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Membrane potential depolarization due to electrogenic peptide transport activity was examined in eel (Anguilla anguilla) intestinal brush-border membrane vesicles (BBMV) by monitoring the fluorescence quenching of the voltage-sensitive dye 3,3'-diethylthiadicarbocyanine iodide. Our experimental approach consisted of generating an internal negative membrane potential mimicking in vivo conditions and measuring membrane potential depolarization due to different extravesicular dipeptides. Peptide-dependent membrane potential depolarization was observed in both the presence and absence of extravesicular Na+ and was inhibited by diethylpyrocarbonate, which is consistent with the involvement of electrogenic, Na+-independent, H+-dependent peptide transport activity. Kinetic analysis indicated that peptide-dependent membrane potential depolarization is a saturable process (K-m,K-app similar to 1.5 mmol L-1) and that within the 0.1-10 mmol L-1 peptide range a single carrier system is involved in the transport process. Our results suggest that a peptide transport activity, kinetically resembling the PepT1(Slc15A1)-type-mediated H+/peptide cotransport action, can be monitored in eel intestinal BBMV using an easy and inexpensive fluorescence assay.

Molecular mechanisms involved in sperm motility initiation in two sparids (Sparus aurata and Lithognathus mormyrus) have been studied. Our comparative study demonstrates that osmolality is the key signal in sperm motility activation in both species, whereas K(+) and Ca(2+) do not have any role. The straight-line velocity that resulted, however, was significantly different when measured in sperm activated with non-ionic and/or calcium-free solutions with respect to that measured in seawater-activated sperm. In both species, motility initiation depends on cAMP-dependent protein phosphorylation. The phosphorylation/dephosphorylation patterns that resulted in gilthead and striped sea bream were quite different. In gilthead sea bream, the phosphorylated proteins have molecular weights of 174, 147, 138, 70, and 9-15 kDa, whereas the dephosphorylated proteins have molecular weights of 76, 57, and 33 kDa. In striped sea bream, phosphorylation after sperm motility activation occurred on proteins of 174, 147, 103, 96, 61, 57, and 28 kDa, whereas only one protein of 70 kDa resulted from dephosphorylation. Matrix-assisted laser desorption ionization-time of flight analyses allowed identification of the following proteins: In gilthead sea bream, the 9-15 kDa proteins that were phosphorylated after motility activation include an A-kinase anchor protein (AKAP), an acetyl-coenzyme A synthetase, and a protein phosphatase inhibitor, and in striped sea bream, 103- and 61-kDa proteins that were phosphorylated after motility activation were identified as a phosphatase (myotubularin-related protein 1) and a kinase (DYRK3), respectively.

The present paper reported postmortem changes in muscle proteins on sea bream (Sparus aurata), during ice storage. The postmortem evolution of protein patterns in farmed sea bream was monitored by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and two-dimension electrophoresis (2DE). Matrix associated laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) was used for identification of proteins. Results of the bio-informatics analysis demonstrate that: (1) the normalized volume of skeletal alpha-actin and tropomyosin is not affected by the time of storage; (2) the normalized volumes of myosin light chain 3 and of major histocompatibility complex (MHC) class II beta1 increase; (3) the normalized volumes of Sec 13-like and parvalbumin significantly decrease.

Malignant gliomas are lethal primary intracranial tumors. To date, little information on the role of deregulated genes in gliomas have been identified. As the involvement of miRNAs in the carcinogenesis is well known, we carried out a pilot study to identify, as potential biomarkers, differentially expressed microRNAs in blood samples of patients affected by glioma. We studied the miRNAs' expression, by means of microarray and Real-Time PCR, in 30 blood samples from glioma patients and in 82 blood samples of patients suffering from: (a) various neurological disorders (n=30), (b) primary B-lymphoma of the Central Nervous System (PCNSL, n=36) and (c) secondary brain metastases (n=16). By quantitative real time reverse-transcriptase polymerase chain reaction (qRT-PCR), we identified significantly increased levels of two candidate biomarkers, miR-15b and miR-21, in blood of patients affected by gliomas. ROC analysis of miR-15b biomarker levels allowed to differentiate patients with tumour from patients without glioma. Furthermore, combined expression analyses of miR15b and miR-21 distinguished between patients with and without glioma (90% sensitivity and 100% specificity). In addition, a decrement in the expression levels of miR-16 characterized glioblastomas compared to low grade and anaplastic gliomas. In conclusion, this pilot study suggest that it's possible to identify the disease state by meaning miR-15b and miR-21 markers in blood, while miR-16 can be used to distinguish glioblastoma from other grade gliomas. They can potentially be used as biomarkers for non-invasive diagnosis of gliomas; further studies are mandatory to confirm our preliminary findings.

In PC Cl3 cells, a rat thyroid cell line, angiotensin (Ang II) activates the atypical protein kinase C-ζ (PKC-ζ) and the extracellular signal-regulated kinase (ERK) pathways. We here studied the Ang II effects on PC Cl3 cell proliferation. It was found that Ang II: (1) induced the phosphorylation of protein kinase B (PKB), (2) induced the growth-related early gene c-fos expression, (3) enhanced the cyclin E and p27kip expression, (4) had no effects on Cdk2, and (5) did not affect the transition from G0/G1 to S phase. Inhibition of phosphoinositide-3kinase by LY294002 further increased the effect of Ang II on p27kip induction, whilst PKCs inhibition by GF109203X decreased such effect. The role of PKC-ζ was recognized by the use of a synthetic myristoylated peptide with sequences based on the endogenous PKC-ζ pseudosubstrate and by PKC-ζ downregulation using the small interfering RNA (siRNA). Insulin had a replicating effect on PC Cl3 cells, induced the phosphorylation of PKB, decreased p27kip expression and had no effect on the PKC-ζ cytosol-to-membrane translocation. PC Cl3 cell proliferation was induced more potently by simultaneous stimulation with insulin and Ang II than by stimulation with insulin alone, and the effect on p27kip expression was similar to that obtained with insulin only. These observations demonstrate that in PC Cl3 cells Ang II causes a block in G1 phase, although both ERK and PKB pathways are activated, and this effect may be due to the upregulation of p27kip and PKC-ζ operativity.

In PC Cl3 cells, a continuous, fully differentiated rat thyroid cell line, P2Y2 purinoceptor activation provoked a transient increase of [Ca2+]i, followed by a decreasing sustained phase. The α and β1 protein kinase C (PKC) inhibitor Gö6976 decreased the rate of decrement to the basal [Ca2+]i level and increased the peak of Ca2+ entry of the P2Y2-provoked Ca2+transients. These effects of Gö 6976 were not caused by an increased permeability of the plasma membrane, since the Mn2+ and Ba2+ uptake were not changed by Gö 6976. Similarly, the Na+/Ca2+ exchanger was not implicated, since the rate of decrement to the basal [Ca2+]i level was equally decreased in physiological and Na+-free buffers, in the presence of Gö 6976. On the contrary, the activity of the sarcoplasmic–endoplasmic reticulum Ca2+ATPase (SERCA) 2b was profoundly affected by Gö 6976 since the drug was able to completely inhibit the stimulation of the SERCA 2b activity elicited by P2-purinergic agonists. Finally, the PKC activator phorbol myristate acetate had effects opposite to Gö 6976, in that it markedly increased the rate of decrement to the basal [Ca2+]i level after P2Y2 stimulation and also increased the activity of SERCA 2b. These results suggest that SERCA 2b plays a role in regulating the sustained phase of Ca2+ transients caused by P2Y2 stimulation.

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.

In the present study we used two-dimensional polyacryl amide gel electrophoresis (2-DE) and matrix-associated laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry to verify whether the protein expression of sea bass sperm was affected by the cryopreservation procedure. The protein profiles differed between fresh and frozen-thawed semen as revealed by visual inspection and by image analysis software. We identified 163 spots in fresh sperm; among these, 13 were significantly decreased and 8 were absent in two-dimensional gel obtained with cryopreserved sperm. Five of these spots were analyzed with MALDI-TOF, but only three showed a significant match in the databases used in bio-informatics analysis (Pept-Ident, Mascot, and MS-Fit). In particular, spot 5 showed homology with a novel protein of zebrafish (similar to SKB1 of human and mouse), spot 13 showed homology with amphibian G1/S-specific cyclin E2, and spot 20 showed homology with the hypothetical protein DKFZp566A1524 of Brachidanio rerio. The present work shows that the use of the cryopreservation procedure causes the degradation of sperm proteins and among these, two could be at least partially responsible for the observed decrease in sperm motility duration and the lower hatching rate of eggs fertilized with cryopreserved sperm.

We demonstrated previously that in serum-starved MCF-7 breast cancer cell line, Ang II increased Na+/K+ATPase activity and activated the protein kinase C zeta (PKC-zeta) (Muscella et al., 2002 J Endocrinol 173:315-323; 2003 J Cell Physiol 197:61-68.). The aim of the present study was to investigate the modulation of the activity of the Na+/K+ATPase by PKC-zeta in MCF-7 cells. Here, using serum-starved MCF-7 cells, we have demonstrated that the effect of Ang II on the Na+/K+ATPase activity was inhibited by a synthetic myristoylated peptide with sequences based on the endogenous PKC-zeta pseudosubstrate region (zeta-PS) and by high doses of GF109203X, inhibitor of PKCs. When MCF-7 cells, grown in 10% fetal bovine serum (FBS), were stimulated with Ang II a dose- and time-dependent inhibition of the Na+/K+ATPase activity was obtained. Under this growth condition we found that mRNAs for AT1, AT2, and for Na+/K+ATPase alpha1 and alpha3 subunits were unchanged; besides both the activity of the Na+/K+ATPase and the level of PKC-zeta also were unaffected by the serum. The atypical PKC-iota level (present in very low abundance in serum-starved MCF-7) was increased and Ang II provoked its translocation from the cytosol to plasma membrane. PKC-zeta was localized to the membrane, and upon Ang II treatment its cellular localization did not change. The Ang II-mediated decrease of the Na+/K+ATPase activity was inhibited by high doses of GF109203X but not by zeta-PS, thus indicating that such effect was not due to PKC-zeta activity. The treatment of cells with PKC-iota antisense oligodeoxynucleotides inhibited the effects of Ang II on the Na+/K+ATPase activity. Additionally, the effect of Ang II on Na+/K+ATPase activity was also blocked by the phosphatidylinositol 3-kinase (PI3K) inhibitors, wortmannin and LY294002, and by the actin depolymerizing agents, cytochalasin D. In conclusion, in MCF-7 cells Ang II modulates the Na+/K+ATPase activity by both atypical PKC-zeta/-iota. The effects of Ang II are opposite depending upon the presence of the serum-sensitive PKC-iota, with the inhibitory effect possibly due to the redistribution of sodium pump from plasma membrane to the inactive intracellular pool.

We have previously shown that HeLa cells express P2Y2 and P2Y6 receptors endogenously and determined the pathways by which the P2Y2 controls proliferation and Na+/K+ATPase activity. Our objective in this study was to investigate the hypothesis that P2Y6 also controls proliferation and Na+/K+ATPase activity; the pathways used in these actions were partially characterised. We found that P2Y6 activation controlled cell proliferation but not the activity of the Na+/K+ATPase. UDP activation of P2Y6 provoked: (a) an increase in free cytosolic calcium; (b) the activation of protein kinase C-alpha, -beta, -delta, -epsilon, and -zeta but not of PKC-iota and -eta; (c) the phosphorylation of the extracellular signal-regulated protein kinases 1 and 2 (ERK1/2); (d) the expression of c-Fos protein. The P2Y6 induced cell proliferation was blocked by the mitogen-activated protein kinase kinase (MAPKK) inhibitor PD098059, thereby indicating that the ERK pathway mediates the mitogenic signalling of P2Y6. PKC and phosphoinositide 3-kinase (PI3K) inhibitors were tested at two different time points of ERK1/2 phosphorylation (10 and 60 min). The results suggest that novel PKCs and PI3K initiate the response but both conventional and atypical PKCs are required for the maintenance of the UDP-induced phosphorylation of ERK1/2. The induction of c-Fos was greatly diminished by conventional or atypical PKC-zeta inhibition, suggesting that it may be due to PKC-alpha/beta and -zeta activity. These observations demonstrate that UDP acts as a proliferative agent in HeLa cells activating multiple signalling pathways involving conventional, novel, and atypical PKCs, PI3K, and ERK. Of these pathways, conventional and atypical PKCs appear responsible for the induction of c-Fos, while ERK is responsible for cell proliferation and depends upon both novel and atypical PKCs and PI3K activities.

The goal of the present study was to evaluate the changes in the cell type composition and ATPase activities (total ATPase, ouabain-sensitive Na+/K(+)-ATPase, furosemide-sensitive Na(+)-ATPase) that occur during the different stages of the moulting cycle in the hepatopancreas of the Marsupenaeus japonicus. The results clearly suggest that the number of resorptive and fibrillar cell types changes significantly during the different stages. An inverse correlation between resorptive and fibrillar cells is observed during moulting (both in normally fed and fasted animals). Fasting, but not the moulting cycle, affects the number of blister-like cells. In the resorptive cells the enzymatic activities (total ATPases and ouabain-sensitive Na+/K(+)-ATPase) also change during the moulting in a cyclical manner. All these results are in agreement with and confirm the different functions carried out by the two cell types within the hepatopancreas.

Objective—Epidemiology suggests that Mediterranean diets are associated with reduced risk of cardiovascular disease. Because monocyte adhesion to the endothelium is crucial in early atherogenesis, we evaluated whether typical olive oil and red wine polyphenols affect endothelial–leukocyte adhesion molecule expression and monocyte adhesion.Methods and Results—Phytochemicals in olive oil and red wine, including oleuropein, hydroxytyrosol, tyrosol, elenolic acid, and resveratrol, with or without antioxidant activity, were incubated with human umbilical vein endothelial cells for 30 minutes, followed by co-incubation with bacterial lipopolysaccharide or cytokines to trigger adhesion molecule expression. At nutritionally relevant concentrations, only oleuropein, hydroxytyrosol, and resveratrol, possessing a marked antioxidant activity, reduced monocytoid cell adhesion to stimulated endothelium, as well as vascular cell adhesion molecule-1 (VCAM-1) mRNA and protein by Northern analysis and cell surface enzyme immunoassay. Reporter gene assays with deletional VCAM-1 promoter constructs indicated the relevance of nuclear factor-κB, activator protein-1, and possibly GATA binding sites in mediating VCAM-1 transcriptional inhibition. The involvement of nuclear factor-κB and activator protein-1 was finally demonstrated at electrophoretic mobility shift assays.Conclusions—Olive oil and red wine antioxidant polyphenols at nutritionally relevant concentrations transcriptionally inhibit endothelial adhesion molecule expression, thus partially explaining atheroprotection from Mediterranean diets.

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.

The effect of angiotensin II (Ang II) on Ca(2+) signalling in human primary cultured breast epithelial cells was investigated by using fura-2 as the Ca(2+) probe. Ang II (0.1-1000 nM) induced an intracellular free calcium ([Ca(2+)](i)) transient peak which was unchanged by external Ca(2+ )removal. In Ca(2+)-free medium pretreatment with thapsigargin abolished Ang II-induced Ca(2+ )release. Suppression of 1,4,5-inositol trisphosphate formation by U73122, a phospholipase C inhibitor, blocked the Ang II-induced Ca(2+) response. Losartan (DuP753), an inhibitor of Ang II type I receptor (AT1), decreased the [Ca(2+)](i) increase evoked by Ang II, while CGP4221A, an inhibitor of Ang II type II receptor (AT2) did not. AT1 desensitisation was demonstrated with respect to the Ca(2+) response after subsequent exposure of cells to Ang II and also after pretreatment for 25 min with 1000 nM phorbol 12-myristate 13-acetate. Staurosporine, an inhibitor of protein kinases C (PKC), inhibited the AT1 desensitisation. Epithelial breast cells expressed PKC-alpha, -beta1, -delta and -zeta isozymes, and Ang II provoked translocation from the cytosol to the membranes of PKC-alpha, -beta1, and -delta (but not -zeta). Ang II was also able to stimulate cell proliferation in a dose-dependent manner; this effect was blocked by Go 6976, a specific inhibitor of PKC-alpha and -beta1, the Ca(2+)-dependent isozymes. The main conclusion of this study is that the the Ang II signalling mechanism in breast epithelial cells is based on the elevation of [Ca(2+)](i )released from intracellular stores through AT1 activation. In addition, Ang II stimulates cell proliferation by the activation of PKC isozymes.

The effect of carbachol (Cch) on intracellular calcium concentration ([Ca2+]i) in eel enterocytes was examined using the fluorescent Ca2+ indicator fura-2. Cch caused a biphasic increase in [Ca2+]i, with an initial spike followed by a progressively decreasing level (over 6 min) to the initial, pre-stimulated, level. The effect of Cch was dose-dependent with a 7.5-fold increase in [Ca2+]i over basal level induced by the maximal dose of Cch (100 microM). In Ca2+-free/EGTA buffer the effect of Cch was less pronounced and the [Ca2+]i returned rapidly to basal levels. The increment of [Ca2+]i was dose-dependently attenuated in cells pre-treated with U73122, a specific inhibitor of phospholipase C, suggesting that the Cch-stimulated increment of [Ca2+]i required inositol triphosphate formation. In the presence of extracellular Ca2+, thapsigargin (TG), a specific microsomal Ca2+-ATPase inhibitor, caused a sustained rise in [Ca2+]i whereas in Ca2+-free medium the increase in [Ca2+]i was transient; in both cases, subsequent addition of Cch was without effect. When 2 mM CaCl2 were added to the cells stimulated with TG or with Cch in Ca2+-free medium, a rapid increase in [Ca2+]i was detected, corresponding to the capacitative Ca2+ entry. Thus, both TG and Cch depleted intracellular Ca2+ stores and stimulated influx of extracellular Ca2+ consistent with capacitative Ca2+ entry. K+ depolarization obtained with increasing concentrations of KCl in the extracellular medium induced a dose-related increase in [Ca2+]i which was blocked by 2 microM nifedipine, a non-specific L-type Ca2+ channel blocker. Nifedipine also changed significantly the height of the Ca2+ transient, and the rate of decrement to the pre-stimulated [Ca2+]i level, indicating that Ca2+ entry into enterocytes also occurs through an L-type voltage-dependent calcium channel pathway. We also show that isolated enterocytes stimulated with increasing Cch concentrations (0.1-1000 microM) showed a dose-dependent inhibition of the Na+/K+-ATPase activity. The threshold decrease was at 1 microM Cch; it reached a maximum at 100 microM (50.5% inhibition) and did not decrease further with the use of higher dose. The effect of Cch on Na+/K+-ATPase activity was dependent on both protein kinase C (PKC) and protein phosphatase calcineurin activation since the PKC inhibitor calphostin C abolished Cch effects, while the calcineurin inhibitor FK506 augmented Cch effect. Collectively, these data establish a functional pathway by which Cch can modulate the activity of the Na+/K+-ATPase through a PKC-dependent (calphostin C-sensitive) pathway and a calcineurin-dependent (FK506-sensitive) pathway.

SUMMARY Carbonic anhydrase (CA) activity was measured in blood, intestine, kidney and gill of two Antarctic teleosts, the haemoglobinless Chionodraco hamatus and the red-blooded Trematomus bernacchii, and of the temperate teleost Anguilla anguilla. In all species, the highest CA activity was in the gills, with the greatest activity in C. hamatus. CA activity in the blood was highest in A. anguilla, but none was detected in the blood of C. hamatus despite the presence of plasma CA inhibitors. The enzyme was present but its activity was low in the intestine and kidney of all three species. The existence of very high CA activity in C. hamatus gills compared with the red-blooded species was investigated further by isolating and characterising the branchial cytosolic CA isoforms. The turnover rate of the C. hamatus isoform was significantly higher than that of T. bernacchii and A. anguilla. The isoforms from both the Antarctic species exhibited lower apparent Km (Km,app) and heat stability than those from A. anguilla. Sensitivity to sulphonamides was similar in all species and was within the range of the mammalian CA II isoform. The branchial CA isoforms of C. hamatus, T. bernacchii and A. anguilla displayed relative molecular masses of 28.9, 29.9 and 31.2 kDa, respectively. The results suggest that the hemoglobinless teleost possesses a different branchial cytosolic CA isoform from that of red-blooded teleosts.

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.

This work reports recent data on mechanisms of cold adaptation exhibited by the Antarctic teleosts Trematomus bernacchii and Chionodraco hamatus. Analysis of fatty acid in intestinal mucosa brush border suggested that an increase in unsaturated fatty acid could be a mechanism for the preservation of cell membrane integrity and functionality. The investigation of several transporters involved in the regulation of cell homeostasis (Na+/K+‐AT‐Pase, Na+‐D‐glucose cotransport, Na+/H+ exchanger and Ca++‐AT‐Pase) showed kinetic characteristics that could explain part of the adaptation of these proteins to work at low temperature. The activity of carbonic anhydrase, involved in pH control both at intra‐cellular and systemic level, was related to plasma buffer capacity.

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.

Immunocytochemistry of paraffin-embedded and cryostat sections of eel (Anguilla anguilla) gill showed that angiotensin II receptors (Ang II-R) were present in chloride cells, uniformly distributed in the cytoplasm and on surface membranes. Computerised image analysis of these preparations showed that gills from sea water (SW)-adapted animals had a significantly (3-fold) higher Ang II-R concentration compared with freshwater (FW)-adapted eel gills. Isoelectric focusing gel electrophoresis revealed two Ang II-R isoforms with pI 6·5 and 6·6 that were differentially modulated by environmental salinity: they were equally abundant in SW while in FW the pI 6·6/pI 6·5 ratio was 1·66. Using catalytic cytochemistry with image analysis, gill chloride cell membrane Na+/K+ATPase activity was shown to increase 4-fold in response to SW adaptation. Additionally, perfusion of gills for 30 min with 0·1, 10 or with 100 nM Ang II provoked a dose-dependent increment in Na+/K+ATPase activity in FW, and a biphasic response in SW gills in which activity was significantly increased at low Ang II concentrations but was reduced to basal values at 100 nM. The data suggest that adaptation to sea water significantly increases Ang II-R concentration in the chloride cell and, together with the effects of Ang II on Na+/K+ATPase activity, suggest a role for this hormone in gill NaCl retention. The different responses of Na+/K+ATPase to Ang II stimulation in FW and SW may be attributed to the presence of two receptor subtypes that are differently modulated by salinity and that have opposing effects on Na+/K+ATPase.

Adaptation of organisms to extreme environments requires proteins to work at thermodynamically unfavorable conditions. To adapt to subzero temperatures, proteins increase the flexibility of parts of, or even the whole, 3D structure to compensate for the lower thermal kinetic energy available at low temperatures. This may be achieved through single-site amino acid substitutions in regions of the protein that undergo large movements during the catalytic cycle, such as in enzymes or transporter proteins. Other strategies of cold adaptation involving changes in the primary amino acid sequence have not been documented yet. In Antarctic icefish ( Chionodraco hamatus ) peptide transporter 1 (PEPT1), the first transporter cloned from a vertebrate living at subzero temperatures, we came upon a unique principle of cold adaptation. A de novo domain composed of one to six repeats of seven amino acids (VDMSRKS), placed as an extra stretch in the cytosolic COOH-terminal region, contributed per se to cold adaptation. VDMSRKS was in a protein region uninvolved in transport activity and, notably, when transferred to the COOH terminus of a warm-adapted (rabbit) PEPT1, it conferred cold adaptation to the receiving protein. Overall, we provide a paradigm for protein cold adaptation that relies on insertion of a unique domain that confers greater affinity and maximal transport rates at low temperatures. Due to its ability to transfer a thermal trait, the VDMSRKS domain represents a useful tool for future cell biology or biotechnological applications.

SummaryMatrix metalloproteinase (MMP)-9 plays an important role in stroke by accelerating matrix degradation, disrupting the blood-brain barrier and increasing infarct size. Dipyridamole is an antiplatelet agent with recognised benefits in ischaemic stroke prevention. In addition to its antiplatelet properties, recent studies have reported that dipyridamole also features anti-inflammatory and anti-oxidant properties. We therefore investigated whether dipyridamole can ameliorate the proinflammatory profile of human monocytes, a source of MMP-9 in stroke, in terms of regulation of MMP-9 activity and expression, and explored underlying mechanisms. Human peripheral blood mononuclear cells (PBMC) and U937 cells were treated with increasing concentrations of dipyridamole (up to 10 µg/ml) for 60 minutes before stimulation with tumour necrosis factor (TNF)-α or phorbol myristate acetate (PMA). Exposure of PBMC and U937 to dipyridamole reduced TNF-α- and PMA-induced MMP-9 activity and protein release as well as MMP-9 mRNA, without significantly affecting the release of TIMP-1. This inhibitory effect was independent of dipyridamole-induced cyclic adeno-sine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) increase. Correspondingly, dipyridamole also significantly inhibited TNF-α-induced nuclear factor (NF)-κB activation and nuclear translocation of the p65 NF-κB subunit through a mechanism involving the inhibition of IkBα degradation and p38 MAPK activation. In conclusion, dipyridamole, at therapeutically achievable concentrations, reduces the expression and release of MMP-9 through a mechanism involving p38 MAPK and NF-κB inhibition. These results indicate that dipyridamole exerts anti-inflammatory properties in human monocytes that may favourably contribute to its actions in the secondary prevention of stroke, independent of its antiplatelet properties.

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 .

Morphological and physiological characteristics of the European eel (Anguilla anguilla) intestine were analyzed in two different stages (yellow and silver) of the fish ontogenic development. Intestine/body (Wi/Wa) and scraped mucosa/intestine (Wsc/Wi) wet weight ratios were significantly (P < 0.01) reduced in seawater acclimated silver eels compared to yellow ones. Electrogenic, Na+-dependent transport rates of a number of nutrients (D-glucose, L-proline, β-alanine, L-phenylalanine, L-methylalanine) were indirectly measured in intestinal brush border membrane vesicles of seawater acclimated eels, by monitoring the fluorescence quenching of the electrical membrane potential sensitive dye 3,3′-diethylthiocarbocyanine iodide [Dis-C2(5)], and were significantly lower (P < 0.05 for L-proline; P < 0.01 for all the others) in silver eels compared to yellow ones. The transepithelial electrical potential difference (Vt) and short-circuit current (Isc), measured by applying the short-circuit current technique to “in vitro” isolated intestine, were significantly higher in silver eels compared to yellow ones in both sea- and freshwater. The addition of 10−5 M bumetanide, inhibitor of the Na+-K+-2Cl− cotransporter, to the mucosal bathing solution almost completely abolished Vt and Isc in either yellow or silver eels intestine acclimated to both seawater and freshwater. Further, by using the microelectrode technique, it was shown that the apical membrane potential (Vm) averaged -27.3 ± 2.3 mV in yellow eels and -42.9 ± 3.3 mV in silver eels (P = 0.02). It is concluded that the changes in morphological (mucosal weight) and physiological (nutrient transport rates and electrophysiological parameters) characteristics found in silver compared to yellow eel intestine could represent adaptative features preparatory to the onset of the prolonged marine migration of the silver eel. © 1996 Wiley-Liss, Inc.

The pH-sensitive fluorescent dye, 2′,7′-bis-carboxy-ethyl-5,6-carboxyfluorescein acetoxymethyl ester, was used to examine the effects of fish or human angiotensin II (Ang II) on the activity of the basolateral located Na+/H+ antiporter in eel intestinal cell suspensions. Exposure of eel enterocytes to either hormone led to an increased activity of the antiporter. This time- and dose-dependent stimulatory effect was inhibited by the specific antiporter inhibitor dimethylamiloride (DMA). Preincubation with a monoclonal antibody (6313/G2), directed against the N-terminal extracellular domain of the mammalian AT1 Ang II receptor, prevented the stimulatory effect of the hormone and inhibited the binding of [3,5-3H]Tyr4-Ile5-Ang II to intestinal cell suspensions, suggesting specific binding of the antibody to the eel Ang II receptor. The results indicate that both fish and human Ang II stimulate the DMA-sensitive Na+/H+ antiporter present in eel intestinal cells by means of a mammalian AT1-like receptor.

We evaluated the presence and variability of oestrogen receptor (ER) isoforms in endometrial cancer by using [3H]oestradiol-labelled ERs and the H222 monoclonal antibody obtained from the Abbott enzyme immunoassay kit. Using isoelectric focusing (IEF), endometrial ER was shown to be composed of four different species, with pI values of 6·1, 6·3, 6·6 and 6·8, indistinguishable from the isoforms found in normal rat uterus, and human breast and larynx carcinomas. The isoforms at pI 6·3, 6·6 and 6·8, all sedimenting at 4S by sucrose gradient fractionation, showed, on two-dimensional SDS electrophoresis, relative masses of 50, 70 and 65 kDa respectively, equal to the masses previously found in breast cancer. These isoforms did not alter their pI values during IEF fractionation performed in a linear gradient of urea, while the pI 6·1, sedimenting at 8S, generated a new isoform at about 9 mol/l urea with pI 7·2 and a relative mass of 65 kDa. The urea-dissociated isoform (pI 7·2) was able approximately to double the antibody binding with respect to the non-dissociated oligomer, which suggested that some epitopes are 'masked', i.e. not accessible to the antibodies when ER is present in its complexed form. The evidence thus suggested that the oligomer at pI 6·1 contained a single 65 kDa ER form which, as a monomer, focused at pI 7·2. The variability in the ER isoform profile found in endometrial cancer was similar to the variability previously reported in breast and larynx carcinomas. The balance between these isoforms could be a dynamic parameter involved in the functionality of this receptor and consequently in cell transformation.

An altered expression of microRNAs (miRNAs) contributes both to the development of cancer and to the progression of the disease. Malignant tumours and tumour cell lines have widespread deregulated expressions of miRNAs compared to normal tissues. In this study, we investigated the expression profiles of 340 mammalian miRNAs in 93 cases of multiform glioblastoma (primary and secondary glioblastoma tumours), by means of DNA microarrays. We show that the expression profiles of 10 miRNAs can distinguish primary from secondary glioblastoma types. Moreover, we found elevated miR-155 levels in primary and secondary glioblastoma tissues as well as in glioblastoma primary cultures. We hypoth- esised that γ-aminobutyric acid A receptor 1 (GABRA1) is a miR-155 target, and studied the correlation between miR-155 up-regulation and the GABRA1 protein in cultured glioblas- toma cells by miRNA silencing. We show that a decrease in miR-155 expression to normal levels restores the expression of GABRA1, making glioblastoma cells sensitive to signals that inhibit cell proliferation mediated by GABRA1. In conclu- sion, the expression patterns of different miRNAs characterise primary and secondary glioblastomas. The aberrant overex- pression of miR-155 contributes to the malignant phenotype of glioblastoma cells removing growth inhibition. * Contributed equally

Diets with high content of antioxidant polyphenols are associated with low prevalence of cardiovascular diseases and cancer. Inflammatory angiogenesis is a key pathogenic process both in cancer and atherosclerosis, and is tightly regulated by the proinflammatory enzyme cyclooxygenase (COX)-2 and the matrix degrading enzymes matrix metalloproteinases (MMPs). We studied the effects of antioxidant polyphenols from virgin olive oil (oleuropein and hydroxytyrosol) and red wine (resveratrol and quercetin) on endothelial cell angiogenic response in vitro, and explored underlying mechanisms. Cultured endothelial cells were pre-incubated with 0.1-50 μmol/L polyphenols before stimulation with phorbol myristate acetate (PMA). All tested polyphenols reduced endothelial cell tube formation on matrigel and migration in wound healing assays. The reduced angiogenesis was associated with the inhibition of PMA-induced COX-2 protein expression and prostanoid production, as well as MMP-9 protein release and gelatinolytic activity. These effects were accompanied by a significant reduction in the stimulated intracellular reactive oxygen species levels and in the activation of the redox-sensitive transcription factor nuclear factor (NF)-κB. Our findings reveal that olive oil and red wine polyphenols reduce inflammatory angiogenesis in cultured endothelial cells, through MMP-9 and COX-2 inhibition, supporting a potential protective role for dietary polyphenols in atherosclerotic vascular disease and cancer.

Humangenesencodingpassivetrans- porters, ion-coupled transporters, and ex- changers are all included in the so-called SoLute Carrier (SLC) gene series (the Hu- man Genome Organization Gene Nomencla- ture Committee; http://www.genenames.org/), consisting of 51 families and at least 378 genes (http://www.bioparadigms.org). Or- tholog genes encoding for transport proteins of the SLC series have comparatively been de- scribed in teleost fish, although their functional properties, in terms of kinetic parameters, sub- strate specificities, and inhibition patterns of theexpressedtransportproteins,haveonlyspo- radically been assessed in vitro. This chapter gives the latest updates (March 2011) for the SLC families and their members in teleost fish as well as relevant links to GenBank database and literature. By using a functional genomics approach, a list (version 1.0) of all currently known SLC families in teleost fish is provided in the form of SLC tables.

Previous studies have shown the effects of angiotensin II (Ang II) in teleosts, and Ang II-binding sites have also been localized in tissues from rainbow trout. The purpose of this study was to extend these findings and to provide an analysis of Ang II receptor (Ang II-R) isoforms in three tissues obtained from European eel (Anguilla anguilla). Ang II-Rs were identified in eel liver, kidney and intestine membranes by the binding of either 0·5 nmol human 125I-labelled Tyr4-lle5-Ang II/l or increasing concentrations (1–120 nmol/l) of [3,5-3H]Tyr4-Ile5-Ang II. Using an isoelectric focusing technique, two Ang II-binding sites were identified in liver membranes. These migrated to isoelectric points (pI values) 6·5 and 6·7. Seventy per cent of binding to both sites was displaced by a 10 000-fold excess of unlabelled human Ang II. In both whole plasma membranes and brush border membranes from intestine, only one form of the Ang II-R was found, with pI 6·5 and high affinity (Kd=3·4 nmol/l) for the [3,5-3H]Tyr4-Ile5-Ang II. Similarly, only the isoform focusing at pI 6·5 was observed in renal tubular epithelial brush border membranes. Reduction of disulphide bridges with dithiothreitol significantly enhanced Ang II binding to the isoform at pI 6·5 in liver (PPP The data suggest the existence in eel liver of multiple forms of Ang II-R, which may have different functions, while one single form appeared to be present in enterocyte plasma membrane and in renal brush border membrane.

8 páginas, 2 figuras, 3 tablas., Our previous studies demonstrated that osmolality is the key signal in sperm motility activation in Sparus aurata spermatozoa. In particular, we have proposed that the hyper-osmotic shock triggers water efflux from spermatozoa via aquaporins. This water efflux determines the cell volume reduction and, in turn, the rise in the intracellular concentration of ions. This increase could lead to the activation of adenylyl cyclase and of the cAMP-signaling pathway, causing the phosphorylation of sperm proteins and then the initiation of sperm motility. This study confirms the important role of sea bream AQPs (Aqp1a and Aqp10b) in the beginning of sperm motility. In fact, when these proteins are inhibited by HgCl2, the phosphorylation of some proteins (174 kDa protein of head; 147, 97 and 33 kDa proteins of flagella), following the hyper-osmotic shock, was inhibited (totally or partially). However, our results also suggest that more than one transduction pathways could be activated when sea bream spermatozoa were ejaculated in seawater, since numerous proteins showed an HgCl2(AQPs)-independent phosphorylation state after motility activation. The role played by each different signal transduction pathways need to be clarified.

It was previously demonstrated that [Pt(O,O'-acac)(γ-acac)(DMS)] exerted toxic effects at high doses, whilst sub-cytotoxic concentrations induced anoikis and decreased cell migration. Aim of this study was to investigate the hypothesis that [Pt(O,O'-acac)(γ-acac)(DMS)] alters the [Ca(2+)](i) and that this is linked to its ability to trigger rapid apoptosis in MCF-7 cells. Thus, cells were treated with [Pt(O,O'-acac)(γ-acac)(DMS)] and its effects on some of the systems regulating Ca(2+) homeostasis were studied, also in cells dealing with the complex changes occurring during the Ca(2+) signalling evoked by extracellular stimuli. [Pt(O,O'-acac)(γ-acac)(DMS)] caused the decrease of PMCA activity (but not SERCA or SPCA) and Ca(2+) membrane permeability. These two opposite effects on [Ca(2+)](i) resulted in its overall increase from 102±12nM to 250±24nM after 15min incubation. The effects of [Pt(O,O'-acac)(γ-acac)(DMS)] were also evident when cells were stimulated with ATP: the changes in Ca(2+) levels caused by purinergic stimulation resulted altered due to decreased PMCA activity and to the closure of Ca(2+) channels opened by purinergic receptor. Conversely, [Pt(O,O'-acac)(γ-acac)(DMS)] did not affect the store-operated Ca(2+) channels opened by thapsigargin or by ATP. [Pt(O,O'-acac)(γ-acac)(DMS)] provoked the activation of PKC-α and the production of ROS that were responsible for the Ca(2+) permeability and PMCA activity decrease, respectively. The overall effect of [Pt(O,O'-acac)(γ-acac)(DMS)] is to increase the [Ca(2+)](i), an effect that is likely to be linked to its ability to trigger rapid apoptosis in MCF-7 cells. These data reinforce the notion that [Pt(O,O'-acac)(γ-acac)(DMS)] would be a promising drug in cancer treatment.

he enzymatic activity (expressed as milliunits per milligram total proteins) of three intestinal brush-border membrane enzymes, leucine aminopeptidase, alkaline phosphatase and maltase, measured over a range of temperatures between 1.5 and 37-degrees-C, has been found to be much higher in the Antarctic fish Pagothenia bernacchii than in the temperate fish Anguilla anguilla. To explain this experimental observation the apparent Michaelis-Menten constant, the maximal velocity, the activation energy values and the thermal stability of these three enzymes were measured. The apparent Michaelis-Menten constant values of leucine amino peptidase and alkaline phosphatase were different in the intestine mucosal homogenate of the two fish at each measured temperature (from a minimum of 2.5 to a maximum of 37-degrees-C). However, the values found at 2.5-degrees-C for the Antarctic species and 15-degrees-C for the eel where comparable. Furthermore, its value was unchanged in eel intestine apical membranes, both in the presence and without enzyme lipid microenvironment. While the maximal enzymatic activities of the leucine aminopeptidase and maltase did not decrease without their enzyme lipid microenvironment, produced by treatment with Triton X-100, the impairment of alkaline phosphatase maximal activity cannot be significantly differentiated from a non-specific inhibitory effect of the detergent. The activation energy values of leucine amino peptidase, alkaline phosphatase and maltase were lower in the Antarctic fish (11.7, 5.6 and 11.8 kcal.mol-1, respectively) than in the eel (13.6, 7.6 and 13.1 kcal . mol-1, respectively). The thermal stability of alkaline phosphatase and maltase is different in Pagothenia bernacchii and Anguilla anguilla intestinal homogenate.

Transport of L-ascorbate by intestinal brush-border membrane vesicles of European eel Anguilla anguilla was stimulated by a transmembrane Na gradient (out > in) but not by a similarly directed gradient of K. Under short-circuited membrane potential conditions, a kinetic analysis of L-ascorbate influx indicated the presence of a single Na-dependent carrier process (Kapp = 0.75 +/- 0.07 mM and Jmax = 0.33 +/- 0.03 nmol.mg protein-1.min-1) and a nonsaturable transfer component with an apparent diffusional permeability (P) of 0.27 +/- 0.02 microliter.mg protein-1.min-1. D-Isoascorbate was a competitive inhibitor of L-ascorbate influx exhibiting a Ki of 8.21 +/- 0.63 mM. The electrogenic nature of +Na-L-ascorbate cotransport was confirmed by a stimulatory effect of an inside-negative membrane potential on vitamin uptake. Hill analysis of L-ascorbate influx over a wide range of external Na concentrations suggested a 2 Na-to-1 L-ascorbate binding ratio. Results indicate that the vitamin L-ascorbate is transported across fish intestinal brush-border membranes by an electrogenic Na-dependent carrier process in conjunction with more than one sodium ion.

Aberrant methylation of CpG islands in the promoter regions of tumour cells results in loss of gene function. In addition to genetic lesions, changes in the methylation profile of the promoters may be considered a factor for tumour-specific aberrant expression of the genes.We investigated the methylation status of E-cadherin gene (CDH1) promoter in low-grade glioma and correlated it with clinical outcome. Eighty-four cases of low-grade glioma (43 diffuse astrocytomas, 27 oligodendrogliomas and 14 oligoastrocytomas) with assessable paraffin-embedded tumour blocks and normal brain tissue, derived from non-cancerous tissue adjacent to tumour and commercially normal brain tissue, were collected, from which we determined CDH1 promoter methylation status and E-cadherin protein expression by methylation-specific polymerase chain reaction (MSP) and immunohistochemistry, respectively. CDH1 promoter was found hypermethylated in 54 out of 84 low grade gliomas (64%) compared with 84 normal brain tissue. CDH1 hypermethylation was found in 65% astrocytomas, 66% oligodendrogliomas and 57% oligoastrocytomas. A significant correlation between hypermethylation status, patient survival and progression-free survival was found (P = 0.04). Survival and progression-free survival were lower in patients with hypermethylated CDH1 promoter. We found that 15 astrocytomas, 9 oligodendrogliomas and 6 oligoastrocytomas were immunoreactive for E-cadherin. The incidence of loss of immunoreactivity for E-cadherin decreased significantly with age, overall survival and progression-free survival (P = 0.001, Kaplan–Meier test). We have demonstrated that CDH1 promoter hypermethylation significantly associated with down-regulated E-cadherin expression and overall survival of patients. This may have a bearing on the prognosis of low-grade glioma.

We developed an array-based subtractive hybridization system for one-step high-throughput subtraction. We printed subtractor RNA up to 10.000 times obtaining an excellent contact surface using a little amount of RNA. During hybridization cDNA, common to subtractor and target samples, remains attached to slide immobilized RNA, leaving free in solution target specific cDNA which after retrieval is cloned.

AIMS: Cyclooxygenase (COX)-2 expression is increased in inflammation and angiogenesis and also in atherosclerotic plaques, where it co-localizes with metalloproteinases (MMPs) involved in the fibrous cap weakening. Insight into the regulation of COX-2 and MMP-9 expression suggests the involvement of a Rho-dependent pathway. Because statins interfere with Rho activation, we investigated the statin effect on COX-2 and MMP expressions in the human endothelium. METHODS AND RESULTS: Simvastatin and atorvastatin were incubated with endothelial cells for 12 h before stimulation with phorbol myristate acetate or tumour necrosis factor-alpha, for times suitable to assess the endothelial tube differentiation on Matrigel and COX-2 and MMPs activities, proteins, and mRNA expressions. At 0.1-10 micromol/L, both statins reduced COX-2 expression and activity, without affecting COX-1. The statin effect was reversed by mevalonate and geranylgeranyl-pyrophosphate and mimicked by the Rho inhibitor C3 transferase, indicating the involvement of Rho in the signal transduction pathway leading to COX-2 expression. In parallel, statins, as well as COX-2 inhibitors, reduced the MMP-9 stimulated release and the endothelial tubular differentiation. CONCLUSION: In the human vascular endothelium, statins reduce COX-2 and MMP-9 expression and activity. Through this mechanism, statins exert an anti-angiogenic effect possibly contributing to the cholesterol-lowering-unrelated protective effects of statins against plaque inflammatory angiogenesis and rupture.

Dysregulated body copper homeostasis can negatively impact neuronal functions, but full knowledge of the mechanisms underlying the cell metal distribution has not been achieved yet. The high-affinity copper transporter 1 (Ctr1) is considered the main route for cell copper entry, while the cellular prion protein (PrP(C)) is presumed to be involved in the same process. Anchored to the outer side of the plasma membrane, this protein has the ability to bind copper ions and undergo internalization. To provide indications about the contribution of Ctr1 and PrP(C) proteins in cell copper transport, we used a fluorimetric method to characterize the kinetic properties of ion internalization in a neuroblastoma cell model, overexpressing prion protein (B104). Biochemical characteristics of intake delineated in the presence of other metal ions and an excess of extracellular potassium were compatible with PrP(C)-mediated endocytotic transport. Accordingly, inhibition of clathrin-dependent endocytosis by hypertonic shock and enzymatic removal of surface prion protein reduced copper influx by the same extent. On the whole, experimental evidence collected in a neuron-like cell model sustains a role for PrP(C) in mediating copper uptake by clathrin-dependent endocytosis.

BACKGROUND: In modern clinical neuro-oncology, the pathologic diagnoses are very challenging, creating significant clinical confusion and affecting therapeutic decisions and prognosis. METHODS: TP53 and PTEN gene sequences were analyzed, and microarray expression profiling was also performed. The authors investigated whether gene expression profiling, coupled with class prediction methodology, could be used to determine the prognosis of gliomatosis cerebri in a more consistent manner than standard pathology. RESULTS: The authors reported the results of a molecular study in 59 cases of gliomatosis cerebri, correlating these results with prognosis. The well-known prognostic factors of gliomas (ie, age, Karnofsky performance status, histology [grade 2 vs 3], and contrast enhancement) were found to be predictive of response or outcome in only a percentage of patients but not in all patients. The authors identified a 23-gene signature that was able to predict patient prognosis with microarray gene expression profiling. With the aim of producing a prognosis tool that is useful in clinical investigation, the authors studied the expression of this 23-gene signature by real-time quantitative polymerase chain reaction. Real-time expression values relative to these 23 gene features were used to build a prediction method able to distinguish patients with a good prognosis (those more likely to be responsive to therapy) from patients with a poor prognosis (those less likely to be responsive to therapy). CONCLUSIONS: The results of the current study demonstrated not only a strong association between gene expression patterns and patient survival, but also a robust replicability of these gene expression–based predictors. Cancer 2009. © 2009 American Cancer Society.

We developed a novel filtration-based method that can eliminate dead or severally damaged Salmonella enterica and Listeria monocytogenes in food samples. This new method can recover all viable bacteria in less than 30 min, and can be coupled with a subsequent bacterial DNA extraction and real-time PCR. No statically significant differences (p < 0.01) were found between real-time PCR results obtained separately from S. enterica and L. monocytogenes when different ratios of living and dead cells were used. The analytical sensitivity in both cases was 1 genome equivalent (GE), and the quantification was linear (R2 > 0.9969) over a 5-log dynamic range with PCR efficiencies >0.9754. When compared with the standard microbiological methods for the detection of these foodborne pathogens, the relative accuracy was excellent ranging from 95.72% to 104.48%. Finally, we applied the pre-treatment method to the direct detection of viable forms of these foodborne pathogens in food samples using yogurt as a model, the results being similar to those obtained using pure cultures.

Epidermal growth factor receptor (EGFR) signal transduction pathway has been reported to play a vital role in the biologic progression of several tumours and as targets for therapeutic intervention. We have investigated the role of EGFR in the thyroid PC Cl3 cells response to the chemo-therapeutic agent cisplatin. It was found that cisplatin provoked (1) the activation (phosphorylation) and internalization of EGFR, (2) the phosphorylation of mitogen-activated protein kinase (MAPK)/p38, (3) the activation of PKC-epsilon, (4) the enhancement of matrix metalloproteinase-2 (MMP-2) expression and activity, (5) the generation of reactive oxygen species (ROS) and (6) the activation of the apoptotic intrinsic pathway. Inhibition or down regulation of EGFR reduced (1) the phosphorylation of MAPK/p38, (2) the cisplatin-provoked activation of PKC-varepsilon, and (3) the activation of caspase-7 and PARP cleavage and the overall cells sensitivity to cisplatin. PKC-varepsilon inhibition achieved by siRNA blocked MAPK/p38 activation and significantly increased the cell resistance to cisplatin. Finally, when the cisplatin-induced ROS generation was blocked by using NAD(P)H oxidase inhibitors, a decrease in cisplatin-induced MMP-2 enhancement, MAPK/p38 and EGFR activation, and caspase-7 proteolysis occurred. In conclusion, these findings supported a model in which cisplatin provokes an oxidant-induced MMP-2-dependent EGFR transactivation responsible for the induction of cell apoptosis, a process ascribable to the intracellular signalling of PKC-epsilon and MAPK/p38.

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.

We have previously demonstrated that in sea bream Sparus aurata motility initiation determined changes in the phosphorylation state of some proteins. This paper describes an investigation of the effect of a freezing–thawing procedure on the protein phosphorylation/dephosphorylation pattern. Proteins extracted from fresh and cryopreserved spermatozoa (before and after motility activation) were separated on SDS–PAGE, blotted on nitrocellulose membrane and treated with anti-phosphotyrosine, anti-phosphothreonine, or anti-phosphoserine antibodies. The results obtained demonstrate that the cryopreservation protocol has a strong effect on the phosphorylation state of proteins. In general, compared to fresh sperm, phosphorylated proteins are most numerous in both activated and non-activated cryopreserved sperm, and in particular we observed a dramatic increase in threonine phosphorylation. However, frozen–thawed sperm showed a minor number of proteins that changed their phosphorylation state after motility activation. Among these, we identified the acetyl-coenzyme A synthetase that plays a role in sperm motility initiation in both fresh and cryopreserved sperm.

Sulfate plays an essential role during growth, development and cellular metabolism. In this study, we characterized the function and structure of the zebrafish ( Danio rerio) Na+-sulfate cotransporter 1 (NaS1) cDNA and gene ( slc13a1). Zebrafish NaS1 encodes a protein of 583 amino acids with 13 putative transmembrane domains. Expression of zebrafish NaS1 protein in Xenopus oocytes led to Na+-sulfate cotransport, which was significantly inhibited by thiosulfate, selenate, molybdate, and tungstate. Zebrafish NaS1 transport kinetics were: Vmax = 1,731.670 ± 92.853 pmol sulfate/oocyte·hour and Km = 1.414 ± 0.275 mM for sulfate and Vmax = 307.016 ± 32.992 pmol sulfate/oocyte·hour, Km = 24.582 ± 4.547 mM and n (Hill coefficient) = 1.624 ± 0.354 for sodium. Zebrafish NaS1 mRNA is developmentally expressed in embryos from day 1 postfertilization and in the intestine, kidney, brain, and eye of adult zebrafish. The zebrafish NaS1 gene slc13a1 contains 15 exons spanning 8,716 bp. Characterization of the zebrafish NaS1 contributes to a greater understanding of sulfate transporters in a well-defined genetic model and will allow the elucidation of evolutionary and functional relationships among vertebrate sulfate transporters.

We studied the expression and the hormonal regulation of the PDS gene product, pendrin, which is, in thyrocytes, responsible for the iodide transport out of the cell. We show that PC C13 cells, a fully differentiated thyroid cell line, grown without TSH and insulin, express very low level of PDS mRNA; such expression is greatly increased after stimulation with insulin or TSH. (125)1 pre-loaded cells showed an (125)1 efflux accelerated in chloride-containing buffer with respect to chloride-free buffer, suggesting that this efflux is chloride dependent. By immunoblotting, pendrin was found in agonists-stimulated cells, whereas it was barely detectable in un-stimulated cells. An increase in both PDS mRNA and protein was also obtained using phorbol ester PMA, or using 8-Br-cAMP and forskolin. Stimulation with insulin ((125)1 mu g/ml: 0-40 min) provoked the cytosol-to-membrane translocation of pendrin and a decrease of intracellular I content in 25 1 pre-loaded cells. Insulin- or PMA-treated cells also showed a cytosol-to-membrane translocation of PKC-delta and -epsilon. Inhibition of both PKC-delta and -epsilon. activities by GF 109203X blocked pendrin translocation, whilst the inhibition of PKA did not. The selective inhibition of PKC-delta by rottlerin did not affect the insulin-provoked translocation of pendrin whilst it was inhibited by a PKC-epsilon, translocation inhibitor peptide and also by PKC-epsilon clownregulation using the small interfering RNA, thus indicating that such translocation was due to PKC-epsilon, activity. In conclusion, our study demonstrates that, in PC C13 cells, pendrin expression and localisation are regulated by insulin and influenced by a PKC-epsilon depenclent intracellular pathway.

L-[3H]lysine uptake was measured in brush-border membrane vesicles prepared from intestinal mucosa of the European eel Anguilla anguilla. Lysine uptake occurred via 1) a nonsaturable component with an apparent diffusional permeability (P) of 0.58 microliter.mg protein-1.min-1,2) a Na-dependent transport system [half-saturation constant (Kapp) 0.16 mM, maximal transport rate (Jmax) 3.57 nmol.mg protein-1.min-1]; 3) a Na-independent transport system (Kapp 0.17 mM, Jmax 2.77 nmol.mg protein-1.min-1). Both carrier-mediated processes were accelerated by the presence of an intravesicular negative membrane potential. Hill analysis of L-lysine influx, over a wide range of external Na concentrations, resulted in a Hill coefficient (n) of approximately 2, suggesting that two or more Na ions may be associated with amino acid transport. The inhibition of lysine uptake by other amino acids was studied. Na-dependent lysine uptake was competitively inhibited by proline [inhibitory constant (Ki) approximately 2 mM] and may occur by a system specific for cationic amino acids. Na-independent lysine uptake was competitively inhibited by alanine (Ki approximately 1 mM) and may occur by a classic L system.

Gill cytoplasmic carbonic anhydrase of the haemoglobinless Antarctic icefish Chionodraco hamatus (Ice-CA) was directly sequenced and consists in 259 residues with an acetylated N-terminus. The molecular mass, deduced from the sequence, was 28.45 kDa, while mass spectrometry analysis of the native protein gave higher values. Treatment with dithiothreitol abolished this difference, indicating possible post-translational modifications. Isoelectric focusing analysis of Ice-CA suggested S-thiolation, which was identified as S-glutathionylation by immunostaining. Deglutathionylated Ice-CA maintained the anhydrase activity but showed higher susceptibility to hydrogen peroxide, suggesting that glutathione binding to Cys residues may have a role in the defence against oxidative damage. Ice-CA is characterized by lower thermal stability, higher activity and lower activation energy than its homologue gill CA of the temperate European eel, confirming the adaptation of the catalytic capacity to low temperatures. Alignment of Ice-CA with homologous enzymes from other fish shows high identity; the enzyme is grouped with a previously described fish CA monophyletic clade although Ice-CA shows several characteristics that can increase protein-solvent interaction and structural flexibility.