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21.
Nishimura K Yoshihara F Tojima T Ooashi N Yoon W Mikoshiba K Bennett V Kamiguchi H 《The Journal of cell biology》2003,163(5):1077-1088
The cell adhesion molecule L1 (L1-CAM) plays critical roles in neurite growth. Its cytoplasmic domain (L1CD) binds to ankyrins that associate with the spectrin-actin network. This paper demonstrates that L1-CAM interactions with ankyrinB (but not with ankyrinG) are involved in the initial formation of neurites. In the membranous protrusions surrounding the soma before neuritogenesis, filamentous actin (F-actin) and ankyrinB continuously move toward the soma (retrograde flow). Bead-tracking experiments show that ankyrinB mediates L1-CAM coupling with retrograde F-actin flow in these perisomatic structures. Ligation of the L1-CAM ectodomain by an immobile substrate induces L1CD-ankyrinB binding and the formation of stationary ankyrinB clusters. Neurite initiation preferentially occurs at the site of these clusters. In contrast, ankyrinB is involved neither in L1-CAM coupling with F-actin flow in growth cones nor in L1-based neurite elongation. Our results indicate that ankyrinB promotes neurite initiation by acting as a component of the clutch module that transmits traction force generated by F-actin flow to the extracellular substrate via L1-CAM. 相似文献
22.
The MER3 protein of Saccharomyces cerevisiae is required for crossover in meiosis and has been suggested to act at the initiation of homologous pairing and the resolution of Holliday junctions. The purified MER3 protein is a DNA helicase that translocates along single-stranded DNA in the 3' to 5' direction displacing annealed DNA fragments. Here, MER3 was found to be able to unwind various double-stranded DNA (dsDNA) substrates, including a 30-bp dsDNA with a 20-nucleotide 3'-overhang, a 30-bp dsDNA with a 20-nucleotide 5'-overhang, a 50-bp dsDNA with blunt ends, and a Holliday junction with 25-bp arms, each of which had a blunt end. Efficient unwinding of the 3'-overhang substrate appeared to initiate by the binding of MER3 to the 3' single-stranded tail in a reaction that required six or more unpaired bases. Unwinding of the blunt end and 5'-overhang substrates appeared to initiate at the blunt ends of these substrates. Unwinding of the Holliday junction was more efficient than the unwinding of the blunt and 5'-overhang substrates and was influenced by Mg(2+) concentrations that cause changes in the structure of the junction. Possible roles for Holliday junction unwinding in meiotic crossover are discussed. 相似文献
23.
Sato Y Nabeta Y Tsukahara T Hirohashi Y Syunsui R Maeda A Sahara H Ikeda H Torigoe T Ichimiya S Wada T Yamashita T Hiraga H Kawai A Ishii T Araki N Myoui A Matsumoto S Umeda T Ishii S Kawaguchi S Sato N 《Journal of immunology (Baltimore, Md. : 1950)》2002,169(3):1611-1618
To investigate the immunogenic property of peptides derived from the synovial sarcoma-specific SYT-SSX fusion gene, we synthesized four peptides according to the binding motif for HLA-A24. The peptides, SS391 (PYGYDQIMPK) and SS393 (GYDQIMPKK), were derived from the breakpoint of SYT-SSX, and SS449a (AWTHRLRER) and SS449b (AWTHRLRERK) were from the SSX region. These peptides were tested for their reactivity with CTL precursors (CTLps) in 16 synovial sarcoma patients using HLA-A24/SYT-SSX peptide tetramers and also for induction of specific CTLs from four HLA-A24(+) synovial sarcoma patients. Tetramer analysis indicated that the increased CTLp frequency to the SYT-SSX was associated with pulmonary metastasis in synovial sarcoma patients (p < 0.03). CTLs were induced from PBLs of two synovial sarcoma patients using the peptide mixture of SS391 and SS393, which lysed HLA-A24(+) synovial sarcoma cells expressing SYT-SSX as well as the peptide-pulsed target cells in an HLA class I-restricted manner. These findings suggest that aberrantly expressed SYT-SSX gene products have primed SYT-SSX-specific CTLps in vivo and increased their frequency in synovial sarcoma patients. The identification of SYT-SSX peptides may offer an opportunity to design peptide-based immunotherapeutic approaches for HLA-A24(+) patients with synovial sarcoma. 相似文献
24.
In vitro gene transfection using dendritic poly(L-lysine) 总被引:3,自引:0,他引:3
Monodispersed dendritic poly(L-lysine)s (DPKs) of several generations were synthesized, and their characteristics as a gene transfection reagent were then investigated. The agarose gel shift and ethidium bromide titration assay proved that the DPKs of the third generation and higher could form a complex with a plasmid DNA, and the degree of compaction of the DNA was increased by the increasing number of the generation. The DPKs of the fifth and sixth generation, which have 64 and 128 amine groups on the surface of the molecule, respectively, showed efficient gene transfection ability into several cultivated cell lines without significant cytotoxity. In addition, the transfection efficiency of the DPK of the sixth generation was not seriously reduced even if serum was added at 50% of the final concentration into the transfection medium. Because we can strictly synthesize various DPK derivatives, which have several types of branch units, terminal cationic groups, and so on, they are expected to be a good object of study regarding the basic information on the detailed mechanism of gene transfection into cells. We also expect to be able to easily construct DPK-based functional gene carriers, e.g., DPKs modified by ligands such as a sugar chain, which can enable advanced gene delivery in vivo. 相似文献
25.
Kagawa HK Yaoi T Brocchieri L McMillan RA Alton T Trent JD 《Molecular microbiology》2003,49(1):143-156
The hyperthermoacidophilic archaeon Sulfolobus shibatae contains group II chaperonins, known as rosettasomes, which are two nine-membered rings composed of three different 60 kDa subunits (TF55 alpha, beta and gamma). We sequenced the gene for the gamma subunit and studied the temperature-dependent changes in alpha, beta and gamma expression, their association into rosettasomes and their phylogenetic relationships. Alpha and beta gene expression was increased by heat shock (30 min, 86 degrees C) and decreased by cold shock (30 min, 60 degrees C). Gamma expression was undetectable at heat shock temperatures and low at normal temperatures (75-79 degrees C), but induced by cold shock. Polyacrylamide gel electrophoresis indicated that in vitro alpha and beta subunits form homo-oligomeric rosettasomes, and mixtures of alpha, beta and gamma form hetero-oligomeric rosettasomes. Transmission electron microscopy revealed that beta homo-oligomeric rosettasomes and all hetero-oligomeric rosettasomes associate into filaments. In vivo rosettasomes were hetero-oligomeric with an average subunit ratio of 1alpha:1beta:0.1gamma in cultures grown at 75 degrees C, a ratio of 1alpha:3beta:1gamma in cultures grown at 60 degrees C and a ratio of 2alpha:3beta:0gamma after 86 degrees C heat shock. Using differential scanning calorimetry, we determined denaturation temperatures (Tm) for alpha, beta and gamma subunits of 95.7 degrees C, 96.7 degrees C and 80.5 degrees C, respectively, and observed that rosettasomes containing gamma were relatively less stable than those with alpha and/or beta only. We propose that, in vivo, the rosettasome structure is determined by the relative abundance of subunits and not by a fixed geometry. Furthermore, phylogenetic analyses indicate that archaeal chaperonin subunits underwent multiple duplication events within species (paralogy). The independent evolution of these paralogues raises the possibility that chaperonins have functionally diversified between species. 相似文献
26.
Riki Okita Diana Wolf Koichiro Yasuda Ai Maeda Takuro Yukawa Shinsuke Saisho Katsuhiko Shimizu Yoshiyuki Yamaguchi Mikio Oka Eiichi Nakayama Andreas Lundqvist Rolf Kiessling Barbara Seliger Masao Nakata 《PloS one》2015,10(10)
IntroductionSeveral cytotoxic anticancer drugs inhibit DNA replication and/or mitosis, while EGFR tyrosine kinase inhibitors inactivate EGFR signalling in cancer cell. Both types of anticancer drugs improve the overall survival of the patients with non-small-cell lung cancer (NSCLC), although tumors often become refractory to this treatment. Despite several mechanisms by which the tumors become resistant having been described the effect of these compounds on anti-tumor immunity remains largely unknown.MethodsThis study examines the effect of the cytotoxic drug Gemcitabine and the EGFR tyrosine kinase inhibitor Gefitinib on the expression of NK group 2 member D (NKG2D) ligands as well as the sensitivity of NSCLC cells to the NK-mediated lysis.ResultsWe demonstrate that Gemcitabine treatment leads to an enhanced expression, while Gefitinib downregulated the expression of molecules that act as key ligands for the activating receptor NKG2D and promote NK cell-mediated recognition and cytolysis. Gemcitabine activated ATM and ATM- and Rad-3-related protein kinase (ATR) pathways. The Gemcitabine-induced phosphorylation of ATM as well as the upregulation of the NKG2D ligand expression could be blocked by an ATM-ATR inhibitor. In contrast, Gefitinib attenuated NKG2D ligand expression. Silencing EGFR using siRNA or addition of the PI3K inhibitor resulted in downregulation of NKG2D ligands. The observations suggest that the EGFR/PI3K pathway also regulates the expression of NKG2D ligands. Additionally, we showed that both ATM-ATR and EGFR regulate MICA/B via miR20a.ConclusionIn keeping with the effect on NKG2D expression, Gemcitabine enhanced NK cell-mediated cytotoxicity while Gefitinib attenuated NK cell killing in NSCLC cells. 相似文献
27.
Mika Baba Takuro Shimbo Masaru Horio Masahiko Ando Yoshinari Yasuda Yasuhiro Komatsu Katsunori Masuda Seiichi Matsuo Shoichi Maruyama 《PloS one》2015,10(6)
Background
Chronic kidney disease is an important concern in preventive medicine, but the rate of decline in renal function in healthy population is not well defined. The purpose of this study was to determine reference values for the estimated glomerular filtration rate (eGFR) and rate of decline of eGFR in healthy subjects and to evaluate factors associated with this decline using a large cohort in Japan.Methods
Retrospective cross-sectional and longitudinal studies were performed with healthy subjects aged ≥18 years old who received a medical checkup. Reference values for eGFR were obtained using a nonparametric method and those for decline of eGFR were calculated by mixed model analysis. Relationships of eGFR decline rate with baseline variables were examined using a linear least-squares method.Results
In the cross-sectional study, reference values for eGFR were obtained by gender and age in 72,521 healthy subjects. The mean (±SD) eGFR was 83.7±14.7ml/min/1.73m2. In the longitudinal study, reference values for eGFR decline rate were obtained by gender, age, and renal stage in 45,586 healthy subjects. In the same renal stage, there was little difference in the rate of decline regardless of age. The decline in eGFR depended on the renal stage and was strongly related to baseline eGFR, with a faster decline with a higher baseline eGFR and a slower decline with a lower baseline eGFR. The mean (±SD) eGFR decline rate was ‒1.07±0.42ml/min/1.73m2/year (‒1.29±0.41%/year) in subjects with a mean eGFR of 81.5±11.6ml/min/1.73m2.Conclusions
The present study clarified for the first time the reference values for the rate of eGFR decline stratified by gender, age, and renal stage in healthy subjects. The rate of eGFR decline depended mainly on baseline eGFR, but not on age, with a slower decline with a lower baseline eGFR. 相似文献28.
29.
Shunsuke Ohashi Tatsuya Iemura Naoki Okada Shingo Itoh Hayato Furukawa Masaaki Okuda Mayumi Ohnishi-Kameyama Takuro Ogawa Hideaki Miyashita Tadashi Watanabe Shigeru Itoh Hirozo Oh-oka Kazuhito Inoue Masami Kobayashi 《Photosynthesis research》2010,104(2-3):305-319
Minor but key chlorophylls (Chls) and quinones in photosystem (PS) I-type reaction centers (RCs) are overviewed in regard to their molecular structures. In the PS I-type RCs, the prime-type chlorophylls, namely, bacteriochlorophyll (BChl) a′ in green sulfur bacteria, BChl g′ in heliobacteria, Chl a′ in Chl a-type PS I, and Chl d′ in Chl d-type PS I, function as the special pairs, either as homodimers, (BChl a′)2 and (BChl g′)2 in anoxygenic organisms, or heterodimers, Chl a/a′ and Chl d/d′ in oxygenic photosynthesis. Conversions of BChl g to Chl a and Chl a to Chl d take place spontaneously under mild condition in vitro. The primary electron acceptors, A 0, are Chl a-derivatives even in anoxygenic PS I-type RCs. The secondary electron acceptors are naphthoquinones, whereas the side chains may have been modified after the birth of cyanobacteria, leading to succession from menaquinone to phylloquinone in oxygenic PS I. 相似文献
30.
GTP binding is essential to the protein kinase activity of LRRK2, a causative gene product for familial Parkinson's disease 总被引:5,自引:0,他引:5
Leucine-rich repeat kinase 2 (LRRK2), a product of a causative gene for the autosomal-dominant form of familial Parkinson's disease (PARK8), harbors a Ras-like small GTP binding protein-like (ROC) domain besides the kinase domain, although the relationship between these two functional domains remains elusive. Here we show by thin-layer chromatographic analysis that LRRK2 stably binds GTP but lacks a GTPase activity in HEK293 and Neuro-2a cells. A ROC domain mutation that converts LRRK2 to a guanine nucleotide-free form (T1348N) abolishes the kinase activity of LRRK2 as well as its phosphate incorporation upon metabolic labeling. The phosphorylation of LRRK2 was inhibited by potential inhibitors for cyclic AMP-dependent protein kinase. These data suggest that binding of GTP to the ROC domain regulates the kinase activity of LRRK2 as well as its phosphorylation by other kinase(s). 相似文献