Search PubMed⌕ Search

Biomedical subjects

S Iwanaga

Publications and source records attributed to S Iwanaga.

At least 145 records · Page 8Linked to original sources
PubMed1993

The limulus clotting reaction.

Our biochemical studies on the hemolymph coagulation-complement system using limulus indicate that the circulating hemocytes contain at least four serine protease zymogens and one clottable protein, coagulogen, which constitute a cascade triggered by bacterial endotoxins and (1,3)-beta-D-glucan. We also found several antimicrobial substances, tachyplesin peptides and anti-lipopolysaccharide factor, in the hemocytes. These clotting factors and antimicrobial substances are released into the hemolymph in response to lipopolysaccharide, where they cooperate in immobilization and killing of invading microorganisms as a host defense.

Amino Acid Sequence↗
PubMed1993

Clinical evaluation of the use-dependent QRS prolongation and the reverse use-dependent QT prolongation of class I and class III antiarrhythmic agents and their value in predicting efficacy.

We measured the QRS duration during a treadmill exercise test and the QT interval using a 24-hour Holter electrocardiogram at various heart rates to identify use-dependent QRS prolongation and reverse use-dependent QT prolongation of class I and III antiarrhythmic drugs. Use-dependent QRS prolongation was detected in 61%, 53%, and 64% of patients receiving disopyramide, mexiletine, and pilsicainide, respectively. Reverse use-dependent QT prolongation was found in 40% and 70% of patients receiving disopyramide and E4031. Drugs suppressed > or = 75% of the total premature ventricular contractions in all patients who had both use-dependent QRS prolongation and reverse use-dependent QT prolongation, in 79% of patients with use-dependent QRS prolongation alone, in 70% with reverse use-dependent QT prolongation alone, and in 11% with neither use-dependent QRS prolongation nor reverse use-dependent QT prolongation. Use-dependent QRS prolongation and reverse use-dependent QT prolongation were identified noninvasively and were useful in evaluating antiarrhythmic efficacy.

Adult↗
PubMed1993

Primary structures of platelet aggregation inhibitors (disintegrins) autoproteolytically released from snake venom hemorrhagic metalloproteinases and new fluorogenic peptide substrates for these enzymes.

A hemorrhagic protein (60 kDa), HR1B, present in the venom of Trimeresurus flavoviridis is a mosaic protein consisting of an NH2-terminal metalloproteinase-domain, a disintegrin (platelet aggregation inhibitor)-like domain, and a unique COOH-terminal Cys-rich domain. Since the gross structures of HR1B and protein precursors of disintegrins, trigramin, and rhodostomin, all of which contain the metalloproteinase domain, are similar, many disintegrins so far detected in snake venoms are assumed to be autoproteolytic fragments released from precursors. In ongoing related experiments, the newly purified hemorrhagic metalloproteinases, HR1A from T. flavoviridis venom and HT-1 from Crotalus ruber ruber venom, in addition to HR1B, were autoproteolyzed, in the absence of Ca2+, at 37 degrees C for 3-12 h. Under these conditions, HR1A, HR1B, and HT-1 each released a single major fragment of 32, 34, and 31 kDa, respectively. The entire amino acid sequences of the isolated fragments indicated the presence of disintegrin-like and Cys-rich domains in the COOH-terminal regions of HR1A, HR1B, and HT-1, respectively. It seems likely that so-called disintegrins probably originate from various metalloproteinases present in venom. On the bases of peptide sequences close to the autoproteolytic cleavage sites of these metalloproteinases and the sites of fibrinogen cleaved by these enzymes, we synthesized new intramolecularly quenched fluorogenic peptide substrates. Among the 10 peptides tested, 2-aminobenzoyl (Abz)-Ser-Pro-Met-Leu-2,4-dinitroanilinoethylamide (Dna) proved to be the best substrate for venom metalloproteinase, as deduced from kinetic analyses.

Amino Acid Sequence↗
PubMed1993

Separation of large and small granules from horseshoe crab (Tachypleus tridentatus) hemocytes and characterization of their components.

We designed a method for separating two types of granules, a smaller (S) but dense and a larger (L) but less dense granule from hemocytes of the horseshoe crab (Tachypleus tridentatus), using continuous sucrose density gradient centrifugation. The isolated L-granules contained at least three clotting factors plus a clottable protein, coagulogen, as the major component. The known anti-lipopolysaccharide factor and 7 additional unknown protein components were also present in the L-granules. Two known natural substrates, Pro-rich protein and 8.6 kDa protein, for limulus transglutaminase [Tokunaga, F., Yamada, M., Miyata, T., Ding, Y.-L., Hiranaga-Kawabata, M., Muta, T., Iwanaga, S., Ichinose, A., & Davie, E.W. (1993) J. Biol. Chem. 268, 252-261] were present in the L-granules. On the other hand, the isolated S-granules contained antimicrobial tachyplesins I and II (17 amino acids in length) as the major component, in addition to 6 unidentified proteins with molecular masses of less than 30 kDa. The structural analyses of tachyplesin analogs indicated that all these peptides of mature form are stored in the S-granules, together with a processing intermediate containing the COOH-terminal Gly-Lys sequence. We also found an Arg-rich protein of 22 kDa and a Leu-rich protein of 30 kDa in S-granules. Based on these observations, we speculate that protein components in L-granules, which probably contain all the factors essential for the limulus clotting system, participate in immobilization of invading microbes and that factors in the S-granules containing tachyplesins contribute to a self-defense system against invaders.

Amino Acid Sequence↗
PubMed1993

Structural analysis of O-linked sugar chains in human blood clotting factor IX.

Type and structural analysis of O-linked sugar chains in human blood clotting factor IX was performed by the pyridylamination method developed for O-linked sugar chains [Kuraya, N. & Hase, S. (1992) J. Biochem. 112, 122-126]. O- and N-linked sugar chains were released with hydrazine, and then N-acetylated, followed by pyridylamination. The type of sugar chain was determined by reducing-end analysis of the pyridylaminated (PA-) sugar chains. Sugar chains with PA-GalNAc at the reducing terminal and that with PA-Fuc [Nishimura, H. et al. (1992) J. Biol. Chem. 267, 17520-17525] were obtained besides known sugar chains with PA-Glc from the Xyl-Glc-Ser type and those with PA-GlcNAc from asparagine-linked sugar chains. The sugar chains with PA-GalNAc were identified as mono- and disialyl Gal beta 1-3GalNAc by two-dimensional HPLC mapping. The structure of the sugar chain with PA-Fuc was Neu5Ac alpha 2-6Gal beta 1-4GlcNAc beta 1-3Fuc, as determined by exoglycosidase digestion, methylation analysis, and Smith degradation.

Carbohydrate Conformation↗
PubMed1993

Purification and characterization of a protein cryoprotective for Vibrio cholerae extracted from the prawn shell surface.

A substance cryoprotective for Vibrio cholerae on the prawn shell surface was purified by ammonium sulfate precipitation and gel filtration. It was a protein of 81 kDa and called cryoprotective protein (CPP). The cryoprotective activity of this protein for V. cholerae was sensitive to heat at 100 C and trypsin treatment. In the presence of Mg ion the protein can bind to the bacterial cell surface. V. cholerae can adhere to the shell surface of the prawn. The number of adhered bacteria was reduced by treating the shell with anti-CPP serum, heat or by trypsin. The presence of Mg ion promoted the adherence. These results suggest that the CPP could serve as an adherence site for V. cholerae on the shell surface.

Amino Acid Sequence↗
PubMed1993

Alveolar soft part sarcoma of the uterine cervix.

A rare case of an alveolar soft part sarcoma of the uterine cervix in an 8 year old girl is presented. The patient was admitted because of genital bleeding lasting for 7 months. A polypoid tumor, 2 x 1.5 cm in diameter, was found in her external uterine os and was surgically resected. Microscopically, the tumor consisted of a uniform sheet of tumor cells in the cytoplasm which contained granules and which were stained with periodic acid-Schiff, both before and after the diastase digestion. Alveolar arrangement of the tumor cells was manifested with reticulin silver impregnation. Dense, membrane bound granules were evident at an ultrastructural level in the cytoplasm of the tumor cells. An immunohistochemical examination demonstrated a positive reaction for anti-desmin, anti-myoglobin, anti-HHF35 and anti-neuron specific enolase in the cytoplasm.

Child↗
PubMed1993

Mode of action of an antimicrobial peptide, tachyplesin I, on biomembranes.

An antimicrobial peptide, tachyplesin I, isolated from hemocytes of the horseshoe crab, Tachypleus tridentatus, increased the K+ permeability of Staphylococcus aureus and Escherichia coli cells, concomitantly reducing cell viability. At a higher concentration range, this peptide also enhanced the permeability of human erythrocytes. Tachyplesin decreased the phase transition temperature of an artificial membrane composed of dipalmitoylphosphatidylglycerol and, further, broadened it extensively, while it did not affect that of dipalmitoylphosphatidylcholine membrane. The latter result related closely to the fact that this peptide acted weakly on erythrocytes in which acidic lipids constitute a minor portion. Tachyplesin altered the normal discoid shape of human erythrocytes to a crenated form, suggesting that the peptide accumulated predominantly in the outer half of the membrane bilayer and destabilized the membrane structure, thus causing the change in permeability. The mode of action of tachyplesin was compared with that of gramicidin S, a peptide forming an amphiphilic structure analogous to tachyplesin.

Amino Acid Sequence↗
PubMed1993

Immature teratoma of the uterine fundus.

An exceedingly rare case of extragonadal immature teratoma, which occurred primarily in the uterus, is described. The tumor developed into the pelvic cavity from the uterine fundus and consisted of ectodermal, mesodermal and endodermal derivatives. There were also significant amounts of immature elements; immature neuroepithelium with brisk mitotic activity, immature mesenchymal tissue, immature cartilage, immature striated muscle and immature hepatic tissue. Histologically, it was classified as a grade 3 immature teratoma. Treatment consisted of total simple hysterectomy followed by 2 courses of combination chemotherapy with vincristine, actinomycin D and cyclophosphamide (VAC). The patient was well and without evidence of recurrence at 5 years post-operatively.

Adult↗
PubMed1993

[Coronary flow dynamics in the right coronary artery providing collateral circulation: comparison before and after successful angioplasty with a Doppler catheter].

Coronary blood flow dynamics were investigated in 9 patients with isolated coronary artery disease of the left anterior descending artery (LAD) with good collateral flow (grade 2 or 3 of Rentrop's classification) from the right coronary artery (RCA) and 20 patients with normal coronary arteries as controls. The coronary flow velocity (Vs: systolic peak, Vd: diastolic peak, Vm: mean) was measured with a Doppler catheter and the diameter (D) by the edge detection method in the proximal portion of the RCA. Vs/Vd was calculated. The areas under the velocity curve during systole (integral of s) and diastole (integral of d) were measured to obtain the ratio (integral of s/integral of d). Peak to resting velocity ratio (PRVR) was obtained as an index of coronary flow reserve, using intracoronary injections of 6 ml contrast medium or 8-12 mg papaverine. These parameters before percutaneous transluminal coronary angioplasty (PTCA), after PTCA, and in control subjects were compared. The collaterals disappeared angiographically immediately after successful PTCA. D (before PTCA: 3.2 +/- 0.1 mm, after PTCA: 3.3 +/- 0.1 mm, controls: 3.2 +/- 0.1 mm) and Vs did not vary between before and after PTCA and in control subjects (NS). The values of Vd, Vm decreased and Vs/Vd and integral of s/integral of d increased after PTCA to the values in the controls. PRVR obtained with contrast medium and papaverine also increased.

Aged↗
PubMed1993

[Lethal arrhythmias in a patient with coarctation of the aorta and severe heart failure: their control by combination of low dose amiodarone with procainamide for 3 years].

In 1970, a 19 year-old man was diagnosed as having coarctation of the aorta (CoA). But the patient and his family rejected further examination for CoA and high blood pressure was treated after that time. When the patient was 37 years old, he was admitted to our hospital because of congestive heart failure. During the 2nd admission for determining the operability of CoA in December, 1988, non-sustained ventricular tachycardia was detected. Immediately, intravenous administration of lidocaine or/and mexiletine were started. However, cardiac arrest occurred. After his recovery, lethal ventricular arrhythmias were still observed frequently despite administration of class Ia or Ib antiarrhythmic drugs. Oral amiodarone administration (600 mg) with procainamide (1000 mg) was started on 1st of May, 1989. Axillo-femoral bypass graft was performed during the 2nd admission because curable operation was abandoned because of severely impaired cardiac function. Subsequently, the patient was admitted 5 times due to exacerbated congestive heart failure. However, lethal arrhythmias were able to be controlled by the combination of low dose amiodarone (100-200 mg) with procainamide until he died of congestive heart failure on 9th of May, 1992. We reported a rare adult case with CoA and severe heart failure. Lethal arrhythmias in this case were well controlled by the combined administration of low dose amiodarone with procainamide regardless of severely impaired cardiac function.

Administration, Oral↗
PubMed1993

[Rest and stress myocardial perfusion imaging on the same day with two injections of 99mTc-tetrofosmin].

99mTc-tetrofosmin myocardial perfusion imagings under different protocols were performed at rest and stress on the same day. In the stress/rest protocol, the exercise study was carried out first, and then the rest one followed. Eight patients were involved in the stress/rest protocol. Seven patients were examined in the reverse, rest/stress protocol. In any protocols, the injection interval was 3 hours, and injection doses in the first and second studies were 370 MBq and 740 MBq, respectively. Myocardial counts were obtained by placing region of interest over the myocardial walls in short axial SPECT images. Based on myocardial counts from the first injection and the wash-out rate of tetrofosmin, we calculated, at the second imaging, counts caused from the first injection. Approximately 20-25% of counts in the second study were found to be caused from the residual radiotracer. The residual radiotracers affected the interpretation of imagings of two patients we examined: one with marked reversible ischemia examined in the stress/rest protocol and the other with mild ischemic change examined in the rest/stress one. Our results suggested that some modifications of studies, such as the increase in the injection intervals or the reduce of the first-to-second dose ratio, might be necessary to conduct the same day protocols.

Exercise Test↗
PubMed1992

Conformation of tachyplesin I from Tachypleus tridentatus when interacting with lipid matrices.

The mode of action of tachyplesin I, an antimicrobial cationic heptadecapeptide amide isolated from the hemocyte debris of a horseshoe crab, Tachypleus tridentatus, toward lipid matrices was studied with synthetic tachyplesin I, its analogs with Phe in place of Trp or Tyr, a linear analog with no disulfide bonds, and two linear short fragments. Circular dichroism spectra showed that tachyplesin I took an antiparallel beta-structure in buffer solution and a certain less ordered structure in acidic liposomes composed of egg phosphatidylcholine and egg phosphatidylglycerol (3:1). Spectrophotometric titration of the peptides with laurylphosphorylcholine revealed that both Trp and Tyr residues orient toward the inside of lipid matrices, suggesting that they are on the same side of the peptide backbone. The carboxyfluorescein leakage experiment and fluorescence data indicated that tachyplesin I interacted strongly with neutral and acidic lipid bilayers and an aromaticity-rich hydrophobic part of the peptide was embedded in lipid membranes. All the peptides except for the short fragments were almost equally active in lipopolysaccharide binding. The energy-transfer experiment showed that a conformational change occurred such that the Tyr and Trp residues are positioned more closely to each other in acidic liposomes than in buffer solution. The present study strongly suggested that amphipathic lipid bilayers induced a conformational change of tachyplesin I from an energetically stable beta-structure to a less ordered, probably more amphipathic structure.

Amino Acid Sequence↗
PubMed1992

Expression of human soluble tissue factor in yeast and enzymatic properties of its complex with factor VIIa.

The extracellular domain of human tissue factor (TF, amino acids 1-217) was expressed in Saccharomyces cerevisiae, using the inducible yeast acid phosphatase promoter and the yeast invertase signal sequence to direct its secretion into the culture broth. Two active soluble forms sTF alpha (high molecular weight form) and sTF beta (low molecular weight form) were purified, the yield being approximately 10 and 1 mg/liter of culture supernatant, respectively. sTF alpha had an apparent molecular mass of 150 kDa on SDS-polyacrylamide gel electrophoresis and contained more than 200 residues of mannose/mol of protein. sTF beta had an apparent molecular mass of 37 kDa and contained 22 residues of mannose/mol of protein. N-Glycosidase F treatments of both rTFs reduced the apparent molecular mass to 35 kDa. The amino-terminal sequences and amino acid compositions of sTF alpha and sTF beta were consistent with those deduced from the cDNA sequence, thereby indicating that the difference in molecular mass is caused by heterogeneity of oligosaccharide structures. Of these recombinant TFs, sTF beta enhanced factor VIIa-amidolytic activity 40-fold toward the chromogenic substrate and 147-fold toward the fluorogenic substrate, affecting mainly the kcat value. The enhancement was comparable with that of TF purified from human placenta. The TF-mediated enhancement of factor VIIa-amidolytic activity was inhibited by heparin-activated antithrombin III, forming a high molecular weight complex. As treatment of sTF beta with denaturants such as guanidine hydrochloride or urea led to a biphasic loss of the activity, the extracellular domain of TF probably consists of two discrete domains. This expression system provides a significant amount of the extracellular domain of TF so that studies of interactions with factor VII are feasible.

Amino Acid Sequence↗
PubMed1992

Molecular mechanism of hemolymph clotting system in Limulus.

Limulus (horseshoe crab) hemolymph is known to be very sensitive to bacterial endotoxin (LPS), which causes a rapid coagulation response. Hemolymph contains a single type of hemocyte that undergoes aggregation, adhesion, and degranulation in response to LPS. The granule contents are released into the hemolymph, where they form an insoluble gel. We have characterized four components involved in this coagulation response that comprise a cascade of three serine protease zymogens (factor C, factor B, and proclotting enzyme) and one clottable protein (coagulogen). Of these components, factor C sensitive to LPS is a protein composed of five complement-related domains ("Sushi" or SCR), an EGF-like domain, and a C-type lectinlike domain as well as a putative amino-terminal LPS-binding domain. This domain structure is very similar to that of selectin family of cell adhesion molecules, suggesting that it might also function as a cell adhesion molecule after the release into the hemolymph. Factor B and the proclotting enzyme share a common Cys-rich motif ("cliplike" domain) in the amino-terminal portions. This domain is also found in a putative serine protease zymogen ("easter") in Drosophila, which is essential for normal embryonic development. All four of the components of the cascade and an antibacterial protein (anti-LPS factor) are localized to a specific type of the hemocyte granule. Another antibacterial peptide (tachyplesins I and II) is localized in a distinct granule population. The contents of both granule populations are released into the hemolymph in response to LPS, where they cooperate in immobilization and killing of Gram-negative bacteria.

Amino Acid Sequence↗
PubMed1992

Human factor IX has a tetrasaccharide O-glycosidically linked to serine 61 through the fucose residue.

We have recently discovered unusual sugar chains (xylose (Xyl)-glucose (Glc) and (Xyl)2-Glc) linked to a serine residue in the epidermal growth factor (EGF)-like domains of human and bovine clotting factors VII (Ser-52), IX (Ser-53), and protein Z (Ser-53), in addition to bovine platelet glycoprotein thrombospondin. We now have evidence of another modification in the first EGF-like domain of human factor IX, which proved to be a tetrasaccharide O-fucosidically linked to Ser-61. Two large peptides containing Ser-61 (positions 44-63), named hIX-GP1 and hIX-GP2, were first isolated from the lysyl endopeptidase-digest of human factor IX, by reversed-phase high performance liquid chromatography (HPLC). Data on the component sugar analysis after pyridylamination (PA) and sialic acid analysis of the isolated peptides indicated that they contained 1 mol each of galactose (Gal), fucose (Fuc), N-acetylglucosamine (GlcNAc), and N-acetylneuraminic acid (NeuAc), in addition to Glc and Xyl. hIX-GP1 was further digested with asparaginyl endopeptidase, and two glycopeptides containing Ser-61, named N-3 (positions 59-63) and N-9 (positions 55-63), were isolated, respectively. These glycopeptides were both composed of 1 mol each of Gal, Fuc, GlcNAc, and NeuAc but did not contain Xyl and Glc. Moreover, the data on beta-elimination for N-9 and of the fast atom bombardment mass spectrometric analysis on peptide N-3 suggested the presence of a tetrasaccharide linked to Ser-61. An analysis of the PA-oligosaccharide released from hIX-GP1 by hydrazinolysis followed by pyridylamination revealed that the reducing end was PA-Fuc. All the results support the proposal that human factor IX has a novel tetrasaccharide consisting of 1 mol each of Gal, Fuc, GlcNAc, and NeuAc, which is O-glycosidically linked to Ser-61 through the Fuc residue.

Amino Acid Sequence↗
PubMed1992

Tissue factor potentiates the factor VIIa-catalyzed hydrolysis of an ester substrate.

We designed a simple and sensitive method to assay the activity of the factor VIIa-tissue factor complex, using as a substrate N alpha-benzyloxycarbonyl-L-arginine p-nitrobenzyl ester (Z-Arg-ONb) (Zur, M., and Nemerson, Y. (1978) J. Biol. Chem. 253, 2203-2209). The principle was to measure the amount of p-nitrobenzyl alcohol released during ester hydrolysis using reversed-phase high performance liquid chromatography. Z-Arg-ONb had a broad specificity for plasma serine proteases and factor IXa. Using this method, we examined the effect of tissue factor on the esterase activity of factor VIIa under various conditions. We found that tissue factor greatly potentiates the factor VIIa-catalyzed hydrolysis of Z-Arg-ONb. Phospholipids were not required for the factor VIIa-catalyzed hydrolysis of Z-Arg-ONb, even in the presence of tissue factor. The Km value of factor VIIa alone toward the ester substrate was six times higher than that of a VIIa-tissue factor complex (3.2 versus 0.54 mM), whereas the kcat value was 12 times lower than that of the VIIa-tissue factor complex (14.3 versus 173 s-1). Thus, tissue factor apparently affects the catalytic site of factor VIIa and enhances hydrolysis of the ester substrate. This enhancing effect of tissue factor disappeared on removal of the gamma-carboxyglutamic acid domain from factor VIIa, whereas the esterase activity in the absence of tissue factor was not affected by this modification. The gamma-carboxyglutamic acid domain is probably required as a potent determinant for interactions with tissue factor, even in the absence of phospholipids in the reaction mixture.

Amino Acid Sequence↗