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To understand the hormonal regulation of plasminogen activators (PAs) in human breast cancer, we have examined the hormonal regulation and properties of PAs in four human breast cancer cell lines that differ markedly in their estrogen receptor (ER) content: MCF-7 cells contain high levels of ER (approx 7 pmol/mg DNA) and their PA activity was increased 3-4-fold by physiological concentrations of estradiol; T47-D and ZR-75-1 cells contain lower levels of ER (0.9 and 2.1 pmol/mg DNA respectively) and their PA activity was also increased 3-4-fold by estradiol. In contrast, MDA-MB-231 cells, which do not contain ER, showed a high level of PA activity that was not modulated by estradiol. SDS-PAGE followed by zymography indicated that MCF-7 cells secreted tissue-type PA (t-PA), T47-D and ZR-75-1 cells secreted urokinase-type PA (u-PA), and MDA-MB-231 cells secreted both types of PAs. The types of PAs secreted by these cell lines did not change upon treatment with estradiol. Dose-response curves for the stimulation of MCF-7 PA activity by different estrogens showed an excellent correlation between affinities of the estrogens for ER and their potency in stimulating PA activity. With a clonal subline of MCF-7 cells, MCF-L, a soluble inhibitor of both t-PA and u-PA was secreted. Incubation of purified t-PA or u-PA with the serum-free conditioned medium from MCF-L cells resulted in a shift in the mobility of t-PA and u-PA in SDS-polyacrylamide gels to forms increased in molecular mass by about 50,000-70,000. The shifts in molecular mass could be prevented by the presence of the competitive inhibitor p-aminobenzamidine, indicating that the active sites of the PAs were involved in the formation of these complexes. Furthermore, co-cultivation, of RT4-D rat neuroblastoma cells, which exhibit high levels of t-PA activity, with MCF-L cells resulted in a marked decrease in the PA activity of the RT4-D cells. Our results were consistent with the following conclusions: t-PA, u-PA or both were secreted by human breast cancer cells. In the ER-containing cell lines, depending upon the specific cell line, t-PA or u-PA was stimulated by estrogens. The unstimulated levels of PA activity and the magnitude of PA stimulation by estrogens were not closely related to ER content.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Plasminogen activators in human breast cancer cell lines: hormonal regulation and properties. 338 80

Human thrombospondin, a 450-kDa glycoprotein isolated from platelets and endothelial cells, specifically interacts with osteonectin, a protein of 30 kDa isolated from bovine bones and human platelets. Using ELISA, purified osteonectin binds to solid-phase-adsorbed thrombospondin with a dissociation constant (Kd) of 0.7 nM. Binding of thrombospondin to solid-phase-adsorbed osteonectin was also observed (Kd = 0.86 nM). The interaction of thrombospondin with solid-phase-adsorbed osteonectin was significantly decreased (81% inhibition) when using an excess of fluid-phase osteonectin. Thrombospondin-osteonectin complex formation was calcium-dependent as shown by a 50-80% inhibition in the presence of EDTA. None of the proteins known to interact with thrombospondin (fibrinogen, fibronectin, collagen, plasminogen) had a significant inhibitory effect on thrombospondin-osteonectin complex formation. This selective interaction was confirmed by affinity chromatography. Iodinated osteonectin, previously incubated with purified thrombospondin, specifically bound to an anti-thrombospondin monoclonal antibody (P10) linked to protein-A--Sepharose 4B. Elution of the anti-thrombospondin antibody from protein A allowed the recovery of the thrombospondin-osteonectin complex in the eluate, as judged by SDS/polyacrylamide gel electrophoresis and autoradiography. Blotting of purified thrombospondin to osteonectin adsorbed onto nitrocellulose further confirmed complex formation. In addition, when released from thrombin-stimulated platelets, thrombospondin and osteonectin bound to anti-thrombospondin IgG-coated plates indicating that osteonectin was complexed to thrombospondin once the platelet-release reaction has occurred.
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PMID:Complex formation of human thrombospondin with osteonectin. 340 55

Ten human ovarian carcinoma cell lines (A121, A121[as], Caov-3, Caov-4, NIH:OVCAR-3, OVCA 420, OVCA 429, OVCA 432, OVCA 433, and SK-OV-3) were examined for secretion of plasminogen activators (PAs) using a chromogenic PA assay and SDS-PAGE zymography. PA activity was detected in conditioned media from all 10 cell lines. PA levels secreted by the 10 individual lines in a 24-hr period spanned a large range, with the extremes being 8 and 5244 milliPloug units (mPU)/10(6) cells for SK-OV-3 and OVCA 420 cells, respectively. Secreted PAs were identified as urokinase (UK)-like or tissue plasminogen activator (tPA)-like using dual criteria of comigration with UK or tPA standards on SDS-PAGE zymography and fibrin-dependence characteristics. Using both criteria, all 10 cell types produced UK-like activity, while tPA-like activity was produced by only 5 of the lines: A121[as], Caov-3, NIH:OVCAR-3, OVCA 429, and OVCA 433. Two additional cell lines produced PA activities that were tPA-like if judged by only one of the two criteria. Thus, Caov-4 cells produced a PA which comigrated with tPA, yet displayed no fibrin-dependent characteristics. Conversely, SK-OV-3 cells produced a fibrin-dependent PA, yet a band comigrating with tPA was not seen on SDS-PAGE zymography. Two lines derived from primary and ascitic sites from the same patient (A121 and A121[as], respectively) produced PAs with markedly different characteristics. Thus, PA produced by A121 cells was 100% UK-like, while that produced by A121[as] cells was greater than 90% tPA-like. Also, the total PA activity secreted by A121 cells was four times that secreted by A121[as] cells. In addition to bands comigrating with UK or tPA, all of the cell lines except Caov-3 and NIH:OVCAR-3 displayed higher molecular weight PA activities suggestive of the SDS-stable complexes between PAs and PA inhibitors reported in other cell types. While our results indicate that PA production may be a general characteristic of ovarian carcinoma cells in culture, individual patterns of UK and tPA production appear to be complex and vary from cell line to cell line. The precise characteristics of PA production in a given cell line may therefore depend on currently unidentified characteristics of the original tumor.
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PMID:Plasminogen activator secretion by established lines of human ovarian carcinoma cells in vitro. 341 Mar 41

The plasminogen activator inhibitor from human endothelial cells (PAI-1) exists in two forms in the culture medium: an active form that binds to and inactivates plasminogen activators and a latent form that in its native state has no anti-activator activity. Inhibitor activity associated with the latent form can be generated by treatment with protein denaturants and makes up more than 98% of the total inhibitor activity in conditioned medium. Plasminogen activator inhibitor activity is also found in cell cytosol. This inhibitor activity is stable to SDS-treatment but is not enhanced by it. We investigated the relationship between this active cell-associated inhibitor and the latent PAI-1 found in the conditioned medium. Both intracellular and extracellular inhibitors were immunoprecipitated by a monoclonal antibody produced against the latent inhibitor from HT1080 fibrosarcoma cells and electrophoresis on SDS gels of various acrylamide concentrations demonstrated that both forms had the same Mr. Incubation of cytosol inhibitor at 37 degrees C resulted in a decline in inhibitor activity with a half-life of approximately 4 hours, a rate of decline similar to that of the active PAI-1 in conditioned medium, with less than 10% of the original activity present after eight hours. This decline is accelerated at higher temperatures and is not affected by the presence of a variety of protease inhibitors. Approximately 90% of the activity can be regenerated after SDS treatment suggesting that the cell associated inhibitor, during incubation at 37 degrees C, converts to a form similar to that found in conditioned medium. Despite these similarities, the apparent Stoke's radii of the active intracellular inhibitor and the latent inhibitor in conditioned medium were significantly different with values of 2.77 nm and 2.40 nm for active and latent PAI-1, respectively. Incubation of the active form at 37 degrees C resulted in the shift of the Stoke's radius to that similar to the latent PAI-1 (2.45 nm). Thus, the active and latent PAI-1, while being immunologically similar and of the same apparent Mr, can be differentiated by their behavior on gel permeation columns. This suggests that the intracellular inhibitor is a precursor to the latent form.
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PMID:Conversion of the active to latent plasminogen activator inhibitor from human endothelial cells. 349 18

We have examined the dissolution of plasminogen-free fibrin clots by proteases in a leukocyte lysate using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to monitor alterations of cross-linked fibrin structure before solubilization and to characterize the structure of soluble derivatives. Progressive clot lysis was also followed by quantitation of fibrin derivatives that were present as solubilized products, as clot-associated fragments, and as residual degrading clot. An estimate of the total fibrinolytic potential of the plasminogen system relative to that of entrapped leukocytes was based on the specific fibrinolytic activities of plasmin and leukocyte lysate, and this indicated that the capacity of leukocyte-mediated fibrinolysis was only 3% of fibrin-bound plasminogen. Early during leukocyte lysate digestion the predominant soluble products were small peptides of Mr less than 20,000, whereas at later times heterogeneous groups of large fragments were present that were distinct from those produced during plasmic degradation. Electrophoresis in nondissociating conditions showed that a later leukocyte lysate digest of cross-linked fibrin contained distinct bands with mobilities indistinguishable from plasmic derivatives DD/E and DY/YD, suggesting a similar assembly in the native state of the leukocyte lysate fragments to those produced by plasmin. During degradation by leukocyte lysate, up to 70% of the degrading, insoluble clot could be solubilized in SDS, indicating that extensive early cleavage of the fibrin matrix failed to release much of the protein into solution. A market difference in the composition of fragments and polypeptide chains in the protein noncovalently bound to clot was seen in comparison with soluble derivatives. This appeared to be caused by the relative resistance to degradation of the C-terminal portions of the gamma-chain of the soluble derivatives, whereas the matrix-associated protein could be more easily cleaved in this region. The results demonstrate a distinct difference in the overall pattern of degradation compared with plasmic fibrinolysis.
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PMID:Degradation of cross-linked fibrin by human leukocyte proteases. 351 76

Immunoblots of proteins extracted from the skin of a small viviparous fish (Xiphophorus) showed that a monoclonal antibody against human urokinase recognizes multiple molecular weight species of antigens. The immunoaffinity-purified antigens had serine-protease activity for the hydrolysis of a chromogenic substrate and could convert human plasminogen to plasmin in a manner similar to that for human urokinase in vitro. Two antigens with apparent molecular weights of 55 and 50 kilodaltons that had been purified on a fibrin-Celite column were separable on SDS-polyacrylamide gels and were characterized as major plasminogen activators on fibrin-agar indicator plates. The 125I-tryptic peptide maps of both antigens were similar to that of human urokinase; therefore, the fish activators and human urokinase are structurally and functionally related.
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PMID:Fish plasminogen activators: their identification and characterization. 355 60

A relatively rapid method for the isolation of complement protein C4 from bovine plasma is described. The method consists of DEAE Sephacel anion exchange chromatography of plasminogen-depleted bovine plasma followed by cation exchange chromatography on CM Sepharose and finally gel filtration on a TSK G3000 SW column. A yield of approximately 20% was obtained. Conventional SDS-PAGE of purified bovine C4 showed the presence of alpha, beta and gamma polypeptide chains, the molecular weights of which were determined from Ferguson plots to be 95,000 +/- 2,500, 80,500 +/- 2,000 and 30,000 +/- 500 daltons, respectively. SDS-PAGE of C4 immunoprecipitated from the plasma of individual cattle in gels with a reduced proportion of crosslinker showed size polymorphism of the alpha chain. The presence of dual alpha chains was confirmed by radiolabelling their reactive thiol ester moiety with 14C methylamine. The difference in size of the two bovine alpha chains is approximately 1,800 daltons. On activation of bovine C4 both alpha chains were cleaved into alpha' chains (87,000 and 85,000 daltons) characteristic of C4b.
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PMID:Purification and characterisation of the fourth component of bovine complement. 356 27

We have investigated here the coordinate expression of both procoagulant (PCA) and fibrinolytic (FA) activity of cells from 16 human ovarian carcinoma cases. To avoid interference of contaminating host cells, we used cells isolated in primary culture from ascitic fluid or from solid tumor. The FA was determined in cellular extracts by an amidolytic assay in the presence of fibrin monomers. FA, which was plasminogen dependent in almost all of the cases, showed a wide range of activity (from less than 0.001 to 2.30 UK units/mg protein). The molecular analysis of plasminogen activator (by SDS-PAGE and fibrin autography) showed a single molecular form of 52,000 daltons, inhibited by an antibody against human urokinase. PCA, studied with a one stage clotting assay in disrupted cells, was of tissue thromboplastin type in all instance and varied from 12.0 to 1300 thromboplastin units/10(4) cells. No simple correlation was found between FA and PCA in the cellular samples studied; moreover, for neither parameter was it possible to find any changes with the staging of the disease.
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PMID:Procoagulant and fibrinolytic activity of human ovarian carcinoma cells in culture. 373 46

Urine samples from 10 species of mammals were analyzed by SDS-PAGE followed by zymography for the presence of both plasminogen activators and plasminogen activator binding proteins. In contrast to results obtained with urine from humans (Homo sapiens), and to a lesser extent urine from baboons (Papio cynocephalous), urine from gorillas (Gorilla gorilla) and orangutans (Pongo pygmaeus) did not exhibit either very high molecular weight plasminogen activators or the presence of plasminogen activator binding proteins. Low levels of very high molecular weight plasminogen activators could be detected in concentrates of urine samples from rabbits (Oryctolagus cuniculus), dogs (Canis familiaris) and sheep (Ovis aries). Very high molecular weight plasminogen activators could be detected in unconcentrated guinea-pig (Cavia porcella) urine, concentrated urine samples from rats (Rattus norvegicus), but not in concentrated samples of urine from mice (Mus musculus). These results indicate that considerable variation between species exists at the level of the plasminogen activators present in urine, a finding that may relate to whether plasminogen activator binding proteins are also present in this fluid.
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PMID:Species differences in the detection of high molecular weight urinary plasminogen activators. 374 21

Plasma kallikrein purified from acetone-activated, plasminogen-free rat plasma yielded in polyacrylamide gel electrophoresis protein bands corresponding to Mr values of 143,000 (main band) and 135,000 (lighter band). After SDS treatment without reduction the protein pattern had changed to two strong bands corresponding to Mr values of 87,000 and 78,000. Gel electrophoresis of kallikrein purified from plasma of rats pretreated with clinical dextran (200 mg/kg intravenously) produced main bands corresponding to Mr values of 120,000-130,000 and 78,000-80,000 for native samples and SDS-treated samples respectively (Johansen & Briseid 1983). Both kinds of kallikrein reduced the capacity of human high molecular weight kininogen to function as a cofactor in the surface-mediated activation of factor XII in a crude plasma preparation. The preparation obtained from plasma of dextran-treated rats was significantly more potent than was the normal kallikrein preparation, both as regards the effect against HMrK, and as an activator of plasminogen.
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PMID:Reduced cofactor function of human high molecular weight kininogen induced by rat plasma kallikrein. 385 Jul 11


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