Effects of 17beta-estradiol and 4-nonylphenol on osmoregulation and hepatic enzymes in gilthead sea bream (Sparus auratus)

Sexually immature Sparus auratus were injected intraperitoneally with coconut oil either alone (control) or containing 17beta-estradiol (E2, 10 microg/g body mass) or 4-nonyphenol (4-NP, 100 and 200 microg/g body mass) and sampled 10 days later. Gill and kidney Na(+),K(+)-ATPase activities, plasma levels of E2 and cortisol, plasma osmolites (osmolality, sodium and chloride) and metabolites (glucose, lactate, proteins and triglycerides) were examined. Livers were used for measuring hepatosomatic index (HSI) and determinations of the activities of antioxidant defences catalase (CAT) and total glutatione peroxidase (t-GPX), the CYP1A-dependent, 7-ethoxyresorufin O-deethylase (EROD) and glutathione S-transferase (GST). HSI and plasma levels of E2 were significantly increased in E2 -treated fish. E2 treatment enhanced plasma osmolality, glucose, triglycerides and proteins, but had no effect on plasma cortisol, and gill and kidney Na(+),K(+)-ATPase activities. Hepatic activities of EROD, GST and CAT were significantly decreased after E2 administration, whereas t-GPX remained unaffected. Treatment with 200 microg/g 4-NP caused a slight increase in plasma E2 relative to the control group. Plasma glucose and protein levels were not affected by 4-NP, while triglycerides were increased. Fish treated with the higher dose of 4-NP displayed a clear reduction in kidney Na(+),K(+)-ATPase activity, together with increases in plasma osmolality, relative to the control group. High 4-NP also caused a significant decrease in EROD and an increase in GST activity. Our results confirm the regulation of the natural estrogen E2 and the weak xenoestrogen 4-NP on osmoregulation and biotransformation enzymes in a partially similar manner. The actions of xenoestrogens on critical physiological processes may have an ecological significance as it can reduce adaptability and capacity to metabolise xenobiotics under stressful conditions.     

Higher contribution of globally rare bacterial taxa reflects environmental transitions across the surface ocean

Microbial taxa range from being ubiquitous and abundant across space to extremely rare and endemic, depending on their ecophysiology and on different processes acting locally or regionally. However, little is known about how cosmopolitan or rare taxa combine to constitute communities and whether environmental variations promote changes in their relative abundances. Here we identified the Spatial Abundance Distribution (SpAD) of individual prokaryotic taxa (16S rDNA‐defined Operational Taxonomic Units, OTUs) across 108 globally‐distributed surface ocean stations. We grouped taxa based on their SpAD shape (“normal‐like”‐ abundant and ubiquitous; “logistic”‐ globally rare, present in few sites; and “bimodal”‐ abundant only in certain oceanic regions), and investigated how the abundance of these three categories relates to environmental gradients. Most surface assemblages were numerically dominated by a few cosmopolitan “normal‐like” OTUs, yet there was a gradual shift towards assemblages dominated by “logistic” taxa in specific areas with productivity and temperature differing the most from the average conditions in the sampled stations. When we performed the SpAD categorization including additional habitats (deeper layers and particles of varying sizes), the SpAD of many OTUs changed towards fewer “normal‐like” shapes, and OTUs categorized as globally rare in the surface ocean became abundant. This suggests that understanding the mechanisms behind microbial rarity and dominance requires expanding the context of study beyond local communities and single habitats. We show that marine bacterial communities comprise taxa displaying a continuum of SpADs, and that variations in their abundances can be linked to habitat transitions or barriers that delimit the distribution of community members.     

Application of 16S rRNA gene-targetted fluorescence in situ hybridization and restriction fragment length polymorphism to study porcine microbiota along the gastrointestinal tract in response to different sources of dietary fibre

A total of 32 pigs of 15+/-0.38 kg body weight were fed for 6 weeks one of four diets differing in their source of dietary fibre. FISH was used to quantify the main bacterial groups in the pig gut using the following probes: Eub338, Bac303, Rfla729, Rbro730, Erec482, Fprau645, Prop853, Str493 and Lab158. FISH counts revealed important differences at four sites along the pig gastrointestinal tract, but we were unable to show differences related to diets. Stomach and jejunal samples gave total bacterial counts of 0.1-5.3 x 10(8) g(-1) of contents. In the stomach, streptococci and lactobacilli were predominant, and the clostridial cluster IX group was abundant (14-41% of total bacterial count). Clostridial cluster IX bacteria were present elsewhere in the gastrointestinal tract at 1-8%. The other groups were generally more abundant in the proximal colon and rectum: Bacteroides/Prevotella (5-10%), clostridial cluster XIVa (10-19%), and cluster IV relatives of Faecalibacterium prausnitzii (1-4%) and ruminococcus (4-10%). Restriction fragment length polymorphism profiles showed changes related to diet, with pigs fed wheat bran having the lowest richness of all diets (P=0.008).     

Efficacy of an inhibitor of adhesion molecule expression (GI270384X) in the treatment of experimental colitis

Modulation of adhesion molecule expression or function is regarded as a promising therapy for inflammatory conditions. This study evaluates the effects of an inhibitor of adhesion molecule expression (GI270384X) in two experimental models of colitis. Colitis of different severity was induced in C57BL/6J mice by administering 1, 2, or 3% dextran sulfate sodium (DSS). GI270384X (3, 10, or 25 mg.kg(-1).day(-1)) was administered as pretreatment or started 3 days after colitis induction. In IL-10-deficient mice, the highest dose was given for 2 wk. The clinical course of colitis, pathological changes, serum inflammatory biomarkers, expression of adhesion molecules, and leukocyte-endothelial cell interactions in colonic venules were measured in mice treated with vehicle or with active drug. In the most severe forms of colitis (2% and 3% DSS and IL-10-deficient mice), the magnitude of colonic inflammation was not modified by treatment with GI270384X. In a less severe form of colitis (1% DSS), GI270384X treatment dose dependently ameliorated the clinical signs of colitis, colonic pathological changes, and serum levels of biomarkers (IL-6 and serum amyloid A). Administration of 25 mg.kg(-1).day(-1) GI270384X abrogated upregulation of ICAM-1 in the inflamed colon but had no effect on VCAM-1 or E-selectin expression. This was associated with a significant reduction in number of rolling and firmly adherent leukocytes in colonic venules. These results indicate that GI270384X is effective in the treatment of experimental colitis of moderate severity. Reduced adhesion molecule expression and leukocyte recruitment to the inflamed intestine contribute to this beneficial effect.     

Comparison of melatonin, vitamin E and L-carnitine in the treatment of neuro- and hepatotoxicity induced by thioacetamide

This study was designed to evaluate and compare the effect of melatonin, vitamin E and L-carnitine on brain and liver oxidative stress and liver damage. Oxidative stress and hepatic failure were produced by a single dose of thioacetamide (TAA) (150 mg kg(-1)) in Wistar rats. A dose of either melatonin (3 mg kg(-1)) vitamin E (20 mg kg(-1) ) or L-carnitine (100 mg kg(-1)) was used. Blood samples were taken from the neck vasculature in order to determine ammonium, blood urea nitrogen (BUN) and liver enzymes. Lipid peroxidation products, glutathione (GSH) content and antioxidative enzymes were determined in cerebral and hepatic homogenates. The results showed a decrease in BUN and in the antioxidant enzymes activities and GSH in the brain and liver. Likewise, TAA induced significant enhancement of lipid peroxidation products levels in both liver and brain, as well as in ammonia values. Melatonin, vitamin E and L-carnitine, although melatonin more significantly, decreased the intensity of the changes produced by the administration of TAA alone. Furthermore melatonin combined with TAA, decreased the ammonia levels and increased the BUN values compared with TAA animals. Also it was more effective than vitamin E or L-carnitine in these actions. These data show the protective effect of these agents, especially melatonin, against oxidative stress and hepatic damage present in fulminant hepatic failure.     

IGF-I binding and receptor signal transduction in primary cell culture of muscle cells of gilthead sea bream: changes throughout in vitro development

We examined the possibility of culturing muscle cells of gilthead sea bream in vitro and assessed variations in insulin-like growth factor-I (IGF-I) binding during myocyte development. The viability of the cell culture was determined by fluorescence-activated cell-sorting analysis, which showed that the percentage of dead cells decreased with cell differentiation. The intracellular reduction of MTT into formazan pigment was preferentially carried out as cells differentiated (from day 4) indicating an increase in metabolic activity. IGF-I-binding assays demonstrated that the number of receptors increased from 190  ±  0.09 fmol/mg protein in myocytes at day 5 to 360 ± 0.09 fmol/mg protein in myotubes at day12. The affinity of IGF-I receptors did not change significantly during cell development (from 0.89 ± 0.09 to 0.98 ± 0.09 nM). The activation of various kinase (ERK 1/2 MAPK and Akt/PKB) proteins by IGFs and insulin was studied by means of Western blot analysis. Levels of MAPK-P increased after IGF and insulin treatment during the first stages of cell culture, with a low response being observed at day 15, whereas IGFs displayed a stimulatory effect on Akt-P throughout the cell culture period, even on day 15. This study thus shows that (1) gilthead sea bream myocytes can be cultured, (2) they express functional IGF-I receptors that increase in number as they differentiate in vitro; (3) IGF signalling transduction through IGF-I receptors stimulates the MAPK and Akt pathways, depending on the development stage of the muscle cell culture. This work was supported by grant AGL2004–06319-C02/ACU from the Ministerio de Educación y Ciencia to I.N. and grant (CRA)-2004 303038/2.2 from the Centre de Referència en Aqüicultura to J.G.     

The structure of the ATP-bound state of S. cerevisiae phosphofructokinase determined by cryo-electron microscopy

Phosphofructokinase (Pfk1, EC 2.7.1.11) plays a key regulatory role in the glycolytic pathway. The combination of X-ray crystallographic and biochemical data has provided an understanding of the different conformational changes that occur between the active and inhibited states of the bacterial enzyme, and of the role of the two bacterial effectors. Eukaryotic phosphofructokinases exhibit a far more sophisticated regulatory mechanism, they are more complex structures regulated by a large number of effectors (around 20). Saccharomyces cerevisiae Pfk1 is an 835 kDa hetero-octamer which shows cooperative binding for fructose-6-phosphate (F6P) and non-cooperative binding for ATP. The 3D structure of the F6P-bound state was obtained by cryo-electron microscopy to 1.1 nm resolution. This electron microscopy structure, in combination with molecular replacement using the bacterial enzyme has helped provide initial phases to solve the X-ray structure of the F6P-bound state 12S yeast truncated-tetramer. Biochemical and small-angle X-ray scattering (SAXS) studies had indicated that Pfk1 underwent a large conformational change upon Mg-ATP binding. We have calculated a reconstruction using reference-based 3D projection alignment methods from 0 degrees images acquired from frozen-hydrated preparations of the enzyme in the presence of Mg-ATP. The ATP-bound structure is more extended or open, and the calculated radius of gyration of 7.33 nm (7.0 nm for F6P) is in good agreement with the SAXS data. There is a substantial decrease in the rotational angle between the top and bottom tetramers. Interestingly, all these changes have arisen from a reorientation of the alpha- and beta-subunits in the dimers. The interface region between the alpha- and beta-subunits is now approximately half the size of the one in the F6P-bound structure. This is the first time that the 3D structure of a eukaryotic Pfk1 has been visualized in its T-state (inhibited-state).     

PI(3)Kgamma has an important context-dependent role in neutrophil chemokinesis

The directional movement of cells in a gradient of external stimulus is termed chemotaxis and is important in many aspects of development and differentiated cell function. Phophoinositide 3-kinases (PI(3)Ks) are thought to have critical roles within the gradient-sensing machinery of a variety of highly motile cells, such as mammalian phagocytes, allowing these cells to respond quickly and efficiently to shallow gradients of soluble stimuli. Our analysis of mammalian neutrophil migration towards ligands such as fMLP shows that, although PtdIns(3,4)P(2) and PtdIns(3,4,5)P(3) accumulate in a PI(3)Kgamma-dependent fashion at the up-gradient leading-edge, this signal is not required for efficient gradient-sensing and gradient-biased movement. PI(3)Kgamma activity is however, a critical determinant of the proportion of cells that can move, that is, respond chemokinetically, in reaction to fMLP. Furthermore, this dependence of chemokinesis on PI(3)Kgamma activity is context dependent, both with respect to the state of priming of the neutrophils and the type of surface on which they are migrating. We propose this effect of PI(3)Kgamma is through roles in the regulation of some aspects of neutrophil polarization that are relevant to movement, such as integrin-based adhesion and the accumulation of polymerized (F)-actin at the leading-edge.     

PI3K delta and PI3K gamma: partners in crime in inflammation in rheumatoid arthritis and beyond?

Dysregulated signal transduction in innate and adaptive immune cells is known to be associated with the development of various autoimmune and inflammatory diseases. Consequently, targeting intracellular signalling of the pro-inflammatory cytokine network heralds hope for the next generation of anti-inflammatory drugs. Phosphoinositide 3-kinases (PI3Ks) generate lipid-based second messengers that control an array of intracellular signalling pathways that are known to have important roles in leukocytes. In light of the recent progress in the development of selective PI3K inhibitors, and the beneficial effects of these inhibitors in models of acute and chronic inflammatory disorders, we discuss the therapeutic potential of blocking PI3K isoforms for the treatment of rheumatoid arthritis and other immune-mediated diseases.