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PubMed1993

Nuclear magnetic resonance solution structure of the pheromone Er-10 from the ciliated protozoan Euplotes raikovi.

The three-dimensional structure in solution of the pheromone Er-10 from the ciliated protozoan Euplotes raikovi has been determined by nuclear magnetic resonance spectroscopy. The structure of this 38-residue protein was obtained from 384 nuclear Overhauser enhancement distance constraints and 78 dihedral angle constraints using the distance geometry program DIANA for the structure calculation and the program AMBER for energy minimization. For a group of 20 conformers used to characterize the solution conformation, the average root-mean-square distance calculated for the backbone heavy atoms relative to the mean structure was 0.33 A. The structure includes three short helices of residues 2 to 8, 12 to 19 and 24 to 33, and a turn in the carboxy-terminal region of residues 34 to 38. These structural elements are held together by three disulfide bridges. The structure is quite stable relative to heat denaturation, since at both pH 4.6 and pH 6.0 only minor changes in the circular dichroism and nuclear magnetic resonance spectra were observed over the temperature range 20 to 80 degrees C. The surface of the Er-10 structure shows an asymmetric charge distribution that results in a predominantly apolar surface on one side of the molecule. There is also a deep cleft in the structure with an asymmetric distribution of charged and apolar residues on the two walls. These surface features may be important for the homologous (autocrine) and heterologous binding of the pheromone to receptors.

Amino Acid Sequence↗
PubMed2001

NMR structure of the Euplotes raikovi pheromone Er-23 and identification of its five disulfide bonds.

The NMR solution structure of the 51 residue pheromone Er-23 from the ciliated protozoan Euplotes raikovi (Er) was calculated with the torsion angle dynamics program DYANA from 582 nuclear Overhauser enhancement (NOE) upper limit distance constraints, 46 dihedral angle constraints and 30 disulfide bond constraints. The disulfide bridges had not been assigned by chemical methods, and initially were assigned tentatively on the basis of inspection of the positioning of the Cys sulfhydryl groups in a bundle of 20 conformers that was calculated without disulfide bond constraints. The assignment of disulfide bridges was then validated by structure calculations that assessed the compatibility of plausible alternative Cys-Cys disulfide combinations with the input of NOE upper distance constraints and dihedral angle constraints. For a group of 20 conformers used to characterize the solution structure, the average pairwise root-mean-square distances from the mean coordinates calculated for the backbone heavy atoms N, C(alpha) and C' of resideus 1-51 is 0.38 A. The molecular architecture consists of a three-dimensional arrangement of five helices comprised of residues 2-8, 14-17, 26-29, 34-36 and 38-47, with five disulfide bridges in the positions 3-24, 6-16, 13-47, 27-40, and 35-51, which has so far not been represented in the Protein Data Bank. Er-23 is unique among presently known Er-pheromones with respect to size, sequence, the number of disulfide bonds and the three-dimensional structure, thus providing a new structural basis for rationalizing the physiological functions of this protein family.

Amino Acid Sequence↗
PubMed1993

Electrical responses of the marine ciliate Euplotes vannus (hypotrichia) to mechanical stimulation at the posterior cell end.

Electrical responses upon mechanostimulation at the posterior cell end were investigated in the marine hypotrichous ciliate Euplotes vannus. A new mechanostimulator was developed to mimic stimuli that are identical with those involved in cell-cell collisions. The receptor potential hyperpolarized by 18-35 mV within 12-25 msec, reached a peak value of -62 mV with a delay of 4-9 msec after membrane deformation, and was deactivated after 50-70 msec. Cirri were stimulated to beat accelerated backward. The corresponding receptor current exerted a similar time course with a peak of 2.4 nA. The shift of the reversal potential by 57.6 mV at a tenfold increase of [K+]o identifies potassium ions as current carriers within the development of the receptor potential. An intracellular K concentration of 355 mmol/liter was calculated for cells in a medium that was composed similar to sea-water. The mechanically activated potassium current was totally inhibited by extracellular TEA and intracellular Cs+, and partially inhibited by extracellular 4-AP. The total inhibition of the current by injected EGTA points to a Ca dependence of the posterior mechanosensitivity. It was confirmed by the increase of the peak current amplitude with rising [Ca2+]o. Sodium presumably repolarizes the receptor potential because the repolarization was delayed and after-depolarizations were eliminated in media without sodium. Since deciliation did not affect mechanosensitivity, the corresponding ion channels reside within the soma membrane.

Animals↗
PubMed1993

Inward rectification by hyperpolarization-activated Na current in the marine ciliate Euplotes vannus.

The ionic mechanisms underlying inward or anomalous rectification have been studied in the marine hypotrichous ciliate Euplotes vannus. Inward-current pulses of moderate amplitude elicited time-dependent rectification that started from a hyperpolarization peak and was expressed as a depolarizing sag towards rest. Voltage-clamp analysis showed that this depolarization is caused by the activation of a complex inward current that does not inactivate with time. The current is carried by a major Na and a minor K component. The Na-current component has been identified by its concentration-dependent reduction in low extracellular Na solutions and the capability of Li+ as Na substitute to carry the current, though with a slightly reduced amplitude. The K-current component has been isolated from the total current after the replacement of Na+ within the experimental solution. It was blocked in media that contained 10 mmol/liter TEA, a well-known blocker for K inwardly rectifying currents. TEA was only effective at membrane potentials close to or negative to the potassium equilibrium potential. The inward current was reduced after the injection of the Ca chelator EGTA into the cell. Also the elimination of the ciliary membrane, by deciliating cells with ethanol, reduced the amplitude of the inwardly rectifying currents. Both experiments indicate a regulatory function of Ca2+ in inward rectification.

Animals↗
PubMed1993

Inaccessibility of the Euplotes telomere binding protein.

The telomere binding protein (TP) from the macronucleus of the ciliate Euplotes eurystomus was purified by removal of tenaciously bound DNA with hydroxylapatite, and the purified TP partially sequenced. Rabbit antiserum was generated against a synthetic peptide of 14 amino acids at the amino-terminus of the TP. This antiserum was employed to examine the accessibility of TP antigenic determinants in nuclei and chromatin. Immunofluorescent staining of isolated macronuclei revealed only weak reactivity with specific antiserum. Reactivity within replication bands was demonstrated, and could be augmented by preparation of nuclear scaffolds. Employing a dot immunoblot analysis, the amino-terminal antigenic determinants of TP were revealed after extraction of histone H1 (and some nonhistones). A different aspect of TP inaccessibility was demonstrated by immunoblot analysis of trypsin-treated macronuclei and chromatin; TP was considerably less susceptible to digestion by trypsin than were histones H1 and H3. The relative inaccessibility of TP was not a consequence of chromatin higher-order structure, since soluble macronuclear chromatin in low salt exhibited the same burying of antigenic determinants by dot blot analysis, and the same decreased susceptibility to trypsin, as did isolated nuclei. Electron microscopy of soluble macronuclear chromatin spread in low salt revealed that most telomeres appear unfolded, without stable higher-order structure. The mechanisms for the relative inaccessibility of TP are not yet known, but probably arise as a consequence of the strong interactions of TP with the telomere nucleotide sequence and additional interactions of TP with various chromatin proteins, perhaps including histone H1.

Amino Acid Sequence↗
PubMed1996

Timing of differential amplification of macronucleus-destined sequences during macronuclear development in the hypotrichous ciliate Euplotes crassus.

The change in copy numbers of macronucleus-destined gene sequences was followed in anlagen DNA during postconjugational development in Euplotes crassus. As noted earlier, copy numbers increase during the polytene stage. During this replication process major differential amplification of different genes is not observed. Instead it is only achieved during or shortly after the fragmentation of the polytene chromosomes. This process is not totally synchronous with respect to different genes. Highly amplified genes are excised earlier than genes with a low final macronuclear copy number. Unexpectedly, the pattern of processing of the newly added oversized telomeres also appears to correlate with the degree of gene amplification. These observations are discussed in terms of a limited replication period after polytene chromosome fragmentation leading to preferential amplification of early excised genes.

Animals↗
PubMed2002

Characterization of the cold-adapted alpha-tubulin from the psychrophilic ciliate Euplotes focardii.

Tubulin dimers of psychrophilic organisms can polymerize into microtubules at temperatures below 4 degrees C, at which non-cold-adapted microtubules disassemble. This capacity requires specificities in the structure and/or in the posttranslational modifications of the tubulin subunits. A contribution to the knowledge of these specificities was provided by the finding that the amino acid sequence of the alpha-tubulin of the Antarctic ciliate Euplotes focardii contains substitutions that, in addition to conferring an increased hydrophobicity to the molecule, modify sites that are involved in alpha-/alpha-tubulin lateral contacts between protofilaments. At the level of the coding sequence, the alpha-tubulin gene of E. focardii revealed an A+T content appreciably higher than in its homologs in ciliates of temperate waters. This was interpreted as an adaptation to favor DNA strand separation in an environment which is energetically adverse.

Adaptation, Physiological↗
PubMed2006

Cytotoxic effects and apoptotic signalling mechanisms of the sesquiterpenoid euplotin C, a secondary metabolite of the marine ciliate Euplotes crassus, in tumour cells.

Most antitumour agents with cytotoxic properties induce apoptosis. The lipophilic compound euplotin C, isolated from the ciliate Euplotes crassus, is toxic to a number of different opportunistic or pathogenic microorganisms, although its mechanism of action is currently unknown. We report here that euplotin C is a powerful cytotoxic and pro-apoptotic agent in mouse AtT-20 and rat PC12 tumour-derived cell lines. In addition, we provide evidence that euplotin C treatment results in rapid activation of ryanodine receptors, depletion of Ca2+ stores in the endoplasmic reticulum (ER), the release of cytochrome c from the mitochondria, activation of caspase-12, and activation of caspase-3, leading to apoptosis. Intracellular Ca2+ overload is an early event which induces apoptosis and is parallelled by ER stress and the release of cytochrome c, whereas caspase-12 may be activated by euplotin C at a later stage in the apoptosis pathway. These events, either independently or concomitantly, lead to the activation of the caspase-3 and its downstream effectors, triggering the cell to undergo apoptosis. These results demonstrate that euplotin C may be considered for the design of cytotoxic and pro-apoptotic new drugs.

Animals↗
PubMed1993

Structures of the Euplotes crassus Tec1 and Tec2 elements: identification of putative transposase coding regions.

The Tec1 and Tec2 transposon-like element families of Euplotes crassus are highly unusual in that all 30,000 copies of each family are excised from the genome during a discrete time period of macronuclear development. Complete nucleotide sequences were generated for the Tec1-1 and Tec2-1 elements, representing the Tec1 and Tec2 families. Open reading frames (ORFs) are conserved in position and sequence between the two elements, although sequences that comprise one ORF (ORF2) of Tec1-1 are split into two overlapping ORFs (ORFs 2A and 2B) in Tec2-1. ORF1 in Tec1-1, its homolog in Tec2-1 and one of the overlapping ORFs from Tec2-1 (ORF2B) contain TGA codons, which may be translated as Cys, as observed for two other Euplotid genes. Sequence analyses of ORFs from other members of each element family indicate that the families are distinct from each other and are highly conserved within each family. Computer searches of sequence databases have revealed sequence similarity between Tec ORF1s and the previously described Tc1-IS630 family of transposases which includes ORFs from bacterial, nematode and insect transposons.

Amino Acid Sequence↗
PubMed1993

The macronuclear gamma-tubulin-encoding gene of Euplotes octocarinatus contains two introns and an in-frame TGA.

The gamma-tubulin (gamma-Tub)-encoding gene (gamma-tub) of Euplotes octocarinatus was amplified from macronuclear DNA with the help of the polymerase chain reaction (PCR) and sequenced. The polypeptide deduced from the gene consists of 462 amino acids (aa). It shares 61% aa identity with the Aspergillus nidulans gamma-Tub. The gene contains an in-frame TGA codon and two small pre-mRNA introns (36 and 26 bp). We suggest that the TGA, like TGA codons in the pheromone-encoding genes of E. octocarinatus, codes for a cysteine. This suggestion is supported by the finding that in the gamma-Tub of other organisms, a cysteine is located at this position. Sequencing the mRNA revealed that the introns are absent from the gamma-tub transcripts. The second intron constitutes the shortest one reported so far. We have also sequenced the 5'- and 3'-flanking regions of the gene up to the telomeres and report here the entire sequence of the macronuclear DNA molecule carrying gamma-tub.

Amino Acid Sequence↗
PubMed1994

Sequence of the macronuclear DNA encoding large subunit ribosomal protein 29 (L29) in Euplotes crassus and cycloheximide sensitivity.

As a first step towards developing a DNA transformation method for the ciliated protozoan Euplotes crassus we determined the minimum inhibitory concentration (MIC) for cell division in the presence of cycloheximide (Chx) for several cell lines and the range of Chx sensitivity for 106 different progeny cell lines derived by mating two lines. All of the cell lines are highly sensitive to Chx. Progeny cell lines show a wider range of sensitivities than the parental lines. Because site-directed mutagenesis of the RPL29 gene encoding the large subunit ribosomal protein 29 (L29) has been used to generate a Chx-resistance marker (ChxR) for another ciliate, Tetrahymena thermophila [Yao and Yao, Proc. Natl. Acad. Sci. USA 88 (1991) 9493-9497], we isolated and sequenced the entire E. crassus macronuclear DNA carrying RPL29. The encoded peptide is 52-73% identical in sequence to L29 sequences from organisms ranging from T. thermophila and Saccharomyces cerevisiae to mouse. In E. crassus, the codon that has been mutated to confer Chx resistance in both S. cerevisiae and T. thermophila already encodes the amino-acid residue of one of the mutant forms identified in these other organisms. Thus, E. crassus RPL29 is not a convenient source of a selectable marker. Notable features of the macronuclear DNA carrying RPL29 are its extremely short non-coding regions and a TAG stop codon.

Amino Acid Sequence↗
PubMed1994

Expression of the pheromone 3-encoding gene of Euplotes octocarinatus using a novel bacterial secretion vector.

The pheromone 3-encoding gene (phr3) of Euplotes octocarinatus was expressed in Escherichia coli using a novel expression-secretion vector. The vector, pExSec1, contains a strong and tightly regulated T7 promoter, the corresponding Shine-Dalgarno sequence and the T7 terminator region. Translation starts at the protein A leader sequence followed by the synthetic ZZ sequence of protein A. The expression-secretion modules are embedded in the high-copy-number plasmid vector, pUK21, which carries a kanamycin-resistance marker (KmR). The produced ZZ-pheromone 3 (Phr3) fusion protein was secreted into the culture medium of the host cells. It was isolated by affinity chromatography and was further purified by gel filtration. After refolding with protein disulfide isomerase (PDI), the fusion protein exhibited the same high activity as the native pheromone.

Animals↗
PubMed2004

Cell cycle and behaviour in Euplotes crassus: an attempt at describing the biological relationship between its cellular and organismic natures.

The two-faced nature of protozoa allowed us to study the relationship between cell cycle and behaviour in Euplotes crassus; the former represents one of the most typical cellular traits, the latter is one of the most significant characteristics of an organism. Dividing cells of E. crassus were isolated on a slide and recorded for 11 h: the classic ethographic parameters were calculated every 60 min. The percentage of mobile cells went from 0-100% in the first 2.5 h. This value was maintained for 6.5 h, but from 9 h the value began to drop, reaching 0% at 11 h. The relative frequency of leftward arcs was very high in the first hour, the radius and length of the arcs increased from 1-7 h; velocity showed a similar trend, peaking at 7 h. All our results showed that the behaviour of this ciliate is heavily affected by its cell-division cycle.

Animals↗
PubMed2005

Expression, characterization and immunolocalization of translation termination factor eRF3 in the ciliate Euplotes octocarinatus.

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.

Animals↗
PubMed2006

Identification of translational release factor eRF1a binding sites on eRF3 in Euplotes octocarinatus.

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.

Amino Acid Sequence↗
PubMed2006

The spectral studies on the effect of Glu 101 to the metal binding characteristic of Euplotes octocarinatus centrin.

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)x10(5)M(-1) according to the modified equation of the single binding process.

Animals↗
PubMed2003

Cross-talk between the autocrine (mitogenic) pheromone loop of the ciliate Euplotes raikovi and the intracellular cyclic AMP concentration.

Cell type-specific protein signals, called pheromones, are constitutively secreted by Euplotes raikovi and bound back in autocrine fashion, with a positive effect on the vegetative (mitotic) cell growth. In cells growing suspended with their secreted pheromone, it was found that any interruption of this autocrine signaling loop was immediately followed by an effective enhancement of the basal intracellular cyclic AMP (cAMP) level. To establish a cause-effect relationship between these pheromone-induced variations in the cytoplasmic cAMP level and cell growth, cells ready to pass from a resting stage to a new growth cycle were conditioned either to incorporate a cAMP analog resistant to phosphodiesterase degradation, or to utilize cAMP released (following cell irradiation) from incorporated "caged" cAMP. Cells responded at every induced increase in their basal cAMP level by markedly decreasing their commitment to start a new growth cycle. It was deduced that the autocrine signaling of E. raikovi pheromones involves cAMP as inhibitor of its mitogenic activity.

Adenylyl Cyclases↗
PubMed2002

Structural characterization of a protein pheromone from a cold-adapted (Antarctic) single-cell eukaryote, the ciliate Euplotes nobilii.

Free-living species of ciliated Protozoa control their vegetative (mitotic) proliferation and mating (sexual) processes by diffusible, cell type-specific protein signals (pheromones). One of these molecules, designated En-2, was isolated from a species, Euplotes nobilii, living in the stably cold marine waters of Antarctica, and its complete amino acid sequence of 60 residues was determined by automated Edman degradation of the whole protein and peptides generated by trypsin digestion. The proposed sequence is: DIEDFYTSETCPYKNDSQLA(20)WDTCSGGTGNCGTVCCGQCF(40)SFPVSQSCAGMADSNDCPNA(60). The En-2 structure appears to be characterized by an adaptive insertion of a glycine-rich motif potentially capable to confer more flexibility to a functionally critical region of the molecule.

Amino Acid Motifs↗