Carnosine modulates the Sp1-Slc31a1/Ctr1 copper-sensing system and influences copper homeostasis in murine CNS-derived cells.

Carnosine (CAR) is an endogenous dipeptide physiologically present in excitable tissues, such as central nervous system (CNS) and muscle. CAR is acknowledged as a substrate involved in many homeostatic pathways and mechanisms and, due to its biochemical properties, as a molecule intertwined with the homeostasis of heavy metals such as copper (Cu). In CNS, Cu excess and dysregulation imply oxidative stress, free-radical production, and functional impairment leading to neurodegeneration. Here, we report that CAR intercepts the regulatory routes of Cu homeostasis in nervous cells and tissues. Specifically, in a murine neuron-derived cell model, i.e., the B104 neuroblastoma cells, extracellular CAR exposure up to 24 h influenced intracellular Cu entry and affected (downregulated) the key Cu-sensing system, consisting of the gene coding for the Slc31a1 transmembrane Cu importer (alias Ctr1), and the gene coding for the Cu-responsive transcription factor Sp1 ( Sp1). Also, CAR exposure upregulated CAR biosynthesis ( Carns1), extracellular degradation ( Cndp1), and transport ( Slc15a4, alias Pht1) genes and elicited CAR intracellular accumulation, contributing to the outline of functional association between CAR and Cu within the cell. Interestingly, the same gene modulation scheme acting in vitro operates in vivo in brains of mice undergoing dietary administration of CAR in drinking water for 2 wk. Overall, our findings describe for the first time a regulatory interaction between CAR and Cu pathways in CNS and indicate CAR as a novel active molecule within the network of ligands and chaperones that physiologically regulate Cu homeostasis.

Therapeutic potential of the dual peroxisome proliferator activated receptor (PPAR)α/γ agonist aleglitazar in attenuating TNF-α-mediated inflammation and insulin resistance in human adipocytes.

Adipose tissue inflammation is a mechanistic link between obesity and its related sequelae, including insulin resistance and type 2 diabetes. Dual ligands of peroxisome proliferator activated receptor (PPAR)α and γ, combining in a single molecule the metabolic and inflammatory-regulatory properties of α and γ agonists, have been proposed as a promising therapeutic strategy to antagonize adipose tissue inflammation. Here we investigated the effects of the dual PPARα/γ agonist aleglitazar on human adipocytes challenged with inflammatory stimuli. Human Simpson-Golabi-Behmel syndrome (SGBS) adipocytes were treated with aleglitazar or - for comparison - the selective agonists for PPARα or γ fenofibrate or rosiglitazone, respectively, for 24h before stimulation with TNF-α. Aleglitazar, at concentrations as low as 10nmol/L, providing the half-maximal transcriptional activation of both PPARα and PPARγ, reduced the stimulated expression of several pro-inflammatory mediators including interleukin (IL)-6, the chemokine CXC-L10, and monocyte chemoattractant protein (MCP)-1. Correspondingly, media from adipocytes treated with aleglitazar reduced monocyte migration, consistent with suppression of MCP-1 secretion. Under the same conditions, aleglitazar also reversed the TNF-α-mediated suppression of insulin-stimulated ser473 Akt phosphorylation and decreased the TNF-α-induced ser312 IRS1 phosphorylation, two major switches in insulin-mediated metabolic activities, restoring glucose uptake in insulin-resistant adipocytes. Such effects were similar to those obtainable with a combination of single PPARα and γ agonists. In conclusion, aleglitazar reduces inflammatory activation and dysfunction in insulin signaling in activated adipocytes, properties that may benefit diabetic and obese patients. The effect of aleglitazar was consistent with dual PPARα and γ agonism, but with no evidence of synergism.

Multiple anti-inflammatory and anti-atherosclerotic properties of red wine polyphenolic extracts: differential role of hydroxycinnamic acids, flavonols and stilbenes on endothelial inflammatory gene expression.

PURPOSE: The aim of the study was to evaluate the vascular anti-inflammatory effects of polyphenolic extracts from two typical South Italy red wines, the specific contribution of individual polyphenols and the underlying mechanisms of action. METHODS: Human endothelial cells were incubated with increasing concentrations (1-50 µg/mL) of Primitivo and Negroamaro polyphenolic extracts (PWPE and NWPE, respectively) or pure polyphenols (1-25 µmol/L), including hydroxycinnamic acids (p-coumaric, caffeic and caftaric acids), flavonols (kaempferol, quercetin, myricetin) or stilbenes (trans-resveratrol, trans-piceid) before stimulation with lipopolysaccharide. Through multiple assays, we analyzed the endothelial-monocyte adhesion, the endothelial expression of adhesion molecules (ICAM-1, VCAM-1 and E-Selectin), monocyte chemoattractant protein-1 (MCP-1) and macrophage colony-stimulating factor (M-CSF), as well as ROS intracellular levels and the activation of NF-κB and AP-1. RESULTS: Both PWPE and NWPE, already at 1 µg/mL, inhibited monocyte adhesion to stimulated endothelial cells, a key event in triggering vascular inflammation. They down-regulated the expression of adhesion molecules, ICAM-1, VCAM-1, E-Selectin, as well as MCP-1 and M-CSF, at mRNA and protein levels. All polyphenols reduced intracellular ROS, and everything, except caftaric acid, inhibited the endothelial expression of adhesion molecules and MCP-1, although with different potency. Flavonols and resveratrol significantly reduced also the endothelial expression and release of M-CSF. The decrease in endothelial inflammatory gene expression was related to the inhibition of NF-κB and AP-1 activation but not to intracellular oxidative stress. CONCLUSIONS: This study showed multiple anti-inflammatory and anti-atherosclerotic properties of red wine polyphenolic extracts and indentified specific bioactive polyphenols which could counteract inflammatory diseases including atherosclerosis.

Protein cold adaptation strategy via a unique seven-amino acid domain in the icefish (Chionodraco hamatus) PEPT1 transporter.

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.

miR-15b and miR-21 as Circulating Biomarkers for Diagnosis of Glioma.

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.

Teleost fish models in membrane transport research: the PEPT1(SLC15A1) H+-oligopeptide transporter as a case study.

Human genes for passive, ion-coupled transporters and exchangers are included in the so-called solute carrier (SLC) gene series, to date consisting of 52 families and 398 genes. Teleost fish genes for SLC proteins have also been described in the last two decades, and catalogued in preliminary SLC-like form in 50 families and at least 338 genes after systematic GenBank database mining (December 2010-March 2011). When the kinetic properties of the expressed proteins are studied in detail, teleost fish SLC transporters always reveal extraordinary 'molecular diversity' with respect to the mammalian counterparts, which reflects peculiar adaptation of the protein to the physiology of the species and/or to the environment where the species lives. In the case of the H+ -oligopeptide transporter PEPT1(SLC15A1), comparative analysis of diverse teleost fish orthologs has shown that the protein may exhibit very eccentric properties in terms of pH dependence (e.g., the adaptation of zebrafish PEPT1 to alkaline pH), temperature dependence (e.g., the adaptation of icefish PEPT1 to sub-zero temperatures) and/or substrate specificity (e.g., the species-specificity of PEPT1 for the uptake of l-lysine-containing peptides). The revelation of such peculiarities is providing new contributions to the discussion on PEPT1 in both basic (e.g., molecular structure-function analyses) and applied research (e.g., optimizing diets to enhance growth of commercially valuable fish).

Comparative analysis and functional mapping of SACS mutations reveal novel insights into sacsin repeated architecture.

Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) is a neurological disease with mutations in SACS, encoding sacsin, a multidomain protein of 4,579 amino acids. The large size of SACS and its translated protein has hindered biochemical analysis of ARSACS, and how mutant sacsins lead to disease remains largely unknown. Three repeated sequences, called sacsin repeating region (SRR) supradomains, have been recognized, which contribute to sacsin chaperone-like activity. We found that the three SRRs are much larger (≥1,100 residues) than previously described, and organized in discrete subrepeats. We named the large repeated regions Sacsin Internal RePeaTs (SIRPT1, SIRPT2, and SIRPT3) and the subrepeats sr1, sr2, sr3, and srX. Comparative analysis of vertebrate sacsins in combination with fine positional mapping of a set of human mutations revealed that sr1, sr2, sr3, and srX are functional. Notably, the position of the pathogenic mutations in sr1, sr2, sr3, and srX appeared to be related to the severity of the clinical phenotype, as assessed by defining a severity scoring system. Our results suggest that the relative position of mutations in subrepeats will variably influence sacsin dysfunction. The characterization of the specific role of each repeated region will help in developing a comprehensive and integrated pathophysiological model of function for sacsin.

Mediterranean diet polyphenols reduce inflammatory angiogenesis through MMP-9 and COX-2 inhibition in human vascular endothelial cells: a potentially protective mechanism in atherosclerotic vascular disease and cancer.

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.

Retrospective protein expression and epigenetic inactivation studies of CDH1 in patients affected by low-grade glioma.

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.

Fluorimetric analysis of copper transport mechanisms in the b104 neuroblastoma cell model: a contribution from cellular prion protein to copper supplying.

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.

Solid phase subtractive cloning in differentially expressed genes identification.

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.

Evidence for the involvement of aquaporins in sperm motility activation of the teleost gilthead sea bream (Sparus aurata).

The expression of aquaporins in the spermatozoa of the marine teleost gilthead sea bream (Sparus aurata) and their involvement in the motility activation process were investigated. Sperm motility was activated by a hyperosmotic shock, but it was completely inhibited by 10 microM HgCl(2), such inhibition being partially recovered by beta-mercaptoethanol (ME). Conventional RT-PCR using primers specific for S. aurata aquaglyceroporin (glp) and aquaporin 1a (aqp1a) demonstrated the presence of both mRNAs in spermatozoa. Heterologous expression in Xenopus laevis oocytes showed that 10 and 100 microM HgCl(2) equally inhibited water and solute transport through S. aurata aquaporin 1a and S. aurata aquaglyceroporin, but treatment with ME only recovered aquaporin 1a-mediated water permeability. Western blot analysis using isoform-specific antisera on protein extracts from spermatozoa revealed bands that corresponded to the predicted molecular mass of S. aurata aquaglyceroporin (31 kDa) and S. aurata aquaporin 1a (28 kDa). The antisera also demonstrated that both aquaporins were localized in the head and flagellum of the spermatozoa. However, the immunoreaction at the plasma membrane of the spermatozoa head was more intense after the hyperosmotic activation, suggesting the translocation of both aquaporin 1a and aquaglyceroporin into the plasma membrane after the osmotic shock. This study therefore provides the first direct demonstration for the presence of aquaporins in fish sperm. The different sensitivities of S. aurata aquaporin 1a and S. aurata aquaglyceroporin to ME may explain the failure of this reducing agent to fully recover the mercurial inhibition of sperm motility, suggesting that these aquaporins may play different physiological roles during the activation and maintenance of sperm motility in sea bream.

A filtration-based real-time PCR method for the quantitative detection of viable Salmonella enterica and Listeria monocytogenes in food samples.

We developed a novel filtration-based method that can eliminate dead or severely 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 (R(2)>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.

Functional and structural characterization of the zebrafish Na+-sulfate cotransporter 1 (NaS1) cDNA and gene (slc13a1).

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: V(max) = 1,731.670 +/- 92.853 pmol sulfate/oocyte.hour and K(m) = 1.414 +/- 0.275 mM for sulfate and V(max) = 307.016 +/- 32.992 pmol sulfate/oocyte x hour, K(m) = 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.

Biochemical characterization of a S-glutathionylated carbonic anhydrase isolated from gills of the Antarctic icefish Chionodraco hamatus.

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.

Analysis of calcium concentration fluctuations in hepatopancreatic R cells of Marsupenaeus japonicus during the molting cycle.

In this study we examined the fluctuations of the intracellular calcium concentration in isolated hepatopancreatic R cells during the four molting stages of the prawn Marsupenaeus japonicus. In addition, we used the Fura-2-AM fluorescence technique to investigate the release of calcium from mitochondria and ATP-sensitive calcium stores (endoplasmic reticulum (ER), Golgi, and nucleus) into cytoplasm during the molting cycle. Results demonstrate that both the cytosolic free calcium concentration and the total cell calcium (free, bound to calcium-binding proteins, and stored in amorphous form) in the R cells strictly depend upon the molting cycle. Interestingly, the total cell calcium was higher (approximately 10 mmol l(-1)) in postmolt than in premolt (approximately 1 mmol l(-1)) and intermolt (approximately 0.3 mmol l(-1)). The calcium released from mitochondria was higher during premolt than during postmolt and intermolt, but the amount of calcium released from ATP-sensitive calcium stores was similar during all four stages. All together, our results suggest that the mitochondria-ATP-sensitive calcium stores system does not play a key role in calcium storage during the molting cycle but that it is involved in transcellular calcium flux. We hypothesize that lysosome or membrane-clad concretion vacuoles could represent the main site of calcium storage in hepatopancreatic R cells.

Di- and tripeptide transport in vertebrates: the contribution of teleost fish models.

Solute Carrier 15 (SLC15) family, alias H+-coupled oligopeptide cotransporter family, is a group of membrane transporters known for their role in the cellular uptake of di- and tripeptides (di/tripeptides) and peptide-like molecules. Of its members, SLC15A1 (PEPT1) chiefly mediates intestinal absorption of luminal di/tripeptides from dietary protein digestion, while SLC15A2 (PEPT2) mainly allows renal tubular reabsorption of di/tripeptides from ultrafiltration, SLC15A3 (PHT2) and SLC15A4 (PHT1) possibly interact with di/tripeptides and histidine in certain immune cells, and SLC15A5 has unknown function. Our understanding of this family in vertebrates has steadily increased, also due to the surge of genomic-to-functional information from 'non-conventional' animal models, livestock, poultry, and aquaculture fish species. Here, we review the literature on the SLC15 transporters in teleost fish with emphasis on SLC15A1 (PEPT1), one of the solute carriers better studied amongst teleost fish because of its relevance in animal nutrition. We report on the operativity of the transporter, the molecular diversity, and multiplicity of structural-functional solutions of the teleost fish orthologs with respect to higher vertebrates, its relevance at the intersection of the alimentary and osmoregulative functions of the gut, its response under various physiological states and dietary solicitations, and its possible involvement in examples of total body plasticity, such as growth and compensatory growth. By a comparative approach, we also review the few studies in teleost fish on SLC15A2 (PEPT2), SLC15A4 (PHT1), and SLC15A3 (PHT2). By representing the contribution of teleost fish to the knowledge of the physiology of di/tripeptide transport and transporters, we aim to fill the gap between higher and lower vertebrates.

High-affinity peptide transporter PEPT2 (SLC15A2) of the zebrafish Danio rerio: functional properties, genomic organization, and expression analysis.

Solute carrier 15 (SLC15) membrane proteins PEPT1 (SLC15A1) and PEPT2 (SLC15A2) have been described in great detail in mammals. In contrast, information in lower vertebrates is limited. We characterized the functional properties of a novel zebrafish peptide transporter orthologous to mammalian and avian PEPT2, described its gene (pept2) structure, and determined mRNA tissue distribution. An expressed sequence tag (EST) cDNA (Integrated Molecular Analysis of Gene Expression; IMAGE) corresponding to zebrafish pept2 was completed by inserting a stretch of 75 missing nucleotides in the coding sequence to obtain a 3,238-bp functional clone. The complete open reading frame (ORF) was 2,160 bp and encoded a 719-amino acid protein. Electrophysiological analysis after cRNA injection in Xenopus laevis oocytes suggested that zebrafish PEPT2 is a high-affinity/low-capacity transporter (K(0.5) for glycyl-L-glutamine approximately 18 microM at -120 mV and pH 7.5). Zebrafish pept2 gene was 19,435 kb, thus being the shortest vertebrate pept2 fully characterized so far. Also, zebrafish pept2 exhibited 23 exons and 22 introns, whereas human and rodent pept2 genes contain 22 exons and 21 introns only. Zebrafish pept2 mRNA was mainly detected in brain, kidney, gut, and, interestingly, otic vesicle, the embryonic structure that develops into the auditory/vestibular organ, homolog to the higher vertebrate inner ear, of the adult fish. Characterization of zebrafish pept2 will contribute to the investigation of peptide transporters using a well-established genetic model and will allow the elucidation of the evolutionary and functional relationships among vertebrate peptide transporters. Moreover, it can represent a useful marker to screen mutations that affect choroid plexus and inner ear development.

Angiotensin II does not stimulate proliferation of rat thyroid PC Cl3 cell line.

In PC Cl3 cells, a rat thyroid cell line, angiotensin (Ang II) activates the atypical protein kinase C-zeta (PKC-zeta) 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 p27(kip) 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 p27(kip) induction, whilst PKCs inhibition by GF109203X decreased such effect. The role of PKC-zeta was recognized by the use of a synthetic myristoylated peptide with sequences based on the endogenous PKC-zeta pseudosubstrate and by PKC-zeta downregulation using the small interfering RNA (siRNA). Insulin had a replicating effect on PC Cl3 cells, induced the phosphorylation of PKB, decreased p27(kip) expression and had no effect on the PKC-zeta 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 p27(kip) 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 p27(kip) and PKC-zeta operativity.

The sarcoplasmic-endoplasmic reticulum Ca2+ ATPase 2b regulates the Ca2+ transients elicited by P2Y2 activation in PC Cl3 thyroid cells.

In PC Cl3 cells, a continuous, fully differentiated rat thyroid cell line, P2Y(2) purinoceptor activation provoked a transient increase of [Ca(2+)](i), followed by a decreasing sustained phase. The alpha and beta1 protein kinase C (PKC) inhibitor Gö6976 decreased the rate of decrement to the basal [Ca(2+)](i) level and increased the peak of Ca(2+) entry of the P2Y(2)-provoked Ca(2+)transients. These effects of Gö 6976 were not caused by an increased permeability of the plasma membrane, since the Mn(2+) and Ba(2+) uptake were not changed by Gö 6976. Similarly, the Na(+)/Ca(2+) exchanger was not implicated, since the rate of decrement to the basal [Ca(2+)](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 Ca(2+)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 [Ca(2+)](i) level after P2Y(2) stimulation and also increased the activity of SERCA 2b. These results suggest that SERCA 2b plays a role in regulating the sustained phase of Ca(2+) transients caused by P2Y(2) stimulation.