Latest PUBLICATIONS

  • Effects of triterpene derivatives from Maytenus rigida on VEGF-induced Kaposi's sarcoma cell proliferation.
    Publication Date: 12/02/2010, on Chemico-biological interactions
    by Martucciello S, Balestrieri ML, Felice F, Estevam Cdos S, Sant'Ana AE, Pizza C, Piacente S
    DOI: 10.1016/j.cbi.2009.12.012

    Betulinic acid (BA) is a naturally occurring lupane-type triterpene which exhibits a variety of biological activities including potent cytotoxic properties. On the basis of the structural similarity to BA, two lupane derivatives namely lup-20(29)-ene-3beta,30-diol (1) and lup-20(29)-ene-3beta,28-diol (2), along with two friedelane derivatives, namely friedelan-3-one (3) and friedelan-3beta-ol (4), isolated from the Brazilian plant Maytenus rigida, have been evaluated for their anti-proliferative effect. Similarly to BA, compounds 1 and 3 at 1 microM concentration significantly inhibited the VEGF-induced Kaposi's sarcoma (KS) cell proliferation by 50%. In contrast, this effect was not found in control endothelial cells (EC). Moreover, compounds 1 and 3 showed a dose-dependent effect on the apoptotic cell death, as detected by FACS analysis and caspase-3 assay. Specifically, at 10 microM concentration, apoptosis was significantly induced (from 45% to 55% of hypodiploid cells vs control cells) and showed the same potency order observed for the anti-proliferative effect at 1 microM, i.e., compound 3>BA>compound 1. Taking into account the interest given rise by BA as anticancer agent, the comparable anti-proliferative activity shown by compounds 1 and 3 and BA, can give an impulse to further investigate lupane and friedelane derivatives as cytotoxic agents.

  • Intra-brain microinjection of human mesenchymal stem cells decreases allodynia in neuropathic mice.
    Publication Date: 01/02/2010, on Cellular and molecular life sciences : CMLS
    by Siniscalco D, Giordano C, Galderisi U, Luongo L, Alessio N, Di Bernardo G, de Novellis V, Rossi F, Maione S
    DOI: 10.1007/s00018-009-0202-4

    Neuropathic pain is a very complex disease, involving several molecular pathways. Current available drugs are usually not acting on the several mechanisms underlying the generation and propagation of pain. We used spared nerve injury model of neuropathic pain to assess the possible use of human mesenchymal stem cells (hMSCs) as anti-neuropathic tool. Human MSCs were transplanted in the mouse lateral cerebral ventricle. Stem cells injection was performed 4 days after sciatic nerve surgery. Neuropathic mice were monitored 7, 10, 14, 17, and 21 days after surgery. hMSCs were able to reduce pain-like behaviors, such as mechanical allodynia and thermal hyperalgesia, once transplanted in cerebral ventricle. Anti-nociceptive effect was detectable from day 10 after surgery (6 days post cell injection). Human MSCs reduced the mRNA levels of the pro-inflammatory interleukin IL-1beta mouse gene, as well as the neural beta-galactosidase over-activation in prefrontal cortex of SNI mice. Transplanted hMSCs were able to reduce astrocytic and microglial cell activation.

  • Dual role of parathyroid hormone in endothelial progenitor cells and marrow stromal mesenchymal stem cells.
    Publication Date: 01/02/2010, on Journal of cellular physiology
    by Di Bernardo G, Galderisi U, Fiorito C, Squillaro T, Cito L, Cipollaro M, Giordano A, Napoli C
    DOI: 10.1002/jcp.21976

    Hematopoietic stem cells derive regulatory information also from parathyroid hormone (PTH). To explore the possibility that PTH may have a role in regulation of other stem cells residing in bone marrow, such as mesenchymal stem cells (MSCs) and endothelial progenitor cells (EPCs) we assessed the effect of this hormone on the in vitro behavior of MSCs and EPCs. We evidenced that MSCs were much more responsive to PTH than EPCs. PTH increased the proliferation rate of MSCs with a diminution of senescence and apoptosis. Taken together, our results may suggest a protective effect of PTH on MSCs that reduces stress phenomena and preserve genome integrity. At the opposite, PTH did not modify the fate of EPCs in culture.

  • Differential neuropsychological profiles in Parkinsonian patients with or without vascular lesions.
    Publication Date: 15/01/2010, on Movement disorders : official journal of the Movement Disorder Society
    by Santangelo G, Vitale C, Trojano L, De Gaspari D, Bilo L, Antonini A, Barone P
    DOI: 10.1002/mds.22893

    The purpose of this study is to compare the neuropsychological profile of patients affected by parkinsonism and vascular lesions to that in patients with PD alone (PD) and to evaluate whether the brain vascular lesion load is associated with neuropsychological variables. Thirty-six nondemented patients with parkinsonism were divided into 3 groups of 12 patients each, according to both clinical history and the presence of brain vascular lesions and/or dopaminergic denervation as revealed by magnetic resonance and dopamine transporter imaging, respectively. The first group had vascular lesions without dopaminergic denervation (VP group); the second group had vascular lesions and dopaminergic denervation (DD) (VP+DD group); and the third group consisted of patients with dopaminergic denervation (PD group) without vascular lesions. All patients underwent neurological and neuropsychological assessments. The groups differed in disease duration, age at onset, and cerebrovascular risk factors. The VP and VP+DD groups performed worse than the PD group on frontal/executive tasks. Regardless of the presence of dopaminergic denervation, cerebrovascular lesions in hemispheric white matter, basal ganglia, and cerebellum have an important effect in determining early onset and severity of cognitive impairment in patients with parkinsonism.

  • Glycoproteome study in myocardial lesions serum by integrated mass spectrometry approach: preliminary insights.
    Publication Date: 01/01/2010, on European journal of mass spectrometry (Chichester, England)
    by Carpentieri A, Giangrande C, Pucci P, Amoresano A
    DOI: 10.1255/ejms.1035

    Bottom up proteomics requires efficient and selective pre-fractionation procedures to simplify the analysis of the enormous number of peptides resulting from the hydrolysis of a cellular extract enabling the detection, identification and the structural characterization of the post-translational modifications. Glycosylation, a well-known post-translational modification, plays a key role in the enormous complexity, and heterogeneity of the human blood serum proteome. Thereby, characterization of glycosylation from serum is a challenging task, even for the existing sophisticated analytical methodologies. Here we report a glycoproteomics study on the identification of even low abundant glycoproteins, including the localization of N-glycosylation sites and the glycan profiling in human sera from healthy and myocarditis affected donors. The strategy is simply based on proteolytic digestion of total serum proteins followed by a single enrichment step of glycopeptides on ConA lectin affinity chromatography. Glycopeptides were then deglycosylated by PNGaseF treatment and nano-liquid chromatography-electrospray ionization tandem mass spectrometry analyses of the free peptides provided the basis for both identification of the individual proteins and elucidation of their modification sites. Moreover, glycan profilings could be obtained by matrix-assisted laser desorption/ionization mass spectrometry analysis of the released oligosaccharides. Our data led to the identification of 68 different glycosylation sites within 49 different proteins. Moreover, the analyses carried out on glycans represent the first picture of a glycosylation pattern in myocardial lesions. As a whole, several differences in the glycosylation patterns from different sera were observed, thus indicating glycan profiling as a possible tool to discriminate among different diseases.

  • Discriminative behavioral assessment unveils remarkable reactive astrocytosis and early molecular correlates in basal ganglia of 3-nitropropionic acid subchronic treated rats.
    Publication Date: 01/01/2010, on Neurochemistry international
    by Cirillo G, Maggio N, Bianco MR, Vollono C, Sellitti S, Papa M
    DOI: 10.1016/j.neuint.2009.09.013

    Reactive astrocytosis seems to be strongly implicated in the development and maintenance of inflammatory and neurodegenerative disorders. We design a new toxic model treatment with 3-nitropropionic acid (3-NP), a mitochondrial complex II irreversible inhibitor, to induce in rats Huntington's disease (HD) like syndrome, characterized by hindlimb dystonia, involuntary choreiform movements and reduced global activity. In an attempt to find out whether molecular and morphological changes in the neuro-glial network could be involved in the pathogenesis of this disease, we developed a protocol of subchronic intra-peritoneal 3-NP intoxication. Moreover we set up specific, highly discriminative, behavioral tests to detect very early mild motor disabilities in 3-NP treated rats. This treatment did not cause severe cell death. However, in the Caudate-Putamen (CPu) of all 3-NP treated animals we found a massive astrogliosis, revealed by increased GFAP levels, paralleled by changes of the glial glutamate transporter GLAST distribution. To these glial changes we detected a transcriptional upregulation of c-fos and Sub-P in the striatal medium spiny neurons (MSN). We propose that this model of 3-NP intoxication along with the designed set of behavioral analyses allow to unmask in a very early phase the motor deficits and the underlying morpho-molecular changes associated to the onset of motor disabilities in the HD-like syndrome. Therefore this model unveil the key role played by the different components of the tripartite synapse in the pathogenesis of the HD, a putative non-cell-autonomous disease.

  • Intrathecal NGF administration reduces reactive astrocytosis and changes neurotrophin receptors expression pattern in a rat model of neuropathic pain.
    Publication Date: 01/01/2010, on Cellular and molecular neurobiology
    by Cirillo G, Cavaliere C, Bianco MR, De Simone A, Colangelo AM, Sellitti S, Alberghina L, Papa M
    DOI: 10.1007/s10571-009-9430-2

    Nerve growth factor (NGF), an essential peptide for sensory neurons, seems to have opposite effects when administered peripherally or directly to the central nervous system. We investigated the effects of 7-days intrathecal (i.t.) infusion of NGF on neuronal and glial spinal markers relevant to neuropathic behavior induced by chronic constriction injury (CCI) of the sciatic nerve. Allodynic and hyperalgesic behaviors were investigated by Von Frey and thermal Plantar tests, respectively. NGF-treated animals showed reduced allodynia and thermal hyperalgesia, compared to control animals. We evaluated on lumbar spinal cord the expression of microglial (ED-1), astrocytic (GFAP and S-100beta), and C- and Adelta-fibers (SubP, IB-4 and Cb) markers. I.t. NGF treatment reduced reactive astrocytosis and the density of SubP, IB4 and Cb positive fibers in the dorsal horn of injured animals. Morphometric parameters of proximal sciatic nerve stump fibers and cells in DRG were also analyzed in CCI rats: myelin thickness was reduced and DRG neurons and satellite cells appeared hypertrophic. I.t. NGF treatment showed a beneficial effect in reversing these molecular and morphological alterations. Finally, we analyzed by immunohistochemistry the expression pattern of neurotrophin receptors TrkA, pTrkA, TrkB and p75(NTR). Substantial alterations in neurotrophin receptors expression were observed in the spinal cord of CCI and NGF-treated animals. Our results indicate that i.t. NGF administration reverses the neuro-glial morphomolecular changes occurring in neuropathic animals paralleled by alterations in neurotrophin receptors ratio, and suggest that NGF is effective in restoring homeostatic conditions in the spinal cord and maintaining analgesia in neuropathic pain.

  • The dilemma: does tissue expression of cathepsin D reflect tumor malignancy? The question: does the assay truly mirror cathepsin D mis-function in the tumor?
    Publication Date: 01/01/2010, on Cancer biomarkers : section A of Disease markers
    by Nicotra G, Castino R, Follo C, Peracchio C, Valente G, Isidoro C
    DOI: 10.3233/CBM-2010-0143

    Three molecular forms of the proteolytic enzyme Cathepsin D (CD) are found in the cell: the precursor (proCD), the intermediate single-chain and the mature double-chain. ProCD, which is found in the Golgi Complex, is enzymatically inactive, while the intermediate and the mature forms, respectively found in endosomes and lysosomes, are enzymatically active. The latter are involved in autophagy and apoptosis pathways thus playing a crucial role in the control of cell and tissue homeostasis. ProCD can be secreted in the extracellular space and, by interacting with membrane receptors, can promote cell proliferation. At slightly acid pH, secreted proCD undergoes partial maturation and becomes active. In the extracellular space, CD can degrade the protein components of the matrix and free growth factors therein embedded, thus favoring tumor growth, invasion and angiogenesis. Based on the multiple tasks performed by CD inside and outside the cell, it is not irrational to hypothesize its involvement in cancer development and progression and a strict link between its tissue expression and the clinico-pathological features of the tumor. Thus, not surprisingly, as many as 519 articles are found in the database of pubmed with the keywords 'cathepsin D, cancer and marker'. Disappointingly, however, in spite of, or because of, this large number of studies, the scientific community has not reached a general agreement on the prognostic value of CD in cancer progression. Here, we will briefly review the relevant literature and offer a possible explanation for the conflicting data.

  • High expression of cathepsin D in non-Hodgkin's lymphomas negatively impacts on clinical outcome.
    Publication Date: 01/01/2010, on Disease markers
    by Nicotra G, Manfroi F, Follo C, Castino R, Fusco N, Peracchio C, Kerim S, Valente G, Isidoro C
    DOI: 10.3233/DMA-2010-0698

    The lysosomal protease Cathepsin D (CD) has been implicated in the homeostasis of lymphatic tissues. We investigated whether the level of CD expression influences the progression and the clinical outcome in Non-Hodgkin's Lymphomas (NHLs). The expression of CD was assessed by immunohistochemistry and immunofluorescence in biopsies of Diffuse Large B Cell Lymphomas (DLBCL, 35 cases), Follicular Lymphomas (FL, 9 cases of grade I-II plus 14 cases of grade IIIB), Chronic Lymphocytic Leukaemias (CLL, 17 cases) and Peripheral T-cell Lymphomas (PTCL, 5 cases). CD staining showed a cytoplasmic punctate pattern compatible with its lysosomal localization. Based on the level of CD expression and the proportion of positive cells, lymphomas were classified as 'low expressing' (< 20% of tumor cells) or 'highly expressing' (>or= 20% of tumor cells). Lymphomas highly expressing CD were associated with a worse stage (III-IV) at diagnosis (31/34 cases; p=0.002) and with a poor clinical outcome (i.e., partial remission and death; 28/34 cases; p=0.03). In the subgroup of aggressive/high grade of malignancy lymphomas (i.e., DLBCL, FL IIIB and PTCL), the Kaplan-Meier curve revealed a very low cumulative overall survival probability (approximately 20% at 5 year) for patients bearing a NHL with > 40% CD-positive cells compared to that of patients bearing a NHL with < 20% CD-positive cells ( approximately 70% at 5 year). This correlation was statistically significant (log-rank test, p=0.01). In Cox multivariate analysis CD failed to be a prognosticator independent of pathologic stage, though the hazard ratio confirmed the association of low expression with a better survival probability. These data indicate that the presence of a high percentage of CD-positive tumor cells negatively reflects on the progression of NHLs.

  • Rehabilitation in peripheral non femoral fractures: a review.
    Publication Date: 01/01/2010, on Clinical cases in mineral and bone metabolism : the official journal of the Italian Society of Osteoporosis, Mineral Metabolism, and Skeletal Diseases
    by Mangone G, Postiglione M, Pasquetti P
    DOI:

    This paper is a short review of the available information on peripheral non femoral fractures (PNFF) which are strictly related to osteoporosis. Particular attention is focused on wrist fractures in view of their frequent occurrence as indicated by world wide statistics. Of special interest is the definition of risk groups (old age), risk areas (developed countries) and risk factors. Reference is made to 2008 WHO guidelines for fracture risk assessment. PNFF are a serious concern to health authorities because their high incidence in constant growth, causes a serious burden on the health budget. The pathway of patient care is described, from initial evaluation (including bone fragility, multi-morbidity and risk factors) to specific diagnosis and treatment. The multiplicity of etiological factors involved requires multidisciplinary approach. This aspect justifies the importance given to rehabilitation, which is the longest part of patient care and is strictly connected to preventive measures. There is ample reference to falls, to exercises, to appropriate sports, to complications and to active and passive mobilization. The paper suggests research in areas related to PNFF, to health economics, epidemiology, prevention, health education, training and multidisciplinary coordination.

  • In AbetaPP-overexpressing cultured human muscle fibers proteasome inhibition enhances phosphorylation of AbetaPP751 and GSK3beta activation: effects mitigated by lithium and apparently relevant to sporadic inclusion-body myositis.
    Publication Date: 01/01/2010, on Journal of neurochemistry
    by Terracciano C, Nogalska A, Engel WK, Askanas V
    DOI: 10.1111/j.1471-4159.2009.06461.x

    Muscle fiber degeneration in sporadic inclusion-body myositis (s-IBM) is characterized by accumulation of multiprotein aggregates, including aggregated amyloid-beta (Abeta)-precursor protein 751 (AbetaPP751), Abeta, phosphorylated tau, and other 'Alzheimer-characteristic' proteins. Proteasome inhibition is an important component of the s-IBM pathogenesis. In brains of Alzheimer's disease (AD) patients and AD transgenic-mouse models, phosphorylation of neuronal AbetaPP695 (p-AbetaPP) on Thr668 (equivalent to T724 of AbetaPP751) is considered detrimental because it increases generation of cytotoxic Abeta and induces tau phosphorylation. Activated glycogen synthase kinase3beta (GSK3beta) is involved in phosphorylation of both AbetaPP and tau. Lithium, an inhibitor of GSK3beta, was reported to reduce levels of both the total AbetaPP and p-AbetaPP in AD animal models. In relation to s-IBM, we now show for the first time that (1) In AbetaPP-overexpressing cultured human muscle fibers (human muscle culture IBM model: (a) proteasome inhibition significantly increases GSK3beta activity and AbetaPP phosphorylation, (b) treatment with lithium decreases (i) phosphorylated-AbetaPP, (ii) total amount of AbetaPP, (iii) Abeta oligomers, and (iv) GSK3beta activity; and (c) lithium improves proteasome function. (2) In biopsied s-IBM muscle fibers, GSK3beta is significantly activated and AbetaPP is phosphorylated on Thr724. Accordingly, treatment with lithium, or other GSK3beta inhibitors, might benefit s-IBM patients.

  • Endoplasmic reticulum stress reduces the export from the ER and alters the architecture of post-ER compartments.
    Publication Date: 01/12/2009, on The international journal of biochemistry & cell biology
    by Amodio G, Renna M, Paladino S, Venturi C, Tacchetti C, Moltedo O, Franceschelli S, Mallardo M, Bonatti S, Remondelli P
    DOI: 10.1016/j.biocel.2009.08.006

    In eukaryotic cells several physiologic and pathologic conditions generate the accumulation of unfolded proteins in the endoplasmic reticulum (ER), leading to ER stress. To restore normal function, some ER transmembrane proteins sense the ER stress and activate coordinated signalling pathways collectively called the Unfolded Protein Response (UPR). Little is known on how the UPR relates to post-ER compartments and to the export from the ER of newly synthesized proteins. Here, we report that the ER stress response induced by either thapsigargin or nitric oxide modifies the dynamics of the intracellular distribution of ERGIC-53 and GM130, two markers of the ER Golgi Intermediate Compartment and of the cis-Golgi, respectively. In addition, induction of ER stress alters the morphology of the ERGIC and the Golgi complex and interferes with the reformation of both compartments. Moreover, ER stress rapidly reduces the transport to the Golgi complex of the temperature sensitive mutant of the Vesicular Stomatitis Virus G Glycoprotein (VSV-G) fused with the Green Fluorescent Protein (ts045G), without apparently decreasing the amount of the protein competent for export. Interestingly, a parallel rapid reduction of the number of Sec31 labelled fluorescent puncta on the ER membranes does occur, thus suggesting that the ER stress alters the ER export and the dynamic of post-ER compartments by rapidly targeting the formation of COPII-coated transport intermediates.

  • Toward an antitumor form of bovine pancreatic ribonuclease: the crystal structure of three noncovalent dimeric mutants.
    Publication Date: 01/12/2009, on Biopolymers
    by Merlino A, Russo Krauss I, Perillo M, Mattia CA, Ercole C, Picone D, Vergara A, Sica F
    DOI: 10.1002/bip.21183

    The cytotoxic action of bovine seminal ribonuclease (BS-RNase) depends on its noncovalent swapped dimeric form (NCD-BS), which presents a compact structure that allows the molecule to escape ribonuclease inhibitor (RI). A key role in the acquisition of this structure has been attributed to the concomitant presence of a proline in position 19 and a leucine in position 28. The introduction of Leu28, Cys31, and Cys32 and, in addition, of Pro19 in the sequence of bovine pancreatic ribonuclease (RNase A) has produced two dimeric variants LCC and PLCC, which do exhibit a cytotoxic activity, though at a much lower level than BS-RNase. The crystal structure analysis of the noncovalent swapped form (NCD) of LCC and PLCC, complexed with the substrate analogue 2 '-deoxycytidylyl(3 ',5 ')-2 '-deoxyguanosine, has revealed that, differently from NCD-BS, the dimers adopt an opened quaternary structure, with the two Leu residues fully exposed to the solvent, that does not hinder the binding of RI. Similar results have been obtained for a third mutant of the pancreatic enzyme, engineered with the hinge peptide sequence of the seminal enzyme (residues 16-22) and the two cysteines in position 31 and 32, but lacking the hydrophobic Leu residue in position 28. The comparison of these three structures with those previously reported for other ribonuclease swapped dimers strongly suggests that, in addition to Pro19 and Leu28, the presence of a glycine at the N-terminal end of the hinge peptide is also important to push the swapped form of RNase A dimer into the compact quaternary organization observed for NCD-BS.

  • Comparison of the structural and functional properties of RNase A and BS-RNase: a stepwise mutagenesis approach.
    Publication Date: 01/12/2009, on Biopolymers
    by Ercole C, Colamarino RA, Pizzo E, Fogolari F, Spadaccini R, Picone D
    DOI: 10.1002/bip.21176

    The original structure of bovine seminal ribonuclease (BS-RNase), solved in 1993, represents a milestone in the story of protein structure, because it represented the first X-ray structure showing two polypeptide chains entangled through their terminal regions. It is generally assumed that this structural feature is the basis of several special biological activities, including a potent antitumor activity, but this has not been yet definitely proved. To assess this hypothesis, in this article we have analyzed the effects of the N-terminal hinge region and/or of Arg80 on the swapping propensity and cytotoxicity in newly designed proteins, using a covalent dimeric variant of bovine pancreatic ribonuclease (RNase A) as scaffold. All the proteins have a very poor cytotoxic activity, independently on the swapping propensity, that can even reach the same value of native BS-RNase. Overall our data suggest that the swapping represents still an essential requisite for the cytotoxic activity, because it keeps the dimeric structure stable even in the reducing cytosolic environment, but other features are essential to design dimeric antitumor ribonucleases, including a strong positive potential at the N-terminal face and a quaternary structure able to evade the cytosolic ribonuclease inhibitor, with or without the interchain disulfide bridges.

  • Proteomic strategies for the identification of proteinaceous binders in paintings.
    Publication Date: 01/12/2009, on Analytical and bioanalytical chemistry
    by Leo G, Cartechini L, Pucci P, Sgamellotti A, Marino G, Birolo L
    DOI: 10.1007/s00216-009-3185-y

    The identification of proteinaceous components in paintings remains a challenging task for several reasons. In addition to the minute amount of sample available, complex and variable chemical composition of the paints themselves, possible simultaneous presence of several binders and contaminants, and degradation of the original materials due to aging and pollution are complicating factors. We proposed proteomic strategies for the identification of proteins in binders of paintings that can be adapted to overcome the requirements and difficulties presented by specific samples. In particular, we worked on (1) the development of a minimally invasive method based on the direct tryptic cleavage of the sample without protein extraction; (2) the use of microwave to enhance the enzymatic digestion yield, followed by the analysis of the peptide mixtures by nanoLC-MS/MS with electrospray ionization (ESI). Moreover, as an additional tool to tackle the problem of contaminating proteins, we exploited the possibility of generating an exclusion list of the mass signals that in a first run had been fragmented and that the mass spectrometer had to ignore for fragmentation in a subsequent run. The methods, tested on model samples, allowed the identification of milk proteins in a sample from paintings attributed to Cimabue and Giotto, thirteenth-century Italian masters, decorating the vaults of the upper church in the Basilica of St. Francis in Assisi, Italy.