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Biomedical subjects

N Sakurai

Publications and source records attributed to N Sakurai.

At least 55 records · Page 3Linked to original sources
PubMed1999

Primary structure, expression, and site-directed mutagenesis of inorganic pyrophosphatase from Bacillus stearothermophilus.

The complete primary structure of inorganic pyrophosphatase [EC 3.6. 1.1] from Bacillus stearothermophilus (ATCC 12016) was determined at the amino acid level by automated Edman degradation. The subunit of the enzyme consists of 164 amino acid residues with a calculated molecular mass of 18,796. The amino acid sequence of the enzyme is almost identical to that of thermophilic bacterium PS-3. Based on the determined primary structure, a PCR-amplified semi-synthetic gene was constructed and expressed in Escherichia coli JM109. The recombinant Bst. PPase showed the same characteristics and activity as the authentic enzyme, and exhibits higher thermostability than the E. coli enzyme. Furthermore, we prepared tyrosine-substituted variants by site-directed mutagenesis to elucidate the role of two highly conserved tyrosines (Y46 and Y130). As a result, two variants, Y46F and Y130F, lost most of their enzyme activity, whereas their conformations were unaffected. However, the wild-type and two variants exhibited different thermostability behaviors in the presence or absence of Mg2+. Therefore, these tyrosines may contribute to the structural integrity of the active site of the enzyme.

Amino Acid Sequence↗
PubMed1999

Site-directed mutagenesis of a possible type 1 copper ligand of bilirubin oxidase; a Met467Gln mutant shows stellacyanin-like properties.

In our previous paper, we reported a mutant of recombinant Myrothecium verrucaria bilirubin oxidase, in which the Met467 residue was replaced by Gly [Shimizu, A. et al. (1999) Biochemistry 38, 3034-3042]. This mutant displayed a remarkable reduction in enzymatic activity and an evident decrease in the intensity of the absorption band around 600 nm (type 1 charge transfer transition). In this study, we report the preparation of three Met467 mutants (Met467Gln, Met467His, and Met467Arg) and characterize their enzymatic activities, midpoint potentials, and absorption and ESR spectra. Met467His and Met467Arg show no enzymatic activity and a great reduction in the intensity of the absorption band around 600 nm. Furthermore, their ESR spectra show no type 1 copper signal, but only a type 2 copper signal; however, oxidation by ferricyanide caused the type 1 copper signal to appear. On the other hand, Met467Gln as expressed shows both type 1 and type 2 copper signals in its ESR spectrum, the type 1 copper atom parameters being very different from usual blue copper proteins but very similar to those of stellacyanin. The enzymatic activity of the Met467Gln mutant for bilirubin is quite low (0.3%), but the activity for potassium ferrocyanide is similar (130%) to that of the wild type enzyme. These results indicate that Met467 is important for characterizing the features of the type 1 copper of bilirubin oxidase.

Amino Acid Sequence↗
PubMed1999

A new UV method for serum gamma-glutamyltransferase assay using recombinant 4-aminobenzoate hydroxylase as a coupling enzyme.

4-aminobenzoate hydroxylase (4ABH) is a flavin-dependent monooxygenase that catalyzes the decarboxylative hydroxylation of 4-aminobenzoate to 4-hydroxyaniline. For use as a clinical reagent, the gene encoding 4ABH from Agaricus bisporus was cloned by the RACE method. Also, the cDNA encoding 4ABH was expressed in Escherichia coli cells as a fusion protein with glutathione S-transferase (GST). The expressed GST-4ABH fusion protein (recombinant 4ABH) in the soluble fraction exhibits decarboxylative hydroxylation and additional NADH oxidation activities.We investigated a new ultraviolet spectrometric method for determining serum gamma-glutamyltransferase (gamma-GT) using recombinant 4ABH as a coupling enzyme. The principle of the method is as follows. Using gamma-glutamyl-3-choloro-4-aminobenzoate (L-gamma-glu-PAClBA) and glycylglycine as the donor and acceptor substrates, 3-choloro-4-aminobenzoate (PAClBA) is formed by the catalysis of serum gamma-GT. PAClBA is stoichiometrically converted to 3-choloro-4-hydroxyaniline (PHClA) and NAD(+) by 4ABH and NADH. However, NADH oxidation results in a high reagent blank, which is considered as a drawback for use as a clinical reagent. Using recombinant 4ABH, we examined the effects of pH and detergents on these two activities, and found that several detergents suppress the additional NADH oxidation activity with little or no effect on hydroxylation activity. The results indicate a promising approach to establishing an ultraviolet spectrophotometric method for determining serum gamma-GT activity using L-gamma-glu-PAClBA as the donor substrate and recombinant 4ABH as a coupling enzyme.

Amino Acid Sequence↗
PubMed1999

Specific targeting immunotherapy of cancer with bispecific antibodies.

In order to enhance cell mediated cytotoxicity, bispecific antibodies (BsAbs), molecules combining two or more antibodies with different antigenic specificities, have been developed as new agents for immunotherapy. Our recent studies revealed that simultaneous administration of two kinds of BsAbs (anti-tumor x anti-CD3 plus anti-tumor x anti-CD28) together with lymphokine activated killer cells with a T cell phenotype (T-LAK cells) inhibited growth of human xenotransplanted tumors in severe combined immunodeficient (SCID) mice, while single BsAb was without effect. Three kinds of BsAbs (anti-tumor x anti-CD3, anti-tumor x anti-CD28, anti-tumor x anti-CD2) showed the highest cytotoxicity against tumor cells when given simultaneously with T-LAK cells or peripheral blood mononuclear cells in vitro and in vivo. BsAbs can be preserved for immediate application, while cytotoxic T lymphocytes (CTLs) must be made-to-order, and are time-consuming to prepare. Tumor associated antigens, such as MAGE antigens, SART antigens, MUC1 antigen, c-erbB 2 antigen or cancer/testis antigens can be served to target antigens for BsAb production. By conjugation with antibodies to effector cells (anti-CD3, anti-CD28, anti-CD16, anti-CD64, anti-CD89 or anti-CD2), many kinds of BsAbs can be produced to cover most types of cancers from different organs. Therefore this strategy might be ubiquitously applicable to most malignancies.

Animals↗
PubMed1999

[The moderating effects of positive appraisal on the burden of family caregivers of older people].

This study focused on the moderating effects of positive appraisal of family caregivers of older people on caregiving burden. In Study 1, 16 original items of the Caregiving Burden Scale and 14 original items of the Positive Appraisal Scale were administered 140 caregivers. By factor analysis, the Caregiving Burden Scale was divided into four subscales named: "Restriction of freedom (Kosoku-kan)"; "Wish to give up caregiving (Genkai-kan)"; "Family trouble (Taijin-katto)"; and "Economic restraint (Keizaiteki-futan)". The positive appraisal scale was also divided into three subscales named "Caregiving satisfaction (Manzoku-kan)", "Caregiver's self-growth (Jiko-seicho-kan)", and "Will to continue care-giving (Kaigo-keizoku-ishi)". In Study 2, 306 caregivers completed the Caregiving Burden Scale and Positive Appraisal Scale. The results indicated that; (a) positive appraisal moderated caregiver's wish to give up caregiving ("Genkai-kan"); (b) Caregiving satisfaction ("Manzoku-kan") was more effective than caregiver's self-growth ("Jiko-seicho-kan"); (c) moderating patterns varied by the combination of stressor, burden and positive appraisal.

Aged↗
PubMed1998

Roles of four iron centers in Paracoccus halodenitrificans nitric oxide reductase.

Reactions of Paracoccus halodenitrificans nitric oxide reductase (NOR) containing four iron centers, a low spin hemec, a low spin heme b, a high spin heme b and a non-heme iron, have been studied to show the roles of each iron center. Soon after reacting the resting (oxidized) NOR with L-ascorbate, the low spin heme c and low spin heme b were reduced to a considerable extent but the high spin heme b was still in the oxidized form and was reduced slowly. When CO acted on the reduced NOR, the high spin heme b center changed to a low spin state. On the other hand, when NO acted on the resting NOR, no apparent spectral change was observed. However, when NO acted on the reduced NOR (a steady state condition, excess dithionite is present), both of the low spin centers changed to be partly in the oxidized form. A small but clear new EPR signal with g = 4.1 appeared together with some new signals at the g = 2 region soon after the action of NO on the reduced NOR. During incubation at room temperature the nitrosyl-heme signal typical of 5-coordination developed. These results suggested that both the high spin-heme b center and the non-heme iron are the reaction centers and their reductions are indispensable for the enzyme process in contrast to the reaction mechanism proposed for the P-450 type NOR(P-450nor).

Ascorbic Acid↗
PubMed1998

Synthesis and structure-activity relationships of 7-(2-aminoalkyl)morpholinoquinolones as anti-Helicobacter pylori agents.

A series of the titled compounds was synthesized and tested for anti-Helicobacter pylori activities. We discovered Y-34867 having the most potent activity against Helicobacter pylori among the quinolones tested along with high photostability. Furthermore, Y-34867 showed an excellent therapeutic effect in the experimental Helicobacter pylori infected Mongolian gerbil model.

4-Quinolones↗
PubMed1998

Effective adoptive immunotherapy by T-LAK cells retargeted with bacterial superantigen-conjugated antibody to MUC1 in xenografted severe combined immunodeficient mice.

To reinforce cytotoxic activity and the targeting ability of lymphokine-activated killer cells with a T-cell phenotype (T-LAK) for adoptive immunotherapy against human bile duct carcinoma (BDC), staphylococcal enterotoxin A (SEA) was conjugated chemically with MUSE11 monoclonal antibody (MUSE11 mAb), directed to the MUC1 antigen, using N-succinimidyl 3-(2-pyridyldithio) propionate and 2-iminothiolane HCl. Both SEA-conjugated MUSE11 mAb (SEA-MUSE11) and the F(ab')2 of MUSE11 mAb (SEA-F(ab')2) showed significant enhancement of T-LAK cell tumor neutralization for MUC1 positive-target tumor cells, even with a concentration of 0.01 microg/ml at an E:T ratio of 5:1 in vitro. In this in vitro test, MUC1-positive BDC cells were observed to attach to surrounding T-LAK cells in the presence of SEA-MUSE11 or SEA-F(ab')2. Remarkable tumor growth inhibition was observed in BDC-grafted severe combined immunodeficient mice to which 2 x 10(7) T-LAK cells preincubated with 2 microg of SEA-MUSE11 or SEA-F(ab')2, together with recombinant interleukin 2 (500 IU), were administered i.v. for 4 consecutive days, when tumor size was 5 mm in diameter. These results point to a promising adoptive immunotherapy for patients with BDC.

Animals↗
PubMed1998

Genomic DNA cloning of the region encoding nitric oxide reductase in Paracoccus halodenitrificans and a structure model relevant to cytochrome oxidase.

The structural genes for the NO reductase in Paracoccus halodenitrificans, norC, norB, and norQ were sequenced. The norC and norB encode the cytochrome c (NorC) and cytochrome b (NorB) subunits, respectively. The matured NorC (17,258 Da, 148 residues) has a binding motif (CXYCH) for heme c, which is axially coordinated by His65 and Met115. NorB (52,337 Da, 451 residues) has twelve putative transmembrane helices and the 19% sequence homology with the subunit I of cytochrome oxidase from Paracoccus denitrificans. Several histidine and glutamate residues were identified as the ligands for two hemes b and a non-heme iron in comparison with the sequence of cytochrome oxidase. The higher-order model structures constructed from the amino acid sequences of NorC and NorB showed the topology of the helical segments and the locations of the metal centers.

Bacterial Proteins↗
PubMed1998

Plasminogen Kanagawa-I, a novel missense mutation, is caused by the amino acid substitution G732R.

A new dysplasminogen, plasminogen Kanagawa-I, was identified in a healthy male with no previous thrombotic episodes. His plasma plasminogen (PLG) activity was 51.4% of that of normal pooled plasma (reference interval 70-130%) and the antigen level was 94.2% of that of normal pooled plasma (reference interval 80-150%). Nucleotide sequencing revealed a heterozygous G to A transition in exon 18, which resulted in an amino acid substitution of G732R. Both the proband's father and paternal grandfather were heterozygous for this mutation. Interestingly, the grandfather was found to be a compound heterozygote for plasminogen Kanagawa-I and Tochigi (A601T), so that his plasminogen activity and antigen level was 7.7% and 87.2% of that of normal pooled plasma, respectively. However, he has never been affected by significant thrombosis.

Adult↗
PubMed1998

Increase in the amount of celA1 protein in tobacco BY-2 cells by a cellulose biosynthesis inhibitor, 2,6-dichlorobenzonitrile.

The biochemical analysis of cellulose biosynthesis by plants has been a difficult problem due to the lack of a reliable assay procedure for cellulose synthase activity. Recently, the celA1 gene was cloned from cotton fiber, and this gene was identified from the rsw1 mutant of Arabidopsis as a catalytic subunit of cellulose synthase (Arioli et al. 1998). The cloning of these genes enables us to obtain specific antibodies against cellulose synthase. A highly specific antibody against celA1 protein was prepared and used to detect the protein from microsomal fraction of tobacco BY-2 cells. The quantity of celA1 protein in microsomal fraction of normal BY-2 cells was under the detection limit, although they contained a large quantity of cellulose. In contrast, cells habituated to 1 microM DCB (a specific inhibitor of cellulose biosynthesis) produced 1/10 of cellulose content of the normal cells, but had much more celA1 protein than the normal cells. The amount of polysaccharides in the EDTA-soluble fraction was relatively increased in habituated cells. The results suggest that celA1 protein is stabilized upon DCB binding and that the crystallization of cellulose microfibrils is inhibited simultaneously.

Amino Acid Sequence↗
PubMed1998

An activity monitor study on the sleep-wake rhythm of healthy aged people residing in their homes.

The aim of this preliminary study was to investigate sleep-wake rhythm in active, healthy elderly people residing in their usual habitat. The subjects were thirty-five male volunteers within an age range of 65-95 for 3-4 days. We measured the sleep-wake rhythm of the subjects with an Actillume which is a combined wrist activity monitor and illumination recorder. Analysis of the Actillume recording showed that 24 of the 35 subjects (69%) kept continuous activity indicating good maintenance of wakefulness with high light exposure. The mean mesor of sleep-wake rhythm, however, significantly decreased in the older subjects (aged 80-95; n=15).

Activity Cycles↗
PubMed1998

Vasoactive effects of cimicifugic acids C and D, and fukinolic acid in cimicifuga rhizome.

Vasoactive effects of cimicifugic acids A-E, fukinolic acid and fukiic acid isolated from Cimicifuga plants were investigated using rat aortic strips. Cimicifugic acid D and fukinolic acid at 3x10(-4) M caused a sustained, slowly developing relaxation of aortic strips precontracted with norepinephrine (NE) in preparations with or without endothelium. Cimicifugic acid C inversely caused a weak contraction. Cimicifugic acids A, B and E and fukiic acid showed no vasoactivity at 3x10(-4) M. Cimicifugic acids A-E and fukinolic acid are esters between cinnamic acids and the hydroxyl group of benzyltartaric acids. For the manifestation of vasoactivity in the rat aorta, it is concluded that in the cinnamic acid moiety, a caffeoyl group might be necessary for the relaxation activity, and the p-coumaroyl group causes contraction. Concentration response curves for the Ca2+-induced contracture of depolarized aortic strips with isotonic high K+ were not affected by cimicifugic acid D or fukinolic acid. The Ca2+-induced contraction of aortic strips, preincubated with 10(-6) M NE in the presence of 10(-6) M nicardipine and 0.01 mM EGTA in Ca2+-free solution, were inhibited by cimicifugic acid D and fukinolic acid. These results indicated that the inhibition by cimicifugic acid D and fukinolic acid of the NE-induced contraction of rat aorta are attributable to the suppression of Ca2+ influx from the extracellular space enhanced by NE.

Animals↗
PubMed1998

Survival of transfused CD18-positive granulocytes and their chemiluminescent response in a heifer with leukocyte adhesion deficiency.

Granulocyte transfusion (GT) was performed in an 8-month-old heifer with leukocyte adhesion deficiency (BLAD) to monitor the changes in transfused CD18-positive neutrophils and associated neutrophil chemiluminescent (CL) response in beta 2-integrin-deficient host. The CD18-positive neutrophils were detected in blood from the BLAD heifer during the first 3 hr after 2.6 x 10(9) cells were infused by GT, and disappeared by 5 hr after GT. The CL response of neutrophils was increased 1.7 to 2.8-fold in the BLAD heifer during the first 3 hr after GT, thereafter CL response decreased gradually from 2 to 5 hr after GT.

Animals↗
PubMed1998

TMC-52A to D, novel cysteine proteinase inhibitors, produced by Gliocladium sp.

New cysteine proteinase inhibitors, TMC-52A, B, C, and D, were isolated from the fungal fermentation broth. On the basis of a taxonomical study, the producing strain, F-2665, was characterized as Gliocladium sp. Spectroscopic analyses and chemical degradation have shown TMC-52A to D to be epoxysuccinyl peptides. TMC-52A to D strongly inhibited cysteine proteinases, in particular, cathepsin L with IC50 values of 13 nM, 10nM, 10nM, and 6nM, respectively.

Cathepsin L↗