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51.
Nishino Y Yamamoto K Kimura S Kikuchi A Shiro Y Iyanagi T 《Archives of biochemistry and biophysics》2007,465(1):254-265
The object of this study was to clarify the mechanism of electron transfer in the human endothelial nitric oxide synthase (eNOS) reductase domain using recombinant eNOS reductase domains; the FAD/NADPH domain containing FAD- and NADPH-binding sites and the FAD/FMN domain containing FAD/NADPH-, FMN-, and a calmodulin-binding sites. In the presence of molecular oxygen or menadione, the reduced FAD/NADPH domain is oxidized via the neutral (blue) semiquinone (FADH(*)), which has a characteristic absorption peak at 520 nm. The FAD/NADPH and FAD/FMN domains have high activity for ferricyanide, but the FAD/FMN domain has low activity for cytochrome c. In the presence or absence of calcium/calmodulin (Ca(2+)/CaM), reduction of the oxidized flavins (FAD-FMN) and air-stable semiquinone (FAD-FMNH(*)) with NADPH occurred in at least two phases in the absorbance change at 457nm. In the presence of Ca(2+)/CaM, the reduction rate of both phases was significantly increased. In contrast, an absorbance change at 596nm gradually increased in two phases, but the rate of the fast phase was decreased by approximately 50% of that in the presence of Ca(2+)/CaM. The air-stable semiquinone form was rapidly reduced by NADPH, but a significant absorbance change at 520 nm was not observed. These findings indicate that the conversion of FADH(2)-FMNH(*) to FADH(*)-FMNH(2) is unfavorable. Reduction of the FAD moiety is activated by CaM, but the formation rate of the active intermediate, FADH(*)-FMNH(2) is extremely low. These events could cause a lowering of enzyme activity in the catalytic cycle. 相似文献
52.
Saito K Dan H Masuda K Katsu T Hanioka N Yamamoto S Miyano K Yamano S Narimatsu S 《Chirality》2007,19(7):550-558
We examined the enzymatic function of recombinant CYP2C19 in enantiomeric hexobarbital (HB) 3'-hydroxylation, and searched the roles of amino acid residues, such as Phe-100, Phe-114, Asp-293, Glu-300, and Phe-476 of CYP2C19 in the stereoselective HB 3'-hydroxylation, using a yeast cell expression system and site-directed mutagenesis method. CYP2C19 wild-type exerted substrate enantioselectivity of (R)-HB>(S)-HB and metabolite diastereoselectivity of 3'(R)<3'(S) in 3'-hydroxylation of HB enantiomers. The substitution of Asp-293 by alanine failed to yield an observable peak at 450 nm in its reduced carbon monoxide-difference spectrum. CYP2C19-E300A and CYP2C19-E300V with alanine and valine, respectively, in place of Glu-300 exerted total HB 3'-hydroxylation activities of 45 and 108%, respectively, that of the wild-type. Interestingly, these two mutants showed substrate enantioselectivity of (R)-HB<(S)-HB, which is opposite to that of the wild-type, while metabolite diasteroselectivity remained unchanged. The replacement of Phe-476 by alanine increased total HB 3'-hydroxylation activity to approximately 3-fold that of the wild-type. Particularly, 3'(S)-OH-(S)-HB-forming activity elevated to 7-fold that of the wild-type, resulting in the reversal of the substrate enantioselectivity. In contrast, the substitution of phenylalanine at positions 100 and 114 by alanine did not produce a remarkable change in the total activity or the substrate enantioselectivity. These results indicate that Glu-300 and Phe-476 are important in stereoselective oxidation of HB enantiomers by CYP2C19. 相似文献
53.
Md. Habibur Rahman Tohru Tsuchiya Keita Suwabe Junna Kohori Rubens Norio Tomita Yasuaki Kagaya Issei Kobayashi Katsuyuki Kakeda Yasuo Kowyama 《Sexual plant reproduction》2007,20(2):63-72
Sporophytic self-incompatibility (SSI) in the genus Ipomoea (Convolvulaceae) is controlled by a single polymorphic S locus. We have previously analyzed genomic sequences of an approximately 300 kb region spanning the S locus of the S
1
haplotype and characterized the genomic structure around this locus. Here, we further define the physical size of the S locus region by mapping recombination breakpoints, based on sequence analysis of PCR fragments amplified from the genomic
DNA of recombinants. From the recombination analysis, the S locus of the S
1
haplotype was delimited to a 0.23 cM region of the linkage map, which corresponds to a maximum physical size of 212 kb. To
analyze differences in genomic organization between S haplotypes, fosmid contigs spanning approximately 67 kb of the S
10
haplotype were sequenced. Comparison with the S
1
genomic sequence revealed that the S haplotype-specific divergent regions (SDRs) spanned 50.7 and 34.5 kb in the S
1
and S
10
haplotypes, respectively and that their flanking regions showed a high sequence similarity. In the sequenced region of the
S
10
haplotype, five of the 12 predicted open reading frames (ORFs) were found to be located in the divergent region and showed
co-linear organization of genes between the two S haplotypes. Based on the size of the SDRs, the physical size of the S locus was estimated to fall within the range 34–50 kb in Ipomoea. 相似文献
54.
Redox-active Langmuir-Blodgett (LB) films containing dihydrophytyl ferrocenoate (DFc) and beta-carotene (betaC) were fabricated by use of 6-O-dihydrophytylcellulose (DHPC) as a matrix. A mixture of DFc-DHPC formed a stable monolayer. Atomic force microscopy images revealed that the DFc molecules were dispersed uniformly throughout the surface in the ratio DFc:DHPC = 2:8 at 30 mN m-1. The DFc-DHPC monolayer was transferred successfully onto a substrate, yielding Y-type LB films. Cyclic voltammograms for the DFc-DHPC LB films on an indium tin oxide (ITO) electrode exhibited a well-defined surface wave. The voltammograms of the DFc-DHPC LB films exhibited 60-40% redox-active ferrocene moieties, whereas those of the DFc-DHPC-betaC LB films exhibited 90-70%. X-ray diffraction patterns indicated that the distance between layers was independent of betaC molecules incorporated into the LB films. Consequently, these results suggested that betaC can function as a molecular wire. 相似文献
55.
56.
57.
Mouse Models for Assessing the Protective Efficacy of Lactobacillus gasseri SBT2055 against Helicobacter suis Infection Associated with the Development of Gastric Mucosa‐Associated Lymphoid Tissue Lymphoma
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58.
Irene TM Arkesteijn Lucas A Smolders Sandra Spillekom Frank M Riemers Esther Potier Bj?rn P Meij Keita Ito Marianna A Tryfonidou 《Arthritis research & therapy》2015,17(1)
IntroductionEarly degenerative changes in the nucleus pulposus (NP) are observed after the disappearance of notochordal cells (NCs). Thus, it has been suggested that NCs play an important role in maintaining the NP and may have a regenerative potential on other cells of the NP. As the number of resident NP cells (NPCs) decreases in a degenerating disc, mesenchymal stromal (stem) cells (MSCs) may be used for cell supplementation. In this study, using cells of one species, the regenerative potential of canine NCs was assessed in long-term three-dimensional coculture with canine NPCs or MSCs.MethodsCanine NCs and canine NPCs or MSCs were cocultured in alginate beads for 28 days under hypoxic and high-osmolarity conditions. Cell viability, cell morphology and DNA content, extracellular matrix production and expression of genes related to NC markers (Brachyury, KRT18) and NP matrix production (ACAN, COL2A1, COL1A1) were assessed after 1, 15 and 28 days of culture.ResultsNCs did not completely maintain their phenotype (morphology, matrix production, gene expression) during 28 days of culture. In cocultures of NPCs and NCs, both extracellular matrix content and anabolic gene expression remained unchanged compared with monoculture groups, whereas cocultures of MSCs and NCs showed increased glycosaminoglycan/DNA. However, the deposition of these proteoglycans was observed near the NCs and not the MSCs. Brachyury expression in the MSC and NC coculture group increased in time. The latter two findings indicate a trophic effect of MSCs on NCs rather than vice versa.ConclusionsNo regenerative potential of canine NCs on canine NPCs or MSCs was observed in this study. However, significant changes in NC phenotype in long-term culture may have resulted in a suboptimal regenerative potential of these NCs. In this respect, NC-conditioned medium may be better than coculture for future studies of the regenerative potential of NCs.
Electronic supplementary material
The online version of this article (doi:10.1186/s13075-015-0569-6) contains supplementary material, which is available to authorized users. 相似文献59.
Khoshgoftar M van Donkelaar CC Ito K 《Computer methods in biomechanics and biomedical engineering》2011,14(2):135-144
The load-bearing capacity of today's tissue-engineered (TE) cartilage is insufficient. The arcade-like collagen network in native cartilage plays an important role in its load-bearing properties. Inducing the formation of such collagen architecture in engineered cartilage can, therefore, enhance mechanical properties of TE cartilage. Considering the well-defined relationship between tensile strains and collagen alignment in the literature, we assume that cues for inducing this orientation should come from mechanical loading. In this study, strain fields prescribed by loading conditions of unconfined compression, sliding indentation and a novel loading regime of compression-sliding indentation are numerically evaluated to assess the probability that these would trigger a physiological collagen architecture. Results suggest that sliding indentation is likely to stimulate the formation of an appropriate superficial zone with parallel fibres. Adding lateral compression may stimulate the formation of a deep zone with perpendicularly aligned fibres. These insights may be used to improve loading conditions for cartilage tissue engineering. 相似文献
60.
Itoh T Terazawa R Kojima K Nakane K Deguchi T Ando M Tsukamasa Y Ito M Nozawa Y 《Free radical research》2011,45(9):1033-1039
This study aimed to examine the roles of reactive oxygen species (ROS) in cisplatin treatment of human prostate cancer cells; hormone-sensitive LNCaP and hormone-refractory PC3 and DU145 cells. Intracellular levels of ROS and H(2)O(2) were measured and visualized using specific fluorescent probes. NADPH oxidase (NOX) activity was detected by lucigenin chemiluminescence assay. Expression levels of NOX isoforms were determined by semi-quantitative RT-PCR. Cisplatin treatment increased the intracellular levels of ROS and H(2)O(2) in three prostate cancer cell lines. The increase was transient and robust in hormone-sensitive LNCaP cells compared with hormone-refractory PC3 and DU145 cells. Consistent with these findings, the NOX activity induced by cisplatin was higher in LNCaP cells than in PC3 and DU145 cells. Expression pattern of NOX isoforms varied among three cell lines and the NOX activity was independent of NOX expression. Taken together, we have shown that cisplatin induces production of ROS and H(2)O(2) via NOX activation in human prostate cancer cell lines, which is most prominent in hormone-sensitive LNCaP cells. 相似文献