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Hydroxytyrosol (HT), the major olive oil antioxidant polyphenol in cardioprotective Mediterranean diets, is endowed with anti-inflammatory and anti-atherosclerotic activity. The production of cyclooxygenase (COX)-2-dependent inflammatory eicosanoids and the functionally linked release of matrix metalloproteinase (MMP)-9 by macrophages likely contribute to plaque instability leading to acute coronary events. Objective of the study was to examine the HT effects on inflammatory markers in human activated monocytes, including MMP-9 and COX-2 activity and expression and explore HT underlying mechanisms.Human peripheral blood mononuclear cells (PBMC) and U937 monocytes were treated with 1-10 μmol/L HT before activation with phorbol myristate acetate (PMA). HT blunted monocyte matrix invasive potential and reduced MMP-9 release and expression at zymography, ELISA and RT-PCR, with an IC50 = 10 μmol/L ( P0.05), without affecting tissue inhibitor of metalloproteinase (TIMP)-1. Moreover, HT inhibited prostaglandin (PG)E2 production and COX-2 expression, without affecting COX-1. These effects were mediated by inhibition of transcription factor nuclear factor (NF)-κB and protein kinase C (PKC)α and PKCβ1 activation.HT, at nutritionally relevant concentrations, reduces MMP-9 and COX-2 induction in activated human monocytes via PKCα and PKCβ1 inhibition, thus featuring novel anti-inflammatory properties. Overall, such results contribute to explaining the vascular protective effects by olive oil polyphenols in Mediterranean diets.

In the present study, sexual gonadal differentiation and first sexual maturation of Meagre ( Argyrosomus regius ) was studied, based upon the annual changes in gonadosomatic index (GSI), gonadal histology, and the plasma steroid hormones, testosterone (T), 11-ketotestosterone (11-KT), and estradiol (E2). In addition, spermatozoa characteristics were evaluated by measuring sperm motility and morphology. Results demonstrated that Meagre completes sex differentiation at 10 to 12 mo of age, and are group-synchronous spawners, which reach puberty at 2 (mean length 26.8 ± 0.7 cm, mean weight 920 ± 75 g; N = 10) and 3 (mean length 35.8 ± 0.8 cm, mean weight 1610 ± 89 g; N = 10) years of age for males and females, respectively. In males, during the sex differentiation period, T levels were significantly higher with respect to those of 11-KT; this suggests that T has a key role in the early phases of the sex differentiation. During the spawning season an increase in plasma concentrations of all hormones was observed with 11-KT levels being significantly higher that those of T. In females, during the sex differentiation period, there was an increase in E2 plasma levels, while during the first spawning season, a significant increase of T and E2 levels were measured. Regarding sperm characteristics, the measured curvilinear velocity (VCL) and straight-linear velocity (VSL), resulted in the same order of magnitude with respect to those measured in other marine fish, while the average path velocity (VAP) was similar to that measured in the European Eel. The head of Meagre spermatozoa presents as oval shaped with a surface area of approximately 3.66 μm 2 and a perimeter of approximately 6.65 μm. All these findings represent an important basis for further investigation on the reproductive biology of this specie and may assist the farmers to improve seed production in aquaculture.

International audience; Peptide transport and expression of SoLute Carrier 15 (SLC15) peptide transporters was assessed in rat thyroid tissue and a rat thyroid cell line (PC Cl3 cells). Peptide transport was studied by monitoring the uptake of the fluorophore-conjugated dipeptide β-Ala-Lys-N-7-amino-4-methyl-coumarin-3-acetic acid (Ala-Lys-AMCA). Expression of SLC15-specific mRNA transcripts was analyzed by RT-PCR. Of the two SLC15 transporters expressed in thyroid follicular cells, namely PEPT2 (SLC15A2) and PHT1 (SLC15A4), only PEPT2 was involved in peptide transport at the plasma membrane, with PHT1 most likely being intracellular. Interestingly, at the mRNA level PEPT2 was up-regulated under TSH stimulation. These findings represent the first evidence that peptide transport occurs in thyroid follicular cells. SLC15 transporters could participate to recycling of peptides derived from extracellular and lysosomal thyroglobulin proteolysis, both essential steps for thyroid hormone synthesis.

The effects of cisplatin ( cis Pt) on the extra cellular signal-regulated kinase (ERK) and the protein kinase B (PKB/Akt), known to play important roles in promoting cell survival and in down regulating apoptosis, were investigated in thyroid cell lines. The cytotoxic effect of cis Pt was highest in normal PC-Cl3 cells, intermediate in dedifferentiated PC-E1A and PC-raf cells and lowest in fully transformed and tumorigenic PC-E1Araf cells. Cis Pt provoked ERK phosphorylation; such phosphorylation was unaltered by Go6976, a conventional PKC inhibitor, whilst blocked by low doses (0.1 μM) or high doses (10 μM) of GF109203X, an inhibitor of all PKC isozymes, in PC-Cl3 and in PC-E1Araf cells, respectively. In PC-E1Araf, but not in PC-Cl3 cells, the cis Pt-provoked ERK phosphorylation was also blocked by a myristoylated PKC-ζ pseudo substrate peptide (PS-ζ). The cytotoxic effects of cis Pt increased when cells were pre-incubated with the mitogen-activated protein kinase (MEK) inhibitor PD98059. Cis Pt provoked the phosphorylation of PKB/Akt and this effect was blocked by LY294002, a PI3K inhibitor. In PC-Cl3 cells pre-incubated with LY294002 the effects of cis Pt on ERK phosphorylation and cell mortality resulted unaffected; conversely, LY294002 reduced the ERK phosphorylation and increased cis Pt cytotoxity of in PC-E1Araf cells. Furthermore, in PC-E1Araf cells pre-incubated with LY294002 and PS-ζ ERK phosphorylation was abolished and cis Pt cytotoxicity was highest. Altogether results highlight a role for PKCs in the upstream regulation of ERK pathway facing the cell response to cis Pt treatments. Understanding the mechanisms by which cells process cis Pt provides important insights for designing more efficient platinum-based drugs.

In this paper we have tested two different procedures (the “three-step” and the “four-step” procedures) for the covalent immobilization of glutamate dehydrogenase (GDH) onto silicon supports. Atomic force microscopy (AFM), Fourier-transform infrared spectroscopy (FT-IR), fluorescence spectroscopy and an enzymatic assay were used to probe the structure and activity of the immobilized enzyme. Our results demonstrate that coupling through the “three-step” procedure does not significantly affect either the fold pattern or the activity of the enzyme, suggesting that this method could be ideally suited to the development of high quality monolayers for use in enzyme-based planar biosensors.

The role of ATP on regulation of the Na(+)/K(+)-ATPase activity in the human cancerous HeLa cells was investigated. HeLa cells stimulated with increasing ATP concentrations showed a dose-dependent inhibition of the Na(+)/K(+)-ATPase activity. These effects were also obtained by UTP. ATP and UTP provoked a rise in intracellular calcium concentration ([Ca(2+)](i)) persisting for at least 4 min. The inhibitor of phospholipase C, U73122, blocked the elevation of [Ca(2+)](i) provoked by ATP/UTP. The expression of mRNA for P2Y2 and P2Y6 receptors was demonstrated by RT-PCR. ATP/UTP activated PKC-alpha, -betaI and -epsilon isoforms, but not PKC-delta and -zeta. The inhibition of the Na(+)/K(+)-ATPase activity by ATP/UTP was blocked by Gö6976, a specific inhibitor of the calcium-dependent PKCs. In conclusion, our results suggest that ATP/UTP modulate Na(+)/K(+)-ATPase activity in HeLa cells through the P2Y2 purinoceptor via calcium mobilisation and activation of calcium-dependent PKCs.

To determine the importance of glucocorticoids in the salt water adaptation of European yellow eel we have evaluated the concentration, affinity and physical properties of glucocorticoid receptors (GR) in gill from both sea water- (SW) and freshwater-adapted (FW) animals. Using ligand binding techniques we demonstrated that high affinity GR were present in both cytosolic and nuclear fractions obtained from whole gill. Isoelectric focusing (IEF) of branchial GR indicated the presence of two distinct species, with pI values of 6.1 and 6.7. The form at pI 6.7 sedimented with a Svedberg constant of 4S on glycerol density gradients while the pI 6.1 sedimented in fractions corresponding to 9S. Treatment of the pI 6.1 form with urea (4 M) resulted in generation of the form with pI 6.7. The evidence thus suggested that the oligomeric urea-sensitive form (pI 6.1) contained a form of GR which, as a monomer, focused at pI 6.7. IEF revealed the same concentrations of the pI 6.7 form in both SW and FW. However, there was significantly more (3-fold) pI 6.1 isoform in FW than in SW, and this form decreased gradually during the course of seawater transfer. A transient increase of the nuclear-bound GR was also observed during SW adaptation. The balance between these forms could represent a dynamic parameter with important implications regarding GR function and gill responses to cortisol in salt water adaptation in teleosts.

Angiotensin II (Ang II) has an important role in cardiovascular regulation and in the control of electrolyte balance, and its role in the regulation of Na+ transcellular movements through its actions on the activity of Na + K + ATPase is well documented. We showed previously that human umbilical vein endothelial cells (HUVEC) express the Ang II type 1 (AT1) receptor, which mediates Ang II modulation of Na + K + ATPase activity (1). We here investigate the effects of Ang II on the activity of the Na + H + exchanger in HUVEC. When compared with controls, incubation of HUVEC for 20 min with different concentrations of Ang II provoked significant increases in Na + H + activity. The stimulation was dose dependent between 1 and 10 nM Ang II and varied with time of incubation up to 20 min. The maximal response, obtained with 10 nM Ang II after 20 min treatment, resulted in a 65% increment in Na + H + activity. Preincubation of HUVEC with 10 μM DuP753 blocked Na + H + activation by Ang II. These results suggest that the effects of Ang II on both the Na + K + ATPase and the Na + H + exchanger may increase the transendothelial flux of Na+ and are mediated by the AT1 receptor.

In a series of 100 breast rumours, oestrogen receptors (ER) were analysed by Scatchard plots, progesterone receptors (PR) by enzyme immunoassay and pS2 by an immunoradiometric assay. Scatchard analysis gave information on receptor heterogeneity in that there was a large variation in K-d values obtained, from 0.001-2.95 nM. This variation was largely confined to tumours containing less than 70 fmol receptor per mg protein, while tumours with higher receptor concentrations were a more homogeneous population with low K-d values. An obvious correlation between ER and PR was found; moreover, pS2 was correlated to both ER and PR. In addition, 20 of the 100 tumours gave biphasic Scatchard plots, indicating the presence of at least two oestrogen-binding moieties, with K-d values and concentrations both in the range of those of receptors, The tumours displaying biphasic Scatchard plots had very low pS2 expression, regardless of ER concentrations; this was not true for PR. It is suggested that variability in responses to endocrine therapy may be related to the heterogeneity of the ER present in breast tumours.

The fatty acid composition of intestinal mucosa of two Antarctic teleosts, Chionodraco hamatus and Trematomus bernacchii , was investigated in comparison with two temperate water species: Anguilla anguilla and Dicentrarchus labrax . Higher amounts of unsaturated and monounsaturated fatty acids were present in the Antarctic species, while the polyunsaturated fatty acid levels were not significantly different in all fish species. A higher content of phosphatidylethanolamine (a phospholipid rich in unsaturated fatty acids and with a relatively low melting point) and a lower content of sphingomyelin (a phospholipid rich in saturated fatty acids and with relatively high melting point) were observed in T. bernacchii in comparison with A. anguilla .

1. 1. In brush border membrane vesicles isolated from eel kidneys, adapted either to sea water or freshwater environments, a Na + /H + antiporter is present. 2. 2. Using a calibration plot it is possible to evaluate the amount of protons that this antiporter can accumulate inside the vesicular space. 3. 3. The activity of the antiporter seems to be affected by the salinity of the water; it is higher in animals adapted to seawater. 4. 4. This adaptation seems to occur by a J max regulation of the antiporter.

Steroid receptors and tamoxifen binding sites (TBS) were assayed in the soluble fraction of 121 primary breast cancers. Scatchard analysis of TBS in high speed supernatant (100 000 g ) showed one population of binding sites; however, biphasic plots were obtained in low speed supernatants (40 000 g ). Isoelectric focussing of supernatants preincubated with radioactive tamoxifen identified two classes of TBS (pI 4.1–4.6) which have different binding affinities and bind neither oestradiol nor diethylstilbestrol. Association between TBS and steroid receptors was: TBS positive/progesterone receptor positive 32.6%, TBS positive/glucocorticoid receptor positive 52.7%, TBS positive/oestrogen receptor positive 60% and TBS positive/androgen receptor positive 72.2%. We conclude that heterogeneous TBS are present in low speed fractions and can be easily separated from the oestrogen receptor by isoelectric focussing. The association between TBS and steroid receptor status could be of clinical value in the management of primary breast cancer.

The aquaporins (AQPs) are a family of small, hydrophobic, integral membrane proteins that allow rapid water transport across the cell membrane. Relatively few reports about AQPs in non-mammalian vertebrates exist. AQP homologous have been reported in some teleosts such as the sea bream, sea bass and eel. At the best of our knowledge, until now there is no information regarding the expression of aquaporins in fish spermatozoa. Here we show that 0.01, 0.1 and 1 mM HgCl2 (known aquaporin inhibitor) completely inhibited sperm motility, a 0.001 mM concentration was ineffective. In addition, using the RT-PCR technique, we demonstrated the presence of aquaglyceroporin mRNA in sea bream and sea bass spermatozoa. Primers used in the RT-PCR were designed for sea bream aquaglyceroporin (AY363261). The sequencing of PCR products of the expected size (213 bp) revealed that this portion of aquaporin of sea bream and sea bass sperm have an homology of 100% with that of gilthead sea bream hind-gut. The presence of AQP in sea bream and sea bass spermatozoa was also confirmed by Western blot experiments. All together our findings allow us to hypothesize that in both sea bream and sea bass sperm AQPs are involved in sperm motility initiation mechanisms.