Experimental Measurements and Mathematical Modeling of Cytosolic Ca(2+) Signatures upon Elicitation by Penta-N-acetylchitopentaose Oligosaccharides in Nicotiana tabacum Cell Cultures.

Plants have developed sophisticated recognition systems for different kinds of pathogens. Pathogen-associated molecular patterns (PAMPs) can induce various defense mechanisms, e.g., the production of reactive oxygen species (ROS) as an early event. Plant defense reactions are initiated by a signal transduction cascade involving the release of calcium ions (Ca(2+)) from both external and internal stores to the plant cytoplasm. This work focuses on the analysis of cytosolic Ca(2+) signatures, experimentally and theoretically. Cytosolic Ca(2+) signals were measured in Nicotiana tabacum plant cell cultures after elicitation with penta-N-acetylchitopentaose oligosaccharides (Ch5). In order to allow a mathematical simulation of the elicitor-triggered C(a2)+ release, the Li and Rinzel model was adapted to the situation in plants. The main features of the Ca2+ response, like the specific shape of the C(a2)+ transient and the dose-response relationship, could be reproduced very well. Repeated elicitation of the same cell culture revealed a refractory behavior with respect to the Ca(2+) transients for this condition. Detailed analysis of the obtained data resulted in further modifications of the mathematical model, allowing a predictive simulation of Ch5-induced C(a2)+ transients. The promising results may contribute to a deeper understanding of the underlying mechanisms governing plant defense.

Toward protein biomarkers for allergy: CD4+ T cell proteomics in allergic and nonallergic subjects sampled in and out of pollen season.

Allergy is an immunological disorder of the upper airways, lung, skin, and the gut with a growing prevalence over the last decades in Western countries. Atopy, the genetic predisposition for allergy, is strongly dependent on familial inheritance and environmental factors. These observations call for predictive markers of progression from atopy to allergy, a prerequisite to any active intervention in neonates and children (prophylactic interventions/primary prevention) or in adults (immunomodulatory interventions/secondary prevention). In an attempt to identify early biomarkers of the "atopic march" using minimally invasive sampling, CD4+ T cells from 20 adult volunteers (10 healthy and 10 with respiratory allergies) were isolated and quantitatively analyzed and their proteomes were compared in and out of pollen season (± antigen exposure). The proteome study based on high-resolution 2D gel electrophoresis revealed three candidate protein markers that distinguish the CD4+ T cell proteomes of normal from allergic individuals when sampled out of pollen season, namely Talin 1, Nipsnap homologue 3A, and Glutamate-cysteine ligase regulatory protein. Three proteins were found differentially expressed between the CD4+ T cell proteomes of normal and allergic subjects when sampled during pollen season: carbonyl reductase, glutathione S-transferase ω 1, and 2,4-dienoyl-CoA reductase. The results were partly validated by Western blotting.

2-D differential membrane proteome analysis of scarce protein samples.

Proteome studies with small sample amounts are difficult to perform, especially when membrane proteins are the focus of interest. In our study a new method for the analysis of scarce membrane protein samples combining large gel 2-D-CTAB/SDS-PAGE with fluorescence dye saturation labelling (satDIGE) was developed, allowing a highly sensitive differential analysis of different cell states. After Triton X-114 phase partitioning, enriched membrane protein samples of T cells were labelled at cysteine residues using fluorescence dyes and separated by large gel 2D-CTAB/SDS-PAGE. For a differential analysis 3 mug protein was found to be sufficient to detect proteins in a widespread well-separated diagonal spot pattern.