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PubMedDate not supplied

Co-infection of the macronucleus of Paramecium caudatum by free-living bacteria together with the infectious Holospora obtusa.

Infection experiments were performed incubating Paramecium caudatum with non-infectious free-living bacteria or weakly infectious intracellular bacteria together with the infectious Holospora obtusa. Two of four non-infectious free-living bacteria (Enterobacter aerogenes and Klebsiella pneumoniae) were found to get into the nuclei when added to Paramecium together with H. obtusa. The endonuclear bacterium Nonospora macronucleata that is weakly infectious by itself increases its infectivity when presented together with the infectious holosporas. The results provide evidence that H. obtusa may facilitate entry of other, non-infectious bacteria into the nuclei of Paramecium.

Animals↗
PubMedDate not supplied

Identification of single nucleotide mutations that prevent developmentally programmed DNA elimination in Paramecium tetraurelia.

The excision of internal eliminated sequences (IESs) occurs during the differentiation of a new somatic macronuclear genome in ciliated protozoa. In Paramecium tetraurelia, IESs show few conserved features with the exception of an invariant 5'-TA-3' dinucleotide that is part of an 8-bp inverted terminal repeat consensus sequence with similarity to the ends of mariner/Tc1 transposons. We have isolated and analyzed two mutant cell lines that are defective in excision of individual IESs in the A-51 surface antigen gene. Each cell line contains a mutation in the flanking 5'-TA-3' dinucleotide of IES6435 and IES1835 creating a 5'-CA-3' flanking sequence that prevents excision. The results demonstrate that the first position of the 5'-TA-3' is required IES excision just as previous mutants have shown that the second position (the A residue) is required. Combining these results with other Paramecium IES mutants suggests that there are few positions essential for IES excision in Paramecium. Analysis of many IESs reveals that there is a strong bias against particular nucleotides at some positions near the IES termini. Some of these strongly biased positions correspond to known IES mutations, others correlate with unusual features of excision.

Animals↗
PubMed1997

Supplemental melatonin increases clonal lifespan in the protozoan Paramecium tetraurelia.

The hypothesis that melatonin supplementation can increase the lifespan of a single-celled organism was tested by the administration of melatonin to the ciliated protozoan Paramecium tetraurelia. Melatonin supplementation in dim red light at a dose of 0.043 mM (10 mg/L) of nutrient media (bacterized Cerophyl) per day, followed by incubation for 23 hr in darkness, increased the mean clonal lifespan of Paramecium tetraurelia in days by percentages ranging from 20.8% (P < 0.01, two-tailed t-test) to 24.2% (P < 0.01, ANOVA) over controls. Maximum clonal lifespan in days was also increased in melatonin-supplemented cells, from 14.8% to 24.0% over controls. Mean clonal lifespan in fissions was not significantly greater in melatonin-supplemented cells, with values ranging from 6.0% to 15.5% over controls. Maximum clonal lifespan in fissions did not differ appreciably, with values ranging from 1.0% to 9.1% over controls, except in the case of cells selected for rapid division rates, in which melatonin-supplemented cells (393 fissions) lived 20.9% longer than controls (325 fissions) in terms of cumulative cell doublings during the clonal lifespan. The finding that melatonin supplementation increased clonal lifespan in Paramecium tetraurelia, an aerobic, single-celled organism, suggests that the mechanism of melatonin's longevity-promoting effects may be intracellular.

Animals↗
PubMed1996

Inhibition of trichocyst exocytosis and calcium influx in Paramecium by amiloride and divalent cations.

Regulated exocytosis of defensive secretory organelles, the trichocysts, as well as a transient Ca(2+)-influx can be induced in Paramecium by aminoethyldextran (Kerboeuf and Cohen, J Cell Biol (1990) 111, 2527). Knoll et al (Febs Lett (1992) 304, 265) reported that veratridine was also a secretagogue for Paramecium. Here we show that, like aminoethyldextran, veratridine induces a transient Ca(2+)-influx. Both aminoethyldextran- and veratridine-induced exocytosis and associated Ca(2+)-influx were: i) blocked in the ndl2 thermosensitive mutant at the non-permissive temperature; and ii) inhibited by amiloride and four divalent cations, Ba2+, Mg2+, Sr2+ and Co2+. This suggests that, although of different chemical nature, aminoethyldextran and veratridine act through the same physiological pathway. In addition, the inhibitory doses are comparable to the ones found to inhibit a hyperpolarization-sensitive Ca(2+)-current described in Paramecium (Preston et al (1992) J Gen Physiol 100, 233). The possibility that the activation of this Ca(2+)-current by the secretagogue represents an early step in the regulation of trichocyst exocytosis is discussed.

Amiloride↗
PubMed1992

Regulation of adenylyl cyclase from Paramecium by an intrinsic potassium conductance.

Hyperpolarization of the cell membrane of Paramecium stimulates adenosine 3',5'-monophosphate (cAMP) formation. Manipulations of the K+ resting conductance of the ciliate by adaptation in different buffers affected excitability of the cAMP generating system. Blockade of K+ channels inhibited hyperpolarization-stimulated cAMP formation. A mutant of Paramecium that is unable to control its K+ resting conductance had a defect in cAMP formation. Purified adenylyl cyclase, when incorporated into an artificial lipid bilayer membrane, revealed properties of a voltage-independent K+ channel. This indicates that the adenylyl cyclase of Paramecium has a secondary function as carrier of the K+ resting conductance. A hyperpolarization-activated K+ efflux appears to directly regulate adenylyl cyclase activity in vivo.

Adenylyl Cyclases↗
PubMed1990

Calmodulin activation of calcium-dependent sodium channels in excised membrane patches of Paramecium.

Calmodulin is a calcium-binding protein that participates in the transduction of calcium signals. The electric phenotypes of calmodulin mutants of Paramecium have suggested that the protein may regulate some calcium-dependent ion channels. Calcium-dependent sodium single channels in excised patches of the plasma membrane from Paramecium were identified, and their activity was shown to decrease after brief exposure to submicromolar concentrations of calcium. Channel activity was restored to these inactivated patches by adding calmodulin that was isolated from Paramecium to the cytoplasmic surface. This restoration of channel activity did not require adenosine triphosphate and therefore, probably resulted from direct binding of calmodulin, either to the sodium channel itself or to a channel regulator that was associated with the patch membrane.

Animals↗
PubMed2006

Multigene family encoding 3',5'-cyclic-GMP-dependent protein kinases in Paramecium tetraurelia cells.

In the ciliate Paramecium tetraurelia, 3',5'-cyclic GMP (cGMP) is one of the second messengers involved in several signal transduction pathways. The enzymes for its production and degradation are well established for these cells, whereas less is known about the potential effector proteins. On the basis of a current Paramecium genome project, we have identified a multigene family with at least 35 members, all of which encode cGMP-dependent protein kinases (PKGs). They can be classified into 16 subfamilies with several members each. Two of the genes, PKG1-1 and PKG2-1, were analyzed in more detail after molecular cloning. They encode monomeric enzymes of 770 and 819 amino acids, respectively, whose overall domain organization resembles that in higher eukaryotes. The enzymes contain a regulatory domain of two tandem cyclic nucleotide-binding sites flanked by an amino-terminal region for intracellular localization and a catalytic domain with highly conserved regions for ATP binding and catalysis. However, some Paramecium PKGs show a different structure. In Western blots, PKGs are detected both as cytosolic and as structure-bound forms. Immunofluorescence labeling shows enrichment in the cell cortex, notably around the dense-core secretory vesicles (trichocysts), as well as in cilia. Immunogold electron microscopy analysis reveals consistent labeling of ciliary membranes, of the membrane complex composed of cell membrane and cortical Ca2+ stores, and of regions adjacent to ciliary basal bodies, trichocysts, and trafficking vesicles. Since PKGs (re)phosphorylate the exocytosis-sensitive phosphoprotein pp63/pf upon stimulation, the role of PKGs during stimulated exocytosis is discussed, in addition to a role in ciliary beat regulation.

Amino Acid Motifs↗
PubMed1994

Non-Mendelian inheritance of macronuclear mutations is gene specific in Paramecium tetraurelia.

Paramecium tetraurelia contains two types of nuclei, a diploid germinal micronucleus and a large transcriptionally active macronucleus. The macronuclear genome is formed from the micronuclear DNA during sexual reproduction. Previous studies have shown that the processing of the A-type variable surface protein gene during formation of a new macronucleus is dependent on the presence of the A gene in the old macronucleus. It is not clear if this is a general feature that controls the formation of the Paramecium macronuclear genome or a unique feature of the A locus. Using micronuclear transplantation, we have constructed a strain that has a wild-type micronucleus but has macronuclear deletions of the A- and B-type surface protein genes. Neither the A nor the B gene is incorporated into the new macronucleus after sexual reproduction. Macronuclear transformation of this strain with the B gene rescues the B-gene deletion after formation of the next macronucleus but has not effect on the A deletion. Similarly, transformation with the A gene shows gene-specific rescue for A but not B. The effect of the old macronucleus on the processing of the new macronucleus results in a pattern of non-Mendelian inheritance of both macronuclear deletions. Progeny from the wild-type exconjugant are all wild type, and progeny from the A- B- exconjugant are mutant. The features of this A- B- non-Mendelian mutant demonstrate that the regulation of macronuclear DNA processing is gene specific, and our results open the possibility that this type of regulation affects many regions of the Paramecium genome.

Animals↗
PubMed1989

Intracellular binding of wheat germ agglutinin by Golgi complexes, phagosomes, and lysosomes of Paramecium multimicronucleatum.

The compartments of the Paramecium digestive system were investigated with wheat germ agglutinin (WGA). By use of cryosectioning or Lowicryl K4M embedding combined with pulse-chase studies and WGA-gold labeling, WGA binding sites were located on membranes of the phagosome-lysosome system, including all four stages of digestive vacuoles, the discoidal vesicles, acidosomes, and lysosomes. In addition, the contents of lysosomes, cisternae at the trans face of Golgi stacks, and coated and uncoated blebs and vesicles at the putative trans Golgi network bind to WGA. Crystal-containing vacuoles characteristic of mid-log to stationary-phase cultures are enclosed by heavily labeled membranes. Alveoli underlying the plasma membrane sometimes contain binding sites, particularly on their outer membranes. Ciliary membranes previously shown to be labeled with WGA-FITC are negative in frozen thin and Lowicryl K4M sections. The presence of WGA binding sites on the trans face of the Golgi stack is the first indication in ciliated protozoa, such as Paramecium, of probable Golgi complex involvement in glycosylation similar to that in higher organisms. WGA-labeled coated vesicles in the endoplasm apparently lose their coats and coalesce to form lysosomes. Our study shows that WGA can be used as a specific intracellular marker of all digestive system membranes and of lysosomal content. These results support and extend our published scheme of membrane flow and recycling in Paramecium by providing another means of demonstrating membrane relationships.

Animals↗
PubMed2002

NSF regulates membrane traffic along multiple pathways in Paramecium.

N-ethylmaleimide (NEM)-sensitive factor (NSF), a regulator of soluble NSF attachment protein receptors (SNAREs), is required for vesicular transport in many eukaryotic cells. In the ciliated protozoon Paramecium, complex but well-defined transport routes exist, constitutive and regulated exocytosis, endocytosis, phagocytosis and a fluid excretory pathway through contractile vacuoles, that can all be studied independently at the whole cell level. To unravel the role of NSF and of the SNARE machinery in this complex traffic, we looked for NSF genes in Paramecium, starting from a partial sequence found in a pilot random sequencing project. We found two very similar genes, PtNSF1 and PtNSF2, which both seem to be expressed. Peptide-specific antibodies (Abs) recognize PtNSF as a 84 kDa band. PtNSF gene silencing results in decreasing phagocytotic activity, while stimulated exocytosis of dense core-vesicles (trichocysts), once firmly attached at the cell membrane, persists. Ultrastructural analysis of silenced cells shows deformation or disappearance of structures involved in membrane traffic. Aggregates of numerous small, smooth vesicles intermingled with branches of ER occur in the cytoplasm and are most intensely labeled with anti-NSF Ab-gold. Furthermore, elongated vesicles of approximately 30 nm diameter can be seen attached at cortical calcium storage compartments, the alveolar sacs, whose unknown biogenesis may thus be revealed. Involvement of PtNSF in some low frequency fusion events was visualized in non-silenced cells by immuno-fluorescence, after cautious permeabilization in the presence of ATP-gamma-S and NEM. Our data document that PtNSF is involved in distinct pathways of vesicle traffic in Paramecium and that actual sensitivity to silencing is widely different, apparently dependent on the turnover of membrane-to-membrane attachment formation.

Amino Acid Sequence↗
PubMed1997

Dynamics of calcium regulation in Paramecium and possible morphogenetic implication.

This paper is the first report of the use of a fluorescent indicator (Dextran-coupled calcium green-1) for imaging of cytosolic free calcium in ciliate cells. Using this technique in Paramecium, we show that a very transient increase in the mean intracellular calcium concentration accompanied exocytosis. It has long been postulated based on indirect experimental evidence, that a calcium wave which would spread across the cortex at the time of cell division, would be the primary event that triggers morphogenesis in these species. We theoretically show that a unifying interpretation can be given for the possible occurrence of a single wave and that of multiple oscillations of cytosolic calcium: both of which correspond to two different behaviors of the same dynamic system. Experimental conditions allowing the visualization of possible calcium periodicities in the interphase Paramecium cell are much more easily fulfilled than those permitting the observation of a single wave at the time of cell division. Hence, experiments were performed on interphase cells. After microinjection of calcium indicator into a mutant strain which is defective in exocytosis, we observed Ca2+ oscillations with a period close to 2 minutes. Hence, we conclude that Paramecium possesses all the dynamic elements required to generate, at the time of cell division, a morphogenetic calcium wave.

Animals↗
PubMed2004

Dynamin-association with agonist-mediated sequestration of beta-adrenergic receptor in single-cell eukaryote Paramecium.

Evidence that dynamin is associated with the sequestration of the Paramecium beta(2)-adrenergic receptor (betaAR) immunoanalogue is presented. We previously reported a dramatic change in the distribution of betaAR analogue in the subcellular fractions upon isoproterenol treatment: it is redistributed from the membraneous to the cytosolic fraction, as revealed by quantitative image analysis of western blots. Here we confirm and extend this observation by laser scanning confocal and immunogold electron microscopy. In the presence of isoproterenol (10 micro mol l(-1)) betaAR translocated from the cell surface into dynamin-positive vesicles in the cytoplasmic compartment, as observed by dual fluorochrome immunolabeling in a series of the confocal optical sections. Colocalization of betaAR and dynamin in the tiny endocytic vesicles was detected by further electron microscopic studies. Generally receptor sequestration follows its desensitization, which is initiated by receptor phosphorylation by G-protein-coupled receptor kinase. We cloned and sequenced the gene fragment of 407 nucleotides homologous to the beta-adrenergic receptor kinase (betaARK): its deduced amino acid sequence shows 51.6% homology in 126 amino acids that overlap with the human betaARK2 (GRK3), and may participate in Paramecium betaAR desensitization. These results suggest that the molecular machinery for the desensitization/sequestration of the receptor immunorelated to vertebrate betaAR exists in unicellular PARAMECIUM:

Adrenergic beta-Agonists↗
PubMed1993

Guanine nucleotides modulate calcium currents in a marine Paramecium.

Voltage-dependent calcium channels play a critical role in many cell functions and in many cell types ranging from protozoa to vertebrates. We have shown previously that guanine nucleotides modulate the calcium action potential and the duration of backward swimming in Paramecium, both indirect measurements of calcium channel function. To determine whether guanine nucleotides to indeed alter calcium currents, the inward calcium current (ICa) in Paramecium calkinsi was studied. First, the calcium current was characterized. The magnitude of ICa increased as the extracellular calcium concentration was increased from 0.5 to 50 mmol l-1, unlike the situation in freshwater species of Paramecium where the inward calcium current magnitude is maximal when extracellular calcium levels reach 1 mmol l-1. Inorganic compounds (NiCl2 at 10 mumol l-1 and CdCl2 at 1 mmol l-1) and organic compounds (naphthalene sulfonamides, W-7 and W-12-Br at 100 and 2 mumol l-1, respectively) reduced ICa. Regardless of the holding membrane potential (from -80 to -20 mV), the threshold activation for ICa was at -10 mV and the maximum value of ICa was reached at +20 mV, suggesting that there is only one type of calcium channel in P. calkinsi. Second, we injected GTP gamma S, GTP and GDP beta S into voltage-clamped cells while monitoring calcium and/or potassium currents. GTP gamma S increased the magnitude of ICa by 42 +/- 6% (mean +/- S.D., N = 5) and the effect was irreversible, GTP increased the magnitude of ICa by 37 +/- 4% (N = 4) in a reversible manner, and GDP beta S decreased ICa by 57 +/- 8% (N = 3) irreversibly. The outward potassium currents did not change when GTP gamma S was injected into the cells. These results support the hypothesis that injection of guanine nucleotides modulates the voltage-dependent calcium channel in P. calkinsi, presumably by activating G-protein-dependent processes.

Animals↗
PubMed2001

ATP-regenerating system in the cilia of Paramecium caudatum.

The energy supply for eukaryotic ciliary and flagellar movement is thought to be maintained by ATP-regenerating enzymes such as adenylate kinase, creatine kinase and arginine kinase. In this study, the energy-supplying system for the ciliary movement of Paramecium caudatum was examined. Arginine kinase and adenylate kinase activities were detected in the cilia. To demonstrate that phosphoarginine satisfactorily supplies high-energy phosphate compounds into the narrow ciliary space, we prepared an intact ciliated cortical sheet from live Paramecium caudatum. These cortical sheets, with an intact ciliary membrane, produced a half-closed system in which each cilium was covered with a ciliary membrane with an opening to the cell body. Ciliary beating on the intact cortical sheets was induced by perfusing not only ATP but also ADP. Addition of phosphoarginine (0.2 mmol l(-1)) increased the beat frequency. A further increase in beat frequency was observed in 0.4 mmol l(-1) phosphoarginine, and this was enhanced when the cilia were reactivated with relatively low concentrations of ATP. We have demonstrated that phosphoarginine supplies energy as a 'phosphagen' for ciliary beating in Paramecium caudatum, suggesting that phosphoarginine functions not only as a reservoir of energy but also as a transporter of energy in these continuously energy-consuming circumstances. http://www.biologists.com/JEB/movies/jeb3123.html

Adenosine Triphosphate↗
PubMed1978

A study on the toxicity of natural food dyes--toxicity and enzyme inhibition in Paramecium caudatum.

The toxicity of 14 commercial natural dyes which are widely used as food additives in Japan was studied on Paramecium caudatum. Laccaic acid and capsanthin were found to be very toxic to Paramecium caudatum. Some of the commercially available carminic acid and crocin were also toxic. The inhibitory effect of natural food dyes on leucine aminopeptidase, acid phosphatase and esterase in vitro was proportional to the toxic effect of the dyes on the survival time of Paramecium caudatum. Analyses of the commercial natural food dyes by high performance liquid chromatography failed to identify the toxic components.

Acid Phosphatase↗
PubMedDate not supplied

[Motor activity of paramecium].

A systemic study of the mechanisms of motor activity of paramecium was carried out. The movements of paramecium responding to various influences were photographed. The analysis of the data revealed the time dependences of the rate of movement, rate of rotation, and the radius and the pitch of the helix trajectory. Mathematical models of the membrane and a unit that transforms the calcium signal to programs of regulating the effectors were constructed. A system of equations for constructing the trajectory of movement was proposed. It is concluded that the biomolecular system that involves calmodulin, calmodulin-dependent ionic channels, adenylate cyclase, guanylate cyclase, phosphodiesterases, Ca(2+)-calmodulin, cAMP, cGMP-dependent protein kinases, and phosphoprotein phosphatases is capable of regulating motor reactions necessary for complex maneuvering of paramecium under various conditions.

Animals↗
PubMed1987

Amino acid sequence of a novel calmodulin from Paramecium tetraurelia that contains dimethyllysine in the first domain.

A class of Paramecium behavioral mutants called pantophobiacs have a deficiency in calcium-dependent potassium efflux, and this deficiency can be corrected by the microinjection of wild-type Paramecium calmodulin (Hinrichsen, R. D., Burgess-Cassler, A., Soltvelt, B. C., Hennessey, T., and Kung, C. (1986) Science 232, 503-506). As a starting point in investigations of which features allow wild-type Paramecium calmodulin to fully restore this behavior while other calmodulins are inactive or poorly effective, we elucidated the amino acid sequence of the wild-type calmodulin. We utilized an approach that combined Edman chemistry with mass spectrometry. This approach resulted in the identification of a new post-translational modification in calmodulin: N epsilon,N epsilon-dimethyllysine at residue 13. This particular modification has not been described for calmodulins studied previously. The only other first-domain modification that has been described for any calmodulin is acetylation of the amino terminus (Watterson, D. M., Sharief, F., and Vanaman, T. C. (1980) J. Biol. Chem. 255, 962-975). These results along with analyses of pantophobiac calmodulin and calmodulin binding proteins will provide insight into calmodulin's role in a well-defined behavioral mutant.

Acetylation↗
PubMed1987

A novel Ca2+-dependent protein kinase from Paramecium tetraurelia.

The ciliated protozoan Paramecium tetraurelia contained two protein kinase activities that were dependent on Ca2+. We purified one of the enzymes to homogeneity by Ca2+-dependent affinity chromatography on phenyl-Sepharose and ion exchange chromatography. The purified enzyme contained polypeptides of 50 and 55 kDa, with the 50-kDa species predominant. From its Stokes radius (32 A) and sedimentation coefficient (3.9 S), we calculated a native molecular weight of 51,000, suggesting that the active form is a monomer. Its specific activity was 65-130 nmol X min-1 X mg-1 and the Km for ATP was 17-35 microM, depending on the exogenous substrate used. Kinase activity was completely dependent upon Ca2+; half-maximal activation occurred at approximately 1 microM free Ca2+ at pH 7.2. Phosphatidylserine and diacylglycerol did not stimulate activity, nor did the addition of purified Paramecium calmodulin. The enzyme phosphorylated casein and histones, forming primarily phosphoserine and phosphothreonine, respectively. It also catalyzed its own phosphorylation in a Ca2+-dependent reaction; the half-maximal rate of autophosphorylation occurred at approximately 1-1.5 microM free Ca2+, and both the 50- and 55-kDa species were autophosphorylated. After separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and renaturation in situ, the 50-kDa protein retained its Ca2+-dependent ability to phosphorylate casein, suggesting that Ca2+ interacts directly with this polypeptide. This was confirmed by direct binding studies; when the enzyme was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis transferred to nitrocellulose, and renatured, there was 45Ca2+-binding in situ to both the 50- and 55-kDa polypeptides. The Paramecium enzyme appears to be a new and unique type of Ca2+-dependent protein kinase.

Animals↗