Methylmercury Effect and Distribution in Two Extremophile Microalgae Strains Dunaliella salina and Coccomyxa onubensis from Andalusia (Spain).

The main entrance point of highly toxic organic Hg forms, including methylmercury (MeHg), into the aquatic food web is phytoplankton, which is greatly represented by various natural microalgal species. Processes associated with MeHg fate in microalgae cells such as uptake, effects on cells and toxicity, Hg biotransformation, and intracellular stability are detrimental to the process of further biomagnification and, as a consequence, have great importance for human health. The study of MeHg uptake and distribution in cultures of marine halophile Dunaliella salina and freshwater acidophilic alga Coccomyxa onubensis demonstrated that most of the MeHg is imported inside the cell, while cell surface adhesion is insignificant. Almost all MeHg is removed from the culture medium after 72 h. Significant processes in rapid MeHg removal from liquid medium are its abiotic photodegradation and volatilization associated with algal enzymatic activity. The maximum intracellular accumulation for both species was in 80 nM MeHg-exposed cultures after 24 h of exposure for D. salina (from 27 to 34 µg/gDW) and at 48 h for C. onubensis (up to 138 µg/gDW). The different Hg intakes in these two strains could be explained by the lack of a rigid cell wall in D. salina and the higher chemical ability of MeHg to pass through complex cell wall structures in C. onubensis. Electron microscopy studies on the ultrastructure of both strains demonstrated obvious microvacuolization in the form of many very small vacuoles and partial cell membrane disruption in 80 nM MeHg-exposed cultures. Results further showed that Coccomyxa onubensis is a good candidate for MeHg-contaminated water reclamation due to its great robustness at nanomolar concentrations of MeHg coupled with its very high intake and almost complete Hg removal from liquid medium at the MeHg levels tested.

Induction of Oxidative Stress by Waterborne Copper and Arsenic in Larvae of European Seabass (Dicentrarchus labrax L.): A Comparison with Their Effects as Nanoparticles.

The aim of this work was to compare the potential induction of oxidative stress and the antioxidant enzymatic response after a short-term waterborne exposure to copper (Cu) and arsenic (As) with that of the nanoparticles (NPs) of these elements (Cu-NPs and As-NPs) in fish larvae of the species Dicentrarchus labrax. Larvae were grouped in several tanks and exposed to different concentrations of contaminants (0 to 10 mg/L) for 24 or 96 h under laboratory conditions. Copper and arsenic concentrations were analysed in larval tissues using ICP-MS. A set of oxidative stress biomarkers, including the levels of hydroperoxides (HPs), and superoxide dismutase (SOD) and catalase (CAT) activities were assessed. The trace element concentrations (mg/kg d.w.) in larvae ranged as follows: 3.28-6.67 (Cu at 24 h) and 2.76-3.42 (Cu at 96 h); 3.03-8.31 (Cu-NPs at 24 h) and 2.50-4.86 (Cu-NPs at 96 h); 1.92-3.45 (As at 24 h) and 2.22-4.71 (As at 96 h); and 2.19-8.56 (As-NPs at 24 h) and 1.75-9.90 (As-NPs at 96 h). In Cu tests, the oxidative damage (ROOH levels) was induced from 0.1 mg/L at both exposure times, while for Cu-NPs, this damage was not observed until 1 mg/L, which was paralleled by concomitant increases in SOD activity. The CAT activity was also increased but at lower metal concentrations (0.01 mg/L and 0.1 mg/L for both chemical forms). No oxidative damage was observed for As or As-NPs after 24 h, but it was observed for As after 96 h of treatment with 0.01 mg/L. A decrease in SOD activity was observed for As after 24 h, but it turned out to be increased after 96 h. However, As-NPs did not alter SOD activity. The CAT activity was stimulated only at 96 h by As and at 24 h by As-NPs. Therefore, the two chemical forms of Cu exhibited a higher bioaccumulation and toxicity potential as compared to those of As. Importantly, the association of both Cu and As in NPs reduced the respective trace metal bioaccumulation, resulting also in a reduction in the toxic effects (mortality and biochemical). Furthermore, the assessment of oxidative stress-related biomarkers in seabass larvae appears to be a useful tool for biomonitoring environmental-occurring trace elements.

Suppression of metastatic organ colonization and antiangiogenic activity of the orally bioavailable lipid raft-targeted alkylphospholipid edelfosine.

Metastasis is the leading cause of cancer mortality. Metastatic cancer is notoriously difficult to treat, and it accounts for the majority of cancer-related deaths. The ether lipid edelfosine is the prototype of a family of synthetic antitumor compounds collectively known as alkylphospholipid analogs, and its antitumor activity involves lipid raft reorganization. In this study, we examined the effect of edelfosine on metastatic colonization and angiogenesis. Using non-invasive bioluminescence imaging and histological examination, we found that oral administration of edelfosine in nude mice significantly inhibited the lung and brain colonization of luciferase-expressing 435-Lung-eGFP-CMV/Luc metastatic cells, resulting in prolonged survival. In metastatic 435-Lung and MDA-MB-231 breast cancer cells, we found that edelfosine also inhibited cell adhesion to collagen-I and laminin-I substrates, cell migration in chemotaxis and wound-healing assays, as well as cancer cell invasion. In 435-Lung and other MDA-MB-435-derived sublines with different organotropism, edelfosine induced G2/M cell cycle accumulation and apoptosis in a concentration- and time-dependent manner. Edelfosine also inhibited in vitro angiogenesis in human and mouse endothelial cell tube formation assays. The antimetastatic properties were specific to cancer cells, as edelfosine had no effects on viability in non-cancerous cells. Edelfosine accumulated in membrane rafts and endoplasmic reticulum of cancer cells, and membrane raft-located CD44 was downregulated upon drug treatment. Taken together, this study highlights the potential of edelfosine as an attractive drug to prevent metastatic growth and organ colonization in cancer therapy. The raft-targeted drug edelfosine displays a potent activity against metastatic organ colonization and angiogenesis, two major hallmarks of tumor malignancy.

Fe (III)-Mediated Antioxidant Response of the Acidotolerant Microalga Coccomyxa onubensis.

Coccomyxa onubensis (C. onubensis) is an acidotolerant microalga isolated from Tinto River (Huelva), which contains high levels of metal cations in solution, mainly Fe (II) and (III), and Cu (II). Fe is more bioavailable at low pH, mainly because Fe (II) and Fe (III) are far more soluble, especially Fe (III). For this reason, this study aims to evaluate both physiological and biochemical responses of C. onubensis when subjected to Fe (III)-induced stress. Changes in growth, photosynthetic viability and antioxidant responses to the induced oxidative stress were determined. The results obtained suggest that the addition of moderate Fe (III) levels to C. onubensis cultures results in improved growth and photosynthetic viability. Increases in the intracellular levels of the enzyme superoxide dismutase (SOD) and flavonoids, used as antioxidant response biomarkers, a point at Fe (III)-mediated oxidative stress induction. The apparent decrease in the content of other phenolic molecules and polyunsaturated fatty acids might be understood as a sign of antioxidant molecules' involvement in reactive oxygen species (ROS) scavenging. In conclusion, a noticeable antioxidant capacity displayed by C. onubensis allows the use of moderate Fe (III) levels to trigger the accumulation of valuable antioxidant molecules, allowing the production of cell extracts with potential anti-inflammatory activity.

CD89 Is a Potent Innate Receptor for Bacteria and Mediates Host Protection from Sepsis.

Direct bacterial recognition by innate receptors is crucial for bacterial clearance. Here, we show that the IgA receptor CD89 is a major innate receptor that directly binds bacteria independently of its cognate ligands IgA and c-reactive protein (CRP). This binding is only partially inhibited by serum IgA and induces bacterial phagocytosis by CD11c+ dendritic cells and monocytes and/or macrophages, suggesting a physiological role in innate host defense. Blood phagocytes from common variable immunodeficiency patients bind, internalize, and kill bacteria in a CD89-dependent manner, confirming the IgA independence of this mechanism. In vivo, CD89 transgenic mice are protected in two different models of sepsis: a model of pneumonia and the cecal ligation and puncture (CLP) polymicrobial model of infection. These data identify CD89 as a first-line innate receptor for bacterial clearance before adaptive responses can be mounted. Fc receptors may emerge as a class of innate receptors for various bacteria with pleiotropic roles.

Early genotoxic response and accumulation induced by waterborne copper, lead, and arsenic in European seabass, Dicentrarchus labrax.

Cu, Pb, and As, which are among the most abundant metals in the aquatic environment, are also among the most health-threatened by causing diverse cellular injuries. The aim of this study was to assess and compare the potential early induction of genotoxic effects after waterborne Cu, Pb, and As exposure in European seabass, Dicentrarchus labrax, a commercial widely cultured fish, using the micronucleus (MN) assay in peripheral blood erythrocytes. Fish were exposed under laboratory conditions to nominal solutions ranging 0-10 mg/L for 24 and 96 h. Furthermore, actual metal ion concentrations were measured by inductively coupled plasma atomic emission spectroscopy (ICP-AES) or differential pulse anodic stripping voltammetry (DPASV) in water and four fish tissues differentially related to environmental exposition and metal accumulation, i.e. the gills, liver, muscle, and brain. Dose-dependent increases of micronuclei (MNi) frequency were observed after these very short exposures; based on measured metal concentrations in water, the genotoxic effect ordered as Cu > As > Pb. Significant genotoxic effect at 0.009 mg/L Cu, 0.57 mg/L Pb, and 0.01 mg/L As was seen. For Cu and Pb these are only slightly higher, but for As it is notably lower than the USEPA criteria of maximum concentration to prevent acute toxicity in aquatic organisms. Furthermore, genotoxicity was differentially related to metal accumulation. MNi frequency correlated positively with the content of Pb in all the organs, with the content of As in liver and gills and only with the content of Cu in the brain. In conclusion, our findings raised environmental concerns because these depicted a genotoxic potential of Cu, Pb, and As after a very short exposure to low but environmentally relevant concentrations, too close to regulatory thresholds. In addition, the MN test in D. labrax could be considered an early biomarker of genotoxicity induced by these metals in fish.

The Expression of the Ubiquitin Ligase SIAH2 (Seven In Absentia Homolog 2) Is Increased in Human Lung Cancer.

OBJECTIVES: Lung cancer is the leading cause of cancer-related deaths worldwide. Overall 5-year survival has shown little improvement over the last decades. Seven in absentia homolog (SIAH) proteins are E3 ubiquitin ligases that mediate proteasomal protein degradation by poly-ubiquitination. Even though SIAH proteins play a key role in several biological processes, their role in human cancer remains controversial. The aim of the study was to document SIAH2 expression pattern at different levels (mRNA, protein level and immunohistochemistry) in human non-small cell lung cancer (NSCLC) samples compared to surrounding healthy tissue from the same patient, and to analyse the association with clinicopathological features. MATERIALS AND METHODS: One hundred and fifty-two samples from a patient cohort treated surgically for primary lung cancer were obtained for the study. Genic and protein expression levels of SIAH2 were analysed and compared with clinic-pathologic variables. RESULTS: The present study is the first to analyze the SIAH2 expression pattern at different levels (RNA, protein expression and immunohistochemistry) in non-small cell lung cancer (NSCLC). We found that SIAH2 protein expression is significantly enhanced in human lung adenocarcinoma (ADC) and squamous cell lung cancer (SCC). Paradoxically, non-significant changes at RNA level were found, suggesting a post-traductional regulatory mechanism. More importantly, an increased correlation between SIAH2 expression and tumor grade was detected, suggesting that this protein could be used as a prognostic biomarker to predict lung cancer progression. Likewise, SIAH2 protein expression showed a strong positive correlation with fluorodeoxyglucose (2-deoxy-2(18F)fluoro-D-glucose) uptake in primary NSCLC, which may assist clinicians in stratifying patients at increased overall risk of poor survival. Additionally, we described an inverse correlation between the expression of SIAH2 and the levels of one of its substrates, the serine/threonine kinase DYRK2. CONCLUSIONS: Our results provide insight into the potential use of SIAH2 as a novel target for lung cancer treatment.

AM404 inhibits NFAT and NF-κB signaling pathways and impairs migration and invasiveness of neuroblastoma cells.

N-Arachidonoylphenolamine (AM404), a paracetamol lipid metabolite, is a modulator of the endocannabinoid system endowed with pleiotropic activities. AM404 is a dual agonist of the Transient Receptor Potential Vanilloid type 1 (TRPV1) and the Cannabinoid Receptor type 1 (CB1) and inhibits anandamide (AEA) transport and degradation. In addition, it has been shown that AM404 also exerts biological activities through TRPV1- and CB1 -independent pathways. In the present study we have investigated the effect of AM404 in the NFAT and NF-κB signaling pathways in SK-N-SH neuroblastoma cells. AM404 inhibited NFAT transcriptional activity through a CB1- and TRPV1-independent mechanism. Moreover, AM404 inhibited both the expression of COX-2 at transcriptional and post-transcriptional levels and the synthesis of PGE2. AM404 also inhibited NF-κB activation induced by PMA/Ionomycin in SK-N-SH cells by targeting IKKß phosphorylation and activation. We found that Cot/Tlp-2 induced NFAT and COX-2 transcriptional activities were inhibited by AM404. NFAT inhibition paralleled with the ability of AM404 to inhibit MMP-1, -3 and -7 expression, cell migration and invasion in a cell-type specific dependent manner. Taken together, these data reveal that paracetamol, the precursor of AM404, can be explored not only as an antipyretic and painkiller drug but also as a co-adjuvant therapy in inflammatory and cancer diseases.

Hypoximimetic activity of N-acyl-dopamines. N-arachidonoyl-dopamine stabilizes HIF-1α protein through a SIAH2-dependent pathway.

The N-acyl conjugates of amino acids and neurotransmitters (NAANs) are a class of endogenous lipid messengers that are expressed in the mammalian central and peripheral nervous system. Hypoxia inducible factor-1α (HIF-1α) is a transcription factor that plays a key role in the cellular adaptation to hypoxia and ischemia, and hypoxic preconditioning through HIF-1α has been shown to be neuroprotective in ischemic models. This study showed that N-acyl-dopamines induce HIF-1α stabilization on human primary astrocytes and neurons as well as in transformed cell lines. N-arachidonoyl-dopamine (NADA)-induced HIF-1α stabilization depends on the dopamine moiety of the molecule and is independent of cannabinoid receptor-1 (CB1) and transient receptor potential vanilloid type I (TRPV1) activation. NADA increases the activity of the E3 ubiquitin ligase seven in absentia homolog-2 (SIAH2), inhibits prolyl-hydroxylase-3 (PHD3) and stabilizes HIF-1α. NADA enhances angiogenesis in endothelial vascular cells and promotes the expression of genes such as erythropoietin (EPO), vascular endothelial growth factor A (VEGFA), heme oxygenase 1 (HMOX-1), hexokinase 2 (HK2) and Bcl-2/E1B-nineteen kiloDalton interacting protein (BNIP3) in primary astrocytes. These findings indicate a link between N-acyl-dopamines and hypoxic preconditioning and suggest that modulation of the N-acyl-dopamine metabolism might prove useful for prevention against hypoxic diseases.

Effect of selenate on viability and selenomethionine accumulation of Chlorella sorokiniana grown in batch culture.

The aim of this work was to study the effect of Se(+VI) on viability, cell morphology, and selenomethionine accumulation of the green alga Chlorella sorokiniana grown in batch cultures. Culture exposed to sublethal Se concentrations of 40 mg · L(-1) (212 µM) decreased growth rates for about 25% compared to control. A selenate EC50 value of 45 mg · L(-1) (238.2 µM) was determined. Results showed that chlorophyll and carotenoids contents were not affected by Se exposure, while oxygen evolution decreased by half. Ultrastructural studies revealed granular stroma, fingerprint-like appearance of thylakoids which did not compromise cell activity. Unlike control cultures, SDS PAGE electrophoresis of crude extracts from selenate-exposed cell cultures revealed appearance of a protein band identified as 53 kDa Rubisco large subunit of Chlorella sorokiniana, suggesting that selenate affects expression of the corresponding chloroplast gene as this subunit is encoded in the chloroplast DNA. Results revealed that the microalga was able to accumulate up to 140 mg · kg(-1) of SeMet in 120 h of cultivation. This paper shows that Chlorella sorokiniana biomass can be enriched in the high value aminoacid SeMet in batch cultures, while keeping photochemical viability and carbon dioxide fixation activity intact, if exposed to suitable sublethal concentrations of Se.

The fungal metabolite galiellalactone interferes with the nuclear import of NF-κB and inhibits HIV-1 replication.

Galiellalactone (GL) is a metabolite produced by the fungus Galiella rufa that presents antitumor and immunomodulatory activities. GL interferes with the binding to DNA of signal transducer and activator of transcription (STAT)-3 and also inhibits other signal pathways such as NF-κB, but the mechanism of action in this pathway remains unknown. In this study we report that GL inhibits vesicular stomatitis virus-recombinant HIV-1 infection and the NF-κB-dependent transcriptional activity of the HIV-LTR promoter. We found that GL prevents the binding of NF-κB to DNA but neither affects the phosphorylation and degradation of NF-κB inhibitory protein, IκBα, nor the phosphorylation and acetylation of the NF-κB p65 subunit. However, GL prevents the association of p65 with the importin α3 impairing the nuclear translocation of this transcription factor. Using a biotinylated probe we found that GL binds to p65 but not to importin α3. Therefore, GL is a dual NF-κB/STAT3 inhibitor that could serve as a lead compound for the development of novel drugs against HIV-1, cancer and inflammatory diseases.

IDENTIFICATION AND PHYSIOLOGICAL ASPECTS OF A NOVEL CAROTENOID-ENRICHED, METAL-RESISTANT MICROALGA ISOLATED FROM AN ACIDIC RIVER IN HUELVA (SPAIN)(1).

A heavy-metal-resistant, carotenoid-enriched novel unicellular microalga was isolated from an acidic river in Huelva, Spain. The isolated ribosomal 18S subunit rDNA sequence showed homology with known sequences from green microalgae, the closest sequence (98% homology) belonging to the genus Coccomyxa. The isolated microalga therefore was an up to now uncultured microalga. The microalga was isolated from Tinto River area (Huelva, Spain), an acidic river that exhibits very low pH (1.7-3.1) with high concentrations of sulfuric acid and heavy metals, including Fe, Cu, Mn, Ni, and Al. Electron micrographs show that the microalga contains a large chloroplast with a presence of lipid droplets, an increased number of starch bodies as well as electron-dense deposits and plastoglobules, the last observed only in iron-exposed cells. Unlike other acidophile microalgae, the isolated microalga showed high growth rates when cultivated photoautotrophycally (up to 0.6 d(-1) ) in a suitable culture medium prepared at our laboratory. The growth was shown to be iron dependent. When the microalga is grown in fluidized bed reactors, the high growth rates resulted in unexpectedly high productivities for being a microalga that naturally grows in acidic environments (0.32 g·L(-1) ·d(-1) ). The microalga also grows optimally on reduced carbon sources, including glucose and urea, and at an optimal temperature of 35°C. The alga pigment profile is particularly rich in carotenoids, especially lutein, suggesting that the microalga might have potential for antioxidant production, namely, xanthophylls.

Acute toxicity of manganese in goldfish Carassius auratus is associated with oxidative stress and organ specific antioxidant responses.

Manganese is a relatively common, yet poorly studied element in freshwater ecosystems, where it can be significantly bioconcentrated. The knowledge about the mechanisms of Mn toxicity on fish health is still limited. The aim of the present study was to assess the potential induction of oxidative stress and the antioxidant response after a 96 h waterborne Mn-exposure (at 0.1 and 1mM) in gill, kidney, liver and brain of goldfish (Carassius auratus). Mn 1mM induced an increase of lipid hydroperoxides, superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities in all tissues with the exception of SOD inhibition in the brain. Particular response of catalase (CAT) was indicated-its inhibition in the liver and kidney, but activation in the gill. Exposure to Mn 0.1mM provoked most prominent changes in the liver and did not change the indexes in brain. These results strongly suggest that Mn exposure caused a generalized oxidative stress in the fish and revealed an organ specific antioxidant response involving a differential modulation of the SOD, CAT and GPx activities.

Q fever: epidemiology, clinical features and prognosis. A study from 1983 to 1999 in the South of Spain.

OBJECTIVES: Clinical polymorphism is a main feature of Q fever and, depending upon the geographic location, differences in its clinical picture have been described. The objective of this study was to determine the epidemiology, clinical features and prognosis of acute Q fever in our area. METHODS: From 1985 to 1999, consecutive cases of Q fever, presented as febrile syndrome and attended in a tertiary teaching hospital in Sevilla, Spain, were included and followed prospectively. RESULTS: Two hundred and thirty-one cases of acute Q fever were included. A non-focalized febrile syndrome lasting from 7 to 28 days (fever of intermediate duration) was the most frequent presentation (n=208, 90%). One hundred and forty-eight patients had hepatitis. Overall, 53% of the cases were urban and contact with animals was referred in 39% of the patients. No relationship between clinical presentation and possible route of infection was observed. Prognosis was excellent (100% cured), although in 18 patients fever was prolonged more than 28 days and three patients developed life-threatening organ affection. Antimicrobial treatment was more effective if it was administered in the first two weeks (median defervescence of fever: 3 days versus 5.5 days, p<0.01). CONCLUSIONS: Acute Q fever is a common cause of fever of intermediate duration, even in urban areas. Elevation of hepatic enzymes was the most frequent laboratory finding. Severe organ affection is uncommon and the overall prognosis of the disease is excellent. Early treatment seems to shorten the duration of the disease.

Somatostatin stimulates GH secretion in two porcine somatotrope subpopulations through a cAMP-dependent pathway.

Somatostatin (SRIF) inhibits GH release from rat somatotropes by reducing adenylate cyclase (AC) activity and the free cytosolic calcium concentration ([Ca(2+)](i)). In contrast, we have reported that SRIF can stimulate GH release in vitro from pig somatotropes. Specifically, 10(-7) and 10(-15) M SRIF stimulate GH release from a subpopulation of high density (HD) somatotropes isolated by Percoll gradient centrifugation, whereas in low density (LD) somatotropes only 10(-15) M SRIF induces such an effect. To ascertain the signaling pathways underlying this phenomenon, we assessed SRIF effects on second messengers in cultured LD and HD cells by measuring cAMP, IP turnover, and [Ca(2+)](i). Likewise, contribution of the corresponding signaling pathways to SRIF-induced GH release was evaluated by blocking AC, PLC, extracellular Ca(2+) influx, or intracellular Ca(2+) mobilization. Both 10(-7) and 10(-15) M SRIF increased cAMP, IP turnover, and [Ca(2+)](i) in HD cells. Conversely, in LD cells 10(-7) M SRIF reduced [Ca(2+)](i), but did not alter cAMP or IP, and 10(-15) M SRIF was without effect. Interestingly, SRIF-stimulated GH release was abolished in both subpopulations by AC blockade, but not by PLC inhibition. Furthermore, SRIF-induced GH release was not reduced by blockade of extracellular Ca(2+) influx through voltage-sensitive channels or by depletion of thapsigargin-sensitive intracellular Ca(2+) stores. Therefore, SRIF stimulates GH secretion from cultured porcine somatotrope subpopulations through an AC/cAMP pathway-dependent mechanism that is seemingly independent of net increases in IP turnover or [Ca(2+)](i). These novel actions challenge classic views of SRIF as a mere inhibitor for somatotropes and suggest that it may exert a more complex, dual function in the control of porcine GH release, wherein molecular heterogeneity of somatotropes would play a critical role.