PURPOSE: We measured the gastric cancer tissue uPA and plasminogen activator inhibitor-1 (PAI-1) levels and compared them to those of the peripheral and portal blood levels to evaluate the correlation. MATERIALS AND METHODS: Tissue uPA and PAI-1 levels were measured by ELISA assay (Monozyme, Netherland) in paired 85 normal and cancer tissues resected from gastric cancer patients. In 50 patients, blood uPA and PAI-1 levels were measured from pre- operative peripheral and portal blood, post-operative portal blood. RESULTS: Gastric cancer tissue uPA and PAI-1 levels increased from the early stage. The elevated cancer-to-normal ratios of the uPA and PAI-1 were constant from stage I to IV. There were correlations of uPA between normal and cancer tissues (r2=0.38) and between peripheral and pre-resection portal blood level (r2=0.64). There were no correlations between tissue PAI-1 level and blood PAI-1 levels. However, there were correlations in PAI- 1/uPA ratio between cancer tissue and peripheral blood (r2=0.25), peripheral blood and pre- resection portal blood (r2=0.60). CONCLUSION: Even if the cancer tissue levels of uPA and PAI-1 increased from the early stage of gastric cancer, only blood uPA level correlated with tissue uPA level. A modest correlation found in PAI-1/uPA ratio between cancer tissue and blood suggests applicability of blood PAI-1/uPA ratio in predicting tissue uPA, PAI-1 expression.
Enzyme-Linked Immunosorbent Assay ; Humans ; Plasminogen Activator Inhibitor 1 ; Plasminogen Activators ; Stomach Neoplasms* ; Urokinase-Type Plasminogen Activator*

Drugs are exogenous compounds for human bodies, and will be metabolized by many enzymes after administration. CYP450 enzyme, as a major metabolic enzyme, is an important phase I drug metabolizing enzyme. In human bodies, about 75% of drug metabolism is conducted by CYP450 enzymes, and CYP450 enzymes is the key factor for drug interactions between traditional Chinese medicine( TCM) -TCM, TCM-medicine and other drug combination. In order to make clear the interaction between metabolic enzymes and TCM metabolism, we generally chose the enzymatic activity as an evaluation index. That is to say, the enhancement or reduction of CYP450 enzyme activity was used to infer the inducing or inhibitory effect of active ingredients and extracts of traditional Chinese medicine on enzymes. At present, the common method for measuring metabolic enzyme activity is Cocktail probe drugs, and it is the key to select the suitable probe substrates. This is of great significance for study drug's absorption, distribution, metabolism and excretion (ADME) process in organisms. The study focuses on the interaction between TCMs, active ingredients, herbal extracts, cocktail probe substrates as well as CYP450 enzymes, in order to guide future studies.
Animals ; Cytochrome P-450 Enzyme Inhibitors ; metabolism ; pharmacology ; Cytochrome P-450 Enzyme System ; chemistry ; metabolism ; Drugs, Chinese Herbal ; metabolism ; pharmacology ; Enzyme Activation ; drug effects ; Enzyme Activators ; metabolism ; pharmacology ; Humans

PURPOSE: Cancer invasion is induced by several proteolytic enzyme systems associated with the destruction of basement membrane and extracellular matrix. Urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor-1 (PAI-1) have been reported as prognostic factors in breast cancer patients and plasminogen activation is regulated by various factor such as uPAR and growth factor. So we examined the tissue levels of urokinase-type plasminogen activator receptor (uPAR) in breast cancer patients. MATERIALS AND METHODS: Tissue uPAR levels were measured by ELISA assay in 268 breast cancer patients. RESULTS: The median and mean values of tissue uPAR level in breast cancer were 3.5 ng/mg and 4.8+-3.6 ng/mg cytosol protein, respectively. Tissue uPAR level was the highest in T1 stage, but there was no statistical significance between T stage (p >0.05). In nodal stage, there was also no difference in the value of uPAR according to progression. And the value of uPAR expression was not associated with estrogen and progesteron receptor status, number of involved node and percent of node involvement. In TNM stage, tissue uPAR levels were higher in patients with stage I-II than in patients with stage III-IV (p=0.027). In univariate analysis, nodal factor (p=0.0023) and TNM stage (p=0.0004) were significantly associated with overall survival. But, multivariate analysis showed that TNM stage was the only significant prognostic factor (p=0.0002). CONCLUSION: These results suggest that uPAR is mainly associated with initial tumor invasion and other factors might be involved in later stages of cancer progression.
Basement Membrane ; Breast Neoplasms* ; Breast* ; Cytosol ; Enzyme-Linked Immunosorbent Assay ; Estrogens ; Extracellular Matrix ; Humans ; Multivariate Analysis ; Plasminogen Activators* ; Plasminogen* ; Urokinase-Type Plasminogen Activator*

BACKGROUND: Impaired fibrinolytic system has been considered to play an important role in the pathogenesis of coronary artery disease, especially associated with thrombus formation. To evaluate the pathogenetic role of fibrinolytic system in coronary artery disease, major determinants of fibrinolytic system, tissue plasminogen activator(t-PA) and palsminogen activator inhibitor-1(PAI-1) levels were measured in control(n=7) and chronic stable angina patients(n=7). METHODS: Blood samplings were done in resting state, venous occlusion and peak exercise. Levels of plasma t-PA antigen and PAI-1 antigen were measured by ELISA method. RESULTS: 1) In resting state, there was no significant difference in plasma level of t-PA(control group : 9.15+/-2.82ng/ml vs, study group ; 9.65+/-3.53ng/ml) and PAI-1(control group ; 20.27+/-9.98ng/ml vs, study group ; 17.43+/-3.53ng/ml) between each group. 2) With venous occlusion test, increment of plasma t-PA level was noted in both groups which was lower in patient group, however, this difference in increment was not statistically significant. 3) Increased plasma t-PA level was noted after exercise in both groups. 4) Plasma level of PAI-1 was not significantly changed after venous occlusion or exercise in both groups. CONCLUSIONS: In patients with chronic stable angina, there was no definite evidence of impaired fibriolytic system although plasma t-PA increased somewhat less after venous occlusion in patients with chronic stable angina than control.
Angina, Stable* ; Coronary Artery Disease ; Enzyme-Linked Immunosorbent Assay ; Humans ; Plasma* ; Plasminogen Activator Inhibitor 1 ; Plasminogen Activators* ; Plasminogen* ; Thrombosis ; Tissue Plasminogen Activator*

PURPOSE: We measured and compared the uPA, plasminogen activator inhibitor-1 (PAI-1) and uPA receptor (uPAR) levels in breast cancer tissues and blood of the patients to evaluate their clinical relevance for biotherapy. MATERIALS AND METHODS: uPA, PAI-1 (Monozyme, Netherland), uPAR (American Diagnostics, USA) levels were measured by ELISA assay in 192 breast cancer tissues, in 18 normal breast tissues and in 163 blood from breast cancer patients. RESULTS: There was a tendency of uPA increment from ductal carcinoma in situ while increment of PAI-1 and uPAR occurred from Ti. With the progression of cancer, uPA, PAI-1, uPAR tended to decrease; however, the uPA/uPAR, uPA/PAI-1 ratios remained unchanged. There was a correlation of uPA expression between normal and cancer tissues ( r(2)= 0.49). Correlation of uPA and PAI-1 was found in normal tissue and stage I cancer tissue while correlation of uPAR and PAI-1 was found with cancer progression. Between cancer tissue and blood significant correlations were found in uPA, PAI-1, uPAR levels. CONCLUSION: uPA, PAI-1, uPAR levels in cancer tissue elevated from the early stage maintaining correlative expressions with cancer progression. A positive correlation between cancer tissue and blood level suggested the applicability of the levels of uPA, PAI-1 or uPAR for detecting patients for biotherapy.
Biological Therapy* ; Breast Neoplasms* ; Breast* ; Carcinoma, Intraductal, Noninfiltrating ; Enzyme-Linked Immunosorbent Assay ; Humans ; Plasminogen Activator Inhibitor 1 ; Plasminogen Activators* ; Plasminogen* ; Urokinase-Type Plasminogen Activator*

BACKGROUND: Insulin resistance is known as the common denominator of risk factors of atheros-clerosis as well as the major pathogenic process of type 2 diabetes mellitus (DM). Recently some investigators indicated the relationship of chronic inflammatory reaction to atherosclerosis and insulin resistance. We examined the relationship between insulin resistance and high sensitivity CRP (hs-CRP) in Koreans. METHODS: Twenty-five patients with type 2 DM and eleven healthy men were examined. Glucose disposal rate (GDR, mg/kg/min) was determined as the index of insulin resistance by the euglycemic insulin clamp test with De Fronzo method. The serum hs-CRP level was determined by Behring nephelometric assay, fibrinogen by functional assay, and plasminogen activator inhibitor-1 (PAI-1) by ELISA. We also included 81 healthy subjects to determine the reference range of hs-CRP. RESULTS: The reference range (median) of hs-CRP was 0-5.20 (0.56) mg/L. The hs-CRP concentration was not significantly different between control and DM groups. The GDR of DM (3.8+/-1.7) showed significantly decreased value compared with normal (8.4+/-1.5) group (P<0.001). In all subjects, there was no significant correlation of GDR and hs-CRP. CONCLUSTIONS: There was no significant correlation of GDR and hs-CRP. We think the interventional prospective study with anti-inflammatory drug is warranted to elucidate the independent relationship between insulin resistance and hs-CRP.
Atherosclerosis ; C-Reactive Protein* ; Diabetes Mellitus, Type 2 ; Enzyme-Linked Immunosorbent Assay ; Fibrinogen ; Glucose ; Humans ; Inflammation ; Insulin Resistance* ; Insulin* ; Male ; Plasminogen Activators ; Reference Values ; Research Personnel ; Risk Factors

BACKGROUND AND PURPOSE: Thrombolytics-induced recanalization fails in a significant portion of patients with ischemic stroke, which is partly due to the resistance of clots to lysis by thrombolytic agents. The pretreatment level of endogenous fibrinolysis inhibitors may affect such thrombolysis failure. METHODS: We studied 43 stroke patients whose arterial recanalization had been evaluated by angiography, and whose blood had been obtained prior to the administration of thrombolytic agents. Plasma samples from 34 healthy volunteers were used as normal controls. Plasminogen activator inhibitor type 1 (PAI-1) and thrombin-activatable fibrinolysis inhibitor (TAFI) levels were quantified using an enzyme-linked immunosorbent assay. RESULTS: Arteries were recanalized [Thrombolysis in Myocardial Infarction (TIMI) grade 2 or 3] in 30 patients, but not (TIMI grade 0 or 1) in the other 13. The plasma PAI-1 level was significantly higher in patients without recanalization (nonrecanalization) than in those with recanalization and in normal controls. The TAFI levels did not differ among the groups. CONCLUSIONS: The pretreatment PAI-1 levels are increased in acute stroke patients with thrombolysis failure.
Angiography ; Arteries ; Carboxypeptidase U ; Enzyme-Linked Immunosorbent Assay ; Fibrinolysis* ; Fibrinolytic Agents ; Healthy Volunteers ; Humans ; Myocardial Infarction ; Plasma* ; Plasminogen Activator Inhibitor 1 ; Plasminogen Activators ; Stroke*

BACKGROUNDHepatitis B virus (HBV) X protein (HBx) and p53 could mutually down-regulate at transcriptional level and HBx could bind with p53 protein within its transactivation domain and inhibit the function of p53 protein. In recent years, effects of arsenic trioxide (As2O3) on the expression of p53 protein have been widely studied, while little is known about the activity of p53 protein. This study was undertaken to delineate the effect of HBV X gene and As2O3 on p53 protein expression (level and activity) in HepG2 cells by small hairpin RNA (shRNA)-mediated RNA interference (RNAi) technique.

METHODSCell line HepG2 and cells with stable expression of HBV X gene (HepG2-X) were treated with 2 micromol/L As2O3, with corresponding untreated cells serving as controls. Cell lysates and nuclear extracts were extracted. Total level and the relative activity of p53 protein were detected by modified enzyme-linked immunosorbent assay (ELISA). HBV X gene sequence-specific shRNA expression vector (pXi-1 and pXi-2) and sequence-unrelated control (pXi-3) were transfected into HepG2-X. Single cell clone with stable expression of shRNA was selected and exposed to propagating culture. The effect of As2O3 on p53 protein expression and activity was re-observed.

RESULTSTotal p53 protein level was up-regulated and its relative activity ratio was enhanced by As2O3 in HepG2 and HepG2-X cells. The total p53 protein level induced by As2O3 was up-regulated by HBV X gene expression, while its relative activity was significantly suppressed. The suppression was removed after HBV X gene expression was repressed by shRNA.

CONCLUSIONSAs2O3 up-regulates p53 protein expression and enhance its activity. HBV X up-regulates As2O3 induced-p53 protein expression while suppresses its activity.

Arsenicals ; pharmacology ; Cell Line, Tumor ; Enzyme-Linked Immunosorbent Assay ; Humans ; Oxides ; pharmacology ; RNA Interference ; Trans-Activators ; genetics ; Tumor Suppressor Protein p53 ; analysis

OBJECTIVETo establish a reliable platform for screening glucokinase activators (GKAs) in vitro.

METHODSPancreatic glucokinase (PGK) protein expressed in a prokaryotic expression system as a histidine-tagged fusion protein from Homo sapiens was produced. Then, response surface methodology (RSM) was used to optimize the microplate-based GKA screening platform. In the first step of optimization with Plackett-Burman design (PBD), initial pH, reaction time and MgCl2 were found to be important factors affecting the activity ratio of GKA (RO-28-1675) significantly. In the second step, a 2(3) full factorial central composite design (CCD) and RSM were applied to the optimal condition determination of each significant variable. A second-order polynomial was determined by a multiple regression analysis of the experimental data.

RESULTSThe following optimal values for the critical factors were obtained: initial pH 0 (7.0), reaction time-0.63 (13.7 min) and MgCl2 0.11 (2.11 mmol/L) with a predicted value of the maximum activity ratio of 34.1%.

CONCLUSIONUnder the optimal conditions, the practical activity ratio is 34.8%. The determination coefficient (R2) is 0.9442, ensuring adequate credibility of the model. LLAE3, extracted from Folium nelumbinis in our laboratory, has prominently activated effects on PGK.

Analysis of Variance ; Drug Discovery ; methods ; Enzyme Activators ; analysis ; Escherichia coli ; genetics ; Genetic Vectors ; Glucokinase ; metabolism ; Humans ; Hydrogen-Ion Concentration ; Hypoglycemic Agents ; analysis ; Kinetics ; Time Factors

Glucokinase (GK) is a new target for the treatment of type II diabetes mellitus (T2DM). In order to find a structure-simplified small molecule GK activator, 19 salicylic acid derivatives were designed and synthesized based on new lead compound (1). Experimental results showed that the potency of compound 8h is superior to control RO-28-0450 in GK activation.
Drug Design ; Enzyme Activation ; drug effects ; Enzyme Activators ; chemical synthesis ; chemistry ; pharmacology ; Glucokinase ; metabolism ; Hypoglycemic Agents ; chemical synthesis ; chemistry ; pharmacology ; Molecular Structure ; Salicylates ; chemical synthesis ; chemistry ; pharmacology ; Thiazoles ; pharmacology