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:3.4.21.1 (chymotrypsin)
10,938 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Fibronectin (Fn) is a high molecular-weight glycoprotein that can influence many aspects of monocyte function. The purpose of this study was to determine whether Fn could stimulate monocyte tumor necrosis factor (TNF) secretion. Monocytes were isolated from the peripheral blood of healthy volunteers by density gradient centrifugation and adherence to plastic (2 h). Plasma Fn was purified from the blood by gelatin-Sepharose affinity chromatography. Monocytes were stimulated with Fn for 18 h and the supernatants were assayed for TNF activity using the L929 bioassay. Intact Fn stimulated the secretion of TNF in a dose-dependent manner. Intact Fn-induced TNF secretion by monocytes was inhibited (50%) but not eliminated by the addition of the R-G-D-containing peptide GRGDSP. Limited proteolysis of the Fn molecule using insoluble chymotrypsin resulted in a fragment preparation that was dramatically more stimulatory than the intact Fn preparation. High-performance liquid chromatographic (HPLC) purification of the fragments demonstrated that at least two fragments were capable of stimulating TNF secretion. Further purification by affinity chromatography and HPLC localized the stimulatory activity to the 120-kd cell-binding fragment. The possibility that the stimulatory activity was the result of endotoxin contamination was ruled out using macrophages from C3H/Hej mice. These results suggest that Fn fragments are potentially important molecules for activation of monocytes and may stimulate monocyte cytotoxic activity.
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PMID:Fibronectin fragments stimulate tumor necrosis factor secretion by human monocytes. 174 Jun 45

We have used 125I-labeled fibronectin (FN) as an extracellular substrate for neutrophils (PMN) in order to investigate the mechanism responsible for FN solubilization by PMN and the effects of recombinant cytokines on this process. Pure active alpha 1-antitrypsin (alpha 1AT), when added to PMN before or during, but not after, adherence to FN, inhibited solubilization of the substrate in a dose-dependent manner, but alpha 1AT that had been inactivated by proteolysis or oxidation and alpha 1AT Pittsburgh (alpha 1AT 358Met-Arg) had no significant effect. The solubilization of FN was also inhibited by the PMN elastase inhibitor N-methoxysuccinyl-alanyl-alanyl-prolyl-valine-chloromethylketone but not by the chymotrypsin and cathepsin G inhibitor N-Cbz-glycyl-glycyl-phenylalanine-chloromethylketone, nor by catalase or superoxide dismutase. The products of solubilization of FN by PMN, analyzed by sodium dodecyl sulphate polyacrylamide electrophoresis, were similar to those produced by pure PMN elastase but not cathepsin G. These results suggest that FN solubilization by PMN is caused largely by the pericellular activity of PMN elastase. The solubilization of FN by PMN was increased significantly by adding tumor necrosis factor-alpha, interleukin-1 alpha, or interferon-gamma to the adherent cells but without a significant general release of elastase into the culture supernatants. Granulocyte/macrophage colony-stimulating factor (GM-CSF) had no significant effect. None of the cytokines had any effect when preincubated with the cells in suspension, and non increased FN solubilization by PMN incubated with the optimal (10(-6) mol/liter) or suboptimal dose (10(-8) mol/liter) of the peptide formylmethionylleucylphenylalanine.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Extracellular proteolysis of fibronectin by neutrophils: characterization and the effects of recombinant cytokines. 201 99

A population of latent (cryptic) receptors for tumor necrosis factor-alpha (TNF) has been characterized in the rat liver plasma membrane (PM). 125I-TNF bound to high (Kd = 1.51 +/- 0.35 nM) and low (Kd = 13.58 +/- 1.45 nM) affinity receptors in PM. Solubilization of PM with 1% Triton X-100 prior to incubation with 125I-TNF increased both high affinity (from 0.33 +/- 0.04 to 1.67 +/- 0.05 pmol/mg of protein) and low affinity (from 1.92 +/- 0.16 to 7.57 +/- 0.50 pmol/mg of protein) TNF binding without affecting the affinities for TNF. Digestion of intact PM with chymotrypsin abolished most of the TNF binding capacity of PM. However, substantial binding activity was recovered by solubilization of chymotrypsin-treated PM with 1% Triton X-100, suggesting the presence of a large latent pool of TNF receptors. The affinities of the high and low affinity sites recovered from chymotrypsin-treated membranes were similar to those of intact PM. Affinity labeling of receptors whether from PM, solubilized PM, or membranes digested with chymotrypsin and then solubilized resulted in cross-linking of 125I-TNF into Mr 130,000, 90,000, and 66,000 complexes. Thus, the properties of the latent TNF receptors were similar to those initially accessible to TNF. To determine if exposure of latent receptors is regulated by TNF, 125I-TNF binding to control and TNF-pretreated membranes was assayed. Specific binding was increased by pretreatment with TNF (p less than 0.05), demonstrating that hepatic PM contains latent TNF receptors whose exposure is promoted by TNF. Homologous up-regulation of TNF receptors may, in part, be responsible for sustained hepatic responsiveness during chronic exposure to TNF.
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PMID:Identification, characterization, and homologous up-regulation of latent (cryptic) receptors for tumor necrosis factor-alpha in rat liver plasma membranes. 217 Apr 16

Monocytes lysing a variety of tumor cells were isolated by adhesion to autologous serum-coated plastic surfaces. When the blood monocytes were co-cultured with K562 cells for 3-24 h, the supernatants contained soluble factors, termed monocyte cytotoxic factors (MCF), capable of lysing K562 and other tumor cells in a 48-h microcytotoxicity assay. The production of MCF was mediated by typical monocytes expressing a surface phenotype of CD11 (+), CD16 (-), LeuM1 (+). When target cells were pretreated with actinomycin D, they showed an increase in their susceptibility to lysis by MCF. Addition of the drug to MCF assays also resulted in an enhancement of MCF-mediated lysis. Thus, the lytic activity of MCF was detectable in an 18-h assay. The presence of interferon (IFN)-alpha or -gamma augmented the biological activity of MCF, while pretreatment of target cells with IFN did not enhance MCF activity. The absorption of MCF activity was not elevated by actinomycin D or IFN. MCF lysed target cells that were resistant to tumor necrosis factor (TNF). One result of importance is that MCF lysed autologous and allogeneic freshly isolated human tumor cells. The lysis of fresh human tumor cells by MCF was not inhibited by monoclonal antibodies directed against TNF, lymphotoxin (LT), IFN-alpha, IFN-gamma, or interleukin 1 (IL-1). Furthermore, TNF, LT, IFNs, and IL-1 did not kill fresh human tumor cells. MCF activity was stable at low temperatures but was destroyed by heating. The biological activity of MCF was reduced or abolished by serum, trypsin, chymotrypsin and proteinase K, indicating the proteinaceous nature of MCF. The lytic activity was resistant to protease inhibitors. These data indicate that MCF is a noble cytokine that acts on human fresh tumor cells.
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PMID:[Production and function of the monocyte cytotoxic factor (MCF)]. 244 Mar 86

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

Mouse tumor necrosis factor (TNF) was purified from serum through a series of steps, and each step was monitored for L-cell cytotoxicity in vitro and tumor-necrotizing activity in vivo. The two activities copurified and could not be dissociated. Purified mouse TNF has a specific activity of 2.2 X 10(7) (L-cell assay in the absence of actinomycin D) and 1 microgram causes necrosis of the standard TNF-sensitive sarcoma Meth A. TNF has a Mr of 39,000 +/- 2000 by gel filtration and a Mr of 16,000-18,000 by NaDodSO4/PAGE. Both molecular weight forms display cytotoxic and necrotizing activities. TNF has a pI of 3.9 and is destroyed by trypsin, protease, elastase, and alpha-chymotrypsin but not by neuraminidase or papain. These characteristics of nonrecombinant mouse TNF clearly resemble those of recombinant human and mouse TNF.
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PMID:Purification, characterization, and antitumor activity of nonrecombinant mouse tumor necrosis factor. 352 May 61

Sulfatides have been established recently as ligands for L-selectin, and we investigated whether they trigger transmembrane signals through ligation of L-selectin. We found that sulfatides trigger the increase of cytosolic free calcium in neutrophils and that this effect was strictly dependent on sulfation of the galactose ring, as non-sulfated galactocerebrosides were not stimulatory. Chymotrypsin and phorbol 12-myristate 13-acetate treatment of neutrophils caused shedding of L-selectin, but not of class I major histocompatibility complex antigens or beta 2 integrins, and blunted the capability of neutrophils to respond to sulfatides with an increase of cytosolic free calcium. Four different anti-L-selectin antibodies (DREG-200, LAM1/3, LAM1/6, and LAM1/10), but not four control antibodies directed against different surface molecules of neutrophils, also triggered an increase of cytosolic free calcium. The anti-L-selectin antibodies were stimulatory both if used in a soluble form, after cross-linking with anti-mouse F(ab')2 fragments, and immobilized to protein A of Staphylococcus aureus through the Fc fragment. With immobilized antibodies, an increase of cytosolic free calcium was found also by plating neutrophils on antibodies bound to protein A-coated coverslips and monitoring the increase of cytosolic free calcium by fluorescence microscopy. Both sulfatides and anti-L-selectin antibody effects were not inhibited by pertussis toxin, thus indicating that a pertussis toxin-sensitive GTP-binding protein was not involved in signal transduction. Sulfatides also triggered an increase of tumor necrosis factor-alpha and interleukin-8 mRNAs in neutrophils. Also to act as stimuli of cytokine mRNA expression, sulfatides required sulfation of the galactose ring, as non-sulfated galactocerebrosides were not stimulatory, and depended on expression of L-selectin, as shedding of this molecules induced by chymotrypsin blunted their effects. These findings suggest that L-selectin can transduce signals activating selective cell function.
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PMID:Sulfatides trigger increase of cytosolic free calcium and enhanced expression of tumor necrosis factor-alpha and interleukin-8 mRNA in human neutrophils. Evidence for a role of L-selectin as a signaling molecule. 750 38

We report the purification of a protease from tumor cells undergoing apoptosis that is involved in activating DNA fragmentation. Initial studies revealed that two inhibitors of serine proteases, N-1-tosylamide-2-phenylethylchloromethyl ketone and carbobenzoxy-Ala-Ala-borophe (DK120), suppressed tumor necrosis factor or ultraviolet (UV) light-induced DNA fragmentation in the U937 histiocytic lymphoma as well as UV light-induced DNA fragmentation in the BT-20 breast carcinoma, HL-60 myelocytic leukemia, and 3T3 fibroblasts. The protease was purified by affinity chromatography with DK120 as ligand and showed high activity on a synthetic substrate preferred by elastase-like enzymes (Ala-Ala-Pro-Val p-nitroanilide), but was inactive on the trypsin substrate, N-alpha-benzyloxycarbonyl-L-lysine thiobenzyl ester, or the chymotrypsin substrate, Ala-Ala-Pro-Phe p-nitroanilide. The activity of the DK120-binding protease purified from U937 cells undergoing apoptosis was increased approximately 10-fold over that recovered from normal cells. Further purification to homogeneity by heparin-Sepharose affinity chromatography followed by reverse phase high-performance liquid chromatography revealed a single band of 24 kD on a silver-stained sodium dodecyl sulfate gel. In addition to protease activity, the purified enzyme induced DNA fragmentation into multiples of 180 basepairs in isolated U937 nuclei. These findings suggest the 24-kD protease is a novel enzyme that activates DNA fragmentation in U937 cells undergoing apoptosis.
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PMID:Purification of a 24-kD protease from apoptotic tumor cells that activates DNA fragmentation. 796 87

A growth-inhibiting activity was identified in supernatants of the neoplastic V79 Chinese hamster cell line based on its ability to inhibit the proliferation of the same cell line. The partially purified activity, provisionally termed "growth inhibiting factor" (GIF) activity, inhibited the growth of a wide variety of human tumor cells, but not various normal human fibroblasts. This species-nonspecific activity was reversible, saturable, and highly potent in tumorigenic cell lines, and was noted in both monolayer culture and in soft agar. The inhibitory activity of GIF was also exhibited in a chemically defined serum-free medium supplemented with insulin and transferrin. GIF activity was stable to acid, heat, trypsin, and dithiothreitol but sensitive to alpha-chymotrypsin. The pattern of growth modulation by GIF on V79 cells was apparently different from those exhibited by bifunctional peptides such as transforming growth factor-beta, tumor necrosis factor-alpha, and interleukin-1-alpha. In addition, GIF activity cannot be ascribed to these cytokines based on the physicochemical and immunologic properties. Although GIF has yet to be purified to homogeneity, these data suggest that GIF might be a novel growth regulator which has a critical role in regulating growth of V79 cells. The growth modulation of tumor cells by this tumor-derived growth inhibiting activity suggested the presence of an autocrine growth regulatory mechanism even in tumor cells.
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PMID:Growth modulation of human tumor cells by a growth-inhibiting activity derived from tumorigenic V79 Chinese hamster cells. 832 Jan 83

The effects of the inflammatory mediators lipopolysaccharide (LPS) and tumor necrosis factor-alpha (TNF) and unstimulated and activated neutrophils (PMNs) on endothelial cell (EC) necrosis were studied using the cultured human EC line (ECV-304) and human PMNs in vitro. LPS and TNF alone or their combination failed to induce EC necrosis. Activated PMNs, as evidenced by augmentations in CD11b expression and respiratory burst, induced significant EC necrosis commencing at 12 hr of coculture, which was strongly dependent on the ratio of PMN:ECs and the duration of PMN:EC coculture. In contrast, unstimulated PMNs induced no significant increases in EC necrosis. To examine the mechanisms of activated PMN-mediated EC necrosis, the oxygen radical scavengers superoxide dismutase (SOD) and catalase, as well as the protease inhibitors phenylmethylsulfonyl fluoride (PMSF), alpha 1-antitrypsin (alpha 1-AT), soybean trypsin-chymotrypsin inhibitor (TCI), and aprotinin, were studied in coculture experiments. EC necrosis induced by activated PMNs could be markedly attenuated by SOD, PMSF, alpha 1-AT, TCI, aprotinin, or their combinations. Although aprotinin enhanced respiratory burst, this agent inhibited necrosis by downregulating PMN CD11b and PMN-EC adhesion. These results demonstrate that the inflammatory mediators LPS and TNF and quiescent PMNs fail to induce EC necrosis. However, PMNs activated by inflammatory mediators can induce EC necrosis through oxidative and nonoxidative mechanisms and this process is dependent on PMN-EC adhesion.
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PMID:Mechanisms involved in the induction of human endothelial cell necrosis. 859 44


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