Copper-induced increased expression of genes involved in photosynthesis, carotenoid synthesis and C assimilation in the marine alga Ulva compressa.

BACKGROUND: The marine alga Ulva compressa is the dominant species in coastal areas receiving effluents from copper mines. The alga can accumulate high amounts of copper and possesses a strong antioxidant system. Here, we performed short-term transcriptomic analyses using total RNA of the alga cultivated with 10 µM of copper for 0, 3, 6, 12 and 24 h by RNA-seq. RESULTS: De novo transcriptomes were assembled using the Trinity software, putative proteins were annotated and classified using Blast2GO. Differentially expressed transcripts were identified using edgeR. Transcript levels were compared by paired times 0 vs 3, 0 vs 6, 0 vs 12 and 0 vs 24 h at an FDR < 0.01 and Log2 Fold Change > 2. Up-regulated transcripts encode proteins belonging to photosystem II (PSII), Light Harvesting II Complex (LHCII), PSI and LHCI, proteins involved in assembly and repair of PSII, and assembly and protection of PSI. In addition, transcripts encoding enzymes leading to ß-carotene synthesis and enzymes belonging to the Calvin-Benson cycle were also increased. We further analyzed photosynthesis and carotenoid levels in the alga cultivated with 10 µM of copper for 0 to 24 h. Photosynthesis was increased from 3 to 24 h as well as the level of total carotenoids. The increase in transcripts encoding enzymes of the Calvin-Benson cycle suggests that C assimilation may also be increased. CONCLUSIONS: Thus, U. compressa displays a short-term response to copper stress enhancing the expression of genes encoding proteins involved in photosynthesis, enzymes involved carotenoids synthesis, as well as those belonging to the Calvin-Benson cycle, which may result in an increase in C assimilation.

Alpinone exhibited immunomodulatory and antiviral activities in Atlantic salmon.

In this study, we seek to identify flavonoids able to regulate the gene expression of a group of cytokines important for the control of infections in Atlantic salmon (Salmo salar). Particularly, we studied the potential immunomodulatory effects of two flavonoids, Alpinone and Pinocembrine, which were isolated and purified from resinous exudates of Heliotropium huascoense and Heliotropium sinuatum, respectively. The transcript levels of TNF-α and IL-1 (inflammatory cytokines), IFN-γ and IL-12 (T helper 1 type cytokines), IL4/13A (Th2-type cytokine), IL-17 (Th17 type cytokine) TGF-ß1 (regulatory cytokine) and IFN-α (antiviral cytokine) were quantified by qRT-PCR in kidneys of flavonoid-treated and control fish. We demonstrated that the administration of a single intramuscular dose of purified Alpinone increased the transcriptional expression of five cytokines, named TNF-α, IL-1, IFN-α, IFN-γ and TGF-ß1 in treated fish compared to untreated fish. Conversely, administration of purified Pinocembrine reduced the transcriptional expression of TNF-α, IL-1 and IL-12 in the kidney of treated fish. No other changes were observed. Interestingly, Alpinone also induced in vitro antiviral effects against Infectious Salmon Anaemia virus. Results showed that Alpinone but not Pinocembrine induces the expression of cytokines, which in vertebrates are essential to control viral infections while Pinocembrine reduces pro-inflammatory cytokines. Altogether results suggest that Alpinone is a good candidate to be further tested as immunostimulant and antiviral drug.

P2X4 Receptor in Silico and Electrophysiological Approaches Reveal Insights of Ivermectin and Zinc Allosteric Modulation.

Protein allosteric modulation is a pillar of metabolic regulatory mechanisms; this concept has been extended to include ion channel regulation. P2XRs are ligand-gated channels activated by extracellular ATP, sensitive to trace metals and other chemicals. By combining in silico calculations with electrophysiological recordings, we investigated the molecular basis of P2X4R modulation by Zn(II) and ivermectin, an antiparasite drug currently used in veterinary medicine. To this aim, docking studies, molecular dynamics simulations and non-bonded energy calculations for the P2X4R in the apo and holo states or in the presence of ivermectin and/or Zn(II) were accomplished. Based on the crystallized Danio rerio P2X4R, the rat P2X4R, P2X2R, and P2X7R structures were modeled, to determine ivermectin binding localization. Calculations revealed that its allosteric site is restricted to transmembrane domains of the P2X4R; the role of Y42 and W46 plus S341 and non-polar residues were revealed as essential, and are not present in the homologous P2X2R or P2X7R transmembrane domains. This finding was confirmed by preferential binding conformations and electrophysiological data, revealing P2X4R modulator specificity. Zn(II) acts in the P2X4R extracellular domain neighboring the SS3 bridge. Molecular dynamics in the different P2X4R states revealed allosterism-induced stability. Pore and lateral fenestration measurements of the P2X4R showed conformational changes in the presence of both modulators compatible with a larger opening of the extracellular vestibule. Electrophysiological studies demonstrated additive effects in the ATP-gated currents by joint applications of ivermectin plus Zn(II). The C132A P2X4R mutant was insensitive to Zn(II); but IVM caused a 4.9 ± 0.7-fold increase in the ATP-evoked currents. Likewise, the simultaneous application of both modulators elicited a 7.1 ± 1.7-fold increase in the ATP-gated current. Moreover, the C126A P2X4R mutant evoked similar ATP-gated currents comparable to those of wild-type P2X4R. Finally, a P2X4/2R chimera did not respond to IVM but Zn(II) elicited a 2.7 ± 0.6-fold increase in the ATP-gated current. The application of IVM plus Zn(II) evoked a 2.7 ± 0.9-fold increase in the ATP-gated currents. In summary, allosteric modulators caused additive ATP-gated currents; consistent with lateral fenestration enlargement. Energy calculations demonstrated a favorable transition of the holo receptor state following both allosteric modulators binding, as expected for allosteric interactions.

ß-1,3/1,6-Glucan-supplemented diets antagonize immune inhibitory effects of hypoxia and enhance the immune response to a model vaccine.

The diets of farmed salmon are usually supplemented with immunostimulants to improve health status. Because ß-glucan is one of the most common immunostimulants used in diets, here we examined the effect of two ß-1,3/1,6-glucan-supplemented diets on the expression of immune response genes of Atlantic salmon. The relative abundances of IFN-α1, Mx, IFN-γ, IL-12, TGF-ß1, IL-10, and CD4 transcripts were evaluated in head kidney by qRT-PCR. We assessed the effects of the diets under normoxia and acute hypoxia, as salmon are especially sensitive to changes in the concentration of dissolved oxygen, which can also affect immunity. These effects were also tested on vaccinated fish, as we expected that ß-1,3/1,6-glucan-supplemented diets would enhance the adaptive immune response to the vaccine. We found that administration of the Bg diet (containing ß-1,3/1,6-glucan) under normoxia had no effects on the expression of the analyzed genes in the kidney of the diet-fed fish, but under hypoxia/re-oxygenation (90 min of acute hypoxia), the ßg diet affected the expression of the antiviral genes, IFN-α1 and Mx, preventing their decrease caused by hypoxia. The Bax diet, which in addition to ß-1,3/1,6-glucan, contained astaxanthin, increased IL-12 and IFN-γ in kidneys. With fish exposed to hypoxia/reoxygenation, the diet prevented the decrease of IFN-α1 and Mx levels observed after hypoxia. When fish were vaccinated, only the levels of IL-12 and CD4 transcripts increased, but interestingly if fish were also fed the Bax diet, the vaccination induced a significant increase in all the analyzed transcripts. Finally, when vaccinated fish were exposed to hypoxia, the effect of the Bax diet was greatly reduced for all genes tested and moreover, inducible effects completely disappeared for IL-12, IFN-α, and Mx. Altogether, these results showed that the tested ß-1,3/1,6-glucan diets increased the levels of transcripts of key genes involved in innate and adaptive immune response of salmon, potentiating the response to a model vaccine and also antagonizing the effects of hypoxia.

Neisseria gonorrhoeae induced disruption of cell junction complexes in epithelial cells of the human genital tract.

Pathogenic microorganisms, such as Neisseria gonorrhoeae, have developed mechanisms to alter epithelial barriers in order to reach subepithelial tissues for host colonization. The aim of this study was to examine the effects of gonococci on cell junction complexes of genital epithelial cells of women. Polarized Ishikawa cells, a cell line derived from endometrial epithelium, were used for experimental infection. Infected cells displayed a spindle-like shape with an irregular distribution, indicating potential alteration of cell-cell contacts. Accordingly, analysis by confocal microscopy and cellular fractionation revealed that gonococci induced redistribution of the adherens junction proteins E-cadherin and its adapter protein ß-catenin from the membrane to a cytoplasmic pool, with no significant differences in protein levels. In contrast, gonococcal infection did not induce modification of either expression or distribution of the tight junction proteins Occludin and ZO-1. Similar results were observed for Fallopian tube epithelia. Interestingly, infected Ishikawa cells also showed an altered pattern of actin cytoskeleton, observed in the form of stress fibers across the cytoplasm, which in turn matched a strong alteration on the expression of fibronectin, an adhesive glycoprotein component of extracellular matrix. Interestingly, using western blotting, activation of the ERK pathway was detected after gonococcal infection while p38 pathway was not activated. All effects were pili and Opa independent. Altogether, results indicated that gonococcus, as a mechanism of pathogenesis, induced disruption of junction complexes with early detaching of E-cadherin and ß-catenin from the adherens junction complex, followed by a redistribution and reorganization of actin cytoskeleton and fibronectin within the extracellular matrix.

Lipopolysaccharide inhibits the channel activity of the P2X7 receptor.

The purinergic P2X7 receptor (P2X7R) plays an important role during the immune response, participating in several events such as cytokine release, apoptosis, and necrosis. The bacterial endotoxin lipopolysaccharide (LPS) is one of the strongest stimuli of the immune response, and it has been shown that P2X7R activation can modulate LPS-induced responses. Moreover, a C-terminal binding site for LPS has been proposed. In order to evaluate if LPS can directly modulate the activity of the P2X7R, we tested several signaling pathways associated with P2X7R activation in HEK293 cells that do not express the TLR-4 receptor. We found that LPS alone was unable to induce any P2X7R-related activity, suggesting that the P2X7R is not directly activated by the endotoxin. On the other hand, preapplication of LPS inhibited ATP-induced currents, intracellular calcium increase, and ethidium bromide uptake and had no effect on ERK activation in HEK293 cells. In splenocytes-derived T-regulatory cells, in which ATP-induced apoptosis is driven by the P2X7R, LPS inhibited ATP-induced apoptosis. Altogether, these results demonstrate that LPS modulates the activity of the P2X7R and suggest that this effect could be of physiological relevance.

Oxidative damage in lymphocytes of copper smelter workers correlated to higher levels of excreted arsenic.

Arsenic has been associated with multiple harmful effects at the cellular level. Indirectly these defects could be related to impairment of the integrity of the immune system, in particular in lymphoid population. To characterize the effect of Arsenic on redox status on this population, copper smelter workers and arsenic unexposed donors were recruited for this study. We analyzed urine samples and lymphocyte enriched fractions from donors to determinate arsenic levels and lymphocyte proliferation. Moreover, we studied the presence of oxidative markers MDA, vitamin E and SOD activity in donor plasma. Here we demonstrated that in human beings exposed to high arsenic concentrations, lymphocyte MDA and arsenic urinary levels showed a positive correlation with SOD activity, and a negative correlation with vitamin E serum levels. Strikingly, lymphocytes from the arsenic exposed population respond to a polyclonal stimulator, phytohemaglutinin, with higher rates of thymidine incorporation than lymphocytes of a control population. As well, similar in vitro responses to arsenic were observed using a T cell line. Our results suggest that chronic human exposure to arsenic induces oxidative damage in lymphocytes and could be considered more relevant than evaluation of T cell surveillance.

The 5'-untranslated region of the mouse mammary tumor virus mRNA exhibits cap-independent translation initiation.

In this study, we demonstrate the identification of an internal ribosome entry site (IRES) within the 5'-untranslated region (5'-UTR) of the mouse mammary tumor virus (MMTV). The 5'-UTR of the full-length mRNA derived from the infectious, complete MMTV genome was cloned into a dual luciferase reporter construct containing an upstream Renilla luciferase gene (RLuc) and a downstream firefly luciferase gene (FLuc). In rabbit reticulocyte lysate, the MMTV 5'-UTR was capable of driving translation of the second cistron. In vitro translational activity from the MMTV 5'-UTR was resistant to the addition of m(7)GpppG cap-analog and cleavage of eIF4G by foot-and-mouth disease virus (FMDV) L-protease. IRES activity was also demonstrated in the Xenopus laevis oocyte by micro-injection of capped and polyadenylated bicistronic RNAs harboring the MMTV-5'-UTR. Finally, transfection assays showed that the MMTV-IRES exhibits cell type-dependent translational activity, suggesting a requirement for as yet unidentified cellular factors for its optimal function.

Neurosteroids differentially modulate P2X ATP-gated channels through non-genomic interactions.

As neuroactive steroids modulate several ionotropic receptors, we assessed whether the ATP-gated currents elicited by P2X(4) receptors are modulated by these compounds. We transfected HEK293 cells or injected Xenopus laevis oocytes with the cDNA coding for rat P2X(4) receptor. Application of 0.1-10 microM alfaxolone potentiated within 60-s the 1 microM ATP-evoked currents with a maximal potentiation of 1.8 and 2.6-fold in HEK293 or oocytes cells respectively. Allopregnalolone or 3alpha, 21-dihydroxy-5alpha-pregnan-20-one (THDOC) also potentiated the ATP-gated currents but with a maximal effect only averaging 1.25 and 1.35-fold respectively. In contrast, 0.3-10 microM pregnanolone, but not its sulfated derivative, inhibited the ATP-gated currents; the maximal inhibition reached 40% in both cell types. THDOC, but not other neurosteroids increased significantly the tau(off) of the ATP-evoked currents, revealing another mode of neurosteroid modulation. Sexual steroids such as 17beta-estradiol or progesterone were inactive revealing explicit structural requirements. Alfaxolone or THDOC at concentrations 30- to 100-fold larger than required to modulate the receptor, gated the P2X(4) receptor eliciting ATP-like currents that were reduced with suramin or brilliant blue G, but potentiated the P2X(4) receptor more than 10-fold by 10 microM zinc. In conclusion, neurosteroids rapidly modulate via non-genomic mechanisms and with nanomolar potencies, the P2X4 receptor interacting likely at distinct modulator sites.