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)

We identified a chymotrypsin-like activity in the granules of IL-2 lymphokine-activated killer (LAK) cells and a NK cell line (YT) that reacted preferentially with the oligopeptide substrate succinyl-Phe-Leu-Phe-thiobenzyl ester (Suc-Phe-Leu-Phe-SBzl). The enzyme was isolated by detergent extraction of sedimented cytotoxic granules and then by a sequence of sieve, hydrophobic, and anion exchange chromatography. On SDS-PAGE, the protein migrated at 42 kDa in nonreduced form and became two bands (31 and 19 kDa, respectively) after reduction. Amino-terminal sequencing of the reduced protein bands revealed 100% homology with cathepsin A-like protective protein (CAPP), a lysosomal enzyme that expresses serine carboxypeptidase and deamidase activities. The carboxypeptidase activity of lymphocyte CAPP was verified by showing that the protease preferred hydrophobic amino acids in the penultimate position of the C terminus (i.e., cleaved arginine from dansyl-Phe-Leu-Arg). The presence of lymphocyte CAPP in secretory lysosomes was demonstrated by showing that Suc-Phe-Leu-Phe-SBzl activity co-migrated with tryptase and Asp-ase activities on Percoll density gradients and that 95% of the Suc-Phe-Leu-Phe-SBzl activity in granule fractions of cavitated YT cells could be immunoprecipitated with an anti-CAPP antiserum. In addition, calcium ionophore-stimulated YT cells were shown to secrete immunoprecipitable CAPP. As proposed for platelets, lymphocyte CAPP may be secreted to function extracellularly by inactivating bioactive peptides.
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PMID:Dominant chymotrypsin-like esterase activity in human lymphocyte granules is mediated by the serine carboxypeptidase called cathepsin A-like protective protein. 796 38

The supernatant of a cell line of squamous cell carcinoma of the head and neck (SCCHN), PCI-50, was previously shown to induce activation, promote proliferation and increase antitumor cytotoxicity of freshly purified human natural killer (NK) cells and CD4+ T lymphocytes [Arch Otolaryngol Head Neck Surg (1994) in press]. This supernatant was found also to promote the growth of a variety of hematopoietic cell lines, including Jurkat, THP-1, K562, NK-92 or Epstein-Barr-virus-transformed B cell lines. The Jurkat cell line was selected as a reporter cell in an 18-h proliferation assay established to measure the growth-promoting activity of PCI-50 supernatant. The presence of soluble tumor-derived factors able to induce proliferation of Jurkat cells was demonstrated in the supernatant produced by several other SCCHN cell lines but not in that produced by a gastric cancer cell line (HR) or renal cell carcinoma line (5117G8). The growth-promoting PCI-50 supernatant was shown to contain 28 +/- 0.5 pg/ml interleukin-6 (IL-6) in vitro but was negative for interferon gamma, IL-1, IL-2, IL-4, tumor necrosis factor alpha, granulocyte/macrophage-colony-stimulating factor and IL-12. The addition of any of these recombinant cytokines to Jurkat cell cultures did not significantly promote growth, while PCI-50 supernatant was consistently growth-stimulatory. This supernatant neither enhanced intracellular Ca2+ concentration in Jurkat cells nor induced up-regulation of activation antigens on the cell surface, although it supported growth of Jurkat cells in the absence of IL-2. The growth-promoting activity in the PCI-50 supernatant was acid-labile at pH 2 for 4 h, heat-resistant at 96 degrees C for 1 h and sensitive to treatments with trypsin and pepsin. Preincubation of the PCI-50 producer cells with tunicamycin or cyclohexamide reduced the level of growth-promoting activity in the supernatant. A partial purification of this activity was achieved using Amicon filtration, chromatography on concanavalin-A-Sepharose and then a hydroxyapatite column and high-pressure liquid chromatography gel filtration. The partially purified glycoprotein had a molecular mass of 50-70 kDa, as determined by gel filtration.
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PMID:Proliferation of hematopoietic cell lines induced by a soluble factor derived from human squamous cell carcinomas of the head and neck. 800 Oct 29

The effect of sera from mice bearing a T cell lymphoid leukaemia (LB) and the supernatants from short term cultures of the tumour cells were studied on cell proliferation using syngeneic and allogeneic normal and tumour cells. An inhibitory activity was demonstrated in 24-48 h supernatants of LB cells in culture and disappeared after 4 days of culture. Inhibitory activity was cytostatic but not cytotoxic and was non-specific since it inhibited the growth of both syngeneic and allogeneic normal and tumour cells. Such activity was found in the 10(5)-1.3 x 10(5) M(r) serum fraction after a Sephacryl S200 chromatography. Though sensitive to protease, trypsin or neuraminidase treatment, which indicated its glycoprotein nature, it remained stable after heating or freezing-thawing cycles as well as after alkaline, acid or hyaluronidase treatment. Addition of exogenous IL-2 abrogated inhibitory activity. ELISA showed the presence of soluble IL-2R both in LB conditioned medium and in above serum fraction. It is demonstrated that the inhibitory factor, soluble IL-2R, is produced by LB leukaemia cells, then secreted into blood and ascitic fluid or released into culture supernatants. Soluble IL-2R exerts inhibitory activity blocking cell proliferation and modulating immune response by binding to free IL-2.
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PMID:Inhibitory activity of soluble IL-2R in sera, ascites and culture supernatants from murine leukaemic cells. 809 Nov 30

mAb against the lymphocyte homing receptor CD44/Hermes up-regulate the proliferation of human T PBL induced by anti-CD3 or anti-CD2 mAb. Moreover, certain anti-CD44 mAb can activate human resting T cells and mouse cytotoxic T cells in the absence of anti-CD3 or anti-CD2 mAb. Here, we show that anti-CD44 mAb trigger proliferation of human CD3+/CD4+ T cell clones in a fashion similar to that observed with mAb to CD3. Such an effect is IL-2-dependent, as shown by IL-2 production induced by anti-CD44 mAb and by complete inhibition of cell proliferation in the presence of anti-IL-2 antibodies or cyclosporin A. Moreover, anti-CD44 mAb trigger human cytolytic T cell clones to lyse Fc gamma-R+ P815 cells in the absence of additional stimuli. The magnitude of the cytolytic response induced by anti-CD44 mAb is comparable to that observed in the presence of anti-CD3 mAb for both CD4+ and CD8+ TCR-alpha/beta+ clones, and for V delta 1 or V delta 2 TCR-gamma/delta+ clones. By contrast, in CD3-/CD16+ NK cell clones, no cytolytic responses to anti-CD44 mAb could be observed. Granule trypsin-like esterase enzyme (granzyme) release by cytolytic T cell clones is induced by plastic-immobilized anti-CD44 mAb. Anti-CD44 mAb-triggered proliferation ([3H]thymidine incorporation) and cytotoxicity are blocked by the protein tyrosine kinase inhibitor, genestein. In addition, ligation of the CD44 molecule induces tyrosine phosphorylation of proteins identical, by molecular mass, to those phosphorylated after anti-CD3 mAb stimulation. Notably, anti-CD44 mAb does not induce tyrosine phosphorylation of a 21-kDa protein (the phosphorylated zeta-chain of the TCR molecular complex) typically observed upon anti-CD3 mAb stimulation. In conclusion, this study shows that the ligated CD44 molecule provides the necessary stimuli for a variety of T cell-mediated functions triggered via protein tyrosine kinase-dependent signal transduction pathways at least in part similar to those that follow stimulation of the CD3/TCR complex.
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PMID:Antibodies to CD44 trigger effector functions of human T cell clones. 809 50

We have shown that certain CD4+ T cell lines can function as suppressor cells in a cell culture system. In this context, the CD4+ T cells (AS-9) cloned from the peripheral blood lymphocytes (PBL) of a melanoma patient are capable of suppressing the induction of cytolytic response in autologous PBL in coculture. Here we show that a trypsin-sensitive cell-free culture supernatant factor from the AS-9 cells, AS-9 SF, interferes with IL-2 synthesis by T cells when they are stimulated. AS-9 SF also selectively blocks the expression of interleukin-2 receptor alpha (IL-2R alpha) on T cells during activation. Expression of transferrin receptors and the CD3 molecules is not down-regulated by this factor. The AS-9 SF consequently blocks proliferation of T cells when they are stimulated by lectin or activated through the T cell receptors. AS-9 SF suppresses the IL-2R alpha induction and the T cell proliferation at the induction phase only because it has no suppressive effect on preactivated T cells. Interleukin-2, IL-2R alpha, and beta messages are not down-regulated by the AS-9 SF and the suppressive effect of the AS-9 SF on IL-2R alpha expression and on T cell proliferation is not neutralized by the addition of exogenous recombinant IL-2. The factor does not appear to be IL-4, IL-10, or TGF-beta, three known cytokines possessing regulatory properties on T cell activation.
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PMID:Inhibition of interleukin-2 synthesis and interleukin-2 receptor alpha expression on T cells by a cell-free factor derived from a CD4+ regulatory T cell clone. 810 19

The basophilic leukaemia cell line KU812 can be induced to differentiate into basophil-like cells in vitro when exposed to supernatant from the Mo T-cell line. KU812 cells express affinity receptors for IgE, produce histamine and tryptase and have the capacity for IgE-mediated histamine release. In this study we have examined the cytokines, produced by the Mo cell line, which are responsible for the observed differentiation-inducing effect in the KU812 cell line. It was shown that interleukin-6 (IL-6) and tumour necrosis factor-alpha (TNF-alpha) induced differentiation in the KU812 cells and that these cytokines were responsible for the differentiation-inducing effect of the Mo supernatant. Other cytokines tested, IL-1 beta, IL-2, IL-4, IL-5, IL-8, granulocyte-macrophage colony-stimulating factor (GM-CSF) and nerve growth factor (NGF) were without effect on the KU812 cells. KU812 was also shown to express receptors for both TNF-alpha and IL-6 after 3 days cultivation with conditioned media from the Mo T-cell line. Untreated cells showed no detectable levels of TNF-alpha or IL-6 receptors indicating induction of these receptors during differentiation. Spontaneous differentiation was shown to occur under serum-free conditions which may be the result of endogenous IL-6 production through an autocrine loop. The activity of TNF-alpha and IL-6 could be blocked by specific monoclonal antibodies (mAb) to the respective cytokine.
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PMID:TNF-alpha and IL-6 induce differentiation in the human basophilic leukaemia cell line KU812. 813 23

Aminopeptidase (AP) activity on rat natural killer (NK) cells was found to have the following characteristics: (1) the activity was surface associated and not secreted, as determined by extracellular location of product and by the cessation of hydrolysis of substrate upon removal of the cells from the medium. (2) The activity was linear with respect to time and cell number. (3) The enzymatic activity on splenocytes and on the NK leukemia cell line CRNK-16, but not on IL-2 activated NK (A-NK) cells, was sensitive to trypsin treatment. (4) The AP activity on intact cells had a broad pH dependency with optimal activity at slightly alkaline pH but lower activity at acidic pH. (5) There was a preference for neutral substrates and essentially no activity towards acidic substrates. (6) Enzymatic activity was inhibited in the presence of the AP inhibitors bestatin and amastatin, and in the presence of the chelator, 1,10 phenanthroline, indicating the involvement of a metalloprotease. (7) Culture of A-NK cells with bestatin resulted in a decrease in cytotoxicity against YAC-1 and P815 targets. Amastatin treatment caused only a slight decrease in cytotoxicity against YAC-1 targets, but a significant decrease in cytotoxicity against P815 targets. (8) Treatment of A-NK cultures with specific inhibitors of APases caused an increase in expression of CD2 (an increase from 20-80% with bestatin and an increase from 25-35% in the presence of amastatin). These results provide the first evidence for the existence of APases on the surface of NK cells and suggest a role for these enzymes in the regulation of cytotoxic activity and of CD2 surface expression.
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PMID:Surface aminopeptidase activity of rat natural killer cells. I. Biochemical and biological properties. 816 43

Blood lymphocytes of cancer patients lysed autologous, freshly isolated tumor cells. The autologous tumor-killing (ATK) activity is strongly associated with postoperative clinical course, indicating that ATK is a meaningful prognostic indicator and provides evidence for immunological control of tumor growth and metastasis. Large granular lymphocytes (LGL) with ATK activity released a soluble cytotoxic factor(s), termed LGL-CF (LGL-derived cytotoxic factor) during interaction with autologous tumor cells. The cytotoxic factor lysed autologous and allogeneic freshly isolated human tumor cells, while they were resistant to any of recombinant cytokines, including tumor necrosis factor (TNF), lymphotoxin (LT), interferon (IFN) alpha, IFN gamma, interleukin (IL)-1 alpha and IL-2. Biological activity of LGL-CF was not abrogated by monoclonal and polyclonal antibodies against these cytokines. LGL-CF also exhibited lysis of a variety of tumor cell lines, but not of nonmalignant cells. Actinomycin D augmented the lysis of LGL-CF. LGL-CF was stable at 56 degrees C, but was destroyed at 100 degrees C. Treatment of LGL-CF with trypsin or proteinase K reduced or abrogated the lytic effect, respectively, while it was resistant to papain, catalase, and superoxide dismutase. These results indicate that LGL produce a novel cytotoxic factor in response to autologous tumor cells that mediates lysis of fresh human tumor cells.
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PMID:Role of NK cell cytotoxic factor against fresh human tumors. 825 31

We have reported that P388D1 cell line murine macrophages stimulated with lipopolysaccharide (LPS) from Actinobacillus actinomycetemcomitans release interleukin-1 (IL-1) inhibitor. The IL-1 inhibitor was purified from conditioned media of P388D1 cells stimulated with A. actinomycetemcomitans LPS for 72 h to homogeneity by a four-step procedure: acetic acid extraction from conditioned media; Bio-Gel P-60 gel filtration chromatography; DEAE-Sepharose CL-6B column chromatography; and reverse-phase high-performance liquid chromatography on a C18 hydrophobic support. The purified IL-1 inhibitor gave a single band of protein with a molecular mass of 26 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified IL-1 inhibitor was a heat- and acid-stable protein that was inactivated by digestion with trypsin and reduction with dithiothreitol. This inhibitory factor suppressed the proliferation of C3H/HeJ mouse thymocytes and the proliferation of IL-1-dependent cell lines, D10.G4.1 and RPMI 1788, induced by IL-1. However, this inhibitor did not affect the proliferation of IL-2-dependent CTLL-2 cells induced by IL-2, the proliferation of C3H/HeJ mouse thymocytes stimulated with a mitogenic dose of concanavalin A, and the proliferation of IL-6-dependent B9 cells induced by IL-6. Furthermore, the IL-1 inhibitor significantly blocked stimulation of bone resorption in organ cultures of newborn mouse calvaria and inhibited the osteoclast-like cell formation in mouse marrow cultures. A monoclonal antibody prepared against the purified IL-1 inhibitor reacted with mouse recombinant IL-1 receptor antagonist (rIL-1ra), and a polyclonal antibody to mouse rIL-1ra reacted with the IL-1 inhibitor by Western blot (immunoblot) analysis. These results indicate that the IL-1 inhibitor is an identical molecule to rIL-1ra, suggesting that the IL-1 inhibitor (IL-1ra) released by macrophages stimulated with LPS from A. actinomycetemcomitans may play an important mediative role in the development of periodontal disease.
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PMID:Mouse interleukin-1 receptor antagonist induced by Actinobacillus actinomycetemcomitans lipopolysaccharide blocks the effects of interleukin-1 on bone resorption and osteoclast-like cell formation. 830 Feb

Rat small intestine epithelial cell (EC) culture conditioned media (ECCM) contains a factor that has inhibitory activity against: a) the response of freshly isolated lymphocytes to Concanavalin A (Con A) or IL-2, b) the proliferative response to antigen of primed lymphocytes, and c) the normal growth of transformed cell lines derived from several species. Inhibition is reversible, and not the result of a cytotoxic effect. The inhibitory activity is enterocyte derived, effective at low concentration (1-2% in growth media), and can be derived from freshly isolated EC. Biochemical analysis indicates the inhibitory activity is associated with a protein with an approximate molecular weight of 32 kd, and an isoelectric point (PI) in the range of 3-5. The protein is trypsin sensitive, and labile with prolonged heating at 56 degrees C. The described activity differs from previously reported mucosally-derived inhibitory activities on the basis of molecular weight, and its ability to inhibit the growth of several cell lines. We suggest that this factor can provide the immunomodulatory activity necessary to produce the low level of intestinal T cell reactivity that is observed in vivo.
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PMID:Characterization of an inhibitory factor derived from epithelial cells of the small intestine. 834 67


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