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Biomedical subjects

S Iwanaga

Publications and source records attributed to S Iwanaga.

At least 163 records · Page 9Linked to original sources
PubMed1992

Prothrombin Salakta: substitution of glutamic acid-466 by alanine reduces the fibrinogen clotting activity and the esterase activity.

Structural studies on a hereditary abnormal prothrombin, prothrombin Salakta, have been performed to identify the difference responsible for its reduced fibrinogen clotting activity and its reduced esterase activity. Amino acid composition and sequence analyses of a peptide isolated from a lysylendopeptidase digest of the abnormal thrombin indicated that Glu-466 had been replaced by Ala. This amino acid substitution can result from a single nucleotide change in the codon for Glu-466 (GAG----GCG). The model building and the molecular dynamics simulation of thrombin Salakta suggest that the Glu-466----Ala substitution would change the proper conformation around the substrate binding site containing Trp-468, which is a unique surface loop on the thrombin molecule. This is the experimental and theoretical evidence supporting the role of the surface loop containing Trp-468 for the proper conformation of the substrate binding site.

Alanine↗
PubMed1992

Coagulation factor X activating enzyme from Russell's viper venom (RVV-X). A novel metalloproteinase with disintegrin (platelet aggregation inhibitor)-like and C-type lectin-like domains.

We determined the complete amino acid sequence of RVV-X, the blood coagulation factor X activating enzyme, isolated from Russell's viper venom and studied structure-function relationships. RVV-X (M(r) 79,000) consists of a disulfide-bonded two-chain glycoprotein with a heavy chain of M(r) 59,000 and a light chain of heterogeneous M(r) 18,000 (LC1) and 21,000 (LC2). These chains were separated after reduction and S-pyridylethylation, and the isolated major component LC1 was used for sequence analysis. The heavy chain consists of 427 residues containing four asparagine-linked oligosaccharides, and its entire sequence was similar to that of the high molecular mass hemorrhagic protein, HR1B, isolated from the venom of Trimere-surus flavoviridis. The heavy chain contains three distinct domains, metalloproteinase, disintegrin (platelet aggregation inhibitor)-like and unknown cysteine-rich domains. On the other hand, light chain LC1 consists of 123 amino acid residues containing one asparagine-linked oligosaccharide and shows sequence homology similar to that found in the so-called C-type (Ca(2+)-dependent) lectins. Therefore, RVV-X is a novel metalloproteinase containing a mosaic structure with distintegrin-like, cysteine-rich, and C-type lectin-like domains. RVV-X potently inhibits collagen- and ADP-stimulated platelet aggregations, probably via its distintegrin-like domain, although this domain does not contain the Arg-Gly-Asp sequence which is conserved in various venom distintegrins and which is thought to be one of the interaction sites for platelet integrins. Our findings also indicate that snake venom factor IX/factor X-binding protein with a C-type lectin structure (Atoda, H., Hyuga, M., and Morita, T. (1991) J. Biol. Chem. 266, 14903-14911) inhibits RVV-X-catalyzed factor X activation; hence, the light chain of RVV-X probably participates in recognizing some portion of the zymogen factor X.

Amino Acid Sequence↗
PubMed1992

cDNA cloning of rat proteasome subunit RC1, a homologue of RING10 located in the human MHC class II region.

The nucleotide sequence of a cDNA that encodes a new subunit, named RCl, of rat proteasomes (multicatalytic proteinase complexes) has been determined. The polypeptide predicted from the open reading frame consisted of 208 amino acid residues with a calculated molecular mass of 23, 130, which is consistent with the size obtained by electrophoretic analysis of purified RCl. The partial amino acid sequences of several fragments of RCl, obtained by protein chemical analyses, were found to be in excellent accordance with those deduced from the cDNA sequence. Surprisingly, the overall structure of RCl was found to be almost identical to that of recently isolated RING10, whose gene is located in the class II region of the human MHC gene cluster. This finding suggests that RCl is a homologue of human RING10, supporting the proposal that proteasomes are involved in the antigen processing pathway.

Amino Acid Sequence↗
PubMed1992

Putative inositol 1,4,5-trisphosphate binding proteins in rat brain cytosol.

In previous works, we synthesized a series of inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) analogs, with a substituent on the second carbon of the inositol ring. Using these analogs, the Ins(1,4,5)P3 affinity media were also synthesized (Hirata, M., Watanabe, Y., Ishimatsu, T., Yanaga, F., Koga, T., and Ozaki, S. (1990) Biochem. Biophys. Res. Commun. 168, 379-386). When the cytosol fraction from the rat brain was applied to an Ins(1,4,5)P3 affinity column, an eluate with a 2 M NaCl solution was found to have remarkable Ins(1,4,5)P3-binding activity. The active fraction was further fractionated with gel filtration chromatography, and two proteins with an apparent molecular mass of 130 or 85 kDa were found to be Ins(1,4,5)P3-binding proteins but with no Ins(1,4,5)P3 metabolizing activities. Partial amino acid sequences determined after proteolysis and reversed-phase chromatography revealed that the protein with an apparent molecular mass of 85 kDa is the delta-isozyme of phospholipase C and that of 130 kDa has no sequence the same as the Ins(1,4,5)P3-recognizing proteins hitherto examined. Ins(1,4,5)P3 at concentrations greater than 1 microM strongly inhibited 85-kDa phospholipase C delta activity, without changing its dependence on the concentrations of free Ca2+ and H+. Among inositol phosphates examined, Ins(3,4,5,6)P4 inhibited the binding of [3H]Ins(1,4,5)P3 to the 130-kDa protein at much the same concentrations as seen with Ins(1,4,5)P3. This report seems to be the first evidence for the presence of soluble Ins(1,4,5)P3-binding proteins in the rat brain, one of which is the delta isozyme of phospholipase C.

Amino Acid Sequence↗
PubMed1992

Analysis of intrinsic fibrinolysis in human plasma induced by dextran sulfate.

The analysis of normal human plasma by fibrin autography revealed four species of plasminogen activator (PA) activity related to tissue-type PA, factor XII, prekallikrein and urokinase-type PA (u-PA). The u-PA activity increased significantly by incubating plasma with dextran sulfate. This increase was coincident with both the cleavage of factor XII and the complex formation of activated factor XII with its plasma inhibitors, which were determined by immunoblotting procedure. The dextran sulfate-dependent activation of u-PA required both factor XII and prekallikrein, but did not require either plasminogen or factor XI. High molecular weight kininogen was required only at a low concentration of dextran sulfate. Thus the results indicate that the factor XII and prekallikrein-mediated activation of single chain u-PA (scu-PA) operates as a major pathway of scu-PA activation in whole plasma in contact with dextran sulfate.

Dextran Sulfate↗
PubMed1992

Evidence for the existence of O-linked sugar chains consisting of glucose and xylose in bovine thrombospondin.

We have recently discovered unusual sugar chains [xylose-glucose and (xylose)2-glucose] linked to a serine residue in the first epidermal growth factor (EGF)-like domains of human and bovine coagulation factors VII, IX, and protein Z. The sequence surrounding this serine residue has a common -Cys-X-Ser-X-Pro-Cys- structure. Since one (residues 533-538) of the three EGF-like domains found in human thrombospondin contains the conserved sequence, we examined the presence of such O-linked sugar chains in bovine thrombospondin (bTSP) and its 210-kDa fragment. Component sugar analysis after pyridylamination (PA) of the acid hydrolysates of the S-aminoethylated proteins revealed that the proteins contain glucose (Glc) and xylose (Xyl). The oligosaccharide moieties released from intact bTSP by hydrazinolysis followed by pyridylamination were separated into two PA-oligosaccharides by high performance liquid chromatography (HPLC). Component sugar analysis of these PA-oligosaccharides indicated that they consist of Glc and Xyl in molar ratios of 1:1 and 1:2 (or 1:3). The reducing ends of both PA-sugar chains were found to be PA-Glc, as judged from the retention time of the HPLC peak of their hydrolysates. The presence of these PA-sugar chains in bTSP was confirmed by HPLC mapping with two different columns, using standard PA-di- or PA-trisaccharide derived from coagulation factors. From these results, we concluded that bTSP contains O-linked sugar chains consisting of Glc and Xyl in one of its three EGF-like domains.

Amino Acid Sequence↗
PubMed1992

Preparation and properties of monoclonal antibodies against lipopolysaccharide-sensitive serine protease zymogen, factor C, from horseshoe crab (Tachypleus tridentatus) hemocytes.

Seventeen murine monoclonal antibodies (mAbs) against horseshoe crab clotting factor, factor C, were prepared and characterized. When the binding sites of these mAbs were analyzed by immunoblotting, ten mAbs recognized nonreduced factor C, five mAbs were directed against the heavy chain, and two mAbs were directed against the B chain. Three mAbs, 1H4, 2C12, and 2A7, one selected from each group, were used for further study. The mAb 1H4, which recognized only nonreduced factor C molecule, inhibited the factor C activity in a dose-dependent manner. It also inhibited lipopolysaccharide (LPS)- and alpha-chymotrypsin-mediated activations of the zymogen factor C, suggesting that 1H4 binds close to the active site and/or the substrate-binding site located in the serine protease domain (B chain) of factor C. On the other hand, 2C12 and 2A7 recognized, respectively, an epitope located in the heavy and the B chains, and inhibited LPS-mediated activation of factor C, but not alpha-chymotrypsin-mediated activation of factor C or factor C activity. Both F(ab')2 and Fab' fragments derived from 2C12 inhibited LPS-mediated activation in the same manner. These three mAbs did not bind with LPS, although a factor C-mAb complex was able to bind LPS, suggesting that the LPS-mediated activation of the zymogen factor C was induced through intermolecular interaction between the LPS-bound factor C molecules. The dissociation constants (Kd) for 1H4, 2C12, and 2A7 binding to factor C were determined as 1.9 x 10(-9), 0.6 x 10(-10), and 1.8 x 10(-10) M, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Production, purification, and characterization of botulinolysin, a thiol-activated hemolysin of Clostridium botulinum.

A hemolysin, botulinolysin, produced by Clostridium botulinum was purified to homogeneity and characterized. First, a strain of C. botulinum type C, strain C-203 Tox, which produced a large amount of hemolysin, was selected, and optimal culture medium and conditions for its production of hemolysin were determined. The hemolysin produced in the culture supernatant of this strain under optimal conditions was purified by a combination of ammonium sulfate precipitation, DEAE-Sepharose CL-6B column chromatography, Sephadex G-75 gel permeation chromatography, and SP-Toyopearl 650 M cation-exchange column chromatography, with a recovery of 12%. The purified hemolysin gave a single protein band in polyacrylamide gel electrophoresis (PAGE) with and without sodium dodecyl sulfate (SDS). The protein in this band in PAGE with SDS was estimated to have a molecular weight of 58,000 and was immunostained with a neutralizing monoclonal antibody. In PAGE without SDS, the hemolytic activity corresponded in position to the single protein band. The pI of the hemolysin was 8.4. Amino acid analysis of the purified hemolysin indicated the presence of four half-cystine residues per molecule. The purified hemolysin had a specific activity of 2,100 hemolytic units per microgram of protein on rabbit erythrocytes. It was activated by SH compounds, inhibited by cholesterol, and heat labile. The optimum pH for hemolysis was 6.0 to 7.0. Rabbit, human, and guinea pig erythrocytes were the most susceptible to the hemolysin, while sheep, mouse, rat, and chicken erythrocytes were much less susceptible. The purified hemolysin had a lethal effect in mice and was cytotoxic for some cultured cells: its 50% lethal dose in mice was 310 ng, and its 50% cytotoxic dose for Vero cells was 120 ng/ml.

Amino Acids↗
PubMed1992

[Establishment and characterization of human ovarian fibrosarcoma cell line and its sensitivity to anticancer agents].

We succeeded in establishing a cell line (KEN-3) for subculture from a fibrosarcoma which originated in the ovary in a girl aged 17 years. Its characteristics and sensitivity to anticancer agents are reported in this paper. 1. Characteristics of established cell line. Lined cells consist of multinucleated giant cells mixed among many spindle-shaped cells. They grow in small colonies and have none of the pavement-like arrangement characteristic of epithelial tumor cells. The number of chromosomes ranged from 45 to 128 (mode: pseudo-triploidy region, 65). The doubling time, cellular density and plating efficiency were 76.9 hours, 5.4 x 10(5)/cm2 and 30.2%, respectively. Concerning tumor markers, CEA and sialyl SSEA-1 were only produced in small quantities. Subculture was possible subcutaneously in the nude mouse with no capacity for the production of ascites. 2. Susceptibility to anticancer agents and GP170 expression. The in vitro susceptibility to about 12 types of anticancer agents was investigated with the MTT assay. IC50/PPC was shown to be less than 1 for Adriamycin only. The sensitivity to CDDP (IC50/PPC: 4.8) was low, and no sensitivity was observed at all to DTIC, which is used frequently for mesenchymal tumors. GP170 (mdr-1 products) was positive in established cells in immunohistochemical stain.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1992

Human fibrinogen heterogeneity: the COOH-terminal residues of defective A alpha chains of fibrinogen II.

Fibrinogen fraction I (340 kDa) and fraction II (305 kDa) were isolated by glycine precipitation. The subunit chains of the two fractions were separated, after reduction, by reverse-phase high performance liquid chromatography. The amino acid compositions of the B beta and tau chains of fibrinogen II were identical with those of fibrinogen I. In contrast, the A alpha chains of fibrinogen II were composed of two populations, one comprising homogeneous, intact A alpha chains and the other consisting of heterogeneous, deficient A alpha chains (A alpha' chains) of lengths varying according to the sizes of their COOH-terminal defects. The molar ratio of the A alpha to the A alpha' chains in fibrinogen II was 1.16:1. The amino acid composition and sequence analyses of the TPCK-trypsin peptides derived from the A alpha' chains revealed that the COOH-terminal residues of the A alpha' chains were mainly Asn-269, Gly-297 and Pro-309. These results indicate that the fibrinogen II molecule is asymmetrical and can be represented by the formula (A alpha) (A alpha')(B beta)2(tau)2 and that fibrinogen II cannot be a plasmin degradation product of fibrinogen I.

Amino Acid Sequence↗
PubMed1992

Interleukin-1 receptor antagonist in inflammatory exudate cells of rabbits. Production, purification and determination of primary structure.

A rabbit interleukin-1 (IL-1) inhibitor in inflammatory peritoneal exudate cells was purified to apparent homogeneity. This inhibitor was extracted from exudate cells of the 24-hr stage of casein-induced peritoneal inflammation and purified using isoelectrofocusing (IEF), gel filtration, followed in this order by high-performance liquid chromatography (HPLC) steps with hydroxylapatite and anionic ion exchanger. The purified factor showed a single band on silver-stained SDS-PAGE. This molecule of MW 19,000 and pI 5.5 inhibited the binding of both IL-1 alpha and beta to receptors on a thymoma cell line, EL-4 and a B-cell line, 70Z/3. We determined its primary structure by a combination of peptide chemistry and molecular cloning. The inhibitor was synthesized as a precursor composed of 177 amino acids and was processed to a mature molecule of 143 amino acids. The N-terminal amino acid of the mature inhibitor was N-acetyl-methionine residue. The deduced amino acid sequence of the inhibitor showed a 77% homology to the human IL-1 receptor antagonist (IL-1Ra) and essentially the same mode of action as seen with human IL-1Ra. We consider that this inhibitor is a rabbit counterpart of human IL-1Ra, although there are differences with respect to the molecular structure; the N-terminus of the mature rabbit IL-1Ra at a position of nine amino acids downstream from that of human IL-1Ra.

Amino Acid Sequence↗
PubMed1992

[Phase I clinical trial of a new myocardial imaging agent, 99mTc-PPN1011].

A new myocardial imaging agent, 99mTc-1,2-bis[bis(2-ethoxyethyl) phosphino]ethane (99mTc-PPN1011) was administered to healthy male volunteers to evaluate its safety and kinetics. No significant drug-related events were observed. After intravenous injection, blood clearance was rapid. Prompt heart uptake (1.8% of the injected dose after 5 minutes) was followed by relatively slow wash-out (1.0% remained after 3 hours). Liver and lung uptake was observed but the clearance was more rapid than in the heart. 4.7-24.0% of the injected 99mTc-PPN1011 was excreted into urine until 4 hours after injection. 99mTc-PPN1011 also seemed to be excreted through hepato-biliary system. Excellent and low-background myocardial images were obtained using either SPECT or planar imaging. We conclude that 99mTc-PPN1011 could be useful for myocardial perfusion imaging.

Adult↗
PubMed1992

[Importance of collateral circulation to preserve the exercise capacity in patients with coronary artery disease].

We studied the effects of coronary collaterals on exercise capacity in relation with the coronary flow reserve. Thirty-one patients with single vessel disease of the left anterior descending artery (LAD) were selected using coronary angiography. Thirteen of them had angiographical collaterals. A Doppler coronary catheter was positioned in the proximal portion of the LAD, and the resting and peak coronary flow velocity was measured using an intracoronary injection of 6 ml of contrast material. Peak to resting velocity ratio was calculated as an index of the coronary flow reserve. Multi-stage treadmill exercise ECG test was performed, and the end point of the exercise was 0.1 mV depression of ST segment. The exercise capacity was expressed as the ratio of maximal to resting double product. In patients without collaterals, peak to resting coronary flow velocity ratio was correlated with double product ratio (r = 0.94, p less than 0.01). But in patients with collaterals, double product ratios were higher than those in patients without collaterals. We conclude that coronary collaterals preserve exercise capacity in spite of the low coronary flow reserve in the recipient coronary artery.

Aged↗
PubMed1992

[Recurrent ischemia after acute myocardial infarction: clinical profile and significance of electrocardiographic ST changes during the attacks].

This study investigated the clinical characteristics of acute myocardial infarction (AMI) complicated by recurrent ischemia, especially relating to the electrocardiographic ST changes during the attacks. Fifty-six patients with AMI were complicated by recurrent ischemia (ischemia group), and 238 were not (non-ischemia group). The ischemia group was preceded by prior episodes of angina or myocardial infarction in 88%, as compared with 65% in the non-ischemia group (p less than 0.05). There were non Q wave infarction in 45% of the ischemia group, and 24% of the non-ischemia group (p less than 0.05). The overall in-hospital mortality rate was similar in the ischemia group (13%) and in the non-ischemia group (17%), although the causes were predominantly pump failure in the former and cardiac rupture in the latter. ST segment elevation occurred in 29 patients and ST depression occurred in 26 patients of the ischemia group during the attacks. Multivessel coronary arterial lesions were more frequently present in the latter subgroup than the former (38% vs 79%, p less than 0.05). The in-hospital cardiac deaths were also more frequently noted in the latter subgroup. Recurrent ischemia after AMI with concomitant electrocardiographic ST depression is a high risk subgroup, and, therefore, aggressive revascularization procedures may be indicated in such cases if suitable.

Coronary Vessels↗
PubMed1991

cDNA and amino acid sequences of bovine tissue factor.

A cDNA encoding bovine tissue factor has been isolated from a lambda gt11 bovine adrenal cDNA library. The cDNA insert was 1877 base pairs with an open reading frame of 876 base pairs that encoded a presequence of 35 amino acids and a mature tissue factor of 257 amino acids. Bovine tissue factor had three potential N-glycosylation sites, four extracellular cysteine residues, a cytoplasmic cysteine residue, and one tripeptide tryptophan-lysine-serine motif. Identities of the amino acid sequences of the mature forms between the bovine tissue factor and each of human, mouse, and rabbit tissue factors were 70.4%, 57.2%, and 74.1%, respectively.

Adrenal Glands↗
PubMed1991

Isolation and characterization of a thermolabile beta-2 macroglycoprotein ('thermolabile substance' or 'Hakata antigen') detected by precipitating (auto) antibody in sera of patients with systemic lupus erythematosus.

A novel thermolabile beta-2 macroglycoprotein ('thermolabile substance' (TLS) or 'Hakata antigen' (HA], which was detected by the precipitating (auto) antibodies of patients with systemic lupus erythematosus, was isolated and characterized. The purification procedure entailed the following steps: isoelectric precipitation in the range between pH 5.2-6.1, hydroxyapatite absorption chromatography, 35% saturated ammonium sulfate precipitation, Sephadex G-200 gel filtration, Pevikon block electrophoresis, lentil lectin affinity chromatography and immobilized rabbit anti-human whole serum IgG column chromatography. Utilizing these procedures, 0.1 mg of HA was purified from 3 1 of pooled human serum. The molecular mass of HA was determined as 650 kDa by Sepharose 4B gel filtration. On SDS-PAGE analysis, HA showed a single band at 35 kDa under reduced conditions and numerous ladder bands between 35 kDa to more than 300 kDa under nonreduced conditions. On analytical ultracentrifugation, HA gave a molecular mass of 520 kDa with a single meniscus and a sedimentation constant of 12.0. The amino acid and carbohydrate analysis of reduced and S-pyridylethylated HA revealed that it contained five residues of hydroxyproline and an N-linked type sugar chain.

Adult↗