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PubMed2006

Inefficient serotype knock down leads to stable coexistence of different surface antigens on the outer membrane in Paramecium tetraurelia.

Expression of surface antigens is usually mutually exclusive, meaning that only one protein is present on the cell surface. With the RNAi feeding technology we induce serotype shifts in Paramecium tetraurelia which are demonstrated to be incomplete, meaning that the cells remain in a shifting state. The coexpression of "old" and "new" protein on the surface can be detected to be stable for more than 15 divisions over a 5-day feeding procedure, a time period different from that reported for temperature-induced shifts. A characteristic heterogenic distribution of the different surface antigens is demonstrated by double indirect-immunofluorescent-staining and we show antigen transport mechanisms related to the tips of cilia. Therefore, we discuss release mechanisms, potential sorting mechanisms for glycosylphosphatidylinositol-anchored proteins and the localizations of surface antigens, which are important for the reported classical immobilization reaction.

Animals↗
PubMed2006

Detection of NADPH-diaphorase activity in Paramecium primaurelia.

Recently, we showed that Paramecium primaurelia synthesizes molecules functionally related to the cholinergic system and involved in modulating cell-cell interactions leading to the sexual process of conjugation. It is known that nitric oxide (NO) plays a role in regulating the release of transmitter molecules, such as acetylcholine, and that the NO biosynthetic enzyme, nitric oxide synthase (NOS), shows nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) activity. In this work, we detected the presence of NADPH-d activity in P. primaurelia. We characterized this activity histochemically by examining its specificity for beta-NADPH and alpha-NADH co-substrates, and sensitivity both to variations in chemico-physical parameters and to inhibitors of enzymes showing NADPH-d activity. Molecules immunologically related to NOS were recognized by the anti-rat brain NOS (bNOS) antibody. Moreover, bNOS immunoreactivity and NADPH-d activity sites were found to be co-localized. The non-denaturing electrophoresis, followed by exposure to beta-NADPH or alpha-NADH co-substrates, revealed the presence of a band of apparent molecular mass of about 124 kDa or a band of apparent molecular mass of about 175 kDa, respectively. In immunoblot experiments, the bNOS antibody recognized a single band of apparent molecular mass of about 123 kDa.

Animals↗
PubMed2004

GABAA receptor subunits identified in Paramecium by immunofluorescence confocal microscopy.

The presence of opioid, beta-adrenergic and cholinergic receptors has been demonstrated in ciliated protozoa, but little is known about gamma-aminobutyric acid (GABA) receptors. In this study we have analyzed the distribution of GABA(A)-type receptor subunits in Paramecium. Confocal laser microscopy using antibodies specific for alpha(1)-, alpha(2)-, alpha(3)-, alpha(6)-, beta(2/3)-, gamma(2)-, epsilon-, lambda-, and theta-subunits showed that most receptors are aggregated in clusters and are distributed both on cell surface and in the cytoplasm. The intensity of labelling of the alpha(6)-, beta(2/3)- and gamma(2)-subunits was more intense than the alpha(1)-, epsilon-, and theta-subunits, suggesting that the former are present in higher concentrations than the latter.

Animals↗
PubMed2004

Differences in gene expression of the ciliate Paramecium caudatum caused by endonuclear symbiosis with Holospora obtusa, revealed using differential display reverse transcribed PCR.

We identified six genes of Paramecium caudatum, which differentially expressed in Holospora obtusa-bearing and H. obtusa-free cells using differential display reverse transcribed PCR (DDRT-PCR). Northern blot analyses revealed that two of the genes, CA10-3 and CA20-2, were expressed extensively in the H. obtusa-free cell, while the other four, AS16-1, CS14, CS21 and CA17-1, were expressed more in the H. obtusa-bearing cell. Putative amino acid sequences of CA10-3, AS16-1 and CA17-1 showed high homologies with known genes related to intracellular signaling, transcription and aerobic metabolism. CS14 and CS21 also showed homologies with some genes whose products are still functionally unknown, but CA20-2 encoded a novel protein. We show in this study that H. obtusa alters multiple gene expression of the host after establishing endosymbiosis.

Adaptation, Physiological↗
PubMed2005

Paramecium caudatum acquires heat-shock resistance in ciliary movement by infection with the endonuclear symbiotic bacterium Holospora obtusa.

Holospora obtusa is a macronucleus-specific bacterium of the ciliate Paramecium caudatum. Three types of P. caudatum cells (H. obtusa-free cells, cells bearing the reproductive form of H. obtusa and cells bearing the predominantly infectious form of H. obtusa) cultured at 25 degrees C were transferred to 4, 10, 25, 35 and 40 degrees C and their swimming velocities were measured by taking photomicrographs with two-second exposures. The H. obtusa-free cells almost ceased swimming at both 4 and 40 degrees C, while cells bearing the reproductive form and those bearing the predominantly infectious form actively swam even at these temperatures. These results show that the host cell can acquire heat-shock resistance when infected by H. obtusa in the macronucleus. This is the first evidence to show that the endonuclear symbiont Holospora contributes to maintain the ciliary movement of the host even at temperatures unsuitable for the host growth.

Animals↗
PubMed2006

A physical model for galvanotaxis of Paramecium cell.

We propose a qualitative physical model of galvanotaxis of Paramecium cells using a bottom-up approach to link the microscopic ciliary motion and the macroscopic behavior of the cells. From the characteristic pattern of ciliary motion called the Ludloff phenomenon, the torque that orients the cell toward the cathode is derived mathematically. Dynamical equations of motion are derived and their stability is discussed. In numerical simulations using our model, cells exhibit realistic behavior, such as U-turns, like real cells.

Animals↗
PubMed2004

An alpha-tocopherol dose response study in Paramecium tetraurelia.

Vitamin E (D,L-alpha-tocopherol) was administered to Paramecium tetraurelia in doses of 10, 100, 1000 and 10,000mg/l throughout its clonal lifespan. ANOVA revealed significant differences in clonal lifespan between groups, whether lifespan was measured in total fissions, or in days (P<0.05). When mean clonal lifespan was measured in fissions the greatest difference was between the 1000mg/l alpha-tocopherol treatment at 382 fissions, and the ethanol control at 255.5 fissions. The greatest difference in mean clonal lifespan in days survived was between the 10,000mg/l alpha-tocopherol treatment at 292.5 days and the ethanol control at 76 days. ANOVA also revealed significant differences (P<0.05) in the initial cell fission rates between groups. At the 1000 and 10,000mg/l concentrations of alpha-tocopherol, a decrease in cell fission rates was apparent early in the lifespan, but these rates began to increase gradually during the late clonal lifespan. Although no clonal toxicity effects were found in terms of decreasing life-expectancy, the 1000 and 10,000mg/l treatment groups exhibited higher background mortality rates throughout their respective lifespans than did the control groups, which could represent a cytotoxic effect.

Animals↗
PubMed2004

A Paramecium tetraurelia mutant that has long autogamy immaturity period and short clonal life span.

We have isolated an unprecedented mutant of Paramecium tetraurelia that has a long immaturity period until autogamy. This mutant stock, d4-RK, screened for 0% autogamy at the age of 27 fissions, began to undergo autogamy around the age of 50 fissions in some clones and underwent autogamy scarcely even after the age of 100 fissions in others. d4-RK expressed its mutant phenotype at 25 degrees C, but resembled the wild-type phenotype at 32 degrees C. Genetic analyses indicated that a single recessive gene, named rie (remote immaturity exit), was responsible for the mutant phenotype. This is the first report to show a gene that elongates the time to sexual maturation in unicellular organisms. The clonal life span was shorter and fission rate was lower in the rie mutant than in the wild-type, both at 25 degrees C and 32 degrees C. Even in the fourth autogamous generation following the third backcross to the wild-type, the progeny with the elongated autogamy immaturity period still had a short clonal life span and low fission rate, while those with the wild-type phenotype in autogamy immaturity period showed the wild-type phenotypes in clonal life span and fission rate, too.

Animals↗
PubMed2005

A new mutation in the timing of autogamy in Paramecium tetraurelia.

We have isolated a new type of Paramecium tetraurelia mutant, named rie-2, that has a long immaturity period until autogamy. We previously isolated such an autogamy mutant, designated rie-1. These two mutants had some additional common features such as dependence of the occurrence of autogamy on the temperature, involvement of a single recessive gene, lower fission rate and shorter clonal life span. However, rie-2 was considered a new type mutant distinguishable from rie-1 because of their different natures of temperature sensitivity. First, the temperature at which they resembled the wild-type phenotype was low (19 degrees C) in rie-2, although it was high (32 degrees C) in rie-1. Second, the clonal life span of rie-2 at 25 degrees C was similar to that of the wild-type, but it was extremely shorter at 32 degrees C than at 25 degrees C, although it was similarly shorter at both temperatures in rie-1. Third, the difference of the fission rate between mutant and wild-type was greater at 32 degrees C than at 25 degrees C in rie-2, although it was similar at both temperatures in rie-1. This report shows that a gene mutation to elongate the period until sexual maturation does not necessarily assure the long life span.

Animals↗
PubMed2006

Alteration of developmental program in Paramecium by treatment with trichostatin a: a possible involvement of histone modification.

In the ciliate Paramecium caudatum, the somatic macronucleus for the next generation differentiates from a zygotic germinal micronucleus during conjugation. Subsequently, progeny are programmed to enter a sexually "immature" period, during which cells do not mate. This programming occurs at a critical period during the fifth cell cycle after conjugation. Here, we show that treatment with trichostatin A, a histone deacetylase inhibitor, leads to a partial recovery of mating reactivity in immature cells, suggesting that histone acetylation can affect the developmental program. In addition, immunostaining demonstrated that Lys 4 methylation of histone H3 was absent from or present at an undetectable level in the nascent macronucleus during the first cell cycle after conjugation. The methylation level gradually increased in proportion to de novo DNA synthesis, until the new macronucleus reached the maximum level of methylation, concomitant with its full maturation. A link between gene expression and Lys 4 methylation is indirectly supported by the observation that a transcriptionally active gene was enriched by chromatin immunoprecipitation with an antibody directed against methyl-Lys 4 H3, whereas a silent gene was not. These results provide evidence that histone modification plays a key role in the regulation of gene expression and the developmental programming that determine sexual immaturity after conjugation.

Amino Acid Sequence↗
PubMed2004

Large-scale genome remodelling by the developmentally programmed elimination of germ line sequences in the ciliate Paramecium.

In Paramecium, during the development of the somatic macronucleus, precise excision of thousands of single-copy non-coding sequences is initiated by specific DNA double-strand breaks, while imprecise elimination of germ-line-limited repeated sequences leads to internal deletions or chromosome fragmentation. Recent data point to a role of non-coding RNAs in the epigenetic programming of these rearrangements.

Animals↗
PubMed1986

Evaluation of chemically-induced phototoxicity to aquatic organism using Paramecium as a model.

Phototoxicity evaluation using Paramecium aurelia as a model revealed that 4 out of 21 pesticides produced lethal toxicity to cells. Four commonly used synthetic dyes (bromophenol blue, rose bengal, benzanthrone and methylene blue) also exhibited toxicity. Well known phototoxic agents like hematoporphyrin, riboflavin, and anthracene produced positive phototoxic response. Psoralen, a DNA cross-linking agent, also produced phototoxicity to the cells. The results clearly demonstrate that the synergistic action of chemical agents and sunlight produce lethal effects to aquatic organism.

Animals↗
PubMed1976

Contractile events in the cilia of Paramecium, Opalina, Mytilus, and Phragmatopoma.

The motion of the abnormal cilia of Opalina and Mytilus can be described by the recently developed model for ciliary motion, provided the activation of the contractility during the effective stroke is reduced by three- to fivefold compared with that in the recovery stroke. The stiffness of the Mytilus cilium during the effective stroke is found several hundred times larger than that predicted by the model, however. The stiffness of the cilia of Paramecium, Opalina, Phragmatopoma, and of Mytilus in the recovery phase, is predicted approximately correctly by the model. The activation of contractility in Mytilus and Phragmatopoma cilia increases with the viscosity of the medium, as the velocity of the ciliary motion slows down. This leads to the equivalent of a force-velocity relation. The velocity of propagation of the bend in the cilia during the recovery stroke is shown to be dependent only on the elastic properties of the ciliary shaft, and to be independent of the contractile activiey.

Animals↗
PubMed1978

Morphometric analysis of macronuclei and macronuclear fragments in autogamous cultures of Paramecium aurelia.

The general pattern of ultrastructural organization of macronuclear fragments which appear during autogamous changes of the nuclear apparatus of Paramecium aurelia is similar to that observed in undisturbed macronuclei. Morphometric analysis of electron-micrographs reveals, however, that only the nucleolar relative surface area remains unchanged in macronuclear fragments as compared with macronuclei, this suggesting the same intensivity of rRNA synthesis in them. Moreover, there exists, between the relative surface area covered by heterochromatin and the nucleoli in macronuclear fragments, an inversely proportional, statistically significant correlation, different than that calculated for macronuclei. The relative surface area of heterochromatin in macronuclear fragments is smaller than in macronuclei, what may be a sign either of enhancement of chromatin activity or of proceeding nuclear lysis.

Animals↗
PubMed2003

Refilling of cortical calcium stores in Paramecium cells: in situ analysis in correlation with store-operated calcium influx.

This is the first thorough study of refilling of a cortical calcium store in a secretory cell after stimulation in which we combined widely different methodologies. Stimulation of dense-core vesicle ("trichocysts") exocytosis in Paramecium involves a Ca(2+) -influx" superimposed to Ca(2+) -release from cortical stores ("alveolar sacs" (ASs)). In quenched-flow experiments, membrane fusion frequency rose with increasing [Ca(2+)](o) in the medium, from approximately 20-25% at [Ca(2+)](o) < or = 0.25 microM to 100% at [Ca(2+)](o) between 2 and 10 microM, i.e. close to the range of estimated local intracellular [Ca(2+)] during membrane fusion. Next, we analyzed Ca(2+)-specific fluorochrome signals during stimulation under different conditions. Treatment with actin-reactive drugs had no effect on Ca(2+) -signaling. In double trigger experiments, with BAPTA in the second secretagogue application (BAPTA only for stimulation and analysis), the cortical Ca(2+) -signal (due solely to Ca(2+) released from cortical stores) recovered with t(1/2) approximately 65 min. When ASs were analyzed in situ by X-ray microanalysis after different trigger times (+Ca(2+)(o)), t(1/2) for store refilling was similar, approximately 60 min. These values are similar to previously measured 45Ca(2+) -uptake by isolated ASs. In sum we find, (i) exogenous Ca(2+) increases exocytosis/membrane fusion performance with EC(50)=0.7 microM, (ii) Ca(2+) -signaling in this system is not sensitive to actin-reactive drugs, and (iii) refilling of these cortical calcium stores goes on over hours and thus is much slower than expected.

Actins↗
PubMed1998

An exocytotic mutant of Paramecium caudatum: membrane fusion without secretory contents release.

This is a detailed characterization of a secretory mutant incapable of releasing secretory contents despite normal exocytotic membrane fusion performance. Trichocyst non-discharge strain tnd1 of Paramecium caudatum and its wildtype (wt) both show a transient cortical [Ca2+]i increase and exocytotic membrane fusion in response to the polyamine secretagogue, aminoethyldextran (AED), or to caffeine. tnd1 cells frequently display spontaneous Ca2+ signals parallelled by spontaneous exocytotic membrane fusion. This remains undetected, unless the trichocyst matrix is shown to be freely accessible to the inert, non-membrane permeable fluorochrome, F2FITC, from the outside. In these tnd1 cells, spontaneous and AED- or caffeine-induced membrane fusion, always without contents expulsion by decondensation (i.e. several-fold stretching), is ascertained by electron microscopy. Exocytotic openings, with condensed trichocysts retained, may persist for hours without impairing cells. Trichocyst decondensation normally requires micromolar [Ca2+]e, but an increase to 10 mM has no effect on tnd1 trichocyst expansion in vivo or in vitro (when isolated and exposed to ionophore A23187 + Ca2+). Paracrystalline packing of the major secretory components (trichynins) does occur, despite incomplete proteolytic precursor processing (according to SDS-PAGE). However, 45Ca(2+)-binding by secretory components is considerably reduced--the likely cause of the non-discharge phenotype. Our findings imply significant untriggered membrane fusion in a system normally following the triggered pathway and clear separation of exocytotic membrane fusion from any later Ca(2+)-dependent steps of the secretory cycle.

Animals↗
PubMed2001

Paramecium genome survey: a pilot project.

A consortium of laboratories undertook a pilot sequencing project to gain insight into the genome of Paramecium. Plasmid-end sequencing of DNA fragments from the somatic nucleus together with similarity searches identified 722 potential protein-coding genes. High gene density and uniform small intron size make random sequencing of somatic chromosomes a cost-effective strategy for gene discovery in this organism.

Animals↗
PubMed2002

RNA interference by feeding in Paramecium.

RNA interference can be induced very efficiently by feeding the ciliate Paramecium with bacteria engineered to express double-stranded RNA, opening the possibility of large-scale functional screening in this unicell.

Animals↗