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)

When Fc receptors (FcR) on normal human peripheral blood lymphocytes were induced to modulate by overnight (18 h) incubation in the presence of soluble or particulate immune complexes, the natural killer (NK) activity of the effector lymphocyte suspension, as measured against the K562 erythroleukemia cell line, was significantly, but only partially, inhibited. The NK activity which remained was always strong, and was not significantly inhibited by inclusion of antigen-antibody complexes in the cytotoxicity assay, nor was it further depleted by adsorbing the modulated cells on plastic surfaces coated with immobilized antigen-antibody complexes. Antibody-dependent cell-mediated cytotoxicity (ADCC) against rabbit antibody-sensitized Chang liver cells was totally abrogated following the modulation process, and could not be restored by exposure of modulated effector cells to trypsin, indicating that the FcR had actually been shed and were not merely being blocked with immune complexes. Although freshly isolated peripheral blood lymphocytes active in natural (or "spontaneous") cytotoxicity have been shown to bear FcR, our data indicate that NK activity against the K562 cell line can be effectively mediated by NK cells which have lost their FcR. This supports the concept that NK activity against K562 is independent of FcR, and, therefore, of IgG.
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PMID:Cytotoxicity against the K562 erythroleukemia cell line by human natural killer (NK) cells which do not bear free Fc receptors for IgG. 29 May 70

The human erythroleukemia cell line (HEL) has been used as a model system for studying signal transduction processes as they might relate to platelet/megakaryocyte function. We were interested in examining the role of thrombin in the regulation of adenylyl cyclase in this cell line. As opposed to its predominantly inhibitory effects on cyclic AMP production in platelets or in membranes from HEL cells, our initial experiments in intact HEL cells revealed that thrombin markedly potentiated the cyclic AMP response to prostaglandin E1 (2.9 +/- 0.2-fold), prostacyclin (1.9 +/- 0.2-fold) and carbacyclin (2.5 +/- 0.5-fold), measured either by radioimmunoassay or by the [3H]adenine preloading procedure. Thrombin, although ineffective alone, also potentiated cyclic AMP production stimulated by vasoactive intestinal peptide (1.6 +/- 0.2-fold), cholera toxin (3.0 +/- 0.6-fold) and AIF4- (2.3 +/- 0.6-fold), but not by forskolin (0.9 +/- 0.1-fold). The thrombin effect 1) produced an increase in the efficacy of the prostaglandins with no change in potency; 2) was long-lived; 3) required the proteolytic activity of thrombin; 4) was insensitive to pertussis toxin; and 5) was at least partially mimicked by trypsin, extracellular ATP and UTP, platelet activating factor and activators of protein kinase C. Down-regulation of protein kinase C or pre-exposure to the protein kinase inhibitor staurosporine blocked the potentiating effect. Together, these results suggest that in HEL cells, the mechanism of thrombin potentiation of cyclic AMP production may involve alterations in the interaction between stimulatory guanine nucleotide binding protein and the catalytic subunit of adenylyl cyclase, possibly involving protein kinase C-mediated phosphorylation.
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PMID:Potentiation of cyclic adenosine monophosphate production by thrombin in the human erythroleukemia cell line, HEL. 133 12

We recently reported that lymphokine activated killer (LAK) cells were stimulated to release both interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) when stimulated by a variety of tumor cells. We proposed then that the released cytokines may play a role in mediating tumor cell regression in vivo. In this paper, we provide further information on the nature of the signals, provided by the tumor cells (K562 erythroleukemia), that stimulate LAK cells to secrete IFN-gamma and TNF-alpha. Using a previously published protocol for coating tumor-membrane molecules onto cell-sized hydrophobic beads (also called pseudocytes), we demonstrate that the signal provided by the tumor cell is membrane associated. Beads coated with K562 membranes stimulated LAK cells to release IFN-gamma and TNF-alpha. The pretreatment of these beads with trypsin and sodium periodate eliminated the ability of these pseudocytes to stimulate cytokine release in LAK cells. The glycoproteins that stimulate LAK cells to secrete IFN-gamma and TNF-alpha were further enriched by their ability to bind concanavalin A (Con A, Jack Bean). To determine if the tumor-associated molecules that stimulate LAK cells to release IFN-gamma and TNF-alpha are also the molecules involved in mediating tumor cell lysis, we tested the ability of the Con A binding and nonbinding proteins to inhibit the LAK cell-mediated lysis of K562 cells. Our results demonstrate that molecules that inhibited LAK cell-mediated cytotoxicity were not enriched by Con A. These results are therefore consistent with the conclusion that different sets of tumor-associated molecules are involved in the stimulation of LAK cells to secrete cytokine and in the induction of LAK cells to mediate tumor cell cytolysis.
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PMID:Ability of cell-sized beads bearing tumor cell membrane proteins to stimulate LAK cells to secrete interferon-gamma and tumor necrosis factor-alpha. 190 21

We have recently demonstrated that many cancer cell lines produce a novel trypsinogen isoenzyme called tumor-associated trypsinogen 2 (TAT-2). It was found during a search of the target protease for tumor-associated trypsin inhibitor (TATI). We now show that degradation of subendothelial cell extracellular matrix (ECM) by four different cell lines (COLO 205 colon carcinoma, K-562 erythroleukemia, CAPAN-1 pancreatic carcinoma, and HT 1080 fibrosarcoma) can be partially inhibited by TATI or neutralizing trypsin antibodies. When cells were cultured in serum-free medium on ECM, TATI and trypsin antibodies inhibited the release of immunoreactive fibronectin fragments from ECM by 47-54 and 40%, respectively. Degradation of isotopically labeled ([3H]serine, [3H]proline, and [35S]sulfate) ECM was also significantly prevented by TATI. At its maximum, it exerted a 57% inhibition on the degradation of [3H]serine-labeled ECM. Plasminogen added exogenously to the culture medium further potentiated the proteolysis of ECM. Interestingly, addition of enteropeptidase, an activator of TAT-2, also enhanced cell-mediated proteolysis as assessed by degradation of purified fibronectin coated onto the surface of wells. Immunoblot analysis showed that enteropeptidase-mediated proteolysis generated a pattern of fibronectin fragments similar to that obtained by digestion of purified fibronectin by TAT-2. These results demonstrate the existence of a proteolytic system in tumor cells which is dependent on the activation of TAT-2. We suggest that TAT-2 is involved in a protease cascade-stimulating tumor cell invasion and degradation of extracellular matrix.
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PMID:Tumor-associated trypsin participates in cancer cell-mediated degradation of extracellular matrix. 200 30

Administration of alpha/beta-interferon (IFN) exerts a marked antitumor effect in DBA/2 mice given injections i.v. of large numbers of IFN-alpha/beta-resistant erythroleukemia cells (FLC). To investigate the possible mechanisms of FLC tumor inhibition in the liver of interferon-treated mice, we developed an in vitro model consisting of a coculture of IFN-alpha/beta-resistant 3Cl8 FLC and syngeneic mouse hepatocytes. Whereas IFN-alpha/beta did not inhibit the multiplication of 3Cl8 FLC cultivated alone, it effectively inhibited the multiplication of 3Cl8 FLC in coculture with hepatocytes. The inhibitory effect was directly proportional to the amount of IFN-alpha/beta added to the cocultures, and more than 90% inhibition of FLC multiplication was noted with 1.6 x 10(5) IU/ml of IFN-alpha/beta on Day 3 of coculture. When FLC were separated from the monolayer of hepatocytes by a pored membrane (0.4 microns), the inhibitory effect on FLC proliferation was unchanged, indicating that a soluble factor(s) released from IFN-treated hepatocytes was most important in the inhibition of FLC multiplication. An inhibitory activity of FLC multiplication was detected only in the conditioned medium of IFN-treated hepatocytes but not in the conditioned medium of control hepatocytes nor in extracts of IFN-treated or control hepatocytes. The inhibitory factor(s) in the conditioned medium of IFN-treated hepatocytes was retained by an ultrafiltration membrane (Mr cut off 10,000), and its activity was completely abrogated by trypsin digestion. Its stability to treatment with 1 M acetic acid as well as lack of correlation between the antiproliferative effect and the amount of L-arginine in the medium distinguished this factor(s) from liver arginase which was also found to be a potent inhibitor of FLC multiplication in vitro. The inhibitory factor(s) was also distinguishable in its biological activity from IFN gamma, interleukin 1 alpha and beta, and transforming growth factor beta 1 and beta 2. These results suggest the possibility that the inhibitory effect of IFN-alpha/beta on the development of 3Cl8 FLC in the livers of IFN-treated mice may be mediated by an IFN-induced inhibitor of FLC multiplication.
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PMID:Inhibition of mouse alpha/beta-interferon of the multiplication of alpha/beta-interferon-resistant Friend erythroleukemia cells cocultured with mouse hepatocytes. 214 Feb 90

Ferrochelatase from bovine kidney mitochondria has been purified 1600-fold with a 6.5% yield, exhibiting a specific activity of 490 nmol mesoheme formed/mg of protein per min. The Km values for mesoporphyrin IX and protoporphyrin IX with iron were 12.5 and 12.7 microM, respectively. The Km values for iron and zinc with mesoporphyrin IX were 3.51 and 3.17 microM, respectively. The purified enzyme showed a single band with an apparent molecular mass of 42,000 daltons (42 kDa) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The rabbit antibody against the purified enzyme markedly inhibited activities of the enzyme from both the kidney and liver. Immunoblot analysis showed that the antibody reacted with the renal as well as the hepatic enzymes showing the same molecular weight. Peptide mapping with trypsin or alpha-chymotrypsin showed that digested peptides of renal enzyme were similar to those of hepatic enzyme. Ferrochelatase activity in mouse erythroleukemia (MEL) cells increased in parallel with an increase of heme synthesis by treatment with dimethylsulfoxide. Using immunoblotting techniques, the amount of the enzyme in the MEL cells has been shown to increase by the induction, showing a molecular mass of 41 kDa which was the same as that of the mouse hepatic enzyme. Comparative structural analysis of the enzyme of MEL cells and that of mouse liver by peptide mapping showed that the partial digestive peptides of both enzymes exhibited a similar pattern. These results strongly suggest that ferrochelatase in kidney, liver and erythroid cells can be of one type.
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PMID:Characterization of ferrochelatase in kidney and erythroleukemia cells. 231 Jul 48

Actinobacillus actinomycetemcomitans leukotoxin permeabilized the plasma membrane of HL-60 promyelocytic leukemia cells, resulting in colloid osmotic lysis. These events were associated with efflux of 51chromium (from prelabeled cells), influx of propidium iodide, and ultrastructural evidence of cellular damage. Target cell lysis was inhibited by procedures which may interfere with the initial interaction of the toxin with the plasma membrane. For example, washing cultures (to dilute and remove toxin) or the addition of monoclonal antibodies (to neutralize toxin) or trypsin (to inactivate toxin) limited lysis when undertaken within the first 5 min of the reaction. The extent of injury was also diminished when radiolabeled HL-60 cells were exposed to toxin in the presence of unlabeled, toxin-sensitive cells (e.g., HL-60 cells or human neutrophils) or certain toxin-resistant target cells (e.g., human K562 erythroleukemia cells). This suggests that the association of the toxin with the cell membrane may not be sufficient to cause lysis without activation of additional effector mechanisms. The addition of specific trivalent (e.g., La3+) or divalent (e.g., Ca2+ and Zn2+) cations to toxin-treated cells appeared to enhance their capacity to repair or minimize the extent of toxin-mediated membrane damage. Depending on size, certain saccharides served as osmotic protectants: maltose almost completely inhibited radiolabel release, while smaller molecules provided correspondingly less protection. The results imply that the leukotoxin has membranolytic activity, producing pores in target cells with a functional diameter approximately the size of maltose (0.96 nm).
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PMID:Effects of cations and osmotic protectants on cytolytic activity of Actinobacillus actinomycetemcomitans leukotoxin. 234 Nov 78

A new hemagglutinating monoclonal antibody, MoAb31, detected glycophorins A and B in Western blots. Results with enzyme-modified erythrocytes indicated the MoAb31 determinants were sialic acid dependent, and resided on glycophorin A on the trypsin-resistant, ficin-sensitive segment, and on glycophorin B on the ficin-sensitive segment. Another new monoclonal antibody, MoAb36, detected the Wrb antigen, located on the non-glycosylated segment of glycophorin A near its insertion into the lipid bilayer. Immunofluorescent staining of normal hematopoietic and leukemia cells with these and other monoclonal antibodies to glycophorin A demonstrated glycophorin A on erythroid cells only. Cytofluorograph analysis showed the majority of cells of the erythroleukemia cell lines K562 and HEL expressed glycophorin A, as indicated by reactivity with the monoclonal glycophorin A antibodies R10, R18, 6A7 and 10F7. However, reactivity with monoclonal antibodies to glycosylated determinants (MoAb31 and R1.3) and to the non-glycosylated segment near the membrane insertion (MoAb36, and R7.1) was reduced or absent. Expression of "missing" glycophorin A antigens on K562 and HEL could not be induced using a variety of chemical and biologically active modifiers. We conclude that glycophorin A of erythroleukemia cell lines K562 and HEL differs from glycophorin A at the surface of normal, mature erythrocytes with respect to reactivity with monoclonal glycophorin A antibodies.
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PMID:Glycophorin A on normal and leukemia cells detected by monoclonal antibodies, including a new monoclonal antibody reactive with glycophorins A and B. 241 9

Cell growth and synchronization, gene expression and regulation, maintenance of epithelial cell polarization are major functional aspects of the nephron which have been difficult to approach with conventional preparations. Certainly, without the introduction of tissue culture techniques, the opportunity to analyze these issues could not have been afforded. Several renal epithelial cell lines with differentiated characteristics of proximal and distal segments of the nephron are already available. LLC-PK1, derived from a normal Hampshire pig kidney, shows multiple differentiated characteristics of in vivo epithelia. Specifically, this cell line has Na+-dependent sugar, amino acid, and phosphate cotransport systems with similar characteristics as those present in the renal proximal tubule. The expression of the Na+-dependent sugar transport system in LLC-PK1 cells depends on the growing conditions of the cells. For instance, isolated cells obtained by trypsin-EDTA treatment of confluent monolayers or exponentially growing cells do not express the Na+-dependent sugar transport system. Full expression occurs after the monolayer reaches confluency. Expression of the transporter can also be modified by agents that affect the differentiation of other cellular systems, such as the Friend erythroleukemia cells. The expression of the Na-sugar cotransport system is also regulated by the concentration of glucose in the growth medium. Low glucose concentration increases the sugar influx through the Na+-coupled apical membrane transporter by increasing the number rather than the affinity of the transporter. This effect appears to be mediated through the sugar metabolism of the cell. The expression of the Na+-amino acid cotransport system in LLC-PK1 cells and the epithelial cell line MDCK derived from a normal dog kidney also responds to regulatory signals associated with cell growth or amino acid deprivation. LLC-PK1 cells and the cell line OK derived from an opposum kidney, shows a Na+-dependent phosphate cotransport system modulated by parathyroid hormone and cyclic nucleotides that will prove to be an excellent model not only to study the mechanisms of the action of the hormone, but to study the mechanisms involved in the expression and modulation of the Na+-dependent phosphate cotransport system.
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PMID:Sodium cotransport processes in renal epithelial cell lines. 242 Nov 46

Four monoclonal antibodies against human erythrocyte membrane antigens were established. The antigenic determinants of KOR-E1, E3, E6 were Pr1h antigen, Wrb antigen, and the trypsin sensitive portion of glycophorin A (EnaTS) respectively. The antigen recognized by KOR-E4 could not be determined. The reactivities of these antibodies with normal hematopoietic cells, malignant hematopoietic cell lines (N = 31), and fresh leukemic cells obtained from 128 patients with various types of leukemias were studied. All antibodies reacted only with erythrocytes among peripheral blood cells, and also KOR-E6 reacted only with erythroid cells among bone marrow cells. KOR-E3 had no reactivity with any cell lines examined, and KOR-E1 and KOR-E4 were reactive with some lymphoid cell lines. However, KOR-E6 had specific reactivities with erythroid (HEL, K562), megakaryocytic (CMK-1), multiphenotypic (KOPM-28), and basophilic (KU-812) cell lines. The antigen (glycophorin A) recognized by KOR-E6 was expressed on a small population of mononuclear cells separated from acute lymphoblastic leukemia (3/70), acute myelogenous leukemia (2/12), monosomy 7-myeloproliferative disorder (1/1), juvenile CML (1/1), and transient myeloproliferative disorder with Down's syndrome (4/12), although it could not be determined whether these cells were leukemic cells or not. KOR-E6 was reactive with a large population of leukemic blasts in erythroleukemia (2/2) and acute megakaryoblastic leukemia (3/6). Thus, KOR-E6 appears to be an erythroid marker of leukemic cells.
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PMID:[Monoclonal antibodies against human erythrocyte membrane antigens and their reactivities with hematopoietic cells]. 261 36


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