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Biomedical subjects

M Moroi

Publications and source records attributed to M Moroi.

At least 55 records · Page 3Linked to original sources
PubMed1990

[Significance of residual coronary artery stenosis after reperfusion therapy in acute myocardial infarction].

We evaluated the efficacy of reperfusion therapy in acute myocardial infarction in terms of postinfarction angina (PIA), reinfarction and coronary reocclusion. In 99 hospitalized patients with acute myocardial infarction within 6 hours after the onset of symptoms, 67 were treated using intracoronary thrombolysis (ICT) alone (Group T) and the remaining 32 using ICT followed by percutaneous transluminal coronary angioplasty (PTCA) (Group T + A). PTCA was performed for the arteries with high grade residual stenosis (TIMI grade 0, 1, 2) after ICT. Recatheterization was performed 28 +/- 12 days after hospitalization in 93% (62/67) of Group T and in all of Group T + A. There were no significant differences in age, sex, time interval from the onset to reperfusion, the extents of coronary artery disease and the Cohn grade of collaterals. However, anteroseptal infarction was more frequent in Group T than in Group T + A (p less than 0.05). Residual stenosis (diameter) at the end of intervention was 81 +/- 14% in Group T, and 48 +/- 15% in Group T + A, (p less than 0.01). Residual stenosis at recatheterization was 70 +/- 23% in Group T, and 55 +/- 22% in Group T + A (p less than NS). The incidence of PIA did not differ between the two groups (20.1% vs 6.2%). However, the incidence was higher in patients with residual stenosis of 70% or more than in those with residual stenosis of less than 70% (23.8% vs 2.9%, p less than 0.05). The incidence of reinfarction (re-elevation of CPK) did not differ between the two groups (7.4% in Group T, 6.2% in Group T + A); and neither did the incidence of coronary reocclusion at the time of recatheterization (14.5% vs 3.1%). We concluded that higher degree of residual stenosis at the end of intervention has a greater risk of PIA and reocclusion. Although differences were not statistically significant, the patients treated with ICT followed by PTCA seemed to have lower incidence of PIA and reocclusion compared with those treated with ICT alone, thus having better hospital prognosis.

Aged↗
PubMed1989

A patient with platelets deficient in glycoprotein VI that lack both collagen-induced aggregation and adhesion.

Molecular level studies on platelets deficient in collagen-induced aggregation provide evidence for identifying possible platelet collagen receptors. We investigated platelets from a patient with mild bleeding time prolongation, but otherwise normal coagulation data. Her platelets lacked collagen-induced aggregation and adhesion, but retained normal aggregation and release by other agonists. Labeling her platelets with 125I or 3H and analysis by SDS-PAGE/autoradiography showed normal levels of glycoproteins Ia, Ib, IIa, IIb, IIIa, and IV. However, there were significantly decreased incorporations of both radioactivities into a 61-kD membrane glycoprotein (GP), which was identified as GPVI from its mobility on unreduced-reduced, two-dimensional SDS-PAGE. Sugiyama et al. (1987. Blood. 69: 1712) reported that the serum from an idiopathic thrombocytopenic purpura (ITP) patient contained an antibody against a 62-kD platelet protein. Our patient's platelets lacked the antigen for the ITP patient's antibody, demonstrating that the ITP serum contains a specific antibody against GPVI. The patient's parents' platelets contained approximately 50% the normal amount of GPVI, but still had normal collagen-induced aggregation and adhesion. The patient's platelets did not bind to types I and III collagen fibrils. Our results suggest that GPVI functions as a collagen receptor.

Adult↗
PubMed1989

Inhibition of development of tolerance to nitroglycerin by preventive administration of N-acetylcysteine in rats.

The efficacy of N-acetylcysteine in reversing nitrate tolerance has been controversial. This study examined whether continuous administration of N-acetylcysteine, a sulfhydryl compound, can prevent the development of tolerance to nitroglycerin; its acute effects on developed tolerance were also assessed. Rats were treated with subcutaneous injections of 1) 100 mg/kg nitroglycerin, 2) 100 mg/kg nitroglycerin and 700 mg/kg N-acetylcysteine, 3 times a day for 3 days. The sensitivity to nitroglycerin was studied in aortic preparations. The degree of developed tolerance to nitroglycerin was partially inhibited by simultaneous injection of N-acetylcysteine. Subsequent in vitro preincubation of aortic strips with nitroglycerin (10(-5) M) reduced the subsequent nitroglycerin sensitivity of vessels from rats treated with nitroglycerin and N-acetylcysteine; sensitivity returned to the initial control level after in vitro preincubation with N-acetylcysteine. The nitroglycerin sensitivity of vessels from rats treated only with nitroglycerin, though, was not affected by in vitro preincubation with N-acetylcysteine. In conclusion, N-acetylcysteine is not effective in reversing the high degree of tolerance developed to nitroglycerin. However, continuous administration of N-acetylcysteine is effective in preventing the development of nitroglycerin tolerance.

Acetylcysteine↗
PubMed1989

Growth and differentiation of a human megakaryoblastic cell line, CMK.

Recently, a human megakaryoblastic cell line, CMK, was established from the peripheral blood of a megakaryoblastic leukemia patient with Down syndrome. Using this cell line, we studied the proliferation and differentiation of megakaryocytic cells in the presence of highly purified human hematopoietic factors and phorbol 12-myristate-13-acetate (PMA). In a methylcellulose culture system, interleukin-3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF) facilitated colony formation by CMK cells in a dose-dependent manner. The maximum stimulating doses of these factors were 10 and 200 U/mL, respectively. These concentrations were comparable to those that stimulate activity in normal hematopoietic cells. In contrast, granulocyte-colony stimulating factor (G-CSF), macrophage-colony stimulating factor (M-CSF), and erythropoietin (EPO) had no effects on the colony formation of CMK cells. In a liquid culture system, 20% of the CMK cells expressed glycoprotein IIb/IIIa (GPIIb/IIIa) antigen without hematopoietic factors, whereas 40% of the cells expressed GPIIb/IIIa with the addition of IL-3 and GM-CSF. EPO also slightly enhanced expression of GPIIb/IIIa. On the other hand, PMA inhibited growth of CMK cells and induced most of them to express the GPIIb/IIIa antigen. Furthermore, PMA induced CMK cells to produce growth activity toward new inocula of CMK cells. This growth factor (GF) contained colony-stimulating activity (CSA) in normal bone marrow (BM) cells. The activity was believed to be attributable mainly to GM-CSF, since 64% of this activity was neutralized by anti-GM-CSF antibodies and a transcript of GM-CSF was detected in mRNA from PMA-treated CMK cells by Northern blot analysis. These observations suggest that GM-CSF, as well as IL-3, should play an important role in megakaryocytopoiesis.

Blotting, Northern↗
PubMed1988

Characterization of an apparently lower molecular weight gamma-chain variant in fibrinogen Kyoto I. The replacement of gamma-asparagine 308 by lysine which causes accelerated cleavage of fragment D1 by plasmin and the generation of a new plasmin cleavage site.

Congenitally abnormal fibrinogen Kyoto I with impaired fibrin monomer polymerization contains a normal gamma-chain and a gamma-chain variant (gamma Kyoto I) that has an apparently lower Mr on sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the Laemmli system (Laemmli, U. K. (1970) Nature 227, 680-685) but migrates with apparently normal Mr in the Weber and Osborn system (Weber, K., and Osborn, M. (1969) J. Biol. Chem. 244, 4406-4412). Reverse-phase high performance liquid chromatographic analyses of the cyanogen bromide or lysyl endopeptidase cleavage fragments of the purified gamma-chains of fibrinogen Kyoto I showed the presence of peptides not seen from normal fibrinogen. Amino acid sequence analysis of these peptides indicated that gamma Asn308 of the gamma-chain variant is replaced by lysine. Purified fragment D1 of fibrinogen Kyoto I also contains two types of D1 gamma-remnants: normal and apparently lower Mr types. Abnormal fragment D1 is cleaved faster to fragments D2 and D3 by plasmin in the presence of [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA) than normal fragment D1, as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by immunoblotting using anti-gamma-chain monoclonal antibody. Analysis of peptides released from fragment D1 by plasmin in the presence of EGTA demonstrated the cleavage of the gamma Lys308-Gly309 bond. Fragment D1 of fibrinogen Kyoto I has normal calcium binding properties. The data suggest that a region or conformation containing gamma Asn308 affects the polymerization of fibrin monomers and that the gamma Asn308----Lys replacement causes a conformational change in the gamma-chain which results in the accelerated cleavage of gamma Lys356-Ala357 and gamma Lys302-Phe303 bonds by plasmin and also results in the generation of a new plasmin cleavage site between Lys308 and Gly309 in the presence of EGTA. During these studies, we found that part of the gamma Lys212-Glu213 bond in fragment D1 is cleaved by plasmin in the presence of EGTA.

Amino Acid Sequence↗
PubMed1988

Platelet cytoskeletal protein distributions in two triton-insoluble fractions and how they are affected by stimulants and reagents that modify cytoskeletal protein interactions.

Actin filament formation during early stages of platelet activation by stimulants was analyzed by determining the distributions of cytoskeletal proteins to a low speed centrifuge pellet (TP), ultracentrifuge pellet (UP), and ultracentrifuge supernatant (US) after lysing platelets with 1% Triton X-100. TP contained actin binding protein, myosin, alpha-actinin and actin, and UP contained these cytoskeletal proteins and other proteins. During thrombin activation, total protein and actin in TP increased with time, while they decreased in UP. Aggregating agents (ADP, PMA, thrombin) and, to a small extent, colchicine and nocodazole, microtubule inhibitors, increased cytoskeletal proteins and total protein in TP, while cytochalsin B, an inhibitor of actin polymerization, had the opposite effect. When the amounts of the respective proteins in TP and UP were summed, these values were not affected by agonists and inhibitors, except in the case of thrombin stimulation. These data suggest that the actin in UP is a form intermediate between the actin filaments in TP and actin monomer in the soluble fraction, and there may be a dynamic conversion between these forms upon platelet activation. UP was also characterized by immunostaining with antibodies against fibrinogen, tubulin, and glycoprotein Ib after SDS-PAGE/electrophoretic transfer to nitrocellulose sheets.

Actins↗
PubMed1988

Establishment of megakaryoblastic cell lines by coinfection of Abelson murine leukemia virus and recombinant SV40-retrovirus.

Murine embryonic cells including yolk sac prepared from 8-day embryos were co-infected with Abelson murine leukemia virus (A-MuLV) and/or a recombinant retrovirus containing large T and small t antigens, and early region of simian virus 40 (M-SV40). By coinfection with A-MuLV and M-SV40, megakaryoblastic cells were obtained in addition to mast cells and fibroblastic cells. However, following infection with A-MuLV or M-SV40 alone, no megakaryoblastic cells were detected, although mast cells and/or fibroblastic cells developed. The same results were obtained in several experiments. By single-cell transfer, 6 acetyl-cholinesterase (AchE)-positive clonal cell lines were established. Characteristics of megakaryocytes, such as AchE, glycoproteins IIb and IIIa, and platelet peroxidase were detected in two representative cells (C1 and C8). More significant changes expressing differentiation were observed following treatment with phorbol myristate acetate or pokeweed mitogen-stimulated murine spleen cell conditioned medium, although release of platelets was not observed. This is the first report showing development of megakaryocytic cells as the result of coinfection with retroviruses.

Abelson murine leukemia virus↗
PubMed1988

An apparently higher molecular weight gamma-chain variant in a new congenital abnormal fibrinogen Tochigi characterized by the replacement of gamma arginine-275 by cysteine.

A gamma-chain variant with an apparently higher molecular weight than the normal gamma-chain was detected in a new congenital abnormal fibrinogen with impaired polymerization of the fibrin monomer and with normal release of fibrinopeptides A and B in a 51-year-old male. Purified fibrinogen analyzed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under the reduced condition in the system of Laemmli contained two protein bands in the gamma-chain region (molecular weight, 50,500 as compared with 50,000 for the normal), both with normal crosslinking ability. The presence of two types of gamma-chains was more clearly detected when reduced and carboxymethylated fibrinogen was analyzed by SDS-PAGE or when reduced fragment D2 was analyzed on SDS-PAGE followed by Western blotting, and identified by positive staining for anti gamma-chain monoclonal antibody. Cyanogen bromide- or lysylendopeptidase-cleavage of purified gamma-chains analyzed on reverse-phase high performance liquid chromatography showed the decrease of one peptide compared with the normal and the appearance of an abnormal peptide peak. Amino acid sequence analysis demonstrated that the gamma arginine-275 of gamma-chain variant was replaced by a cysteine. These data suggest that some regions or conformations containing gamma 275 will affect the polymerization of fibrin monomers. The propositus' two daughters had the same abnormal fibrinogen. This unique inherited abnormal fibrinogen was designated as fibrinogen Tochigi, and the gamma-chain variant as gamma Tochigi.

Arginine↗
PubMed1988

Thrombasthenia with an abnormal platelet membrane glycoprotein IIb of different molecular weight.

We describe an individual with abnormal platelet glycoprotein (GP) IIb of different molecular weight (mol wt), a defect that distinguishes this patient from previously reported thrombasthenics. The patient, a 21-year-old female, has a mild bleeding tendency; her platelets lack adenosine diphosphate (ADP) aggregation and have severely suppressed collagen aggregation but a normal response to ristocetin. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of her platelets indicates that they contain two types of GPIIb molecules: one with an abnormal mol wt (122 kd, unreduced; 128 kd, reduced) and one with a normal mol wt (128 kd, unreduced; 118 kd, reduced). Relative to the amount of GPIIb in normal platelets, her platelets contain approximately 35% abnormal GPIIb and 20% normal GPIIb. Fibrinogen binding assays on the patient's platelets indicated that they contained 25% of the normal amount of fibrinogen receptors. Crossed immunoelectrophoresis of the patient's platelets demonstrated the formation of a GPIIb/IIIa complex that was mainly composed of normal mol wt GPIIb and GPIIIa. The patient's father has decreased ADP aggregability, and his platelets also contained both abnormal and normal GPIIb (about 50% of the normal level and about 50% of the normal number of fibrinogen receptors); her mother has only normal GPIIb. These results indicate that the patient has heterozygous GPIIb molecules with an abnormality of GPIIb at the molecular level. Studies on this abnormal GPIIb should provide information about the function of GPIIb and the mechanism of its biosynthesis.

Adult↗
PubMed1986

Polymorphism of platelet glycoprotein Ib in the United States.

Platelet glycoprotein (GP) Ib from healthy individuals from the United States was analyzed using SDS-polyacrylamide gel electrophoresis and staining with peanut agglutinin-coupled peroxidase or wheat germ agglutinin-coupled peroxidase after transfer of electrophoresed proteins to nitrocellulose. The GPIb from American Caucasians (109 individuals) was found to be polymorphic, showing types A, B, C, and D of GPIb, similar to what was observed in Japanese. The phenotype distribution pattern, however, was very different from that observed in the Japanese population. GPIb heterogeneity was also observed in American Blacks, Asian Americans, and Americans of Hispanic origin. Thus, it appears that the polymorphism of GPIb is a general phenomenon which should be regarded as an important factor when doing studies on this glycoprotein.

Asian People↗
PubMed1986

A lower molecular weight gamma-chain variant in a congenital abnormal fibrinogen (Kyoto).

A gamma-chain variant with a lower molecular weight than the normal gamma chain was detected in a new congenital abnormal fibrinogen with impaired polymerization of the fibrin monomer and with normal release of fibrinopeptides A and B in a 45-year-old male. Purified fibrinogen analyzed on SDS-polyacrylamide gel electrophoresis under the reduced condition contained an abnormal protein band with an apparent molecular weight of 48,000 compared with the gamma chain with a molecular weight of 50,000. This abnormal protein band was found to be a gamma-chain variant from the molar ratio of A alpha chain:B beta chain:gamma chain:abnormal protein (about 2:2:1:1), with positive staining for carbohydrate and crosslinking ability. Crosslinked fibrin contained three types of gamma-gamma dimers with apparent molecular weights of 94,000 (the same as normal major gamma-gamma dimer), 92,000 and 90,000, and the plasmic digests of crosslinked fibrin in the presence of calcium retained three types of gamma-gamma dimer remnants. This suggests that the abnormal gamma-chain variant has a shorter polypeptide chain not in the NH2-terminal but in the COOH-terminal portion, probably at or near the polymerization site. This patient's two daughters had the same abnormal fibrinogen. This unique inherited abnormal fibrinogen was designated as fibrinogen Kyoto, and the gamma-chain variant as gamma Kyoto.

Blood Coagulation Disorders↗
PubMed1984

Selective staining of human platelet glycoproteins using nitrocellulose transfer of electrophoresed proteins and peroxidase-conjugated lectins.

Platelet proteins (0.5-5 micrograms) were electrophoresed in a one-dimensional or an unreduced-reduced, two-dimensional sodium dodecyl sulfate gel system. The separated proteins were then transferred electrophoretically to nitrocellulose and reacted with peroxidase-conjugated lectins. Visualization of specific glycoproteins which bound the lectins was made by the chromogenic reaction catalyzed by peroxidase utilizing 3,3'-diaminobenzidine as the substrate. Wheat germ agglutinin specifically reacted with and allowed the visualization of glycoprotein Ib. Peanut agglutinin also specifically stained glycoprotein Ib after treatment of the nitrocellulose transferred proteins with neuraminidase. Ricinus communis agglutinin I stained thrombospondin, a 260 kDa protein, and factor VIII. Concanavalin A stained mainly glycoproteins IIb, III, IV, and V. Glycoproteins Ia, Ic, IIa, and other minor glycoproteins could be separated by unreduced-reduced, two-dimensional gel electrophoresis and were stained weakly with wheat germ agglutinin conjugates. These techniques were found to be reproducible as well as easily applied to the analysis and identification of platelet glycoproteins, particularly when dealing with a limited amount of platelets.

Animals↗
PubMed1984

Genetic polymorphism of platelet glycoprotein Ib.

Platelet glycoprotein (GP) Ib from 131 healthy Japanese was analyzed using SDS-polyacrylamide gel electrophoresis and specific staining with peroxidase-coupled wheat germ agglutinin after it was transferred to nitrocellulose membranes. Four slightly different species of GPIb were observed and designated as A, B, C, and D for glycoproteins with molecular weights of 168,000, 162,000, 159,000, and 153,000 daltons, respectively. The respective gene frequencies were calculated to be .073, .011, .561, and .355 for A-, B-, C-, D-type GPIb. Portions from each type of GPIb molecule (alpha-chain and glycocalicin) showed heterogeneity with the same molecular weight difference, indicating that the variance would be derived from the polypeptide portion that is exposed to the outer medium. The different types of GPIb were the same with respect to their accessibility to lactoperoxidase, reactivity to lectins, and affinity to TLCK-thrombin. Although Bolin et al reported patients with a bleeding tendency whose platelets have double GPIb bands, here we found that platelets with different GPIb phenotypes showed no significant differences in aggregating activity and platelet retention. Analysis of GPIb phenotype should be important for structural and physiologic studies on GPIb and glycocalicin.

Blood Platelets↗
PubMed1983

Crosslinking of platelet glycoprotein Ib by N-succinimidyl(4-azidophenyldithio)propionate and 3,3'-dithiobis(sulfosuccinimidyl propionate).

To examine the relationship between glycoprotein Ib and other proteins in the platelet membrane and the interaction of this protein with thrombin, platelets were crosslinked by two cleavable reagents, SADP (N-succinimidyl(4-azidophenyldithio)propionate) and DTSSP (3,3'-dithiobis(sulfosuccinimidyl propionate]. Two-dimensional, unreduced-reduced sodium dodecyl sulphate (SDS)-polyacrylamide electrophoresis and staining by silver or wheat germ agglutinin-conjugated peroxidase, after protein transfer to nitrocellulose, demonstrated that SADP intramolecularly crosslinked glycoprotein Ib and formed intermolecular complexes of glycorprotein IIb and some high molecular weight proteins. DTSSP intermolecularly crosslinked glycoprotein Ib, glycoprotein IIb, and other high molecular weight proteins. With a low concentration of 125I-labeled TLCK-thrombin (6 nM), crosslinking with SADP yielded a 200 000 Da complex containing radioactive-labeled thrombin, and high TLCK-thrombin concentration (0.1 microM) gave the complex and a 167 000 band. alpha- and TLCK-thrombin crosslinking with DTSSP also yielded the 200 000 complex formed by reaction with SADP or DTSSP was markedly reduced by preincubation of platelets with excess unlabeled TLCK-thrombin and had a pI similar to glycoprotein Ib. These results suggest that glycoprotein Ib is one of the proteins composing the high affinity receptor for thrombin.

Azides↗
PubMed1982

Isolation of platelet glycocalicin by affinity chromatography on thrombin-sepharose.

Platelet glycocalicin has been purified to homogeneity by a two step procedure involving affinity chromatography on WGA-Sepharose and then on thrombin-Sepharose using selective elution with heparin. The procedure is more rapid (3-4 days), more reproducible and gives about twice the yield (10 mg/40 units platelets) of the previous method (Okumura et al. J. Biol. Chem. 251, 5950-5955, 1976). The two preparations showed identical inhibition of aggregation of gel filtered platelets induced by thrombin and by ristocetin. Glycocalicin was cleaved in a controlled fashion by trypsin-Sepharose over 3hr to yield the macroglycopeptide and peptide "tail" fragments and there was no apparent further degradation with 18 hrs digestion.

Blood Platelets↗
PubMed1979

Inhibitory spectrum of alpha 2-plasmin inhibitor.

alpha 2-Plasmin inhibitor (alpha 2PI) has been recently characterized as a fast-reacting inhibitor of plasmin in human plasma and appears to play an important role in the regulation of fibrinolysis in vivo. We have studied the effect of purified alpha 2PI upon various proteases participating in human blood coagulation and kinin generation. At physiological concentration (50 microgram/ml), alpha 2PI inhibited the clot-promoting and prekallikrein-activating activity of Hageman factor fragments, the amidolytic, kininogenase, and clot-promoting activities of plasma kallikrein, and the clot-promoting properties of activated plasma thromboplastin antecedent (PTA, Factor XIa) and thrombin. alpha 2PI had minimal inhibitory effect on surface-bound activated PTA and activated Stuart factor (Factor Xa). alpha 2PI did not inhibit the activity of activated Christmas factor (Factor IXa) or urinary kallikrein. Heparin (1.5-2.0 units/ml) did not enhance the inhibitory function of alpha 2PI. These results suggest that, like other plasma protease inhibitors, alpha 2PI possesses a broad in vitro spectrum of inhibitory properties.

Blood Coagulation Factors↗