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S Fukui

Publications and source records attributed to S Fukui.

At least 109 records · Page 6Linked to original sources
PubMed1991

Expression of the Tn antigen on T-lymphoid cell line Jurkat.

Expression of the Tn antigen on a T-lymphoid cell line, Jurkat, was investigated using an anti-Tn monoclonal antibody, MLS 128. Immunoprecipitation or immunoaffinity chromatography of a lysate of Jurkat cells led to the isolation of a 120 kDa glycoprotein carrying the Tn antigen. This glycoprotein and leukosialin (CD43) were indistinguishable on SDS-PAGE and as to immunoreactivity with MLS 128. Leukosialin from an erythroid cell line, K562, exhibited no reactivity with MLS 128 despite that this leukosialin has several GalNAc alpha-Ser(Thr) structures. Pulse-chase experiments with the Jurkat leukosialin showed that newly synthesized leukosialin acquired the antigenecity after a lag of about 30 min, whereas incorporation of GalNAc into the leukosialin occurred earlier. These results indicate that the Tn antigen is expressed on leukosialin and that its epitopic structure is more complex than GalNAc alpha-Ser(Thr).

Amino Acid Sequence↗
PubMed1991

Characterization of mucin-type oligosaccharides with the sialyl-Le(a) structure from human colorectal adenocarcinoma cells.

Oligosaccharides with the sialyl-Le(a) structure have been isolated on an affinity column of a monoclonal antibody, MSW 113, from mucin-type glycoproteins derived from the surfaces of SW 1116 and LS 180 cells, and their secretions. The oligosaccharides were polydisperse with respect to molecular size, the oligosaccharides derived from glycoproteins in culture media being larger than those in cell lysates, as assessed by gel filtration. Some of the oligosaccharides were susceptible to degradation by endo-beta-galactosidase (E. freundii), as judged from the change in the gel filtration pattern. These results indicate that oligosaccharides with the sialyl-Le(a) structure derived from mucin-type glycoproteins produced by human colonic cancer cells are extremely large in size and complex in structure, and that some of them contain the poly-N-acetyllactosamine structure.

Adenocarcinoma↗
PubMed1991

Elucidation of an essential structure recognized by an anti-GalNAc alpha-Ser(Thr) monoclonal antibody (MLS 128).

To determine the epitopic structure for an anti-GalNAc alpha-Ser(Thr) (anti-Tn) monoclonal antibody, MLS 128, asialo-ovine submaxillary mucin was digested with various proteases, and the digests were fractionated by immunoaffinity column chromatography and high performance liquid chromatography. From the tryptic digest, a glycopeptide, GP-I, and five other glycopeptides, GP-1-5, were obtained as bound and unbound fractions, respectively, of the immunoaffinity column. By solid phase radioimmunoassaying, it was found that GP-I was strongly immunoreactive, whereas GP-1-5 were poorly immunoreactive. On treatment with V8 protease, GP-I was converted to two glycopeptides, one with poor reactivity and the other with intermediate reactivity. From the thermolysin digest, the smallest fragment, GP-II, was isolated, which was as strongly immunoreactive as GP-I. GP-II corresponded to a part of GP-I, its sequence being Leu-Ser*-Glu-Ser*-Thr*-Thr*-Gln-Leu-Pro-Gly, where asterisks denote amino acids to which an alpha-GalNAc residue is attached. Other anti-Tn monoclonal antibodies, NCC-LU-35 and CA 3239, showed essentially the same reactivity to these glycopeptides as MLS 128 did. The glycopeptides (GP-1-5), which exhibited poor immunoreactivity, contained various GalNAc-containing structures, such as GalNAc-Ser, GalNAc-Thr, GalNAc-Ser-(GalNAc)-Ser, and GalNAc-Thr-(GalNAc)-Thr. These results indicate that a glycopeptide including a cluster structure, Ser*-Thr*-Thr*, is an essential part of the epitope recognized by anti-Tn antibodies.

Amino Acid Sequence↗
PubMed1991

An increase in the transglycosylation activity of Saccharomycopsis alpha-amylase altered by site-directed mutagenesis.

The 84th tryptophan residue in Saccharomycopsis alpha-amylase molecule was replaced by a leucine residue and the resulting site-directed mutant, W84L enzyme, showed an increase in transglycosylation activity. At a 40% digestion point of maltoheptaose (G7), for example, maltooligosaccharide products larger than maltodecaose (G10) amounted to approx. 60% of the total product from the mutant enzyme reaction, whereas no such large products were observed in the native enzyme reaction. Analysis of the reaction products from p-nitrophenyl maltooligosaccharides indicated that these large products were formed by addition of the hydrolysis products on the nonreducing end side to the starting intact substrates. These results suggest that the tryptophan residue located at subsite 3 of the enzyme plays an important role not only to hold the substrate, but also to liberate the hydrolysis products from the substrate binding pocket.

Glycosylation↗
PubMed1991

Novel oligosaccharides with the sialyl-Lea structure in human milk.

We have isolated four novel oligosaccharides with the sialyl-Lea structure from human milk using a monoclonal antibody, MSW 113. These oligosaccharides were purified by affinity chromatography on a column of the immobilized monoclonal antibody and by high-performance liquid chromatography. The results of structural analyses, i.e., 500-MHz 1H NMR spectroscopy, fast atom bombardment mass spectrometry, and binding to specific anticarbohydrate antibodies, are consistent with the following structures. (formula; see text)

Antibodies, Monoclonal↗
PubMed1991

Characterization of mucin antigens recognized by monoclonal antibodies raised against human colon cancer cells.

Two monoclonal antibodies, MLS 102, which recognizes cancer-associated mucin antigens, and MLS 103, which recognizes normal mucin, were used to isolate, by immunoaffinity chromatography, the corresponding antigens from cell lysates and spent medium of a human colorectal carcinoma cell line, LS 180. The MLS 102 antigen contained serine, threonine, and proline as major amino acids. The carbohydrate chains of the MLS 102 antigen were composed of O-linked NeuAc alpha 2----6GalNAc (56%), N-acetylgalactosamine (25%), and longer oligosaccharide chains. The MLS 103 antigen differed from the MLS 102 antigen in both amino acid and carbohydrate composition. Most O-linked oligosaccharides of the MLS 103 antigen were longer than the disaccharide found in the MLS 102 antigen. Immunostaining of LS 180 cells using MLS 102 and MLS 103 revealed that the cells are heterogeneous with respect to the expression of the antigens.

Amino Acids↗
PubMed1991

The Saccharomyces cerevisiae genes (CMP1 and CMP2) encoding calmodulin-binding proteins homologous to the catalytic subunit of mammalian protein phosphatase 2B.

Saccharomyces cerevisiae genomic clones that encode calmodulin-binding proteins were isolated by screening a lambda gt11 expression library using 125I-labeled calmodulin as probe. Among the cloned yeast genes, we found two closely related genes (CMP1 and CMP2) that encode proteins homologous to the catalytic subunit of phosphoprotein phosphatase. The presumed CMP1 protein (62,999 Da) and CMP2 protein (68,496 Da) contain a 23 amino acid sequence very similar to those identified as calmodulin-binding sites in many calmodulin-regulated proteins. The yeast genes encode proteins especially homologous to the catalytic subunit of mammalian phosphoprotein phosphatase type 2B (calcineurin). The products of the CMP1 and CMP2 genes were identified by immunoblot analysis of cell extracts as proteins of 62,000 and 64,000 Da, respectively. Gene disruption experiments demonstrated that elimination of either or both of these genes had no effect on cell viability, indicating that these genes are not essential for normal cell growth.

Alleles↗
PubMed1991

Quantitative in vivo analysis of benzodiazepine binding sites in the human brain using positron emission tomography.

Central-type benzodiazepine binding sites were characterized in a single normal human subject, using positron emission tomography (PET) and the radiolabelled benzodiazepine antagonist, carbon-11 labelled flumazenil ([11C] Ro 15-1788). The subject was scanned using tracer alone and tracer plus 4 different concentrations of unlabelled Ro 15-1788, including one concentration of unlabelled Ro 15-1788, chosen to produce maximum displacement of [11C] Ro 15-1788 from specific binding sites. Concentrations of free, unmetabolized [11C] Ro 15-1788 in plasma were estimated using a simple extraction and ultrafiltration method. Radioactivity in the regional exchangeable pool in brain was estimated under non-saturation conditions from the ratio of radioactivity in brain to plasma, under saturation conditions and the kinetics of free ligand in plasma. The specific binding was, then, estimated by the difference between the total radioactivity in brain and exchangeable pool radioactivity. Scatchard analyses were performed to yield Bmax and Kd values under pseudo-equilibrium conditions, which was observed as an increase of specific binding/free with reduction in specific binding. In cerebral cortex and cerebellum, the Bmax values were about 62-73 nmol/l and the Kd values were 3.6-6 nM in the estimation of free ligand in plasma and 12-15 nM in the estimation of exchangeable pool in brain, as free in brain.

Adult↗
PubMed1991

Subsite structure of Saccharomycopsis alpha-amylase secreted from Saccharomyces cerevisiae.

The kinetic parameters (kcat/Km) and the cleaved-bond distributions for the hydrolysis of linear maltooligosaccharides Gn (3 less than or equal to n less than or equal to 9) by Saccharomycopsis alpha-amylase (Sfamy) secreted from Saccharomyces cerevisiae were determined at pH 5.25 and 25 degrees C. The subsite affinities of Sfamy were also evaluated from these data. The subsite structure of Sfamy is characteristic of the active site of an endo-cleavage type enzyme, consisting of internal repulsive sites with the catalytic residues and external attractive sites. Moreover, the pKa values of the catalytic residues were calculated from the pH dependence plot of the kinetic parameter (kcat/Km). The amino acid residues which contribute to the subsite affinities and the catalytic activity of Sfamy are proposed and compared with those of Taka-amylase A.

Kinetics↗
PubMed1991

Isolation and structural studies of human milk oligosaccharides that are reactive with a monoclonal antibody MSW 113.

We have determined the structures of six oligosaccharides isolated from human milk using a monoclonal antibody, MSW 113. The isolation involved affinity chromatography on a column of the immobilized monoclonal antibody and high-performance liquid chromatography. From the results of 500 and 600 mHz 1H NMR spectroscopy and fast atom bombardment-mass spectrometry their structures were deduced to be: [formula; see text] Two of these oligosaccharides, numbers 4 and 5, have not previously been described. All of them bound to MSW 113, but their reactivities are weaker than those of sialyl-Le(a) oligosaccharides. The results indicate that MSW 113 reacts with oligosaccharides with the mono- and disialyl-Le(a), and other sialyl type 1 structures.

Antibodies, Monoclonal↗
PubMed1991

Inhibition of membrane Ca2(+)-ATPase of Saccharomyces cerevisiae by mating pheromone alpha-factor in vitro.

Plasma membrane Ca2(+)-ATPase of Saccharomyces cerevisiae was solubilized and partially purified by calmodulin-affinity chromatography. The activity of Ca2(+)-ATPase isolated from MATa cells was inhibited by physiological concentrations of the mating pheromone alpha-factor in a dose-dependent manner. The enzyme prepared from a receptor-deficient sterile mutant cells (delta ste-2) was similarly inhibited by alpha-factor, but the enzyme from MAT alpha cells was resistant to the mating pheromone. We suggest that the inhibition may be involved in the alpha-factor-induced increase of Ca2+ uptake reaction of MATa cells.

Bacterial Proteins↗
PubMed1991

Blood-brain barrier transport of kynurenines: implications for brain synthesis and metabolism.

To evaluate the potential contribution of circulating kynurenines to brain kynurenine pools, the rates of cerebral uptake and mechanisms of blood-brain barrier transport were determined for several kynurenine metabolites of tryptophan, including L-kynurenine (L-KYN), 3-hydroxykynurenine (3-HKYN), 3-hydroxyanthranilic acid (3-HANA), anthranilic acid (ANA), kynurenic acid (KYNA), and quinolinic acid (QUIN), in pentobarbital-anesthetized rats using an in situ brain perfusion technique. L-KYN was found to be taken up into brain at a significant rate [permeability-surface area product (PA) = 2-3 x 10(-3) ml/s/g] by the large neutral amino acid carrier (L-system) of the blood-brain barrier. Best-fit estimates of the Vmax and Km of saturable L-KYN transfer equalled 4.5 x 10(-4) mumol/s/g and 0.16 mumol/ml, respectively. The same carrier may also mediate the brain uptake of 3-HKYN as D,L-3-HKYN competitively inhibited the brain transfer of the large neutral amino acid L-leucine. For the other metabolites, uptake appeared mediated by passive diffusion. This occurred at a significant rate for ANA (PA, 0.7-1.6 x 10(-3) ml/s/g), and at far lower rates (PA, 2-7 x 10(-5) ml/s/g) for 3-HANA, KYNA, and QUIN. Transfer for KYNA, 3-HANA, and ANA also appeared to be limited by plasma protein binding. The results demonstrate the saturable transfer of L-KYN across the blood-brain barrier and suggest that circulating L-KYN, 3-HKYN, and ANA may each contribute significantly to respective cerebral pools. In contrast, QUIN, KYNA, and 3-HANA cross the blood-brain barrier poorly, and therefore are not expected to contribute significantly to brain pools under normal conditions.

Animals↗
PubMed1990

A monoclonal antibody directed to Tn antigen.

A murine monoclonal antibody, MLS 128, that was assigned to an anti-Tn antibody has been established by immunizing mice with human colonic cancer cells (LS 180). MLS 128 bound to mucin glycopeptides from LS 180 cells and their asialo forms to the same extent as well as to ovine submaxillary mucin (OSM) and asialo OSM. Special non-sialylated GalNAc residue(s) attached to a certain peptide region in the antigens seems to be involved in the binding since N-acetylgalactosaminidase treatment of the antigen abolished the binding and pronase digestion diminished the binding markedly.

Acetylgalactosamine↗
PubMed1990

Occurrence of tetra- and pentasaccharides with the sialyl-Le(a) structure in human milk.

The occurrence of two novel oligosaccharides in human milk was investigated. These oligosaccharides were purified by affinity chromatography on a column of an immobilized monoclonal antibody, MSW 113. Structural studies, involving 500-MHz 1H NMR spectroscopy and fast atom bombardment-mass spectrometry, indicated the structures of these compounds to be NeuAc alpha 2----3Gal beta 1----3(Fuc alpha 1----4) GlcNAc and NeuAc alpha 2----3Gal beta 1----3(Fuc alpha 1----4) GlcNac beta 1----3Gal. This constitutes the first evidence for the occurrence of N-acetylglucosamine or galactose as the reducing-end residue of human milk oligosaccharides. These two oligosaccharides bound MSW 113 to nearly the same extent as sialyl-Lea hexasaccharide but to another sialyl-Le(a) structure-directed monoclonal antibody, NS-19-9, only weakly.

Antigens, Tumor-Associated, Carbohydrate↗
PubMed1990

Essential histidine residues in coenzyme B12-dependent diol dehydrase: dye-sensitized photooxidation and ethoxycarbonylation.

The apoenzyme of diol dehydrase was inactivated by photoirradiation in the presence of rose bengal or methylene blue, following pseudo-first-order kinetics. The inactivation rates were markedly reduced under a helium atmosphere, suggesting that the inactivation is due to photooxidation of the enzyme under air. The half-maximal rate of methylene blue-sensitized photoinactivation was observed at pH around 7.5. Amino acid analyses indicated that one to two histidine residues decreased upon the dye-sensitized photoinactivation, whereas the numbers of tyrosine, methionine, and lysine did not change. Ethoxyformic anhydride, another histidine-modifying reagent, also inactivated diol dehydrase, with pseudo-first-order kinetics and a half-maximal rate at pH 7.7. It was shown spectrophotometrically that three histidine residues per enzyme molecule were modified by this reagent with loss of enzyme activity. Two tyrosine residues per enzyme molecule were also modified rapidly, irrespective of the activity. The photooxidation or ethoxycarbonylation of the enzyme did not result in dissociation of the enzyme into subunits, but deprived the enzyme of ability to bind cyanocobalamin. The percentage loss of cobalamin-binding ability agreed well with the extent of inactivation. The enzyme-bound hydroxocobalamin showed only partial protecting effect against photoinactivation and resulting loss of the cobalamin-binding ability. These results provide evidence that diol dehydrase possesses essential histidine residues which are required for the coenzyme binding.

Amino Acids↗
PubMed1990

The mutagenic modulating effect of p-phenylenediamine on the oxidation of o- or m-phenylenediamine with hydrogen peroxide in the Salmonella test.

The mutagenicity of o- and m-phenylenediamine (PD) was remarkedly enhanced by oxidation; their major mutagenic oxidation products were 2,3- and 2,7-diaminophenazine, respectively. In order to evaluate the modulation effect of p-PD on the oxidation of m- or o-PD, p-PD and mixtures of m- and p-PD (m-PD/p-PD) and o- and p-PD (o-PD/p-PD) were oxidized with hydrogen peroxide and their mutagenicity was tested in Salmonella typhimurium TA98 in the presence or absence of a mammalian metabolic activation system (S9 mix). The H2O2-oxidized m-PD/p-PD and o-PD/p-PD were potent mutagens with S9 mix, whereas H2O2-oxidized p-PD was slightly mutagenic. The major mutagenic oxidation products of m-PD/p-PD and o-PD/p-PD were identified as 2,7- and 2,3-diaminophenazine, respectively, by TLC and HPLC. 2,8-Diaminophenazine was also found as a reaction product in oxidized m-PD/p-PD, and it was weakly mutagenic. The mutagenic potency of oxidized m-PD/p-PD or o-PD/p-PD was lower than that of singly oxidized m-PD or o-PD. The yield of 2,7- and 2,3-diaminophenazine was obviously decreased with increases in p-PD, and it was concluded that the declined mutagenic potency of oxidized m-PD/p-PD or o-PD/p-PD was due to the decrease in diaminophenazines. But the formation of diaminophenazines was not completely inhibited by the addition of a 9-fold molar ratio of p-PD to m-PD or o-PD, 8.6 nmole of 2,7-diaminophenazine and 1882.4 nmole of 2,3-diaminophenazine were formed from 1 mmole of m-PD and o-PD, respectively.

Animals↗
PubMed1990

Mutagenicity of commercial hair dyes and detection of 2,7-diaminophenazine.

Four commercial oxidative-type hair dye formulations, A, B, C, and D, were treated with hydrogen peroxide (H2O2) to simulate normal conditions of use, and the oxidized hair dyes were tested for their mutagenicity in Salmonella typhimurium TA98 in the presence of a mammalian metabolic activation system (S9 mix). Most of them did not show obvious mutagenicity in the range of 1-25 microliters/plate and all exhibited bactericidal activity at 10 microliters/plate. In order to evaluate the mutagenicity of hair dyes both before and after H2O2 oxidation, rayon linked to a copper-phthalocyanine derivative (blue rayon) was used as an adsorbent for the elimination of interfering bactericidal compounds. Adsorbed compounds on blue rayon were eluted with ammoniacal methanol and eluents were subjected to the Ames test. The mutagenicity of the blue-rayon extracts in TA98 with S9 mix was increased by H2O2 oxidation. The blue-rayon extracts obtained from oxidized A and B were potent mutagens and reverted 334 and 999 colonies/10 microliters of original substance, respectively. In addition, 88 and 249 ng of 2,7-diaminophenazine, which was extremely mutagenic in TA98 with S9 mix, were detected in the extracts of 40 ml of the hair dye formulations A and B, respectively. The mutagenicity in oxidized hair dye formulations was successfully detected by use of blue-rayon extraction. 2,7-Diaminophenazine was only formed in the hair dye formulations containing m-phenylenediamine by H2O2 oxidation. Therefore, attention needs to be paid to the use of m-phenylenediamine as a hair dye component, not only for its own toxicity but also for that of its oxidation products.

Biotransformation↗