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)

Different procedures of enzymatic digestion of rat prostatic tissue and unique sets of mitogenic factors made it possible to culture practically pure populations of epithelial and stromal cells without previous separation of the two types of cells. Keratin-positive epithelial cells dissociated by trypsin and collagenase from adult rat ventral prostate proliferated in medium WAJC 404 supplemented with epidermal growth factor, insulin, cholera toxin, and bovine pituitary extract. Proliferation of epithelial cells was completely inhibited by dexamethasone as low as 30 nM. On the other hand, fibroblast-like stromal cells released by trypsin digestion required a plastic substratum coated with calf serum or fibronectin, and proliferated in Eagle's minimum essential medium supplemented with cholera toxin, bovine pituitary extract, dexamethasone, and bovine serum albumin. Epidermal growth factor and insulin had negligible effect on proliferation of stromal cells. Physiological concentrations of dihydrotestosterone and estradiol showed no effect on proliferation of both types of cells.
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PMID:Differences in growth requirements between epithelial and stromal cells derived from rat ventral prostate in serum-free primary culture. 223 29

Epidermal growth factor (EGF) induces rapid rounding of A-431 human epidermoid carcinoma cells in Ca(++)-free medium. Cell rounding is not induced by a variety of other polypeptide hormones, antiserum to cell membranes, local anesthetics, colchicine, cytochalasin B, or cyclic nucleotides. However, trypsin, like EGF, induces rounding of A- 431 cells in the absence of Ca(++). Both trypsin- and EGF-induced rounding are temperature dependent, appear to be energy dependent, and are inhibited by cytochalasins, suggesting that the active participation of microfilaments in cell rounding. However, a medium transfer experiment suggests that EGF-induced rounding is not attributable to secretion of a protease, and a number of serine protease inhibitors have no effect on the EGF-induced rounding process. Cell rounding is not attributable to the slight stimulation by EGF of the release of Ca(++) that is observed in the Ca(++)-free medium, as stimulation of such release by the ionophore A23187 neither induces cell rounding nor blocks EGF-induced rounding. Cells that have rounded up after treatment with EGF or trypsin spread out upon addition of Ca(++) to the medium, even in the continuing presence of EGF or typsin. Like the cell-rounding process, the cell-spreading process is temperature dependent, appears to be energy dependent, and is inhibited by cytochalasin B. Thus, EGF does not destroy the ability of the cell to spread; rather, in the presence of the EGF (or trypsin), cell spreading and the maintenance of the flattened state become dependent on external Ca(++). Because untreated cells remain flattened in the absence of Ca(++), the data suggest that EGF may disrupt Ca(++)-independent mechanisms of adhesion normally present in A-431 cells.
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PMID:Rapid rounding of human epidermoid carcinoma cells A-431 induced by epidermal growth factor. 625 80

Epidermal growth factor (EGF) initiates a wide variety of events when added to responsive cultured cells. These range from early events requiring only brief exposure to EGF, e.g., stimulation of transport of amino acids or ions, to later events such as commitment of cells to a round of DNA synthesis, a process requiring 6 h or more of continuous exposure to hormone. EGF binding is followed first by phosphorylation of EGF receptors, which can be detected in purified membranes and permeabilized cells, and then by internalization and proteolytic processing of receptors in lysosomes. Native 160,000-dalton EGF receptors contain a site that is not exposed on the cell surface and is highly sensitive to cleavage by an endogenous protease, which yields a 145,000-dalton receptor fragment that retains phosphate acceptor activity. Cleavage of receptor at a trypsin-sensitive site, also not exposed to the cell surface, yields a 115,000-dalton fragment that binds EGF, but contains no phosphorylated species. The data indicate that the phosphate acceptor sites on EGF receptors are localized on a 45,000-dalton cytosolic region.
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PMID:Receptor remodeling and regulation in the action of epidermal growth factor. 629 2

Epidermal growth factor (EGF)-dependent transfer of radiolabeled phosphate from [gamma-32P]ATP to 160-kDa EGF receptor solubilized from human epidermoid carcinoma A431 cell surface membranes was stimulated up to 3-fold by addition of 3',5'-cAMP and purified cAMP-dependent protein kinase. Phosphorylation of EGF receptors was stimulated to the same extent when cAMP-dependent protein kinase catalytic subunit was substituted for 3',5'-cAMP and cAMP-dependent protein kinase. Phosphoamino acid analysis revealed that the extent of phosphorylation of EGF receptor at tyrosine residues was the same regardless of whether cAMP-dependent protein kinase catalytic subunit was present in or omitted from the system. Increased EGF receptor phosphorylation occurring in response to cAMP-dependent protein kinase catalytic subunit was accounted for by phosphorylation at serine or threonine residues. In samples phosphorylated in the presence of cAMP-dependent protein kinase catalytic subunit, phosphate was present in tyrosine, serine, and threonine in a ratio of 32:60:8. Two-dimensional mapping of radiolabeled phosphopeptides produced from EGF receptors by digestion with trypsin revealed the generation of one additional major phosphoserine-containing peptide when cAMP-dependent protein kinase was present with EGF in the EGF receptor kinase system. Degradation of 160-kDa EGF receptors to a 145-kDa form by purified Ca2+-activated neutral protease produced a 145-kDa fragment with phosphoserine content increased over that present initially in the 160-kDa precursor.
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PMID:cAMP-dependent protein kinase stimulates epidermal growth factor-dependent phosphorylation of epidermal growth factor receptors. 632 45

Epidermal growth factor (EGF) undergoes a specific series of alterations during the course of its binding and internalization into cultured fibroblasts. The modified EGF species can be distinguished from each other and from native EGF by their isoelectric points. We employed peptide mapping techniques to determine the nature of these alterations. We found that 125I-EGF with a pI of 4.55 was converted to a pI 4.2 species by removal of 1 or 2 amino acid moieties from the COOH-terminal end of the protein. A pI 4.35 species was generated by a trypsin-like cut between amino acid residues 48 and 49, for a total of 5 amino acid moieties removed from the native EGF. The pI 4.0 species was formed by removal of at least the COOH-terminal arginine from the pI 4.35 species. Thus, upon binding and internalization, EGF was sequentially cleaved in the COOH-terminal region. Removal of the COOH-terminal polypeptide has been shown to dramatically reduce the affinity of EGF for its receptor, raising the possibility that intracellular dissociation of EGF from its receptor may be a direct result of the intracellular processing of EGF.
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PMID:Intracellular processing of epidermal growth factor. II. Intracellular cleavage of the COOH-terminal region of 125I-epidermal growth factor. 660 23

The role of choleragen (CT) and epidermal growth factor (EGF) has been examined in relation to the control of growth and differentiation of adult human cervical epithelial (HCE) cells derived from the ectocervix. Cervical biopsies derived from hysterectomy specimens were trypsin disaggregated and HCE cells were plated at 5 X 10(3)/cm2 in the presence of 2 X 10(4)/cm2 lethally irradiated Swiss 3T3 fibroblasts. Cultures were grown in Liebovitz medium supplemented with 10% fetal bovine serum and hydrocortisone. Epidermal growth factor at 10 ng/ml and choleragen at 10(-10) M were added to cultures either singly or in combination. DNA replication in these cultures was measured autoradiographically after exposing cells to tritiated thymidine for 2 h. Differentiation was assessed histochemically by determining glycogen accumulation using the periodic acid Schiff technique. Choleragen increased colony plating efficiency by at least a factor of two but had no effect on colony size. Epidermal growth factor did not increase plating efficiency but did increase colony size. In EGF treated colonies DNA replication occurred throughout the colony compared to CT treated colonies in which replication was restricted to the periphery. In the absence of EGF, population doublings achieved in culture did not exceed 32 and glycogen accumulation was evident in cells early in culture life. Colonies treated with EGF exhibited glycogen accumulation late in culture life and the EGF treated cells achieved at least 50 population doublings in culture. The results are discussed in relation to the role of EGF and choleragen on cell differentiation.
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PMID:The effect of choleragen and epidermal growth factor on proliferation and maturation in vitro of human ectocervical cells. 660 84

Using separation of total cellular proteins by two dimensional (2-D) gel electrophoresis (isoelectric focusing/SDS-PAGE) we have characterized two regulated proteins, p21 and p19, in dog thyroid cells. We have used the same 2-D gel technique to purify these proteins before their trypsin cleavage and partial sequencing. Three peptides were sequenced in the case of p19 and two peptides in the case of p21. The Swiss-Prot protein sequence database revealed that p19 was identical to destrin/ADF (actin depolymerizing factor) and p21 to cofilin, two closely related and widely distributed actin-binding proteins. This was further verified by cross-reactivity with specific antibodies against brain cofilin and chicken ADF. We have demonstrated, using 2-D gel electrophoresis with a nonequilibrium pH gradient in the first dimension (nonequilibrium pH gradient in the first dimension (nonequilibrium pH gradient electrophoresis/SDS-PAGE) that, in the thyroid cell, cofilin and destrin/ADF were present, under control conditions, in two forms: a phosphorylated and an unphosphorylated one. Thyrotropin (TSH), through cyclic AMP, provoked a very rapid dephosphorylation of these two proteins, which was already maximal after 20 min of action, whereas their dephosphorylation in response to 12-O-tetradecanoylphorbol-13-acetate (TPA) was slower. This suggests that dephosphorylation of cofilin and destrin/ADF by TSH could be implicated in the disruption of actin-containing stress fibers and in the reorganization of microfilaments induced by this hormone. Epidermal growth factor, which does not induce acute morphological changes in thyroid cells, did not affect the state of phosphorylation of cofilin and destrin/ADF except for a delayed decrease (after 24 h) of destrin/ADF phosphorylation. A 10% dimethyl sulfoxide treatment of thyroid cells also induced rapid dephosphorylation of destrin and cofilin. This was accompanied by a reorganization of actin microfilaments that clearly resembles the one induced by TSH and by the appearance of intranuclear cofilin-containing rods. However, these rod structures were not observed in response to TSH, forskolin, or TPA, suggesting that dephosphorylation of cofilin correlates with the reorganization of actin microfilaments but not with the nuclear transport of cofilin. We propose that the dephosphorylation of destrin and cofilin could be involved in the TSH-stimulated macropinocytic activity, a key process in thyroid hormone secretion.
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PMID:Characterization and identification as cofilin and destrin of two thyrotropin- and phorbol ester-regulated phosphoproteins in thyroid cells. 817 42

Bacterial translocation (BT) from the gastrointestinal tract to mesenteric lymph nodes (MLN) and other extraintestinal organs is an important source of infection in acute pancreatitis (AP). Epidermal growth factor (EGF), a peptide hormone with trophic effects on gut mucosa, has decreased intestinal mucosal injury in septic rats and decreased burn-induced BT in mice. The purpose of this study is to examine whether EGF could affect BT in acute necrotizing pancreatitis. Forty-eight male Sprague-Dawley rats (250-350 g) were studied. AP was induced in Group I and Group II by pressure injection of 3% taurocholate and trypsin into the biliopancreatic duct (1 ml/kg of body weight). Group III and Group IV underwent laparotomy without induction of acute pancreatitis. Group I rats received human recombinant EGF (100 micrograms/kg, subcutaneously twice daily) and Group II rats received a similar volume of 0.1% bovine serum albumin as a placebo postoperatively. Group III and Group IV received EGF and placebo, respectively. At 48 hr postoperatively, blood was drawn for culture and amylase determinations. Jejunum and ileum were obtained to measure mucosal protein content, mucosal thickness, villus height, and crypt depth. Specimens from MLN, spleen, liver, pancreas, and cecum were harvested for pathology and culture of gram positive (G+), gram negative (G-), and anaerobic bacteria. Ileal mucosal protein levels were increased significantly in Group I (1.96 +/- 0.14 mg/cm) compared to Group II (0.95 +/- 0.15 mg/cm intestinal segment) (P < 0.01). Jejunal and ileal mucosal thickness, villus height, and crypt depth in Group I were significantly increased when compared to Group II (P < 0.05). All 12 rats in Group II had BT to MLN compared to 58% (7 of 12 rats) in Group I (P < 0.05). Thirty-three percent (4 of 12 rats) had BT to distant sites such as pancreas, spleen, liver, and/or blood in Group I vs 83% (10 of 12 rats) in Group II (P < 0.05). EGF treatment minimizes intestinal damage, decreases BT to MLN and bacterial spread to distant sites, and may be beneficial in preventing septic complications in AP.
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PMID:The effect of epidermal growth factor on the septic complications of acute pancreatitis. 920 65

The M-1 cell line, derived from the mouse cortical collecting duct (CCD), is being used as a mammalian model of the CCD to study Na+ transport. The present studies aimed to further define the role of various hormones in affecting Na+ transport in M-1 cells grown in defined media. M-1 cells on permeable support, in serum-free media, developed amiloride-sensitive current 4-5 days after seeding. As expected for the involvement of epithelial Na+ channels, alpha-, beta-, and gamma-subunits of the epithelial Na+ channel were identified by RT-PCR. Either dexamethasone (Dex, 10-100 nM) or aldosterone (Aldo, 10(-6)-10(-7) M) for 24 h stimulated transport. Cells grown in the presence of Aldo and Dex had higher transport than with Dex alone. Spironolactone added to Dex media decreased transport. The acute effects of hormones reported to inhibit Na+ transport in CCD were also examined. Epidermal growth factor, phorbol esters, and increased intracellular Ca2+ with thapsigargin did not alter transport. Arginine vasopressin caused a transient increase in transport (probably Cl- secretion), which was not amiloride sensitive. Also, the protease inhibitor aprotinin decreased Na+ transport; in aprotinin-treated cells, trypsin stimulated transport. This study demonstrates that adrenal steroids (Dex > Aldo) stimulate Na+ transport in M-1 cells. At least part of this response may represent activation of mineralocorticoid receptors based on an additive effect of Dex and Aldo, as well as inhibition by spironolactone. Responses to immediate-acting hormones is limited. However, an endogenous protease activity, which activates Na+ transport, is present in these cells.
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PMID:Regulation of sodium transport in M-1 cells. 984 18

Oral cancer which comprises about 40% of total cancers in India, has one of the lowest relative survival rates of all cancers. Epidermal growth factor (EGF) has been known to play a role in the proliferation/malignant transformation of oral neoplasms. Since, the somatostatin analog RC-160 is reported to be a potent inhibitor of EGF stimulated cell proliferation, its anti-proliferative activity in the human oral carcinoma cell line KB was investigated, in this study. RC-160 was found to potently inhibit EGF-induced proliferation in KB cells in vitro, suggesting a therapeutic potential of the same in oral carcinoma. However, the therapeutic potential of RC-160 is limited by its short serum half life. To overcome this limitation, fatty acids namely butanoic acid and myristic acid individually were coupled to RC-160. The lipophilized derivatives of RC-160 were synthesized, purified and characterized. The anti-proliferative activity of lipophilized derivatives of RC-160 on KB cells was evaluated in vitro. Myristoyl-RC-160 (0.75 nM) inhibited the growth of KB cells at a 10-fold lower concentration relative to RC-160 (8.8 nM) and at a 100-fold lower concentration relative to butanoyl-RC-160 (0.83 microM) (p<0.001). The affinity of RC-160 towards somatostatin receptors remains unaltered by lipophilization. The signaling pathways underlying the antineoplastic activity of these lipopeptides are similar to RC-160, and do not involve the stimulation of a protein tyrosine phosphatase or a serine threonine phosphatase 1A and 2A. The anti-proliferative activity of the lipopeptides was found to be mediated by somatostatin receptors and correlates with the inhibition of protein tyrosine kinase activity and decrease in intracellular cAMP levels. Myristoyl-RC-160 displayed significantly greater resistance towards trypsin and serum degradation than RC-160 (p<0.01). These findings demonstrate that RC-160 can inhibit the growth of oral cancer cells in vitro. Lipophilization of RC-160 with long chain fatty acids like myristic acid improves its stability and anti-proliferative activity, in human oral carcinoma cells in vitro, thereby enhancing the scope of improving its therapeutic index.
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PMID:Lipophilization of somatostatin analog RC-160 with long chain fatty acid improves its anti-proliferative activity on human oral carcinoma cells in vitro. 1126 86


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