From micro to mesoscale: Understanding the influence of macroalgal communities on Ostreopsis Schmidt blooms.

Harmful Algal Blooms (HABs) are increasing in temperate areas, and the growth rates of benthic harmful dinoflagellates may be favoured in the context of global climate change. Benthic dinoflagellates, including species belonging to the Ostreopsis Schmidt genus, are known to develop on the surface of macroalgae and different macroalgal morphotypes and communities could host higher or lower cell abundances. The physical structure of the macroalgal substrate at the small scale (cm, microhabitat scale) and the structural complexity of the macroalgal community at the medium scale (few m, mesohabitat scale) could play a relevant role in bloom facilitation: the hypothesis that Ostreopsis species could be associated with macroalgal turfs and shrubs, structurally less complex communities than canopy-forming macroalgae, is especially under discussion and, if confirmed, could link bloom occurrence to regime shifts in temperate ecosystems. The present study, performed in two locations of the Ligurian Sea (Rochambeau, France and Vernazzola, Italy) aimed at understanding marine vegetation's role at the micro and mesohabitat scales in controlling the distribution and abundance of Ostreopsis. The abundance of the microalgal cells was quantified at different spatial scales, from cm to a few m, on different macroalgal species and communities, including artificial substrates, to tease apart the micro and mesohabitat effects. The results obtained show a high spatio-temporal variability, potentially hiding habitat-related patterns. The substrate's preferences diminish when cell abundances are very high, as in the case of Rochambeau, while in presence of moderate cell abundances as in Vernazzola or the first phases of blooms, it is possible to appreciate differences in abundances among substrates (in our study, Dictyota fasciola (Roth) Lamouroux supporting higher abundances). Our results open new research topics such as the study of blooms at a larger scale (macrohabitat) and testing different sampling methods to standardise the cells' abundances independently on the substrate.

Decreased pH impairs sea urchin resistance to predatory fish: A combined laboratory-field study to understand the fate of top-down processes in future oceans.

Changing oceans represent a serious threat for a wide range of marine organisms, with severe cascading effects on ecosystems and their services. Sea urchins are particularly sensitive to decreased pH expected for the end of the century and their key ecological role in regulating community structure and functioning could be seriously compromised. An integrated approach of laboratory and field experiments has been implemented to investigate the effects of decreased pH on predator-prey interaction involving sea urchins and their predators. Our results suggest that under future Ocean Acidification scenarios adult sea urchins defence strategies, such as spine length, test robustness and oral plate thickness, could be compromised together with their survival chance to natural predators. Sea urchins represent the critical linkage between top-down and bottom-up processes along Mediterranean rocky reefs, and the cumulative impacts of global and local stressors could lead to a decline producing cascading effects on benthic ecosystems.

Habitat effects on Ostreopsis cf. ovata bloom dynamics.

In the last few decades, Ostreopsis spp., toxic benthic dinolagellates of tropical origin, generated large interest in the Mediterranean Sea, where several bloom events have been observed. Ecology and proliferation dynamics of O. cf. ovata are driven by complex interactions among biotic and abiotic drivers, and understanding mechanisms triggering bloom events is still far from being complete. The aim of the present study is to highlight the role of different habitat conditions, elucidating the effects of i) exposure to hydrodynamic conditions, ii) macroalgal community and iii) urbanisation level, in driving O. cf. ovata bloom dynamics. A significant effect of hydrodynamics was observed only for cells in seawater, with higher abundances in sheltered zones, irrespective of the urbanisation level. Similarly, a significant effect of the dominant macroalgal community, with higher abundances in Corallinales and turf dominated communities, and lower ones in Cystoseira amentacea canopies, has been recorded, consistently in the differently urbanised sites. Additionally, stretches of the coast suffering from a more intense anthropic exploitation are in general more prone to the proliferation of potentially toxic benthic microalgae. All these results imply a larger risk exposure to toxic effects for humans in urban beaches and sheltered areas, usually more attended by swimmers and bathers. These findings underline the need to preserve, and eventually restore, canopy dominated assemblages, which presently are under regression because of human threats, providing a straightforward example that restoration of relevant habitats implies a cascading improvement of human welfare.

Cancer regulator microRNA: potential relevance in diagnosis, prognosis and treatment of cancer.

MicroRNAs (miRNAs) are small (typically 22 nucleotides) non-coding, endogenous, single-stranded RNAs. MiRNA genes are evolutionarily conserved and are located within the introns or exons of protein-coding genes, as well as in intergenic areas. Before the discovery of miRNAs, it had been known that a large part of the genome is not translated into proteins. This so called "junk" DNA was thought to be evolution debris with no function. Recently, the explosive research in this area has established miRNAs as powerful regulators of gene expression. While only about 1,424 human miRNA sequences have been identified so far, genomic computational analysis indicates that as many as 50,000 miRNAs may exist in the human genome, and each may have multiple targets based on similar sequences in the 3'-UTR of mRNA. MiRNAs have been implicated in different areas such as the immune response, neural development, DNA repair, apoptosis, oxidative stress response and others and it is impressive the list of diseases which have recently been found to be associated with abnormal miRNA expression. Here, we focus our attention on the importance of cancer regulator miRNAs. They are divided into oncomiRs and anti-oncomiRs that negatively regulate tumor suppressor genes and oncogenes, respectively. Importantly, the association of miRNAs with cancer has prompted additional functional classification of these short RNAs and their potential relevance in cancer diagnosis, prognosis and treatment.

Toxicity of metal oxide nanoparticles in immune cells of the sea urchin.

The potential toxicity of stannum dioxide (SnO2), cerium dioxide (CeO2) and iron oxide (Fe3O4) nanoparticles (NPs) in the marine environment was investigated using the sea urchin, Paracentrotus lividus, as an in vivo model. We found that 5 days after force-feeding of NPs in aqueous solutions, the three NPs presented different toxicity degrees, depending on the considered biomarkers. We examined: 1) the presence of the NPs in the coelomic fluid and the uptake into the immune cells (coelomocytes); 2) the cholinesterase activity and the expression of the stress-related proteins HSC70 and GRP78; 3) the morphological changes affecting cellular compartments, such as the endoplasmic reticulum (ER) and lysosomes. By Environmental Scanning Electron Microscope (ESEM) analysis, coupled with Energy Dispersive X-ray Spectroscopy (EDS) we found that NPs were uptaken inside coelomocytes. The cholinesterases activity, a well known marker of blood intoxication in vertebrates, was greatly reduced in specimens exposed to NPs. We found that levels of stress proteins were down-regulated, matching the observed ER and lysosomes morphological alterations. In conclusion, this is the first study which utilizes the sea urchin as a model organism for biomonitoring the biological impact of NPs and supports the efficacy of the selected biomarkers.

Senescence and cell death pathways and their role in cancer therapeutic outcome.

Anticancer drug-induced tumor suppression may involve mechanisms of protection against neoplastic transformation that are normally latent in mammalian cells and consist in a genetic program implemented during anti-tumoral defense. This defense program results in the self elimination of cells harboring potentially dangerous mutations by triggering cell death through apoptosis and/or autophagy or in the execution of a program that leads to a permanent growth arrest known as senescence. These responses are considered crucial tumor suppressive mechanisms and their study appears to be essential to develop therapeutical procedures based on the enhancement of the different responses. This review summarizes fundamental knowledge on the underlying mechanisms able to limit excessive or aberrant cellular proliferation and on the prognostic value of both apoptosis and senescence detection. In addition, interesting evidence showing that different drugs induce senescence or cell death depending on the genetic features of the tumor cells as well as on the integrity of the relative pathways is reported.

The toxic benthic dinoflagellate Ostreopsis ovata: quantification of proliferation along the coastline of Genoa, Italy.

Toxic harmful algal bloom (HAB) occurrence is becoming more frequent and problematic in highly urbanized coastal zones. In summer 2005 along the urbanized Genoa coastline (Ligurian Sea, North western Mediterranean Sea), local first aid stations treated about 200 people, who all showed similar symptoms following exposure to marine aerosols. The link with proliferation of Ostreopsis ovata was made, and it highlighted for the first time, the risks that benthic HABs may represent in highly urbanised temperate areas. Subsequently, a specific monitoring plan was designed and implemented in the same area in July 2006, before the first signs of Ostreopsis proliferation were detected. Here we report on this quantification of an Ostreopsis ovata bloom in the Ligurian Sea. Cells were quantified both in the water column and in the epiphytic community on macrophytes. Our results suggest a role of sea water temperature and weather conditions in favouring bloom development.

Perspectives in biomolecular therapeutic intervention in cancer: from the early to the new strategies with type I interferons.

Interferon (IFN) was the first cytokine produced by recombinant DNA technology used in wide-spread clinical treatment of infectious diseases as well as malignancies. The IFN clinical potential was clearly realized from the outset. However, IFN represents one of the most controversial drugs of our time, as remarkable cycles of promise and disappointment have affected its development and use. Considerable evidence regarding anti-tumor activities of IFNs has been reported. In this paper we focus on molecular bases of the IFN system that may relate to its antitumor activities. Many of the numerous genes transcriptionally activated by IFNs have been shown to encode proteins that activate immune recognition of tumor cells, directly or indirectly exert tumor suppressor activity and/or control tumor cell cycle and programmed cell death. In addition, a physiological relevant function for endogenous type I IFN in cancer immunoediting process and a new way to IFN clinical use based on gene therapy or vaccine-like approaches have recently been suggested. The identification of selected tissue-specific and/or tumor-specific target pathways as well as of different type I IFN tumor escape and resistance mechanisms may provide novel approaches in the search for new IFN-based therapeutic strategies to circumvent cancer disease or improve clinical outcome. Promising IFN treatment has been recently defined by using novel pharmaceutical preparations with a more favourable pharmacokinetic response, also in combination with other bioreagents or other modalities of therapy. Translational research, linking both basic and clinical research, will lead to a new rationale for the use of IFN in cancer therapy.

Cholinesterase activities in the Antarctic scallop Adamussium colbecki: tissue expression and effect of ZnCl2 exposure.

Biochemical characterization of cholinesterase activity (ChE) was carried out on the Antarctic scallop Adamussium colbecki collected in winter 2000 from Campo Icaro (Ross Sea, Antarctica) in order to increase its suitability as a sentinel organism for monitoring the Antarctic environment. The digestive gland, gills and adductor muscle were investigated for substrate specificity and inhibitors sensitivity using acetylthiocholine iodide (ASCh) and butyrylthiocholine iodide (BSCh) as substrates and tetra (monoisopropyl)pyrophosphor-tetramide (Iso-OMPA), 1,5-bis(4-allyldimethylammoniumphenyl)-penthan-3-one dibromide (BW284c51) and the insecticide chlorpyrifos as inhibitors. Effect of in vivo exposure to ZnCl(2) was also investigated. All the tissues expressed ChE activity (gill > adductor muscle > digestive gland) and low substrates specificity throughout the hydrolysis of both ASCh and BSCh substrates. Partial (25-29%) and total inhibition (100%) of ChE activity in gills was demonstrated following in vitro incubation with Iso-OMPA and BW284c51 (3 mM), respectively. Concentration-dependent inhibition was also evident with chlorpyrifos in the range 10(-4)-10(-10) M (IC(50) 10(-6)) while in vivo exposure to ZnCl(2) did not seem to affect ChE activity in the scallop. The potential use of ChE in the A. colbecki as biomarker for monitoring water contamination in the marine Antarctic environment is discussed.

Seasonal variations of susceptibility to oxidative stress in Adamussium colbecki, a key bioindicator species for the Antarctic marine environment.

The area of free radical biology is of increasing interest for marine organisms since the enhanced formation of reactive oxygen species (ROS) is a common pathway of toxicity induced by stressful environmental conditions. In polar environments responses of the antioxidant system could be useful as an early detection biomarkers of unforeseen effects of human activities which are progressively increasing in these remote areas. However, the characterization of antioxidant defences in appropriate sentinel species is of particular value also in terms of a possible adaptation to this extreme environment. The scallop, Adamussium colbecki, is a key species for monitoring the Antarctic environment and, besides single antioxidants, the total oxyradical scavenging capacity (TOSC) assay has been recently used for quantifying the overall ability of this organism to neutralize peroxyl radicals (ROO*), hydroxyl radicals (*OH) and peroxynitrite (HOONO). The aim of this work was to obtain a better characterization of these biological responses which can indicate the occurrence of biological disturbance; in this study the total oxyradical scavenging capacity was further analyzed to assess the presence of seasonal fluctuations in the susceptibility to oxidative stress in this species. The capability to neutralize peroxyl radicals and hydroxyl radicals increased at the end of December, while resistance towards peroxynitrite did not show any significant variations during the Antarctic summer. These results suggest the occurrence of metabolic changes which mainly influence intracellular formation of ROO* and *OH, with more limited effects on HOONO. Despite the limited time window analyzed, as a typical constraint in Antarctic research at Terra Nova Bay, an increased resistance to these specific oxyradicals might be related to the period of highest feeding activity, or to other intrinsic factors in the animals' physiology such as the phase of reproductive cycle.

Interferon-beta induces S phase slowing via up-regulated expression of PML in squamous carcinoma cells.

Type I Interferon (IFN) and all-trans retinoic acid (RA) inhibit cell proliferation of squamous carcinoma cell lines (SCC). Examinations of growth-affected cell populations show that SCC lines ME-180 and SiHa treated with IFN-beta undergo a specific slower progression through the S phase that seems to trigger cellular death. In combination treatment RA potentiates IFN-beta effect in SCC ME-180 but not in SiHa cell line, partially resistant to RA antiproliferative action. RA added as single agent affects cell proliferation differently by inducing a slight G1 accumulation. The IFN-beta-induced S phase lengthening parallels the increased expression of PML, a nuclear phosphoprotein specifically up-regulated at transcriptional level by IFN, whose overexpression induces cell growth inhibition and tumor suppression. We report that PML up-regulation may account for the alteration of cell cycle progression induced by IFN-beta in SCC by infecting cells with PML-PINCO recombinant retrovirus carrying the PML-3 cDNA under the control of the 5' LTR. In fact PML overexpression reproduces the IFN-beta-induced S phase lengthening. These findings provide important insight into the mechanism of tumor suppressing function of PML and could allow PML to be included in the pathways responsible for IFN-induced cell growth suppression.

Differences in uptake and metabolism of retinoic acid between estrogen receptor-positive and -negative human breast cancer cells.

PURPOSE: Our previous work had shown that retinoic acid (RA) inhibits cell growth and induces apoptosis in estrogen receptor-positive (ER-positive) MCF-7 and T-47D human breast carcinoma cells, but not in ER-negative human breast carcinoma cells MB-231 and MB-453. The purpose of this work was to determine whether these differences might be due to differences in uptake and metabolism of the drug between ER-positive and ER-negative cells. METHODS: We measured RA uptake in cultured human breast cancer cells and determined its metabolism by high-pressure liquid chromatographic analysis. RESULTS: The two ER-positive cell lines reached maximum RA uptake at about 2 h, followed by a sharp decline, so that most RA had disappeared from the cells and from the medium by 24 h and was found as oxidation products in the culture medium. In contrast, the two ER-negative cell lines showed a pattern of lower accumulation without the sharp increase and subsequent steep decline, so that by 24 h there was more RA in these cells and their culture medium than in the RA-responsive ER-positive cells, even though at 2 h the ER-negative cells had taken up less RA than the ER-positive cells. Kinetic analysis of the uptake of RA in MCF-7 cells was consistent with rapid movement across the cell membranes and the actual rate determined by diffusion of albumin-bound retinoid to the cells. CONCLUSIONS: This study is the first to demonstrate profound differences in RA accumulation and confirms previous results on different rates of RA metabolism between ER-positive and ER-negative human breast cancer cells. The findings reported here, therefore, may introduce additional elements to be considered in the design of new drugs for cancer chemoprevention and therapy.

Retinoic acid is able to induce interferon regulatory factor 1 in squamous carcinoma cells via a STAT-1 independent signalling pathway.

Interferon regulatory factor 1 (IRF-1) transcription factor binds to DNA sequence elements found in the promoters of type I IFN and IFN-inducible genes. Transient up-regulation of the IRF-1 gene by virus and IFN treatment causes the consequent induction of many IFN-inducible genes involved in cell growth control and apoptosis. We reported recently that IFN-alpha and all-trans retinoic Acid (RA) inhibit the cell proliferation of squamous carcinoma cell line ME-180 by inducing apoptotic cell death. IRF-1 expression correlates with the IFN-alpha-induced apoptosis phenomenon and, surprisingly, with the RA-induced apoptosis phenomenon. To study how these two different ligands cross-talk in the regulation of cellular antitumor responses, the signalling pathways involved in IRF-1 induction were analyzed in RA and/or IFN-alpha-treated ME-180 cells. We provide evidence indicating that RA-induced IRF-1 gene expression is independent of the STAT-1 activation pathway, despite the presence of the IFN-gamma activated sequence element in the gene promoter, but involves nuclear factor-kappaB activation. Thus, here we first describe the activation of nuclear factor-kappaB by both IFN-alpha and RA in the ME-180 cell line. The induced IRF-1 protein is successively able to bind the IFN-stimulated responsive element in the promoter of the target gene 2',5'-oligoadenylate synthetase.

Carcinoma cell lines resistant for growth inhibition and apoptosis to retinoic acid are responsive to 4-hydroxy-phenyl-retinamide: correlation with tissue transglutaminase.

Retinoic acid (RA)-resistant cell lines are highly malignant. To inhibit the growth of the RA-resistant cells we used 4-HPR, a synthetic retinoid, which may act through alternative signal transduction pathways. 4-HPR induced cell growth inhibition and apoptosis in all RA-sensitive as well as -resistant cells, demonstrating a wider spectrum of potency over RA. 4-HPR induced tissue TGase activity. A tight correlation between the induction of tissue TGase, the inhibition of cell growth, and apoptosis was evident in all eight RA-sensitive cell lines. However, basal TGase differed in the different cells, suggesting inducibility rather than basal levels as the relevant parameter. In sharp contrast to the RA-sensitive cells, RA-resistant cells showed sporadic response to 4-HPR for tissue TGase. The wider spectrum of activity of 4-HPR in inhibiting cell growth and inducing apoptosis makes it a good candidate for the treatment of RA-resistant cancer cells.

Expression of a dominant-negative retinoic acid receptor construct reduces retinoic acid metabolism and retinoic acid-induced inhibition of NIH-3T3 cell growth.

We have previously reported an unexpected relationship between retinoic acid-induced inhibition of cell growth and the ability of various cell lines to metabolize the retinoid. Here, we report that stable expression of the truncated retinoic acid receptor RAR alpha403, transduced in NIH-3T3 cells by a retroviral vector, rendered the cells resistant to retinoic acid for growth inhibition and reduced their ability to metabolize the retinoid at the same time as it blunted the induction of the target gene transglutaminase II. The data suggest that retinoic acid receptors mediate the growth-inhibitory action of retinoic acid as well as its metabolism and the induction of transglutaminase II.

Aryl hydrocarbon receptor knockout mice (AHR-/-) exhibit liver retinoid accumulation and reduced retinoic acid metabolism.

Livers from aryl hydrocarbon receptor-null mice showed a 3-fold increase in retinoids and a 65% decrease in retinoic acid metabolism. Levels of expression of the retinoic acid 4-hydroxylase, P450RAI, did not change, whereas cytochrome P4501A2 levels were lower in the null mouse, as shown earlier; however, this enzyme was found not to be active toward retinoic acid. These data suggest that aryl hydrocarbon receptor controls retinoic acid catabolism, through modulation of an unidentified target gene. Aldehyde dehydrogenases 1 and 2 were down-regulated markedly in the aryl hydrocarbon receptor-deficient mouse liver. 2,3,7,8-Tetrachlorodibenzo-p-dioxin induced cytochrome P4501A2 but not the aldehyde dehydrogenases in wild-type mice, suggesting that aryl hydrocarbon receptor is not involved directly in the down-regulation of this gene. Transglutaminase II, a retinoic acid-responsive gene product, was increased 2-fold, consistent with the liver fibrosis phenotype observed in the null mice. These findings suggest a molecular connection between xenobiotic-activated receptor signaling and retinoid homeostasis.

TPA induces transglutaminase C and inhibits cell growth in the colon carcinoma cell line SW620.

In contrast to most other systems, TPA induced TGc activity and protein in SW620 human colon carcinoma cells. This induction was accompanied by cell growth inhibition and increased apoptosis. The general protein kinase-C inhibitor GF-109203X blocked the induction of TGc by TPA, whereas the specific inhibitor of the PKC alpha isoform, the indocarbazole Go6976, reduced it by 40%. These PKC inhibitors had similar inhibitory effects on TPA increased apoptosis and inhibition of cell growth, suggesting that the observed actions of TPA are mediated by PKC, and a close connection between TGc activity, increased apoptosis and cell growth inhibition. We conclude that TPA may offer new approaches in the management of colon cancer cell growth.

Non-specific pinocytosis by human endothelial cells cultured as multicellular aggregates: uptake of lucifer yellow and horse radish peroxidase.

We have analyzed the pattern of time-dependent and concentration-dependent incorporation of Lucifer Yellow CH (LY) and Horseradish Peroxidase (HRP) by human umbilical vein endothelial cells cultured on a non-adhesive substratum, where they they become organized into stable, multicellular aggregates. The data were compared with those previously obtained from low-density cultures of non-growing endothelial cells adherent to plastic. While the linear trend of the incorporation kinetics is preserved, the rate of uptake with both time and concentrations is highly dependent on the culture conditions, namely typology of cell-cell and cell-substrate interactions. An at least two-fold increase of the rate of uptake was observed with both markers in the aggregated cells. The extracellular concentration of LY required to saturate the binding capacity of the cell surface shifts from approximately 0.25 mg/ml, with the adherent cells, to approximately 0.5 mg/ml in the aggregated cells; the rate of uptake of three different forms of HRP shows, besides a sharp quantitative increase, also qualitative variations, testified by differential changes of their incorporation rates. These results are entirely consistent with the assumption that the association of the endothelial cells into multicellular aggregates increases the rate of pinocytic uptake by modifying the physicochemical properties of the cell surface, thereby increasing its differential affinity for the extracellular markers.