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)

The complement C4-derived anaphylatoxin, C4a, possesses a strong chemotaxis inhibitory capacity to blood monocytes at concentrations as low as 10(-16) mol/L. In our study, treatment with carboxypeptidase B to convert it to C4a des Arg77 decreased the inhibitory activity to less than 1/1,000. The extraordinary inhibitory capacity of C4a suggests the presence of an amplification mechanism in this inhibition. Indeed, we found that the conditioned media of peripheral blood mononuclear cells or monocyte/macrophage lineage cell lines (U937 and THP-1 cells) preincubated with 10(-16) mol/L C4a for 5 minutes or more at 37 C possessed the inhibitory capacity 100,000-fold stronger than the original activity of C4a. The monocyte-derived chemotaxis inhibitory factor seemed monocyte-specific. This cell-derived factor was sensitive to treatment with trypsin and chymotrypsin and immunologically distinct from C4a. The apparent molecular size of the monocyte factor was estimated to be approximately 20 kd by gel filtration. These results indicate that C4a anaphylatoxin induces the release from monocytes of a protein with inhibitory activity for monocyte chemotaxis.
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PMID:Novel function of C4a anaphylatoxin. Release from monocytes of protein which inhibits monocyte chemotaxis. 850 53

In this study, we show that three proteolytic enzymes of different specificity-pronase, chymotrypsin, and trypsin-induced a dramatic stimulation of neutrophil apoptosis as shown by morphologic characteristics, analysis of cell DNA content, and presence of a characteristic "ladder" pattern of DNA fragmentation. The action of either chymotrypsin or trypsin was completely prevented by the serine protease inhibitor aprotinin, indicating that the proteolytic activity of the enzymes accounts for apoptosis induction. Stimulation of neutrophil apoptosis by proteases was observed in culture medium supplemented with either inactivated fetal calf serum (0.1-50%), autologous serum (0.1-50%), bovine serum albumin (0.1%), or in protein-free medium. Other cell types such as human peripheral blood monocytes and lymphocytes, human leukemic cells from THP-1, HL-60 and K562 lines, murine L929 fibroblasts, and unstimulated murine macrophages harvested from the peritoneal cavity were not induced to undergo apoptosis after the treatment with proteases. In an attempt to determine whether neutrophil serine proteases could induce apoptosis as chymotrypsin and trypsin do, the effect of elastase was assessed. A significant increase in the percentage of apoptotic cells was observed in elastase-treated neutrophils. We propose that the selective stimulation of neutrophil apoptosis by proteolytic enzymes may play an important role in the normal resolution of inflammation by limiting the autotoxic potential of the neutrophil.
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PMID:Neutrophil apoptosis induced by proteolytic enzymes. 860 Mar 21

The receptor for urokinase plasminogen activator (uPAR; CD87) is a 50- to 65-kDa glycosylphosphatidylinositol (GPI)-anchored glycoprotein expressed by leukocytes and tumor cells where it facilitates uPA-dependent, plasmin-mediated pericellular proteolysis during cellular invasion. Because uPAR is inducibly shed into culture supernatants and human body fluids, we tested the hypothesis that soluble uPAR (suPAR) can bind to the plasma membrane of hematopoietic cells where it might modulate their invasive phenotype. As measured by flow cytometry, recombinant biotinylated-suPAR (B-suPAR) bound in a specific fashion to THP-1 leukemia cells and blood PMNs and monocytes (but not to lymphocytes). B-suPAR also demonstrated specific binding to a variety of leukemic lines, including cells that are positive or negative for membrane uPAR expression. Binding of B-suPAR to THP-1 cells was enhanced four- to sevenfold by 24-h exposure of cells to PMA or by co-incubation with uPA ligand (but not its isolated catalytic and binding fragments). Conversely, binding of B-suPAR to PMNs was unaffected by brief exposure to fMLP, and was inhibited by co-incubation with uPA. B-suPAR biding to PMA-differentiated THP-1 cells in the presence of uPA was further enhanced by acid washing (removing endogenous uPA) but was partially inhibited by treatment of cells with trypsin. Pretreatment of PMA-differentiated THP-1 cells and unstimulated PMNs with soluble sugars, calcium chelators, and antibodies specific for integrins or extracellular matrix proteins failed to consistently block the binding of B-suPAR. Whereas the binding of suPAR did not measurably affect cell-associated plasmin activation, suPAR did competitively inhibit the binding of exogenous uPA to membrane-associated uPAR. These observations support the hypothesis that suPAR can bind specifically to trypsin-sensitive receptors expressed by certain normal and neoplastic hematopoietic cells where its binding is variably influenced by uPA ligand.
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PMID:A soluble form of the urokinase plasminogen activator receptor (suPAR) can bind to hematopoietic cells. 971 60

We previously observed that IFN gamma-inducible expression of the human MHC class II, HLA-DR alpha, gene was enhanced by treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA) only in human monocytic leukemia THP-1 cells, but not in HeLa cells. In the HLA-DR alpha gene, three DNase I hypersensitive sites (DHS) are known to be present in the promoter region (DHS-I) and first intron (DHS-II and -III) and are assumed to be involved in HLA-DR alpha gene regulation. In this study, we found a binding factor which recognized a unique palindrome sequence (DHS-22) in the region of the DHS II site of the HLA-DR alpha gene in THP-1 cells and HeLa cells. The binding activity of this factor was decreased by TPA treatment in THP-1 cells, but not in HeLa cells. This binding activity was also detectable in nuclear extracts of bovine brains. Thus, we isolated the DHS-22 binding factor from bovine brain nuclear extracts and finally identified it as NF90 on the basis of molecular mass analysis of Lys-C-digested fragments and amino acid sequences of the two peptides of the trypsin-digested binding protein. The DHS-22 binding protein(s) in THP-1 cells is (are) further confirmed by reactivity to an antibody against NF90, and we have demonstrated that the GST fusion protein of NF90 interacts with DHS-22 by electrophoretic gel mobility shift assay (EMSA). The mRNA of NF90 was decreased by TPA treatment in THP-1 cells but not in HeLa cells. These results suggest that the binding of NF90 to the DNase I hypersensitive site II of HLA-DR alpha gene seems to negatively regulate HLA-DR alpha gene expression.
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PMID:A binding protein to the DNase I hypersensitive site II in HLA-DR alpha gene was identified as NF90. 1007 79

Interleukin-15 (IL-15) is a potent regulator of T-, B-, and natural killer cell proliferation and displays unusually tight controls of secretion. Even though IL-15 mRNA is constitutively expressed in monocytes/macrophages and is upregulated by a variety of stimuli, evidence for IL-15 cytokine secretion is only found exceptionally, eg, conditions of pathological, chronic inflammation. This raises the possibility that monocytes express membrane-bound IL-15 rather than secrete it. The current study explores this hypothesis. We demonstrate here that biologically active IL-15 is indeed detectable in a constitutively expressed, membrane-bound form on normal human monocytes, as well as on monocytic cell lines (MONO-MAC-6, THP-1, and U937), but not on human T or B cells (MT4, M9, C5966, JURKAT, DAUDI, RAJI, and Epstein-Barr virus-immortalized B-cell clones). Furthermore, cell surface-bound IL-15 is upregulated upon interferon-gamma stimulation. Interestingly, monocyte/macrophage inhibitory cytokines such as IL-4 and IL-13 fail to downregulate both constitutive and induced cell-surface expression of IL-15. Membrane-bound IL-15 does not elute with acetate buffer or trypsin treatment, suggesting that it is an integral membrane protein and that it is not associated with the IL-15 receptor complex. Finally, membrane-bound IL-15 stimulates T lymphocytes to proliferate in vitro, indicating that it is biologically active. These findings enlist IL-15 in the fairly small family of cytokines for which the presence of a biologically active membrane-bound form has been demonstrated (eg, IL-1, tumor necrosis factor-alpha, and IL-10) and invites the speculation that most of the biological effects of IL-15 under physiological conditions are exerted by the cell surface-bound form.
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PMID:Human monocytes constitutively express membrane-bound, biologically active, and interferon-gamma-upregulated interleukin-15. 1023 6

Flagella from diverse gram-negative bacteria induce tumor necrosis factor alpha (TNF-alpha) and interleukin-1beta (IL-1beta) synthesis by human monocytes (F. Ciacci-Woolwine, P. F. McDermott, and S. B. Mizel, Infect. Immun. 67:5176-5185, 1999). In this study, we establish that purified flagellin (FliC or FljB), the major filament protein from Salmonella enterica serovar Enteritidis, S. enterica serovar Typhimurium, and Pseudomonas aeruginosa, is an extremely potent inducer of TNF-alpha production by human monocytes and THP-1 myelomonocytic cells. Fifty percent of maximal TNF-alpha production (EC(50)) was obtained with 1.5 x 10(-11) M flagellin (0.75 ng/ml). Mutagenesis studies revealed that the central hypervariable region of flagellin is essential for the TNF-alpha-inducing activity of the protein. Although less active than the wild-type protein, a Salmonella flagellin mutant composed of only the central hypervariable region retained substantial TNF-alpha-inducing activity at nanomolar concentrations. In contrast, the conserved amino- and carboxy-terminal regions are inactive. Mutational analysis of the hypervariable region revealed that it contains two equally active TNF-alpha-inducing domains. The ability of THP-1 cells to respond to purified flagellins is dramatically reduced by mild trypsin treatment of the cells. Taken together, our results demonstrate that the cytokine-inducing activity of flagellins from gram-negative bacteria results from the interaction of these proteins with high-affinity cell surface polypeptide receptors on monocytes.
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PMID:High-affinity interaction between gram-negative flagellin and a cell surface polypeptide results in human monocyte activation. 1099 49

A unique property of the extracellular matrix of J774 and THP-1 cells has been identified, which contributes to the ability of these cells to promote cholesterol efflux. We demonstrate high level apolipoprotein (apo) A-I binding to macrophage cells (THP-1 and J774) and to their extracellular matrix (ECM). However, high level apoA-I binding is not observed on fibroblasts, HepG2 cells, or U937 cells (a macrophage cell line that does not efflux cholesterol to apoA-I or bind apoA-I on their respective ECM). Binding to the ECM of THP-1 or J774 macrophages depends on the presence of apoA-I C-terminal helices and is markedly reduced with a mutant lacking residues 187-243 (apoA-I Delta(187-243)), suggesting that the hydrophobic C terminus forms a hydrophobic interaction with the ECM. ApoA-I binding is lost upon trypsin treatment or with Triton X-100, a preparation method that de-lipidates the ECM. However, binding is recovered with re-lipidation, and is preserved with ECM prepared using cytochalasin B, which conserves the endogenous phospholipid levels of the ECM. We also demonstrate that specific cholesterol efflux to apoA-I is much reduced in cells released from their native ECM, but fully restored when ECM-depleted cells are added back to ECM in the presence of apoA-I. The apoA-I-mediated efflux is deficient in plated or suspension U937 macrophages, but is restored to high levels when the suspension U937 cells are reconstituted with the ECM of J774 cells. The ECM-dependent activity was much reduced in the presence of glyburide, indicating participation of ABCA1 (ATP-binding cassette transporter 1) in the efflux mechanism. These studies establish a novel binding site for apoA-I on the macrophage ECM that may function together with ABCA1 in promoting cholesterol efflux.
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PMID:Trypsin-sensitive and lipid-containing sites of the macrophage extracellular matrix bind apolipoprotein A-I and participate in ABCA1-dependent cholesterol efflux. 1205 Jan 68

Langerhans' cells (LC) of skin are CD4 expressing, dendritic, antigen-presenting cells, that are essential for activation of primary immune responses and are productively infected by HIV. We have shown previously that lymphocytes and monocytes express CD4 both as monomers and covalently linked homodimers. In those cells the 55-kDa monomer structure predominates. LC in un-fractionated human epidermal cell (EC) suspension also expresses both forms of CD4, but in EC the dimer form is predominant. Because isolation of LC into single cell suspension by trypsin, as is routinely used for LC isolation, degrades CD4, a systematic study for an alternate procedure for LC isolation was performed. Thus it was found that collagenase blend F treatment can efficiently release LC into suspension, under conditions of only minimal degradation of control soluble recombinant CD4 or CEM-T4 or THP-1 cell CD4, or importantly of LC surface CD4. SDS-PAGE immunoblotting of purified LC extracted from EC by collagenase confirmed CD4 structure as predominantly 110-kDa dimers, with only minimal 55-kDa monomers. The suitability of LC prepared thus for functional studies was demonstrated with binding of functional ligand HIV gp120. It remains to be determined, however, why tissue embedded LC express mainly CD4 dimers, but single-celled blood lymphocytes and monocytes mainly monomers.
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PMID:CD4 is expressed by epidermal Langerhans' cells predominantly as covalent dimers. 1470 12

In order to better understand the mechanisms governing display of mast cell characteristics in human myeloid cells, we have studied the mast cell phenotype in human promyelocytic (HL-60) and myelocytic (U-937, TPH-1) vs. basophilic (KU-812) and mast cell (HMC-1) lines, in part also in skin mast cells and blood monocytes, at mRNA and protein level before and after stimulation with mast cell growth factors. In unstimulated cells, mRNA for the stem cell factor (SCF) receptor c-kit and the gamma chain of the high-affinity IgE receptor (FcepsilonRI) was noted in all cells studied. Like mast and basophilic cells, THP-1 cells expressed the FcepsilonRIalpha and beta chains and weakly histidine decarboxylase (HDC), but they lacked mRNA for mast cell-specific proteases [tryptase, chymase, carboxypeptidase A (CPA)]. In contrast, HL-60 and U-937 cells lacked FcepsilonRIalpha, but expressed tryptase and chymase, HL-60 cells also CPA. KU-812 cells failed to express the basophil-specific marker 2D7. After a 10-day culture with SCF or fibroblast supernatants, baseline mRNA expression of most mast cell characteristics was upregulated, whereas c-kit mRNA expression decreased in all but THP-1 cells. Differential mRNA expression of FcepsilonRI vs. protease (tryptase) was confirmed at protein level by immunocytochemistry and enzymatic activity. KU-812 cells are thus closest to skin mast cells in that they express all molecules studied, except for chymase, followed by THP-1 cells that lack all mast cell proteases. In contrast, HL-60 and U-937 cells fail to express the FcepsilonRIalpha and beta chains but express most mast cell proteases. The selective and differential expression of mast cell characteristics in human myeloid cell lines suggests that induction of the mast cell phenotype is regulated by several independent genes and that mast cells and basophils branch off at early and distinct points of myeloid development.
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PMID:Differential expression of mast cell characteristics in human myeloid cell lines. 1533 53

A small percentage of women at high risk for human immunodeficiency virus (HIV) exposure remain uninfected for long periods, protected by unknown mechanisms. We hypothesized that one mechanism could be inhibition of interactions between HIV and dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN) in the genital tract. In an analysis of 95 cervicovaginal lavage samples, we found that 12 (12.6%) strongly inhibited the binding of laboratory-adapted and primary HIV-1 isolates to B-THP-1/DC-SIGN cells in a dose-dependent manner, independently of the donor's risk of exposure. Three of 5 primary isolates were also blocked from binding to primary DCs. The inhibitor has a high molecular weight, is heat stable, and is resistant to trypsin. It is sensitive to pronase and periodate, indicating that it is likely a glycoprotein. Mannosidase digestion and concanavalin A adsorption indicate that the terminal residues of the carbohydrate are not mannose. Mechanistic experiments indicate that the inhibitor acts via binding to DC-SIGN. Further study of such inhibitors may help to elucidate the role played by DC-SIGN in HIV transmission.
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PMID:Human cervicovaginal lavage fluid contains an inhibitor of HIV binding to dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin. 1602 32


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