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Biomethylation is an effective means of arsenic detoxification by organisms living in aquatic environments. Ciliated protozoa (including Tetrahymena species) play an important role in the biochemical cycles of aquatic ecosystems and have a potential application in arsenic biotransformation. This study compared arsenic tolerance, accumulation, methylation, and efflux in 11 Tetrahymena species. Nineteen arsenite (As(III)) S -adenosylmethionine (SAM) methyltransferase (arsM) genes, of which 12 are new discoveries, were identified, and protein sequences were studied. We then constructed recombinant cell lines based on the Tetrahymena thermophila (T. thermophila) wild-type SB210 strain and expressed each of the 19 arsM genes under the control of the metal-responsive the MTT1 promoter. In the presence of Cd2+ and As(V), expression of the arsM genes in the recombinant cell lines was much higher than in the donor species. Evaluation of the recombinant cell line identified one with ultra-high arsenic methylation enzyme activity, significantly higher arsenic methylation capacity and much faster methylation rate than other reported arsenic methylated organisms, which methylated 89% of arsenic within 6.5 h. It also had an excellent capacity for the arsenic detoxification of lake water containing As(V), 56% of arsenic was methylated at 250 μg/L As(V) in 48 h. This study has made a significant contribution to our knowledge on arsenic metabolism in protozoa and demonstrates the great potential to use Tetrahymena species in the arsenic biotransformation of aquatic environments. [Display omitted] • 12 of 19 arsM genes identified in 15 Tetrahymena species were new discoveries. • Metal-responsive recombinant cell lines expressing 19 arsM genes were obtained. • The arsM genes in recombinant cell lines could be induced by Cd2+ and As(V). • The recombinant cell line could efficiently methylate As(V) in lake water. [ABSTRACT FROM AUTHOR]

Oxidative stress results from an imbalance between the intracellular antioxidants and the radicals ROS or RNS, in favor of the latter. Oxidative stress is associated with several human diseases and causes significant cellular damage to the structure, nucleic acids (DNA, RNA), proteins and lipids. These consequences can compromise the health and viability of the living cell by causing cell death by necrosis or apoptosis. A variety of antioxidant supplements have been developed in recent years to enrich the endogenous defense systems of living organisms. However, the prevention of chronic diseases through the use of antioxidant supplements remains controversial until now. However, natural products with regard to essential oils are very much in demand in biology research because of their antimicrobial and antioxidant properties. We investigated the protective effect of oil of oregano and sage against the oxidative stress induced by hydrogen peroxide (H 2 O 2) and sodium nitroprusside (SNP) in two cellular models Tetrahymena thermophila and Tetrahymena pyriformis. The results of our study showed that oxidative stress inhibits growth, mobility and changes in cell shape of both species. The application of essential oils has shown that both oils protect cells treated with H 2 O 2 and SNP. However, the intracellular mechanism of SNP inhibition is longer than that of H 2 O 2. Therefore, the use of these essential oils could be important to the prevention of chronic diseases related to oxidative stress in higher organisms. [ABSTRACT FROM AUTHOR]

[Display omitted] • T. thermophila has 121 papain-family cysteine protease encoding genes. • TtPFCPs were divided into four distinct subgroups CATL, CATB, CATC, and CATX. • The majority of true TtPFCPs (96 out of the total) were in TtCATL subgroup. • Phylogenetic analysis showed that ciliate's PFCPs have ancestral features. • The results may help researchers to construct TtPFCP's KO mutants in T. thermophila. Tetrahymena thermophila is a promising host for recombinant protein production, but its utilization in biotechnology is mostly limited due to the presence of intracellular and extracellular papain-family cysteine proteases (PFCPs). In this study, we employed bioinformatics approaches to investigate the T. thermophila PFCP genes and their encoded proteases (TtPFCPs), the most prominent protease family in the genome. Results from the multiple sequence alignment, protein modeling, and conserved motif analyses revealed that all TtPFCPs showed considerably high homology with mammalian cysteine cathepsins and contained conserved amino acid motifs. The total of 121 TtPFCP-encoding genes, 14 of which were classified as non-peptidase homologs, were found. Remaining 107 true TtPFCPs were divided into four distinct subgroups depending on their homology with mammalian lysosomal cathepsins: cathepsin L-like (TtCATLs), cathepsin B-like (TtCATBs), cathepsin C-like (TtCATCs), and cathepsin X-like (TtCATXs) PFCPs. The majority of true TtPFCPs (96 out of the total) were in TtCATL-like peptidase subgroup. Both phylogenetic and chromosomal localization analyses of TtPFCPs supported the hypothesis that TtPFCPs likely evolved through tandem gene duplication events and predominantly accumulated on micronuclear chromosome 5. Additionally, more than half of the identified TtPFCP genes are expressed in considerably low quantities compared to the rest of the TtPFCP genes, which are expressed at a higher level. However, their expression patterns fluctuate based on the stage of the life cycle. In conclusion, this study provides the first comprehensive in-silico analysis of TtPFCP genes and encoded proteases. The results would help designing an effective strategy for protease knockout mutant cell lines to discover biological function and to improve the recombinant protein production in T. thermophila. [ABSTRACT FROM AUTHOR]

There has been an increasing incidence rate of rice false smut in global rice cultivation areas. However, there is a dearth of studies on the environmental concentrations and hazards of ustiloxin A (UA), which is the major mycotoxin produced by a pathogenic fungus of the rice false smut. Here, the concentrations of UA in the surface waters of two paddy fields located in Enshi city, Hubei province, China, were measured, and its toxicity in T. Thermophila was evaluated. This is the first study to detect UA in the surface waters of the two paddy fields, and the measured mean concentrations were 2.82 and 0.26 μg/L, respectively. Exposure to 2.19, 19.01 or 187.13 μg/L UA for 5 days significantly reduced the theoretical population and cell size of T. thermophila. Furthermore, treatment with 187.13 μg/L UA changed the percentages of T. thermophila cells in different cell-cycle stages, and with an increased malformation rate compared with the control, suggesting the disruption of the cell cycle. The expressions of 30 genes involved in the enriched proteasome pathway, 7 cyclin genes (cyc9 , cyc10 , cyc16 , cyc22 , cyc23, cyc26, cyc33) and 2 histone genes (mlh1 and hho1) were significantly down-regulated, which might be the modes of action responsible for the disruption of cell cycling due to UA exposure. Image 1 • For the first time, ustiloxin A was detected in the surface water of paddy fields. • Exposure to ustiloxin A reduced the theoretical population of T. Thermophila and caused cell malformation. • Cell cycle was disrupted by ustiloxin A. • Expressions of genes related to cell cycling were changed due to ustiloxin A exposure. For the first time, it was founded that ustiloxin A (UA) was detected in surface waters of paddy fields, and exposure to UA in T. Thermophila caused toxicity by disrupting cell cycle. [ABSTRACT FROM AUTHOR]

In Tetrahymena , K antigens associate only with mature basal bodies and are expected to play important roles in the morphogenesis and function of the membrane skeleton around basal bodies, but these proteins have not been identified and their functions are unknown. Commercially available anti-human Rho GDP-dissociation inhibitor α (RhoGDIα) antibody (sc-33201) was accidentally found to show very similar immunofluorescence staining patterns to those of anti-K antigen antibodies, such as 424A8 and 10D12 mouse monoclonal antibodies, in Tetrahymena . A 40 kDa protein recognized by this antibody was partially purified and identified as granule lattice protein 1 (Grl1p) by matrix-assisted laser desorption/ionization-tandem time-of-flight mass spectrometry. In immunoblotting experiments this antibody was suggested to recognize endogenous Grl1p. The three-dimensional structure of proGrl1p protein predicted by I-TASSER was similar to a spectrin family protein. Grl1 may be a K antigen and a spectrin-like protein in Tetrahymena . [ABSTRACT FROM AUTHOR]

Metallothioneins (MTs) are low-molecular-weight proteins with high Cys content and high metal-chelating ability. CdMT and CuMT subfamilies present different characteristics in Tetrahymena. To explore the effect of the cysteine arrangement and sequence length of MTs for binding different metal ions, MTT1, truncated MTT1 (TM1), MTT2, and truncated MTT2 (TM2) were expressed in E. coli. The half-maximal inhibiting concentrations (IC50) of Cd2+ and Cu+ for the recombinant strains were different. Furthermore, E. coli cells expressing MTT1 and TM1 exhibited higher accumulating ability for Cd2+ than cells expressing MTT2 and TM2. However, the opposite is true for Cu+. The binding ability of the different recombinant proteins to Cd2+ and Cu+ were also different. MTT1 and truncated mutant TM1 were the preference for Cd2+, whereas MTT2 and truncated mutant TM2 were the preference for Cu+ coordination. These results showed that metal ion tolerance and accumulation ability not only depended on cysteine arrangement pattern but also on sequence length of MT in Tetrahymena. [ABSTRACT FROM AUTHOR]

Cilia are complex and dynamic organelles that have motility and sensory functions. Defects in cilia biogenesis and function are at the origin of human ciliopathies. In motile cilia, a basal body organizes the axoneme composed of nine microtubule doublets surrounding a central pair of singlet microtubules. The distal ends of axonemal microtubules are attached to the membrane by microtubule-capping structures. Little is known about the early steps of cilium assembly. Although cilia grow and resorb from their distal tips, it remains poorly understood where and when the components of the caps are first assembled. By using Atomic Force Microscopy in tapping mode, with resolution at the nanometer range and with minimum sample manipulation, we show that Tetrahymena cilia assembly requires transient assembly of structures, composed of three components that are placed asymmetrically on an early elongating axoneme. In small uncapped axonemes the microtubule central pair was never observed. Additionally, we show that cilia cap assembly is a multi-step process in which structures of different sizes and shapes are put together in close proximity before the axoneme appears capped. We propose that the cap modifies the axoneme microtubule rate of polymerization and present a model for Tetrahymena cilia cap assembly. [ABSTRACT FROM AUTHOR]

N 6 -methyladenine DNA (6mA) modification exists in both prokaryotes and eukaryotes in which it plays a crucial role in regulating numerous biological processes. In prokaryotes, 6mA is a marker to distinguish self from foreign DNA. Its role in eukaryotes, however, remains elusive. Ciliates were among the first eukaryotes reported to contain 6mA. In the model organism T. thermophila , cellular localization and features of 6mA have been extensively studied, especially in ribosomal DNA (rDNA) molecules. Here, we summarize the features and potential functions of 6mA in Tetrahymena thermophila and other ciliates, and the major findings and contributions of the Tetrahymena model in studies of 6mA methylation. We also discuss other questions in order to improve understanding of the function and evolution of 6mA in eukaryotes. [ABSTRACT FROM AUTHOR]

Ciliated protists contain both germline micronucleus (MIC) and somatic macronucleus (MAC) in a single cytoplasm. Programmed genome rearrangements occur in ciliates during sexual processes, and the extent of rearrangements varies dramatically among species, which lead to significant differences in genomic architectures. However, genomic sequences remain largely unknown for most ciliates due to the difficulty in culturing and in separating the germline from the somatic genome in a single cell. Single-cell whole genome amplification (WGA) has emerged as a powerful technology to characterize the genomic heterogeneity at the single-cell level. In this study, we compared two single-cell WGA, multiple displacement amplification (MDA) and multiple annealing and looping-based amplification cycles (MALBAC) in characterizing the germline and somatic genomes in ciliates with different genomic architectures. Our results showed that: 1) MALBAC exhibits strong amplification bias towards MAC genome while MDA shows bias towards MIC genome of ciliates with extensively fragmented MAC genome; 2) both MDA and MALBAC could amplify MAC genome more efficiently in ciliates with moderately fragmented MAC genome. Moreover, we found that more sample replicates could help to obtain more genomic data. Our work provides a reference for selecting the appropriate method to characterize germline and somatic genomes of ciliates. [ABSTRACT FROM AUTHOR]

Recognition of food and, in consequence, ingestion of digestible particles is a prerequisite for energy metabolism in Tetrahymena pyriformis . Understanding why some particles are ingested and digested, whereas others are not, is important for many fields of research, e.g. survival of pathogens in single-celled organisms or establishment of endosymbiotic relationships. We offered T. pyriformis synthetical bovine-serum-albumin (BSA)-methacrylate microparticles of approximately 5.5 μm diameter and studied the ciliates’ ingestion and digestion behaviour. Different staining techniques as well as co-feeding with a transformant strain of Escherichia coli revealed that T. pyriformis considers these particles as natural food source and shows no feeding preference. Further, they are ingested at normal rates and may serve as sole food source. A pivotal advantage of these particles is the convenient modification of their surface by binding different ligands resulting in defined surface properties. Ingestion rate of modified microparticles either increased (additional BSA, enzymes) or decreased (amino acids). Furthermore, we investigated glycosylation patterns by lectin binding. By binding different substances to the surface in combination with various staining techniques, we provide a versatile experimental tool for elucidating details on food recognition and digestion that may allow to study evading digestion by pathogens or potential endosymbionts, too. [ABSTRACT FROM AUTHOR]

Microalgae offer a promising route to the production of high-value products, foods, animal feeds, and biofuels. Efficient algal dewatering strategies are therefore important for minimising energy and costs. Bio-flocculation is a potentially low-cost, low-energy harvesting strategy which can be facilitated by the microbial production of extracellular polymeric substances (EPS), but must be inducible since productive algal culture generally requires cells to be in suspension. Here we show that algal bio-flocculation can be controllably induced using the protozoan Tetrahymena . As little as 1:400 starved Tetrahymena to algal cells can be used to initiate rapid bio-flocculation. We demonstrate that stimulators of ryanodine receptors (caffeine and p- chlorocresol) trigger exocytosis in Tetrahymena and that inexpensive physicochemical stimuli (ammonium ions, shifts in pH and salinity) can also be effective. We suggest that triggered EPS secretion by protozoans in the starved state may explain apparently spontaneous bioflocculation of algae in both natural and artificial systems. [ABSTRACT FROM AUTHOR]

The toxicity of binary mixture of Cu2+ and phenols (phenol; o-nitrophenol; m-nitrophenol; p-nitrophenol) was evaluated using Tetrahymena thermophila as the model organism, by microcalorimetry, optical density, field emission scanning electron microscope (FESEM) and attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR). The growth curves and metabolic properties of Tetrahymena exposed to Cu2+ and phenols were monitored by microcalorimetry. Binary mixture toxicity changed with the concentration of Cu2+/phenols and the order of toxicity was Cu2+/phenol2+/m-nitrophenol2+/o-nitrophenol2+/p-nitrophenol. The results of FESEM and ATR-FTIR also indicated that Cu2+/phenols had a great effect on cell cortex and flagellum. A synergistic effect was noted between Cu2+ and phenols on Tetrahymena. [ABSTRACT FROM AUTHOR]

Summary: Tetrahymena is a ciliated protozoan that can infect a wide range of fish species, although it is most commonly reported in guppies (Poecilia reticulata). The aim of this study was to compare the susceptibility to infection with Tetrahymena of five different ornamental fish species from two different super orders. The species examined were platy (Xiphophorus), molly (Poecilia sphenops) and angelfish (Pterophyllum scalare) of the Acanthopterygii super order (which also includes guppies) and goldfish (Carassius auratus auratus) and koi carp (Cyprinus carpio) of the Ostariophysi super order. These two super orders are phylogenetically distant from each other. Infection with Tetrahymena resulted in parasite invasion of internal organs, skin and muscle in all fish species. A relatively strong inflammatory response was observed in infected goldfish and koi, with negligible response in fish species of the Acanthopterygii super order. Guppies were the most susceptible to Tetrahymena infection, exhibiting a mortality rate of 87% and 100% in two separate experiments. A high mortality rate was also observed in platy (77%), while that of molly and angelfish was significantly lower (23% and 33%, respectively). Goldfish and koi carp were less susceptible to infection compared with guppies (24% and 59% mortality, respectively). Immunization studies revealed that the Tetrahymena are immunogenic, since infection of koi carp increased their Tetrahymena immobilization response by approximately three-fold at 3 weeks post infection, while immunization with Tetrahymena plus adjuvant increased their immobilization response by approximately 30-fold. [ABSTRACT FROM AUTHOR]

Abstract: In Tetrahymena, besides apparent cell polarity generated by specialized cortical structures, several proteins display a specific asymmetric distribution suggesting their involvement in the generation and the maintenance of cell polarization. One of these proteins, a membrane skeleton protein called fenestrin, forms an antero-posterior gradient, and is accepted as a marker of cell polarity during different cellular processes, such as cell division or oral replacement. In conjugating cells, fenestrin forms an intracytoplasmic net which participates in pronuclear exchange. The function of fenestrin is still unknown. To better understand the role of fenestrin we characterized this protein in an amicronuclear Tetrahymena pyriformis. We show that in this ciliate not only does fenestrin localization change in a cell division-dependent manner, but its mRNA and protein level is also cell cycle-regulated. We determine that the two available anti-fenestrin antibodies, 3A7 and 9A7, recognize different pools of fenestrin isoforms, and that 9A7 is the more general. In addition, our results indicate that fenestrin is a phosphoprotein. We also show that the level of fenestrin in the amicronuclear T. pyriformis and the amicronuclear BI3840 strain of T. thermophila is several times lower than in micronuclear T. thermophila. [Copyright &y& Elsevier]

Abstract: Endosymbiosis in ciliates is a common and highly diverse phenomenon in nature, but its development at the mechanistic level and the origins are not easy to understand, since these associations may have arisen at any time during evolution. Therefore a laboratory model is helpful. It could be provided by the interaction of Tetrahymena pyriformis and Escherichia coli. Microscopic analyses with a genetically manipulated fluorescent strain of E. coli show single bacteria leaving food vacuoles and escaping digestion, an important prerequisite for further experiments. Under selective conditions, beneficial for T. pyriformis, the ciliate was shown to internalize E. coli cells. After feeding, bacteria, transformed with the plasmids pBS-neoTet or pNeo4, provide T. pyriformis with the ability to handle toxic conditions, caused by the aminoglykoside antibiotic paromomycin. Axenic cultures or cocultures with untransformed bacteria show lower cell numbers and survival rates compared to cocultures with transformed bacteria after transfer to paromomycin containing media. PCR detects bacterial DNA inside T. pyriformis cells. Additionally, microscopical analysis of selectively grown cocultures reveals fluorescing particles in the cytoplasm of T. pyriformis containing DNA and lipids, corresponding in size to E. coli. This system could be a reasonable model for understanding mechanisms of endosymbiosis establishment in ciliates. [Copyright &y& Elsevier]

Abstract: The importance of Ca2+-signaling for many subcellular processes is well established in higher eukaryotes, whereas information about protozoa is restricted. Recent genome analyses have stimulated such work also with Alveolates, such as ciliates (Paramecium, Tetrahymena) and their pathogenic close relatives, the Apicomplexa (Plasmodium, Toxoplasma). Here we compare Ca2+ signaling in the two closely related groups. Acidic Ca2+ stores have been characterized in detail in Apicomplexa, but hardly in ciliates. Two-pore channels engaged in Ca2+-release from acidic stores in higher eukaryotes have not been stingently characterized in either group. Both groups are endowed with plasma membrane- and endoplasmic reticulum-type Ca2+-ATPases (PMCA, SERCA), respectively. Only recently was it possible to identify in Paramecium a number of homologs of ryanodine and inositol 1,3,4-trisphosphate receptors (RyR, IP3R) and to localize them to widely different organelles participating in vesicle trafficking. For Apicomplexa, physiological experiments suggest the presence of related channels although their identity remains elusive. In Paramecium, IP3Rs are constitutively active in the contractile vacuole complex; RyR-related channels in alveolar sacs are activated during exocytosis stimulation, whereas in the parasites the homologous structure (inner membrane complex) may no longer function as a Ca2+ store. Scrutinized comparison of the two closely related protozoan phyla may stimulate further work and elucidate adaptation to parasitic life. See also “Conclusions” section. [Copyright &y& Elsevier]

This article presents research results of the adaptation of the bioinducative measuring method to evaluate the toxicity of textiles. In the experiment, Tetrahymena pyriformis as a bioindicator and formaldehyde, a well known toxic substance used in some finishing processes, were used as a sample chemical substance. To examine the usefulness of the bioindicative method in the safety evaluation of textiles, formaldehyde harmfulness within the range 1.5 -- 50 mg/L of the solution and extracts of textiles with an analytically determined content of formaldehyde were analysed in the presence of a bioindicator . Changes in cell development in formaldehyde solutions, extracts as well as in group control, were registered by directly measuring the optical density over time (0 - 24 h period), and microscopic observations and absorbance measurements were made in the presence of the alamarBlue cell viability reagent. Empirical data enabled to it logistic curves, on the basis of which the level of formaldehyde toxicity in dependence on time was estimated. The experiment performed showed a high toxicity of formaldehyde as well as the possibility of the application of the bioindicative measuring method based on ciliate to estimate very small amounts of formaldehyde in water solutions and textile extracts. [ABSTRACT FROM AUTHOR]

Aphidicolin (APH), an inhibitor of DNA polymerase α, arrested cell divisions in Tetrahymena thermophila. Surprisingly, low concentrations of APH induced an increase of macronuclear DNA content and cell size in non-dividing cells. In spite of the cell size increase, most proliferation of basal bodies, ciliogenesis and development of new oral primordia were prevented by the APH treatment. The division arrest induced by APH was partly overridden by caffeine (CAF) treatment, which caused the fragmentation (“pulverization”) of the chromosomes in G2 micronuclei. Somatic progeny of dividers with pulverized micronuclei (APH+CAF strains) contained aneuploid and amicronucleate cells. The amicronucleate cells, after losing their oral structures and most of their cilia, and undergoing progressive disorganization of cortical structures, assumed an irregular shape (“crinkled”) and were nonviable. “Crinkled” cells were not formed after APH + CAF treatment of the amicronuclear BI3840 strain, which contains some mic-specific sequences in its macronucleus. Most of the APH +CAF strains had a typical “*”- like conjugation phenotype: they did not produce pronuclei, but received them unilaterally from their mates and retained old macronuclei. However, 4 among 100 APH+CAF clones induced arrest at meiotic metaphase I in their wt mates. It is likely that the origin of such clones was enhanced by chromosome pulverization. [ABSTRACT FROM AUTHOR]

The article presents a study on the regulation of triiodothyronine production in the unicellular Tetrahymena. Tetrahymena content was measured and compared using immunocytochemical flow cytometry. The study found that the thyroid-stimulating hormone (TSH) regulation of thyroid hormone synthesis and troph-hormone overlap can be deduced to a unicellular level. Such finding may suggest that the endocrine mechanisms at a low level of phylogeny are preserved for the higher ranked organisms.

The article presents cases of nematodes and ciliate nasal infection in captive Archey's frogs. One frog of the four captive animals was treated successfully with oral moxidectin for the nematode infection and topical metronidazole for the protozoal infection. The clinical signs of hemorrhagic purulent nasal discharge and weight loss abated after both infections in frogs were cleared. The second frog died before it was treated while the third and fourth frogs were found dead.

Arsenic biomethylation and biovolatilization are thought to be two important metabolic pathways in aquatic and soil environments. Tetrahymena thermophila is a genus of free-living ciliated protozoan that is widely distributed in freshwater environments around the world. In this study, we studied arsenic accumulation, speciation, efflux, methylation and volatilization in this unicellular eukaryote exposed to various concentrations of arsenate. Our results show that T. thermophila accumulated 187 mg.kg−1 dry weight of arsenic when exposed to 40 μM for 48 h, with MMAs(V) (monomethylarsenate) and DMAs(V) (dimethylarsenate) as the dominant species, accounting for 66% of the total arsenic. Meanwhile, arsenate, arsenite, MMAs(V) and DMAs(V) were detected in the culture medium; the last three were released by the cells. The production of volatile arsenic increased with increasing external As(V) concentrations and exposure time. To our knowledge, this is the first study on arsenic metabolism, particularly biomethylation and biovolatilization, in protozoa. [Copyright &y& Elsevier]

POPs, plant organellar DNA polymerases, have been isolated from various photosynthetic eukaryotes. Previously, we purified the native POP of Cyanidioschyzon merolae (CmPOP) from whole cellular extracts and showed that CmPOP has DNA polymerase activity with a high processivity and a 3’–5’ exonuclease activity, and its expression is related to cell proliferation. In rice, the recombinant protein of POP has activities found in CmPOP, and high fidelity of POP has also been demonstrated. These facts suggest that POPs are involved in the replication of organellar genomes. POPs are also conserved in most non-opisthokont eukaryotes, which lack DNA polymerase γ (Polγ), a mitochondrial replication enzyme in opisthokonts (fungi and animals). The ciliate Tetrahymena thermophila contains a single gene for a putative POP (TetPOP). Immunoblot analysis demonstrated that TetPOP is localized in mitochondria, and TetPOP has been purified from mitochondria through a column chromatography series. Sensitivity to phosphonoacetate and dideoxyTTP was examined in POPs (TetPOP and CmPOP) or POP-containing organelles (chloroplasts of Arabidopsis ) and other polymerases (DNA polymerase I and mitochondria of rat liver, which contain Polγ), and the results suggest that high sensitivity to phosphonoacetate is unique to POPs in Family-A DNA polymerases. Finally, we propose a model for the succession of organellar DNA polymerases. [Copyright &y& Elsevier]

DNA barcoding using the mitochondrial cytochrome c oxidase subunit I (cox-1) gene has recently gained popularity as a tool for species identification of a variety of taxa. The primary objective of our research was to explore the efficacy of using cox-1 barcoding for species identification within the genus Tetrahymena . We first increased intraspecific sampling for Tetrahymena canadensis, Tetrahymena hegewischi, Tetrahymena pyriformis, Tetrahymena rostrata, Tetrahymena thermophila, and Tetrahymena tropicalis . Increased sampling efforts show that intraspecific sequence divergence is typically less than 1%, though it may be more in some species. The barcoding also showed that some strains might be misidentified or mislabeled. We also used cox-1 barcodes to provide species identifications for 51 unidentified environmental isolates, with a success rate of 98%. Thus, cox-1 barcoding is an invaluable tool for protistologists, especially when used in conjunction with morphological studies. [ABSTRACT FROM AUTHOR]

Abstract: Tetrahymena thermophila is a model organism for molecular and cellular biology. Previous studies from our group showed that Tetrahymena contains major components of the endocannabinoid system, such as various endocannabinoids and FAAH. In mammalian cells the endocannabinoid 2-arachidonoylglycerol is inactivated mainly by MAGL. In this study we showed that 2-arachidonoylglycerol and 2-oleoylglycerol are hydrolyzed by the combined actions of MAGL and FAAH. MAGL-like activity was examined in the presence of FAAH specific inhibitors, URB597 or AM374 and showed optimum pH of 8–9, apparent K M of 14.1μM and V max of 5.8nmol/min×mg. The enzyme was present in membrane bound and cytosolic isoforms; molecular mass was determined at ∼45 and ∼40kDa. MAGL and FAAH could also inactivate endogenous signaling lipids, which might play an important role in Tetrahymena as suggested in mammals. Tetrahymena could be used as a model system for testing drugs targeting enzymes of the endocannabinoid system. [ABSTRACT FROM AUTHOR]

The endocannabinoid system is a lipid signaling system in mammalian cells. We reported that major components of the endocannabinoid system such as Fatty Acid Amide Hydrolase and Monoacylglycerol Lipase, are present in the protist Tetrahymena, with characteristics similar to those in mammals. Tetrahymena is a model organism for molecular and cellular biology studies as its genome sequence is available. Here we report the presence of N-acylethanolamines (AcEs) and their respective 2-acylglycerols (2-AcGs) in Tetrahymena thermophila for the first time; the former is a new lipid class for the protist. Using LC-MS/MS we identified, N y-linolenoyl, N-eicosenoyl, N-linoleoyl, N-palmitoyl, N-stearoyl and N-oleoylethanolamines as well as the corresponding monoacylglycerols. The levels of 2-acylglycerols were much higher than the corresponding N-acylethanolamines, as reported for mammals. To our knowledge, N-γ-linolenoylethanolamine (GLEA) was found for the first time in nature. Anandamide and 2-AG were present in trace amounts. These results demonstrate the existence of a new lipid class in Tetrahymena, strengthen the conviction that the endocannabinoid system is present in this protist, verifying its importance throughout evolution. Tetrahymena could be used as a model for metabolic studies on the endocannabinoids, as well as for the study of drugs targeted towards biosynthetic and catabolic enzymes of AcEs and 2-AcGs. [Copyright &y& Elsevier]

The effect of road salt on the eating of bacteria or bacterivory by the ciliate, Tetrahymena thermophila, was followed in non-nutrient Osterhout's solution with Escherichia coli expressing green fluorescent protein. Bacterivory was impaired at between 0.025 and 0.050% w/v but the ciliates appeared to have normal morphologies and motilities, whereas at above 0.1%, bacterivory was blocked and many ciliates died. By contrast, E. coli remained viable, suggesting salt could alter predator–prey relationships in microbial communities. In nutrient medium, salt was not toxic and the ciliates grew. After growth in salt, ciliates consumed bacteria in 0.2% salt, indicating the salt acclimation of bacterivory. Bacteria and ciliates were added to urban creek samples to compare their capacity to support exogenous bacterivory. Even though samples were collected weekly for a year and be expected to have fluctuating salt levels as a result of deicing, all creek samples supported a similar level of bacterivory. [Copyright &y& Elsevier]

Metallothioneins are ubiquitous small, cysteine-rich, metal-binding proteins that play important roles in intracellular metal homeostasis and detoxification. Very few data are available on the promoter region and the mechanism of metallothionein transcription in Protozoa. In this study, we focused on Tetrahymena thermophila MTT5 5’-flanking region. To define the sequence elements underlying the metal-responsiveness of this promoter, we constructed a series of deletions and mutations starting with a 1777bp fragment immediately upstream of the start codon of MTT5. As a reporter gene we used the previously tested IAG52B surface antigen from the protozoan fish parasite Ichthyophthirius multifiliis. The results suggest that a region spanning between –300bp and –274bp, dubbed Tetrahymena thermophila Cadmium-Response-Element (TtCdRE), is necessary to elicit high-level expression of the transgene following induction with cadmium. This is the first demonstration by in vivo analyses of a regulatory element essential for Cd-mediated control of protozoan metallothionein gene expression, where the sequence GATA appears to be involved. [Copyright &y& Elsevier]

The acute toxicity of CuO and ZnO nanoparticles in artificial freshwater (AFW) and in natural waters to crustaceans Daphnia magna and Thamnocephalus platyurus and protozoan Tetrahymena thermophila was compared. The L(E)C50 values of nanoCuO for both crustaceans in natural water ranged from 90 to 224 mg Cu/l and were about 10-fold lower than L(E)C50 values of bulk CuO. In all test media, the L(E)C50 values for both bulk and nanoZnO (1.1–16 mg Zn/l) were considerably lower than those of nanoCuO. The natural waters remarkably (up to 140-fold) decreased the toxicity of nanoCuO (but not that of nanoZnO) to crustaceans depending mainly on the concentration of dissolved organic carbon (DOC). The toxicity of both nanoCuO and nanoZnO was mostly due to the solubilised ions as determined by specific metal-sensing bacteria. [Copyright &y& Elsevier]

Although the ciliate Tetrahymena is a good model for the study of chemotaxis, its profound motility makes it difficult to monitor intracellular calcium (Ca2+) changes induced by chemotactic stimuli. In this study, we report a microfluidic-based chemotaxis system generating directional chemotactic gradients under highly viscous conditions, suppressing T. pyriformis motility, and allowing for the stable confocal imaging of changes in intracellular Ca2+ in the ciliate. Once the viscous condition was achieved, directional chemical gradients were formed inside the center chamber via the release of N-methyl-d-aspartate (NMDA), a known chemoattractant, from the surrounding chemical reservoirs into the center chamber. As a result, intracellular Ca2+ in the ciliate increased up to three-fold, and its distribution was skewed in the direction of NMDA stimulation. However, the Ca2+ in ciliates pretreated with phospholipase C (PLC) or phosphatidylinositol-3-kinase (PI3K) blockers did not increase even after stimulation. Additionally, the PI3K blocker induced the secretion of granules, the size of which was dependent on the concentration of the blocker. Collectively, the results indicate that both PLC and PI3K perform pivotal roles in controlling the levels of intracellular Ca2+ in T. pyriformis during chemotaxis. [Copyright &y& Elsevier]

Tetrahymena vorax is a polymorphic ciliate capable of forming two cell types that depend on environmental cues. One type, called the microstomal form, feeds on bacteria and other small particulate matter. The other, the macrostomal cell, is a carnivore and feeds on small ciliates or flagellates. Large numbers of synchronously differentiating macrostomal cells can be induced by suspending microstomal cells in stomatin, an exudate produced by a potential ciliate prey, Tetrahymena pyriformis. The active principle in stomatin has been identified as a ferrous iron chelate of hypoxanthine and uracil (Smith-Somerville et al., 2000). They produced a synthetically derived chelate with strong biological activity equivalent to that of stomatin. The purpose of our study is to use our chemicals and our strain of Tetrahymena vorax, and following the methods of Acevedo-Chavez et al., 1996 as modified by Smith-Somerville et al., 2000 to synthesize an active chelate. Our results show that while lower levels of chelate (1, 5, and 10 ug/ml) had little or no effect on levels of differentiation, chelate concentrations in the range of 100 ug/ml induced macrostomal differentiation equivalent to 78 % of the stomatin control value. Our results confirm findings published by Smith-Somerville et al. 2000. [ABSTRACT FROM AUTHOR]

The aim of this study was to search for a mechanism responsible for the acquisition of cell polarity in a ciliate Tetrahymena. Homologs of the mammalian genes coding for CDC42-GSK3Β- MARK/PAR1-MAPs proteins were found in the Tetrahymena genome (Eisen et al., 2006, and this study). These proteins belong to a pathway which controls assembly and disassembly of microtubule bundles and cell polarity in neural cells. In Tetrahymena, there are two types of morphogenesis: divisional and oral replacement (OR). In divisional morphogenesis, an elongation of longitudinal microtubule bundles (LMs) takes place during cell division. In contrast, in OR type morphogenesis, which occurs in starved non-dividing cells, a polar retraction of LMs occurs. In T. pyriformis, the frequency of developmental switch to OR morphogenesis increases in the presence of wortmannin, an inhibitor of the CDC42-GSK3Β-MARK pathway. In contrast, wortmannin when applied to dividing cells does not affect divisional morphogenesis. Using immunostaining with the antibody against mammalian mitotic phosphoproteins (MPM-2) we show that these proteins co-localize with the LMs and are distributed along the anterior-posterior gradient. In addition, we show that during OR type morphogenesis, the fate of LMs correlates with the anterior-posterior gradient of instability of the cortical structures. We used the conditional mouthless mutant of T. thermophila (Tiedtke et al., 1988) to test if the presence of the oral apparatus is required for the maintenance of cell polarity. We discuss our results in relation to the hypothesis of GSK3-Β-MARK pathway involvement in the acquisition of cell polarity in Tetrahymena. [ABSTRACT FROM AUTHOR]

Because the range of biological mechanisms responsible for the inactivation of viruses in man-made and natural water systems is poorly understood, the involvement of the free-living ciliated protozoan, Tetrahymena thermophila, in viral inactivation was investigated. The ciliate was found to remove the bacteriophage MS2 when the phage and ciliate were co-incubated in a simple salt solution. MS2 was enumerated as plaque forming units (pfus). MS2 removal was achieved only by living and not formalin-fixed ciliates, and was blocked by treatments that impaired the formation of food vacuoles. These treatments were cytochalasin B and low temperature. When fluorescently labelled with SYBR gold prior to co-incubation, MS2 were seen inside Tetrahymena within vesicles that had the shape and size of food vacuoles. The number of pills associated with Tetrahymena was low. This suggests that the engulfment of the phage into food vacuoles led to the inactivation of MS2, which is frequently used as a surrogate for poliovirus in environmental microbiology. In the future, a broader understanding of the capacity of ciliates to inactivate viruses could lead to methods for improving water quality through the manipulation of ciliate populations and activities. [ABSTRACT FROM AUTHOR]

The article describes the purification, properties and role of high-molecular-weight polyphenols, which were referred to as mitochondrial activation factors (MAF), from oolong and black teas. Ciliated protozoan Tetrachymena and rhodamine 123 measured the mitochondrial membrane potential of polyphenols. Delphinidin and cyanidin were detected when the MAFs were hydrolyzed with HCl-BuOH. The study suggests that MAFs are heterogenous polymers of flavan-3-ols and flavan-3-ol gallates.

Abstract: Although sulfide is typically regarded as toxic to eukaryotic cells, it is avidly consumed by Tetrahymena pyriformis. That was observed only when the sulfide concentration was kept below 1μM. Previously concentrations that were too high had been tested. A new device (Sulfidostat) was used to measure sulfide consumption in steady-state concentrations as low as 10−12 M. The technique was validated non-biologically by slowly injecting AgNO3 into buffer and using Ag2S precipitation to mimic sulfide consumption, confirming that rates of sulfide consumption could be measured independently of sulfide concentrations. With T. pyriformis, sulfide consumption was 0.25μmol (gprotein)−1 s−1 in 0.5μM sulfide. Sulfide consumption required O2 and was inhibited by HCN or by too much sulfide. When cells were separated into fractions, sulfide consumption occurred in the particulate (mitochondrial) fraction. Unexpectedly, the soluble cytosolic fraction slowly produced sulfide even when aerated. The observations are consistent with the conjecture that mitochondria evolved from sulfidotrophic symbionts in a sulfidogenic host cell. [Copyright &y& Elsevier]

Bacterivory by dilates in various water ecosystems, both natural and artificial, plays a significant role on the microbial population composition and consequently affects water quality. A convenient, rapid and inexpensive methodology to evaluate the capacity of the ciliate protozoan Tetrahymena therm ophila for bacterivory was developed utilizing fluorescent protein expressing bacteria (FPEB) in a microtitre plate fluorimeter. Bacterivory was correlated with a loss in fluorescence measured in the fluorimeter and confirmed by fluorescence microscopy showing that the FPEB were engulfed during the assay and subsequently lost their fluorescence, whereas Cytochalasin B, a known inhibitor of phagocytosis, prevented a decrease in relative fluorescence units (RFUs). The ciliate bacterivory (CB) assay has a great dynamic range allowing the assay to be performed with a variety of predator:prey concentrations. A model toxicant, CuCl2, known to have a toxicological impact on protozoa and often present in different types of wastewater, resulted in measurable decreases in bacterivory. As well, starvation of Tetrahymena for 24 h resulted in reduced bacterivory. In the future, the CB assay could be developed for water monitoring purposes to rapidly assess water samples for the capacity to support bacterivory as an indicator of ecosystem health. [ABSTRACT FROM AUTHOR]

Three viability assays using fluorescent dyes effectively detected a loss of viability in cultures of three mammalian cell lines (H4IIE, Caco2, and HepG-2), two fish cell lines (RTgill-W1 and RTL-W1), and a ciliated protozoan, Tetrahymena thermophila, after exposure to Triton X-100, used as a model toxicant. The dyes were Alamar Blue (AB), neutral red (NR), and propidium iodide, which respectively monitored energy metabolism, lysosomal activity, and membrane integrity. A fourth fluorescent dye, 5-carboxyfluorescein diacetate acetoxymethyl ester, was problematic. For 2-h Triton X-100 exposures, mammalian cell lines were as susceptible as piscine cell lines, whereas T. thermophila was approximately twofold less sensitive as detected with AB and NR. Despite being less sensitive, cytotoxicity tests on T. thermophila could be done in spring water, which means that unlike animal cells they could be directly exposed to most industrial effluents without osmolality adjustments. Therefore, T. thermophila could be a useful complement to animal cells as alternatives to fish in toxicity testing. [Copyright &y& Elsevier]

The sensitivity of protozoa, and particularly ciliated protozoa, to environmental changes suggested a study on the physiological responses arising from exposure to toxic compounds. Tetrahymena pyriformis was used as a test organism in a set of miniaturized assays. The physiological response of this ciliate was assessed in terms of adenosine-5′-triphosphate content and acid phosphatase activity after exposure of the cultures of T. pyriformis to four toxicants: copper, zinc, Triton X-100, and cycloheximide. In the range of concentrations used, stimulation and inhibition of these two parameters were observed. The correlation between the two parameters is analyzed. [Copyright &y& Elsevier]

Fluorescence in situ hybridization (FISH) was applied to detect ingested natural bacteria within the food vacuoles of ciliates harvested from the natural sediment. In addition to this important qualitative aspect, FISH was also successfully used to measure the bacterivory of a culture of the ciliate Tetrahymena pyriformis on natural field sediment bacteria. In this feeding experiment, we compared the FISH technique with the only available alternative technique using fluorescently stained sediment (FS-sediment). The ingestion rate of unstained sediment bacteria determined by FISH was 4.6 bacteria per ciliate and hour. In contrast, Tetrahymena pyriformis cells that fed on bacteria from FS-sediment ingested 12.7 bacteria per ciliate and hour. Bacterial abundances in the sediment were equal in both sediment types (4 × 108 cells g sediment dry weight-1) when determined by DAPI counts. However, when analyzed using DTAF-counts, the number of bacteria in the FS-sediment increased to 9.7 × 108 cells g sediment dry weight-1. From our findings we conclude that bacterivory by ciliates is overestimated when FS-sediment is used because DTAF stains bacteria as well as protein-containing detritus particles, which are also ingested by many ciliates. In contrast, FISH is a direct, a posteriori method that specifically stains phylogenetic lineages, e.g. eubacteria, after ingestion and thereby avoids a false determination of the number of ingested bacteria. Thus this method can also be used for the study of natural ciliate bacterivory in benthic systems. [ABSTRACT FROM AUTHOR]

Lanthanum (La3+), a Ca-channel blocker, affected proliferation of Tetrahymena concentration-dependently in 2% proteose-peptone (PP) medium. The effect increased with decreasing amount of organic matter (2-0.5% PP). Cell motility was affected slightly in low La3+ concentrations but in high concentrations the cells became motionless with rocking movements; however, most cells regained motility after some hours (restored Ca-homeostasis). Addition of La3+ to the PP media caused formation of a fluffy precipitate which was engulfed by the cells. The capacity to form food vacuoles, was normal in proliferating cells but decreased, or ceased, in growth-inhibited cells. Small refractive granules appeared in all treated cells and most abundantly in growth-inhibited cells. The fine structure of treated cells related to whether they were capable, or not, of proliferation. Electron-dense material (La3+) was abundant in food vacuoles, seen on the surface of cells early after exposure to high concentrations, and present in the dense (small refractive) granules. Hence, La3+ apparently enters the cells. [ABSTRACT FROM AUTHOR]

Concanavalin A (Con A) is a plant lectin that inhibits the persistent swimming behaviour of Tetrahymena cells by increasing ciliary reversals. Con A also increases ciliary reversals following chemosensory adaptation when it acts as a chemoattractant. The Con A effect is inhibited by mannose suggesting that Con A binds to glycoproteins in cellular membranes. In order to isolate these glycoproteins membrane proteins were fractionated by Con A-Sepharose affinity chromatography. A ciliary membrane protein denoted p66 was purified by two-step affinity chromatography from a whole cell Triton X-100 extract of Tetrahymena thermophila. [ABSTRACT FROM AUTHOR]

Ciliary motility in ciliates such as Paramecium or Tetrahymena is the mechanism by which cellular behaviour is regulated and on which survival depends. As a consequence, protozoan cilia have elaborate sensing systems built into them, probably by intraciliary transport (ICT-orthologous to intraflagellar transport). As a response to stimuli, 2nd messenger concentration around the axoneme increases. A unifying hypothesis suggests that for rapid responses the 2nd messengers act on axonemal dynein to change the rates of doublet sliding, which in turn affects ciliary parameters that control behaviour. [ABSTRACT FROM AUTHOR]

The structure of four new MT (metallothionein) genes of Tetrahymena thermophila and T. pigmentosa were characterized. The MT-2 genes from the two species are very similar, differing by 10 out of 2259 sequenced nucleotides, and the deduced amino acid sequences are identical. The MT-1 genes from T. pigmentosa and T. thermophila are also very similar, differing only by 3 nucleotides in the 5-UT region. The promoter regions contain a TATA box and many stretches partially matching some regulatory elements such as metal-responsive (MREs), antioxidant-responsive (AREs), a CAAT box, a Gbox, and AP1 and ACE-1 binding sites. The related coding and amino acid sequences were compared with those previously sequenced in Tetrahymena. This analysis revealed two independent events of duplication occurring in Cd- (MT-1 and MTT1) and Cu- (MT-2) induced MTs. This evolutionary pathway also explains the unusual length of these proteins, which are much longer than many MTs studied so far. Additionally, the orthology and paralogy relationships of the various MTs are presented. Finally, on the basis of phylogenetic analyses of Tetrahymena MTs, two evolutionary hypotheses are proposed. [ABSTRACT FROM AUTHOR]

Summary: Tetrahymena thermophila cells have two types of polarized morphogenesis: divisional morphogenesis and oral reorganization (OR). The aim of this research is the analysis of cortical patterns of immunostaining during cell division and in OR using previously characterized antibodies against fenestrin and epiplasm B proteins. During cell division, the anarchic field of basal body proliferation of the new developing oral apparatus (AF) showed concomitant strong binding of the fenestrin antigen and withdrawal of a signal of the epiplasm B antigen. At a specific stage, the fenestrin antigen also appeared as a character of the anterior cortex pole, with a co-localized decrease in the detected epiplasm B antigen. The fenestrin antigen also showed a polarity of duplicating basal bodies in ciliary rows. Indirect immunofluorescence and immunogold labeling experiments were performed in the absence and presence of an inhibitor of activity of serine/threonine kinases, 6-dimethylaminopurine (6-DMAP) as an inducer of the oral replacement process. In the presence of 6-DMAP, one class of cells started OR, and some others were trapped and affected in cell division. Both types of cells showed an instability of oral structures and formed enlarged primordial oral fields. These anarchic fields (AFs) bind the fenestrin antigen, with disappearance of epiplasmic antigen staining. Only one protein (about 64 kDa) is detected in western blots by the anti-fenestrin antibody and it accumulated in 6-DMAP-treated cells that are involved in uncompleted morphogenetic activity. At a defined stage of oral development, both during cell division and in OR, the fenestrin antigen served as a marker of polarity of the cell of the anterior pole character. [Copyright &y& Elsevier]

The most successful quantitative structure–activity relationships have been developed by separating compounds by their mechanisms of toxic action (MOAs). However, to correctly determine the MOA of a compound is often not easy. We investigated the usefulness of discriminant analysis and logistic regression in determining MOAs. The discriminating variables used were the logarithm of octanol–water partition coefficients (log Kow) and the experimental toxicity data obtained from Pimephales promelas and Tetrahymena pyriformis assays. Small total error rates were obtained when separating nonpolar narcotic compounds from other compounds, however, relatively high total error rates were obtained when separating less reactive compounds (polar, ester, and amine narcotics) from more reactive compounds (electrophiles, proelectrophiles, and nucleophiles). [Copyright &y& Elsevier]

Summary: We have employed monoclonal antibodies to reinvestigate the janus mutants of the ciliate Tetrahymena thermophila, which cause reversal of circumferential polarity on the dorsal surface of the cell. This reversal brings about frequent ectopic expression of ventral cortical landmarks, such as a “secondary” oral apparatus, on the dorsal surface. The principal antibody employed, FXXXIX-12G9, immunolabels both transient cortical structures not directly associated with basal bodies (the fission line and the postoral meridional filament) and more permanent structures (apical band and oral crescent) that are associated with basal bodies. 12G9-immunolabeling of janus cells has revealed additional phenotypes, including disorder of ciliary rows. Further, this labeling has shown that the postoral meridional filament is often expressed and the apical band is frequently interrupted on the mid-dorsal surface of janus cells irrespective of whether or not these cells express a “secondary” oral apparatus. Of the permanent structures revealed by 12G9 immunofluorescence, modifications of the oral crescent (OC) are associated with prior modifications in the development of basal body-containing structures in the secondary oral apparatus. The formation of the apical band (AB) is also commonly abnormal in janus cells; analysis of specific abnormalities shows that the AB depends both on its initiation at a specific site near the anterior basal body of apical basal body couplets and on the normal location of these couplets just posterior to the fission line. We also have uncovered an intriguing difference in the reactivity of apical-band filaments to the 12G9 antibody in the two non-allelic janus mutants (janA1 and janC2)that we have investigated. Taken together, our observations indicate that the formation of new cellular structures at division depends both upon pre-existing cytoskeletal structures and upon the positional information provided by large-scale cellular polarities. [Copyright &y& Elsevier]

Summary: The patterns of expression of two metallothionein (MT) genes, MT-1 and MT-2, previously identified as Cd-MT and Cu-MT, were analysed in Tetrahymena pigmentosa in response to metal inducers cadmium, copper and zinc and to a mixture of copper and cadmium at appropriate concentrations. Co-treatment induces synergistic accumulation of both metals and higher expression of MT-mRNAs in the first few hours. mRNA levels were observed not to completely correlate with MT-protein levels, suggesting that post-transcriptional regulation may be involved in MT induction. MT-1 is induced to higher levels than MT-2. Zinc does not induce any MT expression. The lowest level of mRNA was observed for MT-2, induced only by copper. Cadmium is a powerful inducer of the MT-1 gene, although a very low transcription rate by copper occurs in the first hour. [Copyright &y& Elsevier]

In aquatic ecosystems, the calcium (Ca) concentration varies greatly. It is well known that Ca affects the aggregation of nanoparticles (NPs) and thus their bioaccumulation. Nevertheless, Ca also plays critical roles in various biological processes, whose effects on NP accumulation in aquatic organisms remain unclear. In this study, the effects of Ca on the uptake of polyacrylate-coated hematite NPs (HemNPs) by the aquatic ciliate Tetrahymena thermophila were investigated. At all of the tested Ca concentrations, HemNPs were well dispersed in the experimental medium, excluding the possibility of Ca to influence HemNP bioaccumulation by aggregating the NPs. Instead, Ca was shown to induce the clathrin-mediated endocytosis and phagocytosis of HemNPs. Manipulation of the Ca speciation in the experimental medium as well as the influx and intracellular availability of Ca in T. thermophila indicated that HemNP uptake was regulated by the intracellular Ca level. The results of the proteomics analyses further showed that the binding of intracellular Ca to calmodulin altered the activity of proteins involved in clathrin-mediated endocytosis (calcineurin and dynamin) and phagocytosis (actin). Overall, the biologically inductive effects of Ca on NP accumulation in aquatic organisms should be considered when evaluating the environmental risks of NPs. [Display omitted] • Ca induced the clathrin-mediated endocytosis and phagocytosis of nanoparticles. •Nanoparticle uptake was mainly regulated by the intracellular Ca level. •Intracellular Ca regulated nanoparticle uptake through binding to calmodulin. •Calmodulin altered the activity of various proteins involved in endocytosis. [ABSTRACT FROM AUTHOR]

• T. thermophila can overcome sublethal CDNB dose cell survival strategies. • LD50 value of CDNB is determined as 0.079 mM for T. thermophila. • TtGSTmu19 and TtGSTmu34 were linked with the survival under CDNB exposure. • TtGSTmu19 and TtGSTmu34 mRNA levels increase upon CDNB exposure. • TtGSTmu19 and TtGSTmu34 are highly responsive to CDNB. Industrial xenobiotic pollutants have toxic effects on diverse organisms in their natural environments. This study aims to identify the Glutathione-S-transferases (GST) from Tetrahymena thermophila that are highly responsive to the treatment of synthetic substrate 1-chloro-2,4-dinitrobenzene (CDNB). The LD50 value of CDNB was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test as 0.079 mM at 9 h exposure. The glutathione affinity-purified 22 kDa and 23 kDa GSTs from CDNB-treated cells were identified as GSTm19 and GSTm34 with 2D-gel electrophoresis coupled MALDI-Tof MS/MS analysis. The specific activitiy of the affinity-purified GSTs was upregulated upon the treatment of 0.072 mM CDNB with the decreased cell survival. GSTm19 and GSTm34 had also upregulated the mRNA expression under the highest dose treatment. The high cell survival and elevated total GST enzyme activity at 9 h under CDNB doses could be the result of both transcriptional upregulations as well as post-translational modifications. As a result, the cell survival of Tetrahymena thermophila was significantly affected by CDNB exposure in a concentration-dependent manner with the effect of low-dose stimulation and high-dose inhibition. [ABSTRACT FROM AUTHOR]

The ciliated protist Tetrahymena thermophila is a well-known model organism with typical nuclear dimorphism containing a somatic macronucleus (MAC) and a germline micronucleus (MIC). The presence in the same cell compartment of two nuclei with distinctly different structural and functional properties provides an ideal model system to explore mechanisms of genome maintenance. Although methods for the isolation of MIC have been available for many years, cross-contamination and DNA degradation remain unresolved. Here, we describe a reliable and quick method to isolate MIC with high purity and DNA integrity in T. thermophila. Different factors are examined to optimize the MIC purification. The MAC contamination ratio in purified MIC is about 0.19% and DNA integrity of purified MIC is maintained. We also establish a more accurate method to detect the contamination rate of nuclei including microscopic observation and PCR detection. This study will facilitate further epigenetic research in Tetrahymena. [ABSTRACT FROM AUTHOR]