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:3.4.24.3 (collagenase)
18,340 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Rat liver cells prepared by collagenase treatment and separated by density gradient centrifugation with Ficoll-Hypaque solution (specific gravity 1.120), containing only hepatocytes (80%) and Kupffer cells (20%), were found to stimulate strongly Ag-B-incompatible lumphocytes without 2-mercaptoethanol in culture. The same liver cells could also stimulate Ag-B-compatible lymphocytes in the presence of 2-mercaptoethanol. A positive response was only seen when the liver cell numbers were 1 approximately 2% of the responding lymphocytes. Viable liver cells inhibited the reaction in a two-way culture.
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PMID:Liver and immune responses. IV. Characteristics of the liver cell-lymphocyte interaction. 14 29

Mononuclear cell infiltration and alteration in the connective tissues are prominent features of the inflammatory response in a number of diseases. To determine whether mononuclear cell products can modulate collagen synthesis, human peripheral mononuclear cells from normal donors were isolated by Ficoll-Hypaque gradient centrifugation and then incubated for 48 h with or without phytohemagglutinin. Confluent cultures of normal, human skin fibroblasts were incubated with [14C]proline and various amounts of dialyzed supernates from the mononuclear cell cultures. Labeled, newly synthesized collagen was estimated by [14C]hydroxyproline analysis, collagenase digestion, and chromatography on Agarose A-5m in sodium dodecyl sulfate. The total incorporation of [14C]proline was not significantly affected by addition of the mononuclear cell supernates, but as much as 90% decrease in the synthesis by the fibroblasts of labeled collagen was found relative to controls. Supernates from the phytohemagglutinin-stimulated cultures were more active than those from nonstimulated cells. These results suggest that mononuclear cells can synthesize a factor(s) which can selectively inhibit collagen synthesis.
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PMID:Inhibition of collagen synthesis by mononuclear cell supernates. 22 88

The elaboration of leukocyte chemotactic factors by human fibroblasts was studied. 12 lines of normal fibroblasts obtained by skin biopsy and then cultured in vitro produced chemoattractants (assessed by modified Boyden-chamber techniques) for both peripheral blood polymorphonuclear leukocytes and monocytes (obtained by Hypaque-Ficoll and dextran sedimentation). Chemotactic activity was not present performed in fibroblasts, and cycloheximide blocked its elaboration. The chemotactic activity of crude-culture supernate was heat stable (56 degrees C for 30 min), trypsin- and pronase-sensitive, and neuraminidase resistant. Characterization of the chemotactic activity by gel filtration (Sephadex G-75) showed two active fractions, one with mol wt greater than 100,000 and the other less than 10,000. In studies designed to relate these chemotactic factors to collagen, we have confirmed that type I collagen and alpha 1-chain; are chemotactically active for monocytes but not polymorphonuclear leukocytes. However, the chemotactic activity in fibroblast-culture media was media was distinct from collagen in that it attracted neutrophils, it was not precipitated by 25% ammonium sulfate, and it was resistant to collagenase treatment; ascorbic acid, in concentrations known to stimulate fibroblast collagen synthesis, had no effect on the elaboration of the chemotactic factors. Furthermore, amino acid analysis of Sephadex G-75 fractions with chemotactic activity failed to reveal amino acids such as hydroxyproline characteristic of collagen. In addition to the chemotactic factors secreted by fibroblasts, a heat-resistant factor (30 min at 56 degrees C) which generated the chemotactically active fragment of C5 (C5a) from human serum was also secreted. The elaboration of mediators of the inflammatory and immune responses by fibroblasts may initiate and(or) modulate local skin inflammatory reactions and play a protective role in vivo.
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PMID:Polymorphonuclear leukocyte and monocyte chemoattractants produced by human fibroblasts. 43 25

In an attempt to clarify a mechanism of polymorphonuclear cell and/or macrophage infiltration in alcoholic liver disease, we investigated a novel chemotactic and activating factor generated by rat hepatocytes isolated from the chronically ethanol-fed rats. Hepatocytes and hepatic macrophages were isolated from rat liver by perfusion and digestion with collagenase and subsequently by differential centrifugation on a metrizamide gradient. Rat polymorphonuclear cells were prepared from blood by the dextran sedimentation and Hypaque-Ficoll technique. Chemotactic activity was measured as migration of polymorphonuclear cells or hepatic macrophages using a chemotactic chamber. When hepatocytes isolated from the ethanol-fed rats were cultured in vitro, chemotactic activity for rat polymorphonuclear cells and hepatic macrophages was demonstrated in the culture supernatant. Inhibitors of transcription and protein synthesis reduced generation of chemotactic factor from these hepatocytes. Chemotactic activity of the conditioned medium was reduced after trypsin (0.25%, 37 degrees C, 30 min) or heat (56 degrees C, 30 min) treatment. The chemotactic activity was eluted at molecular weights of 20-25 kDa and 40-45 kDa following Sephadex G-150 chromatography. Superoxide anion production by polymorphonuclear cells and hepatic macrophages under the stimulation of phorbolmyristate acetate was enhanced in the presence of this chemotactic factor. This chemotactic factor may contribute to the pathogenesis of alcoholic liver disease.
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PMID:Generation of chemotactic factor by hepatocytes isolated from chronically ethanol-fed rats. 156 5

During infection or endotoxemia, the immune system is activated and its energy needs increase. Alcohol (ETOH) intoxication on the other hand suppresses the immune system and increases susceptibility to infection. Since the liver is the primary site both for metabolism of ETOH and detoxification of bacterial lipopolysaccharides (LPS), this investigation was directed at studying the effect of acute ETOH intoxication on the LPS-induced enhancement of in vivo glucose utilization in different types of hepatic cells. Rats were given an intravenous (IV) injection of ETOH followed by a constant infusion for 7 hr to maintain blood alcohol levels at about 175 mg/dl. E. coli LPS was administered IV at 4 hr and in vivo glucose utilization by the different types of liver cells was estimated 3 hr later using the 14C-2-deoxyglucose technique. Hepatocytes (HP), Kupffer (KC), and endothelial cells (EC), as well as the sequestered polymorphonuclear leukocytes (PMN), were separated from the liver by collagenase-pronase digestion followed by centrifugal elutriation and Ficoll-Hypaque density gradient centrifugation. The number of PMN in the liver was increased several-fold 3 hr after LPS administration. The presence of ETOH did not inhibit the LPS-induced neutrophil migration into the liver. ETOH depressed the LPS-induced increase in glucose uptake in both EC and KC by 50 to 80%, respectively. It also reduced the LPS-induced increase of plasma tumor necrosis factor activity by 80%. ETOH alone did not produce any significant changes in the parameters studied.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Acute ethanol intoxication suppresses E. coli lipopolysaccharide enhanced glucose utilization by hepatic nonparenchymal cells. 205 1

A method for isolating and characterizing intestinal lymphoid cells from colonoscopic biopsies is presented. Intraepithelial lymphocytes were separated from the lamina propria by incubation in edetic acid (EDTA) and lamina propria lymphoid cells isolated by incubation in collagenase followed by Ficoll-Hypaque density flotation. Quantitation of T lymphocyte helper (OKT4) and suppressor (OKT8) cells was performed using monoclonal antibodies to cell surface markers and analyzed on a flow cytometer. The isolation procedure yielded approximately 400,000 lamina propria cells and 100,000 intraepithelial cells per sample, with better than 90% viability. Surface marker analysis demonstrated significant differences in the ratios of helper to suppressor cells between the intraepithelial lymphocytes and the lamina propria lymphocytes. These demonstrate the feasibility of lymphoid cell isolation from colonoscopic biopsy specimens for surface marker analysis by flow cytofluorimetry. These techniques could prove important in the study of immune mechanisms in inflammatory bowel diseases.
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PMID:Isolation of intestinal mononuclear cells from colonoscopic biopsies for immunofluorescence analysis by flow cytometry. 293 80

The immunologic competence of human placental mononuclear cells was compared to that of adult and cord blood mononuclear cells. Mononuclear cells were isolated from fresh placentas by digestion with collagenase and DNase, followed by Ficoll-Hypaque and discontinuous Percoll separation. Placental cells incubated with phytohemagglutinin (PHA) synthesized significantly more interferon-gamma (IFN-gamma) at 2 days (29 +/- 5.5 IU/ml) and 5 days (46 +/- 8.5 IU/ml) than PHA-activated cord cells (3.6 +/- 0.6 IU/ml at 2 days and 2.7 +/- 0.7 IU/ml at 5 days) but less than PHA-activated adult cells (81 +/- 20 IU/ml at 2 days and 270 +/- 161 IU/ml at 5 days). Placental and adult cells, but not cord cells, also synthesized significant quantities of IFN-gamma following incubation with interleukin-2 (IL-2). There was synergism between IL-2 and PHA activation for IFN-gamma production for some cord samples. After a 5- to 7-day incubation with IL-2, the lymphocyte-activated killer (LAK cell) cytotoxicity of placental cells (measured in a 3-hr chromium-release assay at an E:T ratio of 40:1) was enhanced 13-fold against K562 target cells (6 +/- 2% to 77 +/- 4%) compared to a 4-fold increase in cord cells (16 +/- 4% to 68 +/- 3%) and a 2-fold increase in normal adult cells (35 +/- 4% to 65 +/- 3%. Against the natural killer (NK)-resistant Raji target, placental cells increased their LAK cytotoxic activity (3 +/- 1% to 59 +/- 7%) compared to a 7-fold increase with cord cells (6 +/- 1% to 43 +/- 3%) and a 3-fold increase with adult cells (11 +/- 2% to 38 +/- 4%). A notable degree of cytotoxic activity in the absence of IL-2 against Molt targets was noted in 11 of 14 (79%) placental cell samples at 5 days. Only 10 of 24 (42%) adult and 17 of 37 (40%) cord samples showed spontaneous cytotoxic activity equal to or greater than 10%. Some placental samples actually showed an increase in cytotoxic activity when incubated without IL-2. The ability of placental cells to produce significant levels of IFN-gamma, to develop considerable LAK activity, and to maintain or develop cytotoxic activity in the absence of IL-2 suggests a vigorous, active immune system of the placenta compared to the relatively dormant immune system of the neonate. These observations suggest that placental cells may have a primary role in fetal defense.
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PMID:Enhanced interferon production and lymphokine-activated cytotoxicity of human placental cells. 313 Jan 92

Primary breast adenocarcinomas obtained from ten patients were enzymatically digested using collagenase (1 mg/ml), hyaluronidase (1 mg/ml), elastase (0.1 mg/ml) and DNAse (0.2 mg/ml). The tumor cells were labeled with 3H-thymidine and, in some cases, with 3H-estradiol. The isolated cells were submitted successively to a Ficoll-Hypaque and a bovine serum albumin gradient, from which 12 fractions were obtained. In each fraction, several characteristics were determined: carcinoembryonic antigen (CEA), thymidine (dThd) incorporation, and estrogen receptors (ER). Three main cellular subpopulations were characterized: An intermediate density subpopulation (1.046-1.054 g/ml), in which the proliferating cells are concentrated. In this subpopulation a small number of CEA-positive cells are present, but ER containing cells are virtually absent. A high-density, small cell subpopulation that concentrates most of the ER-containing cells. This subpopulation lacks proliferating cells, but CEA-containing cells are abundant. A low-density subpopulation, lacking proliferating cells and with scarce ER-positive cells, although CEA-positive cells are frequent. These findings strongly suggest that proliferating cells lack ER.
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PMID:Determination of DNA synthesis, estrogen receptors, and carcinoembryonic antigen in isolated cellular subpopulations of human breast cancer. 352 93

Aryl hydrocarbon hydroxylase (AHH) activity mediated by cytochrome P-450 is present in pig hepatic microsomes [10 nmol.3 mg protein-1.hr-1]. AHH activity was detectable in both hepatocytes and Kupffer cells isolated from pig liver biopsy material. These cells were isolated from needle or wedge biopsy material by collagenase perfusion and incubation with collagenase at 37 degrees. The two cell types were separated from the resulting cell suspension as previously described for whole liver. Kupffer cells were enriched by adherence and were cultured for 24 hr prior to harvesting. Cells were harvested, and cell viability was determined. AHH activity was assayed in Kupffer cell and hepatocyte homogenates. Kupffer cell AHH activity was approximately one-eighth the level detected in hepatocytes. To determine whether this enzyme was present in other macrophages, monocytes were isolated from 10 ml of heparinized peripheral blood using Ficoll-Hypaque and were enriched by adherence. After 24 hr in culture, cell viability was assessed and monocytes were identified by by cytochemical staining. AHH activity was detectable in pig monocyte homogenates, and the AHH level was similar to that in pig Kupffer cells. AHH was also easily detectable in human monocytes. This macrophage AHH activity was compared with AHH activity in rat monocytes, mouse Kupffer cells and mouse peritoneal macrophages. Monocyte AHH was relatively stable in cell culture but decreased rapidly upon storage at -70 degrees. Macrophage AHH activity was depressed following phagocytic activation in vitro by latex beads with a concomitant increase in heme oxygenase activity.
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PMID:Drug-metabolizing enzymes in rat, mouse, pig and human macrophages and the effect of phagocytic activation. 368 28

Mononuclear cells were isolated from human peripheral blood by Ficoll-Hypaque centrifugation, and the cells adherent to plastic substrata were cultured in serum-free media supplemented with lactalbumin hydrolysate. These cell cultures, which consisted predominantly of monocyte-macrophages as judged by nonspecific esterase staining, accumulated collagenase in the medium. This collagenase resembled other vertebrate collagenases in that it cleaved native triple-helical type I collagen at a locus 3/4-length away from the amino-terminal end of the molecule. The collagenase activity was inhibited by Na2EDTA, dithiothreitol, and fetal calf serum, while the addition of Ca++ or N-ethylmaleimide enhanced the enzyme activity. The accumulation of collagenase in the culture media was markedly enhanced by the incubation of cells with concanavalin A or phorbol myristic acetate. In the presence of cycloheximide, the levels of collagenase activity were markedly reduced, suggesting that active protein synthesis was required to express the enzyme activity. In additional experiments, monocytes were further purified by counterflow centrifugation-elutriation. The collagenase production was markedly increased in cultures enriched in monocyte-macrophages and devoid of polymorphonuclear leukocytes. The accumulation of collagenase in monocyte cultures incubated for 48 hours in the presence of concanavalin A or phorbol myristic acetate was of the same order of magnitude as in parallel cultures containing the same number of polymorphonuclear leukocytes purified by Ficoll-Hypaque centrifugation and Plasmagel sedimentation.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:The production of collagenase by adherent mononuclear cells cultured from human peripheral blood. 609 71


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