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81.
Reduced sensitivity of Niemann-Pick C1-deficient cells to θ-toxin (perfringolysin O): sequestration of toxin to raft-enriched membrane vesicles 总被引:1,自引:1,他引:0
Ohsaki Y Sugimoto Y Suzuki M Kaidoh T Shimada Y Ohno-Iwashita Y Davies JP Ioannou YA Ohno K Ninomiya H 《Histochemistry and cell biology》2004,121(4):263-272
-Toxin (perfringolysin O) binds to cell surface cholesterol and forms oligomeric pores that cause membrane damage. Both in cytotoxicity and cell survival assays, a mutant Chinese hamster ovary cell line NPC1(–) that lacked Niemann-Pick C1 showed reduced sensitivity to -toxin, compared with wild-type (wt) cells. BC is a derivative of -toxin that retains cholesterol-binding activity but lacks cytotoxicity. Confocal and electron microscopy revealed the presence of multiple vesicles which bound BC, both on the cell surface and in the extracellular space of these cells. BC binding to raft microdomains was verified by its resistance to 1% Triton X-100 at 4°C and recovery of bound BC in floating low-density fractions on sucrose density gradient fractionation. BC-labeled vesicles were abolished when NPC1(–) cells were depleted of lipoproteins and also when treated with a Rho-associated kinase inhibitor Y-27632. In addition, similar vesicles were observed in wt cells treated with progesterone. In parallel with these results, -toxin sensitivity of NPC1(–) cells was increased when cells were depleted of lipoproteins or treated with Y-27632, whereas that of wt cells was decreased by progesterone. Our findings suggest that sequestration of toxin to raft-enriched cell surface vesicles may underlie reduced sensitivity of NPC1-deficient cells to -toxin. 相似文献
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83.
Quantitative PCR with 16S rRNA-gene-targeted species-specific primers for analysis of human intestinal bifidobacteria 总被引:18,自引:0,他引:18
Matsuki T Watanabe K Fujimoto J Kado Y Takada T Matsumoto K Tanaka R 《Applied and environmental microbiology》2004,70(1):167-173
A highly sensitive quantitative PCR detection method has been developed and applied to the distribution analysis of human intestinal bifidobacteria by combining real-time PCR with Bifidobacterium genus- and species-specific primers. Real-time PCR detection of serially diluted DNA extracted from cultured bifidobacteria was linear for cell counts ranging from 10(6) to 10 cells per PCR assay. It was also found that the method was applicable to the detection of Bifidobacterium in feces when it was present at concentrations of >10(6) cells per g of feces. Concerning the distribution of Bifidobacterium species in intestinal flora, the Bifidobacterium adolescentis group, the Bifidobacterium catenulatum group, and Bifidobacterium longum were found to be the three predominant species by examination of DNA extracted from the feces of 46 healthy adults. We also examined changes in the population and composition of Bifidobacterium species in human intestinal flora of six healthy adults over an 8-month period. The results showed that the composition of bifidobacterial flora was basically stable throughout the test period. 相似文献
84.
Morita N Iizuka K Okita K Oikawa T Yonezawa K Nagai T Tokumitsu Y Murakami T Kitabatake A Kawaguchi H 《American journal of physiology. Endocrinology and metabolism》2004,287(6):E1064-E1069
Contraction of skeletal muscle generates pressure stimuli to intramuscular tissues. However, the effects of pressure stimuli, other than those created by electricity or nerve impulse, on physiological and biochemical responses in skeletal muscles are unknown. The purpose of this study is to examine the effects of a pure pressure stimulus on metabolic responses in a skeletal muscle cell line. Atmospheric pressure was applied to L6 myoblasts using an original apparatus. Succinate dehydrogenase (SDH) activity was evaluated by colorimetric assay using tetrazolium monosodium salt. The amounts of 2-deoxy-[(3)H]glucose uptake and lactate release were measured. SDH activity was 2.6- to 2.9-fold higher in pressurized L6 cells than in nonpressurized L6 cells (P < 0.01), and 2-deoxy-[(3)H]glucose uptake was 2.2-fold higher (P < 0.001). In addition, the amount of released lactate decreased from 6.8 to 3.7 mumol/dish when pressure was applied (P < 0.001). In contrast, the intracellular lactate contents of the pressurized cells were higher than those of nonpressurized cells (P < 0.01). However, the total amount of released lactate and intracellular lactate was lower in the pressurized cells than in nonpressurized cells. These findings demonstrate that a pure pressure stimulus enhances aerobic metabolism in L6 skeletal muscle cells and raise the possibility that elevated intramuscular pressure during muscle activity may be an important factor in stimulating oxidative metabolic responses in skeletal muscles. 相似文献
85.
Mizushima T Hirao T Yoshida Y Lee SJ Chiba T Iwai K Yamaguchi Y Kato K Tsukihara T Tanaka K 《Nature structural & molecular biology》2004,11(4):365-370
SCF(Fbs1) is a ubiquitin ligase that functions in the endoplasmic reticulum (ER)-associated degradation pathway. Fbs1/Fbx2, a member of the F-box proteins, recognizes high-mannose oligosaccharides. Efficient binding to an N-glycan requires di-N-acetylchitobiose (chitobiose). Here we report the crystal structures of the sugar-binding domain (SBD) of Fbs1 alone and in complex with chitobiose. The SBD is composed of a ten-stranded antiparallel beta-sandwich. The structure of the SBD-chitobiose complex includes hydrogen bonds between Fbs1 and chitobiose and insertion of the methyl group of chitobiose into a small hydrophobic pocket of Fbs1. Moreover, NMR spectroscopy has demonstrated that the amino acid residues adjoining the chitobiose-binding site interact with the outer branches of the carbohydrate moiety. Considering that the innermost chitobiose moieties in N-glycans are usually involved in intramolecular interactions with the polypeptide moieties, we propose that Fbs1 interacts with the chitobiose in unfolded N-glycoprotein, pointing the protein moiety toward E2 for ubiquitination. 相似文献
86.
The eukaryotic 20S proteasome is the multifunctional catalytic core of the 26S proteasome, which plays a central role in intracellular protein degradation. Association of the 20S core with a regulatory subcomplex, termed PA700 (also known as the 19S cap), forms the 26S proteasome, which degrades ubiquitinated and nonubiquitinated proteins through an ATP-dependent process. Although proteolytic assistance by this regulatory particle is a general feature of proteasome-dependent turnover, the 20S proteasome itself can degrade some proteins directly, bypassing ubiquitination and PA700, as an alternative mechanism in vitro. The mechanism underlying this pathway is based on the ability of the 20S proteasome to recognize partially unfolded proteins. Here we show that the 20S proteasome recognizes the heat-denatured forms of model proteins such as citrate synthase, malate dehydrogenase. and glyceraldehydes-3-phosphate dehydrogenase, and prevents their aggregation in vitro. This process was not followed by the refolding of these denatured substrates into their native states, whereas PA700 or the 26S proteasome generally promotes their reactivation. These results indicate that the 20S proteasome might play a role in maintaining denatured and misfolded substrates in a soluble state, thereby facilitating their refolding or degradation. 相似文献
87.
88.
Kimura Y Toyoshima N Hirakawa N Okamoto K Ishijima A 《Journal of molecular biology》2003,328(4):939-950
Endoplasmic streaming of characean cells of Nitella or Chara is known to be in the range 30-100 microm/second. The Chara myosin extracted from the cells and fixed onto a glass surface was found to move muscle actin filaments at a velocity of 60 microm/second. This is ten times faster than that of skeletal muscle myosin (myosin II). In this study, the displacement caused by single Chara myosin molecules was measured using optical trapping nanometry. The step size of Chara myosin was approximately 19nm. This step size is longer than that of skeletal muscle myosin but shorter than that of myosin V. The dwell time of the steps was relatively long, and this most likely resulted from two rate-limiting steps, the dissociation of ADP and the binding of ATP. The rate of ADP release from Chara myosin after the completion of the force-generation step was similar to that of myosin V, but was considerably slower than that of skeletal muscle myosin. The 19nm step size and the dwell time obtained could not explain the fast movement. The fast movement could be explained by the load-dependent release of ADP. As the load imposed on the myosin decreased, the rate of ADP release increased. We propose that the interaction of Chara myosin with an actin filament resulted in a negative load being imposed on other myosin molecules interacting with the same actin filament. This resulted in an accelerated release of ADP and the fast sliding movement. 相似文献
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90.