PubMed Health⌕ Search

Biomedical subjects

N Fukusen

Publications and source records attributed to N Fukusen.

At least 19 recordsLinked to original sources
PubMed2001

Potent cytolytic response by a CD8+ CTL clone to multiple peptides from the same protein in association with an allogeneic class I MHC molecule.

CTL clone 2C recognizes the allogeneic class I MHC molecule L(d) in association with peptides derived from alpha-ketoglutarate dehydrogenase (oxoglutarate dehydrogenase (OGDH)), a ubiquitous intracellular protein. One of these peptides, QLSPFPFDL (QL9), elicits more vigorous cytolytic responses than two previously identified naturally processed peptides with overlapping sequences, LSPFPFDL (p2Ca) and VAITRIEQLSPFPFDL (p2Cb), from OGDH. In this study, we show that QL9 forms a more stable complex with cell surface L(d) than does p2Ca or p2Cb and is processed from the longer, naturally occurring peptide p2Cb by 20S proteosomes in vitro. The N-terminal cyclized pyroglutaminyl QL9 (pyroQL9), a form of QL9 to which it is converted at the low pH used for peptide isolation from tissue extracts, is even more active than QL9 in cytotoxicity assays with 2C CTL. Overall, the results indicate that along with the abundant natural peptides p2Ca and p2Cb, the QL9 and other OGDH peptides of various lengths, sharing a conserved C-terminal sequence, are also processed and presented with L(d) as allogeneic ligands for T cells expressing 2C TCR. All these peptides, each available in a low amount, could act in concert at the cell surface, resulting in a high density of cognate ligands that accounts for the exceptionally potent cytolytic response by 2C CTL.

Alanine↗
PubMed1996

Purification and characterization of novel trypsin-like serine proteases from mouse spleen.

Novel trypsin-like serine proteases (mouse trypsin-type serine proteases 1 and 2 [MTSP-1 and -2]) were purified to homogeneity from mouse spleen. Each protease consisted of a single polypeptide with a molecular mass of about 29 kDa, as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis under reducing conditions. Both were totally inhibited by diisopropylfluorophosphate, soybean trypsin inhibitor, aprotinin, antipain, and leupeptin and partially inhibited by chymostatin and dithiothreitol, suggesting that they are trypsin-like serine proteases. They hydrolyzed synthetic substrates for trypsin-like proteases but not those for chymotrypsin-like proteases, elastase and kallikrein. MTSP-1 hydrolyzed tert-butyloxycarbonyl (Boc)-Asp(OBzl)Pro-Arg-amino-4-methyl-coumaryl-7-amide (MCA) and Boc-Ile-Glu-Gly-Arg-MCA faster than Boc-Phe-Ser-Arg-MCA. On the other hand, MTSP-2 hydrolyzed Boc-Phe-Ser-Arg-MCA most rapidly, with a specific activity 15 times higher than that of MTSP-1. The N-terminal amino acid sequence of MTSP-1 was Ile-Val-Gly-Gly-Tyr-Thr-His-Leu-Asp-Asn-Gln-Val-Pro-Tyr. This sequence was 71% homologous with the N-terminal of bovine trypsin. The Boc-Phe-Ser-Arg-MCA hydrolyzing activity of mouse spleen significantly (p < 0.01) increased to about 1.5-fold the basal activity 2 weeks after an injection of Freund's complete adjuvant, suggesting that these proteases are involved in the immune response.

Amino Acid Sequence↗
PubMed1994

Characterization of cytochrome P450-monooxygenases of Chinese hamsters with respect to aflatoxin B1 activation.

Monooxygenases of Chinese hamster livers were characterized with respect to aflatoxin B1 activation and cytochrome P450 isozymes. Polychlorinated biphenyl (PCB)-treatment induced higher activity for aflatoxin B1 activation in Chinese hamsters than in rats and this activity was more elevated by the treatment with phenobarbital than with 3-methylcholanthrene, which is contrary to the observation in Syrian hamsters and rather similar to that in rats. Western blot analysis demonstrated that the treatment of Chinese hamsters with phenobarbital markedly elevated the level of cytochrome P450 isozyme belonging to the CYP2A family and only slightly that immuno-related to CYP2B1. In parallel with this, the activities of testosterone 15 alpha-hydroxylase and 7-ethoxycoumarin O-deethylase were significantly induced by phenobarbital treatment. These suggest that phenobarbital mainly induces an isozyme related to CYP2A2 which is responsible for aflatoxin B1 activation in Chinese hamsters.

Aflatoxin B1↗
PubMed1993

A ubiquitous protein is the source of naturally occurring peptides that are recognized by a CD8+ T-cell clone.

We previously isolated from mouse spleen an octapeptide (LSPFPFDL) that in association with the class I major histocompatibility complex protein Ld is recognized by the antigen-specific receptor of an alloreactive CD8+ T-cell clone (2C). Guided by an assay dependent upon the same 2C T-cell receptor, we have now isolated from the same source another naturally occurring peptide. The second peptide (VAITRIEQLSPFPFDL) includes the entire octapeptide sequence and preliminary evidence suggests that it may be a natural precursor of the octapeptide. On finding extensive sequence homology between the 16-mer and part of human 2-oxoglutarate dehydrogenase, we determined the cDNA sequence of mouse 2-oxoglutarate dehydrogenase and found that the deduced amino acid sequence matches precisely the two naturally occurring peptides, indicating their origin by cellular processing of this ubiquitous self protein.

Amino Acid Sequence↗
PubMed1993

Purification of a human cytochrome P-450 isozyme catalyzing lanosterol 14 alpha-demethylation.

An isozyme of cytochrome P-450 catalyzing lanosterol 14 alpha-demethylation was purified from human liver using column chromatography, including immunoaffinity chromatography. The purified protein exhibited a single protein band (53 kDa) on sodium dodecylsulfate-polyacrylamide gel electrophoresis. When reconstituted with NADPH-cytochrome P-450 reductase, the purified protein showed an activity of 14 alpha-demethylation of 24,25-dihydrolanosterol (3.20 nmol/min per mg protein). The apparent Km value for 24,25-dihydrolanosterol was found to be 27 microM. This enzyme converted in the reconstituted system, the oxygenated intermediates of 24,25-dihydrolanosterol 14 alpha-demethylation, 32-hydroxy-24,25-dihydrolanosterol and 32-oxo-24,25-dihydrolanosterol, to the 32-nor compound, 4,4-dimethylcholesta-8,14-dien-3 beta-ol.

Animals↗
PubMed1989

Diacylglycerols enhance the anti-tumor effect of glucocorticoid on L5178Y lymphoblasts in vivo.

1,2-Dioleoyl-rac-glycerol, a potent activator of protein kinase C, was found to enhance the growth-inhibitory effect of triamcinolone acetonide on L5178Y lymphoblasts in adrenalectomized, male DBA/2 mice. On the other hand, in mice without adrenalectomy, it markedly inhibited tumor growth without increasing the plasma level of corticosterone or adrenocorticotropic hormone or reducing the body weight. These results suggest that diacylglycerol enhances the action of endogenous glucocorticoid to a sufficient level to inhibit the growth of lymphoblasts. Of various diacylglycerols with different carbon chain lengths tested, 1,2-dioleoyl-rac-glycerol was the most potent growth inhibitor and was maximally effective at a dose of above 30 micrograms/100 g body weight. This finding suggests that diacylglycerols may be useful for enhancing the antitumor effect of a low dose of glucocorticoid or endogenous glucocorticoid on lymphoblasts without any significant side effect.

Adrenalectomy↗
PubMed1988

Peptide boronic acids, substrate analogs, inhibit chymase, and histamine release from rat mast cells.

Peptide boronic acids, such as methoxysuccinyl-Ala-Ala-Pro-(L)boro-Phe-OH, its pinacol ester, and t-butyloxycarbonyl-Phe-Pro-(L)boro-Phe-pinacol, inhibited the activity of chymase from connective tissue mast cells approximately 40- to 80-fold more than atypical chymase from mucosal mast cells, and did not inhibit trypsin. Only peptide boronic acids containing "L" forms of boronic acids were inhibitory. The Ki values of these peptide boronic acids for chymase were in the 60-170 nM concentration range, like those of the natural inhibitors tested, but all the natural inhibitors tested except Eglin C and chymostatin inhibited both chymase and trypsin. Thus these peptide boronic acids should be useful for selective inhibition of chymase with less inhibitory activity for atypical chymase and without inhibition of trypsin. These peptide boronic acids markedly inhibited histamine release induced by anti-rat immunoglobulin E, suggesting that chymase in connective tissue mast cells plays some role in the process of histamine release. These peptides are assumed to be therapeutically useful for treatment of allergic inflammations catalyzed by chymase.

Animals↗
PubMed1988

Antibodies and inhibitor of chymase are incorporated into mast cell granules and inhibit histamine release.

The release of histamine from rat mast cells induced by anti-immunoglobulin E (IgE) was markedly inhibited by Bowman-Birk soybean protease inhibitor (BBI) and anti-chymase F(ab')2 fragments which inhibit chymase activity [Kido, H., et al. (1985) Biochem. Int. 10, 863-871, and Fukusen, et al. (1987) Biochem. Med. Metab. Biol. 38, 165-169]. When radioiodinated anti-chymase F(ab')2 fragments or BBI were incubated with mast cells at 37 degrees C, they were subsequently recovered in the fractions of mast cell granules and of plasma membranes by Percoll density gradient centrifugation. However, when they were incubated with mast cells at 0 degrees C, they had no effect on histamine release and were subsequently recovered only in the plasma membrane fraction. Histamine release from mast cells was suppressed dose-dependently by the antibodies or BBI accumulated in mast cell granules at 37 degrees C. These results suggest that the antibodies and BBI are incorporated into mast cell granules and inhibit chymase activity, resulting in inhibition of histamine release.

Animals↗
PubMed1988

Role of medullasin in granulocytes in biophylaxis. Elastinolytic activity and the potentiation of cytostatic activity of human monocytes.

Medullasin, a serine protease in bone marrow cells, resembles elastase, but is essentially devoid of elastinolytic activity. The protease revealed elastinolytic activity when small amounts of other proteases such as trypsin, papain, chymotrypsin, or collagenase coexisted in the incubation mixture. In vitro treatment of human monocytes with medullasin caused an increment of their cytostatic activity. Since medullasin failed to increase the cytostatic activity in the supernatant of monocytes, the enhancement of cytostatic activity of monocytes by medullasin is considered to be not mediated through the production of soluble factors from monocytes.

Animals↗
PubMed1987

Epidermal growth factor as a new regulator of induction of tyrosine aminotransferase and tryptophan oxygenase by glucocorticoids.

Epidermal growth factor (EGF) dose-dependently enhanced the induction of tyrosine aminotransferase and tryptophan oxygenase by glucocorticoids in primary cultures of adult rat hepatocytes without itself having any effect on these enzymes in the absence of glucocorticoids. The amplifications were observed even with dexamethasone at high concentrations (10(-6) M-10(-5) M) that had a maximal effect. EGF had no effect on induction of tyrosine aminotransferase by glucagon or Bt2cAMP. The effect of EGF was also observed in adrenal-ectomized and submaxillary gland-ectomized rats. These results suggest that EGF is an endogenous amplifier of the action of glucocorticoids.

Adrenalectomy↗
PubMed1987

Tumor-promoting phorbol ester amplifies the inductions of tyrosine aminotransferase and ornithine decarboxylase by glucocorticoid.

In adrenalectomized rats, the tumor-promoting phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) markedly enhanced the inductions of tyrosine aminotransferase (TAT) and ornithine decarboxylase by glucocorticoids, even with sufficient concentration of glucocorticoids to have a maximal effect, whereas it had no effect on TAT activity and increased ornithine decarboxylase activity only slightly in the absence of glucocorticoids. Phorbol derivatives and components of TPA such as 4 beta-phorbol, phorbol 12-tetradecanoate, phorbol 13-acetate, and 4-O-methylphorbol 12-tetradecanoate 13-acetate, which have no tumor-promoting activity or ability to activate protein kinase C, did not have any effect on TAT induction by glucocorticoid. TPA enhanced the induction of TAT by various glucocorticoids but had no effect on induction of TAT by glucagon or insulin and did not enhance the induction of glucose-6-phosphate dehydrogenase by 17 beta-estradiol. These results suggest that TPA specifically enhances the induction of TAT and ornithine decarboxylase by glucocorticoids. Similar effects of TPA on TAT induction by glucocorticoid were observed in primary cultures of adult rat hepatocytes. Another activator of protein kinase C, rac-1,2-dioctanoylglycerol, was also found to have similar effects on the cells.

Adrenalectomy↗
PubMed1987

Inhibition by 1-(5-isoquinolinesulfonyl)-2-methylpiperazine, an inhibitor of protein kinase C, of enzyme induction by glucocorticoid and of nuclear translocation of glucocorticoid-receptor complexes.

Induction of tyrosine aminotransferase by glucocorticoid in rat hepatocytes was inhibited concentration-dependently by 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7), an inhibitor of protein kinase C, but not by N- [2-(methyl-amino)-ethyl]-5-isoquinolinesulfonamide dihydrochloride, an inhibitor of cyclic nucleotide dependent protein kinases. H-7 also inhibited the accumulation of glucocorticoid-receptor complexes in the nuclear fraction with associated accumulation of these complexes in the cytoplasmic fraction, but did not affect incorporation of glucocorticoid into hepatocytes. These results indicate that protein kinase C may be essential in translocation of glucocorticoid-receptor complexes to the nuclei.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1987

Kinetic studies on the inhibitions of mast cell chymase by natural serine protease inhibitors: indications for potential biological functions of these inhibitors.

We have found that degranulation from mast cells is specifically inhibited by the inhibitors of chymase (10). Among the natural serine protease inhibitors tested, Bowman-Birk soybean protease inhibitor, Eglin C, and human alpha 1-antichymotrypsin inhibited chymase more strongly than did chymostatin, Kunitz soybean protease inhibitor, and phosphatidylserine. Of the inhibitors tested, Bowman-Birk soybean protease inhibitor was the strongest inhibitor of chymase, its Ki value being 13.2 X 10(-9) M. Kinetic studies showed that these inhibitors were all noncompetitive inhibitors of chymase. Bowman-Birk and Kunitz soybean protease inhibitors inhibited both chymotrypsin-type and trypsin-type serine proteases but Eglin C specifically inhibited chymotrypsin-type proteases.

Animals↗
PubMed1986

Diacylglycerol amplifies the induction in vivo of tyrosine aminotransferase and ornithine decarboxylase by glucocorticoid.

In adrenalectomized rats, diacylglycerol, a potent activator of protein kinase C, specifically enhanced the induction of tyrosine aminotransferase and ornithine decarboxylase by even maximally effective doses of dexamethasone phosphate, but itself had no effect on these enzyme inductions in the absence of glucocorticoid. The amplifications of enzyme induction by diacylglycerol was dose-dependent and the time courses of the amplified inductions were similar to those of the inductions by dexamethasone phosphate alone. Since diacylglycerol did not affect the induction of these enzymes by glucagon and insulin, its amplifying effect seemed to be specific for induction by glucocorticoids.

Adrenalectomy↗
PubMed1986

A chymotrypsin-type serine protease in rat basophilic leukemia cells: evidence for its immunologic identity with atypical mast cell protease.

Activity of a chymotrypsin-type serine protease was found in a subline of rat basophilic leukemia (RBL-2H3) cells. The protease was immunologically cross-reactive with anti-atypical mast cell protease immunoglobulin (Ig) G, and its activity was inhibited in a dose-dependent manner by the antibody. The apparent m.w. of the protease that reacted with the antibody was 25,000, which was identical with that of atypical mast cell protease in rat mucosal mast cells. These results show that the chymotrypsin type serine protease in RBL-2H3 cells is immunologically identical with atypical mast cell protease, which was first purified from rat small intestine. Immunohistochemical studies showed that the protease was located not only in intracytoplasmic granules but also in organelles synthesizing protein, such as cisternae of the rough endoplasmic reticulum, perinuclear spaces, and the Golgi apparatus. However, no immunoreactivity was demonstrated in rat basophils. The activity of the protease increased in the exponential phase of growth of RBL-2H3 cells in which some activity was also detected in the medium, and it decreased in the late stationary phase.

Animals↗
PubMed1986

Biological functions of serine proteases in the granules of rat mast cells.

The effects of specific low- and high-molecular weight inhibitors of chymase and tryptase and F(ab')2 of antichymase on histamine release from activated mast cells were examined. The release of histamine induced by anti-rat immunoglobulin E was markedly inhibited by F(ab')2 fragments of antichymase and the low-molecular weight inhibitor of chymase chymostatin, whereas release of histamine induced by calcium ionophore A23187 was inhibited only by chymostatin. Neither the inhibitor nor the antibody affected histamine release induced by compound 48/80. These results suggest that two main chymotrypsin-type proteases are involved in process of degranulation: one is chymase, which acts at a step before calcium entry, and the other is an unidentified protease, which acts at a step after calcium entry. These results are summarized in Figure 8. After degranulation, released chymase remains associated with the cell surface while released tryptase was found in the extracellular milieu. Tryptase converted bovine prothrombin to thrombin, as shown by increase in thrombin activity with a synthetic substrate, t-butyloxy-carbonyl-Val-Pro-Arg-4-methyl-coumaryl-7-amide. The apparent Km value toward prothrombin was relatively low (2.3 microM), suggesting that tryptase contributes to blood coagulation or the process of fibrosis in tissues. The proteolytic products of IgG1 produced by chymase had chemotactic activity for neutrophil leukocytes in vitro and in vivo. These findings indicate the possible functions of these proteases after degranulation.

Animals↗
PubMed1985

Tryptase from rat mast cells converts bovine prothrombin to thrombin.

The effect of tryptase purified from rat peritoneal mast cells on bovine prothrombin was examined. Tryptase activated prothrombin, as evidenced by the increase in thrombin activity with a synthetic substrate, t-butyloxy-carbonyl-Val-Pro-Arg-4-methylcoumaryl-7-amide. The apparent Km value toward bovine prothrombin and the kcat value were 2.3 microM and 46.3 s-1, respectively. Studies on the time course of prothrombin activation by tryptase and by activated factor X (Xa), and analysis of the activation products on sodium dodecyl sulfate gel electrophoresis showed that the process of activation of prothrombin by tryptase was similar to that by Xa except that an intermediate of 67,000 daltons was formed.

Alpha-Globulins↗