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51.
Lerouxel O Mouille G Andème-Onzighi C Bruyant MP Séveno M Loutelier-Bourhis C Driouich A Höfte H Lerouge P 《The Plant journal : for cell and molecular biology》2005,42(4):455-468
A mutant called defective glycosylation1-1 (dgl1-1) was identified in Arabidopsis based on a growth defect of the dark-grown hypocotyl and an abnormal composition of the non-cellulosic cell wall polysaccharides. dgl1-1 is altered in a protein ortholog of human OST48 or yeast WBP1, an essential protein subunit of the oligosaccharyltransferase (OST) complex, which is responsible for the transfer in the ER of the N-linked glycan precursor onto Asn residues of candidate proteins. Consistent with the known function of the OST complex in eukaryotes, the dgl1-1 mutation led to a reduced N-linked glycosylation of the ER-resident protein disulfide isomerase. A second more severe mutant (dgl1-2) was embryo-lethal. Microscopic analysis of dgl1-1 revealed developmental defects including reduced cell elongation and the collapse and differentiation defects of cells in the central cylinder. These defects were accompanied by changes in the non-cellulosic polysaccharide composition, including the accumulation of ectopic callose. Interestingly, in contrast to other dwarf mutants that are altered in early steps of the N-glycan processing, dgl1-1 did not exhibit a cellulose deficiency. Together, these results confirm the role of DGL1 in N-linked glycosylation, cell growth and differentiation in plants. 相似文献
52.
The asymmetric synthesis of isobenzofurane analogues, new potential antiviral agents, is reported. High performance liquid chromatography (HPLC) was the technique chosen to separate the enantiomers. We describe this chiral separation and then determine the enantiomerical excess. The biological results of each tested enantiomer are given. 相似文献
53.
Repressed E-cadherin expression in the lower crypt of human small intestine: a cell marker of functional relevance 总被引:6,自引:0,他引:6
54.
Effect of electric field vectoriality on electrically mediated gene delivery in mammalian cells 总被引:1,自引:0,他引:1
Faurie C Phez E Golzio M Vossen C Lesbordes JC Delteil C Teissié J Rols MP 《Biochimica et biophysica acta》2004,1665(1-2):92-100
Electropermeabilization is a nonviral method used to transfer genes into living cells. Up to now, the mechanism is still to be elucidated. Since cell permeabilization, a prerequired for gene transfection, is triggerred by electric field, its characteristics should depend on its vectorial properties. The present investigation addresses the effect of pulse polarity and orientation on membrane permeabilization and gene delivery by electric pulses applied to cultured mammalian cells. This has been directly observed at the single-cell level by using digitized fluorescence microscopy. While cell permeabilization is only slightly affected by reversing the polarity of the electric pulses or by changing the orientation of pulses, transfection level increases are observed. These last effects are due to an increase in the cell membrane area where DNA interacts. Fluorescently labelled plasmids only interact with the electropermeabilized side of the cell facing the cathode. The plasmid interaction with the electropermeabilized cell surface is stable and is not affected by pulses of reversed polarities. Under such conditions, DNA interacts with the two sites of the cell facing the two electrodes. When changing both the pulse polarity and their direction, DNA interacts with the whole membrane cell surface. This is associated with a huge increase in gene expression. This present study demonstrates the relationship between the DNA/membrane surface interaction and the gene transfer efficiency, and it allows to define the experimental conditions to optimize the yield of transfection of mammalian cells. 相似文献
55.
MutY is an adenine glycosylase in the base excision repair (BER) superfamily that is involved in the repair of 7,8-dihydro-8-oxo-2'-deoxyguanosine (OG):A and G:A mispairs in DNA. MutY contains a [4Fe-4S]2+ cluster that is part of a novel DNA binding motif, referred to as the iron-sulfur cluster loop (FCL) motif. This motif is found in a subset of members of the BER glycosylase superfamily, defining the endonuclease III-like subfamily. Site-specific cross-linking was successfully employed to investigate the DNA-protein interface of MutY. The photoreactive nucleotide 4-thiothymidine (4ST) incorporated adjacent to the OG:A mismatch formed a specific cross-link between the substrate DNA and MutY. The amino acid participating in the cross-linking reaction was characterized by positive ion electrospray ionization (ESI) tandem mass spectrometry. This analysis revealed Arg 143 as the site of modification in MutY. Arg 143 and nearby Arg 147 are conserved throughout the endo III-like subfamily. Replacement of Arg 143 and Arg 147 with alanine by site-directed mutagenesis reduces adenine glycosylase activity of MutY toward OG:A and G:A mispairs. In addition, the R143A and R147A enzymes exhibit a reduced affinity for duplexes containing the substrate analogue 2'-deoxy-2'-fluoroadenosine opposite OG and G. Modeling of MutY bound to DNA using an endonuclease III-DNA complex structure shows that these two conserved arginines are located within close proximity to the DNA backbone. The insight from mass spectrometry experiments combined with functional mutagenesis results indicate that these two amino acids in the [4Fe-4S]2+ cluster-containing subfamily play an important role in recognition of the damaged DNA substrate. 相似文献
56.
Benarroch D Egloff MP Mulard L Guerreiro C Romette JL Canard B 《The Journal of biological chemistry》2004,279(34):35638-35643
Ribavirin is one of the few nucleoside analogues currently used in the clinic to treat RNA virus infections, but its mechanism of action remains poorly understood at the molecular level. Here, we show that ribavirin 5'-triphosphate inhibits the activity of the dengue virus 2'-O-methyltransferase NS5 domain (NS5MTase(DV)). Along with several other guanosine 5'-triphosphate analogues such as acyclovir, 5-ethynyl-1-beta-d-ribofuranosylimidazole-4-carboxamide (EICAR), and a series of ribose-modified ribavirin analogues, ribavirin 5'-triphosphate competes with GTP to bind to NS5MTase(DV). A structural view of the binding of ribavirin 5'-triphosphate to this enzyme was obtained by determining the crystal structure of a ternary complex consisting of NS5MTase(DV), ribavirin 5'-triphosphate, and S-adenosyl-l-homocysteine at a resolution of 2.6 A. These detailed atomic interactions provide the first structural insights into the inhibition of a viral enzyme by ribavirin 5'-triphosphate, as well as the basis for rational drug design of antiviral agents with improved specificity against the emerging flaviviruses. 相似文献
57.
Structural and thermodynamic basis for the binding of TMC114, a next-generation human immunodeficiency virus type 1 protease inhibitor
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King NM Prabu-Jeyabalan M Nalivaika EA Wigerinck P de Béthune MP Schiffer CA 《Journal of virology》2004,78(21):12012-12021
TMC114, a newly designed human immunodeficiency virus type 1 (HIV-1) protease inhibitor, is extremely potent against both wild-type (wt) and multidrug-resistant (MDR) viruses in vitro as well as in vivo. Although chemically similar to amprenavir (APV), the potency of TMC114 is substantially greater. To examine the basis for this potency, we solved crystal structures of TMC114 complexed with wt HIV-1 protease and TMC114 and APV complexed with an MDR (L63P, V82T, and I84V) protease variant. In addition, we determined the corresponding binding thermodynamics by isothermal titration calorimetry. TMC114 binds approximately 2 orders of magnitude more tightly to the wt enzyme (K(d) = 4.5 x 10(-12) M) than APV (K(d) = 3.9 x 10(-10) M). Our X-ray data (resolution ranging from 2.2 to 1.2 A) reveal strong interactions between the bis-tetrahydrofuranyl urethane moiety of TMC114 and main-chain atoms of D29 and D30. These interactions appear largely responsible for TMC114's very favorable binding enthalpy to the wt protease (-12.1 kcal/mol). However, TMC114 binding to the MDR HIV-1 protease is reduced by a factor of 13.3, whereas the APV binding constant is reduced only by a factor of 5.1. However, even with the reduction in binding affinity to the MDR HIV protease, TMC114 still binds with an affinity that is more than 1.5 orders of magnitude tighter than the first-generation inhibitors. Both APV and TMC114 fit predominantly within the substrate envelope, a property that may be associated with decreased susceptibility to drug-resistant mutations relative to that of first-generation inhibitors. Overall, TMC114's potency against MDR viruses is likely a combination of its extremely high affinity and close fit within the substrate envelope. 相似文献
58.
Martin-Chouly CA Astier A Jacob C Pruniaux MP Bertrand C Lagente V 《Life sciences》2004,75(7):823-840
Over-expression of matrix metalloproteinases by lung fibroblasts has been blamed for much of the tissue destruction associated with airway inflammation. Because cyclic AMP is known to regulate fibroblast proliferation, as well as cytokine and extracellular matrix protein production, the current study was designed to evaluate the ability of three selective phosphodiesterase (PDE) type 4 inhibitors, rolipram, cilomilast and CI-1044, to inhibit extracellular matrix degradation. Using zymography and ELISA, we found that pro-MMP-2 release was enhanced following 24 h treatment of human lung fibroblast (MRC-5) with TGF-beta1 (10 ng/ml) or TNF-alpha (10 ng/ml), whereas PMA (0.02 microM) had no effect. One hour of pre-incubation with PDE4 inhibitors (10 microM) induced an inhibition of TNF-alpha-stimulated pro-MMP-2 release. Zymography and immunoblotting revealed that fibroblasts cultured with PMA or TNF-alpha released increased amounts of pro-MMP-1, whereas TGF-beta1 had no effect. Incubation with CI-1044 or cilomilast significantly prevented the TNF-alpha increase in pro-MMP-1. These results suggest that PDE4 inhibitors are effective in inhibiting the pro-MMP-2 and pro-MMP-1 secretion induced by TNF-alpha and might underline a potential therapeutic benefit of selective PDE4 inhibitors in lung diseases associated with abnormal tissue remodelling. 相似文献
59.
Ricolleau G Charbonnel C Lodé L Loussouarn D Joalland MP Bogumil R Jourdain S Minvielle S Campone M Déporte-Fety R Campion L Jézéquel P 《Proteomics》2006,6(6):1963-1975
Novel prognostic biomarkers are imperatively needed to help direct treatment decisions by typing subgroups of node-negative breast cancer patients. The current study has used a proteomic approach of SELDI-TOF-MS screening to identify differentially cytosolic expressed proteins with a prognostic impact in 30 node-negative breast cancer patients with no relapse versus 30 patients with metastatic relapse. The data analysis took into account 73 peaks, among which 2 proved, by means of univariate Cox regression, to have a good cumulative prognostic-informative power. Repeated random sampling (n = 500) was performed to ensure the reliability of the peaks. Optimized thresholds were then computed to use both peaks as risk factors and, adding them to the St. Gallen ones, improve the prognostic classification of node-negative breast cancer patients. Identification of ubiquitin and ferritin light chain (FLC), corresponding to the two peaks of interest, was obtained using ProteinChip LDI-Qq-TOF-MS. Differential expression of the two proteins was further confirmed by Western blotting analyses and immunohistochemistry. SELDI-TOF-MS protein profiling clearly showed that a high level of cytosolic ubiquitin and/or a low level of FLC were associated with a good prognosis in breast cancer. 相似文献
60.
Marie Deghorain Laetitia Fontaine Blandine David Jean-Luc Mainardi Pascal Courtin Richard Daniel Jeff Errington Alexei Sorokin Alexander Bolotin Marie-Pierre Chapot-Chartier Bernard Hallet Pascal Hols 《The Journal of biological chemistry》2010,285(31):24003-24013
Cell wall peptidoglycan assembly is a tightly regulated process requiring the combined action of multienzyme complexes. In this study we provide direct evidence showing that substrate transformations occurring at the different stages of this process play a crucial role in the spatial and temporal coordination of the cell wall synthesis machinery. Peptidoglycan substrate alteration was investigated in the Gram-positive bacterium Lactococcus lactis by substituting the peptidoglycan precursor biosynthesis genes of this bacterium for those of the vancomycin-resistant bacterium Lactobacillus plantarum. A set of L. lactis mutant strains in which the normal d-Ala-ended precursors were partially or totally replaced by d-Lac-ended precursors was generated. Incorporation of the altered precursor into the cell wall induced morphological changes arising from a defect in cell elongation and cell separation. Structural analysis of the muropeptides confirmed that the activity of multiple enzymes involved in peptidoglycan synthesis was altered. Optimization of this altered pathway was necessary to increase the level of vancomycin resistance conferred by the utilization of d-Lac-ended peptidoglycan precursors in the mutant strains. The implications of these findings on the control of bacterial cell morphogenesis and the mechanisms of vancomycin resistance are discussed. 相似文献