[Show abstract][Hide abstract]ABSTRACT: Long-chain fatty acids are internalized by receptor-mediated mechanisms or receptor-independent diffusion across cytoplasmic
membranes and are utilized as nutrients, building blocks, and signaling intermediates. Here we describe how the association
of long-chain fatty acids to a partially unfolded, extracellular protein can alter the presentation to target cells and cellular
effects. HAMLET (human α-lactalbumin made lethal to tumor cells) is a tumoricidal complex of partially unfolded α-lactalbumin
and oleic acid (OA). As OA lacks independent tumoricidal activity at concentrations equimolar to HAMLET, the contribution
of the lipid has been debated. We show by natural abundance 13C NMR that the lipid in HAMLET is deprotonated and by chromatography that oleate rather than oleic acid is the relevant HAMLET
constituent. Compared with HAMLET, oleate (175 μm) showed weak effects on ion fluxes and gene expression. Unlike HAMLET, which causes metabolic paralysis, fatty acid metabolites
were less strongly altered. The functional overlap increased with higher oleate concentrations (500 μm). Cellular responses to OA were weak or absent, suggesting that deprotonation favors cellular interactions of fatty acids.
Fatty acids may thus exert some of their essential effects on host cells when in the deprotonated state and when presented
in the context of a partially unfolded protein.
[Show abstract][Hide abstract]ABSTRACT: In a variety of neurological diseases, pathological progression is cell type and region specific. Previous reports suggest that mass spectrometry imaging has the potential to differentiate between brain regions enriched in specific cell types. Here, we utilized a matrix-free surface mass spectrometry approach, nanostructure initiator mass spectrometry (NIMS), to show that spatial distributions of multiple lipids can be used as a 'fingerprint' to discriminate between neuronal- and glial- enriched brain regions. In addition, glial cells from different brain regions can be distinguished based on unique lipid profiles. NIMS images were generated from sagittal brain sections and were matched with immunostained serial sections to define glial cell enriched areas. Tandem mass spectrometry (LC-MS/MS QTOF) on whole brain extracts was used to identify 18 phospholipids. Multivariate statistical analysis (Nonnegative Matrix Factorization) enhanced differentiation of brain regions and cell populations compared to single ion imaging methods. This analysis resolved brain regions that are difficult to distinguish using conventional stains but are known to have distinct physiological functions. This method accurately distinguished the frontal (or somatomotor) and dorsal (or retrosplenial) regions of the cortex from each other and from the pons region.