5 %). In order to extract biological processes and molecular functions statistically over-represented in SO libraries, we performed a hyper-geometrical test between GO terms from the SO library and those from the AO library, which represents the natural physiological conditions. The p-values were then adjusted

using Bonferroni’s correction. In order to perform a functional enrichment analysis of the unigenes extracted from the SSH, we used the FatiGO web tool [39] against the SO library. With respect to the GO analysis, four different levels of description (3, 4, 6, and 9) were chosen for the biological processes. Quantitative expression by Real-Time RT-PCR Gene expression quantification was performed in whole animal, ovaries, and immune tissues Aurora Kinase inhibitor (hemocytes and hematopoietic organs pooled) Topoisomerase inhibitor of asymbiotic and symbiotic females. RNA extractions For the whole animal condition,

each individual was crushed with pestle and mortar in liquid nitrogen. Total RNA extraction was performed from about 30 mg of powder with TRIzol® reagent according to the manufacturer’s instructions (Invitrogen). For ovaries and immune tissues, total RNA extractions were performed from 25 and 50 females respectively with RNeasy Mini Kit according to the manufacturer’s instructions (QIAGEN). Real-Time RT-PCR First-strand cDNA was synthesized with the SuperScript III kit (Invitrogen) in accordance with manufacturer’s instructions, starting from 1 µg of total RNA using random hexamer primers. For whole animal samples, 0.2 µg of 5 individual extractions were pooled in 1 µg. Three biological replicates of each sample (whole animals, ovaries, and immune tissues) were used. For each gene, DNA Damage inhibitor primer pairs were designed with the Real-time PCR function of PerlPrimer [40]. The Tm and the length of each primer pair were fixed at 60°C and 18-22 bp, respectively.

Primers used for quantitative PCR are summarized in Additional File 1. Quantitative RT-PCR was performed using LightCycler LC480 system (Roche) as follows: 10 min at 95°C, 45 times [10 sec at 95°C, 10 sec at 60°C, 20 sec at 72°C]. A melting curve (65°C to 97°C) was recorded at the end of each reaction in order to check that the PCR product was unique. The reaction mixture consisted of 1.25 µL of each primer (10 µM), 5 µL of Fast SYBR-Green Master Mix (Roche) and 2.5 µL of diluted cDNA (corresponding to 12.5 ng of cDNA). Standard curves were plotted using 4 dilutions (125 ng, 25 ng, 5 ng, 1.25 ng) of pooled cDNAs from whole animals and ovaries. Efficiency of the PCR reaction was calculated. Expression data for each gene were estimated using the efficiency of the primer pair and the crossing point [41]. All gene expressions were normalized by the geometric mean of the expression level of the L8-ribosomal (RbL8) and Elongation Factor 2 (EF2) reference genes. Normalization and statistical pair-wise comparisons have been determined using REST [42].

jejuni 11168 infected mice: from grade 1 in previous experiments to grade 2 after serial passage. The tests for trends were statistically significant for strains 11168 (χ2 = 16.47; d.f. = 1; 0.00001 < P < 0.0001), D0835 (χ2 = 18.25; d.f. = 1; 0.00001 < P < 0.0001), and D2600 (χ2 = 16.90; d.f. = 1; 0.00001 < P < 0.0001). The test was not significant for strain D2586 (χ2 = 2.14; d.f. = 1; 0.14 < P < 0. 15) and could not be conducted for strain NW since there were no NW-infected

mice having histopathology scores in grade 2. DNA:DNA microarrray comparison of C. jejuni strains 11168 and NW (experiment 3) revealed differences between the strains Because strain NW was able to colonize C57BL/6 IL-10-/- mice but did not cause severe enteritis in the initial infection and did not evolve to a higher level of pathogenicity during repeated passages, we elected

to examine its genetic content more closely by comparing it to the selleck inhibitor highly pathogenic strain 11168 using an in-house full open reading frame (ORF) microarray with coverage of 95% of the C. jejuni 11168 genome [50]. The microarray was constructed using PCR products synthesized using primers for sequence-validated ORFs developed by Parrish et al. [51] and genomic DNA from strain 11168 (See NCBIGEO series number GSE13794 for a description of chip NVP-LDE225 solubility dmso manufacture.) We hypothesized that known virulence determinants would be among the genes present in strain 11168 but absent from strain NW. Sixty-nine C. jejuni 11168 ORFs were identified as possibly absent in strain NW by Genomotyping (GACK) analysis of microarray data [52]. Fifty-four of the 69 ORFs were confirmed to be absent or strongly divergent by PCR assay (Additional file 1, Table S2); PCR products of the appropriate size were obtained for thirteen of the remaining ORFs. Many of the ORFs missing in strain NW belong to complex loci encoding surface structures known both to be involved in C. jejuni pathogenesis and to be highly variable in gene content (flagellin, 8 ORFs; capsule, 11 ORFs; LOS, 1 ORF

(gmhA); [53]). Nine additional ORFs may encode membrane proteins; three may encode DNA restriction and modification proteins. Four periplasmic proteins were absent or strongly divergent C-X-C chemokine receptor type 7 (CXCR-7) in strain NW, along with seven ORFs having other known or putative functions and 11 ORFs encoding hypothetical proteins for which no function could be suggested [53]. For two ORFs, Cj 0987c (putative integral membrane protein) and Cj0874c (possible cytochrome c protein), strain NW DNA yielded PCR products smaller than those produced from strain 11168 DNA. Sequencing of the PCR products from strain NW showed that Cj0987c had a 649 bp deletion (nucleotides 121–770 of Cj0987c from strain 11168) compared to strain 11168. ORF Cj0874c in strain NW had a 182 bp deletion (nucleotides 212–393 of Cj0874c from strain 11168) compared to strain 11168.

After the determination of gfp and 16S rRNA gene copies of Gfp-tagged Asaia, total Asaia, and bacteria, the following ratios were calculated: Gfp-labelled Asaia to total Asaia ratio,

Gfp-labelled Asaia to bacteria ratio (GfpABR), and Asaia to bacteria 16S rRNA gene copy ratio (ABR), the latter according to Favia et al. [6]. These ratios were used to estimate the relative abundances of the introduced strain within total Asaia population in S. titanus individuals and of Gfp-labelled Asaia and Asaia sp. in the bacterial community associated with the insect samples. Statistical analyses To compare the Gfp Asaia density detected in co-feeding or venereal transmission experiments for every tested period, q-PCR data relative to the gfp gene INK 128 molecular weight concentration were log-transformed, after Roxadustat manufacturer adding the constant 10, and analyzed by one-way analysis of variance (ANOVA). In addition, means were separated by Tukey test (P<0.05) when variance homogeneity was satisfied (Levene test, P<0.05). Fluorescent in situ hybridization Fluorescent in situ hybridization analysis was carried out on organs dissected in a sterile saline solution from donor and recipient S. titanus individuals that were not used for Real time PCR experiments. The dissected organs were fixed for 2 min at 4°C in 4% paraformaldehyde and washed in

PBS. All hybridization experiment steps were performed as previously described [4] using specific and universal fluorescent probes. For detection of Gfp-labelled Asaia, probes gfp540 (5’-CCTTCGGGCATGGCACTCTT-3’) and gfp875 (5’-GGTAAAAGGACAGGGCCATCGCC-3’) were labelled with Cy5.5 (indodicarbocyanine, absorption/emission at 675-694 nm). Probes Asaia1 and Asaia2, labelled with Cy3 (indocarbocyanine, absorption/emission at 550/570 nm), were used to observe the total

Asaia population hosted by S. titanus individuals [6]. As a positive control for the hybridization experiment, a universal bacterial probe EUB388 labelled with fluorescein isothiocyanate (FITC, absorption/emission at 494/520 nm) was also used [32]. After hybridization, the samples were mounted in antifading medium and then observed in a laser scanning confocal check details microscope SP2- AOBS (Leica). Authors’ contributions EG designed and performed most of the experiments, analyzed data and wrote the manuscript. EC and AR provided the Asaia strain SF2.1(cGfp) and designed the experiments, MM designed FISH experiments and performed confocal microscopy observations. GF gave suggestions and contributed to data analysis. AA and DD designed and supervised all the experiments. All authors have read and approved the final manuscript. Acknowledgements We are grateful to Greg Hurst for English editing of the manuscript.

CrossRef 19. Bertucci M, LeLay G, Manneville selleckchem M, Kern R: Desorption kinetics of condensed phases: two-dimensional phases of silver on Ge(111). Surf Sci 1979, 85:471–492.CrossRef 20. Zandvliet HJW, Louwsma HK, Hegeman PE, Poelsema B: Energetics of Ni-induced vacancy line defects on Si(001).

Phys Rev Lett 1995, 75:3890–3893.CrossRef Competing interests The authors declare that they have no competing interests. Authors’ contributions T-YF conceived of the study and wrote the manuscript. AT was involved in carrying out the experiment and drafting the manuscript. X-LH and J-HL were involved in carrying out the experiment. P-IH and M-KJ analyzed the data. All authors read and approved the final version of the manuscript.”
“Background Colorectal tumors, which are caused by uncontrolled cell

growth in the colon or rectum [1], have constituted the third most commonly diagnosed cancer in the world, especially in developed countries [2]. In screening methods, a stool occult blood test is usually performed when the patient has experienced symptoms such as unusual bowel habits. When the result is positive, flexible sigmoidoscopy, barium enema, or colonoscopy is further applied. Because of discomfort and risks, such as the colonic perforation that can occur in these invasive methods, noninvasive methods [3], such as computed tomography (CT), positron emission tomography (PET), and magnetic resonance imaging (MRI), are alternatively used to image not only the primary colorectal tumor but also metastatic tumors in other organs. Two approaches can enhance buy Maraviroc the sensitivity and specificity of these medical imaging procedures [3]. The first approach is the multimodality of structural imaging and functional imaging, such as the CT/PET and MRI/PET. The second is based on image contrast media using bioprobes. Here, the image contrast media are the radioactive materials for CT and PET Fludarabine supplier and the superparamagnetic materials for MRI. It is well known that these radioactive media and methodologies entail

a biological risk and that the clinically popular gadolinium medium of MRI superparamagnetic materials induces the side effect of kidney disease [4]. Because iron oxide materials have a low risk of toxicity [5], superparamagnetic iron oxide nanoparticles (SPIONPs) coated with bioprobes have been developed for highly specific labeling [6] of targeted tumors in examining [7] and treating [8] tumors. Because carcinoembryonic antigen (CEA) is expressed in colorectal cancer [9], it is a useful indicator for treatment progress according to the decreasing CEA level in plasma [10]. Therefore, anti-CEA SPIONPs were developed as the contrast medium of MRI for colorectal cancer. However, because MRI requires a no-metal and shielded environment, as well as the patient to lie inside a coil, the procedure is limited to preoperative examination rather than intraoperative examination.

By doing so, we found that ALS1, ALS2 and ALS5 were overexpressed in all model systems, but their fold upregulations were more pronounced in both in vitro models and in the in vivo model, compared to the RHE model.

Using mutant strains, it was already demonstrated that Als1p and Als2p are involved in biofilm formation on abiotic surfaces [29, 34]. Furthermore, ALS4 was highly upregulated in the two in vitro models, and was extremely overexpressed in the RHE and in vivo models. However, deletion of ALS4 did not significantly reduce biofilm formation on silicone and neither resulted in reduced biomass on RHE, but it is likely that Als2p compensates for the loss of ALS4 [34]. Our data clearly show high expression levels for ALS4 in biofilms grown on mucosal surfaces as well as on abiotic surfaces in vitro and in vivo, suggesting selleck chemicals a role for Als4p in C. albicans Alectinib molecular weight biofilms. For ALS6 and ALS9, on the other hand, model-dependent up- and downregulations were observed. ALS6 was not overexpressed in the RHE model, which is not surprising as Als6p reduces adhesion of the fungus to buccal epithelial cells [35]. In both in vitro models and in the in vivo model, on the other hand, we observed an upregulation of ALS6. Using RT-PCR, it was previously shown that ALS6 was weakly expressed in biofilms grown on silicone [21]. However, using real-time PCR, we detected low Ct values (i.e. high

absolute mRNA levels) for ALS6 (data not shown). Furthermore, ALS9 is downregulated in the RHE model, in the MTP and in the vivo model, whereas this

gene is slightly upregulated under flow conditions in the CDC reactor. It is possible that shear stress generated in the CDC reactor induces the expression of ALS9, although further research is needed to confirm this hypothesis. We also studied the expression of ALS3 and HWP1, two genes that encode hyphae-specific adhesins [36, 37]. Their expression levels were higher in the CDC reactor than in the MTP, and the percentage of filaments was also higher in biofilms grown in the CDC reactor. Hyphae are known for their increased adhesive properties [13], and presumably shear stress in the CDC reactor triggers the fungus to form more filaments, which this website in turn express more ALS3 and HWP1. We also found that the percentage of filaments gradually decreased during biofilm formation in both in vitro models. It is known that contact-sensing induces filamentation in C. albicans [38], and therefore it is likely that initial contact of the fungus with the silicone results in filamentation. This could explain why young biofilms contain more filaments than mature ones in both in vitro models. Furthermore, ALS3 and HWP1 were highly upregulated in biofilms grown in the RHE model, and we found an increase in the percentage of filaments during biofilm formation in this model system. In order to grow in the RHE model, C.

Of this suspension, 25 μl was used to assay for total glutathione (reduced glutathione Torin 1 datasheet + oxidised glutathione ratio – GSH + GSSG) content, while the other 25 μl was treated with

4-vinylpyridine 0.5 μmol/l, a scavenger of GSH, to assay the GSSG content. One hundred twenty-five microlitres of reaction buffer (PBS 143 mmol/l containing 6.3 mmol/l EDTA at pH 7.4, 229 U/ml GSH reductase, 2.39 mmol/l β-nicotinamide adenine dinucleotide phosphate (NADPH) and 0.01 mol/l 5, 5’-dithiobis (2-nitrobenzoic acid) (DTNB)) was added to each 25-μl suspension. The conversion of DTNB to 5’-thiol-2-nitrobenzoic acid (TNB) by the oxidation of GSH to GSSG was monitored by measuring absorbance at 405 nm every min over 10 min using a Tecan GENios plate reader. The rate of conversion, measured by the slope of the curve, was proportional to the concentration of glutathione in the sample. A standard curve with different concentrations of GSSG was used

to calculate the glutathione contents in the samples. Statistical analysis For all the assays used, we performed three independent experiments with exposures carried out in triplicate for each concentration. The values shown are expressed as mean ± standard error of the mean (SEM). Sigma Plot 12 software (Systat Software Inc, CA, USA) was used for statistical analysis. The normality of the distribution was checked by means of the Shapiro-Wilk test. Equal variance was not assumed by the software and was tested (F test). A one-way repeated measures analysis of GDC-0199 concentration variance (RM-ANOVA) was carried out, followed by a post hoc Dunnett’s test with P < 0.05 or P < 0.01. Results Physico-chemical characterisation of PBH-capped AuNPs The AuNPs were synthesised using PBHs as capping ligands (Figure 1). In a previous study [9], we used PBHs containing cysteine (Cys), tyrosine (Tyr) and glycine (Gly) Celecoxib amino acids to form stable AuNPs: Au[(TrCys)2B] and [(Gly-Tyr-TrCys)2B]. In the present study, we demonstrate that the amino acids methionine (Met) and tryptophan (Trp) are also useful to prepare stable functionalised

AuNPs such as Au[(Met)2B], Au[(Gly-Tyr-Met)2B] and Au[(Gly-Trp-Met)2B]. TEM images of the PBH-capped AuNPs and the corresponding size distribution histograms are shown in Figure 2. The micrographs show isolated near-spherical NPs with diameters of 1.5, 1.6, 2.3, 1.8 and 2.3 nm for Au[(Gly-Tyr-Met)2B], Au[(Gly-Trp-Met)2B], Au[(Met)2B], Au[(Gly-Tyr-TrCys)2B] and Au[(TrCys)2B], respectively. The NPs stabilised with the bulkiest PBHs were smaller. This observation may be attributable to the steric bulk of the ligand controlling NP growth. Figure 2 TEM images and size histograms of PBH-capped AuNPs. (a) Au[(Gly-Trp-Met)2B], (b) Au[(Gly-Tyr-TrCys)2B], (c) Au[(Gly-Tyr-Met)2B], (d) Au[(Met)2B] and (e) Au[(TrCys)2B] [Scale bars: 10 nm for (a) and (b); and 5 nm for (c), (d) and (e)].

05) . P, probiotic group; C, control group; W33, 33rd gestational week (black colour); W37, 37th gestational week (grey colour). Cytokine or chemokine names are reported

in x-axis. Data are expressed as pg of the target cytokine or chemokine per μg of total proteins present in the vaginal sample (y-axis). The diagrams show means with error bars representing the standard deviations. Figure 5 shows women, belonging to P and C groups, who registered significant variations in total levels of immune-mediators during the study period (P < 0.05). Significant changes were found for women N. 18, 19, 20, 21, 23, 24, 25 and 27 (8/12; 67%) of C group and women N. 1, 2, 3, 10, 11 (5/15; 33%) of P group. Figure 5 Women registering significant variations in total levels of immune-mediators. P, probiotic group; C, control group; W33, 33rd gestational AZD5363 in vivo week (black colour); W37, 37th gestational week (grey colour). Identification Talazoparib nmr numbers of women registering

significant variations are reported in x-axis. Data are expressed as pg of total immune-mediators per μg of total vaginal proteins (y-axis). The diagrams show means with error bars representing the standard deviations. Discussion To our knowledge, this is the first study describing the effect of a probiotic mixture, orally consumed during the last trimester of pregnancy, on the vaginal microbiota and immune response. Although several health-promoting activities of probiotics have been described in relation to the gut homeostasis [16, 32], less information is available regarding the interactions between orally administered probiotic bacteria and the vaginal microbial habitat. The first step Sitaxentan in ascertaining the influence of the dietary supplementation with the probiotic VSL#3 on the vaginal microbiota of pregnant women was the characterization of vaginal bacterial communities by using an integrated approach based on PCR-DGGE and qPCR. DGGE population profiling, conducted

with universal primers for bacteria and Lactobacillus-specific primers, allowed us to investigate the variations of the predominant vaginal bacterial communities and Lactobacillus species occurring both physiologically in the last trimester of pregnancy and potentially associated with VSL#3 intake. The influence of the probiotic intake in modulating the predominant bacterial populations and Lactobacillus species could be hypothesized since significant differences between DGGE profiles at W33 and W37 were found only in women belonging to P group. Notably, the lower percentage of women belonging to P group who displayed significant differences in Lactobacillus-specific DGGE profiles between W33 and W37, compared to the universal bacterial DGGE patterns, suggested a major stability of lactobacilli population and a more extended impact of the probiotic VSL#3 on total bacteria than lactobacilli.

The samples used in these experiments were prepared by J. Dekker and collaborators (Dekker et al. 1989, 1990; Eijckelhoff and Dekker 1995; Kwa et al. 1992). They were subsequently diluted in buffer and glycerol to work at low temperature (Den Hartog et al. 1998b). The SD behaviour of the PSII sub-core complexes is compared here with that of B777, the monomer subunit of the LH1 complex of purple bacteria. B777 was obtained from LH1 by adding the detergent n-octyl-β-glucopiranoside (OG) and diluted in buffer and glycerol (Creemers et al. 1999a, and references therein). The B777 complex, in turn, is compared with BChl a embedded

in the same OG detergent (diluted in buffer and glycerol) without the protein, which we call here BChl a in OG-glass (Creemers and Völker 2000). The purpose of this experiment was two-fold, to compare the SD behaviour this website of proteins with that of glasses, and to clear up a long-standing problem: whether the BTK inhibitor chemical structure BChl a molecule in B777 is bound or not to the protein (Sturgis and Robert 1994, and references therein). HB results on SD of B820, the dimer subunit of LH1, at various temperatures and delay

times, and its comparison to glasses, can be found in Störkel et al. (1998). Photosystem II (PSII), the ‘engine of life’, is a large complex embedded in the thylakoid membranes of plants, algae and cyanobacteria. Driven ifenprodil by solar energy, PSII catalyzes the splitting of water into oxygen which is essential for the survival of life on Earth (for a review, see Barber 2008). The events that give rise to the primary and secondary electron-transfer processes, which lead to water oxidation start with the absorption of sunlight by a peripheral light-harvesting complex, called LHCII (Kühlbrandt et al. 1994),

which transfers the excitation energy to the RC within the PSII core complex. The isolated PSII RC, which is the smallest unit that shows photochemical activity (Nanba and Satoh 1987; Rhee et al. 1997), is composed of the D1 and D2 proteins and bound mainly to the CP43 and CP47 complexes (Boekema et al. 1998; Dekker and Boekema 2005). The D1 and D2 proteins contain the cofactors that bring about charge separation. The crystal structures of cyanobacterial PSII, determined by X-ray crystallography at 3.5 Å (Ferreira et al. 2004) and 3 Å (Loll et al. 2005) resolution, confirmed the dimeric organization of the isolated complex and the positioning of the major subunits within each monomer, previously obtained by electron crystallography (Eijckelhoff et al. 1997; Rhee et al. 1997). Loll et al. (2005) concluded that there are about 36 Chl a and 11 β-carotene molecules per PSII core, and that the CP43 and CP47 complexes bind 13 and 16 Chls, respectively, while the RC binds 6 Chls, 2 pheophytin (Pheo) molecules, 2 plastoquinone (PQ) molecules, at least one β-carotene and a non-heme Fe.

aeruginosa SG81ΔlipA, the corresponding complementation strain P. aeruginosa SG81ΔlipA::lipA and the lipA overexpression strain P. aeruginosa SG81lipA + carrying plasmid pBBL7 were used. This vector based on pBBR1MCS [64] and carries the genes lipA and lipH from P. aeruginosa PAO1 [1]. For construction of a ΔlipA-mutant from SG81 a Gmr cassette was cloned into the suicide vector pMEΔAH11 [63] containing a 2.06 kbp KpnI/XbaI-fragment

with Δ(2/3 lipA 1/5 lipH). The resulting vector pMEΔAH::Ω-Gmr was used for homologous recombination. Pirfenidone mouse All plasmids were transferred into P. aeruginosa SG81 via conjungation using Escherichia coli S-17. Table 3 Bacterial strains and plasmids used in this study Strain/plasmids Relevant genotype/ phenotype Reference E. coli S17-1 thi pro hsdR – M +, chromosomally integrated [RP4-2 Tc::Mu:Kmr::Tn7, Tra+ Trir Strr] [65] P. aeruginosa   [38] PABST7.1/pUCPL6A Overexpression of lipA and lipH from pUCPL6A FRD1 Mucoid ΔmucA22 CF-lung isolate [66] FRD1153 ΔalgJ5-mutant derived from FRD1, defect in O-acetylation of alginate [61, 62] SG81 Mucoid biofilm isolate from technical water system [67] SG81MCS Vector control pBBR1MCS [1] SG81ΔlipA Δ(2/3 lipA 1/5 lipH)::Ω-Gmr

, deletion of lipA and lipH This study SG81ΔlipA::lipA Deletion of lipA and lipH complemented in trans from pBBL7 This study SG81lipA+ Expression of lipA and lipH in trans from pBBL7 [1] pBBR1MCS lacZα Cmr mob Plac, PT7 [64] pBBL7 2.8 kbp XmnI/SmaI fragment with lipA/H operon in pBBR1MCS under Plac control   pMEΔAH11 2.06 kbp KpnI/XbaI-fragment with

Δ(2/3 Selleckchem Everolimus lipA 1/5 lipH) in pME3087 [63] pMEΔAH::Ω-Gmr 1.6 kbp SmaI-fragment with Ω-Gmr from pBSL142 in pMEΔAH11 This study Biofilm Pregnenolone cultures were grown for 24 h at 36°C on Pseudomonas Isolation Agar (PIA; Difco) in the form of confluent mucoid lawns. Cell numbers of biofilms, which were scraped from the agar surface and suspended in 0.14 M NaCl, were determined microscopically using a Thoma counting chamber. Cell-free EPS solutions prepared from the biofilm suspensions according to Tielen et al. [1] were used to measure uronic acid (alginate) concentration and lipase activity as described below. For CLSM analysis, biofilms were grown on membrane-filters (polycarbonate, size: 2.5 cm, pore size: 0.4 μm; Millipore, Billerica, Massachusetts) placed on PIA supplemented with 0.1 M CaCl2 for stabilization of the biofilm matrix as described previously [68]. Visualization of lipase activity in situ For visualization of lipase activity in biofilms of P. aeruginosa strains, ELF® 97 palmitate (Molecular Probes, Invitrogen GmbH, Karlsruhe, Germany) was used as a substrate. This enzyme substrate is cleaved by lipases to the water-insoluble ELF® 97 alcohol, which precipitates directly at the site of enzymatic hydrolysis, thus reporting the location of lipase enzyme activity, when visualized by fluorescence microscopy [69].

When probed with antibodies against total p38, the 38 kDa band showed no change at the investigated time Idelalisib in vivo points of OUA treatment, in comparison with that observed in the lysate of untreated cells (Figure 4c). Thus, OUA 100 nM activates p38 MAPK in U937 cells. Then, we investigated the involvement of NCX in the phosphorylation of p38. However, we did not detect a difference in the band of phospho-p38 in the lysate of cells pretreated with KBR and

then with OUA, in comparison with the band observed in the lysate of OUA treated cells (Figure 4c). Thus, these results suggest that, although p38 plays a pro-survival role in OUA treated cells, its activation is NCX independent. Discussion The first aim of our investigation was to Selleck RAD001 evaluate if OUA is cytotoxic for U937 cells and we detected that at concentrations ≥500 nM it causes ROS generation and a large increase of [Ca++]i followed by cell death. We did not explore the link between ROS generation, Ca++ increase and cell demise, as it is not surprising that this intracellular milieu can lead to cell death. We were surprised by the

survival pathway sparked by lower doses of OUA in which a modest rise of Ca++ seems to play an important role. Indeed, U937 cells exposed to ouabain 100 nM were growth arrested in G1 cell cycle phase and escaped from death by activation of a survival pathway, in which were involved the Na+/Ca++-exchanger active in the Ca++ influx mode and p38 MAPK. It is widely accepted that partial inhibition of the cardiac myocyte Na+/K+-ATPase by cardiac glycosides causes a modest increase of [Na+ i, which in turn affects the plasma membrane Na+/Ca++-exchanger, leading to a significant increase of [Ca++ i and in the force of contraction [4–9]. In the present investigation we show that in U937 cells OUA leads to a rise of [Ca++ i through NCX active in the Ca++ influx mode because this event could be prevented by KBR, an inhibitor known to affect only this type of NCX activity [30, 31]. Moreover, OUA became largely cytotoxic after NCX inhibition and not after block of L-type

Ca++ channel by nifedipine. These conclusions were confirmed treating the cells with the Na+ ionophore monensin which, similarly to OUA, causes an increase of [Ca++ Adenosine i through NCX active in the Ca++ influx mode. Finally, the endoplasmic reticulum stressor tunicamycin, not affecting NCX, proved to be a good control because it induced cell death in a low proportion of cells, not increased by KBR. MAPK are central mediators of cellular survival and death pathways [33–36]. To investigate their involvement in the survival pathway activated by OUA, we pretreated the cells with inhibitors at concentrations affecting specifically one MAPK and then analyzed cell viability. These experiments indicated that p38 plays a pro-survival role in OUA treated cells.