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101.
Cryo-electron microscopy of vitreous sections 总被引:10,自引:0,他引:10
Al-Amoudi A Chang JJ Leforestier A McDowall A Salamin LM Norlén LP Richter K Blanc NS Studer D Dubochet J 《The EMBO journal》2004,23(18):3583-3588
Since the beginning of the 1980s, cryo-electron microscopy of a thin film of vitrified aqueous suspension has made it possible to observe biological particles in their native state, in the absence of the usual artefacts of dehydration and staining. Combined with 3-d reconstruction, it has become an important tool for structural molecular biology. Larger objects such as cells and tissues cannot generally be squeezed in a thin enough film. Cryo-electron microscopy of vitreous sections (CEMOVIS) provides then a solution. It requires vitrification of a sizable piece of biological material and cutting it into ultrathin sections, which are observed in the vitrified state. Each of these operations raises serious difficulties that have now been overcome. In general, the native state seen with CEMOVIS is very different from what has been seen before and it is seen in more detail. CEMOVIS will give its full potential when combined with computerized electron tomography for 3-d reconstruction. 相似文献
102.
Davezac N Tondelier D Lipecka J Fanen P Demaugre F Debski J Dadlez M Schrattenholz A Cahill MA Edelman A 《Proteomics》2004,4(12):3833-3844
Cystic fibrosis (CF) is a genetic disease caused by mutations in the CF gene (cftr). Physiologically, CF is characterized by an abnormal chloride secretion in epithelia due to a dysfunction of a mutated cystic fibrosis transmembrane conductance regulator (CFTR). CFTR is a cAMP-dependent chloride channel whose most frequent mutation, deltaF508, leads to an aberrantly folded protein which causes a dysfunction of the channel. However, a growing number of reports suggest that modifier genes and environmental factors are involved in the physiology of CF. To identify proteins whose expression depends on wild-type WT-CFTR or deltaF508-CFTR, we chose a global proteomic approach based on the use of two-dimensional gel electrophoresis (2-DE) and mass spectrometry. The experiments were carried out with HeLa cells stably transfected with WT-CFTR (pTCFWT) or deltaF508-CFTR (pTCFdeltaF508). These experiments unmasked keratin 8 (K8) and 18 (K18) which were differentially expressed in pTCFWT vs. pTCFdeltaF508. An immunoblot of K18 confirmed the 2-DE results. Intracellular localization experiments of WT-CFTR, deltaF508-CFTR, K8, and K18 suggest that the expression of these proteins are linked, and that the concentrations of K8 and K18 and/or their distribution may be involved in the traffic of WT-CFTR/deltaF508-CFTR. A functional assay for CFTR revealed that specifically lowering K18 expression or changing its distribution leads to the delivery of functional deltaF508-CFTR to the plasma membrane. This work suggests a novel function of K18 in CF. 相似文献
103.
The increasingly stringent requirements laid down by regulatory authorities have brought to an end the largely empirical design of vaccines. Vaccines must now be designed rationally, in order that appropriate immune responses are elicited with few or no side effects. The DC plays a pivotal role in determining the type of immune response that ensues following exposure of the host to an Ag. In this review, we identify some of the features and properties of DCs, and how these properties can be exploited in the design of smart vaccines. 相似文献
104.
Prado S Michel S Tillequin F Koch M Pfeiffer B Pierré A Léonce S Colson P Baldeyrou B Lansiaux A Bailly C 《Bioorganic & medicinal chemistry》2004,12(14):3943-3953
Fagaronine and nitidine are natural benzo[c] phenanthridinium alkaloids, which display antileukemic activity. Both act as topoisomerase I and topoisomerase II inhibitors. The objective of the present study was to prepare noncharged isosters of these compounds, with replacement of the aromatic A ring by a pyridine ring, present in other topoisomerase I inhibitors. Various 7,8- and 8,9-dimethoxy and metylenedioxy benzo[c][1,7] and [1,8]phenanthrolines were readily synthesized by benzyne-mediated cyclization of the corresponding substituted N-(2-halobenzyl)-5-quinolinamines or 5-isoquinolinamines. In both series, compounds bearing oxygenated substituents at positions 8 and 9 exhibited cytotoxic properties towards L1210 murine leukemia cells, which may result from their capacities to intercalate into DNA. Topoisomerase I inhibition was observed for all active compounds. 相似文献
105.
The aim of this study was to establish the requirements for macrophage activating factor (MAF) production by sea bass head-kidney leucocytes and the kinetics of macrophage activation when exposed to MAF-containing supernatants and/or lipopolysaccharide (LPS), a known macrophage stimulant. MAF activity was found in culture supernatants of total head-kidney leucocytes pulsed with 5 microg ml(-1)Con A, 5 or 10 ng ml(-1)PMA and 100 ng ml(-1)calcium ionophore, or 10 microg ml(-1)Con A alone, as assessed by the capacity to prime macrophages for enhanced production of reactive oxygen intermediates (ROI). Mixed leucocyte cultures from two or eight fish showed higher MAF activity after stimulation, indicating that a mixed leucocyte reaction was also important for MAF production. MAF-induced activation of macrophage cultures was highest at 18 h of exposure and was lost by 72 h except for MAF induced by Con A-stimulation alone. LPS primed macrophages for increased ROI production at early incubation times and down-regulated ROI production after 24 h. LPS had no effect in further stimulating the MAF-induced priming effect on production of ROI and down-regulated the MAF-priming by 48 h. Sea bass head-kidney macrophages did not show increased nitrite production when exposed to MAF and/or LPS, which may be related to their differentiation status. 相似文献
106.
A novel engineered meganuclease induces homologous recombination in yeast and mammalian cells 总被引:17,自引:12,他引:5
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Epinat JC Arnould S Chames P Rochaix P Desfontaines D Puzin C Patin A Zanghellini A Pâques F Lacroix E 《Nucleic acids research》2003,31(11):2952-2962
Homologous gene targeting is the ultimate tool for reverse genetics, but its use is often limited by low efficiency. In a number of recent studies, site- specific DNA double-strand breaks (DSBs) have been used to induce efficient gene targeting. Engineering highly specific, dedicated DNA endonucleases is the key to a wider usage of this technology. In this study, we present two novel, chimeric meganucleases, derived from homing endonucleases. The first one is able to induce recombination in yeast and mammalian cells, whereas the second cleaves a novel (chosen) DNA target site. These results are a first step toward the generation of custom endonucleases for the purpose of targeted genome engineering. 相似文献
107.
Morin A Huysveld N Braun F Dimova D Sakanyan V Charlier D 《Journal of molecular biology》2003,332(3):537-553
The degree of sequence conservation of arginine repressor proteins (ArgR) and of the cognate operators (tandem pairs of 18 bp imperfect palindromes, ARG boxes) in evolutionarily distant bacteria is unusually high, and the global mechanism of ArgR-mediated regulation appears to be similar. However, here we demonstrate that the arginine repressor from the hyperthermophilic bacterium Thermotoga neapolitana (ArgR(Tn)) exhibits characteristics that clearly distinguish this regulator from the well-studied homologues from Escherichia coli, Bacillus subtilis and B.stearothermophilus. A high-resolution contact map of ArgR(Tn) binding to the operator of the biosynthetic argGHCJBD operon of Thermotoga maritima indicates that ArgR(Tn) establishes all of its strong contacts with a single ARG box-like sequence of the operator only. Protein array and electrophoretic mobility-shift data demonstrate that ArgR(Tn) has a remarkable capacity to bind to arginine operators from Gram-negative and Gram-positive bacteria, and to single ARG box-bearing targets. Moreover, the overall effect of L-arginine on the apparent K(d) of ArgR(Tn) binding to various cognate and heterologous operator fragments was minor with respect to that observed with diverse bacterial arginine repressors. We demonstrate that this unusual behaviour for an ArgR protein can, to a large extent, be ascribed to the presence of a serine residue at position 107 of ArgR(Tn), instead of the highly conserved glutamine that is involved in arginine binding in the E.coli repressor. Consistent with these results, ArR(Tn) was found to behave as a superrepressor in E.coli, inhibiting growth in minimal medium, even supplemented with arginine, whereas similar constructs bearing the S107Q mutant allele did not inhibit growth. We assume that ArgR(Tn), owing to its broad target specificity and its ability to bind single ARG box sequences, might play a more general regulatory role in Thermotoga 相似文献
108.
109.
Small alluvial gravels (some millimetres up to 40 mm) were selected and carried by mesolithic peoples to their settlement where they use them in fire related activities. These small gravels varies in size and quantity between their first appearance, at the end of the azilian period, and their scarceness in the final sauveterrian period. Within particular morphological context, their shape rapidly change and allows us to order occupational layers. 相似文献
110.