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Biomedical subjects

N Sakurai

Publications and source records attributed to N Sakurai.

At least 37 records · Page 2Linked to original sources

[A case of pseudomyxoma peritonei treated successfully with intraperitoneal administration of CBDCA and etoposide, followed by local delivery of dextran with CDDP during surgery].

A 77-year-old woman was diagnosed as having pseudomyxoma peritonei originating in the ovary or appendix. Low-molecular dextran and CDDP 70 mg was administered directly into the peritoneal cavity, followed by initial exploratory laparotomy with total abdominal hysterectomy, bilateral salpingo-oophorectomy, appendectomy, and omentectomy. During surgery, an implantable port system was positioned to facilitate repeated intraperitoneal administration. Dextran was used with mucolytic therapy and augmentable efficacy of drug administered simultaneously. Within three weeks after surgery, four courses of intraperitoneal CBDCA 300 mg and etoposide 100 mg were administered at four-week intervals. The patient has remained in good general condition with no sign of recurrence for one year. This chemotherapy regimen has minimum side effects and seems effective in maintaining quality of life.

Aged↗

[Study of sesquiterpene alkaloids from Tripterygium wilfordii Hook.f].

AIM: To study the chemical composition of Tripterygium wilfordii Hook.f. METHODS: Column chromatography was used to separate the chemical constituents. UV, IR, MS, HRMS, 1HNMR, 13CNMR (COM and OFR), 1H-1H COSY, 1H-13C COSY, 2D-NOESY and 1H-13C COLOC were used to determine the structures of the isolated constituents. RESULTS: Two sesquiterpene alkaloids were isolated and their structures were elucidated as euonine and wilfordconine on the basis of spectral evidence. CONCLUSION: Wilfordconine, a new sesquiterpene alkaloid, was shown to be immunosuppressive.

Alkaloids↗

Crystal structure of NADH-dependent ferredoxin reductase component in biphenyl dioxygenase.

Oxidative biodegradation of aromatic compounds by bacteria usually begins with hydroxylation of the aromatic ring by multi-component dioxygenases like benzene dioxygenase, biphenyl dioxygenase, and others. These enzymes are composed of ferredoxin reductase, ferredoxin, and terminal oxygenase. Reducing equivalents that originate from NADH are transferred from ferredoxin reductase to ferredoxin and, in turn, to the terminal oxygenase, thus resulting in the activation of a dioxygen. BphA4 is the ferredoxin reductase component of biphenyl dioxygenase from Pseudomonas sp. strain KKS102. The amino acid sequence of BphA4 exhibits significant homology with the putidaredoxin reductase of the cytochrome P450cam system in Pseudomonas putida, as well as with various other oxygenase-coupled NADH-dependent ferredoxin reductases (ONFRs) of bacteria. To date, no structural information has been provided for the ferredoxin reductase component of the dioxygenase systems. In order to provide a structural basis for discussing the mechanism of electron transport between ferredoxin reductase and ferredoxin, crystal structures of BphA4 and its NADH complex were solved. The three-dimensional structure of BphA4 is different from those of ferredoxin reductases whose structures have already been determined, but adopts essentially the same fold as the enzymes of the glutathione reductase (GR) family. Also the three-dimensional structure of the first two domains of BphA4 adopts a fold similar to that of adrenodoxin reductase (AdR) in the mitochondrial cytochrome P450 system. Comparing the amino acid sequence with what is known of the three-dimensional structure of BphA4 strongly suggests that the other ONFRs have secondary structural features that are similar to that of BphA4. This analysis of the crystal structures of BphA4 suggests that Lys53 and Glu159 seem to be involved in the hydride transfer from NADH to FAD. Since the amino acid residues around the active site, some of which seem to be important to electron transport, are highly conserved among ONFRs, it is likely that the mechanism of electron transport of BphA4 is quite applicable to other ONFRs.

Amino Acid Sequence↗

Tau neutrinos favored over sterile neutrinos in atmospheric muon neutrino oscillations.

The previously published atmospheric neutrino data did not distinguish whether muon neutrinos were oscillating into tau neutrinos or sterile neutrinos, as both hypotheses fit the data. Using data recorded in 1100 live days of the Super-Kamiokande detector, we use three complementary data samples to study the difference in zenith angle distribution due to neutral currents and matter effects. We find no evidence favoring sterile neutrinos, and reject the hypothesis at the 99% confidence level. On the other hand, we find that oscillation between muon and tau neutrinos suffices to explain all the results in hand.

Journal Article↗

Antitumor-promoting effects of cyclic diarylheptanoids on Epstein-Barr virus activation and two-stage mouse skin carcinogenesis.

Eleven cyclic diarylheptanoids were screened as potential antitumor promoters by examining the ability of the compounds to inhibit Epstein-Barr virus early antigen activation (induced by 12-O-tetradecanoylphorbol-13-acetate) in Raji cells. 13-Oxomyricanol and myricanone showed the highest activity and also exhibited remarkable inhibitory effects on mouse skin tumor promotion in an in vivo two-stage carcinogenesis test. These data suggest that certain diarylheptanoids might be valuable antitumor promoters and/or chemopreventors.

9,10-Dimethyl-1,2-benzanthracene↗

Functional Fv fragment of an antibody specific for CD28: Fv-mediated co-stimulation of T cells.

The most predominant co-stimulation pathway, which is critical for T cell activation and proliferation, is the CD28-B7 pathway. The anti-CD28 monoclonal antibody (mAb) also provides a co-stimulatory signal to T cells. In order to construct a functional Fv fragment (complex of VH and VL domains) of anti-CD28 antibody using a bacterial expression system, cDNA encoding the variable regions of immunoglobulin from 15E8 hybridoma cells was cloned and expressed in Escherichia coli. The Fv fragment was obtained as a soluble protein from the periplasmic fraction and showed a binding pattern similar to parental IgG. The Fv fragment induced proliferation of peripheral blood mononuclear cells in the presence of anti-CD3 or anti-CD2 mAb and enhanced anti-tumor activity of anti-MUC1x(anti)-CD3 bispecific antibody when tested with lymphokine-activated killer cells with T cell phenotype. Thus, the anti-CD28 Fv fragment will be promising not only for the study of co-stimulation, but also for cancer immunotherapy.

Amino Acid Sequence↗

Effects of acids on neural activity elicited by other taste stimuli in the rat Chorda tympani.

The purpose of this study is whether the gustatory neural response of taste cell to a binary mixture with threshold concentration of acid becomes synergistic or antagonistic can be estimated from the whole chorda tympani (CT) nerve in the rat. The present data demonstrate that acids are synergistic enhancer for sugars, and suppressor for NaCl and QHCl, but no effect to glycine and alanine. These results suggest that the acid was modifying the interaction of the other stimulus with its transduction mechanism.

Acids↗

Pancreatic arteriovenous malformation presenting as cysts: Doppler sonographic diagnosis.

A 50-year-old man presented with multiple lesions resembling cysts throughout the pancreas on gray-scale sonography. Pulsatile blood flow detected within the lesions on color Doppler sonography led to the diagnosis of pancreatic arteriovenous malformation (PAVM). Most previously reported PAVMs have been described as having a poorly defined, reticular, or mosaic-like hypoechoic appearance on gray-scale sonography; the cystic appearance of the PAVM seen in our case is unusual.

Arteriovenous Malformations↗

Functional construction of the anti-mucin core protein (MUC1) antibody MUSE11 variable regions in a bacterial expression system.

A bacterial expression system for the variable region fragments (Fvs) of the anti-MUC1 tumor antigen antibody MUSE11 has been constructed. The Fv fragment showed binding specificity toward TFK-1 cells, with slightly reduced affinity compared to its parent IgG. The single-chain Fv fragment was arranged in two orders, VH-linker-VL and VL-linker-VH. However, linking the regions with a flexible peptide linker (GGGGS)(3) or with a shorter linker (GGGGS) led to a dramatic decrease in the biological activity toward the target antigen in both arrangements, suggesting that the MUSE11 antibody loses its activity when the domains are linked with polypeptide linkers. These results indicate that the variable region domains of the anti-MUC1 antibody MUSE11 have specificity only in the Fv form, and that linking the domains strongly reduces the association with its target antigen. Gel filtration analysis indicates that the scFv has a dimeric structure, suggesting that the inactivation of MUSE11 scFv is due to unfavorable intermolecular associations of the scFv chains. To our knowledge, this is the first report of a significant reduction in affinity caused by linking the variable domains in both arrangements, i.e., VH-VL and VL-VH.

Animals↗

Auxin-induced elongation growth and expressions of cell wall-bound exo- and endo-beta-glucanases in barley coleoptiles.

When auxin stimulates rapid cell elongation growth of cereal coleoptiles, it causes a degradation of 1,3:1,4-beta-glucan in hemicellulosic polysaccharides. We examined gene expressions of endo-1,3:1,4-beta-glucanase (EI) and exo-beta-glucanase (ExoII), of which optimum pH are about 5, and molecular distribution of hemicellulosic polysaccharides in barley (Hordeum vulgare L.) coleoptile segments treated with or without IAA. IAA (10(-5) M) stimulated the gene expression of EI, while it did not affect that of ExoII. IAA induced gene expression of EI after 4 h and increased wall-bound glucanase activity after 8 h. The molecular weight distribution of hemicellulosic polysaccharides from coleoptile cell walls was shifted to lower molecular weight region by 2 h of IAA treatment. Fusicoccin (10(-6) M) mimicked IAA-induced elongation growth and the decrease in molecular weight of hemicellulosic 1,3:1,4-beta-glucan of coleoptiles in the first 4 h, but it did not promote elongation growth thereafter. These facts suggest that acidification of barley cell walls by IAA action enhances pre-existing cell wall-bound glucanase activity in the early first phase of IAA-induced growth and the late second phase involves the gene expression of EI by IAA.

Blotting, Northern↗

Construction of a diabody (small recombinant bispecific antibody) using a refolding system.

Diabodies are the recombinant bispecific antibodies (BsAbs), constructed from heterogeneous single-chain antibodies. Usually, diabodies have been prepared from bacterial periplasmic fraction using a co-expression vector (i.e. genes encoding two chains were tandemly located under the same promoter). Some diabodies, however, cannot be expressed as a soluble material owing to inclusion body formation, which limits the utilization of diabodies in various fields. Here we report an improved method for the construction of diabodies using a refolding system. As a model, a bispecific diabody binding to adenocarcinoma-associated antigen MUC1 and to CD3 on T cells was studied. One chain consisted of a VH specific for MUC1 linked to a VL specific for CD3 with a short polypeptide linker (GGGGS). The second was composed of a VL specific for MUC1 linked to a VH specific for CD3. The two hetero scFvs were independently obtained from intracellular insoluble fractions of Escherichia coli, purified, mixed stoichiometrically (at an equivalent molar ratio of 1:1) and refolded. The refolded two hetero scFv has a hetero-dimeric structure, with complete specificity for both target cells [i.e. MUC1 positive cells and CD3 positive lymphokine-activated killer cells with a T cell phenotype (T-LAK)]. Evaluation of the in vitro efficacy of T-LAK with the diabody by growth inhibition assay of cancer cells demonstrated maximum growth inhibition of cancer cells to reach approximately 98% at an effector:target ratio (E:T ratio) of 10, almost identical with that with anti-MUC1xanti-CD3 chemically synthesized BsAbs (c-BsAbs). This is the first report of the construction of a diabody using a refolding system.

Antibodies, Bispecific↗

Protection of porcine oocytes against apoptotic cell death caused by oxidative stress during In vitro maturation: role of cumulus cells.

The present study was conducted to examine the protective effect of cumulus cells on oocyte damage caused by reactive oxygen species (ROS), generated by the hypoxanthine-xanthine oxidase (XOD) system, during in vitro maturation of porcine oocytes. Cumulus-oocyte complexes (COCs) and cumulus-denuded oocytes (DOs) were cultured for 44 h in NCSU37 supplemented with cysteine, gonadotropins, 10% porcine follicular fluid, and hypoxanthine in the presence or absence of XOD. DNA cleavage and damage were analyzed using the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) method and single cell microgel electrophoresis (comet) assay, respectively, and caspase-3 activity and glutathione (GSH) content were measured in each experimental group. Exposure of DOs to ROS resulted in meiotic arrest and the increase of degenerated oocytes. These degenerated DOs underwent apoptosis, as shown by the TUNEL-positive reaction within their germinal vesicles and the activation of caspase-3. The length of DNA migration in DOs treated with XOD was significantly longer than that of untreated DOs (P: < 0.05). However, irreparable cell damage caused by ROS was not observed in COCs, and no difference was observed in the caspase-3 activity of both COCs treated with and without XOD. A significantly (P: < 0.05) high level of GSH was found in COCs after 44 h of culture, compared with that of oocytes freshly isolated from their follicles, whereas GSH content in DOs markedly decreased after treatment with or without XOD. These findings suggest that cumulus cells have a critical role in protecting oocytes against oxidative stress-induced apoptosis through the enhancement of GSH content in oocytes.

Animals↗

Oleanane acid from Myrica cerifera.

From the twigs of Myrica cerifera L. (Myricaceae), a new oleanane triterpenic acid named myrica acid was isolated along with myricalactone and several other known constituents. The structure of the acid was determined as 3beta-hydroxy-1-oxoolean-11,13(18)-dien-28-oic acid on the basis of chemical and spectral evidence.

Molecular Structure↗

[Metachronous bilateral primary malignant lymphoma of the testis: a case report].

A 72-year-old man was referred to our department with the chief complaint of painless swelling of the left scrotum in May 1997. Left high orchiectomy was performed under the diagnosis of primary testicular tumor. Histological findings revealed non-Hodgkin's lymphoma (NHL) of diffuse, mixed type, B cells. No evidence of tumors in any other site was detected by further examinations. About 3 years and a month earlier, he had undergone right high orchiectomy and postoperative radiotherapy (inverted Y irradiation) and chemotherapy (CHOP 5 cycles) for a right testicular tumor whose histological findings were NHL of diffuse, large cell type, B cells. Metachronous bilateral primary malignant lymphoma of the testis is very rare and we discussed each tumor origin by using IgH gene (IgJHDNA) rearrangement as a tumor specific marker of B cell lineage malignant lymphoma. We discussed the clonality of IgJHDNA rearrangement using polymerase chain reaction (PCR) in each paraffin fragment diagnosed pathologically as NHL of B cell origin.

Aged↗

Myrothecium verrucaria bilirubin oxidase and its mutants for potential copper ligands.

Bilirubin oxidase (EC:1.3.3.5) purified from a culture medium of Myrothecium verrucaria MT-1 (authentic enzyme) catalyzes the oxidation of bilirubin to biliverdin in vitro and recombinant enzyme (wild type) was obtained by using an overexpression system of the bilirubin oxidase gene with Aspergillus oryzae harboring an expression vector. The absorption and ESR spectra showed that both bilirubin oxidases are multicopper oxidases containing type 1, type 2, and type 3 coppers similar to laccase, ascorbate oxidase, and ceruloplasmin. Site-directed mutagenesis has been performed for the possible ligands of each type of copper. In some mutants, Cys457 --> Val, Ala, His94 --> Val, and His134.136 --> Val, type 1 and type 2 copper centers were perturbed completely and the enzyme activity was completely lost. Differing from the holoenzyme, these mutants showed type 3 copper signals. However, the optical and magnetic properties characteristic of type 1 copper were retained even by mutating one of the type 1 copper ligands, i.e., a mutant, Met467 --> Gly, showed a weak but apparent enzyme activity. A double mutant His456.458 --> Val had only type 1 Cu, showing a blue band at 600 nm (epsilon = 1.6 x 10(3)) and an ESR signal with very narrow hyperfine splitting (A parallel = 7.2 x 10(-)3 cm-1). Since the type 2 and type 3 coppers are not present, the mutant did not show enzyme activity. These results strongly imply that the peculiar sequence in bilirubin oxidase, His456-Cys457-His458, forms an intramolecular electron-transfer pathway between the type 1 copper site and the trinuclear center composed of the type 2 and type 3 copper sites.

Amino Acid Sequence↗

SEA-scFv as a bifunctional antibody: construction of a bacterial expression system and its functional analysis.

A SEA-antibody single chain Fv (SEA-scFv) fusion protein was produced by bacterial expression system in this study. SEA-scFv has both staphylococcal enterotoxin A (SEA) effects and antibody activity directed at the epithelial mucin core protein MUC1, a cancer associated antigen. It was expressed mostly in the cytoplasm as an insoluble form. The gene product was solubilized by guanidine hydrochloride, refolded by conventional dilution method, and purified using metal-chelating chromatography. The resulting SEA-scFv fusion protein preparation was found to react with MUC1 and MHC class II antigens and had the ability to enhance cytotoxicity of lymphokine activated killer cells with a T cell phenotype against a human bile duct carcinoma cell line, TFK-1, expressing MUC1. This genetically engineered SEA-scFv fusion protein promises to be an important reagent for cancer immunotherapy.

Antibodies, Bispecific↗