Kinetics of violaxanthin de-epoxidation by violaxanthin de-epoxidase, a xanthophyll cycle enzyme, is regulated by membrane fluidity in model lipid bilayers.

This paper describes violaxanthin de-epoxidation in model lipid bilayers. Unilamellar egg yolk phosphatidylcholine (PtdCho) vesicles supplemented with monogalactosyldiacylglycerol were found to be a suitable system for studying this reaction. Such a system resembles more the native thylakoid membrane and offers better possibilities for studying kinetics and factors controlling de-epoxidation of violaxanthin than a system composed only ofmonogalactosyldiacylglycerol and is commonly used in xanthophyll cycle studies. The activity of violaxanthin de-epoxidase (VDE) strongly depended on the ratio of monogalactosyldiacylglycerol to PtdCho in liposomes. The mathematical model of violaxanthin de-epoxidation was applied to calculate the probability of violaxanthin to zeaxanthin conversion at different phases of de-epoxidation reactions. Measurements of deepoxidation rate and EPR-spin label study at different temperatures revealed that dynamic properties of the membrane are important factors that might control conversion of violaxanthin to antheraxanthin. A model of the molecular mechanism of violaxanthin de-epoxidation where the reversed hexagonal structures (mainly created by monogalactosyldiacylglycerol) are assumed to be required for violaxanthin conversion to zeaxanthin is proposed. The presence of monogalactosyldiacylglycerol reversed hexagonal phase was detected in the PtdCho/monogalactosyldiacylglycerol liposomes membrane by 31P-NMR studies. The availability of violaxanthin for de-epoxidation is a diffusion-dependent process controlled by membrane fluidity. The significance of the presented results for understanding themechanism of violaxanthin de-epoxidation in native thylakoid membranes is discussed.     

Life History and Secondary Production of Mayflies (Ephemeroptera) Indicate Disturbance in Two Small Carpathian Streams

The life histories and the secondary production of 9 mayflies (Ephemeroptera) species were studied at two sites in a submountain stream (West Carpathians, Slovakia). The disturbed site has a deforested and converted to meadows and pastures catchment and the undisturbed one is a well‐preserved submountain stream with 60% of the catchment covered by spruce forests. Differences in the forest cover and in the thermal regime of both streams were reflected in the structural as well as the functional measures. At the undisturbed site, the total secondary production of mayfly community was more than two times higher (3568 mg DW m^–2 y^–1) than at the disturbed site (1446 mg DW m^–2 y^–1). Species could be divided according to their affinity to particular disturbance level. Ecdyonurus picteti, Rhithrogena carpatoalpina, Habrophlebia lauta and Habroleptoides confusa appeared to prefer undisturbed conditions. On the contrary, Ecdyonurus aurantiacus and Habrophlebia fusca occurred only at the disturbed site. The dominant species Baetis rhodani and Electrogena samalorum did not show any clear preference regarding the catchment disturbance. However, nymphs of Electrogena samalorum seem to be more successful at the disturbed site, where they reached bigger body sizes (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Erythrocyte Membrane Properties in Patients with Essential Hypertension

In spite of the extensive research efforts that have been conducted over the last decades, it is still very difficult to point out genetic determinants or environmental conditions responsible for the development of essential hypertension. We searched for differences in the RBC membrane skeleton structure and O₂ membrane permeability between RBCs from patients with both essential arterial hypertension and hypercholesterolemia, from patients having only hypercholesterolemia and from healthy donors. The topography of RBCs and the content of various hemoglobin forms were detected using atomic force microscopy and Mössbauer spectroscopy, respectively. We found that the membrane skeleton of RBCs from healthy donors displayed a well-known honeycomb pattern, whereas in patients with essential hypertension and/or hypercholesterolemia, who had never received anti-hypertensive therapy, it displayed a corncob pattern. Hypertensive RBCs had an oval shape and the average lateral to longitudinal diameter ratio for the changed cells (about 70 %) did not exceed 0.80. We observed that after the incubation of RBCs under high nitrogen (low O₂) pressure at room temperature and then their transfer into 85 K, a content of oxyHb (deoxyHbOH) already after 1 h reached a stable level of about 85 ± 3 % (15 ± 3 %) in hypertensives, whereas in healthy individuals it showed a decrease for deoxyHbOH and an increase for oxyHb, which stabilized at a level of about 81 ± 5 % and 19 ± 5 %, respectively, only after 9 h. Quantitative analysis of the Δ(oxyHb) change estimated as the difference between the oxyHb level measured after 9 and 2 h at 85 K under low N₂ pressure (to slow down oxyHb formation) was significantly higher in normotensives than in hypertensive patients with or without hypercholesterolemia, 19.9 versus ?4.2, p < 0.02. Our findings indicate an impaired oxygen release by Hb in RBCs of patients with hypertension under low oxygen pressure which if present in vivo may cause hypoxemia and, in turn, further increase of blood pressure.     

Undifferentiated Bronchial Fibroblasts Derived from Asthmatic Patients Display Higher Elastic Modulus than Their Non-Asthmatic Counterparts

During asthma development, differentiation of epithelial cells and fibroblasts towards the contractile phenotype is associated with bronchial wall remodeling and airway constriction. Pathological fibroblast-to-myofibroblast transition (FMT) can be triggered by local inflammation of bronchial walls. Recently, we have demonstrated that human bronchial fibroblasts (HBFs) derived from asthmatic patients display some inherent features which facilitate their FMT in vitro. In spite of intensive research efforts, these properties remain unknown. Importantly, the role of undifferentiated HBFs in the asthmatic process was systematically omitted. Specifically, biomechanical properties of undifferentiated HBFs have not been considered in either FMT or airway remodeling in vivo. Here, we combine atomic force spectroscopy with fluorescence microscopy to compare mechanical properties and actin cytoskeleton architecture of HBFs derived from asthmatic patients and non-asthmatic donors. Our results demonstrate that asthmatic HBFs form thick and aligned ‘ventral’ stress fibers accompanied by enlarged focal adhesions. The differences in cytoskeleton architecture between asthmatic and non-asthmatic cells correlate with higher elastic modulus of asthmatic HBFs and their increased predilection to TGF-β-induced FMT. Due to the obvious links between cytoskeleton architecture and mechanical equilibrium, our observations indicate that HBFs derived from asthmatic bronchi can develop considerably higher static tension than non-asthmatic HBFs. This previously unexplored property of asthmatic HBFs may be potentially important for their myofibroblastic differentiation and bronchial wall remodeling during asthma development.     

Nanomechanical analysis of pigmented human melanoma cells

Based on hitherto measurements of elasticity of various cells in vitro and ex vivo, cancer cells are generally believed to be much softer than their normal counterparts. In spite of significant research efforts on the elasticity of cancer cells, only few studies were undertaken with melanoma cells. However, there are no reports concerning pigmented melanoma cells. Here, we report for the first time on the elasticity of pigmented human melanoma cells. The obtained data show that melanin significantly increases the stiffness of pigmented melanoma cells and that the effect depends on the amount of melanin inside the cells. The dramatic impact of melanin on the nanomechanical properties of cells puts into question widely accepted paradigm about all cancer cells being softer than their normal counterparts. Our findings reveal significant limitations of the nanodiagnosis approach for melanoma and contribute to better understanding of cell elasticity.     

A facile synthesis of D-ribo-C(20)-phytosphingosine and its C2 epimer from D-ribose

A facile synthetic route to d-ribo-C(20)-phytosphingosine 31 and its C2 epimer 32 is described. The Overman rearrangement of allylic trichloroacetimidates derived from the known ribose derivative 7 has been used as the key step. The subsequent functional group interconversions in rearranged products 14 and 15 followed by Wittig olefination, Pd/C-mediated reduction and the removal of protecting groups successfully constructed the final molecules.     

Scavenging of superoxide generated in photosystem I by plastoquinol and other prenyllipids in thylakoid membranes

We have examined scavenging of a superoxide by various prenyllipids occurring in thylakoid membranes, such as plastoquinone-9, alpha-tocopherolquinone, their reduced forms, and alpha-tocopherol, measuring oxygen uptake in hexane-extracted and untreated spinach thylakoids with a fast oxygen electrode under flash-light illumination. The obtained results demonstrated that all the investigated prenyllipids showed the superoxide scavenging properties, and plastoquinol-9 was the most active in this respect. Plastoquinol-9 formed in thylakoids as a result of enzymatic reduction of plastoquinone-9 by ferredoxin-plastoquinone reductase was even more active than the externally added plastoquinol-9 in the investigated reaction. Scavenging of superoxide by plastoquinol-9 and other prenyllipids could be important for protecting membrane components against the toxic action of superoxide. Moreover, our results indicate that vitamin K(1) is probably the most active redox component of photosystem I in the generation of superoxide within thylakoid membranes.     

A facile synthesis of d-ribo-C20-phytosphingosine and its C2 epimer from d-ribose

A facile synthetic route to d-ribo-C₂₀-phytosphingosine 31 and its C2 epimer 32 is described. The Overman rearrangement of allylic trichloroacetimidates derived from the known ribose derivative 7 has been used as the key step. The subsequent functional group interconversions in rearranged products 14 and 15 followed by Wittig olefination, Pd/C-mediated reduction and the removal of protecting groups successfully constructed the final molecules.