László Kőhidai

Artificial magnetotactic motion control of Tetrahymena pyriformis using ferromagnetic nanoparticles: A tool for fabrication of microbiorobots

We induce artificial magnetotaxis in Tetrahymena pyriformis, a eukaryotic ciliate, using ferro-magnetic nanoparticles and an external time-varying magnetic field. Magnetizing internalized iron oxide particles (magnetite), allows control of the swimming direction of an individual cell using two sets of electromagnets. Real-time feedback control was performed with a vision tracking system, which demonstrated controllability of a single cell. Since the endogenous motility of the cell is combined in one system with artificial magnetotaxis, the motion of artificially magnetotactic T. pyriformis is finely controllable. Thus, artificially magnetotactic T. pyriformis is a promising candidate microrobot for microassembly and transport in microfluidic environments.

Galvanotactic and phototactic control of Tetrahymena pyriformis as a microfluidic workhorse

A eukaryotic ciliate, Tetrahymena pyriformis, has been controlled using galvanotaxis and phototaxis in a low Reynolds number fluidic environment. A cell-tracking algorithm demonstrates the controllability of Tetrahymena pyriformis under two types of external stimuli. Electrical stimulation, in the form of a direct current electric field through the containing fluid, causes a change in swimming direction toward the cathode. Photostimulation, by high intensity broadband light, results in a rotational motion of the cells. The motivation of this work is to progress further with biological microfluidic actuators and sensors for use in engineered systems.

Effects of the aquatic contaminant human pharmaceuticals and their mixtures on the proliferation and migratory responses of the bioindicator freshwater ciliate Tetrahymena

An increasing attention is paid to the potential harmful effects of aquatic contaminant pharmaceuticals exerted on both biosystems and humans. In the present work the effects of 14 pharmaceuticals including NSAIDs, antibiotics, β-blockers and a frequently used X-ray contrast media on the proliferation and migratory behavior of the freshwater ciliate Tetrahymena pyriformis was investigated. Moreover, the mixture toxicity of four selected pharmaceuticals (diclofenac, ibuprofen, metoprolol and propranolol) was evaluated in binary mixtures using full factorial experimental design. Our results showed that the sensitivity of Tetrahymena to NSAIDs and β-blockers (EC(50) ranged from 4.8 mg L(-1) to 308.1 mg L(-1)) was comparable to that of algal or Daphnia bioassays. Based on these elevated EC(50) values acute toxic effects of these pharmaceuticals to T. pyriformis are unlikely. Antibiotics and the contrast agent sodium-diatrizoate had no proliferation inhibiting effect. Chemotactic response of Tetrahymena was more sensible than proliferation as significant chemorepellent action was observed in the environmentally realistic concentration range for acetylsalicylic acid, diclofenac, fenoprofen, paracetamol, metoprolol, propranolol, timolol and trimethoprim (Chemotaxis Index ranged from 63% to 88%). Mixture toxicity experiments resulted in a complex, concentration dependent interaction type pattern with antagonism being the predominant interaction type (59%) followed by additivity (37%) and synergism (4%). Hence the concept of concentration addition validated for NSAIDs in other organisms cannot be adopted for this ciliate. In summary authors suggest Tetrahymena as a sensible model of testing aquatic contaminants as well as underline the significance using more specific endpoints to understand the complex mechanisms investigated.

Vacuum ultraviolet photolysis of diclofenac and the effects of its treated aqueous solutions on the proliferation and migratory responses of Tetrahymena pyriformis

The effects of dissolved O2, phosphate buffer and the initial concentration of diclofenac on the vacuum ultraviolet photolysis of this contaminant molecule were studied. Besides kinetic measurements, the irradiated, multicomponent samples were characterized via the proliferation and migratory responses (in sublethal concentrations) of the bioindicator eukaryotic ciliate Tetrahymena pyriformis. The results suggest that hydroxyl radicals, hydrogen atoms and hydroperoxyl radicals may all contribute to the degradation of diclofenac. The aromatic by-products of diclofenac were presumed to include a hydroxylated derivative, 1-(8-chlorocarbazolyl)acetic acid and 1-(8-hydroxycarbazolyl)acetic acid. The biological activity of photoexposed samples reflected the chemical transformation of diclofenac and was also dependent on the level of dissolved O2. The increase in toxicity of samples taken after different irradiation times did not exceed a factor of two. Our results suggest that the combination of vacuum ultraviolet photolysis with toxicity and chemotactic measurements can be a valuable method for the investigation of the elimination of micropollutants.

Verification of epigenetic inheritance in a unicellular model system: multigenerational effects of hormonal imprinting

The unicellular Tetrahymena has receptors for hormones of higher vertebrates, produces these hormones, and their signal pathways are similar. The first encounter with a hormone in higher dose provokes the phenomenon of hormonal imprinting, by which the reaction of the cell is quantitatively modified. This modification is transmitted to the progeny generations. The duration of the single imprinter effect of two representative signal molecules, insulin and 5‐HT (5‐hydroxytryptamine), in two concentrations (10−6 and 10−15 M) were studied. The effects of imprinting were followed in 5 physiological indices: (i) insulin binding, (ii) 5‐HT synthesis, (iii) swimming behaviour, (iv) cell growth and (v) chemotaxis in progeny generations 500 and 1000. The result of each index was different from the non‐imprinted control functions, growth rate, swimming behaviour and chemotactic activity to insulin being enhanced, while others, e.g. synthesis and chemotactic responsiveness of 5‐HT and the binding of insulin were reduced. This means that a function‐specific heritable epigenetic change during imprinting occurs, and generally a single encounter with a femtomolar hormone concentration is enough for provoking durable and heritable imprinting in Tetrahymena. The experiments demonstrate the possibility of epigenetic effects at a unicellular level and call attention to the possibility that the character of unicellular organisms has changed through to the present day due to an enormous amount of non‐physiological imprinter substances in their environment. The results – together with results obtained earlier in mammals – point to the validity of epigenetic imprinting effects throughout the animal world.

Daunomycin-polypeptide conjugates with antitumor activity

We have developed a group of water-soluble drug conjugates in which daunomycin (Dau) is coupled to cationic, amphoteric or anionic branched polypeptides and a new conjugate containing a cationic polypeptide carrier modified with a cell penetrating octaarginine. We investigated in vitro physiological activity of these conjugates in several aspects: in vitro cytotoxicity and cytostatic effect, adhesion and cellular uptake were examined on murine (J774 and L1210) and human (MonoMac6 and HL-60) leukemia cell lines and on murine bone marrow derived macrophages. We found that these processes are dependent on the properties of the carrier, on experimental conditions like concentration and incubation time. We found that attachment of polypeptide and cell penetrating peptide to the bioactive agent, depending on the cell line, could significantly improve the antitumor activity of the drug.

Metastasis-associated S100A4 is a specific amine donor and an activity-independent binding partner of transglutaminase-2.

Transglutaminase-2 (TG2) is best known as a Ca(2+)-dependent cross-linking enzyme; however, some of its extracellular matrix-related functions are independent of its catalytic activity and include matrix remodelling, adhesion and migration. S100A4 belongs to the Ca(2+)-binding EF-hand S100 protein family and acts both intra- and extra-cellularly through binding to various partners. It regulates cell migration and its overexpression is strongly associated with metastasis and poor survival in various cancers. It has recently been suggested that TG2 mediates S100A4-dependent tumour cell migration. In the present study we provide evidence that S100A4 is an interacting partner and also a specific amine donor of TG2. TG2 incorporates a glutamine donor peptide to Lys(100) in the C-terminal random coil region of S100A4. Importantly, the enzyme activity is not necessary for the interaction: S100A4 also binds to TG2 in the presence of a specific inhibitor that keeps the enzyme in an open conformation, or to an enzymatically inactive mutant. We also found that S100A4 considerably enhances TG2-mediated adhesion of A431 epithelial carcinoma cells to the extracellular matrix. This role is independent of enzyme activity and requires the open conformation of TG2. We propose that S100A4 stabilizes the open conformation of TG2, which binds to its cell-surface receptor in this state and increases cell adhesion.

Adaptive Evolution of Formyl Peptide Receptors in Mammals

The formyl peptide receptors (FPRs) are a family of chemoattractant receptors with important roles in host defense and the regulation of inflammatory reactions. In humans, three FPR paralogs have been identified (FPR1, FPR2, and FPR3) and may have functionally diversified by gene duplication and adaptive evolution. However, the evolutionary mechanisms operating in the diversification of FPR family genes and the changes in selection pressures have not been characterized to date. Here, we have made a comprehensive evolutionary analysis of FPR genes from mammalian species. Phylogenetic analysis showed that an early duplication was responsible for FPR1 and FPR2/FPR3 splitting, and FPR3 originated from the latest duplication event near the origin of primates. Codon-based tests of positive selection reveal interesting patterns in FPR1 and FPR2 versus FPR3, with the first two genes showing clear evidence of positive selection at some sites while the majority of them evolve under strong negative selection. In contrast, our results suggest that the selective pressure may be relaxed in the FPR3 lineage. Of the six amino acid sites inferred to evolve under positive selection in FPR1 and FPR2, four sites were located in extracellular loops of the protein. The electrostatic potential of the extracellular surface of FPR might be affected more frequently with amino acid substitutions in positively selected sites. Thus, positive selection of FPRs among mammals may reflect a link between changes in the sequence and surface structure of the proteins and is likely to be important in the host’s defense against invading pathogens.

Development of Cyclic NGR Peptides with Thioether Linkage: Structure and Dynamics Determining Deamidation and Bioactivity

NGR peptides that recognize CD13 receptors in tumor neovasculature are of high interest, in particular due to their potential applications in drug targeting. Here we report the synthesis and structural analysis of novel thioether bond-linked cyclic NGR peptides. Our results show that their chemostability (resistance against spontaneous decomposition forming isoAsp and Asp derivatives) strongly depends on both sample handling conditions and structural properties. A significant correlation was found between chemostability and structural measures, such as NH(Gly)-CO(Asn-sc) distances. The side-chain orientation of Asn is a key determining factor; if it is turned away from HN(Gly), the chemostability increases. Structure stabilizing factors (e.g., H-bonds) lower their internal dynamics, and thus biomolecules become even more resistant against spontaneous decomposition. The effect of cyclic NGR peptides on cell adhesion was examined in A2058 melanoma cell lines. It was found that some of the investigated peptides gradually increased cell adhesion with long-term characteristics, indicating time-dependent formation of integrin binding isoAsp derivatives that are responsible for the adhesion-inducing effect.

Investigation on chemotactic drug targeting (chemotaxis and adhesion) inducer effect of GnRH-III derivatives in Tetrahymena and human leukemia cell line

GnRH‐III has been shown to exert a cytotoxic effect on the GnRH‐R positive tumor cells. The chemotactic drug targeting (CDT) represents a new way for drug delivery approach based on selective chemoattractant guided targeting. The major goal of the present work was to develop and investigate various GnRH‐III derivatives as potential targeting moieties for CDT. The cell physiological effects (chemotaxis, adhesion, and signaling) induced by three native GnRHs (hGnRH‐I, cGnRH‐II, and lGnRH‐III) and nine GnRH‐III derivatives were evaluated in two model cells (Tetrahymena pyriformis and Mono Mac 6 human monocytes). According to our results, the native GnRH‐III elicited the highest chemoattractant and adhesion inducer activities of all synthesized peptides in micromolar concentrations in monocytes. With respect to chemoattraction, dimeric derivatives linked by a disulfide bridge ([GnRH‐III(C)]2) proved to be efficient in both model cells; furthermore, acetylation of the linker region ([GnRH‐III(Ac‐C)]2) could slightly improve the chemotactic and adhesion effects in monocytes. The length of the peptide and the type of N‐terminal amino acid could also determine the chemotactic and adhesion modulation potency of each fragment. The application of the chemoattractant GnRH‐III derivatives was accompanied by a significant activation of phosphatidylinositol 3‐kinase in both model cells. In summary, our work on low‐level differentiated model cells of tumors has proved that GnRH‐III and some of its synthetic derivatives are promising candidates to be applied in CDT: these compounds might act both as carrier, delivery unit, and antitumor agents. Copyright