Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.21.73 (urokinase-type plasminogen activator)
 10,685 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The effect of sodium pentosan polysulphate (SPP) was investigated in calcium oxalate stone forming rats with respect to the urinary excretion of certain risk factors and enzymes. Calcium oxalate stones were induced by feeding 3% w/w sodium glycollate to the rats. Urinary calcium, oxalate, phosphorus and uric acid levels were increased in stone formers. In contrast magnesium excretion was low in this group. SPP treatment lowered oxalate and calcium levels in both controls and experimental animals. Magnesium levels were increased moderately. Increased excretion of urinary enzymes--LDH, alkaline phosphatase, gamma-GT and beta glucuronidase--in calculogenic rats indicates membranuria and damage to proximal tubules during stone formation. Decreased pyrophosphatase activity was observed in glycollate fed rats. SPP treatment decreased the excretion of the above enzymes in the treated groups. Stone formers exhibited decreased LAP and fibrinolytic (urokinase) activities. SPP being associated with fibrinolytic properties, increased the activities of the above two enzymes to that of control levels in calculogenic rats.
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PMID:Alterations in some risk factors and urinary enzymes in urolithiatic rats treated with sodium pentosan polysulphate. 768 93

We previously reported that urokinase (uPA) is produced by the human prostate cancer cell line, PC-3, and could function as a growth factor for cells of the osteoblast phenotype. To examine the role of uPA in metastasis to the skeleton and to extraskeletal sites, we have developed a homologous model of uPA overexpression in a rat prostate cancer cell line. Full length cDNA encoding rat (r) uPA was isolated and subcloned as a 1.4-kilobase XbaI-BspHI fragment in the sense and antisense orientation into the Moloney murine leukemia retroviral vector pYN. The control (pYN) and experimental (pYN-ruPA, pYN-ruPA-AS) plasmids were transfected into Dunning R 3227, Mat LyLu rat prostate carcinoma cells. Experimental clones expressing at least 5-fold higher (pYN-ruPA) or 3-fold lower (pYN-ruPA-AS) than controls were selected, and control and experimental cells were inoculated into the left ventricles of inbred male Copenhagen rats. Animals were sacrificed at timed intervals to examine the evolution of metastatic lesions. Control animals developed metastases to the lumbar vertebrae resulting in spinal cord compression and hind limb paralysis at 20-21 days postinoculation. Animals inoculated with cells overexpressing uPA developed hind limb paralysis significantly earlier (by day 14-15 postinoculation). Additionally, more widespread skeletal (ribs, scapula, and femora) metastases were seen. Serum from experimental animals showed a progressive elevation in alkaline phosphatase levels, and histological examination of lumbar metastases revealed markedly increased osteoblastic activity over that observed in control animals. In contrast to this, animals inoculated with cells underexpressing uPA developed hind limb paralysis significantly later (days 25-29 postinoculation) and displayed decreased tumor metastasis. These studies support a role for the catalytic domain of uPA in enhancing both skeletal and nonskeletal prostate cancer invasiveness and are consistent with a role for the growth factor domain of uPA in mediating an osteoblastic skeletal response.
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PMID:Urokinase overproduction results in increased skeletal metastasis by prostate cancer cells in vivo. 816 83

Disulfide bonds are rarely found in cytoplasmic proteins. Mutations were selected for in Escherichia coli that allow disulfide bond formation in the cytoplasm. In the presence of these mutations, export-defective versions of alkaline phosphatase and mouse urokinase were able to fold into their enzymatically active conformations in the cytoplasm because their disulfide bonds were formed. The mutations were mapped to the gene for thioredoxin reductase and diminish or eliminate the activity of this enzyme. Thioredoxin itself was found to be unnecessary for this disulfide bond formation. Thioredoxin reductase, but not thioredoxin, is thus implicated in keeping cysteines reduced in cytoplasmic proteins.
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PMID:Mutations that allow disulfide bond formation in the cytoplasm of Escherichia coli. 825 21

The cellular receptor for urokinase-type plasminogen activator (uPAR) binds pro-urokinase (pro-uPA) and facilitates its conversion to enzymatically active urokinase (uPA). uPA in turn activates surface-bound plasminogen to plasmin, a process of presumed importance for a number of biologic processes including cell migration and resolution of thrombi. We have previously shown that uPAR is expressed on the plasma membrane of circulating neutrophils, and we now report that stimulation with phorbol myristate acetate (PMA), FMLP, or tumor necrosis factor-alpha results in a rapid increase in the expression of uPAR. This process is accompanied by an increased cell-associated plasminogen activation after preincubation of neutrophils with pro-uPA in vitro. By subcellular fractionation of unstimulated neutrophils, 50% of uPAR is recovered in fractions containing latent alkaline phosphatase, corresponding to an intracellular compartment of easily mobilizable secretory vesicles distinct from both primary and specific granules, whereas the remaining 50% of uPAR is associated with a compartment eluting close to the specific granules. In contrast, the ligand pro-uPA is primarily (approximately 80%) found in the specific granules, but small amounts of pro-uPA/uPA (approximately 20%) coelute with latent alkaline phosphatase. Stimulation of neutrophils with FMLP results in translocation of uPAR as well as of pro-uPA from the secretory vesicles, whereas stimulation with PMA is required to translocate material from specific granules. Flow cytometry of neutrophils saturated with exogenous diisopropyl fluorophosphate-uPA shows a large excess (approximately 90%) of unoccupied uPAR on resting as well as FMLP- and PMA-stimulated neutrophils, suggesting a possible role for exogenous pro-uPA in providing neutrophils with a potential for plasminogen activation. These processes may be important for neutrophil extravasation and migration through extracellular matrix and for the contribution of neutrophils to resolution of thrombi.
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PMID:The receptor for urokinase-type plasminogen activator and urokinase is translocated from two distinct intracellular compartments to the plasma membrane on stimulation of human neutrophils. 829 41

Phenotyping of cytokeratin (CK)18-positive cells in bone marrow is gaining increasing importance for future prognostic screening of carcinoma patients. Urokinase-type plasminogen activator receptor (uPA-R) is one example of a potential aggressive marker for those cells. However, a valid and reliable double staining method is needed. Using monoclonal antibodies against uPA-R and CK18, we modified an immunogold/alkaline phosphatase double staining protocol. UPA-R/CK18-positive tumor cell controls exhibited black uPA-R staining in 15-80% of cases and red CK18 staining in almost 100% of tumor cells. Isotype- and cross-matched controls were completely negative. Bone marrow from healthy donors was always CK18-negative. Reproducibility of CK18-positive cell detection was estimated in a series of specimens from 61 gastric cancer patients comparatively stained with the single alkaline phosphatase-anti-alkaline phosphatase (APAAP) and our double staining method (10(6) bone marrow cells/patient). In four cases, double staining could not reproduce CK18-positive cells. In 34 cases it revealed fewer or equal numbers, and in 23 cases more CK18-positive cells than the APAAP method. Overall quantitative analysis of detected cell numbers (838 in APAAP, range 1-280 in 10(6); double staining 808, range 0-253) demonstrated relative reproducibility of APAAP results by double staining of 97%. Correlation of results between both methods was significant (p < 0.001, linear regression). Sensitivity of double staining tested in logarithmic tumor cell dilutions was one CK18-positive cell in 300,000. Specific uPA-R staining was seen on CK18-positive cells in bone marrow from 29 of 61 patients, and also on single surrounding bone marrow cells. To test the specificity of this staining, bone marrow cytospins from 10 patients without tumor disease were stained for uPA-R with the APAAP method. uPA-R expression was confirmed in all 10 cases, with a mean of 6.5% uPA-R-positive cells in 1000 bone marrow cells (SEM 1.2%). These results suggest that our double staining protocol is a sensitive, reproducible, and specific method for routine uPA-R phenotyping of disseminated CK18-positive cells in bone marrow of carcinoma patients.
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PMID:Immunocytochemical phenotyping of disseminated tumor cells in bone marrow by uPA receptor and CK18: investigation of sensitivity and specificity of an immunogold/alkaline phosphatase double staining protocol. 901 10

Evidence of dynamic development of cytokeratin (CK) 18-positive disseminated tumor cells in bone marrow of curatively resected cancer patients has implicated a subclinical minimal residual disease as a biologically relevant component in solid cancer. However, differentiation between irrelevant shed cells and those cells potentially capable of causing later recurrence has not yet been made. In parallel, accumulating data show functional association of the urokinase plasminogen activator (uPA) system and the membranous uPA receptor (uPA-R) with the capacity of a tumor cell for invasion and metastasis. The present study was designed to find descriptive evidence in vivo concerning whether uPA-R could be one potential characteristic for metastatically relevant phenotypes of disseminated tumor cells. An immunocytochemical double staining for uPA-R and CK18 (immunogold/alkaline phosphatase anti-alkaline phosphatase) was performed on perioperative and follow-up bone marrow aspirations of 78 curatively resected gastric cancer patients, if positive tumor cell status had been shown previously with the single alkaline phosphatase anti-alkaline phosphatase method. Bone marrow cells (10(6)) were examined in each assay. Postoperative qualitative and quantitative development of uPA-R-expressing disseminated tumor cells was followed in relation to uPA-R-negative cells and correlated with later clinical relapse. Double staining could be performed perioperatively or in follow-up, or both, in 58 of 78 patients. Expression of uPA-R on perioperatively disseminated tumor cells significantly correlated with later quantitative increases of tumor cells (P = 0.0009). Overall median tumor cell numbers with uPA-R expression significantly increased during follow-up from a median value of 5.5 to 10.0 in 10(6) cells (P = 0.008), and the mean relative percentage of uPA-R-positive, compared with uPA-R-negative, disseminated tumor cells also increased, from 47.9% at surgery to 68.6% in follow-up (P < 0.001). This was mainly due to patients with later tumor relapse (increase from 63.9 to 80.7%, P = 0.001). Patients without relapse showed slight increases at lower percentage levels (5.7% at surgery, 7.4% in follow-up). Differences for relapsing patients were significant (surgery, P = 0.006; follow-up, P < 0.001). Our results suggest from an in vivo model that uPA-R may be one antigen that enables identification and follow-up observations of metastatically relevant phenotypes of disseminated tumor cells, differentiating their individual potential for causing relapse.
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PMID:Urokinase plasminogen activator receptor (uPA-R): one potential characteristic of metastatic phenotypes in minimal residual tumor disease. 910 29

ELISAs for urokinase plasminogen activator (uPA) and plasminogen activator inhibitor type-1 (PAI-1) have shown that tumor levels of these molecules are prognostic parameters in breast cancer as well as other types of cancer. These ELISAs measure the total amount of the given component, including preforms, active, inactive, and complex-bound forms. However, the amount of the active forms of a component may more closely reflect the ongoing level of proteolytic activity and thereby be particularly related to prognosis. Since the inactive complex between uPA and PAI-1 can only be formed the active forms of the individual components, we have developed a sensitive and specific uPA:PAI-1 complex ELISA consisting of a sandwich format with two monoclonal antibodies (MAbs) against PAI-1 as capture antibodies and three biotinylated MAbs against uPA as detector antibodies. The data were collected as kinetic measurements of bound alkaline phosphatase activity. A standard of uPA:PAI-1 complex could be specifically measured in the assay with a detection limit of 8 pg/ml and a linear relationship between signal and complex concentration up to 4 ng/ml. Neither free uPA nor free PAI-1 were detected by this assay and the addition to the internal standard of free PAI-1 in amounts up to 20 ng/ml or uPA did not reduce the detection of complex by the assay. This ELISA was applied to extracts from 20 individual breast cancers. Each tumor was extracted in two different buffers and the median concentration of uPA:PAI-1 complex in the optimal extraction buffer was 0.8 ng/mg protein, range 0.4-2.8 ng/mg protein. Extraction of the tumor tissue at a low pH prevented de novo formation of complex from free uPA and PAI-1 in the tissue without destabilizing preformed uPA:PAI-1 complex. During incubation of the assay plate at neutral pH further uPA:PAI-1 complex formation from free components in the extracts was blocked by p-nitrophenyl guanidinobenzoate (NPGB). Thus, the present assay selectively quantifies preformed complex in tumor extracts and will enable us, for the first time, to evaluate the potential prognostic value of the uPA:PAI-1 complex in cancer.
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PMID:The complex between urokinase plasminogen activator and its type-1 inhibitor in breast cancer extracts quantitated by ELISA. 913 30

Prostate cancer (PRCA) cells metastasize to bone with high frequency, inducing typical osteosclerotic lesions. To establish if local stimuli on the bone tissue may derive from metastatic colonies of prostatic origin, we evaluated the biologic activities secreted by human prostatic epithelium and effective on osteoblast-like cells in vitro. Supernatant from short-term tissue cultures of human prostatic tissue samples obtained from PRCA (35 cases) and benign prostatic hyperplasia (BPH, 12 cases) patients were applied to three models of cells with osteoblastic phenotype: two normal [rabbit osteoblasts (OB) and rat periosteal cells (PO)] and one transformed (human osteosarcoma cell line, MG63). Proliferative activity was monitored through enzymatic reduction of tetrazolium salts and expressed as relative mitogenic activities (RMA). Analysis of proliferation and alkaline phosphatase (ALP) activity, a marker of osteoblast function, demonstrates that conditioned media (CM) from PRCA cultures stimulate both growth and activity of osteoblast-like cells to a greater extent compared to CM from BPH. Furthermore, cell growth and activity of osteoblast-like cells are progressively increased by CM derived from patients with stage B (tumor confined within the prostate capsule), stage C (locally invasive tumor), and stage D (invasive tumor with distant metastasis) disease. One of the mechanisms potentially underlying the CM-stimulated effects on bone cells is associated with the urokinase (uPA) enzyme route, whose release progressively increases with the stage of disease. However, antibodies against uPA and p-aminobenzamidine (a low molecular weight urokinase inhibitor) treatment, which both inhibit the proliferative and differentiative effects induced by exogenous urokinase, partially slow down the effects of CM from PRCA tissue cultures, suggesting that additional factors are secreted by prostatic tumor cells in vitro. In conclusion, we show that the mitogenic and differentiative activities for osteoblasts produced by prostatic tumor cells in short-term tissue cultures are related to PRCA stage and may predict the behavior of skeletal metastases in single cases of tumor. In addition, the culture methods used may represent a valid model to study prostatic and bone cellular interactions, which may indicate new therapeutic approaches in metastatic prostate tumors.
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PMID:Human prostatic tumor cells in culture produce growth and differentiation factors active on osteoblasts: a new biological and clinical parameter for prostatic carcinoma. 943 95

1. This study aimed to determine the effect of luminal butyrate on proliferative kinetics, a differentiation marker (alkaline phosphatase), and a molecule that controls cell-substratum adhesion (urokinase) in histologically normal human rectal mucosa. 2. Ten subjects with a colonoscopically normal colon (seven had previous adenomas) were given either butyrate or saline enemas for 4 days in a double-blind cross-over manner. Rectal biopsies were taken before and after each course of enemas. Epithelial proliferative kinetics were measured immunohistochemically using antibodies to proliferating cell nuclear antigen. Urokinase and alkaline phosphatase activities were measured spectrophotometrically in biopsy homogenates. 3. Both saline and butyrate enemas were well tolerated and induced no histological change except for a significant increase in crypt length (P < 0.05). The number of proliferating cells per crypt also increased significantly after butyrate (P = 0.018). 4. Compared with saline enemas, butyrate did not affect kinetic indices nor alkaline phosphatase activities. However, mucosal urokinase activities were significantly lower in butyrate-treated patients (9.5 +/- 2.0 i.u./g) than in saline-treated patients (12.8 +/- 2.0 i.u./g; P = 0.045). 5. Delivering of extra butyrate to the distal colon in healthy subjects may stabilize cell-substratum adhesion in surface epithelium and therefore offer a potential mechanism by which elevating distal colonic luminal butyrate concentrations might be beneficial in patients with colitis or hyperproliferative large bowel epithelium.
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PMID:Effect of topical butyrate on rectal epithelial kinetics and mucosal enzyme activities. 985 67

Butyrate may have paradoxical effects on epithelial cells of similar origin. This study aimed to examine the hypothesis that one mechanism that dictates a cell's response to butyrate is its state of activation. First, the responses to 24 h exposure to butyrate (1-2 mM) of normal and neoplastic human colonic epithelial cells activated by their isolation and primary culture, and of colon cancer cell lines, LIM1215 and Caco-2, were examined. In primary cultures of normal and cancer cells, butyrate had no effect on alkaline phosphatase activities but significantly suppressed urokinase receptor expression by a mean +/- SEM of 30 +/- 12% and 36 +/- 9%, respectively. Interleukin-8 secretion was suppressed by 44 +/- 7% in normal cells (P < 0.05) but was unchanged in cancer cells. In contrast, the cell lines significantly increased alkaline phosphatase activities by >50%, urokinase receptor expression >2-fold and interleukin-8 secretion >3-fold in response to butyrate. Secondly, the effect of butyrate on Caco-2 cells was examined with or without prior exposure to a specific activating stimulus [tumour necrosis factor alpha (TNF alpha)]. Interleukin-8 secretion increased by 145 +/- 23% and 132 +/- 17% on 24 h exposure to 2 mM butyrate or 0.1 microM TNF alpha alone, respectively. However, in cells pre-treated with TNF alpha, butyrate significantly inhibited secretion by 34 +/- 7% below unstimulated levels. The response to butyrate of urokinase receptor, whose expression was not stimulated by TNF alpha, was unchanged. These effects were mimicked by trichostatin A, an inhibitor of histone deacetylase, suggesting that butyrate's paradoxical effects may have been operating by the same mechanism. In conclusion, some of the paradoxical effects of butyrate do not appear to represent inherent differences between normal and transformed cells. Rather, the response may be determined by the state of activation of the cells.
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PMID:Colonic epithelial cell activation and the paradoxical effects of butyrate. 1022 79


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