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)

We have compared the prediction of distant recurrence for S-phase fraction (SPF) and DNA-ploidy, as estimated by flow cytometry, on an epithelial cell population and an unselected cell population from 268 node-negative breast-cancer patients diagnosed between 1985 and 1988. The tumor tissue was mechanically disintegrated and divided for flow cytometric analysis using both gated cells containing cytokeratin 8 and 18 and ungated cells treated with a detergent-trypsin solution. The relationship to distant recurrence was investigated for flow cytometric data, tumor size and estrogen and progesterone receptor content in univariate and multivariate Cox's regression analysis. The regression analyses were performed on 209 cases with S-phase fractions estimated by both methods. In 11 cases, DNA-ploidy classification differed, reflecting increased sensitivity to minor aneuploid peaks but a decreased ability to separate peaks in the near-diploid region for the gated populations. When SPF were used in univariate analysis as a continuous parameter or the upper tertile was used as cut-off value, SPF from the cytokeratin-gated cell population were most closely associated with recurrence and contributed additional prognostic information to SPF from the unselected cell population in the multivariate analysis. Out of the following variables:tumor size, ER and PR status, SPF and DNA ploidy, only SPF from immunoselected cells contributed prognostic information in multivariate analysis. These results indicate that SPF from immunoselected cell populations improves the prediction of recurrence in node-negative breast cancer.
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PMID:S-phase fraction after gating on epithelial cells predicts recurrence in node-negative breast cancer. 752 14

Immunocytochemical study in paraffin sections of human endometrium showed that the receptor contents for both oestrogen and progesterone receptors were lower than in the frozen sections although the staining patterns were similar in these two section types. Pretreating the specimens with proteolytic enzymes like trypsin, DNase and pronase improved the oestrogen receptor staining but a better result with progesterone receptor staining was obtained when no enzymatic pretreatment was applied to the sections.
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PMID:Oestrogen and progesterone receptors in normal human endometrium: comparison of immunocytochemical analysis on frozen and paraffin sections with or without enzymatic pretreatment. 763 70

This review summarizes some recent findings in human sperm which show that progesterone and 17 alpha hydroxyprogesterone are able rapidly (within seconds) to elevate [Ca2+]i and elicit the acrosome reaction (AR) via a non-genomic cell surface receptor. Progesterone promotes a transient elevation in [Ca2+]i which is blocked by extracellular La3+ and Ni2+ and removal of extracellular Ca2+ following chelation with EGTA. Some studies suggest that polyamines, trypsin-like proteases, and progesterone receptor aggregation are involved in progesterone-induced Ca2+ influx and AR. The receptor is not stimulated by the potent synthetic progestigins (e.g. promegestone, norethynodrel, megestrol acetate, cyproterone acetate) and is weakly antagonized by the genomic anti-progestins RU 486 and ZK 98.299. The sedative-hypnotic 3 alpha hydroxyl A-ring reduced pregnane steroids, which are powerful activators of the GABAA Cl- channel, are weak activators of Ca2+ influx and the AR. These data suggest that human sperm have a cell surface steroid receptor which is unlike the genomic progesterone receptor and the GABAA Cl- channel steroid receptor.
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PMID:Rapid non-genomic actions of progesterone stimulate Ca2+ influx and the acrosome reaction in human sperm. 831 30

Human spermatozoa have recently been introduced as a model for the study of rapid, nongenomic effects of steroids on the cell. Indirect evidence suggests the presence of a novel type of progesterone receptor on the cell surface; some cellular responses mediated by the receptor have been shown to be sensitive to protease inhibitors, but the molecular identity and the mode of function of this receptor are not known. Recent biochemical evidence showed that Ca2+ influx and a Ca(2+)-dependent exocytotic event (the acrosome reaction) can be induced in human sperm by antibody-mediated aggregation of the cell-surface progesterone receptor. These data suggested that progesterone receptor aggregation, occurring after ligand binding, may represent an early reaction in the signal transduction pathway. In this study we used cytological methods to examine ligand-induced changes in the distribution of the progesterone receptor in the sperm plasma membrane. We also examined the effects of trypsin and of trypsin inhibitors on the function of the receptor. Under the conditions of this study, neither trypsin nor trypsin inhibitors affected sperm viability, motility, or the acrosome reaction. However, the trypsin treatment completely abolished the ligand-binding activity of the sperm progesterone receptor. On the other hand, trypsin inhibitors did not influence the ligand binding despite their inhibitory effect on the ligand-induced exocytosis. The treatment with trypsin inhibitors was thus used to prevent the exocytotic reaction and so to preserve the plasma membrane for the study of ligand-induced receptor migration. The distribution of ligand-receptor complexes in the sperm acrosomal region remained homogeneous during incubation at 4 degrees C, but warming to 37 degrees C entailed a rapid formation of patches, followed by migration of the complexes towards the sperm equatorial region and ending by a virtually complete disappearance of the complexes from the anterior acrosomal region. It is concluded that aggregation is an early response of the sperm-surface progesterone receptor to ligand binding, that trypsin inhibitors block the function of the receptor downstream of the aggregation, and that some mechanism must exist in the plasma membrane to protect the ligand-binding site from digestion while permitting the protease action in the signal transduction mechanism.
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PMID:Insights into the function of a sperm-surface progesterone receptor: evidence of ligand-induced receptor aggregation and the implication of proteolysis. 845 85

The binding of natural or synthetic ligands to nuclear receptors is the triggering event leading to gene transcription activation or repression. Ligand binding to the ligand binding domain of these receptors induces conformational changes that are evidenced by an increased resistance of this domain to proteases. In vitro labeled receptors were incubated with various synthetic or natural agonists or antagonists and submitted to trypsin digestion. Proteolysis products were separated by SDS-PAGE and quantified. The amount of trypsin-resistant fragments was proportional to receptor occupancy by the ligand, and allowed the determination of dissociation constants (kDa). Using the wild-type or mutated human retinoic acid receptor alpha as a model, kDa values determined by classical competition binding assays using tritiated ligands are in agreement with those measured by the proteolytic assay. This method was successfully extended to human retinoic X receptor alpha, glucocorticoid receptor, and progesterone receptor, thus providing a basis for a new, faster assay to determine simultaneously the affinity and conformation of receptors when bound to a given ligand.
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PMID:Limited proteolysis for assaying ligand binding affinities of nuclear receptors. 963 26

Steroid-induced changes in receptor protein conformation constitute a logical means of translating the variations in steroid structures into the observed array of whole cell biological activities. One conformational change in the rat glucocorticoid receptor (GR) can be readily discerned by following the ability of trypsin digestion to afford a 16-kDa fragment. This fragment is seen after proteolysis of steroid-free receptors but disappears in digests of either glucocorticoid- or antiglucocorticoid-bound receptors. The location of this cleavage site has now been located unambiguously as R651, in helix 6 of the ligand binding domain, by a combination of point mutagenesis, arginine specific protease digestion, and radiochemical sequencing. This 16-kDa species, corresponding to amino acids 652-795, was non-covalently associated with another, approximately 17-kDa species that was determined to be amino acids 518-651 after a comparison of co-immunoprecipitated fragments from wild type and two chimeric receptors. These assignments revise our earlier report of amino acids 537-673 being the 16-kDa fragment and suggest that sequences of the entire ligand binding domain are required for high affinity and specificity binding. This was supported by the observation that trypsin digestion of the steroid-free R651A mutant GR gave rise to the 30-kDa meroreceptor (amino acids 518-795), which displayed wild type affinity. This 30-kDa species is thus the smallest non-associated fragment of GR possessing wild type steroid binding affinity. This suggests that other GR regions do not influence steroid binding affinity. The above results are reminiscent of those observed for the estrogen receptor. However, unlike the estrogen receptor or the more closely related progesterone receptor, the precise proteolytic cleavage points of both the steroid-free and -bound GR fall within regions that are predicted, on the basis of X-ray crystal structures of related receptors, to be alpha-helical and resistant to proteolysis. Thus, the tertiary structure of the GR ligand binding domain may be distinctly different from that of estrogen and progesterone receptors.
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PMID:Steroid-induced conformational changes of rat glucocorticoid receptor cause altered trypsin cleavage of the putative helix 6 in the ligand binding domain. 1058 Aug 42

Progesterone stimulates sperm functions, e.g. hyperactivation, acrosome reaction, binding to oocyte zona pellucida and penetration rate into the hamster oocyte. The physiological relevance of these effects has been shown using female genital tract fluids which modulate sperm function according to their progesterone content. Progesterone interacts with specific sperm binding sites that, unlike the classic nuclear receptors, are located on the plasma membrane of the spermatozoon. Binding studies have revealed the presence of two classes of progesterone receptors in the human spermatozoon, one class has an elevated affinity constant (nanomolar) and is specific for progesterone, whereas the other class has an affinity constant in the micromolar range and binds equally well other hydroxylated progesterone derivatives. Following exposure to progesterone, the main event is a rapid (within seconds) increase of the intracellular free calcium concentration, followed by a sustained rise lasting for several minutes (plateau phase). Both these calcium transients are dependent upon entry of extracellular calcium. The nature of the calcium channel that mediates the effects of progesterone is, currently, unknown. It has been postulated that it may be: (i) part of the progesterone receptor; (ii) voltage-dependent; or (iii) operated by second messengers following activation of the progesterone receptor. Progesterone also modulates sperm function by stimulating a trypsin-like proteolytic activity, the biosynthesis of polyamine (putrescine and spermidine), phospholipase A2 activity and protein tyrosine kinase activity in the sperm cell. Recent studies have shown that chloride ion efflux is vital for progesterone to promote the acrosome reaction. This effect is achieved by interaction with a sperm membrane receptor which resembles the neuronal GABA(A) receptor. Accordingly, GABA(A) receptors have been found in the spermatozoon plasma membrane and GABA stimulates hyperactivation and promotes the acrosome reaction.
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PMID:Effects of progesterone on sperm function: mechanisms of action. 1092 17

We have previously reported the identification of seven in vivo phosphorylation sites in the amino-terminal region of the human progesterone receptor (PR). From our previous in vivo studies, it was evident that several phosphopeptides remained unidentified. In particular, we wished to determine whether human PR contains a phosphorylation site in the hinge region, as do other steroid receptors including chicken PR, human androgen receptor, and mouse estrogen receptor. Previously, problematic trypsin cleavage sites hampered our ability to detect phosphorylation sites in large incomplete tryptic peptides. Using a combination of mass spectrometry and in vitro phosphorylation, we have identified six previously unidentified phosphorylation sites in human PR. Using nanoelectrospray ionization mass spectrometry, we have identified two new in vivo phosphorylation sites, Ser(20) and Ser(676), in baculovirus-expressed human PR. Ser(676) is analogous to the hinge site identified in other steroid receptors. Additionally, precursor ion scans identified another phosphopeptide that contains Ser(130)-Pro(131), a likely candidate for phosphorylation. In vitro phosphorylation of PR with Cdk2 has revealed five additional in vitro Cdk2 phosphorylation sites: Ser(25), Ser(213), Thr(430), Ser(554), and Ser(676). At least two of these, Ser(213) and Ser(676), are authentic in vivo sites. We confirmed the presence of the Cdk2-phosphorylated peptide containing Ser(213) in PR from in vivo labeled T47D cells, indicating that this is an in vivo site. Our combined studies indicate that most, if not all, of the Ser-Pro motifs in human PR are sites for phosphorylation. Taken together, these data indicate that the phosphorylation of PR is highly complex, with at least 14 phosphorylation sites.
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PMID:Identification of a phosphorylation site in the hinge region of the human progesterone receptor and additional amino-terminal phosphorylation sites. 1111 Aug 1

One of the potential therapeutic interventions to hormone-independent breast cancer would be to reactivate the expression of estrogen receptor or progesterone receptor (PR) in the tumor cells so as to render the tumor responsive to the hormones. We have reported previously that progesterone markedly inhibited cell growth and induced remarkable focal adhesions in PR-transfected MDA-MB-231 cells. The aim of this study was to determine the effects of progesterone on the invasive properties and in vivo tumor growth of PR-transfected MDA-MB-231 cells. It was found that progesterone has increased cell resistance to trypsin digestion and increased cell attachment to extracellular matrix proteins, especially laminin and fibronectin. In vitro invasion assays using modified Boyden chambers showed that progesterone increased cell migration through matrix protein-coated membranes. However, Northern blotting analysis demonstrated that progesterone strongly down-regulated (up to 60-fold) the gene expression of urokinase plasminogen activator and increased (up to 5-fold) the expression of tissue-type plasminogen activator in these cells. This pattern of gene regulation suggested an inhibition of cell invasiveness because numerous clinical studies have indicated that low levels of urokinase plasminogen activator and high levels of tissue-type plasminogen activator in breast cancer are associated with favorable prognosis. Furthermore, animal studies showed that progesterone strongly inhibited the tumor formation and growth in Scid mice. After 12 weeks of inoculation, the median weight of tumors in the progesterone-treated group was 25 mg compared with 203 mg in the placebo group (P < 0.001). These results suggest that progesterone may provide effective treatment for estrogen receptor- and PR-negative breast cancer if the PR expression were reactivated. Alternatively, activation of progesterone-mediated molecular pathways in hormone-independent breast cancer may achieve similar therapeutic effects.
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PMID:Effect of progesterone on the invasive properties and tumor growth of progesterone receptor-transfected breast cancer cells MDA-MB-231. 1155 6

We report a woman with ascites, hydrothorax, pancreatic tumor, left cystic ovarian tumor, and an elevated serum cancer antigen 125 level. Exploratory laparotomy was performed to determine peritoneal disseminated carcinoma of unknown origin. Immunohistochemical analysis demonstrated positive staining for carcinoembryonic antigen, trypsin, and progesterone receptor and nonspecific or negative reaction for calretinin, estrogen receptor, amylase, lipase, Wilms tumor gene 1 protein, and inhibin or chromogranin A. These results together with the morphology of tubular structure suggested the pathological diagnosis of adenocarcinoma with pancreatic characteristics and contradicted ovarian cancer or mesothelioma. Immunohistochemistry is an adjunct tool to differentiate the primary site of carcinomatous peritonitis.
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PMID:Immunohistochemistry for the differentiation of peritoneal disseminated carcinoma of unknown origin. 1520 56


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