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 human carcinoma line RPMI 2650 produces autocrine factors; they are detected by the ability of RPMI 2650 conditioned medium (CM) to stimulate growth in soft agar of RPMI 2650 cells plated at low density. The autocrine activity in crude CM can be fractionated by ultrafiltration into a lower molecular weight (MW) fraction (R1-30), which concentrates molecules in the 1000-30,000 Da range; and a higher MW fraction (R30) with molecules greater than 30,000 Da in a more concentrated form. R1-30 is labile to acid, base, and heat treatment, whereas R30 is stable to (and sometimes activated by) these treatments. Boiling of R30, however, renders it labile to acid, base, and trypsin treatments. CM can be separated into a weakly heparin-binding fraction (with stability properties similar, but not identical, to R1-30), and a non-heparin binding fraction (with stability properties similar to R30). RPMI 2650 cells secrete transforming growth factor (TGF)alpha- and TGF beta-like molecules, but the R1-30 fraction can be distinguished from these TGFs, and from most other known growth factors, by its unusual combination of acid lability and weak affinity for heparin. Since the R30/non-heparin binding fraction is rendered labile by boiling or acid treatment, it may represent a bound or conformationally stable form of a growth factor.
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PMID:Production of autostimulatory growth factors by the human carcinoma line, RPMI 2650. 768 48

Streptococcus suis type 2 was evaluated for hemolysin production. Supernatants of S. suis type 2 grown in Todd-Hewitt broth were assayed for hemolytic activity by a photometric assay. Twenty-two additional serotypes of S. suis (1,3 to 22, and 1/2) were evaluated for hemolysin production; nine of them (1/2, 1, 4, 5, 14, 15, 17, 19, and 20) were positive. The effects of temperature, atmosphere, centrifugation, sonication, chemicals, bovine serum albumin, fetal calf serum, and enzymes on S. suis type 2 hemolysin activity were studied. Maximum hemolysis occurred after incubation in RPMI 1640 medium at 40 degrees C in 6% CO2 and after growth in Todd-Hewitt broth at 37 degrees C under anaerobic conditions. Hemolytic activity was absent after the addition of fetal calf serum and decreased after the addition of trypsin or amylase. However, treatment of erythrocytes with amylase or trypsin prior to incubation with supernatant also resulted in a decrease in hemolytic activity. The addition of bovine serum albumin caused increased hemolytic activity. Dipyridyl and EDTA had negligible effects on hemolysis. Hemolytic S. suis type 2 culture supernatant injected intraperitoneally failed to cause death in BALB/c mice. Data from our study indicate that S. suis type 2 hemolysin is a secreted or loosely cell bound, thermolabile molecule whose activity is growth condition dependent.
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PMID:Partial characterization of Streptococcus suis type 2 hemolysin. 805 Dec 53

Hepatocyte growth factor is a recently cloned potent mitogen to hepatocytes, but its extrahepatic roles are not completely defined. It causes proliferation of endothelial and epithelial cells implicating potential action in the glomerulus. We aimed to determine whether cultured human mesangial cells secrete hepatocyte growth factor and the effect of high glucose conditions. Mesangial cells were isolated from the normal cortex of a child's kidney. After differential glomerular sieving and trypsin digestion of glomeruli, mesangial cells were cultured in 20% fetal calf serum/RPMI. Glucose concentration in the medium was adjusted to 5 mmol/l, 11 mmol/l, 25 mmol/l or 5 mmol/l/20 mmol/l mannitol to correct for osmolality. After 0, 24, 48, 72 h incubation, hepatocyte growth factor was measured in the supernatant by enzyme immuno assay using recombinant hepatocyte growth factor and monoclonal antibodies to human hepatocyte growth factor. Hepatocyte growth factor was secreted by cultured mesangial cells. High glucose and hyperosmolar conditions caused a 100-200% increase in hepatocyte growth factor secretion at 48-72 h (p = 0.001). Hepatocyte growth factor secretion at 48 h in 5 mmol/l glucose was 16.46 +/- 1.09 ng/ml (mean +/- SEM), 11 mmol/l glucose: 32.98 +/- 4.54, 25 mmol/l glucose: 33.32 +/- 7.89, 5 mmol/l glucose/20 mmol/l mannitol: 34.05 +/- 3.64; at 72 h in 5 mmol/l glucose: 23.92 +/- 2.85 ng/ml, 11 mmol/l glucose: 28.26 +/- 2.03, 25 mmol/l glucose: 62.04 +/- 12.2, 5 mmol/l glucose/20 mmol/l mannitol: 45.76 +/- 6.25. Trypan blue exclusion demonstrated membrane integrity.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:High glucose and hyperosmolality stimulate hepatocyte growth factor secretion from cultured human mesangial cells. 805 93

This study reports for the first time the establishment of immortalized cell lines from normal adult rat parotid glands. The freshly prepared cellular clumps obtained from parotid glands of isoproterenol-treated rats were incubated in 0.2% trypsin solution without EDTA. These clumps were transfected with plasmid vectors pSV3neo and pSV5neo by electroporation and calcium phosphate-Co-DNA-precipitation techniques. The untransfected and transfected cellular clumps were plated in precoated dishes containing modified MCDB-153 medium. Epithelial cells grew from the clumps that were attached. All epithelial cells from untransfected culture died within 6 to 8 wk. Two cell lines which were isolated from transfected cultures subsequently grew on regular tissue culture dishes. One of them, which was isolated from pSV5neo transfected cultures, exhibited non-epithelial cell morphology, but at confluency, many cells mature to acinar-like cells containing numerous granules. The other cell line (2RS), which was isolated from pSV3neo transfected culture, contained cells of non-epithelial and epithelial morphology. During the initial phase of the growth, MCDB-153 medium was essential; however, at a later time, RPMI medium was better than MCDB-153 or F12 medium for maintaining morphology and growth of these cells. The immortalized cells grew in RPMI with a doubling time of about 25 h, synthesize T-antigen, alpha-amylase mRNAs of 1176 and 702 bp, and alpha-amylase and were non-tumorigenic. These amylase-producing cells can be a useful model to study the mechanisms of regulation of growth and differentiation in these cells.
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PMID:Establishment and characterization of immortalized cell lines from rat parotid glands. 806 58

Indirect ELISA was employed to monitor the serum anti-UEA (urea soluble egg antigen of Schistosoma japonicum) antibody level of mice immunized by: a. UEA pulsed macrophage (Mphi+); b. Cultural supernatant of Mphi+; c. paraformaldehyde fixed M phi (P-Mphi) pulsed with UEA; d. Ammonium chloride treated M phi (NH4Cl-Mphi) pulsed with UEA; e. P-Mphi pulsed with trypsin digested UEA (T-UEA); f. NH4Cl-Mphi pulsed with T-UEA. The normal Mphi, its supernatant and the culture media RPMI 1640 acted as the negative control. The results showed: 1. Serum anti-UEA antibody levels of mice immunized by a and b raised markedly, indicating that the immunogenicity of UEA might be kept up after Mphi processing and the antigenic message could be transferred either by the Mphi+ or by its supernatant; 2. Mice immunized by c and d gave similar results, but the anti-UEA antibody level of the former was higher than that of the latter, suggesting that polyformaldehyde could not alter the UEA binding site on the surface of Mphi; 3. In the case of mice immunized by e and f, the antibody levels were much lower than that of mice immunized by c and d, suggesting that UEA binding sites on Mphi surface as well as UEA immunogenicity could be changed by trypsin.
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PMID:[Role of macrophage in presenting antigens of Schistosoma japonicum]. 817 21

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

An assay of epithelial barrier function was developed to monitor immune-mediated changes in lung permeability that may be occurring during pulmonary allograft rejection and inflammatory lung diseases. Lung tissue was obtained from minipigs, digested with collagenase (1 mg/ml) overnight, and propagated in RPMI 1640 tissue culture medium. Cells with an epithelioid morphology were purified by differential detachment using trypsin-ethylenediaminetetraacetic acid and were characterized as epithelial by positive staining with an anti-cytokeratin monoclonal antibody. Monolayers of these epithelial cells were cultured on porous tissue culture inserts, and transmonolayer resistance values were measured. Transmonolayer resistance values reached a mean of 5487 +/- 2882 omega (mean +/- 95% confidence interval; n = 9) after 5 days in culture. These values indicated the presence of functional intercellular tight junctions between the cells. Addition of cytotoxic immune effector cells to the cultured monolayers caused a rapid reduction in the transmonolayer resistance values, whereas unstimulated splenocytes failed to produce this effect. Comparison of these results with those obtained in parallel experiments performed with standard isotopic cytotoxicity assays indicated the sensitivity of the transmonolayer resistance technique. The assay described in this report will enable in vitro modeling of epithelial permeability damage mediated by both activated lymphoid cells and their soluble products.
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PMID:An in vitro system to model pulmonary epithelial barrier dysfunction mediated by immune effector cells. 832 23

Our studies indicate the effects of in vivo asbestos exposure on the ability of alveolar macrophages (AM) to elaborate a chemoattractant for fibroblast using a rat model of asbestos inhalation. Two groups of rats were exposed by intermittent inhalation (6 hr/day for 5 days/week over a total period of 4 weeks) to either amphibole (crocidolite) or serpentine (chrysotile) asbestos. A group of control rats were sham-exposed to clean air only. The animals were sacrificed 2-5 months after the cessation of exposure. The AM were obtained from the 3 exposure groups in 2 different rat strains by the bronchoalveolar lavage and the cultured in RPMI-1640 medium for 24-96 hr at 37 degrees C. The supernatants from cultured AM were tested for chemotactic activity towards fetal rat skin fibroblasts in a chemotactic assay using 8 microns pore-size filters. The culture supernatants of AM obtained from crocidolite-exposed rats exhibited a significantly greater chemotactic activity towards rat fibroblasts than similar culture supernatants from sham-exposed control animals (p < 0.01) in both rat strains. Significant chemotactic activity was observed after chrysotile exposure (p < 0.05) in ACI rats but not in Fischer-344 rats. Maximal chemoattractant release from AM was noted after 48 hr in culture. Preliminary characterization of the chemoattractant has shown that it is a thermolabile and trypsin sensitive factor whose activity was partially reduced after dialysis. Since AM accumulate at sites of intrapulmonary asbestos deposition, these findings may have relevance to the pathologic accumulation of interstitial lung fibroblasts which occurs during asbestos-mediated lung injury.
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PMID:Enhanced release of an alveolar macrophage-derived chemoattractant for fibroblasts in rats after asbestos inhalation. 839 Mar

The effect of Fasciola hepatica excretory-secretory products (ES products) on the polarization and chemokinesis of sheep and human neutrophils was investigated. Flukes were cultured overnight in RPMI-1640 medium, and the resulting ES products concentrated by dialysis (cut-off 1.2 kDa) before use. At the concentrations used toxicity tests showed no effect on neutrophil viability, but ES products were capable of causing morphological changes and chemokinesis in both sheep and human neutrophils. The polarizing effect of the ES products was found to be greater in the case of sheep neutrophils with 81% of cells polarizing at 80 micrograms total ES protein/ml as opposed to 36% of human neutrophils. Chemokinesis in response to 80 micrograms total ES protein/ml was significant (P < 0.01) in both species with sheep producing the stronger response. Delipidation of the ES products with Lipidex caused no loss of polarizing activity, nor did the purified lipid fraction show any polarizing effect. ES products lost polarizing activity after heat treatment or trypsin digestion. Preliminary fractionation on an S-300 Sephacryl column suggested the presence of at least four fractions capable of causing polarization in sheep neutrophils.
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PMID:Polarization and chemokinesis of ovine and human neutrophils in response to Fasciola hepatica excretory-secretory products. 877 28

Twenty-two Actinomyces pyogenes isolates were recovered from hepatic abscesses in cattle and evaluated for hemolysin production. Hemolysin was collected from supernatant of cultures grown in 6% CO2 in brain heart infusion (BHI) broth. The effect of oxidizing and reducing agents, enzymes, temperatures and pH on hemolytic activity were studied using sheep erythrocytes as the target cells. Our study showed that A. pyogenes hemolysin is oxygen stable; sensitive to treatment by protease, trypsin, and amylase; and destroyed by treatment at extreme temperatures (56 and 100 degrees C) and pH (pH 3 and 11). Production of hemolysin was studied in BHI, RPMI-1640, and a defined serum-free A. pyogenes medium under aerobic and anaerobic conditions. Maximum hemolysin was produced in BHI incubated aerobically in 6% CO2 and to a lesser degree anaerobically in RPMI-1640. No hemolysin was produced in the defined A. pyogenes medium. Differential filtration, isoelectric focusing and sodium dodecyl sulfate polyacrylamide gel electrophoresis identified two hemolysin proteins with pI values of 3.40 and 9.45 and estimated molecular masses of 62 and 58 kDa, respectively. Cell-free supernatant samples positive for hemolysin activity also were screened for leukotoxin activity. Significant levels of leukotoxin were detected in all samples screened.
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PMID:Identification and partial characterization of an Actinomyces pyogenes hemolysin. 881 14


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