Maria Umberta Delmonte Corrado

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.

Detection of cholinesterase activities and acetylcholine receptors during the developmental cycle of Dictyostelium discoideum

Previous results of our study carried out on Dictyostelium discoideum showed the presence in single-cell amoebae of both cholinesterase (ChE) activity, able to hydrolyze the propionyl thiocholine iodide substrate, and “true” acetylcholinesterase (AChE) activity, similar to that of Electrophorus electricus . These activities are sensitive to anti-ChE agents, and to the inhibitors classically used to discriminate different ChEs. In this work, we have investigated the presence of ChE activities and of both acetylcholine (ACh) nicotinic and muscarinic receptors, during the developmental cycle of D. discoideum . The spectrophotometric evaluation showed that slugs displayed the highest enzyme activities, AChE being the main activity, while fruiting bodies exhibited the lowest. The presence of AChE activity was cytochemically detected in all the cell populations of the developmental cycle, except in the fruiting bodies where it was localized exclusively to the level of the spores. Molecules recognized by the anti-butyrylcholinesterase antibody were detected in single-cell amoebae, first fingers, slugs, and culminants, except in the fruiting bodies where they were found exclusively in the spore walls. The ACh nicotinic receptors were cytochemically identified in single-cell amoebae, slugs, and spores; however, molecules immunologically related to the ACh muscarinic receptors were not present in the spores.

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.

Lectin-Binding Sites Involved in Paramecium primaurelia Mating Pair Formation

ABSTRACT. Mating pair formation in Paramecium primaurelia was shown to be inhibited by incubating mating‐competent mating type (mt) I and mt U cells with Limulus polyphemus agglutinin (LPA) or wheat germ agglutinin (WGA). Preincubation of LPA and WGA with their specific binding‐monosaccharides, N‐acetylneuraminic acid (NeuAc) and N‐acetylglucosamine (GlcNAc), respectively, prevented the lectin effect on pair formation. Addition either of NeuAc or GlcNAc resulted in a reversal of cell pairing inhibition by LPA or WGA, respectively. Both NeuAc and GlcNAc monosaccharides inhibited pair formation when their concentration exceeded a threshold value. The pattern of the relative distribution of NeuAc and GlcNAc molecules on the cell surface was analyzed using fluorescence resonance energy transfer techniques combined with imaging systems. Mt I1 cells showed a high lectin‐binding site density localized just on the surface region engaged in conjugation. The pattern of mt I cells, consisting of a quite homogeneous lectin‐binding site density spread on the cell surface, was also common to nonmating‐competent cells and to immature cells. These findings suggest that in P. primaurelia pair formation involves both NeuAc and GlcNAc residues and that the development of mating‐competence is related to a modification in NeuAc and GlcNAc relative distribution on the cell surface of mt 11 cells.

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.

Three-dimensional reconstruction of Paramecium primaurelia oral apparatus through confocal laser scanning optical microscopy

The oral apparatus of the ciliate protozoan Paramecium primaurelia, a single-celled eukaryotic organism, is a highly organized structure whose arrangement is of important taxonomic, phylogenetic and developmental significance. This paper analyses oral structures by means of a confocal laser scanning optical microscope (CLSM), which allows their three-dimensional visualization and measurement. The extraction of the intrinsic three-dimensional information related to the biological objects under investigation can in turn be related to their functional state, according to the classical paradigms of structure to function relationship identification. In our experiments, we acquired different data sets. These are optical slices of the biological sample under investigation, acquired in a confocal situation, through epi-illumination, in reflection. For comparison with conventional microscopy, two-dimensional images were acquired via a standard TV camera coupled to the microscope itself. The volumes obtained by piling up the slices were rendered through different techniques, some of them directly implemented on the workstation controlling the CLSM system, some of them on a SUN SPARCstation 1, where the original data were transferred via an Ethernet link. In this last instance, original software has been developed for the visualization and animation of the three-dimensional structures, under UNIX and X-Window, according to a ray-tracing algorithm.

Ultrastructural aspects of a ‘rejuvenated’ cell line of Colpoda cucullus (Protozoa, Ciliophora)

Abstract A ‘rejuvenated’ cell line of Colpoda cucullus was yielded from an aged culture submitted to a long‐term resting encystment, and was examined ultrastructurally in logarithmic growth phase and reproductive encystment, after recovery of the standard fission rate. In logarithmically growing cells, the macronuclear envelope shows deep invaginations with the appearance of inner ‘cytoplasmic isles’, and the nucleolar units loosely aggregate as in logarithmically growing young cells. On the other hand, new macronuclear features appear in the reproductive cysts, mainly because of the fusion of the nucleolar units. On the whole, the cytoplasm ultrastructure in rejuvenated cells appears not to differ remarkably from that of young cells. This study indicates that rejuvenated cells of C. cucullus show some ultrastructural aspects typical of young cells and others which are commonly observed in aged cells, and are probably derived from the previous senescent condition.

Three-dimensional reconstruction of paramecium primaurelia oral apparatus through confocal laser scanning optical microscopy

Studies on the complementary mating types of Paramecium primaurelia (Protozoa, Ciliates) have shown that cell lines which differ from each other in mating type expression are characterized by different cell contents, organization, and physiology. Referring to these differences and to the differential rates of food vacuole formation, oral apparatuses of the two mating type cells are assumed to possibly differ from each other in some traits, such as, for instance, in their lengths. In our work, the highly organized oral structures are analyzed by means of a laser scanning confocal optical microscope (CLSM), which provides their 3-D visualization and measurement. The extraction of the 3-D intrinsic information related to the biological objects under investigation can be in turn related to their functional state, according to the classical paradigm of structure to function relationships identification. In our experiments, we acquired different data sets. These are optical slices of the biological sample under investigation, acquired in a confocal situation, through epi-illumination, in reflection, and, for comparison with conventional microscopy, 2-D images acquired via a standard TV camera coupled to the microscope itself. Our CLSM system is equipped with a laser beam at 488 and 514 nm and the data have been acquired with various steps of optical slicing, ranging from .04 to .25 micrometers. The volumes obtained by piling-up the slices are rendered through different techniques, some of them directly implemented on the workstation controlling the CLSM system, some of them on a SUN SPARC station 1, where the original data were transferred via an Ethernet link. In this last instance, original software has been developed for the visualization and animation of the 3-D structures, running under UNIX and X-Window, according to a ray-tracing algorithm.