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991.
Axel T. Brunger Pavel Strop Marija Vrljic Steven Chu Keith R. Weninger 《Journal of structural biology》2011,173(3):497-505
Single molecule fluorescence energy transfer experiments enable investigations of macromolecular conformation and folding by the introduction of fluorescent dyes at specific sites in the macromolecule. Multiple such experiments can be performed with different labeling site combinations in order to map complex conformational changes or interactions between multiple molecules. Distances that are derived from such experiments can be used for determination of the fluorophore positions by triangulation. When combined with a known structure of the macromolecule(s) to which the fluorophores are attached, a three-dimensional model of the system can be determined. However, care has to be taken to properly derive distance from fluorescence energy transfer efficiency and to recognize the systematic or random errors for this relationship. Here we review the experimental and computational methods used for three-dimensional modeling based on single molecule fluorescence resonance transfer, and describe recent progress in pushing the limits of this approach to macromolecular complexes. 相似文献
992.
993.
994.
Peter M Huelsmann Andreas D Hofmann Stefanie A Knoepfel Jasmin Popp Pia Rauch Francesca Di Giallonardo Christina Danke Eva Gueckel Axel Schambach Horst Wolff Karin J Metzner Christian Berens 《BMC biotechnology》2011,11(1):4
Background
Regulated expression of suicide genes is a powerful tool to eliminate specific subsets of cells and will find widespread usage in both basic and applied science. A promising example is the specific elimination of human immunodeficiency virus type 1 (HIV-1) infected cells by LTR-driven suicide genes. The success of this approach, however, depends on a fast and effective suicide gene, which is expressed exclusively in HIV-1 infected cells. These preconditions have not yet been completely fulfilled and, thus, success of suicide approaches has been limited so far. We tested truncated Bid (tBid), a human pro-apoptotic protein that induces apoptosis very rapidly and efficiently, as suicide gene for gene therapy against HIV-1 infection.Results
When tBid was introduced into the HIV-1 LTR-based, Tat- and Rev-dependent transgene expression vector pLRed(INS)2R, very efficient induction of apoptosis was observed within 24 hours, but only in the presence of both HIV-1 regulatory proteins Tat and Rev. Induction of apoptosis was not observed in their absence. Cells containing this vector rapidly died when transfected with plasmids containing full-length viral genomic DNA, completely eliminating the chance for HIV-1 replication. Viral replication was also strongly reduced when cells were infected with HIV-1 particles.Conclusions
This suicide vector has the potential to establish a safe and effective gene therapy approach to exclusively eliminate HIV-1 infected cells before infectious virus particles are released.995.
996.
Spyrakis F Bruno S Bidon-Chanal A Luque FJ Abbruzzetti S Viappiani C Dominici P Mozzarelli A 《IUBMB life》2011,63(5):355-362
Nonsymbiotic hemoglobins AHb1 and AHb2 discovered in Arabidopsis thaliana are likely to carry out distinct physiological roles, in consideration of their differences in sequence, structure, expression pattern, and tissue localization. Despite a relatively fast autoxidation in the presence of O(2) , we were able to collect O(2) -binding curves for AHb2 in the presence of a reduction enzymatic system. AHb2 binds O(2) noncooperatively with a p50 of 0.021 ± 0.003 Torr, a value consistent with a recently proposed role in O(2) transport. The analysis of the internal cavities derived from the structures sampled in molecular dynamics simulations confirms strong differences with AHb1, proposed to work as a NO deoxygenase in vivo. Overall, our results are consistent with a role for AHb2 as an oxygen carrier, as recently proposed on the basis of experiments on AHb2-overexpressing mutants of A. thaliana. 相似文献
997.
Appel H Maier R Wu P Scheer R Hempfing A Kayser R Thiel A Radbruch A Loddenkemper C Sieper J 《Arthritis research & therapy》2011,13(3):R95
Introduction
In this study, we analysed the number of IL-17+ cells in facet joints, in the peripheral blood (PB) and synovial fluid (SF) of spondyloarthritis (SpA) patients and compared these results with those of patients with other rheumatic diseases and controls. 相似文献998.
The single and triple helical organization of protein chains strongly influences the mechanical properties of gelatin-based materials. A chemical method for obtaining different degrees of helical organization in gelatin is covalent functionalization, while a physical method for achieving the same goal is the variation of the drying conditions of gelatin solutions. Here we explored how the introduction of desaminotyrosine (DAT) and desaminotyrosyl tyrosine (DATT) linked to lysine residues of gelatin influenced the kinetics and thermodynamic equilibrium of the helicalization process of single and triple helices following different drying conditions. Drying at a temperature above the helix-to-coil transition temperature of gelatin (T > T(c), called v(short)) generally resulted in gelatins with relatively lower triple helical content (X(c,t) = 1-2%) than lower temperature drying (T < T(c), called v(long)) (X(c,t) = 8-10%), where the DAT(T) functional groups generally disrupted helix formation. While different helical contents affected the thermal transition temperatures only slightly, the mechanical properties were strongly affected for swollen hydrogels (E = 4-13 kPa for samples treated by v(long) and E = 120-700 kPa for samples treated by v(short)). This study shows that side group functionalization and different drying conditions are viable options to control the helicalization and macroscopic properties of gelatin-based materials. 相似文献
999.
Pfaff A Schallon A Ruhland TM Majewski AP Schmalz H Freitag R Müller AH 《Biomacromolecules》2011,12(10):3805-3811
The synthesis of galactose-displaying core-shell nanospheres exhibiting both fluorescent and magnetic properties by grafting a glycocopolymer consisting of 6-O-methacryloylgalactopyranose (MAGal) and 4-(pyrenyl)butyl methacrylate (PyMA) onto magnetic silica particles via thiol-ene chemistry is reported. Magnetization measurements indicated that neither the encapsulation of the iron oxide particles into silica nor the grafting of the glycocopolymer chains had a significant influence on the superparamagnetic properties. This not only simplifies the purification of the particles but may facilitate the use of the particles in applications such as hyperthermia or magnetic resonance imaging (MRI). Furthermore, the hydrophilic glycopolymer shell provided solubility of the particles in aqueous medium and enabled the uptake of the particles into the cytoplasm and nucleus of lung cancer cells via carbohydrate-lectin recognition effects. 相似文献
1000.
Guangming Wu Na Liu Ina Rittelmeyer Amar Deep Sharma Malte Sgodda Holm Zaehres Martina Bleidi?el Boris Greber Luca Gentile Dong Wook Han Cornelia Rudolph Doris Steinemann Axel Schambach Michael Ott Hans R. Sch?ler Tobias Cantz 《PLoS biology》2011,9(7)
Using the murine model of tyrosinemia type 1 (fumarylacetoacetate hydrolase [FAH] deficiency; FAH
−/− mice) as a paradigm for orphan disorders, such as hereditary metabolic liver diseases, we evaluated fibroblast-derived FAH
−/−-induced pluripotent stem cells (iPS cells) as targets for gene correction in combination with the tetraploid embryo complementation method. First, after characterizing the FAH
−/− iPS cell lines, we aggregated FAH
−/−-iPS cells with tetraploid embryos and obtained entirely FAH
−/−-iPS cell–derived mice that were viable and exhibited the phenotype of the founding FAH
−/− mice. Then, we transduced FAH cDNA into the FAH
−/−-iPS cells using a third-generation lentiviral vector to generate gene-corrected iPS cells. We could not detect any chromosomal alterations in these cells by high-resolution array CGH analysis, and after their aggregation with tetraploid embryos, we obtained fully iPS cell–derived healthy mice with an astonishing high efficiency for full-term development of up to 63.3%. The gene correction was validated functionally by the long-term survival and expansion of FAH-positive cells of these mice after withdrawal of the rescuing drug NTBC (2-(2-nitro-4-fluoromethylbenzoyl)-1,3-cyclohexanedione). Furthermore, our results demonstrate that both a liver-specific promoter (transthyretin, TTR)-driven FAH transgene and a strong viral promoter (from spleen focus-forming virus, SFFV)-driven FAH transgene rescued the FAH-deficiency phenotypes in the mice derived from the respective gene-corrected iPS cells. In conclusion, our data demonstrate that a lentiviral gene repair strategy does not abrogate the full pluripotent potential of fibroblast-derived iPS cells, and genetic manipulation of iPS cells in combination with tetraploid embryo aggregation provides a practical and rapid approach to evaluate the efficacy of gene correction of human diseases in mouse models. 相似文献