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PubMed1983

Antigenic variation during cellular aging in Paramecium tetraurelia.

Clones of Paramecium tetraurelia undergoing cellular aging were exposed to two antisera, anti-32 and anti-25, raised against surface antigen isolated from cells grown at 32 degrees C or 25 degrees C. When young clones were exposed to the two antisera about 50% were immobilized by anti-32 while others were not immobilized by either antisera. These clones were grouped as group A or B depending upon their sensitivity or nonsensitivity to the two antisera. Young cells of group A were immobilized by anti-32 and during aging these cells either maintained the same antigenic type or transformed to another. Young group B clones were not immobilized by either antisera but gradually became sensitive to anti-32. Some clones from both groups showed sensitivity to both antisera. The possible mechanism of antigenic variation during fission age is discussed.

Animals↗
PubMed1984

Clonal aging in Paramecium tetraurelia. Absence of evidence for a cytoplasmic factor.

Cells of Paramecium tetraurelia show clonal aging with characteristics comparable to the aging seen in tissue culture cell lines. An investigation is underway to determine the relative contributions, if any, of the cytoplasm and the macronucleus to the onset of senescence. Using a microinjection protocol, the importance of the cytoplasm to aging was tested. Cytoplasm was transferred from young cells into old cells to see if the mean age of death of the injected cell lines could be increased, compared with uninjected controls and sham injected controls. Cytoplasm from old cells was transferred into young cells to see if the mean age of death of the injected cell lines could be decreased, compared with sham injected and uninjected controls. In neither case was there any statistically significant change in mean ages of death. Furthermore, no changes can be demonstrated even with a protocol which transplanted 3 times the normal cytoplasmic volume. Thus, a cytoplasmic effect upon vegetative aging in P. tetraurelia cannot be demonstrated. Tests of a macronuclear influence are now underway.

Aging↗
PubMed1984

Resistance of germinal nucleus to aging in Paramecium: evidence obtained by micronuclear transplantation.

A diversity of time when increase in mortality after conjugation occurs during the lifespan was found in subclones of three stocks of Paramecium caudatum. A possible micronuclear contribution to the increase in sterility has been investigated by micronuclear transplantation. We found two classes of micronuclei in aged clones: those that can function normally if the cytoplasmic environment is young, and those that cannot, even in a young cytoplasmic environment. The results indicate that in the former, the age-dependent increase in sterility is due to a deleterious macronucleus and/or cytoplasm, and in the latter it is due to micronuclear damage. The micronuclear damage in aged clones is probably induced by a deleterious cytoplasmic environment because aged clones with transplanted young micronuclei showed an abrupt decrease in progeny survival between 14 and 42 cell divisions after transplantation. Overall, the micronucleus seems not to be a source of age-related damage but rather is subjected to damage from macronuclear and/or cytoplasmic sources.

Animals↗
PubMed1985

Studies on catalase, glutathione peroxidase and superoxidismutase activities in aging cells of Paramecium tetraurelia.

The nature of the aging process has been the subject of considerable speculation. Now, some data indicate that free radical reactions going on continuously in the cells contribute to aging. Considering these data, we have investigated the activity of enzymes (catalase, glutathione peroxidase, superoxidismutase) present physiologically in the cell to limit to tolerable levels, the rate of free radicals or H2O2. These enzymes activities were assayed in Paramecium tetraurelia as clonal age increased. Catalase activity increases slightly during aging of paramecia, i.e. during maturity and senescence phases (20-150 fissions). No significant changes in glutathione peroxidase and superoxidismutase is found. Catalase activity was also assayed as a function of culture conditions. As the cells begin starving and the percentage of autogamous cells increases, catalase activity decreases. After autogamy, a large increase of catalase activity occurs during the sexual immaturity phase, i.e. during the first 20 fissions. By another way, H2O2 added in the culture medium (from 0 to 15 X 10(-5)M) causes an important increase of catalase activity (from 100 U.I. to 250 U.I.). The possible role of O-.2, OH. and H2O2 in aging is discussed.

Animals↗
PubMed1985

Phenotypes associated with early clonal death in Paramecium tetraurelia.

The aging characteristics of various known mutations of Paramecium tetraurelia have been studied. Among the mutations which had been previously isolated on the basis of the inability of the cell to do exocytosis of its trichocysts, one group was identified which had clonal life spans which were indistinguishable from, or were somewhat shorter than, the life span of wild-type. Another group of trichocyst mutations, however, had clonal life spans which were 1/3 to 1/4 that of wild-type. Further study of the phenotypes of these various mutations revealed that members of the short-lived group all had a common phenotypic defect: a frequent inability to properly divide the macronucleus during cell division. Another mutation which has normal trichocysts, but which shows macronuclear division defects, also has an extremely short life span. Thus, the short clonal life spans seen are not associated with the inability to do trichocyst exocytosis but rather are associated with the inability to properly divide the macronucleus. An hypothesis is presented which relates macronuclear misdivision to the short clonal life spans of the mutants expressing it. By extension of the hypothesis, the significance of normal macronuclear divisions to the normal aging process in P. tetraurelia is proposed.

Aging↗
PubMedDate not supplied

Clonal aging in Paramecium tetraurelia. II. Evidence of functional changes in the macronucleus with age.

The contribution of the macronucleus of Paramecium tetraurelia to the long term proliferation potential of a cell line was tested using a nuclear transplantation protocol. Macronuclei from young or old wild-type cells were injected into genetically marked host cells of a standard clonal age. The subsequent proliferation to clonal death of successfully and stably transformed hybrids was compared to the proliferation of injected but untransformed lines (injection controls). Young macronuclear donor material significantly prolonged the proliferation of the hybrid cell lines over that of the injection controls, but old donor material caused only a slight increase in post-injection proliferation of the hybrids. Total cell proliferation was also considered. Comparison of the total life spans of injected, non-transformed lines with uninjected host controls demonstrates that the injection itself has no significant effect upon proliferation potential. The mean life spans of uninjected donor controls and the mean total life spans of the transplanted macronuclear material (donor age at injection plus subsequent hybrid proliferation) are similar, regardless of the age of the donor at the time of transplantation. These results suggest that there is an age-related decrease in the ability of a macronucleus to support subsequent cell growth and division. The results also show that the proliferation potential of the donor macronucleus does not appear to be changed to any great degree by transplantation into a host cell of different clonal age. The macronucleus thus "remembers its age" after transplantation. Coupled with an absence of any detectable cytoplasmic effects upon aging during vegetative growth, these results argue in favor of a macronuclear determination of the proliferation potential of a cell line. The identification of a macronuclear basis for clonal aging in P. tetraurelia should permit a better directed approach for further research in this area.

Cell Division↗
PubMed1984

Characterization and purification of a soluble protein controlling Ca-channel activity in paramecium.

The analysis of the voltage-sensitive Ca++ channel of the unicellular eucaryote, Paramecium has been extended to a biochemical level based on recent observations that the transfer of cytoplasm from wild-type cells into mutants lacking Ca++-channel function ("pawn" in P. tetraurelia and "CNR" in P. caudatum) causes mutant cells to regain Ca++-channel function. We have microinjected various cytoplasmic fractions into mutant cells and measured the restored Ca++-channel function using a convenient behavioral assay. Following the "curing" activity, we characterized and purified the component from wild-type cytoplasm that can restore the function missing in cells carrying mutations in the cnrC gene. The curing factor is not an RNA, but a heat-labile, -SH-containing protein that appears to affect existing mutant channels on the ciliary membrane. We have purified this factor over 500-fold from the soluble cytoplasm using conventional techniques. The protein is of low apparent molecular weight (less than 30,000 daltons), acidic, soluble, and does not have the properties of calmodulin.

Animals↗
PubMed1985

Respective role of microgravity and cosmic rays on Paramecium tetraurelia cultured aboard Salyut 6.

Paramecium tetraurelia cultured aboard Salyut 6 have shown in increase in cell growth rate, cell volume, water content and changes in electrolyte content. Additional experiments, carried out in balloon flight and on earth, showed that the stimulating effect observed on cell proliferation is related to exposure to cosmic rays. Other changes seem to be due to a direct effect of microgravity on cell. Mechanism of gravity action on cell is discussed.

Animals↗
PubMed1995

Subplasmalemmal Ca2+ stores of probable relevance for exocytosis in Paramecium. Alveolar sacs share some but not all characteristics with sarcoplasmic reticulum.

Isolated subplasmalemmal Ca2+ stores ('alveolar sacs') from Paramecium tetraurelia cells sequester 45Ca2+ depending on ATP concentration. 45Ca2+ uptake is sensitive to SERCA-type Ca(2+)-ATPase inhibitors. They cause a slow release of 45Ca2+, as does caffeine. Of some importance are also the negative results we obtained with ryanodine, inositol 1,4,5-trisphosphate (InsP3), cyclic adenosinediphosphoribose (cADPR), 3',5'-cyclic guanosine monophosphate (cGMP, +/- beta-nicotinamide-adenine dinucleotide) or with increased [Ca2+]. These data were corroborated by experiments in vivo, including microinjection studies. Again ryanodine, InsP3, cADPR or cGMP did not trigger exocytosis, the trigger effect of SERCA inhibitors was sluggish, whereas caffeine induced exocytosis in a dose-dependent fashion. We then tested 45Ca2+ release also with isolated cell cortices (cell fragments containing cell membranes with stores and secretory organelles still attached). Under conditions which initiate exocytosis in vitro (depending on [ATP], reduction of [Mg2+] in presence of Ca2+; c.f. Lumpert et al. 1990, Biochem. J. 269, 639) we observed significant 45Ca2+ release with cortices as with isolated alveolar sacs. Our interpretation is as follows. (a) Alveolar sacs have a SERCA-type Ca(2+)-pump. (b) They have some sensitivity to caffeine, but none to ryanodine, InsP3 or cADPR. (c) There might be a direct functional coupling of these subplasmalemmal Ca2+ stores to the plasmalemma to which they are connected via feet-like structures; also like the SR, activation of this store is modulated by Mg2+ and ATP.

Adenosine Triphosphate↗
PubMed1992

The unique structure of the Paramecium caudatum hemoglobin gene: the presence of one intron in the middle of the coding region.

The gene encoding hemoglobin (Hb) in the ciliated protist, Paramecium caudatum, has been cloned and sequenced. A single Hb locus was detected on an isolated genomic fragment with a size of approx. 15 kbp. This gene contained 443 bp. The 5' transcriptional regulatory region contained TATA box- and CAAT box-like sequences which resemble the structure of the promoter region in higher eukaryotic genes. The amino acid sequence deduced from the gene agreed precisely with that of the major Hb component. It was an unusual structure, much smaller than Hbs from many other organisms. Furthermore, all of the untranslated regions in this gene were also small. Particularly, one intervening sequence (intron) being only 23 bp was shorter than any other introns reported for eukaryotic genes encoding protein. The position of the intron in the P. caudatum Hb gene corresponded closely to that of the second intron which had been reported only for plant Hb genes. Possible routes for the evolution of Hb genes were discussed.

Amino Acid Sequence↗
PubMed1987

Biochemical characterization of a genetically altered calmodulin in Paramecium.

Recent evidence proposes that the calcium-binding protein, calmodulin, plays a crucial role in the regulation or modulation of the calcium-dependent potassium conductance in Paramecium tetraurelia (Hinrichsen, R.D., Burgess-Cassler, A., Soltvedt, B.C., Hennessey, T. and Kung, C. (1986) Science 323, 503-506). We purified the calmodulins from both the wild type and pantophobiac A (a mutant lacking the above-mentioned conductance and whose phenotypic defect is traceable to its calmodulin) by hydrophobic interaction and immunoaffinity chromatographies, and examined them biochemically. In this paper we address the preliminary characterization of the two calmodulins and discuss the consequences of the genetic alteration. The differences described here are in their electrophoretic mobilities in polyacrylamide gel electrophoresis and in their binding characteristics to monoclonal antibodies raised against calmodulin from wild-type paramecia. Also, we present data which indicate a difference in the stimulation of the calmodulin-dependent enzyme bovine brain phosphodiesterase under certain conditions.

Amino Acid Sequence↗
PubMed1989

Cyclic AMP-dependent protein kinases of Paramecium. I. Chromatographic and physical properties of the enzymes from cilia.

The cAMP-dependent protein kinases of the cilia of the protozoan Paramecium tetraurelia were resolved and characterized. Two cAMP-dependent activities were present in cilia; the two ciliary kinases resemble types I and II from vertebrate tissues. Part of the ciliary kinase activity (primarily type II) was released by freeze-thawing, but a significant amount remained particulate. Both kinases were found as aggregates of about 220 kDa and of about 70 kDa. A portion of the cAMP-binding activity in ciliary extracts separated from kinase activity, and eluted at 36 kDa during gel filtration. Photoaffinity labeling with 8-azido-cAMP identified cAMP-binding proteins of 45-52 kDa in type II kinase from cilia, and of 43-46 kDa in type I kinase. The type II kinase was apparently autophosphorylated, causing a decrease in mobility during sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Affinity Labels↗
PubMed1984

Effects of gravity and cosmic rays on cell proliferation kinetics in Paramecium tetraurelia.

Space flights resulted in a stimulating effect on kinetics of proliferation in Paramecium tetraurelia. Additional experiments were performed in order to determine the origin of this phenomena. Paramecia were cultivated in balloon flights or in a slow clinostat, or were exposed to different levels of hypergravity. The results suggest that changes in cell proliferation rate are related to cosmic rays and to a direct effect of microgravity.

Animals↗
PubMed1992

Swimming behavior of Paramecium--first results with the low-speed centrifuge microscope (NIZEMI).

The low-speed centrifuge microscope NIZEMI (= Nieder-Geschwindigkeits-Zentrifugen-Mikroskop) is an excellent tool with which to investigate the effects of slightly increased gravity in the fields of biology and material sciences. We investigated the swimming behavior of Paramecium in the NIZEMI, by aid of a computer-controlled image analysis system. In the range of acceleration (1 g to 5 g), cells retained their swimming capability, did not sediment, and even increased the precision of their negative gravitaxis but reduced their mean swimming velocity.

Animals↗
PubMed1984

Blocking of in vitro translation of Paramecium messenger RNAs is due to messenger RNA primary structure.

Paramecium primaurelia mRNAs were translated in vitro in rabbit reticulocyte lysate and the products of translation were analyzed by their size. We show that the large majority of these products are of short but discrete sizes irrespective of the length of the mRNA which directs their synthesis. An illustrative example is given by the translation of mRNA of G surface antigen which directs the synthesis of a 50 kD polypeptide instead of the complete 250 kD protein. Control experiments suggest that the blocking is due to mRNA primary structure.

Animals↗
PubMed1994

Protein processing and morphogenesis of secretory granules in Paramecium.

The ciliated protozoan Paramecium provides a model system for the study of regulated secretion, featuring architecturally complex secretory storage granules-trichocysts-docked at the plasma membrane, ready to respond to an exocytotic stimulus. The trichocysts are characterized by crystalline contents that confer upon the organelle a defined shape which can be altered by single gene mutation. The crystalline trichocyst contents are built up from a heterogeneous set of small acidic polypeptides generated by proteolytic maturation of a family of precursor molecules, suggesting an important role for protein processing in this system. We have recently shown that the primary defect in several secretory mutants lacking functional trichocysts is in intracellular trafficking rather than protein processing. However, analysis of how these defects lead to altered trichocyst shape supports the notion that the protein processing is essential for morphogenesis. Preliminary results of a cloning project reveal that an extensive multigene family (approximately 100 genes) codes for the trichocyst matrix proteins. Deduced amino acid sequences of putative processing sites indicate that (at least) two distinct processing reactions are probably involved in the maturation of these proteins, and allow us to speculate that each reaction may control a key event of trichocyst biogenesis.

Amino Acid Sequence↗
PubMed1975

The evolutionary significance of ultrastructural variations in the micronuclear spindle apparatus in the genus Paramecium.

Ultrastructural variations of micronuclear mitosis in three species of Paramecium have been reviewed for the purpose of hypothesizing possible evolutionary relationships. P. bursaria is considered to be the most primitive species because it has complex kinetochore-equivalents termed microlamellae which attach to microfilaments. P. multimicronucleatum is considered more advanced since it has typical kinetochores which intercalate between the kinetochores and polar microfilaments. P. aurelia is considered the most advanced member of the genus, and has typical diffuse or well defined kinetochores and chromosomal microtubules while it lacks obvious meshworks of microfilaments. A hypothetical model of chromosome movement in the genus involves the interaction of microtubules and microfilaments.

Animals↗
PubMed1988

Regulation of axonemal Mg2+-ATPase from Paramecium cilia: effects of Ca2+ and cyclic nucleotides.

Ciliary activity is regulated by Ca2+ and cyclic nucleotides, but the molecular mechanisms of the regulation are unknown. We have tested the ability of Ca2+ and cyclic nucleotides to alter ciliary Mg2+-ATPase or to stimulate phosphorylation of axonemal dynein. Mg2+-ATPase activity in cilia and axonemes from Paramecium was stimulated 2-fold by micromolar Ca2+, but this Ca2+ sensitivity was lost upon solubilization of the dyneins from the axoneme. The Ca2+-sensitive component of ciliary Mg2+-ATPase activity was inhibited by the dynein inhibitors vanadate and Zn2+, but was insensitive to the calmodulin antagonists calmidazolium and melittin. Dynein activity in the high-salt extract from axonemes was also insensitive to calmidazolium. Calmodulin did not sediment with 22 S or 12 S dyneins on sucrose gradients containing Ca2+, but it did sediment in the region from 19 S to 14 S. Mg2+-ATPase activity in ciliary fractions was unaltered in the presence of cAMP or cGMP. However, polypeptides associated with the 22 S and 12 S dyneins, as well as proteins of 19 S, 15 S, and 8 S, were substrates for endogenous ciliary kinases. High molecular weight polypeptides that sedimented at 22 S and 19 S were phosphorylated in a cyclic nucleotide-stimulated manner.

Animals↗