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Motivation: Long-read sequencing technologies can be employed to detect and map DNA modifications at the nucleotide resolution on a genome-wide scale. However, published software packages neglect the integration of genomic annotation and comprehensive filtering when analyzing patterns of modified bases detected using Pacific Biosciences (PacBio) or Oxford Nanopore Technologies (ONT) data. Here, we present DNA Modification Annotation (DNAModAnnot), a R package designed for the global analysis of DNA modification patterns using adapted filtering and visualization tools. Results: We tested our package using PacBio sequencing data to analyze patterns of the 6-methyladenine (6mA) in the ciliate Paramecium tetraurelia, in which high 6mA amounts were previously reported. We found P. tetraurelia 6mA genome-wide distribution to be similar to other ciliates. We also performed 5-methylcytosine (5mC) analysis in human lymphoblastoid cells using ONT data and confirmed previously known patterns of 5mC. DNAModAnnot provides a toolbox for the genome-wide analysis of different DNA modifications using PacBio and ONT long-read sequencing data., SCOPUS: ar.j, info:eu-repo/semantics/published

Can ecological changes impact somatic genome development? Efforts to resolve this question could reveal a direct link between environmental changes and somatic variability, potentially illuminating our understanding of how variation can surface from a single genotype under stress. Here, we tackle this question by leveraging the biological properties of ciliates. When Paramecium tetraurelia reproduces sexually, its polyploid somatic genome regenerates from the germline genome through a developmental process that involves the removal of thousands of ORF-interrupting sequences known as internal eliminated sequences (IESs). We show that exposure to nonstandard culture temperatures impacts the efficiency of this process of programmed DNA elimination, prompting the emergence of hundreds of incompletely excised IESs in the newly developed somatic genome. These alternative DNA isoforms display a patterned genomic topography, impact gene expression, and might be inherited transgenerationally. On this basis, we conclude that environmentally induced developmental thermoplasticity contributes to genotypic diversification in Paramecium.

Ciliates are unicellular eukaryotes with both a germline genome and a somatic genome in the same cytoplasm. The somatic macronucleus (MAC), responsible for gene expression, is not sexually transmitted but develops from a copy of the germline micronucleus (MIC) at each sexual generation. In the MIC genome of Paramecium tetraurelia, genes are interrupted by tens of thousands of unique intervening sequences called internal eliminated sequences (IESs), which have to be precisely excised during the development of the new MAC to restore functional genes. To understand the evolutionary origin of this peculiar genomic architecture, we sequenced the MIC genomes of 9 Paramecium species (from approximately 100 Mb in Paramecium aurelia species to >1.5 Gb in Paramecium caudatum). We detected several waves of IES gains, both in ancestral and in more recent lineages. While the vast majority of IESs are single copy in present-day genomes, we identified several families of mobile IESs, including nonautonomous elements acquired via horizontal transfer, which generated tens to thousands of new copies. These observations provide the first direct evidence that transposable elements can account for the massive proliferation of IESs in Paramecium. The comparison of IESs of different evolutionary ages indicates that, over time, IESs shorten and diverge rapidly in sequence while they acquire features that allow them to be more efficiently excised. We nevertheless identified rare cases of IESs that are under strong purifying selection across the aurelia clade. The cases examined contain or overlap cellular genes that are inactivated by excision during development, suggesting conserved regulatory mechanisms. Similar to the evolution of introns in eukaryotes, the evolution of Paramecium IESs highlights the major role played by selfish genetic elements in shaping the complexity of genome architecture and gene expression., A comparative genomics study of nine Paramecium species reveals successful invasion of genes by transposable elements in their germline genomes, showing that the internal eliminated sequences (IESs) followed an evolutionary trajectory remarkably similar to that of spliceosomal introns.

Calcium ions (Ca2+) entering cilia through the ciliary voltage-gated calcium channels (CaV) during the action potential causes reversal of the ciliary power stroke and backward swimming in Paramecium tetraurelia. How calcium is returned to the resting level is not yet clear. Our focus is on calcium pumps as a possible mechanism. There are 23 P. tetraurelia genes for calcium pumps that are members of the family of plasma membrane Ca2+ ATPases (PMCAs). They have domains homologous to those found in mammalian PMCAs. Of the 13 pump proteins previously identified in cilia, ptPMCA2a and ptPMCA2b are most abundant in the cilia. We used RNAi to examine which PMCA might be involved in regulating intraciliary Ca2+ after the action potential. RNAi for only ptPMCA2a and ptPMCA2b causes cells to significantly prolong their backward swimming, which indicates that Ca2+ extrusion in the cilia is impaired when these PMCAs are depleted. We used immunoprecipitations (IP) to find that ptPMCA2a and ptPMCA2b are co-immunoprecipitated with the CaV channel α1 subunits that are found only in the cilia. We used iodixanol (OptiPrep) density gradients to show that ptPMCA2a and ptPMCA2b and CaV1c are found in the same density fractions. These results suggest that ptPMCA2a and ptPMCA2b are located in the proximity of ciliary CaV channels.

A human ciliopathy gene codes for Polycystin-2 (Pkd2), a non-selective cation channel. Here, the Pkd2 channel was explored in the ciliate Paramecium tetraurelia using combinations of RNA interference, over-expression, and epitope-tagging, in a search for function and novel interacting partners. Upon depletion of Pkd2, cells exhibited a phenotype similar to eccentric (XntA1), a Paramecium mutant lacking the inward Ca2+-dependent Mg2+ conductance. Further investigation showed both Pkd2 and XntA localize to the cilia and cell membrane, but do not require one another for trafficking. The XntA-myc protein co-immunoprecipitates Pkd2-FLAG, but not vice versa, suggesting two populations of Pkd2-FLAG, one of which interacts with XntA. Electrophysiology data showed that depletion and over-expression of Pkd2 led to smaller and larger depolarizations in Mg2+ solutions, respectively. Over-expression of Pkd2-FLAG in the XntA1 mutant caused slower swimming, supporting an increase in Mg2+ permeability, in agreement with the electrophysiology data. We propose that Pkd2 in P. tetraurelia collaborates with XntA for Mg2+-induced behavior. Our data suggest Pkd2 is sufficient and necessary for Mg2+ conductance and membrane permeability to Mg2+, and that Pkd2 is potentially a Mg2+-permeable channel.

International audience; DNA double-strand breaks (DSBs) induced by genotoxic agents can cause cell death or contribute to chromosomal instability, a major driving force of cancer. By contrast, Spo11-dependent DSBs formed during meiosis are aimed at generating genetic diversity. In eukaryotes, CtIP and the Mre11 nuclease complex are essential for accurate processing and repair of both unscheduled and programmed DSBs by homologous recombination (HR). Here, we applied bioinformatics and genetic analysis to identify Paramecium tetraurelia CtIP (PtCtIP), the smallest known Sae2/Ctp1/CtIP ortholog, as a key factor for the completion of meiosis and the recovery of viable sexual progeny. Using in vitro assays, we find that purified recombinant PtCtIP preferentially binds to double-stranded DNA substrates but does not contain intrinsic nuclease activity. Moreover, mutation of the evolutionarily conserved C-terminal 'RHR' motif abrogates DNA binding of PtCtIP but not its ability to functionally interact with Mre11. Translating our findings into mammalian cells, we provide evidence that disruption of the 'RHR' motif abrogates accumulation of human CtIP at sites of DSBs. Consequently, cells expressing the DNA binding mutant CtIPR837A/R839A are defective in DSB resection and HR. Collectively, our work highlights minimal structural requirements for CtIP protein family members to facilitate the processing of DSBs, thereby maintaining genome stability as well as enabling sexual reproduction.

Paramecium tetraurelia expresses four types of arginine kinase (AK1–AK4). In a previous study, we showed that AK3 is characterized by typical arginine substrate inhibition, where enzymatic activity markedly decreases near a concentration of 1 mM of arginine substrate. This is in sharp contrast to the three other AK types, which obey the Michaelis–Menten reaction curve. Since cellular arginine concentration in another ciliate Tetrahymena is estimated to be 3–15 mM in vivo, Paramecium AK3 likely functions in conditions that are strongly affected by substrate inhibition. The purpose of this work is to find some novel aspect on the kinetic mechanism of the substrate inhibition of Paramecium AK3 enzyme. Substrate inhibition kinetics for AK3 were analyzed using three models and their validity were evaluated with three static parameters (R2, AICc, and Sy.x). The most accurate model indicated that not only ES but also the SES complex reacts to form products, the latter being the complex with two substrates in the active center. The maximum reaction rate for the SES complex, VmaxSES = 30.4 µmol Pi/min/mg protein, was one-eighth of the ES complex, VmaxES = 241.7. The dissociation constant for the SES complex (KiSES: 0.34 mM) was two times smaller than that of the ES complex (KsES: 0.61 mM), suggesting that after the primary binding of the arginine substrate (ES complex formation), the binding of a second arginine to the secondarily induced inhibitory site is accelerated to form an SES complex with a lower VmaxSES. The same kinetics were used for the S79A, S80A, and V81A mutants. The results indicate that the S79 residue is significantly involved in the process of binding the second arginine substrate. Herein, the KiSES value was ten times (3.62 mM) the value for the wild-type (0.34 mM), weakening substrate inhibition. In contrast, VmaxES and VmaxSES values for the mutants decreased by one-third, except for the VmaxSES of the S79A mutant, which had a value that was comparable with the value for the wild-type.

5-methylcytosine DNA methylation regulates gene expression and developmental programming in a broad range of eukaryotes. However, its presence and potential roles in ciliates, complex single-celled eukaryotes with germline-somatic genome specialization via nuclear dimorphism, are largely uncharted. While canonical cytosine methyltransferases have not been discovered in published ciliate genomes, recent studies performed in the stichotrichous ciliate Oxytricha trifallax suggest de novo cytosine methylation during macronuclear development. In this study, we applied bisulfite genome sequencing, DNA mass spectrometry and antibody-based fluorescence detection to investigate the presence of DNA methylation in Paramecium tetraurelia. While the antibody-based methods suggest cytosine methylation, DNA mass spectrometry and bisulfite sequencing reveal that levels are actually below the limit of detection. Our results suggest that Paramecium does not utilize 5-methylcytosine DNA methylation as an integral part of its epigenetic arsenal.

Synthetic Pyrethroids are considered to be safe over other insecticides; however, data indicate that their use may pose risk to environmental biota, especially, aquatic organisms. Therefore, the aim of the present study was to investigate the effect of cypermethrin, a widely used insecticide and one of the most common contaminants in freshwater aquatic system on the oxidative stress biomarkers of the freshwater ciliate Paramecium tetraurelia. After the treatment of paramecium cells with increasing concentrations of cypermethrin (0.05, 0.5,1, 2�g/l), we followed up the growth kinetics, generation time and the response percentage. Also, we studied the variation in biomarkers of stress such as: Proteins, GSH content, GST and CAT activities. Our results showed a significant decrease in the proliferation of cell, we denote a difference of

International audience; Better understanding of uranyl–protein interactions is a prerequisite to predict uranium chemical toxicity in cells. The EF‐hand motif of the calmodulin site I is about thousand times more affine for uranyl than for calcium, and threonine phosphorylation increases the uranyl affinity by two orders of magnitude at pH 7. In this study, we confront X‐ray absorption spectroscopy with Fourier transform infrared (FTIR) spectroscopy, time‐resolved laser‐induced fluorescence spectroscopy (TRLFS), and structural models obtained by molecular dynamics simulations to analyze the uranyl coordination in the native and phosphorylated calmodulin site I. For the native site I, extended X‐ray absorption fine structure (EXAFS) data evidence a short U−Oeq distance, in addition to distances compatible with mono‐ and bidentate coordination by carboxylate groups. Further analysis of uranyl speciation by TRLFS and thorough investigation of the fluorescence decay kinetics strongly support the presence of a hydroxide uranyl ligand. For a phosphorylated site I, the EXAFS and FTIR data support a monodentate uranyl coordination by the phosphoryl group and strong interaction with mono‐ and bidentate carboxylate ligands. This study confirms the important role of a phosphoryl ligand in the stability of uranyl–protein interactions. By evidencing a hydroxide uranyl ligand in calmodulin site I, this study also highlights the possible role of less studied ligands as water or hydroxide ions in the stability of protein–uranyl complexes.

Summary In eukaryotes, small RNAs (sRNAs) have key roles in development, gene expression regulation, and genome integrity maintenance. In ciliates, such as Paramecium , sRNAs form the heart of an epigenetic system that has evolved from core eukaryotic gene silencing components to selectively target DNA for deletion. In Paramecium , somatic genome development from the germline genome accurately eliminates the bulk of typically gene-interrupting, noncoding DNA. We have discovered an sRNA class (internal eliminated sequence [IES] sRNAs [iesRNAs]), arising later during Paramecium development, which originates from and precisely delineates germline DNA (IESs) and complements the initial sRNAs ("scan" RNAs [scnRNAs]) in targeting DNA for elimination. We show that whole-genome duplications have facilitated successive differentiations of Paramecium Dicer-like proteins, leading to cooperation between Dcl2 and Dcl3 to produce scnRNAs and to the production of iesRNAs by Dcl5. These innovations highlight the ability of sRNA systems to acquire capabilities, including those in genome development and integrity.

We report the detailed evaluation of crucial parameters for chromatin immunoprecipitation (ChIP) of macronuclear DNA in the unicellular eukaryote Paramecium tetraurelia. Optimized parameters include crosslinking conditions, chromatin sonication and antibody titration thus providing a detailed protocol for successful ChIP in P. tetraurelia. As this ciliate is bacterivorous and RNAi by feeding represents a powerful tool for analysis of gene function, we moreover determined the effects of ingested nucleic acids by food bacteria. Feasibility of our protocol is demonstrated by characterisation of chromatin remodelling at promoters of cytosolic HSP70 isoforms during transcriptional activation under heat shock conditions by analyzing RNA abundance, nucleosome occupancy and levels of H3 lysine 9 acetylation.

The ciliate Paramecium tetraurelia contains four arginine kinase genes (AK1-4). We detected cDNA for only three of the AKs (AK1-3) via PCR. Recombinant AK1-4 were expressed in Escherichia coli and their kinetics parameters determined. AK3 showed typical substrate inhibition toward arginine, and enzymatic activity markedly decreased when arginine concentration increased. This is the first example of substrate inhibition in wild-type phosphagen kinases. To explore the substrate inhibition mechanism, site-directed mutations were generated, targeting the amino acid sequence D-D-S-Q-V at positions 77-81 in P. tetraurelia AK3. Among the mutants, substrate inhibition was lost remarkably in the S79A mutant. In spite of high amino acid sequence identity (91%) between P. tetraurelia AK3 and AK4, the enzymatic activity of AK4 was less by 3% than that of AK3. We noticed that the conservative G298 was unusually replaced by R in P. tetraurelia AK4, and we constructed two mutants, R298G/AK4 and G298R/AK3. Enzymatic activity of the former mutant was comparable with that of the wild-type AK3, whereas that of the latter mutant was dramatically reduced. Thus, we concluded that the significantly low activity of P. tetraurelia AK4 is due to the residue R298.

Summary In the ciliated protozoan Paramecium tetraurelia, Piwi-associated small RNAs are generated upon the elimination of tens of thousands of short transposon-derived DNA segments as part of development. These RNAs then target complementary DNA for elimination in a positive feedback process, contributing to germline defense and genome stability. In this work, we investigate the formation of these RNAs, which we show to be transcribed directly from the short (length mode 27 bp) excised DNA segments. Our data support a mechanism whereby the concatenation and circularization of excised DNA segments provides a template for RNA production. This process allows the generation of a double-stranded RNA for Dicer-like protein cleavage to give rise to a population of small regulatory RNAs that precisely match the excised DNA sequences. Video Abstract, Graphical Abstract, Highlights • In Paramecium, pieces of deleted DNA are transcribed to form regulatory RNAs • Ultra-short DNA segments are concatenated and circularized, allowing transcription • This concatenation is carried out by Ligase IV, which also repairs DNA ends • Concatenation is random, which leads to diversity in the resulting sRNA population, “Junk” DNA can be ligated into circles and transcribed to generate regulatory RNAs.

F1Fo-ATP synthases are universal energy-converting membrane protein complexes that synthesize ATP from ADP and inorganic phosphate. In mitochondria of yeast and mammals, the ATP synthase forms V-shaped dimers, which assemble into rows along the highly curved ridges of lamellar cristae. Using electron cryotomography and subtomogram averaging, we have determined the in situ structure and organization of the mitochondrial ATP synthase dimer of the ciliate Paramecium tetraurelia. The ATP synthase forms U-shaped dimers with parallel monomers. Each complex has a prominent intracrista domain, which links the c-ring of one monomer to the peripheral stalk of the other. Close interaction of intracrista domains in adjacent dimers results in the formation of helical ATP synthase dimer arrays, which differ from the loose dimer rows in all other organisms observed so far. The parameters of the helical arrays match those of the cristae tubes, suggesting the unique features of the P. tetraurelia ATP synthase are directly responsible for generating the helical tubular cristae. We conclude that despite major structural differences between ATP synthase dimers of ciliates and other eukaryotes, the formation of ATP synthase dimer rows is a universal feature of mitochondria and a fundamental determinant of cristae morphology.

Heavy metals constitute a source of environmental pollution. Here, novel functional hybrid biomaterials for specific interactions with heavy metals are designed by bioengineering consensus sequence repeats from spider silk of Nephila clavipes with repeats of a uranium peptide recognition motif from a mutated 33-residue of calmodulin protein from Paramecium tetraurelia. The self-assembly features of the silk to control nanoscale organic/inorganic material interfaces provides new biomaterials for uranium recovery. With subsequent enzymatic digestion of the silk to concentrate the sequestered metals, options can be envisaged to use these new chimeric protein systems in environmental engineering, including to remediate environments contaminated by uranium.

For using an organism for ecotoxicological studies, it is important to understand its model of growth under some physicochemical parameters that influence with a direct manner its development. The knowledge of the optimal parameters for the paramecia development leads to realize a model of growth with clear stages. For that, we tried to study the influence of temperature, pH, nutrient density in the culture medium and pollution on the growth ofParamecium tetraurelia. Obtained results showed that paramecia prefer definite conditions. However, the growth was better in temperatures included between 25 and 30°C, with preference to increased temperatures and a slightly acidic pH. The culture medium wasprepared from the mixture of several vegetables which the rates were studied depending to the growth. The combined effects of azoxystrobin and cyproconazole on the growth kinetics were studied. Results showed that treatment affected the population growth, the generation time and the velocity of generation. Key words: Paramecium tetraurelia, growth kinetics, physicochemical parameters, azoxystrobin, cyproconazole.

5-methylcytosine DNA methylation regulates gene expression and developmental programming in a broad range of eukaryotes. However, its presence and potential roles in ciliates, complex single-celled eukaryotes with germline-somatic genome specialization via nuclear dimorphism, are largely uncharted. While canonical cytosine methyltransferases have not been discovered in published ciliate genomes, recent studies performed in the stichotrichous ciliate Oxytricha trifallax suggest de novo cytosine methylation during macronuclear development. In this study, we applied bisulfite genome sequencing, DNA mass spectrometry and antibody-based fluorescence detection to investigate the presence of DNA methylation in Paramecium tetraurelia. While the antibody-based methods suggest cytosine methylation, DNA mass spectrometry and bisulfite sequencing reveal that levels are actually below the limit of detection. Our results suggest that Paramecium does not utilize 5-methylcytosine DNA methylation as an integral part of its epigenetic arsenal.

Paramecium tetraurelia displays a behavioral response to environmental stimuli that involves changes in the direction and speed of swimming. This paper demonstrates that there are periodic variations in the frequency of behavioral responses of individual cells. The frequency of the backward swimming response undergoes repeated oscillations with a period (tau) between 40 and 50 minutes; these oscillations are only slightly affected by the temperature at which the cells have been grown. These data indicate the presence of an ultradian clock that underlies this cellular process. However, growing cells in the presence of low concentrations of LiCl perturbs the ultradian rhythm, with the periodicity being reduced to approximately 11 minutes. The LiCl effect is seen within 10–45 minutes, depending on the concentration of LiCl used, and is completely reversible. In order to determine if the LiCl effect was due to a depletion of inositol, cells were grown in 10 mM myo-inositol for 24 hours prior to the addition o...

Programmed genome rearrangements drive functional gene assembly in ciliates during the development of the somatic macronucleus. The elimination of germline sequences is directed by noncoding RNAs and is initiated by DNA double-strand breaks, but the enzymes responsible for DNA cleavage have not been identified. We show here that PiggyMac (Pgm), a domesticated piggyBac transposase, is required for these rearrangements in Paramecium tetraurelia. A GFP-Pgm fusion localizes in developing macronuclei, where rearrangements take place, and RNAi-mediated silencing of PGM abolishes DNA cleavage. This is the first in vivo evidence suggesting an essential endonucleolytic function of a domesticated piggyBac transposase.

Paramecium tetraurelia is a unicellular eukaryotic ciliate. Certain strains of paramecia have been shown to contain endosymbiotic bacteria. Furthermore, some of these endosymbiotic bacteria confer a killing ability on the host paramecia, known as killer paramecia, because the endosymbiont can be lethal to other paramecia, known as sensitive paramecia, if it is released into the environment. This death has been connected to a protein found within the P. tetraurelia known as an R body. Interestingly, the R body-related-death resembles apoptosis in that it causes membrane blebbing, a characteristic of apoptosis. Furthermore, paramecia contain another marker of apoptosis, a mechanism to degrade DNA which is used in normal paramecia conjugation. Therefore, exposure to the R body and toxin may be activating some existing pathways in the paramecia, leading to apoptosis. To determine whether the mode of cell death induced by the R bodies is apoptotic, a flow cytometer was used to examine DNA fragmentati...

Classical studies in Metabolic Control Theory have shown that metabolic fluxes usually exhibit little sensitivity to changes in individual enzyme activity, yet remain sensitive to global changes of all enzymes in a pathway. Therefore, little selective pressure is expected on the dosage or expression of individual metabolic genes, yet entire pathways should still be constrained. However, a direct estimate of this selective pressure had not been evaluated. Whole-genome duplications (WGDs) offer a good opportunity to address this question by analyzing the fates of metabolic genes during the massive gene losses that follow. Here, we take advantage of the successive rounds of WGD that occurred in the Paramecium lineage. We show that metabolic genes exhibit different gene retention patterns than nonmetabolic genes. Contrary to what was expected for individual genes, metabolic genes appeared more retained than other genes after the recent WGD, which was best explained by selection for gene expression operating on entire pathways. Metabolic genes also tend to be less retained when present at high copy number before WGD, contrary to other genes that show a positive correlation between gene retention and preduplication copy number. This is rationalized on the basis of the classical concave relationship relating metabolic fluxes with enzyme expression.

Chemorepellents are compounds that cause ciliated protozoans to reorient their swimming direction. A number of chemorepellents have been studied in the ciliated protozoans, Paramecium and Tetrahymena. Chemorepellents, such as polycations, cause the organism to exhibit "avoidance behavior," a swimming behavior characterized by jerky movements and other deviations from normal forward swimming, which result from ciliary reversal. One well-characterized chemorepellent pathway in Tetrahymena is that of the proposed polycation receptor that is activated by lysozyme and pituitary adenylate cyclase activating polypeptide (PACAP). In this study, we compare the response of Paramecium to the chemorepellents lysozyme, vasoactive intestinal peptide (VIP), and PACAP to the previously studied polycation response in Tetrahymena. Our results indicate that lysozyme, VIP, and PACAP are all chemorepellents in Paramecium, just as they are in Tetrahymena. However, the signaling pathways involved appear to be different. While previous pharmacological characterization indicates that G-proteins are involved in polycation signaling in Tetrahymena, we present evidence that similar reception in Paramecium involves activation of a tyrosine kinase pathway in order for lysozyme avoidance to occur. Polycation responses of both organisms are inhibited by neomycin sulfate. While PACAP is the most effective of the three chemorepellents in Tetrahymena, lysozyme is the most effective chemorepellent in Paramecium.

The ciliates Tetrahymena and Paramecium respond to strong depolarizing stimuli with Ca(2+)-based action potentials, ciliary reversals, and consequent bouts of backward and forward swimming called "avoidance reactions" (ARs). We found that several representative tastants and odorants cause repetitive ARs in Tetrahymena and Paramecium at low (nM to microM) concentrations. Tetrahymena responded well to capsaicin, quinine, quinacrine, denatonium benzoate, eugenol, piperine, chloroquine, carvacrol, allyl isothiocyanate (AITC), and menthol. Chemosensory adaptation was seen with carvacrol, eugenol, quinacrine, and capsaicin. Cross-adaptation was seen between some of these compounds, suggesting possible similarities in their chemosensory transduction or adaptation pathways. Paramecium only responded well to AITC, quinacrine, piperine, and eugenol (with the effective concentration for 50% response [EC(50)] values in the microM range) while chemosensory adaptation was only seen to eugenol in Paramecium, suggesting possible species differences. Tetrahymena and Paramecium may have primitive receptors that can recognize these and other compounds or some of these compounds can act independently of specific receptors.

Precise analysis of the aquatic cells and their responses to the toxic chemicals, i.e., water disinfective agents, is of crucial importance due to their role in the ecosystem. We demonstrate the application of the droplets based millifluidic tool for isolating and longtime monitoring of single Paramecium tetraurelia cells using a large number of water-in-oil emulsion droplets. Due to the automated monitoring of the fluorescence signal, the droplets containing cells are distinguished from the empty reservoirs. A viability indicator is used to follow the metabolic dynamic of the cells in every single droplet. Finally, we perform ecotoxicity tests in droplets, exposing the encapsulated paramecia cells to silver nitrate for determination of EC50 levels, and compare the output with the conventional microtiter plate assay.

Tetrahymena thermophila and Paramecium tetraurelia are ciliates that reassign TAA and TAG from stop codons to glutamine codons. Because of the lack of full genome sequences, few studies have concentrated on analyzing the effects of codon reassignment in protein evolution. We used the recently sequenced genome of these species to analyze the patterns of amino acid substitution in ciliates that reassign the code. We show that, as expected, the codon reassignment has a large impact on amino acid substitutions in closely related proteins; however, contrary to expectations, these effects also hold for very diverged proteins. Previous studies have used amino acid substitution data to calculate the minimization of the genetic code; our results show that because of the lasting influence of the code in the patterns of substitution, such studies are tautological. These different substitution patterns might affect alignment of ciliate proteins, as alignment programs use scoring matrices based on substitution patterns of organisms that use the standard code. We also show that glutamine is used more frequently in ciliates than in other species, as often as expected based on the presence of the 2 new reassigned codons, indicating that the frequencies of amino acids in proteomes is mostly determined by neutral processes based on their number of codons.

Inhibitors of SERCA (sarcoplasmic/endoplasmie reticulum Ca 2+ -dependent ATPase) calcium pumps were used to investigate the involvement of internal Ca2+ stores in the GTP response in Paramecium. External application of these inhibitors was found to dramatically alter the typical behavioral and electrophysiological responses of Paramecium to extracellular chemical stimulation. In particular, 2,5-di-tert-butylhydroquinone (BHQ) strongly inhibited the backward swimming response of paramecia to externally applied GTP, though it did not inhibit the associated whirling response. BHQ also prolonged the normally brief electrophysiological response of these cells to GTP BHQ completely blocked the behavioral and electrophysiological responses of Paramecium to extracellular Ba 2+ , but had no measurable effect on the behavioral or electrophysiological responses of these cells to another depolarizing stimulus, elevated external K + concentration. These results suggest the involvement of nonciliary. Ca 2+ ions in the GTP and Ba 2+ responses.

Photoreactivation (PR) is an efficient survival mechanism that helps protect cells against the harmful effects of solar-ultraviolet (UV) radiation. The PR mechanism involves photolyase, just one enzyme, and can repair DNA damage, such as cyclobutane-pyrimidine dimers (CPD) induced by near-UV/blue light, a component of sunlight. Although the balance of near-UV/blue light and far-UV light reaching the Earth's surface could be altered by the atmospheric ozone layer's depletion, experiments simulating this environmental change and its possible effects on life have not yet been performed. To quantify the strength of UVB in sunlight reaching the Earth's surface, we measured the number of CPD generated in plasmid DNA after UVB irradiation or exposure to sunlight. To simulate the increase of solar-UV radiation resulting from the ozone layer depletion, Paramecium tetraurelia was exposed to UVB and/or sunlight in clear summer weather. PR recovery after exposure to sunlight was complete at a low dose rate of 0.2 J/m2 x s, but was less efficient when the dose rate was increased by a factor of 2.5 to 0.5 J/m2 x s. It is suggested that solar-UV radiation would not influence the cell growth of P. tetraurelia for the reason of high PR activity even when the ozone concentration was decreased 30% from the present levels.

Actions of cismethrin versus deltamethrin were compared using two functional attributes of rat brain synaptosomes. Both pyrethroids increased calcium influx but only deltamethrin increased Ca(2+)-dependent neurotransmitter release following K(+)-stimulated depolarization. The action of deltamethrin was stereospecific, concentration-dependent, and blocked by omega-conotoxin GVIA. These findings delineate a separate action for deltamethrin and implicate N-type rat brain Ca(v)2.2 voltage-sensitive calcium channels (VSCC) as target sites that are consistent with the in vivo release of neurotransmitter caused by deltamethrin. Deltamethrin (10(-7) M) reduced the peak current (approx. -47%) of heterologously expressed wild type Ca(v)2.2 in a stereospecific manner. Mutation of threonine 422 to glutamic acid (T422E) in the alpha(1)-subunit results in a channel that functions as if it were permanently phosphorylated. Deltamethrin now increased peak current (approx. +49%) of T422E Ca(v)2.2 in a stereospecific manner. Collectively, these results substantiate that Ca(v)2.2 is directly modified by deltamethrin but the resulting perturbation is dependent upon the phosphorylation state of Ca(v)2.2. Our findings may provide a partial explanation for the different toxic syndromes produced by these structurally-distinct pyrethroids.

SUMMARYParamecium tetraurelia is attracted to acetate and biotin by swimming smoothly and fast up gradients of these attractants, and turning immediately and slowing down when leaving these stimuli. We use a group of mutants, each with a different defect in an identified ion conductance, to show that these two stimuli open different ion channels, and the behaviors that occur upon application of stimulus (on-response) and removal of stimulus(off-response) have different roles in attraction to these two stimuli. The most important parameters for successful attraction to acetate are the on-response behaviors of fast swimming with few turns, and the mutants'behavior suggests that IK(Ca,h) is the conductance involved that initiates this behavior. IK(Ca,h or d)appears to be important to the on-response in biotin; the results with mutants suggest that the biotin off-response depolarization is initiated by an ICa, which can be large enough or close enough to channels to open IK(Ca,d), INa(Ca) and IMg(Ca).

Paramecium is known to proliferate faster under microgravity conditions, and slower under hypergravity. Experiments using axenic culture medium have demonstrated that hypergravity affected directly on the proliferation of Paramecium itself. In order to assess the mechanisms underlying the physiological effects of gravity on cell proliferation, Paramecium tetraurelia was grown under clinorotation (2.5 rpm) and the time course of the proliferation was investigated in detail on the basis of the logistic analysis. On the basis of the mechanical properties of Paramecium , this slow rate of the rotation appears to be enough to simulate microgravity in terms of the randomization of the cell orientation with respect to gravity. P. tetraurelia was cultivated in a closed chamber in which cells were confined without air bubbles, reducing the shear forces and turbulences under clinorotation. The chamber is made of quartz and silicone rubber film; the former is for the optically-flat walls for the measurement of cell density by means of a non-invasive laser optical-slice method, and the latter for gas exchange. Because of the small dimension for culture space, Paramecium does not accumulate at the top of the chamber in spite of its known negative gravitactic behavior. We measured the cell density at regular time intervals without breaking the configuration of the chamber, and analyzed the proliferation parameters by fitting the data to a logistic equation. As a result, P. tetraurelia showed reduced proliferation under slow clinorotation. The saturation of the cell density as well as the maximum proliferation rate decreased, although we found no significant changes on the half maximal time for proliferation. We also found that the mean swimming velocity decreased under slow clinorotation. These results were not consistent with those under microgravity and fast rotating clinostat. This may suggest that randomization of the cell orientation performed by slow rotating clinostat has not the same effect on Paramecium as that under microgravity that may affect the proliferation as the result of the reduced cost of propulsion.

Calmodulin (CaM) is the primary calcium signaling protein in eukaryotes and has been extensively studied using various biophysical techniques. Prior crystal structures have noted the presence of ambiguous electron density in both hydrophobic binding pockets of Ca2+-CaM, but no assignment of these features has been made. In addition, Ca2+-CaM samples many conformational substates in the crystal and accurately modeling the full range of this functionally important disorder is challenging. In order to characterize these features in a minimally biased manner, a 1.0 Å resolution single-wavelength anomalous diffraction data set was measured for selenomethionine-substituted Ca2+-CaM. Density-modified electron-density maps enabled the accurate assignment of Ca2+-CaM main-chain and side-chain disorder. These experimental maps also substantiate complex disorder models that were automatically built using low-contour features of model-phased electron density. Furthermore, experimental electron-density maps reveal that 2-methyl-2,4-pentanediol (MPD) is present in the C-terminal domain, mediates a lattice contact between N-terminal domains and may occupy the N-terminal binding pocket. The majority of the crystal structures of target-free Ca2+-CaM have been derived from crystals grown using MPD as a precipitant, and thus MPD is likely to be bound in functionally critical regions of Ca2+-CaM in most of these structures. The adventitious binding of MPD helps to explain differences between the Ca2+-CaM crystal and solution structures and is likely to favor more open conformations of the EF-hands in the crystal.

In Paramecium, the regeneration of a functional somatic genome at each sexual event relies on the elimination of thousands of germline DNA sequences, known as Internal Eliminated Sequences (IESs), from the zygotic nuclear DNA. Here, we provide evidence that IESs' length and sub-terminal bases jointly modulate IES excision by affecting DNA conformation in P. tetraurelia. Our study reveals an excess of complementary base pairing between IESs' sub-terminal and contiguous sites, suggesting that IESs may form DNA loops prior to cleavage. The degree of complementary base pairing between IESs' sub-terminal sites (termed Cin-score) is positively associated with IES length and is shaped by natural selection. Moreover, it escalates abruptly when IES length exceeds 45 nucleotides (nt), indicating that only sufficiently large IESs may form loops. Finally, we find that IESs smaller than 46 nt are favored targets of the cellular surveillance systems, presumably because of their relatively inefficient excision. Our findings extend the repertoire of cis-acting determinants for IES recognition/excision and provide unprecedented insights into the distinct selective pressures that operate on IESs and somatic DNA regions. This information potentially moves current models of IES evolution and of mechanisms of IES recognition/excision forward.

A 33-amino acid peptide corresponding to the helix-loop-helix motif of the calcium binding site I of the protein calmodulin from Paramecium Tetraurelia has been synthesized its binding properties with heavy metal ions have been studied. Herein, we demonstrate that two mutations of two aspartic acid residues in the peptide sequence gave access to a new peptide, which was selective for the uranyl ion UO2 2+. This new peptide can be useful for the development of selective uranyl biosensors to monitor the presence of uranium in contaminated environments.

From an mRNA differential-display analysis of the encystment-excystment cycle of the ciliate Sterkiella histriomuscorum , we have isolated an expressed sequence tag encoding a plasma membrane-type Ca 2+ -ATPase (PMCA). PMCAs are located either in the plasma membranes or in the membranes of intracellular organelles, and their function is to pump calcium either out of the cell or into the intracellular calcium stores, respectively. The S. histriomuscorum macronuclear PMCA gene ( ShPMCA ) and its corresponding cDNA were cloned; it is the first member of the Ca 2+ -ATPase family identified in Sterkiella . The predicted protein of 1,065 amino acids exhibits 37% identity with PMCAs of diverse organisms. A phylogenetic analysis showed its relatedness to homologs of two alveolates: the ciliate Paramecium tetraurelia and the apicomplexan Toxoplasma gondii . Overexpression of the protein ShPMCA failed to rescue the wild-type phenotype of three Ca 2+ -ATPase-defective mutant strains of Saccharomyces cerevisiae ; this failure contrasts with the reported ability of the PMCAs of parasites to complement defects in yeast. ShPMCA mRNA is markedly accumulated during encystment and in resting cysts, suggesting a function during excystment. To address the possibility of a signaling role for calcium at excystment, the capacity of calcium to induce excystment was examined.

Although Au(I) complexes have been used to treat rheumatoid arthritis for over 75 years, their mechanism of action is still poorly understood. A family of enzymes responsible for joint destruction in rheumatoid arthritis, the cathepsins, has been discussed as a possible biological target of Au(I). In this study, inhibition of the cathepsins by known Au(I) drugs and related compounds was investigated. The compounds tested inhibited cathepsin activity with IC50 values as low as 600 nM. More typical IC50 values were in the 50–200 μM range. Although the gold complexes are not extremely potent cathepsin inhibitors, it is likely that this inhibition is biologically relevant given the high concentrations of Au(I) in the serum and joints of patients undergoing chrysotherapy. While it is likely that there are multiple targets of Au(I) in vivo, inhibition of the cathepsins would provide protection against the joint destruction that is a hallmark of rheumatoid arthritis and is one possible mechanism for Au(I) antiarthritic activity.

Protein expression during the excystment of Colpoda cucullus was studied by SDS-PAGE. The expression levels of 60-, 50- and 49-kDa proteins were markedly changed from the early to later stage of excystment. The 60-kDa protein (p60) was temporarily expressed first, and its expression was inhibited by actinomycin D. LC-MS/MS analysis showed that the amino acid sequences of p60 partially coincided with those of the Paramecium tetraurelia unnamed protein homologous to DEAD-box RNA helicase. These results suggest that p60 expression is enhanced by transcriptional regulation and may be involved in initiating the molecular events leading to cellular morphogenesis.

Calmodulin (CaM) regulates a variety of cellular processes by interacting with a large number of proteins in a Ca(2+)-dependent manner. Conformational flexibility plays a key role in CaM function, although the full extent and detailed features of this flexibility are not fully characterized. Here, the 1.75 A resolution crystal structure of Pb(2+)-bound Paramecium tetraurelia CaM crystallized in a previously unobserved monoclinic lattice is reported. Pb(2+)-CaM is disordered in this new lattice and only a portion of each of the two molecules in the asymmetric unit can be modeled. Comparison of the structures of Ca(2+)-CaM and Pb(2+)-CaM show close agreement in the C-terminal domain but significant structural differences in the N-terminal domain. In addition, translation-libration-screw (TLS) refinement and Rosenfield difference analysis reveal inter-helical flexibility in the metal-bound N-terminal domain of the protein that is absent in the metal-bound C-terminal domain and indicates that the two structurally similar domains of CaM are dynamically distinct. These results demonstrate that TLS refinement and Rosenfield difference analysis allow detailed information about macromolecular flexibility to be extracted from X-ray diffraction data even when the crystal lattice prohibits full manifestation of this flexibility.

Peptides (33-34 amino acids long) corresponding to the helix-turn-helix (EF-hand) motif of the calcium binding site I of Paramecium tetraurelia calmodulin have been synthesized. The linear sequence was unable to acquire a native-like conformation and calcium binding. However, incorporation of a well-positioned disulfide bond bridging the two putative helical regions greatly improved the ordered structure and binding properties. Analyzed by electrospray mass spectrometry, circular dichroism and time-resolved laser-induced fluorescence, such a disulfide-stabilized peptide is shown to acquire a calcium-dependent helical conformation and exhibits native-like affinity for calcium, terbium and europium ions with 30+/-1, 3.5+/-0.6 and 0.6+/-0.1 microM dissociation constants, respectively. Comparable affinities were calculated within the biological construct comprising the entire domain I of Arabidopsis taliana calmodulin. Single sequence mutation (Glu25Asp) in the binding loop of the peptide abolishes calcium affinity, but preserves lanthanide affinity, showing that metal selectivity can be modulated by specific mutations. Such disulfide-stabilized peptides represent useful models to engineer metal specificity in new calmodulin proteins, facilitating the development of new systems to monitor metal pollution in biosensors and to increase metal binding capability of bacterial and plant cells in bioremediation techniques.

Summary Crystalline cytoplasmic inclusions were isolated by differential centrifugation from mass cultures of Paramecium tetraurelia feeding on Klebsiella pneumonia. Physical and chemical measurements of intact and solubilized crystals determined that they consist primarily of guanine and hypoxanthine with traces of xanthine. Crystals from the mutant sombre consist primarily of xanthine, suggesting there is a disorder of purine metabolism in this mutant.

Nonsense-mediated decay recognises mRNAs containing premature termination codons. One of its components, UPF3, is a molecular link bridging through its binding to the exon junction complex nonsense-mediated decay and splicing. In protists UPF3 has not been identified yet. We report that Paramecium tetraurelia bears an UPF3 gene and that it has a role in nonsense-mediated decay. Interestingly, the identified UPF3 has not conserved the essential amino acids required to bind the exon junction complex. Though, our data indicates that this ciliate bears genes coding for core proteins of the exon junction complex.

Summary Low concentrations (4 to 10 μM) of the trivalent cation gadolinium raised the input resistance and altered the membrane potential of Paramecium tetraurelia suggesting an interference with membrane channels. Current-clamp conditions revealed a concentration-dependent reduction of membrane rectification, in particular in the hyperpolarizing direction. The graded action potential and the early inward current seen under voltage-clamp conditions were depressed. Standardized applications of probes for focal mechanostimulation showed a reduction in mechanoreceptor potentials. Reversal responses of downward swimming cells in gadolinium transiently rose to higher frequency and settled at a reduced level thereafter explaining observations of a transient increase in gravitaxis. Vertical swimming speed was reduced, whereas the horizontal speed was largely unchanged. Gravity-induced modulation of swimming speed (gravikinesis) was depressed. The data indicate that gadolinium at micromolar concentrations effectively interferes with various types of membrane channels in Paramecium and is therefore no specific channel inhibitor to characterize graviresponses in this ciliate.

We analyzed preparative and analytical aspects of the dynamic localization of Ca(2+) during cell stimulation, using a combination of quenched flow and energy-dispersive X-ray microanalysis (EDX). Calcium (or Sr, as a substitute) was retained as fluorides during freeze-substitution, followed by epoxide embedding. The quenched-flow used allowed analyses, during stimulation, in the subsecond time range. Sections of 500 nm were analyzed and no artificial Ca or Sr leakage was recognizable. We calculated a primary beam spread from 63 to 72 nm that roughly indicated the resolution of EDX/structure correlation. These values are quite compatible with the size of potential structures of interest, e.g., Ca stores (approximately 100-nm thickness) or cilia (approximately 250-nm diameter). We used widely different standards to calibrate the ratio of CaK(alpha) net counts in relation to actual ¿Ca. Calibration curves showed a linear relationship and a detection limit of ¿Ca = 2 mM, while ¿Ca in cytosol was 3 mM and in stores was 43 mM, both in nonactivated cells. Eventually Sr(2+) can rapidly be substituted for Ca(2+) in the medium before and during stimulation, thus allowing one to determine Me(2+) fluxes. With our "model" cell, Paramecium, we showed that, upon stimulation (causing rapid Ca(2+) mobilization from subplasmalemmal stores), Ca was immediately exchanged for Sr in stores.

Type-II pyrethroids, including deltamethrin, are highly toxic to Paramecium tetraurelia, an organism that does not possess a voltage-sensitive sodium channel. Previous research has established that deltamethrin is toxic to P. tetraurelia in mortality bioassays at concentrations as low as 10−10 and 10−11 M under resting and depolarizing conditions, respectively. Deltamethrin, likewise, stimulated P. tetraurelia backward-swimming behavior, an avoidance behavioral response that is controlled exclusively by Ca2+ uptake via the voltage-sensitive calcium channels associated with the cilia. We have now characterized the action of various calcium channel agonists and antagonists on the avoidance behavior and Ca2+ influx in P. tetraurelia and have determined that the voltage-sensitive calcium channel associated with the cilia is blocked by the divalent cation Ni2+ but is insensitive to octanol and amiloride. Radioisotope tracer experiments, using whole cells under resting conditions, established that the toxic 1R isomer of deltamethrin resulted in increased Ca2+ influx, while the nontoxic 1S enantiomer produced no significant effect. Pawn mutants, which lack a functional voltage-sensitive calcium channel, were unaffected by deltamethrin. Fluorescent bioassays, under depolarizing conditions, corroborated behavioral and radioisotope experiments. Specifically, these experiments established that deltamethrin stimulated Ca2+ uptake in a stereospecfic manner and that this uptake was blocked by the phenethylamine-type calcium channel blocker D595 under physiological conditions. Deltamethrin treatment resulted in a dose-dependent increase in Ca2+ uptake and membrane depolarization with concentrations as low as 10−11 M. Electrophysiological recordings of whole cells showed that treatment of 10−9 M deltamethrin resulted in membrane destabilization, increased number of spontaneous action potentials, prolonged repetitive discharges following stimulation, membrane depolarization, and death by osmotic lysis. Our findings establish that the toxic effect of deltamethrin is structurally related, dose dependent, and enhanced by depolarization and provide substantial evidence that Type-II pyrethroids, specifically deltamethrin, act as potent calcium channel agonists on the ciliary voltage-sensitive calcium channel of P. tetraurelia.

A Paramecium cell responded to heat and cold stimuli, exhibiting increased frequency of directional changes in its swimming behavior. The increase in the frequency of directional changes was maintained during heating, but was transient during cooling. Although variations were large, as expected with this type of electrophysiological recording, results consistently showed a sustained depolarization of deciliated cells in response to heating. Depolarizations were also consistently observed upon cooling. However, these depolarizations were transient and not continuous throughout the cooling period. These depolarizations were lost or became small in Ca2+-free solutions. In a voltage-clamped cell, heating induced a continuous inward current and cooling induced a transient inward current under conditions where K+ currents were suppressed. The heat-induced inward current was not affected significantly by replacing extracellular Ca2+ with equimolar concentrations of Ba2+, Sr2+, Mg2+, or Mn2+, and was lost upon replacing with equimolar concentration of Ni2+. On the other hand, the cold-induced inward current was not affected significantly by Ba2+, or Sr2+, however the decay of the inward current was slowed and was lost or became small upon replacing with equimolar concentrations of Mg2+, Mn2+, or Ni2+. These results indicate that Paramecium cells have heat-activated Ca2+ channels and cold-activated Ca2+ channels and that the cold-activated Ca2+ channel is different from the heat-activated Ca2+ channel in the ion selectivity and the calcium-dependent inactivation.

Paramecium tetraurelia locate their␣foodsource by detecting bacterial metabolites and altering swimming behavior to congregate near bacterial populations on which they feed. Several attractants, such as folate, glutamate, cAMP and acetate have been identified and various aspects of chemoreception, signal transduction and effector mechanisms have been described. Here we characterize the Paramecium chemoresponse to biotin. An essential enzymatic cofactor in all cells, biotin is secreted by a large number of bacterial species during growth phase. P. tetraurelia are strongly attracted to biotin with a half-maximal behavioral response at 0.3 mmol · 1−1 in T-maze assays. Physiological recordings from whole cells show that cells hyperpolarize in a concentration-dependent manner in biotin. Whole-cell binding assays utilizing 3H-biotin identify a saturable and specific binding site with an apparent dissociation constant of 0.4 mmol · l−1. The biotin analogs desthiobiotin and biotin methyl ester are also strong attractants. Diaminobiotin fails to attract P. tetraurelia at 1 mmol · l−1, but does interfere with the biotin chemoresponse and displaces 3H-biotin from whole cells. We hypothesize that the keto group and/or fidelity of the ureido ring of biotin are necessary for biotin chemoresponse.

Considering increasing interest in calcium stores in protozoa, including parasitic forms, and specifically in subplasmalemmal stores in higher eukaryotes, we have isolated subplasmalemmal calcium stores (alveolar sacs) from the ciliated protozoan, Paramecium tetraurelia. Using antibodies against established sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCAs) we detected in Western blots of subcellular fractions a band of approximately 106 kDa size selectively in alveolar sacs--but not, for example, in plasma membranes--and concomitant restriction of immunofluorecence labelling to the cell cortex of permeabilised cells. These results are the same as with ABs against a peptide derived from a cloned SERCA-like gene from Paramecium [Hauser K., Pavlovic N., Kissmehl R., Plattner H. Molecular characterization of a sarco(endo)plasmic reticulum Ca(2+)-ATPase gene from Paramecium tetraurelia and localisation of its gene product to subplasmalemmal calcium stores. Biochem J 1998; 334: 31-38]. When such isolated alveolar sacs were now tested for phosphoenzyme intermediate (EP) formation, a phosphoprotein of the same apparent molecular mass (approximately 106 kDa) as in blots could be identified in gel autoradiograms. This EP corresponds to that formed in the reaction cycle of different SERCA-types, with dependency on Ca2+ and Mg2+, sensitivity to La3+ or insensitivity towards calmodulin, calmodulin antagonists and vanadate. However, EP formation in alveolar sacs is not inhibited by established SERCA inhibitors (e.g. thapsigargi[ci]n tested up to 100 microM). Surprisingly, caffeine, which is frequently used to mobilise Ca2+ from intracellular stores, strongly inhibits EP formation. In parallel experiments, we did not find any similar effect with sarcoplasmic reticulum isolated from skeletal muscle. We conclude that the approximately 106 kDa protein of alveolar sacs in Paramecium may represent a SERCA-like Ca(2+)-ATPase with some unorthodox features, which might be relevant also for some other protozoan systems. In this case, the established Ca(2+)-mobilizing effect of caffeine may be amplified by inhibiting store refilling.

The purine nucleotide GTP, when added extracellularly, induces oscillations in the swimming behaviour of the protist Paramecium tetraurelia. For periods as long as 10 min the cell swims backwards and forwards repetitively. The oscillations in swimming behaviour are driven by changes in membrane potential of the cell, which in turn are caused by periodic activation of inward Mg2+- and Na+-specific currents. We screened for and isolated mutants that are defective in this response, exploiting the fact that the net result of GTP on a population of cells is repulsion. One mutant, GTP-insensitive (gin A), is not repelled by GTP. In addition, GTP fails to induce repetitive backwards swimming in gin A mutants, although they swim backwards normally in response to other stimuli. GTP fails to evoke oscillations in membrane potential or Mg2+ and Na+ currents in the mutant, although the Mg2+ and Na+ conductances are not themselves measurably affected. A small, oscillating Ca2+ current induced by GTP in the wild type, which might be part of the mechanism that generates oscillations, is also missing from gin A cells. To our knowledge, gin A is the first example of a mutant defective in a purinergic response. We discuss the possibility that the gin A lesion affects the oscillator itself.

Paramecium tetraurelia contains high concentrations of ethanolamine sphingolipids, especially in its ciliary membrane. Three ethanolamine sphingophospholipids with different long chain bases (dihydrosphingosine, sphingosine and phytosphingosine), and their phosphonyl analogs, were previously identified and characterized. In the present study, radiolabeling experiments on lag- and log-phase cells were performed to investigate the extent of sphingolipid biosynthetic capacities of the ciliate. Long chain bases of sphingolipids are formed by an initial condensation reaction of serine with a fatty-coenzyme A. Thus, radiolabeled palmitic acid, stearic acid and serine were used as precursor compounds in these experiments. The results indicated that (1) sphingolipid precursors were incorporated into every major lipid fraction, (2) ethanolamine sphingophosphonolipids accumulated faster than the ethanolamine sphingophospholipids, (3) in contrast to these sphingolipids, the glycerolipid, phosphatidyethanolamine, accumulated faster than its phosphono analog, and (4) palmitic acid, but not stearic acid, was incorporated into the long chain bases of ethanolamine sphingophospho- and sphingophosphonolipids, consistent with an earlier report demonstrating that these lipids contain only C 18 long chain bases. Since P. tetraurelia takes up serine and other water-soluble substrates very slowly, and catabolizes fatty acids rapidly, label is randomized in intact cells. Thus, cell-free protocols provide useful experimental systems for studies of sphingolipid biosynthesis than do intact organisms, when the uptake of precursor substrates are slow.

Caffeine causes a [Ca2+]i increase in the cortex of Paramecium cells, followed by spillover with considerable attenuation, into central cell regions. From [Ca2+]resti approximately 50 to 80 nm, [Ca2+]acti rises within/=3 sec to 500 (trichocyst-free strain tl) or 220 nm (nondischarge strain nd9-28 degrees C) in the cortex. Rapid confocal analysis of wildtype cells (7S) showed only a 2-fold cortical increase within 2 sec, accompanied by trichocyst exocytosis and a central Ca2+ spread during the subsequent/=2 sec. Chelation of Ca2+o considerably attenuated [Ca2+]i increase. Therefore, caffeine may primarily mobilize cortical Ca2+ pools, superimposed by Ca2+ influx and spillover (particularly in tl cells with empty trichocyst docking sites). In nd cells, caffeine caused trichocyst contents to decondense internally (Ca2+-dependent stretching, normally occurring only after membrane fusion). With 7S cells this usually occurred only to a small extent, but with increasing frequency as [Ca2+]i signals were reduced by [Ca2+]o chelation. In this case, quenched-flow and ultrathin section or freeze-fracture analysis revealed dispersal of membrane components (without fusion) subsequent to internal contents decondensation, opposite to normal membrane fusion when a full [Ca2+]i signal was generated by caffeine stimulation (with Ca2+i and Ca2+o available). We conclude the following. (i) Caffeine can mobilize Ca2+ from cortical stores independent of the presence of Ca2+o. (ii) To yield adequate signals for normal exocytosis, Ca2+ release and Ca2+ influx both have to occur during caffeine stimulation. (iii) Insufficient [Ca2+]i increase entails caffeine-mediated access of Ca2+ to the secretory contents, thus causing their decondensation before membrane fusion can occur. (iv) Trichocyst decondensation in turn gives a signal for an unusual dissociation of docking/fusion components at the cell membrane. These observations imply different threshold [Ca2+]i-values for membrane fusion and contents discharge.