Language：Japanese   Publishing type：Research paper (scientific journal)  

researchmap

Language：Japanese   Publishing type：Research paper (bulletin of university, research institution)  

researchmap

Authorship：Lead author   Language：Japanese   Publishing type：Research paper (scientific journal)  

CiNii Books

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：PLENUM PRESS DIV PLENUM PUBLISHING CORP  

Web of Science

researchmap

Language：English   Publishing type：Research paper (international conference proceedings)   Publisher：ELSEVIER SCIENCE PUBL B V  

Web of Science

researchmap

Language：English   Publisher：LIPPINCOTT WILLIAMS & WILKINS  

DOI： 10.1038/labinvest.3780461

Web of Science

researchmap

Language：English   Publisher：AMER PHYSIOLOGICAL SOC  

In the liver, function of the bile salt export pump (Bsep), a major canalicular exporter of bile salts, is complemented by activity of the multidrug resistance protein 2 (Mrp2), a canalicular organic anions transporter. Mrp2 was found capable of transporting various anticancer drugs out of cells, eventually undermining their therapeutic potential and contributing to multidrug resistance. We employed a RT-PCR, immunoblotting, and immunofluorescence to examine their gene, protein expression, and distribution of antigenic sites in the rat liver during development from 16-day-old fetus to adult animal. Bsep mRNA was almost undetectable before birth. It was first clearly expressed in the liver of newborn rats. On the contrary, Mrp2 mRNA was seen before birth, although at low levels. In concert with mRNA expression, Bsep protein was undetectable before birth, while Mrp2 protein was already expressed. Both proteins were clearly detectable in the postnatal period. Confocal immunofluorescent microscopy showed asynchronous appearance of Bsep and Mrp2 proteins during development but their colocalization in the bile canaliculi once each one is expressed. During the gestational period, a weak immunofluorescence for Mrp2 was observed only in livers of 16-day-old embryos. No fluorescence for Bsep was seen. Both proteins were clearly visualizable after birth, although the pattern of immunostaining varied. These findings provide molecular evidence that expression of both Bsep and Mrp2 during development is transcriptionally regulated. They also point out the differences in relevance to the liver function of the systems responsible for canalicular transport of bile salts versus organic anions.

DOI： 10.1152/ajpgi.00405.2001

Web of Science

researchmap

Language：English   Publisher：Japanese Society for Hygiene  

We were able to isolate a lysosomal fraction from rat brain which had a higher degree of purity than in previous studies with good recovery. This has been made possible by using percoll gradients following the swelling of mitochondria in the presence of calcium which would eliminate contamination from small amounts of mitochondria. By using percoll density gradient centrifugation after the swelling of mitochondria in the presence of calcium, cerebral lysosomes were purified 312-fold with the recovery of approximately 22%, which is the highest reported for any cerebral lysosomal preparation. The most effective procedure for the separation was achieved by using 1.25 mM calcium incubation with post nuclear fraction. As brain lysosomes may play a major role not only in degrading macromolecules but also in their transport to the deposition site, obtaining purified rat cerebral lysosomes represents an important step in the study of the generation of macromolecules which accumulate in the brain.

DOI： 10.1007/BF02931261

Scopus

researchmap

Language：English   Publisher：PORTLAND PRESS  

The A and J series of prostaglandins (PGs) accumulate in the nuclei to suppress the proliferation of cancer cells. Here we report that Delta(7)-PGA(1) methyl ester, a synthetic anti-cancer PG, increased the level of mRNA for the cyclin-dependent kinase inhibitor p21 in human leukaemia HL-60 cells. The induction of p21 was associated with the accumulation of hypophosphorylated retinoblastoma protein (pRB) and the suppression of c-myc gene expression. Since the p53 gene is deleted in HL-60 cells, the anticancer PG is suggested to inhibit cancer cell growth by inducing p21 via a p53-independent pathway. Unlike HL-60 cells, cisplatin-resistant HL-60/R-CP cells were insensitive to Delta(7)-PGA(1) methyl ester, While c-myc expression was transiently suppressed, neither G(1) arrest nor hypophosphorylation of pRB was observed with the anti-cancer PG. Plasma membrane vesicles from HL-60/R-CP cells showed an enhanced level of GS-X pump (ATPdependent glutathione S-conjugate export pump) activity towards the glutathione S-conjugate of Delta(7)-PGA(1) methyl ester (K-m 110 nM), GIF-0019 {N-carbomethoxy-S-[5-(4-benzoylphenyl)pentyl]glutathione dimethyl ester}, a specific inhibitor of the GS-X pump, dose-dependently enhanced the cellular sensitivity of HL-60/R-CP cells to Delta(7)-PGA(1) methyl ester and induced G(1) arrest. The GS-X pump is suggested to play a pivotal role in modulating the biological action of the anti-cancer PG.

Web of Science

researchmap

Language：English   Publisher：NATL INST INDUSTRIAL HEALTH  

It is known that some organophosphates produce not only well-known acute toxicity but also characteristic delayed neurotoxicity. Tri-ortho-tolyl phophate (TOTP), which was formerly named Tri-ortho-cresyl phosphaete (TOCP), was first noticed in an incident of poisoning as the compound which produced organophosphate induced delayed neurotoxicity (OPIDN), It is said that triphenyl phosphite (TPP) is also one of the organophosphates which possesses OPIDN, However, it is thought that TPP-induced delayed neurotoxicity (TPP-DN) is not identical with classical OPIDN, An intermediate syndrome was later proposed as the third neurotoxicity caused by organophosphates, We think that TPP is a model chemical of the third neurotoxicity, We compared TOTP with TPP using Japanese quails. We measured cholinesterase (ChE) activity and clearly demonstrated the difference between the two chemicals, that is to say, the activity recovered after 72 hrs from the administration of TPP, whereas the inhibition continued for more than 11 days after the administration of TOTP.

DOI： 10.2486/indhealth.36.376

Web of Science

PubMed

researchmap

Language：English   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

We recently reported that GS-X pump activity, as assessed by ATP-dependent transport of the glutathione-platinum complex and leukotriene C-4, and intracellular glutathione (GSH) levels were remarkably enhanced in cis-diamminedichloroplatinum(II) (cisplatin)-resistant human leukemia HL-60 cells (Ishikawa, T,, Wright, C, D,, and Ishizuka, H, (1994) J, Biol, Chem, 269, 29085-29093), Now, using Northern hybridization and RNase protection assay, we provide evidence that the multidrug resistance-associated protein (MRP) gene, which encodes a human GS-X pump, is expressed at higher levels in cisplatin-resistant (HL-60/R-CP) cells than in sensitive cells, whereas amplification of the MRP gene is not detected by Southern hybridization, Culturing HL-60/R-CP cells in cisplatin-free medium resulted in reduced MRP mRNA levels, but these levels could be induced to rise within 30 h by cisplatin and heavy metals such as arsenite, cadmium, and zinc, The increased levels of MRP mRNA were closely related with enhanced activities of ATP-dependent transport of leukotriene C-4 (LTC(4)) in plasma membrane vesicles, The glutathione-platinum (GS-Pt) complex, but not cisplatin, inhibited ATP-dependent LTC(4) transport, suggesting that the MRP/GS-X pump transports both LTC, and the GS-Pt complex, Expression of gamma-glutamylcysteine synthetase in the cisplatin-resistant cells was also co induced within 24 h in response to cisplatin exposure, resulting in a significant increase in cellular GSH level, The resistant cells exposed to cisplatin were cross-resistant to melphalan, chlorambucil, arsenite, and cadmium, These observations suggest that elevated expression of the MRP/GS-X pump and increased GSH biosynthesis together may be important factors in the cellular metabolism and disposition of cisplatin, alkylating agents, and heavy metals.

DOI： 10.1074/jbc.271.25.14981

Web of Science

PubMed

researchmap

Language：English   Publisher：ACADEMIC PRESS INC JNL-COMP SUBSCRIPTIONS  

To increase the efficiency of association of tumor necrosis factor (TNF), a hydrophilic model protein, with liposomes, an N-myristoylation signal sequence was linked to the N-terminus of TNF by gene fusion. A DNA sequence coding for the N-myristoylation signal of Rasheed leukemia virus-gag protein was fused to the 5'-end of the cDNA coding for the mature domain of TNF to give N-myristoylated fusion TNF cDNA. In vitro translation of the mRNA coding for this fusion cDNA using rabbit reticulocyte lysate gave rise to an N-myristoylated fusion TNF with a molecular mass of 18 kDa as determined by the incorporation of [H-3]myristic acid and by immunoprecipitation with anti-TNF antibody. Replacement of Gly(2) in the myristoylation signal with Ala entirely inhibited the incorporation of [H-3]myristic acid into the fusion protein. A liposome binding assay using Ficoll density gradient centrifugation revealed that incubating the N-myristoylated fusion TNF with dipalmitoyl phosphatidylcholine-liposomes caused the complete binding of the protein to the liposomes, whereas much less of the nonmyristoylated counterpart bound. Thus, N-myristoylated fusion TNF, with high affinity for liposomes, was synthesized by the in vitro transcription/translation system. (C) 1996 Academic Press, Inc.

DOI： 10.1006/abbi.1996.0063

Web of Science

PubMed

researchmap

researchmap

Language：English   Publisher：KLUWER ACADEMIC PUBL  

We provide evidence that the expression of the human MRP/GS-Xpump encoded by the MRP (multidrug resistance associated protein) gene is induced by cisplatin in human leukemia HL-60/R-CP (cisplatin-resistant) cells and modulates cell growth inhibition by Delta(7)-prostaglandin A(1) (PGA(1)) methyl ester. The MRP mRNA level in HL-60/R-CP cells increased remarkably after a 24-h incubation with 20 mu M cisplatin; interestingly, however, no amplification of the MRP gene was detected. In cisplatin-sensitive HL-60 cells, which express the MRP/GS-X pump at low levels, c-myc expression was substantially suppressed by Delta(7)-PGA(1) methyl ester and the cell cycle was arrested in G1 phase. By contrast, in HL-60/R-CP cells overexpressing the MRP/GS-X pump, c-myc expression and cell proliferation were much less affected by Delta 7-PGA(1) methyl ester. This suggests that induction of the MRP/GS-X pump may confer on cancer cells resistance to anticancer prostaglandins and that the resistance mechanism may involve the increased efflux of PG-glutathione conjugates, as active intermediates, from the cells via the MRP/GS-X pump.

DOI： 10.1007/BF00744216

Web of Science

PubMed

researchmap

Language：English   Publisher：AMER SOC MICROBIOLOGY  

Human pro-tumor necrosis factor (pro-TNF) is a type II transmembrane protein with a highly conserved 76-residue leader sequence. We have analyzed the behavior, both in a microsomal translocational system and by transfection, of a series of mutants with deletions from the cytoplasmic, transmembrane, and linking domains. Cytoplasmic deletions included the Arg doublet at -49 and -48 and/or the Lys doublet at -58 and -57; additional mutants included deletion of residues -73 to -55 and -73 to -55, -49, and -48. The transmembrane and linking domain mutants included deletions in the -42 to -35 region, combined with the deletion of residues -32 to -1. Two hybrid mutants combined the cytoplasmic deletions with the deletion of residues -32 to -1. All of the cytoplasmic deletion mutants were properly translocated, as were the transmembrane deletion mutants with deletions up to residues -36, -35, -32 to -1, although the last one exhibited reduced efficiency; further incremental deletions, including deletions of residues -38 to -35 and -32 to -1, completely blocked translocation. Both hybrid mutants were effectively translocated; furthermore, transfection analysis revealed competent expression and maturation of both the cytoplasmic and hybrid mutants. Thus, proper expression and maturation of human pro-TNF can be accomplished with as few as similar to 12 of the 26 residues of the native transmembrane domain and with a net negative charge in the cytoplasmic domain flanking the transmembrane region.

Web of Science

researchmap

Language：English   Publishing type：Rapid communication, short report, research note, etc. (scientific journal)   Publisher：NATL CANCER INSTITUTE  

Web of Science

researchmap

Language：English   Publisher：MARTIN DUNITZ LTD  

The tumor necrosis factor (TNF) mutant TNF-SAM(2) has previously been shown to have a therapeutic profile superior to parental TNF. To initially evaluate the characteristics of liposomal formulations of TNF-SAM(2), it was modified with the N-hydroxysuccinimide ester of caprylic acid to increase its hydrophobic binding to multilamellar and small unilamellar vesicles (MLVs and SUVs). Native PAGE and fluorescamine analysis of acetylated parental TNF and TNF-SAM(2), indicated that these proteins both displayed trimeric structures based on crosslinking/SDS-PAGE analysis and behaved similarly with respect to reactivity of their amino functions. Limited N-terminal sequencing analysis of partially acetylated (approx 3 acetyl groups per trimer) TNF-SAM(2) indicated that the N-terminal Val was not modified; this was also concluded based on HPLC/mass spectrometric (LC-MS) analysis of Glu C digests. LC-MS analysis of tryptic digests of the acetylated TNFSAM(2) indicated that Lys-98 was unreactive. Molecular ions corresponding to acetylated Lys-containing peptides for all five other Lys residues could be detected; none appeared hyperreactive, but Lys-11 appeared hyporeactive. MLVs composed of DMPC/DMPG (7 : 3) and SUVs composed of DPPC/DSPC (1 : 1) displayed high capacity for binding to caprylated TNF-SAM(2). These formulations of caprylated TNF-SAM(2) displayed tumor necrotizing and growth-inhibitory activity in a syngeneic tumor model, and may be candidates for clinical development.

Web of Science

researchmap

Language：English   Publisher：LIPPINCOTT-RAVEN PUBL  

We have evaluated the hemodynamic effects of systemically administered recombinant human tumor necrosis factor (TNF)-alpha, TNF-SAM(2) and liposome-bound TNF-SAM(2) in an anesthetized mongrel dog model. A dose of 10 mu g TNF protein/kg of each formulation was injected in a peripheral vein and mean systemic arterial pressure (SAP), heart rate (KR) and cardiac output (GO) were measured. TNF-a induced a marked drop in SAP in all three dogs (mean decrease = 59.3 +/- 5.2 mm Hg; to 61.5% of baseline; p = 0.008); whereas TNF-SAM(2) caused a smaller and transient drop in SAP in four dogs (mean decrease = 25.5 +/- 10.1 mm Hg; to 81.2% of baseline; p = 0.086). In three dogs administered liposome-bound TNF-SAM(2), which retains antitumor activity in vivo, a net slight hypertensive phase and sustained elevated CO occurred, followed by a return to an essentially normotensive state (101.0% of baseline SAP). This model demonstrates that the principal acute systemic toxicity of TNF, i.e., hypotension, can be markedly attenuated by liposomal formulation of a second-generation TNF.

Web of Science

researchmap

researchmap

researchmap

Language：English   Publisher：MARCEL DEKKER INC  

Free and liposomal formulations of caprylated TNF-SAM(2), a TNF mutant, were evaluated in both a canine model for TNF-induced hypotension and in the murine Meth A sarcoma model for efficacy. The liposomal formulations consisted of caprylated TNF-SAM(2) either bound to the outer leaflet of small unilamellar vesicles (SUVs) or encapsulated in multilamellar vesicles (MLVs). Whereas systemic administration of parental TNF-alpha to anesthesized mongrel dogs resulted in acute shock with a precipitous fall in systemic arterial pressure (SAP), the SUV-TNF-SAM(2) resulted in no net hypotension. Both the free and liposomal-TNF-SAM(2) elicited a hyperdynamic response with tachycardia and elevated cardiac output and SAP, which was not observed with parental TNF-alpha. Two tail-vein injections of these formulations were administered 4-5 days apart to BALB/c mice in which Meth A sarcoma cells were previously implanted subcutaneously or intradermally. Free TNF-SAM(2) at 4 x 10(4) U/mouse caused highly significant tumor control compared to PBS on days 5 and 12; however, substantial toxicity (similar to 24% lethality) was observed at this level. The liposomal formulations of caprylated TNF-SAM(2) demonstrated significant tumor control at all dose levels (up to 8 x 10(4) U/mouse) on day 12 (p less than or equal to 0.025) with either no or reduced lethality (0-15%); the tumor responses were comparable to those with free TNF-SAM(2). We conclude that the liposomal caprylated-TNF-SAM(2) formulations exhibit a therapeutic index superior to that of free parental TNF-alpha or free TNF-SAM(2).

DOI： 10.3109/08982109509010238

Web of Science

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

Language：English   Publisher：ACADEMIC PRESS INC JNL-COMP SUBSCRIPTIONS  

DOI： 10.1016/0003-9861(92)90264-W

Web of Science

PubMed

researchmap

Language：English   Publisher：ACADEMIC PRESS INC JNL-COMP SUBSCRIPTIONS  

DOI： 10.1016/0003-9861(92)90469-D

Web of Science

PubMed

researchmap

Language：English   Publisher：ACADEMIC PRESS INC JNL-COMP SUBSCRIPTIONS  

DOI： 10.1016/0003-9861(92)90091-A

Web of Science

PubMed

researchmap

Language：Japanese   Publishing type：Research paper, summary (national, other academic conference)   Publisher：(一社)日本放射線影響学会  

J-GLOBAL

researchmap

Language：English   Publisher：ACADEMIC PRESS INC JNL-COMP SUBSCRIPTIONS  

DOI： 10.1016/0003-9861(92)90168-V

Web of Science

PubMed

researchmap

researchmap

researchmap

Language：Japanese   Publisher：Kochi University  

We have investigated the effect of tumor necrosis factor-α (TNF-α) and granulocyte colony stimulating factor (G-CSF) on human peripheral neutrophils (HPPMN). TNF-α and G -CSF enhanced fonylmethionyl-1eucyl-phenylalanine (FMLP) -stimiulated O^∸_2 generation of HPPMN in concentration and time dependent manners. The enhancement of 0^∸_2 generation was inhibited by tyrosine kinase inhibitors. These cytokins also enhanced the tyrosyl phosphorylation of neutrophil proteins, such as 115,110,98,83,72,70,60,54 kDa and many of low molecular weight proteins. Both tyrosine kinase and phosphotyr-osine phosphatase might regulate the protein tyrosyl phosphorylation since the phosphorylation inhibited by tyrosine kinase inhibitor, genistein. The TNF-α enhanced tyrosyl phosphorylation of 115 and 108 kDa proteins, which distributed in the membrane fraction, was increased in manners of incubation and concentration dependent. The kinetics of TNF-α enhanced FMLP -dependent O^∸_2 generation was quite similar to that of tyrosyl phosphorylation of neutrophil proteins by TNF-α Furthermore, cepharanthine specifically inhibited either TNF-α or G-CSF enhanced FMLP-stimulated 0^∸_2 generation and tyrosyl phophorylation of some neutrophil proteins, 115 kDa. Taken together with this finding, it is concluded that substrate proteins for tyrosin kinases might act as critical effector molecules in the mechanism for priming in neutrophils.

CiNii Books

researchmap

Language：Japanese   Publisher：Kochi University  

We have investigated the effect of tumor necrosis factor-α (TNF-α) and granulocyte colony stimulating factor (G-CSF) on human peripheral neutrophils (HPPMN). TNF-α and G -CSF enhanced fonylmethionyl-1eucyl-phenylalanine (FMLP) -stimiulated O^∸_2 generation of HPPMN in concentration and time dependent manners. The enhancement of 0^∸_2 generation was inhibited by tyrosine kinase inhibitors. These cytokins also enhanced the tyrosyl phosphorylation of neutrophil proteins, such as 115,110,98,83,72,70,60,54 kDa and many of low molecular weight proteins. Both tyrosine kinase and phosphotyr-osine phosphatase might regulate the protein tyrosyl phosphorylation since the phosphorylation inhibited by tyrosine kinase inhibitor, genistein. The TNF-α enhanced tyrosyl phosphorylation of 115 and 108 kDa proteins, which distributed in the membrane fraction, was increased in manners of incubation and concentration dependent. The kinetics of TNF-α enhanced FMLP -dependent O^∸_2 generation was quite similar to that of tyrosyl phosphorylation of neutrophil proteins by TNF-α Furthermore, cepharanthine specifically inhibited either TNF-α or G-CSF enhanced FMLP-stimulated 0^∸_2 generation and tyrosyl phophorylation of some neutrophil proteins, 115 kDa. Taken together with this finding, it is concluded that substrate proteins for tyrosin kinases might act as critical effector molecules in the mechanism for priming in neutrophils.

CiNii Books

researchmap

Language：English   Publisher：PHYSIOL CHEM PHYSICS MED NMR  

Preincubation of human peripheral blood polymorphonuclear leukocytes (HPPMN) with recombinant human tumor necrosis factor-alpha (rHuTNF-alpha) enhanced the formylmethionyl-leucylphenylalanine (FMLP)-induced superoxide (O2BAR) generation in a concentration- and preincubation time-dependent manner. The enhancement was very high for the FMLP- or opsonized zymosan (OZ)-induced O2BAR generation, but was low for arachidonic acid (AA)- and phorbol myristate acetate (PMA)-induced O2BAR generation. The rHuTNF-alpha has no effect on the steady state of intracellular calcium ion concentration ([Ca2+]i) nor on the membrane potential of neutrophils. The rHuTNF-alpha-primed FMLP-induced O2BAR generation was inhibited by nicotineamide (NA), pertussis toxin (PT), and by the tyrosine kinase (TK) inhibitor, genistein, but was enhanced by the protein kinase C (PKC) inhibitor, H-7 (1-(5-isoquinolinesulfonyl)-3-methyl-piperazine). The inhibitory actions of NA and PT were also observed in in vivo primed guinea pig peritoneal neutrophils (GPtPMN). However, FMLP-induced O2BAR generation of GPtPMN was enhanced by genistein, but was inhibited by H-7. These data indicate that TNF-alpha does not induce changes in [Ce+]i nor in membrane potential of HPPMN, and that TNF-alpha-primed FMLP-induced O2BAR generation of HPPMN is coupled with ADP-ribosylation and activation of G-proteins, and that protein kinases, especially TK, seem to exert an important role in the priming action of TNF.

Web of Science

researchmap

Language：English   Publisher：JAPAN SOC CELL BIOLOGY  

The mechanism by which nonsteroidal antiestrogen inhibits Ca2+- and phospholipid-dependent protein kinase (PKC) activity was investigated. Antiestrogenic agents, clomiphene and tamoxifen, inhibited the PKC-dependent phosphorylation of histone and r-annexin I in a dose-dependent manner. Ki values for the agents were different for two substrate proteins. The inhibitory action of the agents depended on the membrane-substrate protein interaction. Phosphorylation of cytoplasmic proteins obtained from rat uterus and mammary gland, including annexin I, by endogenous PKC was also inhibited by low concentrations of these agents. These results suggest that the inhibitory action of nonsteroidal antiestrogens occurs through their inhibitory effect on the membrane-substrate protein interaction.

DOI： 10.1247/csf.16.273

Web of Science

PubMed

researchmap

Language：Japanese   Publisher：Kochi University  

Healthy human peripheral neutrophils (HPPMN) are not primed, and have weak responses to stimuli which activate HPPMN through their membrane receptors. Recombinant human tumor necrosis factor-α (rHuTNF) and recombinant granulocyte colony stimulation factor (rG-CSF) primed HPPMN. Superoxide (O^&minusd;_2) generation by formylmethionyl-leucylphenylalanine (FMLP) or opsonized zymosan (OZ) was enhanced by these primers. However, O^&minusd;_2 generation induced by phorbol myristate acetate (PMA) or dioctanoylglycerol (DOG) was not enhanced by the primers. Receptor mediated O^&minusd;_2 generation in rHuTNF primed neutrophils was inhibited by the genistein or alpha-cyan0-3-ethoxy-4-hydroxy-5phenylthiomethylcinnamamide (ST 638), inhibitors of tyrosine kinase (TK). But it was enhanced by 1-(5-isoquinoline sulfonyl)-3-methyl-piperazine (H-7) or staurosporine, inhibitors of Ca^<++>_ and phospholipid-dependent protein kinase (PKO, in their concentration dependent manner. On the contrary, O^&minusd;_2 generation of HPPMN at higher concentration of PMA or DOG was rather stimulated by genistein or ST 638 and was inhibited by H-7 or staurosporine. These results suggest that protein kinases participates in the NADPH-oxidase activation of neutrophils and that two pathways exist in the NADPH-oxidase activation : one in PMA- or DOG-stimulated PKC-dependent pathway and the other in FMLP-stimulated TK-dependent pathway. Moreover, it suggests that TK might be involved in the reaction of neutrophil priming with various ligands.

CiNii Books

researchmap

Language：English   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)   Publisher：SOC FERMENTATION BIOENGINEERING, JAPAN  

A new CGTase-producing moderate thermophile was isolated from soil, and was identified as Bacillus coagulans which have not previously been listed as cyclodextrin producing bacteria. The culture filtrate of the isolate as the CGTase source converted about 60% of soluble starch to CD's in 20 h at 50-degrees-C, and the ratio of alpha-: beta-: gamma-CD produced was 1.0: 0.9: 0.3.

DOI： 10.1016/0922-338X(91)90305-Z

Web of Science

researchmap

researchmap

Language：English   Publisher：SOC FERMENTATION BIOENGINEERING, JAPAN  

Cyclomaltodextrin glucanotransferase (CGTase), produced in a culture filtrate by Bacillus coagulans, was purified to a single, homogeneous protein. It has a monomeric structure with a molecular weight of 65,000, isoelectric point of 4.6, and contains 2 mol of Ca2+ per mol of the enzyme. The enzyme was most active at pH 6.0 and at 70-degrees-C. It did not lose its activity by heat treatment at 70-degrees-C for 10 min in the presence of CaCl2 in the pH range of 5.5 approximately 9.5, and by incubation in the pH range of 5.0 approximately 10.5 at 4-degrees-C for one month. The enzyme converted about 60% of potato starch to cyclodextrins for 20 h at 50-degrees-C, and the ratio of alpha-: beta-: gamma-cyclodextrin produced was 8.1:8.9:1.0. B. coagulans CGTase was compared with B. macerans CGTase which was purified by the same method.

DOI： 10.1016/0922-338X(91)90344-G

Web of Science

researchmap

researchmap

Language：Japanese   Publisher：Kochi University  

Healthy human peripheral neutrophils (HPPMN) are not primed, and have weak responses to stimuli which activate HPPMN through their membrane receptors. Recombinant human tumor necrosis factor-α (rHuTNF) and recombinant granulocyte colony stimulation factor (rG-CSF) primed HPPMN. Superoxide (O^&minusd;_2) generation by formylmethionyl-leucylphenylalanine (FMLP) or opsonized zymosan (OZ) was enhanced by these primers. However, O^&minusd;_2 generation induced by phorbol myristate acetate (PMA) or dioctanoylglycerol (DOG) was not enhanced by the primers. Receptor mediated O^&minusd;_2 generation in rHuTNF primed neutrophils was inhibited by the genistein or alpha-cyan0-3-ethoxy-4-hydroxy-5phenylthiomethylcinnamamide (ST 638), inhibitors of tyrosine kinase (TK). But it was enhanced by 1-(5-isoquinoline sulfonyl)-3-methyl-piperazine (H-7) or staurosporine, inhibitors of Ca^<++>_ and phospholipid-dependent protein kinase (PKO, in their concentration dependent manner. On the contrary, O^&minusd;_2 generation of HPPMN at higher concentration of PMA or DOG was rather stimulated by genistein or ST 638 and was inhibited by H-7 or staurosporine. These results suggest that protein kinases participates in the NADPH-oxidase activation of neutrophils and that two pathways exist in the NADPH-oxidase activation : one in PMA- or DOG-stimulated PKC-dependent pathway and the other in FMLP-stimulated TK-dependent pathway. Moreover, it suggests that TK might be involved in the reaction of neutrophil priming with various ligands.

CiNii Books

researchmap

J-GLOBAL

researchmap

Language：Japanese   Publisher：Kochi University  

Since reactive oxygen species have been postulated to underlie the pathogenesis of various diseases, measurement of luminol chemiluminescence (LCL) has recently been used to quantitate active oxygen production. However, the precise mechanism of LCL and active oxygen species which react with luminol have not been elucidated. Oxidation of xanthine by xanthine oxidase (X-XO system) is one of the main superoxide generating reactions in vivo, in which the superoxide is changed to several reactive oxygen species, such as hydrogen peroxide, hypochlorite, singlet oxygen and hydroxy radicals by enzymatic or nonenzymatic reactions. In this experiment, we clarified the fact that LCL by X-XO system is mainly dependent on uric acid-inhibitable hydroxy radicals which are produced from superoxide radicals and hydrogen peroxide by chemical reaction.

CiNii Books

researchmap

Language：Japanese   Publisher：Kochi University  

Salivary neutrophils (SPMN) were isolated from the oral cavity and stimulation-coupled responses were examined. From the morphological study and content of neutrophil specific 39kDa protein, these cells seems to be similar with those of peritoneal neutrophils. Most of the neutrophils were young stage cells. Stimulation-coupled responses of these cells were observed in superoxide (O^&minusd;_2) generation, Iuminol chemiluminescence (LCL) response, membrane deporalization and changes in intracellular calcium ions. However, strong O^&minusd;_2 generation was observed without addition of any stimulants. This LCL was inhibited by azide but not by uric acid or superoxide dismutase (SOD). The endogenous LCL decreased from the time after isolation from the oral cavity accompanied by the appearance of FMLP-coupled response. The rate of active oxygen generation by PMA was similar with that of peritoneal neutrophils, but opsonized zymosan (OZ) induced LCL was lower than that of peripheral neutrophils. Furthermore, the activity of active oxygen generation was maintained for a long period (more than 10 hours). From these evidences, it is concluded that the salivary neutrophils have the specific characteristics of stimulation-coupled responses, and play an important role in the defense mechanism of the oral cavity.

CiNii Books

researchmap

Language：Japanese   Publisher：Kochi University  

Since reactive oxygen species have been postulated to underlie the pathogenesis of various diseases, measurement of luminol chemiluminescence (LCL) has recently been used to quantitate active oxygen production. However, the precise mechanism of LCL and active oxygen species which react with luminol have not been elucidated. Oxidation of xanthine by xanthine oxidase (X-XO system) is one of the main superoxide generating reactions in vivo, in which the superoxide is changed to several reactive oxygen species, such as hydrogen peroxide, hypochlorite, singlet oxygen and hydroxy radicals by enzymatic or nonenzymatic reactions. In this experiment, we clarified the fact that LCL by X-XO system is mainly dependent on uric acid-inhibitable hydroxy radicals which are produced from superoxide radicals and hydrogen peroxide by chemical reaction.

CiNii Books

researchmap

Language：Japanese   Publisher：Kochi University  

Salivary neutrophils (SPMN) were isolated from the oral cavity and stimulation-coupled responses were examined. From the morphological study and content of neutrophil specific 39kDa protein, these cells seems to be similar with those of peritoneal neutrophils. Most of the neutrophils were young stage cells. Stimulation-coupled responses of these cells were observed in superoxide (O^&minusd;_2) generation, Iuminol chemiluminescence (LCL) response, membrane deporalization and changes in intracellular calcium ions. However, strong O^&minusd;_2 generation was observed without addition of any stimulants. This LCL was inhibited by azide but not by uric acid or superoxide dismutase (SOD). The endogenous LCL decreased from the time after isolation from the oral cavity accompanied by the appearance of FMLP-coupled response. The rate of active oxygen generation by PMA was similar with that of peritoneal neutrophils, but opsonized zymosan (OZ) induced LCL was lower than that of peripheral neutrophils. Furthermore, the activity of active oxygen generation was maintained for a long period (more than 10 hours). From these evidences, it is concluded that the salivary neutrophils have the specific characteristics of stimulation-coupled responses, and play an important role in the defense mechanism of the oral cavity.

CiNii Books

researchmap