Effects of pyraclostrobin on growth,oxidative stress,and gene expression in relation to stress and ATP-binding cassette transporters in Tetrahymena thermophila

Pyraclostrobin(PYR),a widely used fungicide,has negative effects on fish and algae,but its toxicity in protozoa remains unclear.In this study,the effects of PYR on the growth,oxidative stress,and gene expression related to stress and ATP-binding cassette(ABC)transporters in Tetrahymena thermophila were investigated.The result showed that the 96-h IC_(50)of PYR against T.thermophila was 17.2 mg/L.Moreover,PYR inhibited the growth of T.thermophila in concentration-or time-dependent manner.A morphological study revealed that the shape and size of T.thermophila changed,and damage of cell membrane surface was observed by scanning electron microscopy after 96 h of PYR exposure.The activities of superoxide dismutase(SOD)and catalase(CAT)increased throughout the experiment.In contrast,the glutathione(GSH)content was increased at 24 h and 48 h of exposure and decreased at 96 h.Moreover,a significant increase in malondialdehyde(MDA)level was observed in T.thermophila after96 h of exposure.Furthermore,PYR upregulated the HSP703,HSP705,GPx2,and ABAC15 gene expression in the 0.1–5-mg/L groups and downregulated the HSP704,HSP90,TGR,and ABCC52 mRNA levels at 96 h of exposure.These results suggest that PYR may exert adverse effects on T.thermophila by inducing oxidative stress and changing the gene expression related to ABC transporters and stress,which may enrich the understanding of the toxicity mechanism of PYR in aquatic organisms and provide reference data for aquatic ecological risk assessments.

Effects of L-carnitine on Population Growth of Nannochloropsis oculata and Tetrahymena sp.

[Objective] The aim was to study the effects of L-carnitine on population growth of Nannochloropsis oculata and Tetrahymena sp..[Method] When the concentration of L-carnitine was 0,50,100 and 1 000 mg/L,population densities of Nannochloropsis oculata and Tetrahymena were determined respectively.[Result] Adding high-dose L-carnitine had a significant inhibition effect on the population growth of Nannochloropsis oculata （P0.05）.Adding L-carnitine had a significant proliferation promoting effect on the population growth of Tetrahymena （P0.05）.[Conclusion] The research provides theoretical basis for the application of L-carnitine as feed additive in aquaculture.

利用微量热法研究Cd^(2+)和Cu^(2+)对嗜热四膜虫(Tetrahymena thermophila)的毒性效应

利用微量热法研究Cd2 + 和Cu2 + 对嗜热四膜虫BF5(TetrahymenathermophilaBF5)生长代谢毒性效应 ,结果表明 :①低浓度的Cd2 + (0～ 0 .4mgL-1)和Cu2 + (0～ 10mgL-1)对四膜虫的生长有促进作用 ,而高浓度的Cd2 + (0 .8～ 3.2mgL-1)和Cu2 + (2 0～ 2 0 0mgL-1)则产生抑制作用 ;②Cd2 + 和Cu2 + 的半抑制浓度IC50 分别为 2 .0 4mg/L和15 5 .5mg/L ;③联合毒性为协同作用 ;④微量热法所得Cd2 + 和Cu2 + 单独作用时的IC10 分别为 0 .96mg/L和 19.9mg/L ,高于种类损伤法结果 ;⑤微量热法所得Cd2 + 和Cu2 + 单独作用时的IC10 均高于II级饮用水水质标准 .而Cd2 + 和Cu2 +间的协同作用使Cd2 + 的IC10 降低为 0 .31mg/L ,高于水质标准 ;Cu2 + 的IC10 降低为 1.16mg/L ,与水质标准相近 .总之 ,微量热法不仅是一种快速评价污染物毒性效应的新方法 ,而且可用于相关的环境毒理学理论研究 .图 1表 5参

关键环境因子对嗜肺军团菌(Legionella pneumophila)在嗜热四膜虫(Tetrahymena thermophila)细胞内增殖的影响

嗜肺军团菌是一种生活在人工水环境中的条件性致病菌,环境条件对该菌的存活和胞内增殖具有重要的影响.本文用作者建立的军团菌-四膜虫模型,研究了空调水池水、Fe3+和感染温度3种关键性环境因子对嗜肺军团菌在嗜热四膜虫宿主细胞内增殖的影响.结果发现,尽管空调水中Fe3+浓度大大低于培养基中的Fe3+浓度,但适合嗜肺军团菌在嗜热四膜虫宿主胞内的存活和增殖;感染缓冲液中的Fe3+能够被嗜热四膜虫富集,并被胞内的军团菌加以利用,缓冲液中浓度0.05×10-6的Fe3+即可显著促进胞内军团菌的增殖,但0.1×10-6和0.5×10-6的Fe3+促进军团菌胞内增殖的效果差异不显著;较高的温度对军团菌的胞内增殖也有明显促进作用,35℃时,即使感染复数等于10,军团菌也可进行胞内复制扩增,并最终裂解宿主细胞.

QSAR Study of Halogen Phenols Toxicity to Tetrahymena Pyriformis

Structural parameters of 22 halogen phenols were computed at four levels using Hartree-Fock and DFT methods. Based on the experimental data of the acute toxicity to Tetrahymena pyriformis （-lgEC_50）, three-parameter （energy of the lowest unoccupied molecular orbital （E_LUMO）, the molecular volume （V）, and the lowest negative charge （Q_min）） dependent equations were developed using structural parameters as theoretical descriptors. Especially, -lgEC_50dependent equation calculated at the B3LYP/6-31G^＊＊ level is more advantageous than the others in view of their correlation and predictive abilities. This dependent equation was validated by variance inflation factors （VIF） and t-test methods. Upon comparison, the predictive abilities of our work are all more advantageous than those calculated from the semi-empirical PM3 method.

QSAR Studies on the Toxicity of Nitrobenzenes to Population Growth of Tetrahymena Pyriformis

The IGC 50 (50% inhibitory growth concentration) values of 27 nitrobenzenes were determined for the population growth endpoint of Tetrahymena pyriformis . The toxicity order of the observed compounds was found to be as follows: dinitro compounds>mononitro compounds, dichloronitrobenzenes>monochloronitrobenzenes, meta substituted nitrobenzenes>ortho /para substituted nitrobenzenes(NT, NPh, NAnis) except for the dinitrobenzenes and nitroanilines(DNB, NAn). Quantitative structure activity relationships(QSARs) were developed with the logarithm of the reciprocal of IGC 50 [lg(IGC 50 ) -1 ] in mole liter as dependent variable and six molecular descriptors lg P , 1 X Ⅴ, I, 1K a, ∑σ - and E LUMO as independent variables. Via multiple regression analysis, one best equation was obtained: lg(IGC 50 ) -1 =3.029+0.860∑ σ -+0.341I n=27, r=0.924, r 2=0.854, s=0.265, f=70.44 , Pr> f =0.000 1 The equation was used to estimate IGC 50 for five analogues.

Autophagy prevents autophagic cell death in Tetrahymena in response to oxidative stress

Autophagy is a major cellular pathway used to degrade long-lived proteins or organelles that may be damaged due to increased reactive oxygen species（ROS） generated by cellular stress. Autophagy typically enhances cell survival, but it may also act to promote cell death under certain conditions. The mechanism underlying this paradox, however, remains unclear. We showed that Tetrahymena cells exerted increased membranebound vacuoles characteristic of autophagy followed by autophagic cell death（referred to as cell death with autophagy） after exposure to hydrogen peroxide. Inhibition of autophagy by chloroquine or 3-methyladenine significantly augmented autophagic cell death induced by hydrogen peroxide. Blockage of the mitochondrial electron transport chain or starvation triggered activation of autophagy followed by cell death by inducing the production of ROS due to the loss of mitochondrial membrane potential. This indicated a regulatory role of mitochondrial ROS in programming autophagy and autophagic cell death in Tetrahymena. Importantly, suppression of autophagy enhanced autophagic cell death in Tetrahymena in response to elevated ROS production from starvation, and this was reversed by antioxidants. Therefore, our results suggest that autophagy was activated upon oxidative stress to prevent the initiation of autophagic cell death in Tetrahymena until the accumulation of ROS passed the point of no return, leading to delayed cell death in Tetrahymena.

四膜虫(Tetrahymena thermophila)核纤层的研究

采用非树脂包进埋去包埋超薄切片电镜技术,并结合选择性生化抽提方法以及间接免疫荧光染色方法显示,在四膜虫(Tetrahymena thermophila)细胞内存在典型的核纤层结构。蛋白分离纯化与免疫印迹法分析结果说明四膜虫细胞核纤层可能主要由分子量为66KD的多肽构成。蛋白分离纯化结果表明四膜虫细胞的核纤层蛋白可能不如高等动物细胞的丰富。

不同生长期上海四膜虫（Tetrahymena shanghaiensis S1）的同工酶的比较研究

用高分辨力的聚丙烯酰胺凝胶电泳分析和比较了不同生长期的上海四膜虫的五种同工酶。发现对数期细胞和衰老期细胞的酸性磷酸酶同工酶，酯酶同工酶和苹果酸脱氢酶同工酶及其它的同工酶的蛋白条带有明显差异。对数生长期细胞的同工酶活力和数量明显高于衰老期细胞的同工酶的活力和数量。说明同工酶不但是细胞代谢的调节者，亦能作为细胞衰老的一种指标。

Metabolomic Fingerprint of the Model Ciliate, Tetrahymena thermophila Determined by Untargeted Profiling Using Gas Chromatography-Mass Spectrometry

The ciliate Tetrahymena is a valuable model organism in the studies of ecotoxicology. Changes in intracellular metabolism are caused by exogenous chemicals in the environment. Intracellular metabolite changes signify toxic effects and can be monitored by metabolomics analysis. In this work, a protocol for the GC-MS-based metabolomic analysis of Tetrahymena was established. Different extraction solvents showed divergent effects on the metabolomic analysis of Tetrahymena thermophila. The peak intensity of metabolites detected in the samples of extraction solvent Formula 1(F1) was the strongest and stable, while 61 metabolites were identified. Formula 1 showed an excellent extraction performance for carbohydrates. In the samples of extraction solvent Formula 2(F2), 66 metabolites were characterized, and fatty acid metabolites were extracted. Meanwhile, 57 and 58 metabolites were characterized in the extraction with Formula 3(F3) and Formula 4(F4), respectively. However, the peak intensity of the metabolites was low, and the metabolites were unstable. These results indicated that different extraction solvents substantially affected the detected coverage and peak intensity of intracellular metabolites. A total of 74 metabolites(19 amino acids, 11 organic acids, 2 inorganic acids, 11 fatty acids, 11 carbohydrates, 3 glycosides, 4 alcohols, 6 amines, and 7 other compounds) were identified in all experimental groups. Among these metabolites, amino acids, glycerol, myoinositol, and unsaturated fatty acids may become potential biomarkers of metabolite set enrichment analysis for detecting the ability of T. thermophila against environmental stresses.

Toxic Effect of a Pesticide, Diflubenzuron on Freshwater Microinvertebrate (Tetrahymena pyriformis)

The impact of diflubenzuron (DFB) on Tetrahymena pyriformis, a ciliated protiste was studied. The treatment with DFB at concentrations of 10 and 20 μg/mL could appreciably reduce the growth of the protiste. The surveillance of protozoa′s respiratory metabolism with polarography technique showed a sensitive inhibition of its oxygen consumption, and at 20 μg/mL concentration the oxygen consumption was increased. For any malformation of morphology and perturbation in swimming trajectory, the neutral red coloration was done. This technique (neutral red) showed that there was a toxic effect of DFB on T. pyriformis, especially at the highest concentration; this toxicity was translated by rotatory swimming in same place and the increasing of coloration quantity in digestive vacuoles. Fig 5, Ref

Biological Effect Studies of Norfloxacin Complexes on Tetrahymena and Escherichia Coli by Microcalorimetry

Norfloxacin complexes Co（nor）2.8H2O （nor=norfloxaein） and [Co（nor）（phen）]NO3.2H2O （phen=1,10-phenanthroline） were synthesized and their biological activity on Tetrahymena and Escherichia coli （E.coli） was assayed by means of microcalorimetry. Massive experimental parameters such as the growth constant k, inhibitory ratio I, half inhibition concentration IC5o and generation time TG were obtained. Data showed that with the increase of exposing complex concentration, both complexes exhibited strong inhibition during the growth of organisms. While toxic degrees were quite different on Tetrahymena and E.coli, due to various toxic mechanisms. And complex molecular volume and the ability of the complex penetrating into cells may be the keys.

上海四膜虫(Tetrahymena shanghaiensis)线粒体DNA的酶切分析

该文以六种限制性内切酶对上海四膜虫的mtDNA进行单酶完全酶切 ,EcoRⅠ、HindⅢ、HhaⅠ酶切均产生 4个片段 ,PstⅠ、HpaⅡ、HaeⅢ酶切分别产生 5、6、7个片段。不同的酶切片段大小及总和有一定的差异 ,酶切位点分布不均匀 ,片段长度多态性明显。与其它四膜虫mtDNA的同种酶切结果比较 ,有较大的差异。同其它方法如四膜虫大核rDNA限制性内切酶分析、同工酶分析及皮层细胞骨架组分分析结果一致 ,进一步证明上海四膜虫是梨形四膜虫复合种的一种。

稀土金属离子对梨形四膜虫(Tetrahymena pyriformis)的生长毒性

采用S1上海梨形四膜虫（Tetrahymenapyriformis）进行金属离子La3+.Sm3+.Y3+.Gd3+的24h和96h生长毒性试验．结果表明La3+，Sm3+．Y3+在低剂量下均表现出对四膜虫的刺激效应．但在高剂量时上述四种稀土金属离子都表现出抑制效应．而且随着浓度的升高．抑制作用增强．其毒性大小顺序为Gd3+＞Y3+＞Sm3+＞La3+，并且重稀土的毒性要高于中稀土．轻稀土的毒性．

Characterization of Deciliation-Regeneration Process of Tetrahymena Pyriformis for Cellular Robot Fabrication

magnetotactic Tetrahymena pyriformis GL （T. pyriformis） cells were created by the internalization of iron oxide nano particles and became controllable with a time-varying external magnetic field. Thus, T. pyriformis can be utilized as a cellular robot to conduct micro-scale tasks such as transportation and manipulation. To complete these tasks, loading inorganic or organic materials onto the cell body is essential, but functionalization of the cell membrane is obstructed by their motile organelles, cilia. Dibucaine HC1, a local anesthetic, removes the cilia from the cell body, and the functional group would be absorbed more efficiently during cilia regeneration. In this paper, we characterize the recovery of artificial magnetotactic T. pyriformis after the deciliation process to optimize a cellular robot fabrication process. After sufficient time to recover, the motility rate and the average velocity of the deciliated cells were six and ten percent lower than that of non-deciliated cells, respectively. We showed that the motile cells after recovery can still be controlled using magnetotaxis, making T. pyrfformis a good candidate to be used as a celIular robot.

Evolutionary Mode of Metallothioneins Inferred from Cd-MT Genes of Tetrahymena

From Tetrahymena thermophila （strain BF5）, the coding region of Cd-MT gene was cloned and sequenced. and identified as MTT1 isoform. A serial duplicate structure is discovered in its amino acid sequence, which separates the coding region into three parts （Part 1：7-61; Part 2：64-118; Part 3：122-162）. The alignments among them and comparison with the corresponding parts of MT1 isoform suggest that MT1 and MTT1 isoforms both come from the same ancient gene that is homologous to Part 1, and Cd-MTs of Tetrahymena are aroused by such ancient gene duplication. The prediction of secondary structures and the analysis of the disulfide-bonding state of cysteine show that there are a lot of differences between MT1 and MTT1 isoforms, which maybe relate to their function mechanism.

Preparation of Ag/AgBr/TiO_2 as Catalyst Carriers and Its Damage to Plasmid DNA and Tetrahymena

The composites based on the Ti O2 are potentially used in wetland pollution control. In this work, the biological effect of the Ag/Ag Br/Ti O2/Active carbon(AC) composites was studied on the plasmid DNA and Tetrahymena membrane. The atomic force micrograph(AFM) images showed that, in the presence of the composites under illumination, most p UC18 DNA molecules showed quite different topography and were opened and relaxed circle shapes. After DNA was catalyzed for 40 min, all supercoiled and circular DNA were changed into the linear DNA molecules. The gel electrophoresis experiment confirmed the results and demonstrated the dynamic process of DNA degradation. ATR-FTIR spectra revealed that amide groups and PO2-of the phospho-lipid phospho-diester on Tetrahymena surface were oxidized in the presence of the composites under illumination. An increase in the fluorescence polarization of DPH was observed, reflecting a significant decrease in membrane fluidity of Tetrahymena.

Association of DNA with nuclear matrix in in vitro assembled nuclei induced by rDNA from Tetrahymena shanghaiensis in Xenopus egg extracts

The nuclei assembled from exogenous DNA or chromatin in egg extracts resemble their in vivo counterparts in many aspects. However, the distribution pattern of DNA in these nuclei remains unknown. We introduced rDNA from the macronuclei of Tetrabymena into Xenopus cellfree extracts to examine the association of specific DNA sequences with nuclear matrix (NM) in the nuclei assembled in vitro. Our previous works showed the 5’NTS (nontranscription sequences) of the rDNA specifically bind to the NM system in the macronuclei. We show now the rDNA could induce chromatin assembly and nuclear formation in Xenopus cell-free system. When we extracted the NM system and compared the binding affinity of different regions of rDNA with the NM system, we found that the 5’NTS still hold their binding affinity with insoluble structure of the assembled nuclei in the extracts of Xenopus eggs.

The Ciliate Protist <i>Tetrahymena pyriformis</i>as a Cellular Adhesion Model for the Pathogenic Bacterium <i>Staphylococcus aureus</i>

Staphylococcus aureus is one of the main pathogenic agents responsible for nosocomial and community-acquired bacterial infections. The pathogenicity of this Gram-positive bacterium is ensured by its different adhesion factors. Collagen and the extracellular glycoprotein adhesin are among the Staphylococcus most important virulence factors. It has been shown that most of the S. aureus strains carry the ica operon, responsible for biofilm production. However, the coexpression of the icaA and the icaD genes is necessary for complete biofilm synthesis. The aim of our study was to study a collection of 15 clinical strains of S. aureus from different sources for the presence of can and icaD genes coding intercellular adhesion proteins. We also intended to estimate the strains’ ability to form biofilms by the red Cong method and to test the adhesion ability of S. aureus to the ciliated protist Tetrahymena pyriformis, which we used as a novel cellular adhesion model. Finally, we checked the adhesion’s inhibition capacity of some plants extracts. The molecular detection of adhesion genes revealed that 80% of strains are cna positive, and 73% are icaD positive. Qualitative biofilm production of S. aureus revealed that 66.6% of strains were slime producers. The adhesion test revealed that 20% of strains are strongly adhering to T. pyriformis and that the Clematis cirrhosa extract has an anti-adhering effect of S. aureus to the ciliate T. pyriformis.

Cell Stress by Phosphate of Two Protozoa Tetrahymena thermophila and Tetrahymena pyriformis

Phosphorus is one of the bioelements most needed as a compound cell by living organisms. Phosphorus is involved in several pathologies: in human with bone and kidney diseases, in mammals with metabolism disorder (glucose, insulin···), in microorganisms whose phosphorus is involved in cell growth. Phosphorus has various forms including pyrophosphate, a by-product of multiple pathways of biosynthesis. Enzymes that hydrolyze pyrophosphate are called inorganic pyrophosphatases (PPases). Two major types of inorganic pyrophosphatases are distinguished: the soluble pyrophosphatases (sPPases) and the membrane pyrophosphatases (mPPases or H+/Na+-PPases). They play a key role in the control of intracellular inorganic pyrophosphate level and produce an important ions gradient (H+ or Na+) to the cells. In this work, we primarily focused on the physiological study in a phosphate-poor medium of two models Tetrahymena thermophile and Tetrahymena pyriformis, following the mobility, the growth and the morphology of cells. Secondly, we evaluated the enzymatic activity of soluble and membrane pyrophosphatases in both species grown in the same complex medium. A decrease of cell growth is correlated with unusual morphologies and different mobility in the stress medium. The measurement of soluble and membrane inorganic pyrophosphatases activities also shows a decrease which illustrates the lack of phosphate found in the stress medium. Deficiency of phosphate is a limiting factor for protozoan growth. These results indicate that Tetrahymena can be used as a model of cellular stress and consists of a target to study inorganic pyrophosphatases for a better understanding of phosphate cycle in higher organisms.