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La Sclerosi Multipla (SM) è una malattia infiammatoria cronica demielinizzante, a carattere autoimmune, che colpisce il Sistema Nervoso Centrale (SNC). Nonostante siano stati effettuati una serie di studi allo scopo di chiarire le cause della patologia, alcuni meccanismi patogenetici restano ancora oscuri e l’eziologia rimane sconosciuta. Il modello murino della malattia, l'encefalopatia autoimmune, ha tuttavia permesso di ricondurre il processo di demielinizzazione all'azione dei linfociti T CD4+ che, una volta attivati, sarebbero in grado di penetrare attraverso la barriera ematoencefalica e di causare danno assonale con conseguente morte di cellule neuronali. L’attivazione delle cellule T è accompagnata da uno shift metabolico che prevede il passaggio da un metabolismo ossidativo energeticamente efficiente ad un programma prettamente glicolitico; nelle malattie autoimmuni i dati in letteratura concernenti il fenotipo metabolico dei linfociti T sono piuttosto esigui e, tuttora, non vi sono lavori condotti a tal riguardo nella SM.

The arising of a number of neurodegenerative disorders in mammals has been correlated to the impairment of copper homeostasis in the CNS that is far from being completely understood. In the attempt to give a contribution to clarify some molecular aspects of this relationship, we used a rat neuroblastoma cell line (B104) as a cell model aiming to examine how extracellular copper availability can affect the protein expression pattern and Cu fluxes. Briefly, B104 cells were cultured under Cu depletion for rising time periods (48, 96 hours). To reveal possible changes in the protein expression pattern (CTR1, DCT1, PRPc, ATP7A) we conducted Real Time PCR and Immunoblotting analysis, that enlightened an expected complexity of cuproprotein genes expression and regulation. Surprisingly the activation of a transcriptional adaptive response involving the cellular prion protein and the high affinity copper transporter CTR1 was evidenced, that hasn’t been reported before. We tried to estimate possible alterations of transmembrane copper fluxes in ion deprived cells by the employment of a fluorimetric method based on the use of the Cu-sensitive probe Phen Green SK. To verify the suitability of such a method with respect to our intent a preliminary study was carried out to verify the dependence of the rate of copper influx on external potassium and the presence of metal ions (Ag, Cd, Mn, Zn), typically associated to CTR1-mediated Cu entry. A confocal microscopy approach was additionally developed to confirm the specificity of the response of the intracellular Phen Green SK probe to copper intake. In conclusion, our results support the hypothesis that the adapting strategies developed by B104 neuroblastoma cell model in response to reduced copper availability pass through the activation of a wide transcriptional adaptive response with suspected implications in the pathophysiology of neurodegenerative disorders.

Mechanisms of copper transport in mammalian nervous cells have not been completely defined. A pivotal role in Cu uptake is ascribed to the high-affinity membrane transporter CTR1 although we can’t exclude a contribute of DCT1 carrier and prion protein. In this study a neuroblastoma cell model was used in order to investigate how cell culture media, low or high in copper content, can affect the rate of ion influx. Methods: B104 rat neuroblastoma cells were pre-treated for 48-96 h with cuprizone, a selective copper chelator, and with an higher than physiological amount of copper. Then the effect of media Cu concentrations on the rate of copper uptake in suspended cells was determined by a fluorimetric method employing the Cu(I) sensitive dye Phen Green SK (PG). An additional approach consisting of a confocal microscopy method applied to PG loaded cells was devised to pursue the same purposes. Results: Fluorimetric measurements of copper uptake in 48- 96 h Cu-depleted cells didn’t allow us to detect any change in kinetic parameters. Analogously no significant differences were observed in the rate of Cu influx in cells treated with an excess of copper chloride. In order to uncover possible inadequacy of the fluorimetric technique respect to our intents, Cu transport phenomena were evaluated by confocal microscopy in cells grown on pre-treated cover glasses. A diffuse decrease in the cell internalized PG dye signal was observed as a consequence of its binding to copper entering the cells. Conclusion: Exposure of B104 neuroblastoma cells to variable copper availability in culture media didn’t affect the kinetics of Cu cellular intake, thus enlightening strictly regulated mechanisms of copper homeostasis. Meeting FIRB - Lecce http://

Several types of K+ channels have been identified in epithelial cells. Among them high conductance Ca2+- activated K+ channels (BK channels) are of relevant importance for their involvement in regulatory volume decrease (RVD) response following hypotonic stress. The aim of the present work was to investigate the functional and molecular expression of BK in the eel intestine, which is a useful experimental model for cell volume regulation research. In the present paper using rat BK channel-specific primer, a RT-PCR signal of 696 pb cDNA was detected in eel intestine, whole nucleotide sequence showed high similarity (83%) to the alpha subunit of BK channel family. BK channel protein expression was verified by immunoblotting and confocal microscopy, while the functional role of BK channels in epithelial ion transport mechanisms and cell volume regulation was examined by electrophysiological and morphometric analysis on the intact tissue. BKCa channels appeared to be localized along all the plasma membrane of the enterocytes; the apical part of the villi showed the most intense immunostaining. These channels were silent in basal condition, but were activated on both membranes (apical and basolateral) by increasing intracellular Ca2+ concentration with the Ca2+ ionophore ionomycin (1 µM). BKCa channels were also activated on both membranes by hypotonic swelling of the epithelium and their inhibition by 100 nM iberiotoxin (specific BKCa inhibitor) abolished the Regulatory Volume Decrease (RVD) of the intestinal cells after hypotonic swelling. In conclusion, our results demonstrated the molecular and functional expression of high conductance Ca2+ -activated K+ channels in eel intestine; the physiological role of these channels is mainly related to the RVD response of the epithelial cells following hypotonic swelling.

Aim: Copper is an essential trace metal required as a cofactor in a broad range of enzymatic functions. Copper transporter-1 (Ctr1), ubiquitously expressed, is believed to play a major role in Cu uptake into the cell. In the present study we used a rat neuroblastoma cell line (B104) as a cell model aiming to examine how extracellular copper availability can modulate the expression of Ctr1 and Cu fluxes. Methods: B104 cells were cultured under Cu depletion by exposure to cuprizone, a specific Cu chelating agent, and Cu supplementation for rising time periods (48, 96 hours). Ctr1 protein levels were assessed by Western Blotting analysis. Changes in transmembrane copper fluxes were measured by a fluorimetric method employing the Cu+-sensitive fluorophore Phen Green SK. Results: Treatment of cells with 50 µM CuCl2 for 48 h determined a significant increase in Ctr1 expression, while Cu supplementation prolonged for 96 h was found to restore basal protein levels. Exposure to over-physiological Cu level didn’t alter ion transmembrane fluxes as measured by the fluorescence method, indicating that excess protein was unfunctional. Analogously to Cu supplementation, exposure of cells to ion depletion for 48 h induced an increase in protein synthesis, probably as an immediate adaptive response to inadequate copper levels in basal culture medium. No change in protein expression was detected after 96 h treatment. Conclusion: In summary, our results suggest that short-term adapting strategies of B104 cells to varying Cu availability in culture medium involve directly Ctr1, even if we are not able to exclude other possibilities. For a longer time period treatment, presumably other mechanisms can be involved, such as Ctr1 co-regulation with other ion transporters (i.e. DMT1, Divalent Metal Transporter 1).

Aim: Physiological processes are impaired by low temperature. The aim of our research was: i) to understand the molecular aspects of the physiologic and phylogenetic adaptation of functional mechanisms of life to the cold, which allow poikilotherms to survive well in thermodynamically disadvantageous conditions; ii) to provide the scientific background for the biotechnological exploitation of cold-adapted functional mechanisms. Methods: Functional protein (such as carbonic anhydrase and the dipeptide carrier) were isolated from Antarctic teleosts, purified, sequenced and processed for the biochemical and the physiological characterisation. The analysis of the protein lipid microenvironment was performed to assess the role of the lipid-protein interactions on the overall functionality of the protein. FPLC and HPLC were used for protein isolation and purification. Sequencing was carried out by direct protein and cDNA sequencing and mass spectrometry. Isoelectric focusing and Western blot analysis were used to show post-translational modifications. Fluorimetry, spectrophotometry, isotope labelled substrates were used for the biochemical/physiological characterisation of enzymes and transporters. Gas-chromatography, TLC, fluorimetry and spectrophotometry allowed the analysis of the lipid microenvironment. Bioinformatics was applied to perform the phylogenetic analyses. Results: Some molecular aspects of the capability of protein to perform well at low temperature have been identified. The phylogenetic relationships with analogues from higher temperature have been assessed. The lipid analysis showed the adaptive contribution of the membrane lipid microenvironment to the protein adaptation to cold. Conclusions: The results obtained extend our knowledge about the key molecular characteristics and mechanisms which contrast the thermodynamic disadvantages of low temperature toward any physiological process.

Aim: A cytosolic carbonic anhydrase (I-CA) isoform, showing higher catalytic rate with respect to temperate counterparts, was isolated from the gills of the haemoglobinless, circulating CA deprived Antarctic teleost Chionodraco hamatus. To understand the molecular basis of the high I-CA activity, a structural characterisation has been undertaken. Methods: I-CA was purified from gill epithelium by sulphanilamide sepharose gel FPLC. The amino acid sequence of I-CA was established by direct protein sequencing. Isoelectric focusing (IEF) and Western blot analysis were used to show post-translational modifications of I-CA. Thermodynamic parameters of CA were calculated by measuring carbonic anhydrase activity in the 0-40 °C range by a radioactive assay. Sequence analysis and computational procedures were performed. Results: I-CA consists of 259 residues and its molecular mass deduced from the amino acid sequence (28449 Da) was higher than the value obtained by mass spectrometry (2873829225 Da). Treatment with dithiothreitol (DTT) abolished this discrepancy, indicating possible post-translational modifications also confirmed by IEF analysis. S-glutathionylation of I-CA was demonstrated by using specific anti-GSH antibody. Deglutathionylated form of I-CA maintained its activity despite a higher susceptibility to H2O2. Thermodynamic parameters of I-CA showed some traits of cold-adaptation as described for other enzymes from Antarctic fish. The I-CA sequence also suggests specific adaptive features related to the environmental temperature. Conclusion: A novel enzyme variant with high turnover rate was found in the respiratory epithelium of the icefish. The evolution of this branchial isoform could correlate with cold adaptation and the absence of haemoglobin and circulating CA in this fish.

A member of peptide transporter family, well characterised in higher vertebrates, was found in the intestine of the Antarctic waters haemoglobinless teleost Chionodraco hamatus (PepT-Ice; Maffia et al. J. Exp. Biol. 206:705, 2003). PepT-Ice shares high similarity to the low-affinity mammalian PepT1, but also possesses cold-adapted features. To better clarify the molecular basis of PepT-Ice evolutive adaptation, we studied its structural characteristics and tissue distribution. The full-length nucleotide sequence encoding for PepT-Ice was obtained by RT-PCR and 5’-/3’-RACE. PepT-Ice cDNA was 2509 bp long, with an orf of 2232 bp encoding for a putative protein of 743 amino acids. Hydropathy analysis predicted at least 12 potential transmembrane domains (TMD) with a large extracellular loop between TMD IX and X. Six putative extracellular N-glycosilation sites and nine and three intracellular consensus regions for protein kinase C and protein kinase A were identified. PepT-Ice exhibited higher percentage of identity with mammalian PepT1 (59-61%) than PepT2 (48-50%). Also, highest identity (69%) was found with zebrafish PepT1. Phylogenetic analysis clustered PepT-Ice to the PepT1 branch of the phylogenetic tree, In PepT-Ice, the presence of such hydrophobic amino acids as Gly-599, Leu-160 and Gly-271 in substitution of two Glu and one Asp, respectively, could play a relevant role in adaptive mechanisms to cold, possibly reducing intramolecular interaction and increasing protein flexibility. Using PepT-Ice specific primers, 630 bp RT-PCR amplification products were detected in mRNA extracted from C. hamatus intestine, kidney, liver, gills, brain and heart. The molecular characterization of PepT-Ice has been achieved that might elucidate the low temperature adaptation mechanisms of membrane transporters and functional relationships among vertebrate peptide transporters.

Prion diseases are a group of neurodegenerative pathologies that recognize, as aetiopathologic agent, an aberrant isoform of the prion protein (PrPc), named PrPsc [1]. Recently, a correlation between the structural state of the prion protein and its neurotoxicity has been demonstrated and, by using different prion peptide fragments of the structured portion of the protein, the molecular mechanisms involved in the pathogenesis of prion diseases begin to be elucidated [2]. In this study, we examined the neurotoxicity of the prion protein fragment PrP[173-195] (Ac-NNFVHDCVNITIKQHTV-TTTTKG-NH2) on a neuronal cell line (B104 neuroblastoma cells). PrP[173-195] corresponds to a highly conserved region that could provide a nucleation site for the sequence-dependent unfolding and may be implicated in the conformational transition from PrPC to PrPSc. Stock cultures of B104 cells were maintained in Eagle's minimal essential medium (MEM) as described elsewhere [3]. B104 cells were plated into 96-well trays and maintained in the medium containing 10% fetal bovine serum (FBS) for at least 24 h, then, a cell aliquot was switched to a medium containing 0.1% FBS. Then, all cells were treated with increasing concentrations of PrP[173-195] up to 12 µg/ml and cell viability was assessed by MTT (3,[4,5dimethylthiazol-2-yl]-2,5diphenyltetrazolium bromide) test after 18, 24 and 48 h of incubation with the peptide fragment. After 18 h incubation, a maximum of 30% reduction of cell viability was observed in all cells at peptide concentrations of 2 µg/ml. After 24 h incubation, the same concentration of peptide induced a maximum of reduction of almost 55% cell viability in 0.1% FBS, while in cell maintained in 10% FBS the toxic effect was maintained at the same level observed after 18 h incubation. After 48 h incubation, a continuous, concentration-dependent reduction of cell viability up to 90% (10% FBS) and 80% (0.1% FBS) was measured up to 12 µg/ml. To assess the specificity of the neurotoxic effects of the fragment, was assayed by performing the same analyses on human MCF7 breast cancer cell line.

The presence of a low affinity type H+/peptide transporter on the intestinal apical membranes of the Antarctic teleost Chionodraco hamatus, has been previously demonstrated (Ice-PepT; Maffia et al., J. Exp. Biol. 206: 705-714, 2003). The icefish peptide transporter even if kinetically resembled the mammalian PepT-1 isoform, exhibited specific cold adaptive features. In the present study the structural characteristics and the tissutal distribution of the transporter were investigated using RT-PCR and real-time RT-PCR analyses. An incomplete nucleotide sequence of 1744 pb was obtained, encoding for a protein region of 576 amino acid residues with an higher identity (57%) with hPepT1 in respect to hPepT2 (46%). The partial Ice-PepT amino acid sequence corresponded to nine of the twelve transmembrana (TMS) segments (II-X) reported in hPepT1, with a large extracellular loop between TMS IX and X, containing some potential N-linked glycosylation sites. The predicted protein also contained one potential site for protein kinase C-dependent phosphorylation in the putative intracellular loop between TMS X and XI (Ser 576) and one potential site for protein kinase A-dependent phosphorylation in the intracellular loop between TMS VIII and IX (Thr 330). The phylogenetic reconstruction among vertebrate H+/peptide transporters clustered Ice-PepT to the PepT1 branch of the phylogenetic tree and indicated early divergence of the fish protein sequence with respect to those of the tetrapod group. Ice-PepT mRNA was widely expressed in various tissues of C. hamatus, at highest levels in the intestinal mucosa and at a lesser extent in kidney, brain, liver, heart, and gills. In summary a novel member of a proton/oligopeptide transporter family has been partially characterised which can help to elucidate the evolutionary and functional relationship among H+/peptide transporters in vertebrates.

Introduction Prion diseases or Transmissible Spongiform Encephalopathies (TSEs) are a group of unusual neurodegenerative disorders affecting both animals and humans [1]. The agent believed to be responsible for these pathologies is a protein whose name is PrPsc, which is a conformational variant of the normal cellular prion protein, PrPc, a 33-35 KDa glycosyl-phosphatidylinositol-anchored protein, expressed at high levels in neuronal cells. Unlike PrPc, PrPsc is characterized by a higher β-sheet content and is partially resistant to proteolysis [2],[3]. The amino terminus of PrPc contains four octapeptide repeats, which are implicated in the binding of divalent metal ions and in particular copper; because of this property, PrPc could be reasonably involved in copper metabolism and in the defense mechanism of the cell against oxidative stress, possibly through the regulation of the Cu,Zn superoxide dismutase activity [4],[5]. Regarding the mechanisms of conversion of the cellular isoform in the aberrant one, a number of studies have been performed about a short peptide based on the sequence of PrPc, PrP[106-126]. It exhibits a prevalent β-sheet structure and forms amyloid fibrillar aggregates, being reminiscent of PrPsc; since it has been shown to induce apoptosis in cultured cells, it might constitute the toxic core of PrPsc [6]. In parallel, other peptides reproducing different portions of PrPc have been analysed, but their toxicity was found to be much less than with PrP[106-126] [7]. In the present work, we evaluated the effect of several synthetic versions of specific prion protein fragments, such as PrP[173-195] (NNFVHDCVNITIKQHTVTTTTKG and N- and C-blocked form) on B104 neuroblastoma cells and MCF7 breast cancer cells, since this peptide, corresponding to α2 helic, could be involved in prion molecular rearrangement because of its structural instability. Results and Discussion The peptide fragment PrP[173-195], both in the acetylated-amidate and free form, produced a toxic effect on B104 neuroblastoma cells, with a higher effect of PrP[Ac173-195NH2], which remarkably reduced cell viability after 48 h of incubation in cells. Both forms of the prion fragments produced a less pronounced effect on human breast cancer cells, suggesting however a lower unspecific effect on cell proliferation. The higher toxicity of the acetylated/amidated form with respect to the free one could derive from the charge neutralization induced at both N- and C-termini of the peptide, which should facilitate the helix formation thus better reproducing the conformational conditions observed in the native protein. Conclusions The PrPsc domain corresponding to α-helic 2 could be reasonably responsible for the interaction with the cellular prion protein and its conversion in the scrapie isoform. It exhibits a short sequence with a strong β-sheet forming propensity, which may affect the whole protein structure and mediate protein aggregation, characteristic of prion diseases.

E-cadherin is the core protein of the epithelial adherens junction. Through its cytoplasmic domain, E-cadherin interacts with additional proteins among which catenins that bridge it to the actin cytoskeletal. Loss of E-cadherin expression is considered a crucial step in the epithelial–mesenchymal transition process (EMT) and it is implicated with the capacity of cancer cells to invade and metastasize.

Surface-enhanced Raman spectroscopy (SERS) has received renewed interest in recent years in fields such as trace analysis, biorelated diagnosis, and living cell study. However, the interference of impurities left on the surface from the preparation process of substrates limits to some extent the application of SERS. In the present paper, we propose a method to prepare clean SERS substrates by a combined method of hydrothermal green synthesis and thermal treatment to obtain a clean and impurity-free surface for SERS measurements, suitable for cells growth. The goal of such activity was the study of the membrane proteome, with special attention to prion protein (PrPC), in its physiological ambient. SERS has been used to evidence the PrPC-Cu(II) interaction in a rat neuroblastoma cell line (B104), known to overexpress the cellular prion protein PrPC.

Classical Hodgkin lymphoma (cHL) is a malignancy with complex pathogenesis. The hallmark of the disease is the presence of large mononucleated Hodgkin and bi- or multinucleated Reed/Sternberg (H/RS) cells. The origin of HRS cells in cHL is controversial as these cells show the coexpression of markers of several lineages. Using a proteomic approach, we compared the protein expression profile of cHL models of T- and B-cell derivation to find proteins differentially expressed in these cell lines. A total of 67 proteins were found differentially expressed between the two cell lines including metabolic proteins and proteins involved in the regulation of the cytoskeleton and/or cell migration, which were further validated by western blotting. Additionally, the expression of selected B- and T-cell antigens was also assessed by flow cytometry to reveal significant differences in the expression of different surface markers. Bioinformatics analysis was then applied to our dataset to find enriched pathways and networks, and to identify possible key regulators. In the present study, a proteomic approach was used to compare the protein expression profiles of two cHL cell lines. The identified proteins and/or networks, many of which not previously related to cHL, may be important to better define the pathogenesis of the disease, to identify novel diagnostic markers, and to design new therapeutic strategies.

Uterine leiomyoma is a benign smooth muscle tumor characterized by a high incidence in women of reproductive age. The aetiology of this tumor is still unknown but established risk factors include high levels of female hormones, family history, African ancestry, early age of menarche and obesity. Here, to identify proteomic features associated with this tumor type, we performed a liquid cromatography-mass spectrometry (LC-MS/MS) analysis of uterine myomas. The identified proteins were subjected to a gene ontology analysis to generate biological functions, molecular processes, and protein networks that were relevant to the uploaded dataset. Pathway-based analysis was an effective approach to investigate the molecular mechanisms underlying the disease and to create biological hypotheses about regulation of our proteins including the identification of upstream regulators and main protein nodes. Moreover, proteomic and in silico data were combined with immunohistochemistry and western blotting to identify a group of proteins representative of some selected pathways, with a dysregulated expression in in myoma, pseudocapsule, and normal myometrium samples. Based on these results, we confirmed the over-expression of extracellular matrix components, and estrogen and progesterone receptors in uterine myomas, and proposed biological networks, canonical pathways and functions that may be relevant to the pathophysiology of this tumor.

Purpose: The aim of this study was to investigate the angiogenic role of the hepatocyte growth factor (HGF)/cMET pathway and its inhibition in bone marrow endothelial cells (EC) from patients with multiple myeloma versus from patients with monoclonal gammopathy of undetermined significance (MGUS) or benign anemia (control group). Experimental Design: The HGF/cMET pathway was evaluated in ECs from patients with multiple myeloma (multiple myeloma ECs) at diagnosis, at relapse after bortezomib- or lenalidomide-based therapies, or on refractory phase to these drugs; in ECs from patients with MGUS (MGECs); and in those patients from the control group. The effects of a selective cMET tyrosine kinase inhibitor (SU11274) on multiple myeloma ECs' angiogenic activities were studied in vitro and in vivo. Results: Multiple myeloma ECs express more HGF, cMET, and activated cMET (phospho (p)-cMET) at both RNA and protein levels versus MGECs and control ECs. Multiple myeloma ECs are able to maintain the HGF/cMET pathway activation in absence of external stimulation, whereas treatment with anti-HGF and anti-cMET neutralizing antibodies (Ab) is able to inhibit cMET activation. The cMET pathway regulates several multiple myeloma EC activities, including chemotaxis, motility, adhesion, spreading, and whole angiogenesis. Its inhibition by SU11274 impairs these activities in a statistically significant fashion when combined with bortezomib or lenalidomide, both in vitro and in vivo. Conclusions: An autocrine HGF/cMET loop sustains multiple myeloma angiogenesis and represents an appealing new target to potentiate the antiangiogenic management of patients with multiple myeloma. Clin Cancer Res; 20(22); 5796–807. ©2014 AACR.

The prion proteins and their interaction with copper ion represent a suitable marker in neurodegenerative disorders. A SERS based optical sensor has been developed in order to detect and quantify the prion proteins (PrPC) onto the cell membrane using the higher binding affinity of such proteins for copper ions. A combined method of hydrothermal “green” synthesis and thermal treatment allows us to obtain impurity-free surfaces for SERS measurement, suitable for cell growth. The plasmon absorption of the gold nanostructures was monitored by UV–vis spectrometry. The most significant red shift in the longitudinal plasmon resonance absorption of gold nanostructures was maximized in order to achieve the highest electromagnetic enhancement in Raman measurements. Our SERS based optical sensor has been used to detect and quantify the PrPC–Cu2+ interaction in vitro as a function of copper concentration and time in a rat neuroblastoma cell line (B104) and in three other cell models (SH-SY5Y, GN11, HeLa), expressing PrPC at different levels. The proposed methodology can be engineered in order to obtain an extremely fast and low-cost diagnostic tool to evaluate the subject's proneness to incur neurodegenerative processes.

Multiple sclerosis (MS) is a chronic inflammatory autoimmune demyelinating disease of the central nervous system. There are four clinical forms of MS, the most common of which is characterized by a relapsing remitting course (RRMS). The etiology of MS is unknown, but many studies suggested that genetic, environmental and infectious agents may contribute to the development of this disease. In experimental autoimmune encephalomyelitis (EAE), the animal model for MS, it has been shown that CD4(+) T cells play a key role in MS pathogenesis. In fact, these cells are able to cross the blood-brain barrier and cause axonal damage with neuronal death. T cell activation critically depends on mitochondrial ATP synthesis and reactive oxygen species (ROS) production. Interestingly, lots of studies linked the oxidative damage arising from mitochondrial changes to neurodegenerative disorders, such as MS. Based on these evidences, this work focused on the metabolic reprogramming of CD4(+) T cells in MS subjects, being this cell population directly implicated in pathogenesis of disease, paying attention to mitochondrial function and response to oxidative stress. Such aspects, once clarified, may open new opportunities for a therapeutic metabolic modulation of MS disorder.

E-cadherin is the core protein of the epithelial adherens junction. Through its cytoplasmic domain, E-cadherin interacts with several signaling proteins; among them, ?- and ?-catenins mediate the link of E-cadherin to the actin cytoskeleton. Loss of E-cadherin expression is a crucial step of epithelial-mesenchymal transition (EMT) and is involved in cancer invasion and metastatization. In human tumors, down-regulation of E-cadherin is frequently associated with poor prognosis. Despite the critical role of E-cadherin in cancer progression, little is known about proteome alterations linked with its down-regulation. To address this point, we investigated proteomics, biophysical and functional changes of epithelial breast cancer cell lines upon shRNA-mediated stable knockdown of E-cadherin expression (shEcad). shEcad cells showed a distinct proteomic signature including altered expression of enzymes and proteins involved in cytoskeletal dynamic and migration. Moreover, these results suggest that, besides their role in mechanical adhesion, loss of E-cadherin expression may contribute to cancer progression by modifying a complex network of pathways that tightly regulate fundamental processes as oxidative stress, immune evasion and cell metabolism. Altogether, these results extend our knowledge on the cellular modifications associated with E-cadherin down-regulation in breast cancer cells.

Multiple sclerosis (MS) is a chronic inflammatory autoimmune demyelinating disease of the central nervous system. There are four clinical forms of MS, the most common of which is characterized by a relapsing remitting course (RRMS). The etiology of MS is unknown, but many studies suggested that genetic, environmental and infectious agents may contribute to the development of this disease. In experimental autoimmune encephalomyelitis (EAE), the animal model for MS, it has been shown that CD4+ T cells play a key role in MS pathogenesis. In fact, these cells are able to cross the blood-brain barrier and cause axonal damage with neuronal death. T cell activation critically depends on mitochondrial ATP synthesis and reactive oxygen species (ROS) production. Interestingly, lots of studies linked the oxidative damage arising from mitochondrial changes to neurodegenerative disorders, such as MS. Based on these evidences, this work focused on the metabolic reprogramming of CD4+ T cells in MS subjects, being this cell population directly implicated in pathogenesis of disease, paying attention to mitochondrial function and response to oxidative stress. Such aspects, once clarified, may open new opportunities for a therapeutic metabolic modulation of MS disorder.

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.

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.

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.

Copper transporter 1 (CTR1), cellular prion protein (PrP(C)), natural resistance-associated macrophage protein 2 (NRAMP2) and ATP7A proteins control the cell absorption and efflux of copper (Cu) ions in nervous tissues upon physiological conditions. Little is known about their regulation under reduced Cu availability, a condition underlying the onset of diffused neurodegenerative disorders. In this study, rat neuron-like cells were exposed to Cu starvation for 48 h. The activation of Caspase-3 enzymes and the impairment of Cu,Zn superoxide dismutase (Cu,Zn SOD) activity depicted the initiation of a pro-apoptotic program, preliminary to the appearance of the morphological signs of apoptosis. The transcriptional response related to Cu transport proteins has been investigated. Notably, PrP(C) transcript and protein levels were consistently elevated upon Cu deficiency. The CTR1 protein amount was stable, despite a two-fold increase in the transcript amount, meaning the activation of post-translational regulatory mechanisms. NRAMP2 and ATP7A expressions were unvaried. The up-regulated PrP(C) has been demonstrated to enhance the cell Cu uptake ability by about 50% with respect to the basal transport, and so sustain the Cu delivery to the Cu,Zn SOD cuproenzymes. Conclusively, the study suggests a pivotal role for PrP(C) in the cell adaptation to Cu limitation through a direct activity of ion uptake. In this view, the PrP(C) accumulation observed in several cancer cell lines could be interpreted as a molecular marker of cell Cu deficiency and a potential target of therapeutic interventions against disorders caused by metal imbalances.

Surface-enhanced Raman spectroscopy (SERS) allows a new insight into the analysis of cell physiology. In this work, the difficulty of producing suitable substrates that, besides permitting the amplification of the Raman signal, do not interact with the biological material causing alteration, has been overcome by a combined method of hydrothermal green synthesis and thermal annealing. The SERS analysis of the cell membrane has been performed with special attention to the cellular prion protein PrP(C). In addition, SERS has also been used to reveal the prion protein-Cu(II) interaction in four different cell models (B104, SH-SY5Y, GN11, HeLa), expressing PrP(C) at different levels. A significant implication of the current work consists of the intriguing possibility of revealing and quantifying prion protein expression in complex biological samples by a cheap SERS-based method, replacing the expensive and time-consuming immuno-assay systems commonly employed.

An important goal of biomedical research is the development of tools for high-throughput evaluation of drug effects and cytotoxicity tests. Here we demonstrate EIS cell chips able to monitor cell growth, morphology, adhesion and their changes as a consequence of treatment with drugs or toxic compounds. As a case study, we investigate the uptake of copper ions and its effect on two cell lines: B104 and HeLa cells. For further understanding, we also carried out in parallel with EIS studies, a complete characterization of cell morphology and changes induced by copper ions through complementary methodologies (including state-of-the-art AFM, viability test and Western blot). Our results reveal a strong correlation between EIS data and both MTT test and AFM characterization so our chip can be used as powerful tools in all biology lab in combination with other standard methods giving additional information that can be useful in a complete and deep investigation of a biological process. This chip can be used even alone replacing in vitro drug tests based on conventional biochemical methods, being very cheap and reusable and allowing to perform cytotoxicity tests without using any expensive reagent or equipment.