A bioanalytical method for the proteome wide display and analysis of protein complexes from whole plant cell lysates

While protein interaction studies and protein network modeling come to the forefront, the isolation and identification of protein complexes in a cellular context remains a major challenge for plant science. To this end, a nondenaturing extraction procedure was optimized for plant whole cell matrices and the combined use of gel filtration and BN‐PAGE for the separation of protein complexes was studied. Hyphenation to denaturing electrophoresis and mass spectrometric analysis allows for the simultaneous identification of multiple (previously unidentified) protein interactions in single samples. The reliability and efficacy of the technique was confirmed (i) by the identification of well‐studied plant protein complexes, (ii) by the presence of nonplant interologs for several of the novel complexes (iii) by presenting physical evidence of previously hypothetical plant protein interactions and (iv) by the confirmation of found interactions using co‐IP. Furthermore practical issues concerning the use of this 2‐D BN/SDS‐PAGE display method for the analysis of protein–protein interactions are discussed.

Biomarker discovery in asthma‐related inflammation and remodeling

Asthma is a complex inflammatory disease of airways. A network of reciprocal interactions between inflammatory cells, peptidic mediators, extracellular matrix components, and proteases is thought to be involved in the installation and maintenance of asthma‐related airway inflammation and remodeling. To date, new proteic mediators displaying significant activity in the pathophysiology of asthma are still to be unveiled. The main objective of this study was to uncover potential target proteins by using surface‐enhanced laser desorption/ionization‐time of flight‐mass spectrometry (SELDI‐TOF‐MS) on lung samples from mouse models of allergen‐induced airway inflammation and remodeling. In this model, we pointed out several protein or peptide peaks that were preferentially expressed in diseased mice as compared to controls. We report the identification of different five proteins: found inflammatory zone 1 or RELMα (FIZZ‐1), calcyclin (S100A6), clara cell secretory protein 10 (CC10), Ubiquitin, and Histone H4.

Proteomic analysis of chicory root identifies proteins typically involved in cold acclimation

Chicory ( Cichorium intybus ) roots contain high amounts of inulin, a fructose polymer used as a storage carbohydrate by the plant and as a human dietary and prebiotic compound. We performed 2‐D electrophoretic analysis of proteins from root material before the first freezing period. The proteins were digested with trypsin and the peptides analyzed by MS (MALDI‐TOF/TOF). From the 881 protein spots analyzed, 714 proteins corresponded to a database accession, 619 of which were classified into functional categories. Besides expected proteins ( e.g . related to metabolism, energy, protein synthesis, or cell structure), other well‐represented categories were proteins related to folding and stability (49 spots), proteolysis (49 spots), and the stress response (67 spots). The importance of abiotic stress response was confirmed by the observation that 7 of the 21 most intense protein spots are known to be involved in cold acclimation. These results suggest a major effect of the low temperature period that preceded root harvesting.

MALDI‐TOF MS‐based drug susceptibility testing of pathogens: The example of Candida albicans and fluconazole

MALDI‐TOF MS can be used for the identification of microorganism species. We have extended its application to a novel assay of Candida albicans susceptibility to fluconazole, based on monitoring modifications of the proteome of yeast cells grown in the presence of varying drug concentrations. The method was accurate, and reliable, and showed full agreement with the Clinical Laboratory Standards Institute's reference method. This proof‐of‐concept demonstration highlights the potential for this approach to test other pathogens.

A bioanalytical method for the proteome wide display and analysis of protein complexes from whole plant cell lysates

Changes in lung function in young cystic fibrosis patients between two courses of intravenous antibiotics against Pseudomonas aeruginosa

Background and Aims Repeated intravenous antibiotic therapy (IVAT) against chronic pulmonary infection with Pseudomonas aeruginosa is often necessary in cystic fibrosis patients (CF). The aim of this study was to monitor kinetics of degradation of pulmonary and nutritional status after IVAT in CF patients. Methods Lung function, nutritional status and physical activity (PA) were measured for 21 CF patients (mean ± SD age, 16 ± 1.9 years; 9 boys) who were chronically colonized by P. aeruginosa . Each parameter was measured every 15 days during 3–6 months. Results Nutritional status as well as PA did not change in the interval of 2 IVAT. In contrary, lung function worsened with a decrease of −14.6% of the FEV 1 ( P  < 0.05). Conclusion In CF, there is a progressive decrease in lung function without any evident deterioration of nutritional status and PA between two IVAT. Pediatr Pulmonol. 2009; 44:464–471. © 2009 Wiley‐Liss, Inc.

Detailed insights from microarray and crystallographic studies into carbohydrate recognition by microneme protein 1 (MIC1) of Toxoplasma gondii

The intracellular protozoan Toxoplasma gondii is among the most widespread parasites. The broad host cell range of the parasite can be explained by carbohydrate microarray screening analyses that have demonstrated the ability of the T. gondii adhesive protein, TgMIC1, to bind to a wide spectrum of sialyl oligosaccharide ligands. Here, we investigate by further microarray analyses in a dose‐response format the differential binding of TgMIC1 to 2‐3‐ and 2‐6‐linked sialyl carbohydrates. Interestingly, two novel synthetic fluorinated analogs of 3′SiaLacNAc 1–4 and 3′SiaLacNAc 1–3 were identified as highly potent ligands. To understand the structural basis of the carbohydrate binding specificity of TgMIC1, we have determined the crystal structures of TgMIC1 micronemal adhesive repeat (MAR)‐region (TgMIC1‐MARR) in complex with five sialyl‐ N ‐acetyllactosamine analogs. These crystal structures have revealed a specific, water‐mediated hydrogen bond network that accounts for the preferential binding of TgMIC1‐MARR to arrayed 2‐3‐linked sialyl oligosaccharides and the high potency of the fluorinated analogs. Furthermore, we provide strong evidence for the first observation of a CF···HO hydrogen bond within a lectin‐carbohydrate complex. Finally, detailed comparison with other oligosaccharide‐protein complexes in the Protein Data Bank (PDB) reveals a new family of sialic‐acid binding sites from lectins in parasites, bacteria, and viruses.

Identification of a human estrogen receptor α‐derived antiestrogenic peptide that adopts a polyproline II conformation

Polyproline II (PPII) helix is an extended secondary structure present in a number of proteins. PPII‐containing sequences mediate specific protein–protein interactions with partners containing appropriate cognate domains called PPII‐recognizing domains (PRDs) and are involved in the activation of intracellular signaling pathways. Thus, the identification of PPII structures in proteins is of great interest, not only to explore molecular and physiological mechanisms, but also to elaborate new potential drugs. By revisiting X‐ray crystal structures of liganded α‐type human estrogen receptor (ERα), we have identified an 11‐residue PPII‐helical sequence (D 321 AEPPILYSEY 331 ) in the ligand‐binding domain of the receptor. The data recorded by far‐ultraviolet circular dichroism (far‐UV CD), vibrational Raman optical activity (ROA) and differential scanning calorimetry (DSC) show that the corresponding peptide (Ac‐DAEPPILYSEY‐NH 2 ) is particularly well structured in PPII, with the same proportion of PPII as observed from X‐ray structures (∼85%). In addition, studies carried out on ERα‐negative Evsa‐T breast cancer cells transiently co‐transfected with a pcDNA3‐ERα plasmid and a Vit‐tk‐Luc reporter gene revealed that the peptide antagonizes the estradiol‐induced transcription providing perspectives for researching new molecules with antagonistic properties. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.

Optical beam induced current measurements: principles and applications to SiC device characterization

This paper deals with the characterization of SiC devices by optical beam induced currents (OBIC). OBIC is a technique that measures a photocurrent in response to a fine UV laser beam that is scanned laterally over the surface of the device. In this way a number of important material and device parameters can be derived. We concentrate here on three aspects, the field profile in reverse biased diodes with particular emphasis on field terminating issues at the device edges under high reverse voltages; the minority carrier lifetimes in 6H‐SiC; and the determination of ionization coefficients for electrons and holes in 6H‐SiC. The latter are important material parameters for the modelling of high power devices and determine their break‐through voltage. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

Helium implantation into 4H‐SiC

The paper provides the properties of single crystalline 4H‐SiC under helium implantation at temperatures of implantation up to 750 °C and fluences in the range 5 × 10 15 –1 × 10 17  cm −2 . The microstructure evolution was studied by transmission electron microscopy cross‐section and X‐ray diffraction experiments. The mechanical property changes were investigated by using nanoindentation tests followed by atomic force microscopy observations and by using tribological tests. At elevated temperature of implantation and/or in the low fluence regime at room temperature where only the strained state of SiC is obtained, SiC becomes more resistant to crack formation but no significant change in mechanical properties is seen. At room temperature with increasing fluence the damage accumulation leads to the amorphous state for which a strong degradation of the mechanical properties is observed. At elevated temperature of implantation, amorphization is avoided and a thermally activated saturation of the strain is observed in the near surface region whereas defect accumulation occurs near the maximum of damage. Upon annealing subsequent to room temperature implantation, the near surface strain progressively relaxes while the helium ions agglomerate into platelets around the maximum of strain. These platelets evolve into bubble clusters at temperatures where the vacancies become mobile. Under particular conditions of implantation (high fluence and elevated temperature) the swelling of the surface increases during annealing due to the growth of bubbles and the formation of stacking faults resulting from the migration of interstitials towards the maximum of damage.