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Abstract Background Euplotes, a ciliated protozoan, is a useful unicellular model organism. Studies on Euplotes have provided excellent insights into various basic biological principles. We have recently sequenced the macronuclear genome of the common freshwater species Euplotes octocarinatus to provide novel insights into Euplotes genetics and molecular biology. Results In this study, we present the E. octocarinatus Genome Database (EOGD), a functional annotation and analysis platform for the global study of the Euplotes genome. EOGD includes macronuclear genomic and transcriptomic data, predicted gene models, coding sequences, protein sequences, and functional annotations. The GBrowser and BLAST tools are embedded in EOGD to enable the search, visualization and analysis of E. octocarinatus genomic and transcriptomic data. Conclusions EOGD is a useful resource for the research community, particularly for researchers who conduct genome-scale analysis and molecular biology studies of Euplotes or other ciliates. EOGD will be continuously updated to integrate more datasets and analytical tools. EOGD is freely available at http://ciliates.ihb.ac.cn/database/home/#eo .

Programmed ribosomal frameshifting (PRF) exists in all branches of life that regulate gene expression at the translational level. The single-celled eukaryote Euplotes exhibit high frequency of PRF. However, the molecular mechanism of modulating Euplotes PRF remains largely unknown. Here, we identified two novel eIF5A genes, eIF5A1 and eIF5A2 , in Euplotes octocarinatus and found that the Eo-eIF5A2 gene requires a −1 PRF to produce complete protein product. Although both Eo-eIF5As showed significant structural similarity with yeast eIF5A, neither of them could functionally replace yeast eIF5A. Eo-eIF5A knockdown inhibited +1 PRF of the η-tubulin gene. Using an in vitro reconstituted translation system, we found that hypusinated Eo-eIF5A (Eo-eIF5AH) can promote +1 PRF at the canonical AAA_UAA frameshifting site of Euplotes. The results showed eIF5A is a novel trans -regulator of PRF in Euplotes and has an evolutionary conserved role in regulating +1 PRF in eukaryotes. • Eo-eIF5A2 gene needs a −1 PRF to produce complete protein. • Eo-eIF5A1 and Eo-eIF5A2 promote +1 PRF in E. octocarinatus , respectively. • +1 PRF regulated by eIF5A is evolutionary conserved in eukaryotes. [ABSTRACT FROM AUTHOR]

The xeroderma pigmentosum group C protein (XPC) and centrin2 are the primary initiators of global genome nucleotide excision repair (NER). Centrin, acts as a member of the EF-hand super family of calcium-binding proteins, playing roles in reconstitution of the vitro NER reaction. To understand the possible molecular and structural properties of the multiprotein process, the interactions of Euplotes octocarinatus centrin (EoCen), melittin, and DNA are described. EoCen shares a sequence identity of 66% with centrin2. Melittin possesses inverse direction hydrophobic triads-leucine-leucine-tryptophan (LLW) which are responsible for centrin binding. It is applied as a natural peptide to mimic centrin target peptide. As a result, it is proved that the integrated protein shows an endonuclease-like activity to DNA. Melittin is capable of interaction with both EoCen and DNA. More importantly, it is found that melittin displays an inhibitory effect on the endonuclease-like activity of centrin when it co-exists with EoCen and DNA in solution. Meanwhile, the DNA-melittin-EoCen ternary complex forms in the process. Quantitative analyses demonstrated by extensive biophysical assays reveal that binding of the peptide to DNA or centrin modulates the binding properties of it to another component. Furthermore, a possible positioning model of DNA and EoCen on melittin is proposed. This finding may constitute a model for that existing between centrin and its target peptide in NER process. [ABSTRACT FROM AUTHOR]

Centrin is a member of the EF-hand super family of calcium-binding proteins, which can behave as a part of damage detector initiated the initiation of nucleotide excision repair (NER). Its self-assembly plays a causative role in fiber contraction associated with the cell division cycle and ciliogenesis. To explore the possible role of DNA in the process of centrin self-assembly, the aggregation properties of N-terminal domain of Euplotes octocarinatus centrin (N-EoCen) in the presence of DNA with or without metal ions are investigated. It is verified that metal ions, such as Ca 2 + and Tb 3 + , can bind to N-EoCen with 2:1 stoichiometry by isothermal titration calorimetry (ITC). Importantly, this study reports that double strand DNA (dsDNA) is capable of binding N-EoCen, changing conformation of protein and modulating centrin aggregation, as demonstrated by extensive biophysical assays. Interestingly, the open conformation of protein induced by metal ions may be favour of the interaction of protein with dsDNA. Nevertheless, the randomly coiled single strand DNA (ssDNA) is completely inefficient to the aggregation regulation. Furthermore, results reveal that hydrophobic site could play important role in the process. This finding may link to the potent roles of centrin in the NER process. [ABSTRACT FROM AUTHOR]

Background: Euplotes, a ciliated protozoan, is a useful unicellular model organism. Studies on Euplotes have provided excellent insights into various basic biological principles. We have recently sequenced the macronuclear genome of the common freshwater species Euplotes octocarinatus to provide novel insights into Euplotes genetics and molecular biology. Results: In this study, we present the E. octocarinatus Genome Database (EOGD), a functional annotation and analysis platform for the global study of the Euplotes genome. EOGD includes macronuclear genomic and transcriptomic data, predicted gene models, coding sequences, protein sequences, and functional annotations. The GBrowser and BLAST tools are embedded in EOGD to enable the search, visualization and analysis of E. octocarinatus genomic and transcriptomic data. Conclusions: EOGD is a useful resource for the research community, particularly for researchers who conduct genome-scale analysis and molecular biology studies of Euplotes or other ciliates. EOGD will be continuously updated to integrate more datasets and analytical tools. EOGD is freely available at http://ciliates.ihb.ac.cn/ database/home/#eo. [ABSTRACT FROM AUTHOR]

Analysis of transcriptome revealed that a membrane occupation and recognition nexus (MORN) repeat protein-encoding gene ofEuplotes octocarinatus(Eo-morn-9-31) was a candidate for programmed +1 ribosomal frameshifting (+1 PRF). In this study, a dual-luciferase assay was performed to detect its expression. The result showed that the MORN repeat protein (Eo-MORN-9-31) could be produced by the +1 PRF event during the process of translation in yeast and the frameshifting efficiency was about 4–5%. We further confirmed its reality by western blot and mass spectrometry. This study provided experimental evidence indicating that the expression of the Eo-MORN-9-31 ofE. octocarinatusrequired the +1 PRF. TheEo-morn-9-31 could produce a MORN repeat protein containing nine MORN repeats with an apparent molecular weight of 41.2 kD, or only encode a truncated protein (34.8 kD) depending +1 PRF. [ABSTRACT FROM PUBLISHER]

The adsorption behavior of apo-ciliate Euplotes octocarinatus centrin (EoCen) and N(C)-terminal domain of EoCen (N(C)-EoCen) at a glassy carbon (GC) electrode is studied by electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). Interestingly, the adsorption isotherms of C-EoCen, N-EoCen and EoCen at a GC surface differ from one another. It is considered to be associated with the different aggregation properties of three proteins. Furthermore, we analyze the metal-binding properties of centrin and the followed changes in protein structure upon metal-binding. Corresponding to the four binding sites of EoCen, it shows four no-equiv signals of CV or EIS change. It indicates that the four different binding sites of EoCen can be discriminated by the EIS titration curves, which is in contrast with conventional use of spectral method. According to the electrostatic potential at the molecular surface of proteins, the favored orientation of N-EoCen and C-EoCen on the GC surface is modeled. These models can well explain the results of titration curves of Eu3+ to N(C)-EoCen. This work has established the electrochemical methodology which can be used to measure metal-binding sites involved and reveal the contribution of each metal site to the whole protein conformational change. [ABSTRACT FROM AUTHOR]

The eukaryotic acid ribosomal P0, P1, and P2 proteins share a conserved flexible C-terminal tail that is rich in acidic residues, which are involved in the interaction with elongation factor 2 during protein synthesis. Our previous work suggested that the acidic ribosomal P proteins from Euplotes octocarinatus have a special C-terminal domain. To further understand this characteristic feature, both P2 and elongation factor 2 from E. octocarinatus were overexpressed, for the first time, in Escherichia coli in this study. GST pull-down assay indicated that P2 protein from E. octocarinatus (EoP2) interacted specifically with the N-terminal domain of elongation factor 2 from E. octocarinatus (EoEF-2) in vitro. The interacting part of EoP2 is in the C-terminal domains, consistent with the observation in other organisms. Phosphorylation of the recombinant EoP2 was performed in vitro using multiple methods such as 31P-NMR spectroscopy, native PAGE, and Phos-tagTM SDS-PAGE. Results showed that ribosomal protein EoP2 was phosphorylated by casein kinase II at serine 21 located at the N terminus. This phosphorylation site identified in EoP2 is quite different from that of P2 from other organisms, in which the phosphorylation site is located in the conserved C-terminal region. [ABSTRACT FROM AUTHOR]

One factor involved in eukaryotic translation termination is class 1 release factor in eukaryotes (eRF1), which functions to decode stop codons. Variant code species, such as ciliates, frequently exhibit altered stop codon recognition. Studies revealed that some class-specific residues in the eRF1 N-terminal domain are responsible for stop codon reassignment in ciliates. Here, we investigated the effects on stop codon recognition of chimeric eRF1s containing the N-terminal domain of Euplotes octocarinatus and Blepharisma japonicum eRF1 fused to Saccharomyces cerevisiae M and C domains using dual luciferase read-through assays. Mutation of class-specific residues in different eRF1 classes was also studied to identify key residues and motifs involved in stop codon decoding. As expected, our results demonstrate that 3 pockets within the eRF1 N-terminal domain were involved in decoding stop codon nucleotides. However, allocation of residues to each pocket was revalued. Our data suggest that hydrophobic and class-specific surface residues participate in different functions: modulation of pocket conformation and interaction with stop codon nucleotides, respectively. Residues conserved across all eRF1s determine the relative orientation of the 3 pockets according to stop codon nucleotides. However, quantitative analysis of variant ciliate and yeast eRF1 point mutants did not reveal any correlation between evolutionary conservation of class-specific residues and termination-related functional specificity and was limited in elucidating a detailed mechanism for ciliate stop codon reassignment. Thus, based on isolation of suppressor tRNAs from Euplotes and Tetrahymena, we propose that stop codon reassignment in ciliates may be controlled by cooperation between eRF1 and suppressor tRNAs. [ABSTRACT FROM AUTHOR]

Abstract: The aim of the present work was to study the dynamics of the interactions between human adenovirus and ciliates under both experimental and field conditions. Experimental co-cultures of the ciliated protozoan Euplotes octocarinatus and human adenovirus (HAdV) type 2 were established and virus internalization was investigated using nested PCR and direct immunofluorescence (IF). In addition, to study protozoa-virus interactions in the field, wild ciliates were isolated from active sludges of a wastewater treatment plant and analyzed for the presence of adenovirus using direct IF. In vitro experiments revealed HAdV type 2 inside Euplotes cells after 15min of contact and its persistence until at least 35 days post infection. In addition, our results showed the adsorption of adenovirus on the surface of wild ciliates. We conclude that HAdV is taken up by ciliates, however more studies are necessary in order to better investigate the mechanisms, the infectivity of internalized virus and the protective effects of internalization against disinfection. [Copyright &y& Elsevier]

The binding of both factors (eRF1 and eRF3) is essential for fast kinetics of the termination of protein translation. The C-terminal domain of eRF1 is known to interact with the C domain of eRF3. Eo-eRF1b contains two highly conserved tryptophan residues (W-11 and W-373), W-11 located in the Eo-eRF1b N domain and W-373 located in the EoeRF1b C domain. Fluorimetry was used to study the interactions of the proteins. When binding with Eo-eRF3Cm6, the emission peak of Eo-eRF1b is blue shifted, while the emission peak of Eo-eRF1bC has no notable change. Our results suggest that the eRF1-eRF3 interaction induces the N and C domain of eRF1b to become closer to each other. [ABSTRACT FROM AUTHOR]

Abstract: Centrin is a low molecular mass (20KDa) protein that belongs to the EF-hand superfamily. In this work, the interaction between the Tb3+-saturated C-terminal domain of Euplotes octocarinatus centrin (Tb2-C-EoCen) and 2-p-toluidinylnaphthalene-6-sulfonate (TNS) was investigated using difference UV–vis spectra and the fluorescence spectra methods. In 100mM N-2-hydroxy-ethylpiperazine-N-2-ethanesulfonic acid (Hepes) at pH 7.4, with the addition of Tb2-C-EoCen, four new peaks were observed at 265nm, 278nm, 317nm and 360nm by absorptivity compared with blank solution of TNS. At the same time, the reaction could be measured by fluorescence spectra. The fluorescence emission of TNS was shifted from 480nm to 445nm in the presence of Tb2-C-EoCen. Meanwhile, its fluorescence intensity was increased markedly. The 1:1 stoichiometric ratio of C-EoCen to TNS was confirmed by fluorescence titration curves. The conditional binding constants of TNS with C-EoCen and Tb2-C-EoCen were calculated to be log K(C-EoCen-TNS)=5.32±0.04 M−1 and log K(Tb2-C-EoCen-TNS)=5.58±0.12M−1, respectively. In addition, the protein of Tb2-C-EoCen binding with melittin was also studied. Based on the fluorescence titration curves, the 1:1 stoichiometric ratio of Tb2-C-EoCen to melittin was confirmed. And the conditional binding constant of C-EoCen with melittin was calculated to be log Ka′=6.79±0.17M−1. [Copyright &y& Elsevier]

Abstract: Centrin, a member of calcium-binding proteins, is an essential component for microtubule-organizing center (MTOC). Lanthanide (Ln) ions can increase amounts, enhance stability and orderliness of microtubules which is an important component of cytoskeleton. To investigate the structural basis of the effect of Ln ions on orderliness of microtubules, we focused on the interactions between the isolated N-terminal domain of Euplotes centrin (N-EoCen) and Ln by some combined biophysical and biochemical methods. Our results suggest that Ln ions may bind to the canonical calcium binding sites on N-EoCen. Taking advantage of ligand competition, we first determined the metal-binding affinities of Nd3+, Eu3+, Gd3+ and Tm3+ with N-EoCen. Major changes of N-EoCen in secondary and tertiary structure are noted while Ln ions bind with N-EoCen through CD spectra and 2-p-toluidinylnaphthalene-6-sulfonate (TNS) fluorescence. N-EoCen exists in the form of monomer and dimer in the presence of Ln ions. These results can provide some insights into the structural basis of how Ln ions achieve biological effect in cell through the centrin protein. [Copyright &y& Elsevier]

Abstract: The binding of Mg2+ with the Euplotes octocarinatus centrin (EoCen) and the effect of Mg2+ on the binding of EoCen with the peptide melittin were examined by spectroscopic methods. In this study, it was found that Mg2+ may bind with Ca2+-binding sites, at least partly, on EoCen, which displays ∼10-fold weaker affinity than Ca2+. In the presence of Mg2+, Ca2+-saturated EoCen undergoes significant conformational changes resulting in decreased exposure of hydrophobic surfaces on the protein. Additionally, excess Mg2+ did not change the stoichiometry, but rather reduced the affinity of EoCen to melittin. The Mg2+-dependent decrease in the affinities of EoCen to melittin is an intrinsic property of Mg2+, rather than a nonspecific ionic effect. The inhibitory effect of Mg2+ on the formation of complexes between EoCen and melittin may contribute to the specificity of EoCen in target activation in response to cellular Ca2+ concentration fluctuations. [Copyright &y& Elsevier]

Ciliate Euplotes octocarinatus centrin (EoCen) is an EF-hand calcium-binding protein closely related to the prototypical calcium sensor protein calmodulin. The first amino acid of the Ca2+-binding loops found in the EF-hand calcium-binding proteins is a highly conserved aspartic acid residue. The D37K mutant was produced to elucidate the metal binding role of the first aspartic acid of the EF-loop I of EoCen. Aromatic-sensitized Tb3+ fluorescence results indicated that the metal binding ability of loop I was lost due to the D37K mutation. Based on fluorescence titration curves of Lu2-D37K, the conditional binding constants of the EoCen loop II were quantitatively found to be KII = (1.61 ± 0.04) x 105 L mol-1 and KII = (3.52 ± 0.08) x 10² L-1 mol with Tb3+ and Ca2+, respectively. Using 2-p-toluidinylnaphthalene-6-sulfonate as a hydrophobic probe, exposure of the hydrophobic surface upon metal binding was found to be significantly reduced for the metal ion-saturated EoCen D37K mutant. [ABSTRACT FROM AUTHOR]

Abstract: A new species was formed when protein P23 (one segment of ciliate Euplotes octocarinatus centrin) was added to a solution of Eu3+. The interaction between P23 and Eu3+ was investigated by cyclic voltammetry, pulse voltammetry and electrochemical impedance spectroscopy in 10mM N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES) buffer (pH 7.4) using a pyrolytic graphite electrode. The formal potential (E o′) of Eu3+ shifted from −0.61 to −0.84V (versus saturated calomel electrode) after P23 was added to the Eu3+ solution. The diffusion coefficient (D), the charge-transfer coefficient (α) and the electron transfer standard rate constant (k s ) were obtained in the absence and the presence of P23. The affinity constant of Eu3+ and P23 was determined to be (1.89±0.51)×104 M−1. The electrochemical investigation of europium bound to the protein provided useful data for the studies of calcium-binding proteins. [Copyright &y& Elsevier]

Using laboratory microcosms, we studied direct and indirect interactions among different components of bi- and tritrophic communities. Filamentous cyanobacteria ( Phormidium sp.) and autotrophic flagellates ( Chlorogonium elongatum) were primary producers. The second trophic level was represented by ciliates Furgasonia blochmanni and Pseudomicrothorax dubius grazing on the filamentous cyanobacteria and two filter feeders, Euplotes octocarinatus and Stylonychia pustulata, feeding on the autotrophic flagellates. An oligochaete, Chaetogaster sp., was used as the top predator. An experiment was carried out for all combinations of two factors: (1) the identity of the cyanobacteria consumer ( Furgasonia or Pseudomicrothorax) and (2) the presence or absence of the top predator. Significant effects of the treatments on both the abundance of cyanobacteria and filter feeding ciliates and predator-induced defense in Phormidium and Euplotes were observed in a 36-day experiment. The experiment showed that the substitution of one species ( Furgasonia) for another ( Pseudomicrothorax) seemingly playing the same ecological role may lead to significant changes in the whole community. [ABSTRACT FROM AUTHOR]

Abstract: Centrin, an EF-hand calcium-binding protein with high homology to calmodulin (CaM), is an essential component for microtubule-organizing center (MTOC) in organisms ranging from algae and yeast to human. It plays an important structural role by contributing to the formation of Ca2+-sensitive contractile filaments and some super-molecular assemblies. Previous work suggests that the N-terminal domain of centrin especially its first 20-residue fragment, is required for the self-assembly of protein. Native polyacrylamide gel electrophoresis (native-PAGE), pull-down assay, fluorescence resonance light scattering (RLS) and yeast two-hybrid assay indicate that the C-terminal domain of Euplotes octocarinatus centrin (EoCen) also contributes to the centrin self-assembly besides its N-terminal domain in vivo and in vitro. On the basis of our results, a self-assembly mode of centrin, which is C-to-C as well as N-to-N (between C- and C-terminal domains as well as between N- and N-terminal domains), is put forward providing maybe some insights into the molecular mechanism of centrin functions in the cell. [Copyright &y& Elsevier]

Rab GTPases are crucial in the regulation of intracellular vesicular trafficking. A novel Rab GTPase gene, EoRab11a (GenBank accession no. ), was isolated and identified from Euplotes octocarinatus cells in this study. It contains an ORF of 696-bp nucleotides, encoding 231 amino acids with a calculated molecular weight of 26.8 kDa. Alignment of EoRab11a with other Rab11 proteins from other eukaryotes demonstrated that these proteins shared 53–61% identity at the amino acid level. The recombinant EoRab11a was expressed in Escherichia coli and purified by immobilized metal chelate affinity chromatography and iron chromatography. The GTPase activity of EoRab11a was 0.0024 min−1 detected by HPLC at 30 °C. Three mutations were generated at amino acids Ser21 and Gly22 positions in the G1 domain of EoRab11a. All three mutants, S21P, S21G and G22R, increased the GTPase activity in vitro. Immunofluorescence microscopy results indicated that EoRab11a was localized on the phagosomal membrane during phagocytosis of E. octocarinatus. These data show that EoRab11a possesses GTP hydrolysis activity and may participate in vesicle transport events during phagocytosis of E. octocarinatus. [ABSTRACT FROM AUTHOR]

Abstract: Centrin is a member of the EF-hand superfamily that plays critical role in the centrosome duplication and separation. In the present paper, we characterized properties of metal ions binding to Euplotes octocarinatus centrin (EoCen) by fluorescence spectra and circular dichroism (CD) spectra. Changes of fluorescence spectra and α-helix contents of EoCen proved that Tb3+ and Ca2+ induced great conformational changes of EoCen resulting in exposing hydrophobic surfaces. At pH 7.4, Ca2+ (and Tb3+) bond with EoCen at the ratio of 4:1. Equilibrium experiment indicated that Ca2+ and Tb3+ exhibited different binding capabilities for C- and N-terminal domains of protein. C-terminal domain bond with Ca2+ or Tb3+ ∼100-fold more strongly than N-terminal. Aromatic residue-sensitized Tb3+ energy transfer suggested that site IV bond to Tb3+ or Ca2+ more strongly than site III. Based on fluorescence titration curves, we reckoned the conditional binding constants of EoCen site IV quantitatively to be K IV =(1.23±0.51)×108 M−1 and K IV =(6.82±0.33)×105 M−1 with Tb3+ and Ca2+, respectively. Metal ions bond to EoCen in the order of IV>III>II, I. [Copyright &y& Elsevier]

Abstract: The interaction between 2-p-toluidinylnaphthalene-6-sulfonate (TNS) and ciliate Euplotes Octocarinatus centrin (Cen) has been studied by fluorescence spectroscopy. The binding constants of TNS with Cen were measured at different temperature in the 0.01M Hepes, pH 7.4. The binding process is exothermic and involves a positive entropy change. The negative value of enthalpy predominately contributes to the negative free energy of binding between TNS and Cen. The salt (KCl) increases the association constant of TNS and Cen. These results and resonance light scattering experiment suggest that the binding force between TNS and Cen is hydrophobic. The distance (r) between TNS and tryptophan of mutant G115W, which sheds more insight into the binding of TNS to Cen, was determined as 4.85nm based on Förster non-radiative energy transfer theory. [Copyright &y& Elsevier]

Abstract: In the presence of 1.0mM Ca2+, the interaction between Euplotes octocarinatus centrin (EoCen) and melittin (ME) was studied by means of fluorescence spectra. In 0.1M N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (Hepes) and 150mM NaCl at pH 7.4, fluorescence peak of ME was observed at about 353nm indicating that micro-environment of Tryptophan (Trp) residue in ME was hydrophilic. With the addition of 3.2×10−4 M calcium saturated EoCen (holoEoCen), the peak of ME was blue-shifted to 339nm, which may be resulted from micro-environmental changes of the peptide. At the same time, fluorescence emission of ME was increased significantly suggesting that new complex of ME−holoEoCen was formed under the experimental conditions. Based on the fluorescence titration curves, the 1:1 stoichiometric ratio of holoEoCen to ME was confirmed. In addition, the conditional binding constant of holoEoCen with ME was calculated to be log K ME−holoEoCen =6.59±0.14. [Copyright &y& Elsevier]

Abstract: Class II polypeptide release factor (eRF3), a ribosome and eRF1-dependent GTPase, is an important factor, which acts cooperatively with eRF1 to promote hydrolysis of the ester bond linking the polypeptide chain with the peptidyl site tRNA in process of termination of protein synthesis. We prepared antibodies against eRF3 of Euplotes octocarinatus, and performed localization studies by immunoelectron microscopy in the ciliate. Our results indicate that eRF3 is present both in the cytoplasm and the two types of nuclei of this organism. The functions of eRF3 in these nuclei were analyzed by RNA interference methods. The nuclei loose their shape in eRF3 gene-interfered Euplotes cells, suggesting that eRF3 is probably involved in the morphological organization of nuclei. This suggests that eRF3 is a multifunctional protein with roles additionals to its function in the process of termination of protein synthesis. [Copyright &y& Elsevier]

Interactions between model target peptide melittin (ME) and Euplotes octocarinatus centrin (EoCen) were investigated by fluorescence spectra, circular dichroism (CD) spectra and native polyacrylamide gel electrophoresis (PAGE). In 0.1 mol/L N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (Hepes) and 150 mmol/L NaCI at pH 7.4, EoCen and isolated short C-terminal domain of EoCen (SC-EoCen) form 1:1 peptide: protein complexes. However, no detectable signal changes can be observed while isolated N-terminal domain of EoCen (N-EoCen) or isolated long C-terminal domain of EoCen (LC-EoCen) was added into solution of ME. The interaction between EoCen and ME is specified exclusively for the short C-terminal domain of EoCen. On the basis of fluorescence titration curves, the conditional binding constants of ME with EoCen and SC-EoCen were calculated to be logKME-EoCen = 6.81±0.33 and logKME-EoCen = 6.51±0.45, respectively. [ABSTRACT FROM AUTHOR]

Abstract: Glu is highly conserved as the first amino acid of E-helix of the EF-hand protein. In this paper, Glu 101, the first amino acid of E-helix of the third EF-hand motif in Euplotes octocarinatus centrin (EoCen) was mutated to be Lys by the method of site direct mutation. Tb3+ and TNS were used as fluorescence probes in the study of the effect of this mutation to the metal binding characteristic of EoCen by fluorescence spectra. Results indicate that compared with EoCen, the mutation protein (E101K) displays a different Tb3+ binding characteristic and an increased hydrophobic exposure surface. Polyacrylamide gels electrophoresis indicated that the electrophoretic mobilities of EoCen and E101K are distinctly different. It can be deduced that the conformation of EoCen has been altered by this mutation. The general conditional binding constant of Tb3+ to the three loops of EF-hand sites I–III in E101K was calculated to be (5.64±0.57)×105 M−1 according to the modified equation of the single binding process. [Copyright &y& Elsevier]

Abstract: In 10mM N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (Hepes), pH 7.4, 25°C, the conformational change of the truncated form of ciliate Euplotes Octocarinatus centrin (P23) induced by metal ions were investigated using 2-p-toluidinylnaphthalene-6-sulfonate (TNS) as a probe. The results show that upon metal ions binding, P23 undergo a conformational change and the contributions to the conformational change from the two EF-hands are different, and Tb3+ has more larger influence than Ca2+ with the same concentration metal ions, which provide possible the evidence that the different EF-hands play distinct biological functions. Meanwhile, the conditional binding constants of TNS and Ca2-loaded or Tb2-loaded P23 were obtained, K (Ca2-P23 +TNS)=(7.49±0.88)×105 mol−1 L, K (Tb2-P23 +TNS)=(8.24±0.49)×105 mol−1 L. [Copyright &y& Elsevier]

Abstract: Translation termination in eukaryotes is mediated by two polypeptide chain-release factors, eRF1 and eRF3. eRF1 recognizes stop signals, while eRF3 is a ribosome-dependent and eRF1-dependent GTPase. eRF1 forms a stable complex with eRF3 in vivo and in vitro. In the present study, a variety of truncated forms of Euplotes octocarinatus eRF3 were created, and systematic analysis of the interaction between E. octocarinatus eRF1a and these eRF3 mutants was performed by employing both in vivo a yeast two-hybrid assay and in vitro a pull-down assay. The results demonstrated that a short portion of the C-terminal domain of eRF3 is sufficient for eRF1a binding in E. octocarinatus. Specifically, the eRF1a-binding sites on eRF3 are located at a region containing amino acid residues 640-723 in E. octocarinatus eRF3. Amino acid sequence analysis of eRF3 from E. octocarinatus, humans and yeast showed that the eRF1a binding domain on E. octocarinatus eRF3 was similar to that of yeast eRF3 but different from that of human eRF3. [Copyright &y& Elsevier]

Abstract: Class II polypeptide release factor eRF3 stimulates translation termination in a GTP-dependent manner. Recent data show that eRF3 is also involved in cell cycle regulation apart from its function in translation. Here we show that recombinant Eo-eRF3 from the ciliate Euplotes octocarinatus expressed in Escherichia coli can be tested for its ribosome-dependent GTPase activity using an in vitro system. Moreover, polyclonal antibodies against recombinant Eo-eRF3 were used to determine the localization of Eo-eRF3 using immunofluorescence and immunoelectron microscopy, which showed that the Eo-eRF3 is distributed not only around the macronucleus, but also in basal bodies in the cortex of E. octocarinatus cells. The results suggest that Eo-eRF3 may be involved in cytoskeleton organization in addition to its function in translation termination. [Copyright &y& Elsevier]

Rab proteins belong to the largest family of the Ras superfamily of small GTPase that play an important role in intracellular vesicular traffic. So far, almost 60 members of Rab family have been identified in mammalian cells. To further study the diversity and function of Rab protein in evolution, unicellular protozoa ciliates, Euplotes octocarinatus , were used in this study, Rab genes were screened by PCR method from macronuclear DNA of E. octocarinatus . Sixteen Rab genes were obtained. They share 87.6–99.5% identities. Highly conserved GTP-binding domains were found. There are some hot regions that diverse sharply in these genes as well. [ABSTRACT FROM AUTHOR]

Discusses the cloning and sequence analysis of the micronuclear and macronuclear gene encoding the Rab protein of Euplotes octocarinatus. Overview of Rab guanosine triphosphatases; Characteristics of Euplotes octocarinatus; Structure of the micronuclear version of the Rab gene.

Abstract We describe one of the first examples of reciprocal phenotypic plasticity in a predator–prey system: the interaction between an inducible defence and an inducible offence. When confronted with the predatory ciliate Lembadion bullinum , the hypotrichous ciliate Euplotes octocarinatus develops protective lateral wings, which inhibit ingestion by the predator. We show that L. bullinum reacts to this inducible defence by expressing an inducible offence – a plastic increase in cell size and gape size. This counteraction reduced the effect of the defence, but did not completely neutralize it. Therefore, the defence remained beneficial for E. octocarinatus . From L. bullinum 's point of view, the increase in feeding rate because of the offence was not larger than the increase in mean cell volume and apparently, did not increase the predator's fitness. Therefore, the inducible offence of L. bullinum does not seem to be an effective counter-adaptation to the inducible defence of E. octocarinatus . [ABSTRACT FROM AUTHOR]

Ciliates show two forms of reproduction: asexual (vegetative) and sexual reproduction (conjugation). Conjugation in Euplotes octocarinatus is induced by pheromones secreted by cells of one mating type binding to corresponding receptors on cells of a different mating type. In this study, we used heterologously-expressed pheromone 2 (Phr2) in affinity chromatography to isolate pheromone 2 binding proteins in Euplotes octocarinatus syngen 1. Using this approach, we isolated a 42-kDa protein (Phr2B) that bound Phr2. A tryptic fragment of Phr2B was used to construct degenerate oligonucleotides for PCR amplification of the macronuclear gene. The gene is predicted to encode a 58 amino acid protein. The N-terminus of the deduced protein shows significant similarity to membrane bound protein kinases of such diverse organisms as Xenopus laevis, Mus musculus and Homo sapiens, while the C-terminus contains a region which is similar to an extracellular region within chemosensory receptors of Caenorhabditis elegans. Western blotting analysis demonstrated that Phr2B specifically recognizes Phr2 and preliminary results indicate that antibodies raised against a C-terminal peptide of Phr2B inhibit cell mating. Further studies should be undertaken to investigate the precise role that Phr2B plays in preconjugative intercellular communication in E. octocarinatus. [Copyright &y& Elsevier]