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61.
62.
The translocation of single-stranded DNA (ssDNA) across membranes of two cells is a fundamental biological process occurring in both bacterial conjugation and Agrobacterium pathogenesis. Whereas bacterial conjugation spreads antibiotic resistance, Agrobacterium facilitates efficient interkingdom transfer of ssDNA from its cytoplasm to the host plant cell nucleus. These processes rely on the Type IV secretion system (T4SS), an active multiprotein channel spanning the bacterial inner and outer membranes. T4SSs export specific proteins, among them relaxases, which covalently bind to the 5' end of the translocated ssDNA and mediate ssDNA export. In Agrobacterium tumefaciens , another exported protein—VirE2—enhances ssDNA transfer efficiency 2000-fold. VirE2 binds cooperatively to the transferred ssDNA (T-DNA) and forms a compact helical structure, mediating T-DNA import into the host cell nucleus. We demonstrated—using single-molecule techniques—that by cooperatively binding to ssDNA, VirE2 proteins act as a powerful molecular machine. VirE2 actively pulls ssDNA and is capable of working against 50-pN loads without the need for external energy sources. Combining biochemical and cell biology data, we suggest that, in vivo, VirE2 binding to ssDNA allows an efficient import and pulling of ssDNA into the host. These findings provide a new insight into the ssDNA translocation mechanism from the recipient cell perspective. Efficient translocation only relies on the presence of ssDNA binding proteins in the recipient cell that compacts ssDNA upon binding. This facilitated transfer could hence be a more general ssDNA import mechanism also occurring in bacterial conjugation and DNA uptake processes. 相似文献
63.
Anne-Ccile Buisson Jean-Marie Zahm Myriam Polette Denis Pierrot Georges Bellon Edith Puchelle Philippe Birembaut Jean-Marie Tournier 《Journal of cellular physiology》1996,166(2):413-426
Following epithelial injury, extracellular matrix undergoes imposing remodelings. We examined the contribution of matrix metalloproteinases, gelatinases A and B, in an in vitro wound repair model of human respiratory epithelium. Confluent human surface respiratory epithelial (HSRE) cells cultured from dissociated surface cells of human nasal polyps were chemically injured. Over the next 3 to 5 days, cells migrated onto the injured area to repair the circular wound. Repair kinetics of these wounds was monitored until wound closure occurred. Gelatinolytic activities were analysed in culture supernates and in cell protein extracts derived from repairing migratory and non repairing stationary cells. Small amounts of gelatinase A were expressed by HSRE cells, and variations of this gelatinase remained very weak for the time of the wound repair. In contrast, gelatinase B was upregulated during the wound repair process, with a maximum peak observed at wound closure. A marked gelatinase B activation occurred only in cells involved in the repair process. Gelatinase B was localized in some migratory basal cells, recognized by an anti-cytokeratin 14 antibody and located around the wound. We could not detect any gelatinase A in repairing or in stationary HSRE cells. Addition of the 6-6B monoclonal antibody, known to inhibit gelatinase B activation, to the culture medium during the repair process resulted in a dose-dependent decrease of the wound repair speed. These results suggest that gelatinase B, produced by epithelial cells, actively contributes to the wound repair process of the respiratory epithelium. © 1996 Wiley-Liss, Inc. 相似文献
64.
Luciane M.R. Neto Maristela H. Andraus Myriam C. Salvadori 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》1996,678(2):211-218
A method is described for the qualiitative and quantitative determination of phenylbutazone and oxyphenbutazone in horse urine and plasma samples viewing antidoping control. A horse was administered intravenously with 3 g of phenylbutazone. For the qualitative determination, a screening by HPLC was performed after acidic extraction of the urine samples and the confirmation process was realized by GC-MS. Using the proposed method it was possible to detect phenylbutazone and oxyphenbutazone in urine for up to 48 and 120 h, respectively. For the quantitation of these drugs the plasma was deproteinized with acetonitrile and 20 gml were injected directly into the HPLC system equipped with a UV detector and LiChrospher RP-18 column. The mobile phase used was 0.01 M acetic acid in methanol (45:55, v/v). The limit of detection was 0.5 μg/ml for phenylbutazone and oxyphenbutazone and the limit of quantitation was 1.0 μg/ml for both drugs. Using the proposed method it was possible to quantify phenylbutazone up to 30 h and oxyphenbutazone up to 39 h after administration. 相似文献
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66.
Myriam M. Ouberai Juan Wang Marcus J. Swann Celine Galvagnion Tim Guilliams Christopher M. Dobson Mark E. Welland 《The Journal of biological chemistry》2013,288(29):20883-20895
There is increasing evidence for the involvement of lipid membranes in both the functional and pathological properties of α-synuclein (α-Syn). Despite many investigations to characterize the binding of α-Syn to membranes, there is still a lack of understanding of the binding mode linking the properties of lipid membranes to α-Syn insertion into these dynamic structures. Using a combination of an optical biosensing technique and in situ atomic force microscopy, we show that the binding strength of α-Syn is related to the specificity of the lipid environment (the lipid chemistry and steric properties within a bilayer structure) and to the ability of the membranes to accommodate and remodel upon the interaction of α-Syn with lipid membranes. We show that this interaction results in the insertion of α-Syn into the region of the headgroups, inducing a lateral expansion of lipid molecules that can progress to further bilayer remodeling, such as membrane thinning and expansion of lipids out of the membrane plane. We provide new insights into the affinity of α-Syn for lipid packing defects found in vesicles of high curvature and in planar membranes with cone-shaped lipids and suggest a comprehensive model of the interaction between α-Syn and lipid bilayers. The ability of α-Syn to sense lipid packing defects and to remodel membrane structure supports its proposed role in vesicle trafficking. 相似文献
67.
Wilde Chris De; Rycke Riet De; Beeckman Tom; Neve Myriam De; Montagu Marc Van; Engler Gilbert; Depicker Ann 《Plant & cell physiology》1998,39(6):639-646
For the further optimization of antibody expression in plants,it is essential to determine the final accumulation sites ofplant-made antibodies. Previously, we have shown that, uponsecretion, IgG antibodies and Fab fragments can be detectedin the intercellular spaces of leaf mesophyil cells of transgenicArabidopsis thaliana plants. However, immunofluorescence microscopyshowed that this is probably not their final accumulation site.In leaves, IgG and Fabfragments accumulate also at the interiorside of the epidermal cell layers and in xylem vessels. Theseaccumulation sites correspond with the leaf regions where waterof the transpiration stream is entering a space impermeableto the proteins or where water is evaporating. In roots, plant-madeFab fragments accumulate in intercellular spaces of cortex cells,in the cytoplasm of pericycle and, to a lesser extent, endodermiscells, and in cells of the vascular cylinder. In other words,antibody accumulation occurs at the sites where water passeson its radial pathway towards and within the vascular bundle.Taken together, our results suggest that, upon secretion ofplant-made antibodies or Fab fragments, a large proportion ofthese proteins are transported in the apoplast of A. thaliana,possibly by the water flow in the transpiration stream.
4Corresponding author. Fax 32-9-2645349; e-mail: anpic{at}gengenp.rug.ac.be 相似文献
68.
Evaluation of electrochemically synthesized sulfadimethoxine‐imprinted polymer for solid‐phase microextraction of sulfonamides 下载免费PDF全文
Myriam Díaz‐Álvarez Elisabetta Mazzotta Cosimino Malitesta Antonio Martín‐Esteban 《Journal of molecular recognition : JMR》2014,27(6):415-420
Solid‐phase microextraction (SPME) is widely used in analytical laboratories for the analysis of organic compounds, thanks to its simplicity and versatility. In the present work, the synthesis and evaluation of imprinted films for SPME by electropolymerisation of pyrrole alone or in the presence of ethylene glycol dimethacrylate is proposed. Sulfadimethoxine (SDM), a sulfonamide antibiotic, was used as template molecule. Initially, a molecularly imprinted polymer film was prepared by electropolymerisation of pyrrole onto a platinum foil, using SDM as template. The SDM template was removed by overoxidation. The behaviour of SDM on imprinted and non‐imprinted polymers was investigated by differential pulse voltammetry, and a clear imprinting effect was observed, which was confirmed by rebinding experiments using both conventional and electrochemically enhanced‐SPME. However, in general, the extraction efficiency was rather low (<6%) and unspecific interactions are too high. Attempts to increase extraction efficiency were unsuccessful, but the incorporation of ethylene glycol dimethacrylate to the films reduced unspecific interactions to a certain extent. Copyright © 2014 John Wiley & Sons, Ltd. 相似文献
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70.
Wu TY Juteau H Ducharme Y Friesen RW Guiral S Dufresne L Poirier H Salem M Riendeau D Mancini J Brideau C 《Bioorganic & medicinal chemistry letters》2010,20(23):6978-6982
Microsomal prostaglandin E(2) synthase (mPGES-1) represents a potential target for novel analgesic and anti-inflammatory agents. High-throughput screening identified several leads of mPGES-1 inhibitors which were further optimized for potency and selectivity. A series of inhibitors bearing a biaryl imidazole scaffold exhibits excellent inhibition of PGE(2) production in enzymatic and cell-based assays. The synthesis of these molecules and their activities will be discussed. 相似文献