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.

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.

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.

Effects of L-alanine and L-alanine peptides on the chemotaxis of tetrahymena: evolutionary conclusions.

L-alanine and its peptides (L-Ala-2-6) do not attract or repulse Tetrahymena in a 10(-8) M concentration. In 10(-10) M concentration there is a consistent repellent effect. Twenty four hours after L-alanine or L-alanine-peptides' pretreatment (imprinting) the progeny generation of the cells react differently to the same materials. L-Alanine, L-alanine penta- and hexapeptide in both concentrations are chemoattractant, while L-alanine tetrapeptide is repellent. L-Alanine dipeptide is inert in 10(-10) M and repellent at 10(-8) M concentrations, while L-alanine tripeptide is strongly repellent at 10(-10) M and attractant at 10(-8) M concentrations. This means, that the first encounter (imprinting) with an exogenous amino acid or peptide is decisive to the later reaction of the protozoan cell. The chain length is important in the imprinting, however the reaction is not consistent. The experiments call the attention to the significance of imprinting in the receptor and hormone evolution.

Chemotaxis of the unicellular green alga Dunaliella salina and the ciliated Tetrahymena pyriformis--effects of glycine, lysine, and alanine, and their oligopeptides.

Chemotactic properties of amino acids (L-alanine, glycine and L-lysine) and their oligopeptides (10(-6) M) and binding sites to these ligands were investigated in two unicellular models, the heterotrophic Tetrahymena pyriformis and the auxotrophic Dunaliella salina. Chemotaxis of Dunaliella induced by simple amino acids and their derivatives demonstrated that binding sites (receptors) for food molecules are not only present in the membrane but are also able to induce their basic physiological response. In Tetrahymena, substances with special molecular structure and properties (polar, hydrophilic character of the signal peptide chain)-5-L-Lys, 5-Gly- were required for chemoattraction, other peptides tested, lacking the required structure, were repellent. Divergences in chemotaxis and binding assays of both species suggest that trends of functional and binding parameters do not run parallel at this level of evolution.

Effect of adrenocorticotropic hormone (ACTH) and insulin on the phagocytic capacity of Tetrahymena.

Adrenocorticotropic hormone (ACTH) and insulin negatively influenced the phagocytic activity of Tetrahymena. The two hormones had diverse effects after 4 hr of treatments on no-test-particle containing, "0-cells". At this time the number of "0 cells" was significantly lower in the ACTH-treated groups, while in the insulin-treated groups there was an increase of "0-cells" compared to the control and to the results of the starting experiment. Considering previous results, when small molecular weight hormones, if did at all, positively influenced phagocytosis in Tetrahymena, the experiments call the attention to the differences caused by the size of the signal molecules. In the light of the literary data on hormone effects to phagocytosis in mammals and men, the similarity of the effects in species being very far from each other in evolution, could be concluded.

Adaptation of the ASTRIN staining technique for quantitative determination of the functional state of the cellular nucleus. Experiments on the nucleoli of CHO cells.

Cytophotometric determination between 516 less than or equal to lambda less than or equal to 745 nm of the nucleolar spectra of cells stained with the ASTRIN technique has enabled the quantitative assessment of stain binding affinity and its changes. The hormone-like active molecule gamma-L-glutamyl-taurine, which acts at the nuclear level, gave rise to nucleolar activation, which could be precisely followed up by ASTRIN staining. It appears that the ASTRIN staining technique is suitable for the quantitative determination of changes in the functional state of the nucleus and nucleolus.

Effects of the mammalian vasoconstrictor peptide, endothelin-1, on Tetrahymena pyriformis GL, and the immunocytological detection of endogenous endothelin-like activity.

The vasoconstrictor endothelin-1 (ET-1) is shown to have significant physiological effects on a unicellular organism, Tetrahymena pyriformis. These responses include: (1) A significant increase in intracellular [Ca2+] induced by 10(-10) M ET-1; (2) Increased chemotaxis, maximal at 10(-10) M; and (3) A small inhibition of proliferation at the 10(-13)-10(-12) M concentration range. Immunocytochemical detection of endogenous ET-1 using rabbit antibodies directed against human or porcine ET-1 indicates that this is a further example of the widening group of vertebrate hormones now known to be synthesized by Tetrahymena. These observations suggest that hormones are of considerable antiquity in their phylogenetic appearance and have been highly conserved throughout evolution.

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.

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).

Effects of insulin and histamine in themselves and in combination on the glucose metabolism of Tetrahymena.

The glucose metabolism and the related intracellular processes of Tetrahymena pyriformis GL cells are measurably influenced by several hormones of higher organisms, among others by the endocytosis stimulating hormone histamine, and the glucose metabolism regulating hormone insulin. Histamine does not interfere with the glucose metabolizing action of insulin, but markedly enhances the utilization of glucose, to judge from a significant decrease in the PAS-positive (hexose) component of histamine-exposed Tetrahymena pyriformis GL cells.

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.

Method for determination of chemoattraction in Tetrahymena pyriformis.

Earlier works presented a variety of techniques for detection of chemoattractants in ciliates. The present agar layer model and the used double-P cutter give an easy, reproducible way of quantitative evaluation of positive chemotaxis. Beside the two-channel chamber, the multi-channel one seems to be also useful in characterization of different taxons or different chemoattractants.

Overlap of concanavalin-A and insulin imprinting in rat thymocytes.

The thymocytic insulin binding in rats treated with the hormone neonatally on a single occasion increased considerably compared to the untreated control by 3 months of age. Treatment with Concanavalin-A also accounted for an increase in adult insulin binding, whereas neonatal treatment with insulin did not alter the binding relations of Concanavalin-A in adulthood.

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.

Different and selective chemotactic responses of Tetrahymena pyriformis to two families of signal molecules: lectins and peptide hormones.

Tetrahymena chemotactically responds to the presence of different signal molecules. The response elicited is different to peptide hormones and to lectins however the cell distinguishes the individual members of the two groups. The negative chemotactic effect is characteristic of the hormones tested and it is especially true for oxytocin and vasopressin, however Tetrahymena distinguishes also these two hormones. While the effect of hormones was mainly independent to the concentration tested, lectins elicit an increasing chemoattractant effect above 10(-9) M concentration. Below 10(-9) M the sugar specific, receptor level effect of lectins is probable. The experiments draw attention to the perceptional selectivity of Tetrahymena embodied in chemotaxis.

Influence of enkephalins on the ACTH-induced hormonal imprinting in tetrahymena.

Both adrenocorticotrop hormone (ACTH) and the synthetic enkephalins investigated evoked imprinting in Tetrahymena and led to increased hormone binding at further contact with ACTH. Neither molecule evoked, however, imprinting for the enkephalins. The pentapeptide enkephalin containing also proline had the most pronounced imprinting effect and, when given together with ACTH, it increased the imprintatory effect of ACTH considerably. In all the situations investigated the enkephalin tetrapeptide inhibited the positive effect of the enkephalin pentapeptide, whereas it did not influence the imprintatory effect of ACTH. Similarities can be found between the pharmacological and imprinting effects of enkephalin in mammals, and the effects seen in the present investigations.

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.