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31.
Hayashi Y Okino N Kakuta Y Shikanai T Tani M Narimatsu H Ito M 《The Journal of biological chemistry》2007,282(42):30889-30900
Using C6-NBD-glucosylceramide (GlcCer) as a substrate, we detected the activity of a conduritol B epoxide-insensitive neutral glycosylceramidase in cytosolic fractions of zebrafish embryos, mouse and rat brains, and human fibroblasts. The candidates for the enzyme were assigned to the Klotho (KL), whose family members share a beta-glucosidase-like domain but whose natural substrates are unknown. Among this family, only the KL-related protein (KLrP) is capable of degrading C6-NBD-GlcCer when expressed in CHOP cells, in which Myc-tagged KLrP was exclusively distributed in the cytosol. In addition, knockdown of the endogenous KLrP by small interfering RNA increased the cellular level of GlcCer. The purified recombinant KLrP hydrolyzed 4-methylumbelliferyl-glucose, C6-NBD-GlcCer, and authentic GlcCer at pH 6.0. The enzyme also hydrolyzed the corresponding galactosyl derivatives, but each k(cat)/Km was much lower than that for glucosyl derivatives. The x-ray structure of KLrP at 1.6A resolution revealed that KLrP is a (beta/alpha)8 TIM barrel, in which Glu(165) and Glu(373) at the carboxyl termini of beta-strands 4 and 7 could function as an acid/base catalyst and nucleophile, respectively. The substrate-binding cleft of the enzyme was occupied with palmitic acid and oleic acid when the recombinant protein was crystallized in a complex with glucose. GlcCer was found to fit well the cleft of the crystal structure of KLrP. Collectively, KLrP was identified as a cytosolic neutral glycosylceramidase that could be involved in a novel nonlysosomal catabolic pathway of GlcCer. 相似文献
32.
Miyanishi N Nishi N Abe H Kashio Y Shinonaga R Nakakita S Sumiyoshi W Yamauchi A Nakamura T Hirashima M Hirabayashi J 《Glycobiology》2007,17(4):423-432
Galectin-9 (Gal-9) is a tandem-repeat-type member of the galectin family associated with diverse biological processes, such as apoptosis, cell aggregation, and eosinophil chemoattraction. Although the detailed sugar-binding specificity of Gal-9 has been elucidated, molecular mechanisms that underlie these functions remain to be investigated. During the course of our binding study by affinity chromatography and surface plasmon resonance (SPR) analysis, we found that human Gal-9 interacts with immobilized Gal-9 in the protein-protein interaction mode. Interestingly, this intermolecular interaction strongly depended on the activity of the carbohydrate recognition domain (CRD), because the addition of potent saccharide inhibitors abolished the binding. The presence of multimers was also confirmed by Ferguson plot analysis of result of polyacrylamide gel electrophoresis and matrix-assisted laser-desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS). Moreover, this intermolecular interaction was observed between Gal-9 and other galectin members, such as Gal-3 and Gal-8, but not Gal-1. Because such properties have not been reported yet, they may explain an unidentified mechanism underlying the diverse functions of Gal-9. 相似文献
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35.
Tanaka Y Nakamura S Kawamukai M Koizumi N Nakagawa T 《Bioscience, biotechnology, and biochemistry》2011,75(4):804-807
We made two series of Gateway binary vectors, pGWBs and R4pGWBs, possessing a UDP-N-acetylglucosamine: dolichol phosphate N-acetylglucosamine-1-P transferase (GPT) gene driven by the nopaline synthase promoter (Pnos) as a tunicamycin resistance marker for the transformation of Arabidopsis thaliana. The reporters and tags employed in this system are sGFP, GUS, LUC, EYFP, ECFP, G3GFP, mRFP, TagRFP, 6xHis, FLAG, 3xHA, 4xMyc, 10xMyc, GST, T7, and TAP. Selection of transformants was successful on plates containing 0.15 mg/L of tunicamycin. These vectors were compatible with existing pGWB and R4pGWB vectors for kanamycin, hygromycin B, and BASTA? selection, and are useful new tools for making transgenic Arabidopsis. 相似文献
36.
Arisa Miyagawa‐Yamaguchi Takuma Okami Nozomu Kira Haruo Yamaguchi Kouhei Ohnishi Masao Adachi 《Phycological Research》2011,59(2):113-119
A nuclear transformation system for the centric diatom Chaetoceros sp. has been established using two plasmids pTpfcp/nat and pTpNR/green fluorescent protein (GFP) that had been used for Thalassiosira pseudonana transformation. These contain the nourseothricin resistance gene (nat) with the fucoxanthin chlorophyll a/c binding protein (fcp) promoter/terminator from T. pseudonana and the enhanced green fluorescent protein gene (egfp), with the nitrate reductase (NR) promoter/terminator from T. pseudonana, respectively. Transformants were recovered in the presence of the antibiotic nourseothricin. One to four copies of both nat and egfp genes were integrated into genomic DNA of the transformants. Transformation efficiency was 1.5–6.0 transformants per 108 cells. This work is the first report of stable genetic transformation of Chaetoceros, which is important as not only a constituent member of marine ecosystem but also feed for aquaculture. 相似文献
37.
Tetsuji Nakabo Kouji Nakayama Nozomu Muto Masayuki Miyazawa 《Ichthyological Research》2011,58(2):180-183
Oncorhynchus kawamurae (Osteichthyes: Salmonidae) (common name “Kunimasu”), a species endemic to Lake Tazawa, Akita Prefecture, Japan, was believed
to have been extinct since 1940. However, nine specimens were discovered in March and April 2010 in Lake Saiko, Yamanashi
Prefecture, one of the lakes to which eyed eggs of the species were introduced in 1935. These were identified as O. kawamurae because of having 47–62 pyloric caeca, 37–43 gill-rakers, a black-colored body, and spawning at 30–40 m depth in early spring,
which are unique characteristics within Oncorhynchus. Furthermore, the distinctiveness of Kunimasu from sympatric kokanee (O. nerka) was supported by microsatellite DNA data. 相似文献
38.
12-oxo-phytodienoic acid triggers expression of a distinct set of genes and plays a role in wound-induced gene expression in Arabidopsis 下载免费PDF全文
39.
Rev1p in yeast is essential for the translesion of abasic sites and 6-4 photoproducts. It plays a role as a translesion polymerase, but also supports translesion catalyzed by other polymerases. The protein has two domains, BRCT and Y-family polymerase. A point mutation in the BRCT domain is known to abolish the second function. In the present research, we have studied the effects of deletion of the BRCT domain and a point mutation at the two amino acids in the putative polymerase active center. We have introduced an abasic site, its tetrahydrofuran analog, and a 6-4 thymine-thymine photoproduct using the oligonucleotide transformation assay. Translesion efficiencies were estimated from the transforming activities of the oligonucleotides with a lesion, and the mutation spectra were analyzed by DNA sequencing of the transformants. Results showed that the lack of the BRCT domain reduced translesion efficiencies, but that substantial translesion synthesis took place. The mutation spectra of the lesions were not greatly affected. Therefore, the BRCT domain may be important, but dispensable for translesion synthesis. In contrast, the polymerase mutation, rev1AA, has only small effects on the translesion efficiencies, but the mutation spectra were greatly affected; the incorporation of dCMP opposite the lesions was specifically lost. This clearly shows that the polymerase domain is responsible for the dCMP incorporation. The effect of Poleta was also analyzed. From all the results DNA polymerases other than these two translesion polymerases, too, seem to initiate the translesion synthesis. 相似文献
40.
Shoji H Ikenaka K Nakakita S Hayama K Hirabayashi J Arata Y Kasai K Nishi N Nakamura T 《Glycobiology》2005,15(7):709-720
We have identified members of the Xenopus cortical granule lectin (xCGL) family as candidate target glycoproteins of Xenopus galectin-VIIa (xgalectin-VIIa) in Xenopus embryos. In addition to the original xCGL, we also identified a novel member of the xCGL family, xCGL2. Expression of the mRNAs of xCGL and xCGL2, as well as that of xgalectin-VIIa, was observed throughout early embryogenesis. Two and three potential N-glycosylation sites were deduced from the amino acid sequences of xCGL and xCGL2, respectively, and xgalectin-VIIa recognizes N-glycans linked to a common site in xCGL and xCGL2 and also recognizes N-glycans linked to a site specific to xCGL2. However, interaction between xgalectin-Ia and xCGLs was not detectable. We also obtained consistent results on surface plasmon resonance analysis involving xCGLs as ligands and xgalectins as analytes. The Kd value of the interaction between xgalectin-VIIa and xCGLs was calculated to be 35.9 nM. The structures of the N-glycans of xCGLs, which were recognized by xgalectin-VIIa, were analyzed by the two-dimensional sugar map method, and three kinds of N-acetyllactosamine type, biantennary N-glycans were identified as the major neutral N-glycans. The binding specificity of oligosaccharides for xgalectin-VIIa was analyzed by frontal affinity chromatography (FAC). The oligosaccharide specificity pattern of xgalectin-VIIa was similar to that of the human homolog galectin-3, and it was also shown that the N-acetyllactosamine type, biantennary N-glycans exhibit high affinity for xgalectin-VIIa (Kd = 11 microM). These results suggest that xgalectin-VIIa interacts with xCGLs through binding to N-acetyllactosamine type N-glycans and that this interaction might make it possible to organize a lectin network involving members of different lectin families. 相似文献