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111.
Recent technological advances enabled high-throughput collection of Small Angle X-ray Scattering (SAXS) profiles of biological macromolecules. Thus, computational methods for integrating SAXS profiles into structural modeling are needed more than ever. Here, we review specifically the use of SAXS profiles for the structural modeling of proteins, nucleic acids, and their complexes. First, the approaches for computing theoretical SAXS profiles from structures are presented. Second, computational methods for predicting protein structures, dynamics of proteins in solution, and assembly structures are covered. Third, we discuss the use of SAXS profiles in integrative structure modeling approaches that depend simultaneously on several data types. 相似文献
112.
Vasilj A Gentzel M Ueberham E Gebhardt R Shevchenko A 《Journal of proteome research》2012,11(7):3680-3689
Label-free methods streamline quantitative proteomics of tissues by alleviating the need for metabolic labeling of proteins with stable isotopes. Here we detail and implement solutions to common problems in label-free data processing geared toward tissue proteomics by one-dimensional gel electrophoresis followed by liquid chromatography tandem mass spectrometry (geLC MS/MS). Our quantification pipeline showed high levels of performance in terms of duplicate reproducibility, linear dynamic range, and number of proteins identified and quantified. When applied to the liver of an adenomatous polyposis coli (APC) knockout mouse, we demonstrated an 8-fold increase in the number of statistically significant changing proteins compared to alternative approaches, including many more previously unidentified hydrophobic proteins. Better proteome coverage and quantification accuracy revealed molecular details of the perturbed energy metabolism. 相似文献
113.
In the fields of proteomics, metabolic engineering and synthetic biology there is a need for high-throughput and reliable cloning methods to facilitate construction of expression vectors and genetic pathways. Here, we describe a new approach for solid-phase cloning in which both the vector and the gene are immobilized to separate paramagnetic beads and brought into proximity by magnetic force. Ligation events were directly evaluated using fluorescent-based microscopy and flow cytometry. The highest ligation efficiencies were obtained when gene- and vector-coated beads were brought into close contact by application of a magnet during the ligation step. An automated procedure was developed using a laboratory workstation to transfer genes into various expression vectors and more than 95% correct clones were obtained in a number of various applications. The method presented here is suitable for efficient subcloning in an automated manner to rapidly generate a large number of gene constructs in various vectors intended for high throughput applications. 相似文献
114.
Estronca LM Silva JC Sampaio JL Shevchenko A Verkade P Vaz AD Vaz WL Vieira OV 《PloS one》2012,7(4):e34822
Background
Atherosclerosis starts by lipid accumulation in the arterial intima and progresses into a chronic vascular inflammatory disease. A major atherogenic process is the formation of lipid-loaded macrophages in which a breakdown of the endolysomal pathway results in irreversible accumulation of cargo in the late endocytic compartments with a phenotype similar to several forms of lipidosis. Macrophages exposed to oxidized LDL exihibit this phenomenon in vitro and manifest an impaired degradation of internalized lipids and enhanced inflammatory stimulation. Identification of the specific chemical component(s) causing this phenotype has been elusive because of the chemical complexity of oxidized LDL.Methodology/Principal Findings
Lipid “core aldehydes" are formed in oxidized LDL and exist in atherosclerotic plaques. These aldehydes are slowly oxidized in situ and (much faster) by intracellular aldehyde oxidizing systems to cholesteryl hemiesters. We show that a single cholesteryl hemiester incorporated into native, non-oxidized LDL induces a lipidosis phenotype with subsequent cell death in macrophages. Internalization of the cholesteryl hemiester via the native LDL vehicle induced lipid accumulation in a time- and concentration-dependent manner in “frozen" endolysosomes. Quantitative shotgun lipidomics analysis showed that internalized lipid in cholesteryl hemiester-intoxicated cells remained largely unprocessed in those lipid-rich organelles.Conclusions/Significance
The principle elucidated with the present cholesteryl hemiester-containing native-LDL model, extended to other molecular components of oxidized LDL, will help in defining the molecular etiology and etiological hierarchy of atherogenic agents. 相似文献115.
M Mattiazzi Y Sun H Wolinski A Bavdek T Petan G Anderluh SD Kohlwein DG Drubin I Križaj U Petrovič 《PloS one》2012,7(7):e40931
Background
Presynaptically neurotoxic phospholipases A2 inhibit synaptic vesicle recycling through endocytosis.Principal Findings
Here we provide insight into the action of a presynaptically neurotoxic phospholipase A2 ammodytoxin A (AtxA) on clathrin-dependent endocytosis in budding yeast. AtxA caused changes in the dynamics of vesicle formation and scission from the plasma membrane in a phospholipase activity dependent manner. Our data, based on synthetic dosage lethality screen and the analysis of the dynamics of sites of endocytosis, indicate that AtxA impairs the activity of amphiphysin.Conclusions
We identified amphiphysin and endocytosis as the target of AtxA intracellular activity. We propose that AtxA reduces endocytosis following a mechanism of action which includes both a specific protein–protein interaction and enzymatic activity, and which is applicable to yeast and mammalian cells. Knowing how neurotoxic phospholipases A2 work can open new ways to regulate endocytosis. 相似文献116.
Liu F Park JE Qian WJ Lim D Scharow A Berg T Yaffe MB Lee KS Burke TR 《ACS chemical biology》2012,7(5):805-810
In an effort to develop improved binding antagonists of the polo-like kinase 1 (Plk1) polo-box domain (PBD), we optimized interactions of the known high affinity 5-mer peptide PLHSpT using oxime-based post solid-phase peptide diversification of the N-terminal Pro residue. This allowed us to achieve up to two orders of magnitude potency enhancement. An X-ray crystal structure of the highest affinity analogue in complex with Plk1 PBD revealed new binding interactions in a hydrophobic channel that had been occluded in X-ray structures of the unliganded protein. This study represents an important example where amino acid modification by post solid-phase oxime ligation can facilitate the development of protein-protein interaction inhibitors by identifying new binding pockets that would not otherwise be accessible to coded amino acid residues. 相似文献
117.
Andrej Pavlovi? 《Plant signaling & behavior》2012,7(2):295-297
Carnivorous pitcher plants of the genus Nepenthes have evolved a great diversity of pitcher morphologies. Selective pressures for maximizing nutrient uptake have driven speciation and diversification of the genus in a process known as adaptive radiation. This leads to the evolution of pitchers adapted to specific and often bizarre source of nutrients, which are not strictly animal-derived. One example is Nepenthes ampullaria with unusual growth pattern and pitcher morphology what enables the plant to capture a leaf litter from the canopy above. We showed that the plant benefits from nitrogen uptake by increased rate of photosynthesis and growth what may provide competitive advantage over others co-habiting plants. A possible impact of such specialization toward hybridization, an important mechanism in speciation, is discussed. 相似文献
118.
Henderson R Sali A Baker ML Carragher B Devkota B Downing KH Egelman EH Feng Z Frank J Grigorieff N Jiang W Ludtke SJ Medalia O Penczek PA Rosenthal PB Rossmann MG Schmid MF Schröder GF Steven AC Stokes DL Westbrook JD Wriggers W Yang H Young J Berman HM Chiu W Kleywegt GJ Lawson CL 《Structure (London, England : 1993)》2012,20(2):205-214
This Meeting Review describes the proceedings and conclusions from the inaugural meeting of the Electron Microscopy Validation Task Force organized by the Unified Data Resource for 3DEM (http://www.emdatabank.org) and held at Rutgers University in New Brunswick, NJ on September 28 and 29, 2010. At the workshop, a group of scientists involved in collecting electron microscopy data, using the data to determine three-dimensional electron microscopy (3DEM) density maps, and building molecular models into the maps explored how to assess maps, models, and other data that are deposited into the Electron Microscopy Data Bank and Protein Data Bank public data archives. The specific recommendations resulting from the workshop aim to increase the impact of 3DEM in biology and medicine. 相似文献
119.
Brvar M Perdih A Hodnik V Renko M Anderluh G Jerala R Solmajer T 《Bioorganic & medicinal chemistry》2012,20(8):2572-2580
Bacterial DNA gyrase is an established and validated target for the development of novel antibacterials. In our previous work, we identified a novel series of bacterial gyrase inhibitors from the class of 4-(2,4-dihydroxyphenyl) thiazoles. Our ongoing effort was designated to search for synthetically more available compounds with possibility of hit to lead development. By using the virtual screening approach, new potential inhibitors were carefully selected from the focused chemical library and tested for biological activity. Herein we report on a novel class of 5-(2-hydroxybenzylidene) rhodanines as gyrase B inhibitors with activity in low micromolar range and moderate antibacterial activity. The binding of the two most active compounds to the enzyme target was further characterised using surface plasmon resonance (SPR) and differential scanning fluorimetry methods (DSF). 相似文献
120.
Cokl A 《Journal of insect physiology》2008,54(7):1113-1124
In solitary plant-dwelling stink bug species, success depends crucially on efficient mate location and recognition, mediated by signals transmitted through the plant. All stink bugs investigated so far communicate with species and sex-specific narrow-band calling and courtship song signals produced by abdomen vibration. Calling songs of lower specificity are characterized by readily repeated units emitted with regular repetition rate from the same place on a plant, while courtship songs take place at shorter distances in the process of species and sex recognition, together with signals of other modalities. Signal spectra with about 100Hz fundamental frequency and harmonics below 1000Hz are tuned to the resonant properties of their green host plants. The majority of the identified leg vibratory receptor cells and the underlying ventral cord interneurons respond best in the frequency range below 500Hz. Green plants with low pass filtering properties transmit optimally signals with a dominant frequency around 100Hz and strongly attenuate vibrations above 600Hz. Accurate tuning of signal spectral properties with the plant's mechanical characteristics enables communication over several meter distances, with dispersive bending waves running through the plant's rod-like structures under standing wave conditions. 相似文献