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

In halothane-anesthetized and -ventilated cynomologus macaque monkeys, the effects of administering vehicle (n = 3) or the neutral endopeptidase inhibitor N-[L-(1-carboxy-2-phenyl)ethyl]-L-phenylalanyl-beta-alanine (16 mg/kg, n = 5; or 100 mg/kg, n = 3, intravenously) was examined. Cisternal CSF aliquots were examined by radioimmunoassay: 1) for Met enkephalin; 2) after trypsin and carboxypeptidase B treatment for encrypted enkephalin (X-ENK); 3) for substance P; and 4) for unmetabolized drug. Similar measures were carried out in femoral artery and femoral venous plasma, except that substance P was not assayed. In CSF, prior to drug, low, but measurable levels of enkephalin (61 pg/ml), X-ENK (285 pg/ml) and substance P (16 pg/ml) were observed. Vehicle-injected animals showed no change from baseline levels over a 4-hr sampling period in either plasma or CSF levels. In contrast, following 16 mg/kg, in CSF, there was a significant 9-fold increase in MET and 11-fold increase in X-ENK at 30 min. CSF-substance P levels rose also by a factor of 2, with the peak effect observed at 60 min. All levels displayed a significant reduction by 4 hr. There was no statistical difference between the maximum effects observed with either the 16- or 100-mg/kg dose. Plasma peptide levels of enkephalin and X-ENK were not altered by drug. CSF displayed significant drug levels by 30 min, which were between 0.1 and 1% of levels observed concurrently in plasma.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Effects of [N-(L-(1-carboxy-2-phenyl)ethyl]-L-phenylalanyl-beta-alanine (SCH32615), a neutral endopeptidase (enkephalinase) inhibitor, on levels of enkephalin, encrypted enkephalins and substance P in cerebrospinal fluid and plasma of primates. 170 28

We have recently shown that nerve growth factor (NGF) promotes human granulopoiesis, specifically augmenting basophilic cell differentiation observed in methylcellulose hematopoietic colony assays of human peripheral blood. Because the NGF effect was seen in the presence of conditioned medium derived from a human T-cell line (Mo-CM) containing granulocyte-macrophage colony-stimulating factor (GM-CSF), we examined interactions of purified NGF and recombinant human GM-CSF (rhGM-CSF) on granulocyte growth and differentiation. rhGM-CSF stimulated a dose-dependent increase in methylcellulose colony growth at concentrations between 0.1 U/mL and 10 U/mL, and in the presence of NGF at 500 ng/mL this effect was enhanced. The number of basophilic cell colony-forming units (CFU-Baso) and histamine-positive colonies increased synergistically when NGF was added to rhGM-CSF. Furthermore, because Mo-CM acts with sodium butyrate to promote basophilic differentiation of alkaline-passaged myeloid leukemia cells, HL-60, we also examined the interaction of NGF and Mo-CM or rhGM-CSF using this assay. In the presence of NGF, Mo-CM at concentrations of 0.5% to 20% vol/vol, and rhGM-CSF at concentrations of 0.1 U/mL to 100 U/mL synergistically increased histamine production by butyrate-induced, alkaline-passaged HL-60 cells; this was associated with the appearance of metachromatic, tryptase-negative, IgE receptor-positive cells. The effects of rhGM-CSF or Mo-CM were completely abrogated by a specific anti-rhGM-CSF neutralizing antibody in methylcellulose, with or without NGF; the NGF synergy with rhGM-CSF in the HL-60 assay was also inhibited by either anti-rhGM-CSF or anti-NGF antibody. These studies support the notion that differentiation in the basophilic lineage may be enhanced by NGF acting to increase the number of GM-CSF-responsive basophilic cell progenitors.
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PMID:Synergistic effects of nerve growth factor and granulocyte-macrophage colony-stimulating factor on human basophilic cell differentiation. 199 3

Adhesion of lymphocytes to high endothelial venule (HEV) cells is the first step in the migration of these cells from blood into lymph nodes and Peyer's patches (PP). In the present study, we isolated and cultured HEV cells from PP of the rat and assessed their capacity to interact with lymphocytes. Flow cytometric analysis with a rat HEV-specific mAb KJ-4 revealed that greater than 90% of the cultured cells were stained by the antibody. Furthermore, confluent monolayers of PP HEV cells retained the capacity to support the adhesion of lymphocytes from spleen, thoracic duct, and lymph nodes but not binding of immature cells from thymus and bone marrow, which are deficient in cells capable of binding to HEV in vivo. In addition, intraepithelial lymphocytes that preferentially migrated into mucosal lymphoid tissues were also enriched in cells that adhered to the endothelial monolayers. The binding process required energy, was calcium-dependent, and could be inhibited by cytochalasin D, trypsin, and mixed glycosidase. Interestingly, pretreatment of PP HEV cells with rTNF, IFN-gamma, or granulocyte-macrophage CSF significantly increased the endothelial adhesiveness for thoracic duct lymphocytes in a time- and dose-dependent manner. In contrast, stimulation of lymphocytes with phorbol ester or TNF resulted in the rapid modulation of the surface expression of the PP homing receptor and decrease in lymphocyte binding to normal or TNF-stimulated HEV cells. The adhesion of lymphocytes to normal or cytokine-stimulated HEV cells can be blocked by pretreatment of lymphocytes, but not HEV cells, with the PP homing receptor-specific 1B.2.6 antibody. Taken together, these experiments provide strong evidence that the interaction between lymphocytes and cultured HEV cells are mediated by adhesive mechanisms that regulate lymphocyte entry into PP in vivo and that cytokines can promote HEV adhesiveness for lymphocytes through increased expression of organ-specific ligands on HEV cells.
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PMID:Lymphocyte adhesion to cultured Peyer's patch high endothelial venule cells is mediated by organ-specific homing receptors and can be regulated by cytokines. 212 24

A reversed-phase HPLC system was used to concentrate and separate components of substance P-like immunoreactivity (SP-LI) from human CSF. When CSF was injected and fractions collected, no SP-LI could be detected by radioimmunoassay (RIA) at the retention time of SP or SP-sulfoxide. Instead, SP-LI was detected in later eluting fractions. This SP-LI reacted with two different antisera raised against the C-terminal part of SP, but not with an antiserum against the N-terminal part. A compound with similar properties was also found to be present in neutral extracts of rat dorsal spinal cord. When the late-eluting compound from human CSF was treated with trypsin and rechromatographed on HPLC, an immunoreactive component eluting at the position of SP could be detected with both the C- and N-terminally directed SP antisera. These results suggest that an N-terminally extended form of SP is present in human CSF. Trypsinization also gave two other compounds with affinity for the N- but not the C-terminally directed antisera. This may indicate that N-terminal fragments of SP extended at the N-terminus or SP molecules extended at both the N- and the C-terminus (i.e., preprotachykinins) also are present in human CSF. In 32 CSF samples from depressed patients, SP-LI was determined with a C-terminally directed antiserum with and without prior HPLC separation. SP itself could not be detected, but the late-eluting form of SP-LI could be quantitated in all samples by combined HPLC-RIA. In most samples, there was a relatively good agreement between the SP-LI levels measured with and without HPLC.
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PMID:Detection of N-terminally extended substance P but not of substance P in human cerebrospinal fluid: quantitation with HPLC-radioimmunoassay. 245 10

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

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

Past studies have shown that contact between tumor cells and fibroblasts results in stimulation of collagenase production by the fibroblasts. Membrane fractions prepared by differential centrifugation of sonicated B-16 melanoma cells were shown here to contain a collagenase stimulatory factor(s) (CSF). Trypsin treatment of intact B-16 cells prior to membrane fractionation led to loss of 90% of the total activity, indicating that CSF is localized on the outer surface of the cells. Stimulation of fibroblast collagenase production was also observed with dialyzed octylglucoside extracts of the B-16 membranes. Additional of exogenous lipid, ie, a mixture of phosphatidylcholine and phosphatidylserine, to the detergent extract of the membranes followed by dialysis and centrifugation at 100,000g resulted in 80% recovery of the factor activity in the pellet containing reconstituted lipid vesicles. Fractionation of tritium-labeled, reconstituted lipid vesicles on a Sephacryl S-300 column revealed that the collagenase stimulatory factor coeluted with the radioactive lipid vesicles. The fractionated lipid vesicles lost stimulatory activity completely after trypsin treatment or heating at 65 degrees C, indicating that the factor is a protein.
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PMID:Membrane association of collagenase stimulatory factor(s) from B-16 melanoma cells. 282 6

The chemical nature of peptides in human CSF with the enkephalin core sequence from proenkephalin A and proenkephalin B, was investigated. Direct measurements with radioimmunoassay (RIA) were run on enkephalin, enkephalin hexapeptides, dynorphin A, dynorphin A1-8 and dynorphin B. The hexapeptides occurred in about 3 times higher concentration than the corresponding enkephalins. The only dynorphin RIA which gave positive results was the one for dynorphin A. However, most dynorphin A immunoactive material showed higher apparent molecular weight (MW; 3 and 5 kdalton) than the standard (2 kdalton). To identify and quantitate every possible proenkephalin derived peptide with the enkephalin sequence, chromatographic fractions were treated with trypsin. The products, Leu-enkephalin-Arg6 (from proenkephalin B) and Met-enkephalin-Lys6/Arg6 (from proenkephalin A) were measured by specific RIAs and identified by HPLC. In the higher (greater than 5 kdalton) MW interval, there was about 10-fold higher yield of Met-enkephalyl than Leu-enkephalyl hexapeptides. In the intermediate 1-3 kdalton MW interval, most activity derived from proenkephalin B. Finally, from the low MW region, there was about 5 times more proenkephalin A peptides. The main dynorphin A peak at 5 kdalton was transferred to a major Leu-enkephalin-Arg6 peak by trypsin degradation. The data indicate the presence of a whole family of peptides from the two proenkephalin genes in human CSF. Precursors to the peptides supposed to be the active members in the proenkephalin families occur in relatively high concentrations and may provide good markers for activity in these peptide systems.
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PMID:Enkephalin-containing polypeptides in human cerebrospinal fluid. 287 Jul 78

The inhibitory effect by hairy cell conditioned medium (HCCM) on the growth of granulocyte and erythrocyte colony forming cells was studied in vitro. The percent inhibition of CFU-C formation by HCCM from four hairy cell leukemia (HCL) patients ranged from 36% to 76%, while no inhibition was observed with conditioned medium (CM) obtained from three B-cell chronic lymphocytic leukemia (B-CLL) patients nor from two normal controls. HCCM inhibited specially the growth of rG-CSF responding stem cells. The hairy cell-derived colony inhibitory factor from HCCM was nondialyzable, fairly stable to heat treatment, and trypsin sensitive. Its maximal inhibitory activity against granulopoiesis was observed in the fractions of 5,000 to 6,000 daltons. Moreover HCCM inhibited CFU-E colony formation but not BFU-E. These results indicate that hairy cells produce a factor that inhibits granulopoiesis and erythropoiesis in vitro. This factor may play a role in neutropenia and anemia observed in HCL.
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PMID:Inhibition against CFU-C and CFU-E colony formation by soluble factor(s) derived from hairy cells. 292 Feb 12


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