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Biomedical subjects

N Sakurai

Publications and source records attributed to N Sakurai.

At least 73 records · Page 4Linked to original sources
PubMed1997

Isolation and characterization of nitric oxide reductase from Paracoccus halodenitrificans.

Nitric oxide reductase was isolated from the membrane fraction of a denitrifying bacterium, Paracoccus halodenitrificans, in the presence of n-dodecyl beta-D-maltoside. A relatively simple and effective procedure to purify NO reductase using DEAE-Toyopearl and hydroxyapatite (ceramic) chromatographies has been developed. The enzyme consisted of two subunits with molecular masses of 20 and 42 kDa associated with the c-type heme and two b-type hemes, respectively. The optical and magnetic circular dichroism (MCD) spectra of the oxidized (as isolated) and reduced enzymes indicated that the heme c is in the low-spin state and the hemes b are in the high- and low-spin states. The EPR spectrum also showed the presence of the split high-spin component (g perpendicular = 6.6, 6.0) and two low spin components (gz,y,x = 2.96, 2.26, 1.46, gz = 3.59). Although the presence of an extra iron was suggested from atomic absorption spectroscopy, a non-heme iron could not be detected by colorimetric titrations using ferene and 2-(5-nitro-2-pyridylazo)- 5-(N-propyl-N-sulfopropylamino)phenolate (PAPS). One of the extra signals at g = 4.3 and 2.00 might come from a non-heme iron, while they may originate from an adventitious iron and a certain nonmetallic radical, respectively. When CO acted on the reduced enzyme, both of the low-spin hemes were not affected, and when NO acted on the reduced enzyme, the optical and MCD spectra were of a mixture of the oxidized and reduced enzymes. Consequently, the reduction of NO was supposed to take place at the high-spin heme b. The heme c and the low-spin heme b centers were considered to function as electron mediators during the intermolecular and intramolecular processes.

Circular Dichroism↗
PubMed1997

Overexpression in Escherichia coli of chemically synthesized gene for active 0.19 alpha-amylase inhibitor from wheat kernel.

A synthetic gene encoding 0.19 alpha-amylase inhibitor (alpha-AI) from wheat kernel was obtained by enzymatic assembly of 18 oligodeoxynucleotides which were chemically synthesized. The synthetic gene was introduced into vector pET15b for expression in Escherichia coli BL21(DE3) under the control of T7 promoter. However, in SDS-PAGE and Western blotting analyses of the E. coli cell lysate, the expression product could not be detected. Expression analysis for various partially deleted gene fragments suggested that the putative hairpin-like structure of mRNA in the 5'-terminal coding region might interrupt efficient expression. When the hairpin structure was eliminated by using degenerate codons, the resulting gene could be overexpressed in E. coli. Although the gene product was accumulated in an insoluble fraction as inclusion bodies, its inhibitory activity could be recovered by solubilization with 8 M urea, followed by refolding through two successive steps of dialysis at alkaline pH. After purification, the recombinant 0.19 alpha-AI showed the same characteristics as the authentic inhibitor in terms of N-terminal amino acid sequence, peptide mapping on reverse-phase HPLC, far-UV circular dichroism (CD) spectrum and have inhibition of human salivary alpha-amylase. Thus, we have established an overexpression system in E. coli for active recombinant 0.19 alpha-AI.

Amino Acid Sequence↗
PubMed1997

Purification and characterization of wall-bound exo-1,3-beta-D-glucanase from barley (Hordeum vulgare L.) seedlings.

A beta-D-glucanase activity hydrolyzing 1,3:1,4-beta-D-glucan was released from the cell walls of barley by 3 M LiCl treatment. It was purified by sequential cation-exchange, gel-filtration and hydrophobic chromatography. The molecular mass of the glucanase was 66 kDa as determined by SDS-polyacrylamide gel electrophoresis. Sequence determination of the first thirty amino acids of the N-terminus revealed a high homology of this enzyme to the Pseudomonas 1,4-beta-D-glucosidase (56.5%). The purified beta-D-glucanase has a pH optimum at 5.0, and hydrolyzes oligosaccharides containing beta-D-1,3 or beta-D-1,4 linkage. The glucanase showed maximum hydrolytic activity toward laminaritetraose, the rate being about two times that of cellotetraose and about four times that of gentiobiose. Polysaccharides such as lichenan, 1,3:1,4-beta-D-glucan (from barley), laminarin and pustulan are also hydrolyzed, but not carboxylmethyl-curdlan, carboxymethyl-cellulose, xyloglucan and maltose. The purified beta-D-glucanase yielded monomeric glucose from laminarihexaose, and exhibited characteristics of an exo-1,3-beta-D-glucanase (EC 3.2.1.58). The activity and biochemical characteristics of this enzyme suggest that it is an exo-1,3-beta-D-glucanase involved in the rapid turnover of 1,3:1,4-beta-D-glucan in barley cell walls during seedling growth.

Amino Acid Sequence↗
PubMed1997

Genomic diversity in the pfoA region of the theta-toxin-deficient strains of Clostridium perfringens.

The genomic structure of the pfoA-colA region in six theta-toxin-deficient strains of Clostridium perfringens was examined by Southern hybridization using the pfoR, pfoA, pbg, arcABDC and colA genes, encoding regulator for pfoA, theta-toxin, beta-galactosidase, arginine metabolism enzymes and kappa-toxin, respectively, as gene probes. It is suggested that the productivity of theta-toxin in these strains is diverse because of the multiple genetic backgrounds including single deletion of pfoA, large deletion of the pfoA-colA region and the putative point mutations.

Bacterial Toxins↗
PubMed1997

[Long-term follow-up of the Le Duc-Camey antireflux technique].

BACKGROUND: An effective ureteroileal reimplantation without reflux and obstruction is indispensable for lower urinary reconstruction or diversion using an ileal segment. Based on our earlier experience of afferent nipple valve malfunction of the Kock pouch, we performed Le Duc-Camey antireflux ureteroileal implantation. In the present study, we evaluated the long-term outcome of the Le Duc-Camey technique on the upper urinary tract in patients with a postoperative observation period of 2 years or more. PATIENTS AND METHODS: From 1989 to 1994, 39 patients (73 ureters) underwent Le Duc-Camey technique for prevention of reflux of a Kock pouch. In 28 patients the ileal reservoir was connected to the skin for cutaneous urinary diversion, and in 11 it was connected to the urethra for lower urinary reconstruction. The ureters were implanted into the reservoir through the anterior wall window of the reservoir. In most patients, excretory urography (DIP) was performed once a year after the first year. Retrograde cystography or voiding cystourethrography was performed within 12 months postoperatively. After the first year, retrograde study was performed once during the follow-up period in the majority of the patients. The mean follow-up period was 44.8 months (range 24-71 months). RESULTS: There was no urinary tract dilatation in 68 renal units (93.1%), while slight dilatation was noted in 3 (4.1%), moderate dilatation in 1 (1.4%), and marked dilatation due to stenosis of ureteroileal anastomosis in 1 (1.4%). Retrograde studies revealed absence of reflux in all patients. Another complication that related to this procedure was not observed in any patients. CONCLUSION: We conclude that Le Duc-Camey technique is effective in reflux prevention and protection of the upper urinary tract, and recommend it for use with detubularized ileal reservoir.

Adult↗
PubMed1997

[Improvement of inter-assay for the standardization of PT and TT--clinical significance of local standardization method].

We performed a nationwide Inter-assay including 112 laboratories for the standardization of prothrombin time (PT) and thrombotest (TT). The data were expressed as seconds, percentile and INR. INR was expressed by 2 methods; Method I (conventional method): INR was expressed using each ISI assigned for reagent or reagent-instrument at the respective laboratories and Method II (local standardization method): INR was expressed using each reference curve created with INR assigned standard plasmas at the respective laboratories. (1) Sample distribution of PT as well as TT was the smallest with the data expressed by Method II followed by Method I and then by percentile. The data expressed by seconds was widely distributed and not useful for the standardization of PT and TT. (2) Even the sample distribution obtained by Method II was dependent on the different ISI of the reagents, as it was found that the larger the ISI of the reagents, the wider the distribution of data. (3) The difference between PT and TT of each test plasma was analysed by t-test. It was found that the difference was insignificant when both data were expressed by Method II, but significant when expressed by Method I, suggesting that PT and TT were interchangeable with the use of Method II. (4) Sample distribution of percentile expression and INR with the use of method II was compared. It was revealed that the sample distribution of INR was smaller than that of percentile. It was concluded that INR expressed by the local standardization method was most useful for the standardization of PT and TT.

Humans↗
PubMed1997

Repair of bone defect in primate mandible using a bone morphogenetic protein (BMP)-hydroxyapatite-collagen composite.

The purpose of this study was to evaluate a hydroxyapatite (HA)-collagen (Col)-bone morphogenetic protein (BMP) composite as an osteoinductive bone substitute. Partially-purified BMP from bovine bone was mechanically mixed with highly purified type I collagen from calf dermis and then subsequently mixed with pure synthetic hydroxyapatite granules or block. The HA-Col-BMP composite, or the HA-Col composite as a control, was implanted in a surgically-induced mandible bone defect (6 x 7 x 10 mm) in an adult Japanese monkey. The mandible was excised three months after implantation and studied histologically. The BMP-containing implant induced much more new bone than the control implant in all experimental animals of each group, with either HA granules or HA block. Newly formed bone was attached tightly to HA and infiltrated deeply into the pores of the HA of the BMP-containing implant, while fibrous tissue existed between the host bone and HA in the control implant. Thus, we conclude that an HA-Col-BMP composite could be a superior biomaterial for a bone substitute.

Animals↗
PubMed1996

MUC1-specific targeting immunotherapy with bispecific antibodies: inhibition of xenografted human bile duct carcinoma growth.

For the purpose of establishing a new adoptive immunotherapy for bile duct carcinoma (BDC), we synthesized two bispecific antibodies (BsAbs), MUC1 x CD3 BsAb constructed with MUSE11 (anti-MUC1 tumor antigen) and OKT-3 (anti-CD3), and MUC1 x CD28 BsAb constructed with MUSE11 and 15E8 (anti-CD28) antibodies. These two BsAbs reacted well with both MUC1-positive target tumor cells and effector lymphokine-activated killer (LAK) cells. Investigation of in vitro cytotoxicity [3-(4,5-dimethylthiazo-2-yl)-2,5-diphenyltetrazolium bromide assay] revealed that the MUC1 x CD3 BsAb could antigen-specifically enhance the cytotoxicity of LAK cells. Addition of the two BsAbs (MUC1 x CD3 BsAb plus MUC1 x CD28 BsAb) in vitro resulted in a 60% cytotoxicity, similar to that obtained with BsAb (MUC1 x CD3) alone. Interleukin 12-induced LAK cells demonstrated far greater cytotoxicity (50%) than their interleukin 2-induced counterparts (LAK cells), and this was also enhanced by the BsAbs. When 2 x 10(7) LAK cells sensitized with both kinds of BsAbs were administered four times i.v. to BDC-grafted severe combined immunodeficient mice (tumor size 5 mm in diameter), inhibition of tumor growth was observed. Thus, BsAb-LAK therapy for control of BDC warrants clinical trials.

Animals↗
PubMed1996

Primary structure of mannuronate lyases SP1 and SP2 from Turbo cornutus and involvement of the hydrophobic C-terminal residues in the protein stability.

The complete amino acid sequences of two isoforms, SP1 and SP2, of mannuronate lyase from a wreath shell, Turbo cornutus, were determined to elucidate amino acid residues responsible for causing the more stable protein conformation of SP2. The sequences of the two isoforms were identical except for two hydrophobic C-terminal amino acid residues of SP2, Ile and Leu, which were additionally attached to Thr of the C-terminal residue of SP1 (253 residues in total). The molecular weight of SP2 was calculated to be 28,912 from the amino acid sequence data. Two disulfide bond cross-linkages were found to be between 106 and 115 and between 145 and 150, and a partially buried single SH group was located at 236. A carbohydrate chain that consisted of 3 GlcNAc, 3 Fuc, and 1 Man was anchored on Asn-105 in a typical carbohydrate-binding motif of Asn-X-Ser. This is the first evidence of the primary structure of mannuronate lyase, and no significant homology of the amino acid sequence among other proteins was found. The C-terminal truncated SP2, which was produced by digestion with carboxypeptidase Y and corresponded structurally to SP1, showed a thermal stability identical to that of SP1. These results indicate that the higher stability of SP2 than SP1 arises from the presence of the C-terminal two hydrophobic amino acid residues.

Amino Acid Sequence↗
PubMed1996

Complete sequence and organization of the Serratia marcescens biotin operon.

The nucleotide sequence of the biotin (bio) operon of wild-type Serratia marcescens Sr41 was determined. Five ORFs were identified to encode BioA (7,8-diaminopelargonic acid aminotransferase), BioB (biotin synthase), BioF (7-keto-8-aminopelargonic acid synthase), BioC (an enzyme catalysing the synthesis of pimeloyl-CoA) and BioD (dethiobiotin synthase), in this order. The operon was deduced to be transcribed divergently to the left into bioA and to the right into the bioBFCD genes. The promoters and a common predicted operator for both bioA and bioBFCD genes were located between the bioA and bioB genes. The predicted amino acid sequences of these enzymes were similar to the sequences of the corresponding enzymes of Escherichia coli. Analysis of expression of the lacZ structural gene fused with the bioA and bioB promoters revealed that the biotin operon was subject to biotin-mediated feedback repression.

Amino Acid Sequence↗
PubMed1996

Effects of a prescription of Chinese herbal medicine on snake venom-induced nephropathy in mice.

A prescription of Chinese herbal medicine, tentatively named P-19, was examined for its inhibitory effect and its mechanism using an experimental model of nephropathy induced by purified snake venom proteinase, Ac(1)-proteinase (Ac(1)-P). The treated mice were injected with 0.1 ml of crude extract of P-19 intraperitoneally every other day beginning 2 d before to 1 week after the injection of Ac(1)-P. The non-treated mice were injected with saline instead of the medicine P-19. The physiological condition and histopathological observation of the mice at one week after Ac(1)-P injection were better in the treated group than in the non-treated group. This indicates that P-19 inhibited the production of glomerular lesions induced in mice by Ac(1)-P. The physiological condition and histopathological changes in the mice were better with P-19 treatment than with P-3 treatment. Differences in the mechanism of action between the crude extract of P-3 and P-19 are not only in diuretic action but also in the changes in the glomerular basement membrane. On the basis of spectrophotometric studies, phenolic carboxylates were confirmed to be contained in the crude extract of P-19, having a different chemical structure of caffeic acid, which is the effective component in P-3. Immunohistochemical observation revealed a difference between the groups. In the non-treated mice, deposits of the venom were clearly observed in the glomerular tuft and Bowman's capsule, corresponding to the histopathological changes, within 2.5 min after the injection of Ac(1)-P. In the treated mice, the deposits were indistinct in the Bowman's capsule. The difference was considered to be caused by changes in the glomerular basement membrane after P-19 treatment.

Agkistrodon↗
PubMed1996

[Chemical constituents of original plants of Cimicifugae rhizoma in Chinese medicine].

Cimicifugae Rhizoma have been used as an anti-inflammatory, analgesic and antipyretic remedy in the traditional Chinese medicines. Many 9,19-cyclolanostane glycosides have been isolated from Cimicifuga and related genera. Two biogenetically key compounds, acetylshengmanol xyloside and cimicifugoside H-1, were isolated and their chemical structures were elucidated by our group. The former compound seems to be the parent component of the other glycosides such as cimigenol xyloside from C. dahurica, C. iaponica and C. acerina. The latter glycoside, cimicifugoside H-1 was isolated together with cimicifugosides H-2-H-6 from commercial Cimicifugae Rhizoma. They are novel glycosides having a hydroxyl group ay C-11, and cimicifugosides H-3, H-4 and H-6 were trinor-triterpenol glycosides. Cimicifugoside H-1 changed into H-2, H-3 and H-4 under acidic or alkaline conditions. In this review, the structure elucidation of the above glycosides and their chemical transformation into other Cimicifuga glycosides are described.

Animals↗
PubMed1996

[Efficacy of granisetron rescue therapy for nausea and vomiting induced by cancer chemotherapies in urogenital malignant tumor].

In this study, the usefulness of granisetron rescue therapy for nausea and vomiting induced by cancer chemotherapies, including CDDP, was examined. Granisetron was given to twenty patients with urogenital malignant tumor by iv infusion for thirty minutes after the onset of nausea or vomiting. Nausea disappeared in 15 out of 20 patients (75%), 8 of whom (40%) experienced its disappearance while the granisetron was being administered. Vomiting was perfectly controlled in 5 out of 20 patients (25%) for 24 hours after the granisetron administration. No adverse event seemingly due to granisetron was observed. The result of this study confirmed the speedy effect granisetron on nausea induced by cancer chemotherapy including CDDP, but it stopped short of demonstrating sufficient efficacy for vomiting. Prophylactic use, therefore, seems more desirable in view of the patient's QOL, when a highly emetogenic anti-tumor drug, such as CDDP, is used.

Adult↗
PubMed1995

Instability of the mutated biotin operon plasmid in a biotin-producing mutant of Serratia marcescens.

The growth of a d-biotin-producing strain of Serratia marcescens (SB412) was strongly inhibited by the introduction of pLGM412, a low-copy-number plasmid containing the complete biotin (bio) operon derived from SB412, whereas the wild-type strain was not inhibited by the plasmid. SB412 carrying pLGM412 was genetically unstable; large colonies appeared spontaneously from the background small colonies. When the plasmids from the large colonies were transformed into the SB412 host, all of the resultant transformants showed a large-colony phenotype, suggesting that the large-colony phenotype is due to mutations in the plasmid-born bio genes. Some of these plasmids were structurally altered and the others were not. Furthermore, the structurally altered plasmids were classified into a deleted and an elongated type. All of the mutated pLGM412 derivatives reduced or lacked the bio gene expression, indicating that the high expression of bio gene(s) causes the growth inhibition. By subcloning experiments, biotin synthase (the bioB gene product) was responsible for the growth inhibition.

Bacterial Proteins↗
PubMed1995

Gastrointestinal dysfunction in Parkinson's disease detected by electrogastroenterography.

Electrogastroenterography (EGEG) is a method to record electrical activities of the stomach and the intestine using skin electrodes. We investigated whether this method could be used to detect gastrointestinal dysfunction in patients with idiopathic Parkinson's disease. EGEG recordings were done with ten patients with idiopathic Parkinson's disease and ten control subjects before and after a meal. The patients showed changes in EGEG that were markedly similar to those of acute stage of vagotomized patients reported previously. Patients' increase rate in amplitude of gastric activity after the meal (median: 1.19) was significantly (P < 0.05, Mann-Whitney test) smaller than that of the controls (median: 2.84), and normal temporal frequency decrease of gastric activity after the meal was not seen in the patient group. These results suggest vagal nerve dysfunction of patients with Parkinson's disease, though other possibilities could not be denied. EGEG may be useful to assess patients' gastrointestinal dysfunction though we need further study to elucidate the relation between pathophysiology of their symptoms and EGEG findings.

Aged↗
PubMed1995

Therapeutic drama activity for the cognitively impaired elderly in a nursing home.

The use of drama as a nightly activity for the cognitively impaired elderly patients at the Sakubara skilled nursing home commenced in 1989. Although it was started for the pleasure of the patients, and not for a therapeutic purpose, positive behavioral changes were observed. The therapeutic effects of the activity were measured and reported. Based on our 7-year experience and research, the definition, goals, structure, and conceptual framework of therapeutic drama activity were developed; the skills used in this activity and its cultural background are discussed. Finally, the philosophical background of the nursing home that promotes the therapeutic effects is presented.

Aged↗