Ciliate Microtubule Diversities: Insights from the EFBTU3 Tubulin in the Antarctic Ciliate Euplotes focardii.

Protozoans of the Phylum Ciliophora (ciliates) assemble many diverse microtubular structures in a single cell throughout the life cycle, a feature that made them useful models to study microtubule complexity and the role of tubulin isotypes. In the Antarctic ciliate Euplotes focardii we identified five ß-tubulin isotypes by genome sequencing, named EFBTU1, EFBTU2, EFBTU3, EFBTU4 and EFBTU5. By using polyclonal antibodies directed against EFBTU2/EFBTU1 and EFBTU3, we show that the former isotypes appear to be involved in the formation of all microtubular structures and are particularly abundant in cilia, whereas the latter specifically localizes at the bases of cilia. By RNA interference (RNAi) technology, we silenced the EFBTU3 gene and provided evidence that this isotype has a relevant role in cilia regeneration upon deciliation and in cell division. These results support the long-standing concept that tubulin isotypes possess functional specificity in building diverse microtubular structures.

The macronuclear genome of the Antarctic psychrophilic marine ciliate Euplotes focardii reveals new insights on molecular cold adaptation.

The macronuclear (MAC) genomes of ciliates belonging to the genus Euplotes species are comprised of numerous small DNA molecules, nanochromosomes, each typically encoding a single gene. These genomes are responsible for all gene expression during vegetative cell growth. Here, we report the analysis of the MAC genome from the Antarctic psychrophile Euplotes focardii. Nanochromosomes containing bacterial sequences were not found, suggesting that phenomena of horizontal gene transfer did not occur recently, even though this ciliate species has a substantial associated bacterial consortium. As in other euplotid species, E. focardii MAC genes are characterized by a high frequency of translational frameshifting. Furthermore, in order to characterize differences that may be consequent to cold adaptation and defense to oxidative stress, the main constraints of the Antarctic marine microorganisms, we compared E. focardii MAC genome with those available from mesophilic Euplotes species. We focussed mainly on the comparison of tubulin, antioxidant enzymes and heat shock protein (HSP) 70 families, molecules which possess peculiar characteristic correlated with cold adaptation in E. focardii. We found that α-tubulin genes and those encoding SODs and CATs antioxidant enzymes are more numerous than in the mesophilic Euplotes species. Furthermore, the phylogenetic trees showed that these molecules are divergent in the Antarctic species. In contrast, there are fewer hsp70 genes in E. focardii compared to mesophilic Euplotes and these genes do not respond to thermal stress but only to oxidative stress. Our results suggest that molecular adaptation to cold and oxidative stress in the Antarctic environment may not only be due to particular amino acid substitutions but also due to duplication and divergence of paralogous genes.

An In-Silico Comparative Study of Lipases from the Antarctic Psychrophilic Ciliate Euplotes focardii and the Mesophilic Congeneric Species Euplotes crassus: Insight into Molecular Cold-Adaptation.

Cold-adapted enzymes produced by psychrophilic organisms have elevated catalytic activities at low temperatures compared to their mesophilic counterparts. This is largely due to amino acids changes in the protein sequence that often confer increased molecular flexibility in the cold. Comparison of structural changes between psychrophilic and mesophilic enzymes often reveal molecular cold adaptation. In the present study, we performed an in-silico comparative analysis of 104 hydrolytic enzymes belonging to the family of lipases from two evolutionary close marine ciliate species: The Antarctic psychrophilic Euplotes focardii and the mesophilic Euplotes crassus. By applying bioinformatics approaches, we compared amino acid composition and predicted secondary and tertiary structures of these lipases to extract relevant information relative to cold adaptation. Our results not only confirm the importance of several previous recognized amino acid substitutions for cold adaptation, as the preference for small amino acid, but also identify some new factors correlated with the secondary structure possibly responsible for enhanced enzyme activity at low temperatures. This study emphasizes the subtle sequence and structural modifications that may help to transform mesophilic into psychrophilic enzymes for industrial applications by protein engineering.

Transcriptomic responses to silver nanoparticles in the freshwater unicellular eukaryote Tetrahymena thermophila.

Currently, silver nanoparticles (AgNPs) are being increasingly used as biocides in various consumer products and if released in the environment they can affect non-target organisms. Therefore, understanding the toxicity mechanism is crucial for both the design of more efficient nano-antimicrobials and for the design of nanomaterials that are biologically and environmentally benign throughout their life-cycle. Here, the ciliate Tetrahymena thermophila was used to elucidate the mechanisms of action of AgNPs by analysing the gene expression profile by RNA-seq and the transcriptomic effects of AgNPs were compared to those induced by soluble silver salt, AgNO3. Exposure to AgNPs at sublethal concentrations for 24 h induced phagocytosis, transport pathways, response to oxidative stress, glutathione peroxidase activity, response to stimulus, oxidation-reduction, proteolysis, and nitrogen metabolism process. Based on gene set enrichment analysis (GSEA), some biological processes appeared targets of both toxicants. In addition to many similarities in affected genes, some effects were triggered only by NPs, like phagocytosis, glutathione peroxidase activity, response to stimulus, protein phosphorylation and nitrogen metabolism process. This research provides evidence that AgNPs compared to AgNO3 at the same concentration of dissolved silver ions dysregulate a higher number of cellular pathways. These findings confirm that AgNPs can induce toxicity not only due to soluble silver ions released from the particles but also to particle intrinsic features.

Horizontal gene transfer and silver nanoparticles production in a new Marinomonas strain isolated from the Antarctic psychrophilic ciliate Euplotes focardii.

We isolated a novel bacterial strain from a prokaryotic consortium associated to the psychrophilic marine ciliate Euplotes focardii, endemic of the Antarctic coastal seawater. The 16S rDNA sequencing and the phylogenetic analysis revealed the close evolutionary relationship to the Antarctic marine bacterium Marinomonas sp. BSw10506 and the sub antarctic Marinomonas polaris. We named this new strain Marinomonas sp. ef1. The optimal growth temperature in LB medium was 22 °C. Whole genome sequencing and analysis showed a reduced gene loss limited to regions encoding for transposases. Additionally, five genomic islands, e.g. DNA fragments that facilitate horizontal gene transfer phenomena, were identified. Two open reading frames predicted from the genomic islands coded for enzymes belonging to the Nitro-FMN-reductase superfamily. One of these, the putative NAD(P)H nitroreductase YfkO, has been reported to be involved in the bioreduction of silver (Ag) ions and the production of silver nanoparticles (AgNPs). After the Marinomonas sp. ef1 biomass incubation with 1 mM of AgNO3 at 22 °C, we obtained AgNPs within 24 h. The AgNPs were relatively small in size (50 nm) and had a strong antimicrobial activity against twelve common nosocomial pathogenic microorganisms including Staphylococcus aureus and two Candida strains. To our knowledge, this is the first report of AgNPs biosynthesis by a Marinomonas strain. This biosynthesis may play a dual role in detoxification from silver nitrate and protection from pathogens for the bacterium and potentially for the associated ciliate. Biosynthetic AgNPs also represent a promising alternative to conventional antibiotics against common pathogens.

Synthesis of Bioactive Silver Nanoparticles by a Pseudomonas Strain Associated with the Antarctic Psychrophilic Protozoon Euplotes focardii.

The synthesis of silver nanoparticles (AgNPs) by microorganisms recently gained a greater interest due to its potential to produce them in various sizes and morphologies. In this study, for AgNP biosynthesis, we used a new Pseudomonas strain isolated from a consortium associated with the Antarctic marine ciliate Euplotes focardii. After incubation of Pseudomonas cultures with 1 mM of AgNO3 at 22 °C, we obtained AgNPs within 24 h. Scanning electron (SEM) and transmission electron microscopy (TEM) revealed spherical polydispersed AgNPs in the size range of 20-70 nm. The average size was approximately 50 nm. Energy dispersive X-ray spectroscopy (EDS) showed the presence of a high intensity absorption peak at 3 keV, a distinctive property of nanocrystalline silver products. Fourier transform infrared (FTIR) spectroscopy found the presence of a high amount of AgNP-stabilizing proteins and other secondary metabolites. X-ray diffraction (XRD) revealed a face-centred cubic (fcc) diffraction spectrum with a crystalline nature. A comparative study between the chemically synthesized and Pseudomonas AgNPs revealed a higher antibacterial activity of the latter against common nosocomial pathogen microorganisms, including Escherichia coli, Staphylococcus aureus and Candida albicans. This study reports an efficient, rapid synthesis of stable AgNPs by a new Pseudomonas strain with high antimicrobial activity.

Rational Engineering of a Cold-Adapted α-Amylase from the Antarctic Ciliate Euplotes focardii for Simultaneous Improvement of Thermostability and Catalytic Activity.

The α-amylases are endo-acting enzymes that hydrolyze starch by randomly cleaving the 1,4-α-d-glucosidic linkages between the adjacent glucose units in a linear amylose chain. They have significant advantages in a wide range of applications, particularly in the food industry. The eukaryotic α-amylase isolated from the Antarctic ciliated protozoon Euplotes focardii (EfAmy) is an alkaline enzyme, different from most of the α-amylases characterized so far. Furthermore, EfAmy has the characteristics of a psychrophilic α-amylase, such as the highest hydrolytic activity at a low temperature and high thermolability, which is the major drawback of cold-active enzymes in industrial applications. In this work, we applied site-directed mutagenesis combined with rational design to generate a cold-active EfAmy with improved thermostability and catalytic efficiency at low temperatures. We engineered two EfAmy mutants. In one mutant, we introduced Pro residues on the A and B domains in surface loops. In the second mutant, we changed Val residues to Thr close to the catalytic site. The aim of these substitutions was to rigidify the molecular structure of the enzyme. Furthermore, we also analyzed mutants containing these combined substitutions. Biochemical enzymatic assays of engineered versions of EfAmy revealed that the combination of mutations at the surface loops increased the thermostability and catalytic efficiency of the enzyme. The possible mechanisms responsible for the changes in the biochemical properties are discussed by analyzing the three-dimensional structural model.IMPORTANCE Cold-adapted enzymes have high specific activity at low and moderate temperatures, a property that can be extremely useful in various applications as it implies a reduction in energy consumption during the catalyzed reaction. However, the concurrent high thermolability of cold-adapted enzymes often limits their applications in industrial processes. The α-amylase from the psychrophilic Antarctic ciliate Euplotes focardii (named EfAmy) is a cold-adapted enzyme with optimal catalytic activity in an alkaline environment. These unique features distinguish it from most α-amylases characterized so far. In this work, we engineered a novel EfAmy with improved thermostability, substrate binding affinity, and catalytic efficiency to various extents, without impacting its pH preference. These characteristics can be considered important properties for use in the food, detergent, and textile industries and in other industrial applications. The enzyme engineering strategy developed in this study may also provide useful knowledge for future optimization of molecules to be used in particular industrial applications.

Microbial Consortium Associated with the Antarctic Marine Ciliate Euplotes focardii: An Investigation from Genomic Sequences.

We report the characterization of the bacterial consortium associated to Euplotes focardii, a strictly psychrophilic marine ciliate that was maintained in laboratory cultures at 4 °C after its first isolation from Terra Nova Bay, in Antarctica. By Illumina genome analyser, we obtained 11,179 contigs of potential prokaryotic origin and classified them according to the NCBI's prokaryotic attributes table. The majority of these sequences correspond to either Bacteroidetes (16 %) or Proteobacteria (78 %). The latter were dominated by gamma- (39 %, including sequences related to the pathogenic genus Francisella), and alpha-proteobacterial (30 %) sequences. Analysis of the Pfam domain family and Gene Ontology term variation revealed that the most frequent terms that appear unique to this consortium correspond to proteins involved in "transmembrane transporter activity" and "oxidoreductase activity". Furthermore, we identified genes that encode for enzymes involved in the catabolism of complex substance for energy reserves. We also characterized members of the transposase and integrase superfamilies, whose role in bacterial evolution is well documented, as well as putative antifreeze proteins. Antibiotic treatments of E. focardii cultures delayed the cell division of the ciliate. To conclude, our results indicate that this consortium is largely represented by bacteria derived from the original Antarctic sample and may contribute to the survival of E. focardii in laboratory condition. Furthermore, our results suggest that these bacteria may have a more general role in E. focardii survival in its natural cold and oxidative environment.

Distinct functional roles of ß-tubulin isotypes in microtubule arrays of Tetrahymena thermophila, a model single-celled organism.

BACKGROUND: The multi-tubulin hypothesis proposes that each tubulin isotype performs a unique role, or subset of roles, in the universe of microtubule function(s). To test this hypothesis, we are investigating the functions of the recently discovered, noncanonical ß-like tubulins (BLTs) of the ciliate, Tetrahymena thermophila. Tetrahymena forms 17 distinct microtubular structures whose assembly had been thought to be based on single α- and ß-isotypes. However, completion of the macronuclear genome sequence of Tetrahymena demonstrated that this ciliate possessed a ß-tubulin multigene family: two synonymous genes (BTU1 and BTU2) encode the canonical ß-tubulin, BTU2, and six genes (BLT1-6) yield five divergent ß-tubulin isotypes. In this report, we examine the structural features and functions of two of the BLTs (BLT1 and BLT4) and compare them to those of BTU2. METHODOLOGY/PRINCIPAL FINDINGS: With respect to BTU2, BLT1 and BLT4 had multiple sequence substitutions in their GTP-binding sites, in their interaction surfaces, and in their microtubule-targeting motifs, which together suggest that they have specialized functions. To assess the roles of these tubulins in vivo, we transformed Tetrahymena with expression vectors that direct the synthesis of GFP-tagged versions of the isotypes. We show that GFP-BLT1 and GFP-BLT4 were not detectable in somatic cilia and basal bodies, whereas GFP-BTU2 strongly labeled these structures. During cell division, GFP-BLT1 and GFP-BLT4, but not GFP-BTU2, were incorporated into the microtubule arrays of the macronucleus and into the mitotic apparatus of the micronucleus. GFP-BLT1 also participated in formation of the microtubules of the meiotic apparatus of the micronucleus during conjugation. Partitioning of the isotypes between nuclear and ciliary microtubules was confirmed biochemically. CONCLUSION/SIGNIFICANCE: We conclude that Tetrahymena uses a family of distinct ß-tubulin isotypes to construct subsets of functionally different microtubules, a result that provides strong support for the multi-tubulin hypothesis.

Structural thermal adaptation of ß-tubulins from the Antarctic psychrophilic protozoan Euplotes focardii.

Tubulin dimers of psychrophilic eukaryotes can polymerize into microtubules at 4°C, a temperature at which microtubules from mesophiles disassemble. This unique capability requires changes in the primary structure and/or in post-translational modifications of the tubulin subunits. To contribute to the understanding of mechanisms responsible for microtubule cold stability, here we present a computational structural analysis based on molecular dynamics (MD) and experimental data of three ß-tubulin isotypes, named EFBT2, EFBT3, and EFBT4, from the Antarctic protozoon Euplotes focardii that optimal temperature for growth and reproduction is 4°C. In comparison to the ß-tubulin from E. crassus, a mesophilic Euplotes species, EFBT2, EFBT3, and EFBT4 possess unique amino acid substitutions that confer different flexible properties of the polypeptide, as well as an increased hydrophobicity of the regions involved in microtubule interdimeric contacts that may overcome the microtubule destabilizing effect of cold temperatures. The structural analysis based on MD indicated that all isotypes display different flexibility properties in the regions involved in the formation of longitudinal and lateral contacts during microtubule polymerization. We also investigated the role of E. focardii ß-tubulin isotypes during the process of cilia formation. The unique characteristics of the primary and tertiary structures of psychrophilic ß-tubulin isotypes seem responsible for the formation of microtubules with distinct dynamic and functional properties.

The yeast Wickerhamomyces anomalus (Pichia anomala) inhabits the midgut and reproductive system of the Asian malaria vector Anopheles stephensi.

While symbiosis between bacteria and insects has been thoroughly investigated in the last two decades, investments on the study of yeasts associated with insects have been limited. Insect-associated yeasts are placed on different branches of the phylogenetic tree of fungi, indicating that these associations evolved independently on several occasions. Isolation of yeasts is frequently reported from insect habitats, and in some cases yeasts have been detected in the insect gut and in other organs/tissues. Here we show that the yeast Wickerhamomyces anomalus, previously known as Pichia anomala, is stably associated with the mosquito Anopheles stephensi, a main vector of malaria in Asia. Wickerhamomyces anomalus colonized pre-adult stages (larvae L(1)-L(4) and pupae) and adults of different sex and age and could be isolated in pure culture. By a combination of transmission electron microscopy and fluorescent in situ hybridization techniques, W. anomalus was shown to localize in the midgut and in both the male and female reproductive systems, suggesting multiple transmission patterns.

A novel protein kinase from the ciliate Euplotes raikovi with close structural identity to the mammalian intestinal and male-germ cell kinases: characterization and functional implications in the autocrine pheromone signaling loop.

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.

Asaia, a versatile acetic acid bacterial symbiont, capable of cross-colonizing insects of phylogenetically distant genera and orders.

Bacterial symbionts of insects have been proposed for blocking transmission of vector-borne pathogens. However, in many vector models the ecology of symbionts and their capability of cross-colonizing different 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 invasion mechanisms independent from host-specific biological characteristics. This versatility is an important property for the development of symbiont-based control of different vector-borne diseases.

Triggering of transient receptor potential vanilloid type 1 (TRPV1) by capsaicin induces Fas/CD95-mediated apoptosis of urothelial cancer cells in an ATM-dependent manner.

Herein, we provide evidence on the expression of transient receptor potential vanilloid type 1 (TRPV1) on human urothelial cancer (UC) cells and its involvement in the apoptosis induced by the selective agonist capsaicin (CPS). We analyzed TRPV1 messenger RNA and protein expression on human UC cell lines demonstrating its progressive decrease in high-grade UC cells. Treatment of RT4 cells with CPS induced cell cycle arrest in G(0)/G(1) phase and apoptosis. These events were associated with rapid co-ordinated transcription of pro-apoptotic genes including Fas/CD95, Bcl-2 and caspase families and ataxia telangiectasia mutated (ATM)/CHK2/p53 DNA damage response pathway. CPS induced Fas/CD95 upregulation, but more importantly Fas/CD95 ligand independent, TRPV1-dependent death receptor clustering and triggering of both extrinsic and intrinsic mitochondrial-dependent pathways. Moreover, we observed that CPS activates ATM kinase that is involved in Ser15, Ser20 and Ser392 p53 phosphorylation as shown by the use of the specific inhibitor KU55933. Notably, ATM activation was also found to control upregulation of Fas/CD95 expression and its co-clustering with TRPV1 as well as RT4 cell growth and apoptosis. Altogether, we describe a novel connection between ATM DNA damage response pathway and Fas/CD95-mediated intrinsic and extrinsic apoptotic pathways triggered by TRPV1 stimulation on UC cells.

Molecular cold-adaptation of protein function and gene regulation: The case for comparative genomic analyses in marine ciliated protozoa.

Euplotes focardii is a marine ciliated protozoan discovered in the Ross Sea near Terra Nova Bay, Antarctica. This organism is strictly psychrophilic, survives and reproduces optimally at 4-5 °C, and has a genome rich in A/T base pairs. Like other ciliated protozoans, Euplotes spp. are characterized by nuclear dimorphism: 1) the germline micronucleus contains the entire genome as large chromosomes; and 2) the somatic macronucleus (∼50 megabases, or 5% of the micronuclear genome) contains small linear DNA nanochromosomes [1-12 kilobases], each of which constitutes a single genetic unit. These characteristics make E. focardii an ideal model for genome-level analysis to understand the evolutionary mechanisms that determine the adaptation of organisms to cold environments. Here we describe two examples that are controlled by phylogenetically appropriate comparison with mesophilic and psychrotolerant Euplotes species: 1) the genes and encoded proteins of the E. focardii tubulin superfamily, including α-, ß-, and γ-tubulins; and 2) the genes of the heat-shock protein (Hsp) 70 family. The tubulins provide particular insight into protein-level structural changes that are likely to facilitate microtubule nucleation and polymerization in an energy poor environment. By contrast, the hsp70 genes of E. focardii and of its psychrotolerant relative E. nobilii reveal adaptive alterations in the regulation of gene expression in the cold. The unique characteristics of the E. focardii genome and the results that we present here argue strongly for a concerted effort to characterize the relatively low complexity macronuclear genome of this psychrophilic organism.

Different roles of two gamma-tubulin isotypes in the cytoskeleton of the Antarctic ciliate Euplotes focardii: remodelling of interaction surfaces may enhance microtubule nucleation at low temperature.

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.

Biomonitoring of Lake Garda: Identification of ciliate species and symbiotic algae responsible for the "black-spot" bloom during the summer of 2004.

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.

Combination of two regulatory elements in the Tetrahymena thermophila HSP70-1 gene controls heat shock activation.

The induction of heat shock genes (HSPs) 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.

Transient receptor potential vanilloid type 2 (TRPV2) expression in normal urothelium and in urothelial carcinoma of human bladder: correlation with the pathologic stage.

OBJECTIVE: To evaluate the expression of transient receptor potential vanilloid type 2 (TRPV2) in normal human bladder and urothelial carcinoma (UC) tissues. METHODS: Bladder specimens were obtained by transurethral resection or radical cystectomy. TRPV2 mRNA expression in normal human urothelial cells (NHUCs), UC cell lines, and formalin-fixed paraffin-embedded normal (n=6) and cancer bladder tissues (n=58) was evaluated by polymerase chain reaction (PCR) and quantitative real-time PCR (RT-PCR). TRPV2 protein expression was assessed by cytofluorimetric and confocal microscopy analyses in NHUCs and UC cells and by Western blotting and immunohistochemistry in normal and UC tissues. RESULTS: Enhanced TRPV2 mRNA and protein expression was found in high-grade and -stage UC specimens and UC cell lines. Both the full-length TRPV2 (hTRPV2) and a short splice-variant (s-TRPV2) were detected in NHUC and normal bladder specimens, whereas a progressive decline of s-TRPV2 in pTa, pT1, and pT2 stages was observed, up to a complete loss in pT3 and pT4 UC specimens. CONCLUSIONS: Normal human urothelial cells and bladder tissue specimens express TRPV2 at both the mRNA and protein levels. A progressive loss of s-TRPV2 accompanied by a marked increase of hTRPV2 expression was found in high-grade and -stage UC tissues.

Cell cycle-dependent expression of gamma-tubulin in the amicronuclear ciliate Tetrahymena pyriformis.

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.