Platelet impedance adhesiometry: A novel technique for the measurement of platelet adhesion and spreading.

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.

Effect of aliphatic, monocarboxylic, dicarboxylic, heterocyclic and sulphur-containing amino acids on Leishmania spp. chemotaxis.

In the sand-fly mid gut, Leishmania promastigotes are exposed to acute changes in nutrients, e.g. amino acids (AAs). These metabolites are the main energy sources for the parasite, crucial for its differentiation and motility. We analysed the migratory behaviour and morphological changes produced by aliphatic, monocarboxylic, dicarboxylic, heterocyclic and sulphur-containing AAs in Leishmania amazonensis and Leishmania braziliensis and demonstrated that L-methionine (10-12 m), L-tryptophan (10-11 m), L-glutamine and L-glutamic acid (10-6 m), induced positive chemotactic responses, while L-alanine (10-7 m), L-methionine (10-11 and 10-7 m), L-tryptophan (10-11 m), L-glutamine (10-12 m) and L-glutamic acid (10-9 m) induced negative chemotactic responses. L-proline and L-cysteine did not change the migratory potential of Leishmania. The flagellum length of L. braziliensis, but not of L. amazonensis, decreased when incubated in hyperosmotic conditions. However, chemo-repellent concentrations of L-alanine (Hypo-/hyper-osmotic conditions) and L-glutamic acid (hypo-osmotic conditions) decreased L. braziliensis flagellum length and L-methionine (10-11 m, hypo-/hyper-osmotic conditions) decreased L. amazonensis flagellum length. This chemotactic responsiveness suggests that Leishmania discriminate between slight concentration differences of small and structurally closely related molecules and indicates that besides their metabolic effects, AAs play key roles linked to sensory mechanisms that might determine the parasite's behaviour.

The facile generation of two-dimensional stiffness maps in durotactic cell platforms through thickness projections of three-dimensional submerged topography.

An innovative platform that aims to facilitate studies of how adherent cells migrate in response to rigidity gradients or durotaxis has been developed. Soft polyacrylamide gel-based cell culture scaffolds are used to fabricate flat surfaces containing elasticity gradients through changes in the underlying patterned features. Moreover, this inert gel surface supports long-term cell viability and offers a tunable stiffness. By manipulating the thickness of the gel substrate through the embedded patterns, this system is also capable of directing collective cell patterning.

Effect of tuftsin and oligotuftsins on chemotaxis and chemotactic selection in Tetrahymena pyriformis.

The chemotactic properties of tuftsin (H-TKPR-OH), tuftsin derivatives (H-KPR-OH, H-TKPKG-NH(2), Ac-TKPKG-NH(2)) and TKPKG-based oligotuftsins (T20, T30, T40) were investigated in Tetrahymena pyriformis GL. In contrast to its effects on Mammalia, tuftsin elicited chemorepellent or neutral responses; truncation of the N-terminal part (KPR) led to similar results, though with more neutral effects. The significance of the C-terminal part of the molecule was revealed by the chemoattractant properties of TKPKG, which are nevertheless abolished by acylation. Among the oligotuftsins, T20 and T40 were chemoattractants at higher concentrations (10(-9)-10(-6) M), while T30 had a wide-ranging chemorepellent effect, indicating that chemotaxis is elicited in Tetrahymena only by ligands with optimal physicochemical characters (mass, conformation, etc.). The chemotactic selection data indicated that tuftsin-induced chemotaxis results from fairly short-term signalling in Tetrahymena.

Induction of melatonin synthesis in Tetrahymena pyriformis by hormonal imprinting--a unicellular "factory" of the indoleamine.

Melatonin is present in Tetrahymena and its synthesis can be enhanced by pretreatment (imprinting) with melatonin. Two days after imprinting melatonin level is elevated in the cells and more elevated in the supematant. Such a minute quantity, as 10(-12) M melatonin for 1 hour is able to provoke imprinting, however the effect is more expressed using 10(-6) M. Maintenance in light conditions further elevated the amount of melatonin in the cells and supematant alike, related to the melatonin content of cells kept in darkness. The experiments call attention to the light-sensitivity of imprinting-provoked melatonin production in Tetrahymena and to the possibility of using this property for important physiological functions in higher grades of phylogeny.

Chemotactic-range-fitting of amino acids and its correlations to physicochemical parameters in Tetrahymena pyriformis--evolutionary consequences.

Amino acids are considered the oldest organic substances of the prebiotic evolution. Chemotactic effects of amino acid L-isomers investigated in the protozoan model Tetrahymena show that the chemotactic properties of amino acids are complex and depend on multiple physicochemical characteristics of the investigated ligands. The range of effectiveness is significantly wider for chemoattractant ligands than for chemorepellent ones. This phenomenon provides the basis of the "chemotactic-range-fitting" theory. The validity of this theory is supported by a decreased pK (-COOH), an increased pK (-NH2), and a decrease in solvent exposed areas and hydropathy indexes in chemoattractant amino acids compared to chemorepellent ones. Chemotactic selection has proven the activity of long-term (I, H, T) and short-term (P, A, Q, S) selector amino acids and their characteristic diversities in values of the pK and SEA (surface exposed area). Comprehensive studies of the chemotaxis data with the results of consensus analysis of amino acids suggests that chemotactic activity was one of the most primordial physiological activities and had a prospective significance not only in the molecular evolution of ligands, but also in the evolution of signalling.

Melatonin in the unicellular Tetrahymena pyriformis: effects of different lighting conditions.

Melatonin content in the cellular fraction and medium of Tetrahymena pyriformis GL cultures was measured at different time points of light and dark exposures. Tetrahymena produced, stored and secreted immunoreactive melatonin, which in displacement and HPLC studies, behaved like synthetic melatonin. There was not a continuous secretion of melatonin produced by the cells. In contrast to this, storage of melatonin was observed, which was more expressed in dark conditions. Prolonged light exposure suppressed melatonin production and secretion alike, however it did not block it completely.

A short-run new analytical ultracentrifugal micromethod for determining low-density lipoprotein sub-fractions using Schlieren refractometry.

We have developed a new analytical ultracentrifugal micromethod for the determination of serum low-density lipoprotein (LDL) subclasses directly from ultracentrifugal Schlieren scans. We have used special software for the analysis of this type of single-spin density-gradient ultracentrifugation. The flotation of LDL patterns was obtained by underlayering a physiological salt solution with serum or isolated lipoprotein fractions raised to a density of 1.3 g/mL in the spinning ultracentrifugation capillary band-forming cell. The repeated analysis of Schlieren curves of the same sample from 10 to 100 microL in the 60-100 min full-speed interval time resulted in quite reproducible results. We obtained quantitative results by measuring the Schlieren areas between the sample curves and the reference baseline curve by using computerised numerical and graphic techniques. The decomposition of the integrated curve was carried out using a nonlinear regression program followed by deconvolution algorithm analysis in order to determine the parameters of the composing Gaussian subclasses. The LDL particle concentrations were calculated from the area under the integral of the Gaussian curve using a calibration data constant. The flotation range of the LDL Schlieren curves in the cell was identified with serum from which LDL had been removed by means of precipitation reagents and with centrifugation of isolated LDL aliquots. With this technique, we measured the concentration of LDL and analysed its polydispersity without the need for preceding sequential isolation of the LDL. On the basis of the Schlieren curves, the LDL samples were either physically paucidisperse, having a symmetrical peak within a narrow density range, or were polydisperse, showing an asymmetrical pattern distributed over a broader density region. The described method proved to be useful for a clear and immediate visual presentation of the concentration values of the LDL and for the identification of the heterogeneity of LDL variants without the need for the preparative isolation of that density class.

Chemotactic selection with insulin, di-iodotyrosine and histamine alters the phagocytotic responsiveness of Tetrahymena.

Chemotactic selection is a method by which populations of cells exposed to ligands can be isolated and subsequently cultivated. We used Tetrahymena pyriformis GL cultures selected by chemotactic selection to insulin (10 nM), histamine (0.1 nM) and di-iodotyrosine (T2, 10 nM) to study the phagocytotic capacity under the induction of selector hormones. Our results show a long-lasting link between chemotactically selected cultures and phagocytotic activity. Cells selected to histamine produced the highest phagocytotic activity upon a second exposure to the selector hormone. T2 selection was also strongly effective, however, the phagocytosis stimulation was not specific to the hormone given later. Insulin selected sub-populations had different phagocytotic responses to the control substance itself, whereas histamine selected sub-populations seem to be heterogeneous in the phagocytotic response to histamine. For insulin, the increased endocytotic or metabolic activity was demonstrated by the lack of non-phagocytotic cells. These experiments call attention to the evolutionary role of selection in the later developing receptor-hormone relationship.

Effect of vasoactive peptides on Tetrahymena. chemotactic properties of endothelins (ET-1, ET-2, ET-3, fragment 11-21 of ET-1 and big endothelin-1): a short-term inducible signalling mechanism of chemotaxis.

The unicellular Tetrahymena is a sensitive model for the study of chemotaxis induced by endothelins. In short-term chemotactic responses, ET-2 and ET-3 were chemorepellent, compared to the referent control (culture medium) and chemoattractant, ET-1. These differences suggest that the change of some aromatic residues in the loop region (residues 5-9) of the ET-1 abrogates its chemoattractant character. The response of Tetrahymena is highly selective, since substitution of two amino acids are enough to cause this alteration in (behavioural) response. Such a change seems to be more important than the loss of the entire first 10 amino acids (in ET-1 fragment 11-21), since after this it acquires some chemoattractive effect of ET-1. Selection with ET-3 rigorously stimulated the cell's responsiveness to the medium, this ability was abolished by the repeated ET-3 treatment. Big endothelin-1 was repellent in all concentrations. These experiments demonstrate the very sensitive discriminating capacity of chemotactic responsiveness at a low level of phylogeny. Chemotactic selection with endothelins underlines the possibility that there may be separate mechanisms responsible for the short-term chemotactic responses and the long-lasting effects of chemotactic selection.

Chemotactic selection of Tetrahymena pyriformis GL induced with histamine, di-iodotyrosine or insulin.

It has been hypothesized that in phylogeny the encounter between potential signalling molecules and the continously changing cell membrane could result in the formation of a ligand specific receptor. This chemical (hormonal) imprinting is then transmitted to the progeny generations. It is, however, very difficult to know whether the selection of cells with receptor-like patterns or amplification of complete receptor-like patterns led to the formation of the receptor-hormone complex. The new technique of 'chemotactic selection provides a physiological response-guided selection of cells. It also enables the testing of subpopulations with the characteristic selector ligand. We show here that of three chemotactic ligands (histamine, di-iodotyrosine (T2) and human insulin), insulin and T2 selected subpopulations express a significantly high chemotactic response. Since the control medium has a selector capacity itself, we introduced a chemotactic selection coefficient (Chsel) which facilitates the comparison of all groups. Using this factor we found that insulin (Chsel = 1.57), functions as a strong selector and T2 (Chsel = 0.98), was a weak selector. Morphometric evaluation of the cells showed a good correlation between chemotactic responsiveness and morphometric characteristics of subpopulations selected with insulin and histamine. T2 data suggest that the long lasting responsiveness is not general, but might be subpopulation specific.

Presence, uptake and localization of an immunoreactively interleukin 6 (IL-6)-like molecule in Tetrahymena pyriformis.

The unicellular Tetrahymena and its medium contain immunoreactively interleukin 6 (IL-6)-like molecules (hereinafter IL-6) in a measurable quantity in the 24 h-old cultures. This protozoan takes up exogenously supplied IL-6 very quickly, and this can be found in similar amounts in both the cells and the media after 1 h. After 24 h (48 h cultures), an equal amount of IL-6 is present in the control and IL-6-treated cells and their media. By 120 h, cells which have not had their medium changed retained the same quantity of IL-6 as the control; however less than half was found in IL-6-treated cells. In the medium of 120 h-old cultures, there was a reduction of IL-6 content relative to the 24 h content in the control; however, in the IL-6-treated cell culture medium, less than half of the level in the controls was found. Confocal microscopy demonstrated the localization of IL-6 in/on the oral apparatus and basal bodies, and the nuclear envelope also showed moderate labelling. IL-6 antibody binding was enhanced after IL-6 pretreatment (hormonal imprinting). The experiments call attention to the presence of an IL-6-like molecule and its uptake at a very low level of phylogeny.

Prolonged elevation of insulin content in the unicellular tetrahymena after insulin treatment: induction of insulin production or storage?

In the unicellular organism, Tetrahymena, the first encounter with an exogeneously given hormone results in hormonal imprinting. This causes an increase of the binding capacity of receptors and the production of the appropriate hormone in the progeny generations of the treated cell. In the present experiments the quantity (using radioimmunoassay) and localization (using confocal laser scanning microscopy) of the immunologically insulin-like material (hereafter insulin) were studied for 10 days after 4 h or 24 h 10(-6) M insulin treatment (hormonal imprinting). Forty-eight hours after both insulin treatments a high quantity of insulin was present in the cells. This value was also significantly increased after 96 h. After 8 days the difference to the control was significant only in the 24 h treated group. Confocal microscopy (using antibody to pig insulin) localized insulin in the cell body. The oral field contained extremely high quantities of the endogeneous hormone. Insulin treatment (after 48 and 96 h) caused an elevation of insulin content in general, and specific accumulation in the posterior sections of the cell, around the nucleus and in the periphery were observed. Ten days after both treatments only the peripheral region of the cell body and the ciliary row contained more insulin than the control. This means that after insulin treatment the quantity of insulin increases for a lengthy time period which is followed by the expression of insulin in the peripheral region. Insulin contained by Tetrahymena 48 h after imprinting stimulated glucose uptake of rat diaphragm.

Effect of C2 ceramide on the inositol phospholipid metabolism (uptake of 32P, 3H-serine and 3H-palmitic acid) and apoptosis-related morphological changes in Tetrahymena.

Sphingomyelin metabolites have significant role in the regulation of many life processes of mammalian cells. In the present experiments the influence of phospholipid turnover and apoptosis related morphologic signs by one of this metabolite, C2 ceramide was studied, and compared to the control, untreated cells, in the unicellular Tetrahymena. The incorporation of phospholipid head group components (serine, phosphorus) show a clear time-dependence; while the incorporation of fatty acid component (palmitic acid) is very fast: no significant alterations were found between 5- and 60-min incubations. C2 ceramide treatment didn't alter 3H-palmitic acid incorporation into phospholipids, however 3H-serine incorporation was mainly inhibited. The amount of total incorporated 32P was also decreased, on the other hand the lover concentration C2 ceramide (10 microM) elevated the synthesis of inositol phospholipids. The higher concentration of C2 ceramide (50 microM) had inhibitory effect on the synthesis of each phospholipids examined. This means that in the presence of the C2 ceramide the synthesis, recovery and turnover of phospholipids, participating in signal transduction, are altered. However these observations were based the uptake of labeled phospholipid precursors, which gives information on the dynamics of the process, without using lipid mass measurements. C2 ceramide also caused the rounding off the cells, DNA degradation and nuclear condensation. These latter observations point to morphological signs of apoptosis. The results call attention to the role of sphingomyelin metabolites on signalization of unicellulars, to the cross-talk between the inositol phospholipids and sphingomyelin metabolites, and the role of these molecules in the apoptotic processes at a low evolutionary level.

Chemotaxis: the proper physiological response to evaluate phylogeny of signal molecules.

In this review we summarize our results gained on the investigations focused to characterize ligand and signaling mechanisms required for the chemotaxis in the unicellular model Tetrahymena. Our data show that short chain signal molecules (amino acids, oligopeptides) are distinguished upon their physicochemical characteristics - lipophilicity, residual volumes and statistical distribution of side-chain distances (e.g. in proline containing dipeptides), while the vertebrate hormones have also specific attractant or repellent effects in the model (FSH vs. TSH). Hormonal imprinting developed by pretreatments has also special, signal molecule dependent effect (histamine vs. serotonin). It is shown that "chemotactic selection" of cells, by the new probe developed by us is a suitable tool to provide subpopulations possessing enhanced chemotactic receptor-effector mechanisms with respect to the selector signal molecules (IL-8, TNF-alpha).

Chemotaxis and chemotactic selection induced with cytokines (IL-8, RANTES and TNF-alpha) in the unicellular Tetrahymena pyriformis.

Three representative cytokines interleukin (IL-8), RANTES and tumour necrosis factor alpha (TNF-alpha) have a concentration-dependent chemotactic effect on the unicellular Tetrahymena. Maximal effective concentrations of IL-8 (1 ng/ml) and RANTES (75 ng/ml) are in the same range as in mammals, which indicates an evolutionary background of physiological effects elicited. Progeny generations of cells selected for their affinity to cytokines (IL-8 and TNF-alpha) show an enhanced positive chemosensory reaction to the cytokines. The changed reaction of these cells to the chemoattraction of the culturing medium was also observed. The results call attention to the presence of cytokine-dependent processes at a low phylogenetic level.

Effects of tumor necrosis factor alpha (TNF alpha) on the phospholipid metabolism of Tetrahymena pyriformis.

The effect of (0.05 ng ml-1 and 0.1 ng ml(-1)) TNF alpha on the phospholipid metabolism of Tetrahymena pyriformis was studied. The amount of phosphatidyl choline (PC), phosphatidyl inositol (PI), phosphatidic acid (PA), phosphatidyl ethanolamine (PE), diacylglycerol (DAG), arachidonic acid (AA) and ceramide was higher, but the phosphatidyl inositol 4 phosphate (PIP) and phosphatidyl inositol bis-phosphate (PIP2) as well, as sphingomyelin (SM) content was lower in TNF alpha-treated cells than in the controls. In the culture medium (secreted forms) this situation was reversed. There were differences in the results gained by incorporation of [3H]-palmitic acid or 32P into the phospholipids. To control the functional effects of TNF alpha in Tetrahymena, the rate of cell division, the condensation of chromatin, the viability of cells and morphometrical values have been studied. The cytokine reduced cell growth, altered morphometric indices and increased chromatin condensation, however cell viability was not influenced. The results demonstrate the effects of TNF alpha at a low level of evolution, what is realized by changes in the phospholipid metabolism participating in signalling pathways.

Effect of 3-amino-1-propanol on the phosphatidylinositol (PI) and glycosyl phosphatidylinositol (GPI) systems of Tetrahymena.

3-Amino-1-propanol (AP), a substance replacing ethanolamine in phosphatidylethanolamine (PE) significantly reduced 32P incorporation to phosphatidylinositol (PI) and glycosyl-phosphatidylinositol (GPI) in the unicellular organism Tetrahymena pyriformis. At 10 mM, AP completely inhibited the incorporation of 32P into PI. 3H-arachidonate incorporation into PI was also inhibited, while that into diacylglycerol (DAG) was high. The experiments indicate the presence and metabolism of inositol phospholipids and GPI in T. pyriformis.

Effects of dipeptides containing the amino acid, proline on the chemotaxis of Tetrahymena pyriformis. Evolutionary conclusions on the formation of hormone receptors and hormones.

Our investigations demonstrate that proline-containing dipeptides can provoke a chemosensory response from the unicellular Tetrahymena pyriformis. The chemotactic effects of the dipeptides have a close relationship with the side chain and the lipophilicity of the amino-terminal amino acid. Comparison of 'mirror' variants of proline-containing dipeptides points to the fact that dipeptides with small side chain and non-polar character amino acids (Gly-Pro, Ala-Pro) are preferred on the amino-terminal end. In the case of amino acids with very variable side chains, small (Pro-Gly) and the large side chain and non-polar character amino acids (Pro-Leu, Pro-Phe) on the carboxyl-terminal end can induce significant chemotactic responses. With valine on any terminus the proline-containing dipeptide induced a weak repellent effect.

Mitochondrial dehydrogenases in different taxa of tetrahymena: effect of insulin.

Mitochondrial dehydrogenase activity was measured in seven taxa of Tetrahymena (T. pyriformis G1. T. hegewishi, T. malaccensis, T. pigmentosa, T. shapiro, T. thermophila CU-399, T. thermophila MS-1). Enzyme activity was different in the taxa investigated. Insulin reduced enzyme activity in six of the seven taxa studied. The duration of activity reduction was relatively long (5-10 min.) in most of the cases, and in T. hegewishi this lasted up to the end of the measurements (30 min.). There was no interrelation between the basic dehydrogenase activity of the taxon and the effect of insulin. There was also no correlation between the degree of relationship (of the taxa) and the dehydrogenase profile after insulin treatment.