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J Mimuro

Publications and source records attributed to J Mimuro.

At least 37 records · Page 2Linked to original sources
PubMed1996

A gamma Gly-268 to Glu substitution is responsible for impaired fibrin assembly in a homozygous dysfibrinogen Kurashiki I.

A new type of gamma Gly-268 (GGA) to Glu (GAA) substitution has been identified in a homozygous dysfibrinogen by analyses of the affected polypeptide and its encoding gene derived from a 58 year-old man manifesting no major bleeding or thrombosis. The functional abnormality was characterized by impaired fibrin assembly most likely due to failure to construct properly aligned double-stranded fibrin protofibrils. This presumption was deduced from the following findings: (1) Factor XIIIa-catalyzed cross-linking of the fibrin gamma-chains progressed in a normal fashion, indicating that the contact between the central E domain of one fibrin monomer and the D domain of another took place normally; (2) Nevertheless, factor XIIIa-catalyzed cross-linking of the fibrinogen gamma-chains was obviously delayed, suggesting that longitudinal association of D domains of different fibrin monomers, ie, D:D association was perturbed; (3) Plasminogen activation catalyzed by tissue-type plasminogen activator was not as efficiently facilitated by polymerizing fibrin monomer derived from the patient as by the normal counterpart. Therefore, gamma Gly-268 would not be involved in the 'a' site residing in the D domain, which functions as a complementary binding site with the thrombin-activated 'A' site in the central E domain, but would be rather involved in the D:D self association sites recently proposed for human fibrinogen. Thus, the gamma Glu-268 substitution newly identified in this homozygous dysfibrinogen seems to impair proper alignment of adjacent D domains of neighboring fibrin molecules in the double-stranded fibrin protofibril, resulting in delayed fibrin gel formation.

Afibrinogenemia↗
PubMed1996

Expression of plasminogen activator inhibitor 2 in the adult and embryonic mouse tissues.

We have studied the expression of plasminogen activator inhibitor 2 (PAI-2) in the adult mouse and during embryonic development using immunohistochemistry and the polymerase-chain reaction. Mouse PAI-2 mRNA was mainly expressed in the skin, bone-marrow, spleen, lung, thymus, and urinary bladder. Immunohistochemical studies suggested that PAI-2 was synthesized in macrophages and epithelial cells such as skin epithelial cells, transitional cells of the urinary bladder, and mesothelial cell of peritoneum and pleura. PAI-2 mRNA and antigen expression was observed only in the skin of 15 day-old mouse embryos. These data suggest that PAI-2 may play a role in protecting the mouse embryo from the protease attack in the amniotic fluid and also in maintaining and/or protecting the structure of various surfaces in the mouse.

Aging↗
PubMed1995

Effect of staphylokinase concentration of plasminogen activation.

Activation of Glu- and Lys-plasminogen by various concentrations of recombinant staphylokinase (SAK) were studied by the generation of amidolytic activity from the chromogenic substrate S-2251(H-D-Val-Leu-Lys-pNA) and by SDS-PAGE analysis. Surprisingly, excess SAK decreased and fixed the rate of S-2251 hydrolysis in a mixture of Lys-plasminogen and SAK. Since the effect of SAK on S-2251 hydrolysis by plasma was similar, the hydrolysis kinetics by free plasmin and plasmin-SAK complex were studied. Hydrolysis by either enzyme form followed Michaelis-Menten kinetics with a Km of 0.38 mM for plasma and 3.74 mM for SAK-plasmin complex. The catalytic rate constant was 22.7 s-1 for plasmin and 21.0 s-1 for the SAK-plasmin complex. With excess SAK and vigorous removal of plasmin activity from plasminogen, the pre-activation lag period differed greatly between Glu- and Lys-plasminogen. Based on the different substrate specificity of plasmin and plasmin-SAK complex, we analyzed the Glu-plasminogen activation with either catalytic or excess SAK. With excess SAK, almost no Lys-plasminogen was detectable and whole Glu-plasminogen was converted directly to Glu-plasmin, then gradually to Lys-plasmin. In contrast, Lys-plaminogen appeared rapidly with catalytic amount of SAK. These results suggest that inhibition of Glu-plasminogen to Lys-plasminogen to Lys-plasminogen conversion in the plasminogen-SAK complex in the presence of excess SAK prolonged the initial lag phase of activation.

Dose-Response Relationship, Drug↗
PubMed1994

Effect of glycosidase inhibitors on the biosynthesis of alpha 2-plasmin inhibitor and antithrombin III in Hep G2 cells.

We studied the effect of the glucosidase I inhibitor, N-methyl-1-deoxynojirimycin (MdN) and the mannosidase inhibitor, 1-deoxymannojirimycin (dMM) on the biosynthesis and secretion of alpha 2-plasmin inhibitor (alpha 2-PI) and antithrombin III (ATIII) in cultures of human hepatoma (Hep-G2) cells. Incubation with 1 mM MdN decreased secreted alpha 2-PI activity and antigen levels by about 40%, whereas those of ATIII were not affected. Neither inhibitor affected the messenger RNA levels as determined by Northern blotting. Pulse-chase studies using [35S]-methionine showed that MdN decreased alpha 2-PI and ATIII secretion rates. By the 18 h chase, MdN had decreased secreted alpha 2-PI to 50-60%, with little effect on ATIII. Intracellular forms of alpha 2-PI or ATIII synthesized by cells treated with 1 mM MdN were sensitive to endoglycosidase H (Endo H), whereas almost all the secreted forms were resistant, suggesting the presence of complex-type oligosaccharides. In the presence of 1 mM dMM, cells synthesized Endo H-sensitive alpha 2-PI and ATIII with similar secretion rates. These results suggest that retention of glucose on N-linked oligosaccharides not only retards the exit of alpha 2-PI and ATIII, but also changes the catabolic rate of alpha 2-PI in the endoplasmic reticulum.

1-Deoxynojirimycin↗
PubMed1993

Identification of the plasminogen activator inhibitor-1 binding heptapeptide in vitronectin.

We have shown that a heptapeptide which resides in the middle part of vitronectin (VN) is responsible for binding to plasminogen activator inhibitor-1 (PAI-1). A single PAI-1 binding peptide was isolated from human VN after limited proteolysis with protease V8. The amino acid sequence of the fragment corresponded to residues Gly-115-Glu-121 of VN. A murine monoclonal antibody (JYV-1) raised against human VN bond to the same fragment and inhibited binding of PAI-1 to VN. A synthetic peptide (V-115), comprising residues Gly-115-Glu-121 of human VN, competed with VN for both PAI-1 and JYV-1 in a dose-dependent manner. Synthetic peptide V-111 (Ser-111-Glu-121) had a stronger inhibitory effect than V-115 on binding of PAI-1 or JYV-1 to VN. V-111 also inhibited the binding of human PAI-1 to mouse and rabbit VN. The functional half-life of PAI-1 activity was prolonged approximately 2-fold in the presence of V-111 (1 mM). This stabilizing effect of V-111 was equivalent to intact VN, although a 1000-fold higher molar concentration of V-111 over VN was required. These data indicated that VN residues Gly-115-Glu-121 contain a PAI-1 binding site.

Amino Acid Sequence↗
PubMed1993

Fibrinolytic components in fetal membranes and amniotic fluid.

OBJECTIVE: We evaluated fibrinolytic components in plasma and amniotic fluid of pregnant women and in postpartum fetal membranes. STUDY DESIGN: Fibrinolytic parameters in amniotic fluid and plasma were measured by means of enzyme-linked immunosorbent assays. Fetal membranes collected after spontaneous labor at term were analyzed by immunohistochemical methods with immunospecific antibodies against fibrinolytic components. RESULTS: Amniotic fluid contained high plasminogen activator inhibitor-1 concentrations but had low activity. Strong staining for plasminogen activator inhibitor-1 and vitronectin was observed in chorionic trophoblasts and moderate staining in decidual connective tissue. Strong staining for plasminogen activator inhibitor-2 was seen in decidual cells. Although prominent staining of plasminogen activators and plasminogen were observed in the amniotic epithelium, virtually no plasminogen activator inhibitor-1, plasminogen activator inhibitor-2, or alpha 2-plasmin inhibitor staining was detected. CONCLUSION: The delicate balance of fibrinolytic activators and inhibitors in fetal membranes and amniotic fluid may contribute to the triggering of membrane rupture at term.

Amniotic Fluid↗
PubMed1993

[Refractory bilateral pneumothoraces complicated with interstitial pneumonitis after bone marrow transplantation].

We report here a case of 33 year-old-man with refractory bilateral pneumothoraces during the treatment for interstitial pneumonitis 6 months after bone marrow transplantation (BMT). He was diagnosed as having acute myelogenous leukemia (AML) M1. He was treated with chemotherapy, and cerebral irradiation. BMT was performed in August 1989 from a sibling donor whose human leukocyte antigen was matched, ABO blood type mismatched. Preconditioning regimen was cyclophosphamide and total body irradiation (TBI). BMT was successful without major graft versus host disease. Thereafter he complained of respiratory symptom and was admitted on June 14 1990. Computed tomogram (CT) scan showed interstitial and alveolar shadows. We started the treatment against bacterial infection, Pneumocystis carinii, cytomegalovirus (CMV) and against interstitial pneumonitis with bolus dose of steroid. The transbronchial lung biopsy specimen revealed interstitial pneumonitis without typical CMV nor pneumocystis carinii pneumonia. Although a CT scan showed improvement of pneumonitis, bilateral pneumothoraces occurred. The adhesion therapy became successful after the reduction of steroid dosage. A pneumothorax rarely occurs after BMT. In this case it is speculated that TBI might be responsible for interstitial pneumonitis, and the steroid might have inhibited the adhesion therapy of pneumothorax.

Adult↗
PubMed1993

An abnormal protein C (protein C Yonago) with an amino acid substitution of Gly for Arg-15 caused by a single base mutation of C to G in codon 57 (CGG-->GGG). Deteriorated calcium-dependent conformation of the gamma-carboxyglutamic acid domain relevant to a thrombotic tendency.

Protein C Yonago is a dysfunctional protein C characterized by defective anticoagulant activity determined by a coagulation assay and normal amidolytic activity measured on a synthetic substrate S-2366 (Iijima et al., Thromb Res 1991;63:249-257). We have identified a single point mutation of C to G in codon 57 (CGG-->GGG) of the gene for protein C Yonago by genetic analysis utilizing the polymerase chain reaction. The mutation should have resulted in an amino acid substitution of Gly for Arg at position 15 of the light chain of mature protein C. No mutations were found in nucleotides spanning the putative gamma-carboxylase recognition site or gamma-carboxyglutamic acid residues of protein C. Protein C Yonago was non-reactive to monoclonal antibodies JTC-1 and -3 that solely recognized the calcium-dependent conformation of the gamma-carboxyglutamic acid domain. This indicated that the mutation had critically perturbed the highly conserved structure of the gamma-carboxyglutamic acid domain. Thus, the calcium-dependent conformation required for the phospholipids binding to exert the physiological functions of protein C may not have been elicited normally in this abnormal protein C, resulting in defective generation of anticoagulant activities in plasma. Consequently, no anticoagulant activities may have been generated in vivo.

1-Carboxyglutamic Acid↗
PubMed1992

Reversible interactions between plasminogen activators and plasminogen activator inhibitor-1.

We have shown that the urokinase (UK) kringle domain contains a high-affinity plasminogen activator inhibitor-1 (PAI-1) binding site, responsible for the 10-fold faster complex formation between UK and PAI-1 than between PAI-1 and low-molecular-weight urokinase (LMWUK). Complex formation between UK and PAI-1, but not between LMWUK and PAI-1, was suppressed 10-fold in the presence of peptide U-107 derived from the UK kringle domain. Peptide U-373 derived from the UK catalytic domain slowed complex formation between UK and PAI-1 and also LMWUK and PAI-1. Inactivation of tissue-type plasminogen activator (tPA) by PAI-1 was slowed 10-fold in the presence of peptides derived from the tPA finger and kringle-2 domains. DFP-inactivated (DIP) UK and both forms of DIP-tPA inhibited PAI-1 binding to U-107 and to U-373 whereas single-chain urokinase-type PA (scuPA) was unable to compete with either peptide for PAI-1 binding. These data suggest that the reversible PAI-1 binding site in the UK A-chain plays a role in the rapid association with PAI-1 as important as those that reside in the tPA A-chain and that reversible PAI-1 binding sites are expressed on the surface of UK upon conversion from scuPA, in contrast to tPA.

Amino Acid Sequence↗
PubMed1992

Interactions between the finger and kringle-2 domains of tissue-type plasminogen activator and plasminogen activator inhibitor-1.

We have shown that plasminogen activator inhibitor-1 (PAI-1) inhibits the fibrin binding of both the single chain and two chain forms of tissue-type plasminogen activator (tPA) through two different mechanisms. PAI-1 inhibits the finger domain-dependent fibrin binding of diisopropylfluorophosphate-inactivated single chain tPA and the kringle-2 domain-dependent fibrin binding of diisopropylfluorophosphate-inactivated two chain tPA. In accordance with the data, preformed complexes of single chain tPA/PAI-1 and of two chain tPA/PAI-1 lost the fibrin binding abilities mediated by the finger and kringle-2 domains, respectively. These effects of PAI-1 appear to be mediated by steric hindrance of the fibrin binding sites after PAI-1 binding to adjacent regions in the functional domains of tPA. We thus propose a model in which a PAI-1 binding site resides in the finger domain of a single chain, and plays a role in the reversible association of single chain tPA and PAI-1. Conformational changes may take place during the conversion of single chain tPA to two chain tPA, resulting in burying of the original PAI-1 binding site and exposure of an alternate PAI-1 binding site on the surface of the kringle-2 domain.

DNA-Binding Proteins↗
PubMed1992

Gene analyses of abnormal fibrinogens with a mutation in the gamma chain.

In four abnormal fibrinogens with a point mutation in the gamma chain, all characterized by impaired fibrin polymerization, we identified single base exchanges in the respective mutant gamma chain genes by polymerase chain reaction followed by DNA sequence analysis. These base exchanges accounted for the amino acid substitutions previously reported from our laboratory. They were exchanges of C to T (CGC for gamma Arg-275 to TGC for Cys) in fibrinogen Osaka II, T to G (AAT for gamma Asn-308 to AAG for Lys) in fibrinogen Kyoto I, T to C (ATG for gamma Met-310 to ACG for Thr) in fibrinogen Asahi, and G to T (GAT for gamma Asp-330 to TAT for Tyr) in fibrinogen Kyoto III. These base exchanges were found to reside in exon VIII of the gamma chain gene. Since many abnormal molecules are associated with polymerization defects, unless associated with the impaired release of fibrinopeptides A and/or B, exon VIII of the gamma chain gene may deserve careful study to define the structural alterations.

DNA Mutational Analysis↗
PubMed1991

The plasminogen activator inhibitor-1 binding site in the kringle-2 domain of tissue-type plasminogen activator.

We have shown that synthetic peptides containing the amino acid sequence Asn-Arg-Arg-Leu, derived from the amino acid sequence of the inner loop of the kringle-2 domain of tissue-type plasminogen activator (tPA), inhibited complex formation between two chain tPA and plasminogen activator inhibitor-1 (PAI-1) by binding to PAI-1. This binding was reversible and was inhibited by not only tPA but also by enzymatically inactive tPA. Quantitative analyses of the interaction of PAI-1 with the peptide containing the Asn-Arg-Arg-Leu sequence indicated that the PAI-1 binding site residues in the inner loop of the kringle-2 domain and is preferentially expressed in two chain tPA.

Amino Acid Sequence↗
PubMed1991

Inhibitors of urokinase and thrombin in cultured neural cells.

Recent studies have suggested important roles for certain proteases and protease inhibitors in the growth and development of the CNS. In the present studies, inhibitors of urokinase or thrombin in cultured neural cells and serum-free medium from the cells were identified by screening for components that formed sodium dodecyl sulfate-stable complexes with 125I-urokinase or 125I-thrombin. Rinsed glioblastoma possessed two components that complexed 125I-urokinase. One was type 1 plasminogen activator inhibitor (PAI-1), because the 125I-urokinase-containing complexes were immunoprecipitated with anti-PAI-1 antibodies. The other component formed complexes with 125I-urokinase that were not recognized by antibodies to PAI-1 or protease nexin-1 (PN-1). Its identity is unknown. In addition to these cell-bound components, the glioblastoma cells also secreted two inhibitors that formed complexes with 125I-urokinase; one was PAI-1, and the other was PN-1. The secreted PN-1 also formed complexes with 125I-thrombin. It was the only thrombin inhibitor detected in these studies. Human neuroblastoma cells did not contain components that formed detectable complexes with either 125I-urokinase or 125I-thrombin. However, human neuroblastoma cells did contain very low levels of PN-1 mRNA and PN-1 protein. Added PN-1 bound to the surface of both glioblastoma and neuroblastoma cells. This interaction accelerated the inhibition of thrombin by PN-1 and blocked the ability of PN-1 to form complexes with 125I-urokinase. Thus, cell-bound PN-1 was a specific thrombin inhibitor.(ABSTRACT TRUNCATED AT 250 WORDS)

Amyloid beta-Protein Precursor↗
PubMed1991

[Type 1 plasminogen activator inhibitor: its role in biological reactions].

The endothelial cells (ECs) are antithrombotic in the physiological states and maintains the integrity of blood circulation. However, ECs turn to be thrombotic upon being stimulated by various physiological mediators. These functions are mainly achieved by changing specific protein synthesis in ECs. Type 1 plasminogen activator inhibitor (PAI-1) is a serine protease inhibitor synthesized by ECs and thought to play a crucial role in the regulation of fibrinolysis. Basic research as well as clinical studies support this hypothesis. PAI-1 is a physiological inhibitor of both tissue-type plasminogen activator and urokinase-type plasminogen activator, key enzymes in the initiation of fibrinolysis. Thus PAI-1 regulates not only blood clot lysis but also a wide variety of biological reactions occurring in extracellular matrices such as tumor metastasis, neovascularization, inflammation, and cell migration. PAI-1 is a glycoprotein, of which molecular weight is approximately 50,000. Molecular biological analyses indicate that PAI-1 is synthesized as a single polypeptide composed of 402 amino acids containing a signal peptide. After post-translational modification, PAI-1 is secreted from ECs as a polypeptide composed of 379 amino acids and three N-linked carbohydrates. PAI-1 lacks Cys residues, indicating that PAI-1 may not be rigid and thus thermolabile. In fact, PAI-1 is unstable even at 37 degrees C decaying into an inactive form with a biological half life of 2-3 hours. PAI-1 binds to a cell adhesion molecule, vitronectin. The association of PAI-1 with vitronectin appears to stabilize PAI-1. PAI-1 in complex with vitronectin is still accessible to plasminogen activators.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Interactions between type 1 plasminogen activator inhibitor, extracellular matrix and vitronectin.

Regulation of plasminogen activation is a key process in controlling proteolytic events in the extracellular matrix (ECM) and this regulation is achieved through the action of specific plasminogen activator (PA) inhibitors (PAIs). Type I PAI (PAI-1) is the physiological inhibitor both of urinary-type PA (u-PA) and tissue-type PA (t-PA) (Loskutoff et al., 1989) and is a major component of the ECM of cultured cells. This inhibitor may protect ECM constituents against cellular proteases and thus influence the cell migration and tissue destruction that occurs during development, inflammation and tumor metastasis. In this review, we discuss the properties of PAI-1 and the evidence that the binding of PAI-1 to ECM is mediated by serum-derived vitronectin (Vn).

Animals↗
PubMed1990

The majority of type 1 plasminogen activator inhibitor associated with cultured human endothelial cells is located under the cells and is accessible to solution-phase tissue-type plasminogen activator.

The interactions between exogenously added tissue-type plasminogen activator (t-PA) and the active form of type 1 plasminogen activator inhibitor (PAI-1) produced by and present in cultured human umbilical vein endothelial cells (HUVECs) were investigated. Immunoblotting analysis of the conditioned media obtained from monolayers of HUVECs treated with increasing concentrations of t-PA (less than or equal to 10 micrograms/ml) revealed a dose-dependent formation of both t-PA/PAI-1 complexes, and of a 42,000-Mr cleaved or modified form of the inhibitor. Immunoradiometric assays indicated that t-PA treatment resulted in a fourfold increase in PAI-1 antigen present in the conditioned media. This increase did not result from the release of PAI-1 from intracellular stores, but rather reflected a t-PA-dependent decrease in the PAI-1 content of the Triton X-100 insoluble extracellular matrix (ECM). Although the rate of t-PA-mediated release of PAI-1 was increased by the removal of the monolayer, similar quantities of PAI-1 were removed in the presence or absence of the cells. These results suggest that the cells only represent a semipermeable barrier between ECM-associated PAI-1 and exogenous t-PA. Treatment of HUVECs with t-PA (1 microgram/ml, 2 h) to deplete the ECM of PAI-1 did not affect the subsequent rate of PAI-1 production and deposition into the ECM. Immunogold electron microscopy of HUVECs not only confirmed the location of PAI-1 primarily in the region between the culture substratum and ventral cell surface but failed to demonstrate significant (less than 1%) PAI-1 on the cell surface. Thus, the majority of PAI-1 associated with cultured HUVEC monolayers is present under the cells in the ECM and is accessible to solution-phase t-PA.

Cell Division↗