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PubMed1979

Purification and characterization of the myoglobins of Paramecium tetraurelia.

1. The molecular variants of the myoglobins of Paramecium tetraurelia have been purified as five separate components and designated Mb 1 to Mb 5. 2. The five molecular forms are homogeneous on polyacrylamide gel electrophoresis (PAGE) and isoelectric focusing (IF). 3. The myoglobin species appear to have identical molecular weights and amino acid compositions and differ only in their isoelectric points and relative concentrations in vivo. 4. The myoglobin species have an apparent molecular weight of 15,000 +/- 500 and possess a single heme group per mole which appears to be protoporphyrin IX. 5. The amino acid composition of the five species is: lys12, his3, arg4, asp17, thr9, ser6, glu16, pro4, gly11, ala15, cys0, val8, met2, ile7, leu10, tyr5, phe7. 6. The spectra of several ferrous and ferric derivatives of the mixture Mb 1-Mb 5 are presented.

Animals↗
PubMed1979

Control of ciliary activity in paramecium--IV. Ca2+ modification of Mg2+ dependent dynein ATPase activity.

1. Dynein proteins were solubilized from demembranated cilia of Paramecium by extraction at high ionic strength. 2. Mg2+-dependent ATPase (EC 3.6.1.3) activity of crude dynein extracts was inhibited by micromolar concentrations of Ca2+ ions. 3. Sepharose 4B chromatography of the crude extracts yields three dynein fractions. The major fraction contains a single protein and is insensitive to Ca2+ ions. Two other fractions, both heterogeneous in composition, show opposing Ca2+ ion sensitivity expressed as a Ca2+ dependent alteration in MgATP2- dependent ATPase activity. The Ca2+ ion sensitive forms show altered electrophoretic mobility on native polyacrylamide gels in the presence and absence of Ca2+ ions. 4. The data provides evidence for a Ca2+ ion dependent concomitant alteration in both molecular form and hydrolytic activity of the dyneins. The results are discussed in terms of a possible molecular mechanism for Ca ion regulation of ciliary activity in terms of the sliding microtubule model.

Adenosine Triphosphatases↗
PubMed1980

Release of Paramecium immobilization antigen to the non-nutrient medium.

The instability of Paramecium aurelia surface components has been shown: after 60 min incubation of dense cell suspension ((1-2 . 10(6) cells per ml) in Tris-Ca buffer at 4 degrees C or 23 degrees C the surface coat was partially stripped off and the proteins were released to the medium. The electrophoretic analysis of the released proteins shows one major band of mol. wt. 280,000--300,000 and some minor bands. The major released protein is the immobilization antigen as proved using immunodiffusion test and antibody precipitation technique.

Animals↗
PubMed1983

Decoupling of exocytotic membrane fusion from protein discharge in Paramecium cells.

Under certain conditions it is possible in Paramecium cells to induce selectively the fusion of the secretory organelle membrane with the cell membrane without the involvement of any further steps (release of secretory contents, etc.). A Ca2+-mobilizing fusogen was used in the presence of components which inhibit the discharge of the secretory contents (Mg2+ and EGTA, mainly). One can thus produce many exocytotic openings with the secretory contents (which are normally vigorously discharged) still retained.

Dibucaine↗
PubMed1987

Stimulation of Paramecium phagocytosis by phorbol ester and forskolin.

Phorbol ester (PMA) exerted a dose- and time- dependent stimulating effect on phagocytosis in axenic Paramecium aurelia. When cells were exposed to 200-800 nM PMA in the presence of latex beads, the phagocytic coefficient was enhanced 2.25 to 3.14 times, during 10 min of continuous treatment and then rapidly declined. A similar effect was observed when the cells were exposed to a forskolin treatment, which resulted in nearly a twofold increase in phagocytic activity after a 10 min pulse. Both PMA and forskolin strongly stimulated phagocytosis (i.e. fivefold and threefold, respectively) in cells in which such activity had been completely inhibited by pre-exposure to the beta-receptor antagonist 1-propranolol.

Animals↗
PubMed1980

Cloning and characterization of Paramecium mitochondrial DNA replication initiation regions.

Fragments containing the replication-initiation region of mitochondrial (mt) DNA from four species of Paramecium aurelia were ligated to the pBR322 plasmid and used to transform Escherichia coli. The criteria for identifying the desired mitochondrial DNA restriction fragments were based on the palindromic dimer structure of the replicative intermediate of this DNA. The nature of the cloned sequences was verified by hybridization to mitochondrial DNA fragments containing the initiation region, by determining the size of snapback renaturation products, and by determining the symmetry of restriction-endonuclease sites within the clones. Heterologous hybridization experiments showed that one species' initiation region is not homologous to the other three. Cleavage patterns of each of the clones by 18 different restriction enzymes were determined. There are no sites within approx. 250 bp of the center of the insert of any clone although there are numerous cleavages in more distal regions. These sites are generally symmetric about the center of the insert as expected for a palindromic structure. The characteristics of the cloned sequences support a proposed replication model for all four species studied.

Animals↗
PubMed1993

Structure of the Paramecium caudatum gene encoding the B-type of the major hemoglobin component.

We have found the gene (Hb) encoding the B-type of the major hemoglobin (Hb) component in the ciliated protist, Paramecium caudatum, stock K33 of syngen 3. Gene Hb, which encodes a protein consisting of 116 amino acids (aa), had the same sequence as the A-type Hb in this species, with the exception of 10 nucleotides (nt) reflecting 5 single-aa differences. The coding region was interrupted at the position between codons 62 and 63 by one short intron, which was comparable with the second intron of plant Hb in position, phase and nt sequence. The maximum alignment of the P. caudatum Hb aa sequence with the other globin sequences from prokaryotes and lower eukaryotes indicates that these globins can be classified into two groups: one consists of protist and cyanobacterial globins of about 120 aa, and the other consists of fungal and other bacterial globins of about 150 aa having a significant homology with higher eukaryotic Hbs. These proteins share more than 25% homology with one another in each group. Our finding of the P. caudatum Hb intron corresponding to the second intron of plant Hb suggests that all of the globin-encoding genes have a common origin, and that at least two groups had already existed in the globin-encoding gene before prokaryote-eukaryote radiation.

Amino Acid Sequence↗
PubMed1994

The identification of a complex family of low-molecular-weight GTP-binding protein homologues from Paramecium tetraurelia by PCR cloning.

We have used the polymerase chain reaction to clone a large number of gene fragments encoding low-molecular-weight GTP-binding proteins (LMW G-proteins) from Paramecium tetraurelia. All clones were subjected to a computer-assisted search of the GenBank databases to assign putative homologues. On the basis of several conserved features, we place these genes in the rab/ypt and rho subfamilies of the LMW G-proteins. Northern blot analyses indicated that all clones were from active genes. One surprising feature of the cloned genomic sequences was the presence of unusually short introns (20-30 nucleotides).

Amino Acid Sequence↗
PubMed1988

Vitamin E supplementation and intense selection increase clonal life span in Paramecium tetraurelia.

Vitamin E added to standard Cerophyl medium at 0.025 mg/ml significantly increased the mean clonal life span of 32 lines of Paramecium tetraurelia from 52.5 days and 187 fissions to 59.9 days and 209 fissions (p less than .05) when compared to unsupplemented, paired controls. The age-specific death rates increased exponentially. Regression analyses found a significantly lower rate of increasing mortality for the supplemented lines compared to controls. Weekly fission rates of supplemented lines declined linearly; more slowly than controls, but not significantly so. A second experiment tested two higher levels of supplemental vitamin E (0.10 and 1.00 mg/ml). Sublines receiving 1.00 mg/ml of supplemental vitamin E had higher rates of mortality and lower fission rates initially, but the mortality rates increased and the fission rates decreased more slowly than in sublines receiving 0.10 mg/ml supplemental vitamin E, resulting in maximum clonal life spans of 141 days and 330 fissions (1.00 mg/ml sublines), compared to 74 days and 271 fissions (0.10 mg/ml sublines). Survivorship of the last individual cells (nondividing) of each clone followed an exponential decline, with a significant increase in mean survival time for supplemented compared to unsupplemented cells (p less than .05).

Animals↗
PubMed1979

Preliminary results of Cytos experiment flown in Salyut VI: investigations on Paramecium aurelia.

The purpose of the Cytos experiment was to investigate a possible effect of space flight on cell proliferation kinetics. Cultures of Paramecium aurelia successively maintained at a temperature of 8 degrees and 25 degrees C were fixed every twelve hours during the orbital flight. Space flight resulted in a stimulating effect on cell proliferation, an increase in cell volume and changes in the ionic components of the culture medium.

Animals↗
PubMed2005

Quaternary assembly and crystal structure of GDP-D-mannose 4,6 dehydratase from Paramecium bursaria Chlorella virus.

GDP-D-mannose 4,6 dehydratase is the first enzyme in the de novo biosynthetic pathway of GDP-L-fucose, the activated form of L-fucose, a monosaccharide found in organisms ranging from bacteria to mammals. We determined the three-dimensional structure of GDP-D-mannose 4,6 dehydratase from the Paramecium bursaria Chlorella virus at 3.8A resolution. Unlike other viruses that use the host protein machinery to glycosylate their proteins, P. bursaria Chlorella virus modifies its structural proteins using many glycosyltransferases, being the first virus known to encode enzymes involved in sugar metabolism. P. bursaria Chlorella virus GDP-D-mannose 4,6 dehydratase belongs to the short-chain dehydrogenase/reductase protein superfamily. Accordingly, the family fold and the specific Thr, Tyr, and Lys catalytic triad are well conserved in the viral enzyme.

Animals↗
PubMed2005

Biotreatment of wastewater using aquatic invertebrates, Daphnia magna and Paramecium caudatum.

A number of major changes have occurred over the past few years, which give cause for a re-examination of conventional wastewater treatment methods. Among these are growing problems of worldwide energy and food shortages and nutrients not removed by conventional secondary processes causing algal blooms and other problems in the receiving waters. The global increase in wastewater calls for innovative low cost technology approaches to its recycling. Biotreatment systems, utilizing living organisms are receiving growing attention since they are ecologically sound, cheap and applicable in areas without land constraints. Filter feeders (both invertebrates and vertebrates) are promising in this area since they can remove suspended organic matter and bacteria, even in the size range of microns. In the present study biological treatment of municipal wastewater using two invertebrates--Paramecium caudatum, a protozoan and Daphnia magna, a cladoceran was investigated. Analysis at pre-experimental and post-experimental stages revealed the potential of these species in abatement of water pollution. D. magna was more efficient than P. caudatum in laboratory-scale studies.

Animals↗
PubMed2004

One-way calcium spill-over during signal transduction in Paramecium cells: from the cell cortex into cilia, but not in the reverse direction.

We asked to what extent Ca(2+) signals in two different domains of Paramecium cells remain separated during different stimulations. Wild-type (7S) and pawn cells (strain d4-500r, without ciliary voltage-dependent Ca(2+)-channels) were stimulated for trichocyst exocytosis within 80 ms by quenched-flow preparation and analysed by energy-dispersive X-ray microanalysis (EDX), paralleled by fast confocal fluorochrome analysis. We also analysed depolarisation-dependent calcium signalling during ciliary beat rerversal, also by EDX, after 80-ms stimulation in the quenched-flow mode. EDX and fluorochrome analysis enable to register total and free intracellular calcium concentrations, [Ca] and [Ca(2+)], respectively. After exocytosis stimulation we find by both methods that the calcium signal sweeps into the basis of cilia, not only in 7S but also in pawn cells which then also perform ciliary reversal. After depolarisation we see an increase of [Ca] along cilia selectively in 7S, but not in pawn cells. Opposite to exocytosis stimulation, during depolarisation no calcium spill-over into the nearby cytosol and no exocytosis occurs. In sum, we conclude that cilia must contain a very potent Ca(2+) buffering system and that ciliary reversal induction, much more than exocytosis stimulation, involves strict microdomain regulation of Ca(2+) signals.

Animals↗
PubMed2004

Ca2+ oscillations mediated by exogenous GTP in Paramecium cells: assessment of possible Ca2+ sources.

We applied exogenous guanosine trisphosphate, GTP, to Paramecium tetraurelia cells injected with Fura Red for analysing changes of free intracellular Ca(2+) concentrations, [Ca(2+)](i), during periodic back-/forward swimming thus induced. Strain ginA (non-responsive to GTP) shows no Ca(2+) signal upon GTP application. In strain nd6 (normal Ca(2+) signalling) an oscillating [Ca(2+)](i) response with a prominent first peak occurs upon GTP stimulation, but none after mock-stimulation or after 15 min adaptation to GTP. While this is in agreement with previous electrophysiological analyses, we now try to identify more clearly the source(s) of Ca(2+). Stimulation of nd6 cells, after depletion of Ca(2+) from their cortical stores (alveolar sacs), shows the same Ca(2+) oscillation pattern but with reduced amplitudes, and a normal behavioural response is observed. Stimulation with GTP, supplemented with the Ca(2+) chelator BAPTA, results in loss of the first prominent Ca(2+) peak, in reduction of the following Ca(2+) amplitudes, and in the absence of any behavioural response. Both these observations strongly suggest that for the initiation of GTP-mediated back-/forward swimming Ca(2+) from the extracellular medium is needed. For the maintenance of the Ca(2+) oscillations a considerable fraction must come from internal stores, probably other than alveolar sacs, rather likely from the endoplasmic reticulum.

Animals↗
PubMed2006

Sub-second calcium coupling between outside medium and subplasmalemmal stores during overstimulation/depolarisation-induced ciliary beat reversal in Paramecium cells.

As amply documented by electrophysiology, depolarisation in Paramecium induces a Ca(2+) influx selectively via ciliary voltage-dependent Ca(2+)-channels, thus inducing ciliary beat reversal. Subsequent downregulation of ciliary Ca(2+) has remained enigmatic. We now analysed this aspect, eventually under overstimulation conditions, by quenched-flow/cryofixation, combined with electron microscope X-ray microanalysis which registers total calcium concentrations, [Ca]. This allows to follow Ca-signals within a time period (> or =30ms) smaller than one ciliary beat ( approximately 50ms) and beyond. Particularly under overstimulation conditions ( approximately 10(-5)M Ca(2+) before, 0.5mM Ca(2+) during stimulation) we find in cilia a [Ca] peak at approximately 80ms and its decay to near-basal levels within 110ms (90%) to 170ms (100% decay). This [Ca] wave is followed, with little delay, by a [Ca] wave into subplasmalemmal Ca-stores (alveolar sacs), culminating at approximately 100ms, with a decay to original levels within 170ms. Also with little delay [Ca] slightly increases in the cytoplasm below. This implies rapid dissipation of Ca(2+) through the ciliary basis, paralleled by a rapid, transient uptake by, and release from cortical stores, suggesting fast exchange mechanisms to be analysed as yet. This novel type of coupling may be relevant for some phenomena described for other cells.

Animals↗
PubMed2006

Cryopreservation of Holospora undulata, a bacterial parasite of the ciliate Paramecium caudatum.

We investigated cryopreservation of horizontal transmission stages of Holospora undulata, a micronucleus-specific bacterial parasite of Paramecium caudatum. Unlike in previous studies on related Holospora species, protocols using glycerol as cryoprotectant failed entirely. In contrast, freezing with dimethyl sulfoxide (Me2SO) conserved infectiousness of nearly all replicate inocula, although infection success was considerably lower than that of fresh inocula. Infection probability was enhanced by increasing the Me2SO concentration from 5 to 10%, and by freezing at -196 degrees C rather than -80 degrees C. Prolonged storage of up to 3 months had no significant effect on the viability of the inocula.

Animals↗
PubMed2005

Nowa1p and Nowa2p: novel putative RNA binding proteins involved in trans-nuclear crosstalk in Paramecium tetraurelia.

BACKGROUND: The germline genome of ciliates is extensively rearranged during development of a new somatic macronucleus from the germline micronucleus, a process that follows sexual events. In Paramecium tetraurelia, single-copy internal eliminated sequences (IESs) and multicopy transposons are eliminated, whereas cellular genes are amplified to approximately 800 n. For a subset of IESs, introduction of the IES sequence into the maternal (prezygotic) macronucleus specifically inhibits excision of the homologous IES in the developing zygotic macronucleus. This and other homology-dependent maternal effects have suggested that rearrangement patterns are epigenetically determined by an RNA-mediated, trans-nuclear comparison, involving the RNA interference pathway, of germline and somatic genomes. RESULTS: We report the identification of novel developmentally regulated RNA binding proteins, Nowa1p and Nowa2p, which are required for the survival of sexual progeny. Green fluorescent protein (GFP) fusions show that Nowa1p accumulates into the maternal macronucleus shortly before meiosis of germline micronuclei and is later transported to developing macronuclei. Nowa1p/2p depletion impairs the elimination of transposons and of those IESs that are controlled by maternal effects, confirming the existence of distinct IES classes. CONCLUSIONS: The results indicate that Nowa proteins are essential components of the trans-nuclear-crosstalk mechanism that is responsible for epigenetic programming of genome rearrangements. We discuss implications for the current models of genome scanning in ciliates, a process related to the formation of heterochromatin by RNA interference in other eukaryotes.

Amino Acid Sequence↗
PubMed2005

Centrin deficiency in Paramecium affects the geometry of basal-body duplication.

BACKGROUND: Ciliary or flagellar basal bodies and centrioles share the same architecture and remarkable property of duplicating once per cell cycle. Duplication is known to proceed by budding of the daugther organelle close to and at right angles to the mother structure, but the molecular basis of this geometry remains unknown. Among the handful of proteins implicated in basal-body/centriole duplication, centrins seem required in all eukaryotes tested, but their mode of action is not clear. We have investigated centrin function in Paramecium, whose cortical organization allows detection of any spatial or temporal alteration in the pattern of basal-body duplication. RESULTS: We have characterized two pairs of genes, PtCEN2a and PtCEN2b as well as PtCEN3a and PtCEN3b, orthologs of HsCEN2 and HsCEN3, respectively. GFP tags revealed different localization for the two pairs of gene products, at basal bodies or on basal-body-associated filamentous arrays, respectively. Centrin depletion induced by RNAi caused mislocalization of the neoformed basal bodies: abnormal site of budding (PtCen2ap) or absence of separation between mother and daughter organelles (PtCen3ap). Over successive divisions, new basal bodies continued to be assembled, but internalization of the mispositionned basal bodies led to a progressive decrease in the number of cortical basal bodies. CONCLUSIONS: Our observations show that centrins (1) are required to define the site and polarities of duplication and to sever the mother-daughter links and (2) play no triggering or instrumental role in assembly. Our data underscore the biological importance of the geometry of the duplication process.

Amino Acid Sequence↗