Poster Session II (Topics 13-14 & 16-27)

Thursday, 24 September, 11:30-15:00

13. Membrane trafficking, adhesion

Total number of abstracts in this session: 5



Gliadin peptide P31-43 enhances IL15 activity by interfering with its intracellular trafficking
D. Zanzi, M. Nanayakkara, S. Santagata, G. Lania, L. Iaffaldano, V. Discepolo, M. ten Eikeider, Roberta Kosova, M.V. Barone
Dept Pediatrics and ELFID, Univ. Federico II, Naples, Italy

Background: We previously observed that A-gliadin peptide P31-43 induces proliferative effects similar to Epidermal growth factor (EGF) both in cultured cell lines and enterocytes from celiac disease (CD) patients. The effect is mediated by delayed EGF degradation and prolonged EGF receptor (EGFR) activation in endocytic vesicles due to P31-43 mediated interference with endocytic maturation. Aims: To test P31-43 effects on IL15 induction at level of transcription, translation, intracellular trafficking and its role in P31-43 induced proliferation. Results: In Caco2 cells P31-43 slightly increases IL15 mRNA levels. IL 15 protein was found increased only on the cell surface together with markers of recycling vesicles, as transferrin receptor and Lamp2, implying a P31-43-mediated interference with IL15 vesicular trafficking. On the cell surface IL15 is linked to the receptor, its increase is not dependent on new protein synthesis and functions as a growth factor for CTLL 2 cells. Stat5 and the IL15Ra are activated after P31-43 treatment. Anti-IL15 blocking antibodies can prevent P31-43 induced increase of proliferation in Caco2 cells and in enterocytes of biopsies from CD patients. Conclusion: P31-43 induces enhanced presentation of IL15 in trans to the neighboring cells, interfering with its vesicular trafficking. Justacrine signaling of the IL15/IL15-receptor-alfa contributes both to cell proliferation and activation of innate immunity.



Molecular basis of Charcot-Marie-Tooth type 2b disease
L. Cogli, C. Progida, C. Bucci
Dept Biological and Environmental Sciences and Technologies (DiSTeBA), Univ. of Salento, Lecce, Italy

Charcot-Marie-Tooth type 2B (CMT2B) neuropathy is an autosomal dominant axonal neuropathy characterized by distal weakness, wasting and prominent sensory loss complicated by foot ulcerations that often lead to amputation of toes. It is caused by mutations of the Rab7 protein, a small GTPase controlling late endocytic trafficking. It is still unknown how these mutations, that target highly conserved amino acids, cause the neuropathy, and this issue is particularly challenging considering that Rab7 is a ubiquitous protein. We have demonstrated that the Rab7 mutant proteins causative of CMT2B are predominantly in the GTP-bound form, having altered Koff for nucleotides and impaired GTP hydrolysis, and are able to rescue Rab7 function. We are now investigating the effects of these mutant proteins on neuronal functions. Interestingly, we observed a marked inhibition of neurite outgrowth upon expression of all the mutants in PC12 and Neuro2A cells. Inhibition was confirmed by slower up-regulation of growth-associated protein 43 in PC12 cells and of NeuN in Neuro2A cells. These data suggest that CMT2B disease could be due to alteration of this pathway during neuronal regeneration.



Membrane association of Rac and E-cadherin in FRT thyroid epithelial cells
A. Corteggio1, M. Santoriello1, M. Nitti1, A. Mascia2, V. D’Oriano1, G. Calì2, M. Chiariello1, S. Paladino1, C. Garbi1, L. Nitsch1
1Dept Biologia e Patologia Cellulare e Molecolare, Univ. Federico II, Napoli, Italy
2IEOS, CNR, Napoli, Italy

We are interested in determining the molecular mechanism by which Rac affects the expression of the polarized phenotype in FRT thyroid epithelial cells. To this aim an inducible, constitutively-active form of Rac, ER-Rac(QL), and the inducible, dominant-negative ER-Rac(N17) were stably expressed in FRT cells. By immunofluorescence analysis and cell fractionation we determined that upon tamoxifen treatment of FRT clones expressing ER-Rac(QL), the protein moves from the cytosol to the plasma membrane. The same is true for the dominant-negative ER-Rac(N17) after tamoxifen treatment. The bulk of endogenous Rac is also localized on the plasma membrane of wild-type FRT cells. Treatment with the specific Rac inhibitor NSC23766, removes endogenous Rac from the plasma membrane. Strikingly, E-cadherin is correspondingly removed from the membrane, likely by endocytosis. Chelation of calcium in the culture medium, in a Ca++ switch assay, also causes internalization of E-cadherin from the plasma membrane and the partial removal of Rac. Intracellular E-cadherin and Rac do not colocalize. The coordinate regulation of the association of both proteins to the plasma membrane is under investigation.



Diacylglycerol kinase alpha regulates Rab-coupling protein-driven integrin recycling and cell migration on 3D matrices
E. Rainero1, P.T. Caswell2, A. Graziani1, J.C. Norman2
1Clinical and Experimental Medicine Dept, Univ. of Piemonte Orientale, Novara, Italy
2Beatson Institute for Cancer Research, Glasgow, Scotland, UK

A2780 cells can be stimulated to move through 3D matrices either by the addition of osteopontin or by the expression of Rab25. We have shown that this type of migration requires the recycling of integrins from endosomes to the plasma membrane, regulated by RCP. RCP contains a C2 domain, which can bind to phosphatidic acid (PA) the lipid product of diacylglycerol kinases (DGKs) and phospholipase Ds (PLDs).
Here we show that Diacylglycerol kinase alpha (DGKα) is necessary for the recycling of α5β1 integrin and EGFR1, a step previously regulated by RCP. We then found that DGKα was required for the extension of long pseudopods and for the accumulation of RCP-containing vesicles at their tips when cells were stimulated with osteopontin on a 3D fibrillar matrix. Although the expression of Rab25 induces a similar phenotype, it was not affected by DGKα downregulation, but was completely dependent on PLD activity. Furthermore, the expression of a constitutive-active form of DGKα (myr-DGKα) was sufficient to induce pseudopodial elongation and RCP was concentrated at the tips of these. Interestingly, RCP knock-down abrogated myr-DGKα-induced extension of pseudopods.
These data demonstrate that DGKα has a pivotal role in the regulation of the specific signalling pathway induced by osteopontin which leads to α5β1 integrin and EGFR1 recycling and eventually to tumour cell invasion through 3D microenvironments.



A constitutively active Rac impairs the acquisition of epithelial cell polarity
M. Santoriello1, A Corteggio1, M Nitti1, A Mascia2, V.a D’Oriano1, G. Calì2, M. Chiariello1, S. Paladino1, L. Nitsch1, C. Garbi1
1Dept Biologia e Patologia Cellulare e Molecolare, Univ. Federico II, Napoli, Italy
2IEOS, CNR, Napoli, Italy

We demonstrated that treatment of FRT thyroid epithelial cells with a Rac-specific inhibitor causes an impairment in the acquisition of polarity. FRT-β8i cells, in which the signal transduction from the β1A integrin is impaired, manifest a similar defect. We tested here the hypothesis that an active Rac might correct the FRT-β8i polarity defect. FRT-β8i cells that stably expressed an inducible, constitutively active Rac, ER-Rac(QL), were obtained. Upon tamoxifen treatment the ER-Rac(QL) protein became active, localized at the plasma membrane and in confluent cells it was mostly found on the lateral plasmamembrane, at sites of cell-cell contact. In these cultured cells cytokinesis was progressively impaired. Furthermore, activation of ER-Rac(QL) interfered with the acquisition of transepithelial resistance by confluent monolayers on filters, impaired cyst formation by cells in suspension culture and reduced the wound healing efficiency in a scratch test. Similar results were obtained with wild-type FRT cells expressing the same ER-Rac(QL). We conclude that a constitutively active Rac does not promote, but rather hampers, the acquisition of cell polarity in epithelial cells.


14. Microbial biotechnologies

Total number of abstracts in this session: 7



A region of the N-terminal domain of meningococcal factor H-binding protein that elicits bactericidal antibody across antigenic variant groups
A. Angiolillo3, P.T. Beernink2, C. Lo Passo1, J.A. Welsch2, L. Zippilli3, D.M. Granoff2, F. Felici3
1Dept Life Sciences, Messina Univ., Messina, Italy
2Children’s Hospital Oakland Research Institute, Oakland, CA, USA
3Dept S.T.A.T., Molise Univ., Pesche (IS), Italy

Meningococcal factor H-binding protein (fHbp) is a promising vaccine antigen. Previous studies described three fHbp antigenic variant groups and identified amino acid residues between 100 and 255 as important targets of variant-specific bactericidal antibodies. JAR 4 is an IgG2a MAb from a mouse immunized with fHbp from variant 1 group. The anti- factor H-binding protein mAb, JAR 4, elicits cooperative human complement-mediated bactericidal activity with other mAbs specific for fHbp in the variant 1 or 2 groups, which individually are not bactericidal. We have identified the residues affecting the JAR 4 epitope, demonstrating that the N-terminal domain of meningococcal fHbp is important for eliciting cross-reactive bactericidal antibodies against strains expressing fHbp from different antigenic variant groups, this finding may contribute toward understanding of the basis of cooperative cross-reactive anti-fHbp mAb bactericidal activity.



Antibacterial activity against Gram positive and Gram negative bacteria of a porphyrin photoactivated by laser light
M. Bondi, A. Mazzini, G. Marcato, S. de Niederhausern
Dept of Biomedical Sciences, Univ. of Modena and Reggio Emilia, Modena

The increasing of antibiotic-resistance is a very topical problem that lead researchers to try new strategies to treat microbial infections. The photodynamic therapy (PDT) that utilizes a combination of light, a chemical known as a photosensitizer and oxygen to achieve a cytotoxic effect, could be an alternative method as their mode of action is unrelated to the antibiotic-resistance mechanisms.
We studied the antibacterial activity of a porphyrin activated by laser light (635nm, 50mW, optical fibre 300nm diameter) against four bacteria (S.aureus ATCC 6538, E.faecalis ATCC 29212, E.coli ATCC 25922, P.aeruginosa ATCC 27859) employing different porphyrin concentrations and exposition times. The Gram positives showed a great susceptibility (reduction of about 4 and 5 log CFU/ml for S.aureus and E.faecalis with 10mcg/ml porphyrin and 60’’of photosensitization). In the same conditions the Gram negatives demonstrated a very low susceptibility presumably to their cell wall less permeable to the porphyrin. This difficulty could be overcome using porphyrins in conjunction with compounds that open channels in the bacterial membrane. PDT is proposed as a potential low-cost approach to the treat locally occurring infections as periodontal diseases.



Bioremediation of crude oil in contaminated seawater: a bioaugmentation pilot experiment
M. Hassanshahian1, F. Smedile2, M. Genovese3, R. Denaro3, G. Emtiazi1, M.M. Yakimov3, S. Cappello3
1Dept of Biological Sciences, Univ. of Isfahan, Iran
2Dept Biologia Animale ed Ecologia Microbica, Univ. of Messina, Italy
3Istituto per l'Ambiente Marino Costiero (IAMC), CNR of Messina, Italy

Bioaugmentation can be defined as the addition of pre-grown microbial cultures in order to enhance microbial populations in a site for improving contaminant clean up and reducing clean up time and cost.
In this study, we evaluated the bacterial efficiency to remove crude oil from a simulated contaminated area (mesocosms) of the marine environment using bioaugmentation treatments.
Assays of bioaugmentation were carried out by the introduction, in the reaction tanks, of i) an inoculum of Alcanivorax borkumensis SK2 and ii) a consortium of Alkanivorax borkumensis SK2 and Thalassolituus oleivorans MIL-1. One assay of biostimulation, with the addition of inorganic nutrients, was also carried out.
For a period of 15 days, the bioremediation process was monitored by observing the abundance of bacterial population, by measuring the respiration rate and the rate oil degradation.
Surprisingly, at the end of experimentation, the best rate of oil degradation (75%) was observed when only A. borkumensis was added to the system in study. The rate of oil degradation was significantly lower during assays of bioaugmentation with the consortium (60%) and during assays of biostimulation (45%).



Peptide mimics of two pneumococcal capsular polysaccharide serotypes (6B and 9V) protect mice from a lethal challenge with Streptococcus pneumoniae
C. Lo Passo1, C. M. Smith2, A. Scuderi1, I. Pernice1, J. Kolberg3, H. Baxendale4, D. Goldblatt4, M. R. Oggioni5, P.W. Andrew2, F. Felici6
1Dip. di Scienze della Vita, Unive di Messina, Messina, Italy
2Dept of Infection, Immunity and Inflammation, Univ. of Leicester, Leicester, UK
3Norwegian Institute of Public Health, Torshov, Oslo, Norway
4Infectious Diseases and Immunobiology Units, UCL Institute of Child Health, London, UK
5L.A.M.M.B., Dip. di Biologia Molecolare, Univ. di Siena, Policlinico Le Scotte, Siena, Italy
6Dip. S.T.A.T., Univ. del Molise, Pesche (IS), Italy

Anti-polysaccharide immunity is a key facet of protection against several bacterial pathogens. Problems exist with current polysaccharide vaccines and alternative strategies that deliver a protective response are needed. We have identified immunological peptide mimics of type 6B and 9V pneumococcal capsular polysaccharides (CPS) that could be used as vaccine antigens. Peptides mimicking antigenic properties of serotype 6B CPS were obtained from a phage-displayed peptide library using a human mAb (Db3G9). A murine mAb (206,F-5), against the serotype 9V CPS, identified four peptide mimotopes from random peptide libraries. In ELISA, binding of 206,F-5 and Db3G9 to phage displaying the selected mimotopes was significantly inhibited by type-specific pneumococcal polysaccharide. Peptides were conjugated to KLH and were used to immunise mice. Two peptides, MP13 and MP7, induced specific anti-6B and 9V polysaccharide antibodies, respectively. Mice immunised with MP7-KLH or MP13-KLH conjugate were specifically protected against a lethal challenge with pneumococci of the appropriate serotype. This study provides strong in vivo evidence that peptide mimics are alternatives to polysaccharide vaccines.



Electricity generation of bacterial communities using microbial fuel cells
S. Mocali1, C.Galeffi1, A.Florio1, M.Migliore1, F.Canganella2, G.Bianconi2, E.Di Mattia2, E. Perrin3, R. Fani3, A. Benedetti1
1CRA- Centro di Ricerca per lo Studio delle Relazioni tra Pianta e Suolo, Roma, Italy
2Univ. della Tuscia, Dip. di Agrobiologia e Agrochimica, Viterbo, Italy
3Univ. degli Studi di Firenze , Dip. di Biologia Evoluzionistica, Firenze, Italy

Microbial fuel cells (MFCs) represent a new method for directly generating electricity from the oxidation of dissolved organic matter through the catalytic activities of electrochemically active bacteria. Although various types of MFC have been compared in terms of their performance relating to power generation and energy source, little is known about the ecological role of electrigenic bacteria in the environment. In this preliminary study MFCs have been inoculated directly with natural soil (S), the most important natural source of bacterial diversity, and an organic matrix (A) in order to accomplish a selection of environmental microbial communities involved in the electrigenic process while, at the same time, correlating their genetic and functional diversity with organic matter composition of the biomass.
The experiments were conducted using two-chambered MFCs containing graphite electrodes. As expected, in both the systems a high internal resistance was observed; however, a lag time with low power generation a maximum of about 300mV voltage was detected after 30 days, confirming the presence of electrochemically active bacteria. For each sampling, conducted at initial time (T0) and after 30 days (Tf), soil organic fraction was characterized by Differential Thermal Analysis while DGGE, ARDRA and RAPD analysis were applied to assess the genetic diversity of both uncultivable and cultivable bacterial communities.



Functional characterization of the most protective vaccine candidate against ExPEC
I. Pastorello1, I. Bertoldi1, D. Gomes Moriel1, B. Nesta1, A. Spagnuolo1, S. Marchi1, S. Liberatore1, F. Tonello2, S. Savino1, M. Soriani1, M. Pizza1, L. Serino1
1Novartis Vaccines and Diagnostics, Research Center, Siena, Italy
2CNR, Istituto di Neuroscienze, Dip. di Scienze Biomediche, Padova, Italy

Extraintestinal pathogenic E. coli (ExPEC) are able to cause disease outside the host intestinal tract and are responsible for various disorders that cause considerable morbidity and increased healthcare costs. An increasing antimicrobial resistance leads to the need of an efficacious ExPEC vaccine. By a subtractive reverse vaccinology approach a promising vaccine candidate, NOV043, was identified. It is secreted by a type 2 secretion system and each single component of this system is essential for its secretion. FACS analysis and electron microscopy indicate that NOV043 is also surface-exposed, that could be explained by an amino-terminal lipidation motif. We demonstrated that secreted NOV043 is processed after a glycine-rich region and this catalytic process is under investigation. The surface-exposure and secretion of NOV043 are usually associated with ExPEC strains: nonpathogenic strains can express NOV043, but they are not able to secrete it. The presence of several motifs in NOV043 sequence indicates that it could be a metalloprotease, and its biding to zinc has been confirmed by atomic adsorption. The determination of the possible NOV043 substrate is under investigation.



Plasminogen- and fibronectin–binding protein B is involved in the adherence of Streptococcus pneumoniae to human epithelial cell
S. Papasergi1, M. Garibaldi1, G. Tuscano1, S. Ricci2, S. Peppoloni3, I. Pernice4, C. Lo Passo4, G. Teti1, F. Felici5, C.a Beninati1
1The Elie Metchnikoff Dept, Univ. di Messina, Messina, Italy
2Dipartimento di Biologia Molecolare, Univ. di Siena, Siena, Italy
3Dipartimento di Scienze di Sanità Pubblica, Univ. di Modena e Reggio Emilia, Modena, Italy
4Dipartimento di Scienze della Vita M. Malpighi, Univ. di Messina, Messina, Italy
5Dipartimento S.T.A.T., Univ. del Molise, Pesche, Italy

Streptococcus pneumoniae is a major cause of morbidity and mortality worldwide. The ability of this bacterium to adhere to epithelial cells is considered as an essential early step in colonization and infection. By screening a whole genome phage display library with sera from infected patients, we previously identified three antigenic fragments matching the spr0075 ORF of the strain R6 genome. This locus encodes for a c.120 kDa protein, herein referred to as plasminogen- and fibronectin-binding protein B (PfbB), which displays an LPXTG cell wall anchoring motif and 6 repetitive domains. In this study, by using isogenic pfbB-deleted mutants of the encapsulated D39 and of the unencapsulated DP1004 type 2 pneumococcal strains, we show that PfbB is involved in S. pneumoniae adherence to various epithelial respiratory tract cell lines. Our data suggest that PfbB directly mediates bacterial adhesion, since fluorescent beads coated with the recombinant PfbB sp17 fragment (encompassing one of the six repetitive domains and the C terminal region) efficiently bound to epithelial cells. Mutants lacking PfbB bound to fibronectin and plasminogen considerably less efficiently than wild type bacteria, while sp17-coated beads specifically bound to both of these substrates. Taken together, our data suggests that, by directly interacting with Fn, PfbB significantly increases the ability of S. pneumoniae to adhere to human epithelial cells.


16. Oncogenes and tumor suppressors

Total number of abstracts in this session: 10



The in vivo tumor antagonizing role of human RNASET2 is mediated by host stromal cells recruitment
F. Acquati1, S. Bertilaccio2, L. Monti1, P. Campomenosi1, F. Sanvito3, R. Cinquetti1, P. Bonetti1, M. Lualdi1, A. Grimaldi1, D. Vigetti4, S. Mantero7, C. Riva5, A. Passi4, D. Noonan6, P. Ghia2, C. Doglioni3, C. Capella5, A. Sica7, R. Taramelli1
1Dept Biotechnology and Molecular Sciences, Insubria Univ., Varese, Italy
2Dept Oncology - Lymphoid Malign. Unit, San Raffaele Hospital, Milan, Italy
3Dept of Pathology, San Raffaele Hospital, Milan, Italy
4Dept of Experim Clinical and Biomedical Sciences, Insubria Univ., Varese, Italy
5Dept of Pathology, Ospedale di Circolo, Varese, Italy
6Dept of Clinical and Biological Sciences, Insubria Univ., Varese, Italy
7Humanitas Clinical Institute, Milan, Italy

The human RNASET2 gene, encoding for an extracellular Rh/T2/S ribonuclease, has been recently described by our group as a tumor-antagonizing gene, due to its ability to suppress the tumorigenic phenotype in vivo but not in vitro. A detailed analysis of ovarian cancer-derived human tumors grown in vivo in a nude mice model suggested the involvement of host stromal cells in RNASET2-mediated tumor suppression. In particular, a panel of immunohistochemical assays on the tumor sections revealed a diffuse host stromal cells infiltrate within RNASET2-overexpressing tumors. Significantly, a gene expression survey by real-time qPCR showed that some critical cytokines were overexpressed both in RNASET2-expressing tumors and in the clones from which these tumors were derived. Finally, the in vivo tumor-antagonizing role of RNASET2 was further investigated by assessing the tumor growth in rag-gamma chain mice which have been depleted for specific cell lineages. The preliminary data from this experiment further support the role of the host stromal cells in RNASET2-mediated tumor suppression and allow us to draw a pathogenetic model for the biological mechanisms triggered by this protein.



Constitutively active Stat3 enhances Neu-mediated mammry tumor via upregulation of Cten
I. Barbieri1, S. Pensa1, T. Pannellini2, P. Provero1 P. Musiani2, V. Poli1
1Dept of Genetics,Biology and Biochemistry, Univ. of Turin, Turin, Italy
2CeSi Ageing Research Center, "G. D'Annunzio" Univ., Chieti, Italy

The transcription factor STAT3 is constitutively activated in tumors of different origin but the molecular bases for STAT3 addiction of tumor cells have not yet been clearly identified. We generated knock/in mice carrying the constitutively active Stat3 allele, Stat3C, and showed that Stat3C could enhance Neu oncogenic power, triggering the production of earlier onset, more invasive mammary tumors. Tumor-derived cell lines displayed higher migration and invasion and disrupted distribution of cell-cell junction markers. The tensin family member Cten (C-Terminal Tensin-like), known to mediate EGF-induced migration and highly expressed in inflammatory breast cancer, was up-regulated in both Neu;Stat3C cells and tumors. Both Cten expression and enhanced migration were strictly dependent on Stat3, and Cten silencing normalized cell migration and rescued cell-cell contact defects. Importantly, the pro-inflammatory cytokine IL-6 could mediate Cten induction in MCF10 cells, in an exquisitely Stat3-dependent way. This model allowed us to shed some light on the oncogenic role of Stat3 in the breast, suggesting moreover a mechanism through which inflammatory signals can cooperate with EGF receptors in inflammatory breast cancer.



Functional analysis of germline CDKN2A 5’UTR mutations associated with history of melanoma
A. Bisio1, S. Nasti2, J. Jordan3, S. Gargiulo2, L. Pastorino2, A. Provenzani3, A. Quattrone3, G. Bianchi-Scarrà2, P. Ghiorzo2, A. Inga1
1Unit of Molecular Mutagenesis and DNA Repair, National Institute for Cancer Research IST, Genoa, Italy
2Laboratory of Genetics of Rare Hereditary Cancers, DOBIG, University of Genoa, Genoa, Italy
3Centre for Integrative Biology (CIBIO), Univ. of Trento, Trento, Italy

The CDKN2A gene is an established high-penetrance melanoma susceptibility gene. Previously, we had identified four Italian melanoma patients that were heterozygous for non-coding mutations in the CDKN2A (p16) 5’UTR (-21C>T; -25C>T; -56G>T; -67G>C). To address the functional consequences of these mutations, we cloned the wild-type and mutant 5’UTR in different types of reporter vectors. Plasmids were transiently transfected into WM266-4 (metastatic melanoma) and MCF7 (breast cancer) cells. Luciferase activity and mRNA levels were quantified to assess the impact of the mutations both at transcriptional and post-transcriptional levels. We also measured allele unbalance in polysomal RNA obtained from patients’-derived heterozygous cells or in cells transfected to mimic the p16 5’UTR heterozygous state. Polysomal and subpolysomal RNAs were separated on sucrose gradients followed by p16 5’UTR amplification, sequencing and relative allele quantification. Our results indicate that these germline mutations, particularly the -21C>T, negatively impact on p16/INK4a 5’UTR activity, acting mainly at a post-transcriptional level, and can thus be of clinical significance in the melanoma proneness.



Molecular typing from archival paraffin-embedded synovial sarcomas
A. Parafioriti1, S. Del Bianco1, E. Armiraglio1, P.A. Daolio2, S. Mapelli2
1Pathology Dept, Orthopaedic Institute Gaetano Pini, Milan, Italy
2Oncologic Orthopaedic Surgery Center, Orthopaedic Institute Gaetano Pini, Milan, Italy

Synovial sarcomas are mesenchimal tumours with undefined histogenesis characterized by t(X;18)(p11;q11) translocation which joins SYT gene with a member of SSX gene family. We developed an efficient method to detect the two main fusion transcripts SYT-SSX1 and SYT-SSX2 based on RT-PCR or Real-Time PCR applied to archival paraffin-embedded samples. This study includes 52 patients surgically treated for synovial sarcoma. In 40 subjects out of 52 we could find a specific fusion transcript and, in particular, 32 patients were carriers of SYT-SSX1 translocation. Interestingly we could find 8 patients who were carriers of both SYT-SSX1 and SYT-SSX2 transcripts. In 5 patients we didnt\'t detect any fusion transcript and in 7 samples RNA was degradated. We selected 12 samples for Real-Time PCR analysis and we could quantify the reciprocal ratio between the two fusion transcripts when they were both present in the same sample. The use of molecular techniques such as RT-PCR represents a sensitive and reliable tool as an aid to histopathologic diagnosis of synovial sarcoma. Moreover, Real-Time PCR enormously enhances sensibility and enables to dose single transcript quantity when both SYT-SSX1 and SYT-SSX2 are present in the same sample.



Tumoral growth suppression by Prion protein silencing in murine glioma model
S. Palumbo1, K. Gabrusiewicz2, G. Barbieri1, E. Sbalchiero1, B. Kaminska2, S. Comincini1
1Dept of Genetics and Microbiology, Univ. of Pavia, Pavia, Italy
2Dept of Cell Biology, Nencki Institute of Experimetal Biology, Warsaw, Poland

A murine glioma model was used to evaluate the tumoral mass reduction after Prion protein (PrPC) silencing. Recently, PrPC has been shown to have a neuroprotective function and cell viability and proliferation were affected by its silencing in vitro. In this work, EGFP-GL261 murine glioma cells were trasfected with anti-Prion antisense phosphorothionate oligonucleotides (ODNs) and implanted into the brains of syngeneic DBA/2J mice. Fourteen days after the implantation, tumor volumes and proliferation analysis showed significative reductions after antisense treatment. Consequently, apoptotic and autophagic analysis were performed to decipher the involved cell death pathways: TUNEL analysis, PARP and caspase-3 immunoblotting depicted a minimal apoptotic contribution, while LC3 expression was increased in the reduced tumoral mass, suggesting possibile autophagic-mediated mechanisms. These results indicate that Prion silencing seems to be crucial to suppress glioma growth, suggesting novel molecular strategies to interfere with tumor progression.



Optimizing EGFR inhibitor treatment in non small cell lung cancer
R. Alfieri1, A. Cavazzoni1, M. Galetti1., S. La Monica1, M. Mor2, C. Carmi2, A. Lodola2, M. Tiseo3, A. Ardizzoni3, P.G. Petronini1
1Dept Experimental Medicine, Univ. Parma. Italy
2Pharmaceutical Dept Univ. Parma, Italy
3Oncologia Medica Azienda Ospedaliero-Universitaria di Parma, Italy

Epidermal Growth Factor Receptor (EGFR) is an established new target for the treatment of epithelial tumors, including non-small cell lung cancer (NSCLC). Both EGFR intracellular tyrosine kinase (TKI) small molecules inhibitors, as erlotinib and gefitinib, and extracellular domain-directed monoclonal antibodies, as cetuximab, have been proven to be a useful addition to standard therapy in advanced NSCLC.
However, tumor cells often acquire resistance to these EGFR inhibitors. In this study we have investigated in a panel of NSCLC cell lines the mechanisms underlying primary or acquired resistance to gefitinib and new therapeutic approaches to circumvent gefitinib resistance.
We tested whether the intracellular concentration of gefitinib may affect the EGFR autophosphorylation. Moreover, we explored the potential of a therapeutic approach based on treatment with EGFR inhibitors combined with drugs inhibiting deregulated components of downstream pathways. Finally, we focused on the design and synthesis of a new generation of EGFR/ErbB-2 irreversible inhibitors with an improved in vitro potency and selectivity.



MicroRNA-based, p53 dependent post-transcriptional circuits: mechanisms, targets and inter-individual variation
F. Sparapani1*, A. Bisio1,2*, V. Del Vescovo1, C. Tonelli1, Y. Ciribilli1,2, V. De Sanctis1, A. G. Jegga3, M. A. Denti1, A. Inga2
*These author equally contributed to this work
1Center for Integrative Biology, CIBIO, Univ. of Trento, Mattarello, TN, Italy
2Unit of Molecular Mutagenesis and DNA repair, National Institute for Cancer Research, IST, Genoa, Italy
3Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA

The tumor suppressor p53 is a sequence specific master regulatory transcription factor that regulates the expression of many target genes linked, among others, to the control of cell cycle, apoptosis, DNA repair and angiogenesis. Recent studies identified direct p53 regulation of miRs and few related regulatory circuits. miRs are small non-coding RNAs typically 21-25nt long, able to regulate gene expression either by inhibiting translation or repressing stability of target messenger RNAs. Differences in miR expression profiles are associated with various cancers and can contribute to tumorigenesis.
Using bioinformatics approaches, we have identified an additional group of candidate miRs for direct p53 transcriptional control. Furthermore, some of these miRs were predicted to target mRNAs in genes relevant to p53-mediated responses. Notably, we found examples of miR seed binding sequences at target 3’UTRs that contain single nucleotide polymorphisms predicted to modulate miR binding. Our work aims at the validation of p53-mediated control of the newly predicted miR genes and related circuitries that would provide additional negative and/or positive feedback loops for p53 regulation.



Prostate cancer invasion is dependent on EphA2 mediated amoeboid motility
M.L. Taddei1, M. Parri1, A. Angelucci2, C. Marconi1, E. Giannoni1, G. Raugei1, M. Bologna2 P. Chiarugi2
1Dept of Biochemical Sciences, Univ. of Florence, Florence, Italy
2Depts of Experimental Medicine, Basic and Applied Biology, Univ. of L’Aquila, L’Aquila, Italy

The tyrosine kinase EphA2 is frequently overexpressed in several human cancers but its role for in vivo metastasis spreading is still a critical question. Several papers demonstrate that EphA2 has a crucial role as motility factor. Recently, we reported that prostate carcinoma cells expressing EphA2 display a proteolysis-independent amoeboid motility style. Therefore, we investigated the involvement of EphA2 overexpression in prostate carcinoma metastatic dissemination, generating PC3 cells in which EphA2 is stably knocked down by RNA interference. We demonstrate that elimination of EphA2 expression reduces bone and visceral metastases, such as PC3 anchorage independent cell growth, while is accompanied with an increase in CXCR4 expression. In contrast, we did not observe any effect of EphA2 expression on both prostate carcinoma cell survival, adhesion to extracellular matrix, as well as their trans-endothelial migration. EphA2-silenced PC3 cells show an increased production of MMPs, RhoA inhibition and Rac1 activation, in line with loss of amoeboid motility style and a de novo acquired mesenchymal strategy, as confirmed by confocal 3D-invasion analysis. In conclusion, we show that EphA2 silencing is able to eliminate the skill of PC3 cells to move through an amoeboid motility style, thereby strongly inhibiting their ability to spread metastatic colonies.



Role of endocytic trafficking in cell polarity and migration of melanocytes in a zebrafish model of melanoma
C. Santoriello1, Y. Feng2, M. Distel3, V. Anelli1, P. Martin2, R. Koster3, M. Mione1
1IFOM, the Firc Institute of Molecular Oncology, Milan, Italy
2Tissue Repair and Morphogenesis Lab, Bristol Univ., UK
3Helmholtz Zentrum München IDG, Institute of Developmental Genetics, Munich/Neuherberg, Germany

Melanoma is the most aggressive and lethal form of skin cancer and the cascade of events leading to cell invasion during melanoma development is still poorly understood. The transparent zebrafish is an ideal system to study and image directly dynamically melanoma progression. Using the GAL4 -UAS system, we have developed a zebrafish transgenic line that express oncogenic Ras under the kit-a promoter and develop melanomas by 1-3 months of age. Transgenic larvae show already at 3 day post fertilization an over-pigmentation phenotype, due to an increased number and size of melanophores, coupled to abnormal migration. We therefore plan to use the transgenic larvae to screen for pathways and/or mechanisms that revert the abnormal growth and migration phenotype of transformed melanophores. Our goals are: 1) establish if the polarity of melanophores is altered in response to HRASV12 transformation, and how this influences migration and invasiveness of melanoma, 2) establish the role of endocytic trafficking in the localization of cytoskeletal components involved in migration and invasion in transformed melanocytes.



Identification of microRNAs regulating the p53 pathway
A. Bisso1,2, M. Faleschini1, R. Agami3, G. Del Sal1,2
1LNCIB, Area Science Park, Padriciano 99, Trieste
2Dipartimento di Scienze della Vita, Univ. of Trieste, Trieste, Italy
3Division of Tumour Biology, The Netherlands Cancer Institute, Amsterdam, The Netherlands

p53 tumor suppressor protein plays a central role in maintaining genomic stability and in controlling cell cycle, cellular senescence and apoptosis. Indeed, tumors strongly select for functional inactivation of the p53 pathway either mutating the p53 gene (in almost half of tumors) or altering the expression or functions of key p53 regulators or effectors. MicroRNAs are small RNAs able to finely post?transcriptionally regulate gene expression, that have been shown to be clearly involved in cellular transformation acting as oncogenes or tumor suppressors. Taking into account the relevance of the p53 pathway as well as microRNAs in cancer, we aimed at investigating whether microRNAs may modulate p53 functions. To this purpose, we set up a screening based on a reporter luciferase-3'UTR chimera, to identify microRNAs able to target the 3'UTR of some of key p53 regulators such as ATM, Pin1, HIPK2 and iASPP. We tested candidate microRNAs, among those predicted by available algorithms (TargetScan, Miranda, PicTar, PITA), and we isolated positive hits for HIPK2-3'UTR, ATM-3'UTR and iASPP-3'UTR. Furthermore, we confirmed the ability of these microRNAs to modulate target protein levels and we are now characterizing their biological functions. These microRNAs could unveil a new regulatory mechanism of p53 functions, providing a better understanding of normal and aberrant roles of this essential pathway in cancer.


17. Plant biotechnology

Total number of abstracts in this session: 5



Improved saccharification of plant cell wall through in muro pectin modification
V. Lionetti, F. Francocci, S. Ferrari, D. Bellincampi, R. Galletti, G. De Lorenzo, F. Cervone
Dip. Biologia Vegetale, Univ. di Roma "La Sapienza", Roma, Italy

Plant cell walls comprise a significant proportion of the lignocellulosic biomass and are an abundant potential source of ethanol. A key process for the production of ethanol is the degradation of the cell wall polysaccharides into fermentable sugars (saccharification). The major bottleneck for the industrial scale-up of the saccharification process is the recalcitrance of the cell walls to digestion and the need of harsh pre-treatment. We have developed a technology that by modifying the cell wall structure reduce pretreatments and improve saccharification efficiency.
It is well known that intermolecular links of pectin influence wall plasticity and that the acidic form of homogalacturonan (HGA) is cross-linked by calcium ions to form rigid “egg-box” structures. We therefore hypothesized that saccharification could be improved by reducing the de-esterified HGA regions of pectin. To test this hypothesis we used plants expressing a polygalacturonase from Aspergillus niger and plants overexpressing an inhibitor of pectin methylesterases. Treatment of transgenic plants with commercial cellulase resulted in a higher release of soluble sugars than in control plants. The degradability of the cellulose is therefore favoured in PG and PMEI expressing plants and does not require an acid pre-treatment. In principle, modification of crop plants or dedicated energy plants through the expression of PGs and PMEIs may be used to improve the saccharification process.



Expression pattern of genes involved in ethylene signalling in Melting-Flesh and Non-Melting Flesh peaches
F. Baldin, A. Ghiani, N. Negrini, S. Morgutti, F.F. Nocito, D. Bassi, M. Cocucci
DiProVe, Univ. degli Studi di Milano, Milan, Italy

Ethylene plays a pivotal rule in the control of several developmental process in plants, among which of main importance is the ripening process of climacteric fruits. The climacteric peach fruits are classified by different flesh texture and firmness, which affects fruit shelf-life, as well as by ethylene evolution, finally determining the organoleptic quality of the ripe fruit. Concerning texture, peach fruits can be classified as melting flesh (MF), non-melting flesh (NMF) and stony hard. The former two phenotypes are characterized, other by different texture and firmness, also by a different ethylene evolution which is often higher in non-melting fruits. Increased expression of an ethylene-stimulated endo-PG gene and synthesis of the corresponding protein accompany the extreme peach fruit softening in MF phenotypes. This apparently paradoxical behaviour suggests possible genotype-related differences in ethylene signal perception and transduction.
For this reason, we have investigated, by Northern analysis and real-time PCR, the changes, at different ripening and softening stages of two selected MF (‘Bolero’) and NMF (‘Oro A’) peach cultivars, in the expression of genes encoding several components of the ethylene signalling chain trying to relate these changes to the pattern of expression of genes involved in ripening-related cell wall disassembly.



Is Cu-EDDS complex taken up by Brassica carinata roots?
B. Cestone, M.F. Quartacci, F. Navari-Izzo
Dept of Chemistry and Agricultural Biotechnologies, Univ. of Pisa, Italy

Brassica carinata was incubated with 0.12, 30 or 150 μM Cu2+ or the same concentrations of Cu complexed with EDDS. EDDS, removing ions from the plasma membrane, destroys the physiological barriers and impairs root selectivity. The chelant or the metal complex freely enter into the roots, reach the xylem and are translocated to the above-ground part by the transpiration flow. This study was addressed to investigate the uptake of the metal complex in order to understand whether the differences found among free Cu2+ ion, free EDDS and Cu-EDDS uptake kinetics were due to a simple inhibition caused by the presence of the chelant in solution or to the different characteristics of the solutes.
By inhibiting the ATPase proton pumps of the plasma membrane it is possible to obtain a preliminary indication on the uptake mechanism and the characteristics of the absorbed compound. For this, Cu2+, EDDS and Cu-EDDS uptake kinetics by B. carinata excised roots grown in hydroponics and incubated in the presence of increasing concentrations of either the metal, the chelant and the complex were determined in the presence or not of ATPase inhibitors.



A point mutation in the Medicago sativa GSA-AT gene provides an efficent selectable marker for plant genetic engineering
D. Rosellini, N. Ferradini, A. Nicolia, S. Capomaccio, F. Veronesi
Dip. di Biologia Applicata, Univ. degli Studi di Perugia, Perugia, Italy

Selectable marker genes (SMGs) conferring antibiotic resistance are a valuable tool in plant genetic engineering. Consumer concerns and regulatory requirements have stimulated the development of alternative SMGs. A mutated form of the Synechococcus elongatus hemL gene encoding glutamate 1-semialdehyde aminotransferase (GSA-AT) is efficient a SMG in alfalfa (Medicago sativa L). The GSA-AT enzyme catalyses the conversion of glutamate-1-semialdehyde into aminolevulinic acid and is irreversibly inhibited by gabaculine (3-amino-2,3-dihydrobenzoic acid). The mutated bacterial gene has a point mutation resulting in the substitution of a methyonine with an isoleucine in the catalytic site.
We cloned and point-mutated the M. sativa GSA-AT, so to reproduce the M to I substitution. This mutated GSA-AT gene was assessed as a SMG in tobacco and alfalfa Agrobacterium-mediated transformation. Transformation efficiency was 40.3-46.5% in tobacco and 92.3 % in alfalfa, with no escapes. These results suggest that this new SMG can be applied with success to other plant species, as a favourable alternative to currently used SMGs.



In planta production of dermatophagoides pteronyssinus allergens
G. Marconi1, G. Sgaravizzi2, I. Raggi1, H. Lancioni2, A. Palomba2, L. Lucentini2, A. Porceddu3, F. Panara2, E. Albertini1
1Dip. di Biologia Applicata, Univ. di Perugina, Perugia, Italy
2Dip. Biologia cellulare ed ambientale, Univ. di Perugina, Perugina, Italy
3Dip. Scienze agronomiche e genetica vegetale agraria, Univ. di Sassari, Italy

In the last years a remarkable increase in allergic and inflammatory diseases was highlighted. In industrialized countries about 20-25% of living people suffer for IgE mediated allergic diseases. Today allergy immunotherapy is usually performed with natural allergen extracts composed of complex mixtures of several proteins, difficult to standardize and causing cross reactivity. Recombinant allergens allow to determine the exact sensitization profile of certain individual and this is a prerequisite to select those allergens against which a patient is sensitized for setting up the specific immunotherapy. House dust mites of Dermatophagoides species (e.g. Dermatophagoides pteronyssinus) are associated with various allergic disease. Der p 1 of the D. pteronyssinus is an allergenic protein considered to be one of the major allergens. On the contrary, even if Der p 10 is a minor allergen its high cross-reactivity with allergens found in a variety of sea foods make it very interesting to be studied. The expression of Der p1 and Der p 10 allergens mediated by potato virus X (PVX) in Nicotiana benthamiana plants is here reported together with a strategy of Flag and 6his-tag sequences fusion to the N and C-terminus region of Der p1 and Der p 10 cDNAs. The use of Agroinfiltration system to infect N. benthamiana plants is also reported. RT-PCR analysis carried out on cDNAs obtained from mRNA extracted from infected plants and Western blotting with monoclonal anti-Flag and anti-6his antibodies is reported and discussed.


18. Plant development and diseases

Total number of abstracts in this session: 8



Soil microbial diversity positively affects organic matter decomposition and resistance to pathogens invasion
Giuliano Bonanomi, Vincenzo Antignani, Manuela Capodilupo, Felice Scala
Dept Arboricoltura, Botanica e Patologia Vegetale, Univ. of Naples Federico II, Italy

The study of relationships between species diversity and ecosystem processes has received growing attention in recent years, and it is now well recognized that biodiversity strongly affects some ecosystem functions such as productivity, stability and invasibility. However, available studies focused mostly on terrestrial plants and only little attention has been paid to soil microbial communities, despite their importance both in natural and human managed ecosystems. Aim of this work is to investigate the role of microbial diversity in plant litter decomposition and resistance to species invasions, with particular attention to plant pathogens. Different methods have been proposed to understand the role of microbial diversity on soil functions. In our approach, synthetic microbial communities with different diversity levels, ranging from 1 to 64 species, were assembled in laboratory microcosms. Thereafter, the functionality of the different microcosms were determined by measuring their capability to decompose different organic materials and to resist to the invasion of selected plant pathogens. The number of species present in the microcosms positively affects the rates of these processes, with a significant increase of organic matter decay rate. Moreover, microbial diversity significantly reduce the variability of microcosm functions, suggesting a greater stability of ecosystem properties in presence of more diverse microbial communities.



Fluorescence in situ hybridization as a tool for studying phytoplasma-endophytes interaction in plant
D. Bulgari1, P. Casati1, F. Quaglino1, P.A. Bianco1,2, F. Faoro1,2
1Dip. di Produzione Vegetale, Univ. di Milano,
2Istituto di Virologia Vegetale, CNR, U.O. Milano, Milano

Bacteria residing in plant tissues without inducing symptoms of diseases are defined as endophytes. They can protect plants from pathogens through three different mechanisms: competition; production of allelochemic inhibitors and induction of systemic resistance (ISR). Recently statistical analysis, carried out on LH-PCR (Length Heterogeneity-PCR) profiles, highlighted a different composition of endophytic bacterial community associated with healthy, GY diseased, and recovered grapevine plants.
In this study we experimented fluorescence in situ hybridization (FISH) to study the interaction between phytoplasmas and endophytic bacteria in the model plant Catharanthus roseus L.. Both phytoplasmas and bacteria were co-localized by confocal microscopy using different probes to target 16SrV phytoplasmal and bacterial 16S rDNA, respectively. To avoid interference with autofluorescence of tissue constituents, bacterial probes were labelled with fluorophores emitting in the far-red (i.e. CY5). Phytoplasmal probes were labelled with FAM and they prove to be able in discriminating genetically different phytoplasmas, localized only in the phloem tissues. Endophytic bacteria were instead detected in the phloem, xylem and leaf parenchyma. These results, though preliminaries, show the great potentiality of FISH in studying the interaction between phytoplasmas and endophytic bacteria.



A chimeric receptor approach reveals a role of WAK1 in oligogalacturonide sensing
G. De Lorenzo1, F. Sicilia1, A. Brutus1, E. Marchetti2, A. Macone3, F. Cervone1
1Dept Plant Biology, Univ. of Rome “Sapienza”, Rome, Italy
2Dept of Genetics and Molecular Biology “C. Darwin”, Univ. of Rome “Sapienza”, Rome, Italy
3Dept Biochemistry “A. Rossi Fanelli”, University of Rome “Sapienza”, Rome, Italy

Pectin, a main component of the cell wall that is continually modified and remodelled during plant growth and development. A main component of pectin is homogalacturonan, which is capable of binding calcium to form the structures called “egg-box”. These structures may be broken and fragmented by microbial hydrolases and release oligogalacturonides (OGs) that have elicitor or regulator activity. In Arabidopsis, OGs induce the expression of defense genes and proteins and protect the plant against fungal diseases. However, the perception system of OGs is still unknown. Since the response of Arabidopsis to OGs overlaps that of the microbe-associated molecular patterns flagellin and the elongation factor-Tu, it has been hypothesized that the receptors of OGs are similar to their corresponding receptors FLS2 and EFR. On the other hand, candidate receptors of OGs are some members of the Wall-Associated Kinase (WAK) family. WAKs are receptor-like kinases that display the typical eukaryotic Ser/Thr kinase signature and an extracellular domain containing several epidermal growth factor (EGF)-like repeats. Among them, WAK1 is the most characterized: it binds in vitro to nonmethylesterified
homogalacturonans, to elicitor active OGs and the structurally related alginates. However, the detailed role of single WAK receptors remains largely unknown. In order to define the function of WAK1, we have used an approach based on chimeric LRR-RLKs.



Analysis of the sirtuins, NAD dependent deacetylase genes, in Arabidopsis thaliana
M.L. Mauro, P. Costantino
Dept Genetics and Molecular Bilogy, Univ. La Sapienza, Roma, Italy

The Sirtuins gene family, whose name derived from SIR2 (silencing informator regulator), the first gene identified in the yeast, are deacetylase enzymes, mainly involved in chromatin remodelling and in particular in the transcriptional repression process.
All living organisms present sirtuins that, differently from the other deacetylases, are characterised by their NAD-dependent activity. In the last years, sirtuins from several species, including human, have emerged as important regulators of metabolism, defense and ageing.
In arabidopsis thaliana by sequence analysis two genes homologue to yeast SIR2, have been identified, named SRT1 and SRT2.
This study will report the characterization of these two genes at the expression level, with and without treatment with specific inhibitor and activator molecules, known regulate their enzymatic activity.
Moreover, the analysis of mutant plants of the two genes, obtained by insertional technique, was performed in controlled growth condition and in stress induced context.
Expression of the genes that could be involved in the arabidopsis sirtuins pathway has been tested and the results commented also in relation to the sirtuins effects observed in other organisms.



The cerato-platanins: non-catalytic proteins with functions in the relational life of fungi
L. Pazzagli1, I. Baccelli2, R. Bernardi2, G. Cappugi1, L. Carresi3, D.O. Cicero4, C. Comparini3, F. Martellini1, T.A. Pertinhez5, A. Spisni5, A. Scala3
1Dip. di Scienze Biochimiche, Univ. degli Studi di Firenze, Firenze, Italy
2Dip. di Biologia delle Piante Agrarie, Sezione di Genetica, Univ. di Pisa, Pisa, Italy
3Dip. di Biotecnologie Agrarie, Laboratorio di Patologia Vegetale Molecolare, Univ. degli Studi di Firenze, Sesto Fiorentino, Italy
4Dip. di Scienze e Tecnologie Chimiche, Univ. di Roma “Tor Vergata”, Roma, Italy
5Dip. di Medicina Sperimentale, Univ. di Parma, Parma, Italy

Plant pathogenic fungi secrete a lot of non-catalytic proteins, which are involved in various aspects of pathogenesis. These proteins are classified in seven fungal protein families, including the cerato-platanin (CP) family (PF07249). Since a single family contains proteins with different functions in the plant pathogenesis, and different families may contain proteins with similar functions, we aimed to investigate why this happens. For this purpose we developed a study model represented by two proteins belonging to the CP family: CP, the founder protein produced by Ceratocystis platani, and the CP-orthologous cerato-populin (Pop1) produced by C. populicola. Both CP and Pop1 have been purified, cloned in P. pastoris, expressed, and characterized for their ability to aggregate. They are well-structured α/β proteins (the 3D-structure of CP has been recently determined), and have only the 70.1% of the amino acids identical or highly conserved. Moreover, they behave as PAMPs, since it stimulated plants to activate defense responses able to reduce consistently the fungal growth. Their capability to elicit differently defense events and their role in fungal physiology is under study.



A new hydrophobin that affects the interaction between Trichoderma harzianum and the plant
M. Ruocco 1, S. Lanzuise 2, S.L. Woo 2, M. Reverberi3, R. Marra2, F. Vinale2, V. Aloj2, F. Scala2, M. Lorito2
1Institute for Plant Protection (IPP), CNR, Portici (NA), Italy
2Dip. di Arboricoltura, Botanica e Patologia Vegetale sez. Patologia Vegetale, Univ. of Naples, Portici (NA), Italy
3Univ. La Sapienza, Roma, Italy

Trichoderma harzianum T22, a fungal antagonist, is one of the most widely used active ingredients in commercial bio-fungicides and bio-fertilizers. It has not only mycoparasitic activity, but it can also activate extensive metabolic changes in treated plants, resulting in a systemic resistance to a pathogen attack, thus indirectly altering plant-pathogen interactions. By analyzing the T. harzianum T22 “secretome” a few key “effectors” involved in Trichoderma-plant cross-talk were identified. One of these effectors secreted was a class II hydrophobin HYTRA1. This protein was tested for its antimicrobial activity and its ability to induce a defence response in tomato plants. In in vitro and in vivo assays, HYTRA1 directly inhibited pathogen development. It induced in tomato plants, depending upon the concentration, a multiplicity of effects. HYTRA1 activated oxidative burst, the antioxidant system, and ISR with the accumulation of defence-related compounds important in plant defence. Further physiological effects included the induction of de novo rhizogenesis, and an epinastic phenotype in HYTRA1-transformed tomato. The involvement of HYTRA1 in the T. harzianum molecular crosstalk with the plant was confirmed when HYTRA1 disruptants were found unable to induce root growth promotion and ISR on tomato.



Phospholipase A2 and Lipoxygenase role in durum wheat (Triticum durum Desf.) response to infection by leaf rust fungus Puccinia triticina
A. Verlotta, V. De Simone, L. Cattivelli, D. Trono
C.R.A., Cereal Research Centre, Foggia, Italy

In plants, the release of polyunsatured fatty acids from membrane phospholipids by Phospholipase A2 (PLA2) provides substrate for lipoxygenase (LOX) and subsequent synthesis of jasmonate that plays a key role in signal regulation under biotic stress. The aim of this work was to investigate how the expression and the activity of PLA2 and LOX are modulated in leaves of durum wheat exposed to Puccinia triticina, the causal agent of leaf rust. To do this, comparison was made between a resistant (Creso) and a susceptible (Pedroso) genotype. As far as the expression analysis, one of the PLA2 genes investigated was found to be constitutively expressed at higher levels in the resistant cv. with respect to the susceptible one; among LOX genes, LOX A was found to be up-regulated by stress both in Creso and Pedroso, while no differences were observed in the transcript levels of LOX B and LOX C. In line with these findings, PLA2 activity was found to be twice higher in Creso than in Pedroso, and LOX activity increased under stress more in Creso than in Pedroso. In the whole, the results obtained suggest a possible involvement of both PLA2 and LOX in durum wheat response to pathogen attack.



Flowering time pathway: naturally occurring polymorphisms in Populus nigra candidate genes for phenology
G. Zaina1, S. Giacomello1,2, M. Morgante1,2
1Dept Agriculture and Environmental Sciences, Univ. of Udine, Udine, Italy
2Institute of Applied Genomics, Udine, Italy

The seasonal cycle of growth and dormancy is a distinct character of perennial plants and is tightly linked to the yearly dates of bud flush and bud set. Both these traits are under strong selection and show genetic differentiation along geographical clines.
To investigate the molecular-genetic bases of such traits in Populus nigra, we assessed the level and geographic distribution of genomic diversity in candidate genes for phenology. Looking at the flowering time pathway in Arabidopsis, emphasis was given to 15 genes belonging to the Phytochrome and Cryptochrome families and the signal transduction pathway. By re-sequencing 96 alleles from 10 populations distributed across Europe, naturally occurring polymorphic nucleotide sites (SNPs) were detected. A picture of the patterns of nucleotide variation was obtained, as well as, the extent of the linkage disequilibrium. Through these data, we will first infer the evolutionary factors governing the variation detected and identify areas of the genome where natural selection has been especially important. Then, we will examine the genomic context of the diversity by association studies between different SNPs and phenology phenotypes.


19. Plant metabolism and environmental stresses

Total number of abstracts in this session: 13



Metabolite profiles in cauliflower: discrimination among farming systems
M.G. Annunziata1, G. Massaro1, F. Iannuzzi1, P. Carillo1, A. Troccoli2, A. Fuggi1
1Seconda Univ. degli Studi di Napoli, Dip. di Scienze della Vita, Caserta, Italy
2CRA-CER Centro di Ricerca per la Cerealicoltura di Foggia

Metabolite profiles, as phenotipic expression from single cells up to organisms, contain trace information of their individual “true” life. Suitable group of metabolite traits, can help to better characterise living materials. Here is reported the use of some classes of metabolites to analyse cauliflowers grown under different farming systems. Cauliflowers (Brassica oleracea subsp. botrytis cv Atalaya) were grown under traditional tillage farming and minimum tillage farming. Five individual plants were used as replicates from each cultivar. The analyses were done on the immature flowers and the corymb stem separated at the harvest. The samples powdered in liquid nitrogen were used to determine the content of protein, carbohydrates, ascorbate and glutathione, ions, free amino acids and glucosinolates. Multivariate analysis evidenced that different distributions of such metabolites. In the PCA plot the organs were separated along the first components, while the farming conditions along the second component. It can be suggested that metabolite derived indices could be used to control and trace food products. The work was financed by SUN and MIUR (Progetto PRIN 2006077008).



Physiological responses of spinach leaves to low oxygen conditions: a clearer picture
S. Fornaciari, E. Anceschi, L. Arru
Dept of Agricultural and Food Sciences, Univ. of Modena and Reggio Emilia, Italy

Investigation on O2 deficiency stress is generally related to root response. We are studying the effects in leaves of anoxia and hypoxia imposed to roots. In a 12 h experiment, anoxia leads to an early 4-fold increase in ADH activity, stabilized for the rest of the treatment. On the contrary, the same increase is reached gradually in hypoxia after 12 h. These results are also coherent with ADH activity in roots. By means of Q-PCR, we also studied the Adh gene expression: it slowly reaches a 30-fold increase during 12 h of anoxia; while it arrests at 5-fold increase in hypoxia. There seems to be a lack of proportional correlation between gene transcription and enzyme activity, that can suggest a post-transcriptional regulation. Also in roots Adh gene expression does not correlate with the enzyme activity, since in anoxia it reaches a peak of 13-fold increase after 2h, while in hypoxia it needs 12h to increase to 6-fold. In order to understand how leaves can perceive root O2 stress, leaf disks have been incubated with molecules that might be a signal from roots to leaves. Adh gene has not been induced in any way, while in some cases ADH enzyme showed a strongly enhanced activity.



Plant litter phytotoxicity: dynamical patterns, causal mechanisms and chemical characterization by 13C NMR spectroscopy
G. Bonanomi1, V. Antignani1, E. Barile2, M. Capodilupo1, V. Lanzotti2, S. Mazzoleni1, F. Scala1
1Dept Arboricoltura, Botanica e Patologia Vegetale, Univ. Naples Federico II, Italy
2Dept Scienze e Tecnologie Agroalimentari, Ambientali e Microbiologiche, Univ Molise, Italy

Internal recycling of nutrients from decomposing plant litter provides key resources for ecosystems productivity. However, it is well known that decomposing litter may have allelopathic effects that hamper root proliferation and consequently plant nutrition. This work investigated the dynamical patterns of litter phytotoxicity and the mechanisms through which plant litter is converted to a suitable nutrient source for plant growth. Twenty-five litter types were selected and decomposed under controlled condition according to the litterbag method. Litterbags were harvested after 30, 90 and 180 days of decomposition and their phytotoxicity was assessed by a bioassay with Lepidium sativum. Litter chemical changes were characterized by 13C NMR obtained in solid state by a Bruker AV-300 spectrometer. Litter phytotoxicity was widespread among tested species, but all materials showed a rapid phytotoxicity decrease during decomposition. The addition of mineral nutrients to phytotoxic litter showed no positive effect on plant growth, whereas the application of activated charcoal significantly improved root growth, thus providing a strong support for the allelopathic hypothesis. NMR analysis revealed consistent chemical changes occurring during decomposition: carbohydrates steadily decreased, while aliphatic and lignin associated carbons increased. Litter phytotoxicity showed the strongest correlation with the degree of hydrophobicity of the organic materials.



The role of vacuolar H+ pumps in cytosolic pH homeostasis of Fe deficient roots
M. Dell'Orto, G. Zocchi
Dept of Produzione Vegetale, Univ. degli Studi di Milano, Italy

The variable degree of tolerance to Fe shortage in dicots depends on their different ability to induce the biochemical responses in root cells, mainly: 1) increased reduction of extra cellular Fe(III) by a Fe(III)-chelate reductase; 2) increased H+ extrusion in the rhizosphere by the PM H+-ATPase. Moreover, in Fe-deficient cucumber roots a 31P-NMR analysis revealed the increase of vacuolar pH and the decrease of vacuolar [Pi], while in soybean the opposite was found. These evidences suggest a role for H+ and Pi movements across the tonoplast in the homeostasis of cytosolic pH, which could be differently affected by the different degree of response to Fe deficiency in the two species. The role of vacuole in cytosolic pH-stat is well established and should involve the V-ATPase and V-PPase activities. These H+ pumps generate the ΔpH and Δψ mediating the transport of solutes, among which Pi, across the tonoplast. Here, tonoplast enriched fractions were extracted from cucumber and soybean roots grown with or without Fe. Both V-ATPase and V-PPase activities were analysed by enzymatic assays and by Western blot and their role in vacuolar [Pi] changes was investigated.



Natural variation in the locus LOC_Os06g01260 encoding an isoform of phytochelatin synthase in rice
B. Dendena1, B. Wozniak2, C. Lancilli1, G. Bruschi3, P. Piffanelli2, F. F. Nocito1, E. Lupotto3, G. A. Sacchi1
1Dip. di Produzione Vegetale, Univ. degli Studi di Milano, Italy
2Parco Tecnologico Padano, Lodi, Italy
3CRA-RIS Vercelli, Italy

Among cereals, rice and wheat present the highest risk of heavy metal accumulation in grains due to agricultural and genetic reasons. Cd is the element most likely to enter the food chain due to its high mobility in the soil-plant system. To comply with the market standards set by the European Community, Cd content in rice grains must be below 0.2 mg kg-1, whereas for the other cereals the limit is 0.1 mg kg-1. It is therefore important to adopt specific strategies to assure food safety, by selecting rice genotypes preventing Cd accumulation in tissues. It has been shown some intraspecific variation for the trait “Cd allocation in tissues”. The high capacity of Cd retention in cereal roots is usually associated with the biosynthesis of phytochelatins (PCs), a class of heavy metal binding peptides synthesized by phytochelatin synthase (PCS). We investigated the role of PCs in reducing Cd translocation in rice by searching for natural variations at the locus Os06g01260 encoding a PCS isoform. Nine allelic variants were identified by an eco-TILLING approach on a germplasm collection of 96 rice genotypes, and partially characterized with respect to their effect on root Cd retention.



Effects of chronic ozone exposure on lipidic metabolites of bean seeds
A. Ruggiero1, S. Bernasconi2, N. Burlini1, A. Daghetti2, F. Faoro3, M. Iriti3
1Dept Biomolecular & Biotechnol. Sci., Univ. of Milan, Milan, Italy
2Dept Organic & Ind. Chemistry, Univ. of Milan
3Dept Plant Production, Univ. of Milan, Milan, Italy

Chronic exposure of plants to atmospheric pollutants induces metabolic changes both at level of primary and secondary metabolism. In crops, this impact on nutritional traits of foodstuffs, modifying the content of macro/micronutrients and bioactive phytochemicals. In this work, we analyzed the lipid profile of bean (Phaseolus vulgaris L.) seeds collected from plants grown in charcoal filtered (F-) and non filtered open-top chambers (NF-OTCs), located in Curno (Bergamo). During the growing season (summer 2006), the cumulative exposures of plants over an ozone concentration of 40 ppb (AOT40) were about 535 and 4675 ppb h and stomatal fluxes (AFST0, a measurement of the amount of ozone effectively absorbed by plants) were 9055 and 20548 mmol O3 m-2 in F-OTCs and NF-OTCs, respectively. Ozone exposure improved the content of free phytosterols compared to conjugated forms (steryl esters, steryl glycosides and acylated steryl glycosides). Among free phytosterols, β-sitosterol was the most abundant form, followed by stigmasterol. The level of palmitic acid increased after the exposure, whereas linolenic and linoleic acid decreased. Finally, tocopherols (α, β, γ and δ) increased in O3-exposed bean seeds. Further studies are still in progress to ascertain the effects of O3 exposure on bean antinutritional factors and allergenic proteins.



Tropospheric ozone: a novel plant pathogen
G. Lorenzini1,2, C. Nali1,2
1Dept Tree Science, Plant Pathology and Entomology Giovanni Scaramuzzi of the University of Pisa, Italy
2Centro Enrico Avanzi of the University of Pisa, Italy

Ozone (O3) is a naturally occurring chemical present in the troposphere. In addition to its natural sources, man-related emissions (namely nitrogen oxides and volatile hydrocarbons) are responsible for the formation of this gas via a chain of reactions. Long-distance and even intercontinental transport has resulted in a significant presence of O3 in rural and forest areas hundreds and thousands of kilometres from the original sources of its precursors. Besides its role as a direct greenhouse gas, O3 is regarded as the major phytotoxic air pollutant. Future scenarios indicate that global background concentrations are expected to increase. Several studies have been conducted on the impact of O3 pollution on vegetation, ranging from effects at the subcellular level to predicting impacts on a regional scale. Damages include visible leaf injury, decreased photosynthesis and increased senescence, which have significant repercussions for quantity and quality of the yield of major agricultural crops, biodiversity and forest health. Host-parasite relationships may be modified as well. Under the physiological level, O3 is a well recognised elicitor of plant stress, inducing defence responses.



Metabolite profiles of broccoli vegetables during the postharvest storage
G. Massaro, M. G. Annunziata, F. Iannuzzi, P. Carillo, A. Fuggi
Seconda Univ. degli Studi di Napoli, Dip. di Scienze della Vita, Caserta, Italy

The harvest stress in vegetables activate physiological processes that change metabolism and ultimately lead to loss of nutritional and nutraceutic properties. The aim of this work was to study the changes of groups of metabolites occurring in broccoli vegetables during their postharvest storage kept in different atmosphere packaging at different temperatures. Broccoli from Brassica rapa cv. sylvestris (cime di rapa) were stored at 10°C in air, 4°C in air and 4°C in a modified atmosphere (5% O2; 2%CO2) up to 20 days from the harvest. The collected samples were divided in florets, stem and leaf blade and petiole, powdered in liquid nitrogen and used to determine the contents of pigments, protein, carbohydrates, ascorbate, inorganic and organic ions, free amino acids, glucosinolates. Peroxidase activities and isoforms were also determined. The analyses were dane in triplicate. Multivariate analysis of metabolite profiles allowed to discriminate among the different plant organs and samplings at different temperature and packaging The changes of the various metabolites were also analysed and discussed The work was financed by SUN and MIUR (Progetto PRIN 2006077008).



Interaction ammonium-nitrate: response to oxidative stress in chicory plants
M.A. Russo, R. Iacona, F. Badalà, A. Belligno
Dept Agrochemistry, Agricultural Univ., Catania, Italy

The aim of this work was to study, as a function of the different availability of nitrogen in the reduced form, mineral and organic, the induction of the synthesis of some ROS-scavenging molecules and the evolution of some enzymatic activities such as ascorbate peroxidase (APX) and polyphenoloxidase (PPO).
Chicory seedlings were grown in nutritive solution for 35 days in controlled conditions. On the 14th day, one third of the plants was transferred into a nutritive solution containing (NH4)2SO4 60 mM, one third was transferred into a medium containing Urea 60 mM, and the remaining was let grow into the nutrition solution, as a control. Three samplings of leaves were performed, respectively after 21, 28 and 35 days of growth.
The urea and ammonium sulphate-treated samples showed higher ascorbic acid and polyphenol contents than the control, together with a lower anthocyanins content. The most represented antocyanin, not depending of the treatment, is cyanidin-3-glucosyde, which amounts averagely to 95% of the total anthocyanin content.
APX showed the highest activity in the urea-treated samples, while the highest PPO activity was to refer to samples treated with ammonium sulphate.
The variations of the organic components showed the incidence of the nitrogen supply in the reduced form on the cell redox potential, confirming the importance of fertilization for obtaining high amounts of antioxidant molecules.



Effects induced by Diflufenican on Sinapis arvensis
N. Tedde, R. Izzo
Dept Chemistry and Agricultural Biotechnology, Univ. of Pisa, Italy

In winter 2009 Triticum durum Desf., cv. Claudio, was sown in pots (30x30 cm) with clay soil, at density of 600 kernels/m2. Weed seeds (Sinapis arvensis L.) too were sown in the same pots with a crop/weed ratio of 3/1. Pots were fertilised with 50 g/m2 of ammonium sulphate 18-46. Two treatments were performed in post-emergence with Stopper® 500g/L: 250ml/ha (T1) and 350 ml/ha (T2) when wheat plants were at the third leaf stage and weed had well developed cotyledons. After one and two weeks from the treatments plant material was collected and used for fluorescence analyses, chlorophyll and total carotenoids determination and antioxidants such as ascorbate and glutathione. The results show in the weed a significant fluorescence decrease accompanied by a decrease in pigments content showing, after two weeks, a well marked bleaching effect at T1 and a complete life stop at T2. On the contrary, wheat did not show any significant damage depending on the treatments. As concerns ascorbate and glutathione in the treated plants, they might not have been sufficient to overcome the noxious oxidative stress imposed in weed by the herbicide, which leads the plant to death in 15 days.



Metabolic responses to iron deficiency in Strategy I plants
G. Vigani, E. Orsini, G. Zocchi
Dip. di Produzione Vegetale, Univ. degli Studi di Milano, Milano, Italy

The responses to iron deficiency stress in dicotyledonous plants (Strategy I) show the increase in different activities (FC-R, IRT1, H+-ATPase), in order to improve the uptake of iron from the soil. Along with these it has been found that metabolism is involved in sustaining the production of reducing equivalents [NAD(P)H] and ATP. Glycolisys and many metabolic enzymes are increased, in particular phosphoenolpyruvate carboxylase (PEPC) in order to accelerate the glycolitic flux. However, lack of Fe greatly impaired the mithocondrial activity decreasing the reduced equivalent turnover and consequently the amount of ATP. In this contest it is interesting to understand how carbon and nitrogen metabolism respond to Fe deficiency in Cucumber sativus and Arabidopsis thaliana. In this work by using a molecular biology approach we will test, under Fe deficiency condition, iron homeostasis and nitrogen metabolism regulation at the root level.



Isolation and characterization of expressed NBS-LRR resistance gene candidate from Hydrangea macrophylla
P. Woodrow, I. Kafantaris, G. Pontecorvo
Dept of Life Science, II Univ. of Naples, Caserta, Italy

As the largest class of disease resistance R genes, the genes encoding nucleotide binding site leucine-rich repeat (‘‘NBS-LRR genes’’) proteins play a critical role in defending plants from a multitude of pathogens and pests. NBS domains related to R-genes show a highly conserved backbone of an amino acid motif, which makes it possible to isolate resistance gene analogues (RGAs) by degenerate primers. We have isolated from the Hydrangea macrophylla L. genome a new resistance gene (we named RES1) by two PCR-based amplification strategies (direct amplification and overlap extension amplification) with degenerate primers designed to the conserved P-loop, kinase-2, and Gly-Leu-Pro-Leu (GLPL) motifs within the NBS domain of plant resistance gene (R gene) products. BLAST searches in GenBank revealed that they shared significant identity to well-characterized R genes from other plants. The predicted amino acid sequences showed that RES1 contained all the conserved motifs (P-loop, kinase-2, kinase-3a, GLPL, and MHD) present in the majority of other known plant TIR-NBS-LRR resistance genes. Southern analysis indicated that the RES1 gene is present in multiple copies arranged in tandem in H. macrophylla genome. Moreover, RT-PCR showed that RES1 is transcribed at low levels and exhibited tissue-specific expression pattern.



Alteration in growth and antioxidant metabolism in response to heat stress in TBY-2 cells
N. Dipierro1, C. Gadaleta1, S. Dipierro1, L. De Gara1,2, M.C. de Pinto1
1Dipartimento di Biologia e Patologia Vegetale, Univ. degli Studi di Bari, Bari, Italy
2CIR, Univ. Campus Bio-Medico, Rome, Italy

One of the major environmental factors affecting plant growth and productivity is high temperature. Heat stress induces significant changes in normal physiological processes and generates reactive oxygen species (ROS). Plant cells exposed to ROS show temporary cell cycle arrest, slowed DNA replication, and delay in the transitions from G1 to S and from G2 to M. Such delays at specific check points allow time for repairing damages. In order to limit the oxidative damage occurring under stress plants have developed detoxification systems that scavenge the highly toxic ROS. Interestingly up-regulation of defence genes during oxidative stress seems to be correlated with a down-regulation of cell cycle genes. In this work the effect of short and long term heat stress on redox homeostasis as well as on cell growth, mitotic index and cell viability have been analysed. In order to clarify if different cell cycle progression is responsible for the alterations in cell growth, the expression of cyclins and CDK has also been analysed. Finally the possibility that increased antioxidant levels could correlate with a better growth capability of heat stressed cells is also discussed.


20. Plant nutrition

Total number of abstracts in this session: 8



Effect of increased S availability on the efficiency to accumulate Fe in Strategy II plants
S. Astolfi1, S. Zuchi1, S. Cesco2, R. Pinton2
1DABAC, Univ. of Viterbo, Viterbo, Italy
2DISA, Univ. of Udine, Udine, Italy

The metabolic links between S and Fe nutrition are well documented in Strategy II plants (e.g. barley and maize). Here we investigated whether high S availability in nutrient solution (NS) could improve plant efficiency to take up and accumulate Fe.
Durum wheat (Triticum durum L. cv. Svevo) plants were grown for 11 days in NS under three different S supply (0, 1.2 and 2.5 mM, deficient, adequate and high, respectively) and two FeIII-EDTA levels (10 and 80 μM, deficient and sufficient, respectively).
Leaf Fe content increased with increasing S supply and this event was associated with higher rates of both synthesis and release of phytosiderophores. Also S content and the activity of two enzymes involved in S assimilation (ATPS and OASTL) increased with increasing S supply in wheat leaves, with the highest levels being reached when plants were grown at low Fe availability.
Taken together, results show that a high S availability can improve wheat ability to take up and accumulate Fe, thus confirming a close relationship between S nutritional status and Fe use efficiency.



Organic matter dynamics in soil: land use and global change
L. Celi1, C.Cerli1, A. Agnelli2
1Dept Valorisation and Protection of Agroforestry Resources, Turin Univ., Turin, Italy
2Dept Agricultural and Environmental Science, Perugia Univ., Perugia, Italy

Efforts to counteract climate change through protection of agroforest systems have high priority on the agenda of the European Union. Among the different compartments, the most relevant pool of terrestrial organic C is soil, which contains three times as much C as the biomass and more than twice as much as the atmosphere.
Carbon stored in soil, even though accumulated more slowly than in the biomass, is generally considered to be more resistant both to mineralization and to sudden changes than C stored in biomass. Carbon incorporation into soil is result of many different processes such as organic material input, decomposition and mineralisation, physical and chemical transformations, interaction with mineral fractions, translocation in deeper horizons. The delicate equilibrium of these processes as tuned by climate, topography and parent material, determines whether a soil is behaving as a C source or sink.
Changes in land use and agroforest practices can easily affect these processes. Deforestation and soil use for agriculture are expected to cause significant C losses. However, a change in land use from agriculture or even more exploited soils to forestry does not mean necessarily the recovery of soil function as C sink. The effectiveness of soil C uptake in forests and in crops systems depends on different parameters, which drive organic matter transformation and stabilisation, its mobility and translocation in depth, and then the ability of soil to accumulate C.



Impacts of sulphur deficiency on nitrate content and expression levels of nitrate-related genes in wheat
C. Rizzardo1,2, P. Buchner2, S. Parmar2, R.Pinton1, S. Cesco1, M.J. Hawkesford2
1DISA, Univ. of Udine, Udine, Italy
2Rothamsted Research, Harpenden, Herts, UK

Coordination of the uptake, transport and metabolic pathways of S and N is needed in plants, in particular for protein synthesis: deficiency of either S or N reduces the uptake and assimilation of the other, but the mechanisms of this coordination are not understood.
Nitrate content and expression levels of nitrate-related genes have been examined in wheat plants, which have been constantly supplied with nitrate and deprived of S for 7 days. These plants showed a transient increase in shoot nitrate content followed by a decrease; in parallel, nitrate content decreased by 50% in the roots after 7 days of S-deficiency. The expression level of the high affinity nitrate transporter (NRT2.1) was unchanged in S-deficient roots but increased in S-sufficient plants. The expression level of the nitrate-reductase gene was higher in shoots of S-deficient plants. Both these responses demonstrated a biphasic trend, with an early phase of imbalance for the S-starved plants followed by an adaptation phase involving an enlargement of the difference between S-sufficient and control plants. These results confirm that there is a negative regulation mechanism for N acquisition when S is limiting.



Nitrate availability affects roots and leaves proteomes of Zea mays plants
B. Prinsi1, A.S. Negri1, P Pesaresi2, M Cocucci1, L. Espen1
1Dept of Plant Production, Milan Univ., Milano, Italy
2Dept of Biomolecular Sciences and Biotechnology, Milan Univ., Milano, Italy

The growth and production of crop plants strictly depend on nitrogen availability. This nutrient, in fact, affects many processes, such as development, architecture, flowering, senescence and photosynthesis. Up to now, scrappy information about the changes at translational and post-translational levels in response to nitrogen availability are available. In order to overcome this gap, we investigated the protein changes induced by NO3- in both roots and leaves of maize plants by using two-dimensional gel electrophoresis (2-DE).
The 2-DE analysis revealed 20 and 18 spots in roots and leaves, respectively, that significantly changed their amount in response to nitrate. The spot identifications through LC-ESI-MS/MS revealed that in the roots, the changes were referred to enzymes involved in nitrate assimilation and in pathways implicated in the balance of the energy and redox status. In the leaves, most of the characterized proteins were related to regulation of photosynthesis. Finally, two different post-translational modifications of phosphoenolpyruvate carboxylase consisting in monoubiquitination and phosphorylation in roots and leaves, respectively, were found.



Physiological and molecular characterization of nitrate uptake mechanisms in roots of grapevine (Vitis vinifera L.) cultivars
R. Monte1, N. Tomasi1, S. Cesco1, A. Zamboni2, Z. Varanini2, R. Pinton1
1DISA, Univ. Udine, Udine, Italy
2DISTEMEV, Univ. Verona, S.Floriano, Italy

The uptake of nitrate was studied in two grapevine cultivars - Chardonnay (R8 and VCR4 clones) and Sauvignon (R3 and CL297 clones) - using cuttings grafted on different rootstocks. We showed that, like in other plant species already characterized for this phenomenon, exposure of roots to nitrate caused the development of a higher uptake rate (induction), with time and entity strongly dependent on the clone considered. This behavior was not influenced by the rootstock. Expression analysis of genes involved in nitrate influx (NRT1 and NRT2) and efflux (NAXT), performed in root tissues, showed that changes in expression were not correlated with changes in net nitrate influx; on the other hand, NAXT transcript levels increased in clones showing low or delayed induction of nitrate uptake. Results show that modulation of nitrate uptake rate and expression of nitrate transporters in grapevine graftlings is mainly determined by the characteristics of the grafted genotypes.



Carbon dioxide uptake, partitioning and storage in Mediterranean fruit orchards
A. Sofo, A. M. Palese, G. Celano, C. Xiloyannis
Dept Scienze dei Sistemi Colturali, Forestali e dell’Ambiente, Univ. of Basilicata, Potenza, Italy

Agricultural practices can play an important role in atmospheric CO2 emission and fixation. In this study, we present results on carbon fluxes in the biomass of two typical Mediterranean orchards indicating that proper canopy management coupled with other agricultural techniques could increase the absorption of atmospheric CO2 and its storage.We also discuss the potential environmental contribution of the orchards to enhancement of both soil and air quality. Trials were carried out in southern Italy on olive (Olea europaea L.) and peach orchards (Prunus persica L.) at different age and plant densities. At the end of each vegetative season, values of fixed atmospheric CO2 were calculated
by measuring dry matter accumulation and partitioning in the different plant organs. In the early years, sequestered CO2 was primarily distributed in the permanent structures and in the root system while in mature orchards the fixed CO2 was distributed in leaves, pruning materials and fruit. Significant differences in amounts of fixed CO2 were observed in peach orchards cultivated using different planting and training strategies. The results underline the importance of training system, plant density and cultivation techniques in the absorption of atmospheric CO2 and its storage as organic matter in the soil.



Effects of water-extractable humic substances on molecular physiology of nitrate uptake in two maize inbred lines with different nitrogen use efficiency
N. Tomasi1, R. Monte1, C. Rizzardo1, S. Venuti1, A. Zamboni2, S. Cesco1, R. Pinton1, Z. Varanini2
1Dept Agricultural and Environmental Sciences, Univ. of Udine
2Dept Science, Technology and Market of Grapevine and Wine, Univ. of Verona

Soil humic substances are known to positively influence plant growth and nutrition. In particular, low-molecular weight fractions have been shown to increase NO3- uptake and PM H+-ATPase activity and alter expression of related genes. In this work, a water-extractable low-molecular weight humic fraction (WEHS) has been tested for its ability to affect molecular physiology of nitrate uptake in two maize inbred lines with different NUE. WEHS causes an acceleration of the increase in net nitrate uptake rate in both lines, almost halving the time needed to reach the maximal uptake capacity after the first contact between roots and the anion. Transcriptional analyses indicate that WEHS positively modulate some genes involved in nitrate uptake (NRT2.1 and MHA2) and assimilation (NR2). It appears that WEHS can favour nitrogen acquisition by improving the plant responsiveness to variation of nitrate availability in modulating uptake and assimilation. Results point out the importance of considering the interactions between roots and soil components in order to get a better understanding of nutrient use by plants and to improve agricultural practices aiming at reducing input of fertilizers.



Molecular characterization of nitrate uptake mechanisms in roots and leaves of cucumber
A. Zamboni1, N. Tomasi2, S. Gottardi2, R. Monte2, R. Pinton2, S. Cesco2, Z. Varanini1
1Dept Science, Technology and Market of Grapevine and Wine, Univ. of Verona, Italy
2Dept Agricultural and Environmental Sciences, Univ. of Udine

The uptake of nitrate from leaf apoplastic space into the cells is a relevant process in the entire economy of plant nitrogen nutrition. In spite of the detailed knowledge about this phenomenon in roots, little information is available on this process in leaves.
Using leaf discs of cucumber plants we have demonstrated that also in these tissues nitrate uptake is an inducible process. However, different values of kinetic constants (Km, Vmax) of uptake process between root and leaf tissues suggest the operativity of different mechanisms. Changes in gene expression levels of CsNRT2 gene (encoding a high-affinity nitrate transporter) after the contact between roots and the anion, indicate that this gene is involved in the increase in uptake of the anion only in this tissues thus suggesting the activity of other transporter(s) in leaf cells. The existence of different mechanisms in the two kind of tissues is confirmed by the differential expression of two genes encoding for PM H+-ATPases.
The identification of new genes encoding transporters involved in leaf nitrate uptake is in progress using degenerate primers in RT-PCR and 3’- and 5’-RACE experiments.


21. Plant pathogenic microbes

Total number of abstracts in this session: 9



Tomato transcriptome analysis: looking for stably expressed reference genes under viral infection
T. Mascia1, E. Santovito2, F. Cillo1, D. Gallitelli1,2
1Istituto di Virologia Vegetale del CNR, Unità Organizzativa di Bari, c/o DPPMA, Bari, Italy
2Dip. di Biologia e Patologia Vegetale, Univ. degli studi di Bari, Bari, Italy

In the area of plant-virus interactions, transcript and miRNA profiling is providing new perceptions into the mechanisms of pathogenesis, disease symptoms development and basal defence. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) is being largely used in transcriptome analyses but the reproducibility of the results obtained by qRT-PCR depends on accurate transcript normalization using stably expressed genes, known as housekeeping or reference genes. The stability of the expression level in tomato of β-tubulin (TUB), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), elongation factor 1α (EF1α), ubiquitin 3 (UBI3), actin (ACT), 18S ribosomal RNA (18S), urydilate kinase (UK) and cyclophilin (CYP) has been evaluated under infection of Cucumber mosaic virus (CMV), Tomato mosaic virus (ToMV), Tomato spotted wilt virus (TSWV), Potato virus Y (PVY) Tomato yellow leaf curl virus (TYLCV) and Potato spindle tuber viroid (PSTVd). Parallel analyses by dedicated algorithms GeNorm, NormFinder and BestKeeper, showed that GAPDH, ACT and UBI3 had satisfactory levels of stability in tomato infected by different viruses.



Control of patulin accumulation in apples: the potential of phenolic antioxidants
S.M. Sanzani1, L. Schena2, A. De Girolamo3, F. Nigro1, M. Solfrizzo3, A. Ippolito1
1Dept of Plant Protection and Applied Microbiology, Univ. of Bari, Bari, Italy
2Dept of Agricultural and Forest Systems Management, Mediterranean Univ. of Reggio Calabria, Reggio Calabria, Italy
3Institute of Sciences of Food Production, National Research Council, Bari, Italy

Patulin is a mycotoxin chiefly associated to Penicillium expansum Link infection of apples and, owing to its solubility in water, easily transferred into juices during processing. Due to its cytotoxic, immunotoxic, and neurotoxic properties the European Commission has imposed strict regulatory limits on patulin content that has become one of the main quality criteria for apple juice. In this investigation the activity of natural phenolic compounds quercetin and umbelliferone on patulin accumulation was tested. The in vitro trials revealed an anti-toxigenic activity that was confirmed in vivo on two apple cultivars by wound or dipping application. A higher efficacy was observed when the two phenolics were applied in combination in wounds of Golden Delicious as compared to Granny Smith apples. Transcriptomic analyses of selected patulin biosynthetic genes revealed the down-regulation of two cytocrome P450 monooxygenases in P. expansum isolates grown in presence of either quercetin or umbelliferone. When both phenolics were present, the expression of three additional genes coding a 6-methylsalicilic acid synthase, an isoepoxydon dehydrogenase and an ABC transporter was also reduced.



Biocontrol yeasts and low rate of fungicides prevent efficiently fungal rots of apples and table grapes
G. Lima, V. De Cicco

Grey mould (Botrytis cinerea) and blue mould (Penicillium expansum) are the main postharvest rots of table grapes and pome fruit, respectively. P. expansum is also responsible for the production of the mycotoxin patulin in infected fruits and derived products. The use of few authorized fungicides for postharvest treatment involves toxicological risks and they are often ineffective for the onset of fungicide-resistant strains of the pathogens in packinghouses. For these reasons, new effective and safer control strategies are necessary. Combined application of some selected biocontrol yeasts and low rate of fungicides can be an efficient and safer strategy to reduce fungal rots, fungicide residues and mycotoxins accumulation in fruit. In this work representative data regarding the effect of combined application of biocontrol yeasts and low rate of fungicides on the main postharvest rots of apples and table grapes are reported and discussed. The results of our studies evidence that integration of biocontrol agents with low rate of fungicides can prevent rots more efficiently, display synergistic activity and manage both sensitive and resistant isolates of the fungal pathogens.



Characterization of thiabendazole-resistant isolates of Penicillium expansum associated with blue mould on Italian pears
I. Donati, D. Mazzoni, P.Bertolini, M. Mari
Dept Agri-food Protection and Improvement, Bologna Univ., Italy

Seventy three isolates of Penicillium expansum, causing blue mould in pome fruit, were collected in Emilia Romagna region from stored pears (cv ‘Abate Fetel’, ‘Kaiser’, ‘Decana’ and ‘Conference’). The sensitivity to thibendazole (TBZ), was determined in vitro on fungicide amended medium. The resistant isolates were the 67% showing ED50 and ED95 values more than 555μg ml−1and 6095 μg ml−1 respectively, while the ED50 and ED95 values of sensitive isolates were over 4.1 and 11.3 μg ml−1 respectively. For the first the sensitivity of P. expansum with respect to technical or commercial TBZ was also tested in vitro on conidia germination; a stimulatory effect of technical or commercial TBZ was observed in some R-isolates. For the in vivo trials ‘Conference’ pears were inoculated with the isolates; after 7 days of incubation at 20°C fruits inoculated with R isolates showed significantly wider lesion diameters than fruits inoculated with S isolates. Our data also showed that in the presence of R isolates, a TBZ based treatment could increase the incidence of blue mould on fruits with respect to untreated fruit. All isolates of P. expansum grown on MEA were a consistent producer of patulin, however a considerable reduction in mycotoxin production was observed in fruit samples with respect samples obtained from in vitro trials.



Proteomic analysis of grapevine leaves infected by Plasmopara viticola
A. Milli1, A. Persi2, F. Desario2, L. Zolla3, S. Rinalducci3, A.M. Timperio3, D. Cecconi1, A. Polverari2
1Dip. Di Biotecnologie, Univ.di Verona, Verona, Italy
2Dip Scienze Tecnologie e Mercati della Vite e del Vino, Univ. di Verona, San Floriano (VR), Italy
3Dip. di Scienze ambientali, Univ. della Tuscia, Viterbo, Italy

All cultivated grapevine genotypes are susceptible to the downy mildew pathogen, Plasmopara viticola. Resistant sources are available, but breeding efforts are still in progress. Molecular aspects of the infection process are still poorly understood and, up to date, proteomic data about this interactions are completely missing. In this study, infected and control leaves were collected at 24, 48 and 96 hours post-inoculation; protein extraction efficiency was optimized in a “home-made” protocol. Two-dimensional gel patterns were matched by PDQuest software; about 100 differentially expressed proteins were identified by nanoHPLC-ION TRAP-MS/MS analysis. Most of the regulated proteins are involved in oxidative stress response, defense response, and photosynthesis. In particular, a general decrease in photosynthesis-related proteins expression and a concomitant overexpression of carbohydrate metabolism enzymes, oxidative stress-related proteins, and pathogenesis related (PR) proteins was observed. This work provides a first insight into the proteomic changes occurring in grapevine under downy mildew attack.



An oxyilipin-based cross talk occurs during the interaction between a. ochraceus and triticum durum seeds
M. Reverberi1, M. Scarpari1, F. Punelli1, S. Zjalic1, A. Ricelli2, A.A. Fabbri1, C. Fanelli1
1Dept of Plant Biology, Rome Univ. Sapienza, Roma, Italy
2ICB, CNR, Roma, Italy

The oxylipin metabolism controls mycotoxin biosynthesis, conidiogenesis, sclerotia formation and the interaction with the host in Aspergillus nidulans, A. flavus and A. parasiticus. Recent evidences indicate that in A. ochraceus resveratrol, inhibitor of lipoxygenase and cicloxygenase activity, reduces the formation of oxidized lipids and hampers ochratoxin A (OTA) biosynthesis. A lox-like gene has been also found in the genome of A. ochraceus The AoloxA deleted mutant (ΔAoloxA) displays a different colony morphology (delayed conidia formation and induction of sclerotia) and an oxylipins biosynthetic pathways switching from 13-HPODE to a prevalent formation of 7,8 and 8,13-DiHODE. A large number of sclerotia are formed in vitro by ΔAoloxA, possibly due to an incretion of diols formation. Further, the reduced oxylipin formation in ΔAoloxA induces a strong reduction of OTA biosynthesis in comparison with the wild type. The seeds of T. durum cv Ciccio contaminated with ΔAoloxA did not accumulate 9-HODE acid and did not express the pathogenesis related protein PR1 mRNA whereas WT stimulated both these events. The generalized down-regulation of oxylipins synthesis in ΔAoloxA grown on wheat seeds also confirms the existence of a cross-talk between wheat seeds and A. ochraceus mediated by oxylipins.



Horizontal transfer of the Ophiostoma gene encoding cerato-ulmin into unrelated species of the genus Geosmithia
A. Scala1, C. Comparini1, L. Carresi1, P. Bettini2, A. Santini3, A.L. Pepori3, G. Cappugi4, L. Pazzagli4, F. Martellini4, F. Scala5
1Dept Agricultural Biotechnology, Univ Florence, Sesto Fiorentino, Italy
2Dept Evolutionary Biol “Leo Pardi”, Univ Florence, Florence, Italy
3Inst Plant Protection, CNR, Sesto Fiorentino, Italy
4Dept Biochemical Science, Univ. Florence, Florence, Italy
5Dept ArBoPaVe, Univ Naples ‘‘Federico II’’, Portici, Italy

Horizontal gene transfer (HGT) is a relevant evolutionary mechanism by which plant pathogens have emerged in agro-ecosystems over different time scales. In the prokaryotes HGT is generally considered a major factor for the evolution of genomes, whereas in fungi HGT is assumed to play a minor role, just to justify unusual features of genetic elements such as single genes or gene clusters. In the present work we demonstrated that strains belonging to Geosmithia pallida and G. langdonii possess and express the cu gene coding the cerato-ulmin (CU) hydrophobin. CU is produced by various Ophiostoma species (a taxon distant from the genus Geosmithia), and to give the Ophiostomas causing Dutch elm disease key advantages in parasitic fitness and virulence. We are working to understand how and why a portion of the genome of Ophiostoma novo-ulmi, including the cu gene, has been transferred in Geosmithia strains, and what advantages, in terms of fungal fitness, there are for the Geosmithias by acquiring this gene. Moreover, we are characterizing the CUs produced by the Geoesmithias and investigating how they behave towards the elms.



Role of hydrolytic enzymes in the mechanisms of action of yeast antagonists against postharvest pathogens of fruit
D. Spadaro1,2, D. Zhang2, A.Garibaldi2, M.L. Gullino2
1DiVaPRA – Plant Pathology, Univ. of Torino, Italy
2AGROINNOVA, Univ. of Torino, Italy

Among the main postharvest pathogens of fruit, Botrytis cinerea, Penicillium expansum, and Monilinia laxa are included. The interactions between the antagonistic yeast or yeast-like fungi Metschnikowia pulcherrima, Aureobasidium pullulans and Pichia guiliermondii, and the three postharvest pathogens were studied in vitro and/or in vivo in order to highlight their possible modes of action. The yeast strain M. pulcherrima MACH1 produced pulcherrimin in presence of iron, suggesting that iron depletion under low iron conditions could reduce the growth of postharvest pathogens. The yeast was able to produce chitinases and beta-1,3-glucanases and a chitinase gene was amplified using PCR reactions. In addition, a greater accumulation of defense enzymes was registered in apples treated with the strain MACH1 and challenge inoculated with B. cinerea. A. pullulans strain PL5, when cocultured in vitro with M. laxa, B. cinerea and P. expansum, showed beta-1,3-glucanase, exo-chitinase, and endo-chitinase activities. Direct attachment, secretion of hydrolytic enzymes and competition for nutrient were multiple modes of action of P. guilliermondii strain M8 against B. cinerea on apples.



Induced resistance for the control of plant diseases: grapevine/Bois noir and strawberry/postharvest decay as case studies
G. Romanazzi, L. Landi, S. Murolo, M. Santini
Dept of Environmental and Crop Sciences, Plant Protection Section, Marche Polytechnic Univ., Ancona, Italy

The use of resistance inducers is a novel and low impact strategy for control of plant diseases, for which an increasing volume of information is now available. Several compounds on the market have little or no effects on the pathogen while inducing plant defenses. Bois noir (BN) is the main phytoplasma disease of grapevine in Italy; it is widespread and results in severe losses. Five commercial resistance inducers (Aliette, Bion, Chito Plant, Kendal and Olivis) were applied weekly to BN symptomatic plants in the vineyard in mid-spring to mid-summer. These field treatments increased symptom remission, better known as recovery. Strawberries are affected by gray mold in the field and even more so after harvest and, to a lesser extent, by Rhizopus rot. These decay can cause severe losses during transport, storage and shelf life. The Postharvest application of resistance inducers (Algition, Bion, Chito Plant, Fitocalcio and Xedabio) greatly improved control of decay of strawberries stored 10 days at 0 °C, and then exposed to 2 days of shelf life. Several physiological changes occurred in leaves of recovered grapevines and in strawberry fruits treated with these resistance inducers.


22. Protein synthesis

Total number of abstracts in this session: 1



PIM1 oncoprotein is destabilized by ribosomal stress and inhibits cell cycle progression
V. Iadevaia1, S. Caldarola1, L. Biondini1, A. Gismondi1, S. Karlsson2, I. Dianzani3, F. Loreni1
1Dept Biology, Univ. of Rome Tor Vergata, Italy
2Dept of Molecular Medicine and Gene Therapy, Lund Univ. Hospital, Sweden
3Dept Medical Sciences, Univ. of Piemonte Orientale, Novara, Italy

We have recently found that the oncogenic kinase PIM1 interacts with ribosomal protein (RP)S19. A number of RP genes are mutated in Diamond-Blackfan anemia (DBA) patients that exhibit an excess of apoptosis of erythroid precursors. To explore the possible implication of PIM1 in DBA mechanism we have analyzed its expression in cultured cell model systems. We found that depletion of an RP or treatment with drugs known to interfere with nucleolar functions causes a drastic destabilization of PIM1. Moreover, we observed that the lower level of PIM1 in RPS19-deficient cells is associated to an increase of the cell cycle inhibitor p27Kip and to a block in cell proliferation even in the absence of p53. To demonstrate that the decrease of PIM1 could be the cause of the block of cell proliferation we transfected PIM1 in RPS19-deficient cells. Overexpression of PIM1 in these cells induces a recovery from the proliferation arrest caused by RPS19 deficiency. All these data suggest that PIM1 could play a role in the alteration of growth and apoptosis observed in hematopoietic cells from DBA patients.


23. Regulation of transcription

Total number of abstracts in this session: 10



Dual mode of proteasome association to actively transcribed genes in S.cerevisiae
A.A. Alagia1, S. Loreti1, A. Parente1, C. Mannironi2, T. Rinaldi1, M. Esposito1, V. Licursi1, A. Peserico1, R. Negri1
1Dipartimento di biologia cellulare e dello sviluppo, Laboratorio di Genomica Funzionale e Proteomica dei Sistemi Modello, Univ. "Sapienza" Roma, Italia
2Istituto di Biologia Cellulare, Consiglio Nazionale delle Ricerche, Roma, Italia

Several subunits of the 26S proteasome have been physically localized at active transcription units in eucaryotic genomes. Although some hypothesis have been proposed, the mechanism of targeting and the role of this association remein elusive. In yeast, ribosomal protein genes appear particularly enriched in both regulatory lead\'s and catalytic cylinder\'s components. We have analysed the association Pre1 and Rpn11 with a typical RP divergent promoter in different conditions of transcriptionale activity and genetic background. Based on the experimental evidences, we present a model in which th 26S proteasome is recruited by ubiquitylated H2B histone and, in condition of efficient transcription, subsequently loaded on the RNA polymerase II elongation complex. The implications for proteasomal function in transcription are discussed.



NRF-2 controls the expression of mitochondrial transcription and replication proteins
F. Bruni, P. Loguercio Polosa, M. N. Gadaleta, P. Cantatore, M. Roberti

NRF-2 (nuclear respiratory factor 2), also named GABP, is a transcription factor controlling the expression of many genes involved in cell cycle progression and protein synthesis, as well as in mitochondrial biogenesis. We have analyzed the role of NRF-2 in the regulation of genes acting in mtDNA transcription and replication. By a combination of bioinformatics and biochemical approaches, we found that the factor binds to the proximal promoter region of the genes coding for the transcription termination factor mTERF, the RNA polymerase POLRMT, the B subunit of the DNA polymerase-gamma, the DNA helicase TWINKLE and mtSSB. The role of NRF-2 in activating the expression of those genes was further established by RNAi and overexpression strategies. On the contrary, we found that NRF-2 does not bind to the promoter of the genes for the subunit A of DNA polymerase-gamma and for the transcription repressor MTERF3; we suggest that these genes are under regulatory mechanisms that do not involve NRF protein factors. Finally, we developed a more stringent consensus with respect to the general consensus of NRF-2/GABP, when searching for NRF-2 binding sites in the promoter of mitochondrial proteins.



The RNA-helicase BELLE is a new partner of Drosophila melanogaster CRYPTOCHROME
E. Carbognin1, M. Mason1, G.M. Mazzotta1, F. Sandrelli1, M.P. Bozzetti2, R. Costa1
1Dept of Biology, Univ. of Padova, Italy
2Dept of Biological and Environmental Sciences and Technologies (DiSTeBA), Univ. of Salento, Italy

In Drosophila melanogaster CRYPTOCHROME (dCRY) is the blue light photoreceptor involved in the photic input pathway to the circadian clock. It mediates the daily resetting of the clock by light by binding to TIMELESS (dTIM), a cardinal component of the clock machinery, and targeting it to degradation via proteasome. The light-dependent activity of this photoreceptor is specifically regulated by its C-terminus region, which has been recently shown to bear several putative sites for protein-protein interaction.
The research for new partners for dCRY, performed by co-immunoprecipitation, led to the identification of BELLE, an ATP-dependent RNA helicase. The expression of this RNA-helicase seems to be controlled by the circadian clock, as both mRNA and protein show a circadian oscillation either in light:dark cycles or in constant darkness. Moreover, flies mutant for this gene do not show the canonical rhythmic activity profile, suggesting an impairment of the circadian clock. Our data suggest that BELLE is a new protein involved in the circadian pacemaker in Drosophila, where it could act in the post-transcriptional control of circadian genes.



An FLT1 promoter SNP coordinates cis-regulation via DNA damage-specific p53 responses and ligand-dependent Estrogen Receptors
Y. Ciribilli1, V. Andreotti1, D. Menendez 2, J.S. Langen3, G. Schoenfelder3, M.A. Resnick2, A.Inga1
1Unit of Molecular Mutagenesis and DNA Repair, National Cancer Research Institute, IST, Genoa, Italy
2Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, NIEHS, NIH, RTP, NC, USA,
3Institute of Clinical Pharmacology and Toxicology, Charité-Univ.ersitaetsmedizin Berlin, Berlin, Germany

Recently we established that a C>T SNP in the promoter of FLT1 gene generates a half-site p53 response element (RE-T) that results in p53 responsiveness of the promoter. The transcriptional control required two estrogen receptor (ER) half-site RE located respectively 225bp upstream (ERE1) and 145bp downstream (ERE2) the RE-T. Both EREs can impact p53-mediated FLT1-T transactivation in a manner that is cell type and ER level dependent. Gene reporter assays and ChIP experiments revealed that both EREs were sufficient for p53-mediated ERalpharecruitment and transactivation of the FLT1-T promoter/reporter construct and contributed to the recruitment of p53 at the RE-T site. Surprisingly, doxorubicin and 5-fluoro-uracil differentially affected p53-mediated transactivation of FLT1-T constructs, expression of the FLT1 gene as well as binding of p53 and ERs at the promoter. Moreover ER activity was differentially affected by ER ligands. Thus, we establish a new dimension to the p53 master regulatory network where p53-mediated transcription from a half site RE can be determined by ER binding in a way that is dependent on ER protein level and the types of ER ligands and p53-inducing agent.



TAB2 as a shuttle protein of ERa and erbB4 signalling
S. Cutrupi1,2, L. Macrì2, L. Caizzi2, A. Panetto1, S. Reineri1, G. Gambarotta1,2, I. Perroteau1,2, M. De Bortoli1,3
1Center for Complex System in Molecular Biology & Medicine Univ., Turin, Italy
2Dept of Animal & Human Biology Univ., Turin, Italy
3Dept of Oncological Sciences Univ., Turin, Italy
4Bioindustry Park del Canavese, Colleretto Giacosa, Turin, Italy

The protein TAB2 was shown to shuttle NCoR to the cytoplasm in response to inflammatory signals, causing resistance to antiandrogen in prostate and antiestrogen in breast cancer cells. In addition, the complex of Tab2 and NCoR play a role in erbB4 signalling of neuronal precursors. On this way we have studied Tab2 as modulator of differet pathways in two cellular models, breast cancer cells and neuronal precursors. In tamoxifen resitant breast cancer cells, in all clones retaining ERα expression, NCoR was aberrantly localized in the cytoplasm as wild-type tamoxifen-sensitive MCF7 cells after treatment with IL1β. Following these observations, we directly assessed the role of TAB2 in NCoR delocalization, by down-regulating TAB2 in TAMR cells using siRNA. Downregulation of TAB2 was accompanied by recovery of NCoR nuclear localization and cell growth inhibition upon tamoxifen treatment, demonstrating the primary role of TAB2 in gene derepression in this system. In the neuronal precursor lines ST14 the inhibition of TAB2 with RNA interferance modulated the migration of different clones expressing the cleavable erbB4 form. TAB2 was co-immunoprecipitated with erbB4 after NRG1 stimulation. In addition, the localization of TAB2 to the nucleus is induced by NRG1 treatment. Altogether these data support the notion of TAB2 as a main shuttle protein of corepressor complexes between cytosol and nucleus in response to different stimuli.



Regulatory networks and regulatory sequences in familial combined hyperlipidemia patients
M. Coiro1, F. Vuolo1, L. de Magistris1, M. Oliviero1, P. Pauciullo2, M. Gentile2, P. Rubba2, V. de Simone1
1Dept of Biochemistry and Medical Biotechnology, 2 Dept of Clinical and Experimental Medicine, Univ. of Naples "Federico II", Napoli, Italy

We have employed “network biology” computational tools to identify regulatory “nodes” or “modules” that are altered in the familial combined hyperlipidemia (FCHL), the most frequent multifactorial and genetically complex dislipidemic syndrome in our population.
By microarrays transcriptome analysis, we find 1913 genes hyper- or hypo-expressed) in FCHL patients, and 688 genes significantly altered after statin (the elective drugs for FCHL) treatment. Out of the 97 genes present in the intersection of these two lists, the majority are hypo-expressed in FCHL patients and become normo- or hyper-expressed after statin treatment. These genes have been selected for promoter analysis.
MEME, Jaspar and CisRed analysis of the -400/+1 regions of these genes, reveal seven most significantly enriched motifs. Many promoters contain two or more motifs. Network analysis reveals a transcriptional regulatory circuitry connecting these genes. This circuitry is a specific node of a wider transcriptional network of genes involved in FCHL syndrome, and is a promising target for a specific regulatory “interference” approach to FCHL.



G-quadruplex structures within a regulatory element of the human telomerase (hTERT) promoter
M. Martufi1, E. Micheli1,2, M. Savino1
1Dept Genetics and Mol. Biol., Sapienza Univ. of Rome
2Dept Chemistry, Sapienza Univ. of Rome

G-rich nucleic acid sequences have the potential to fold into four stranded structures, called G-quadruplex. Telomeric DNA comprises the best-studied source of quadruplex-forming nucleic acids, but there has been increasing interest in non-telomeric regions of G-rich DNA. There are satisfactory evidences for their significant role in telomeres functioning and for the transcription regulation of a number of protooncogenes (c-myc, c-kit and KRAS, Rb and VEGF).
We are studying the structural features of the promoter of hTERT gene, responsible for the expression of telomerase, that occurs in most cancer cells. We have found, by theoretical analysis, that the hTERT promoter is characterized by the presence of nine putative G-quadruplex sequences (PQS) belonging to the most accessible G-rich region of the hTERT promoter, that is unfavourable for nucleosome formation.
We show using a combination of PAGE, polymerase stop assay, UV/Vis and circular dichroism spectroscopy that the promoter presents two sequences that do have the capability to fold into an intramolecular G-quadruplex, inhibiting the DNA polymerase catalyzed reaction.
The possible equilibrium of this promoter region between the canonical duplex and the G-quadruplex structure could represent an interesting element for the gene transcription regulation by means of suitable chemical ligands and/or nuclear proteins binding.



Study of the role of PARP-1 in immediate early response genes activation during G0-G1 transition of quiescent cells
C. Mostocotto1, A. Notari1, M. Carbone1, MN. Rossi1, P. Amati1, R. Maione1
1Dept of Cellular Biotechnology and Haematology, Univ. of Rome “Sapienza”, Italy

Poly(ADP-ribosyl)ation is a post-translational modification involved in a variety of physiological processes, including cell cycle regulation. It’s catalysed by a family of ADP-ribose polymerases (PARPs), among which PARP-1 is the founding member. We have previously demonstrated that PARP-1 activity is involved in the exit of cells from a quiescent state (G0). Indeed this modification is required for the upregulation of immediate early response genes (IEGs), such as c-Fos and c-Myc after mitogens stimulation.
PARP-1 activity may participate in the regulation of chromatin organization modulating the accessibility of transcription factors. To investigate changes in chromatin status upon mitogen stimulation, the accessibility of the c-myc promoter to DNaseI digestion was measured in presence of PARP-1 inhibitors. The chromatin was more condensed in absence of poly(ADP-ribosyl)ation and this data allowed us to study the direct implication of PARP-1 in the chromatin structure at the IEGs promoters. Preliminary ChIP experiments showed that PARP-1 binds to c-Myc promoter in G0 phase and that its activation results in chromatin poly(ADP-ribosyl)ation.



The ubiquitin ligase Bre1 regulates the accumulation of mRNAs coding for the biosynthetic apparatus in S. cereviasie
A. Peserico1, V. Licursi1, M. Deligios2, A. Alagia1, S. Loreti1, M. Esposito1, T. Rinaldi1, C. Mannironi3, R. Negri1
1Dip. di Biologia Cellulare e dello Sviluppo Univ. "Sapienza", Roma, Italia
2Dip. di Scienze Biomediche, Univ. degli Studi, Sassari, Italia
3Istituto di Biologia Cellulare, Consiglio Nazionale delle Ricerche, Roma, Italia

The BRE1 gene in S. cerevisiae codes for an E3 ubiquitin ligase which is required for the ubiquitination of histone H2B and subsequent methylation of histone H3. Deletion of the gene leads to severe defects in transcription elongation. We show that a bre1 deleted strain has a completely different mRNA abundance rank as compared with the isogenic wild type. Moreover, this strain shows an altered response to rapamycin which can be explained with an increased stability of the mRNAs coding for components of the biosynthetic apparatus. Both features seem not strictly dependent on defect in histone H2B ubiquitylation. The implications of these findings, which could explain the previously reported resistance of this bre1 deleted strain to rapamycin, are discussed.



Simultaneous quantitative and allele-specific expression analysis
S. Radovic1, D. Copetti1,2, M. Morgante1,2
1Dept Agricultural and Environmental Sciences, Univ. of Udine, Italy
2Istituto di Genomica Applicata, Udine, Italy

One of the vital goals of biology is to decipher the genome’s regulatory code buried within the genome and elucidate how genetic and transcriptional variation influences phenotypic variation. It is thus essential to have a powerful and reliable method to easily test the influence of DNA polymorphisms on gene expression. We developed a sensitive expression profiling method that with accuracy simultaneously quantifies the relative expression levels of two alleles of the same gene as well as the genes overall expression in a given sample by comparing the expression levels of the alleles to those of an internal reference gene. RT-PCR, primer extension and capillary gel electrophoresis are used to quantify changes in steady-state mRNA levels of each allele expressing them relatively to the levels of an internal control RNA. Our approach allows a targeted survey of regulatory variation in any collection of interest. In addition, the ability to simultaneously evaluate both, variation in gene expression and variation in allelic expression, establishes this rapid and reliable technique as adequate for investigating physiological changes in allelic and gene expression levels.


24. RNA biology

Total number of abstracts in this session: 9



Antisense-RNA-induced exon-skipping for the gene therapy of Frontotemporal Dementia and Parkinsonism associated with chromosome 17 (FTDP-17)
G. Covello1, V. Del Vescovo1, M. A. Denti1
1Centre for Integrative Biology (CIBIO), Univ. of Trento, Trento, Italy

Tau protein is a cytoskeletal component expressed in the nervous system, with a role in neurogenesis, axonal maintenance and transport.
The deposition of tau in Neurofibrillary Tangles (NT) in the brain is a pathological hallmark of several neurodegenerative disorders, named “tauopathies”, among which Frontotemporal Dementia with Parkinsonism linked to chromosome 17 (FTDP-17). FTDP-17 is an autosomal dominant condition caused by an aberrant inclusion of exon 10 (E10) in the tau mRNA leading to the accumulation of the tau protein in neurons.
We are exploring the feasibility of an antisense-RNA-based gene therapy to correct tau splicing in FTDP-17.
We first tested whether various Antisense Oligonucleotides (AONs) are capable of altering the splicing behaviour of tau. By RT-PCR and Western blot analyses we showed that the transfection of specific AONs in PC-12 cell lines is able to prevent inclusion of E10 into the mature mRNA during the splicing reaction, with variable efficiencies depending on the concentration of the AON and on the targeted sequence.
In order to obtain persistent effects we then embedded the antisense sequences in chimeric snRNAs. We will test whether, upon transfection of plasmids coding for these antisense RNA constructs, the splicing behaviour of tau is corrected in cell lines and in primary neurons.



HUVEC cells respond to radiation by inducing the expression of pro-angiogenic microRNAs
M.P. Etna1, S. Vincenti2, N. Brillante2, V. Lanza2, I. Bozzoni2, C. Presutti2, F. Chiani1, R. Negri1
1Dip. di Biologia Cellulare e dello Sviluppo, Laboratorio di Genomica Funzionale e Proteomica dei Sistemi Modello. Univ. “La Sapienza”-Rome, Italy
2Dip. di Genetica e Biologia Molecolare, Laboratorio di Genomica Funzionale e Proteomica dei Sistemi Modello, Univ. “La Sapienza”-Rome, Italy

In this work we analyzed the effects of a dose of ionizing radiation in the clinical range (1Gray=Gy) on microRNA expression in HUVEC primary cells. In order to analyze the expression profile of microRNAs after short term x-ray exposure (30’ and 1h) we performed microarray experiments and validation of obtained data by Northern Blot and RT-PCR analyses. We found remarkable variations in microRNA expression, previously related to the angiogenesis process. These changes in microRNA expression are strictly related to the kind of radiation used, higher doses of radiations or different stressful treatments have different effects on microRNA expression All together these findings suggest that the early response to moderate radiation exposure in Huvec cells implies a commitment toward angiogenic pathway and open a new key to the reading of microRNA involvement in angiogenesis.



Identification and characterization of tissue-specific alternative transcripts of Tfam gene by both computational and experimental approaches
C. Pousis , C. De Virgilio, G. Gadaleta
Dept Biochemistry and Molecular Biology "Ernesto Quagliariello", Univ. of Bari, Bari, Italy

Recent analyses indicate that 92-94% of human genes undergo alternative splicing and that different tissues show different patterns of alternative splicing as well as alternative cleavage and polyadenilation events. These transcripts can differ both in untranslated (UTR) and in coding regions.
We used both the computational tool ASPic to predict alternatively spliced transcripts of human Tfam and RT-PCR and DNA sequencing for the experimental validation in different human tissues such as breast, brain, lung, skeletal muscle.
Commercially available total RNA from normal and cancerous tissues was used as template for the cDNA synthesis. The cDNA pool was amplified in the PCR reaction using primer pairs designed for the selective identification of specific transcripts.
RACE method was also applied to define the UTRs extensions. Some of the studied isoforms resulted to be tissue-specific. All RT-PCR products corresponding to each specific isoform were sequenced.
The data obtained allowed us not only to validate software predictions but also to highlight the existence of new Tfam isoforms which had not been predicted in silico and to correct the structure predicted for the Tr6 transcript.



Small satellite III RNAs transcribed from repetitive DNA elements in pericentromeric heterochromatic portions of the human genome
M. Giordano1, C. Ghigna1, G. Biamonti1
1Istituto di Genetica Molecolare, Pavia, Italia

The cell response to heat shock entails activation and binding of heat shock factor 1 HSF1 to heat shock elements HSE in the promoter of heat shock genes, leading to the expression of heat shock proteins. In human cells activated HSF1 binds also to HSE-like sequences in Satellite III repetitive DNA elements that form long tandem arrays in the long pericentromeric heterochromatic region of human chromosome 9. Binding of HSF1 triggers transcription of Satellite III DNA and the production of long non-coding SatIII RNAs L-SatIII. These RNAs remain associated with or in close proximity of sites of transcription giving rise to the so-called nuclear stress bodies nSBs. L-SatIII RNAs are stable and are detectable for more than 1 day from stress even though their level steadily decreases after 6 h of recovery. We now show that L-SatIII RNAs are inherited by daughter cells thanks to their association with chromosomes during mitosis. Beginning at 24 h of recovery, L-Sat III RNAs are processed in to a ladder of small s-SatIII RNAs ranging in size from 25 to 70 nt .Similarly long molecules, s-SatIII RNAs are associated to chromatin and they remain stable until 4 days after the stress treatment. This is the first time that small RNAs derived from a pericentromeric DNA region are described in human cells.



Regulation of CDK5R1 gene expression by miR-103/107
S. Moncini1, A. Salvi2, M. Venturin1, G. De Petro2, S. Barlati2, P. Riva1
1Dept of Biology and Genetics for Medical Sciences, Univ. of Milan, Milan, Italy
2Division of Biology and Genetics, Dept of Biomedical Sciences and Biotechnologies, Univ. of Brescia, Brescia, Italy

CDK5R1 encodes for p35, an activator of CDK5, which is involved in neuronal migration and differentiation during CNS development. We recently reported that the large 3’UTR of CDK5R1 contains regulatory elements affecting transcript stability. Twenty miRNAs are predicted to bind CDK5R1: considering their expression profile and prediction score we performed a RealTime PCR on 5 miRNAs. An inverse correlation between miR-103/107 levels and p35 expression was detected, suggesting a negative effect of the two miRNAs on CDK5R1 expression. We overexpressed miR-107 by transfecting the specific precursor in neuroblastoma cells and observed a 75% reduction in p35 expression, while the transfection of anti-miR-107 led to a 2.3 times increase of p35, indicate that miR-107 regulates CDK5R1/p35 expression. miR-103 is under study. Luciferase assays on 3’UTR constructs cotransfected with miR-103/107 showed that 4 out of 9 predicted miR-103/107 target sites are functional; this data will be confirmed by mutagenesis of the binding sites. Our findings on CDK5R1 regulation by miRNAs allow us to hypothesize that a new pathogenetic miRNA-mediated mechanism might influence CDK5 activity and its pathway.



RNA metabolism in Mycobacteria
V. Taverniti1, H. Putzer2, F. Forti1, D. Ghisotti1
1Dip. di Scienze Biomolecolari e Biotecnologie, Univ. degli Studi di Milano, Milano, Italia
2Institut de Biologie Physico-Chimique, CNRS-UPR9073, Paris, France

Mycobacterium smegmatis and Mycobacterium tuberculosis have orthologues for both ribonucleases E and J. However, their roles in the RNA metabolism are not known.
It has been demonstrated that in M. tuberculosis only RNaseE is essential, but not RNaseJ. In order to investigate the importance of these ribonucleases in M. smegmatis, we are constructing a conditional mutant for RNaseE, using a novel inducible system and inactivating the rnaseJ gene.
The effect of these mutants on the transcription pattern of furA-katG genes has to be analyzed. Infact, it has been proposed that the katG mRNA is not transcribed from a dedicated promoter but stems from endonucleolytic processing of the dicistronic transcript. In addition, it was shown that the cleavage site has to be in a single stranded region in order to obtain processing.
We also cloned the M. smegmatis furA-katG region into wild-type and Rnases J1-J2 mutant strains of B. subtilis in order to test whether these ribonucleases are involved in its maturation.



Functional analysis of miRNAs involved in the regulation of the transcription factors Hoxa9, Hoxa10, Hoxa11 and Pax8 in epithelial ovarian cancer
C. Tonelli, V. Del Vescovo, G. Covello, M.A. Denti

Epithelial Ovarian Cancer (EOC), the most lethal of the gynaecological neoplasms, is more differentiated than cells of the likely precursor, the ovarian surface epithelium (OSE). Moreover, the major subtypes of EOCs show morphologic features that resemble those of the müllerian duct‐derived epithelia of the reproductive tract.
Some transcription factors (Hoxa9, Hoxa10, Hoxa11 and Pax8) which normally regulate müllerian duct differentiation, are not expressed in normal OSE, but are expressed in different EOC subtypes according to the pattern of müllerian-like differentiation of these cancers. Our work is aimed at testing the hypothesis that Hoxa9-11 and Pax8 genes are silenced in OSE cells, while differently expressed in the various subtypes of EOC.
We are analysing the levels of Hoxa9-11 and Pax8 mRNAs and proteins in a panel of 15 available EOC cell lines, and comparing these levels with those in cells representative of normal ovarian surface epithelium (IOSE).
Furthermore, we are testing the hypothesis that microRNAs normally silence Hoxa9-11 and Pax8 genes in OSE cells, and are differently de-regulated in different subtypes of EOC, taking advantage of a miRNAs microarray profiling.



A GC-rich minisatellite drives transcription of human telomeres
V. Vitelli1, S.G. Nergadze1, L. Khoriauli1, M. Lupotto1, C.M. Azzalin2, E. Giulotto1
1Dip. di Genetica e Microbiologia "A. Buzzati-Traverso", Univ. degli Studi di Pavia, Pavia, Italy
2Institute of Biochemistry (IBC), ETHZ-Eidgenössische Technische Hochschule Zürich, Zürich, Switzerland

The longstanding dogma that telomeric ends are transcriptionally inactive have been overturned by the discovery, in several eukaryotes, of a heterogeneous population of telomeric repeats-containing RNAs (TERRA) (Azzalin et al, Science 2007). These RNA molecules, ranging from 100 nt to 10.000 nt, are transcribed by RNA Polymerase II starting from different subtelomeric loci. TERRA is a component of shelterin, the nucleoproteic complex of the telomeric tract, and it may function as a regulator of the stability of the chromosome ends and as a suppressor of telomerase activity. Here we show that in human cells, a CpG dinucleotide-rich island, cloned from chromosome X subtelomere, sustains the expression of a reporter gene (eGFP). Further molecular dissection of the region showed that a 29 bp minisatellite alone, comprised within this region, is endowed with promoter activity. In addition, a 61 bp repeat element acts as insulator between this promoter and subtelomeric genes. An in-silico analysis showed that sequences >93% identical to this promoter are present at several subtelomeres, and possibly contribute to the transcription of TERRA molecules.



Identification of an mRNA destabilizing hairpin element in CDK5R1 3’UTR
P. Zuccotti1, S. Moncini1, A. Nicolin2, M. Venturin1, P. Riva1
1Dept Biology and Genetics for Medical Sciences, Univ. of Milan, Milan, Italy
2Dept Pharmacology, Chemotherapy and Medical Toxicology, Univ. of Milan, Milan, Italy

CDK5R1 is implicated in CNS functioning and neurodegenerative disorders. The size and the high degree of conservation of CDK5R1 3’UTR suggest a role in post-transcriptional regulation of its expression. The insertion of CDK5R1 3’UTR into the luciferase gene caused decreased luciferase activity in 4 transfected cell lines. A 65 bp region of 3’UTR leading to an accelerated mRNA degradation was identified. A hairpin motif was predicted in silico inside this region. A 703 bp 3’UTR portion deleted of the 65 bp region was able to restore the luciferase activity. Four constructs with mutated/deleted hairpin showed unchanged luciferase levels in contrast with the wild-type construct, suggesting a negative regulatory role for this putative element. Since complementary mutations restoring the hairpin did not affect luciferase activity, both sequence and structure may be essential to reduce transcript stability. The 65 bp element was further divided in 3 portions; none of them had the same destabilizing effect as the whole region. Partial deletions of this region and constructs mutated outside the hairpin are under study, to investigate if the full fragment is required for destabilization.


25. Signal transduction

Total number of abstracts in this session: 8



Silencing HD-PTP enhances J82 cancer cell migration and invasivity
E. Baldoli, S. Castiglioni, J.AM Maier and M. Mariotti
Dept of Preclinical Sciences, Univ. of Milan medical school, Milan, Italy

HD-PTP is a tyrosine phosphatase which maps on chromosome 3p21.3, a large area frequently deleted in aggressive bladder cancers. Interestingly, the overexpression of the rat homologue PTP-TD14 suppresses the Ha-Ras-mediated transformation in NIH3T3 cells.
Cell migration and invasivity, which are modulated by the reversible phosphorylation of tyrosine residues on target proteins, are fundamental for tumor progression and metastasis. Here we report that the tyrosine phosphatase HD-PTP has a role in modulating the motility and invasivity of J82 bladder carcinoma cells. Indeed, HD-PTP silencing by RNA interference induced the acquisition of a migratory phenotype and enhanced migration. In addition, silencing HD-PTP increased cell invasivity in matrigel. This is paralleled by the enhanced activity of secreted MMP-9 and MMP-2, key regulators of extracellular matrix degradation. Pharmacological inhibitors of ERK1/2, p38, JNK and PI3K were used to identify the signal transduction pathway involved in HD-PTP dependent invasion, but no inhibitory effect was detected. It is noteworthy that an increased tyrosine phosphorylation of FAK and EGFR was found in cells silencing HD-PTP, suggesting that a MAPK-indipendent signal transduction may be implicated in regulating motility and invasivity of J82 bladder cancer cells.



c-ABL modulates MAP kinases activation downstream of VEGFR-2 signaling by direct phosphorylation of the adaptor proteins GRB2 and NCK1
F. Galvagni, F. Anselmi, M. Orlandini, M. Rocchigiani, C. De Clemente, A. Salameh, S. Oliviero

VEGF-A is a key molecule in normal and tumor angiogenesis and the activation of its main receptor VEGFR-2 has been extensively studied. However, several aspects related to the VEGFR-2 downstream signaling pathway remain to be explored. In the current study, we have identified the non-receptor tyrosine kinase c-ABL as a novel target of activation by VEGF-A in endothelial cells. Recently, has been reported that activated c-ABL enhanced bFGF-induced angiogenesis. On the contrary, we showed that c-ABL down-modulates VEGF-A-dependent activation of MAPKs. Inhibiting c-Abl by STI571 and over-expressing GRB2 mutant in tyrosine 209, we showed that the adaptor protein was phosphorylated by c-ABL after VEGF-A stimulus and this phosphorylation was responsible of lower ERK1/2 and JNKs activation. Furthermore, we identified tyrosine 268 of the adaptor NCK1 as a new target of c-ABL kinase activity and described the negative effect of this phosphorylation on p38 pathway. These findings supported the model in which c-ABL differentially mediates bFGF- and VEGF-A-dependent angiogenesis and revealed a new mechanism by which c-ABL regulates p38 activation.



Bimodal pattern of chemokine induced Rap1 activation: dissecting the molecular mechanisms
A. Giammarresi1,2, M. Fabbri1, R. Molteni1, R. Pardi1
1Unit of Leukocyte Biology Vita-Salute Univ. School of Medicine DIBIT-Scientific Institute San Raffaele, Milan, Italy
2Dip. Di Medicina Sperimentale (Dimes) Sez. Anatomia Umana, Univ. degli Studi di Genova, Centro IFOM Di Oncologia Cellulare E Strutturale, Genova, Italy

The multi-step leukocyte extravasation process is governed by adhesion molecules and chemotactic factors. Responsiveness to chemotactic ligands is mediated by G protein-coupled receptors which are finely regulated by cytosolic proteins, beta-arrestins. They play a regulatory role in GPCR desensitization and internalization, and may contribute to GPCR signaling by functioning as scaffolds for the recruitment of signaling proteins into complexes with agonist-occupied receptors. On this basis, we investigated the physiological role of beta-arrestin 2 and 1 in chemokine-driven dynamics associated with leukocyte extravasation, with special interest to the activation of the Rap1 small GTPase, a pivotal regulator of integrin function. The analysis of KC (Keratinocyte-derived Chemokine) induced Rap1 activation profile in RBL (Rat Basophilic Leukemia) cells expressing mCXCR2 shows a bimodal kinetic, with the first peak at 30\'\'/1\' and the second at 5\' after stimulation. RNA interference-mediated depletion of both beta-arrestins specifically inhibit Rap1 activation, although with differrent kinetics as beta-arrestin1 depletion seems to cause a delay in the formation of Rap1-GTP.



Diacylglycerol Kinase alpha mediates HGF-induced Rac activation, ruffling and cell migration by regulating atypical Protein Kinase C zeta/iota and RhoGDI
F. Chianale1, E. Rainero1, P.E. Porporato1, C. Cianflone1, V. Bettio1, I. Locatelli1, N. Filigheddu1, G. Serini2,3, G. Baldanzi1, F. Sinigaglia1, A. Graziani1

Diacylglycerol kinases (DGKs) convert diacylglycerol (DG) into phosphatidic acid (PA), acting as molecular switches between DG- and PA-mediated signalling. We previously showed that Src-dependent activation and plasma membrane recruitment of DGKα are required for growth factor-induced cell migration and ruffling, through the control of Rac small GTPase activation and plasma membrane localization. Herein we unveil a novel signalling pathway through which DGKα coordinates the activation of Rac. We show that upon Hepatocyte Growth Factor-stimulation, DGKα, by producing PA, provides a key signal to recruit atypical PKCζ/ι in complex with RhoGDI and Rac at ruffling sites of colony-growing epithelial cells. There, the release of Rac from the inhibitory complex with RhoGDI allows its activation, leading to formation of membrane ruffles, which constitute essential requirements for cell migration. These findings highlight DGKα as the central element of a novel lipid signalling pathway linking tyrosine kinase growth factor receptors to regulation of aPKC and RhoGDI, and providing a positional signal regulating Rac association to the plasma membrane.



Evidence for a novel calcium channel in yeast involved in response to nutrient and osmotic stress
S. Groppi, I. Mascheretti, F. Belotti, E. Martegani, R. Tisi
Dept of Biotechnology and Biosciences, Univ. of Milano-Bicocca, Milan, Italy

Different stimuli induce an influx of calcium from the extracellular environment in budding yeast, such as glucose addition or hypotonic and hypertonic shocks. The response induced by both types of stimuli suggest that glucose-induced Ca2+ influx is mediated by two different carrier systems, a High Affinity Calcium System (HACS), involving Mid1/Cch1 transporters, and a Low Affinity Calcium System (LACS), involving Fig1 protein. We report evidences that at least another carrier exists, not yet identified at the molecular level, that can substitute HACS and LACS when they are inactivated. This channel appears to constitute a low affinity, high capacity system. Like HACS system, this calcium carrier is activated both by nutrients and by hypotonic shock. Differently from Mid1/Cch1 system, this calcium channel is resistant to Mg2+ and Ni2+, but sensitive to Zn2+ and Mn2+. Furthermore, we have characterized the vacuolar channel Yvc1 involvement in the amplification of calcium signalling.



Sumoylation affects EGF induced Egr-1 expression and stability
G. Manente, G. Pinton, I. Morra, E. Brunelli, L. Moro
DISCAFF and DFB Center, Univ. of Piemonte Orientale “A. Avogadro”, Novara, Italy

Human early growth response-1 (Egr-1) is a member of the zing-finger family of transcription factors induced by a range of molecular and environmental stimuli including epidermal growth factor (EGF). Previously we demonstrated that integrin/EGFR cross-talk is required for expression of Egr-1 through activation of Erk1/2 and PI3K/Akt/Forkhead pathways. The aim of this work was to assess the influence of sumoylation, a more recently described post-translational modification, on EGF induced Egr-1 expression and stability. We demonstrated that in basal conditions and after EGF treatment a fraction of endogenous Egr-1 was mono-sumoylated. Quantitative Real Time PCR experiments were performed to investigate whether sumoylation could affect EGF induced EGR-1 gene transcription. Here, we report that EGF induced Egr-1 mRNA levels were increased by SUMO-1/Ubc9 over-expression. Conversely, Egr-1 protein levels were strongly reduced in SUMO-1/Ubc9 transfected cells. Data obtained from protein expression and ubiquitination analysis in the presence of the proteaosome inhibitor MG132, suggested that sumoylation increased Egr-1 ubiquitination enhancing its degradation.



Fibroblasts activation and tumour progression induced by mitochondrial oxidative stress
C. Marconi1, M.L. Taddei1, G. Raugei1*, S. Papa2 , P. Chiarugi1
1Dept of Biochemical Sciences, Univ. of Florence, Florence, Italy
2Depts of Medical Biochemistry, Biology and Physics, Univ. of Bari, Italy

Increased cellular reactive oxygen species (ROS) are characteristic of both fibrosis and tumour development. Indeed ROS induce the trans-differentiation to myofibroblasts, the activated form of fibroblasts, involved in repair of epithelial injuries. In addition, myofibroblast are found in the reactive stroma of tumour microenvironment, promoting cancer progression through specific communication with malignant cells. Our interest is to assess the role of ROS produced in response to mitochondrial dysfunction in fibroblast activation and in tumour progression and metastatic dissemination. Indeed, mitochondrial produced ROS have been recently involved in metastatic dissemination of cancer cells, probably through a redox dependent stabilization of HIF-1α. To this end, we used as models human fibroblasts carrying mitochondrial dysfunctions of complex I. We demonstrated that ROS level produced by these fibroblasts correlate with their activation. The increase of ROS in these cells provides a greater ability to remodel the ECM leading to an increased motility and invasiveness. Furthermore, we evidentiated that in hypoxic condition these fibroblasts cause HIF-1α stabilization and promote a pro-invasive phenotype of A375 melanoma cells. All together, these data suggest a possible role of deregulated mitochondrial ROS production in fibrosis evolution as well as in cancer progression and invasion.



ATM kinase activity modulates FLIP protein stability and death receptor sensitivity through the ubiquitin-proteasome pathway
S. Santini1, V. Stagni1, R. Giambruno1, M. Mingardi1, D. Giaccari1, M. Pellegrini2, M.T. Cencioni3, L. Battistini3, Daniela Barila’1
1Dept of Biology, Univ. of Tor Vergata and Laboratory of Cell Signaling, Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS) Fondazione Santa Lucia, Rome, Italy
2Dep of Human Anatomy, Univ. of Tor Vergata Rome, Italy
3Laboratory of Immunology, Centro Europeo per la Ricerca sul Cervello (CERC)/ Fondazione Santa Lucia, Rome, Italy

Ataxia Telangiectasia Mutated kinase (ATM) is a Ser/Thr kinase that plays a central role in DNA damage response. As shown recently, ATM kinase is involved in the modulation of protein stability. In response to DNA damage ATM impinges on p53 stability through the phosphorylation of the E3 ubiquitin ligase complex Mdm2/Mdm4.
We have recently demonstrated that ATM kinase activity modulates FLIP protein stability enhancing death receptor sensitivity in both Fas- and TRAIL- (tumour necrosis factor-TNF- related apoptosis inducing ligand) induced apoptosis. We could show that ATM kinase activation promotes FLIP protein degradation and death receptor induced apoptosis. Conversely genetic or pharmacological ATM inhibition results in FLIP protein accumulation and triggers death receptor resistance in several in vitro models (Stagni et al. 2008, Blood; Mingardi et al. 2009, Submitted).
Here, we provide evidence that support the presence of this circuit also in mouse primary T-cells derived from wt and ATM deficient mice, supporting the in vivo relevance of this pathway.
Furthermore we will present data supporting the hypothesis that ATM modulates FLIP protein stability through the ubiquitin-proteasome pathway. Our preliminary data support that ATM may downregulate FLIP levels through the E3 Ubiquitin ligase ITCH. Future experiments will further clarify this issue.


26. Stem cells, iPS, cancer stem cells

Total number of abstracts in this session: 1



Adiponectin is a stem cell factor for muscle progenitor cells
T. Fiaschi1, E. Giannoni1, G. Cossu2, P. Chiarugi1,3
1Dept of Biochemical Science, Univ. of Florence, Italy
2Division of Regenerative Medicine, San Raffaele Scientific Institute, Milan, Italy
3Istituto Interuniversitario di Miologia (IIM)

Adiponectin is an adipocyte-derived hormone with anti-diabetic, anti-inflammatory and anti-atherogenic properties and exerting insulin-sensitizing metabolic effects. After the observation that globular adiponectin (gAd) induces muscle differentiation, we focused our study on muscle progenitors, namely mesoangioblasts and muscle satellite cells. We observed that gAd has a pleiotropic effect on mesoangioblasts affecting their activation, protecting them from apoptotic death induced both by growth factor withdrawal and extracellular matrix detachment. In addition, gAd increases the migration of mesoangioblasts towards myotubes and increases the fusion of mesoangioblasts with myoblasts, thus enhancing the myogenic properties of these stem cells. On the other hand, our preliminary results on muscle satellite cells show that gAd is produced in an autocrine manner and is able to activate their proliferation, to protect them from apoptosis and to increase their migration towards differentiated myotubes. All together, these results demonstrate that gA has a dual role in muscle physiology acting both as a metabolic hormone and as a stem cell factor. In vivo experiments are ongoing to confirm this hypothesis.


27. Virology

Total number of abstracts in this session: 1



Identification of independent domains important for the physical and functional interaction of the HIV-1 auxiliary protein vif with the HIV-1 reverse transcriptase
A. Kataropoulou1*, C. Bovolenta2, S. Trabatti1, A. Garbelli1, S. Porcellini2, R. Lupo2, G. Maga1
1Istituto di Genetica Molecolare – Consiglio Nazionale delle Ricerche
2MolMed S.p.A., Milano, Italy
*Università degli Studi di Pavia, Dottorato di Ricerca in Scienze Genetiche e Biomolecolari – XXIII Ciclo

The Viral Infectivity Factor (Vif), is a basic phosphoprotein (≈23 kDa) essential for efficient viral replication in natural target cells. It is an accessory protein that plays a dual role as it counteracts the antiviral activity of the natural restriction factors APOBEC3G and 3F and ensures efficient retrotranscription of the HIV-1 RNA genome. We previously demonstrated that Vif acts as an auxiliary factor for HIV-1 reverse transcriptase (RT) by increasing the rate of association of the enzyme to RNA or DNA templates. Here, by conducting mutational analysis on seven different mutants, we provide in vitro evidence that Vif stimulates HIV-1 RT through direct protein-protein interaction, mediated by its C-terminal domain. The proline-rich region comprised between amino acid (aa) 161 and 164 appears to be essential for the physical interaction, whereas the stimulatory activity requires, in addition, the extreme C-terminal region (aa 169-192) of the Vif protein. Neither the RNA interaction domain, nor the Zn++-binding domain of Vif are required for its interaction with the viral RT. Pseudotyped lentivirus vectors bearing Vif mutants deleted in the RNA- or RT-binding domains show defects in retrotranscription/integration processes in both permissive and nonpermissive cells. These results broaden our knowledge on how three important functions of Vif (RNA binding, RT binding and stimulation and Zn++-binding) are coordinated by different domains.



  • Abstract
    17 July 2009
  • Grant
    17 July 2009
  • Registration
    17 July 2009
  • Payment
    17 July 2009

Important dates

  • Abstract
    22 June
  • Accepted Posters available on website
    3 August