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)

In inflammatory demyelinating diseases such as multiple sclerosis and experimental allergic encephalomyelitis, myelin destruction occurs in the vicinity of infiltrating mononuclear cells. The observations that myelin can be altered prior to phagocytosis and in areas not contiguous with inflammatory cells suggests a common mechanism for the initial stages of demyelination. Because stimulated macrophages secrete several neutral proteases, including plasminogen activator, we have investigated the possibility that myelinolysis could be mediated directly or indirectly by these enzymes. Isolated myelin was incubated with conditioned media from cultures of thioglycollate-stimulated mouse peritoneal macrophages in the presence and absence of plasminogen. Myelin appeared to be vulnerable to attack by at least two proteolytic activities secreted by the macrophages, a plasminogen-dependent and a plasminogen-independent activity; of the major proteins in myelin, the basic protein was most susceptible. The direct myelinolytic activity of macrophage-conditioned media was abolished by EDTA, and the plasminogen-dependent hydrolysis was abolished by p-nitrophenylguanidinobenzoate, an inhibitor of plasminogen activator and plasmin. These results suggest that the plasminogen activator released by the stimulated macrophages generated plasmin which hydrolyzed basic protein in intact myelin. This interpretation was confirmed by the observation that urokinase, a plasminogen activator, in the presence of plasminogen brought about marked degradation of basic protein in myelin. We propose that the release of neutral proteases by stimulated macrophages involved in cell-mediated reactions, and its amplification by the plasminogen-plasmin system, may play a significant role in the demyelination observed in several inflammatory demyelinating diseases.
 ...
PMID:Degradation of basic protein in myelin by neutral proteases secreted by stimulated macrophages: a possible mechanism of inflammatory demyelination. 14 51

A chromogenic tripeptide - H-D-Val-Leu-Lys-p-nitroanilide-substrate of plasmin, can be used to follow plasminogen activation by an activator such as urokinase or the activator secreted by mouse peritoneal macrophages (thioglycolate-elicited). The acceleration of p-nitroaniline production is proportional to the initial rate of plasmin formation from plasminogen. Thus, at a given plasminogen concentration, this acceleration is proportional to the activator concentration. The acceleration can be evaluated from the spectrophotometer trace recording at 405 nm the appearance of p-nitroaniline, either by means of a computer program or by a plot of delta A405 vs.t2. The sensitivity of this assay allows detection of 0.003 CTA units of urokinase. Thioglycollate-elicited mouse peritoneal macrophages secrete plasminogen activator into the extracellular medium during in vitro cultivation only after a contact with serum.
 ...
PMID:Regulation of plasminogen activator secretion in mouse peritoneal macrophages. I. - Role of serum studied by a new spectrophotometric assay for plasminogen activators. 48 77

Mouse resident peritoneal macrophages display sufficient 5'-nucleotidase activity to hydrolyze 58 nm AMP/min per cell protein. This activity increases approximately 163 nm AMP/min per mg after 72 h in culture. The enzyme is renewed in unstimulated cells with a half-time of 13.9 h. The activity is not reduced by treatment of intact cells with a variety of proteolytic enzymes, including trypsin, pronase, urokinase, and plasmin. Cells obtained from an inflammatory exudate have diminished or absent levels of enzyme activity. Endotoxin-elicited cells display enzyme activitiy of 20.9 nm AMP/min per mg, while thioglycollate-stimulated macrophages have no detectable activity. The reduced level of activity in endotoxin-stimulated cells is due to their elevated rate of enzyme degradation, with a half-time of 6.9 h. Their rate of enzyme synthesis is essentially normal. No evidence for latent enzyme activity could be obtained in thioglycollate-stimulated cells, nor do these cells produce any inhibition of normal cell enzyme activity. Serum deprivation reduces the enzyme activity of resident cells to about 45% of control activity. These conditions do not significantly affect the rate of enzyme synthesis, but again are explainable by an increase in the rate of enzyme degradation. Pinocytic rate is elevated in endotoxin-stimulated cells which show a more rapid rate of enzyme degradation than unstimulated cells do. However, in serum-free conditions, the rate of enzyme degradation is doubled with no change in the pinocytic rate of the cells.
 ...
PMID:5'-Nucleotidase activity of mouse peritoneal macrophages. I. Synthesis and degradation in resident and inflammatory populations. 100 5

Exposure of mouse resident and thioglycollate-elicited peritoneal macrophages to IFN-gamma leads to a marked increase in the TNF-alpha (tumor necrosis factor/cachectin), IL-1 and u-PA (urokinase-type plasminogen activator) mRNA levels. Nuclear run-on experiments show that IFN-gamma acts by enhancing the transcription of these three genes. Transcription of these three genes is also rapidly and transiently induced by cycloheximide, an inhibitor of protein synthesis, indicating that they are under the control of short-lived repressors.
 ...
PMID:Gamma interferon enhances macrophage transcription of the tumor necrosis factor/cachectin, interleukin 1, and urokinase genes, which are controlled by short-lived repressors. 309 40

Plasminogen activator (PA), a neutral protease whose primary function is to convert plasminogen to plasmin, is produced by various cells including macrophages, monocytes, endothelial cells, and tumor cells. This study reports the use of the chromogenic tripeptide substrate D-Val-Leu-Lys-p-nitroanilide (S-2251) and an automated microtiter plate reader spectrophotometer for the determination of PA activity in cells and fluids. There was a linear relationship between the time of incubation at 37 degrees C and the square root of the absorbance measured at 405 nm when urokinase was incubated with the substrate in the presence of plasminogen. There was no activity in the absence of plasminogen. The slopes of the lines (square root A 405/time) were directly related to the concentrations of urokinase, up through 0.05 CTA units. Using this assay, we determined the cellular activity of PA in human promyelocytic cells HL-60 (1.33 +/- 0.12 CTA units/mg), human monocytoid cells U937 (1.27 +/- 0.12 CTA units/mg), mouse myeloid leukemia cells RFM/UN (0.70 +/- 0.07 CTA units/mg), freshly isolated normal human monocytes (0.00 +/- 0.00 CTA units/mg), and human monocytes after 7 days in culture (5.66 +/- 0.38 CTA units/mg). There was a variable amount of activity expressed in freshly isolated cells or cell lysates of peritoneal macrophages from normal mice, or mice that had gotten intraperitoneal injections of peptone, thioglycollate, or NaIO4, but after 24 or 48 h of culture, these activities, in general, increased. Using this assay, PA levels in the euglobulin precipitates from human plasma prepared without venous occlusion (0.03 +/- 0.02 CTA units/mg protein) or after 5 min of venous occlusion of the arm (0.18 +/- 0.01 CTA units/mg) were comparable to those reported by others using different assays. Thus, this represents a simple, rapid, accurate assay of PA that should be useful to those in immunology, cell biology, and clinical medicine.
 ...
PMID:Microassay for the photometric quantitation of cell-associated plasminogen activator using a chromogenic tripeptide substrate. 659 19

Transforming growth factor-beta (TGF-beta) is secreted by most cells as a biologically inactive complex, called the large latent TGF-beta complex. The complex is comprised of latent TGF-beta binding protein (LTBP) and latent TGF-beta, which is mature TGF-beta associated noncovalently with its amino-terminal propeptides. LTBP is disulfide-linked to the amino-terminal propeptide of latent TGF-beta. Active TGF-beta is generated by release of TGF-beta from the complex. Generation of active TGF-beta by macrophages has been reported, but the activation mechanism has not been described. Latent TGF-beta activation by macrophages was characterized using serum-free cultures of resident and thioglycollate-elicited murine peritoneal macrophages that were either unstimulated or LPS-stimulated in vitro. Serum-free conditioned medium was assayed for TGF-beta using a quantitative luciferase-based bioassay. LPS-stimulated thioglycollate-elicited macrophages activated endogenous latent TGF-beta, whereas non-LPS-stimulated thioglycollate-elicited and resident macrophages generated undetectable levels of TGF-beta. Latent TGF-beta activation required plasmin and urokinase (uPA), uPA binding to the uPA receptor, interaction with the cation-independent mannose 6-phosphate/insulin-like growth factor type II receptor, tissue type II transglutaminase, and LTBP. A time-course analysis of latent TGF-beta activation revealed that maximal TGF-beta was generated after 24 h (25 +/- 5 pg/ml). TGF-beta formed within the initial 24 h modulated the plasminogen activator system by down-regulating uPA, suggesting that TGF-beta temporally modulated its own formation by regulating cell-associated uPA.
 ...
PMID:Characterization of latent TGF-beta activation by murine peritoneal macrophages. 763 10

1. Immunotrap assays that can measure the activities of urokinase-type plasminogen activator (uPA) and its inhibitors (PAIs) were characterized. 2. Both human plasma and mouse macrophages in culture were found to contain much higher inhibitor activity than uPA-like activity. 3. The balance between pro- and anti-fibrinolytic activities was quantitatively changed in the murine macrophages after the injection of thioglycollate. uPA-like materials were synthesized by the macrophages and secreted to the conditioned medium continuously, while PAI activity was unchanged during the same time period.
 ...
PMID:Characterization of immunotrap assays for urokinase plasminogen activator and its inhibitors and measurements of these molecules in human plasma and mouse macrophage in culture. 834 18

Mice with combined homozygous deficiency of tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA) (T-U-), of t-PA and plasminogen activator inhibitor-1 (PAI-1) (T-P-), of u-PA and PAI-1 (U-P-) or of t-PA, u-PA, and PAI-1 (T-U-P-) were generated by inbreeding of mice with the respective deficiencies. Homologous recombination at the t-PA, u-PA and PAI-1 locus was verified by Southern blot analysis of genomic tail tip DNA, and confirmed by measurement of antigen levels in plasma or urine. T-P- and U-P- mice were apparently healthy and fertile. T-U- mice showed extensive fibrin deposition with calcification in the liver, whereas T-U-P- mice were significantly (p < 0.001) less affected. Spontaneous in vivo clot lysis measured 4 h after injection of a 125I-fibrin-labeled clot prepared from plasma of wild-type (WT) mice into the jugular vein, was (mean +/- SEM of n experiments) 2 +/- 1% (n = 8) for T-P-, 49 +/- 6% (n = 9) for U-P-, 1 +/- 1% (n = 4) for T-U- and 3 +/- 3% (n = 3) for T-U-P- mice, as compared to 32 +/- 4% (n = 10) for WT, 1 +/- 0% (n = 7) for T-, 30 +/- 5% (n = 5) for U- and 58 +/- 10% (n = 6) for P- mice. Plasminogen-dependent lysis of 125I-fibrin-labeled matrix and of 3H-proline-labeled subendothelial matrix (mean +/- SEM; n = 4 to 6) was lower with thioglycollate-stimulated macrophages obtained from U-P- mice (22 +/- 7% and 5 +/- 1%, respectively), as compared to WT mice (57 +/- 14% and 18 +/- 5%, respectively) and T-P- mice (87 +/- 6% and 27 +/- 4%, respectively). A similar decrease was previously observed with U- mice, but not with T- or P- mice. Thus, the phenotype of mice with combined deficiency of t-PA and PAI-1 or of u-PA and PAI-1 is similar to the phenotype observed in mice with single deficiency of the plasminogen activator. Additional deletion of PAI-1 does not affect viability, fertility, macrophage function or thrombolytic potential of the single deficient mice. Additional deletion of PAI in mice with combined deficiency of t-PA and u-PA does not restore the deficient in vivo fibrinolytic capacity, but significantly reduces the thrombotic phenotype, as revealed by fewer, smaller and less calcified fibrin deposits in the liver.
 ...
PMID:Biological effects of combined inactivation of plasminogen activator and plasminogen activator inhibitor-1 gene function in mice. 856 Apr 24

Mice homozygously deficient for the urokinase-type plasminogen activator (u-PA) receptor (u-PAR-1-) were generated by homologous recombination in D3, embryonic stem cells. The genomic sequences comprising exon 2 through 5 of the u-PAR gene were replaced by the neomycin resistance gene, resulting in inactivation of both u-PAR splice variants. The inactivated u-PAR allele was transmitted via mendelian inheritance, and fertility. Inactivation of u-PAR was confirmed by the absence of binding of rabbit anti-murine u-PAR or of an aminoterminal fragment of murine u-PA (mu-PA.1-48) to u-PAR-1- embryonic fibroblasts and macrophages. u-PAR-1- mice displayed normal lysis of a murine plasma clot injected via the jugular vein. Invasion of macrophages into the peritoneal cavity after thioglycollate stimulation was similar in u-PAR-1- and u-PAR-1- mice. u-PAR-1- peritoneal macrophages had a threefold decreased initial rate of u-PA-mediated plasminogen activation in vitro but degraded extracellular matrix proteins in vitro as efficiently as u-PAR-1- macrophages.
 ...
PMID:Generation and characterization of urokinase receptor-deficient mice. 860 47

Proteolytic cleavage of single-chain, high molecular weight kininogen (HK) by kallikrein releases the short-lived vasodilator bradykinin and leaves behind a two-chain, high molecular weight kininogen (HKa) reported to bind to the beta2-integrin Mac-1 (CR3, CD11b/CD18, alphaMbeta2) on neutrophils and exert antiadhesive properties by binding to the urokinase receptor (uPAR) and vitronectin. We define the molecular mechanisms for the antiadhesive effects of HK related to disruption of beta2-integrin-mediated cellular interactions in vitro and in vivo. In a purified system, HK and HKa inhibited the binding of soluble fibrinogen and ICAM-1 to immobilized Mac-1, but not the binding of ICAM-1 to immobilized LFA-1 (CD11a/CD18, alphaLbeta2). This inhibitory effect could be attributed to HK domain 5 and to a lesser degree to HK domain 3, consistent with the requirement of both domains for binding to Mac-1. Accordingly, HK, HKa, and domain 5 inhibited the adhesion of Mac-1 but not LFA-1-transfected K562 human erythroleukemic cells to ICAM-1. Moreover, adhesion of human monocytic cells to fibrinogen and to human endothelial cells was blocked by HK, HKa, and domain 5. By using peptides derived from HK domain 5, the sequences including amino acids H475-G497 (and to a lesser extent, G440-H455) were identified as responsible for the antiadhesive effect, which was independent of uPAR. Finally, administration of domain 5 into mice, followed by induction of thioglycollate-provoked peritonitis, decreased the recruitment of neutrophils by approximately 70% in this model of acute inflammation. Taken together, HKa (and particularly domain 5) specifically interacts with Mac-1 but not with LFA-1, thereby blocking Mac-1-dependent leukocyte adhesion to fibrinogen and endothelial cells in vitro and in vivo and serving as a novel endogenous regulator of leukocyte recruitment into the inflamed tissue.
 ...
PMID:Regulation of leukocyte recruitment by polypeptides derived from high molecular weight kininogen. 1168 62


1 2 Next >>