Search PubMed⌕ Search

Biomedical subjects

S Iwanaga

Publications and source records attributed to S Iwanaga.

At least 181 records · Page 10Linked to original sources
PubMed1991

Limulus factor C. An endotoxin-sensitive serine protease zymogen with a mosaic structure of complement-like, epidermal growth factor-like, and lectin-like domains.

Factor C is an endotoxin-sensitive, intracellular serine protease zymogen which initiates the coagulation cascade system in the limulus hemolymph. We have determined the entire amino acid sequence of factor C using recombinant DNA technique. The zymogen consisted of 994 amino acid residues with a calculated molecular mass of 109,648 Da. Most interestingly, factor C has five repeating units ("Sushi" domain or short consensus repeat) of about 60 amino acid residues each, which have been found in many proteins participating in the mammalian complement system. In addition to a typical serine protease domain in the carboxyl-terminal portion, characteristic segments with an epidermal growth factor-like, a lectin-like, a cysteine-rich, and a proline-rich domain were also found, revealing a unique mosaic protein structure. The serine protease domain was most analogous to human thrombin. Factor C was identified to localize in large granules in the cell, indicating that it is released from the cell by lipopolysaccharide stimulation. Furthermore, we identified a transcript possibly derived by alternative splicing of factor C mRNA, which encodes a protein sharing the amino-terminal portion of factor C. We suggest that factor C, a newly discovered type of serine protease zymogen, is a "coagulation-complement factor" which may play important roles in both hemostasis and host defense mechanisms.

Amino Acid Sequence↗
PubMed1991

Further studies on lipopolysaccharide-sensitive serine protease zymogen (factor C): its isolation from Limulus polyphemus hemocytes and identification as an intracellular zymogen activated by alpha-chymotrypsin, not by trypsin.

An intracellular serine protease zymogen, factor C, is an initiator in the hemolymph coagulation system of horseshoe crab. We purified this zymogen from the hemocytes of the American horseshoe crab, Limulus (L.) polyphemus, the objective being to compare its properties with those of the Japanese horseshoe crab, Tachypleus (T.) tridentatus, factor C. The purified zymogen L.-factor C showed similar properties to those of T.-factor C, in terms of molecular mass (123,000), amino acid composition (1,011 residues), subunit structure (two chains), and antigenicity. Like the zymogen T.-factor C, this zymogen was also activated autocatalytically in the presence of bacterial lipopolysaccharide (LPS) and its synthetic lipid A analogue. A most interesting finding is that both protease zymogens are rapidly activated by alpha-chymotrypsin or rat mast cell chymase, but not by trypsin. The active enzyme factor C showed alpha-thrombin-like specificity toward synthetic tripeptide substrates. This factor C was also strongly inhibited by an alpha-thrombin inhibitor, D-Phe-Pro-Arg-chloromethyl ketone. Thus, the enzymatic properties of factor C are similar to those of mammalian alpha-thrombin. On the other hand, the coagulation cascade system present in the hemocyte lysate was activated when chymotrypsin, free from LPS, was added to the lysate used to detect the endotoxins. The implication of our findings is that the chymotrypsin-catalyzed initiation of the horseshoe crab coagulation system is unique, since all known mammalian coagulation, fibrinolysis and complement systems are initiated by trypsin-like enzymes.

Amino Acid Sequence↗
PubMed1991

gamma-Carboxyglutamic acid (Gla)-domainless blood coagulation factor IXa species: preparation and properties.

To investigate the function of the gamma-carboxyglutamic acid (Gla) residues of factor IXa in the activation of factor X, a new species of bovine factor IXa, designated "factor IXa beta'," and its corresponding Gla-domainless form, designated "Gla-domainless factor IXa beta'," were prepared under controlled conditions and characterized. First, bovine factor IXa alpha was converted by alpha-chymotrypsin in the presence of calcium ions to factor IXa beta' (Mr 47,000). Compared with factor IXa beta, factor IXa beta' had essentially identical activities towards a synthetic substrate, benzoyl-L-arginine ethylester (BAEE), towards an active site titrant, p-nitrophenyl-p'-guanidinobenzoate, and towards protein substrate, namely, factor X. Next, the Gla-rich region (residues 1-41) of the light chain was removed from factor IXa beta' by additional selective cleavage by alpha-chymotrypsin in the absence of calcium ions. Gla-domainless factor IXa beta' was purified to homogeneity on a column of DEAE-Sepharose CL-6B. The heavy chain was not altered by either chymotryptic digestion. Functional comparisons of the three activated forms, namely, factor IXa alpha, factor IXa beta', and Gla-domainless factor IXa beta', with factor IXa beta revealed that all four activated forms of factor IX had one active-site residue per molecule and essentially identical specific esterase activity towards BAEE. However, the clotting activity of Gla-domainless factor IXa beta' was less than 0.5% of that of factor IXa beta'.(ABSTRACT TRUNCATED AT 250 WORDS)

1-Carboxyglutamic Acid↗
PubMed1991

Inhibitory effect of tachyplesin I on the proliferation of human immunodeficiency virus in vitro.

An antimicrobial peptide, tachyplesin I, isolated from hemocytes of the Japanese horseshoe crab (Tachypleus tridentatus) was examined for its inhibitory effects on human immunodeficiency virus (HIV) infection in vitro. At a concentration of 7.5 micrograms/ml, tachyplesin I suppressed the development of cytopathic effects (CPE) by more than 70% in MT-4 cells infected with HIV (lymphadenopathy-associated virus). This inhibitory effect was observed only when the drug was added during the adsorption period of the virus to the cells. In cocultures of MOLT-4 and persistently HIV-infected cells (MOLT-4/HIV), tachyplesin I at the same concentration completely inhibited multinucleated giant cell formation. Infectivity of HIV was reduced by 10(-2.5) in medium free from fetal calf serum containing tachyplesin I at a concentration of 200 micrograms/ml. Tachyplesin I did not show any inhibitory effect on reverse transcriptase activity of HIV at concentrations of 9-80 micrograms/ml at which tachyplesin I inhibited HIV infection. These results suggest that the anti-HIV action of tachyplesin I was due to the inhibition of virus adsorption.

Animals↗
PubMed1991

Direct virus inactivation of tachyplesin I and its isopeptides from horseshoe crab hemocytes.

Direct virus inactivation of tachyplesin I and related isopeptides, which are antimicrobial peptides isolated from the hemocytes of the horseshoe crab (Tachypleus tridentatus and Limulus polyphemus), was examined against several viruses. Vesicular stomatitis virus (VSV) was inactivated by incubation with tachyplesin I and its isopeptides. Influenza A (H1N1) virus was slightly inactivated by tachyplesin I, whereas herpes simplex virus 1 and 2, adenovirus 1, reovirus 2 and poliovirus 1 were resistant to inactivation. The inactivation of VSV by tachyplesin I depended on the concentration, the time and the temperature of incubation. Pretreatment of tachyplesin I with trypsin or lipopolysaccharide of gram-negative bacteria entirely abolished the antiviral activity. Electron microscopy of VSV treated with tachyplesin I showed naked and damaged virions. These data suggest that tachyplesin I directly inactivates the VSV by destroying its envelope subunits.

Amino Acid Sequence↗
PubMed1991

Medical treatment or surgical intervention? A cooperative retrospective study on infective endocarditis--timing of operation.

Two hundred and five patients treated for infective endocarditis over the last 10 years were reviewed. There were 185 cases of native valve endocarditis (NVE) and 20 of prosthetic valve endocarditis (PVE). In the NVE group there were 175 clinically active patients and 10 non-active patients. The mortalities among 108 non-surgical and 57 surgical patients were 15.7% and 14.0%, respectively. Leading causes of deaths in the former were cardiac failure, embolism and cerebral hemorrhage. Patients with embolism showed significantly higher mortality. Culture negative endocarditis resulted in almost the same incidence of hospital death and urgent operation as staphylococcal endocarditis, and a higher incidence than streptococcal endocarditis. In 9 of 33 patients operated at our hospital, surgery was performed on an urgent basis and one NYHA class IV patient died. Indications for operation were intractable cardiac failure, uncontrollable infection and angina. In the PVE group, 3 of 4 patients operated in the active stage died of severe cardiac failure generated preoperatively. The only survivor was a patient operated early under stable hemodynamics. These results suggest that culture negative endocarditis should be observed as closely as staphylococcal endocarditis and early operation should be considered for patients with progressive cardiac failure, embolism and uncontrollable infection.

Anti-Bacterial Agents↗
PubMed1991

Long-term results of immunochemotherapy for advanced ovarian cancer.

Fifty-one patients with stage III and IV ovarian adenocarcinoma underwent initial cytoreductive surgery at Kurume University Hospital between January 1982 and October 1985. They were assigned postoperatively by randomized trials to immunochemotherapy or chemotherapy alone. Of all 51 patients, long-term results of immunochemotherapy for 43 patients were evaluated versus radicality of initial cytoreductive surgery. As a result, the group of patients treated with immunochemotherapy tended to have a better prognosis than the group of patients treated with chemotherapy alone, whereas no statistical difference was observed between the two groups. Moreover, no significant difference was observed in the monitoring of OKT 4/8 ratio between the two groups. However, immunochemotherapy produced a favorable prognosis in the patients with residual disease of greater than 2 cm in diameter at initial surgery. In conclusion, these data suggested that immunochemotherapy may have some impact on survival of patients with ovarian adenocarcinoma.

Adenocarcinoma↗
PubMed1991

[Coronary flow characteristics in hypertrophic cardiomyopathy--a study with Doppler catheter].

UNLABELLED: We compared the pattern and reserve of coronary flow in 8 cases of hypertrophic non-obstructive cardiomyopathy (H) with those in 20 cases of chest pain not accompanied by organic heart disease (N). A catheter-tip Doppler velocimeter was positioned in the proximal portion of the left anterior descending (LAD), circumflex (LCX) and right coronary (RCA) arteries. Coronary flow velocity (Vs: systolic peak, Vd: diastolic peak, Vm: mean) was recorded and the area under the velocity curve was divided into systole (* s) and diastole (* d). The time interval between the dicrotic notch in aortic pressure and the peak of diastolic flow velocity was measured (Tpv). Vm was measured before and after intracoronary injection of 6 ml of contrast media, and peak to resting velocity ratio (PRVR) was calculated as an index of coronary flow reserve. RESULT: In LAD, N showed diastolic predominant coronary flow pattern without backward flow. In H, diastolic predominance was more prominent with systolic backward flow, resulting in decrease in * s/* d(H: 0.07 +/- 0.04, N: 0.25 +/- 0.02, p less than 0.01). In H, Vd (H: 20.1 +/- 2.8, N: 9.2 +/- 1.4 cm/sec, p less than 0.05) and Vm(H: 9.5 +/- 1.3, N: 4.9 +/- 0.7 cm/sec, p less than 0.05) were higher, while PRVR was lower (H: 1.7 +/- 0.1, N: 2.6 +/- 0.1, p less than 0.05). In both N and H, the flow pattern of LCX was diastolic predominant with two peaks (one in systole and the other in diastole).(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1991

[Phase I clinical study on 99mTc-MIBI].

A phase I clinical study on 99mTc-hexakis 2-methoxy isobutylisonitrile (99mTc-MIBI) was carried out in 6 normal volunteers. There was no significant change in vital signs and laboratory parameters attributing to the reagent other than complaint of slight and transient metallic taste immediately after the injection in 4 volunteers. The highest dosimetry was calculated as 1.1 mGy/37 MBq at lower large intestine, which was within the acceptable range. 99mTc-MIBI was rapidly cleared from the blood and accumulated in the heart immediately after the injection with 1.4% dose and 1.8% dose at 5 min at rest and at stress, respectively. The retention of radioactivity in the heart well continued for at least several hours. The heart-to-lung ratio was over 2.00 at 5 min and heart-to-liver ratio was over 1.00 at 60 min. Myocardial planar and SPECT images were obtained with high quality. In conclusion, 99mTc-MIBI is a useful myocardial perfusion imaging agent.

Adult↗
PubMed1991

[Real-time coronary blood flow measurement in patients with atrial fibrillation: a study using a catheter-tip Doppler velocimeter].

To clarify the influence of changes in the cardiac cycle length (R-R) and aortic pressure on the coronary blood flow, a catheter-tip Doppler velocimeter was applied for 16 patients with chronic atrial fibrillation (11 with valvular heart disease, 2 with coronary artery disease, 2 with cardiomyopathy and one with atrial septal defect). An area under the coronary flow velocity curve during systole (integral of S), diastole (integral of D) and one cardiac cycle (integral of T) for the proximal portion of the left anterior descending artery (LAD: 12 cases) or the right coronary artery (RCA: 10 cases) was calculated in beat-by-beat. Then, the correlations between each area and the R-R, systolic period (S), diastolic period (D) and aortic pressure were assessed. In both the LAD and RCA, prolongation of R-R associated with prolonged D increased integral of D, which caused an increase of integral of T. Integral of D correlated with D (p < 0.05), but integral of S did not correlate with S, and the degree of change in integral of S or S was much less than that in integral of D or D. R-R or D of the preceding beat correlated inversely (p < 0.05) with integral S in 11 of 12 LAD cases. In the RCA, positive correlations between R-R or D of the preceding beat and integral of S were observed in cases with mitral stenosis (n = 6) or coronary heart disease (n = 1), but not in other cases; a case with aortic regurgitation or hypertrophic cardiomyopathy, negative, and dilated cardiomyopathy, no correlation.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1991

Fibrinogen Kanazawa: a congenital dysfibrinogenaemia with delayed polymerization having a replacement of proline-18 by leucine in the A alpha-chain.

Congenital dysfibrinogenaemia was found in a 39-year-old female and her two children. The proposita, apparently heterozygous for this abnormality, had no episode of bleeding or thrombosis. The abnormal fibrinogen showed normal release of fibrinopeptides A and B but impaired polymerization of the fibrin monomer. Amino acid sequence analysis of the whole A alpha-chain isolated from fibrinogen Kanazawa showed a substitution of Leu for Pro at position 18 in the A alpha-chain. This substitution was corroborated by the analysis of the amino acid sequence which demonstrated the lysyl endopeptidase peptides derived from the A alpha-chain of fibrinogen Kanazawa. The minimal genetic exchange responsible for this substitution was a C----T transition in the middle position of the Pro codon. We conclude that Pro-18 in the A alpha-chain is crucial for the polymerization of the fibrin monomer.

Adult↗
PubMed1990

Proclotting enzyme from horseshoe crab hemocytes. cDNA cloning, disulfide locations, and subcellular localization.

Proclotting enzyme is an intracellular serine protease zymogen closely associated with an endotoxin-sensitive hemolymph coagulation system in limulus. Its active form, clotting enzyme, catalyzes conversion of coagulogen to insoluble coagulin gel. We present here the cDNA and amino acid sequences, disulfide locations, and subcellular localization of proclotting enzyme. The isolated cDNA for proclotting enzyme consists of 1,501 base pairs. The open reading frame of 1,125 base pairs encodes a sequence comprising 29 amino acid residues of prepro-sequence and 346 residues of the mature protein with a molecular mass of 38,194 Da. Three potential glycosylation sites for N-linked carbohydrate chains were confirmed to be glycosylated. Moreover, the zymogen contains six O-linked carbohydrate chains in the amino-terminal light chain generated after activation. The cleavage site that accompanies activation catalyzed by trypsin-like active factor B, proved to be an Arg-Ile bond. The resulting carboxyl-terminal heavy chain is composed of a typical serine protease domain, with a sequence similar to that of human coagulation factor XIa (34.5%) or factor Xa (34.1%). The light chain has a unique disulfide-knotted domain which shows no significant homology with any other known proteins. Thus, this proclotting enzyme has a mammalian serine protease domain and a structural domain not heretofore identified in coagulation and complement factors. Immunohistochemical studies showed that the proclotting enzyme is localized in large granules of hemocytes.

Amino Acid Sequence↗
PubMed1990

Antimicrobial tachyplesin peptide precursor. cDNA cloning and cellular localization in the horseshoe crab (Tachypleus tridentatus).

The hemocytes of the horseshoe crab have been found to contain a new family of Arthropodous antibiotics, termed the "tachyplesin family." These peptides are composed of 17-18 amino acid residues with a carboxyl-terminal arginine alpha-amide. We report here the entire cDNA sequence coding for the tachyplesin precursors and their distribution in various tissues of the horseshoe crab. Sequence analysis of the cloned cDNAs revealed that the tachyplesin precursors consist of 77 amino acids with 23 residues in a presegment, and that there are two types of mRNAs corresponding to the isopeptides tachyplesins I and II. Both precursors contain a putative signal peptide, a processing peptide sequence and a carboxyl-terminal amidation signal "Gly-Lys-Arg" connected to the mature tachyplesin peptide. Moreover, an unusual acidic amino acid cluster, Asp-Glu-Asp-Glu-Asp-Asp-Asp-Glu-Glu-COOH, is present in the carboxyl-terminal portions of both precursors. These results suggest that the two types of tachyplesin precursors are first synthesized as preproproteins and are then incorporated into the intracellular organelle, accompanied by various processing events. Northern blot analysis on a total RNA from various tissues of the horseshoe crab revealed that the tachyplesin precursors are expressed mainly in hemocytes and cardiac and brain tissues. Tachyplesin was immunohistochemically localized in the smaller dense granules rather than the typical large granules present in abundance in the hemocytes.

Amino Acid Sequence↗
PubMed1990

Amino acid sequence of human protein Z, a vitamin K-dependent plasma glycoprotein.

Protein Z is a vitamin K-dependent glycoprotein isolated and characterized from human and bovine plasma. A cDNA coding for human protein Z has been obtained by the isolation of phage clones from a liver cDNA library and in vitro amplification of two other liver libraries. Protein Z is synthesized with a prepro-leader sequence of 40 amino acids. The mature protein is composed of 360 residues including a Gla domain of 13 carboxyglutamic acid residues, two epidermal growth factor domains, and a carboxyl terminal region which is highly homologous to the catalytic domain of serine proteases. Human protein Z, however, contains an Asp instead of Ser and a Lys instead of His in the catalytic triad of the active site.

Amino Acid Sequence↗
PubMed1990

Blood clotting factor IX Nagoya 3: the molecular defect of zymogen activation caused by an arginine-145 to histidine substitution.

Factor IX Nagoya 3 (IX Nagoya 3) is a natural mutant of factor IX recognized in a patient with moderately severe hemophilia B. The patient had 0.60 units/ml of factor IX antigen and 2-5% of clotting activity. IX Nagoya 3 was purified from the patient's plasma by immunoaffinity chromatography with an anti-factor IX monoclonal antibody column. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that the treatment of IX Nagoya 3 with factor XIa/calcium ions resulted in cleavage only at the Arg180-Val181 bond. The amino acid sequence analysis of one of the lysyl endopeptidase peptides derived from IX Nagoya 3 revealed that Arg-145 is replaced by His. This substitution impairs the cleavage between the light chain and the activation peptide by factor XIa/calcium ions.

Amino Acid Sequence↗
PubMed1990

Importance of the alpha 3-fragment of complement C4 for the binding with C4b-binding protein.

The human regulatory complement component C4b-binding protein (C4BP) is a multimeric plasma protein, which regulates the classical pathway of the complement system. C4BP functions as a cofactor to factor 1 in the degradation of C4b and accelerates the decay rate of the C4b2a complex. Previously, we have demonstrated that monoclonal antibodies (C4-2 and 9) directed against the alpha'-chain of C4b inhibit the binding of C4b to C4BP. In order to identify the structural domain of C4b that binds C4BP, proteolytic fragments of C4 were generated with trypsin and Staphylococcus aureus V8 protease. Sodium dodecyl sulfate polyacrylamide gel electrophoresis, immunoblotting and amino acid sequence analysis of the proteolytic fragments reactive with the anti-C4 mAb's revealed that the residues Ala738-Arg826 of the alpha 3-fragment of C4b are important for the interaction with C4BP.

Amino Acid Sequence↗
PubMed1990

The complete amino acid sequence of the high molecular mass hemorrhagic protein HR1B isolated from the venom of Trimeresurus flavoviridis.

Hemorrhage is a common occurrence in a victim bitten by crotalid and viperid snakes, and hemorrhagic components in these various venoms have been isolated and characterized. Previously, we have shown that a low molecular weight hemorrhagic protein (HR2a, 202 amino acid residues) isolated from the venom of Trimeresurus flavoviridis is a member of a new subfamily of metalloproteinases. We now report the complete amino acid sequence of a high molecular mass hemorrhagic protein isolated from the same venom. This protein, HR1B, is a mosaic protein composed of 416 residues containing four asparagine-linked oligosaccharide chains. The amino-terminal half (residues 1-203) of HR1B contains a metalloproteinase domain, the sequence of which is 62% identical to that of HR2a and 52% identical to that of hemorrhagic toxin d isolated from Crotalus atrox venom. The most interesting finding is that the middle region (residues 204-300) of HR1B shows a striking similarity to disintegrins, Arg-Gly-Asp-containing platelet aggregation inhibitors, recently found in several viper venoms. Interestingly, however, this region of HR1B does not contain the Arg-Gly-Asp sequence which is known to be a putative binding site in the disintegrins for the platelet fibrinogen receptor, the glycoprotein IIb-IIIa complex. We also found that the carboxyl-terminal region (residues 213-336) of the middle part of HR1B shows 30% identity to residues 1543-1656 of von Willebrand factor and that the remaining region at the carboxyl-terminal end is unique and has a cysteine-rich sequence. These results suggest that the middle portion of HR1B, which shows structural similarities to the disintegrins and von Willebrand factor, may be important in synergistically stimulating hemorrhagic activity in the NH2-terminal metalloproteinase domain.

Amino Acid Sequence↗