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Biomedical subjects

Y Usami

Publications and source records attributed to Y Usami.

At least 37 records · Page 2Linked to original sources
PubMed1994

Direct detection using the Drosophila DNA-repair test and isolation of a DNA-damaging mycotoxin, 5,6-dihydropenicillic acid, in fungal culture.

The Drosophila DNA-repair test was used in an attempt to detect fungal production of DNA-damaging mycotoxins without an extraction process. 29 species of fungi, 13 Aspergillus, 12 Penicillium and four Fusarium were inoculated directly to a Drosophila medium, and the larvae were then bred in the mouldy medium. Production of DNA-damaging mycotoxin was detected directly by counting the decrease in the survival of DNA-repair-deficient flies. With the direct detection method, Aspergillus ochraceus, A. parasiticus and A. versicolor produced DNA-damaging mycotoxins. The same results were obtained with the mouldy medium extract using the standard DNA-repair test. The direct detection method was convenient for surveying the fungal production of DNA-damaging mycotoxins. The extracts of A. parasiticus and A. versicolor contained aflatoxin B1 and sterigmatocystin, respectively. The DNA-damaging compound in the extract of A. ochraceus was isolated and purified to clear, colourless 'needles'. With nuclear magnetic resonance-mass spectroscopy spectra, the compound was confirmed to be 5,6-dihydropenicillic acid, the DNA-damaging potency of which has not been previously reported.

Animals↗
PubMed1994

Phytogrowth-inhibitory activities of 2-thiophenecarboxylic acid and its related compounds.

2-Thiophenecarboxylic acid (I) exhibited growth-inhibitory activity in five kinds of plants. In particular, I strongly inhibited the growth of the roots of Lactuca sativa L. var. longifolia LAM and Echinochloa utilis OHWI et YABUNO, even at the low concentration of 5.0 x 10(-3) M. Furthermore, all of the I-related compounds (II-V and VII-X) except for VI, showed more or less obvious inhibitory activity on the seeds of Sesamum indicum L. Compounds VII-X, in which the carboxyl group of I was replaced by acetic acid, propionic acid, butyric acid and acrylic acid, and exhibited more potent phytogrowth-inhibitory activity than I. Among these compounds, 2-thiophenebutyric acid (IX) showed the strongest activity. Esterification of the carboxyl group in I increased the inhibitory activity relative to that of I, while amidation and reduction of this group markedly decreased its inhibitory activity. The radicles of the plants treated with each of the compounds except for VI showed negative geotropism, even though germination occurred.

Carboxylic Acids↗
PubMed1994

Migration of canine neutrophils to chitin and chitosan.

Suspension of chitin and chitosan particles (mean size of 1 micron) were found to attract canine neutrophils chemotactically as determined by a checkerboard assay through polycarbonate filter with 5 microns pore size in Blind well chamber. Suspension of chitin induced chemokinetic migrations of the neutrophils. These evidences might reflect accumulation of neutrophils to chitin- and chitosan-implanted regions in dogs.

Animals↗
PubMed1993

Alboaggregin-B and botrocetin, two snake venom proteins with highly homologous amino acid sequences but totally distinct functions on von Willebrand factor binding to platelets.

Alboaggregin-B (AL-B) (Peng et al., Biochemistry (1991) 30, 11529-11536) was highly purified from the snake venom of Trimeresurus albolabris and characterized structurally and functionally, comparing with botrocetin, another snake venom protein recently characterized (Usami et al., Proc. Natl. Acad. Sci. USA (1993) 90, 928-932). Both the venom proteins are a heterodimer and show a high degree of sequence homology to each other and also to C-type lectins. Botrocetin specifically binds to von Willebrand factor (vWF), whereas AL-B binds to platelet glycoprotein (GP) Ib without affecting the binding of botrocetin to vWF. The binding of AL-B to GPIb does not potentiate the platelet aggregation even by exogenous fibrinogen, suggesting that AL-B binding to GPIb does not activate GPIIb/IIIa complex.

Amino Acid Sequence↗
PubMed1993

Primary structure of two-chain botrocetin, a von Willebrand factor modulator purified from the venom of Bothrops jararaca.

The complete amino acid sequence and location of the disulfide bonds of two-chain botrocetin, which promotes platelet agglutination in the presence of von Willebrand factor, from venom of the snake Bothrops jararaca are presented. Sequences of the alpha and beta subunits were determined by analysis of peptides generated by digestion of the S-pyridylethylated protein with Achromobacter protease I or alpha-chymotrypsin and by chemical cleavage with cyanogen bromide or 2-(2'-nitrophenylsulfenyl)-3-methyl-3-bromoindolenine. Two-chain botrocetin is a heterodimer composed of the alpha subunit (consisting of 133 amino acid residues) and the beta subunit (consisting of 125 amino acid residues) held together by a disulfide bond. Seven disulfide bonds link half-cystine residues 2 to 13, 30 to 128, and 103 to 120 of the alpha subunit; 2 to 13, 30 to 121, and 98 to 113 of the beta subunit; and 80 of the alpha subunit to 75 of the beta subunit. In terms of amino acid sequence and disulfide bond location, two-chain botrocetin is homologous to echinoidin (a sea urchin lectin) and other C-type (Ca(2+)-dependent) lectins.

Amino Acid Sequence↗
PubMed1993

Binding of human xanthine oxidase to sulphated glycosaminoglycans on the endothelial-cell surface.

Much evidence has suggested that the superoxide generated by xanthine oxidase (XOD) within the endothelial cell triggers characteristic free-radical-mediated tissue injuries. Although it has been reported that XOD exists not only in the cytoplasm, but also on the outside surface of the endothelial cell membrane, it is not clear how XOD localizes on the outside of the plasma membrane. Purified human xanthine oxidase (h-XOD) had an affinity for heparin-Sepharose. The binding was largely independent of the pH over the physiological range, whereas it tended to increase at lower pH and to decrease at higher pH. Exposure of h-XOD to the lysine-specific reagent trinitrobenzenesulphonic acid or the arginine-specific reagent phenylglyoxal caused it to lose its affinity for heparin-Sepharose. The binding of h-XOD to heparin is apparently of electrostatic nature, and both lysine and arginine residues are involved in the binding. h-XOD was found to bind to cultured porcine aortic endothelial cells, and this binding was inhibited by the addition of heparin or pretreatment of the cells with heparinase and/or heparitinase. Intravenous injection of heparin into two healthy persons led to a prompt increase in plasma h-XOD concentration. These results suggest that XOD localizes on the outside surface of endothelial cells by association with polysaccharide chains of heparin-like proteoglycans on the endothelial-cell membranes. Superoxide extracellularly generated by XOD may injure the source-endothelial-cell membrane and also attract and activate closely appositional neutrophils, which themselves actually cause progressive oxidative damage.

Animals↗
PubMed1993

Pulpal response to a new light-activated fluoride releasing liner.

The purpose of this study was to investigate the degree of pulp irritation of a newly developed light-activated fluoride-releasing adhesive resin liner using canine teeth compared to the amount caused by a negative control, zinc oxide eugenol cement (ZOE), and a positive control, silicate cement (silicate). In the cases of unexposed pulp, this experimental liner showed none, slight or moderate pulpal changes at 3 d postoperatively. At 30 and 90 d, no pulpal response to the experimental liner was detected in almost all cases. These findings were similar to the ZOE and better than the silicate. There was less evidence of pulpal irritation produced by the experimental liner. This material seems to be safe to the pulp under the conditions of this study.

Analysis of Variance↗
PubMed1992

The interaction of botrocetin with normal or variant von Willebrand factor (types IIA and IIB) and its inhibition by monoclonal antibodies that block receptor binding.

We have recently shown the existence of two distinct forms of botrocetin (one-chain and two-chain), and demonstrated that the two-chain species is approximately 30 times more active than the one-chain in promoting von Willebrand factor (vWF) binding to platelet glycoprotein (GP) Ib. The N-terminal sequence of two-chain botrocetin is highly homologous to sea-urchin Echinoidin and other Ca(2+)-dependent lectins (Fujimura et al., Biochemistry 1991; 30: 1957-64). Present data indicate that purified two-chain botrocetin binds to vWF from plasmas of patients with type IIA or IIB von Willebrand disease and its interaction is indistinguishable from that with vWF from normal individuals. However, an "activated complex" formed between botrocetin and IIB vWF expresses an enhanced biological activity for binding to GP Ib whereas the complex with IIA vWF has a decreased binding activity. Among several anti-vWF monoclonal antibodies (MoAbs) which inhibit ristocetin-induced platelet aggregation and/or vWF binding to GP Ib, only two MoAbs (NMC-4 and RFF-VIII RAG:1) abolished direct binding between purified botrocetin and vWF. This suggests that they recognize an epitope(s) on the vWF molecule in close proximity to the botrocetin binding site.

Antibodies, Monoclonal↗
PubMed1992

A new light-activated adhesive composite: shear bond strength and microleakage.

A new light-activated adhesive composite has been formulated. Its shear bond strength and marginal microleakage were compared with glass ionomer cements: Fuji Ionomer Type II, Ketac-Fil and Ketac-Bond. The new material showed significantly (p < 0.05) higher bond strength to non-etched enamel (7.9 MPa) and dentin (7.3 MPa) than commercially available materials. It also performed significantly better (p < 0.05) in the microleakage test. Ease of use and the excellent marginal sealing ability of the new material are expected to allow considerable innovation in restorations of root caries, cervical erosion and other conditions where the marginal seal of the gingival cavosurface is difficult.

Aluminum Silicates↗
PubMed1992

A new light-activated adhesive cavity liner: an in vitro bond strength and microleakage study.

A new formulation of a light-activated calcium hydroxide-based cavity liner was developed for this study. Its shear bond strength and marginal microleakage in a Class V cavity preparation were compared to those of commercially available light-activated liners and conventional glass ionomer liners: GC Lining Cement (GC), Ketac-Bond (ESPE), Time Line (Caulk), Vitrabond (3M), XR lonomer (Kerr), and Zionomer (DenMat). The new formulation had significantly higher shear bond strength to dentin than the commercially available materials. Its microleakage along gingival (cemento-dentin) walls was statistically equivalent to one of the other materials and significantly better than all the others.

Analysis of Variance↗
PubMed1992

Application of L-DNA to the study of the specific DNA recognition mechanism of bleomycin.

The hexadeoxynucleotide analog, L-d(CGCGCG) composed of L-deoxyribose was synthesized and clearly shown to have the same conformation and dynamic properties with natural D-d(CGCGCG) except for chirality with CD spectra. This unnatural hexanucleotide was not cleaved by bleomycin, an antitumor DNA cleaving drug, but was able to bind to the DNA binding domain of bleomycin to a similar extent with the natural one. These results strongly suggest the importance of the other moiety than the DNA binding domain for the specific DNA recognition of bleomycin. Thus, L-oligonucleotides are useful for the study of DNA-drug interactions.

Binding Sites↗
PubMed1991

Isolation and chemical characterization of two structurally and functionally distinct forms of botrocetin, the platelet coagglutinin isolated from the venom of Bothrops jararaca.

Two distinct forms of botrocetin, the von Willebrand factor (vWF)-dependent platelet coagglutinin isolated from the venom of the snake Bothrops jararaca, were purified and characterized structurally and functionally. The apparent molecular mass of the one-chain botrocetin was 28 kDa before and 32 kDa after reduction of disulfide bonds, while that of the two-chain botrocetin was 27 kDa before and 15/14.5 kDa after reduction. Amino acid composition of the two species revealed a similar high content of potentially acidic residues (greater than 60 Asx and Glx residues/molecule) but significant differences in the content of Cys and Phe residues. The NH2-terminal sequence of the one-chain botrocetin was Ile-Ile/Val-Ser-Pro-Pro-Val-Cys-Gly-Asn-Glu-. Two constituent polypeptides of the two-chain botrocetin showed similar but different NH2-terminal sequences, distinct from that of the one-chain species: (alpha) Asp-Cys-Pro-Ser-Gly-Trp-Ser-Ser-Tyr-Glu- and (beta) Asp-Cys-Pro-Pro-Asp-Trp-Ser-Ser-Tyr-Glu-. The carbohydrate content of both species was less than 2% of the total mass, and the pI was 4.0-4.1 for the one-chain species, and 4.6, 5.3-5.4, and 7.7-7.8 for the two-chain species. No free sulfhydryl group was detected in each species. Both types of botrocetin were resistant to proteolysis at neutral pH. Incubation of 125I-labeled one-chain botrocetin with the crude venom solution resulted in no detectable structural change. On a weight basis, the two-chain botrocetin was 34 times more active than the one-chain form in promoting vWF binding to platelets.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1991

Studies on anti-von Willebrand factor (vWF) monoclonal antibody NMC-4, which inhibits both ristocetin- and botrocetin-induced vWF binding to platelet glycoprotein Ib.

Anti-von Willebrand factor (vWF) monoclonal antibody NMC-4 completely inhibited vWF binding to platelet glycoprotein (GP) lb induced by either ristocetin or botrocetin at an IgG concentration of approximately 10 micrograms/mL, and also blocked binding of asialo-vWF to GP lb. NMC-4 coupled beads isolated a 97-Kd fragment (Fr) from a whole tryptic digest of vWF. The N-terminal sequencing of the nonreduced 97-Kd Fr, in combination with amino acid analysis, showed it to be a homodimer of residues 449 through 728 of the constituent subunit. Present data, together with the results obtained from previous studies, confirm the existence of one or three possible inter-subunit disulfide bonds between cysteine residues 459, 462, and 464. NMC-4 bound to reduced vWF Fr(s) more weakly than to nonreduced Fr(s), but it did not react with Fr III-T2 of vWF, a disulfide-linked twin heterodimer of residues 273 through 511 and 674 through 728 (Marti et al, Biochemistry 26:8099, 1987). Fr III-T2 completely inhibited ristocetin-induced vWF binding at a concentration of 100 mumol/L but had no effect on botrocetin-induced binding. In addition, both the N- and C-terminal polypeptides, residues 449 through 549 and 674 through 728, generated by subdigestion of the 52/48-Kd Fr (Fujimura et al, J Biol Chem 261:381, 1986), inhibited preferentially ristocetin-induced vWF binding without affecting to botrocetin-induced vWF binding. These findings suggest that amino acid residues 512 through 673 of the vWF subunit are involved in botrocetin-induced vWF binding.

Antibodies, Monoclonal↗
PubMed1991

Spectroscopic study on interaction of nucleic acid base with tryptophan-containing tripeptides: acetyl-Trp-X-Trp-NHCH3 (X = Gly, Asn, Asp, Gln and Glu).

As part of a series of peptides designed to have binding ability selective for each of the nucleic acid bases, five tripeptides consisting of N-acetyl-Trp-X-Trp-NHCH3 (X = Gly, Asn, Asp, Gln and Glu) were synthesized, and their abilities to form complexes with four different nucleotides were examined by the fluorescence and phase distribution methods. The association constants obtained indicated that, depending on the sort of X residue, the peptides showed a variation in their interaction with guanosine monophosphate (GMP), while no noticeable selectivity was observed for other nucleotides adenosine monophosphate (AMP), uridine monophosphate (UMP) and cytidine monophosphate (CMP). The binding mode of N-acetyl-Trp-Asp-Trp-NHCH3 for the guanine base was further investigated using the proton nuclear magnetic resonance (1H-NMR) method. The mode was suggested to involve intimate cooperation of (1) the hydrogen bond formation between the carboxyl group of the Asp side chain and the guanine C2-amino group, and (2) the stacking interaction of the base with two terminal Trp residues of the peptide. Such interaction was strengthened by the protonation of the guanine base. A tentative binding mode is proposed based on these results.

Molecular Structure↗
PubMed1991

[Domain structure of human von Willebrand factor, and its modulators involved in the platelet adhesion process in vitro].

von Willebrand factor (vWF), a macromolecular glycoprotein synthesized in endothelial cells and megakaryocytes circulates in blood as heterogenous multimers and plays a critical role in the formation of platelet plugs. vWF is composed of an identical subunit with a molecular weight (MW) of 270 kDa, which is held together by disulfide bonds. Functional domains of vWF for factor VIII, heparin, platelet glycoprotein (GP) Ib, collagen, and GP IIb/IIIa locate in this order from the N-to C-terminus of subunit. The GP Ib binding domain is cryptic in normal circulation and only becomes expressed when vWF is associated with the subendothelial matrix or with fibrin. In vitro, an antibiotic ristocetin or the snake venom botrocetin isolated from Bothrops jararaca mimics the active component of subendothelial matrix and causes the binding of vWF to GP Ib. To elucidate the mechanism of in vitro process of vWF-GPIb binding, we describe here the isolation and characterization of two distinct forms of botrocetin. Since ristocetin is a heavily glycosylated proteoid, it is hard to characterize the structure on the basis of amino acid analysis. The apparent MW of the one-chain botrocetin was 28 kDa before and 32 kDa after reduction of disulfide bonds, while that of the two-chain botrocetin was 27 kDa before and 15/14.5 kDa after reduction. Amino acid composition of the two species revealed a similar high content of potentially acidic residues (greater that 60 Asx and Glx residues/molecules) but significant differences in the content of Cys and Phe residues. The N-terminal sequence of the one-chain was Ile-Ile/Val-Ser-Pro-Pro-Val-Cys-Gly-Asn-Glu-.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1990

Enhanced botrocetin-induced type IIB von Willebrand factor binding to platelet glycoprotein Ib initiates hyperagglutination of normal platelets.

Botrocetin, a protein isolated from the venom of the snake Bothrops jararaca, induces platelet aggregation/agglutination by von Willebrand factor (vWF) binding to the membrane glycoprotein (GP) Ib, an action resembling that of ristocetin. However, some differences in the interaction between vWF and platelet GPIb induced by these two substances have been reported. We have recently shown that the GPIb binding domain on the vWF molecule, in both instances, resides in the tryptic 52/48 kDa fragment extending from amino acid residue 449 to 728 of the constituent subunit. In the present report, we demonstrate that botrocetin does not induce agglutination of formalin-fixed platelets from a patient with Bernard-Soulier syndrome congenitally lacking GPIb and GPIX as well as GPV, a finding similar to that shown with ristocetin. A monoclonal antibody against GPIb (AP-1) inhibits either ristocetin- or botrocetin-dependent vWF binding to formalin-fixed platelets from normal individuals. Therefore, botrocetin-induced vWF binding to formalin-fixed platelets may reflect the interaction between vWF and platelet GPIb. To strengthen this concept, we have now found that heightened botrocetin-induced type IIB vWF binding to platelet GPIb causes hyperagglutination of normal platelets.

Antibodies, Monoclonal↗
PubMed1990

Suprapopliteal flap.

To investigate the cutaneous artery in the popliteal region, cadaver dissections were performed on 16 lower limbs. Two types of the cutaneous vessel have been found. One is the direct cutaneous vessel from the popliteal vessels; the other is the cutaneous vessel branching from the muscular vessel to the semimembranous muscle from the popliteal vessels. In our cadaver dissections, the latter was found consistently in the 16 lower limbs. The location of the vascular pedicle is 12.5 to 24.0 cm from the tibial condyle. The newly developed suprapopliteal flap supplied by this vessel is described. We applied this flap in four clinical patients: in one as a free flap, in the others as an island flap. Three (one free, two pedicled) flaps survived completely. The upper part of a pedicled flap partially necrosed. The operative procedure and the characteristics of the suprapopliteal flap are discussed. This flap can be used as a free flap and is also a versatile way to reconstruct soft tissue defects around the knee joint.

Adult↗