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991.
992.
A quantitative histochemical method for assaying cytochrome c oxidase (COX) has been validated with two new findings concerning the optimal tissue thickness and a suitable substrate. The kinetics of a COX-catalysed reaction coupled to the oxidation of diaminobenzidine (DAB) were followed at 37 degrees C in single muscle fibres in unfixed sections of mouse gastrocnemius using a real-time image analysis system. The optimum composition of the substrate medium for the reaction was 0.1 mM reduced cytochrome c, 4 mM DAB, 2% dimethylsulphoxide, 2% polyvinyl alcohol and 0.1 mM HEPES buffer, final pH 7.5. The absorbances at 451 nm of the final reaction products, DAB polymer oxides, deposited in the intermyofibrillar mitochondria increased linearly as a function of incubation time for at least 80 s after the start of incubation. The initial velocities (v(i)) of the COX reaction calculated from the gradients of the linear regression best fits for times between 40 and 60 s were reproducible. The v(i) determined in single muscle fibres at a saturated concentration of cytochrome c (0.1 mM) were proportional to section thickness for thicknesses less than 3 microns, but they decreased exponentially when the thickness was greater than 4 microns. Thus, for the quantitative assay, unfixed sections 3 microns thick must be used. The Michaelis constants (Km) determined for commercial cytochrome c in the range of 20-26 microM for COX in three types of skeletal muscle fibres of mouse gastrocnemius were higher than the corresponding in situ Km (12-13 microM) for reduced cytochrome c. However, the Km values for commercial cytochrome c were in good agreement with the value previously determined with homogenates of rat hind limb muscle. Therefore, reduced cytochrome c is a more suitable substrate for the kinetic study and assay of COX in situ. 相似文献
993.
Hara K Maruki Y Long X Yoshino K Oshiro N Hidayat S Tokunaga C Avruch J Yonezawa K 《Cell》2002,110(2):177-189
mTOR controls cell growth, in part by regulating p70 S6 kinase alpha (p70alpha) and eukaryotic initiation factor 4E binding protein 1 (4EBP1). Raptor is a 150 kDa mTOR binding protein that also binds 4EBP1 and p70alpha. The binding of raptor to mTOR is necessary for the mTOR-catalyzed phosphorylation of 4EBP1 in vitro, and it strongly enhances the mTOR kinase activity toward p70alpha. Rapamycin or amino acid withdrawal increases, whereas insulin strongly inhibits, the recovery of 4EBP1 and raptor on 7-methyl-GTP Sepharose. Partial inhibition of raptor expression by RNA interference (RNAi) reduces mTOR-catalyzed 4EBP1 phosphorylation in vitro. RNAi of C. elegans raptor yields an array of phenotypes that closely resemble those produced by inactivation of Ce-TOR. Thus, raptor is an essential scaffold for the mTOR-catalyzed phosphorylation of 4EBP1 and mediates TOR action in vivo. 相似文献
994.
Assembly of G Protein-Coupled Receptors onto Nanosized Bacterial Magnetic Particles Using Mms16 as an Anchor Molecule 总被引:2,自引:3,他引:2 下载免费PDF全文
Tomoko Yoshino Masayoshi Takahashi Haruko Takeyama Yoshiko Okamura Fukuichi Kato Tadashi Matsunaga 《Applied microbiology》2004,70(5):2880-2885
G protein-coupled receptors (GPCRs) play a central role in a wide range of biological processes and are prime targets for drug discovery. GPCRs have large hydrophobic domains, and therefore purification of GPCRs from cells is frequently time-consuming and typically results in loss of native conformation. In this work, GPCRs have been successfully assembled into the lipid membrane of nanosized bacterial magnetic particles (BMPs) produced by the magnetic bacterium Magnetospirillum magneticum AMB-1. A BMP-specific protein, Mms16, was used as an anchor molecule, and localization of heterologous Mms16 on BMPs was confirmed by luciferase fusion studies. Stable luminescence was obtained from BMPs bearing Mms16 fused with luciferase at the C-terminal region. D1 dopamine receptor (D1R), a GPCR, was also efficiently assembled onto BMPs by using Mms16 as an anchor molecule. D1R-BMP complexes were simply extracted by magnetic separation from ruptured AMB-1 transformants. After washing, the complexes were ready to use for analysis. This system conveniently refines the native conformation of GPCRs without the need for detergent solubilization, purification, and reconstitution after cell disruption. 相似文献
995.
Microbial communities from methane hydrate-bearing deep marine sediments in a forearc basin 总被引:11,自引:0,他引:11
Reed DW Fujita Y Delwiche ME Blackwelder DB Sheridan PP Uchida T Colwell FS 《Applied and environmental microbiology》2002,68(8):3759-3770
Microbial communities in cores obtained from methane hydrate-bearing deep marine sediments (down to more than 300 m below the seafloor) in the forearc basin of the Nankai Trough near Japan were characterized with cultivation-dependent and -independent techniques. Acridine orange direct count data indicated that cell numbers generally decreased with sediment depth. Lipid biomarker analyses indicated the presence of viable biomass at concentrations greater than previously reported for terrestrial subsurface environments at similar depths. Archaeal lipids were more abundant than bacterial lipids. Methane was produced from both acetate and hydrogen in enrichments inoculated with sediment from all depths evaluated, at both 10 and 35 degrees C. Characterization of 16S rRNA genes amplified from the sediments indicated that archaeal clones could be discretely grouped within the Euryarchaeota and Crenarchaeota domains. The bacterial clones exhibited greater overall diversity than the archaeal clones, with sequences related to the Bacteroidetes, Planctomycetes, Actinobacteria, Proteobacteria, and green nonsulfur groups. The majority of the bacterial clones were either members of a novel lineage or most closely related to uncultured clones. The results of these analyses suggest that the microbial community in this environment is distinct from those in previously characterized methane hydrate-bearing sediments. 相似文献
996.
997.
Tanaka H Miake J Notsu T Sonyama K Sasaki N Iitsuka K Kato M Taniguchi S Igawa O Yoshida A Shigemasa C Hoshikawa Y Kurata Y Kuniyasu A Nakayama H Inagaki N Nanba E Shiota G Morisaki T Ninomiya H Kitakaze M Hisatome I 《Biochemical and biophysical research communications》2005,331(4):1001-1006
ATP-sensitive K+ channels (K(ATP):SUR2A+Kir6.2) play a pivotal role in cardiac protection against ischemia and reperfusion injury. When expressed in COS cells, Kir6.2 was short-lived with a half-life time of 1.9 h. The half-life time of Kir6.2 was prolonged by proteasome inhibitors MG132, ALLN, proteasome inhibitor 1, and lactacystine, but not at all by a lysosomal inhibitor chloroquine. MG132 also increased the level of ubiquitinated Kir6.2 without affecting its localization in the endoplasmic reticulum and Golgi apparatus. In electrophysiological recordings, MG132 augmented nicorandil-activated K(ATP) currents in COS cells expressing SUR2A and Kir6.2 as well as the same currents in neonatal rat cardiomyocytes. Like MG132, a Na+ channel blocker aprindine prolonged the half-life time of Kir6.2 and augmented K(ATP). Finally, both aprindine and MG132 inhibited the 20S proteasome activity in vitro. These results suggest a novel activity of aprindine to enhance K(ATP) currents by inhibiting proteasomal degradation of Kir 6.2 channels, which may be beneficial in the setting of cardiac ischemia. 相似文献
998.
Mikio Yagi Shinichiro Kato Yoshiko Kobayashi Kazuo Kubo Shinichi Oyama Toshiyuki Shimizu Tsuyoshi Nishitoba Toshiyuki Isoe Kazuhide Nakamura Hideya Ohashi Nami Kobayashi Noriko Iinuma Tatsushi Osawa Rie Onose Hiroyuki Osada 《Experimental cell research》1997,234(2):285
This report describes the biological effects of our original compound, Ki6783 ((3,4-dimethoxy)-4-phenoxy-6,7-dimethoxyquinoline), a potent and selective inhibitor of platelet-derived growth factor (PDGF) receptor autophosphorylation. This compound strongly inhibited autophosphorylation of the PDGF β-receptor in cultured rat glomerular mesangial cells (MC) bearing this receptor (IC500.1 μM), although it did not inhibit autophosphorylation of other growth factor receptors even at 100 μM.In a cell-free kinase experiment, it showed selective inhibition of PDGF β-receptor tyrosine kinase. A kinetic study of the compound to this tyrosine kinase revealed a competitive mode of action to ATP. [3H]Thymidine incorporation and cell proliferation of MC were inhibited by Ki6783 in a dose-dependent manner after Ki6783 and PDGF-BB were added to the culture medium. Furthermore, this compound normalized the fibrotic cell shape of v-sis-transformed NIH3T3 cells, which grow in an autocrine manner via the PDGF receptor. These effects could be explained by the inhibition of intracellular signal transduction triggered by PDGF receptor autophosphorylation, in which activation of mitogen-activated protein kinase occurs. These results suggest that Ki6783 is one of the more potent and selective inhibitors of PDGF receptor autophosphorylation and that it may be useful in ameliorating cell abnormalities due to excess action of PDGF and its receptor systems in several diseases. 相似文献
999.
Down-regulation of procaspase-8 expression by focal adhesion kinase protects HL-60 cells from TRAIL-induced apoptosis 总被引:1,自引:0,他引:1
Tamagiku Y Sonoda Y Kunisawa M Ichikawa D Murakami Y Aizu-Yokota E Kasahara T 《Biochemical and biophysical research communications》2004,323(2):445-452
We have demonstrated that focal adhesion kinase (FAK)-overexpressed (HL-60/FAK) cells have marked resistance against various apoptotic stimuli such as hydrogen peroxide, etoposide, and ionizing radiation compared with the vector-transfected (HL-60/Vect) cells. HL-60/FAK cells are highly resistant to TRAIL-induced apoptosis, while original HL-60 or HL-60/Vect cells were sensitive. TRAIL at 500 ng/ml induced significant DNA fragmentation, activation of caspase-8 and 3, the processing of a proapoptotic BID, and mitochondrial release of cytochrome c in HL-60/Vect cells, whereas no such events were observed in the HL-60/FAK cells. In particular, the expression of procaspase-8 gene and subsequent cleavage of caspase-8 were markedly reduced in HL-60/FAK cells, while expression of TRAIL-receptor 2 and 3, TRADD, and FADD was equivalent in both types of cells. In HL-60/FAK cells, the phosphoinositide 3 (PI3)-kinase/Akt survival pathway was constitutively activated, accompanied by significant induction of inhibitor-of-apoptosis proteins, XIAP, RIP, and Bcl-XL. The introduction of FAK siRNA in HL-60/FAK cells sensitized them against TRAIL-induced apoptosis, confirming that overexpressed FAK downregulates procaspase-8 expression, which subsequently inhibits downstream apoptosis pathway in the HL-60/FAK cells. 相似文献
1000.
Kikuchi R Sobue S Murakami M Ito H Kimura A Iwasaki T Shibayama S Takagi A Kojima T Suzuki M Banno Y Nozawa Y Murate T 《FEBS letters》2007,581(9):1800-1804
1α,25-Dihydroxyvitamin D3 (VitD3) increases protein and gene expression of phospholipase D1 (PLD1), but not PLD2, in HaCaT human keratinocytes. We show that VitD3 increases PLD1 gene expression through a vitamin D responsive element (VDRE) on the 5′ PLD1 promoter (−260 bp to −246 bp from exon 1). Similar results were obtained by transfecting VitD3 receptor (VDR) into HEK293 cells, which are originally VitD3-unresponsive. Electrophoresis mobility shift assays (EMSA) and chromatin immunoprecipitation (CHIP) assays showed that the complex of VitD3, VDR and retinoid X receptor α (RXRα) binds to the VDRE and increases PLD1 gene expression in HaCaT cells. 相似文献