Proteomic analysis of a spring wheat cultivar in response to prolonged cold stress

Cold represents one of the major abiotic factors influencing plant growth and development worldwide. We analysed the long-term responsiveness of an Iranian spring wheat (cv. Kohdasht) to cold from a proteomic point of view, in order to unravel the molecular mechanisms helping a cold-sensitive cultivar to survive exposure to suboptimal temperatures. Plants were grown at 20 or 4°C until entering the reproductive stage and a cross-comparison on the leaf proteomes was performed. Quantitative analyses on protein alterations occurring upon low-temperature exposure showed a reinforcement in ascorbate recycling (dehydroascorbate reductase, ascorbate peroxidase) and protein processing (proteasome subunit, cysteine proteinase), as well as the accumulation of the enzyme devoted to tetrapyrrole resynthesis (glutamate semialdehyde aminomutase). In contrast, among proteins down-regulated after cold stress, we could identify some key Krebs cycle enzymes (isocitrate dehydrogenase, malate dehydrogenase), together with many photosynthesis-related proteins (oxygen-evolving complex proteins, ATP synthase subunits, ferredoxin NADPH oxidoreductase and some Calvin cycle enzymes). Physiological and biochemical parameters (such as shoot apex dissection, chlorophyll, proline and sugar content determination) sustained proteomics findings allowing the present research to contribute to the current knowledge on these long-term responses, which may be crucial to stress adaptation under field conditions.     

Chemically enhanced liquid chromatography/tandem mass spectrometry determination of glutamic acid in the diffusion medium of retinal cells

A rapid, reproducible and highly sensitive method, based on liquid chromatography mass spectrometry, was developed for the determination of the excitatory amino acid glutamic acid released in the diffusion medium of control, ischemic and mutant cells from retinas. Signal intensity of glutamic acid was enhanced by dansyl chloride derivatization giving rise to a detection limit in the order of pmol/mL. Further, in HPLC-ESI-MS detection an MS-friendly dansyl group to glutamic acid enhanced both ionization efficiency in the ESI source and collision-activated dissociation in the collision cell. The sample processing procedure included liquid-liquid extraction, derivatization with dansyl chloride and a final cation-exchange extraction to generate clean extracts for LC/MS/MS analysis. This approach has been validated as sensitive, linear (20-300 ng/mL), accurate and precise for the differential quantification of glutamic acid in the diffusion medium of retina cells. This is the first report of using chemical derivatization to enhance MS/MS detection of the glutamic acid released in the diffusion medium of wild-type and mutant retina cells, under ischemic conditions.     

Recombinant clotting factor VIII concentrates: Heterogeneity and high‐purity evaluation

Factor VIII is an important glycoprotein involved in hemostasis. Insertion of expression vectors containing either the full‐length cDNA sequence of human factor VIII (FLrFVIII) or B‐domain deleted (BDDrFVIII) into mammalian cell lines results in the production of recombinant factor VIII (rFVIII) for therapeutic usage. Three commercially available rFVIII concentrates (Advate^®, Helixate NexGen^® and Refacto^®), either FLrFVIII or BDDrFVIII, were investigated by 1‐ and 2‐DE and MS. The objective of this study was to compare the heterogeneity and the high purity of both rFVIII preparations before and after thrombin digestion. In particular, the 2‐D gel was optimized to better highlight the presence of contaminants and many unexpected proteins. Recombinant strategies consisting of insertion of expression vectors containing BDDrFVIII and FLrFVIII resulted in homogeneous and heterogeneous protein products, respectively, the latter consisting in a heterogeneous mixture of various B‐domain‐truncated forms of the molecule. Thrombin digestion of all the three rFVIII gave similar final products, plus one unexpected fragment of A2 domain missing 11 amino acids. Regarding the contaminants, Helixate NexGen^® showed the presence of impurities, such as Hsp70 kDa, haptoglobin and proapolipoprotein; Refacto^® showed glutathione S‐transferase and β‐lactamase, whereas Advate^® apparently did not contain any contaminants. The proteomic approach will contribute to improving the quality assurance and manufacturing processes of rFVIII concentrates. In this view, the 2‐DE is mandatory for revealing the presence of contaminants.     

Native analysis of plasma-derived clotting factor VIII concentrates: "sponge effect" and contaminants

In the present study, we analysed two commercially available plasma-derived FVIII preparations, Beriate and Emoclot, through native gel-based approaches (CN-PAGE). The rationale behind this study was to assess whether protein complexes from plasma resisted the aggressive manufacturing processes. A preliminary analysis of plasma complexes was performed focusing on the molecular weight range between 45 kDa and 1 MDa. As a result, we could evidence in both preparations the presence of complexes, which resisted the cryoprecipitation, chromatographic and heat treatment processing steps. These complexes behaved in a "sponge-effect"-like fashion through the enrichment of otherwise less abundant contaminants. The use of an alternative electrophoretic approach such as CN-PAGE allowed us to confirm 2DE-based observations and expand the list of non-FVIII proteins which contaminate the preparations.     

High-performance liquid chromatography coupled on-line with electrospray ionization mass spectrometry for the simultaneous separation and identification of the Synechocystis PCC 6803 phycobilisome proteins

The complete resolution of the protein components of phycobilisome from cyanobacterium Synechocystis 6803, together with their detection and determination of molecular mass, has successfully been obtained by the combined use of HPLC coupled on-line with electrospray ionization mass spectrometry. The method proposed consists of the isolation of the light-harvesting apparatus of cyanobacterium, by simply breaking cells in low-ionic-strength buffer, and subsequent injection of the total mixture of phycobilisomes into a C4 reversed-phase column. Identification of proteins was performed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the samples collected from HPLC or by measuring the protein molecular mass coupling HPLC with mass spectrometry. The latter method allows the simultaneous separation of the phycobiliproteins, phycocyanin and allophycocyanin, from linker proteins and their identification, which due to their similar amino acid sequence and their similar hydrophobicity, might not be detected by denaturing SDS-PAGE. Under the experimental conditions used, the pigment phycobilin is not removed from the polypeptide backbone, determining the hydrophobicity of the phycoproteins and hence their interaction with the reversed-phase column as well as in determining the protein-protein interaction into the phycobilisome aggregation. Removal of the pigment, in fact, abolishes HPLC separation, emphasizing the essential role that the pigments play in maintaining the unusual tertiary structure of these proteins.     

Pure currents in foliated waveguides

We give an explicit characterization of obliquely propagatingelectric modes within a multilayered planar dielectric waveguideof cross-section [0, 1]. This foliated strip, with fixed metallicboundaries at 0 and 1, consists of an integer number N of periodiccells of thickness      

Fingerprinting of Antenna Proteins of Photosystem I by Reversed Phase High Performance Liquid Chromatography

The paper reports the results of a study that has been performed to investigate the suitability of reversed phase high performance liquid chromatography (RP-HPLC) at producing confident fingerprints for rapid and simple identification of the light-harvesting proteins (Lhca) comprising the photosystem I (PSI) complex. The paper shows that Lhca proteins are completely resolved and separated from the other proteins comprising the core-complex by subjecting to RP-HPLC the entire PSI preparation. In each of all examined species the four antenna proteins elute in the same order, namely Lhca1 < Lhca3 < Lhca4 < Lhca2, with significant differences in retention times among the four proteins, indicating that each PSI light harvesting protein exhibits a unique chromatographic pattern reflecting hydrophobicity and stoichiometry of the PSI proteins. A good correlation between experimental retention times in RP-HPLC of proteins and their predicted values based on the overall hydrophobicity and polypeptide chain length of the examined antenna proteins is reported. These findings and the observation that the amino acid sequence of Lhca proteins are highly conserved among species, as well as the chromatographic behaviour in RP-HPLC of the PSI proteins are equivalent in all investigated species, endorse the possibility of using RP-HPLC as a highly confident fingerprint supportive of unequivocal assignment of the proteins in preparations from different species without the necessity of protein identification by additional methods and useful for comparison within a single and among different species for future studies of the PSI proteins.Acknowledgements We are grateful to Dr. Maria Bianchetti and Dr. Corrado Ciambella for technical assistance.     

Leaky modes in twisted microstructured optical fibers

The purpose of this paper is to investigate the effect of a twist on the losses of leaky modes in microstructured optical fibers (MOF). A helicoidal system of coordinates is introduced to define the structure and to set up the problem. These coordinates, albeit non-orthogonal, preserve the translational invariance in a way that allows a two-dimensional finite element model similar to that of classical straight waveguides. The Perfectly Matched Layer (PML) technique is used to compute the leaky modes in the fibers. Helicoidal coordinates and PML are unified under the point of view of geometrical transformations which allows the design of 'twisted PML' that provides the right tool to determine the leaky modes in the twisted structures.     

Electromagnetic analysis of cylindrical invisibility cloaks and the mirage effect

We present a finite-element analysis of a diffraction problem involving a coated cylinder enabling the electromagnetic cloaking of a lossy object with sharp wedges located within its core. The coating consists of a heterogeneous anisotropic material deduced from a geometrical transformation as first proposed by Pendry [Science 312, 1780 (2006)]. We analyze the electromagnetic response of the cloak in the presence of an electric line source in p polarization and a loop of magnetic current in s polarization. We find that the electromagnetic field radiated by such a source located a fraction of a wavelength from the cloak is perturbed by less than 1%. When the source lies in the coating, it seems to radiate from a shifted location.     

Asymptotic modelling of weakly twisted electrostatic problemsModélisation asymptotique de problèmes électrostatiques faiblement torsadés

We present an analysis of an electrostatic field within a helicoidal structure with a twist, which is small compared to the characteristic size of the cross-section. The asymptotic results are checked against exact computations thanks to helicoidal coordinates, which preserve the intrinsically two-dimensional nature of the problem. The numerical studies are performed using the finite elements. To cite this article: A. Nicolet et al., C. R. Mecanique 334 (2006).