Spruce versus Arabidopsis: different strategies of photosynthetic acclimation to light intensity change.

The acclimation of higher plants to different light intensities is associated with a reorganization of the photosynthetic apparatus. These modifications, namely, changes in the amount of peripheral antenna (LHCII) of photosystem (PS) II and changes in PSII/PSI stoichiometry, typically lead to an altered chlorophyll (Chl) a/b ratio. However, our previous studies show that in spruce, this ratio is not affected by changes in growth light intensity. The evolutionary loss of PSII antenna proteins LHCB3 and LHCB6 in the Pinaceae family is another indication that the light acclimation strategy in spruce could be different. Here we show that, unlike Arabidopsis, spruce does not modify its PSII/PSI ratio and PSII antenna size to maximize its photosynthetic performance during light acclimation. Its large PSII antenna consists of many weakly bound LHCIIs, which form effective quenching centers, even at relatively low light. This, together with sensitive photosynthetic control on the level of cytochrome b6f complex (protecting PSI), is the crucial photoprotective mechanism in spruce. High-light acclimation of spruce involves the disruption of PSII macro-organization, reduction of the amount of both PSII and PSI core complexes, synthesis of stress proteins that bind released Chls, and formation of "locked-in" quenching centers from uncoupled LHCIIs. Such response has been previously observed in the evergreen angiosperm Monstera deliciosa exposed to high light. We suggest that, in contrast to annuals, shade-tolerant evergreen land plants have their own strategy to cope with light intensity changes and the hallmark of this strategy is a stable Chl a/b ratio.

Effect of the natural flavonoids myricetin and dihydromyricetin on the wound healing process in vitro.

Myricetin (MYR) and dihydromyricetin (DHM) are classified as natural flavonoids. Both substances are known for their anti-inflammatory and antioxidant properties. In this study, an in vitro model of inflammation was demonstrated on monolayers of scratched fibroblasts or keratinocytes exposed to LPS from Pseudomonas aeruginosa for six hours. MYR and DHM were subsequently applied to the cells for 24 hours at sub toxic concentrations (5-15 µM). Inflammatory parameters were analysed in collected cell medium and lysate after the incubation period using the Enzyme-Linked ImmuneSorbent Assay (ELISA) and Western blot. Both flavonoids inhibit the production of pro-inflammatory cytokines (IL-6, IL-8) in LPS-stimulated skin cells as well as the decreased level of MMP-1 in fibroblasts. However, the application of MYR and DHM dose dependently increased the level of MMP-1 in keratinocytes. In our experiments, we focused on the anti-glycation activity of MYR and DHM, where the higher concentration of MYR seems to be more effective.

Electrocatalytic artificial carbonylation assay for observation of human serum albumin inter-individual properties.

Human serum albumin (HSA) is a multifunctional protein with ligand binding, transporting and buffering properties. Posttranslational modifications and ligand binding processes are closely related to albumin final functional status. In the last few decades, HSA has been characterized using a broad spectrum of methods, but quantitative data on the HSA's modifications among individuals have not been reported. The investigations presented here are based on the non-denaturing electrocatalytic screening of HSA samples isolated from the blood serum of healthy subjects. The electrocatalytic responses of the native protein (Rnat) varied depending on its modifications among individuals, which enable us to express the inter-individual variability. Consequently, the native HSA samples were subjected to ex vivo carbonylation with 50 mM methylglyoxal for 36 h. The differences between Rnat and the responses of artificially carbonylated protein (Rmod) corresponded with inter-individual binding capacity variations (ΔR = Rnat-Rmod). The coefficients of variation for the Rnat and ΔR values of purified HSA samples were estimated to be 8.5 and 23.2%, respectively. A sensitive non-denaturing electrocatalytic assay was utilized to provide new data about albumin inter-individual variations and evaluate its oxidative modifications and binding capacity, which could be used for further studies targeting not only on HSA but also other clinically important proteins.

Na+/K+-ATPase interaction with methylglyoxal as reactive metabolic side product.

Proteins are subject to oxidative modification and the formation of adducts with a broad spectrum of reactive species via enzymatic and non-enzymatic mechanisms. Here we report that in vitro non-enzymatic methylglyoxal (MGO) binding causes the inhibition and formation of MGO advanced glycation end-products (MAGEs) in Na+/K+-ATPase (NKA). Concretely, MGO adducts with NKA amino acid residues (mainly Arg) and Nε-(carboxymethyl)lysine (CML) formation were found. MGO is not only an inhibitor for solubilized NKA (IC50=91±16µM), but also for reconstituted NKA in the lipid bilayer environment, which was clearly demonstrated using a DPPC/DPPE liposome model in the presence or absence of the NKA-selective inhibitor ouabain. High-resolution mass spectrometric analysis of a tryptic digest of NKA isolated from pig (Sus scrofa) kidney indicates that the intracellular α-subunit is naturally (post-translationally) modified by MGO in vivo. In contrast to this, the ß-subunit could only be modified by MGO artificially, and the transmembrane part of the protein did not undergo MGO binding under the experimental setup used. As with bovine serum albumin, serving as the water-soluble model, we also demonstrated a high binding capacity of MGO to water-poorly soluble NKA using a multi-spectral methodology based on electroanalytical, immunochemical and fluorimetric tools. In addition, a partial suppression of the MGO-mediated inhibitory effect could be observed in the presence of aminoguanidine (pimagedine), a glycation suppressor and MGO-scavenger. All the results here were obtained with the X-ray structure of NKA in the E1 conformation (3WGV) and could be used in the further interpretation of the functionality of this key enzyme in the presence of highly-reactive metabolic side-products, glycation agents and generally under oxidative stress conditions.