[The detection of activity of components and factors of complement according its effect on infusorians].

The article considers the developed techniques of detection of functional activity of components C1, C2, C3, C4 and membrane attacking complex of classical pathway and also factors B and D of alternative pathway of human complement using automated device measurement of immobilizing effect on infusorians Tetrahymena pyriformis. The techniques are based on application of artificially produced reagents containing all necessary components of compliment besides testing one. The mathematical mode of activities calculation is developed. The linear dependency of velocity of infusorians immobilizing from functional activities of testing component. The relevance of techniques is demonstrated by correlation of results derived using the proposed mode with the developed earlier hemolytic mode.

In silico prediction of toxicity of non-congeneric industrial chemicals using ensemble learning based modeling approaches.

Ensemble learning approach based decision treeboost (DTB) and decision tree forest (DTF) models are introduced in order to establish quantitative structure-toxicity relationship (QSTR) for the prediction of toxicity of 1450 diverse chemicals. Eight non-quantum mechanical molecular descriptors were derived. Structural diversity of the chemicals was evaluated using Tanimoto similarity index. Stochastic gradient boosting and bagging algorithms supplemented DTB and DTF models were constructed for classification and function optimization problems using the toxicity end-point in T. pyriformis. Special attention was drawn to prediction ability and robustness of the models, investigated both in external and 10-fold cross validation processes. In complete data, optimal DTB and DTF models rendered accuracies of 98.90%, 98.83% in two-category and 98.14%, 98.14% in four-category toxicity classifications. Both the models further yielded classification accuracies of 100% in external toxicity data of T. pyriformis. The constructed regression models (DTB and DTF) using five descriptors yielded correlation coefficients (R(2)) of 0.945, 0.944 between the measured and predicted toxicities with mean squared errors (MSEs) of 0.059, and 0.064 in complete T. pyriformis data. The T. pyriformis regression models (DTB and DTF) applied to the external toxicity data sets yielded R(2) and MSE values of 0.637, 0.655; 0.534, 0.507 (marine bacteria) and 0.741, 0.691; 0.155, 0.173 (algae). The results suggest for wide applicability of the inter-species models in predicting toxicity of new chemicals for regulatory purposes. These approaches provide useful strategy and robust tools in the screening of ecotoxicological risk or environmental hazard potential of chemicals.

Microcalorimetry: a powerful and original tool for tracking the toxicity of a xenobiotic on Tetrahymena pyriformis.

Fighting against water pollution requires the ability to evaluate the toxicity of pollutants such as herbicides. Tetrahymena pyriformis are ubiquitous ciliated protozoans commonly used in ecotoxicological research. Microcalorimetry can be used in many biological investigations as a universal, non-destructive and highly sensitive tool that provides a continuous real-time monitoring of the metabolic activity. This technique based on the thermal power output was applied to evaluate the influence of herbicide diuron on cultures of T. pyriformis. The heat flux produced upon addition of 0, 3.5, 7.0, 14.0, 28.0, and 56.0 µg mL⁻¹ of diuron was monitored. The biomass change during the growth was also determined by flow cytometry. The results confirmed that the growth of T. pyriformis is progressively inhibited as the concentration of diuron increases and revealed that the state of the living system is severely altered at a concentration of 56.0 µg mL⁻¹. The IC50 was estimated at 13.8 µg mL⁻¹ by microcalorimetry and at 18.6 µg mL⁻¹ by flow cytometry. It was shown that microcalorimetry is not only a very effective tool for the determination of the growth rate constant but that it is also a valuable probe for a rapid detection of the metabolic perturbations and, in ultimate cases, of the critical alterations of the living system under the action of a toxic agent.

PLS-optimal: a stepwise D-optimal design based on latent variables.

Several applications, such as risk assessment within REACH or drug discovery, require reliable methods for the design of experiments and efficient testing strategies. Keeping the number of experiments as low as possible is important from both a financial and an ethical point of view, as exhaustive testing of compounds requires significant financial resources and animal lives. With a large initial set of compounds, experimental design techniques can be used to select a representative subset for testing. Once measured, these compounds can be used to develop quantitative structure-activity relationship models to predict properties of the remaining compounds. This reduces the required resources and time. D-Optimal design is frequently used to select an optimal set of compounds by analyzing data variance. We developed a new sequential approach to apply a D-Optimal design to latent variables derived from a partial least squares (PLS) model instead of principal components. The stepwise procedure selects a new set of molecules to be measured after each previous measurement cycle. We show that application of the D-Optimal selection generates models with a significantly improved performance on four different data sets with end points relevant for REACH. Compared to those derived from principal components, PLS models derived from the selection on latent variables had a lower root-mean-square error and a higher Q2 and R2. This improvement is statistically significant, especially for the small number of compounds selected.

Does rational selection of training and test sets improve the outcome of QSAR modeling?

Prior to using a quantitative structure activity relationship (QSAR) model for external predictions, its predictive power should be established and validated. In the absence of a true external data set, the best way to validate the predictive ability of a model is to perform its statistical external validation. In statistical external validation, the overall data set is divided into training and test sets. Commonly, this splitting is performed using random division. Rational splitting methods can divide data sets into training and test sets in an intelligent fashion. The purpose of this study was to determine whether rational division methods lead to more predictive models compared to random division. A special data splitting procedure was used to facilitate the comparison between random and rational division methods. For each toxicity end point, the overall data set was divided into a modeling set (80% of the overall set) and an external evaluation set (20% of the overall set) using random division. The modeling set was then subdivided into a training set (80% of the modeling set) and a test set (20% of the modeling set) using rational division methods and by using random division. The Kennard-Stone, minimal test set dissimilarity, and sphere exclusion algorithms were used as the rational division methods. The hierarchical clustering, random forest, and k-nearest neighbor (kNN) methods were used to develop QSAR models based on the training sets. For kNN QSAR, multiple training and test sets were generated, and multiple QSAR models were built. The results of this study indicate that models based on rational division methods generate better statistical results for the test sets than models based on random division, but the predictive power of both types of models are comparable.

DNA content alterations in Tetrahymena pyriformis macronucleus after exposure to food preservatives sodium nitrate and sodium benzoate.

The toxicity, in terms of changes in the DNA content, of two food preservatives, sodium nitrate and sodium benzoate was studied on the protozoan Tetrahymena pyriformis using DNA image analysis technology. For this purpose, selected doses of both food additives were administered for 2 h to protozoa cultures and DNA image analysis of T. pyriformis nuclei was performed. The analysis was based on the measurement of the Mean Optical Density which represents the cellular DNA content. The results have shown that after exposure of the protozoan cultures to doses equivalent to ADI, a statistically significant increase in the macronuclear DNA content compared to the unexposed control samples was observed. The observed increase in the macronuclear DNA content is indicative of the stimulation of the mitotic process and the observed increase in MOD, accompanied by a stimulation of the protozoan proliferation activity is in consistence with this assumption. Since alterations at the DNA level such as DNA content and uncontrolled mitogenic stimulation have been linked with chemical carcinogenesis, the results of the present study add information on the toxicogenomic profile of the selected chemicals and may potentially lead to reconsideration of the excessive use of nitrates aiming to protect public health.

Predicting Michael-acceptor reactivity and toxicity through quantum chemical transition-state calculations.

The electrophilic reactivity of Michael acceptors is an important determinant of their toxicity. For a set of 35 α,ß-unsaturated aldehydes, ketones and esters with experimental rate constants of their reaction with glutathione (GSH), k(GSH), quantum chemical transition-state calculations of the corresponding Michael addition of the model nucleophile methane thiol (CH(3)SH) have been performed at the B3LYP/6-31G** level, focusing on the 1,2-olefin addition pathway without and with initial protonation. Inclusion of Boltzmann-weighting of conformational flexibility yields intrinsic reaction barriers ΔE(‡) that for the case of initial protonation correctly reflect the structural variation of k(GSH) across all three compound classes, except that they fail to account for a systematic (essentially incremental) decrease in reactivity upon α-substitution. By contrast, the reduction in k(GSH) through ß-substitution is well captured by ΔE(‡). Empirical correction for the α-substitution effect yields a high squared correlation coefficient (r(2) = 0.96) for the quantum chemical prediction of log k(GSH), thus enabling an in silico screening of the toxicity-relevant electrophilicity of α,ß-unsaturated carbonyls. The latter is demonstrated through application of the calculation scheme for a larger set of 46 Michael-acceptor aldehydes, ketones and esters with experimental values for their toxicity toward the ciliates Tetrahymena pyriformis in terms of 50% growth inhibition values after 48 h exposure (EC(50)). The developed approach may add in the predictive hazard evaluation of α,ß-unsaturated carbonyls such as for the European REACH (Registration, Evaluation, Authorization and Restriction of Chemicals) Directive, enabling in particular an early identification of toxicity-relevant Michael-acceptor reactivity.

Epoxide and thiirane toxicity in vitro with the ciliates Tetrahymena pyriformis: structural alerts indicating excess toxicity.

The 48 h toxicity of 18 organic narcotics, 13 epoxides, and 2 thiiranes toward the ciliates Tetrahymena pyriformis was determined in terms of 50% growth inhibition EC(50). Nominal EC(50) was corrected for volatilization and sorption to quantify the freely dissolved compound fraction in solution. The derived baseline narcosis model served to evaluate toxicity enhancements T(e) as ratios of narcosis-predicted over experimental EC(50) values. Among the nine heterocycles with aliphatic side chains that include two thiiranes, three compounds yielded T(e) > 10, suggesting their covalent binding at nucleophilic protein sites such as -OH, -NHR, and -SH through S(N)2-type ring-opening. As a general trend of this group, T(e) decreases with increasing alkyl group size. Moreover, four of the six nonaliphatic epoxides exerted substantial excess toxicities with T(e) > 10, which could be rationalized by ring-opening activation through negative inductive effect, benzylic stabilization, and phenyl ring H-bonding. By contrast, 1,2 substituted epoxides showed narcosis-level toxicity, despite the opportunity of side-chain Schiff-base formation with protein amino groups. The resulting structural alerts enable an in silico screening of epoxides and thiiranes for their potential to exert excess toxicity. Note that observed differences in T(e) sensitivity between ciliates, bacteria and fish should be taken into account when designing in vitro alternatives to fish toxicity studies.

Listeria monocytogenes virulence factor Listeriolysin O favors bacterial growth in co-culture with the ciliate Tetrahymena pyriformis, causes protozoan encystment and promotes bacterial survival inside cysts.

BACKGROUND: The gram-positive pathogenic bacterium Listeria monocytogenes is widely spread in the nature. L. monocytogenes was reported to be isolated from soil, water, sewage and sludge. Listeriolysin O (LLO) is a L. monocytogenes major virulence factor. In the course of infection in mammals, LLO is required for intracellular survival and apoptosis induction in lymphocytes. In this study, we explored the potential of LLO to promote interactions between L. monocytogenes and the ubiquitous inhabitant of natural ecosystems bacteriovorous free-living ciliate Tetrahymena pyriformis. RESULTS: Wild type L. monocytogenes reduced T. pyriformis trophozoite counts and stimulated encystment. The effects were observed starting from 48 h of co-incubation. On the day 14, trophozoites were eliminated from the co-culture while about 5 x 104 cells/ml remained in the axenic T. pyriformis culture. The deficient in the LLO-encoding hly gene L. monocytogenes strain failed to cause mortality among protozoa and to trigger protozoan encystment. Replenishment of the hly gene in the mutant strain restored toxicity towards protozoa and induction of protozoan encystment. The saprophytic non-haemolytic species L. innocua transformed with the LLO-expressing plasmid caused extensive mortality and encystment in ciliates. During the first week of co-incubation, LLO-producing L. monocytogenes demonstrated higher growth rates in association with T. pyriformis than the LLO-deficient isogenic strain. At latter stages of co-incubation bacterial counts were similar for both strains. T. pyriformis cysts infected with wild type L. monocytogenes caused listerial infection in guinea pigs upon ocular and oral inoculation. The infection was proved by bacterial plating from the internal organs. CONCLUSIONS: The L. monocytogenes virulence factor LLO promotes bacterial survival and growth in the presence of bacteriovorous ciliate T. pyriformis. LLO is responsible for L. monocytogenes toxicity for protozoa and induction of protozoan encystment. L. monocytogenes entrapped in cysts remained viable and virulent. In whole, LLO activity seems to support bacterial survival in the natural habitat outside of a host.

An Electronic-structure Informatics Study on the Toxicity of Alkylphenols to Tetrahymena pyriformis.

Alkylphenols (APs) dissolved in water are known to be toxic to animals including humans. In this study, regression models describing the toxicity of the 33 AP molecules were investigated for reproducing and thereby making it possible to predict a quantitative structure-activity relationship (QSAR). For this purpose, we tried to derive regression models for the experimental IGC50 (growth inhibition concentration at 50 %) to Tetrahymena pyriformis by using various descriptor sets consisting of electronic and shape descriptors. By applying the multiple linear regression (MLR) analysis, it was successful to derive a quantitative regression model for the IGC50 values. In this analysis, the size parameters of the molecules were found important, suggesting that bulky molecules should be less toxic. We could also obtain, when the size descriptors were excluded, an MLR model indicating that the electron affinity (EA) should be important, which is consistent with the previous QSAR studies. Through the correlation analysis among the descriptors, it was shown that, in the present set of molecules, EA and a size parameter are highly correlated. Since EA was calculated to be negative, indicating that the related process would be energetically unfavourable, it was concluded that the size of the molecules should be a dominant factor determining IGC50 . This implies that a molecular recognition process would play a critical role in the mode of action for the toxicity.

Causal relations among cell cycle processes in Tetrahymena pyriformis. An analysis employing temperature-sensitive mutants.

Utilization of temperature-sensitive mutants of Tetrahymena pyriformis affected in cell division or developmental pathway selection has permitted elucidation of causal dependencies interrelating micronuclear and macronuclear replication and division, oral development, and cytokinesis. In those mutants in which cell division is specifically blocked at restrictive temperatures, micronuclear division proceeds with somewhat accelerated periodicity but maintains normal coupling to predivision oral development. Macronuclear division is almost totally suppressed in an early acting mutant (mola) that prevents formation of the fission zone, and is variably affected in other mutants (such as mo3) that allow the fission zone to form but arrest constriction. However, macronuclear DNA synthesis can proceed for about four cycles in the nondividing mutant cells. A second class of mutants (psm) undergoes a switch of developmental pathway such that cells fail to enter division but instead repeatedly carry out an unusual type of oral replacement while growing in nutrient medium at the restrictive temperature. Under these circumstances no nuclei divide, yet macronuclear DNA accumulation continues. These results suggest that (a) macronuclear division is stringently affected by restriction of cell division, (b) micronuclear division and replication can continue in cells that are undergoing the type of oral development that is characteristic of division cycles, and (c) macronuclear DNA synthesis can continue in growing cells regardless of their developmental status. The observed relationships among events are consistent with the further suggestion that the cell cycle in this organism may consist of separate clusters of events. with a varying degree of coupling among clusters. A minimal model of the Tetrahymena cell cycle that takes these phenomena into account is suggested.

Nonidentity of ribosomal structural proteins in growing and starved Tetrahymena.

We have examined the ribosomal structural proteins isolated from vegetatively growing Tetrahymena pyriformis and from cells that had been starved of all nutrients for 24 h. Reproducible, nonartifactual differences in protein complement, primarily associated with the large ribosomal subunit, were found. The kinetics of change in ribosomal protein complement were followed both in refed and in newly starved cells. Furthermore, attempts at correlating a certain protein "phenotype" with a particular functional state of the ribosome were made. It was concluded that the alterations seen could not be correlated with a specific stage in the normal ribosome cycle. We did show, however, that the change in protein complement could occur as a result of altering preexisting ribosomes. In addition, we showed that the change correlates with a decrease in growth rate rather than being caused by the starvation conditions themselves. Speculations as to the functional significance of the protein changes are presented.

Ribosome biosynthesis in Tetrahymena pyriformis. Regulation in response to nutritional changes.

Ribosome contents of growing and 12-h-starved Tetrahymena pyriformis (strain B) were compared. These studies indicate that (a) starved cells contain 74% of the ribosomes found in growing cells, (b) growing cells devote 20% of their protein synthetic activity to ribosomal protein production, and (c) less than 3% of the protein synthesized in starved cells is ribosomal protein. Ribosome metabolism was also studied in starved cells which had been refed. For the first 1.5 h after refeeding, there is no change in ribosome number per cell. Between 1.5 and 2 h, there is an abrupt increase in rate of ribosome accumulation but little change in rate of cell division. By 3.5 h, the number of ribosomes per cell has increased to that found in growing cells. At this time, the culture begins to grow exponentially at a normal rate. During the first 2 h after refeeding, cells devote 30-40% of their protein synthetic activity to ribosomal protein production. We estimate that the rate of ribosomal protein synthesis per cell increases at least 80-fold during the first 1-1.5 h after refeeding, reaching the level found in exponentially growing cells. This occurs before any detectable change in ribosome number per cell. The transit time for the incorporation of these newly synthesized proteins into ribosomes is from 1 to 2 h during early refeeding, whereas in exponentially growing cells it is less than 30 min. The relationship between ribosomal protein synthesis and ribosome accumulation is discussed.

Regulation of microtubules in Tetrahymena. I. Electron microscopy of oral replacement.

The coupled resorption and redifferentiation of oral structures which occurs in Tetrahymena pyriformis under conditions of amino acid deprivation has been studied by transmission electron microscopy. Two patterns of ciliary resorption have been found, (a) in situ, and (b) after withdrawal into the cytoplasm. No autophagic vacuoles containing cilia or ciliary axonemes have been seen. Stomatogenic field basal bodies arise by a process of rapid sequential nucleation, with new ones always appearing next to more mature ones, even though the latter may not be fully differentiated. Accessory radial ribbons of microtubules develop immediately adjacent to oral field basal bodies as a late step in their maturation. It can be seen that the formation of basal bodies and their orientation within the oral complex are separate processes. This is true for about 130 of the approximately 170 oral basal bodies; the remaining 40 or so form within the patterned groups of ciliary units as a later event. Clusters of randomly oriented thin-walled microtubules are found surrounding oral basal bodies at all times during stomatogenesis. They may either represent stores of microtubule subunit protein, or serve as effectors of basal body movement during their orientation into pattern.

Effect of cell population density on G2 arrest in Tetrahymena.

The ciliated protozoan, Tetrahymena pyriformis strain GL-C, has been used to study the effect of cell population density during starvation on the synchrony obtained after refeeding and on the number of cells arrested in G2 phase of the cell cycle. At high cell densities two peaks of division indices were observed after refeeding while only one was observed at low cell densities. Cell division began earlier in cultures starved at high cell densities. Most importantly, the proportion of cells in G2 was considerably higher in populations starved at high cell densities. When tritiated thymidine was present during the refeeding period, radioautographs of cell samples at different times showed that the first cells to exhibit division furrows contained unlabeled nuclei. The first peak in the division index after refeeding was observed only at higher cell densities and is attributed to the cells arrested in G2. These results suggest that Tetrahymena is an excellent organism to study the concept of resting stages in the cell cycle and their control.

A feedback control of cell cycle parameters in Tetrahymena.

Protein and RNA contents of individual cells were measured cytophotometrically and related to the duration of individual generation times. Constant amounts of RNA per cell at division, and generation time-dependent protein contents, resulted in generation time-specific RNA/protein ratios. Experimental reduction of these ratios by inhibition of RNA synthesis stimulated premature macronuclear S phases.

The synthesis of microtubule and other proteins of the oral apparatus in Tetrahymena pyriformis.

Several proteins, including microtubule proteins, have been isolated from the oral apparatus of the ciliate Tetrahymena. The synthesis of these proteins has been studied in relation to formation of this organelle system by the cell. Electron microscopy has shown that the isolated oral apparatus consists primarily of basal bodies, pellicular membranes, and a system of subpellicular microtubules and filaments. Cilia were removed during the isolation; therefore none of the proteins studied was from these structures. Evidence was obtained from the study of total oral apparatus protein which indicates that at least some of the proteins involved in formation of this organelle system may be synthesized and stored in the cytoplasm for use over long periods. This pattern of regulation was found for three individual proteins isolated from the oral apparatus fraction after extraction with a phenol-acetic acid solvent. A different pattern of regulation was found for microtubule proteins isolated from the oral apparatus of Tetrahymena. The data suggest that microtubule proteins, at least in logarithmically growing cells, are not stored in a cytoplasmic pool but are synthesized in the same cell cycle in which they are assembled into oral structures.

Regulation of microtubules in Tetrahymena. II. Relation between turnover of microtubule proteins and microtubule dissociation and assembly during oral replacement.

Experiments are reported which were designed to test for induced synthesis of microtubule proteins associated with the rapid proliferation of basal bodies and associated intracytoplasmic microtubules which occurs during oral replacement in Tetrahymena. None was found. Instead, it is shown that these structures can be formed with de novo synthesis of as little as 6% of their microtubule proteins. It is suggested that basal body proliferation may be controlled by synthesis of morphogenetic regulator proteins.

Accumulation of replicative intermediates of mitochondrial DNA in Tetrahymena pyriformis grown in ethidium bromide.

The effect of growth of Tetrahymena pyriformis in ethidium bromide (EthBr) on the structure and synthesis of mitochondrial DNA (mtDNA) has been investigated. During the first 5 h of growth in EthBr, mtDNA synthesis is inhibited 95% or more. After 10-15 h, this block is partially released and large numbers of replicating molecules accumulate, indicating that inhibition by EthBr primarily affects the rate of chain growth and not the initiation of new rounds of replication. The accumulated molecules sediment more rapidly than normal Tetrahymena mtDNA and do not contain enough single-strand regions to distinguish them from normal Tetrahymena mtDNA when banded in buoyant CsCl or NaI gradients. Electron microscopy shows that the predominant species in this rapidly sedimenting DNA is a linear molecule containing one symmetrical double-stranded replication loop of varying size located at its center. No degradation of mtDNA from cells grown in EthBr was detected in alkaline velocity gradients.

Evidence for a temporal incompatibility between DNA replication and division during the cell cycle of Tetrahymena.

The mechanism of coordination between DNA replication and cell division was studied in Tetrahymena pyriformis GL-C by manipulation of the timing of these events with heat shocks and inhibition of DNA synthesis. Preliminary experiments showed that the inhibitor combination methotrexate and uridine (M + U) was an effective inhibitor of DNA synthesis. Inhibition of the progression of DNA synthesis with M + U in exponentially growing cells, in which one S period usually occurs between two successive divisions, or in heat-shocked cells, when successive S periods are known to occur between divisions, resulted in the complete suppression of the following division. In further experiments in which the division activities were reassociated with the DNA synthetic cycle by premature termination of the heat-shock treatment, it was shown that (a) the completion of one S period during the treatment was sufficient for cell division, (b) the beginning of division events suppressed the initiation of further S periods, and (c) if further S periods were initiated while the heat-shock treatment was continued, division preparations could not begin until the necessary portion of the S period was completed, even though DNA had previously been duplicated. It was concluded that a temporal incompatibility exists between DNA synthesis and division which may reflect a coupling mechanism which insures their coordination during the normal cell cycle.