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At least 631 records · Page 35Linked to original sources
PubMed1999

Immunolabeling analysis of biosynthetic and degradative pathways of cell surface components (glycocalyx) in Paramecium cells.

Biosynthetic and degradative pathways of glycocalyx components are largely unknown in Paramecium and in some related parasitic protozoa. We isolated cell surface (glyco-)proteins, i.e., surface antigens (SAg) and used them in the native (nSAg) or denatured (dSAg) state to produce antibodies (AB) for immunolocalization by confocal imaging and by quantitative immunogold EM-labeling of ultrathin sections or of freeze-fracture replicas. Antibodies against nSAg or dSAg, respectively, yield different labeling densities over individual structures, thus indicating biosynthetic or degradative pathways, respectively. We derive the following biosynthetic way: ER --> Golgi apparatus --> non-regulated/non-dense core vesicle transport --> diffusional spread over non-ciliary (somatic) and ciliary cell membrane. For degradation we show the following pathways: Concentration of nSAg in the cytostome --> nascent digestive vacuole --> mature vacuoles --> release of dSAg at cytoproct, with partial retrieval by "discoidal vesicles". A second internalization pathway proceeds via coated pits ("parasomal sacs") --> early endosomes ("terminal cisternae") --> digestive vacuoles. Dense packing of SAg in the glycocalyx may drive them into the endo-/phagocytic pathway. Still more intriguing is the site of nSAg integration into the cell membrane by unstimulated exocytosis. We consider unconspicuous clear vesicles relevant for nSAg export, probably via sites which most of the time are occupied by coated pits. This could compensate for membrane retrieval by coated pits, while scarcity of smooth profiles at these sites may be explained by the much longer time period required for coated pit formation as compared to exocytosis.

Animals↗
PubMedDate not supplied

Structural inheritance in Paramecium: ultrastructural evidence for basal body and associated rootlets polarity transmission through binary fission.

One main difference between basal bodies and centrioles resides in the expression of their polarity: centrioles display a structural nine-fold radial symmetry, whereas basal bodies express a circumferential polarity, thanks to their asymmetric set of rootlets. The origin of this polarity during organelle duplication still remains under debate: is it intrinsic to the nine-fold structure itself (i.e. the nine microtubular triplets are not equivalent) or imposed by its immediate environment at time of assembly? We have reinvestigated this problem using the Ciliate Paramecium, in which the pattern of basal body duplication is well known. In this cell, all basal bodies produced within ciliary rows appear immediately anterior to parental ones. Observations on cells fixed with the tannic acid protocol suggest that, to be competent for basal body assembly, parental basal bodies have to be individually associated with a complete set of rootlets (monokinetid structure). During pro-basal body assembly, full microtubular triplets were detected according to a random circumferential sequence; during the whole process, the new basal body and its associated rootlets maintained structural relations with the parental monokinetid structure by way of specific links. These results strongly suggest that basal body and associated rootlets (kinetid) polarity is driven by its immediate environment and provide a basis for the structural heredity property observed by Sonneborn some decades ago.

Animals↗
PubMed1999

Longevity in space; experiment on the life span of Paramecium cell clone in space.

Life span is the most interesting and also the most important biologically relevant time to be investigated on the space station. As a model experiment, we proposed an investigation to assess the life span of clone generation of the ciliate Paramecium. In space, clone generation will be artificially started by conjugation or autogamy, and the life span of the cell populations in different gravitational fields (microgravity and onboard 1 x g control) will be precisely assessed in terms of fission age as compared with the clock time. In order to perform the space experiment including long-lasting culture and continuous measurement of cell division, we tested the methods of cell culture and of cell-density measurement, which will be available in closed environments under microgravity. The basic design of experimental hardware and a preliminary result of the cultivation procedure are described.

Animals↗
PubMed2001

Regulation of Paramecium primaurelia glycosylphosphatidyl-inositol biosynthesis via dolichol phosphate mannose synthesis.

A set of glycosylinositol-phosphoceramides, belonging to a family of glycosylphosphatidyl-inositols (GPIs) synthesized in a cell-free system prepared from the free-living protozoan Paramecium primaurelia has been described. The final GPI precursor was identified and structurally characterized as: ethanolamine-phosphate-6Man alpha 1-2Man alpha 1-6(mannosylphosphate) Man alpha 1-4glucosamine-inositol-phospho-ceramide. During our investigations on the biosynthesis of the acid-labile modification, the additional mannosyl phosphate substitution, we observed that the use of the nucleotide triphosphate analogue GTP gamma S (guanosine 5-O-(thiotriphosphate)) blocks the biosynthesis of the mannosylated GPI glycolipids. We show that GTP gamma S inhibits the synthesis of dolichol-phosphate-mannose, which is the donor of the mannose residues for GPI biosynthesis. Therefore, we investigated the role of GTP binding regulatory 'G' proteins using cholera and pertussis toxins and an intracellular second messenger cAMP analogue, 8-bromo-cAMP. All the data obtained suggest the involvement of classical heterotrimeric G proteins in the regulation of GPI-anchor biosynthesis through dolichol-phosphate-mannose synthesis via the activation of adenylyl cyclase and protein phosphorylation. Furthermore, our data suggest that GTP gamma S interferes with synthesis of dolichol monophosphate, indicating that the dolichol kinase is regulated by the heterotrimeric G proteins.

Animals↗
PubMedDate not supplied

Developmentally programmed excision of internal DNA sequences in Paramecium aurelia.

The development of a new somatic nucleus (macronucleus) during sexual reproduction of the ciliate Paramecium aurelia involves reproducible chromosomal rearrangements that affect the entire germline genome. Macronuclear development can be induced experimentally, which makes P. aurelia an attractive model for the study of the mechanism and the regulation of DNA rearrangements. Two major types of rearrangements have been identified: the fragmentation of the germline chromosomes, followed by the formation of the new macronuclear chromosome ends in association with imprecise DNA elimination, and the precise excision of internal eliminated sequences (IESs). All IESs identified so far are short, A/T rich and non-coding elements. They are flanked by a direct repeat of a 5'-TA-3' dinucleotide, a single copy of which remains at the macronuclear junction after excision. The number of these single-copy sequences has been estimated to be around 60,000 per haploid genome. This review focuses on the current knowledge about the genetic and epigenetic determinants of IES elimination in P. aurelia, the analysis of excision products, and the tightly regulated timing of excision throughout macronuclear development. Several models for the molecular mechanism of IES excision will be discussed in relation to those proposed for DNA elimination in other ciliates.

Animals↗
PubMed2002

Detection of molecules related to the GABAergic system in a single-cell eukaryote, Paramecium primaurelia.

Gamma-aminobutyric acid (GABA)-related molecules were identified in Paramecium primaurelia by immunocytochemical methods, and GABA(A) receptors by their histochemical BODIPY-binding sites. Confocal microscope analysis showed different localizations according to the stages of the developmental cycle. A comparison was made with the cholinergic molecules, such as the acetylcholine biosynthetic enzyme (choline acetyltransferase), in double-labelled cells by confocal microscopy. In vivo experiments suggested the involvement of GABA-related molecules in cell-cell interaction.

4-Aminobutyrate Transaminase↗
PubMed2001

Organization and expression of the Paramecium caudatum gene encoding nucleosome assembly protein 1.

The complete genomic and partial complementary DNAs encoding the ciliate Paramecium caudatum nucleosome assembly protein 1 (NAP1) have been sequenced. The nap1 gene is situated 1.2 kbp from the hemoglobin (hb) gene, with the 3' end of both genes facing each other. The nap1 gene contains no introns, and encodes a protein of 369 amino acid residues with a calculated molecular weight of 42,627. The P. caudatum NAP1 amino acid sequence shares only 23-27% identity with NAP1 amino acid sequences from other eukaryotes. Although the nap1 transcript was detected in the P. caudatum cells at both the logarithmic and stationary phases, its level increased during the stationary phase. Southern blot analysis and polymerase chain reaction amplification revealed that the P. caudatum macronucleus has a heterogeneous composition at genomic regions around the nap1 gene. The present studies indicate the nap1 and hb genes are closely arranged in the macronucleus with the intergenic region between their sequences heterogeneously composed.

Amino Acid Sequence↗
PubMed2002

Transformation of Paramecium caudatum with a novel expression vector harboring codon-optimized GFP gene.

We have developed a novel expression vector, pTub-tel3, for transformation in Paramecium caudatum. The vector was constructed by cloning P. caudatum alpha-tubulin 5' and 3' non-coding regions. To examine transformation with the pTub-tel3 construct, we chose the green fluorescent protein (GFP) as a selection marker. When a linearized pTub-tel3 vector containing a GFP open reading frame was injected into the macronucleus, the GFP transcript was expressed in many clones whereas protein expression was detected only after extensive optimization of original GFP codons. GFP-derived fluorescence was distributed throughout the nuclei and cytoplasm except for contractile and food vacuoles. Upon continuous cell division, notable heterogeneity of GFP fluorescence among descendants from the same transformant has emerged. This expression vector can be applied to the analysis of protein trafficking and localization in addition to exogenous gene expression in P. caudatum.

Animals↗
PubMed2000

Changes in the endoplasmic reticulum structure of Paramecium primaurelia in relation to different cellular physiological states.

The fluorochrome 3,3'-dihexyloxacarbocyanine iodide [DiOC6(3)], a vital dye utilized to stain the endoplasmic reticulum (ER) of animal and plant cells, has been used to visualize the ER-type structures of Paramecium primaurelia under confocal laser scanning microscopy (CLSM). The morphology of the ER has been studied in paramecia in different physiological conditions. Cells are analysed in early and late logarithmic growth phases, in stationary and in death phases, during shift-up by refeeding after starvation and shift-down by using a starvation medium. In log-phase growing paramecia, the ER constitutes an anastomosing membrane system consisting of short tubules and flattened sacs forming a peripheral network, which is abundant in the cortical region around the trichocysts and the ciliary basal bodies. The tubular network and cytoplasmic membranes are reduced in stationary-phase cells; the original conditions are restored in starved cells after refeeding. The analysis of serial optical sections collected by CLSM at 0.5 microm intervals and three-dimensional reconstruction from these sections allow us to visualize differences between differently growing cells.

Animals↗
PubMed2000

An unusual complementation in non-excitable mutants in Paramecium.

A non-excitable behavioural mutant, d4-662, was previously characterized as the fourth pawn locus mutant pwD in Paramecium tetraurelia. We now provide data demonstrating that d4-662 is in fact controlled by a pwB allele that has the unusual feature of complementing other pwB alleles in heterozygous F1 progeny. Neither the cytoplasm nor the nucleoplasm of d4-662 cured the mutational defects of pwB and in the reverse combination of d4-662 and pwB, the result was the same. On the other hand, pwA, another non-excitable mutant, was cured upon cross-injection with d4-662 and mutants carrying trichocyst non-discharge marker genes were also cured. This evidence suggests that d4-662 is a new mutant belonging to pwB, and would be better designated as pwB662. Extensive crossbreeding analyses, however, showed an unusual genetic relationship between d4-662 and pwB (pwB95 or pwB96). When d4-662 was crossed with pwB mutants, many progeny expressing wild-type phenotype or mixed clones of wild-type and pawn cells were obtained in the F1. Less than 12.5% expressed the pawn phenotype. The appearance of wild-type progeny in this F1 strongly suggests that an inter-allelic interaction between pwB662 and other pwB alleles may occur during development of the macronucleus.

Animals↗
PubMed2001

Paramecium calmodulin mutants defective in ion channel regulation associate with melittin in the absence of calcium but require it for tertiary collapse.

Calmodulin (CaM) is a small acidic protein essential to calcium-mediated signal transduction. Conformational change driven by calcium binding controls its selective activation of myriad target proteins. In most well characterized cases, both homologous domains of CaM interact with a target protein. However, physiologically separable roles for the two domains were demonstrated by mutants of Paramecium tetraurelia [Kung, C. et al. (1992) Cell Calcium 13, 413], some of which have altered calcium affinities [Jaren, O. R. et al. (2000) Biochemistry 39, 6881]. To determine whether these mutants can associate with canonical targets in a calcium-dependent manner, their ability to bind melittin was assessed using analytical gel permeation chromatography, analytical ultracentrifugation, and fluorescence spectroscopy. The Stokes radius of wild-type PCaM and 11 of the mutants decreased dramatically upon binding melittin in the presence of calcium. Fluorescence spectra and sedimentation velocity studies showed that melittin bound to wild-type PCaM and mutants in a calcium-independent manner. However, there were domain-specific perturbations. Mutations in the N-domain of PCaM did not affect the spectrum of melittin (residue W19) under apo or calcium-saturated conditions, whereas most of the mutations in the C-domain did. These data are consistent with a calcium-dependent model of sequential target association whereby melittin (i) binds to the C-domain of PCaM in the absence of calcium, (ii) remains associated with the C-domain upon calcium binding to sites III and IV, and (iii) subsequently binds to the N-domain upon calcium binding to sites I and II of CaM, causing tertiary collapse.

Amino Acid Sequence↗
PubMed2003

Spatiotemporal sites of DNA replication in macro- and micronuclei of the ciliate Paramecium caudatum.

Spatiotemporal sites of DNA replication in macro- and micronuclei of the ciliated protozoan Paramecium caudatum were analyzed by confocal laser scanning microscopy following incorporation of the thymidine analogue BrdU and indirect immunofluorescence. In the macronucleus, replication sites were localized to numerous small domains and scattered throughout the nucleoplasm. This pattern persisted during all periods of the S phase. A single constant pattern with discrete replication foci was also observed in the micronucleus. No obvious differences were seen between the two kinds of nuclei. Pulse-chase-pulse double-labeling experiments with two thymidine analogues (CldU and IdU) revealed that dispersed sites of replication were activated at different times during the S phase and a replication site takes about 2h to complete replication in the macronucleus. When cells were labeled by BrUTP to examine transcriptional activity in the two kinds of nuclei, incorporation of BrUTP into the macronucleus occurred throughout the cell cycle, whereas there was no detectable RNA synthesis in the micronucleus. From these findings, we conclude that, despite large differences in structure and function of macro- and micronuclear genomes, both nuclei show a similar replication pattern with discrete subnuclear foci scattered throughout the nucleoplasm at all times during the S phase.

Animals↗
PubMed2003

DNA replication and transcription in new macronuclei of Paramecium caudatum exconjugants.

We analyzed the onset and location of replicational and transcriptional activity during the first cell cycle in new macronuclei of the ciliate Paramecium caudatum exconjugants. Synchronous exconjugants were pulse labeled with 5-bromo-2'-deoxyuridine or 5-bromouridine-5'-triphosphate to visualize replication or transcription sites, respectively. The first morphological change after macronuclear determination is the appearance of heterochromatic aggregates. Confocal microscopic examination revealed that DNA replication started at the stage when new macronuclear chromatin was partially decondensed, and that replication sites were located in a large number of small spot-like areas excluding the heterochromatic regions. Transcriptional activation in the new macronuclei also took place in the same developmental stage and in the same region that replication started. As macronuclear development progressed, heterochromatic aggregates disappeared, and replication and transcription sites were scattered throughout the nucleoplasm. Moreover, studies on aphidicolin-treated exconjugants demonstrated that inhibition of the DNA replication did not hinder transcriptional activation in the new macronuclei. On the other hand, replicational and transcriptional activity were also detected in old macronuclear fragments irrespective of their morphology and size, and length and timing of the replication corresponded to those in the new macronuclei.

Animals↗
PubMed1977

Separation of membrane currents using a Paramecium mutant.

The net membrane current of the electrically excitable membrane of Paramecium during step depolarisations was measured using voltage-clamp techniques. The mutant, pawn B, lacks a functional Ca channel. Thus the difference between the total current measured in the wild type and the leakage and rectification currents measured in the pawn mutant is the Ca current. These findings were confirmed by ion substitution experiments. The possibility that inactivation may be apparent only, and caused by a hypothetical, superposed, Ca-induced, rectifying K+ current was eliminated.

Animals↗
PubMed1977

Determination of the relatedness of the soluble surface proteins of Trypanosoma brucei and Paramecium aurelia.

The soluble surface proteins isolated by Cross (1975) from Trypanosoma brucei are compared to those which occur on Paramecium aurelia. Calculations based on the amino acid residue data and the molecular weights of these surface antigens indicate that there is a considerable degree of similarity between the T. brucei and the P. aurelia proteins, and that in neither case can simple mutation be responsible for the differences of molecules within each family of antigens. The analyses are consistent with the suggestion that the P. aurelia system can serve as a model for the phenomenon of phase transformation (relapse syndrome) which occurs in trypanosomiasis.

Amino Acids↗
PubMed2006

Global trends of whole-genome duplications revealed by the ciliate Paramecium tetraurelia.

The duplication of entire genomes has long been recognized as having great potential for evolutionary novelties, but the mechanisms underlying their resolution through gene loss are poorly understood. Here we show that in the unicellular eukaryote Paramecium tetraurelia, a ciliate, most of the nearly 40,000 genes arose through at least three successive whole-genome duplications. Phylogenetic analysis indicates that the most recent duplication coincides with an explosion of speciation events that gave rise to the P. aurelia complex of 15 sibling species. We observed that gene loss occurs over a long timescale, not as an initial massive event. Genes from the same metabolic pathway or protein complex have common patterns of gene loss, and highly expressed genes are over-retained after all duplications. The conclusion of this analysis is that many genes are maintained after whole-genome duplication not because of functional innovation but because of gene dosage constraints.

Animals↗
PubMed2006

Multiple-axis tomography: applications to basal bodies from Paramecium tetraurelia.

BACKGROUND INFORMATION: Transmission electron tomography is becoming a powerful tool for studying subcellular components of cells. Classical approaches for electron tomography consist of recording images along a single-tilt axis. This approach is being improved by dual-axis reconstructions and/or high-tilt devices (tilt angle>+/-60 degrees) on microscopes to compensate part of the information loss due to the 'missing wedge' phenomena. RESULTS: In the present work we have evaluated the extension of the dual-axis technique to a multiple-axis approach, and we demonstrate a freely available plug-in for the Java-based freeware image-analysis software ImageJ. Our results from phantom and experimental data sets from Paramecium tetraurelia epon-embedded sections have shown that multiple-axis tomography achieves results equivalent to those obtained by dual-axis approach without the requirement for high-tilt devices. CONCLUSIONS: This new approach allows performance of high-resolution tomography, avoiding the need for high-tilt devices, and therefore will increase the access of electron tomography to a larger community.

Algorithms↗
PubMed1984

Incorporation of serine into Paramecium ethanolamine phospholipid and phosphonolipid head groups.

Ethanolamine phospholipid head groups in Paramecium were synthesized directly from ethanolamine. As in other cell types, radioactivity from ethanolamine failed to incorporate significantly into head groups of ethanolamine phosphonolipids, indicating that the phosphonolipids are not derived from their phospholipid analogues. Unlike other systems previously examined, radioactivity from serine is incorporated into both ethanolamine phospholipid and phosphonolipid head groups of glycerolipids and sphingolipids in this ciliate. These observations suggest that synthesis of ethanolamine phosphonolipids involves synthesis de novo of free phosphonoserine, which is then incorporated into lipids, and then lipid-bound phosphonoserine intermediates (glycerolipids or sphingolipids) undergo decarboxylation, forming lipidbound phosphonoethanolamine compounds.

Animals↗