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Biomedical subjects

Y Usami

Publications and source records attributed to Y Usami.

At least 19 recordsLinked to original sources
PubMed1999

The cleavage site specificity of human prostate specific antigen for insulin-like growth factor binding protein-3.

The cleavage site of human insulin-like growth factor binding protein-3 by urinary prostate specific antigen was examined. Human insulin-like growth factor binding protein-3 was incubated with urinary prostate specific antigen at 37 degrees C and its proteolyzed fragments were separated by a reversed phase HPLC followed by N-terminal amino acid sequence analysis, demonstrating that the cleavage mainly occurred at Tyr-159. The synthetic peptide including Tyr-159 was also cleaved at the same site, although its reaction rate was relatively low. These results indicate that human insulin-like growth factor binding protein-3 is specifically cleaved at Tyr-159 by prostate specific antigen. Human insulin-like growth factor binding protein-3 was previously reported to be cleaved at five sites including Arg-97, Arg-132, Tyr-159, Phe-173 and Arg-179 by another group, however, prostate specific antigen preparation is possibly contaminated by trypsin-like protease. In contrast, our purified urinary prostate specific antigen had only a chymotrypsin-like activity, demonstrating that prostate specific antigen has the high substrate specificity for human insulin-like growth factor binding protein-3.

Amino Acid Sequence↗
PubMed1998

Chitin and chitosan stimulate canine polymorphonuclear cells to release leukotriene B4 and prostaglandin E2.

The effects of chitin and chitosan on the release of arachidonic acid products were investigated in this study. Supernatants of canine polymorphonuclear cell (PMN) suspensions incubated with chitin and chitosan contained a leukotriene B4 (LTB4) concentration high enough to induce canine PMN migration in vitro. The supernatants also contained the same concentration of prostaglandin E2 (PGE2) as that normally found in the peripheral blood of dogs. Intraperitoneal administration of chitosan to dogs induced peritoneal exudative fluid (PEF), but chitin did not. The PEF contained numerous PMNs and macrophages. The supernatant of PEF contained both heat-stable and heat-labile chemotactic factors for canine PMNs. It also contained enough LTB4 to attract the canine PMNs in vitro.

Animals↗
PubMed1998

Induction of necrosis by zinc in prostate carcinoma cells and identification of proteins increased in association with this induction.

Zinc exhibits inhibitory effects on apoptosis, and a deficiency in this metal generally causes this type of cell death to occur. In the present study, we found that exposure to zinc results in necrosis of prostate carcinoma cells. When zinc acetate was added to LNCaP or PC-3 cells in monolayer culture, they began to detach from the culture dishes, and viability was lost after 4-8 h. Most of the cell death was found to be due to necrosis as determined by double staining with fluorescein-isothiocyanate-labeled annexin V and ethidium bromide, and by detection of hypodiploid cells. Associated with the induction of necrosis was an increase in low molecular-mass proteins, identified by HPLC analysis to be thymosin beta10, parathymosin and GAGE in LNCaP cells, and thymosin beta4, parathymosin and metallothionein in PC-3. The time course of the increase of thymosin beta10 in LNCaP cells and thymosin beta4 in PC-3 cells was consistent with that of appearance of cell detachment and dead cells. These results indicate that zinc can induce necrosis and suggest that production of proteins including beta-thymosins is involved in induction of processes leading to cell detachment.

Adenocarcinoma↗
PubMed1998

Cytotoxic substances produced by a fungal strain from a sponge: physico-chemical properties and structures.

Five novel metabolites, trichodenones A-C (1-3), harzialactone A (4) and B (5), have been isolated together with known R-mevalonolactone (6) from the culture broth of a strain of Trichoderma harzianum OUPS-N115 originally separated from the sponge Halichondria okadai. Their structures have been elucidated by spectral evidence. Among them, 1-3 exhibited significant cytotoxicity against cultured P388 cells.

Animals↗
PubMed1997

Purification and characterization of pranlukast hydrolase from rat liver microsomes: the hydrolase is identical to carboxylesterase pI 6.2.

Two carboxylesterases with pI 6.0 and 6.2 derived from rat liver microsomes were purified. The two isozymes were remarkably different in substrate specificity, but they had equal enzymatic activity for alpha-naphthyl acetate and were inhibited equally by phenylmethylsulfonyl fluoride (PMSF) and bis-(4-nitrophenyl) phosphate (BNPP). Carboxylesterases pI 6.0 and 6.2 are identical to the enzymes referred to as hydrolase A and B, respectively, from the results of amino acid sequence analyses. Pranlukast was effectively hydrolyzed by carboxylesterase pI 6.2 but not by the pI 6.0 enzyme, and the difference in the pranlukast metabolism between the human and the rat could be explained by the substrate specificity of carboxylesterase. Furthermore, prodrugs of angiotensin converting enzyme inhibitors were found to be converted to the active drugs after hydrolysis by the carboxylesterases pI 6.0 and 6.2. Carboxylesterases generally catalyze the hydrolysis of ester-type drugs preferentially rather than amide-type drugs.

Amino Acid Sequence↗
PubMed1997

Hydrolytic profile for ester- or amide-linkage by carboxylesterases pI 5.3 and 4.5 from human liver.

Carboxylesterases (EC 3.1.1.1) from human liver were purified using Q-Sepharose, Sephadex G-150, isoelectrofocusing and Con A-Sepharose. The calculated molecular mass of the pI 5.3 enzyme was 120 kDa and 61 kDa from the results of Sephadex G-150 gel filtration and SDS-polyacrylamide gel electrophoresis (PAGE), respectively, suggesting that this enzyme is a dimer. On the other hand, carboxylesterase pI 4.5, with a molecular mass of 64 kDa, was a monomer. The activities of both enzymes were inhibited by typical serine enzyme inhibitors. Amino acid sequence analysis of the purified enzymes pI 5.3 and 4.5 showed high homology with rabbit carboxylesterase form 1 and 2, respectively. The results also suggested that carboxylesterase pI 5.3 is identical to the deduced amino acid sequence from cDNA for HU1, and that carboxylesterase pI 4.5 is identical to the deduced amino acid sequence from the cDNA registered as human carboxylesterase (hCE-2) in GenBank. We first purified carboxylesterase pI 4.5 and investigated its hydrolytic activity upon various drugs. The two enzymes differed in substrate specificity. Prodrugs of angiotensin-converting enzyme inhibitors, such as delapril and imidapril, were converted to active metabolites by carboxylesterase pI 5.3, but not by carboxylesterase pI 4.5. The hydrolysis velocity of temocapril by carboxylesterase pI 5.3 was 12-fold faster than by carboxylesterase pI 4.5. In contrast, aspirin, oxybutynin and procaine were hydrolyzed by only carboxylesterase pI 4.5. We also found that an amide-linkage in drugs, except for that in aniracetam, was not a good substrate for the two enzymes. Consequently, carboxylesterases pI 5.3 and 4.5 may be involved in the metabolism of various drugs containing an ester-linkage.

Amino Acid Sequence↗
PubMed1996

Complete amino acid sequence and identification of the platelet glycoprotein Ib-binding site of jararaca GPIb-BP, a snake venom protein isolated from Bothrops jararaca.

Jararaca GPIb-BP, a snake venom protein composed of alpha and beta subunits purified from Bothrops jararaca, binds to platelet glycoprotein (GP)Ib and functions as a receptor blocker for von Willebrand factor binding to GPIb (Fujimura, Y., Ikeda, Y., Miura, S., Yoshida, E., Shima, H., Nishida, S., Suzuki, M., Titani, K., Taniuchi, Y., and Kawasaki, T. (1995) Thromb. Haemostasis 74, 743-750). We present here the entire 142- and 123-residue amino acid sequence of the respective alpha and beta subunits and also demonstrate that the platelet GPIb-binding site resides on the beta and not on the alpha subunit based on an enzyme-linked immunosorbent assay using biotin-labeled jararaca GPIb-BP and competing ligands. Sequences of the alpha and beta subunits were determined by analysis of the intact S-pyridylethylated proteins and their peptides generated by digestion with Achromobacter protease I, Staphyloccocus aureus V8 protease, pepsin, endoproteinase Asp-N, or L-1-tosylamino-2-phenylethyl chloromethyl ketone-trypsin. A 38-39% identity of amino acid sequence between the alpha and beta subunits of jararaca GPIb-BP was observed, as well as a high degree of sequence identities (38-64%) with the respective subunits of botrocetin (Usami, Y., Fujimura, Y., Suzuki, M., Ozeki, Y., Nishio, K., Fukui, H., and Titani, K (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 928-932) and the beta-chain of echicetin (Peng, M., Holt, J. C., and Niewiarowski, S. (1994) Biochem. Biophys. Res. Commun. 205, 68-72).

Amino Acid Sequence↗
PubMed1996

Primary structure of alboaggregin-B purified from the venom of Trimeresurus albolabris.

The complete amino acid sequences of alpha and beta subunits of alboaggregin-beta are presented. The alpha and beta subunits were separated by reversed-phase HPLC after reduction and S-pyridylethylation, and their sequences were determined by analysis of peptides generated by enzymatic or chemical digestion. The alpha and beta subunits consist of 133 and 123 amino acid residues, respectively. The sequences are highly homologous to each other (41.4% identity) and also to those of the alpha and beta subunits of botrocetin (a von Willebrand factor modulator) and the A and B chains of factor IX/X binding protein from other snake venoms. It is also homologous to C-type lectins with a homodimeric structure, but it shows no lectin-like activity.

Amino Acid Sequence↗
PubMed1996

Purification and partial characterization of an indomethacin hydrolyzing enzyme from pig liver.

PURPOSE: Indomethacin is well known to be metabolized via O-demethylation and N-deacylation. In this paper we found an enzyme involved in the hydrolysis of amide-linkage of indomethacin and partially characterized it as well as its substrate specificity. METHODS: An indomethacin hydrolyzing enzyme was purified to homogeneity from pig liver microsomes using columns of Q-Sepharose, Red-Sepharose and Blue-Sepharose. The enzyme activity was assayed by measuring of rho-chlorobenzoic acid liberated from indomethacin by HPLC. RESULTS: The purified enzyme effectively hydrolyzed the amide linkage in indomethacin but not those in alpha-naphthylacetate and rho-nitrophenylacetate, which are typical substates for carboxylesterase. The subunit molecular mass of the enzyme was 65 kDa according SDS-polyacrylamide gel electrophoresis. The Michaelis constant (K(m)) and maximum velocity (Vmax) values for indomethacin were 67.8 microM and 9.02 nmol/min/mg protein, respectively. The amino acid sequence analysis of the enzyme after cyanogen bromide cleavage showed high homology with a mouse carboxylesterase isozyme designated as ES-male. The activity of indomethacin hydrolysis was relatively high in the pig, rabbit and human liver homogenate, but not in those from rat and mouse. On the other hand, purified human liver carboxylesterases pl 5.3 and 4.5, and pig liver carboxylesterases have no catalytic activity for indomethacin. CONCLUSIONS: These results indicate that the hydrolysis of amide-linkage of indomethacin in humans would be associated with an enzyme similar to the indomethacin hydrolyzing enzyme from pig liver microsomes described here.

Animals↗
PubMed1996

Variable glyceryl dinitrate formation as a function of glutathione S-transferase.

Nitroglycerin (GTN) has been used as the drug of choice in the treatment of angina pectoris. It has been shown that some glutathione S-transferases (GSTs) catalyze the metabolic conversion from GTN to glyceryl dinitrates (GDNs). In this study, we examined the substrate specificity of GSTs for GTN. Alpha and mu GSTs were isolated from porcine liver and intestinal mucosa by means of CM-cellulose and glutathione-affinity column chromatography. Mu GSTs degraded GTN time-dependently and formed 1,3-GDN in preference to 1,2-GDN as a ratio (1,2-GDN/ 1,3-GDN) of 0.61, whereas alpha GSTs formed twice as much 1,2-GDN as 1,3-GDN. These results showed that two GST families participate in the metabolic conversion of GTN at different hydrolyzing portions of the nitrogroups.

Amino Acid Sequence↗
PubMed1996

Inhibition of purified human sucrase and isomaltase by ethanolamine derivatives.

Sucrase-isomaltase complex was purified from human intestinal mucosa. Immunostaining shows that sucrase-isomaltase is confined to the area of the striated cell borders of human small intestinal absorptive cells of the villus. Inhibition of sucrase and isomaltase activity by ethanolamine derivatives was investigated. Tris inhibits both types of enzyme activity and is the strongest inhibitor of the ethanolamine derivatives investigated. Bis-Tris inhibited sucrase more than isomaltase. On the other hand, mono-, di- and tri-ethanolamine were weak inhibitors of sucrase but not isomaltase.

Electrophoresis, Polyacrylamide Gel↗
PubMed1995

Gastrointestinal and skin lesions in piglets naturally infected with pseudorabies virus.

Pseudorabies virus (PRV) infection was diagnosed in 4 piglets from a litter by immunohistopathologic examination and virus isolation. Three piglets had moderate to severe neuronal degeneration, and PRV antigen was detected in Auerbach's myenteric plexus and Meissner's submucosal plexus of the gastrointestinal tract. One piglet had 2 types of skin lesions. One lesion appeared on the hip and ear and was characterized by ballooning degeneration, necrosis of epithelial cells, and intranuclear inclusion bodies. The other was found on the ear and hematoma-like lesion was composed of fibrinoid exudation and degenerative connective tissue. PRV antigen was clearly demonstrated in both skin lesions. These results suggested that degeneration of myenteric plexuses might be another characteristic of lesions in PRV-infected pigs and that the virus spreads by interaction between the skin and myenteric plexuses to the central nervous system.

Animals↗
PubMed1994

A 28 kDa-protein with disintegrin-like structure (jararhagin-C) purified from Bothrops jararaca venom inhibits collagen- and ADP-induced platelet aggregation.

A 28 kDa-protein with inhibitory activity on collagen- and ADP-induced platelet aggregation was purified from the venom of the snake Bothrops jararaca. Its complete amino acid sequence corresponded to the carboxyl-terminal region consisting of disintegrin-like and cysteine-rich domains of jararhagin, a high molecular weight hemorrhagic metalloprotease. Sequence homology of the protein to other disintegrins and disintegrin-like proteins from various snake venoms is also presented.

Adenosine Diphosphate↗
PubMed1994

Purification and characterization of bothrombin, a fibrinogen-clotting serine protease from the venom of Bothrops jararaca.

A fibrinogen-clotting enzyme (bothrombin) was purified from the venom of Bothrops jararaca. Bothrombin showed M(r) values of 33,000 under nonreducing and 35,000 under reducing conditions on SDS polyacrylamide gel electrophoresis and specific fibrinogen-clotting activity equivalent to 814-904 NIH alpha-thrombin units/mg. Diisopropyl fluorophosphate totally abolished its activity, but hirudin, a specific alpha-thrombin inhibitor, had negligible effect on bothrombin activity. Unlike alpha-thrombin, bothrombin split off fibrinopeptide A without releasing fibrinopeptide B. Bothrombin activated blood coagulation factor VIII, but its activity was about 950 times less than that of alpha-thrombin. Bothrombin did not induce aggregation or serotonin release of washed normal platelets by itself, but did aggregate platelets in the presence of exogenous fibrinogen. This latter activity was completely inhibited by either anti-glycoprotein (GP) IIb/IIIa monoclonal antibody (which blocks fibrinogen binding to GP IIb/IIIa) or anti-GP Ib monoclonal antibody (which specifically inhibits alpha-thrombin binding to GP Ib). Prostaglandin E1 (1 microM) and EDTA (10 mM) also abolished platelet aggregation without affecting clotting activity. Washed platelets from a patient with Bernard-Soulier syndrome did not respond to bothrombin even in the presence of exogenous fibrinogen, suggesting that the initial binding of bothrombin on platelets is GP Ib, but not a recently cloned thrombin receptor. The complete amino acid sequence of bothrombin was determined by analysis of (S)-pyridylethylated protein and peptides generated by digestion with cyanogen bromide and Achromobacter protease I, respectively. Bothrombin is composed of 232 amino acid residues and contains three Asn-linked oligosaccharide chains.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
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↗