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PubMed1983

Effects of protease inhibitors on early events in the conjugation of paramecium caudatum.

The effects of 11 different protease inhibitors on mating reactivity and pair formation in the conjugation process of the ciliated protozoan Paramecium caudatum were investigated. Of the 11 inhibitors tested, six microbial and three synthetic inhibitors showed no effect on either pair formation or mating reactivity even at their highest concentrations. Soybean trypsin inhibitor and ovomucoid also showed no effect on pair formation even though they temporarily repressed mating reactivity. These results indicate that the process of pair formation during the conjugation process does not involve an extracellular proteolytic reaction.

Animals↗
PubMed1989

Free amino acids and cell volume regulation in the euryhaline ciliate Paramecium calkinsi.

Paramecium calkinsi was isolated from a tidal marsh in which the salinity fluctuated widely on a daily basis. In the laboratory, this ciliate survived for days in sea water ranging in osmotic strength from 10 to 2,000 mOsm and divided in nutritive media of 1,000 mOsm or less. When transferred from 750 to 250 mOsm, cells swelled but regained 78% of the original volume within 60 min and the original volume within 1 day. Cells acclimated to 250 mOsm and transferred to 750 mOsm shrank, regained 40% of the original volume in 60 min, and regained little more volume during the next 24 hr. Free amino acids (FAA), principally proline and alanine, are osmolytes in P. calkinsi. In cells that have been acclimated for more than 1 month, Pro is undetectable at 10 mOsm but at 250 mOsm is present in substantial amounts and is still higher at 750 mOsm. Ala is found in cells at all three salinities and increases dramatically with increasing salinity. A complex pattern of amino acid changes occurs during the 4 hr following a transfer from 250 to 750 mOsm, resulting in a marked increase in Ala but no change in Pro. Thus the metabolic changes that lead to the increased FAA levels of acclimated cells are apparently long-term and complex. After transfer of cells from 750 to 250 mOsm there is a rapid and selective loss of Pro and Ala from the cells to the medium.

Amino Acids↗
PubMed1976

A mutant of Paramecium with increased relative resting potassium permeability.

Fast-2, a membrane mutant of Paramecium aurelia, is due to a single-gene mutation and has behavioral abnormalities. Intracellular recordings through changes of external solutions were made. The mutant membrane hyperpolarized when it encountered solutions with low K+ concentration. This hyperpolarization and other associated activities were best observed in Ca- or Na-solutions devoid of K+. Membrane potential was plotted against the concentration of K+ (0.5 to 16 mM) in solutions of fixed Na+ or Ca++ concentration. The slopes of the curves for the mutant membrane were steeper than those for the wild type at the lower concentrations of K+. Inclusion of 2 mM tetraethylammonium chloride (TEA-Cl) counteracted the mutational effects. Spontaneous action potentials in Ba-solution and the electrically evoked action potentials in various solutions are normal in this mutant. We conclude that the resting permeability to K+ relative to the permeabilities to Na+ and Ca++ has been increased by the mutation.

Animals↗
PubMed1994

Chromosome end formation and internal sequence elimination as alternative genomic rearrangements in the ciliate Paramecium.

Programmed chromosome end formation resulting from DNA fragmentation and telomeric repeat addition as well as programmed internal sequence elimination occur extensively during differentiation of macronuclear genomes from micronuclear chromosomal sets in ciliated protozoa. Major macronuclear telomere addition sites were previously identified some 5 kb downstream from the G gene that encodes the G surface antigen in Paramecium primaurelia strain 156. Here I report the existence of minor telomeric addition sites some 2.8 kb downstream from the G gene. I show that major and minor macronuclear chromosomal ends arise by micronuclear DNA fragmentation, since none of them matches a micronuclear chromosomal end. Three DNA segments absent from the previously established macronuclear sequence map around the minor fragmentation sites in the micronuclear DNA. I show that one of these internal eliminated sequences is retained on some macronuclear chromosome copies and that all retained copies are found at minor telomeres. These results suggest that chromosome fragmentation and internal sequence elimination are alternative outcomes of a single DNA processing pathway.

Animals↗
PubMed2000

Complete sequence of the mitochondrial genome of Tetrahymena pyriformis and comparison with Paramecium aurelia mitochondrial DNA.

We report the complete nucleotide sequence of the Tetrahymena pyriformis mitochondrial genome and a comparison of its gene content and organization with that of Paramecium aurelia mtDNA. T. pyriformis mtDNA is a linear molecule of 47,172 bp (78.7 % A+T) excluding telomeric sequences (identical tandem repeats of 31 bp at each end of the genome). In addition to genes encoding the previously described bipartite small and large subunit rRNAs, the T. pyriformis mitochondrial genome contains 21 protein-coding genes that are clearly homologous to genes of defined function in other mtDNAs, including one (yejR) that specifies a component of a cytochrome c biogenesis pathway. As well, T. pyriformis mtDNA contains 22 open reading frames of unknown function larger than 60 codons, potentially specifying proteins ranging in size from 74 to 1386 amino acid residues. A total of 13 of these open reading frames ("ciliate-specific") are found in P. aurelia mtDNA, whereas the remaining nine appear to be unique to T. pyriformis; however, of the latter, five are positionally equivalent and of similar size in the two ciliate mitochondrial genomes, suggesting they may also be homologous, even though this is not evident from sequence comparisons. Only eight tRNA genes encoding seven distinct tRNAs are found in T. pyriformis mtDNA, formally confirming a long-standing proposal that most T. pyriformis mitochondrial tRNAs are nucleus-encoded species imported from the cytosol. Atypical features of mitochondrial gene organization and expression in T. pyriformis mtDNA include split and rearranged large subunit rRNA genes, as well as a split nad1 gene (encoding subunit 1 of NADH dehydrogenase of respiratory complex I) whose two segments are located on and transcribed from opposite strands, as is also the case in P. aurelia. Gene content and arrangement are very similar in T. pyriformis and P. aurelia mtDNAs, the two differing by a limited number of duplication, inversion and rearrangement events. Phylogenetic analyses using concatenated sequences of several mtDNA-encoded proteins provide high bootstrap support for the monophyly of alveolates (ciliates, dinoflagellates and apicomplexans) and slime molds.

Animals↗
PubMed2002

Crystal structure analysis of the exocytosis-sensitive phosphoprotein, pp63/parafusin (phosphoglucomutase), from Paramecium reveals significant conformational variability.

During exocytosis of dense-core secretory vesicles (trichocysts) in Paramecium, the protein pp63/parafusin (pp63/pf) is transiently dephosphorylated. We report here the structures of two crystal forms of one isoform of this protein which has a high degree of homology with rabbit phosphoglucomutase, whose structure has been reported. As expected, both proteins possess highly similar structures, showing the same four domains forming two lobes with an active-site crevice in between. The two X-ray structures that we report here were determined after crystallization in the presence of sulfate and tartrate, and show the lobes arranged as a closed and an open conformation, respectively. While both conformations possess a bound divalent cation, only the closed (sulfate-bound) conformation shows bound sulfate ions in the "phosphate-transfer site" near the catalytic serine residue and in the "phosphate-binding site". Comparison with the open form shows that the latter dianion is placed in the centre of three arginine residues, one contributed by subunit II and two by subunit IV, suggesting that it causes a contraction of the arginine triangle, which establishes the observed conformational closure of the lobes. It is therefore likely that the closed conformation forms only when a phosphoryl group is bound to the phosphate-binding site. The previously published structure of rabbit phosphoglucomutase is intermediate between these two conformers. Several of the known reversible phosphorylation sites of pp63/pf-1 are at positions critical for transition between the conformations and for binding of the ligands and thus give hints as to possible roles of pp63/pf-1 in the course of exocytosis.

Adenosine Triphosphate↗
PubMed1999

Quantitative energy-dispersive X-ray microanalysis of calcium dynamics in cell suspensions during stimulation on a subsecond time scale: preparative and analytical aspects as exemplified with paramecium cells.

We analyzed preparative and analytical aspects of the dynamic localization of Ca(2+) during cell stimulation, using a combination of quenched flow and energy-dispersive X-ray microanalysis (EDX). Calcium (or Sr, as a substitute) was retained as fluorides during freeze-substitution, followed by epoxide embedding. The quenched-flow used allowed analyses, during stimulation, in the subsecond time range. Sections of 500 nm were analyzed and no artificial Ca or Sr leakage was recognizable. We calculated a primary beam spread from 63 to 72 nm that roughly indicated the resolution of EDX/structure correlation. These values are quite compatible with the size of potential structures of interest, e.g., Ca stores (approximately 100-nm thickness) or cilia (approximately 250-nm diameter). We used widely different standards to calibrate the ratio of CaK(alpha) net counts in relation to actual ¿Ca. Calibration curves showed a linear relationship and a detection limit of ¿Ca = 2 mM, while ¿Ca in cytosol was 3 mM and in stores was 43 mM, both in nonactivated cells. Eventually Sr(2+) can rapidly be substituted for Ca(2+) in the medium before and during stimulation, thus allowing one to determine Me(2+) fluxes. With our "model" cell, Paramecium, we showed that, upon stimulation (causing rapid Ca(2+) mobilization from subplasmalemmal stores), Ca was immediately exchanged for Sr in stores.

Animals↗
PubMed1986

Extrachromosomal elements of extrachromosomal elements of Paramecium and their extrachromosomal elements.

Expression of killer traits in Paramecium is due to a complex interaction between the lower eukaryote host and two or three elements that can be viewed either as extrachromosomal elements or as endosymbionts. In all cases, the determinants of the killer trait are carried by obligate bacterial endosymbionts belonging to the genus Caedibacter. However, the actual genetic determinants for expression of these traits are not an integral part of the symbiont genome. They are located on extrachromosomal genetic elements (plasmids or bacteriophages) which essentially are molecular endosymbionts of Caedibacter. In the case of the plasmids, they are associated with yet another set of extrachromosomal genetic elements, which are transposons. These transposons have been observed to move into new sites in the plasmids and even to disrupt expression of R body production and the killer trait. Thus, the transposons can be considered either as extrachromosomal elements of extrachromosomal elements (plasmids) of extrachromosomal elements (C. taeniospiralis) of paramecia, or as molecular parasites of molecular endosymbionts (plasmids) of bacterial endosymbionts of paramecia.

Animals↗
PubMed1994

Correlation between circadian periods and cellular activities in Paramecium bursaria.

Paramecium bursaria shows a circadian rhythm of photoaccumulation: photoaccumulation is stronger during the day than at night. We obtained five strains of P. bursaria having different circadian periods under continuous light conditions, ranging from 20.9 to 27.9 h. Various physiological activities were compared in the cells of these strains. The periods of contractile vacuole contraction were in the range 10-15 s, which was almost proportional to the periods of the circadian rhythm in each strain. Swimming velocities were inversely proportional to the circadian period; i.e. swimming velocities were high in strains whose circadian periods were short. Resting membrane potential was more depolarized in strains with longer circadian periods. Finally, the membrane resistance of the resting state was reduced in proportion to the increase of the circadian period. Such correlation between the cellular properties and the circadian period suggests that the circadian clock mechanism is associated with various physiological activities of the cell.

Animals↗
PubMed1994

Oxidants act as chemorepellents in Paramecium by stimulating an electrogenic plasma membrane reductase activity.

Paramecium is a valuable eukaryotic model system for studying chemosensory transduction, adaptation and cellular sensory integration. While millimolar amounts of many attractants hyperpolarize and cause faster forward swimming, oxidants are repellents that depolarize and cause backward swimming at micromolar concentrations. The non-permeant oxidants cytochrome c, nitro blue tetrazolium and ferricyanide are repellents with half maximal concentrations of 0.4 microM, 2.2 microM and 100 microM respectively. In vivo reductase activities follow the same order of potencies. The concentration dependence of the cytochrome c reductase activity is well correlated with cytochrome c-induced depolarizations. This suggests that plasma membrane reduction of external cytochrome c is electrogenic, causing membrane depolarization and chemorepulsion. The reductase activity also appears to be voltage dependent. Depolarization by either K+, Na+, Ca++ or Mg++ correlates with inhibition of both in vivo reductase activities and cytochrome c-induced membrane potential changes. These responses were also seen in deciliated cells, showing that the body plasma membrane is sufficient for the response. Both chloroquine and diphenyleneiodonium inhibited reductase activities but only at unusually high concentrations. This activity showed no pH dependence in the physiological range. We propose that a plasma membrane bound NA-DPH-dependent reductase controls oxidant-induced depolarizations and consequent chemorepulsion.

Animals↗
PubMed1977

Studies on the surface coat of Paramecium aurelia. II. Relationship to the immobilization antigen.

Correlations between the presence of surface coat and immobilization antigen of Paramecium tetraurelia were studied. Supravital, partial removal of the surface coat resulted in accelerated response of monobacterially and axenically grown cells to the homologous antiserum. Ciliates pretreated with trypsin or pronase (0.5 mg/ml for 45 min at 0-4 degrees C) were immobilized approximately twice as fast as untreated control cells. The probable localization of at least part, of the immobilization antigen within the surface coat of P. tetraurelia is discussed.

Animals↗
PubMed1994

Inhibition of Mg2+ current by single-gene mutation in Paramecium.

"Eccentric" is a newly-isolated mutant of Paramecium tetraurelia that fails to swim backwards in response to Mg2+. In the wild type, this backward swimming results from Mg2+ influx via a Mg(2+)-specific ion conductance (IMg). Voltage-clamp analysis confirmed that, as suspected, step changes in membrane potential over a physiological range fail to elicit IMg from eccentric. Further electrophysiological investigation revealed a number of additional ion-current defects in eccentric: (i) The Ca2+ current activated upon depolarization inactivates more slowly in eccentric than in the wild type, and it requires longer to recover from this inactivation. (ii) The Ca(2+)-dependent Na+ current deactivates significantly faster in the mutant. (iii) The two K+ currents observed upon hyperpolarization are reduced by > 60% in eccentric. It is difficult to envision how these varied pleiotropic effects could result from loss of a single ion current. Rather, they suggest that the eccentric mutation affects a global regulatory system. Two plausible hypotheses are discussed.

Action Potentials↗
PubMed1978

Regulation of surface antigens expression in Paramecium primaurelia: genetic and physiological factors involved in allelic exclusion.

In paramecium aurelia, allelic exclusion can be considered as a basic feature of the surface antigens system in the same way as intergenic exclusion. Our studies on allelic exclusion in G156/G168 heterozygotes show that (1) allelic exclusion does not depend on discrete regulatory genes dispersed throughout the genome; (2) it does not seem to be influenced by cytoplasmic factors; (3) it occurs regardless of the surface antigen expressed by the parental strains at the time of the cross. These results are discussed in relation to both intergenic and interallelic exclusion for which a common basis is proposed.

Alleles↗
PubMed1980

Genetic interactions in the control of mitochondrial functions in Paramecium. I. Interactions between nuclear genes.

The genetic and physiological properties of two nuclear mutants of Paramecium tetraurelia affecting mitochondrial properties, and first screened as resistant to tetrazolium (TTC) are described. The mutant TTC64-1R is strongly deficient in cytochrome c and the mutant TTC66pR is partially deficient in cytochrome aa3; both mutants display cyanide insensitive respiration in exponential growth phase. In the double mutant TTC64-1R -- TTC66pR/TTC64-1R -- TTC66pR the deficiency in cytochrome aa3 due to the TTC66pR mutation is suppressed. The mutation TTC64-1R does not suppress cytochrome aa3 deficiencies due to mitochondrial mutations, but does interact with another nuclear mutation, cl1, (compatible only with mitochondria deficient in cytochrome oxidase) in such a way that the double mutant TTC64-1R -- cl1/TTC64-1R -- cl1 displays a normal amount of cytochrome aa3. The possible mechanisms and physiological significance of these suppressive effects are discussed.

Animals↗
PubMed1992

Quantitative immuno-gold labelling and ultrastructural preservation after cryofixation (combined with different freeze-substitution and embedding protocols) and after chemical fixation and cryosectioning. Analysis of the secretory organelle matrix of Paramecium trichocysts.

Among the variety of parameters affecting immuno-gold labelling efficiency, mainly the effects of different preparative protocols were tested. Preservation of ultrastructure and of antigenicity are the salient features of this study. We have labelled insoluble components of the secretory matrix of Paramecium trichocysts with specific antisera, using 10 nm colloidal gold particles. The highest labelling efficiency was obtained with fast freezing (cryofixation, either by sandwich or spray-freezing), freeze-substitution in methanol (without added fixatives) and hydrophilic Lowicryls, particularly when applied at low temperatures (K11M at 193 K). The presence of different chemical fixatives always reduced the labelling density and some recommendations from the literature do not appear advisable. Methods commencing with fixation at greater than or equal to 0 degree C, such as "progressive lowering of temperature" (PLT) or preparation of cryostat sections, i.e. with chemical pretreatments, always resulted in lower labelling density. Our data appear, therefore, relevant for optimal immuno-gold labelling of insoluble antigens and emphasize the potential of cryofixation as a primary preparation step. In addition, ultrastructural preservation was also superior after cryofixation.

Acrylic Resins↗
PubMed1980

Mitochondrial chromatin in Paramecium aurelia.

Chromatin-like structures have been observed in material extracted from the mitochondria of Paramecium aurelia and evidence is presented which establishes that these structures do not originate from nuclear contamination of mitochondrial preparations but are exclusively of mitochondrial origin. Extraction of these chromatin-like structures with dilute acid followed by gel electrophoresis of the extract shows the presence of five major basic proteins which are of similar electrophoretic mobility to histones. Digestion of mitochondrial extracts with endogenous nuclease, followed by electrophoresis of the extracted DNA, demonstrated that the mitochondrial genome is protected from nuclease digestion by regular repeating units very similar to those observed in the nuclease digestion of chromatin.

Animals↗
PubMed1979

Internuclear control of DNA synthesis in exconjugant cells of Paramecium caudatum.

An exconjugant cell of Paramecium caudatum has two kinds of macronuclei, fragmented prezygotic macronuclei and postzygotic new macronuclei (anlagen). Although the DNA synthesis in the fragmented prezygotic macronucleus continues until the third cell cycle after conjugation, selective suppression of the DNA synthesis in the prezygotic macronucleus takes place at the fourth cell cycle. The inhibition of DNA synthesis in prezygotic fragmented macronuclei is due to the presence of a postzygotic macronucleus (anlage) in the same cytoplasm because the inhibition does not occur when the postzygotic macronucleus (anlage) is removed by micromanipulation during the third or fourth cell cycle. Well-developed postzygotic macronuclei (anlagen) with full ability to divide have the ability to depress the DNA synthesis of prezygotic macronuclear fragments. The suppression of DNA synthesis in prezygotic macronuclear fragments seems to be irreversible. Competition for the limited amount of DNA precursors also plays an important role in the onset of the selective suppression of the DNA synthesis.

Animals↗