Latest PUBLICATIONS

  • Sequence analysis of retinoic acid receptor alpha, beta and gamma isoforms in the lizard, Podarcis sicula.
    Publication Date: 01/05/2007, on The Journal of steroid biochemistry and molecular biology
    by Esposito T, Caccavo M, Cianci A, Cardone A, Angelini F, Varriale B
    DOI: 10.1016/j.jsbmb.2007.03.005

    Vitamin A and its principal biologically active derivative, retinoic acid (RA), play a fundamental role in diverse processes, such as proliferation, differentiation, morphogenesis, metabolism and apoptosis of many types of cells. In addition, RA has been shown to be involved in the regulation of testicular function. These effects are mediated by interaction with two families of nuclear receptors, retinoic acid receptor (RAR) and retinoid X receptor (RXR), each with three subtypes alpha, beta and gamma. The physiological involvement of retinoids in testicular function has been conducted mainly in mammals. Recently, we found that exogenous all-trans-retinoic acid impairs spermatogenesis and enhance testicular germ cell apoptosis in the lizard, Podarcis sicula, a seasonal breeder. To further investigate the role of retinoic acid in lizard, we focus this work principally on the characterization of lizard retinoic acid receptors (alpha, beta and gamma isoforms). RARalpha is 2720 bp long with a putative ORF between 699 and 2133. A Kozac sequence is present at 696 and a putative poly-adenilation site is present in position 2612. The RARalpha sequence shares 87% homology with mouse RARalpha mRNA while it has 76 and 80% homology with lizard RARbeta and gamma mRNAs. RARbeta is 2478 bp long showing a putative ORF between 196 and 1543. A canonical Kozac sequence is present at 193 and a putative poly-adenilation site is present at 2294. RARbeta shares 91% homology with mouse RARbeta mRNA and has 76% homology with both RARalpha and gamma. RARgamma is 2416bp long. With a putative ORF between 444 and 1818. A Kozac sequence is present at 441 and a putative poly-adenilation site is present at 2288. RARgamma shares 86% homology with mouse RARbeta mRNA and has 80 and 76% homology with both RARalpha and beta respectively. It is worth to note that, as in mouse, the 5'UTR of all isoforms is TATA and CAAT less. Both Northern blot and PCR analyses indicate that lizard testis expresses only RARalpha and RARbeta mRNAs, while RARgamma mRNA transcript was not found. In the period analysed, RARbeta was expressed during the gonadal full activity (May) and RARalpha was present in the post-reproductive period (August). During the autumnal recrudescence (October) RARalpha and RARbeta are co-expressed and, as indicated by quantitative PCR analysis, RARbeta mRNA levels are lower than RARalpha ones. Thus, the appearance and abundance of each receptor correspond to a specific phase of lizard reproductive cycle, allow us to hypothesize that each RAR subtype could play a specific role in the regulation of spermatogenetic activity. The results of the present study show, for the first time, the characterization of RAR mRNAs in the testis of lizard P. sicula, whose expression is related to the different phase of reproductive cycle. Moreover, the gamma form, is principally expressed in the skin during the March-July period, having probably a role in regulating skin homeostasis and colour livery, which are important factor in mating during the reproductive cycle.

  • Resveratrol induces cell death in colorectal cancer cells by a novel pathway involving lysosomal cathepsin D.
    Publication Date: 01/05/2007, on Carcinogenesis
    by Trincheri NF, Nicotra G, Follo C, Castino R, Isidoro C
    DOI: 10.1093/carcin/bgl223

    In human colorectal cancer cells, the polyphenol resveratrol (RV) activated the caspase-dependent intrinsic pathway of apoptosis. This effect was not mediated via estrogen receptors. Pepstatin A, an inhibitor of lysosomal cathepsin D (CD), not (2S,3S)-trans-epoxysuccinyl-L-leucylamido-3-methylbutane ethyl ester, an inhibitor of cathepsins B and L, prevented RV cytotoxicity. Similar protection was attained by small interference RNA-mediated knockdown of CD protein expression. RV promoted the accumulation of mature CD, induced lysosome leakage and increased cytosolic immunoreactivity of CD. Inhibition of CD or its post-transcriptional down-regulation precluded Bax oligomerization, permeabilization of mitochondrial membrane, cytosolic translocation of cytochrome c, caspase 3 activation and terminal deoxinucleotidyl transferase-mediated dUTP-biotin nick end labeling positivity occurring in RV-treated cells. The present study identifies the lysosome as a novel target of RV activity and demonstrates a hierarchy of the proteolytic pathways involved in its cytotoxic mechanism in which the lysosomal CD acts upstream of the cytosolic caspase activation. Our data indicate that metabolic, pharmacologic or genetic conditions affecting CD expression and/or activity could reflect on the sensitivity of cancer cells to RV.

  • Axonal and presynaptic RNAs are locally transcribed in glial cells.
    Publication Date: 01/05/2007, on Rivista di biologia
    by Giuditta A, Chun JT, Eyman M, Cefaliello C, Bruno AP, Crispino M
    DOI:

    In the last few years, the long-standing opinion that axonal and presynaptic proteins are exclusively derived from the neuron cell body has been substantially modified by the demonstration that active systems of protein synthesis are present in axons and nerve terminals. These observations have raised the issue of the cellular origin of the involved RNAs, which has been generally attributed to the neuron soma. However, data gathered in a number of model systems indicated that axonal RNAs are synthesized in the surrounding glial cells. More recent experiments on the perfused squid giant axon have definitively proved that axoplasmic RNAs are transcribed in periaxonal glia. Their delivery to the axon occurs by a modulatory mechanism based on the release of neurotransmitters from the stimulated axon and on their binding to glial receptors. In additional experiments on squid optic lobe synaptosomes, presynaptic RNA has been also shown to be synthesized locally, presumably in nearby glia. Together with a wealth of literature data, these observations indicate that axons and nerve terminals are endowed with a local system of gene expression that supports the maintenance and plasticity of these neuronal domains.

  • FLUOXETINE modifies the expression of serotonergic markers in a differentiation-dependent fashion in the mesencephalic neural cell line A1 mes c-myc.
    Publication Date: 27/04/2007, on Brain research
    by Di Lieto A, Leo D, Volpicelli F, di Porzio U, Colucci-D'Amato L
    DOI: 10.1016/j.brainres.2007.01.076

    Serotonin (5-HT) is a neurotransmitter involved in a variety of CNS functions during development and in adulthood. 5-HT neurons are also involved in the pathogenesis of a number of psychiatric disorders. FLUOXETINE (FLX), a prototypic antidepressant, is a selective 5-HT uptake inhibitor (SSRI) with a demonstrated clinical efficacy in these disorders. SSRI, in a short-term period, binds 5-HT transporter (SERT) raising 5-HT levels at the synapse. Nevertheless, clinical improvement is observed only after 3-4 weeks of treatment. Recently, it has been shown that antidepressants, besides interfering with neurotransmission, can also display an effect on neural cells' proliferation and differentiation. Therefore it has been proposed that antidepressant may exert their clinical effects also acting on cellular functions other then neurotransmission. Here we show that a mesencephalic neural cell line, mes-c-myc A1 (A1) produces 5-HT and expresses SERT and both peripheral (TPH1) and CNS-specific (TPH2) form of tryptophan hydroxylase, the limiting enzyme in 5-HT biosynthesis. Cyclic AMP-dependent neuronal differentiation of A1 cells modulates the expression of TPHs. FLX, as well as citalopram (CIT), another SSRI inhibitor, modulates expression of serotonergic markers depending on the differentiation status of the cells. Interestingly, long-term but not short-term FLX treatment selectively modulates mRNA levels of TPH2, only in differentiated A1 cells. Finally, FLX and citalopram selectively decrease the proliferation rate of undifferentiated A1 cells, whereas have no effects on NIH-3T3 fibroblasts proliferation. In conclusion, neuronal differentiation of A1 cells not only modulates the expression of serotonergic markers, but appears to affect the response to FLX.

  • On the genesis of unilateral micrographia of the progressive type.
    Publication Date: 09/04/2007, on Neuropsychologia
    by Barbarulo AM, Grossi D, Merola S, Conson M, Trojano L
    DOI: 10.1016/j.neuropsychologia.2007.01.002

    We report a patient who, following a focal ischemic lesion of the left basal ganglia, developed right hand micrographia characterised by progressive reduction of letter size during writing (progressive micrographia). The patient did not show relevant cognitive impairments, but achieved pathological scores in tests for verbal fluency, and cognitive flexibility and monitoring. A systematic investigation of the writing performances demonstrated that micrographia showed a clear length effect in whatever writing style or task, while it was not observed in drawing, or in left hand writing to a comparable extent. Right hand progressive micrographia was not affected by a concurrent motor and cognitive load; instead, switching between two kinds of allographic responses and presenting one letter at a time in copying tasks reduced severity of micrographia significantly. These findings support the view that progressive micrographia in our patient could be ascribed to a defect in regulating the motor output on the basis of self-generated strategies. This conclusion would be consistent with neuroimaging evidence about the role of the basal ganglia in the control of motor sequencing, and could suggest that progressive micrographia might be associated with specific executive defects.

  • Driving forces in the delivery of penetratin conjugated G protein fragment.
    Publication Date: 05/04/2007, on Journal of medicinal chemistry
    by Albrizio S, Giusti L, D'Errico G, Esposito C, Porchia F, Caliendo G, Novellino E, Mazzoni MR, Rovero P, D'Ursi AM
    DOI: 10.1021/jm060935b

    A42 is a chimera peptide consisting of Galphas(374-394)C379A--the 21-mer C terminus of the Galphas protein, able of adenosine inhibitory activity--and penetratin--the 16 residue fragment, derived from the homeodomain of the Drosophila transcription factor Antennapedia. A42 is able to cross cell membranes and to inhibit A2A and A2B adenosine and beta-adrenergic receptor stimulated camps (D'Ursi et al. Mol. Pharmacol. 2006, 69, 727-36). Here we present an extensive biophysical study of A42 in different membrane mimetics, with the objective to evaluate the molecular mechanisms which promote the membrane permeation. Fluorescence, CD, and NMR data were acquired in the presence of negatively charged and zwitterionic sodium dodecyl sulfate and dodecylphosphocholine surfactants. To validate the spectroscopic results in a larger scale, fluorescence microscopy experiments were performed on negatively charged and zwitterionic dipalmitoylphosphatidylglycerol and dipalmitoylphosphatidylcholine vesicles. Our results show that the internalization of A42 is mainly driven by electrostatic interactions, hydrophobic interactions playing only a secondary, sinergistic role. The distribution of the charges along the molecule has an important role, highlighting that internalization is a process which requires a specific matching of peptide and membrane properties.

  • Autobiographical recall training in elderly adults with subjective memory complaint: a pilot study.
    Publication Date: 01/04/2007, on Perceptual and motor skills
    by Grossi D, Postiglione A, Schettini B, Trojano L, Barbarulo AM, Giugliano V, Ambron E, Aiello A
    DOI: 10.2466/pms.104.2.621-628

    Subjective memory complaint is a self-reported memory impairment which affects elderly people. This problem does not interfere with daily living activities but could decrease quality of life. This study's purpose was to verify whether a specific, newly developed, autobiographical recall training could modify self-perception of memory of subjects with subjective memory complaint. Seven elderly subjects (4 women and 3 men; mean age 65.5 yr., SD=11) with such complaint, evidenced through a specific questionnaire, attended the training course and were prospectively assessed on standard neuropsychological tests, depressive symptomatology, and self-perception of memory. Self-perception of memory, as assessed by scores on a formalized questionnaire, improved significantly after the training, while depressive symptoms did not change. Neuropsychological performances were normal before and after the training, but a statistically significant improvement was observed only on the phonological fluency test. Thus the present pilot study suggested that the training may be effective in improving self-perception of memory and metamnestic capacity in elderly people with subjective memory complaints but not in changing subjects' depressive symptoms. This requires replication of this work with a much larger sample so statistical power is adequate.

  • Progressive topographical disorientation: a case of focal Alzheimer's disease.
    Publication Date: 01/04/2007, on Neurological sciences : official journal of the Italian Neurological Society and of the Italian Society of Clinical Neurophysiology
    by Grossi D, Fasanaro AM, Cecere R, Salzano S, Trojano L
    DOI: 10.1007/s10072-007-0797-x

    We describe a follow-up study of a patient with a selective, progressive impairment of topographical orientation. The patient's topographical difficulties were evident only in unfamiliar surroundings at the beginning of the observation period but later on they were observed even at home. Serial neuropsychological tests demonstrated a progressive impairment of visuospatial abilities with sparing of the other cognitive domains; only at the last assessment, about six years after early disturbances and three years after the first evaluation, the patient developed the typical cognitive impairments of Alzheimer's disease (AD). This case represents a focal variant of AD not previously described and suggests that the neuronal pathways underlying spatial orientation may be selectively damaged by the degenerative process.

  • Comparison between total endothelial progenitor cell isolation versus enriched Cd133+ culture.
    Publication Date: 01/04/2007, on Journal of biochemistry
    by Casamassimi A, Balestrieri ML, Fiorito C, Schiano C, Maione C, Rossiello R, Grimaldi V, Del Giudice V, Balestrieri C, Farzati B, Sica V, Napoli C
    DOI: 10.1093/jb/mvm060

    Endothelial progenitor cells (EPCs) play a role in endogenous neovascularization of ischaemic tissues. Isolation and characterization of EPCs from circulating mononuclear cells are important for developing targeted cellular therapies and reproducibility of data are the major scientific goals. Here we compared two currently employed isolation methods, i.e. from total peripheral blood mononuclear cells (PBMCs) and from enriched CD133(+) cells, by defining the cell morphology and functional activities. We show that EPCs from cultured PBMCs resulted in an adherent population of 23% +/- 4% merged cells positive for Dil-Ac-LDL and lectin, whereas the percentage of double positive cells in cultured CD133(+) enriched cells was 50% +/- 7% (P < 0.01). These data were obtained through a novel and a more complete method of analysis of cell calculations (specifically by dividing each microscope field into 120 subfields). When stimulated with tumour necrosis factor alpha (TNF)-alpha and glucose, cell number was reduced in EPCs from total PBMCs and, more consistently, in CD133(+) enriched cells. However, both cultured total PBMCs and CD133(+) enriched cells respond similarly to TNF-alpha or glucose-induced p38-phosphorylation. EPCs from both procedures show similar results in terms of phenotype and response to modulators of their functional activities. However, when the cell phenotype of CD133(+) enrichment-derived cells was compared with that of cells from the total PBMC, a significant increase in CD133(+) expression was observed (P < 0.01) This may have relevance during intervention studies using cultured EPCs.

  • Comparative evaluation of different DNA extraction procedures from food samples.
    Publication Date: 01/03/2007, on Biotechnology progress
    by Di Bernardo G, Del Gaudio S, Galderisi U, Cascino A, Cipollaro M
    DOI: 10.1021/bp060182m

    Five methodologies for extracting DNA from food samples are described. The food products analyzed are from either soybean or maize. They were selected on the basis of the mechanical, thermal, and chemical treatments that they had been subjected to during industrial processing. DNA preparations were evaluated for purity, yield, and average fragment size. Two endogenous genes, soybean lectin gene and alcohol dehydrogenase gene (adh1), were used to assess the degree of DNA degradation at different stages of the transformation chain. The goal of this study was to determine the role that extraction methods play in DNA amplification in order to select the best protocol for a food sample. This comparative evaluation can be specifically useful for detection of genetically modified ingredients in a variety of food matrices.

  • Bidimensional tandem mass spectrometry for selective identification of nitration sites in proteins.
    Publication Date: 01/03/2007, on Analytical chemistry
    by Amoresano A, Chiappetta G, Pucci P, D'Ischia M, Marino G
    DOI: 10.1021/ac0620361

    Nitration of protein tyrosine residues is very often regarded as a molecular signal of peroxynitrite formation during development, oxidative stress, and aging. However, protein nitration might also have biological functions comparable to protein phosphorylation, mainly in redox signaling and in signal transduction. The major challenge in the proteomic analysis of nitroproteins is the need to discriminate modified proteins, usually occurring at substoichiometric levels from the large amount of nonmodified proteins. Moreover, precise localization of the nitration site is often required to fully describe the biological process. Existing methodologies essentially rely on immunochemical techniques either using 2D-PAGE fractionation in combination with western blot analyses or exploiting immunoaffinity procedures to selectively capture nitrated proteins. Here we report a totally new approach involving dansyl chloride labeling of the nitration sites that rely on the enormous potential of MSn analysis. The tryptic digest from the entire protein mixture is directly analyzed by MS on a linear ion trap mass spectrometer. Discrimination between nitro- and unmodified peptide is based on two selectivity criteria obtained by combining a precursor ion scan and an MS3 analysis. This new procedure was successfully applied to the identification of 3-nitrotyrosine residues in complex protein mixtures.

  • Oligomerization is a specific requirement for apical sorting of glycosyl-phosphatidylinositol-anchored proteins but not for non-raft-associated apical proteins.
    Publication Date: 01/03/2007, on Traffic (Copenhagen, Denmark)
    by Paladino S, Sarnataro D, Tivodar S, Zurzolo C
    DOI: 10.1111/j.1600-0854.2006.00522.x

    Protein apical sorting in polarized epithelial cells is mediated by two different mechanisms, raft dependent and raft independent. In Madin-Darby canine kidney (MDCK) cells, an essential step for apical sorting of glycosyl-phosphatidylinositol (GPI)-anchored proteins (GPI-APs) is their coalescence into high-molecular-weight (HMW) oligomers. Here we show that this mechanism is also functional in Fischer rat thyroid cells, which possess a different sorting phenotype compared with MDCK cells. We demonstrate that, as in MDCK cells, both apical and basolateral GPI-APs associate with detergent-resistant microdomains, but that only the apical proteins are able to oligomerize into HMW complexes during their passage through the medial Golgi. We also show that oligomerization is a specific requirement for apical sorting of GPI-APs and is not used by transmembrane, non-raft-associated apical proteins.

  • RB and RB2/P130 genes cooperate with extrinsic signals to promote differentiation of rat neural stem cells.
    Publication Date: 01/03/2007, on Molecular and cellular neurosciences
    by Jori FP, Galderisi U, Napolitano MA, Cipollaro M, Cascino A, Giordano A, Melone MA
    DOI: 10.1016/j.mcn.2006.11.009

    Mechanisms governing commitment and differentiation of the cells of the nervous system begin to be elucidated: how extrinsic and intrinsic components are related remains poorly understood. To investigate this issue, we overexpressed genes of the retinoblastoma (Rb) family RB and RB2/p130, which play an important role during nerve cell maturation, in rat neural stem cells (NSCs). Immunostaining of neurons, astrocytes and oligodendrocytes in cultures overexpressing pRb or pRb2/p130 revealed that these genes affect lineage specification of differentiating NSCs. We observed modifications in percentage of differentiated cells indicating a shift towards the phenotype induced by culture conditions. Results were confirmed by detection of the expression levels of differentiation markers by RT-PCR. Analysis of BrdU incorporation and detection of an early marker of apoptosis suggest that the effect of pRb and pRb2/p130 overexpression is not dependent on the inhibition of cell proliferation, nor does it rely on the regulation of cell survival. Our findings suggest that Rb family genes are involved in fate determination of the cells of the nervous system. However, their role seems subsidiary to that of the extrinsic signals that promote lineage specification and appear to be mediated by a direct effect on the acquisition of a specific phenotype.

  • Trigeminal perineural spread of head and neck tumors.
    Publication Date: 28/02/2007, on The neuroradiology journal
    by Bartiromo F, Cirillo L, Caranci F, Elefante A, D'Amico A, Tortora F, Brunetti A, Cirillo S
    DOI: 10.1177/197140090702000119

    Perineural tumor spread (PNS) of head and neck malignancies is a well-known form of metastatic disease in which a lesion can migrate away from the primary site along the endoneurium or perineurium. MR imaging is considered the primary method for evaluating patients with symptoms related to the trigeminal nerve in most clinical settings. Both CT and MR imaging can detect perineural spread, but MRI is the modality of choice because of its capability to detect direct signs (nerve enlargement and enhancement) and indirect signs (neuropathic muscular atrophy, obliteration of fat planes). In addition, MRI is more sensitive because of its superior soft-tissue contrast, its multiplanar capability and decreased artifacts from dental hardware. Fat suppression images after contrast injection are mandatory to better detect nerve enhancement. CT is useful in detecting foraminal enlargement or more destructive bone patterns. Nerve function can be perserved until later in the course of the disease: patients with perineural spread demonstrated at radiologic or pathologic examination may have normal or nonspecific nerve function at clinical examination (patients are misdiagnosed with Bell's palsy or trigeminal neuralgia). Hence MRI assessment of perineural tumor location and extension is important.

  • A new mutant of bovine seminal ribonuclease with a reversed swapping propensity.
    Publication Date: 27/02/2007, on Biochemistry
    by Ercole C, Spadaccini R, Alfano C, Tancredi T, Picone D
    DOI: 10.1021/bi0613630

    Bovine seminal ribonuclease (BS-RNase) is made up of two identical subunits bridged through two disulfide bonds. In solution, it exists as a 2:1 equilibrium mixture between two forms, with (MxM) and without swapping (M=M) of the N-terminal arms. The swapping endows BS-RNase with some special biological functions, including antitumor activity, since MxM retains a dimeric structure even under reducing conditions, thus evading the cytosolic ribonuclease inhibitor. To investigate the structural basis of domain swapping in BS-RNase, we have obtained several mutants by replacing selected residues with the corresponding ones of its monomeric counterpart, bovine pancreatic ribonuclease (RNase A). We have already shown that, in contrast with all other cases of swapped proteins, the swapping propensity of BS-RNase does not depend on the specific sequence of the 16-22 hinge loop, which connects the main body to the dislocating arm. In this paper we report the design, the expression, and the structural characterization of two mutants obtained by replacing Arg80 with Ser either in BS-RNase or in the mutant already containing the 16-22 hinge sequence of RNase A. NMR and circular dichroism data indicate that, in the monomeric form of the latter mutant, Ser80 acts as a switch for the conformation of the hinge region. Accordingly, in the dimeric form of the same mutant the MxM:M=M equilibrium ratio is inverted to 1:2. Overall, these data suggest that the presence of Arg80 triggers the swapping of N-terminal ends and plays a relevant role in the stability of the swapped form of BS-RNase.