Climacostol inhibits <Emphasis Type="Italic">Tetrahymena</Emphasis> motility and mitochondrial respiration

Climacostol is a resorcinol derivative that is produced by the ciliate Climacostomum virens. Exposure to purified climacostol results in lethal damage to the predatory ciliate Dileptus margaritifer and several other ciliates. To elucidate the mechanism of climacostol toxic action, we have investigated the effects of this compound on the swimming behavior of Tetrahymena thermophila and the respiratory system of isolated rat liver mitochondria. When added to living T. thermophila cells, climacostol markedly increased the turning frequency that was accompanied by a decrease in swimming velocity and subsequently by cell death. Observations by DIC and fluorescence microscopy showed morphological alterations in climacostol treated T. thermophila, indicating that climacostol might exert cytotoxic action on this organism. In the experiment with isolated rat liver mitochondria, climacostol was found to inhibit the NAD-linked respiration, but had no apparent effect on succinate-linked respiration. This finding indicates that climacostol specifically inhibits respiratory chain complex I in mitochondria. The combination of results suggest that the inhibition of mitochondrial respiration may be the cytotoxic mechanism of climacostol’s defenses against predatory protozoa.     

Bioenergetic Investigation of the Effects of La(III) and Ca(II) on the Metabolic Activity of Tetrahymena thermophila BF5

The heat flux of Tetrahymena thermophila BF5 during growth and the effects of La³⁺ and Ca²⁺ on them were investigated with microcalorimetry; simultaneously, morphological changes of T. thermophila were obtained by light microscope. La³⁺ in low concentration (0–5.0 × 10^–4 mol/l) remarkably stimulated T. thermophila metabolism, but high dose of La³⁺ (5.8–8.6 × 10^–4 mol/l) restrained it in a linear manner with IC₅₀ being 7.2 × 10^–4 mol/l. In contrast, low concentration of Ca²⁺ did not manifest obvious stimulation on T. thermophila metabolism; moreover, the IC₅₀ of Ca²⁺ was much higher than that of La³⁺. Low concentration of La³⁺ did not lead to changes in appearance of T. thermophila, but low dose of Ca²⁺ clearly promoted the cell proliferation. In addition, the morphological changes of T. thermophila evoked by high concentrations of La³⁺ and Ca²⁺ were consistent with relevant microcalorimetric results. It is concluded that La and Ca influence T. thermophila via different pathways, and La represents toxic action rather than Ca analogy.     

Expression,secretion and surface display of a human alkaline phosphatase by the ciliate <Emphasis Type="Italic">Tetrahymena thermophila</Emphasis>

Background  

Tetrahymena thermophila possesses many attributes that render it an attractive host for the expression of recombinant proteins. Surface proteins from the parasites Ichthyophthirius multifiliis and Plasmodium falciparum and avian influenza virus antigen H5N1 were displayed on the cell membrane of this ciliate. Furthermore, it has been demonstrated that T. thermophila is also able to produce a functional human DNase I. The present study investigates the heterologous expression of the functional human intestinal alkaline phosphatase (hiAP) using T. thermophila and thereby presents a powerful tool for the optimization of the ciliate-based expression system.     

Effects of butyltins and inorganic tin on chemotaxis of aquatic bacteria

Summary Tributyltin (TBT) and its degradation products dibutyltin (DBT), monobutyltin (MBT) and Sn^IV were toxic toPseudomonas fluorescens SHC-6 andSerratia sp. Gil-1 with EC₅₀ values in the range of 10^–3 to 10^–4M. These four compounds were negative chemotactic agents forP. fluorescens, and the butyltins were negatively chemotactic forSerratia sp. at concentrations over four orders of magnitude lower than the EC₅₀ values.l-Aspartate was a positive chemotactic agent for both organisms. TBT, DBT and MBT negated the effect ofl-aspartate onP. fluorescens but not onSerratia sp. Thus, TBT has the potential to affect microbial populations at concentrations much lower than those which prevent growth, and degradation of TBT does not always detoxify it. SnCl₄ was less toxic than TBT or DBT to these organisms and it was not chemotactic forSerratia sp. Gil-1. Tributylamine and tributylphosphate were less than 1/10th as toxic as TBT and they did not have a chemotactic effect on either organism at concentrations at which TBT had a significant effect. Therefore, both the Sn-and butyl-moieties contribute to the toxic and chemotactic properties of TBT.     

Identification of lncRNA‐associated differential subnetworks in oesophageal squamous cell carcinoma by differential co‐expression analysis

Differential expression analysis has led to the identification of important biomarkers in oesophageal squamous cell carcinoma (ESCC). Despite enormous contributions, it has not harnessed the full potential of gene expression data, such as interactions among genes. Differential co‐expression analysis has emerged as an effective tool that complements differential expression analysis to provide better insight of dysregulated mechanisms and indicate key driver genes. Here, we analysed the differential co‐expression of lncRNAs and protein‐coding genes (PCGs) between normal oesophageal tissue and ESCC tissues, and constructed a lncRNA‐PCG differential co‐expression network (DCN). DCN was characterized as a scale‐free, small‐world network with modular organization. Focusing on lncRNAs, a total of 107 differential lncRNA‐PCG subnetworks were identified from the DCN by integrating both differential expression and differential co‐expression. These differential subnetworks provide a valuable source for revealing lncRNA functions and the associated dysfunctional regulatory networks in ESCC. Their consistent discrimination suggests that they may have important roles in ESCC and could serve as robust subnetwork biomarkers. In addition, two tumour suppressor genes (AL121899.1 and ELMO2), identified in the core modules, were validated by functional experiments. The proposed method can be easily used to investigate differential subnetworks of other molecules in other cancers.     

Characterization and evolutionary analysis of tributyltin‐binding protein and pufferfish saxitoxin and tetrodotoxin‐binding protein genes in toxic and nontoxic pufferfishes

Understanding the evolutionary mechanisms of toxin accumulation in pufferfishes has been long‐standing problem in toxicology and evolutionary biology. Pufferfish saxitoxin and tetrodotoxin‐binding protein (PSTBP) is involved in the transport and accumulation of tetrodotoxin and is one of the most intriguing proteins related to the toxicity of pufferfishes. PSTBPs are fusion proteins consisting of two tandem repeated tributyltin‐binding protein type 2 (TBT‐bp2) domains. In this study, we examined the evolutionary dynamics of TBT‐bp2 and PSTBP genes to understand the evolution of toxin accumulation in pufferfishes. Database searches and/or PCR‐based cDNA cloning in nine pufferfish species (6 toxic and 3 nontoxic) revealed that all species possessed one or more TBT‐bp2 genes, but PSTBP genes were found only in 5 toxic species belonging to genus Takifugu. These toxic Takifugu species possessed two or three copies of PSTBP genes. Phylogenetic analysis of TBT‐bp2 and PSTBP genes suggested that PSTBPs evolved in the common ancestor of Takifugu species by repeated duplications and fusions of TBT‐bp2 genes. In addition, a detailed comparison of Takifugu TBT‐bp2 and PSTBP gene sequences detected a signature of positive selection under the pressure of gene conversion. The complicated evolutionary dynamics of TBT‐bp2 and PSTBP genes may reflect the diversity of toxicity in pufferfishes.     

Effects of Artemisinin Derivative on the Growth Metabolism of <Emphasis Type="Italic">Tetrahymena thermophila</Emphasis> BF5 Based on Expression of Thermokinetics

The toxic effects of artesunate and dihydroartemisinin on the growth metabolism of Tetrahymena thermophila BF5 were studied by microcalorimetry. The results showed that: (1) low concentrations of artesunate (≤1 mg L⁻¹) and dihydroartemisinin (≤ 2 mg L⁻¹) promoted the growth metabolism of T. thermophila BF5, whereas high concentrations of artesunate (1–60 mg L⁻¹) and dihydroartemisinin (2–60 mg L⁻¹) inhibited its growth; (2) the half inhibition concentrations IC₅₀ of artesunate and dihydroartemisinin were 17.5817 and 9.5089 mg L⁻¹, respectively. It was concluded that the inhibition of dihydroartemisinin was stronger than that of artesunate.     

Some but not All Tetrahymena Species Destroy Monolayer Cultures of Cells from a Wide Range of Tissues and Species

The activities of Tetrahymena corlissi, Tetrahymena thermophila, and Tetrahymena canadensis were studied in coculture with cell lines of insects, fish, amphibians, and mammals. These ciliates remained viable regardless of the animal cell line partner. All three species could engulf animal cells in suspension. However, if the animal cells were monolayer cultures, the monolayers were obliterated by T. corlissi and T. thermophila. Both fibroblast and epithelial monolayers were destroyed but the destruction of human cell monolayers was done more effectively by T. thermophila. By contrast, T. canadensis was unable to destroy any monolayer. At 4 °C T. thermophila and T. corlissi did not carryout phagocytosis and did not destroy monolayers, whereas T. canadensis was able to carryout phagocytosis but still could not destroy monolayers. Therefore, monolayer destruction appeared to require phagocytosis, but by itself this was insufficient. In addition, the ciliates expressed a unique swimming behavior. Tetrahymena corlissi and T. thermophila swam vigorously and repeatedly into the monolayer, which seemed to loosen or dislodge cells, whereas T. canadensis swam above the monolayer. Therefore, differences in swimming behavior might explain why T. corlissi has been reported to be a pathogen but T. canadensis has not.     

The unique N‐terminal insert in the ribosomal protein,phosphoprotein P0, of Tetrahymena thermophila: Bioinformatic evidence for an interaction with 26S rRNA

Phosphoprotein P0 (P0) is part of the stalk complex of the eukaryotic large ribosomal subunit necessary for recruiting elongation factors. While the P0 sequence is highly conserved, our group noted a 15–16 residue insert exclusive to the P0s of ciliated protists, including Tetrahymena thermophila. We hypothesized that this insert may have a function unique in ciliated protists, such as stalk regulation via phosphorylation of the insert. Almost no mention of this insert exists in the literature, and although the T. thermophila ribosome has been crystallized, there is limited structural data for Tetrahymena's P0 (TtP0) and its insert. To investigate the structure and function of the TtP0 insert, we performed in silico analyses. The TtP0 sequence was scanned with phosphorylation site prediction tools to detect the likelihood of phosphorylation in the insert. TtP0's sequence was also used to produce a homology model of the N‐terminal domain of TtP0, including the insert. When the insert was modeled in the context of the 26S rRNA, it associated with a region identified as expansion segment 7B (ES7B), suggesting a potential functional interaction between ES7B and the insert in T. thermophila. We were not able to obtain sufficient data to determine whether a similar relationship exists in other ciliated protists. This study lays the groundwork for future experimental studies to verify the presence of TtP0 insert/ES7 interactions in Tetrahymena, and to explore their functional significance during protein synthesis. Proteins 2015; 83:1078–1090. © 2015 Wiley Periodicals, Inc.     

Fetotoxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) for rhesus macaques (Macaca mulatta)

The fetotoxic and teratogenic potential of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) for rhesus macaques (Macaca mulatta) was tested through oral administration to monkeys early in pregnancy. A single or divided dose, 1 μg of TCDD/kg of body weight, was followed by abortion in 13 of 16 pregnant monkeys treated between days 20 and 40 of gestation. One of four aborted at 0.2 μg/kg, and two of two at 5 μg/kg. None of the mothers given 0.2 μg/kg showed signs of toxicity. Eight of the monkeys aborting at 1 μg/kg showed clinical toxicity 44 to 111 days after aborting, and three died. Both given 5 μg/kg became toxic soon after abortion and died. No malformations except for two minor palatal abnormalities of questionable significance were found in the six fetuses that were not aborted at doses of 0.2 and 1.0 μg/kg. These results indicate (1) that TCDD is fetotoxic at doses that frequently have delayed toxicity to the mother, but that conclusions about teratogenicity cannot be drawn, and (2) that pregnant rhesus females are more sensitive to the toxic effects of TCDD than any species tested but the guinea pig.     

Optimal culture conditions for keratinase production by a novel thermophilic Myceliophthora thermophila strain GZUIFR‐H49‐1

Aims: To investigate the effect of medium compositions and culture conditions on keratinase production by a novel thermophilic fungus Myceliophthora thermophila (Apinis) Oorschot strain GZUIFR‐H49‐1. Methods and Results: The thermophilic strain GZUIFR‐H49‐1 with keratinolytic ability was characterized and identified as a strain of M. thermophila on the basis of its morphological characters and molecular analysis of ITS1‐5.8S‐ITS2 rDNA sequence. Among the medium compositions tested, the soluble starch (SS), urea, sodium thiosulfate and CaCl₂ were the most effective C‐source, N‐source, S‐source and mineral ion, respectively, by employing the single‐factor experiment. The urea and pH value were the significant factors (P < 0·05) for the keratinase production in this experiment condition using Plackett–Burman factorial design. The conditions of keratinase production were further optimized by Box–Behnken design. Consequently, there was a 6·4‐fold increase (5100 U l^?1) in the keratinase activity than the initial value (800 U l^?1) by this optimal process. Conclusions: This study indicated that the optimization design proved a useful and powerful tool for the development of optimal medium compositions and culture conditions. Myceliophthora thermophila strain GZUIFR‐H49‐1 was a promising fungus strain for keratinase production. Significance and Impact of the Study: This study characterized a novel thermophilic M. thermophila strain GZUIFR‐H49‐1 with potential applications for keratinase production. These conditions of keratinase production obtained by means of optimization design will be accumulated as potential information for exploration and utilization to the new fungus isolate.     

Stalking the wild Tetrahymena

We live on a microbial planet. Microorganisms dominate in terms of numbers of lineages, numbers of organisms, biomass and evolutionary innovations. Yet much remains to be learned about our microbial neighbours. We have gotten to know a few species that have been transformed into ‘laboratory rats’ (i.e. model organisms), but even here our understanding of the natural history of these lineages remains inadequate as there are few data from populations living in natural habitats. Zufall et al. (2013) move beyond this trend by providing insights into the natural history of Tetrahymena thermophila, a ciliate that has been used in many studies of cellular and molecular biology. Characterization of T. thermophila sampled from numerous ponds across this ciliate's range in Eastern North America reveals the following: (i) considerable differentiation among isolates, with the greatest diversity among lineages in New England, and (ii) a relatively small effective population size for this model ciliate. Such population data are fundamental for inferences about the origins of the numerous remarkable features of T. thermophila.     

2,3,7,8-tetrachlorodibenzo-p-dioxin mechanism of action to reduce lipoprotein lipase activity in the 3T3-L1 preadipocyte cell line

Lipoprotein lipase (LPL) is important in the process of triglyceride storage in adipose tissue. Depression of LPL activity in adipose tissue is associated with 2,3,7,8-tetrachlorodibenzo-p -dioxin (TCDD)-induced wasting syndrome and may have a role in the associated serum hyperlipidemia produced by TCDD. The 3T3-L1 cell line was used as an in vitro model, independent of hormonal, nutritional, or other interfering factors associated with in vivo studies, in order to systematically examine the mechanism of action of TCDD. TCDD produced a statistically significant (P < 0.05) time- and dose-dependent decrease in LPL activity. Results of experiments with Ah-receptor blockers and structure activity studies with different polychlorinated biphenyl (PCB) and dioxin congeners were consistent with reduction of LPL activity being mediated by the Ah receptor. Culturing of 3T3-L1 cells without glucose or with cytochalasin B, a blocker of facilitative glucose transporters (GLUT), was effective in reducing LPL activity (P < 0.05). TCDD did not further reduce LPL activity in cytochalasin B pretreated 3T3-L1 cells or in 3T3-L1 cells cultured in glucose-free media. Dexamethasone pretreatment, which is known to increase GLUT expression in 3T3-L1 cells, prevented the reduction of LPL activity by TCDD. Protein tyrosine kinase activities, assayed using γ-³²P-ATP and RR-SRC, a src specific peptide substrate, were significantly increased (P < 0.05) over control levels by both TCDD and glucose deprivation. Furthermore, results of experiments treating 3T3-L1 cells with either insulin, EGF, 8-Br-cAMP, TPA, or genistein, alone or in combination with TCDD, were generally consistent with the hypothesis that lowered intracellular glucose and altered cellular kinase activities may be involved in reduction of LPL activities by TCDD. Further work is needed to confirm and better understand the role protein phosphorylation plays in TCDD-mediated alteration of glucose disposition and LPL activity. In summary, TCDD reduced LPL activity in 3T3-L1 cells as seen in vivo. Manipulation of glucose transport through a number of experimental approaches produced changes in 3T3-L1 LPL activity consistent with results of previous investigators showing glucose to be a positive regulator of LPL activity and consistent with our hypothesis that TCDD-mediated reduction of glucose transport is an important factor in the down regulation of LPL activity by TCDD. © 1997 John Wiley & Sons, Inc. J Biochem Toxicol 12: 29–39, 1998     

From heavy metal‐binders to biosensors: Ciliate metallothioneins discussed

Metallothioneins (MTs) are ubiquitous proteins with the capacity to bind heavy metal ions (mainly Cd, Zn or Cu), and they have been found in animals, plants, eukaryotic and prokaryotic micro‐organisms. We have carried out a comparative analysis of ciliate MTs (Tetrahymena species) to well‐known MTs from other organisms, discussing their exclusive features, such as the presence of aromatic amino acid residues and almost exclusive cysteine clusters (CCC) present in cadmium‐binding metallothioneins (CdMTs), higher heavy metal‐MT stoichiometry values, and a strictly conserved modular–submodular structure. Based on this last feature and an extensive gene duplication, we propose a possible model for the evolutionary history of T. thermophila MTs. We also suggest possible functions for these MTs from consideration of their differential gene expressions and discuss the potential use of these proteins and/or their gene promoters for designing molecular or whole‐cell biosensors for a fast detection of heavy metals in diverse polluted ecosystems.     

PCR with degenerate primers amplifies a subgenomic DNA fragment from the endoglucanase gene(s) of Torula thermophila, a thermophilic fungus

The aim of this study was to enable the polymerase chain reaction (PCR) amplification of DNA fragments within endoglucanase gene(s) of Torula thermophila, by using degenerate primers so that the amplified fragment(s) could be used as homologous probe(s) for cloning of full-length endoglucanase gene(s). The design of the degenerate PCR primers was mainly based on the endoglucanase sequences of other fungi. The endoglucanase gene sequence of Humicola insolens was the only sequence from a thermophilic fungus publicly available in the literature. Therefore, the endoglucanase sequences of the two Trichoderma species, Trichoderma reesei and Trichoderma longibrachiatum, were used to generalize the primers. PCR amplification of T. thermophila genomic DNA with these primers resilied in a specific amplification. The specificity of the amplified fragment was shown by Southern hybridization analysis using egl3 gene of T. reesei as probe. This result suggested that the degenerate primers used in this study may be of value for studies aimed at cloning of endoglucanase genes from a range of related fungi.     

Adaptive characteristics of a ciliateTetrahymena thermophila in endosymbiotic association with a green algaChlorella vulgaris derived in a long-term microcosm culture

We investigated directly an early stage of the development of an endosymbiosis between an alga and a ciliate, beginning from a non-associated stage by conducting a long-term microcosm culture composed of a green alga (Chlorella vulgaris), a bacterium (Escherichia coli) and a ciliate (Tetrahymena thermophila) for three years. During this culture,Chlorella-harboringTetrahymena appeared and increased in frequency. We examined the adaptive characteristics of theT. thermophila in association with theC. vulgaris derived from a well-established microcosm culture maintained for 1164–1400 days, and compared it with the original culture ofT. thermophila. TheT. thermophila with the associated, intracellularC. vulgaris grew at a lower density ofE. coli than the originalT. thermophila with the originalC. vulgaris, and the former survived longer than the latter in the absence ofE. coli. These results suggest that this induced algal-ciliate association confers some fitness advantage at least to the host under the conditions of less or no available food such asE. coli. Although the algal-ciliate association is not completely stable, this quasi-stable association may enable the host to exploit a new niche through the advantages of mixotrophy.