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)

A convenient procedure has been developed for preparing a suspension of isolated rat anterior pituitary cells which retains responsiveness to secretagogues. Rat anterior pituitaries were dispersed with collagenase and hyaluronidase followed by mechanical dispersion by means of a Pasteur pipette. Immediately after dispersion, the cells showed only slight responses to secretagogues, whereas after short-term culture (20-22 h) in the presence of sera, the cells recovered their ability to respond to synthetic LH-releasing hormone (LHRH) and synthetic thyrotropin-releasing hormone (TRH). During a 3-h incubation, cells prepared from pituitaries of male rats released LH and FSH, or TSH and prolactin (PRL) in amounts directly related to the dose of synthetic LHRH or TRH, respectively. The minimum effective concentrations of hypophysiotropic hormones lay between 10(-10) and 10(-9)M, although it was observed that cells originating from female rats usually gave quicker and larger responses to LHRH. No significant net increase in the total hormonal content (cells + medium) of radioimmunoassayable LH or FSH in response to LHRH, or of TSH or PRL in response to TRH, was observed during the 3-h incubation period. The cells released significant amounts of PRL, TSH, and to a lesser extent, LH, in response to 1-5 X 10-3M N6,O2'-dibutyryl cyclic AMP, accompanied by remarkable elevation in total content (cells + medium) of PRL and TSH but not of LH. The response of the cells to theophylline or high [K+] was similar to that usually observed in previous hemipituitary experiments. These results demonstrate the viability of this in vitro cell system and its suitability for further study of the regulation of the secretion of pituitary hormones.
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PMID:Enzymatic dissociation and short-term culture of isolated rat anterior pituitary cells for studies on the control of hormone secretion. 17 97

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

Isolated sheep thyroid follicles release specific insulin-like growth factor-binding proteins (IGFBPs). Since IGFBPs can modulate IGF bioactivity, at least in vitro, their presence in thyroid tissue may influence synergistic interactions between TSH and endogenous IGF-I or -II which are known to control both thyroid growth and function. We have examined the hormonal control of IGFBP release in relation to iodine organification. Sheep thyroid follicles were isolated by incubation with collagenase and differential centrifugation, grown in Coon's modified Ham's F12M medium with the addition of transferrin, glycylhistidyl-lysine, somatostatin (3H), TSH, cortisol and insulin (6H), and maintained in OH (hormone-free) or 3H medium with or without further supplements for 48 h. Conditioned culture medium was separated by 8% sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis, transferred to nitrocellulose and incubated with 125I-labelled IGF-II followed by autoradiography (ligand blot). Additionally, the radioactive bands were cut from the filters and quantified by gamma-spectrometry. Iodine organification was assessed by incubation of follicles with 10(6) c.p.m. Na125I for 3 h before washing, solubilization in 0.1 mol NaOH/l and the precipitation of organified radioisotope with 10% (v/v) trichloroacetic acid. Cells conditioned in OH or 3H medium released specific IGFBPs of 46, 34, 28 and 19 kDa on ligand blot analysis. The proteins of 34 and 19 kDa were immunopositive on Western blot analysis using anti-bovine IGFBP-2 antiserum. The 46-kDa IGFBP was retained by Concanavalin A-Sepharose chromatography and demonstrated to be glycoprotein. This is probably ovine IGFBP-3. The addition of TSH, or TSH plus cortisol to OH or 3H medium significantly decreased the 125I-labelled IGF-II associated with the 34- and 28-kDa IGFBP species. All IGFBP species were substantially reduced in 6H medium, which was predominantly due to the effects of TSH and cortisol. When total 125I-labelled IGF-II associated with IGFBPs was considered, a significant (P less than 0.01) inverse correlation existed between IGFBP activity and iodine organification in the same cultures; the latter being greatest in OH or 3H medium supplemented with TSH and cortisol. None of these hormone additions altered the endogenous release of IGF-II by the cells. These results suggest that endogenous IGFs, under hormonal control, may modulate the action of endogenous IGF in the regulation of thyroid function.
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PMID:Hormonal regulation of insulin-like growth factor (IGF)-binding proteins secreted by isolated sheep thyroid epithelial cells: relationship with iodine organification. 171 78

Interactions of receptor-bound bovine thyrotropin (bTSH) and immunoglobulins G from sera of patients with Graves' (G-IgG) or Hashimoto's (H-IgG) disease with porcine thyrocytes were studied by immunocytochemistry. Porcine thyroid fragments were fixed and prepared for immunoreaction or enzymatically dissociated with collagenase and dispase II. The dispersed cells were cultured in primary monolayer in a hormone-free medium or in a medium with bTSH (150 micrograms/ml) for 7 days. After immunostaining the thyrocytes in fragments and monolayers were stained with periodic acid Schiff (PAS) or with PAS and haemalum. Cultivation of the isolated thyrocytes in bTSH-enriched medium leads to a monolayer with globular aggregates, i.e., reconstructed three-dimensional follicles. Follicular cells in these monolayers and in fragments give a weak to moderate immunoreaction to anti-bTSH and a strong reaction to G-IgG and H-IgG (vs. control IgG). Precipitate is found particularly in the perinuclear area and to a lesser degree throughout the cytoplasm. Cells cultured in the absence of bTSH show minimal immunoreaction to anti-bTSH, but moderate reaction to G-IgG and H-IgG. Preincubation with bTSH leads to a strong reduction of immunoreaction to G-IgG but does not affect reaction to H-IgG. Morphological results indicate that G-IgG and H-IgG interact with the same cellular sites as bTSH. Hashimoto's disease antibodies bind to a determinant on the TSH receptor separate from the one on which TSH and Graves' IgG bind.
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PMID:Immunocytochemical localization of bovine thyrotropin and thyroid auto-antibodies in porcine thyrocytes. 197 9

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

Rat thyroid follicles were isolated by collagenase digestion and cultured in suspension on agarose for 1-12 days with 0-0.1-1 mU/ml thyrotropin (TSH). After a 4 h exposure to Na125I they were processed for light and electron microscopy, autoradiography and biochemical analysis. Follicular 125I accumulation (A) and organification (PBI) were measured. Thyroglobulin (Tg) content of follicles and 125I-labelled amino acids in Tg were analyzed by high-performance liquid chromatography (HPLC). Without TSH, follicular lumina and cell polarity persisted. From day 3, the rough endoplasmic reticulum (RER) and ribosomes disappeared while autophagic vacuoles appeared: 125I accumulation and PBI were significantly reduced. From day 6, ultrastructural cell dedifferentiation occurred. At day 12, autoradiographic labelling was found over very few lumina; half of the 125I accumulated was still organified. With 1 mU TSH, follicles formed aggregates with narrow densely labelled lumina lined by tall cells. The RER was well developed up to day 12. 125I accumulation, PBI and iodothyronine (T3, T4) formation in Tg remained significantly higher than in follicles cultured without TSH, showing a transient decrease at days 6 and 9. Monoiodotyrosine/diiodotyrosine (MIT/DIT) and T3/T4 ratios in Tg were not modified, suggesting the persistence in the follicles of a significant iodine pool available for iodination. With 0.1 mU TSH, alterations of cell morphology and reduction of functional properties occurred later than without TSH. In the presence of TSH, morphological signs of new follicle formation were seen. These data demonstrate that closed follicles keep their follicular structure up to 12 days of culture, even without TSH. However, TSH is necessary to maintain iodine accumulation and organification.
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PMID:Correlated morphological and functional study of isolated rat thyroid follicles in suspension culture. 237 85

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

Previous studies of human thyroid cells in culture (mostly from pathological tissues) failed to demonstrate a mitogenic effect of TSH, leading to the proposal that the growth effect of TSH in vivo might be indirect. To reexamine the influence of TSH on DNA synthesis and cell proliferation, we established primary cultures of normal thyroid tissue from nine subjects. When seeded in a 1% serum-supplemented medium, thyroid follicles released by collagenase/dispase digestion developed as a cell monolayer that responded to TSH by rounding up and by cytoplasmic retraction. When seeded in serum-free medium, the cells remained associated in dense aggregates surrounded by few slowly spreading cells. In the latter condition, the cells responded to TSH and other stimulators of cAMP production, such as cholera toxin and forskolin, by displaying very high iodide-trapping levels. Exposure to serum irreversibly abolished this differentiated function. TSH stimulated the proliferation (as shown by DNA content per culture dish) of 1% serum cultured cells (doubling times were reduced from 106 to 76 h) and increased by 100% the [3H]thymidine labeling indices. In serum-free cultured cells (dense aggregates or cell monolayers after initial seeding with serum), control levels of DNA synthesis were lower, and up to 8-fold stimulation of DNA synthesis occurred in response to 100 mU/L TSH (stimulation was consistently detected with 20 mU/L), based on measurements of [3H]thymidine incorporation into acid-precipitable material and counts of labeled nuclei on autoradiographs (up to 40% labeled nuclei within 24 h). The mitogenic effect of TSH required a high insulin concentration (8.3 X 10(-7) mol/L) or a low insulin-like growth factor I concentration. The mitogenic effects of TSH were mimicked in part by cholera toxin, forskolin, and dibutyryl cAMP. Epidermal growth factor and phorbol myristate ester also stimulated thyroid cell proliferation and DNA synthesis, but they potently inhibited TSH-stimulated iodide transport. We conclude that TSH, acting at least in part through cAMP, is a potent growth factor for human thyroid cells and thus provide an experimental basis in vitro for the well established in vivo goitrogenic action of TSH.
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PMID:Mitogenic effects of thyrotropin and adenosine 3',5'-monophosphate in differentiated normal human thyroid cells in vitro. 283 70

We have used a retroviral vector carrying the adenovirus E1A oncogene and the neomycin phosphotransferase gene to establish a human thyroid-derived cell line that exhibits TSH-mediated cAMP generation as well as the differential expression of HLA class II antigens in response to recombinant gamma-interferon. Twenty-two-week gestation, histologically confirmed, human fetal thyroid was collagenase digested, cultured as a monolayer, and infected directly with 12S or 13S E1A-containing retrovirus constructs. Infected clones (n = 30) were selected in a hormone-supplemented medium containing bovine TSH (bTSH; 1 mU/ml), 10% fetal bovine serum, and 0.5 mg/ml G418 antibiotic. A rapidly growing clone (designated 12S) was chosen for detailed analysis over 18 months of continuous culture. The 12S clone was sensitive to less than 10 microU/ml bTSH when assessed by extracellular accumulation of cAMP, but TSH had no influence on 72-h incorporation of [3H]thymidine. Clone 12S responded to recombinant human gamma-interferon (1-10(4) U/ml) by induction of HLA DR alpha-chain-specific mRNA and the surface expression of HLA-DR antigen detected by fluorescein isothiocyanate-labeled monoclonal antibody to nonpolymorphic HLA-DR regions using flow cytometry. These studies indicate the potential for immortalizing human thyroid cells for use as targets of anti-TSH receptor immune responses and for long term studies of human throcyte HLA gene regulation.
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PMID:HLA-DR gene expression in a proliferating human thyroid cell clone (12S). 284 57

It is currently believed that the thyroid stimulating immunoglobulin (TSI) of Graves' disease is involved in the pathogenesis of hyperthyroidism through the stimulation of the adenylate cyclase-cyclic AMP system. To evaluate this mechanism, TSI in the serum of patients with Graves' disease was determined by its ability to generate cyclic AMP (cAMP) in monolayer cells prepared from a normal thyroid gland. The thyroid tissue was digested with collagenase, and the liberated follicles were collected from the supernatant and cultured for 7 days. One gram of thyroid tissue yielded more than 1 X 10(7) monolayer cells which were stored in aliquots at -80C. Cells (1 approximately 2 X 10(4)/0.28 cm2 microtiter well) were incubated for 4 hours in 0.2 ml Hanks solution poor in NaCl, with various amounts of bovine TSH (bTSH) or 1.5 mg/ml Graves' serum IgG extracted by polyethylene glycol. cAMP accumulated in medium and cells was measured by RIA. Total cAMP (both medium and cells) was about 4 times higher when NaCl was deleted from Hanks solution. Moreover, as more than 90% of the cAMP was released into the medium, it was possible to omit the measurement of cellular cAMP, which requires extraction. The increase in medium cAMP concentration was dependent upon the number of cells, incubation time, and dose of bTSH. Time course and dose response curves in medium cAMP stimulated by IgG from 3 Graves' patients paralleled those of bTSH equivalent units. Accordingly, TSI activity could be expressed in bTSH equivalent units (bTSH microUeq). The assay could detect 1.0 or 3.3 microU/ml of bTSH and was highly reproducible. TSI activity in all of 16 IgGs from normal subjects was under 3.3 bTSH microUeq/ml, while it was greater than 3.3 bTSH microUeq/ml in IgGs from 33 of 37 (89%) untreated patients with Graves disease. Of the 13 patients followed for 2 to 7 months while on antithyroid drugs, 12 had greater than 3.3 bTSH microUeq/ml and, with the exception of one, all showed a decrease in their TSI activity. Moreover, 5 of 12 patients treated continuously for more than 1 year were TSI negative (less than 3.3 bTSH microUeq/ml), and except for one case, all had TSI values below 8 bTSH microUeq/ml (a value found in only 25% of untreated patients). This in vitro bioassay for TSI is simple and sensitive. It detects the presence of TSI in virtually 90% of untreated patients with Graves' disease. TSI activity showed a clear decrease during the course of antithyroid drug therapy.
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PMID:[Thyroid stimulating immunoglobulin bioassay using cultured normal human thyroid cells]. 286 82


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