Autofluorescence as a Signal to Sort Developing Glandular Trichomes by Flow Cytometry.

The industrial relevance of a number of metabolites produced in plant glandular trichomes (GTs) has spurred research on these specialized organs for a number of years. Most of the research, however, has focused on the elucidation of secondary metabolite pathways and comparatively little has been undertaken on the development and differentiation of GTs. One way to gain insight into these developmental processes is to generate stage-specific transcriptome and metabolome data. The difficulty for this resides in the isolation of early stages of development of the GTs. Here we describe a method for the separation and isolation of intact young and mature type VI trichomes from the wild tomato species Solanum habrochaites. The final and key step of the method uses cell sorting based on distinct autofluorescence signals of the young and mature trichomes. We demonstrate that sorting by flow cytometry allows recovering pure fractions of young and mature trichomes. Furthermore, we show that the sorted trichomes can be used for transcript and metabolite analyses. Because many plant tissues or cells have distinct autofluorescence components, the principles of this method can be generally applicable for the isolation of specific cell types without prior labeling.

Novel insights in the dysfunction of human blood-brain barrier after glycation.

The blood-brain barrier (BBB) provides a dynamic and complex interface consisting of endothelial cells, pericytes and astrocytes, which are embedded in a collagen and fibronectin-rich basement membrane. This complex structure restricts the diffusion of small hydrophilic solutes and macromolecules as well as the transmigration of leukocytes into the brain. It has been shown that carbonyl stress followed by the formation of advanced glycation endproducts (AGE=glycation) interfere with the BBB integrity and function. Here, we present data that carbonyl stress induced by methylglyoxal leads to glycation of endothelial cells and the basement membrane, which interferes with the barrier-function and with the expression of RAGE, occludin and ZO-1. Furthermore, methylglyoxal induced carbonyl stress promotes the expression of the pro-inflammatory interleukins IL-6 and IL-8. In summary, this study provides new insights into the relationship between AGE formation by carbonyl stress and brain microvascular endothelial barrier dysfunction.

Glycation of the high affinity NGF-receptor and RAGE leads to reduced ligand affinity.

AGEs are posttranslational modifications generated by irreversible non-enzymatic crosslinking reactions between sugars and proteins - a reaction referred to as glycation. Glycation, a feature of ageing, can lead to non-degradable and less functional proteins and enzymes and can additionally induce inflammation and further pathophysiological processes such as neurodegeneration. In this study we investigated the influence of glycation on the high affinity NGF-receptor TrkA and the AGE-receptor RAGE. We quantified the binding affinity of the TrkA-receptor and RAGE to their ligands by surface plasmon resonance (SPR) and compared these to the binding affinity after glycation. At the same time, we established a glycation procedure using SPR. We found that glycation of TrkA reduced the affinity to NGF by a factor of three, which could be shown to lead to a reduction of NGF-dependent neurite outgrowth in PC12 cells. Glycation of RAGE reduced binding affinity of AGEs by 10-fold.