Post-translational modifications of arabinogalactan-peptides of Arabidopsis thaliana. Endoplasmic reticulum and glycosylphosphatidylinositol-anchor signal cleavage sites and hydroxylation of proline

We have developed a method for separating the deglycosylated protein/peptide backbones of the small arabinogalactan (AG)-peptides from the larger classical arabinogalactan-proteins (AGPs). AGPs are an important class of plant proteoglycans implicated in plant growth and development. Separation of AG-peptides enabled us to identify eight of 12 AG-peptides from Arabidopsis thaliana predicted from genomic sequences. Of the remaining four, two have low abundance based on expressed sequence tag databases and the other two are only present in pollen (At3g20865) or flowers (At3g57690) and therefore would not be detected in our analysis. Characterization of AG-peptides was performed using matrix-assisted laser desorption ionization-time of flight mass spectrometry and tandem mass spectrometry protein sequencing. These data provide (i) experimental evidence that AG-peptides are processed in vivo for the addition of a glycosylphosphatidylinositol (GPI) anchor, (ii) cleavage site information for both the endoplasmic reticulum secretion signal and the GPI-anchor signal for eight of the 12 AG-peptides, and (iii) experimental evidence that the Gly-Pro motif is hydroxylated in vivo. Furthermore, we show that AtAGP16 is GPI-anchored despite its unusually long hydrophobic C-terminal GPI-signal sequence. Prior to this work, the GPI-anchor cleavage site for only two plant proteins, NaAGP1 from Nicotiana alata and PcAGP1 from Pyrus communis, had been determined experimentally. Characterization of the post-translational modifications of AG-peptides contributes toward obtaining the complete primary structure of this class of biologically important plant proteoglycans and provides a greater understanding of post-translational modifications of plant proteins.     

Caspase-specific and nonspecific in vivo protein processing during Fas-induced apoptosis

We generated a comprehensive picture of protease substrates in anti-Fas-treated apoptotic human Jurkat T lymphocytes. We used combined fractional diagonal chromatography (COFRADIC) sorting of protein amino-terminal peptides coupled to oxygen-16 or oxygen-18 differential labeling. We identified protease substrates and located the exact cleavage sites within processed proteins. Our analysis yielded 1,834 protein identifications and located 93 cleavage sites in 71 proteins. Indirect evidence of apoptosis-specific cleavage within 21 additional proteins increased the total number of processed proteins to 92. Most cleavages were at caspase consensus sites; however, other cleavage specificities suggest activation of other proteases. We validated several new processing events by immunodetection and by an in vitro assay using recombinant caspases and synthetic peptides containing presumed cleavage sites. The spliceosome complex appeared a preferred target, as 14 of its members were processed. Differential isotopic labeling further revealed specific release of nucleosomal components from apoptotic nuclei.     

Preparation of protein extracts from recalcitrant plant tissues: an evaluation of different methods for two-dimensional gel electrophoresis analysis

This study focuses on the specific problems of protein extraction from recalcitrant plant tissues and evaluates several methods to bypass them. Sample preparation is a critical step in a two-dimensional gel electrophoresis proteome approach and is absolutely essential for good results. We evaluated four methods: the classical trichloroacetic acid (TCA)/acetone precipitation, TCA/acetone precipitation and fractionation, an alternative based on fractionation and without precipitation, and phenol extraction methanol/ammonium acetate precipitation. We optimized the phenol extraction protocol for small amounts of tissue, which is essential when the study material is limited. The protocol was optimized for banana (Musa spp.) and was subsequently applied to two other plant species: apple (Malus domestica L.) and potato (Solanum tuberosum L.). Banana (Musa spp.) is a good representative of a "difficult" plant species since it contains many interfering metabolites. Only classical TCA/acetone precipitation and phenol extraction methods proved useful as standard methods. Both methods are associated with a minor but reproducible loss of proteins. Every extraction method and the subsequent analytical procedure have their physicochemical limitations; both methods should be investigated before selecting an appropriate protocol. The study, which is presented in this paper, is useful for guiding the experimental setup of many other nonmodel species, containing various interfering elements.     

Calcineurin initiates smooth muscle differentiation in neural crest stem cells

The process of vascular smooth muscle cell (vSMC) differentiation is critical to embryonic angiogenesis. However, despite its importance, the vSMC differentiation program remains largely undefined. Murine gene disruption studies have identified several gene products that are necessary for vSMC differentiation, but these methodologies cannot establish whether or not a factor is sufficient to initiate the differentiation program. A gain-of-function system consisting of normal vSMC progenitor cells would serve as a useful complement to whole animal loss-of-function studies. We use such a system here, namely freshly isolated rat neural crest stem cells (NCSCs), to show that activation of the calcineurin signaling pathway is sufficient to drive these cells toward a smooth muscle fate. In addition, we present data suggesting that transforming growth factor (TGF)-beta1, which also causes NCSCs to differentiate into smooth muscle, activates calcineurin signaling in NCSCs, leading to a model in which activation of calcineurin signaling is the mechanism by which TGF-beta1 causes SMC differentiation in these cells.     

Role of Oxidative Phosphorylation in Histatin 5-Induced Cell Death in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

The succeptibility of Saccharomyces cerevisiae to the anti-microbial peptide, histatin 5, was tested after pre-growth in fermentable and non-fermentable carbon sources and in the absence or presence of the uncoupler of oxidative phosphorylation, carbonyl cyanide m-chlorophenylhydrazone (CCCP). S. cerevisiae was more resistant to histatin 5 when grown on a fermentable carbon source compared to growth on a non-fermentable carbon source, indicating an important role for oxidative phosphorylation in histatin 5-induced cell death. Oxidative phosphorylation is a pre-requisite for histatin 5-induced cell death in Candida albicans but this is not the case in S. cerevisiae. Incubation of CCCP-treated S. cerevisiae cells with histatin 5 still resulted in cell death. These results suggest that histatin 5-induced cell death in S. cerevisiae differs from that in C. albicans.Revisions received 28 September 2004     

DNA vaccines: designing strategies against parasitic infections

The complexity of parasitic infections requires novel approaches to vaccine design. The versatility of DNA vaccination provides new perspectives. This review discusses the use of prime-boost immunizations, genetic adjuvants, multivalent vaccines and codon optimization for optimal DNA vaccine design against parasites.     

Construction of a two-dimensional gel electrophoresis protein database for the Nicotiana tabacum cv. Bright Yellow-2 cell suspension culture

Using two-dimensional gel electrophoresis (2-DE) and electrospray-tandem mass spectrometry (ESI-MS/MS), we have started the proteome analysis of the cell line Nicotiana tabacum cv. Bright Yellow-2 (tobacco BY-2). The BY-2 cell suspension culture is widely used as a model system to study the growth and development of plant cells. We present a protocol describing the sample preparation and 2-DE, enabling us to separate and display more than 1000 proteins from this cell culture. A reference gel was generated, using immobilized pH gradient isoelectric focusing in a linear gradient from pH 3 to 10 and 12% Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Although the tobacco genome is not sequenced yet, a range of protein spots from this reference map was identified by means of a semi-automated liquid chromatography-ESI-quadrupole time of flight-tandem MS (LC-ESI-QTOF-MS-MS) setup and cross-species matching. These data were integrated in a database, which can be accessed at http://tby2-www.uia.ac.be/tby2/. On the on-line reference map, the identified protein spots are hyperlinked to individual protein entries. Each protein entry contains all identification information, as well as links to relevant entries in other on-line databases. Comprehensive search functions are implemented. Especially for an unsequenced but widespread model organism like tobacco BY-2, such a reference database is a convenient source for protein information that brings protein identification within reach without the need for extensive MS. This publicly accessible database provides a solid basis for tobacco BY-2 proteomics in the future.     

Synthesis and biological evaluation of the major metabolite of atomoxetine: elucidation of a partial kappa-opioid agonist effect

The major human metabolite of atomoxetine (4-hydroxyatomoxetine) was tested against a panel of receptors and enzymes, and was found to interact with the mu, delta, and kappa-opioid receptors based upon studies involving both binding and functional assays. 4-hydroxyatomoxetine was determined to be a partial agonist of the kappa-opioid receptor.     

Vinculin--a dynamic regulator of cell adhesion

The ability of cells to tightly adhere to one another and to the extracellular matrix is fundamentally important in numerous biological processes, including embryogenesis, wound healing and maintenance of tissue integrity. Vinculin, a protein localized at the cytoplasmic face of cell-matrix and cell-cell adhesions, is required for strong cell adhesion. Two new crystal structures reveal that vinculin exhibits a high degree of structural plasticity upon ligand binding that might promote rapid changes in cell adhesion.     

Shin-byung (Divine Illness) in a Korean woman

Culture, Medicine, and Psychiatry -