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At least 523 records · Page 29Linked to original sources
PubMed1998

Immunolocalization of the exocytosis-sensitive phosphoprotein, PP63/parafusin, in Paramecium cells using antibodies against recombinant protein.

We have localized a structure-bound fraction of the exocytosis-sensitive phosphoprotein, PP63/parafusin (PP63/pf), in Paramecium cells by widely different methods. We combined cell fractionation, western blots, as well as light and electron microscopy (pre- and post-embedding immunolabeling), applying antibodies against the recombinant protein. PP63/pf is considerably enriched in certain cortical structures, notably the outlines of regular surface fields (kinetids), docking sites of secretory organelles (trichocysts) and the membranes of subplasmalemmal Ca2+-stores (alveolar sacs). From our localization studies we tentatively derive several potential functions for PP63/pf, including cell surface structuring, assembly of exocytosis sites, and/or Ca2+ homeostasis.

Animals↗
PubMed2005

Symbiotic Chlorella sp. of the ciliate Paramecium bursaria do not prevent acidification and lysosomal fusion of host digestive vacuoles during infection.

Each symbiotic Chlorella sp. of the ciliate Paramecium bursaria is enclosed in a perialgal vacuole derived from the host digestive vacuole, and thereby the alga is protected from digestion by lysosomal fusion. Algae-free cells can be reinfected with algae isolated from algae-bearing cells by ingestion into digestive vacuoles. To examine the timing of acidification and lysosomal fusion of the digestive vacuoles and of algal escape from the digestive vacuole, algae-free cells were mixed with isolated algae or yeast cells stained with pH indicator dyes at 25+/-1 degrees C for 1.5 min, washed, chased, and fixed at various time points. Acidification of the vacuoles and digestion of Chlorella sp. began at 0.5 and 2 min after mixing, respectively. All single green Chlorella sp. that had been present in the host cytoplasm before 0.5 h after mixing were digested by 0.5 h. At 1 h after mixing, however, single green algae reappeared in the host cytoplasm, arising from those digestive vacuoles containing both nondigested and partially digested algae, and the percentage of such cells increased to about 40% at 3 h. At 48 h, the single green algae began to multiply by cell division, indicating that these algae had succeeded in establishing endosymbiosis. In contrast to previously published studies, our data show that an alga can successfully escape from the host's digestive vacuole after acidosomal and lysosomal fusion with the vacuole has occurred, in order to produce endosymbiosis.

Animals↗
PubMed2005

Distinct localization of a beta-tubulin epitope in the Tetrahymena thermophila and Paramecium caudatum cortex.

Many of the highly organized microtubular arrangements in ciliates are located in the cortical area containing membrane vesicles and vacuoles. In Tetrahymena thermophila and Paramecium caudatum, immunofluorescence microscopy with the monoclonal antibody TU-06, directed against beta-tubulin, revealed distinct staining of this cortical region alone, while the cilia and other microtubular structures were unstained. The specificity of the antibody was confirmed by immunoblotting and by preabsorption of the antibody with purified tubulin. Double-label immunofluorescence with antibodies against gamma-tubulin, detyrosinated alpha-tubulin, and centrin showed that the TU-06 epitope is localized outside the basal body region. This was also confirmed by immunogold electron microscopy of thin sections. Proteolytic digestion of porcine brain beta-tubulin combined with a peptide scan of immobilized, overlapping peptides disclosed that the epitope was in the beta-tubulin region beta81-95, a region which is phylogenetically highly conserved. As known posttranslational modifications of beta-tubulin are located outside this area, the observed staining pattern cannot be interpreted as evidence of subcellular sequestration of modified tubulin. The limited distribution of the epitope could rather reflect the dependence of TU-06 epitope exposition on conformations of tubulin molecules in microtubule arrangements or on differential masking by interacting proteins.

3T3 Cells↗
PubMed2005

Nocodazole inhibits macronuclear infection with Holospora obtusa in Paramecium caudatum.

Holospora obtusa is a Gram-negative bacterium inhabiting the macronucleus of the ciliate Paramecium caudatum. Experimental infection with H. obtusa was carried out under nocodazole treatment. Nocodazole has been shown to cause disassembly of the cytoplasmic microtubules radiating from the cytopharynx and postoral fibers in P. caudatum. Treatment with this drug did not prevent the ingestion of both prey bacteria and H. obtusa, but it reduced the phagosome number and affected cyclosis. In situ hybridization revealed infectious forms of this endobiont very close to the macronucleus, but never inside it. These results indicate that disassembly of microtubules does not impair transportation of the infectious forms of H. obtusa in the cytoplasm, but that it completely blocks the invasion of the nucleus by the bacteria.

Actin Cytoskeleton↗
PubMed2006

Symbiotic alga Chlorella vulgaris of the ciliate Paramecium bursaria shows temporary resistance to host lysosomal enzymes during the early infection process.

Paramecium bursaria free of symbiotic Chlorella species can be experimentally reinfected with algae isolated from algae-bearing cells by ingestion into digestive vacuoles. Isolated symbiotic algae were cloned, mixed with the algae-free P. bursaria at 25 +/- 1 degrees C for 1.5 min, washed and chased, with or without fixation 3 h after mixing. Though genetically identical, a few of the algae were not digested but coexisted with the digested ones in the same vacuole after lysosomal fusion. Light microscopy showed that algal fate did not depend on cell cycle stage or location in the vacuole. Electron microscopy showed that the nondigested algae were not protected by a perialgal vacuole membrane in the digestive vacuole. Moreover, this phenomenon was also observed in the presence of cycloheximide and puromycin, which are known to inhibit algal and host protein synthesis, respectively. These observations suggest that a few algae can acquire temporary resistance to host lysosomal enzymes in order to establish endosymbiosis without algal protein synthesis.

Animals↗
PubMed2006

Graviresponses of Paramecium biaurelia during parabolic flights.

The thresholds of graviorientation and gravikinesis in Paramecium biaurelia were investigated during the 5th DLR (German Aerospace Center) parabolic-flight campaign at Bordeaux in June 2003. Parabolic flights are a useful tool for the investigation of swimming behaviour in protists at different accelerations. At normal gravity (1 g) and hypergravity (1 g to 1.8 g), precision of orientation and locomotion rates depend linearly on the applied acceleration as seen in earlier centrifuge experiments. After transition from hypergravity to decreased gravity (minimal residual acceleration of <10(-2) g), graviorientation as well as gravikinesis show a full relaxation with different kinetics. The use of twelve independent cell samples per flight guarantees high data numbers and secures the statistical significance of the obtained data. The relatively slow change of acceleration between periods of microgravity and hypergravity (0.4 g/s) enabled us to determine the thresholds of graviorientation at 0.6 g and of gravikinesis at 0.4 g. The gravity-unrelated propulsion rate of the sample was found to be 874 microm/s, exceeding the locomotion rate of horizontally swimming cells (855 microm/s). The measured thresholds of graviresponses were compared with data obtained from earlier centrifuge experiments on the sounding rocket Maxus-2. Measured thresholds of gravireactions indicate that small energies, close to the thermal noise level, are sufficient for the gravitransduction process. Data from earlier hypergravity experiments demonstrate that mechanosensitive ion channels are functioning over a relative wide range of acceleration. From this, we may speculate that gravireceptor channels derive from mechanoreceptor channels.

Acceleration↗
PubMed1979

The respiratory chain of Paramecium tetraurelia in wild type and the mutant Cl1. I. Spectral properties and redox potentials.

1. Purified mitochondria have been prepared from wild type Paramecium tetraurelia and from the mutant Cl1 which lacks cytochrome aa3. Both mitochondrial preparations are characterized by cyanide insensitivity. Their spectral properties and their redox potentials have been studied. 2. Difference spectra (dithionite reduced minus oxidized) of mitochondria from wild type P. tetraurelia at 77 K revealed the alpha peaks of b-type cytochrome (s) at 553 and 557 nm, of c-type cytochrome at 549 nm and a-type cytochrome at 608 nm. Two alpha peaks at 549 and 545 nm could be distinguished in the isolated cytochrome c at 77 K. After cytochrome c extraction from wild type mitochondria, a new peak at 551 nm was unmasked, probably belonging to cytochdrome c1. The a-type cytochrome was characterized by a split Soret band with maxima at 441 and 450 nm. The mitochondria of the mutant Cl1 in exponential phase of growth differed from the wild type mitochondria in that cytochrome aa3 was absent while twice the quantity of cytochrome b was present. In stationary phase, mitochondria of the mutant were characterized by a new absorption peak at 590 nm. 3. Cytochrome aa3 was present at a concentration of 0.3 nmol/mg protein in wild type mitochondria and ubiquinone at a concentration of 8 nmol/mg protein both in mitochondria of the wild type and the mutant Cl1. Cytochrome aa3 was more susceptible to heat than cytochromes b and c,c1.

Animals↗
PubMed1979

The respiratory chain of Paramecium tetraurelia in wild type and the mutant Cl1. II. Cyanide-insensitive respiration. Function and regulation.

1. The cyanide-insensitive respiration in Paramecium tetraurelia was found to be located in mitochondria. 2. Sensitivity of the mitochondrial respiration to cyanide depended on growth conditions. Under standard conditions of growth, 15--20% of respiration was insensitive to 1 mM cyanide. Full resistance to 1 mM cyanide was observed by growing cells in the presence of erythromycin (100--400 microgram/ml) 0.2 mM cyanide. The mitochondrial respiration of the mutant Cl1 harvested during the exponential phase of growth was largely insensitive to cyanide (more than 80%). 3. Pyruvate was oxidized at the same rate by wild type mitochondria and mitochondria of the mutant Cl1. In contrast, succinate oxidation was 2--3 times faster in mitochondria of the mutant Cl1 than in wild type mitochondria. 4. The cyanide-insensitive respiration was inhibited by 1 mM salicylhydroxamic acid to nearly 100%. Other efficient respiratory inhibitors included amytal and heptylhydroxyquinoline. Antimycin was not inhibitory even at concentrations as high as 5 microgram/mg protein, a finding consistent with the lack of antimycin binding sites.

Animals↗
PubMed1976

Biochemical studies of the excitable membrane of Paramecium aurelia. I. 45Ca2+ fluxes across resting and excited membrane.

The characteristics of Ca2+ transport across the excitable membrane of Paramecium aurelia were studied by measuring 45Ca2+ influx and efflux. The intracellular concentration of free Ca2+ in resting P. aurelia was at least ten times less than the extracellular concentration. Ca2+ influx was easily measurable at 0 degrees C, but not at 23 degrees C. The influx of 45Ca2+ was stimulated by the same conditions which cause membrane depolarization and ciliary reversal. Addition of Na+ and K+ (which stimulate ciliary reversal) resulted in a 10-fold increase in the rate of Ca2+ influx. An externally applied, pulsed, electric field (1-2 mA/cm2 of electrode surface), caused the rate of Ca2+ influx to increase 3-5 times, with the extent of stimulation dependent on the current density and the pulse width. Ca2+ influx had the characteristics of a passive transport system and was associated with the chemically or electrically triggered Ca2+ "gating" mechanism, which has been studied electrophysiologically. In contrast, Ca2+ efflux appeared to be catalyzed by an active transport system. With cells previously loaded at 0 degrees C with 45Ca2+, Ca2+ efflux was rapid at 23 degrees C, but did not occur at 0 degrees C. This active Ca2+ efflux mechanism is probably responsible for maintaining the low internal Ca2+ levels in unstimulated cells.

Azides↗
PubMed1981

Biochemical studies of the excitable membrane of Paramecium tetraurelia. V. Effects of proteases on the ciliary membrane.

The swimming behavior of Paramecium is regulated by an excitable membrane that covers the body and cilia of the protozoan. In order to obtain information on the topology and function of ciliary membrane proteins, Paramecia were treated with trypsin, chymotrypsin or pronase and the effects of these proteases were analyzed using electron microscopy, gel electrophoresis of ciliary fractions and behavioral tests. At the concentrations used, trypsin and chymotrypsin had little or no effect on the cells while pronase removed the cell surface coat, visible as fuzzy material covering the cell membrane. The same pronase treatment caused the specific removal of a high molecular weight protein (250 000), as judged by sodium dodecyl sulfate polyacrylamide gel electrophoresis. This protein, the 'immobilization antigen', constitutes the major protein of the ciliary membrane. Although the immobilization antigen was removed (or markedly decreased), no marked and reproducible difference was observed in the swimming behavior of the treated cells. We also determined the effects of proteases on isolated ciliary fractions to explore the sidedness of ciliary membrane proteins. A set of proteins relatively resistant to protease digestion was identified; they may be intrinsic membrane proteins.

Cell Membrane↗
PubMed1982

A Ca2+-activated ATPase specifically released by Ca2+ shock from Paramecium tetraurelia.

Deciliation of Paramecium tetraurelia by a Ca2+ shock procedure releases a discrete set of proteins which represent about 1% of the total cell protein. Marker enzymes for cytoplasm (hexokinase), endoplasmic reticulum (glucose-6-phosphatase), peroxisomes (catalase), and lysosomes (acid phosphatase) were not released by this treatment. Among the proteins selectively released is a Ca2+-dependent ATPase. This enzyme has a broad substrate specificity which includes GTP, ATP, and UTP, and it can be activated by Ca2+, Sr2+, or Ba2+, but not by Mg2+ or by monovalent cations. The crude enzyme has a specific activity of 2-3 mumol/min per mg; the optimal pH for activity is 7.5. ATPase, GTPase, and UTPase all reside in the same protein, which is inhibited by ruthenium red, is irreversibly denatured at 50 degrees C, and which has a sedimentation coefficient of 8-10 S. This enzyme is compared with other surface-derived ATPases of ciliated protozoans, and its possible roles are discussed.

Acid Phosphatase↗
PubMed1986

Characterization of Ca2+- or Mg2+-ATPase of the excitable ciliary membrane from Paramecium tetraurelia: comparison with a soluble Ca2+-dependent ATPase.

We have characterized divalent-cation-stimulated nucleoside triphosphate hydrolase activity of the excitable ciliary membrane and compared it with a soluble Ca2+-ATPase released upon deciliation of Paramecium. The membrane-bound activity is strongly dependent on a divalent cation; calcium stimulates the basal activity of this enzyme at least 10-fold; magnesium and manganese stimulate less well, and strontium and barium, although less effective, also give measurable stimulation. This membrane-bound activity prefers ATP and GTP as substrates but also hydrolyzes UTP and CTP at measurable rates. The maximum velocity at saturating ATP concentrations and optimal calcium concentrations is 0.3 mumol/min per mg. The pH optimum for the membrane-bound activity is broad and centers around pH 7. From the temperature dependence of ATP hydrolysis, we calculate activation energies of 14 and 11 kcal/mol for the Ca2+- and Mg2+-stimulated activities, respectively. The Arrhenius plot is linear over the temperature range of 4 to 25 degrees C. The membrane ATPase is relatively insensitive to ouabain, oligomycin, N,N'-dicyclohexylcarbodiimide, vanadate, Ruthenium red and two calmodulin antagonists. Polyclonal antisera raised against the purified soluble ATPase from the deciliation supernatant show low reactivity with the membrane-bound ATPase. We conclude from the comparison of properties of the two activities that the ciliary membrane-bound ATPase is distinct from the soluble ATPase released by deciliation.

Animals↗
PubMed1990

Characterization of a putative Ca2(+)-transporting Ca2(+)-ATPase in the pellicles of Paramecium tetraurelia.

In Paramecium, no Ca2(+)-ATPases with the properties of Ca2+ pumps have been identified. Here we report a pellicle associated Ca2(+)-ATPase activity and a corresponding phosphoprotein intermediate characteristic of a pump. The Ca2(+)-ATPase activity requires 3 mM Mg for optimal Ca2+ stimulation (KCa = 90 nM) and is specific for ATP as substrate (Km = 75 microM). Vanadate and calmidazolium inhibit Ca2(+)-stimulated activity with an EC50 of about 2 microM and 0.5 microM, respectively. Likewise, 10 microM trifluoperazine inhibits 80% of Ca2(+)-ATPase activity, but bovine calmodulin fails to stimulate. The Ca2(+)-ATPase is not inhibited by sodium azide (10 mM), oligomycin (10 micrograms/ml) or ouabain (0.2 mM). Incubation of pellicles with [gamma-32P]ATP specifically labels a 133 kDa protein in a Ca2(+)-dependent, hydroxylamine-sensitive manner, and the level of phosphorylation is increased by 100 microM La3+. Phosphorylation of an endoplasmic reticulum-enriched fraction labels a Ca2(+)-dependent protein different from the pellicle protein, being lower in molecular mass and unaffected by La3+. Ca2+ uptake by the alveolar sacs, integral components of the pellicle membrane complex, is poorly coupled to Ca2(+)-stimulated ATP hydrolysis (Ca2+ transported/ATP hydrolysed less than 0.2) and is much less sensitive to vanadate inhibition (EC50 approx. 20 microM) compared to the total Ca2(+)-ATPase activity. Therefore, the majority of the Ca2(+)-ATPase activity is likely to be plasma membrane associated.

Adenosine Triphosphate↗
PubMed1992

Isolation of the membranes from secretory organelles (trichocysts) of Paramecium tetraurelia.

We present for the first time a method for isolation of the membranes of extrusive organelles (trichocysts) from sterile culture of different strains of Paramecium tetraurelia. First, trichocysts are isolated according to a new method (Glas-Albrecht, R. and Plattner, H. (1990) Eur. J. Cell Biol. 53, 164-172) with high purity and yield. Then the organelles are subjected to osmotic swelling. Since trichocysts then easily 'decondense' and entangle membranes, these cannot be isolated directly by centrifugation, but only by passage through a filter and subsequent centrifugation. Purity of membrane fractions is analysed by electron microscopy and SDS-PAGE, combined with silver staining or, after biotinylation, by avidin-peroxidase labelling. Molecular masses resolved in our gels are in a range from less than or equal to 15 to greater than or equal to 105 kDa. Main bands obtained with nd9-28 degrees C trichocyst membranes (most bands also being common to wild type trichocysts) are of about 16.5, 19-21, 27-29, 33-34, 44-45 (strong), 47-48 (strong), 57, 61, 65 (strong), 68-71, 75, 81, 94-95 (strong), 104 and greater than or equal to 110 kDa, from a total of approx. 23 bands resolved. There is no remarkable occurrence of dominant protein bands from trichocyst contents ('trichynins'), though these might represent up to 10(3)-times more of the total trichocyst proteins. The ratio of phospholipid/protein is approx. 0.2 mg/mg. The methodology developed might also be valuable for the isolation of extrusome membranes from some other protozoan species.

Animals↗
PubMed1996

Caffeine inhibits Ca2+ uptake by subplasmalemmal calcium stores ('alveolar sacs') isolated from Paramecium cells.

Caffeine inhibits 45Ca2+ sequestration by subplasmalemmal calcium stores ('alveolar sacs') of low thapsigargicin sensitivity which we have isolated from the ciliated protozoan, Paramecium tetraurelia. Inhibition depends on caffeine concentration, with an IC50 of 31.8 mM. According to kinetic evaluation this is compatible with non-competitive inhibition of Ca2+ uptake, rather than with superimposed 45Ca2+ release during sequestration. It remains to be analysed whether this mechanism might be of possible relevance also for Ca2+-mediated activation in vivo in this or in any other secretory system. Such an effect could also operate indirectly, e.g., by Ca2+-release induction via sequestration inhibition. This is the first description of caffeine-mediated inhibition of Ca2+ uptake by calcium stores from a secretory system. Our data are compatible with some observations with sarcoplasmic reticulum from striated muscle fibers.

Adenosine Triphosphate↗
PubMed1980

Biochemical studies of the excitable membrane of Paramecium. IV. Protein kinase activities of cilia and ciliary membrane.

Two protein kinases (ATP: protein phosphotransferase, EC 2.7.1.37) were detected in disrupted cilia of Paramecium tetraurelia. One of the enzymes exhibited maximum activity at pH 6.0, required 4 mM Mg2+ for its maximum activity and was stimulated by cyclic AMP and cyclic GMP. Histone was a good exogenous protein substrate for this enzyme, but protamine sulfate was not. The other protein kinase showed a peak of activity at pH 8.0, required 10 mM Mg2+ for its maximum activity and was slightly inhibited by cyclic AMP and cyclic GMP. Protamine sulfate was a good exogenous substrate for this enzyme. The pH 8.0 activity partitioned preferentially with the axonemes, but the pH 6.0 activity was divided almost equally between the axonemes and the membranes. We also found indirect evidence for the presence in cilia of phosphoprotein phosphatase (phosphoprotein phosphohydrolase, EC 3.1.3.16) and adenyl cyclase (ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1) activity.

Adenosine Triphosphate↗
PubMed1984

Fatty acid metabolism in Paramecium. Oleic acid metabolism and inhibition of polyunsaturated fatty acid synthesis by triparanol.

Paramecium requires oleic acid for growth and can grow in media containing no other fatty acids. In the present study, we have shown that this ciliate utilized oleate mainly as a carbon and energy source, even though this fatty acid was the only substrate available for synthesis of polyunsaturated fatty acids. Culture growth was inhibited by the addition of the drug triparanol. Triparanol decreased the formation of polyunsaturated fatty acids from oleate by preventing desaturation to form the dienoic acid, linoleate. Triparanol inhibition resulted in an altered phospholipid fatty acyl composition, an increased fragility and an altered behavioral response of the cells to a depolarizing stimulation solution. Therefore, although most of the dietary oleate was not used by the cells for polyunsaturated fatty acid synthesis, the desaturation of oleic acid was critical for normal culture growth, cell integrity and swimming behavior, all of which are expected to be dependent on normal membrane lipid composition.

Animals↗
PubMed1976

Transfer RNA methyltransferase activity in paramecium aurelia.

The tRNA methyltransferases from Paramecium aurelia were investigated. The effects of varying the Mg2+ and NH4+ concentrations, pH, and temperature on the methylation of Escherichia coli B tRNA using extracts from P. aurelia were determined. Optimum tRNA methyltransferase activity was observed at pH 7.8 and 37 degrees C. The Mg2+ optimum occurred at 0.66 mM in the absence of NH4+ while the NH4+ optimum occurred at 100 mM in the absence of Mg2+. Analysis of the bases methylated in (E. coli B) tRNA by extracts of P. aurelia showed the presence of 1-methyladenine, 1-methylguanine, N2-methylguanine, N2,N2-dimethylguanine and methylated pyrimidine nucleotides. In comparison, an analysis of the in vivo methylation of tRNA from P. aurelia showed the presence of 1-methyladenine, 6-methyladenine, 6,6-dimethyladenine, 1-methylguanine, N2-methylguanine, N2,N2-dimethylguanine, 7-methylguanine, and methylated pyrimidine nucleotides. The pattern of methylation of tRNA in P. aurelia is similar to that observed in other eukaryotes.

Ammonia↗