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)

Epidermal growth factor (EGF), phorbol esters (PEs), and retinoic acid (RA) inhibit differentiated functions of thyrocytes. In the present study the inhibitory effects of these growth-promoting factors on hormone synthesis were studied in thyroid follicles cultured in type-I collagen gel, and morphologic alteration by these factors was examined by light and electron microscopy (EM). Porcine open thyroid follicles obtained by treatment with 0.1% collagenase were embedded in collagen gel and cultured in Ham's F12 medium supplemented with 6H (insulin, hydrocortisone, somatostatin, transferrin, glycyl-his-lys, and thyrotropin) + 0.5% fetal bovine serum (FBS). After 1 week these open follicles developed to closed follicles, and the medium was changed to one containing 6H + 0.5% FBS + 0.1 microM sodium iodide (NaI). Some media were supplemented with either EGF, phorbol 12-myristate 13-acetate (PMA), or all-trans RA. The closed follicles retained ability for hormone synthesis for 2 weeks after the medium change in the presence of 6H + FBS + NaI. The amounts of T4 and T3 secreted into the culture medium from day 9 to day 12 after the medium change were 60% and 45% of those from day 0 to day 4, respectively. EGF reduced production of T4 and T3 by 61% and 69%, respectively; PMA, by 87% and 99%; and RA, by 55% and 44%. In the medium supplemented with 6H + 0.5% FBS, the follicles exhibited intact polarity. Apical surfaces with microvilli were oriented to the follicular lumen and tight junctions were on the apical side of cell-to-cell contacts. Desmosomes were found on both the apical and basal halves of the cell contacts.(ABSTRACT TRUNCATED AT 250 WORDS)
Thyroid 1992
PMID:Effects of epidermal growth factor, phorbol ester, and retinoic acid on hormone synthesis and morphology in porcine thyroid follicles cultured in collagen gel. 149 78

We have characterized the insulin-like growth factor-binding proteins (IGF-BPs) released by isolated sheep thyroid epithelial cells. Thyroid follicles were isolated with collagenase and cultured in Coon's modified F-12 M (0H medium) supplemented with insulin, cortisol, transferrin, glycyl-histidyl-lysine and somatostatin (5H medium) and TSH (6H medium). Conditioned 0H medium specifically bound both 125I-labelled IGF-I and -II, although binding capacity was reduced following acid-gel filtration to separate endogenous IGF-BP complexes, suggesting some destruction of BPs. The binding of 125I-labelled IGF-I or -II to conditioned (0H) medium was progressively displaced by increasing amounts of unlabelled homologous peptides, while fractionation on concanavalin A-Sepharose showed that the IGF-BPs consisted of both glycoprotein and non-glycoprotein components. The molecular sizes of the IGF-BPs were resolved by separation of 0H medium on SDS-PAGE and ligand blot analysis with 125I-labelled IGF-I or -II. Conditioned medium contained four specific binding species for IGF-II of 19, 30, 38 and 46 kDa; all but the smallest also binding radiolabelled IGF-I. Prior fractionation on concanavalin A-Sepharose showed that the 46 kDa binding species was a glycoprotein. Competition studies with increasing concentrations of unlabelled IGF-I or -II during ligand blotting suggested that the 46 and 30 kDa binding species had a greater affinity for IGF-II than IGF-I, while the 38 kDa had a greater relative affinity for IGF-I. Incubation of cells in 5H medium reduced the abundance of the 46 kDa binding protein, while incubation in 6H medium decreased the release of all binding protein species. Results show that isolated thyroid follicles released several forms of IGF-BP with differing relative affinities for IGF-I and -II. Gross changes seen in the presence of BPs between 0H, 5H and 6H media suggest acute hormonal control of release.
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PMID:Characterization of insulin-like growth factor-binding proteins secreted by isolated sheep thyroid epithelial cells. 169 63

A method for isolation of C-cells from rat fetuses was developed, and the morphological plasticity of the cells in primary culture systems was tested. Thyroid-parathyroid-ultimobranchial body (UB) complexes from 16-day rat fetuses were treated with 0.1% collagenase and 1000 PU/ml Dispase at 37 degrees C for 1 h. After dissociation by pipetting, UBs were obtained as remaining cell aggregates with diameters of 150-200 microns. The isolated UBs were cultured on untreated, fibronectin-coated, or laminin-coated substratum in Dulbecco's modified Eagle's medium/Ham's nutrient mixture F-12 (1:1) supplemented with 5% fetal calf serum. In some experiments, the medium was changed to serum-free medium after 24 h of incubation, until the UBs had formed cell sheets. At Day 4 in vitro, the cultures were subjected to immunostaining using anti-calcitonin antiserum. On untreated or fibronectin-coated substratum, most of the C-cells exhibited polygonal or ovoid shapes, and 5-8% of them were found to project processes. On laminin-coated substratum, the ratio of process-bearing C-cells to total C-cells was 23% in serum-supplemented medium and 51% in serum-free medium. The longest processes reached 150 microns in length. The processes were intensely reactive with anti-alpha-tubulin antibody and were completely disintegrated by colcemid, suggesting that the microtubule cytoskeleton participated in the maintenance of the processes. Thus it was demonstrated that fetal rat C-cells are still responsive to environmental signals, such as laminin, and extend neuritic processes.
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PMID:Laminin-induced process outgrowth from isolated fetal rat C-cells. 172 30

We describe a method of culturing intact porcine thyroid follicles for physiological de novo thyroid hormone formation; the roles of cAMP and protein kinase-C in thyroid hormone formation were also studied. Thyroid follicles were obtained by digesting minced porcine thyroid tissue with 0.04% collagenase and cultured in Coon's Modified Ham's F-12 medium supplemented with 0.5% calf serum, 0.5 mU/ml TSH, other standard hormones, and 3 antibiotics (6H medium). On the fourth day of culture, 6000-8000 follicles/well were plated in 12-well culture dishes. On the sixth day, thyroid hormone formation was carried out by incubating thyroid follicles with 0.5 microM KI in the presence of 6H medium for 2 days in a 5% CO2-95% air incubator at 37 C. To examine the effects of cAMP and protein kinase-C on de novo thyroid hormone formation, follicles were incubated with KI in the presence of 1-2.5 mM (Bu)2cAMP, 10 microM forskolin, 2 microM prostaglandin E2 (PGE2), or 0.5-1 microM 12-O-tetradecanoylphorbol-13-acetate in TSH-free medium for 2 days. The amount of newly formed thyroid hormone was measured by RIA of T3 content in the Pronase digest of thyroid follicular cells. Thyroid follicles cultured in 6H medium had normal polarity of the membrane, determined by electron microscope, and thyroid cAMP was responsive to the alteration of TSH. In this culture system cAMP alone was sufficient to form thyroid hormone. 12-O-Tetradecanoylphorbol-13-acetate, a protein kinase-C stimulator, disrupted thyroid follicles and inhibited cAMP-mediated thyroid hormone formation. The integrity of follicular structure was also required for thyroid hormone formation in this culture system. This study introduces perhaps the most physiological culture system for de novo thyroid hormone formation. Our data provide direct evidence that thyroid hormone formation is linked to cAMP and that the protein kinase-C system acts as an inhibitor of thyroid hormone formation.
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PMID:Physiological de novo thyroid hormone formation in primary culture of porcine thyroid follicles: adenosine 3',5'-monophosphate alone is sufficient for thyroid hormone formation. 215 8

Thyroid enlargement in response to chronic hypersecretion of TSH reflects the coordinated growth of both parenchyma and stroma. Because Wollman et al. observed in propylthiouracil-fed rats that enlargement and remodeling of thyroid capillaries were strictly localized around follicles, they hypothesized that growth of perifollicular blood vessels is stimulated by angiogenic factors secreted by neighboring follicular epithelial cells. In support of this hypothesis, we report that media conditioned by rat thyroid cells were very active in an in vitro angiogenesis bioassay that measures stimulation of endothelial cell migration through chemotaxis membranes in microwell Boyden chamber assemblies. Primary cultures of thyroid cells from collagenase-dispersed glands from male or female Holtzman rats fed 0.01% propylthiouracil in the drinking water released activity that produced up to 5-fold increases in endothelial cell migration rates relative to those in identical unconditioned medium. Thyroid-derived activity was primarily chemotactic (i.e. only weakly chemokinetic) to both rabbit aortic and microvascular endothelial cells. That endotheliotropic activity is derived from thyroid parenchyma is indicated by the finding that media conditioned by FRTL cells, a clonally derived thyroid follicular epithelial cell line, produced parallel chemoattractant responses. Thyroid-conditioned media were also chemoattractant to mouse BALB/c-3T3 cells, which have endothelial cell characteristics. In contrast, thyroid-conditioned media did not increase the high spontaneous migration rate of Walker rat sarcoma (WR256) cells. T4, T3, thyroglobulin, bovine fibroblast growth factor (alpha and beta), and media conditioned by rabbit endothelial cells were inactive. Chemoattractant activity in serum containing conditioned media was retained by both 10,000 and 30,000 mol wt cut-off (MWCO) ultrafilters. Activity in serum-free thyroid-conditioned media was largely retained by 10,000 MWCO filters, but only partially retained by 30,000 MWCO filters; activity in the 30,000 filtrate was recoverable in a 10,000 MWCO retentate. These findings support the hypothesis that capillary growth during thyroid enlargement occurs, at least in part, as a result of a parenchymal-stromal (epithelial-mesenchymal) paracrine interaction mediated by specific endotheliotropic (angiogenic) factors released by follicular epithelial cells and distinct from T3, T4, and thyroglobulin.
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PMID:Thyroid angiogenesis: endotheliotropic chemoattractant activity from rat thyroid cells in culture. 244 58

The present study was undertaken to examine whether thyrocytes possess phagocytic activity and whether the phagocytic activity is influenced by cytokines, such as interleukin 1, 2 (IL 1, IL 2) and interferon-alpha, -beta, and -gamma (IFN-alpha, beta, and gamma), and drugs, such as methimazole and dexamethasone. Thyroid glands were obtained from patients with Graves' disease. Thyrocytes were prepared by collagenase digestion. Thyrocytes were pre-incubated in the presence or absence of cytokines and drugs at 37 degrees C for 20 h and were further incubated with fluoresceinated latex beads at 37 degrees C for 60 min. The number of phagocytic thyrocytes was determined by FACS IV. Phagocytosis of latex beads was indeed seen within thyrocytes and gradually increased in a time-dependent manner. The rate of phagocytosis in thyrocytes was extremely slow as compared with that in macrophages. Phagocytic activity was detected in thyrocytes from patients with Graves' disease and from normal thyroid tissue adjacent to thyroid cancer. Phagocytosis was inhibited by IL 1, but was enhanced by IL 2. Although the enhanced phagocytosis with IFN-beta was consistently seen, little effect was detected with IFN-alpha and -gamma. Both methimazole and dexamethasone markedly inhibited phagocytosis. These results indicated that thyrocytes had phagocytic properties and that their phagocytic activity was modulated by cytokines, antithyroidal drugs and dexamethasone.
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PMID:The effects of cytokines, antithyroidal drugs and glucocorticoids on phagocytosis by thyroid cells. 246 Oct 40

Thyroid hormones influence Ca2+ homeostasis in both skeletal and cardiac muscle. Since secretory cells, like muscle cells, store and use Ca2+ in stimulus-response coupling, we have studied the effects of thyroid status on Ca2+ mobilization and secretion in a model secretory tissue, the pancreatic acinar cell. Hyperthyroidism was induced by rats by daily, subcutaneous injections of triiodothyronine for 8 days and hypothyroidism by adding 6-n-propyl-2-thiouracil to the drinking water for 14 days. Pancreatic acini were prepared by collagenase digestion of pancreatic tissue from hyper- and hypo-thyroid animals and from euthyroid controls. Ca2(+)-mobilization was assessed using Quin-2 fluorescence and secretion by assaying amylase release. The data indicate that the amount of Ca2+ mobilized by the muscarinic agonist carbachol or by cholecystokinin octapeptide increases with increasing thyroid hormone concentrations. Only in hypothyroidism was this change in Ca2+ homeostasis reflected by a parallel change in amylase secretion. This implies the existence of some compensatory mechanism which stabilizes secretory rate in the face of stimulus-evoked increases in intracellular Ca2+ concentration.
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PMID:Influence of thyroid status on Ca2+ mobilization and amylase secretion in rat pancreatic acini. 248 94

A bioassay for thyroid stimulating immunoglobulins (TSI) of patients with Graves' disease was developed by porcine thyroid monolayer cells. Thyroid cells were prepared by dispersion using collagenase and trypsin. Aliquots of the cell suspension (2 X 10(6) cells/1.5 ml/dish) in Ham's F-12 medium (pH 7.2) containing 10% calf serum and 1.5 mM Hepes were seeded and cultured in air at 36 C. On day 6 of culture, cells were incubated with test samples (IgG or bTSH) in 1 ml of serum-free, 0.5 mM IMX-included fresh medium for an additional time, and cAMP in the cells was measured by radioimmunoassay. Intracellular cAMP was increased within 5 minutes after the addition of bTSH and the maximal increase was observed after 30 min. Responses of cAMP were in a dose-related manner up to 10 mU/ml of bTSH. With the addition of IgG from untreated Graves' patients, dose-related increases in cAMP were also observed up to 10 mg/ml IgG and the maximal response was seen at 2 hours incubation. Thyroid stimulating activity in IgG's from normal subjects and patients with Graves' disease was tested with a dose of 10 mg/ml and 2 hours incubation and the activity was expressed as a percent of the control (incubated in the same experiment without IgG). One hundred forty one of 145 untreated patients showed higher activity (228 +/- 51.8%, mean +/- SD; 127-393%, range) than normal subjects (103 +/- 13.3%, mean +/- SD, n = 24; 80-129%, range). Sequential changes in TSI activity in 27 patients after initiating thionamide drugs were studied for 24 months. Initially all 27 patients showed positive TSI and 6 months later 15 remained positive. At 6 months after that, 10 of 23, 4 of 16, and 2 of 6 followed patients showed positive TSI. These results indicate that this bioassay is clinically useful for detecting TSI.
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PMID:A bioassay for thyroid stimulating immunoglobulins of patients with Graves' disease using porcine thyroid monolayer cells. 301 50

We have prepared thyroid follicles in suspension culture to use as a model system in vitro for investigation of some properties of the thyroid gland. The follicles were free of endothelial cells, fibroblasts, and other nonepithelial cells. They were prepared by collagenase treatment of minced rat thyroid glands followed by differential filtration of the suspension through nylon meshes. Small clusters of principal thyroid epithelial cells were separated from large fragments and single cells. They were cultured in dilute suspension in Coon's modified F-12 medium in dishes coated with agarose to avoid having the cells attach to the dishes. By culture day 3, most of the clusters formed closed follicles containing a periodic acid-Schiff-positive colloid but without a basal lamina. Follicle walls contained an occasional C cell. The epithelium resembled that in the thyroid of a recently hypophysectomized rat, with normal polarity and organelle complement normal with respect to position and abundance, with basally located lysosomes, no pseudopods, and no colloid droplets. The cells were responsive to thyroid-stimulating hormone (thyrotropin) and to dibutyryl cyclic AMP. Thyroid-stimulating hormone at 10 munits/ml resulted in apical migration of lysosomes and formation of pseudopods and colloid droplets within 30 min; longer exposure resulted in depletion of luminal colloid. The results indicate that the suspended follicles resemble follicles in vivo with respect to morphology and responsiveness to thyroid-stimulating hormone in the absence of other cell types.
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PMID:Suspension culture of separated follicles consisting of differentiated thyroid epithelial cells. 624 62

A microcytotoxicity assay for detection of thyroid-specific complement-dependent cytotoxic antibody (cytotoxic antibody), antibody-dependent cell-mediated cytotoxicity (ADCC) and direct lymphocyte cytotoxicity was developed using human, thyroid epithelial cells as targets. Thyroid tissue was obtained from patient with Graves' disease and was treated with collagenase and then trypsin. The red blood cells, interfollicular fibroblasts and infiltrating lymphoid cells in thyroid tissue from patients with Graves' disease could be removed by this procedure. To obtain entirely single cells as target cells, the suspension of dispersed thyroid epithelial cells was allowed to stand for 1 h at 4 degree C in culture medium to allow cell clumps to settle. For assay of cytotoxic antibody, a mixture of the single target cells, patient's serum and human complement was incubated in microwells for 18 h. After removing the detached cells, remaining target cells in the wells were fixed, stained and counted to assess cytotoxicity. For assay of ADCC and of direct lymphocyte cytotoxicity, target thyroid cells were precultured in the microwells for 18 h. Then effector cells with or without patient's serum were added and cultured further for 24-72 h. Cytotoxicity was assessed as described above. Adherence of effector cells to target thyroid cells sometimes disturbed the enumeration of target cells when the effector/target cell ratio was high. With this microassay system 3 different cytotoxic immune reactions against human thyroid cells could be measured quantitatively at the same time on 2-3 ml blood samples.
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PMID:A microcytotoxicity assay for thyroid-specific cytotoxic antibody, antibody-dependent cell-mediated cytotoxicity and direct lymphocyte cytotoxicity using human thyroid cells. 689 61


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