Labeling of Phosphatidylinositol Lipid Products in Cells through Metabolic Engineering by Using a Clickable myo-Inositol Probe.

Phosphatidylinositol (PI) lipids control critical biological processes, so aberrant biosynthesis often leads to disease. As a result, the capability to track the production and localization of these compounds in cells is vital for elucidating their complex roles. Herein, we report the design, synthesis, and application of clickable myo-inositol probe 1 a for bioorthogonal labeling of PI products. To validate this platform, we initially conducted PI synthase assays to show that 1 a inhibits PI production in vitro. Fluorescence microscopy experiments next showed probe-dependent imaging in T-24 human bladder cancer and Candida albicans cells. Growth studies in the latter showed that replacement of myo-inositol with probe 1 a led to an enhancement in cell growth. Finally, fluorescence-based TLC analysis and mass spectrometry experiments support the labeling of PI lipids. This approach provides a promising means for tracking the complex biosynthesis and trafficking of these lipids in cells.

Free radicals induced by sunlight in different spectral regions - in vivo versus ex vivo study.

Sunlight represents an exogenous factor stimulating formation of free radicals which can induce cell damage. To assess the effect of the different spectral solar regions on the development of free radicals in skin, in vivo electron paramagnetic resonance (EPR) investigations with human volunteers and ex vivo studies on excised human and porcine skin were carried out. For all skin probes, the ultraviolet (UV) spectral region stimulates the most intensive radical formation, followed by the visible (VIS) and the near infrared (NIR) regions. A comparison between the different skin models shows that for UV light, the fastest and highest production of free radicals could be detected in vivo, followed by excised porcine and human skin. The same distribution pattern was found for the VIS/NIR spectral regions, whereby the differences in radical formation between in vivo and ex vivo were less pronounced. An analysis of lipid composition in vivo before and after exposure to UV light clearly showed modifications in several skin lipid components; a decrease of ceramide subclass [AP2] and an increase of ceramide subclass [NP2], sodium cholesterol sulphate and squalene (SQ) were detectable. In contrast, VIS/NIR irradiation led to an increase of ceramides [AP2] and SCS, and a decrease of SQ. These results, which are largely comparable for the different skin models investigated in vivo and ex vivo, indicate that radiation exposure in different spectral regions strongly influences radical production in skin and also results in changes in skin lipid composition, which is essential for barrier function.

Characterization of atopic skin and the effect of a hyperforin-rich cream by laser scanning microscopy.

Atopic dermatitis (AD) is a multifactorial inflammatory skin disease that affects both children and adults in an increasing manner. The treatment of AD often reduces subjective skin parameters, such as itching, dryness, and tension, but the inflammation cannot be cured. Laser scanning microscopy was used to investigate the skin surface, epidermal, and dermal characteristics of dry and atopic skin before and after treatment with an ointment rich in hyperforin, which is known for its anti-inflammatory effects. The results were compared to subjective parameters and transepidermal water loss, stratum corneum moisture, and stratum corneum lipids. Using biophysical methods, in particular laser scanning microscopy, it was found that atopic skin has distinct features compared to healthy skin. Treatment with a hyperforin-rich ointment resulted in an improvement of the stratum corneum moisture, skin surface dryness, skin lipids, and the subjective skin parameters, indicating that the barrier is stabilized and improved by the ointment. But in contrast to the improved skin surface, the inflammation in the deeper epidermis/dermis often continues to exist. This could be clearly shown by the reflectance confocal microscopy (RCM) measurements. Therefore, RCM measurements could be used to investigate the progress in treatment of atopic dermatitis.

Fluorescently labeled substrates for monitoring α1,3-fucosyltransferase IX activity.

Fucosylation is often the final process in glycan biosynthesis. The resulting glycans are involved in a variety of biological processes, such as cell adhesion, inflammation, or tumor metastasis. Fucosyltransferases catalyze the transfer of fucose residues from the activated donor molecule GDP-ß-L-fucose to various acceptor molecules. However, detailed information about the reaction processes is still lacking for most fucosyltransferases. In this work we have monitored α1,3-fucosyltransferase activity. For both donor and acceptor substrates, the introduction of a fluorescent ATTO dye was the last step in the synthesis. The subsequent conversion of these substrates into fluorescently labeled products by α1,3-fucosyltransferases was examined by high-performance thin-layer chromatography coupled with mass spectrometry as well as dual-color fluorescence cross-correlation spectroscopy, which revealed that both fluorescently labeled donor GDP-ß-L-fucose-ATTO 550 and acceptor N-acetyllactosamine-ATTO 647N were accepted by recombinant human fucosyltransferase IX and Helicobacter pylori α1,3-fucosyltransferase, respectively. Analysis by fluorescence cross-correlation spectroscopy allowed a quick and versatile estimation of the progress of the enzymatic reaction and therefore this method can be used as an alternative method for investigating fucosyltransferase reactions.

One- and two-dimensional high-performance thin-layer chromatography as an alternative analytical tool for investigating polyphenol-protein interactions.

INTRODUCTION: Polyphenols and simple phenolic compounds are able to react with other food constituents during processing and storage. In the past, it has been shown that their reaction with proteins can lead to changes of the technofunctional or even physiological properties of both compound classes. However, identification of specific binding sites of small molecules within a protein sequence (and the corresponding conformational position) is still challenging. OBJECTIVE: Investigating the reaction between different food proteins and phenolic compounds in alkaline medium with one- and two-dimensional high-performance thin-layer chromatography (HPTLC) coupled to matrix-assisted laser desorption/ionisation (MALDI) with time-of-flight (TOF) MS for analysing the peptide profiles after tryptic digestion. METHODS: After modification with phenolic compounds, protein derivatives were digested and peptides were separated with one- and two-dimensional HPTLC. Peptide profiles were detected with visible and UV wavelengths as well as with fluorescamine, ninhydrin and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid staining. In order to perform mass spectrometric measurements, peptides separated in the first dimension were analysed by MALDI/TOF/MS. RESULTS: Results show that the phenolic acids applied in this study show different specificity and susceptibility when modifying proteins resulting in changes of the peptide profiles, peptide quantity, polarity, UV-activity, radical-scavenging activity and molecular mass. CONCLUSION: One- and two-dimensional HPTLC supported by mass spectrometric detection represents an innovative, alternative tool for investigating and understanding polyphenol-protein interactions. This approach enables the identification of binding sites inside the protein chain and contributes to understanding the mechanism of polyphenol-protein interactions in vitro and in vivo.