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International audience; Gamma-tubulin belongs to the tubulin superfamily and plays an essential role in the nucleation of cellular microtubules. In the present study, we report the characterization of gamma-tubulin from the psychrophilic Antarctic ciliate Euplotes focardii. In this organism, gamma-tubulin is encoded by two genes, gamma-T1 and gamma-T2, that produce distinct isotypes. Comparison of the gamma-T1 and gamma-T2 primary sequences to a Euplotesgamma-tubulin consensus, derived from mesophilic (i.e. temperate) congeneric species, revealed the presence of numerous unique amino acid substitutions, particularly in gamma-T2. Structural models of gamma-T1 and gamma-T2, obtained using the 3D structure of human gamma-tubulin as a template, suggest that these substitutions are responsible for conformational and/or polarity differences located: (a) in the regions involved in longitudinal 'plus end' contacts; (b) in the T3 loop that participates in binding GTP; and (c) in the M loop that forms lateral interactions. Relative to gamma-T1, the gamma-T2 gene is amplified by approximately 18-fold in the macronuclear genome and is very strongly transcribed. Using confocal immunofluorescence microscopy, we found that the gamma-tubulins of E. focardii associate throughout the cell cycle with basal bodies of the non-motile dorsal cilia and of all of the cirri of the ventral surface (i.e. adoral membranelles, paraoral membrane, and frontoventral transverse, caudal and marginal cirri). By contrast, only gamma-T2 interacts with the centrosomes of the spindle during micronuclear mitosis. We also established that the gamma-T1 isotype associates only with basal bodies. Our results suggest that gamma-T1 and gamma-T2 perform different functions in the organization of the microtubule cytoskeleton of this protist and are consistent with the hypothesis that gamma-T1 and gamma-T2 have evolved sequence-based structural alterations that facilitate template nucleation of microtubules by the gamma-tubulin ring complex at cold temperatures.

The ciliate Euplotes raikovi produces a family of diffusible signal proteins (pheromones) that function as prototypic growth factors. They may either promote cell growth, by binding to pheromone receptors synthesized by the same cells from which they are secreted (autocrine activity), or induce a temporary cell shift from the growth stage to a mating (sexual) one by binding to pheromone receptors of other, conspecific cells (paracrine activity). In cells constitutively secreting the pheromone E r -1, it was first observed that the expression of the E r -1 receptor “p15,” a type II membrane protein of 130 amino acids, is quantitatively correlated with the extracellular concentration of secreted pheromone. p15 expression on the cell surface rapidly and markedly increased after the removal of secreted E r -1 and gradually decreased in parallel with new E r -1 secretion. It was then shown that p15 is internalized through endocytic vesicles following E r -1 binding and that the internalization of p15/E r -1 complexes is specifically blocked by the paracrine p15 binding of E r -2, a pheromone structurally homologous to, and thus capable of fully antagonizing, E r -1. Based on previous findings that the p15 pheromone-binding site is structurally equivalent to E r -1 and that E r -1 molecules polymerize in crystals following a pattern of cooperative interaction, it was proposed that p15/E r -1 complexes are internalized as a consequence of their unique property (not shared by p15/E r -2 complexes) of undergoing clustering.

Density separated trout erythrocytes, using a discontinuous Percoll gradient, yielded three distinct subfractions (top, middle and bottom) since older cells are characterized by increasing density. Cells from each subfraction were incubated with mitochondria-specific fluorescent probe Mitotracker and JC-1 in order to assess mitochondrial mass and membrane potential by means of cytofluorimetric analysis, confocal microscopy and subsequent computer-aided image analysis allowing a detailed investigation at single cell level. Both cytofluorimetric data and image analysis revealed changes in size and redistribution of mitochondria starting from the light fraction to the bottom. In particular in young erythrocytes small mitochondria were detected localized exclusively around the nucleus in a crown-like shape, the middle fraction revealed enlarged mitochondria partially scattered throughout the cytosol, whereas the last fraction represented again mitochondria with reduced size being distinctly dispersed throughout the cytosol in the cells. Concerning membrane potential considerations, our study revealed a dramatic decrease of DeltaPsi(m) in the bottom layer cell mitochondria compared to the top and unusual membrane potential increase of a subpopulation of enlarged mitochondria. DeltapH was also investigated in the three fractions by pretreating the cells with nigericin, allowing to confirm a mitochondrial energetic impairment in older cells.

In the free-living ciliate Euplotes raikovi, we identified (and designated as Er-MAPK1) a protein kinase of 631 amino acids, that appears to be constantly phosphorylated in cells which are in growth stage and interact in autocrine fashion with their water-soluble signal pheromones. Er-MAPK1 is specified by a gene that requires a+1 translational frame-shift to be expressed. Its amino-terminal region represents a canonical catalytic domain and carries an activation loop distinctive of the mitogen-activated protein kinases, with the Thr-Asp-Tyr motif deputed to be site of double phosphorylation. In contrast, the carboxy-terminal region appears to be structurally unique. It shows a strongly basic amino acid composition, is very rich in glycine repetitions, and contains a bipartite signal for translocation of Er-MAPK1 into the nucleus.

Summary Bacterial symbionts of insects have been proposed for blocking transmission of vector-borne pathogens. However, in many vector models the ecology of sym- bionts and their capability of cross-colonizing differ- ent hosts, an important feature in the symbiotic control approach, is poorly known. Here we show that the acetic acid bacterium Asaia, previously found in the malaria mosquito vector Anopheles stephensi, is also present in, and capable of cross-colonizing other sugar-feeding insects of phylogenetically distant genera and orders. PCR, real-time PCR and in situ hybridization experiments showed Asaia in the body of the mosquito Aedes aegypti and the leafhopper Scaphoideus titanus, vectors of human viruses and a grapevine phytoplasma respectively. Cross- colonization patterns of the body of Ae. aegypti, An. stephensi and S. titanus have been documented with Asaia strains isolated from An. stephensi or Ae. aegypti, and labelled with plasmid- or chromosome-encoded fluorescent proteins (Gfp and DsRed respectively). Fluorescence and confocal microscopy showed that Asaia, administered with the sugar meal, efficiently colonized guts, male and female reproductive systems and the salivary glands. The ability in cross-colonizing insects of phylogenetically distant orders indicated that Asaia adopts body inva- sion mechanisms independent from host-specific bio- logical characteristics. This versatility is an important property for the development of symbiont-based control of different vector-borne diseases.

At the end of July 2004, a "black-spot" appeared in the western portion of Lake Garda, an oligomictic lake classified as meso-oligotrophic. A few days later, this phenomenon spread throughout the lake. A first analysis by optical microscopy revealed that the origin of the black spot was a ciliated protozoan. Ciliates represent a small percentage of the total zooplanktonic community of Lake Garda and have never produced bloom episodes. Using morphological and molecular analysis, we characterized the protozoan responsible for the bloom as Stentor amethystinus and its symbiotic algae as a Chlorella sp. Continuous monitoring of the northeast of Lake Garda showed that the apex of the S. amethystinus bloom took place during the first 20 days of August, and the highest density of S. amethystinus occurred in the euphotic zone. During this period, high chlorophyll a values were obtained in water samples collected from the euphotic zone due to the presence of the endosymbiont Chlorella. After early September, the black spot completely disappeared, and the causative organism was detected at low concentration only in the southern basin of the lake. The results obtained on the progress of the black spot phenomenon led us to hypothesize that: (i) S. amethystinus was recently introduced in Lake Garda by anthropogenic activities or it was already a member of the zooplanktonic community but at a very low concentration; (ii) S. amethystinus blooms may have been driven by an unusual high availability of total phosphorous in the euphotic zone and (iii) Lake Garda is not the preferred habitat for S. amethystinus.

The induction of heat shock genes ( HSP s) is thought to be primarily regulated by heat shock transcription factors (HSFs), which bind target sequences on HSP promoters, called heat shock elements (HSEs). In this study, we investigated the 5′ untranslated regions of the Tetrahymena thermophila HSP70-1 gene, and we found, in addition to the canonical and divergent HSEs, multiple sets of GATA elements that have not been reported previously in protozoa. By means of in vivo analysis of a green fluorescent protein reporter transgene driven by the HSP70-1 promoter, we demonstrate that HSEs do not represent the minimal regulatory elements for heat shock induction, since the HSP70-1 is tightly regulated by both HSE and GATA elements. Electrophoretic mobility shift assay also showed that HSFs are constitutively bound to the HSEs, whereas GATA elements are engaged only after heat shock. This is the first demonstration by in vivo analysis of functional HSE and GATA elements in protozoa. Furthermore, we provide evidence of a functional link between HSE and GATA elements in the activation of the heat shock response.

In ciliates, different microtubular structures are nucleated from diverse Microtubule Organizing Centers (MTOCs). gamma-Tubulin is a tubulin superfamily member that plays an essential role in microtubule nucleation at the MTOCs. However, little is known about mechanisms regulating the activity of gamma-tubulin on different MTOCs and during the cell cycle. In Tetrahymena thermophila, the alpha- and beta-tubulin expression is regulated mainly at the transcriptional level, and changes in the ratio of polymerized/unpolymerized tubulin dimers lead to an increase or decrease of alpha- and beta-tubulin transcription. This study deals with the characterization of gamma-tubulin in the amicronuclear ciliate Tetrahymena pyriformis. Sequence analysis revealed some specific substitutions in nucleotide-binding loops characteristic of the Tetrahymena genus and putative conserved phosphorylation sites located on the external surface of the gamma-tubulin molecule. gamma-Tubulin expression during the cell cycle, in the presence of microtubular poisons and after deciliation, was also characterized. We found that gamma-tubulin mRNA levels are correlated with basal body proliferation and gamma-tubulin nuclear localization. We also found that gamma-tubulin expression changes during anti-microtubular drugs treatment, but does not changes during reciliation. These findings suggest a relationship between the level of unpolymerized tubulin dimers and gamma-tubulin transcription.

A series of new alpha1-adrenoreceptor antagonists (5-18) was prepared by introducing various substituents (Topliss approach) into the ortho, meta, and para positions of the benzyloxy function of the phendioxan open analogue 4 ("openphendioxan"). All the compounds synthesized were potent antagonists and generally displayed, similarly to 4, the highest affinity values at alpha1D- with respect to alpha1A- and alpha1B-AR subtypes and 5-HT1A subtype. By sulforhodamine B (SRB) assay on human PC-3 prostate cancer cells, the new compounds showed antitumor activity (estimated on the basis of three parameters GI50, TGI, LC50), at low micromolar concentration, with 7 ("clopenphendioxan") exhibiting the highest efficacy. Moreover, this study highlighted for the first time alpha1D- and alpha1B-AR expression in PC3 cells and also demonstrated the involvement of these subtypes in the modulation of apoptosis and cell proliferation. A significant reduction of alpha1D- and alpha1B-AR expression in PC3 cells was associated with the apoptosis induced by 7. This depletion was completely reversed by norepinephrine.

Four different tubulin genes were identified in the somatic nucleus (macronucleus) of Euplotes focardii, a strictly cold-adapted, Antarctic ciliate: one of 1,800 bp for alpha-tubulin and three of 2,150, 1,900, and 1,600 bp, respectively, for beta-tubulin. Preliminarily analysed for restriction fragment length polymorphisms, these genes showed remarkable differences in organisation from tubulin genes of other ciliates which live in temperate areas and were analysed in parallel with E. focardii. The complete coding sequence of the 1,600 bp beta-tubulin gene was then determined and shown to contain unique structural features of potential importance for E. focardii microtubule organization and activity. Of eight unique substitutions detected, seven were concentrated in the large amino terminal domain of the molecule that directly interacts with the carboxy terminal region of alpha-tubulin for heterodimer formation. Sequence analysis of the cloned gene revealed, in addition, a potential new exception in the use of the genetic code by ciliates. A TAG codon was aligned in correspondence with Trp-21 which is strictly conserved in every tubulin sequence so far determined.

Previous literature reports have demonstrated that nucleated trout erythrocytes in condition of oxidative stress are subjected to DNA and membrane damage, and inactivation of glutathione peroxidase. The present study was undertaken to investigate if mitochondrial membrane potential in stressed conditions was also influenced. Density-separated trout erythrocyte fractions, obtained using a discontinuous Percoll gradient, were submitted to stress conditions and the mitochondrial membrane potential was determined by means of cytofluorimetric analysis after incubation of each subfraction with JC-1, a mitochondrial specific fluorescent probe. The results clearly show that the mitochondrial membrane potential decreased significantly in all erythrocyte fractions, also if the oxidative effect on mitochondria is more severe with increased density (age) of the cell. Ebselen was very effective in preventing mitochondrial depolarization in young as well as in old erythrocytes.

SUMMARY We recently discovered, in mating-competent Paramecium primaurelia, the presence of functionally related molecules of the cholinergic system: the neurotransmitter acetylcholine (ACh), both its nicotinic and muscarinic receptors and its lytic enzyme acetylcholinesterase (AChE). Our results on the inhibition of mating-cell pairing in vivo in mating-competent cells treated with cholinomimetic drugs support the hypothesis that the cholinergic system plays a role in cell-to-cell adhesion. To investigate the possible function of the signal molecule ACh in conjugation in P. primaurelia, we attempted to detect the intracellular sites of ACh synthesis by localizing the ACh biosynthetic enzyme choline acetyltransferase (ChAT). Using immunocytochemical and histochemical methods, we have demonstrated the presence and activity of ChAT principally on the surface membrane of mating-competent cells and of mature but non-mating-competent cells. No evidence for ChAT activity was found in immature cells. Immunoblot analysis revealed the presence of immunoreactive bands, ranging in molecular mass from 42 to 133kDa, as reported for ChAT isolated from higher organisms. In vivo experiments showed that inhibition of ChAT activity by Congo Red, known to be a potent competitive inhibitor of acetyl coenzyme A, did not affect mating-cell pairing. Conversely, inhibition of AChE with BW284c51 or eserine, which block enzyme activity by reacting with a specific serine within the catalytic centre, significantly inhibited mating-cell pairing. Our results suggest that ACh has a negative modulating effect on conjugation in P. primaurelia.