[Effect of Aflatoxin B1 on Paramecium caudatum and Paramecium bursaria].
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Aflatoxins↗
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[Biologic action of natural irradiation: study of the growth of Paramecium aurelia and Paramecium caudatum in a subterranean laboratory].
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Animals↗
Interaction of blood sera with the culture of Paramecium caudatum. II. Individual sensitivity to medium lethal x-ray doses determined by the consumption of complement by the culture of Paramecium caudatum.
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Animals↗
Method for the simultaneous establishment of many axenic cultures of Paramecium.
A method is described for the simultaneous treatment of 42 (or more) stocks of Paramecium, and their adaptation to growth in axenic culture. Samples of dense cultures of these ciliates growing with Enterobacter aerogenes are rendered bacteria-free by migration through 2 sets of tubes containing Adaptation Medium (Peters' salts solution, stigmaterol, vitamins, and autoclaved E. aerogenes). The 2nd set of tubes contains Adaptation Medium plus antibiotics. Bacteria-free samples containing approximately 100 animals are then transferred to test tubes containing Adaptation Medium without antibiotics. This medium also serves as a growth medium. It supports indefinite growth of all Paramecium stocks tested. After adaptation to this medium, the ciliattes can be grown in the axenic medium developed by Soldo, Godoy & van Wagtendonk. On a single trial at least half of the stocks can be expected to produce axenic cultures within 5 to 10 days by these procedures. The method has been applied successfully to several of the species of the Paramecium aurelia complex, to all syngens of Paramecium multimicronucleatum, to several stocks of Paramecium jenningsi, and to 1 stock of Paramecium caudatum and Paramecium calkinsi. A modification of the method also works for Didinium nasutum.
Animals↗
Protein phosphatase 2C is involved in the cAMP-dependent ciliary control in Paramecium caudatum.
Forward swimming of the Triton-extracted model of Paramecium is stimulated by cAMP. Backward swimming of the model induced by Ca(2+) is depressed by cAMP. Cyclic AMP and Ca(2+) act antagonistically in setting the direction of the ciliary beat. Some ciliary axonemal proteins from Paramecium caudatum are phosphorylated in a cAMP-dependent manner. In the presence of cAMP, axonemal 29- and 65-kDa polypeptides were phosphorylated by endogenous A-kinase in vitro. These phosphoproteins, however, were not dephosphorylated after in vitro phosphorylation, presumably because of the low endogenous phosphoprotein phosphatase activity associated with isolated axonemes. We purified the protein phosphatase that specifically dephosphorylated the 29- and 65-kDa phosphoproteins from Paramecium caudatum. The molecular weight of the protein phosphatase was 33 kDa. The protein phosphatase had common characteristics as protein phosphatase 2C (PP2C). The characteristics of the protein phosphatase were the same as those of the PP2C from Paramecium tetraurelia (PtPP2C) [Grothe et al., 1998: J. Biol. Chem. 273:19167-19172]. We concluded that the phosphoprotein phosphatase is the PP2C from Paramecium caudatum (PcPP2C). The PcPP2C markedly accelerated the backward swimming of the Triton-extracted model in the presence of Ca(2+). On the other hand, the PcPP2C slightly depressed the forward swimming speed. This indicates that the PP2C plays a role in the cAMP-dependent regulation of ciliary movement in Paramecium caudatum through dephosphorylation of 29- and/or 65-kDa regulatory phosphoproteins by terminating the action of cAMP.
Aniline Compounds↗
Comparison of the evolutionary distances among syngens and sibling species of Paramecium.
The morphospecies of the genus Paramecium have several mating type groups, so-called syngens, composed of cells of complementary mating types. The Paramecium aurelia complex is composed of 15 sibling species assigned to the species from the syngen. To increase our understanding of the evolutionary relationships among syngen and sibling species of the genus Paramecium, we investigated the gene sequences of cytosol-type hsp70 from 7 syngens of Paramecium caudatum and 15 sibling species of P. aurelia. Molecular phylogenetic trees indicated that the P. aurelia complex could be divided into four lineages and separated into each sibling species. However, we did not find any obvious genetic distance among syngens of P. caudatum, and they could only be separated into two closely related groups. These results indicated that the concept of syngens in P. caudatum differs quite markedly from that of the P. aurelia complex. In addition, we also discuss the relationships among these species and other species, Paramecium jenningsi and Paramecium multimicronucleatum, which were once classified as varieties of P. aurelia.
Animals↗
A 63 kDa phosphoprotein undergoing rapid dephosphorylation during exocytosis in Paramecium cells shares biochemical characteristics with phosphoglucomutase.
We have enriched phosphoglucomutase (PGM; EC 5.4.2.2) approximately 20-fold from Paramecium tetraurelia cells by combined fractional precipitation with (NH4)2SO4, gel filtration and anion-exchange chromatography yielding two PGM peaks. Several parameters affecting PGM enzymic activity, molecular mass and pI were determined. Phosphorylation studies were done with isolated endogenous protein kinases. Like the 63 kDa phosphoprotein PP63, which is dephosphorylated within 80 ms during synchronous trichocyst exocytosis [Höhne-Zell, Knoll, Riedel-Gras, Hofer and Plattner (1992) Biochem. J. 286, 843-849], PGM has a molecular mass of 63 kDa and forms of identical pI. Since mammalian PGM activity depends on the presence of glucose 1,6-bisphosphate (Glc-1,6-P2) (which is lost during anion-exchange chromatography), we analysed this aspect with Paramecium PGM. In this case PGM activity was shown not to be lost, due to p-nitrophenyl phosphate-detectable phosphatase(s) (which we have separated from PGM), but also due to loss of Glc-1,6-P2. Like PGM from various vertebrate species, PGM activity from Paramecium can be fully re-established by addition of Glc-1,6-P2 at 10 nM, and it is also stimulated by bivalent cations and insensitive to chelating or thiol reagents. The PGM which we have isolated can be phosphorylated by endogenous cyclic-GMP-dependent protein kinase or by endogenous casein kinase. This results in three phosphorylated bands of identical molecular mass and pI values, as we have shown to occur with PP63 after phosphorylation in vivo (forms with pI 6.05, 5.95, 5.85). In ELISA, antibodies raised against PGM from rabbit skeletal muscle were reactive not only with original PGM but also with PGM fractions from Paramecium. Therefore, PGM and PP63 seem to be identical with regard to widely different parameters, i.e. co-elution by chromatography, molecular mass, phosphorylation by the two protein kinases tested, pI values of isoforms, and immuno-binding. Recent claims that PP63 ('parafusin') would not be identical with PGM specifically in Paramecium are critically evaluated. Since some glycolytic enzymes are discussed as being associated with the Ca(2+)-release channel in muscle sarcoplasmic reticulum, and since sub-plasmalemmal Ca2+ stores in Paramecium closely resemble sarcoplasmic reticulum, a possible function of PP63/PGM in exocytosis regulation is discussed, particularly since dephosphorylation strictly parallels exocytosis.
Animals↗
Ca2+-dependent protein kinases of Paramecium--cloning provides evidence of a multigene family.
Two genes for Ca2+-dependent protein kinases, PCaPK-alpha and PCaPK-beta, were isolated from a Paramecium genomic DNA library. The coding region of PCaPK-alpha encoded 481 amino acids and that of PCaPK-beta encoded 493 amino acids, predicting molecular masses of 55603 Da and 57131 Da for each putative protein. The sequences of the protein kinase catalytic domains of PCaPK-alpha and PCaPK-beta were closely related to those of the Ca2+-dependent protein kinases (CDPKs) from Plasmodium, Eimeria, and several plants, and the catalytic region of the Ca2+/calmodulin-dependent protein kinase family (35-48% identity). In the junction region between the catalytic and regulatory regions, only 9 of 31 amino acid residues are the same in the two Paramecium genes, and the sequences encoded in the Paramecium genes differ from those in the plant CDPK genes in about 20 of 31 residues in the junction region. The C-terminal region of the Paramecium kinases shared sequence similarity with Paramecium calmodulin (30-34% identity). Two Ca2+-dependent protein kinases previously characterized from Paramecium (52 kDa CaPK-1, and 50 kDa CaPK-2) are activated by Ca2+ in the micromolar concentration range and they directly bind Ca2+ in a 45Ca2+ overlay blot assay. The size predicted from the genes, the presence of four putative Ca2+-binding motifs encoded in PCaPK-alpha and PCaPK-beta, and the immunological cross-reaction of expressed cloned fragments of these genes with CaPK-2, suggest that they encode proteins of the same family.
Amino Acid Sequence↗
A mutant Paramecium with a defective calcium-dependent potassium conductance has an altered calmodulin: a nonlethal selective alteration in calmodulin regulation.
The Paramecium mutant, pantophobiac A, has a defect that results in an in vivo loss of calcium-dependent potassium efflux channel activity. This defect is corrected fully by the microinjection of wild-type Paramecium calmodulin into pantophobiac A cells and is partially restored by calmodulins from other organisms, but it cannot be restored by microinjection of pantophobiac calmodulin. Overall, these results suggested that wild-type Paramecium calmodulin has unique features that allow it to restore fully a normal phenotype and that the defect in pantophobiac A might be an altered calmodulin molecule. Previous studies established the amino acid sequence of wild-type calmodulin and showed that Paramecium calmodulin has several differences from other calmodulins, including the presence of dimethyllysine at residue 13. To test directly the possibility that calmodulin from the pantophobiac mutant might be altered, we purified the mutant calmodulin and compared its properties to those of wild-type Paramecium calmodulin. We found one amino acid sequence difference between the two Paramecium calmodulins: a phenylalanine in the mutant protein, instead of a serine, at residue 101. This change is at a calcium-liganding residue in the third calcium-binding loop. These and previous studies demonstrate that comparatively subtle changes in the structure of calmodulin can result in quantitative alterations in in vivo activity, provide insight into the in vivo roles of calmodulin and the regulation of ion channels, and demonstrate that functional alterations of calmodulin are not necessarily lethal.
Amino Acid Sequence↗
Ca(2+)-binding proteins of cilia and infraciliary lattice of Paramecium tetraurelia: their phosphorylation by purified endogenous Ca(2+)-dependent protein kinases.
We purified two small, acidic calcium-binding proteins (Paramecium Ca(2+)-binding proteins, PCBP-25alpha and PCBP-25beta) from Paramecium tetraurelia by Ca(2+)-dependent chromatography on phenyl-Sepharose and by anion-exchange chromatography. The proteins were immunologically distinct. Monoclonal antibodies against PCBP-25beta did not react with PCBP-25alpha, and antibodies against centrin from Chlamydomonas reacted with PCBP-25alpha but not with PCBP-25beta. Like the centrins described previously, both PCBPs were associated with the infraciliary lattice (ICL), a fibrillar cytoskeletal element in Paramecium. Both were also present in isolated cilia, from which they could be released (with dynein) by a high-salt wash, and both PCBPs cosedimented with dynein in a sucrose gradient. PCBP-25beta was especially prominent in cilia and in the deciliation supernatant, a soluble fraction released during the process of deciliation. The results of immunoreactivity and localization experiments suggest that PCBP-25alpha is a Paramecium centrin and that PCBP-25beta is a distinct Ca(2+)-binding protein that confers Ca(2+) sensitivity on some component of the cilium, ciliary basal body or ICL. We characterized these proteins and Paramecium calmodulin as substrates for two Ca(2+)-dependent protein kinases purified from Paramecium. PCBP-25alpha and calmodulin were in vitro substrates for one of the two Ca(2+)-dependent protein kinases (CaPK-2), but only PCBP-25alpha was phosphorylated by CaPK-1. These results raise the possibility that the biological activities of PCBP-25alpha and calmodulin are regulated by phosphorylation.
Animals↗
Preferential cleavage of Paramecium DNA mediated by the C. elegans Tc1 transposase in vitro.
In the ciliate Paramecium aurelia complex, thousands of internal eliminated sequences (IESs) are excised from the germline micronuclear DNA during macronuclear differentiation. Based on the resemblance of Paramecium IES end sequences to Tc1 transposon termini, it has been proposed that Paramecium IESs might have degenerately evolved from Tc1 family transposons, and still be removed by an enzyme homologous to a Tc1 transposase. In this study, we found that transposase preferentially cleaved (or nicked) 58 sites near the IESs in Paramecium DNA, at sequences consisting of TT or TCTA. Since one excision junction of the P. primaurelia W2 IES was included in such sites, this suggests that a Tc1-like transposase is involved in the IES excision process, although it is probably not a sole factor responsible for the precise cleavage. In addition, unmethylated substrate DNA appeared to decrease the cleavage specificity, suggesting an involvement of DNA methylation in the cleavage. Although these results do not directly address the transposon origin of Paramecium IESs, it is likely that the enzymatic machinery responsible for the initial cleavage is derived from a Tc1-like transposase. The mechanism necessary for precise excision is discussed, in relation to recent knowledge of IES excision obtained in Tetrahymena and Paramecium.
Animals↗
Okadaic acid, an inhibitor of protein phosphatase 1 in Paramecium, causes sustained Ca2(+)-dependent backward swimming in response to depolarizing stimuli.
Backward swimming is a stereotypic behavioural response of Paramecium. It is triggered by depolarizing stimuli, which open calcium channels in the excitable ciliary membrane. The influx of Ca2+ causes the reversal of ciliary beat and initiates backward swimming. Here, we demonstrate that the protein phosphatase inhibitor okadaic acid does not affect the normal forward swimming pattern of Paramecium, but greatly extends the duration of backward swimming as initiated by depolarization caused by a rise in extracellular K+. Chelation of external Ca2+ results in an immediate resumption of forward swimming. The results suggest that the voltage-operated calcium channel is inactivated by a dephosphorylation event, and that okadaic acid blocks this dephosphorylation without any effect on the motile apparatus of the cilia. In addition, Paramecium is unique among eukaryotic cells, in that okadaic acid inhibits just one protein phosphatase, namely a type 1 enzyme, 75% of which is tightly associated with the excitable ciliary membrane. The type 2A protein phosphatases in Paramecium are unaffected by okadaic acid. The results indicate that protein phosphatase 1 is the enzyme responsible for the dephosphorylation and closure of the calcium channel in Paramecium.
Animals↗
Pharmacological prevention of acute lead poisoning in Paramecium.
To understand how lead (Pb2+) and other metals and chelating agents effect living cells, behavioral experiments in the marine ciliate Paramecium calkinsi were carried out. The duration of Backward Swimming Behavior (BSB) of Paramecium was partially reduced when cells were exposed to 100 microM of Ni2+, CD2+ and Co2+. In contrast, Pb2+ increased Paramecium BSB in a dose-dependent manner. Thus, 1, 10, 20, 50 and 100 microM of Pb2+ increased the duration of BSB by 20.4, 83.9, 143.2, 163.2 and 185.2%, respectively. The naphthalenesulphonamide W-7, a calcium channel blocker in lower organisms, abolished the increase of Paramecium BSB initially produced by Pb2+. Paramecium, poisoned with 10 MicroM of Pb2+, were also treated with putative Pb2+ chelating agents, such as meso-2-3-dimercaptosuccinic acid (DMSA), Ca-Na2-EDTA and ascorbic acid. These compounds inhibited the increase of the duration of BSB initially produced by Pb2+ in a dose-dependent manner. The potency of these antidotes in blocking the effects of Pb2+ was as follows: DMSA >> Ca-Na2-EDTA > ascorbic acid. These results provide evidence for a membrane-based mechanism of lead poisoning and support the use of DMSA as a lead antidote.
Animals↗
Biological effect of paramecium in diffused ultrasonic fields.
Since the cell structure or biophysical reaction includes in the interaction between ultrasound and living matter. When multi-cell creature is exposed to ultrasound, this reaction will lead the biological effect becomes complex. Therefore, in this paper, a single cell creature is chosen to study the biological effects induced by ultrasound exposure. The paramecium, which possesses many features typical of higher-order animal cells, was considered an appropriate choice for this study. The ability shown by ultrasound in promoting and/or accelerating many reactions has been shown to be a useful field. The growth phase of paramecium by using the ultrasound irradiation is an important parameter in this study. In our experiment, the exponential and stationary phases were employed. Three important factors must be taken into account when an ultrasonic biological reaction is investigated: the medium system, the bubbles' field, and the acoustic field. The medium system involves the physical parameters of the medium field. The bubbles' field includes the reacting bubble size of the liquid and the resonance frequency of the reaction bubbles. The oscillation of the cells in response to the ultrasound radiation is simulated using Rayleigh-Plesset's bubble activation theory. The resonance frequency of the unicellular creature is then calculated. The acoustic field is about using the diffuse field theory of Sabine to create a uniform sound field for the radiation experiment. The resonance frequency of the paramecium vacuole is among 0.54-1.09 MHz. When the 0.25 and 0.5 MHz frequencies of ultrasound was irradiated in the stationary phase of the paramecium, the relative growth rate was about 20% lower than that of unexposed sample. Therefore, the phenomenon of inhibition and destruction appeared during irradiation. The exponential phase of the paramecium samples appear to be different when irradiated with 1 MHz ultrasound. As can be seen in the results, the maximum relative growth rate was increasing 18% with 1 MHz ultrasound exposure.
Animals↗
Succinate dehydrogenase (SDH) activity in single Paramecium caudatum cells.
We measured in situ the activity of succinate dehydrogenase (SDH), one of the mitochondrial marker enzymes, in single Paramecium cells. SDH activity was detected with nitroblue tetrazolium (Nitro BT). Images of cells were captured every 30 sec at 590 nm, nearly the isosbestic wavelength of two reduction products of Nitro BT, by using a microphotometric system for image analysis. Activity was estimated by the slope of linear regression lines representing the relationship between total absorbance of the processed image and delta reaction time (real reaction time minus 30 sec). To investigate individual differences in Paramecium cell populations, SDH activity was measured in cells at various succinate concentrations. Paramecium SDH showed bimodal activity distribution patterns at three of four succinate concentrations tested. This result suggests that there are two groups of Paramecium populations with different SDH activity under control culture conditions. On the basis of the relationship between SDH activity and succinate concentration, mean Vmax and apparent Km values were estimated. A Km of 3.2 mM was found for Paramecium.
Animals↗
Dense-core secretory vesicle docking and exocytotic membrane fusion in Paramecium cells.
Work with Paramecium has contributed to the actual understanding of certain aspects of exocytosis regulation, including membrane fusion. The system is faster and more synchronous than any other dense-core vesicle system described and its highly regular design facilitates correlation of functional and ultrastructural (freeze-fracture) features. From early times on, several crucial aspects of exocytosis regulation have been found in Paramecium cells, e.g. genetically controlled microdomains (with distinct ultrastructure) for organelle docking and membrane fusion, involvement of calmodulin in establishing such microdomains, priming by ATP, occurrence of focal fusion with active participation of integral and peripheral proteins, decay of a population of integral proteins ("rosettes", mandatory for fusion capacity) into subunits and their lateral dispersal during fusion, etc. The size of rosette particles and their dispersal upon focal fusion would be directly compatible with proteolipid V(0) subunits of a V-ATPase, much better than the size predicted for oligomeric SNARE pins (SCAMPs are unknown from Paramecium at this time). However, there are some restrictions for a straightforward interpretation of ultrastructural results. The rather pointed, nipple-like tip of the trichocyst membrane could accommodate only one (or very few) potential V(0) counterpart(s), while the overlaying domain of the cell membrane contains numerous rosette particles. Particle size is compatible with V(0), but larger than that assumed for the SNARE complexes. When membrane fusion is induced in the presence of antibodies against cell surface components, focal fusion is seen to occur with dispersing rosette particles but without dispersal of their subunits and without pore expansion. Clearly, this is required for completing fusion and pore expansion. After cloning SNARE and V(0) components in Paramecium (with increasing details becoming rapidly available), we may soon be able to address the question more directly, whether any of these components or some new ones to be detected, serve exocytotic and/or any other membrane fusions in Paramecium.
Animals↗
Isolation and characterization of libraries of monoclonal antibodies directed against various forms of tubulin in Paramecium.
Ciliates are very good models for studying post-translationally generated tubulin heterogeneity because they exhibit highly differentiated microtubular networks in combination with reduced genetic diversity. We have approached the analysis of tubulin heterogeneity in Paramecium through extensive isolation and characterization of monoclonal antibodies using various antigens and several immunization protocols. Eight monoclonal antibodies and 10 hybridoma supernatants were characterized by: i) immunoblotting on ciliate and pig brain tubulins as well as on peptide maps of Paramecium axonemal tubulin; ii) immunoblotting on ciliate tubulin fusion peptides generated in E coli, a procedure which allows in principle to discriminate antibodies that are directed against tubulin sequence (reactive on fusion peptides) from those directed against a post-translational epitope (non-reactive); and iii) immunofluorescence on Paramecium, 3T3 and PtK2 cells. Twelve antibodies labeled all microtubules in Paramecium cells and were found to be directed against tubulin primary sequences (nine of them being located in the alpha N-terminal domain, one in the beta C-terminal one, and two in alpha and beta central stretches). The remaining ones decorated only a specific subset of microtubules within the cell and were presumably directed against post-translational modifications. Among these, three antibodies are directed against an N-terminal acetylated epitope of alpha-tubulin whereas the epitopes of three other ones (TAP 952 degrees, AXO 58 and AXO 49 degrees) apparently correspond to still unidentified post-translational modifications, located in the C-terminal domain of both alpha- and beta-tubulins. The AXO 49 degrees specificity is similar to that of a previously described polyclonal serum raised against Paramecium axonemal tubulin [2]. The results are discussed in terms of identification and accessibility of the epitopes and immunogenicity of ciliate tubulin with reference to mammalian and ciliate tubulin sequences.
Amino Acid Sequence↗
Comparative studies of the graviresponses of Paramecium and Loxodes.
Gravitactic protozoa offer advantages in studying how the gravity stimulus is perceived on the cellular level. By means of a slow rotating centrifuge microscope in space the acceleration thresholds for gravitaxis of Loxodes striatus and Paramecium biaurelia were determined: < or = 0.15 x g for Loxodes and 0.3 x g for Paramecium, indicating different sensitivities of these species. Neutral-buoyant densities of immobilized cells were determined using media of different densities, revealing densities of 1.03 to 1.035 g/cm3 for Loxodes and 1.04 g/cm3 to 1.045 g/cm3 for Paramecium. Behavioral studies revealed that gravitaxis of Loxodes persisted independent of the density of the medium. In contrast, negative gravitaxis of Paramecium was no longer measurable if the density of the medium approached the density of the cell. The results suggest that in the case of Loxodes gravity is perceived by an intracellular receptor and, in the case of Paramecium by its own mass via the pressure on the lower cell membrane.
Animals↗