Eszter Illyés

The role of nanoparticle concentration-dependent induction of cellular stress in the internalization of non-toxic cationic magnetoliposomes.

Magnetoliposomes (MLs), built up of ultrasmall iron oxide cores each individually surrounded by a lipid bilayer, have emerged as highly biocompatible nanoparticles and promising tools in many biomedical applications. To improve cell uptake, cationic amphiphiles are inserted into the ML coat, but this often induces cytotoxic effects. In the present work, we synthesized and tested a cationic peptide-lipid conjugate (dipalmitoylphosphatidylethanolamine-succinyl-tetralysine [DPPE-succ-(Lys)4]) which is entirely composed of biodegradable moieties and specifically designed to exert minimal cytotoxic effects. Uptake studies with both murine 3T3 fibroblasts and C17.2 neural progenitor cells shows 95.63 +/- 5.83 pg Fe and 87.46 +/- 5.62 pg Fe per cell after 24 h, respectively, for 16.66% DPPE-succ-(Lys)4-containing MLs, with no effect on cell viability. However, these high intracellular nanoparticle concentrations transiently affect actin cytoskeleton architecture, formation of focal adhesion complexes and cell proliferation, returning to control levels after approximately 7 days post ML-incubation in both cell types. This study points out the great need for thorough characterization of cell-nanoparticle interactions as subtle time-dependent effects are hard to monitor and commonly used viability and functionality assays are not sufficient to address the broad spectrum of possible interferences of the nanoparticle with normal cell functioning.

Semi-dry grasslands along a climatic gradient across Central Europe: Vegetation classification with validation

Abstract Question: What is the variation in species composition of Central European semi-dry grasslands? Can we apply a training-and-test validation approach for identifying phytosociological associations which are floristically well defined in a broad geographic comparison; can we separate them from earlier described associations with only a local validity? Location: A 1200 km long transect running along a gradient of increasing continentality from central Germany via Czech Republic, Slovakia, NE Austria, Hungary to NW Romania. Methods: Relevés with > 25% cover of Brachypodium pinnatum and/or Bromus erectus were geographically selected from a larger database. They were randomly split into two data sets, TRAINING and TEST, each with 422 relevés. Cluster analysis was performed for each data set on scores from significant principal coordinates. Different partitions of the TRAINING data set were validated on the TEST data set, using a new method based on the comparison of % frequencies of species occurrence in clusters. Clusters were characterized by statistically defined groups of diagnostic species and values of climatic variables. Results: Species composition changed along the NW-SE gradient and valid clusters were geographically well separated. Optimal partition level was at 11 clusters, six being valid: two clusters Germany and the Czech Republic corresponded to the Bromion erecti; two clusters from the Czech Republic and Hungary to the Cirsio-Brachypodion, and two clusters were transitional between these two alliances. Conclusion: The training-and-test validation method used in this paper proved to be efficient for discriminating between robust clusters, which are appropriate candidates for inclusion in the national or regional syntaxonomic overviews, and weak clusters, which are specific to the particular classification of the given data set. Nomenclature: Ehrendorfer (1973).

A grid-based, satellite-image supported, multi-attributed vegetation mapping method (MÉTA)

In this paper we present the main characteristics of a new, grid-based, landscape-ecology-oriented, satellite-image supported, field vegetation mapping method, called MÉTA (MÉTA stands for Magyarországi Élőhelyek Térképi Adatbázisa: GIS Database of the Hungarian Habitats). The goals of the MÉTA method based vegetation mapping program (MÉTA mapping) include the following: (1) to map the actual (semi-)natural vegetation of Hungary; (2) to evaluate Hungarian (semi-)natural vegetation heritage for conservation purposes; (3) to evaluate the present state of Hungarian landscapes from a vegetation point of view; (4) to collect vegetation and landscape ecological data for the prognosis of future changes of vegetation and the landscape. Spatial resolution, mapped attributes and mapping methods were developed to meet these goals.The MÉTA method uses a hexagon grid with cells of 35 hectares. In the hexagons, habitat types are listed, then the area, naturalness-based habitat quality, spatial pattern in the hexagon, effect of the neighbourhood, connectedness, and threats are recorded for each habitat type. Other attributes are recorded in the hexagons: potential natural vegetation, area occupied by invasive plant species, area of old fields, land use of grasslands, and landscape health status (naturalness and regeneration potential of the landscape in general). One hundred hexagons form a quadrat — mainly for practical, organizational reasons, but also for collecting certain vegetation data at this spatial scale. For standardization of mapping, three different pre-printed data sheets and two different kinds of guides have been composed (Mapping Guide and Habitat Guide) and field trainings were organized. For standardization of estimation of naturalness-based habitat quality and regeneration potential field examples were prepared for each habitat type and each category of these attributes.

Anti-influenza virus activity and structure-activity relationship of aglycoristocetin derivatives with cyclobutenedione carrying hydrophobic chains

Abstract Previous studies have demonstrated that glycopeptide compounds carrying hydrophobic substituents can have favorable pharmacological (i.e. antibacterial and antiviral) properties. We here report on the in vitro anti-influenza virus activity of aglycoristocetin derivatives containing hydrophobic side chain-substituted cyclobutenedione. The lead compound 8e displayed an antivirally effective concentration of 0.4μM, which was consistent amongst influenza A/H1N1, A/H3N2 and B viruses, and a selectivity index ≥50. Structural analogues derived from aglycovancomycin were found to be inactive. The hydrophobic side chain was shown to be an important determinant of activity. The narrow structure–activity relationship and broad activity against several human influenza viruses suggest a highly conserved interaction site, which is presumably related to the influenza virus entry process. Compound 8e proved to be inactive against several unrelated RNA and DNA viruses, except for varicella-zoster virus, against which a favorable activity was noted.

Two functional active conformations of the integrin α2β1, depending on activation condition and cell type

For several integrins, the existence of multiple conformational states has been studied intensively. For the integrin α2β1, a major collagen receptor on platelets and other cell types, however, no such experimental data were available thus far. Recently, our group has developed a monoclonal antibody IAC-1 sensitive to the molecular conformation of α2β1 because it only binds to the activated state of α2β1 on platelets, induced upon inside-out signaling. By investigating IAC-1 binding in combination with collagen binding after inside-out stimulation and outside manipulation, we demonstrated the existence of three different conformations of α2β1 on platelets and Chinese hamster ovary cells as follows: (i) a nonactivated, resting state with no collagen nor IAC-1 binding; (ii) an intermediate state, induced by outside manipulation, with collagen but no IAC-1 binding; and (iii) a fully activated state, induced after inside-out stimulation, with both collagen and IAC-1 binding. Moreover, these different conformational states of α2β1 are dependent on the cell type where α2β1 is expressed, as IAC-1 binding to peripheral blood mononuclear cells and Jurkat cells could also be induced by outside manipulation, in contrast to platelets and α2β1-expressing Chinese hamster ovary cells. Finally, we revealed a functional relevance for these different conformational states because the conformation of α2β1, induced after outside manipulation, resulted in significantly more cell spreading on coated collagen compared with nonactivated or inside-out stimulated cells.

Characterization of chemotactic ability of peptides containing N‐formyl‐methionyl residues in Tetrahymena fMLP as a targeting ligand

The chemotactic effects of six formylated, putatively bacterial peptides (fMLP, fMLPP, fMMM, fMP, fMV, and fMS) were studied. From the set of six peptides, only fMLP (one of the most effective chemoattractant peptides in mammals) elicited a significant positive chemotactic response in the eukaryotic ciliate Tetrahymena pyriformis, while the other formylated ligands, e.g. fMMM (which is also effective in mammals), had neutral or antagonistic effects in Tetrahymena. A study of their amino acid sequences points to an, as yet obscure, interaction between C‐terminal f‐Met and N‐terminal aromatic Phe. Some optimal physicochemical characteristics (e.g. solvent exposed area, solubility) of the molecule may be responsible for this special feature of f‐MLP at such a low level of phylogeny. This means that the unicellular Tetrahymena is able to select between related molecules, giving high priority to the molecule that is the most chemoattractive in mammals. The results call attention to the possible presence of f‐Met receptors at a unicellular level and to the evolutionary conservation of chemotaxis‐activating processes.

Synthesis and Studies on Cell-Penetrating Peptides

The ability of different synthetic cell penetrating peptides, as Antennapedia (wild and Phe(6) mutated penetratins), flock house virus, and integrin peptides to form complexes with a 25mer antisense oligonucleotide was compared and their conformation was determined by circular dichroism spectroscopy. The efficiency for oligonucleotide delivery into cells was measured using peptides labeled with a coumarin derivative showing blue fluorescence and the fluorescein-labeled antisense oligonucleotide showing green fluorescence. Fluorescence due to the excitation energy transfer confirmed the interaction of the antisense oligonucleotide and cell-penetrating peptides. The most efficient oligonucleotide delivery was found for penetratins. Comparison of the two types of penetratins shows that the wild-type penetratin proved to be more efficient than mutated penetratin. The paper also emphasizes that the attachment of a fluorescent label may have an effect on the conformation and flexibility of cell-penetrating peptides that must be taken into consideration when evaluating biological experiments.

Coloured peptides: synthesis, properties and use in preparation of peptide sub-library kits.

Several methods were developed for the solid‐phase synthesis (SPPS) of coloured peptides and peptide libraries. At first a bifunctional red compound, 4‐(4‐(N‐ethyl‐N‐(3‐(tert‐butyloxycarbonyl)aminopropyl)amino)phenylazo)benzoic acid (Boc‐EPAB), was coupled with chloromethyl resin to obtain a new solid support suitable for SPPS using Boc chemistry. Peptides synthesized on this coloured resin had the chromophore at their C‐termini. N‐terminally coloured peptides were synthesized on a traditional solid support, coupled with chromophoric carboxylic acid before cleavage. A model pentapeptide, Phe‐Ala‐Val‐Leu‐Gly, and its ten derivatives were synthesized and their properties studied. It was found that the presence of chromophores decreases the water solubility of peptides. However, insertion of solubilizing tags (penta‐lysine sequences or polyoxyethyl chains) into the molecule of any coloured derivative resulted in enhancement of the solubility. The RP‐HPLC hydrophobicity indexes (φ0) of the coloured peptides were also determined because φ0 values are closely related to their water solubility. A coloured pentapeptide library was synthesized using the portioning‐mixing method. Each component of this library contained the red azo dye (EPAB) and the penta‐lysine tag. Before the last coupling step the samples were not mixed. All of the 19 sub‐libraries obtained after cleavage were readily soluble in water, giving intense red solutions.

Fluorescent derivatives of bovine neurotensin 8-13 fragment

Fluorescent derivatives of bovine neurotensin 8–13 fragment were prepared. For N-terminal labelling, 4-[7-hydroxycoumaryl]acetic acid (Hca), 4-[7-methoxycoumaryl]acetic acid (Mca) and 2-amino-3-[4-[7-methoxycoumaryl]]propionic acid (Amp) were used while the C-terminus of the peptide chain was elongated with Amp. The fluorescence excitation and emission spectra of the peptide derivatives were studied. Hca- and Mca/Amp-derivatives were easily distinguishable because of the 60 nm shift of their emission maxima. Compared with the natural sequence, the presence of an N-terminal label did not influence the biological potency in a longitudinal muscle strip of guinea-pig ileum, while labelling at the C-terminus considerably reduced the activity of the peptide.

Chemotaxis induced by SXWS tetrapeptides in Tetrahymena-overlapping chemotactic effects of SXWS sequences and their identical amino acids

The chemotactic potential of SXWS peptides and the components of the extracellular domain of cytokine receptors were investigated in Tetrahymena as a functional index of substitution with different amino acids in the position ‘X’ of the tetrapeptide. Data obtained demonstrate that position X plays a special determining role in the ligand, SEWS and STWS possess extremely strong chemoattractant ability, and aromatic amino acids result in chemorepellent ligands. Diverse effects of structurally related molecules, for example, SNWS–SDWS, demonstrate a highly sensitive discrimination potential in the applied model system. Physicochemical characteristics (hydropathy, residue size, and solvent‐exposed area) of the amino acids were correlated with the chemotactic activity. Data obtained by computer‐assisted conformation analysis of SXWS peptides and the highly overlapping chemotactic effects of the investigated SXWS peptides as well as the presence of the amino acids in the ‘X’ position indicate that member ‘X’ of the SXWS sequence performs a special role in interactions with the chemotaxis receptors in the membrane. Copyright