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121.
Tadashi Satoh Eri Sakata Shunsuke Yamamoto Yoshiki Yamaguchi Akira Sumiyoshi Koichi Kato 《Biochemical and biophysical research communications》2010,400(3):329-333
Lys48-linked polyubiquitin chains serve as a signal for protein degradation by 26S proteasomes through its Ile44 hydrophobic patches interactions. The individual ubiquitin units of each chain are conjugated through an isopeptide bond between Lys48 and the C-terminal Gly76 of the preceding units. The conformation of Lys48-linked tetraubiquitin has been shown to change dynamically depending on solution pH. Here we enzymatically synthesized a wild-type Lys48-linked tetraubiquitin for structural study. In the synthesis, cyclic and non-cyclic species were obtained as major and minor fractions, respectively. This enabled us to solve the crystal structure of tetraubiquitin exclusively with native Lys48-linkages at 1.85 Å resolution in low pH 4.6. The crystallographic data clearly showed that the C-terminus of the first ubiquitin is conjugated to the Lys48 residue of the fourth ubiquitin. The overall structure is quite similar to the closed form of engineered tetraubiquitin at near-neutral pH 6.7, previously reported, in which the Ile44 hydrophobic patches face each other. The structure of the second and the third ubiquitin units [Ub(2)-Ub(3)] connected through a native isopeptide bond is significantly different from the conformations of the corresponding linkage of the engineered tetraubiquitins, whereas the structures of Ub(1)-Ub(2) and Ub(3)-Ub(4) isopeptide bonds are almost identical to those of the previously reported structures. From these observations, we suggest that the flexible nature of the isopeptide linkage thus observed contributes to the structural arrangements of ubiquitin chains exemplified by the pH-dependent closed-to-open conformational transition of tetraubiquitin. 相似文献
122.
Ryo Nagao Tatsuya Tomo Eri Noguchi Takehiro Suzuki Yasuhiro Kashino Masahiko Ikeuchi 《BBA》2010,1797(2):160-166
Oxygen-evolving Photosystem II particles (crude PSII) retaining a high oxygen-evolving activity have been prepared from a marine centric diatom, Chaetoceros gracilis (Nagao et al., 2007). The crude PSII, however, contained a large amount of fucoxanthin chlorophyll a/c-binding proteins (FCP). In this study, a purified PSII complex which was deprived of major components of FCP was isolated by one step of anion exchange chromatography from the crude PSII treated with Triton X-100. The purified PSII was still associated with the five extrinsic proteins of PsbO, PsbQ', PsbV, Psb31 and PsbU, and showed a high oxygen-evolving activity of 2135 μmol O2 (mg Chl a)− 1 h− 1 in the presence of phenyl-p-benzoquinone which was virtually independent of the addition of CaCl2. This activity is more than 2.5-fold higher than the activity of the crude PSII. The activity was completely inhibited by 3-(3,4)-dichlorophenyl-(1,1)-dimethylurea (DCMU). The purified PSII contained 42 molecules of Chl a, 2 molecules of diadinoxanthin and 2 molecules of Chl c on the basis of two molecules of pheophytin a, and showed typical absorption and fluorescence spectra similar to those of purified PSIIs from the other organisms. In this study, we also found that the crude PSII was significantly labile, as a significant inactivation of oxygen evolution, chlorophyll bleaching and degradation of PSII subunits were observed during incubation at 25 °C in the dark. In contrast, these inactivation, bleaching and degradation were scarcely detected in the purified PSII. Thus, we succeeded for the first time in preparation of a stable PSII from diatom cells. 相似文献
123.
Eri Suzuki Hiroto Okuda Kentaro Nishida Sadaki Fujimoto Kazuki Nagasawa 《Life sciences》2010,86(17-18):676-682
AimPoly(ADP-ribose) polymerase-1 (PARP-1) is a DNA repair enzyme, and its excessive activation, following ischemia, trauma, etc., depletes cellular nicotinamide adenine dinucleotide (NAD+) as a substrate and eventually leads to brain cell death. Nicotinamide, an NAD+ precursor and a PARP-1 inhibitor, is known to prevent PARP-1-triggered cell death, but there is no available information on the mechanisms involved in its transport. Here we clarified the transport characteristics of nicotinamide in primary cultured mouse astrocytes.Main methodsUptake characteristics of [14C]nicotinamide were assessed by a conventional method with primary cultured mouse astrocytes. Cell viability and PARP-1 activity were determined with intracellular LDH activity and immunocytochemical detection of PAR accumulation, respectively.Key findingsPARP-1 activation was induced by treatment of astrocytes with N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), an alkylating agent. MNNG-triggered astrocyte death and PAR accumulation were completely inhibited by treatment with nicotinamide as with DPQ (3,4-dihydro-5-(4-(1-piperidinyl)butoxy)-1(2H)-isoquinolinone), a second generation PARP inhibitor. The uptake of [14C]nicotinamide was time-, temperature-, concentration- and pH-dependent, and was inhibited and stimulated by co- and pre-treatment with N-methylnicotinamide, a representative substrate of an organic cation transport system, respectively. Co-treatment of astrocytes with nicotinamide and N-methylnicotinamide resulted in a decrease in PAR accumulation and absolute prevention of cell death.SignificanceThese findings suggest that nicotinamide has a protective effect against PARP-1-induced astrocyte death and that its transporter-mediated uptake, which is extracellular pH-sensitive and common to N-methylnicotinamide, is critical for prevention of PARP-1-triggered cell death. 相似文献
124.
Vif counteracts a cyclophilin A-imposed inhibition of simian immunodeficiency viruses in human cells
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Takeuchi H Buckler-White A Goila-Gaur R Miyagi E Khan MA Opi S Kao S Sokolskaja E Pertel T Luban J Strebel K 《Journal of virology》2007,81(15):8080-8090
Vif is a primate lentiviral accessory protein that is crucial for viral infectivity. Vif counteracts the antiviral activity of host deaminases such as APOBEC3G and APOBEC3F. We now report a novel function of African green monkey simian immunodeficiency virus (SIVagm) Vif that promotes replication of SIVagm in human cells lacking detectable deaminase activity. We found that cyclophilin A (CypA) was excluded from wild-type SIV particles but was efficiently packaged into vif-deficient SIVagm virions. The presence of CypA in vif-defective SIVagm was correlated with reduced viral replication. Infection of CypA knockout Jurkat cells or treatment of Jurkat cells with cyclosporine A eliminated the Vif-sensitive inhibition and resulted in replication profiles that were similar for wild-type and vif-deficient SIVagm. Importantly, the inhibitory effect of CypA was restricted to virus-producing cells and was TRIM5alpha independent. The abilities of SIVagm Vif to inhibit encapsidation of CypA and to increase viral infectivity were shared by rhesus macaque SIV Vif and thus seem to be general properties of SIV Vif proteins. Exclusion of CypA from SIVagm particles was not associated with intracellular degradation, suggesting a mode of Vif action distinct from that proposed for APOBEC3G. This is the first report of a novel vif-sensitive antiviral activity of human CypA that may limit zoonotic transmission of SIV and the first demonstration of CypA encapsidation into a virus other than human immunodeficiency virus type 1. 相似文献
125.
A longer polyalanine expansion mutation in the ARX gene causes early infantile epileptic encephalopathy with suppression-burst pattern (Ohtahara syndrome) 总被引:1,自引:0,他引:1
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Kato M Saitoh S Kamei A Shiraishi H Ueda Y Akasaka M Tohyama J Akasaka N Hayasaka K 《American journal of human genetics》2007,81(2):361-366
Early infantile epileptic encephalopathy with suppression-burst pattern (EIEE) is one of the most severe and earliest forms of epilepsy, often evolving into West syndrome; however, the pathogenesis of EIEE remains unclear. ARX is a crucial gene for the development of interneurons in the fetal brain, and a polyalanine expansion mutation of ARX causes mental retardation and seizures, including those of West syndrome, in males. We screened the ARX mutation and found a hemizygous, de novo, 33-bp duplication in exon 2, 298_330dupGCGGCA(GCG)9, in two of three unrelated male patients with EIEE. This mutation is thought to expand the original 16 alanine residues to 27 alanine residues (A110_A111insAAAAAAAAAAA) in the first polyalanine tract of the ARX protein. Although EIEE is mainly associated with brain malformations, ARX is the first gene found to be responsible for idiopathic EIEE. Our observation that EIEE had a longer expansion of the polyalanine tract than is seen in West syndrome is consistent with the findings of earlier onset and more-severe phenotypes in EIEE than in West syndrome. 相似文献
126.
LAT and NTAL mediate immunoglobulin E-induced sustained extracellular signal-regulated kinase activation critical for mast cell survival
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Yamasaki S Ishikawa E Sakuma M Kanagawa O Cheng AM Malissen B Saito T 《Molecular and cellular biology》2007,27(12):4406-4415
Immunoglobulin E (IgE) induces mast cell survival in the absence of antigen (Ag) through the high-affinity IgE receptor, Fcepsilon receptor I (FcepsilonRI). Although we have shown that protein tyrosine kinase Syk and sustained extracellular signal-regulated kinase (Erk) activation are required for IgE-induced mast cell survival, how Syk couples with sustained Erk activation is still unclear. Here, we report that the transmembrane adaptors LAT and NTAL are phosphorylated slowly upon IgE stimulation and that sustained but not transient Erk activation induced by IgE was inhibited in LAT(-/-) NTAL(-/-) bone marrow-derived mast cells (BMMCs). IgE-induced survival requires Ras activation, and both were impaired in LAT(-/-) NTAL(-/-) BMMCs. Sos was preferentially required for FcepsilonRI signals by IgE rather than IgE plus Ag. Survival impaired in LAT(-/-) NTAL(-/-) BMMCs was restored to levels comparable to those of the wild type by membrane-targeted Sos, which bypasses the Grb2-mediated membrane recruitment of Sos. The IgE-induced survival of BMMCs lacking Gads, an adaptor critical for the formation of the LAT-SLP-76-phospholipase Cgamma (PLCgamma) complex, was observed to be normal. IgE stimulation induced the membrane retention of Grb2-green fluorescent protein fusion proteins in wild-type but not LAT(-/-) NTAL(-/-) BMMCs. These results suggest that LAT and NTAL contribute to the maintenance of Erk activation and survival through the membrane retention of the Ras-activating complex Grb2-Sos and, further, that the LAT-Gads-SLP-76-PLCgamma and LAT/NTAL-Grb2-Sos pathways are differentially required for degranulation and survival, respectively. 相似文献
127.
Analysis of risk epitopes of anti-neutrophil antibody MPO-ANCA in vasculitis in Japanese population 总被引:1,自引:0,他引:1
Suzuki K Kobayashi S Yamazaki K Gondo M Tomizawa K Arimura Y Nakabayashi K Ozaki S Yoshida M Yoshida T Tsusaka N Muso E Okazaki T Hashimoto H 《Microbiology and immunology》2007,51(12):1215-1220
Autoantibodies to myeloperoxidase (MPO) are a subset of anti-neutrophil cytoplasmic antibody (ANCA, MPO-ANCA) detected in the sera of some patients with primary systemic vasculitis. The titer of MPO-ANCA does not always reflect disease activity and this inconsistency may be attributable to differences in epitopic specificity by MPO-ANCA among various patients with vasculitis. Epitope analysis may also explain the occurrence of MPO-ANCA in different vasculitic syndromes. We screened the sera of 148 MPO-ANCA positive patients from six vasculitic syndromes: rapidly progressive gromerulonephritis (RPGN), microscopic polyangiitis (MPA), idiopathic crescentic glomerulonephritis (I-CrGN), classic polyangiitis nodosa (cPAN), Churg-Strauss syndrome (CSS), Kawasaki disease (KD); and from patients with rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). The sera were collected by the Intractable Vasculitis Research Project Group in Japan. No serum showed epitopes La and Lb of light chain of MPO, and sera with 68.6% of patients showed a positive reaction to one or more epitopes in heavy chain of MPO. Analysis of binding level showed that RPGN, I-CrGN and MPA sera mainly reacted to the Ha epitope at the N-termimus of the MPO heavy chain, CSS sera reacted to Ha and the Hf epitope close to the C-terminus of the MPO heavy chain, KD reacted mainly to Hf, while SLE and RA sera reacted to all epitopes. These results suggest that MPO-ANCA recognizing specific regions of the N-terminus of the MPO H-chain confer an increased risk of vasculitis RPGN, I-CrGN, MPA and CSS. Furthermore, the epitopic specificity of MPO-ANCA differentiates vasculitic from non-vasculitic syndromes associated with MPO-ANCA positivity and differentiates in the cirtain type of vasculitis from various vasculitic syndromes. In particular, vasculitic syndromes associated with kidney involvement had similar epitopic reactivity which suggests that this pattern confers an increased risk of vasculitis. 相似文献
128.
RNA interference (RNAi) has been used to suppress gene expression in various eukaryotic organisms. In plants, RNAi can be
induced by introduction of an RNAi vector that transcribes a self-complementary hairpin RNA. Most basic RNAi constructs have
an inverted repeat interrupted with a spacer sequence. To test silencing capability of RNAi constructs, we developed an in
vivo assay that is based on the RNAi-mediated changes of the α-linolenic acid content in hairy roots. A tobacco endoplasmic
reticulum ω-3 fatty acid desaturase (NtFAD3) is the main enzyme for production of α-linolenic acid of root membrane lipids.
Tobacco hairy roots transformed with the RNAi vectors against the NtFAD3 gene showed a decrease in α-linolenic acid content. The frequency of RNA silencing was more affected by spacer sequence than
by spacer length, at least between 100 and 1800 bp. Since significant amounts of hairpin RNA against the NtFAD3 gene remained in the transgenic plants displaying a weak silencing phenotype, low degree of silencing was attributed to low
efficiency of hairpin RNA processing mediated by Dicer-like proteins. Our results show the possibility of producing a broad
range of the RNAi-induced silencing phenotypes by replacing the spacer sequence of RNAi construct. 相似文献
129.
Hirokawa YS Takagi A Uchida K Kozuka Y Yoneda M Watanabe M Shiraishi T 《Cellular & molecular biology letters》2007,12(3):370-377
In this paper, we describe the isolation and characterization of two PC3 subclones. One subclone, mr, showed an epithelial
phenotype, the other, M1, showed a sarcomatous morphology. Transplanted into nude mice, mr developed tumors at a dramatically
faster rate than M1. Comparing the two subclones, differentially expressed genes were identified, including E-cadherin, IL-8
and STAG1/PMEPA1. These genes were expressed at higher levels in mr than in M1. 相似文献
130.