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H Fukao

Publications and source records attributed to H Fukao.

50 records · Page 3Linked to original sources
PubMed1991

Plasminogen activator inhibitor in stomach and colorectal carcinomas.

The content of PAI was measured in carcinoma tissues from the stomach and colorectum divided macroscopically into three portions: the central part of the carcinoma, the marginal part of the carcinoma containing some normal mucosa, and the normal mucosa. The PAI-1 antigen was highest in the central part of the carcinoma. On the other hand, no PAI-1 antigen or activity was observed in the normal mucosa. The PAI-1 in the carcinoma tissues showed a nonlytic zone with a molecular weight of 54 kd by reverse fibrin autography, and this 54 kd band reacted with anti-PAI-1 immunoglobulin G (IgG) on an immunoblotted nitrocellulose membrane. The contents of PAI-2 in the carcinoma tissues were not significantly different from those in the normal mucosa of the stomach and colorectum, respectively. In both the stomach and colorectal carcinomas, the highest value of u-PA/total PA (sum of u-PA and t-PA) was observed in the central part of the carcinoma, followed by the marginal part of the carcinoma, and was lowest in the normal mucosa. We conclude that increased levels of PAI-1 in malignant tissue of the stomach and colorectal tract may serve to modulate extracellular proteolysis by u-PA.

Adult↗
PubMed1991

[Levels of tissue-type plasminogen activator and plasminogen activator inhibitor 1 in disseminated intravascular coagulation syndrome].

Since disseminated intravascular coagulation (DIC) may directly reflect the abnormal regulation of the fibrinolytic system by endothelial cells, we have measured the levels of tissue-type plasminogen activator (t-PA), type 1 PA inhibitor (PAI-1) and t-PA . PAI-1 complex which is formed as a result of interaction on the two factors, in the plasma of patients with DIC (n = 51) and healthy controls (n = 42). Antigens of t-PA, PAI-1 and t-PA . PAI-1 complex were significantly increased in the DIC plasma (36.4 +/- 25.1, 106.8 +/- 54.7 and 46.6 +/- 34.5 ng/ml, respectively) compared with those in normal plasma (8.5 +/- 4.3, 54.4 +/- 21.2 and 8.6 +/- 3.5 ng/ml, respectively). The molar ratio of t-PA to PAI-1 was much higher in the DIC plasma (1:3) than in normal plasma (1:6), which caused enhancement of the whole fibrinolytic activity in the DIC plasma. These changes resulted in significant consumption of plasminogen, alpha 2-plasmin inhibitor (alpha 2-PI) and a significant increase of plasmin . alpha 2-PI complex (PPI) and D-dimer. These results suggest that t-PA and its specific inhibitor PAI-1 both of which are secreted from endothelial cells into blood, play an important role on the progress of DIC.

Adult↗
PubMed1990

Thrombolytic properties of staphylokinase.

We evaluated the properties of recombinant staphylokinase in comparison with those of tissue-type plasminogen activator (t-PA) and streptokinase (SK). The presence of fibrin(ogen) fragment FCB-2 in the reaction mixture increased plasminogen activation by staphylokinase more than 20-fold. Such characteristics are similar to those of t-PA. On the other hand, SK was not affected by the presence of FCB-2. The thrombolytic properties of staphylokinase were studied in a system consisting of a radioactive human plasma clot (125I-fibrinogen-labeled) suspended in the circulating citrated plasma. Significant thrombolysis (50% in 3 hours) was obtained with 2 micrograms/mL of staphylokinase and 4.45 micrograms/mL t-PA, as compared with 12 micrograms/mL for SK. The relative molar potency of staphylokinase, calculated from the molecular weight, was about two times more effective than that of SK, but about half of that of t-PA. Systemic fibrinolytic activation and fibrinogen breakdown was not observed with staphylokinase or t-PA, but was observed with SK. The thrombolytic efficiency of staphylokinase, which was calculated as the ratio of the degree of thrombolysis/the degree of fibrinogenolysis, was about five times greater than that of SK, and about half of that of t-PA. These findings suggest that staphylokinase has higher specific thrombolytic properties and lesser fibrinogenolytic properties than those of SK.

Amino Acid Sequence↗
PubMed1990

Suppression of plasminogen activator inhibitor 1 release by fibrin from human umbilical vein endothelial cells.

The effect of fibrin stimulation on the fibrinolytic potential in cultured human umbilical vein endothelial cells (HUVEC) was investigated in the normal state and aged state. The amount of antigen of the two fibrinolytic factors, tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor 1 (PAI-1), was determined using ELISA and the ABC method, respectively. When a fibrin clot was overlayered on the normal HUVEC, the secretion of t-PA or PAI-1 from the HUVEC was greatly changed. That is, PAI antigen was decreased 3-fold and t-PA antigen increased slightly in the conditioned medium. On the other hand, when the aged HUVEC were stimulated by a fibrin clot, PAI antigen was increased 3-fold and t-PA antigen did not change in the conditioned medium. When the level of fibrinolytic activity in the conditioned medium was expressed as the molar ratio of PAI and t-PA (PAI/t-PA), the value in the fibrin-stimulated normal HUVEC was markedly reduced (a 3.5-fold decrease) when compared with that of the non-stimulated normal HUVEC, reflecting a profibrinolytic state. On the other hand, the value in the fibrin-stimulated aged HUVEC was markedly increased (a 5-fold increase) when compared with that of the non-stimulated aged HUVEC, reflecting an antifibrinolytic state. Actinomycin D- or cycloheximide-treated HUVEC showed no response to the fibrin stimulation. We conclude that the level of HUVEC-mediated fibrinolytic activity was regulated mainly by the production and secretion of PAI from the HUVEC to protect against the generation of thrombi. In the aged HUVEC, the regulatory mechanism acts in an opposite manner and a thrombotic process may be induced.

Cell Survival↗
PubMed1990

Purification and characterization of a plasma factor which cleaves single-chain form of t-PA and u-PA.

Low molecular weight heparin (LMW-heparin) enhanced the amidolytic activity of plasma when the chromogenic substrate, H-D-Ile-Pro-Arg-pNA (S-2288), was used. The amidolytic activity increased in a time-dependent manner as the LMW-heparin concentration increased and reached its peak at around 15 mu/ml. Factor XII-deficient plasma increased the S-2288 amidolytic activity by LMW-heparin. In order to clarify the mechanism of the heparin-induced enhancement of the amidolytic activity, a plasma factor was purified. The plasma factor was obtained from human normal plasma by ammonium sulfate fractionation, followed by successive column chromatography with heparin-Sepharose, zinc chelate-Sepharose, aprotinin-Sepharose and protein A-Sepharose. The plasma factor so purified revealed a major band (88% of total protein) at 80 kD with several minor bands on analysis by SDS-PAGE. The plasma factor exhibited an intrinsic amidolytic activity, which was enhanced by heparin. The plasma factor further enhanced the amidolytic activity of sct-PA and scu-PA, the enhancement of which was of much greater degree than that for LMW-heparin. However, when the two-chain form of t-PA or u-PA was reacted with the plasma factor and LMW-heparin, no enhancement of the amidolytic activity of these enzymes was observed. The plasma factor cleaved a peptide bond of sct-PA and scu-PA and induced a structural change from a single-chain to a two-chain form. The amidolytic activity of the plasma factor was not inhibited by anti-t-PA IgG, anti-u-PA IgG, anti-plasminogen IgG, anti-factor XII IgG or anti-plasma prekallikrein IgG. These findings suggest an important role for the plasma factor in the activation of sct-PA and scu-PA in heparin-dependent fibrinolysis.

Amides↗
PubMed1989

Production and characterization of single-chain tissue-type plasminogen activator produced by an established cell line from human uterine muscle.

This report describes the purification and characterization of single-chain tissue-type plasminogen activator (sct-PA) present in tissue culture medium of a cell line established from human uterine muscle. The cell line used for the experiment, KW, had estrogen receptor. The PA fraction (KW-PA) was purified from the tissue culture medium of KW employing several steps of affinity chromatography and gel filtration in the presence of aprotinin. The final product (KW-PA) of purification, which predominantly contained the inactive form of sct-PA as well as active sct-PA to a lesser extent, revealed a single band with a molecular weight of 70,000 on sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis both in the absence and presence of reducing agent. Electrophoretic enzymography demonstrated a single lytic zone at Mr 70,000. When KW-sct-PA was treated with plasmin, SDS-polyacrylamide gel electrophoresis revealed two bands of Mr 37,000 and 33,000 under reduced conditions. Such plasmin treatment of KW-sct-PA enhanced the enzymatic activity as well as the [3H]DFP incorporation significantly. The KW-sct-PA demonstrated a higher affinity for lysine than did melanoma-t-PA, but the fibrin affinity of KW-sct-PA was identical with that of melanoma-t-PA. Circular dichroism (CD) analysis showed that the CD spectra of KW-sct-PA were different from those of melanoma-t-PA. These results suggest that the single-chain inactive form of t-PA which was obtained from the tissue culture medium of the cell line from human uterine muscle is activated to a two-chain form on plasmin treatment, with an accompanying significant increase in enzymatic activity.

Cell Line↗
PubMed1989

Effect of monensin on secretion of t-PA from melanoma (Bowes).

The secretion of tissue-type plasminogen activator (t-PA) from melanoma cells (Bowes) was investigated with or without monensin treatment. Monensin inhibited secretion of t-PA from the cells to the medium in a dose-and time-dependent manner. The inhibition was accompanied by an intracellular accumulation of t-PA. Electrophoretic enzymography of the cell homogenate showed the main lytic zone at 72 kDa, which reacted with the IgG of anti-t-PA. Analysis of the cell organelles using ultracentrifugation with a discontinuous sucrose density gradient revealed that the activity and the antigen of t-PA were observed near the discontinuous phase of the sucrose gradient. Analysis of 3H-mannose- and 35S-methionine-labeled t-PA in the cell organelles revealed that the radioactivity of each was increased by monensin treatment, and that such treatment increased the ratio of 3H-mannose-related glycoprotein to 35S-methionine-related protein. The sugar chain of intracellular t-PA was analyzed with endoglycosidase H and N-glycanase, which reduced the molecular weight of t-PA by 4.5-10 kDa, indicating the intracellular presence of a high-mannose type sugar chain and a complex-type sugar chain of t-PA. t-PA secreted from the monensin-treated cells possesses a high-mannose type sugar chain only. Therefore, monensin alters the secretion of t-PA by abnormal glycosylation.

Biological Transport, Active↗
PubMed1989

Assay method for single-chain urokinase-type plasminogen activator.

A new assay method for measuring single-chain urokinase-type plasminogen activator (scu-PA) activity by quantitative calorimetric assay method is described. The serine enzyme u-PA activates plasminogen to plasmin, and scu-PA is a precursor protein obtained from a tissue culture of human kidney cells. The scu-PA solution was activated by plasmin to two-chain u-PA, which releases p-nitroaniline from pyro-Glu-Gly-Arg-pNA, and the optical density at 405 nm was measured. One unit of scu-PA was defined on the basis of 1 nmol of p-nitroaniline released from the substrate per second. One unit of scu-PA was found to be equivalent to 335 u-PA (two-chain) units (IU).

Calorimetry↗
PubMed1988

Monoclonal antibody interferes with fibrin binding of t-PA.

Tissue-type plasminogen activator (t-PA) has a high affinity for fibrin, which is in contrast to urokinase-type plasminogen activator (u-PA). The relation between the structure and function of t-PA was investigated using monoclonal antibodies and plasmin digested t-PA fragments. The results obtained indicated that the three dimensional structure of kringle 2 is necessary for t-PA to develop its fibrin affinity. The monoclonal antibodies which interfered with the fibrin binding ability of native t-PA had two separate epitopes, one in kringle 2 and other in the N-terminal region of the light chain of t-PA.

Antibodies, Monoclonal↗
PubMed1988

Characterization of plasminogen activator produced by an established cell line from human ovary.

An established cell line (OC-1) was obtained from human ovarian tissue, which yielded a high concentration of plasminogen activator (PA) in the culture medium. The PA (OC-1-PA) produced by the cell line was purified and compared with urokinase (UK), proform of UK (pro-UK), and tissue-type PA (t-PA) purified from human melanoma cells (Bowes). OC-1-PA was purified by Zn chelate-Sepharose affinity chromatography followed by high-performance liquid chromatography with a Zn chelate-5PW column and with a p-amino-benzamidine-5PW column, giving a yield of 58.3% and a purification factor of 15,439. This purified material revealed a single band of Mr 55,000 on sodium dodecylsulfate polyacrylamide gel electrophoresis in the presence or absence of reducing agents. Electrophoretic enzymography demonstrated that the Mr 55,000 protein band had a plasminogen-dependent fibrinolytic activity. Treatment with plasmin did not change the Mr even in the presence of reducing agents. These results suggest that OC-1-PA has a single-chain structure protected from protease degradation, which is completely different from UK. The activator had higher affinities for lysine and fibrin than those of UK or pro-UK. An immunological study demonstrated that OC-1-PA cross-reacted with anti-UK IgG but not with anti-t-PA IgG. All these findings indicate that OC-1-PA belongs immunologically to the UK type, but its structure differs from that of UK.

Cell Line↗
PubMed1988

New type of plasminogen activator produced by an established cell line from human ovary.

An established cell line (OC-1) was obtained from human ovarian tissue, which yielded a high concentration of plasminogen activator (PA) in the culture medium. The PA (OC-1-PA) produced by the cell line was purified and compared with urokinase (u-PA), proform of UK (scu-PA), and tissue-type PA (t-PA) purified from human melanoma cells (Bowes). OC-1-PA was purified by Zn chelate-Sepharose affinity chromatography followed by high performance liquid chromatography with a Zn chelate-5PW column and with a p-aminobenzamidine-5PW column, giving a yield of 58.3% and a purification factor of 15,439. This purified material revealed a single band of Mr 55,000 on sodium dodecylsulfate polyacrylamide gel electrophoresis in the presence or absence of reducing agents. Electrophoretic enzymography demonstrated that the Mr 55,000 protein band had a plasminogen-dependent fibrinolytic activity. Treatment with plasmin did not change the Mr even in the presence of reducing agents. These results suggest that OC-1-PA has a single-chain structure protected from protease degradation, which is completely different from UK. The activator had higher affinities for lysine and fibrin than those of u-PA or scu-PA. An immunological study demonstrated that OC-1-PA cross-reacted with anti-u-PA IgG but not with anti-t-PA IgG. All these findings indicate that OC-1-PA belongs immunologically to the u-PA type, but its structure differs from that of u-PA.

Cell Line↗
PubMed1986

High-performance chromatographic method for the purification of tissue-type plasminogen activator.

High-performance affinity chromatography was performed on five ligand-bound columns in an attempt to purify tissue-type plasminogen activator (t-PA), which is a glycoprotein with a high affinity for fibrin and also has two Kringle structures and finger-domain in its molecule. The five columns were concanavalin A-5PW, p-aminobenzamidine-5PW, imidinodiacetic acid-5PW, boric acid-5PW and lysine-5PW. All five were able to rapidly separate t-PA from contaminating proteins, with high resolution and recovery.

Animals↗
PubMed1984

Sakyomicins A, B, C and D: new quinone-type antibiotics produced by a strain of Nocardia. Taxonomy, production, isolation and biological properties.

Actinomycete strain M-53, a new soil isolate, was found to produce four quinone-type antibiotics. Antibiotic sakyomicin components, A, B, C and D were isolated from the fermentation broth of strain M-53 by XAD-2 column chromatography, silica gel column chromatography and Sephadex LH-20 column chromatography. The components are active against Gram-positive bacteria. Strain M-53 was identified as a strain of genus Nocardia.

Anti-Bacterial Agents↗