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Positional distribution of fatty acids in the major glycerophospholipids of Paramecium tetraurelia.

Fatty acid positional distributions and the fatty acid compositions of diacyl and alkyl acyl analogs of the three major glycerophospholipids of Paramecium tetraurelia were examined. Phosphatidylcholine and the phospho- and phosphonyl ethanolamine glycerolipids were separated into diacyl and alkyl acyl species by thin-layer chromatography after phospholipase C digestion, and acetylation. Phosphatidylcholine and the ethanolamine phosphonolipid were predominantly in the alkyl acyl form and phosphatidylethanolamine was predominantly in the diacyl form. The three major glycerophospholipids were also subjected to hydrolysis by phospholipase A2. Complete and efficient hydrolyses of all three phospholipids were accomplished with the enzyme from porcine pancreas. Sodium tetraborate prevented acyl migration when added to reaction mixtures. Fatty acids released by phospholipase A2 action, and the lysoderivatives were separated by thin-layer chromatography. Fatty acid methyl esters were prepared and analyzed by gas-liquid chromatography. Saturated acids were predominantly at C-1 of diacyl phospholipids. Polyunsaturated fatty acids were mainly at C-2, particularly in the alkyl acyl species. An exception was gamma-linolenate which was a major acid found esterified to C-1 in the three diacyl phospholipids. Identification of this acid at that position supports the idea that in some ciliates there may be an acyltransferase, specific for gamma-linolenate, that esterifies this acid to the glycerophospholipids.

Animals↗

Acid phosphatase activity in mating type I and mating type II cell lines of Paramecium primaurelia.

The cellular acid phosphatase content, a marker enzyme for lysosomal activity, in Paramecium primaurelia mating type I and mating type II cells was determined by optical laser scanning microscopy. The naphthol AS-TR phosphatase-hexazotized pararosaniline method was used to visualize acid phosphatase activity by the light microscopy. Cell lines of both mating types were tested during culture life, from the early log phase to the death phase. The amount of acid phosphatase was higher in mating type II than in mating type I until the onset of the stationary phase, and then the values reversed. Indeed, during the log phase of growth, mating type II cells formed a higher number of food vacuoles, so that, by taking up a higher amount of bacteria, they sooner became deprived of food. It is suggested that, by lacking nutrients, their synthesis activities and acid phosphatase content were reduced as compared with mating type I cells.

Acid Phosphatase↗

Paramecium triaurelia and other species of the aurelia complex in the Kaczawskie Mts and Plateau (western Sudeten).

The present investigations revealed the occurrence of Paramecium triaurelia on the territory of the Kaczawskie Plateau, while its presence was confirmed in the Kaczawskie Mts. In Poland, the presence of this species was recorded only in the Western Sudeten, in spite of many years of investigation. As the result of the studies also the occurrence of P. biaurelia and P. novaurelia was recorded on the territory of the Kaczawskie Mts and the Kaczawskie Plateau.

Animals↗

Effects of atmospheric pollution on Paramecium sp. cells.

The toxicity level of heterocyclic compound fractions obtained from dust in Silesia for Paramecium primaurelia was studied. For the estimation of their toxicity a special scale elaborated for this material was used. It was found that the level of toxicity of investigated fractions differed, depending on the sampling place and season of the year.

Air Pollutants↗

The upstream region is required but not sufficient to control mutually exclusive expression of Paramecium surface antigen genes.

Paramecium tetraurelia stock 51 can express at least 11 different surface antigens, yet only one type is found on the surface of a cell at any given time. The mechanism that controls this mutually exclusive expression is unknown. A previous study has shown that the 51A surface antigen gene is regulated at the level of transcription (Gilley, D., Rudman, B. M., Preer, J. R., Jr., and Polisky, B. (1990) Mol. Cell. Biol. 10, 1538-1544). We show here that the 51B surface antigen gene is also transcriptionally regulated, and when the 51A and 51B genes are cotransformed into an A-, B- mutant, the 51A antigen is dominant at 27 degrees C just as in wild type cells. We have utilized this cotransformation system to experimentally determine that 273 base pairs of DNA upstream of the 51A gene is sufficient to allow the dominant expression of A, but 150 base pairs is not adequate. A hybrid gene that contains the upstream region of 51B attached to the 51A transcribed region was cotransformed with the 51B gene into the A-, B- mutant. Despite containing the same upstream sequences, only the hybrid 51B/A was transcribed at 27 degrees C. These results suggest the upstream region is required but not sufficient for mutually exclusive transcriptional control.

Animals↗

[Effects of hypergravity on Paramecium tetraurelia].

Previous space experiments carried out in Paramecium tetraurelia have shown that exposure to microgravity results in an enhancement of cell multiplication. An opposite effect occurs when paramecia are exposed to hypergravity. Changes in cell growth rate observed in hypergravity cannot be ascribed to the bacteria present in the culture medium, the same effect being observed when paramecia grow in sterile medium.

Animals↗

Age-related changes in neutral lipid content of Paramecium primaurelia as revealed by nile red.

Cellular neutral lipid content of Paramecium primaurelia was measured during culture and clonal life using nile red (9-diethylamino-5H-benzo[alpha] phenoxazine-5-one), a dye utilized for lipid analysis in both mammalian cells and in ciliated protozoa. Lipid droplets in P. primaurelia are concentrated in the anterior pole of the cell; their number is maximum in early log phase cells and decreases in late log phase cells. The quantitative determination of neutral lipids was obtained measuring the fluorescence from the excitation and emission spectra of 480 nm and 540 nm, respectively. Neutral lipid content decreases linearly during the log phase of growth while the decline is minimum during the stationary phase. In the late log phase, the amount is 30% of that of the early log-phase cells. Though the cell size declines too, cell area and lipid content decreases are not correlated in the middle log phase, because the maximum lipid reduction is obtained when the cell size is relatively constant. The cellular lipid content also changes during the clonal life. Neutral lipids decrease discontinuously (r = -0.75, P < 0.05) as the fission age increases. No relationship was found between lipid content and food vacuole formation during both culture and clonal life.

Animals↗

The major ciliary membrane proteins in Paramecium primaurelia are all glycosylphosphatidylinositol-anchored proteins.

Using a strategy based upon specific features of membrane proteins linked to the plasma membrane by a glycosylphosphatidylinositol (GPI) anchor, we have studied in the strain 513 of Paramecium primaurelia the glycosylphosphatidylinositol (GPI) proteins both in their membrane-bound and soluble forms. 35S-Labeling associated with bacterial phosphatidylinositol-specific phospholipase C treatment of purified cilia allowed the identification of soluble GPI proteins (devoid of their lipid moiety), released from cilia. By labeling with 88[33P]phosphoric acid and [3H]ethanolamine, respectively, we identified membrane-bound GPI proteins, when anchored in the cilia membrane via the lipid of their GPI tail. We demonstrated that, in addition to the SAg (surface antigen) which is a high molecular weight protein implicated in the antigenic variation phenomenon, three other ciliary membrane proteins were also GPI-anchored. The membrane-bound and soluble forms of these GPI proteins had apparent molecular weights, in unreduced conditions, of 30,000 and 40 to 50,000, respectively. We named these surface GPI proteins SGPs, SGP1 to SGP3, SGP1 (surface GPI protein 1) and SGP2 (surface GPI protein 2) were the most abundantly expressed. Only SGP2 displayed a rapid turnover. By phosphatidylinositol-specific phospholipase C treatment of the membrane proteins recovered in the detergent phase, following partitioning with Triton X-114, we demonstrated that the SAg and SGPs are the major ciliary membrane proteins of P.primaurelia.

Animals↗

High resolution view of the true cytosolic membrane surface of phagosomes of known ages purified from Paramecium.

Techniques were used for viewing the true cytosolic surfaces of the membranes of intracellular organelles by field emission scanning electron microscopy (SEM). Cells of Paramecium multimicronucleatum were fed briefly with magnetic beads followed by a chase which advanced the newly formed digestive vacuoles (DVs) to predetermined ages. These bead-containing phagosomes were isolated from the whole cell homogenates with a magnet and were determined to be intact by fluorescence microscopy. Antigenically, these DVs were similar to those in situ. The DVs prepared for transmission electron microscopy or SEM showed extensive adherence of cellular debris. The use of 0.2 M KCl in the wash buffer eliminated much of this debris and exposed the true vacuolar surfaces. Three populations of tightly bound vesicles and numerous globular particles of 10 to 20 nm became visible on the DV surfaces. The attached vesicles, having diameters of approximately 300 and approximately 200 nm each, corresponded to the acidosomes and lysosomes that are known to be associated with the DV-I and DV-II, respectively. High resolution SEM also revealed a third set of small vesicles (50-150 nm), which were previously not known to be associated with DVs. The 10 to 20 nm globular particles were judged to be the cytosolic extensions of transmembrane protein complexes as their patterns of distribution on DVs of various ages corresponded to the transmembrane particles previously seen in these membranes in freeze-fracture studies.

Acid Phosphatase↗

Trichocyst membrane fate in living Paramecium primaurelia visualized by multimodal confocal imaging.

Trichocysts are secretory organelles located at the surface of several ciliates, docked at the plasma membrane. Their secretion is similar to other exocytic processes: the trichocyst membrane fuses with the plasma membrane, its content is released outside the cell, and the membrane is retrieved back into the cell. The fate of the trichocyst membrane in living Paramecium primaurelia was investigated by inducing massive synchronous exocytosis in the presence of fluorescein isothiocyanate-conjugated lectins or of cationized ferritin. The marker is trapped within the retrieved trichocyst membrane sac, and many regularly spaced, fluorescent ghosts are formed. As time proceeds, the number of labeled ghosts decreases, and few fluorescent vacuoles appear within the cell. The relationship between trichocyst ghosts and the vesicles of the phagosome-lysosome system was examined by labeling cells with Texas Red-conjugated bovine serum albumin, a fluorescent marker for phagocytosis. Starting from two confocal images of the same cell labeled with the two fluorescent probes, a new single image was generated by associating each image with a different red or green value. This multimodal analysis showed that trichocyst ghosts fuse with secondary lysosomes or are incorporated into digestive vacuoles. The vacuolar content is degraded and fluorescence is then found in the vesicles of the phagosome-lysosome system, and, at last, in small weakly labeled vesicles located on the cell surface.

Animals↗

Application of fluorescence resonance energy transfer techniques to the study of lectin-binding site distribution on Paramecium primaurelia (Protista, Ciliophora) cell surface.

Fluorescence resonance energy transfer (FRET) is a photophysical phenomenon occurring between the molecules of two fluorochromes with suitable spectral characteristics (donor-acceptor dye pair), and consisting in an excitation energy migration through a non-radiative process. Since the efficiency of the process is strictly dependent on the distance and reciprocal orientation of the donor and acceptor molecules, FRET-based techniques can be successfully applied to the study of biomolecules and cell component organisation and distribution. These techniques have been employed in studying Paramecium primaurelia surface membrane for the reciprocal distribution of N-acetylneuraminic acid (NeuAc) and N-acetylglucosamine (GlcNAc) glycosidic residues, which were found to be involved in mating cell pairing. NeuAc and GlcNAc were detected by their specific binding lectins, Limulus polyphemus agglutinin (LPA) and wheat germ agglutinin (WGA), respectively. Microspectrofluorometric analysis afforded the choice of fluorescein isothiocyanate and Texas red conjugated with LPA and WGA, respectively, as a suitable donor-acceptor couple efficiently activating FRET processes. Studies performed both in solution and in cells allowed to define the experimental conditions favourable for a FRET analysis. The comparative study carried out both on the conjugating-region and the non conjugating region of the surface membrane, indicates that FRET distribution appears quite homogeneous in mating-competent mating type (mt) I, whereas, in mating-competent mt II cells, FRET distribution seems to be preferentially localised on the conjugating-region functionally involved in mating cell pairing. This difference in the distribution of lectin-binding sites is suggested to be related to mating-competence acquisition.

Animals↗

Membrane potential fluctuation in Paramecium.

The fluctuation of the membrane potential in Paramecium caudatum cells was measured. The fluctuation was very large and composed of slow and periodic components and fast and random ones. The power of fast fluctuation depended on the environmental salt condition, particularly on the ratio of the potassium ion concentration to the square root of the calcium ion concentration, [K+]o/[Ca2+]o(1/2), in the medium. The power greatly increased with the decrease of this ratio. The power density spectrum of fluctuation was of the 1/(1 + (f/fc)2) type when the total power was large. There, the fluctuation arose mainly from the local current of potassium ions. Some cooperation among potassium channels to increase the fluctuation was suggested. The spectrum became of the 1/f type when the total power was small.

Journal Article↗

Ultrastructure of Chilomonas paramecium and the phylogeny of the cryptoprotists.

The ultrastructure of the cryptoprotist Chilomonas paramecium is reviewed and compared to earlier accounts. Distinctive features include a complex cytoskeleton which defines the cell organization and interconnects cell components; trichocysts which resemble those in other cryptoprotists; and two non-photosynthetic plastids. During mitosis there is partial dispersal of the nuclear envelope early in prophase but some remains at the nuclear periphery throughout mitosis. At metaphase chromosomes are arranged on the longitudinal axis of an elongated elliptical nucleus. During telophase the chromosomes decondense and the nuclear envelope reforms. Cell structure is compared with that in other cryptoprotists, and origin of this taxon of algae is discussed.

Animals↗

K-resistant mutants and "adaptation" in Paramecium.

A class of mutants of Paramecium tetraurelia has been isolated by adding a critical amount of K(+) to mutagenized, autogamized cultures. Adding 35 mM K(+) to the medium kills the wild type. The mutants can grow at 35 mM K(+) and some survive an addition of up to 80 mM K(+). Thirty-three lines of such mutants have been studied. These mutants have few or no behavioral abnormalities in culture medium.Paramecia can "adapt." Hildebrand and Dryl showed that P. caudatum incubated in 15 mM K(+) for over 15 min fail to give avoiding reactions when challenged with Ba(2+) or a thermal gradient [Hildebrand, E. & Dryl, S. (1976) Bioelectrochem. Bioenerg. 3, 543-544]. We have observed a similar phenomenon in wild-type P. tetraurelia. However, the K(+)-resistant mutants show little or no such "adaptation," i.e., they perform avoiding reactions when challenged with Ba(2+) after prolonged incubation in 15 mM K(+). The K(+) resistance and the lack of "adaptation" are strictly correlated. Because all 33 lines of mutants show this correlation, "adaptation" and killing by K(+) must share a mechanism. This mechanism may be studied by finding the basis of the K(+) resistance in the mutants. The possible ionic bases of "adaptation" are discussed.

Journal Article↗

Characterization of a novel cis-syn and trans-syn-II pyrimidine dimer glycosylase/AP lyase from a eukaryotic algal virus, Paramecium bursaria chlorella virus-1.

Endonuclease V from bacteriophage T4, is a cis-syn pyrimidine dimer-specific glycosylase. Recently, the first sequence homolog of T4 endonuclease V was identified from chlorella virus Paramecium bursaria chlorella virus-1 (PBCV-1). Here we present the biochemical characterization of the chlorella virus pyrimidine dimer glycosylase, cv-PDG. Interestingly, cv-PDG is specific not only for the cis-syn cyclobutane pyrimidine dimer, but also for the trans-syn-II isomer. This is the first trans-syn-II-specific glycosylase identified to date. Kinetic analysis demonstrates that DNAs containing both types of pyrimidine dimers are cleaved by the enzyme with similar catalytic efficiencies. Cleavage analysis and covalent trapping experiments demonstrate that the enzyme mechanism is consistent with the model proposed for glycosylase/AP lyase enzymes in which the glycosylase action is mediated via an imino intermediate between the C1' of the sugar and an amino group in the enzyme, followed by a beta-elimination reaction resulting in cleavage of the phosphodiester bond. cv-PDG exhibits processive cleavage kinetics which are diminished at salt concentrations greater than those determined for T4 endonuclease V, indicating a possibly stronger electrostatic attraction between enzyme and DNA. The identification of this new enzyme with broader pyrimidine dimer specificity raises the intriguing possibility that there may be other T4 endonuclease V-like enzymes with specificity toward other DNA photoproducts.

Binding Sites↗

Paramecium bursaria chlorella virus-1 encodes an unusual arginine decarboxylase that is a close homolog of eukaryotic ornithine decarboxylases.

Paramecium bursaria chlorella virus (PBCV-1) is a large double-stranded DNA virus that infects chlorella-like green algae. The virus encodes a homolog of eukaryotic ornithine decarboxylase (ODC) that was previously demonstrated to be capable of decarboxylating l-ornithine. However, the active site of this enzyme contains a key amino acid substitution (Glu for Asp) of a residue that interacts with the delta-amino group of ornithine analogs in the x-ray structures of ODC. To determine whether this active-site change affects substrate specificity, kinetic analysis of the PBCV-1 decarboxylase (PBCV-1 DC) on three basic amino acids was undertaken. The k(cat)/K(m) for l-arginine is 550-fold higher than for either l-ornithine or l-lysine, which were decarboxylated with similar efficiency. In addition, alpha-difluoromethylarginine was a more potent inhibitor of the enzyme than alpha-difluoromethylornithine. Mass spectrometric analysis demonstrated that inactivation was consistent with the formation of a covalent adduct at Cys(347). These data demonstrate that PBCV-1 DC should be reclassified as an arginine decarboxylase. The eukaryotic ODCs, as well as PBCV-1 DC, are only distantly related to the bacterial and plant arginine decarboxylases from their common beta/alpha-fold class; thus, the finding that PBCV-1 DC prefers l-arginine to l-ornithine was unexpected based on evolutionary analysis. Mutational analysis was carried out to determine whether the Asp-to-Glu substitution at position 296 (position 332 in Trypanosoma brucei ODC) conferred the change in substrate specificity. This residue was found to be an important determinant of substrate binding for both l-arginine and l-ornithine, but it is not sufficient to encode the change in substrate preference.

Amino Acid Sequence↗

Photosynthetic Shutdown in Chlorella NC64A Associated with the Infection Cycle of Paramecium bursaria Chlorella Virus-1.

The effects of the algal virus Paramecium bursaria Chlorella virus-1 on the photosynthetic physiology of its host, Chlorella NC64A, was studied by observing changes in Chl fluorescence quenching and O2 exchange. Metabolic changes were calibrated against electron microscopic analysis of the morphological changes that occur during the infection cycle. It takes approximately 10 h from attachment of the virus to final lysis of the host cell, so a complete infection cycle can be observed continuously in one experiment. During the early stages of the infection cycle many rapid changes occurred in the host cell's metabolism and these were reflected in changes of photosynthetic and respiratory rates. The dramatic inhibition of photosynthesis in Chlorella NC64A cells by P. bursaria Chlorella virus-1 has facilitated the use of fluorescence quenching as an accurate measure of the first phase of viral infection (attachment and penetration of the host cell) and the extent to which a population of host cells is infected. Effects of temperature and cation requirement of the infection cycle are described. The relevance of our observations to the events observed during viral infection of higher plants is discussed.

Journal Article↗

The ejectisomes of the flagellate Chilomonas paramecium: visualization by freeze-fracture and isolation techniques.

Freeze-fracture procedures were used to visualize ejectisomes and adjacent plasma membrane specializations in the flagellate protozoan Chilomonas paramecium. The ejectisomes are membrane-bounded, cylindrically rolled, extrusive organelles. Small ones occur in large number beneath the plasma membrane of the body and considerably larger ones are located around the gullet membrane. The intra-membrane particle distribution is different in each type. In small ejectisomes, the portion of the membrane in contact with the plasma membrane of the body has a P-face rosette of five particles while the plasma membrane has not been observed with a rosette. Small ejectisomes and plasma membrane both contain aggregations of particles a short distance from the contact or docking site. Slightly beneath the plasma membrane is the periplastic sheet with which we speculate the small ejectisomes interact during the docking phenomenon. No obvious rosettes have been observed in large ejectisomes. Some other ejectisomal structures are presented and discussed.

Animals↗