Francesca Trielli

Evidence for the presence of a mammalian-like cholinesterase in Paramecium Primaurelia (Protista, Ciliophora) developmental cycle

By histochemical and immunohistochemical methods, the presence of cholinergic-like molecules has previously been demonstrated in Paramecium primaurelia, and their functional role in mating-cell pairing was suggested. In this work, both true acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) activities were electrophoretically investigated, and the presence of molecules immunologically related to BuChE was checked by immunoblotting. The AChE activity, shown in the membrane protein fraction of mating-competent cells and in the cytoplasmic fraction of immature cells, is due to a 260-kDa molecular form, similar to the membrane-bound tetrameric form present in human erythrocytes. This AChE activity does not appear in either the cytoplasmic fraction of mating-competent cells or in the membrane protein fraction of immature cells. No evidence was found for the presence or the activity of BuChE-like molecules. The role of AChE in P. primaurelia developmental cycle is discussed.

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.

Organic matter recycling in a beach environment influenced by sunscreen products and increased inorganic nutrient supply (Sturla, Ligurian Sea, NW Mediterranean)

The beaches are sites where the human influence may be strong and the beach ecosystems have often shown a high sensibility to environmental alterations. These zones may be affected by a large series of anthropogenic-derived pressures, such as unbalanced inorganic nutrient input, that may cause anomalous development of primary production, altering the structure of the trophic webs. Furthermore, the utilisation of cosmetic sunscreen products is reaching unexpected levels, thus assuming a potentially important as well as unknown role in the contamination of marine environments. The present study was planned to test the response of the beach ecosystem to increases in inorganic nutrients (nitrate and phosphate) and to the input of a widely used cosmetic sunscreen product. A short-term laboratory experiment was carried out on microsystems consisting of sediments and seawater from the swash zone of a Ligurian city beach (Sturla). The processes related to organic matter (OM) recycling and some microbial food web components (bacteria and micro-autotrophic organisms) were analysed. The multivariate statistical analysis of the results showed that the increase in inorganic nutrients and sunscreen caused only a transient alteration in the OM recycling processes in the seawater. The sedimentary processes, instead, were different in the different systems, although starting from the same condition. In the sediment, surprisingly, an increase in inorganic nutrients did not lead to an increase in the primary biomass nor to significantly higher bacterial abundance, while the sunscreen caused increased OM recycling, especially devoted to protein and lipid mobilisation, supporting a growing bacterial and autotrophic community by reducing the bottom-up pressure. Additional toxicity tests performed on protozoa highlighted that, while the inorganic nutrients seemed to show no effects, sunscreen decreased the protozoan viability, thus likely favouring microautotrophic and bacterial increases by reducing the top-down pressure.

Action Mechanisms of the Secondary Metabolite Euplotin C : Signaling and Functional Role in Euplotes

ABSTRACT. Among secondary metabolites, the acetylated hemiacetal sesquiterpene euplotin C has been isolated from the marine, ciliated protist Euplotes crassus, and provides an effective mechanism for reducing populations of potential competitors through its cytotoxic properties. However, intracellular signaling mechanisms and their functional correlates mediating the ecological role of euplotin C are largely unknown. We report here that, in E. vannus (an Euplotes morphospecies that does not produce euplotin C and shares with E. crasssus the same interstitial habitat), euplotin C rapidly increases the intracellular concentration of both Ca2+ and Na+, suggesting a generalized effect of this metabolite on cation transport systems. In addition, euplotin C does not induce oxidative stress, but modulates the electrical properties of E. vannus through an increase of the amplitude of graded action potentials. These events parallel the disassembling of the ciliary structures, the inhibition of cell motility, the occurrence of aberrant cytoplasmic vacuoles, and the rapid inhibition of phagocytic activity. Euplotin C also increases lysosomal pH and decreases lysosomal membrane stability of E. vannus. These results suggest that euplotin C exerts a marked disruption of those homeostatic mechanisms whose efficiency represents the essential prerequisite to face the challenges of the interstitial environment.

Changes in the ultrastructure and glycoproteins of the cyst wall of Colpoda cucullus during resting encystment

Summary Our previous studies on long-term resting encystment of Colpoda inflata showed transcriptional activity of the macronucleus and the occurrence of protein synthesis. In this work we have investigated the structure, the glycoprotein composition, and the lectin-binding site localization of the cyst wall of C. cucullus at increasing cyst ages, by means of scanning and transmission electron microscopy, confocal microscopy, and cytochemical and lectin blotting techniques. During an 18-day resting encystment, the cyst wall undergoes structural modifications resulting in the compaction of its previously-formed layers and the production of new material that is placed on the inner side of the cyst wall. Changes in the localization of ConA-/WGA-binding sites have been checked by comparing 7-day-old cysts to 1-day-old ones. Moreover, differences in the electrophoretic and ConA-/WGA-positive patterns have been found in 18-day-old cysts when compared to 1-day-old ones. The results of in vivo ConA and WGA assays suggest that ConA promotes encystment and delays excystment.

Variations in macronuclear chromatin structure andchromatin extrusion in excystment from resting cysts of Colpoda inflata

The macronucleus of Colpoda inflata was studied by image analysis to determine variations that occur in chromatin structure during excystment from resting cysts. Morphometric and densitometric parameters, 22 Markovian chromatin texture variables, and the presence of chromatin extrusion bodies were examined in 1-, 5-, and 10-day-old excysting cells. Marked variations in densitometric values and Markovian variables were found only in 1-day-old excysting cells, thus suggesting that the chromatin extrusion process, where not already accomplished in early encystment, is triggered by excystment. Comparative study of the mean light absorption histograms computed on optical microscopic images of excysting cells, derived from 2- and 25-day-old cysts of a standard culture and from 1-year-old cysts of a senescent culture, showed three and two classes of histograms, respectively, characterised by differently condensed chromatin. Moreover, as excystment progresses, the percentage of macronuclei with decondensed chromatin that also show an extrusion body of condensed chromatin increases, thus suggesting that the macronucleus is somehow renewed by extruding chromatin that is unable to decondense. This event appears as a possible mechanism responsable for the ‘rejuvenescence’ of senescent cell lines.