The use of an amphiphilic aryleneethynylene fluorophore as a plasma membrane marker in fixed and living mammalian cells and liposome model systems is demonstrated. We show here that the optical properties of the novel dye are almost independent on pH, in the range 5.0–8.0. Spectroscopic characterization performed on unilamellar liposomes ascertained that the fluorescence intensity of the aryleneethynylene fluorophore greatly increases after incorporation in lipidic membranes. Experiments performed on different mammalian cells demonstrated that the novel membrane marker exhibits fast staining and a good photostability that make it a suitable tool for live cell imaging. Importantly, the aryleneethynylene fluorophore was also shown to be a fast and reliable blue membrane marker in classical multicolor immunofluorescence experiments. This study adds new important findings to the recent exploitation of the wide class of aryleneethynylene molecules as luminescent markers for biological investigations.

Cadmium, a toxic environmental pollutant, affects the function of different organs such as lungs, liver and kidney. Less is known about its toxic effects on the gastric mucosa. The aim of this study was to investigate the mechanisms by which cadmium impacts on the physiology of gastric mucosa. To this end, intact amphibian mucosae were mounted in Ussing chambers and the rate of acid secretion, short circuit current (Isc), transepithelial potential (Vt) and resistance (Rt) were recorded in the continuous presence of cadmium. Addition of cadmium (20 μM to 1 mM) on the serosal but not luminal side of the mucosae resulted in inhibition of acid secretion and increase in NPPB-sensitive, chloride-dependent short circuit current. Remarkably, cadmium exerted its effects only on histamine-stimulated tissues. Experiments with TPEN, a cell-permeant chelator for heavy metals, showed that cadmium acts from the intracellular side of the acid secreting cells. Furthermore, cadmium-induced inhibition of acid secretion and increase in Isc cannot be explained by an action on: 1) H2 histamine receptor, 2) Ca2+ signalling 3) adenylyl cyclase or 4) carbonic anhydrase. Conversely, cadmium was ineffective in the presence of the H+/K+-ATPase blocker omeprazole suggesting that the two compounds likely act on the same target. Our findings suggest that cadmium affects the functionality of histamine-stimulated gastric mucosa by inhibiting the H+/K+-ATPase from the intracellular side. These data shed new light on the toxic effect of this dangerous environmental pollutant and may result in new avenues for therapeutic intervention in acute and chronic intoxication.

The MTERF protein family comprises members from Metazoans and plants. All the Metazoan MTERF proteins characterized to date, including the mitochondrial transcription termination factors, play a key role in mitochondrial gene expression. In this study we report the characterization of Drosophila MTERF5 (D-MTERF5), a mitochondrial protein existing only in insects, probably originated from a duplication event of the transcription termination factor DmTTF. D-MTERF5 knock-down in D.Mel-2 cells alters transcript levels with an opposite pattern to that produced by DmTTF knock-down. D-MTERF5 is able to interact with mtDNA at the same sites contacted by DmTTF, but only in the presence of the termination factor. We propose that the two proteins participate in the transcription termination process, with D-MTERF5 engaged in relieving the block exerted by DmTTF. This hypothesis is supported also by D-MTERF5 homology modeling, which suggests that this protein contains protein-protein interaction domains. Co-regulation by DREF (DNA Replication-related Element binding Factor) of D-MTERF5 and DmTTF implies that expression of the two factors needs to be co-ordinated to ensure fine modulation of Drosophila mitochondrial transcription.

FAD is a redox cofactor ensuring the activity of many flavoenzymes mainly located in mitochondria but also relevant for nuclear redox activities. The last enzyme in the metabolic pathway producing FAD is FAD synthase (EC 2.7.7.2), a protein known to be localized both in cytosol and in mitochondria. FAD degradation to riboflavin occurs via still poorly characterized enzymes, possibly belonging to the NUDIX hydrolase family. By confocal microscopy and immunoblotting experiments, we demonstrate here the existence of FAD synthase in the nucleus of different experimental rat models. HPLC experiments demonstrated that isolated rat liver nuclei contain ∼300 pmol of FAD·mg(-1) protein, which was mainly protein-bound FAD. A mean FAD synthesis rate of 18.1 pmol·min(-1)·mg(-1) protein was estimated by both HPLC and continuous coupled enzymatic spectrophotometric assays. Rat liver nuclei were also shown to be endowed with a FAD pyrophosphatase that hydrolyzes FAD with an optimum at alkaline pH and is significantly inhibited by adenylate-containing nucleotides. The coordinate activity of these FAD forming and degrading enzymes provides a potential mechanism by which a dynamic pool of flavin cofactor is created in the nucleus. These data, which significantly add to the biochemical comprehension of flavin metabolism and its subcellular compartmentation, may also provide the basis for a more detailed comprehension of the role of flavin homeostasis in biologically and clinically relevant epigenetic events.

Following its uptake from outside, in the cell riboflavin undergoes an ATP-dependent phosphorylation catalyzed by riboflavin kinase (RFK, E.C. 2.7.1.26) to form FMN, most of which is further converted by FAD synthase (FADS, E.C. 2.7.7.2) into FAD, the cofactor for many flavoenzymes involved in several biological processes in different cellular compartments. We previously demonstrated that beside a cytosolic enzyme (i.e. isoform 2 of human FLAD1 gene product) a mitochondrial isoform of FADS exists (isoform 1) (1, 2), presumably responsible for the biogenesis of mitochondrial flavoenzymes. Interestingly, the possibility of unexpected localizations for FADS has arisen (3) and, meanwhile, at least two new isoforms of FADS have been deposited in databases. Among FAD-dependent enzymes, lysine-specific demethylase-1 (E.C. 1.14.11.B1) is a nuclear protein recently shown to regulate cellular energy balance depending on FAD availability (4). These observation prompted us to investigate on the presence of flavin coenzymes in the nucleus and on a possible metabolism of FAD in this organelle. To this aim HPLC analysis of acid-precipitable flavins in nuclei isolated from rat liver were performed. The observation of a time-dependent decrease of FAD suggested us the existence of a hydrolytic activity, as confirmed by spectrofluorimetric measurements revealing an increase in fluorescence due to conversion of FAD into its more fluorescent precursor FMN/riboflavin (5). Immunoblotting experiments performed on rat liver nuclei using a home-made antibody against FADS revealed the enrichment of an immunoreactive band in respect to the other sub-cellular fractions. Confocal microscopy on several mammalian cells confirmed the existence of FADS co-localizing with nuclear markers. 1. Barile et al., Eur J Biochem, 2000, 15, 4888-900. 2. Torchetti et al., Mitochondrion, 2010, 10, 263-73. 3. Lin et al., J Neurol, 2009, 256, 774-82. 4. Hino et al., Nat Commun, 2012, 3, 758. 5. Brizio et al., Eur J Biochem, 1997, 3, 777-85.

Secretory granules of pancreatic β-cells contain high concentrations of Ca2+ ions that are co-released with insulin in the extracellular milieu upon activation of exocytosis. As a consequence, an increase in the extracellular Ca2+ concentration ([Ca2+]ext) in the microenvironment immediately surrounding β-cells should be expected following the exocytotic event. Using Ca2+-selective microelectrodes we show here that both high glucose and non-nutrient insulinotropic agents elicit a reversible increase of [Ca2+]ext within rat insulinoma (INS-1E) β-cells pseudoislets. The glucose-induced increases in [Ca2+]ext are blocked by pretreatment with different Ca2+ channel blockers. Physiological agonists acting as positive or negative modulators of the insulin secretion and drugs known to intersect the secretory machinery at different levels also induce [Ca2+]ext changes as predicted on the basis of their described action on insulin secretion. Finally, the glucose-induced [Ca2+]ext increase is strongly inhibited after disruption of the actin web, indicating that the dynamic [Ca2+]ext changes recorded in INS-1E pseudoislets by Ca2+-selective microelectrodes occur mainly as a consequence of exocytosis of Ca2+-rich granules. In conclusion, our data directly demonstrate that the extracellular spaces surrounding β-cells constitute a restricted domain where Ca2+ is co-released during insulin exocytosis, creating the basis for an autocrine/paracrine cell-to-cell communication system via extracellular Ca2+ sensors

The carnitine/acylcarnitine transporter is a transport system whose function is essential for the mitochondrial β-oxidation of fatty acids. Here, the presence of carnitine/acylcarnitine carrier (CACT) in nervous tissue and its sub-cellular localization in dorsal root ganglia (DRG) neurons have been investigated. Western blot analysis using a polyclonal anti-CACT antibody produced in our laboratory revealed the presence of CACT in all the nervous tissue extracts analyzed. Confocal microscopy experiments performed on fixed and permeabilized DRG neurons co-stained with the anti-CACT antibody and the mitochondrial marker MitoTracker Red clearly showed a mitochondrial localization for the carnitine/acylcarnitine transporter. The transport activity of CACT from DRG extracts reconstituted into liposomes was about 50 % in respect to liver extracts. The experimental data here reported represent the first direct evidence of the expression of the carnitine/acylcarnitine transporter in sensory neurons, thus supporting the existence of the β-oxidation pathway in these cells.

FAD synthetase or ATP:FMN adenylyl transferase (FADS or FMNAT, EC 2.7.7.2) is a key enzyme in the metabolic pathway that converts riboflavin into the redox cofactor FAD. We face here the still controversial sub-cellular localization of FADS in eukaryotes. First, by western blotting experiments, we confirm the existence in rat liver of different FADS isoforms which are distinct for molecular mass and sub-cellular localization. A cross-reactive band with an apparent molecular mass of 60 kDa on SDS–PAGE is localized in the internal compartments of freshly isolated purified rat liver mitochondria. Recently we have identified two isoforms of FADS in humans, that differ for an extra-sequence of 97 amino acids at the N-terminus, present only in isoform 1 (hFADS1). The first 17 residues of hFADS1 represent a cleavable mitochondrial targeting sequence (by Target-P prediction). The recombinant hFADS1 produced in Escherichia coli showed apparent Km and Vmax values for FMN equal to 1.3 ± 0.7 lM and 4.4 ± 1.3 nmol min1 mg protein1, respectively, and was inhibited by FMN at concentration higher than 1.5 lM. The in vitro synthesized hFADS1, but not hFADS2, is imported into rat liver mitochondria and processed into a lower molecular mass protein product. Immunofluorescence confocal microscopy performed on BHK-21 and Caco-2 cell lines transiently expressing the two human isoforms, definitively confirmed that hFADS1, but not hFADS2, localizes in mitochondria.

Extracellular matrix (ECM) degradation is a critical process in tumor cell invasion and requires membrane and released proteases focalized at membrane structures called invadopodia. While extracellular acidification is important in driving tumor invasion, the structure/function mechanisms underlying this regulation are still unknown. Invadopodia are similar in structure and function to osteoclast podosomes responsible for bone degradation, and extracellular acidification is central to podosome action, suggesting that it could also be for invadopodial function. Here, utilizing a novel system for in situ zymography in native matrices, we show that the Na(+)/H(+) exchanger (NHE1) and NHE1-generated extracellular acidification are localized at and necessary for invadopodial-dependent ECM degradation, thereby promoting tumor invasion. Stimulation with EGF increased both NHE1-dependent proton secretion and ECM degradation. Manipulation of the NHE1 expression by RNA interference or activity via either transport-deficient mutation or the specific inhibitor cariporide confirmed that NHE1 expression and activity are required for invadopodia-mediated ECM degradation. Taken together, our data show a concordance among NHE1 localization, the generation of a well-defined acidic extracellular pH in the nanospace surrounding invadopodia, and matrix-degrading activity at invadopodia of human malignant breast carcinoma cells, providing a structural basis for the role of NHE1 in invasion and identifying NHE1 as a strategic target for therapeutic intervention.-Busco, G., Cardone, R. A., Greco, M. R., Bellizzi, A., Colella, M., Antelmi, E., Mancini, M. T., Dell'Aquila, M. E., Casavola, V., Paradiso, A., Reshkin, S. J. NHE1 promotes invadopodial ECM proteolysis through acidification of the peri-invadopodial space

The primary role of the water-soluble vitamin B2 (riboflavin) in cell biology is connected with its conversion into FMN and FAD, the cofactors of a large number of dehydrogenases, oxidases and reductases involved in a broad spectrum of biological activities, among which energetic metabolism and chromatin remodeling. Subcellular localisation of FAD synthase (EC 2.7.7.2, FADS), the second enzyme in the FAD forming pathway, is addressed here in HepG2 cells by confocal microscopy, in the frame of its relationships with kinetics of FAD synthesis and delivery to client apo-flavoproteins. FAD synthesis catalyzed by recombinant isoform 2 of FADS occurs via an ordered bi-bi mechanism in which ATP binds prior to FMN, and pyrophosphate is released before FAD. Spectrophotometric continuous assays of the reconstitution rate of apo-D-aminoacid oxidase with its cofactor, allowed us to propose that besides its FAD synthesizing activity, hFADS is able to operate as a FAD "chaperone." The physical interaction between FAD forming enzyme and its clients was further confirmed by dot blot and immunoprecipitation experiments carried out testing as a client either a nuclear lysine-specific demethylase 1 (LSD1) or a mitochondrial dimethylglycine dehydrogenase (Me2GlyDH, EC 1.5.8.4). Both enzymes carry out similar reactions of oxidative demethylation, in which tetrahydrofolate is converted into 5,10-methylene-tetrahydrofolate. A direct transfer of the cofactor from hFADS2 to apo-dimethyl glycine dehydrogenase was also demonstrated. Thus, FAD synthesis and delivery to these enzymes are crucial processes for bioenergetics and nutri-epigenetics of liver cells.

Cardiac hypertrophy is a complex remodeling process of the heart induced by physiological or pathological stimuli resulting in increased cardiomyocyte size and myocardial mass. Whereas cardiac hypertrophy can be an adaptive mechanism to stressful conditions of the heart, prolonged hypertrophy can lead to heart failure which represents the primary cause of human morbidity and mortality. Among G protein-coupled receptors, the α1-adrenergic receptors (α1-ARs) play an important role in the development of cardiac hypertrophy as demonstrated by numerous studies in the past decades, both in primary cardiomyocyte cultures and genetically modified mice. The results of these studies have provided evidence of a large variety of α1-AR-induced signaling events contributing to the defining molecular and cellular features of cardiac hypertrophy. Recently, novel signaling mechanisms have been identified and new hypotheses have emerged concerning the functional role of the α1-adrenergic receptors in the heart. This review will summarize the main signaling pathways activated by the α1-AR in the heart and their functional implications in cardiac hypertrophy.

The current interest of the scientific community for research in the field of calcium sensing in general and on the calcium-sensing Receptor (CaR) in particular is demonstrated by the still increasing number of papers published on this topic. The extracellular calcium-sensing receptor is the best-known G-protein-coupled receptor (GPCR) able to sense external Ca2+changes. Widely recognized as a fundamental player in systemic Ca2+homeostasis, the CaR is ubiquitously expressed in the human body where it activates multiple signalling pathways. In this review, old and new notions regarding the mechanisms by which extracellular Ca2+microdomains are created and the tools available to measure them are analyzed. After a survey of the main signalling pathways triggered by the CaR, a special attention is reserved for the emerging concepts regarding CaR function in the heart, CaR trafficking and pharmacology. Finally, an overview on other Ca2+sensors is provided.

The kinetics of the p-nitrophenyl butyrate hydrolysis reaction, catalyzed by Candida rugosa lipase in the water-in-oil microemulsion cetyltrimethylammonium bromide/water/pentanol/hexane, was investigated. The results described in the present manuscript reveal two peculiar characteristics of the reaction: (i) the initial rate of hydrolysis is very fast and (ii) by decreasing the water content of the microemulsion, the reaction rate approaches the typical behavior of reactions performed in aqueous solution. In particular, for microemulsion systems with a high water content, the end points of the reactions are dictated by the shape stability of the microemulsion. For these systems, our methodological approach shows that the process follows a second-order kinetics equation, indicative of the dual role played by water, which is involved both as a component of the microemulsion, i.e., relevant for the microemulsion stability and as a reagent of the hydrolysis reaction. In contrast, for microemulsions containing a small amount of water, after the hydrolysis reaction the system seems to fall in the no existence range of the microemulsion. Accordingly, the kinetics results are more complex: in the initial stage, the reaction follows a zero-order kinetics equation, while for longer reaction times a first-order kinetics equation fits the experimental data, as would be expected for an enzymatic reaction in a homogeneous system. (c) 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:360-366, 2014

Store-Operated Cyclic AMP Signaling (SOcAMPS) represents a novel signaling mechanism in which depletion of Ca2+ in the endoplasmic reticulum (ER) leads to a STIM1- dependent (Stromal Interaction Molecule 1) increase in cAMP levels, independently of cytosolic Ca2+. Here we aimed to evaluate whether SOcAMPS was manifest in neonatal rat ventricular myocytes (NRVM) and human "iCardiomyocytes" and exploit its potential role in cardiac cell hypertrophy. cAMP levels and ER [Ca2+]were monitored by live cell fluorescence imaging after transfection with the EPAC H30 and D1ER cameleon probes, respectively. The existence of SOcAMPS in NRVM was first assessed by using the low affinity Ca2+ chelator TPEN, able to induce a reduction of SR Ca2+ levels without affecting cytosolic [Ca2+]. TPEN (1mM) was shown to induce significant cAMP increases both in the absence and presence of 5 M Forskolin (FRSK). Depletion of SR by ionomycin (10 M) was found to exert similar effects. Similar data were obtained in human "iCardiomyocytes". The participation of STIM1 in the observed phenomenon was proven in NRVM by the 47% reduction of the [cAMP] response obtained after shRNA-mediated knockdown of STIM1. Interestingly, a significant increase of the TPEN+FRSK induced response was found after "in vitro" induced cell hypertrophy. These data establish, for the first time, the existence of SOcAMPS in the two cardiac cell models analyzed and suggest a potential role for this new signaling mechanism in cardiac cell hypertrophy.

Background and Aims: Store-Operated Cyclic AMP Signaling (SOcAMPS) represents a recently identified mechanism of cross-talk between Ca2+ and cAMP signals. In this process, depletion of Ca2+ in the endoplasmic reticulum (ER) leads to increases in cAMP levels, independently of cytosolic Ca2+ changes. Expression and functionality of STIM1 (Stromal Interaction Molecule 1), a transmembrane ER Ca2+ sensor protein, is necessary for SOcAMPS to occur. Interestingly, recent reports have demonstrated a critical role for STIM1 in the development of cardiac hypertrophy, a process notoriously controlled both by Ca2+ and cAMP signaling. Here we aimed to evaluate whether SOcAMPS was manifest in neonatal rat cardiomyocytes and its potential role in cardiac cell hypertrophy. Methods: To monitor changes in cAMP levels, real time imaging experiments were performed on neonatal rat cardiomyocytes transiently transfected with an EPAC-based fluorescent probe for [cAMP], EPAC H30. Fura-2 and Fluo-4 were used to monitor cytosolic Ca2+ levels and an ER/SR targeted probe, D1ERcameleon, was used to measure ER [Ca2+]. Long term incubation (48h) of cardiomyocytes with angiotensin II (1 μM) and aldosterone (1 μM) was used to induce "in vitro" cell hypertrophy. Increases in cell size and/or sarcomere alignment were monitored microscopically after labeling with phalloidin-TRITC. Results: To verify the existence of SOcAMPS in neonatal rat cardiomyocytes, cells were stimulated in Ca2+-free Ringer's solutions with the low affinity membrane permeant Ca2+ chelator TPEN (1mM), able to induce a reduction of SR Ca2+ levels ([Ca2+]SR) without affecting cytosolic [Ca2+]. SR Ca2+ measurements demonstrated that under these experimental conditions, 1 mM TPEN led to a reduction in intraluminal [Ca2+] that was 50,5±2,4% (8 exp, 11 cells, p<0.001) of the maximal store depletion. Parallel experiments performed with the EPAC H30 cAMP sensor showed increases in [cAMP] that were 26,5±3% (13 exp, 13 cells, p<0.001) of the maximum delta ratio. In the presence of 5 μM Forskolin (FRSK) the TPEN-induced cAMP augmentation resulted 63,7±3,9% of the maximal response (16 exp, 19 cells, p<0.001). Also depletion of SR by the Ca2+ ionophore ionomycin (10 μM) was found to induce significant cAMP increases both in the absence and presence of FRSK. The participation of STIM1 in the observed phenomenon was proven by the 47 % reduction of the TPEN+FRSK induced [cAMP] signal after transfection of cells with a shRNA against STIM1 (6 exp, p<0,01). To evaluate the putative role of SOcAMPS in cardiac hypertrophy, cAMP measurements were performed on angio+aldo treated cells and compared to control cardiomyocytes. Under these experimental conditions a 20% increase of the TPEN+FRSK induced response was observed in hypertrophic myocytes (16 exp, p<0,01). Conclusions: These data straightforwardly establish, for the first time, the existence of SOcAMPS in the neonatal cardiomyocyte cell model. Also, a significantly increased SOcAMP signalling was shown to exist in hypertrophic cardiomyocytes. Further experiments to ascertain whether a causeand- effect relationship exists between SOcAMPS and cardiac cell hypertrophy are in progress.

Organic thin film transistor (OTFT) technology can be implemented to develop cost-effective and label-free bio-affinity sensor chips, having a field-effect transport directly coupled to a bio-sensing process, useful to high-throughput testing and point-of-care applications. Biological recognition elements such as antibodies or other proteins can be integrated in OTFT devices to confer specificity. In this study the use of lipid bilayers as support for biomolecules immobilization is investigated. Preliminary results in terms of electrical resistance and capacitance of the lipid bilayers are presented.