressed. PSF activity is not due to insulin, insulin-like growth factor-1 (IGF-1), growth hormone, glucocorticoids, or combinations of these hormones. PSF activity was not seen with a number of growth factors including colony-stimulating factor (CSF-1), GM-CSF, interleukins 1, 2, and 3, neuroleukin, tumor necrosis factor, and others. PSF is distinct from the low molecular weight (4-8 kDa) differentiation factor present in rat serum, FCS, and human serum that promotes the adipogenic conversion and cellular differentiation of 3T3-L1, 3T3-F442A, and Ob17 cells. PSF appears to be a new differentiation factor for rat preadipocytes, has properties suggestive of a highly glycosylated protein, and may be highly species specific.
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PMID:Preadipocyte stimulating factor in rat serum: evidence for a discrete 63 kDa protein that promotes cell differentiation of rat preadipocytes in primary cultures. 268 98


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