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The Human Central Nervous System (CNS) is governed by electrochemical networks forming a delicate interplay between the different regions of the brain. The objective of the present experiment is to investigate the phylogenetic background of this electrochemical network by creating a comparable binary and ternary interplay of interactions between different neurotransmitters (noradrenaline, histamine, serotonin, acetylcholine, glutamate, and dopamine) in the unicellular eukaryote Tetrahymena pyriformis. Tetrahymena – as a protozoon – has no nervous system; however, it has been shown that it has not only the ability to store, synthesize and secrete biogenic amines but it also bears binding sites for the corresponding receptors of some of these molecules. The chemotactic responsiveness elicited by the neurotransmitters was examined in Tetrahymena cells, using a modified version of Leick’s two-chamber capillary chemotaxis assay with 20-minute incubation times. The concentration course of each neurotransmitter was determined and the concentration eliciting the strongest effect was further used to examine the chemotactic response of the neurotransmitters when used in pairs and in groups of three. Adequate cellular responses (chemoattractant and chemorepellent) were detected in both cases when the neurotransmitters were used alone and in combinations. A pattern detected in these responses was related to the neurotransmitters’ physicochemical characteristics (XlogP, TPSA). These provide evidence that the chief regulatory molecules of the CNS can be identified even in lower, eukaryotic unicellular levels of phylogeny and possibly alter the basic functions of these organisms. In summary, our results support the theory that any evolved nervous system-like interplay could stem from a common origin. Therefore, identifying the “ancient” function of a molecule or its receptor effect can open new windows in the advancement of therapeutic interventions.

Introduction Thrombogenesis plays an important role in today's morbidity and mortality. Antithrombotics are among the most frequently prescribed drugs. Thorough knowledge of platelet function is needed for optimal clinical care. Platelet adhesion is a separate subprocess of platelet thrombus formation; still, no well-standardized technique for the isolated measurement of platelet adhesion exists. Impedimetry is one of the most reliable, state-of-art techniques to analyze cell adhesion, proliferation, viability, and cytotoxicity. We propose impedimetry as a feasible novel method for the isolated measurement of 2 significant platelet functions: adhesion and spreading. Methods Laboratory reference platelet agonists (epinephrine, ADP, and collagen) were applied to characterize platelet functions by impedimetry using the xCELLigence SP system. Platelet samples were obtained from 20 healthy patients under no drug therapy. Standard laboratory parameters and clinical patient history were also analyzed. Results Epinephrine and ADP increased platelet adhesion in a concentration-dependent manner, while collagen tended to have a negative effect. Serum sodium and calcium levels and age had a negative correlation with platelet adhesion induced by epinephrine and ADP, while increased immunoreactivity connected with allergic diseases was associated with increased platelet adhesion induced by epinephrine and ADP. ADP increased platelet spreading in a concentration-dependent manner. Conclusion Impedimetry proved to be a useful and sensitive method for the qualitative and quantitated measurement of platelet adhesion, even differentiating between subgroups of a healthy population. This novel technique is offered as an important method in the further investigation of platelet function.

The wide range of cellular target reactions (e.g., antitumor) of gonadotropin-releasing hormone (GnRH) variants provides the possibility to develop multifunctional GnRH conjugates. The aim of our work was to compare the cytotoxic/apoptotic activity of different GnRH-based, daunorubicin (Dau)-linked conjugates with or without butyrated Lys in position 4 (4Lys(Bu)) at a molecular level in a human colorectal carcinoma cell line. Cell viability was measured by impedimetry, cellular uptake and apoptosis were studied by flow cytometry, and the expression of apoptosis-related genes was analyzed by qRT-PCR. The modification with 4Lys(Bu) resulted in an increased cytotoxic and apoptotic effects and cellular uptake of the GnRH-I and GnRH-III conjugates. Depending on the GnRH isoform and the presence of 4Lys(Bu), the conjugates could regulate the expression of several apoptosis-related genes, especially tumor necrosis factor (TNF), tumor protein p53 (TP53) and the members of growth-factor signaling. The stronger cytotoxicity of GnRH-I and GnRH-III conjugates containing 4Lys(Bu) was associated with a stronger inhibitory effect on the expression of growth-factor signaling elements in comparison with their 4Ser counterparts, in which the upregulation of TP53 and caspases (e.g., CASP9) seemed to play a more important role. We were able to provide further evidence that targeting the GnRH receptor could serve as a successful therapeutic approach in colon cancer, and GnRH-III-[4Lys(Bu),8Lys(Dau=Aoa)] proved to be the best candidate for this purpose.

Chemotaxis is one of the most essential cell physiological responses, which was developed in parallel the molecular evolution of signal molecules. Previously good correlations were found between chemotactic moieties and physicochemical properties (SEA, solubility, pKa) of peptide type ligands in Tetrahymena model. However, references are rather weak in eukaryotic chemotaxis about significance of simple carbohydrates. In the present work our goal is (i) to investigate the chemotactic effect of 10 mono- and disaccharides in the eukaryotic Tetrahymena pyriformis; (ii) to describe effective ligands with physicochemical parameters; (iii) to test whether sugars are acting via induction of metabolic pathways. Our results are: (i) the tested sugars can trigger both significant attractant (D-glucose, D-mannose) and significant repellent (D-glucosamine, D-fructose, N-acetyl-D-galactosamine, D-arabinose) effects, while some of the sugars (maltose, lactose, sucrose, D-galactose) had no effect. (ii) Correlations were described between the chemotactic effectiveness of the ligands and their physicochemical characters (TPSA, XLogP), which are supposed to influence the internalization of the sugars. (iii) All ligands proved to have low selection potential, which refers to a 'short-term' receptor moiety or influencing specific metabolic pathways. (iv) Starvation elicited modified, strong chemoattractive responsiveness towards glucose; however, it was independent of concentration while 1 h insulin treatment resulted in an increased and concentration dependent chemotaxis induced by glucose.

BACKGROUND Experiments on porcine heart scaffold represent significant assays in development of immunoneutral materials for cardiac surgery. Characterization of cell-cell and cell-scaffold interactions is essential to understand the homing process of cardiac cells into the scaffolds. MATERIAL AND METHODS In the present study, the highly sensitive and real-time impedimetric technique of xCELLigence SP was used to monitor cell adhesion, which is the key process of recellularization in heart scaffolds. Our objectives were: (i) to characterize the effect of decellularized porcine heart scaffold on cell adhesion of human cardiovascular cells potentially used in the recellularization process; and (ii) to investigate cell-extracellular matrix element interactions for building artificial multi-layer systems, applied as cellular models of recellularization experiments. Human fibrosarcoma, endothelial, and cardiomyocyte cells were investigated and the effect of decellularized porcine heart scaffold (HS) and fibronectin on cell adhesion was examined. Adhesion was quantified as slope of curves. RESULTS Heart scaffold had neutral effect on cardiomyocytes as well as on endothelial cells. Adhesion of cardiomyocytes was increased by fibronectin (1.480±0.021) compared to control (0.745±0.029). The combination of fibronectin and HS induced stronger adhesion of cardiomyocytes (2.407±0.634) than fibronectin alone. Endothelial and fibrosarcoma cells showed similarly strong adhesion profiles with marked enhancer effect by fibronectin. CONCLUSIONS Decellularized porcine HS does not inhibit adhesion of human cardiovascular cells at the cell biological level, while fibronectin has strong cell adhesion-inducer effect, as well as an enhancer effect on activity of HS. Consequently, decellularized porcine hearts could be used as scaffolds for recellularization with cardiomyocytes and endothelial cells with fibronectin acting as a regulator, leading to construction of working bioartificial hearts.

The unicellular Tetrahymena distinguishes structure-related vertebrate hormones by its chemosensory reactions. In the present work, the selectivity of hormone receptors was evaluated by analyzing the effects of various gonadotropin-releasing hormone (GnRH) analogs (GnRH-I, GnRH-III) as well as truncated (Ac-SHDWKPG-NH2) and dimer derivatives ([GnRH-III(C)]2 and [GnRH-III(CGFLG)]2) of GnRH-III on (i) locomotory behaviors, (ii) cell proliferation, and (iii) intracellular hormone contents of Tetrahymena pyriformis. The migration, intracellular hormone content, and proliferation of Tetrahymena were investigated by microscope-assisted tracking analysis, flow cytometry, and a CASY TT cell counter, respectively. Depending on the length of linker sequence between the two GnRH-III monomers, the GnRH-III dimers had the opposite effect on Tetrahymena migration. [GnRH-III(CGFLG)]2 dimer had a slow, serpentine-like movement, while [GnRH-III(C)]2 dimer had a rather linear swimming pattern. All GnRH-III derivatives significantly induced cell growth after 6 h incubation. Endogenous histamine content was uniformly enhanced by Ac-SHDWKPG-NH2 and GnRH-III dimers, while some differences between the hormonal activities of GnRHs were manifested in their effects on intracellular levels of serotonin and endorphin. The GnRH peptides could directly affect Tetrahymena migration and proliferation in a structure-dependent manner, and they could indirectly regulate these reactions by paracrine/autocrine mechanisms. Present results support the theory that recognition ability and selectivity of mammalian hormone receptors can be deduced from a phylogenetically ancient level like the unicellular Tetrahymena.

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 © 2012 European Peptide Society and John Wiley & Sons, Ltd.

As the unicellular ciliate, Tetrahymena has insulin receptors and produces insulin itself, which can regulate its glucose metabolism and other cell functions, in the present experiments the feed-back, the effect of glucose on the insulin binding and insulin production was studied. The cells were kept partly in tryptone-yeast medium, partly in Losina salt solution. The duration of treatment (in 0.1, 1.0, 10.0 mg/ml glucose) in the binding study was 10 min, in the hormone production study 30 min. FITC-insulin binding was significantly decreased only by 0.1 mg/ml glucose treatment in medium and by 10 mg/ml glucose in salt. The insulin production was significantly lower only in cells treated with 10 mg/ml glucose in medium. The insulin binding in salt was always higher and the insulin production always lower, than in medium. Earlier results demonstrated that the hormonal system (presence of hormones, receptors and signal pathways) of higher ranked animals can be deduced to a unicellular level, however, the feed-back mechanism is not really present here, only the traces can be observed in these protozoa.

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.

In a previous experiment thyrotropin (TSH) increased the triiodothyronine (T3) production of Tetrahymena and chorionic gonadotropin (HCG) moderately overlapped the effect. At present the production of three amino acid type (histamine, serotonin, epinephrine) and one peptide (endorphin) hormones were studied under the effect of TSH or HCG, in tryptone-yeast (TY) or salt (Losina-Losinsky) medium. The duration of the effect was 10 min. TSH significantly (with almost 20%) decreased epinephrine production in TY medium and HCG similarly decreased epinephrine and increased histamine level. In salt solution TSH as well as HCG decreased the level of serotonin. The results show that at this low level of phylogeny TSH effect is not completely thyroxine-specific, however it is not general. HCG overlaps TSH effect on epinephrine and serotonin production, however its effect is broader. The experiments also demonstrate that the effect of pituitary trop-hormones can be bidirectional in Tetrahymena, as histamine level was increased and epinephrine level was decreased by HCG, in the same cells.

In order to approach their natural conditions, populations of Tetrahymena were kept in Losina-Losinky's salt solution for 1 h, than in the tryptone+yeast medium. During this time they were treated with histamine, serotonin or insulin, or with the combinations of these hormones. Effect of the combined treatments on the production of serotonin (5HT), or adrenocorticotropic hormone (ACTH) or triiodothyronine (T₃) by the cells was compared to the effect of single-hormone treatments. Significant differences were seen between the results obtained following the single or combined treatments. There was no summation of the effects, however an elevation or diminution of the hormone production was observed after the combined treatment, as compared with the untreated controls or with the use of one of the hormones in the samples. The experiments demonstrate that there is a hormonal regulation between the Tetrahymena cells and the hormones influence each other's effect.

Tetrahymena pyriformis was maintained in TYM (tryptone-yeast medium) as well as in Losina salt solution. One hour treatment of 10 ―15 M histamine, serotonin or insulin was given before the histamine, serotonin, triiodothyronine and adrenocorticotropin contents of the cells were measured by flow cytometry after immunocytochemical staining. Maintenance in salt solution increased the hormone level in the cells, and use of the treatment hormone treatments further increased the endogenous hormone content relative to that in medium. The cells in salt mimic better the natural conditions, which means that the effects of hormones under more natural conditions are expressed to a greater extent than the exogenously given hormones in TYM typically used under laboratory conditions. Intercellular hormonal communication between the cells of a Tetrahymena population might assist in the survival of the individual cells.

The unicellular Tetrahymena pyriformis was stressed by 37°C heat for 1 h and its hormone (serotonin, histamine, triiodothyronine) content was measured by immunocytochemical flow cytometry in different time points (immediately after treatment and after 1, 2, 8, 16 weeks). The treatment increased each hormone level for two weeks, however, after 8 weeks the hormone concentration inside the cells decreased and in case of serotonin this was similar in the 16th week, while the other two hormones’ level was similar to the control. Insulin further increased the hormone production during treatment, but this effect was not durable. After one week the cells behave similar to those, subjected to heath shock only. The results show that a single stress causes deep and durable changes in the hormone household of Tetrahymena which is influenced by exogenously given insulin only in the acute phase.

Here we report on the design, synthesis and biochemical characterization of multifunctional bioconjugates containing two chemotherapeutic agents, daunorubicin and methotrexate, coupled to the GnRH-III decapeptide, which served as a targeting moiety. This represents a possible approach to increase the receptor mediated tumor targeting and consequently the cytostatic effect of anticancer drug-peptide bioconjugates. The multifunctional bioconjugates were prepared according to two drug design approaches recently developed by our group. Both bifunctional GnRH-III derivatives, [(4)Lys]-GnRH-III (Glp-His-Trp-Lys-His-Asp-Trp-Lys-Pro-Gly-NH(2)) and [(8)Lys(Lys)]-GnRH-III (Glp-His-Trp-Ser-His-Asp-Trp-Lys(Lys)-Pro-Gly-NH(2)), contain two free amino groups suitable for the attachment of two anticancer drugs, such as methotrexate and daunorubicin. The drugs were chosen with respect to their different mechanisms of action, with the goal of increasing the antitumor effect of the bioconjugates. The in vitro cytostatic effect of the bioconjugates was determined on MCF-7 human breast, HT-29 human colon and LNCaP human prostate cancer cells by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Their in vitro stability/degradation in human serum and in the presence of rat liver lysosomal homogenate was investigated by liquid chromatography in combination with mass spectrometry. The influence of the multifunctional bioconjugates on the cell adhesion and cell proliferation was studied on Mono Mac 6 human leukemic monocytes. It was found that (1) all synthesized bioconjugates had in vitro cytostatic effect; (2) they were stable in human serum for at least 24 h; (3) they were hydrolyzed in the presence of lysosomal homogenate and (4) they exerted a moderate cell-cell adhesion inducing effect. These results demonstrate that multifunctional bioconjugates containing two different anticancer drugs attached to the same GnRH-III targeting moiety could be successfully prepared and resulted in higher in vitro cytostatic effect than the monofunctional bioconjugates containing either methotrexate or daunorubicin, in particular on HT-29 human colon cancer cells.