Transient N-acetylgalactosaminylation of mannosyl phosphate side chain in Paramecium primaurelia glycosylphosphatidylinositols.

The surface antigens of the free-living protozoan Paramecium primaurelia belong to the family of glycosylphosphatidylinositol (GPtdIns)-anchored proteins. Using a cell-free system prepared from P. primaurelia, we have described the structure and biosynthetic pathway for GPtdIns glycolipids. The core glycans of the polar glycolipids are modified by a mannosyl phosphate side chain. The data suggest that the mannosyl phosphate side chain is added onto the core glycan in two steps. The first step involves the phosphorylation of the GPtdIns trimannosyl conserved core glycan via an ATP-dependent kinase, prior to the addition of the mannose linked to the phosphate group. We show that dolichol phosphate mannose is the donor of all mannose residues including the mannose linked to phosphate. Furthermore, we were able to identify in vitro a hydrophilic intermediate containing an additional N-acetylgalactosamine linked to the mannosyl phosphate side chain. The addition of this purified hydrophilic radiolabelled intermediate into the cell-free system leads to a loss of the GalNAc residue and its conversion to the penultimate intermediate having only mannosyl phosphate as a side chain. Together the data indicate that the GalNAc-containing intermediate is a transitional intermediate. We suggest that the GalNAc-containing intermediate is essential for biosynthesis and maturation of GPtdIns precursors. It is hypothesized that this oligosaccharide processing in the course of GPtdIns biosynthesis is required for the translocation of GPtdIns from the cytoplasmic side of the endoplasmic reticulum to the luminal side.     

Nitric oxide production inhibited by xenobiotic compounds in the protozoan Paramecium primaurelia

The notable increase in agricultural and industrial activities over the last decades has caused a considerable increase in anthropogenic waste and, consequently, the presence of pollutants in both water and sediments. For this reason, there is great interest in identifying alternative models and bioassays complying with the 3Rs strategy (aimed at Reducing, Refining and Replacing tests on vertebrate organisms in toxicological studies). Protozoa seem to be well suited to this strategy and it is widely accepted that assays with protozoa are relevant to the study of environmental modifications due to the presence of xenobiotic compounds.Recently, we detected the presence of nitric oxide synthase (NOS)-related NADPH-diaphorase activity and neuronal NOS-related molecules, immunologically recognized by the anti-rat brain NOS antibody, in a single-cell freshwater eukaryote, Paramecium primaurelia. In this work we have looked for the basal NO production in living cells of P. primaurelia using the specific fluorescent probe 4,5-diaminofluorescein diacetate (DAF-2 DA) and measuring the intracellular NO levels with image analysis. The NO production was sensitive to compounds modulating NOS activity such as: S-methyl-tiocitrulline, an NOS activity inhibitor, l-NAME, an analogue of arginine that inhibits NO production, arginine, an NOS substrate, or sodium nitroprusside, an NO donor. The NO production in P. primaurelia was also shown to be sensitive to μM concentrations of heavy metals (HgCl₂ and CdCl₂), or μM concentrations of pesticides (diazinon and AFD 25), thus representing a potential biomarker for environmental biomonitoring. The possible involvement of cellular Ca²⁺ concentration, assayed by the fluorescent probe chlortetracycline hydrochloride, in NO production was examined after xenobiotic exposure.     

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.     

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.     

Organization and closing of mitochondrial deoxyribonucleic acid from Paramecium tetraaurelia and Paramecium primaurelia.

Previously we showed that the mitochondrial deoxyribonucleic acid (DNA) from Paramecium aurelia consists of a linear genome and that replication of this genome is initiated at one terminus and proceeds unidirectionally to the other terminus. Analyses of mitochondria from four closely related species (1, 4, 5, and 7) indicated that the species 1, 5, and 7 DNAs are essentially completely homologous but that the species 4 mitochondrial DNA is only 40 to 50% homologous with that from species 1. The major regions of homology are those containing the genes for ribosomal ribonucleic acid (RNA). To understand the replication and organization of the linear mitochondrial genome better, we compared species 1 (Paramecium primaurelia) and 4 (Paramecium tetraaurelia) DNAs with regard to restriction fragment mapping and homology between initiation regions; we also identified the sites of the genes for ribosomal RNA. In general, the structures of the species 1 and 4 mitochondrial genomes were quite similar. Each ribosomal RNA gene was present in one copy per genome, with the large ribosomal RNA gene located near the terminal region of replication and the small ribosomal RNA gene located more centrally. These two genes were separated by about 10 kilobases in the species 1 genome and by about 12 kilobases in the species 4 genome. In contrast to our previous findings, by using nonstringent hybridization conditions we detected homology between the species 1 and 4 DNA fragments containing the initiation regions. We constructed recombinant DNA clones for many fragments, especially those containing the initiation region and the ribosomal RNA genes. We also constructed restriction enzyme maps for six enzymes for both P. primaurelia and P. tetraaurelia.     

Telomeric site position heterogeneity in macronuclear DNA of Paramecium primaurelia.

In Paramecium primaurelia, the macronuclear gene encoding the G surface protein is located near a telomere. In this study, multiple copies of this telomere have been isolated and the subtelomeric and telomeric regions of some of them have been sequenced. The telomeric sequences consist of tandem repeats of the hexanucleotides C4A2 or C3A3. We show that the location where these repeats are added, which we call the telomeric site, is variable within a 0.6-0.8-kb region. These results are discussed in relation with the formation of macronuclear DNA.     

Food ingestion and egestion in mating reactive populations of Paramecium primaurelia.

The study of food ingestion and egestion carried out on Paramecium primaurelia mating reactive cells shows that, after their transfer into a medium with suspended particles, the complementary mating type cells exhibit very significant differences in the food vacuole formation and egestion rate. Under the same external environmental conditions, the mating type II cells form and egest a higher number of food vacuoles when compared with mating type I cells. The higher rate of food vacuole formation shown by the mating type II cells is related to their faster growth rate.     

Lectin-binding glycoconjugates in Paramecium primaurelia: changes with cellular age and starvation

 Lectins with different sugar specificities and binding to phagosome-lysosome systems as well as cell surface constituents were used to study glycoconjugate variation throughout culture and clonal life in Paramecium primaurelia, particularly during the transition period from logarithmic to stationary growth phase and in relation to clonal decline, respectively. These lectins include Griffonia simplicifolia agglutinin II (GS II), Ricinus communis agglutinin (RCA₁₂₀), Arachis hypogea agglutinin (PNA), succinyl concanavalin A (succinyl-con A), and Triticum vulgaris agglutinin (WGA). The labeling obtained varies both according to the lectin used and to the culture and clonal age of the cells. Negative results were obtained in logarithmic growth phase cells and in clonal young cells by using lectin GS II. Conversely, lectins RCA₁₂₀ and PNA bind to the cell surface, the oral region as well as cilia, and do not undergo modifications with culture or clonal age and after permeabilization. WGA binds to constituents of the cell surface, trichocyst tips, food vacuoles, the oral region, and cilia but the extent of labeling decreases as culture age increases; during clonal decline, cells show the same labeling pattern as starved cells. Finally, the lectin succinyl-con A shows a large amount of binding sites on the cell surface, on trichocyst tips, and in the oral region of logarithmic-phase cells, whereas the number of sites decreases in late stationary phase. The data obtained partly differ from those reported in the literature and the differences can be attributed to the culture conditions and species examined. Nevertheless, the assumption that a rearrangement of some glycoconjugates of membrane throughout culture and clonal life of Paramecium is confirmed. Accepted: 25 November 1996     

The effect of temperature change on gene expression in Paramecium primaurelia

When paramecia grown at 24 degrees C are transferred rapidly to 32 degrees C, DNA and protein synthesis continue uninterrupted but at higher rates. Electron microscopic observations indicate that more of the macronuclear chromatin is transcribed at the elevated temperature. This interpretation is supported by hybridization experiments which show that the percentage of the macronuclear genome transcribed into poly(A)+RNA is 24 degrees C and 35% at 32 degrees C. Kinetic analysis of cDNA-poly(A)+RNA hybridizations reveals three abundance classes of poly(A)+RNA and indicates that the number of genes expressing low abundance sequences is about 9000 at 24 degrees C and 13000 at 32 degrees C. The intermediately abundant and highly abundant classes are represented by 100-200 and 1-3 different kinds of RNA sequence, respectively. Cross hybridization shows that changes occur throughout the distribution of abundance classes of poly(A)+RNA with increase in temperature.     

Regulation of surface antigen expression in Paramecium primaurelia. II. Role of the surface antigen itself.

In the wild-type strains, 156 and 168, of Paramecium primaurelia, the alleles G156 and G168 expressed at medium temperature specify two immunologically distinguishable surface antigens 156G and 168G, whose phenotypic expression shows allelic exclusion, the majority of heterozygotes being phenotypically [156G] while a small minority is phenotypically [156G-168G]. At high temperature, the antigens coded by another locus, generally the D locus, are expressed. This system, displaying both intergenic and interallelic exclusion, provides favourable material to analyze the respective roles of the genome, of the antigens expressed and of the environmental conditions, in particular temperature, on the regulation of the expression of surface antigens. This analysis was carried out by studying the variations of the expression of surface antigens as a function of temperature, culture medium and previously expressed antigens in different genetic situations (a) in homozygotes: the wild-type strains 156 and 168, and the isogenized strains "G156 isogenic 168 carrying the G156 allele in a 168 genetic background; (b) in heterozygotes of the two phenotypic classes of heterozygotes, [156G] and [156G-168G]. The results show that (1) the thermal stability of the expression of a given surface antigen and its rate of re-appearance at the cell surface depend on its own specificity; (2) in heterozygotes [156G-168G], the stability of the expression of the antigen 156G is modified and "adjusted" to that of the less stable surface antigen 168G, and (3) the surface antigen itself exerts a positive control on the maintenance of its own expression. An interpretative model of "transmembranous control" is proposed to account for the regulation of the expression of surface antigens in Paramecium.     

Fluid phase and receptor-mediated endocytosis in Paramecium primaurelia by fluorescence confocal laser scanning microscopy

In ciliated protozoa, most nutrients are internalized via phagocytosis by food vacuole formation at the posterior end of the buccal cavity. The uptake of small-sized molecules and external fluid through the plasma membrane is a localized process. That is because most of the cell surface is internally covered by an alveolar system and a fibrous epiplasm, so that only defined areas of the cell surface are potential substance uptake sites. The purpose of this study is to analyze, by fluorescence confocal laser scanning microscopy, the relationship between WGA (Triticum vulgaris agglutinin) and dextran internalization in Paramecium primaurelia cells blocked in the phagocytic process, so that markers could not be internalized via food vacuole formation. WGA, which binds to surface constituents of fixed and living cells, was used as a marker for membrane transport and dextran as a marker for fluid phase endocytosis. After 3 min incubation, WGA-FITC is found on plasma membrane and cilia, and successively within small cytoplasmic vesicles. After a 10-15 min chase in unlabeled medium, the marked vesicles decrease in number, increase in size and fuse with food vacuoles. This fusion was evidenced by labeling food vacuoles with BSA-Texas red. Dextran enters the cell via endocytic vesicles which first localize in the cortical region, under the plasma membrane, and then migrate in the cytoplasm and fuse with other endocytic vesicles and food vacuoles. When cells are fed with WGA-FITC and dextran-Texas red at the same time, two differently labeled vesicle populations are found. Cytosol acidification and incubation in sucrose medium or in chlorpromazine showed that WGA is internalized via clathrin vesicles, whereas fluid phase endocytosis is a clathrin-independent process.     

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

Summary 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.     

Cytoplasmic inheritance of temperature sensitivity in a wild stock of Paramecium primaurelia

One stock, GBC, has a maximum temperature of growth about 5 degrees C lower than other recently collected stocks of Paramecium primaurelia. The resistant stocks (R) are able to grow continuously at 35 degrees C while the sensitive stock (S) cells die within 48 h. The F1s of R X S crosses exhibited a cytoplasmic pattern of inheritance and all F2-by-autogamy lines derived from the S cytoplasmic parent are sensitive. The F2-by-autogamy lines cytoplasmically descended from the R parent were predominantly (93%) R in the initial assay. Upon reinvestigation one year later, only 64% of these lines were R, 9% were S, and 27% had a new phenotype, weak (W), intermediate between R and S. Backcrosses of W lines to both R and S strongly suggest that the W lines have normal cytoplasm (i.e. R) but also have nuclear gene(s) for temperature sensitivity that are derived from the original S stock. The delayed manifestation of the W phenotype is not understood.     

Variety of serotypes of Paramecium primaurelia: single epitopes are responsible for immunological differentiation

Paramecium primaurelia expresses three major types of surface antigens. We report here the identification of the gene for serotype S, which completes the sequence data of expressed serotypes of P. primaurelia. The complete open reading frame of surface antigen S was identified using a novel technique, based upon the presence of conservative regions in the non-coding areas of the multigene family. We were able to isolate the 7194-bp-long open reading frame from the macronuclear DNA for Serotype 156S. The corresponding mRNA was detected in the two serotype S-expressing stocks, 60 and 156, of P. primaurelia, which clarifies that both stocks are using the same S allele. Comparisons of the nucleic acid and the deduced amino-acid sequence showed high identity to surface antigen 51B of P. tetraurelia, sufficient to cause an immunological cross-reaction in vivo. Immunologically relevant epitopes in vivo were identified in the central regions of the genes, constructed of nearly perfect tandem repeats.