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PubMed1991

Cysteine residue periodicity is a conserved structural feature of variable surface proteins from Paramecium tetraurelia.

The DNA sequences of the entire coding regions of the A and C type variable surface protein genes from Paramecium tetraurelia, stock 51 have been determined. The 8151 nucleotide open reading frame of the A gene contains several tandem repeats of 210 nucleotides within the central portion of the molecule as well as a periodic structure defined by cysteine residues. The 6699 nucleotide open reading frame of the C gene does not contain any identifiable tandem repeats or internal similarity but maintains a periodicity based on the cysteine residue spacing. The deduced amino acid sequences encoded by the two genes are most similar within the 600 amino-terminal and 600 carboxyl-terminal amino acid residues, the central portions show only limited sequence similarity. We conclude that internal repeats are not a conserved feature of variable surface proteins in Paramecium and discuss the possible importance of the regular pattern of cysteine residues.

Amino Acid Sequence↗
PubMed1990

Clonal lifespans cultured in chemically defined medium and conventional bacterized medium in Paramecium octoaurelia.

The unicellular ciliate, paramecium, reproduces by binary fission, but can not continue to divide unlimitedly without sexual reproduction. We examined the clonal life span of Paramecium octaurelia stock 299 cultured in conventional bacterized medium (BM) and a chemically defined medium (DM). The cells that lived in BM divided 300 times. Although the cells in DM divided more slowly, some cells continued to divide more than 100 times. The mean life span of 90 cell lines cultured in BM was 151 +/- 49 fissions and that of 84 cell lines in DM was 68 +/- 28. When some older cells, which had been cultured in DM, were transferred to BM, most of them showed much longer life spans than those remaining in DM. The results showed that the life spans of cell clones were affected by the culture conditions.

Animals↗
PubMedDate not supplied

In vivo Paramecium mutants show that calmodulin orchestrates membrane responses to stimuli.

Paramecium generates a Ca2+ action potential and can be considered a one-cell animal. Rises in internal [Ca2+] open membrane channels that specifically pass K+, or Na+. Mutational and patch-clamp studies showed that these channels, like enzymes, are activated by Ca(2+)-calmodulin. Viable CaM mutants of Paramecium have altered transmembrane currents and easily recognizable eccentricities in their swimming behavior, i.e. in their responses to ionic, chemical, heat, or touch stimuli. Their CaMs have amino-acid substitutions in either C- or N-terminal lobes but not the central helix. Surprisingly, these mutations naturally fall into two classes: C-lobe mutants (S101F, I136T, M145V) have little or no Ca(2+)-dependent K+ currents and thus over-react to stimuli. N-lobe mutants (E54K, G40E+D50N, V35I+D50N) have little or no Ca(2+)-dependent Na+ current and thus under-react to certain stimuli. Each mutation also has pleiotropic effects on other ion currents. These results suggest a bipartite separation of CaM functions, a separation consistent with the recent studies of Ca(2+)-ATPase by Kosk-Kosicka et al. [41, 55]. It appears that a major function of Ca(2+)-calmodulin in vivo is to orchestrate enzymes and channels, at or near the plasma membrane. The orchestrated actions of these effectors are not for vegetative growth at steady state but for transient responses to stimuli epitomized by those of electrically excitable cells.

Amino Acid Sequence↗
PubMed1993

A calcium influx is neither strictly associated with nor necessary for exocytotic membrane fusion in Paramecium cells.

Exocytosis of trichocysts in Paramecium cells was generally believed to depend on extracellular Ca, since it is accompanied by a Ca influx and not seen in the absence of Ca. However, by short term removal of Ca we showed recently that only extrusion of secretory contents, but not membrane fusion after stimulation with aminoethyldextran (AED), depends on extracellular Ca. We have now extended these studies to longer times and shown that membrane fusion is stimulated by AED even after 1 min at low Ca (< or = 30 nM). At prolonged times membrane fusion was induced by sole removal of Ca. In the presence of AED, trichocyst contents were slowly extruded followed by resealing of the fused membranes, indicating independency of endocytotic membrane fusion from extracellular Ca (though we observed aberrant resealing). Later on, Ca removal is followed by cell death. By using videomicroscopy, we further provide the first evidence that exocytosis is not necessarily accompanied by an influx of Ca in the presence of the usual high concentrations (1 mM), since local exocytosis at the rear end of the cells is not followed by ciliary reversal which is triggered by Ca influx. We conclude that a Ca influx is neither regularly associated with, nor necessary for, induction of exocytotic membrane fusion in Paramecium cells. As a source for a possible alternative intracellular liberation of calcium during exocytosis, we analyzed the subplasmalemmal alveolar sac system by electron spectroscopic imaging and found indications for Ca redistributions shortly after stimulation.

Animals↗
PubMed1990

Alkaline phosphatase from Paramecium cilia and cell bodies: purification and characterization.

A soluble alkaline phosphatase was purified 10 000-fold in an overall yield of 8% from both of the cilia and cell bodies of the protozoan Paramecium tetraurelia. The concentration in cilia (1.7 microM) was 6-fold higher than in cell bodies, although the latter contained most of the activity due to their much greater volume. The purified protein showed a single (36 kDa) protein staining band on SDS-PAGE. This value, in conjunction with the apparent molecular mass of 66 kDa for the native enzyme (gel filtration) suggests a dimeric structure. The specific activity of the purified phosphatase ranged from 10 to 70 mumols.min-1.mg-1 at the pH-optimum of 8.0 and the Km for p-nitrophenyl phosphate was 81 microM. Basal enzyme activity was inhibited by metal chelators and stimulated up to 12-fold by addition of divalent cations. Mg2+ acted as a non-essential mixed-type activator with a half-maximal effect at 7 microM. Ca2+ was inhibitory, the extent of inhibition was dependent on the concentration of Mg2+ in the assay. Furthermore, the kinetics of inhibition by Ca2+ varied with the Mg2+ concentration. Phosphate, pyrophosphate, and SH-group blocking agents also strongly inhibited. The enzyme did not dephosphorylate Tyr- or Ser-/Thr-phosphoproteins. The Paramecium enzyme is not of lysosomal origin and its properties are quite different from all known phosphatases. It is a novel type of phosphatase since it (i) shows F(-)-inhibition like Ser/Thr-phosphatases but (ii) is inhibited by vanadate and molybdate like Tyr-phosphatases, and (iii) inhibition by Ca2+ has not been reported for any other phosphatase.

Alkaline Phosphatase↗
PubMed1987

Correlations between cyclic AMP binding and chemoreception in Paramecium.

Paramecium tetraurelia is attracted to cyclic AMP, which probably, as other attractants, signifies the presence of food. Attraction to cyclic AMP was specific, saturable, and, therefore, likely to be receptor-mediated. In these studies, we measured the binding of cyclic [3H]AMP to whole cells and found it to be saturable, reversible, and displaying specificity similar to that of attraction. An HPLC method of separating nucleotides was devised and used to determine that external cyclic AMP was degraded in the absence of IBMX, a phosphodiesterase inhibitor, and that cyclic AMP was taken into the cells in small amounts. Since binding and attraction were subsequently measured in the presence of IMBX, it was cyclic AMP and not a degradation product that served as the attractant stimulus for Paramecium.

1-Methyl-3-isobutylxanthine↗
PubMed1996

A theory of gravikinesis in Paramecium.

The archaic eukaryote unicellular microorganism, Paramecium, is propelled by thousands of cilia, which are regulated by modulation of the membrane potential. Ciliates can successfully cope with gravity, which is the phylogenetically oldest stimulus for living things. One mechanism for overcoming sedimentation is negative gravitaxis, an orientational response antiparallel to the gravity vector. We have postulated the existence of a negative gravikinesis in Paramecium, i.e. a modulation of swimming speed as a function of cellular orientation in space. With negative gravikinesis, an upward oriented cell actively augments the rate of forward swimming and depresses active locomotion during downward orientation. A brief outline of the gravikinesis hypothesis is given on a quantitative basis and experimental data are presented which have confirmed the major assumptions.

Animals↗
PubMed1995

Induction of the thigmotaxis in Paramecium caudatum.

Induction of thigmotaxis in Paramecium caudatum was examined. We succeeded in the induction by changing the ionic concentration and the Ja-value ([K+]/[Ca2+]1/2) of the surrounding medium. P. caudatum showed thigmotaxis stably in the solution containing a relatively high ionic concentration but a lower Ja-value. We found that the Paramecium cell touched the object with the tip of its oral groove and the cilia touching the object scarcely beat when the organism showed thigmotaxis.

Animals↗
PubMed1976

Fluorimetric determination of the resting potential changes associated with the chemotactic response in Paramecium.

(1) Evidence is presented which indicates that the carbocyanine dye (3,3'-dipropyl thiadicarbocyanine) can be used as a spectroscopic probe for monitoring the resting potential across tha plasma membrane of the ciliated protozoan Paramecium. (2) The dye at low concentrations (less than or equal to 1 muM) does not affect either the viability or the motility of the cells, nor does it induce a chemotactic response. (3) The fluorescence of the dye bound to the cells alters as the potential across the membrane is changed by increasing the external cation concentration. (4) The absorbance of the bound dye also changes in response to an alteration of the membrane potential. (5) The membrane potential changes as measured by the fluorescence method have been correlated with the measurements of the potential estimated by microelectrode methods. (6) Both cations which induce a negative chemotactic response in Paramecium (K+, Na+, Ba2+) and several non-toxic cations bring about a rapid depolarization of the plasma membrane. The significance of these rapid changes in relation to the swimming behaviour of the ciliate is discussed.

Animals↗
PubMed1988

Purification and properties of dyneins from Paramecium cilia.

Dynein ATPases were purified from Paramecium cilia by salt extraction followed by sucrose density gradient centrifugation and anion exchange chromatography. The two major dyneins sedimented in sucrose gradients as species of 22 S and 12 S. After purification by anion exchange chromatography, their specific activities were about 0.4 and 0.5 mumol/min per mg, respectively. The dyneins could be distinguished by subunit composition and immunological crossreactivity. Sucrose density gradient centrifugation revealed additional ATPase activity in the region between the 22 S and 12 S dyneins, including a 19 S activity. Mg2+-ATPase activities of the dyneins and the 19 S activity were inhibited by vanadate and Zn2+, and were activated by Triton X-100. Antibodies against the 22 S dynein from Paramecium reacted on immunoblots with most of the polypeptides of 22 S dynein, and showed that the heavy chains of 22 S dynein are not identical to those that sediment at 19 S and 12 S. Several minor ATPase activities were revealed by anion exchange chromatography of fractions from the 22 S, 19 S and 12 S regions of sucrose gradients. These minor activities were stimulated by Mg2+, inhibited by vanadate, and could be distinguished from each other by their elution positions and polypeptide compositions.

Adenosine Triphosphatases↗
PubMed1994

Purification and characterization of calmodulin (lysine 115) N-methyltransferase from Paramecium tetraurelia.

Calmodulin (lysine 115) N-methyltransferase was purified from the cytosolic fraction of Paramecium tetraurelia by sequential dialysis, cellulose phosphate chromatography, Reactive Red 120 agarose chromatography, and calmodulin-Sepharose affinity chromatography. The enzyme was purified 6800-fold with a 15% yield. SDS-PAGE analysis of the purified enzyme invariably revealed a major protein of 37 kDa that was reproducibly obtained and minor proteins of 35 and 28 kDa that were sometimes obtained in variable yields. The enzyme formed a mixture of mono-, di-, and trimethyllysine residues at lysine 115 of calmodulin in vitro, had a Km for the methyl donor, S-adenosyl methionine (AdoMet), of about 1 microM and a pH optimum of about 7.5. The purified enzyme had an absolute requirement for the reductant DTT for activity, whereas the enzyme in crude fractions did not. The enzyme is a monomer with an estimated molecular mass of 33 kDa. Ca2+, Mg2+, Mn2+, and Ni2+ stimulated calmodulin N-methyltransferase activity but Zn2+ did not. Calmodulin N-methyltransferase was inhibited by its reaction product S-adenosyl homocysteine (SAH), but not by sinefungin and tubercidin. The calmodulin antagonists calmidazolium and mellitin were inhibitory but W7 was not. The enzyme was not stimulated by Triton X-100 nor by NaCl. Only calmodulins with an unmethylated lysine at residue 115, including cam2 calmodulin, were substrates. Histones and calcium-binding proteins from Paramecium other than calmodulin did not act as substrates for the purified calmodulin N-methyltransferase and no other substrates in the cytosolic fraction were observed.

Animals↗
PubMed1992

Effects of mutagens on the clonal lifespan of Paramecium tetraurelia.

There has been interest in the phenomenon that a cell cannot undergo unlimited reproduction under adequate conditions and undergoes senescence. In holotrichous ciliates, Paramecium has a limit of vegetative reproduction without sexual reproduction but Tetrahymena does not always have a limited lifespan. Comparing the two species would increase our knowledge of the mechanism of cellular clonal aging. We previously showed that mutations induced by X-rays shorten clonal lifespan. In this study, we examined whether mutagens shorten the clonal lifespan of Paramecium tetraurelia. P. tetraurelia was exposed to the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), 0.045 mg/ml, for 30 min. The animal was exposed to MNNG 6 times in total while young (under 80 divisions from the start of a clonal life cycle) or 4 times during the senescent stage. MNNG shortened the clonal lifespan as expressed by the decrease in fission number from 186 +/- 55 (4 cell lines) to 136 +/- 21 (6 cell lines) with the first two treatments but with further exposures the lifespan increased to 182 +/- 15 (5 cell lines). MNNG had no effect when administered at the older age. Exposure of P. tetraurelia to 4-nitroquinoline-N-oxide at 0.021 mg/ml twice for 12 and 15 min at the younger age reduced the mean clonal lifespan from 143 +/- 28 to 125 +/- 21 and the maximum lifespan from 263 +/- 33 to 175 +/- 25.

4-Nitroquinoline-1-oxide↗
PubMed1990

Purification of the temperature-specific surface antigen of Paramecium primaurelia with its glycosyl-phosphatidylinositol membrane anchor.

The membrane form of the temperature-specific G surface antigen of Paramecium primaurelia strain 156 has been purified by a novel procedure utilizing solubilization by detergent, ammonium sulfate precipitation, and high-performance liquid chromatography. The surface antigen, which was prepared in a nondenatured state containing a glycosyl-phosphatidylinositol membrane anchor, migrated as a single band upon electrophoresis in sodium dodecyl sulfate-polyacrylamide gels. Following cleavage of the purified surface antigen by a phosphatidylinositol-specific phospholipase C from Bacillus thuringiensis, the soluble form was released with the unmasking of a particular glycosidic immunodeterminant called the cross-reacting determinant. The purification protocol described here will now permit further biochemical and biophysical characterization of the nondenatured membrane form of Paramecium surface antigens.

Animals↗
PubMedDate not supplied

Molecular aspects of rapid, reversible, Ca2+-dependent de-phosphorylation of pp63/parafusin during stimulated exo-endocytosis in Paramecium cells.

Ca2+ signalling governs stimulated exocytosis and exocytosis-coupled endocytosis also in Paramecium cells. Upon stimulation, the < or =10(3) dense-core exocytotic organelles (trichocysts) can be synchronously (80 ms) released, followed by endocytotic membrane resealing (350 ms) and retrieval. Paramecium is the most synchronous dense-core exocytotic system known, allowing to dissect rapidly reversible Ca2+-dependent phenomena. This holds for the reversible de-/re-phosphorylation cycle of a 63 kD phosphoprotein, pp63/parafusin (pf), which we have cloned, immuno-localised, and characterised as phosphoglucomutase, the enzyme funneling glucose into the glycolytic pathway. It was isolated ex vivo, followed by MALDI analysis, while X-ray structure analysis was performed after heterologous expression. We found multiple phosphorylation of superficial Ser/Thr residues. Although present also in exo(-) mutants, pp63/pf is selectively de-phosphorylated only in exo(+) strains during synchronous exocytosis (80 ms) and re-phosphorylated within approximately 20 s, i.e., the time required to re-establish [Ca2+] homeostasis. We have isolated relevant protein phosphatases and kinases and probed their activity on pp63/pf in vitro. We consider Ca2+/calmodulin-activated PP2B (calcineurin, whose subunits have been cloned) relevant for de-phosphorylation. Re-phosphorylation can be achieved by two protein kinases that also have been cloned. One is activated by cGMP (PKG) which in turn is formed by Ca2+-activated guanylate cyclase. Another kinase, casein kinase 2, is inhibited by Ca2+ and, hence, activated with some delay in parallel to decreasing [Ca2+] after exocytosis. In total, several Ca2+-sensitive cycles cooperate whose protein components have been localised to the cell cortex. Regulation of the phosphorylation degree of pp63/pf may affect structure binding on a microscale and/or its enzymatic activity. All this may serve fueling substrate into glycolysis with increased ATP re-formation (compromised in exo(-) mutants) and NADH formation, with effects on Ca2+ signalling including mobilisation from cortical stores (alveolar sacs) and overall effects on ATP and Ca2+ dynamics during synchronous exo- and endocytosis.

Animals↗
PubMed2004

Functional chimeras between the catalytic domains of the mycobacterial adenylyl cyclase Rv1625c and a Paramecium guanylyl cyclase.

The class IIIa adenylyl cyclase (AC) Rv1625c from Mycobacterium tuberculosis forms homodimers with two catalytic centres, whereas the Paramecium guanylyl and mammalian ACs operate as pseudoheterodimers with one catalytic centre. The functional and structural relationship of the catalytic domains of these related class III cyclases was investigated. Point mutations introduced into Rv1625c to engineer a forskolin-binding pocket created a single heterodimeric catalytic centre, yet did not result in forskolin activation. Chimerization of these Rv1625c point mutants with corresponding mammalian AC domains was impossible. However, it was successful using a complemental Paramecium guanylyl cyclase domain and resulted in an AC. The data signify a divergence of structural and functional evolution in class III Acs.

Adenylyl Cyclases↗
PubMed2026

Algae-to-host horizontal gene transfer in Paramecium bursaria is associated with host adaptation during endosymbiosis.

Paramecium bursaria maintains a stable endosymbiosis with green algae, yet the evolutionary consequences of this association remain unclear. Here, we screened the host genome for algal-derived horizontally transferred genes (HTGs) using a lineage-aware workflow designed to detect horizontal gene transfer (HGT) between two defined lineages. We identified 16 candidate HTGs, including four putative newly transferred genes and 12 homologous transferred genes, most of which were functionally associated with redox homeostasis and metabolism. Five HTGs showed symbiosis-dependent expression. RNAi knockdown of GH32s and SATs reduced host proliferation, total cell area, and motility, while GH32s knockdown also reduced endosymbiont load. Duplication patterns suggest that most transfers may have occurred after the P. bursaria lineage diverged from the sampled Paramecium species but before its lineage-specific whole-genome duplication (WGD). The HTGs also showed host-associated shifts in GC content and gene length, while representative HTGs retained conserved domains and functional motifs. Together, our results support algae-to-host HGT in P. bursaria and suggest that some transferred genes may contribute to metabolic integration during endosymbiosis.

Gene Transfer, Horizontal↗
PubMed1997

A calcium-activated, large conductance and non-selective cation channel in Paramecium cell.

A non-selective cation channel was found in mutant Paramecium cells (K115). This cell had been selected as a resistant mutant in a high-K+ solution. In patch clamp studies of these cells in the inside-out configuration, this channel was activated by bath applications of elevated Ca2+ concentrations. The channels became very active when the Ca2+ concentration was above 3.2 microM. The channel was also activated by depolarization. The voltage dependency was steep upon depolarization, whereas upon hyperpolarization the channel activity barely changed. This channel had poor selectivity for monovalent alkali cations. Using the Goldman-Hodgkin-Katz equation for the reversal potential, the permeability ratios with respect to K+ for Na+, Rb+, Cs+ and Li+ were nearly 1. Although the permeability ratios were similar for each cation, the single channel conductances differed. The single channel conductances were 467 pS with K+ as the charge carrier, 406 pS with Na+, 397 pS with Rb+, 253 pS with Cs+ and 198 pS with Li+ upon depolarization in 100 mM cation solutions. A similar calcium-activated large conductance channel was observed in the wild-type (G3) Paramecium cells but was very rare.

Animals↗
PubMed1992

Molecular cloning of the cDNA for the major hemoglobin component from Paramecium caudatum.

Nucleotide sequence of the cDNA for the major hemoglobin component of Paramecium caudatum was determined. An oligonucleotide was synthesized on the basis of the amino acid sequence, and the Paramecium cDNA library constructed in phage lambda gt11 was screened with it. Three positive clones, of which insert sizes were 0.4, 0.6, and 0.9 kbp, were obtained. Sequence analysis made clear that the 0.4-kbp cDNA retains a full length of the nucleotides encoding 116 amino acid residues, and that in the coding region it contains four TAA codons which are known to encode glutamine.

Amino Acid Sequence↗