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61.
Sakaidani Y Ichiyanagi N Saito C Nomura T Ito M Nishio Y Nadano D Matsuda T Furukawa K Okajima T 《Biochemical and biophysical research communications》2012,419(1):14-19
O-linked-β-N-acetylglucosamine (O-GlcNAc) modification is a unique cytoplasmic and nuclear protein modification that is common in nearly all eukaryotes, including filamentous fungi, plants, and animals. We had recently reported that epidermal growth factor (EGF) repeats of Notch and Dumpy are O-GlcNAcylated by an atypical O-GlcNAc transferase, EOGT, in Drosophila. However, no study has yet shown whether O-GlcNAcylation of extracellular proteins is limited to insects such as Drosophila or whether it occurs in other organisms, including mammals. Here, we report the characterization of A130022J15Rik, a mouse gene homolog of Drosophila Eogt (Eogt 1). Enzymatic analysis revealed that Eogt1 has a substrate specificity similar to that of Drosophila EOGT, wherein the Thr residue located between the fifth and sixth conserved cysteines of the folded EGF-like domains is modified. This observation is supported by the fact that the expression of Eogt1 in Drosophila rescued the cell-adhesion defect caused by Eogt downregulation. In HEK293T cells, Eogt1 expression promoted modification of Notch1 EGF repeats by O-GlcNAc, which was further modified, at least in part, by galactose to generate a novel O-linked-N-acetyllactosamine structure. These results suggest that Eogt1 encodes EGF domain O-GlcNAc transferase and that O-GlcNAcylation reaction in the secretory pathway is a fundamental biochemical process conserved through evolution. 相似文献
62.
Takekawa Y Saito M Wang C Qin T Ogawa M Kanemaru T Yoshida S 《Canadian journal of microbiology》2012,58(2):179-183
Legionella oakridgensis occasionally causes pneumonia in humans. We report here the characteristic morphology of intracellular microcolonies of L. oakridgensis OR-10 in infected epithelial cells. By light microscopy after Gimenez staining, the bacteria showed serpentine-like chain, disk-like conglomerate, and granular forms when they grew intracellularly in Vero cells, HeLa cells, and A549 cells. In a time-lapse study, we observed the progressive change from a serpentine-like chain form to a conglomerate form in Vero cells. Transmission electron microscopy showed that L. oakridgensis OR-10 proliferated both inside membrane structures and in the cytoplasm. Such highly serpentine chain growth has not been reported in any intracellular bacteria. Furthermore, these results imply that L. oakridgensis OR-10 may be proliferating inside the endoplasmic reticulum. 相似文献
63.
Ansai S Ochiai H Kanie Y Kamei Y Gou Y Kitano T Yamamoto T Kinoshita M 《Development, growth & differentiation》2012,54(5):546-556
Zinc-finger nucleases (ZFNs) are artificial enzymes that create site-specific double-strand breaks and thereby induce targeted genome editing. Here, we demonstrated successful gene disruption in somatic and germ cells of medaka (Oryzias latipes) using ZFN to target exogenous EGFP genes. Embryos that were injected with an RNA sequence pair coding for ZFNs showed mosaic loss of green fluorescent protein fluorescence in skeletal muscle. A number of mutations that included both deletions and insertions were identified within the ZFN target site in each embryo, whereas no mutations were found at the non-targeted sites. In addition, ZFN-induced mutations were introduced in germ cells and efficiently transmitted to the next generation. The mutation frequency varied (6-100%) in the germ cells from each founder, and a founder carried more than two types of mutation in germ cells. Our results have introduced the possibility of targeted gene disruption and reverse genetics in medaka. 相似文献
64.
Hiroyuki Kawano Shutaro Katsurabayashi Yasuhiro Kakazu Yuta Yamashita Natsuko Kubo Masafumi Kubo Hideto Okuda Kotaro Takasaki Kaori Kubota Kenichi Mishima Michihiro Fujiwara N. Charles Harata Katsunori Iwasaki 《PloS one》2012,7(10)
The astrocyte is a major glial cell type of the brain, and plays key roles in the formation, maturation, stabilization and elimination of synapses. Thus, changes in astrocyte condition and age can influence information processing at synapses. However, whether and how aging astrocytes affect synaptic function and maturation have not yet been thoroughly investigated. Here, we show the effects of prolonged culture on the ability of astrocytes to induce synapse formation and to modify synaptic transmission, using cultured autaptic neurons. By 9 weeks in culture, astrocytes derived from the mouse cerebral cortex demonstrated increases in β-galactosidase activity and glial fibrillary acidic protein (GFAP) expression, both of which are characteristic of aging and glial activation in vitro. Autaptic hippocampal neurons plated on these aging astrocytes showed a smaller amount of evoked release of the excitatory neurotransmitter glutamate, and a lower frequency of miniature release of glutamate, both of which were attributable to a reduction in the pool of readily releasable synaptic vesicles. Other features of synaptogenesis and synaptic transmission were retained, for example the ability to induce structural synapses, the presynaptic release probability, the fraction of functional presynaptic nerve terminals, and the ability to recruit functional AMPA and NMDA glutamate receptors to synapses. Thus the presence of aging astrocytes affects the efficiency of synaptic transmission. Given that the pool of readily releasable vesicles is also small at immature synapses, our results are consistent with astrocytic aging leading to retarded synapse maturation. 相似文献
65.
C Terao K Ohmura K Ikari Y Kochi E Maruya M Katayama K Yurugi K Shimada A Murasawa S Honjo K Takasugi K Matsuo K Tajima A Suzuki K Yamamoto S Momohara H Yamanaka R Yamada H Saji F Matsuda T Mimori 《PloS one》2012,7(7):e40067
HLA-DRB1, especially the shared epitope (SE), is strongly associated with rheumatoid arthritis (RA). However, recent studies have shown that SE is at most weakly associated with RA without anti-citrullinated peptide/protein antibody (ACPA). We have recently reported that ACPA-negative RA is associated with specific HLA-DRB1 alleles and diplotypes. Here, we attempted to detect genetically different subsets of ACPA-negative RA by classifying ACPA-negative RA patients into two groups based on their positivity for rheumatoid factor (RF). HLA-DRB1 genotyping data for totally 954 ACPA-negative RA patients and 2,008 healthy individuals in two independent sets were used. HLA-DRB1 allele and diplotype frequencies were compared among the ACPA-negative RF-positive RA patients, ACPA-negative RF-negative RA patients, and controls in each set. Combined results were also analyzed. A similar analysis was performed in 685 ACPA-positive RA patients classified according to their RF positivity. As a result, HLA-DRB1*04:05 and *09:01 showed strong associations with ACPA-negative RF-positive RA in the combined analysis (p = 8.8×10−6 and 0.0011, OR: 1.57 (1.28–1.91) and 1.37 (1.13–1.65), respectively). We also found that HLA-DR14 and the HLA-DR8 homozygote were associated with ACPA-negative RF-negative RA (p = 0.00022 and 0.00013, OR: 1.52 (1.21–1.89) and 3.08 (1.68–5.64), respectively). These association tendencies were found in each set. On the contrary, we could not detect any significant differences between ACPA-positive RA subsets. As a conclusion, ACPA-negative RA includes two genetically distinct subsets according to RF positivity in Japan, which display different associations with HLA-DRB1. ACPA-negative RF-positive RA is strongly associated with HLA-DRB1*04:05 and *09:01. ACPA-negative RF-negative RA is associated with DR14 and the HLA-DR8 homozygote. 相似文献
66.
67.
Furuichi M Suzuki N Dhakshnamoorhty B Minagawa H Yamagishi R Watanabe Y Goto Y Kaneko H Yoshida Y Yagi H Waga I Kumar PK Mizuno H 《Journal of molecular biology》2008,378(2):436-446
l-Lactate oxidase (LOX) belongs to a family of flavin mononucleotide (FMN)-dependent α-hydroxy acid-oxidizing enzymes. Previously, the crystal structure of LOX (pH 8.0) from Aerococcus viridans was solved, revealing that the active site residues are located around the FMN. Here, we solved the crystal structures of the same enzyme at pH 4.5 and its complex with d-lactate at pH 4.5, in an attempt to analyze the intermediate steps. In the complex structure, the d-lactate resides in the substrate-binding site, but interestingly, an active site base, His265, flips far away from the d-lactate, as compared with its conformation in the unbound state at pH 8.0. This movement probably results from the protonation of His265 during the crystallization at pH 4.5, because the same flip is observed in the structure of the unbound state at pH 4.5. Thus, the present structure appears to mimic an intermediate after His265 abstracts a proton from the substrate. The flip of His265 triggers a large structural rearrangement, creating a new hydrogen bonding network between His265-Asp174-Lys221 and, furthermore, brings molecular oxygen in between d-lactate and His265. This mimic of the ternary complex intermediate enzyme-substrate-O2 could explain the reductive half-reaction mechanism to release pyruvate through hydride transfer. In the mechanism of the subsequent oxidative half-reaction, His265 flips back, pushing molecular oxygen into the substrate-binding site as the second substrate, and the reverse reaction takes place to produce hydrogen peroxide. During the reaction, the flip-flop action of His265 has a dual role as an active base/acid to define the major chemical steps. Our proposed reaction mechanism appears to be a common mechanistic strategy for this family of enzymes. 相似文献
68.
Shimazawa M Inokuchi Y Okuno T Nakajima Y Sakaguchi G Kato A Oku H Sugiyama T Kudo T Ikeda T Takeda M Hara H 《Journal of neurochemistry》2008,107(1):279-290
Here, we examined whether amyloid-beta (Abeta) protein participates in cell death and retinal function using three types of transgenic (Tg) mice in vivo [human mutant amyloid precursor protein (APP) Tg (Tg 2576) mice, mutant presenilin-1 (PS-1) knock-in mice, and APP/PS-1 double Tg mice]. ELISA revealed that the insoluble form of Abeta(1-40) was markedly accumulated in the retinas of APP and APP/PS-1, but not PS-1 Tg, mice (vs. wild-type mice). In APP Tg and APP/PS-1 Tg mice, immunostaining revealed accumulations of intracellular Abeta(1-42) in retinal ganglion cells and in the inner and outer nuclear layers. APP Tg and APP/PS-1 Tg, but not PS-1 Tg, mice had less NMDA-induced retinal damage than wild-type mice, and the reduced damage in APP/PS-1 Tg mice was diminished by the pre-treatment of N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester, a gamma-secretase inhibitor. Furthermore, the number of TUNEL-positive cells was significantly less in ganglion cell layer of APP/PS-1 Tg mice than PS-1 Tg mice 24 h after NMDA injection. The phosphorylated form of calcium/calmodulin-dependent protein kinase IIalpha (CaMKIIalpha), but not total CaMKIIalpha or total NMDA receptor 1 (NR1) subunit, in total retinal extracts was decreased in non-treated retinas of APP/PS-1 Tg mice (vs. wild-type mice). CaMKIIalpha and NR2B proteins, but not NR1, in retinal membrane fraction were significantly decreased in APP/PS-1 Tg mice as compared with wild-type mice. The NMDA-induced increase in p-CaMKIIalpha in the retina was also lower in APP/PS-1 Tg mice than in wild-type mice. In electroretinogram and visual-evoked potential recordings, the implicit time to each peak from a light stimulus was prolonged in APP/PS-1 mice versus wild-type mice. Hence, Abeta may impair retinal function by reducing activation of NMDA-receptor signaling pathways. 相似文献
69.
Tahara M Takeda M Shirogane Y Hashiguchi T Ohno S Yanagi Y 《Journal of virology》2008,82(9):4630-4637
Measles is one of the most contagious human infectious diseases and remains a major cause of childhood morbidity and mortality worldwide. The signaling lymphocyte activation molecule (SLAM), also called CD150, is a cellular receptor for measles virus (MV), presumably accounting for its tropism for immune cells and its immunosuppressive properties. On the other hand, pathological studies have shown that MV also infects epithelial cells at a later stage of infection, although its mechanism has so far been unknown. In this study, we show that wild-type MV can infect and produce syncytia in human polarized epithelial cell lines independently of SLAM and CD46 (a receptor for the vaccine strains of MV). Progeny viral particles are released exclusively from the apical surface of these polarized epithelial cell lines. We have also identified amino acid residues on the MV attachment protein that are likely to interact with a putative receptor on epithelial cells. All of these residues have aromatic side chains and may form a receptor-binding pocket located in a different position from the putative SLAM- and CD46-binding sites on the MV attachment protein. Thus, our results indicate that MV has an intrinsic ability to infect both polarized epithelial and immune cells by using distinctive receptor-binding sites on the attachment protein corresponding to each of their respective receptors. The ability of MV to infect polarized epithelial cells and its exclusive release from the apical surface may facilitate its efficient transmission via aerosol droplets, resulting in its highly contagious nature. 相似文献
70.
During the course of studying selenocysteine (Sec) synthesis mechanisms in mammals, we prepared selenophosphate synthetase
(SPS) from bovine liver by 4-step chromatography. In the last step of chromatography of hydroxyapatite, we found a protein
band of molecular mass 33 kDa on SDS-PAGE, consistent with the pattern of SPS activity that was indirectly manifested by [75Se]Sec production activity; however, we could not detect significant Se content in this active fraction. We also found a clear
band of 33 kDa by Western blotting with antibody against a common peptide (387-401) in SPS2. We detected selenophosphate as
the product of this active enzyme in the reaction mixture, composed of ATP, [75Se]H2Se and SPS. Chemically synthesized selenophosphate plays a role in Sec synthesis, not the addition of this enzyme. These results
support that the product of SPS2 is selenophosphate itself. During this investigation, the probable sequence of bovine SPS2
not having Sec was reported in the blast information and the molecular mass was near with the protein in this report. Thus,
bovine active SPS2 of molecular mass 33 kDa does not contain Sec.
K. Furumiya and K. Kanaya contributed equally to this work. 相似文献