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Y Takada

Publications and source records attributed to Y Takada.

At least 541 records · Page 30Linked to original sources
PubMed1988

[The studies on the loss and recovery of the steroid binding ability of rat liver glucocorticoid receptor].

We examined the mechanism of loss and recovery of steroid binding activity (inactivation and reactivation) of the rat liver glucocorticoid receptor (GR) in the cell free condition. Male Sprague-Dawley rats were adrenectomized 2 days before the hepatectomy for the experiments, and were fed with normal diet and saline. The cytosol was prepared in 10mM phosphate buffer (pH 7.4) containing 10% glycerol (PG buffer). The nuclei were isolated by centrifuging at 800 x g for 20 min at 4 degrees C in 10mM phosphate buffer (pH 7.4) containing 0.32M sucrose, 3mM MgCl2, then centrifuged again at 40,000 x g for 20 min at 4 degrees C in 10mM phosphate buffer (pH 7.4) containing 1.8M sucrose, 3mM MgCl2. The glucocorticoid receptor was partially purified by affinity chromatography with DOC-Sepharose 6B, and column chromatography with Sephacryl S-300 and DEAE-cellulose in the presence of molybdate throughout the procedure. The steroid binding activity of the cytosol prepared in PG buffer was inactivated by following procedures; either 1) treatment with 1% activated charcoal, 2) dialysis against 500 volumes of PG buffer, 3) incubation at 37 degrees C for 30 min, or 4) incubation in high salt condition (0.4M KCl) at 0 degrees C for 16h. When the steroid binding activity was inactivated by incubation at 0 degrees C for 16hr and charcoal treatment, dithiothreitol (DTT) and NADPH were effective on reactivation, while glutathione (reduced form) was ineffective. Antipain increased the reactivated steroid binding activity in these cases. The steroid binding activity, which was inactivated by dialysis, was slightly recovered by DTT or NADPH, but antipain was ineffective. We could not reactivate steroid binding activity which was inactivated by incubation at 37 degrees C for 30 min or high salt (0.4M KCl) treatment with any reagent we tested. Leupeptin, however, partially prevented the high salt inactivation. When the cytosol prepared in PG buffer was labeled with 3H-dexamethasone (Dex) and then incubated at 25 degrees C for 30 min, 3H-Dex labeled steroid receptors was transformed and it also lost its steroid binding activity partially. DTT or NADPH increased steroid binding activity, but did not increase nuclear binding activity. Interestingly, by dialysis, the cytosol glucocorticoid receptors was not transformed in the PG buffer. We also examined the reactivation of steroid receptor using the partially purified receptor. To remove molybdate, the partially purified receptor was dialyzed against PG buffer. Large part of steroid binding activity was lost during the dialysis. DTT, however, reactivated steroid binding activity without molybdate, and also S-value of reactivated receptor was undistinguishable from the untransformed (molybdate stabilized) receptor.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1988

Relationship between smoking and fibrinolytic system with special reference to t-PA and PA inhibitor.

Recently tissue plasminogen activator (t-PA) has been clinically applied to the thrombolytic therapy of myocardial infarction. We investigated relationship between cigarette smoking and fibrinolytic system, namely the plasma level of t-PA antigen, plasminogen activator inhibitor (PAI), and PA activity. Nineteen healthy volunteers were asked to smoke for 10 min. The plasma levels of t-PA antigen, PAI activity, PA activity and catecholamine were measured together with measurement of blood pressure and heart rate before, soon after or 30 min after cigarette smoking. Plasma t-PA antigen after cigarette smoking increased to 8.83 +/- 3.11 ng per ml, significantly higher (p less than 0.005) than 6.35 +/- 1.7 ng/ml before cigarette smoking. Plasma PAI activity after cigarette smoking was 5.52 +/- 2.03 u/ml, significantly higher (p less than 0.05) than 4.18 +/- 1.06 u/ml before smoking. Plasma PA activity after smoking was 6.28 +/- 3.85 u/ml significantly higher (p less than 0.05) than 4. 49 +/- 2.74 u/ml. Furthermore, plasma epinephrine level after smoking increased to 59.1 +/- 52.4 pg/ml (p less than 0.1), compared with 36.2 +/- 22.5 pg/ml before smoking. There was a positive correlation between the rate of increase in plasma t-PA antigen and the rate of increase in plasma epinephrine after smoking. It is suggested that plasma epinephrine was related to the mechanism of increased plasma levels of t-PA in cigarette smoking.

Adult↗
PubMed1988

Multiple very late antigen (VLA) heterodimers on platelets. Evidence for distinct VLA-2, VLA-5 (fibronectin receptor), and VLA-6 structures.

After removal of very late antigen (VLA) 2 material from a radiolabeled detergent lysate of platelets, another VLA heterodimer was precipitated using antibody to the common VLA beta subunit. This structure was identified as VLA-5 because it contained VLA beta plus an alpha subunit that was (i) recognized by anti-alpha 5 antibodies and (ii) cleaved by V8 protease to yield a characteristic alpha 5-like pattern of peptide fragments. Besides VLA-2 and VLA-5, a third heterodimer, here named VLA-6, was also present on platelets. VLA-6 (an alpha 6 beta complex) was defined using the monoclonal antibody GoH3 (Sonnenberg, A., Janssen, H., Hogervorst, F., Calafat, J., and Hilgers, J. (1987) J. Biol. Chem. 262, 10376-10383). Although it resembled VLA-5 in size, VLA-6 was different from VLA-5 because (i) removal of the alpha 5 subunit did not remove alpha 6, (ii) removal of alpha 6 by the GoH3 antibody did not remove alpha 5, (iii) the alpha 5 and alpha 6 subunits had very distinct one-dimensional V8 peptide maps, and (iv) the alpha 6 and alpha 5 subunits had distinct migration patterns on two-dimensional O'Farrell gels. The beta subunit of VLA-6 was identified as the common VLA beta subunit because (i) it was recognized by anti-VLA beta antibody and (ii) it yielded a V8 protease cleavage map characteristic of beta. VLA-6 was not readily seen in anti-VLA beta immunoprecipitations, apparently because the alpha 6 subunit is only loosely or partially associated with the VLA beta subunit. Because VLA-5 and VLA-6 both closely resemble the previously defined Ic-IIa platelet protein complex, it is likely that there is more than one platelet "Ic" protein complexed with IIa.

Antibodies, Monoclonal↗
PubMed1988

Kinetic analyses of the enhancement of the activities of t-PA induced by the presence of monoclonal antibody (C9-5).

Nine monoclonal antibodies (Mab) against two chain tissue plasminogen activator (t-PA(W] were obtained. The effects of these Mabs on the enzymatic activities of one chain t-PA (t-PA(TD] and two chain t-PA (t-PA(W] were examined by incubating t-PA and Mabs with S-2288, or with plasminogen (plg) and S-2251 in the presence or absence of fibrin. One of Mabs called C9-5 significantly enhanced the activities of t-PA, which were kinetically analyzed. The kinetic analyses of the hydrolysis of S-2288 by t-PA showed increase in kcat from 5.17/sec to 7.75/sec in the presence of 75 micrograms/ml of C9-5 without change in Km. When Glu-plg was activated by t-PA(TD) in the absence of fibrin, Km decreased from 2 microM to 0.17 microM in the presence of C9-5 without change in Vmax. The addition of fibrin resulted in further decrease in Km from 0.133 microM to 0.077 microM. When Lys-plg was activated by t-PA(TD) in the absence of fibrin, Km decreased from 0.408 microM to 0.185 microM in the presence of C9-5, and kcat increased from 0.131 sec-1 to 0.465 sec-1. The presence of fibrin further increased kcat of the activation of Lys-plg. Similar results were obtained when plasminogen was activated by t-PA(W). Mab, C9-5, was shown to bind to B-chain of t-PA by immunoblotting. These results suggest that a dimolecular complex of t-PA and C9-5 has a higher affinity to plasminogen in the presence or absence of fibrin.

Animals↗
PubMed1988

The urokinase type of plasminogen activator in cancer of digestive tracts.

The amounts of urokinase (UK) antigen and tissue plasminogen activator (t-PA) antigen were determined in plasma, urine and tissues of patients with cancer of digestive tracts. Urinary levels of UK, but not those of t-PA increased in patients with cancer, and generally decreased after the removal of cancer by operation. Urinary UK levels kept increasing in patients with recurrence of cancer or with metastasis into liver or peritoneum. Plasma levels of t-PA, but not those of UK decreased in patients with cancer. When the amounts of UK were compared in cancer tissues and their adjacent normal mucosal layer, cancer tissues always had higher levels of UK, but t-PA levels were same between tumor tissues and normal mucosa. The results suggest that the type of plasminogen activator was UK-type in cancer of digestive tracts.

Adenocarcinoma↗
PubMed1988

Degradation of Glu- and Lys-plasminogen by elastase in the presence or absence of tranexamic acid.

Glu-plasminogen (Glu-plg) was degraded by elastase in the presence or absence of tranexamic acid. Glu-plg was degraded faster in the presence of tranexamic acid. Increase in the concentration of tranexamic acid resulted in increase in the appearance of degradation products, reaching a plateau level at 1 mM of tranexamic acid. Fifty percent increase in the concentration of one of degradation products was obtained at 0.22 mM of tranexamic acid, which is similar to a dissociation constant (Kd) of low affinity lysine binding sites (LBS) with tranexamic acid. As to the degradation rate of two isozymes of Glu-plg (Glu-plg I and II), Glu-plg II containing one carbohydrate chain was degraded faster than Glu-plg I containing two carbohydrate chains. Comparison of the degradation rates of Glu-plg and Lys-plg indicated that Lys-plg was degraded faster.

Cyclohexanecarboxylic Acids↗
PubMed1988

Autoradiographic and immunoblotting analyses of the activation pathways of plasminogen by urokinase and tissue plasminogen activator in the plasma or clotted plasma.

The activation pathway of Glu-plasminogen (Glu-plg) was analyzed by using immunoblotting and autoradiography. Glu-plg was slowly activated to Glu-plasmin by urokinase (UK) and tissue plasminogen activator (t-PA) in the plasma. Significant amounts of a complex of alpha 2AP with plasmin were found. On the other hand, UK and t-PA rapidly activated Glu-plg in the clotted plasma. The major form of plasmin was Lys-form, and significant amounts of alpha 2M complexed with plasmin were found. A complex of alpha 2AP with plasmin was not so significantly formed in the clot, confirming ineffective inhibition of plasmin by alpha 2AP in the presence of fibrin. Glu-plg was directly activated by UK or t-PA to Glu-plasmin and further to Lys-plasmin in the plasma or clotted plasma.

Autoradiography↗
PubMed1988

Activation of Val442-plasminogen (mini-plasminogen) by urokinase, streptokinase and tissue plasminogen activator.

Glu-plasminogen (Glu-plg), Lys-plg and Val442-plg (mini-plg) were activated by urokinase (UK), streptokinase (SK) or tissue plasminogen activator (t-PA). Their activation rates were kinetically analyzed. UK activated Lys-plg with smaller Km and nearly identical Vmax as Glu-plg. Mini-plg was activated by UK with higher Vmax but with the same Km as Glu-plg. On the other hand, t-PA activated Glu-plg, Lys-plg and mini-plg with the same Vmax, but Km was in the order of Glu-plg greater than mini-plg greater than Lys-plg. Fibrin hardly enhanced the UK activation of mini-plg, and fibrinogen, fragment D or E did not enhance the activation of mini-plg by UK. Only D domain, but not E domain, complexed with K5 of mini-plg and SK hydrolysed S-2251 faster than a dimolecular complex of mini-plg and SK, thus a trimolecular mixture of D, SK and mini-plg having a better activator activity than SK-mini-plg complex. In the t-PA activation of mini-plg, fibrin and fibrinogen enhanced the activation, but fragment D or E was ineffective, suggesting that a molecule containing more than one domain was required for the enhanced activation of mini-plg by t-PA.

Fibrin↗
PubMed1988

Extracellular matrix receptors, ECMRII and ECMRI, for collagen and fibronectin correspond to VLA-2 and VLA-3 in the VLA family of heterodimers.

The Very Late Activation Antigen (VLA) proteins are a family of five related heterodimers, which also are part of the integrin superfamily of cell adhesion molecules. Except for the identification of VLA-5 as a fibronectin receptor structure, the functions of the VLA proteins have remained unclarified. In this paper, immunoprecipitation experiments with both anti-alpha and anti-beta subunit antibodies showed that the previously identified cell adhesion receptor for collagen, extracellular matrix receptor II (ECMRII), is equivalent to VLA-2. At the same time a previously described multispecific cell adhesion receptor for collagen, fibronectin, and laminin (ECMRI) has been shown to be identical to VLA-3. Although the mAb 12F1 and P1H5 both recognized VLA-2 (ECMRII), they appeared to define distinct epitopes on the alpha 2 subunit. On the other hand, the mAb P1B5 and J143 recognized the alpha 3 subunit of VLA-3 (ECMRI) at or near the same site. Consistent with the collagen receptor functions of VLA-2 (ECMRII) and VLA-3 (ECMRI), anti-VLA beta antiserum blocked cell attachment to collagen.

Antibodies, Monoclonal↗
PubMed1988

Measurements of the concentration of free plasminogen activator inhibitor (PAI-1) and its complex with tissue plasminogen activator in human plasma.

A new assay system to measure free plasminogen activator inhibitor (PAI-1) and its complex with tissue plasminogen activator (t-PA) was developed. A silicone piece coupled with anti-PAI-1 monoclonal antibody was incubated with human plasma or human plasma which had previously been incubated with t-PA in order to convert all the free PAI-1 to t-PA-PAI-1 complex. After incubation, the amounts of t-PA bound on each silicone piece were measured by enzyme immunoassay. The amounts of t-PA on a silicone piece incubated with plasma represent the amounts of t-PA-PAI-1 complex in the original plasma, and the amounts of t-PA on a silicone piece incubated with plasma previously added with t-PA represent total PAI-1 (the complex plus free PAI-1). Using the technique, the concentration of t-PA-PAI-1 complex seems to increase with age and there was a good correlation between the concentration of free PAI-1 and t-PA-PAI-1 complex in the plasma.

Antibodies, Monoclonal↗
PubMed1988

Changes in various parameters of fibrinolysis in persons infused with tissue plasminogen activator: special reference to plasminogen activator inhibitor.

Tissue plasminogen activator (t-PA) purified from culture of human lung cells was infused over 30 min at the dose of 7.2 mg or 14.4 mg per person into human male volunteers. T-PA declined rapidly after the cessation of infusion with T1/2, alpha of about 4 min and T1/2, beta of about 40 min. The plasma fibrinogen and plasminogen did not change during and after the infusion of t-PA. The concentration of alpha 2antiplasmin (alpha 2AP) did not change but that of alpha 2AP-plasmin complex increased to about 200 nM at the infusion of 14.4 mg/person. Fragment D was hardly found at the infusion of 7.2 mg but some increase in the concentration of D was observed at the infusion of 14.4 mg. The concentration of free plasminogen activator inhibitor (PAI-1) rapidly declined and kept low up to 150 min, and returned to normal the following day. The concentration of t-PA-PAI-1 complex increased rapidly, and slowly declined after the end of the infusion of t-PA, suggesting no significant release of PAI-1 in response to increase in plasma concentration of t-PA or t-PA-PAI-1 complex. The addition of thrombin to the blood after withdrawal resulted in the significant formation of D-dimer, even at 150 min after the infusion. There was not hyperaggregability of platelets during and after the infusion of t-PA. These results suggest that the infusion of t-PA at the dose of 7.2 mg or 14.4 mg/person for 30 min resulted in high fibrinolytic potential in the blood without the breakdown of fibrinogen or increase in PAI-1.

Adult↗