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71.
Toshiharu Nagatsu Kazuhiro Oka Takeshi Kato 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》1979,163(3):247-252
A highly sensitive assay for tyrosine hydroxylase (TH) activity by high-performance liquid chromatography (HPLC) with amperometric detection was devised based on the rapid isolation of enzymatically formed DOPA by a double-column procedure, the columns fitted together sequentially (the top column of Amberlite CG-50 and the bottom column of aluminium oxide). DOPA was adsorbed on the second aluminium oxide column, then eluted with 0.5 M hydrochloric acid, and assayed by HPLC with amperometric detection. d-Tyrosine was used for the control. α-Methyldopa was added to the incubation mixture as an internal standard after incubation. This assay was more sensitive than radioassays and 5 pmol of DOPA formed enzymatically could be measured in the presence of saturating concentrations of tyrosine and 6-methyltetrahydropterin. The TH activity in 2 mg of human putamen could be easily measured, and this method was found to be particularly suitable for the assay of TH activity in a small number of nuclei from animal and human brain. 相似文献
72.
Diacyl derivatives of 2-methylmercaptobenzimidazole undergo the tautomerization 2 1 2′. Thermodynamic predominancy of one isomer over the others depends on the substituents on carbonyl groups. It has been found that electron-withdrawing substituents tend to favor 2-type compounds, whereas electron-releasing substituents make 1-type compounds more stable. The migration has been extended to include the carboethoxy group, and the results are discussed in relation to the mechanism of biotin-dependent enzymic carboxylation. 相似文献
73.
Plasma membranes were islotaed from rat liver mainly under isotonic conditions. As marker enzymes for the plasma membrane, 5′-nucleotidase and (Na++K+)-ATPase were used. The yield of plasma membrane was 0.6–0.9 mg protein per g wet weight of liver. The recovery of 5′-nucleotidase and (Na++K+)-ATPase activity was 18 and 48% of the total activity of the whole-liver homogenate, respectively. Judged from the acitvity of glucose-6 phosphatase and succinate dehydrogenase in the plasma membrane, and from the electron microscopic observation of it, the contamination by microsomes and mitochondria was very low. A further homogenization of the plasma membrane yielded two fractions, the light and heavy fractions, in a discontinuous sucrose gradient centrifugation. The light fraction showed higher specific activities of 5′-nucleotidase, alkaline phosphatase, (Na++K+)-ATPase and Mg2+-ATPase, whereas the heavy one showed a higher specific activity of adenylate cyclase. Ligation of the bile duct for 48 h decreased the specific activities of (Na++K+)-ATPase and Mg2+-ATPase in the light fraction, whereas it had no significant influence on the activities of these enzymes in the heavy fraction. The specific activity of alkaline phosphatase was elevated in both fractions by the obstruction of the bile flow. Electron microscopy on sections of the plasma membrane subfractions showed that the light fraction consisted of vesicles of various sizes and that the heavy fractions contained membrane sheets and paired membrane strips connected by junctional complexes, as well as vesicles. The origin of these two fractions is discussed and it is suggested that the light fraction was derived from the bile front of the liver cell surface and the heavy one contained the blood front and the lateral surface of it. 相似文献
74.
Plasma membranes were isolated from rat liver mainly under isotonic conditions. As marker enzymes for the plasma membrane, 5'-nucleotidase and (Na+ + K+)-ATPase were used. The yield of plasma membrane was 0.6-0.9 mg protein per g wet weight of liver. The recovery of 5'-nucleotidase and (Na+ +K+)-ATPase activity was 18 and 48% of the total activity of the whole-liver homogenate, respectively. Judged from the activity of glucose-6-phosphatase and succinate dehydrogenase in the plasma membrane, and from the electron microscopic observation of it, the contamination by microsomes and mitochondria was very low. A further homogenization of the plasma membrane yielded two fractions, the light and heavy fractions, in a discontinuous sucrose gradient centrifugation. The light fraction showed higher specific activities of 5'-nucleotidase, alkaline phosphatase, (Na+ +K+)-ATPase and Mg2+-ATPase, whereas the heavy one showed a higher specific activity of adenylate cyclase. Ligation of the bile duct for 48 h decreased the specific activities of (Na2+ +K+)-ATPase and Mg2+-ATPase in the light fraction, whereas it had no significant influence on the activities of these enzymes in the heavy fraction. The specific activity of alkaline phosphate was elevated in both fractions by the obstruction of the bile flow. Electron microscopy on sections of the plasma membrane subfractions showed that the light fraction consisted of vesicles of various sizes and that the heavy fractions contained membrane sheets and paired membrane strips connected by junctional complexes, as well as vesicles. The origin of these two fractions is discussed and it is suggested that the light fraction was derived from the bile front of the liver cell surface and the heavy one contained the blood front and the lateral surface of it. 相似文献
75.
76.
Summary Chromosome variants were evaluated on the basis of their DNA-replication pattern (LBA). The size of late-replicating centromeric heterochromatin of chromosomes 2, 5, 6, 7, 8, 10, 11, 12, 17, 18, 19, and 20, i.e., pairs without Q or C (qh) variants, was measured by means of a microdensitometer. The results were expressed in area, related to that of a euchromatic segment of a given chromosome, and were assigned into five classes based on the difference in standard deviation from an average relative size. LBA variants in each of 12 pairs were found in 29%–42% of the chromosomes. 相似文献
77.
Masakazu Goto Chisato Ito Mohammed Sani Yahaya Yoshihito Wakai Shiro Asano Yukari Oka 《Marine and Freshwater Behaviour and Physiology》2013,45(2):99-107
The number of microorganisms in the hindgut of dugongs (Dugong dugon) were estimated and their in vitro volatile fatty acid (VFA) production and degradation of eelgrass measured. Scanning electron microscopy showed that some rod bacteria attached to the surface of plant tissue degraded and eroded the cell walls. Number of starch-, lactate-, cellobiose-, pectin-, xylan- and cellulose-utilizing bacteria, sulfate-reducing bacteria and methane-producing bacteria were estimated at 109 ~ 1010 colony forming units g?1. Microorganisms degraded the cellulose and noncellulolytic components of the eelgrass, and about 47.3% of dry matter was degraded after 36?h in vitro incubation. The total VFA concentration was 10.5?mmol?dL?1 at 36?h incubation, which included 55.7?mol% acetate, 18.0?mol% n-butyrate and 15.1?mol% propionate. The gas composition of in vitro fermentation was 68.4% carbon dioxide, 22.2% methane and 9.4% hydrogen. 相似文献
78.
Chihiro Motozono John J. Miles Zafrul Hasan Hiroyuki Gatanaga Stanley C. Meribe David A. Price Shinichi Oka Andrew K. Sewell Takamasa Ueno 《PloS one》2013,8(6)
Antigen cross-reactivity is an inbuilt feature of the T cell compartment. However, little is known about the flexibility of T cell recognition in the context of genetically variable pathogens such as HIV-1. In this study, we used a combinatorial library containing 24 billion octamer peptides to characterize the cross-reactivity profiles of CD8+ T cells specific for the immunodominant HIV-1 subtype B Nef epitope VY8 (VPLRPMTY) presented by HLA-B*35∶01. In conjunction, we examined naturally occurring antigenic variations within the VY8 epitope. Sequence analysis of plasma viral RNA isolated from 336 HIV-1-infected individuals revealed variability at position (P) 3 and P8 of VY8; Phe at P8, but not Val at P3, was identified as an HLA-B*35∶01-associated polymorphism. VY8-specific T cells generated from several different HIV-1-infected patients showed unique and clonotype-dependent cross-reactivity footprints. Nonetheless, all T cells recognized both the index Leu and mutant Val at P3 equally well. In contrast, competitive titration assays revealed that the Tyr to Phe substitution at P8 reduced T cell recognition by 50–130 fold despite intact peptide binding to HLA-B*35∶01. These findings explain the preferential selection of Phe at the C-terminus of VY8 in HLA-B*35∶01+ individuals and demonstrate that HIV-1 can exploit the limitations of T cell recognition in vivo. 相似文献
79.
Kosuke Murakami Chie Kurihara Tomoichiro Oka Takashi Shimoike Yoshiki Fujii Reiko Takai-Todaka YoungBin Park Takaji Wakita Tsukasa Matsuda Ryota Hokari Soichiro Miura Kazuhiko Katayama 《PloS one》2013,8(6)
Human noroviruses (NoVs) are a major cause of non-bacterial gastroenteritis. Although histo-blood group antigens (HBGAs) have been implicated in the initial binding of NoV, the mechanism of that binding before internalization is not clear. To determine the involvement of NoVs and HBGAs in cell binding, we examined the localization of NoV virus-like particles (VLPs) and HBGAs in a human intestinal cell line and the human ileum biopsy specimens by immunofluorescence microscopy. The localizations of Ueno 7k VLPs (genogroup II.6) and each HBGA (type H1-, H2- and Leb-HBGAs) on the human intestinal cell line, Caco-2, were examined by confocal laser-scanning microscopy. To explore any interactions of NoVs and HBGAs in vivo, fresh biopsy specimens from human ileum were directly incubated with NoV VLPs and examined by immunofluorescence microscopy. We found that VLP binding depended on the state of cell differentiation, but not on the presence of HBGAs. In differentiated Caco-2 cells, we detected no type H1 HBGAs, but VLPs bound to the cells anyway. We incubated fresh biopsies of human ileum directly with VLPs, a model that better replicates the in vivo environment. VLPs mainly bound epithelial cells and goblet cells. Although the incubations were performed at 4°C to hinder internalization, VLPs were still detected inside cells. Our results suggest that VLPs utilize molecule(s) other than HBGAs during binding and internalization into cells. 相似文献
80.
Takeshi Nishijima Hiroyuki Gatanaga Hirokazu Komatsu Misao Takano Miwa Ogane Kazuko Ikeda Shinichi Oka 《PloS one》2013,8(8)