La ricerca dei Dottorandi e dei Post Doc al Vallisneri

Transcript

Bio
Ph. D. &
Post Doc
Day '09
La ricerca dei Dottorandi
e dei Post Doc al Vallisneri
April 3rd
Abstracts
ABSTRACT DOTTORANDI
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Rigers Bakiu
Nicola Barison
Filippo Bernante
Francesca Bortolin
Lucia Brunello
Elena Casanova
Cristina Cerqua
Mariana Cintra Francischinelli
Gaia Codolo
Chiara Compagnin
Alessandra De Nadai
Valentina Degasperi
Alice Domenichini
Ryan Dosselli
Nicola Facchinello
Stefano Fogal
Claudio Forcato
Lorenzo Gesiot
Alessandra Gianoncelli
Paul Gutla
Gaia Litteri
Semenzato Martina
Eva Masiero
Stefano Montelli
Leonardo Morsut
Elena Palma
Viviana Pignataro
Surachai Pikulkaew
Odra Pinato
Matteo Pizzolon
Chiara Rigobello
Alberto C Rossi
Roberta Sartori
Nicola Sassi
Giovanna Sattin
Sandra Maria Soligo
Alice Tadiello
Alessandra Tondello
Dania Vecchia
Andrea Venerando
Enrico Zampese
BIO PhD & PostDoc Day ’09, Complesso Biologico “A. Vallisneri”, Università di Padova
Scuola di Dottorato in Bioscienze
Indirizzo Biologia evoluzionistica
MOLECULAR EVOLUTION OF METALLOTHIONEINS IN ANTARCTIC TELEOST FISH
(NOTOTHENIOIDEI)
Rigers Bakiu*, Paola Irato, Ester Piccinni
*Dipartimento di Biologia, Università degli Studi di Padova, Viale G. Colombo 3, 35131 Padova,
Italy; Tel: +390498276315; Email: [email protected]
ABSTRACT
Metallothioneins (MTs) are low-molecular-weight and sulfur-rich molecules, widely distributed in
nature. They play a homeostatic role in the control and detoxification of heavy metals. Previous
research indicates that MTs have also the capacity to scavenge reactive oxygen species metabolites
(ROS). Whilst studying the evolution of metallothioneins in teleost, we have investigated MT
evolution in a group of teleost fish, namely the Notothenioidei.
Using molecular biology techniques to characterise the coding region and untranslated regions (5'
UTR and 3' UTR) of metallothionein isoforms, we have examined many species of notothenioids:
seven species of the Nototheniidae family (Trematomus hansoni, T. newnesi, T. eulepidotus, T.
pennelli, T. lepidorhinus, Pleuragramma antarcticum e Gobbionotothen gibberifrons), one species
from the Artedidraconidae family (Histiodraco velifer) and the last species from the
Bathydraconidae family (Cygnodraco mawsoni). The relationships among the different isoforms
were inferred by using Maximum Likelihood (ML) and Bayesian methods. Other teleost fish MT
sequences (present in GenBank) were considered in the phylogenetic analyses.
These results indicate that two paralogous MT genes (MT-1 and MT-2) are present in
notothenioids. We found several discrepancies between MT gene genealogy and species phylogeny
by the comparison of notothenioid MTs tree topology and the phylogeny of the considered species.
Using PAML, we accepted the hypothesy that these genes have been subject to selection pressure
different from each other and from the ancestor gene. Additional research projects, using different
methodologies, are in progress with the aim of achieving a more complete picture of MT evolution
within this group of organisms.
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Scuola di Dottorato in Scienze Molecolari
Indirizzo Scienze Chimiche
STRUCTURAL STUDIES OF FACTORS INVOLVED IN THE STOMACH COLONIZATION
BY H. PYLORI
Nicola Barison*1,2, Laura Cendron1,2, Lorenza Sisinni1,2, Sandra Quarantini1,2, Munan Shaik1,2,
Alessandro Angelini1,2, Thomas F. Meyer3, Markus Stein4, Giuseppe Zanotti1,2
*1. Dipartimento di Chimica Biologica, Università degli Studi di Padova
Viale G. Colombo 3, 35131 Padova, Italy; Tel: +390498276286; Email: [email protected]
2. Venetian Institute of Molecular Medicine (VIMM), Padova, Italia
Via G. Orus 2, 35129 Padova, Italia
3. Max Planck Institute for Infection Biology, Berlin, Germany
4. Department of Medical Microbiology and Immunology, University of Alberta, Alberta, Canada
ABSTRACT
Helicobacter pylori is a gram-negative bacterial pathogen specialized in the colonization of the
human stomach. It establishes a life-long chronic infection in more than half of the human
population: most infected people are asymptomatic, but, sometimes, H. pylori causes gastritis,
stomach and duodenal ulcers, adenocarcinomas and stomach lymphomas (1). Aim of this project is
to determine the structure of pathogenicity factors of H. pylori and, eventually, to find their
function. The most pathogenic strains of the bacterium code for a type four secretion system
(T4SS), an apparatus composed of about 30 genes that translocates the CagA toxin into gastric cells
(1). The structures of three proteins, CagZ (2), CagS (3) and CagD (4), belonging to the T4SS, have
been solved. Recently, new factors important for stomach colonization and pathogenicity have been
identified (5). Among them, the structures of HP1287, HP1286, HP0496 have been solved and
crystals of HP1028, HP1454, HP0797 and CagV have been obtained.
REFERENCES
1) Montecucco C et al (2001) Living dangerously: how Helicobacter pylori survives in the human stomach. Molecular
Cell Biology 2, 457-466.
2) Cendron L et al (2004) Crystal structure of CagZ, a protein from the Helicobacter pylori pathogenicity island that
encodes for a type IV secretion system. Journal of Molecular Biology 340, 881-889.
3) Cendron L et al (2007) The crystal structure of CagS from the Helicobacter pylori pathogenicity island. Proteins 69,
440-443.
4) Cendron L et al (2009) The Helicobacter pylori CagD (HP0545, Cag24) protein is essential for CagA translocation
and maximal induction of interleukin-8 secretion. Journal of Molecular Biology 386, 204-217.
5) Baldwin DN et al (2007) Identification of Helicobacter pylori genes that contribute to stomach colonisation.
Infection and Immunity 75, 1005-1016.
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Indirizzo Genetica e Biologia Molecolare Dello Sviluppo
KNOWLEDGEBASES OF TRANSCRIBED SEQUENCES FROM TWO MODEL
INVERTEBRATES: MYTILUS GALLOPROVINCIALIS AND EUPHAUSIA SUPERBA
Filippo Bernante*, Cristiano De Pittà, Laura Varotto, Gabriella Mazzotta, Rodolfo Costa, Paola
Venier, Alberto Pallavicini, Gerolamo Lanfranchi
*Dipartimento di Biologia, Università degli Studi di Padova
Viale G. Colombo 3, 35131 Padova, Italy; Tel: +390498276162; E-mail:[email protected]
ABSTRACT
Due to their economical and ecological role, many studies have been conducted on bivalves
and in particular on mussels. As food-filtering organisms, they substantially accumulate toxic
contaminants and can be used in coastal water biomonitoring. Nonetheless, very little information is
available on their genome.
We have constructed and sequenced seventeen cDNA libraries from different Mediterranean mussel
tissues: gills, digestive gland, foot, anterior and posterior adductor muscle, mantle and haemocytes.
From a total of 24,939 sequenced clones, 18,788 high-quality ESTs have been generated and
assembled into 2,446 overlapping clusters and 4,666 singletons, obtaining a total of 7,112 nonredundant sequences of mussel transcripts. Among the mussel libraries, the construction of one
high-quality normalized cDNA library (Nor01) determined a high rate of gene discovery (65.6%).
Bioinformatic screening of the non-redundant M. galloprovincialis sequences also identified 159
microsatellite-containing ESTs.
The entire bioinformatic process leading to Mytibase involved publicly available ready-to-use
packages as Trace2dbest (http://www.nematodes.org/bioinformatics/trace2dbEST) and Partigene
(http://www.nematodes.org/PartiGene), but many operations were conducted by custom
applications based on bioinformatic libraries as Bioperl and Bioruby. In addition we performed a
microsatellite prediction using Misa (http://pgrc.ipk-gatersleben.de/misa) and a classification based
on GO-slims annotation. A growing Mysql database containing all information’s about sequence,
putative translation, BlastX, GO, and Interpro annotation, is available through a php web interface
at http://mussel.cribi.unipd.it (1).
Little is known about the genome sequences of Euphausiacea (krill) although these
crustaceans are abundant components of the pelagic ecosystems in all oceans and used for
aquaculture and pharmaceutical industry.
We have constructed and sequenced five cDNA libraries from different Antarctic krill tissues: head,
abdomen, thoracopods and photophores. We have identified 1.770 high-quality ESTs which were
assembled into 216 overlapping clusters and 801 singletons resulting in a total of 1.017 nonredundant sequences. In addition, bioinformatic screening of the non-redundant E. superba
sequences identified 69 microsatellite containing ESTs. Clusters, consensuses and related similarity
and gene ontology searches were organized in a dedicated E. superba database at
http://krill.cribi.unipd.it. Our catalogue provides for the first time a genomic tool to investigate the
biology of the Antarctic krill (2).
REFERENCES
1) Venier P, De Pittà C, Bernante F, Varotto L, De Nardi B, Bovo G, Roch P, Novoa B, Figueras A, Pallavicini A,
Lanfranchi G. (2009) MytiBase: a knowledgebase of mussel (M. galloprovincialis) transcribed sequences. BMC
Genomics 10, 72.
2) De Pittà C, Bertolucci C, Mazzotta GM, Bernante F, Rizzo G, De Nardi B, Pallavicini A, Lanfranchi G, Costa R.
(2008) Systematic sequencing of mRNA from the Antarctic krill (Euphausia superba) and first tissue specific
transcriptional signature. BMC Genomics 9, 45.
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Indirizzo Biologia Evoluzionistica
POST-GERM-BAND NEUROGENESIS IN LITHOBIUS FORFICATUS (CHILOPODA)
Francesca Bortolin*, Marta Pierobon, Leandro Drago, Giuseppe Fusco
Via U. Bassi 58/B, 35131 Padova, Italy; Tel. +390498276238; E-mail: [email protected]
ABSTRACT
In the embryogenesis of the centiped Lithobius forficatus, during germ-band formation, early
neurogenesis is based on an invariant set of invagination groups of neural precursor in each hemisegment, which detach from the ventral neuroectoderm. This is similar to the corresponding
processes in other myriapods and chelicerates, but it is quite different from malcostracan and
hexapod neurogenesis, that involves instead stem cells, the neuroblasts.
However, in L. forficatus, as in many other arthropods, only the most anterior trunk segments are
produced during embyogenesis. As typical of lithobiomorphs, L. forficatus hatches with only seven
well-formed leg-bearing segments, developing over five stages a complete trunk with fifteen legbearing segments.
We carried out an histological study on L. forficatus late-embryonic and post-embryonic
segmentation.
Not all the neuromeres of a L. forficatus hatchling are formed during germ-band stage. The most
posterior ones are formed after blastokinesis, during the dorsal closure of the embryo. In both lateembryonic and post-embryonic neuromeres (collectively, post-germ-band neuromeres) the
ingression of neural precursor occurs in two phases, through mechanistically distinct processes. For
each hemi-segment, a first phase of precursor ingression gives rise to a solid mass of cells, while a
later phase of ingression gives rise to an epithelial vesicles. The latter mode of neural precursor
ingression represents the primitive condition for the so called ventral organs, described in the
neurogenesis of other myriapods and onycophorans as well. This two-phase precursor ingression
recalls the neurogenesis in the spider Cupiennius, where pro-neural cells invaginate in two times,
with different modality.
Whether in germ-band neuromeres, after invagination of the groups, there is a later ingression of
neurogenic material, mirroring post-germ-band secondary ingression, and whether the first
ingression of neural precursor in post-germ-band segments follows the same route as in the germband stage, this remains to be determined. This assessment could be relevant for elucidating
evolutionary pathways in arthropod neurogenesis.
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Indirizzo Neurobiologia
SERCA PUMPS ARE THE MAIN TARGET OF FAMILIAL ALZHEIMER’S DISEASELINKED PRESENILIN-2-T122R IN REDUCING ENDOPLASMIC RETICULUM Ca2+ LEVEL
Lucia Brunello*, Enrico Zampese, Cristina Florean, Tullio Pozzan, Paola Pizzo, Cristina Fasolato
Dipartimento di Scienze Biomediche Sperimentali, Università degli Studi di Padova
*Viale G. Colombo 3, 35131 Padova, Italy; Tel: +390498276067; Email: [email protected]
ABSTRACT
Alzheimer’s disease (AD) is a very common neurodegenerative pathology. Although the aethiology
of most AD cases is sporadic, less than 5% of AD patients inherit the disease in a dominant pattern.
The familial forms of AD (FAD) have been traced to mutations in genes for three proteins: the
amyloid precursor protein (APP), presenilin-1 and -2 (PS1 and 2). PSs are the catalytic core of the
γ-secretase complex which, in turn, by cleaving APP in concert with β-secretase, produces the
neurotoxic β-amyloid peptide (Aβ).
It has been reported that some FAD-linked PS mutants cause a dysregulation of cellular Ca2+
homeostasis, which may be an early event in the pathogenesis of FAD; however, the mechanisms
through which FAD-linked PS mutants affect cellular Ca2+ are still controversial (5-7). It has been
suggested that wild type (wt) PSs form a Ca2+ permeable channel in the endoplasmic reticulum
(ER) membrane, while mutated PSs loose this property, leading to an ER Ca2+ overload eventually
resulting in the AD neuronal degeneration (5). On the other hand, different studies have described
either no alteration or reduced ER Ca2+ stores in cells expressing FAD-mutant PSs (2, 7). In
particular, we demonstrated that the FAD-linked PS2 mutations M239I and T122R reduce rather
than enhance Ca2+ release and ER Ca2+ content in fibroblasts from FAD patients and in cell lines
stably or transiently expressing the PS2 mutants (2-4).
Starting from these results, we investigated the mechanism(s) by which mutated PSs affect
intracellular Ca2+ homeostasis and their possible target(s). ER Ca2+ steady state level is maintained
by the equilibrium between the uptake of the ion by the sarco-endoplasmic reticulum Ca2+-ATPase
(SERCA) and its release. Cells expressing mutated PSs showed an increased Ca2+ leak from the ER,
but we ruled out the contribution of the ribosome translocon complex, as a novel Ca2+ leak pathway
that could be affected by PSs. Afterward, we focused our attention on the interaction between PSs
and SERCA pumps, using multiple model cells and different targeted aequorins, as Ca2+ probes. We
found that PS2-T122R impairs SERCA activity, reducing its maximal uptake rate and thus leading
to a decreased store Ca2+ content (1).
REFERENCES
1) Brunello et al (2009) Presenilin-2 dampens intracellular Ca2+ stores by increasing Ca2+ leakage and reducing Ca2+
uptake. JCMM accepted manuscript.
2) Zatti et al (2006) Presenilin mutations linked to familial Alzheimer's disease reduce endoplasmic reticulum and Golgi
apparatus calcium levels. Cell Calcium 39, 539-550.
3) Giacomello et al (2005) Reduction of Ca2+ stores and capacitative Ca2+ entry is associated with the familial
Alzheimer's disease presenilin-2 T122R mutation and anticipates the onset of dementia. Neurobiology of Disease 18,
638-648.
4) Zatti et al (2004.) The presenilin 2 M239I mutation associated with familial Alzheimer's disease reduces Ca2+ release
from intracellular stores. Neurobiology of Disease 15, 269-278.
5) Tu et al (2006) Presenilins form ER Ca2+ leak channels, a function disrupted by familial Alzheimer's disease-linked
mutations. Cell 126, 981-993.
6) Green et al (2008) SERCA pump activity is physiologically regulated by presenilin and regulates amyloid beta
production. J Cell Biol 181, 1107-1116.
7) Cheung et al (2008) Mechanism of Ca2+ disruption in Alzheimer's disease by presenilin regulation of InsP3 receptor
channel gating. Neuron 58, 871-883.
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Scuola di Dottorato in Biochimica e Biotecnologie
Indirizzo Biochimica e Biofisica
ROLE OF THE BUD32 PROTEIN KINASE IN A NEW SIGNALING PATHWAY IN YEAST
Elena Casanova*, Caterina Peggion, Raffaele Lopreiato, Giovanna Carignani, Geppo Sartori
*Dipartimento di Chimica Biologica, Università degli Studi di Padova, Viale G. Colombo 3, 35131
Padova, Italy; Tel: +390498276141; Email:[email protected]
ABSTRACT
The yeast KEOPS/EKC complex is conserved throughout evolution and is involved in transcription
regulation and telomere maintenance (1). The complex is composed of five proteins: the
hypothetical endonuclease Kae1, the protein kinase Bud32, and the still uncharacterized Cgi121,
Pcc1 and Pcc2. Bud32 is an atypical protein kinase essential for normal cell growth, that interacts
with many other proteins, in addition to the components of the EKC⁄KEOPS complex. Among these
Bud32 interactors, the glutaredoxin Grx4, which is an in vitro substrate of the protein kinase, is
readily phosphorylated by recombinant, purified Bud32 at Ser134. Several data demonstrated that
Grx4 is able to interact with Bud32 in yeast cells, and that it is also an in vivo substrate of the
kinase. This relationship is influenced by the phosphorylation state of Bud32. In fact, the kinase
displays a highly conserved C-terminal sequence, that fulfils the consensus recognized by the
mammalian Akt/PKB protein kinase. It has already been demonstrated that, in mammalian cells,
Akt/PKB phosphoryates PRPK, the human homologue of Bud32. This prompted us to investigate
whether a similar kinase interaction could exist also in yeast cells, between Bud32 and the yeast
homologue of Akt/PKB, the Sch9 protein kinase. We observed that the activity of the Bud32
protein kinase on Grx4 was modulated by phosphorylation on its Ser258 residue, mediated by Sch9.
On the basis of the in vitro data, we originated a series of unphosphorylatable mutants (S134A,
S133A, SS133-134AA), as well as the phospho-mimic substitution S134D, in order to compare
their behaviour with that of the wild-type GRX4 sequence. We observed that while the substitution
S133A did not affect the in vivo functionality of Grx4, the mutation S134A slightly affected yeast
growth; this impairment was completely restored by the S134D substitution. We also observed that
overexpression of the wild-type, as well as of the S134D mutant, was toxic to yeast cells, while the
overexpression of the S134A or SS133-134AA Grx4 mutants had no effects. Then we purified the
SS133-134AA mutant version of recombinant, His-tagged, Grx4 and used it as a substrate for an in
vitro phosphorylation test with the endogenous, immunoprecipitated Bud32 kinase. We could
observe that Bud32 phosphorylated the wild-type, but not the mutant Grx4. These data confirm that
the phosphorylation of Grx4 by Bud32 specifically involves the Ser134 residue and that this event
is important, even if dispensable, for Grx4 functionality. Finally, we investigated whether the
phosphorylation of Bud32 at Ser258 by the Sch9 kinase is able to modulate the activity of the whole
KEOPS/EKC complex. We first analyzed telomeres length in different strains. Catalitically inactive
or null BUD32 mutations (bud32-K52A; bud32-D161A; bud32Δ) display telomeres shortening in
comparison to the wild-type, whereas telomere length of the bud32-S258A mutant is unaffected.
Then we compared the transcriptional activation of the GAL1 gene in wild-type and BUD32 mutant
strains. We observed the reduction of mRNA levels in kinase-dead or null mutants, whereas there
was no difference between the wild-type and the S258A mutant strain. These results indicate that the
phosphorylation of Bud32 at Ser258A is unrelated to its function within the EKC/KEOPS complex
(2).
REFERENCES
1) Downey M, et al (2006) A genome-wide screen identifies the evolutionarily conserved KEOPS complex as a
telomere regulator. Cell 124, 1155–1168.
2) Peggion C, Lopreiato R, Casanova E, Ruzzene M, Facchin S, Pinna LA, Carignani G, Sartori G. (2008)
Phosphorylation of the Saccharomyces cerevisiae Grx4p glutaredoxin by the Bud32p kinase unveils a novel signaling
pathway involving Sch9p, a yeast member of the Akt / PKB subfamily. FEBS J 275, 5919-5933.
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Indirizzo Biologia cellulare
TRICHOPLEIN/MITOSTATIN, A NOVEL PROTEIN AT THE CROSSROAD BETWEEN
MITOCHONDRIA AND INTERMEDIATE FILAMENTS
Cristina Cerqua*1, Vasiliki Anesti1, Kai S. Dimmer1, Raffaele Baffa2, Luca Scorrano1,3
*1. Dulbecco-Telethon Institute, Venetian Institute of Molecular Medicine, Via Orus 2, I-35129
Padova, Italy
2. Kimmel Cancer Center and Department of Pathology, Thomas Jefferson University,
Philadelphia, PA 19107, USA
3. Department of Cell Physiology and Metabolism, University of Geneva Medical School, Geneva,
Switzerland
ABSTRACT
Trichoplein/mitostatin is a novel protein that interacts in vitro with the intermediate filament keratin
and contains a trichohyalin/plectin homology domain (TPHD). It has been recently described as a
tumour suppressor gene frequently deleted in bladder and prostate cancers. Fractionation
experiments indicated that a large fraction of trichoplein is retrieved on mitochondria. We therefore
explored the possibility that trichoplein participates in mitochondrial dynamics and morphology.
Fusion to GFP of different fragments of trichoplein showed that the first 111 aa are sufficient for a
punctuate distribution that partially overlaps with mitochondria. Subcellular fractionation
experiments indicated that trichoplein is exclusively localized in mitochondria-associated
membranes (MAM) and that keratin 8 is almost completely accumulated in this fraction as well.
Like other proteins that reside in MAMs, even trichoplein seems to regulate ER-mitochondria
interactions, since high levels of trichoplein dissociate the two organelles. Levels of trichoplein
influence mitochondrial morphology, as its overexpression causes fragmentation of the
mitochondrial network, which is independent of Drp-1, a protein that regulates mitochondrial
fission. Given the binding of trichoplein to keratin we explored the possibility that it could be a role
in mitochondrial movement, since It has been proposed that intermediate filaments could contribute
to anchor mitochondria at specific cellular sites. Real time confocal microscopy experiments
demonstrated that mitochondrial motility was inversely related to levels of trichoplein. Since
mitochondrial fragmentation is commonly associated with apoptosis, we are investigating a possible
role of trichoplein in the death cascade. Preliminary results show that levels of trichoplein correlate
with spontaneous apoptosis. Our results indicate that the crosstalk between intermediate filaments
and mitochondria can be crucial to determine morphology of the organelle and its participation in
the apoptotic cascade.
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Indirizzo Biologia Cellulare
NAPA OF BORRELIA BURGDORFERI DRIVES TH17 INFLAMMATION IN LYME
ARTHRITIS
Gaia Codolo*1,2, Amedeo Amedei3, Allen C. Steere4, Elena Papinutto2,5, Alessandra Polenghi2,
Cosima Tatiana Baldari6, Giuseppe Zanotti2,5, Cesare Montecucco1, Mario Milco D’Elios3,7, Marina
de Bernard2,8
*1. Department of Biomedical Sciences, University of Padua, Viale G. Colombo 3, 35131 Padova,
2. Venetian Institute of Molecular Medicine, Via Orus 2, 35131 Padova
3. Department of Internal Medicine, University of Florence, Viale Morgagni 45, Florence, Italy
4. Center for Immunology and Inflammatory Diseases, Division of Rheumatology, Allergy and
Immunology, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts
5. Department of Chemistry, University of Padua, and ICB-CNR, Section of Padua, Padua, Italy
6. Department of Evolutionary Biology, University of Siena, Siena, Italy
7. Department of Biomedicine, Azienda Ospedaliera Universitaria Careggi, Florence, Italy
8. Department of Biology, University of Padua, Viale G. Colombo 3, 35131 Padua, Italy
ABSTRACT
Background. Human Lyme arthritis caused by Borrelia burgdorferi is characterized by an
inflammatory infiltrate, consisting mainly of neutrophils and T cells (1,2).
Objective. This study was undertaken to evaluate the role of the innate and acquired immune
responses elicited by the Neutrophil Activating Protein A (NapA) of Borrelia burgdorferi in Lyme
arthritis.
Methods. Serum anti-NapA antibodies were measured in 30 patients with Lyme arthritis, and in 30
healthy control subjects. The cytokine profile of synovial fluid T cells specific for NapA was
investigated in 5 patients with Lyme arthritis. It was also evaluated the cytokine profile induced by
NapA in neutrophils and monocytes.
Results. Serum anti-NapA antibodies were found in 54% of patients with Lyme arthritis, whereas
they were undetectable in healthy controls. T cells from synovial fluid of patients with Lyme
arthritis produced interleukin (IL)-17 in response to NapA. Moreover, NapA was able to induce the
expression of IL-23 in neutrophils and monocytes, and IL-6, IL-1β and transforming growth
factor(TGF)-β in monocytes, via Toll-Like Receptor-2.
Conclusion. These findings indicate that NapA of B. burgdorferi is able to drive the expression of
IL-6, IL-1β, IL-23 and TGF-β by cells of the innate immunity and to elicit a synovial T helper 17
response that might play a crucial role in the pathogenesis of Lyme arthritis (3).
REFERENCES
1) Steere AC, Schoen RT, Taylor E (1987) The clinical evolution of Lyme arthritis. Ann Intern Med 107, 725-731.
2) Georgilis K, Noring R, Steere AC, Klempner MS (1991) Neutrophil chemotactic factors in synovial fluids of patients
with Lyme disease. Arthr Rheum 34, 770-775.
3) Codolo G, Amedei A, Steere AC, Papinutto E, Cappon A, Polenghi A, Benagiano M, Rossi Paccani S, Del Prete G,
Baldari CT, Zanotti G, Montecucco C, D’Elios MM, de Bernard M (2008) NapA of Borrelia burgdorferi drives Th17
inflammation in Lyme Arthritis. Arthr and Rheum 58, 3609-3617.
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Scuola di Dottorato in Territorio, Ambiente, Risorse e Salute
Indirizzo Medicina Ambientale: Nutrizione e Inquinamento
NANOPARTICLES LOADED WITH META-TETRA(HYDROXYPHENYL)CHLORIN FOR
IMPROVING PHOTODYNAMIC THERAPY
Chiara Compagnin*, Francesca Moret, Maddalena Mognato, Fabrizio Mancin, Elena Reddi, Lucia
Celotti
Via U. Bassi 58/B, 35131 Padova, Italy; Tel: +390498276335; Email:[email protected]
ABSTRACT
In oncology, nanostructures are receiving considerable attention as injectable nanovectors for the
selective delivery of drugs to cancer cells and as tumour imaging agents.
In photodynamic therapy (PDT) various types of nanosystems are investigated as carriers for
photosensitizers, especially for those with a high degree of hydrophobicity that cannot be
administered in aqueous formulations. PDT is a modality of cancer treatment based on the use of a
photosensitizing agent that, after activation with appropriate wavelengths of light, leads to cell
death through the production of reactive oxygen species (ROS).
We investigated the use of organically modified silica nanoparticles (ORMOSIL NPs) for the
delivery of meta-tetra(hydroxyphenyl)chlorin (mTHPC), an hydrophobic second generation
photosensitizer approved for the palliative treatment of advanced head and neck cancers. These NPs
are easy to prepare, biocompatible, photochemically inert and their surface can be functionalized
with ligands specifically recognized by receptors overexpressed in tumour cells.
The dark cytotoxicity, photodynamic efficacy and intracellular localization of mTPHC entrapped in
NPs were compared to those of mTHPC delivered by the standard solvent ethanol/PEG 400/water
(20:30:50 by vol.). Our results showed that cell viability measured 24 hours after PDT experiments
(irradiation with 0.12 J/cm2 of red light) was the same independently of the modality of drug
delivery. Furthermore, mTHPC in standard solvent or loaded in NPs was rapidly internalized by
cells and accumulated in the Golgi apparatus and the endoplasmic reticulum. Because of the very
similar results obtained by delivering mTHPC by the two modalities, we investigated the possible
release of the photosentizer from NPs by i) fluorescence energy-transfer experiments performed
with NPs loaded with non covalently entrapped mTHPC (donor) and a cyanine derivative
covalently bound (acceptor) to the NP matrix and ii) ultracentrifugation of the NPs suspended in
water or in cell culture medium containing serum proteins. A rapid release of mTHPC from NPs to
serum proteins could be observed.
The release of the drug from NPs could be strongly diminished with the synthesis of “stealthy
particles” coated with a hydrophilic PEG layer (Figure 1). PEGylated NPs are very attractive
because of their prolonged circulation in the blood stream and the property to escape the
macrophages of reticulo-endothelial system. However PEGylation does not prevent completely the
release of mTHPC from NPs, therefore the photosensitizer will be linked covalently to the core of
NPs. The dark cytotoxicity, intracellular uptake and localization of PEGylated silica NPs loaded
with mTHPC will be determined in vitro, in A549 (derived from human lung carcinoma) and CCD34Lu (normal human lung fibroblasts) cells (Figure 2).
Figure 1: TEM image of PEGylated
NPs loaded with mTHPC.
Figure 2: localisation of mTHPC
loaded in PEGylated NPs.
Acknowledgements: This project received research funding from EU’s Seventh Framework Programme, Grant 201031
NANOPHOTO.
11
Scuola di Dottorato in Biologia e Medicina della Rigenerazione
Indirizzo Endocrinologia Comparata
ANALYSIS OF THE CONSERVATION OF INTRONIC SEQUENCES IN GENES CODIFYING
FOR ESTROGEN RECEPTOR ALPHA IN MAMMALS AND TELEOSTS.
Alessandra De Nadai*, Paola Belvedere, Lorenzo Colombo, Luisa Dalla Valle
ABSTRACT
My thesis focuses on the analysis of estrogen receptor alpha (ERα) intronic sequences in mammals and
teleosts. Intronic sequences of 9 mammals (Homo sapiens, Macaca mulatta, Canis familiaris, Mus
musculus, Bos taurus, Monodelphis domestica, Oryctolagus cuniculus, Echinops telfairi and Ochotona
princeps) and 3 species of teleosts (Danio rerio, Takifugu rubripes and Oryzia latipes) were taken from
the Ensembl database, whereas the sequences of the intron placed at the 5’ terminus of the receptor
DNA binding region from 12 different teleosts (Atherina boyeri, Barbus plebejus, Chondrostoma genei,
Rutilus aula, Rutilus rutilus, Phoxinus phoxinus, Rhodeus amarus, Gasterosteus aculeatus, Psetta
maxima, Scorpaena porcus, Polyprion americanus and Sparus aurata) were obtained in our laboratory.
Both mammalian and fish sequences were subjected to bioinformatic scrutiny. Since the examined
intron sequences are extremely long and have hardly comparable lengths, we decided to use an
alignment software called MLAGAN (1). A great number of fragments that share more than 70%
identity were found. Even comparing not closely related species (for example, M. domestica, infraclass:
Metatheria vs. C. familiaris, infraclass: Eutheria) fragments of great similarity were discovered.
Further analyses were performed on conserved sites involved in intron excision: one sequence at the
branching site necessary for the formation of the lariat and the 5’- e 3’- termini. The latter were always
conserved, whereas the branching site exists only in the majority of the considered introns, and in some
cases, multiple copies are present.
The occurrence of transcription factor binding sites was investigated with the software TFSEARCH
(2). A great deal of putative binding sites was found, with different trends among different introns.
Cdxa and SRY binding sites were overexpressed, the latter being probably related to ERα function.
The application of Mreps (3), a bioinformatic software that is able to find repetitive DNA sequences,
showed that tandem repeats are widely expressed in non-conserved regions of all the considered
introns, while, for mammals only, they are also represented in conserved regions.
Finally, the presence of potential microRNA precursors was checked with the ProMiRII program (4)
and other similar softwares. This analysis revealed some putative precursors, although they are not
conserved among the considered species. These and other bioinformatic results provide important cues
about putative regulatory regions in intronic sequences. Currently, gel shift experiments are being
performed in organs expressing ERα (brain, liver and ovary) of zebrafish in order to find possible
interactions between nuclear proteins and introns.
REFERENCES
1) Brudno M et al (2003) LAGAN and Multi-LAGAN: efficient tools for large-scale multiple alignment of genomic DNA.
Genome Res 13, 721-731.
2) Wingender E et al (2000) TRANSFAC: an integrated system for gene expression regulation. Nucleic Acids Res 28, 316319.
3) Kolpakov R et al (2003). Mreps: efficient and flexible detection of tandem repeats in DNA. Nucleic Acids Res 31, 36723678.
4) Nam JW et al (2006) ProMiR II: a web server for the probabilistic prediction of clustered, nonclustered, conserved and
nonconserved microRNAs. Nucleic Acids Res 34, W455–W458.
12
GENE EXPRESSION DURING BODY MUSCLE DIFFERENTIATION IN ASCIDIANS
(TUNICATA) AND THE EVOLUTION OF MUSCLE IN CHORDATES
Valentina Degasperi*1, Sebastian M. Shimeld2, Fabio Gasparini1, Lucia Manni1, Paolo Burighel1
*1. Dipartimento di Biologia, Università degli Studi di Padova, Viale G. Colombo 3, 35131 Padova,
2. Department of Zoology, University of Oxford, UK
ABSTRACT
Ascidians present three types of muscles in their life: striated in the larval tail and heart, and smooth
in the post-metamorphic sessile phase. The larval and cardiac muscles have an arrangement of
myofilaments like the striated muscle of vertebrates. Instead, the smooth body-wall musculature has
intermediate characters between smooth and striated muscle of vertebrates. A great number of
ascidians form colonies constituted of clonal individuals (blastozooids) originated by asexual
reproduction. We studied the musculature in the colonial ascidian Botryllus schlosseri (Fig. 1)
analysing its organization, differentiation and gene expression during development of blastozooids,
beginning from the early bud stage to adult and regression stage. We isolated and characterised two
transcripts from colonies that resulted homologous to muscle genes of solitary ascidians: a muscletype actin (BsMA2), a cytoplasmic-type actin (BsCA1) and a troponin T (BsTnT-c). Moreover, we
obtained also the genomic sequences coding for BsMA2 and BsCA1.
Phylogenetic analyses showed a close relationship between urochordates and vertebrates muscle
genes. The BsMA2 and BsCA1 genomic sequences were compared in the exon-intron organization
with other muscle and cytoplasmic–type actin genes of both invertebrates and vertebrates. Our data
revealed that intron positions are conserved in ascidians and in the other deuterostomes.
We detected the expression of the two genes by in situ hybridization on section (ISH; Fig. 2), in
order to follow the muscle development throughout the blastogenetic cycle of B. schlosseri. The
ISH, in parallel with phalloidin staining experiments, showed that the first diffuse signal of BsMA2
and BsTnT-c labels differentiating muscles which appear in the intersiphonal area of young buds.
Then, the muscle fibres differentiate into the body-wall, while an intense expression only of BsMA2
marks the myocardium just when it begins contractions. The organization and differentiation of
striated cardiac and smooth muscle cells were also investigated at ultrastructural level (Fig. 2)
during all the phases of the blastogenetic cycle from early bud to zooid regression.
a
oral
siphon
b
cerebral
ganglion
tunic
À
À
muscle
fibres
velum
À
cloacal
siphon
Fig. 1. B. schlosseri colony; system
composed of 6 adults (asterisks) with
primary (arrowheads) and secondary buds
(arrows).
pericoronal band
BsMA2 (ISH)
Fig. 2. (a). Expression of BsMA2 mRNAs; marked oral siphon,
velum and pericoronal bands. (b). Neuromuscular junction.
Small vesicles (arrows); dense material in the junctional space
(arrowheads). In transverse section: smooth fibre (mf), with no
regular disposition of filaments.
13
Indirizzo Genetica e Biologia Molecolare dello Sviluppo
GENETIC AND MOLECULAR ANALYSIS OF A PUTATIVE MATERNAL EFFECT ALLELE
INVOLVED IN THE DEVELOPMENT OF NEUROANATOMICAL AND VISCERAL
ASYMMETRIES IN ZEBRAFISH (DANIO RERIO).
Alice Domenichini*, Francesco Argenton
Viale G. Colombo 3, 35131 Padova, Italy, Tel: +390498276349; Email: [email protected]
ABSTRACT
The vertebrate body plan displays distinct left-right asymmetries in the disposition of visceral
organs. This asymmetrical organization extend the vertebrate brain that is both anatomically and
functionally asymmetric. The development of left-right patterning and cerebral lateralization are
thought to be regulated by evolutionary conserved genes.
It has been demonstrated that in vertebrates, the initiation of left-right patterning depend on the
asymmetrical, left-sided, activation of a Nodal mediated gene expression cascade (1).
Moreover studies have found that Nodal-related signals also regulate asymmetric gene expression in
the forebrain of zebrafish and are involved in early establishment of left-right asymmetries in the
epithalamic region of the dorsal diencephalon (2).
In a recent study Facchin et al. (3) showed that lines of zebrafish artificially selected for the right
eye preference in scrutiny their own image had a significant increase in the frequency of reversed
left-right asymmetry in the epithalamus. In the present study it is proposed that Facchin’s selection
for behavioral lateralization promote the isolation of a putative mutant allele responsible for
disruption of normal left-right patterning in zebrafish neuroanatomical structures. Our findings
suggest that this mutation involves a maternal effect gene. We performed selective crosses on
females identified as mutant carriers to test the hypothesis of a recessive maternal effect mutation.
The mutation is expressed in the next generation with a frequency that seems to be influenced by
temporal factors, suggesting that this allele is incomplete penetrant and/or has variable expressivity.
The analysis revealed that in most cases these homozygous mutant mothers generate a percentage of
progeny with reversed diencephalic asymmetries that progressively increase in the subsequent
crosses, suggesting that the penetrance of this putative allele could be affected by the age of the
mother.
Moreover we investigated the signaling pathway responsible for the establishment of visceral and
diencephalic left-right asymmetries. The aim was to determine the involvement of this mutation in
the Nodal-related asymmetric, normally left-sided, gene expression. In situ hybridizations analysis
of mutant females’ progeny showed that at least three genes involved in Nodal signaling pathway
revealed an altered gene expression pattern (either bilateral or right-sided) in a large percentage.
Further experiment showed that a large majority of fish with reversed parapineal organ have also
reversed pancreas indicating that this putative mutation more likely acts on the specification of both
visceral and neuroanatomical left-right asymmetries.
REFERENCES
1) Burdine RD and Schier AF (2000) Conserved and divergent mechanism in left-right axis formation. Genes Dev 16,
2339-2344.
2) Bisgrove BW, Essner JJ and Yost HJ (2000) Multiple pathways in the midline regulate concordant brain, heart and
gut left-right asymmetries. Development 127, 3567-3579.
3) Facchin L, Argenton F and Bisazza A (2009) Lines of Daniorerioselected for opposite behavioural lateralization
show differences in anatomical left–right asymmetries. Behav Brain Res 197, 157-165.
14
IDENTIFYING PRIMARY MOLECULAR TARGETS OF PHOTODYNAMIC THERAPHY
AGAINST BACTERIA: A PROTEOMIC APPROACH
Ryan Dosselli*1, Renato Millioni2, Lucia Puricelli2, Paolo Tessari2, Giorgio Arrigoni3, Anna
Segalla1 & Elena Reddi1.
*1. Dept. of Biology, University of Padova, Viale G. Colombo 3, 35131 Padova, Italy; Tel:
+390498276335; Email: [email protected]
2. Dept. of Clinical and Experimental Medicine, Chair of Metabolism, 35128 Padova, Italy
3. Dept. of Biological Chemistry and VIMM, Venetian Institute for Molecular Medicine, Padova
ABSTRACT
The worldwide rise in bacterial resistance to antibiotics requires the development of new
antibacterial strategies; one of the most promising ones is photodynamic therapy (PDT).
PDT utilizes a visible light absorbing molecule, called photosensitizer (PS), that in the presence of
oxygen generates cytotoxic reactive species, that kill bacterial cells previously loaded with the PS
without harming human cells.
In our study we use a new photosensitizer, namely XF-73, which has been shown to be extremely
efficient in killing a large range of Gram(+) bacteria; moreover, with experimental conditions
yielding a 5-log10 reduction of bacterial survival, only little harmful effects were observed on
eukaryotic cells.
Our research aims to identify some of the primary molecular targets of PDT with XF-73 focusing
on cytoplasmic membrane proteins of an MRSA strain of Staphylococcus aureus using a proteomic
approach.
Preliminary analyses were performed with different PDT doses, thus selecting a sublethal treatment
giving 60 to 80% bacterial survival (low PDT treatment). Protein extracts from S. aureus cells
untreated and PDT treated were resolved by 2-DE (two-dimensional gel electrophoresis) on 4-7 pH
range. Nine 2-DE protein maps per experimental group were produced and stained with Coomassie.
Stained gels were submitted to image analyses that allowed us to match 225 spots, 54 of which
significantly changed their expression level between the two experimental groups: 27 spots were
more expressed in treated than in control gel maps, and 27 were less. In order to get a more
comprehensive analysis, a new set of experiments was performed using samples from bacteria
subjected to a PDT treatment giving 3 to 5% survival (high PDT treatment). Also this new set of
gels were submitted to image analyses, and this analyses confirmed the changing in protein spots
intensity and revealed 12 additional proteins changing significantly their expression levels; these
spots are at present under MS/MS analyses.
All the 54 protein spots have been analyzed with tandem mass spectrometry (MALDI TOF/TOF),
and 52 over 54 of them were identified as unique proteins.
The identified proteins were then searched in protein databases to understand precisely their
function in the bacterial cell, in order to organize them in functional classes (i.e. energy metabolism,
detoxification an so on) to gain a more comprehensive overview of the impact of PDT on S. aureus.
Other software analyses were performed, using program tools available online at the ExPASy
proteomic server, to further characterize all these proteins by their physical and chemical properties,
such as theoretical pI and MW, presence of transmembrane regions and presence of signal peptides.
At present we are completing the MS/MS analyses, and performing further research on online
databases to fully understand our results.
In the future we will complete this study repeating our experimental design on the 6 to 11 pH range,
to reach a more complete overview of the effects of PDT with XF-73 on S. aureus.
15
Indirizzo Biotecnologie
CONDITIONAL INACTIVATION OF COL6A1 AND EMILIN1 GENES
Nicola Facchinello*, Kosjenka Frka, Claudia Del Vecchio, Andrea Carpi, Rina Venerando, Alessia
Angelin, Paolo Bonaldo, Paolo Bernardi, Dino Volpin, Paola Braghetta, Giorgio M. Bressan
*Dipartimento di Istologia, Microbiologia e Biotecnologie Mediche, Università degli Studi di
Padova, Viale G. Colombo 3, 35131 Padova, Italy; Tel: +390498276085;
Email: [email protected]
ABSTRACT
Conditional gene expression methods are important approaches for examining the function of
particular genes in development and disease. In particular, I used a system for lentiviral-mediated
RNAi in vivo silencing of Col6a1 and for conditional-inducible knockout (KO) of Emilin1 gene.
The use of lentiviral vector can accelerate the generation of animals with substantial suppression of
gene expression in an inducible way. Some authors have observed limitations of the technique due
to low efficiency of transgenesis and mosaicism in transgenic mice. Here we analyze how different
factors influence RNAi mediated silencing of Col6a1, a gene of the extracellular matrix with a
complex pattern of tissue-specific expression and (1). Our results, obtained with vectors pLVTHM
and pLVPT-rtTRKRAB, point out three parameters as major determinants of the efficiency of
interference: the choice of interfering sequence, the number of proviral copies integrated into the
mouse genome and the site of integration of the provirus. A lentiviral vector (pLVPT-rtTRKRAB)
with doxycycline inducible production of shRNA was also tested. Control of expression by the drug
was stringent in many tissues; however, in some tissues turning off of shRNA synthesis was not
complete. The data support the application of lentiviral vectors used here in transgenesis.
To gain insight into the function of EMILINs, mice disrupted at the Emilin1 gene locus have been
generated. These animals display increased blood pressure, and reduced arterial vessel size.
Emilin1 is expressed at early stages both in extraembryonic and embryonic tissues, suggesting a
role of the gene in vascular development (2). As it may be possible that the function of Emilin1 is
limited to the early stages of development and that the vascular phenotype of Emilin1 KO is a
consequence of the altered embryogenesis of blood vessels rather than the direct effect of the
protein on blood pressure regulation it is important to verify the effect of the lack of Emilin in adult
animals after a normal development in the presence of the protein. Through site-specific
recombination using Cre-recombinase-estrogen-Receptor-T2 (Cre-ERT2) and loxP sites, Emilin1
can be inactivated in a tissue-specific and time-controlled manner. A targeting vector was prepared
in which both exon1-2 and the Neo-cassette were flanked with loxP Cre target sites ("floxed
allele"). The neo cassette was removed by transient expression of the Cre recombinase in ES cells.
Ten homologous recombinants ES cell clones were characterized by Southern Blot, PCR and tested
for a stable and normal diploid karyotype. Four of these clones have been injected into blastocysts
to generate chimeric mice. Mice carrying the Emilin1 “floxed” allele will be bred with animals
expressing the CreERT2 recombinase under the control of the Emilin1 promoter and Emilin1 gene
inactivation induced by tamoxifen administration. Overall, the analysis of these animals models
should allow the precise definition of the function of genes and a better understanding of the
molecular mechanisms involved.
REFERENCES
1) Bonaldo P et al (1998) Collagen VI deficiency induces early onset myopathy in the mouse: an animal model for
Bethlem myopathy. Hum Mol Genet, 7, 2135-2140.
2) Braghetta P et al (2002) Expression of the EMILIN-1 gene during mouse development. Matrix Biol. 21, 603-612.
16
ENANTIOSELECTIVE HYDROLYSIS FOR A MOXIFLOXACIN BINDING BLOCK
PREPARATION
Stefano Fogala*, Riccardo Motterleb, Giancarlo Arvottib, Marco Galvagnib, Andrea Castellinb,
Elisabetta Bergantinoa
a
Department of Biology, University of Padova, Viale G. Colombo 3, 35121 Padova, Italy. Tel:
390498276342; Email: [email protected]
b
Fabbrica Italiana Sintetici, Viale Milano 26, 36075 Alte di Montecchio Maggiore – Vicenza, Italy
ABSTRACT
The compound C in synthesis of Moxifloxacin represents an expensive binding block because of its
enantiomeric configuration. Enzymes became recently promising and available tools for chemical
synthesis. In particular lipases and esterases are often used to resolve racemic mixture of esters (13).
We propose an enzymatic step in order to prepare B, an enatiopure precursor of C. The process
consists in preparation of racemic mixture of water-soluble A, enantioselective hydrolysis catalyzed
by Candida antarctica lipase B and separation of product from unreact substrate. This last was used
as precursor of C. The process, patent pending, is going to be develop.
O
OH
O
O
N
OH
O
N
O
OH
O
CalB
O
O
OH
O
O
O
N
O
OH
O
N
O
O
A
O
H
N
O
NH
O
B
C
O
O
O
N
N
NH
F
O
Moxifloxacin
REFERENCES
1) Liljeblad A, Lindborg J, Kanerva A, Katajisto J, Kanerva LT (2002) Enantioselective lipase-catalysed reactions of
methyl pipecolinate: transesterification and N-acylation. Tetr. Lett. 43, 2471-2474.
2) Ng-Youn-Chen MC, Serreqi AN, Huang Q, Kazlauskas RJ (1994) Kinetic resolution of pipecolic acid using
partially-purified lipase from Aspergillus niger. J. Org. Chem. 59, 2075-2081.
3) Toone EJ, Bryan Jones J (1987) Enzymes in organic synthesis. Evaluatin of the enantioselectivity of the pig liver
esterase catalized hydrolyses of racemic piperidine carboxylic acid esters. Can. J. Chem. 65, 2722-2726.
17
Scuola di Dottorato in Scienze e Produzione Vegetali
Indirizzo Agrobiotecnologie
GENE PREDICTION AND ANNOTATION PLATFORM: A CASE-STUDY ON THE VITIS
VINIFERA GENOME
Claudio Forcato*, Nicola Vitulo, Albiero A., Davide Campagna, Caniato E., Bilardi A., Giorgio
Valle
*CRIBI, Università degli Studi di Padova
Via U. Bassi 58/B, 35131 Padova, Italy; Tel: +390498276165; Email: [email protected]
ABSTRACT
A genome annotation project is composed by three different phases: the first, gene prediction, the
second, functional annotation, and the final, annotation validation and refinement. Gene prediction
goal is the identification of the intron-exon structure and the localization of genes in the genome.
Functional annotation is the process that assign a biological function, a metabolic role or structural
properties to predicted genes. The last step is the validation of gene structures and functions through
further reliable data, as transcriptome or proteome evidences.
At CRIBI we implemented a bioinformatic platform for gene prediction and functional annotation,
developing an automated system to obtain annotation starting from the raw nucleotide sequence.
The platform has been developed as a part of the V. vinifera sequencing project, a french-italian
collaborative effort (1). For gene prediction step, we adopted three different approaches: a) ab-initio
predictors, based on HMM technology, b) comparative genomics, based on the alignment of three
different plant genomes against V. vinifera genome, c) EST/protein alignment, to obtain direct
evidences of the transcribed or coding portions of the genome. We integrate all this information
using JIGSAW software (2) creating a sort of consensus structure, in order to produce the final
prediction on the V. vinifera 12X assembly. The genome seems to possess about 30000 genes.
The functional annotation is mainly based on similarity approach. In the world wide web a great
number of databases and resources are available to infer gene properties and functions: databases of
protein sequences, functional and structural domains and profiles, sub-cellular localization patterns
(UniProt, PFAM, Prosite), etc. All this information is organized in a dedicated database.
To extract and collect data from the database, we developed a XML-based query interface. The user
can interrogate the database according to selected criteria through a query page, built from welldefined XML files. For this reason, we design an XML language to describe all the information
necessary to define the query environment. This language allows to extend, reduce or modify the
platform in few simple operations, without any code modification. So, the system is extremely
modular and easy to set-up. Another important feature of this query platform is the presence of a
ranking system. In this way, query results are shown in decreasing order on the base of satisfied
criteria, allowing a direct estimation of result significance.
In the last step, to evaluate and validate predictions, we used transcriptome data from leaf and root
of V. vinifera, sequenced with SOLiD technology. We map 150millions 35bp-long reads on the
genome, analyzing the read distribution on four different regions of the genome: UTR, CDS,
INTRON and INTERGENE. As expected, most of the predicted exons (CDS+UTR) are covered
(~60%), confirming the good quality of gene predictions. Interestingly, there are some percentage of
coverage in intronic and intergenic regions. A possible explanation for intronic coverage is the
presence of unspliced mRNAs in the starting libraries or splicing variants. Moreover, we analyzed
intergenic regions covered by SOLiD reads. Most of the covered nucleotides are grouped in
clusters. Some of these are similar to transposable elements (presence of integrase or
retrotranscriptase domains).The remainder could represent not-predicted genes or non-coding RNA.
REFERENCES
1) Jaillon O et al (2007) The grapevine genome sequence suggests ancestral hexaploidization in major angiosperm
phyla. Nature 449, 463.
2) Allen JE et al (2005) JIGSAW: integration of multiple sources of evidence for gene prediction. Bioinformatics 21, 18.
18
Indirizzo Biologia cellulare
CALCIUM IMAGING OF MUSCLE CELLS TREATED WITH SNAKE MYOTOXINS
Mariana Cintra-Francischinelli1*, Paola Pizzo1, Léa Rodrigues-Simioni2, Luis A. Ponce-Soto3,
Ornella Rossetto1, Bruno Lomonte4, José M. Gutiérrez4, Tullio Pozzan1, and Cesare Montecucco1.
1. Dipartimento di Scienze Biomediche, Università di Padova, Padova, Italy
*Viale G. Colombo 3, 35131 Padova, Italy; Tel: +390498276056;
2. Departamento de Farmacologia and 3. Departamento de Bioquímica, Universidade Estadual de
Campinas, Campinas (SP), Brazil
4. Instituto Clodomiro Picado, Facultad de Microbiologí́a, Universidad de Costa Rica, San José,
Costa Rica.
ABSTRACT
Snake venom myotoxins have a relevant impact on human health worldwide. Most of them adopt a
phospholipase A2 fold and occur in two forms which often co-exist in the same venom: the Asp49
toxins hydrolyse phospholipids, whilst Lys49 toxins are enzymatically inactive. However, both
classes of myotoxins cause muscle necrosis (1-3). To gain insights into their mechanism of action,
C2C12 cells (a skeletal muscle cell line) at different stages of development were exposed to
myotoxins isolated from Bothrops snakes. The cytosolic Ca2+ changes and the cytotoxic effects
elicited by the toxins were measured in parallel. In both myoblasts and myotubes, the myotoxins
induced a rapid and transient rise in cytosolic [Ca2+], which derives primarily from intracellular
stores. Subsequently, the toxins induce a large and irreversible increase in cytosolic [Ca2+] only in
myotubes, followed by extensive cell death. This second [Ca2+] rise derives from the extracellular
medium. At variance, myoblasts viability is unaffected. In addition, we found that Asp49 and Lys49
myotoxins act synergistically to increase the plasma membrane permeability to calcium and to
induce cell death of myotubes. These data indicate that these myotoxins may bind to acceptor(s)
coupled to intracellular calcium mobilization in both myoblasts and myotubes. However, only in
myotubes the toxins proceed by altering plasma membrane permeability. The synergic effect of
PLA2 active and inactive toxins on mature myotubes suggests that specific inhibitors of
enzymatically active myotoxins could be considered as preferential because they would impair
synergism among the two classes of toxins.
REFERENCES
1) Arni RK, Ward RJ (1996) Phospholipase A2- a structural review. Toxicon 34, 827-841.
2) Lomonte B, Angulo Y, Calderón L (2003) An overview of lysine-49 phospholipase A2 myotoxins from crotalid
snake venoms and their structural determinants of myotoxic action. Toxicon 42, 885-901.
3) Lomonte B, Angulo Y, Sasa M, Gutiérrez JM (2008) The phospholipase A2 homologues of snake venoms: biological
activities and their possible adaptive roles. Prot. Pept. Lett. In press.
19
NMR STUDIES CONCERNING E. COLI GRX4 DIMERIZATION
Lorenzo Gesiot*1, Massimo Bellanda1, Malin Fladvad2, Aristi P.Fernandes2, Stefano Mammi1
*1. Dipartimento di Scienze Chimiche, Università degli Studi di Padova
Via Marzolo 1, 35131 Padova, Italy; Tel: +390498275740; Email: [email protected]
2. Department of Biochemistry, Medical Biochemistry and Biophysics, Karolinska Institutet,
Stockholm SE17177, Sweden
ABSTRACT
The so called monothiolic Glutaredoxins belong to a recently discovered class of putative
Glutaredoxins and are characterized by a Cys-X1-Phe/Tyr-X2 site which corresponds, by alignment,
to the active site of the well known dithiolic Glutaredoxins. Up till now, neither the physiological
reductant nor the substrate of these proteins are definitely proved, and this implies that their
functional role is actually elusive. Nevertheless, the extent of conservation of these proteins
amongst prokaryotes and eukaryotes, and the poor viability of some knock-outs, suggest a decisive
importance in a very central process within the cells, and a role which is not redundant with
dithiolic Glutaredoxins. Defective mutants seem to show an accentuated sensibility to oxidative
stress.
The Glutaredoxin we are interested in is the E. coli Grx4 (grxD gene), a 115 residue protein with a
thioredoxin-like fold. An NMR structure obtained on 13C and 15N isotope labelled samples is
available (1). It is remarkable that Grx4 has a well defined patch of spatially co-localized residues
on the contact surface with the solvent which is perfectly conserved in other monothiolic
Glutaredoxins of different pathogens. This portion of surface is contiguous to the monothiolic site
and is very close to a hydrophobic, exposed region. The amino acids involved in the interaction
between glutathione and dithiolic Glutaredoxins are partially conserved in Grx4, both for charge
distribution and for steric outline of the lodging groove. It is not clear if the structural gap which
renders Grx4 inactive in traditional dithiolic Glutaredoxins functional assays could be overcome by
an interacting partner.
While performing other NMR studies over Grx4, we noticed a marked tendency to self-aggregation,
a propriety already underlined for other proteins of the thioredoxin superfamily and proposed also
for Glutaredoxins (2).
In this work, we report some data concerning Grx4 oligomerization obtained monitoring NMR
variations with time and concentration. Through 15N-HSQC we were able to identify the backbone
amides most affected by the process and to propose a probable superficial region involved in the
interaction. Then, we tried to predict the translational diffusion coefficient of the monomer and of a
hypothetic dimeric structure, with the purpose of verifying these calculations through DOSY
experiments at different sample concentrations.
REFERENCES
1) Fladvad M et al (2005) Molecular mapping of functionalities in the solution structure of reduced Grx4, a monothiol
glutaredoxin from Escherichia coli. Journal of Biological Chemistry 280, 24553-24561.
2) Noguera V et al (2005) NMR reveals a novel Glutaredoxin-Glutaredoxin interaction interface. Journal of Molecular
Biology 353, 629-641.
20
SYNTHESIS AND CHARACTERIZATION OF NEW DERIVATIVES OF UROLITHIN AS
INHIBITORS OF PROTEIN KINASE CK2
Alessandra Gianoncelli*1,Giorgio Cozza1, Diana Tegazzini2, Giuseppe Zagotto2, Stefano Moro2,
Flavio Meggio1
*1. Dipartimento di Chimica Biologica, Università di Padova, Viale G. Colombo 3, 35131 Padova,
2. Dipartimento di Scienze Farmaceutiche, Università di Padova, Italy
ABSTRACT
The complex interplay of signal-transduction pathways often complicates the search of new
molecules able to target single mechanisms. We are day by day exploring the chemical universe,
substances made by chemical synthesis or extracted, to search for new hits completing the stunning
and wonderful puzzle of the signal-transduction. The “kinome” world, and in particular the Ser/Thr
kinases, attracts our attention.
In the past few years, we have performed an intensive search for new ATP-site directed inhibitors
potentially active against the multifunctional protein kinases CK1 and CK2.
Recently, ellagic acid, Ki = 20 nM(1), was found to be a very efficient and selective inhibitor of
CK2 competing with the phosphodonor nucleotide.This plant derived natural compound was in our
library, but it was also identified using an in silico approach based on a virtual screening of
available databases aiming at discovering novel potent and selective CK2 inhibitors.
During these studies we have also found another natural compound, urolithin A IC50=0.4μM,
which is revealed good and selective inhibitor of CK2
OH
O
HO
OH
OH
O
O
HO
O
O
O
OH
Ellagic acid
Urolithin A
So we have synthesized new derivatives of this family to better understand the Structure-Activity
Relationships. None of the new derivatives outperforms apparently the ellagic acid, but few of them
show a comparable inhibitory activity.
The urolithin A, as the ellagic acid, frame fits to the binding pocket filling it almost completely, so
any substituent bulkier than the hydroxy group seems to reduce the inhibitory activity. In order to
further improve the efficacy and selectivity of this family of inhibitors we are now searching for the
position of the tricyclic structure of these compounds that allows a substituent not to be interacting
with but rather to get out of the catalytic pocket.
REFERENCES
1) Cozza G et al (2006) Identification of Ellagic Acid as Potent Inhibitor of Protein Kinase CK2: A Successful Example
of a Virtual Screening Application. J. Med. Chem. 49, 2363-2366.
21
FLAVONOID NARINGENIN MODIFIES THE TRANSPORT PROPERTIES OF THE SLOW
VACUOLAR ION CHANNEL IN PLANTS
Paul Vijay Kanth Gutla*1,2, Armando Carpaneto1, Fiorella Lo Schiavo2, Franco Gambale1
1. Istituto di Biofisica-CNR, Via De Marini 6, Genova, 16149, Italy
Italy, Tel: +39049827 6247, Email: [email protected]
ABSTRACT
The Slow Vacuolar (SV) channels are ubiquitous in all tissues of higher plants. The SV channel is a
non-selective cation channel permeable to both monovalent and divalent cations. It is well known
that SV currents recorded in a typical patch-clamp experiment require unphysiologically high
cytosolic and low vacuolar calcium concentrations for full activation. We aim at looking for
endogenous plant substances which might be able to modify or shift the voltage activation threshold
of this channel towards more physiological
conditions i.e. more negative membrane potentials.
Flavonoid Naringenin [Nar] is present in all plant
species where it plays a central role in the flavonoid
biosynthetic pathway. Nar is stored in the vacuoles in
glycosylated form called Naringin. When Naringin
was applied to cytosolic as well as to the vacuolar
side it did not show any effect on SV channel
activity. However when Naringenin was added to the
cytosolic bath solution, we recorded a dosedependent reversible decrease in SV channel current
described by a Hill equation with n=1.4 and a half
block concentration of 0.44 mM. Surprisingly Nar
affects the voltage dependence of the channel by
shifting the activation threshold towards more
positive voltages and does not change the single
channel conductance. Experiments are in progress to
identify appropriate conditions in which the SV
channel opens at more negative membrane potentials.
We also tested the effects of Nar at the luminal side:
1 mM [Nar] induced a reversible inhibition of the SV
currents of about 40%. Our group has evidences that
approximately 10% of the total SV current at hight
(e.g. > 50 mV) positive voltages is mediated by
calcium (1). Therefore, in order to verify whether
Nar affects both potassium and calcium conductance, we performed experiments by combining the
patch clamp technique with fluorescence measurements using the fluorophore fura-2: both SV
currents and calcium signals were abolished by 1 mM cytosolic [Nar] (see figure). At the moment
we are performing fast perfusion experiments to investigate the mechanism of interaction between
Naringenin and the SV channel.
REFERENCES
1) Gradogna A, Scholz-Starke J, Gutla, PV, Carpaneto A (2009) Fluorescence combined with excised patch: measuring
calcium currents in plant cation channels. The Plant Journal In Press (doi: 10.1111/j.1365-313X.2008.03762x).
22
REDUNDANCY
AND
GENETIC
COMPENSATION
OF
EMILINS
IN
THE
CARDIOVASCULAR SYSTEM
Gaia Litteri*, Alessandra D’Urso, Paola Braghetta, Patrizia Sabatelli, Giuseppe Lembo, Giorgio
Bressan
*Dipartimento di Istologia, Microbiologia, Biotecnologie Mediche, Università di Padova
ABSTRACT
Emilin1 and Multimerin2, two members of the Emilin/Multimerin extracellular protein family, are
characterized by a common structural arrangement (1). In the cardiovascular system, the proteins
have a typical expression pattern. In blood vessels, Emilin1 is expressed in endothelial cells (ECs),
vascular smooth muscle cells (VSMCs) and adventitial fibroblasts; Multimerin2 is expressed
specifically in the endothelium. Emilin1 knockout mice have cardiovascular phenotype with
ultrastructural alterations of endothelium and elastic fibers in aorta and systolic hypertension,
caused by disregulation of TGFß1 processing (2;3). Also Multimerin2 knockout mice have
hypertension, caused by disregulation of TGFß1 signalling. Considering similar knockout
phenotypes, involvement of TGFß1 signalling and structural similarities, we set out to study genetic
redundancy and functional compensation of Emilin1 and Multimerin2 in the cardiovascular system.
In Multimerin2-/- mice, physiological expression of Emilin1 in the endothelium is not sufficient to
compensate Multimerin2 deficiency. Generation of transgenic mice, in which Emilin1 cDNA is
expressed by an endothelial specific promoter (Tie2_Emilin1 transgene), has allowed to observe
that Emilin1 overexpression in the ECs can not rescue cardiovascular phenotype of Multimerin2
knockout mice. Also in Emilin1-/- mice, endogenous expression of Multimerin2 does not
compensate Emilin1 deficiency. We do not known if overexpression of Multimerin2 by Tie2
promoter (Tie2_Multimerin2 transgene) in the endothelium, the only cell type expressing
Multimerin2, can rescue the cardiovascular phenotype of Emilin1-/- mice. Howewer, studying
Emilin1-/- mice in which Emilin1 cDNA is specifically expressed only in the ECs (Tie2_Emilin1
transgene) or only in the VSMCs (SM22_Emilin1 transgene) by tissue specific promoters, we have
found that, when Emilin1 is expressed in VSMCs, knockout phenotype is rescued. It is possible that
Emilin1 is necessary in the VSMCs to perform its functions. Therefore, to analyse if Multimerin2
can replace Emilin1 functions, we will generate Emilin1-/- mice with a transgene in which the
Emilin1 promoter regulates Multimerin2 cDNA expression. This promoter has been already used to
express Emilin1 cDNA in Emilin1 knockout mice. We have demonstrated that it expresses Emilin1
in wild type pattern and this is sufficient to rescue cardiovascular phenotype of Emilin1 knockout
mice. In summary, actual data lead to conclude that, although both Emilin1 and Multimerin2
regulate TGFß1 through a common biochemical mechanism, each protein fulfils distinct functions
in blood vessels. By expressing Emilin1/Multimerin2 chimeric constructs in transgenic mice, it
should be possible to identify in each protein the domain(s) responsible for specific functions.
REFERENCES
1) Braghetta P et al (2004) Overlapping, complementary and site-specific expression pattern of genes of the
EMILIN/Multimerin family. Matrix Biol. 22, 549-556.
2) Zanetti M et al (2004) EMILIN-1 deficiency induces elastogenesis and vascular cell defects. Mol. Cell. Biol. 24, 638650.
3) Zacchigna L et al (2006) Emilin-1, a cysteine-rich glycoprotein of elastic fibers, is required for blood pressure
homeostasis by regulation TGF-β maturation. Cell 124, 929-942.
23
AUTOPHAGY IS REQUIRED FOR THE CONTROL OF MUSCLE MASS
Eva Masiero*1,2, Cristina Mammucari1,2, Bert Blaauw2, L. Agatea2, Stefano Schiaffino2,3, Marco
Sandri1,2,3
*1. Dulbecco Telethon Institute
2. Venetian Institute of Molecular Medicine, via Orus 2, 35129 Padova, Italy
3. Department of Biomedical Science, University of Padova, Padova, Italy
ABSTRACT
The ubiquitin-proteasome and autophagy-lysosome pathways are the two major routes for protein
and organelle clearance in eukaryotic cells. Autophagy is an evolutionary conserved mechanism
that allows cell survival during starvation. Autophagy deficient mice die soon after birth during the
critical starvation period when transplacental nutrient supply is suddenly interrupted. However, the
contribution of autophagy to homeostasis of organelle and proteins in skeletal muscles and its role
in muscle wasting are not yet understood.
We have generated a muscle specific autophagy knockout mice. Deletion of Atg7 gene completely
blocked autophagosome formation in skeletal muscles but not in heart and other tissues. These mice
develop normally and postnatal growth is unaffected. In adulthood skeletal muscles show signs of
degeneration while remaining myofibers are atrophic. Accumulation of abnormal mitochondria,
dilated SR, disorganization of sarcomere and aberrant concentric membranous structure have been
revealed by electron microscope. The critical genes of muscle atrophy are induced and muscle
strength is strongly decreased.
Thus maintenance of autophagy flux is important to preserve myofiber integrity. Inhibition or
alteration of autophagy can contribute to muscle degeneration in some dystrophies.
24
Indirizzo Genetica e Biologia molecolare dello sviluppo
THE DROSOPHILA CIRCADIAN OSCILLATOR IN THE WILD
Stefano Montelli*, Pamela Menegazzi, Stefano Vanin, Ioannis Kasioulis, Elena Carbognin,
Federica Sandrelli, Rodolfo Costa
Dipartimento di Biologia, Università degli Studi di Padova
*Viale G. Colombo 3, 35131 Padova, Italy; Tel: +390498276228; Email:[email protected]
ABSTRACT
In Drosophila melanogaster many key players of the circadian clock network have been identified
and characterized over the last two decades. The core of the clock is made up of interconnected
positive and negative feedback loops. The first loop is composed of the transcription factors
CLOCK (CLK) and CYCLE (CYC) and the second of PERIOD (PER) and TIMELESS (TIM) (1).
In particular, period (per) and timeless (tim), two clock genes which are essential for the genesis of
the circadian rhythmicity, “respond” to external stimuli and play an important role in the processes
which entrain the clock. Drosophila’s clock relies on about 75 clock neurons per brain hemisphere,
bilaterally clustered in 6 groups: 3 groups of dorsal neurons (DN1s, DN2s, DN3s) and 3 groups of lateral
neurons (LNs) (2).
Different per and tim isoforms have been reported. In the case of per, splicing of 89 bases in the 3’
UTR of the per mRNA is enhanced at low temperatures. The splicing affects per mRNA stability
and efficiency of translation and causes flies to exhibit an earlier evening activity. This is correlated
with short photoperiods as well as cold temperatures as in autumn and winter in Europe. If the level
of splicing remains low, then the flies initiate locomotion in the late afternoon/early night and avoid
the higher temperatures of midday/early afternoon (3) (4). At low temperatures, timcold is a splice
variant in which the last intron of 858 bp is not removed, and a premature stop codon within the
unspliced intron shortens the TIM product by 33 amino acids (5).
The Drosophila circadian clock has been widely studied in laboratory conditions while no data are
available so far for natural conditions. Our work aims to investigate the fly circadian clock in the wild.
We have analyzed the behaviour, the transcriptional profiles of per and tim (via RT-PCR) and PER
protein oscillation (via immunoassay) in the wild-type ALA strain of Drorophila. WT-ALA flies
were exposed to the natural light-dark cycles and temperature throughout the year. Flies maintained in lab
conditions at different temperatures (10ºC, 18ºC and 23ºC) were also analyzed as a control.
Our results show that morning and evening locomotor activity in natural conditions largely depends
on temperature. The proportion of the perspliced and timcold isoforms increase with decreasing
temperature. Also PER expression within clock neurons is temperature dependent. Indeed, no PER
protein is detectable when temperature is lower than 15ºC both in laboratory and natural conditions.
Moreover, the peak of PER in all clock neurons occurs after lights-on in long and hot days
(summer) but before lights-on in autumn, when temperature is lower and nights are longer.
REFERENCES
1) Yu W, Hardin EP ( 2006) Circadian oscillators of Drosophila and mammals. Journal of Cell Science 119, 47934795.
2) Helfrich-Förster C (2003) The neuroarchitecture of the circadian clock in the brain of Drosophila melanogaster.
Microscopy Research and Tecnique 62, 94-102.
3) Collins HB, Rosato E, Kyriakou CP (2004) Seasonal behaviour in Drosophila melanogaster requires the
photoreceptors, the circadian clock, and phospholipase C. Proc. Natl. Acad. Sci USA 101, 1945-1950.
4) Majercak J, Chen W-F, Edery I (2004) Splicing of the period gene 3’-terminal intron is regulated by light, circadian
clock and phospholipase C. Molecular and Cellular Biology 24, 3359-3372.
5) Boothroyd CE et al (2007) Integration of light and temperature in the regulation of circadian gene expression in
Drosophila. PloS Genetics 3, 492-506.
25
Indirizzo Genetica e Biologia molecolare dello sviluppo
ECTODERMIN CONTROLS SMAD4 ACTIVITY IN THE EARLY MOUSE EMBRYO
Leonardo Morsut*, Sirio Dupont, Elena Enzo, Sandra Soligo, Michelangelo Cordenonsi, Stefano
Piccolo
Dipartimento di Istologia, Microbiologia e Biotecnologie Mediche, Sezione di Istologia, Università
degli Studi di Padova
ABSTRACT
TGF-β family signaling molecules play fundamental roles in several physiological and pathological
processes. Among these, early embryonic development is a paradigmatic example of the
multifaceted role of the TGF-β signaling. Yet, to date many of the details remain elusive to the
analysis. Our laboratory has recently cloned Ectodermin/Tif1γ (Ecto), a molecule involved in
Smad4 ubiquitination (1). According to the present model, Ecto operates by promoting Smad4
ubiquitination and this step limits the TGF-β effectiveness (2). In order to analyze the relevance of
Ecto in vivo, we generated, with the help of collaborators, the knock out mice (Ecto-/-). Ecto-/animals die around the seventh day of gestation (E7.0). In order to understand which are the
processes impaired in Ecto-/- mice, we investigate the expression of marker genes of the tissues
present at that time using the in situ hybridization technique, which allows us to monitor both
quantitative and qualitative alterations. Nodal/GDF proteins are the TGF-β family members that are
expressed in these early stages of mouse embryonic development. We test whether the inductive
signals delivered by these signaling molecules are amplified in Ecto-/- embryos. Our first analysis
focused on Nodal capacity of mesoderm induction. Unexpectedly, E6.5 Ecto-/- mice reveal a
complete absence of the mesoderm markers Wnt3, T, Eomes. This led us to shift the analysis to
earlier inductive functions of Nodal ligands. The earliest proven inductive function of Nodal
signaling in mouse embryo is the induction of the AVE signaling center at E5.5. The AVE is an
extraembryonic tissue that expresses TGF-β extracellular inhibitors, and is thought to direct the
anterior-posterior axis establishment by restricting nodal signaling in the prospective posterior side
of the conceptus, in order to let the gastrulation start. In Ecto-/- embryos, AVE marker genes (Cerl,
Lefty1, Lim1) are markedly upregulated at E5.5. These results led us to develop an explanation for
the lack of mesoderm in Ecto-/- mice: the magnified AVE provides an unbounded anti-mesoderm
signal that is able to push away the mesoderm field outside the embryo. Next, we investigated the in
vivo epistatic relationship between Ecto and Smad4, which is the target of Ecto inhibition. Smad4 is
a central player of the pathway, and in Smad4-/- mice we identify several marker genes impairment
well before the beginning of gastrulation. In particular, we show that AVE markers are absent in
Smad4-/- E5.5 embryos. Strikingly, when we perform the same experiment in double knock-out
embryos (Smad4-/-; Ecto-/-) we obtain embryos that are totally devoid of AVE markers. This
allows us to demonstrate in vivo that Ecto is epistatic to Smad4. In order to further analyze our
model we tried to rescue the defects of Ecto-/- embryos by lowering the dose of Nodal signaling.
Preliminary data of Ecto-/-;NodalΔ600/- embryos suggest that the magnified AVE is considerably
reduced, and the mesoderm induction is rescued. This provide insight in the actual events that
underlie the earliest defects of Ecto-/- embryos, and underscore the relevance, for the mouse
embryo, of intracellular negative regulators of the TGF-β pathway.
REFERENCES
1) Dupont S et al (2005) Germ-layer specification and control of cell growth by Ectodermin, a Smad4 ubiquitin ligase.
Cell 121, 87-99.
2) Dupont S et al (2009) FAM/USP9x, a deubiquitinating enzyme essential for TGFbeta signaling, controls Smad4
monoubiquitination. Cell 136, 123-135.
26
GENETIC ABLATION OF CYCLOPHILIN D RESCUES MITOCHONDRIAL DEFECTS AND
PREVENTS MUSCLE APOPTOSIS IN COLLAGEN VI MYOPATHIC MICE
Elena Palma1*, Tania Tiepolo2, Alessia Angelin1, Patrizia Sabatelli3, Nadir M. Maraldi3, Emy
Basso1, Michael A. Forte4, Paolo Bernardi1, Paolo Bonaldo2
*1. Department of Biomedical Sciences University of Padova
2. Department of Histology, Microbiology & Medical Biotechnologies, University of Padova
3. IGM-CNR, Unit of Bologna c/o IOR, 40136 Bologna, Italy
4. Vollum Institute, Oregon Health and Science University, Portland, OR 97239-3098, USA
* Viale G. Colombo 3, 35131 Padova, Italy; Tel: +390498276064; Email: [email protected]
ABSTRACT
Ullrich congenital muscular dystrophy (UCMD) and Bethlem myopathy are inherited muscle
disorders caused by mutations of genes encoding the extracellular matrix protein collagen VI. Mice
lacking collagen VI (Col6a1–/–) display a myopathic phenotype associated with ultrastructural
alterations of mitochondria and sarcoplasmic reticulum, mitochondrial dysfunction with abnormal
opening of the permeability transition pore (PTP), and increased apoptosis of muscle fibers.
Treatment with cyclosporin (Cs) A, a drug that desensitizes the PTP by binding to cyclophilin
(Cyp) D, was shown to rescue myofiber alterations in Col6a1–/– mice and in UCMD patients,
suggesting a correlation between PTP opening and pathogenesis of collagen VI muscular
dystrophies.
Here we show that inactivation of the gene encoding for Cyp-D rescues the disease phenotype of
collagen VI deficiency. In the absence of Cyp-D, Col6a1–/– mice show negligible myofiber
degeneration, rescue from mitochondrial dysfunction and ultrastructural defects, and normalized
incidence of apoptosis. These findings (i) demonstrate that lack of Cyp-D is equivalent to its
inhibition with CsA at curing the mouse dystrophic phenotype; (ii) establish a cause-effect
relationship between Cyp-D-dependent PTP regulation and pathogenesis of the collagen VI
muscular dystrophy; and (iii) validate Cyp-D and the PTP as pharmacological targets for the
therapy of human collagen VI myopathies.
REFERENCES
1) Bonaldo P, Braghetta P, Zanetti M, Piccolo S, Volpin D, Bressan GM (1998) Hum. Mol. Genet. 7, 2135-2140.
2) Basso E, Fante L, Fowlkes J, Petronilli V, Forte MA, Bernardi P (2005) J. Biol. Chem. 280, 18558-18561.
3) Palma E, Tiepolo T, Angelin A, Sabatelli P, Maraldi NM, Basso E, Forte MA, Bernardi P, Bonaldo P. (2009) Hum.
Mol. Genet In Press.
27
Indirizzo Genetica e Biologia Molecolare dello Sviluppo
PGL1 FOUNDER EFFECT IN TRENTINO
Viviana Pignataro*1,2, Francesca Schiavi2, Francesca Boaretto2, Serena Demattè3, Enrica Cecchini3,
Maria Luisa Mostacciuolo1, Giuseppe Opocher2.
Italy; Tel: +390498276213; Email:[email protected]
2. Unità Tumori Ereditari IOV (Istituto Oncologico Veneto), Via Gattamelata 64, 35128 Padova;
Tel:+390498215503
3. Medicina II Ospedale Santa Chiara, Trento, Italy
ABSTRACT
Paraganglioma syndrome type 1 (PGL1) is an autosomic dominant disease with maternal genomic
imprinting characterized by head and neck paragangliomas (HNPs) and pheochromocytoma (Pheo),
due to mutations of the SDHD gene.
In Trentino we have identified and characterized a PGL1 founder effect, due to the SDHD
c.341G>C p.Y114C mutation.
We studied 448 individuals from 63 kindred: 233 carried the SDHD mutation and 215 resulted wild
type. Among the 233 mutation carriers, 108 were affected, 9 resulted asymptomatic after a complete
clinical screening, 18 are under investigation, while in 66 subjects the mutation were transmitted by
the maternal branch and 32 were asymptomatic being unknown the parent of origin of the mutation.
The penetrance resulted nearly complete (92%).
To evaluate the prevalence of this mutation in the Trentino population we have collected DNA from
4 thousand native of three contigous Trentino valleys (Val dei Mocheni, Altopianò di Pinè, Val di
Cembra). All samples were analyzed with allelic discrimination with TaqMan probes and DHPLC
and positive samples were then confirmed with direct sequencing.
We have detected the SDHD p.Y114C mutation in 59 subjects. The overall prevalence of the
mutation was 1.5% with highest prevalence in Val dei Mocheni, and progressively lower
prevalence in Altopiano di Pinè and in Val di Cembra. This may suggest the origin of the founder
mutation in Val dei Mocheni and the following spread to the other two valleys. The living
population of this area is more than 70,000 people and we can estimate the presence of 1,000
carriers.
The founder effect in an isolated population provides the explanation of the spread of SDHD
c.341G>C mutation in Trentino. The peculiar transmission of the disease rather than the incomplete
penetrance are responsible of the awareness in carriers of the presence of PGL1 in relatives or
ancestors.
28
BIO PhD & PostDoc Day ’09, Complesso Biologico “A. Vallisneri, Università di Padova
Scuola di Dottorato in Biologia e Medicina della Rigenerazione
Indirizzo Endocrinologia Comparata
EXPRESSION OF STEROID HORMONE RECEPTOR mRNAS IN ZEBRAFISH (DANIO
RERIO) EMBRYOS AT DIFFERENT DEVELOPMENTAL STAGES
Surachai Pikulkaew*
ABSTRACT
The yolk of ovulated oocytes in teleost fish, as in other oviparous vertebrates, contains large
amounts of liposoluble hormones, such as steroid, thyroid and retinoid hormones (MacCormick,
1999), taken up from the maternal circulation or the follicular envelope. Studies on several fish
species have shown that maternal steroid hormones may be involved in the early development of
the offspring (Feist et al., 1990; Shiraishi et al., 1999). Experimental changes of steroid
concentrations were shown to cause distinct effects on offspring development. Moreover, the
treatment of rainbow trout, Oncorhynchus mykiss, oocytes with cortisol before fertilization was
correlated with depressed postmetamorphic body growth of the offspring, without alteration in
premetamorphic growth (Belvedere et al., 1999).
In this study, we have analyzed by RT/PCR and/or qRT-PCR the abundance and degradation rate of
maternal mRNAs for nine steroid hormone receptors and their possible replacement by
corresponding embryonic transcripts in both ovulated oocytes and embryos of zebrafish collected at
0, 1, 2, 4, 8, 12, 24 and 48 h post-fertilization (hpf). The mRNAs encoded the nuclear receptors for
progesterone (PR), androgen (AR), estrogen (ERα, ERβ1 and ERβ2), glucocorticoids (GR),
mineralocorticoids (MR) and the membrane progestin receptor-α and β (mPRα and β). GR mRNA
was the most abundant maternal transcript in oocytes and early embryos followed by ERβ2 and AR
mRNAs. They declined during the first 8 hpf, being replaced, thereafter, by the embryonic
messengers. ERβ1 and MR transcript levels were low till 8 hpf, but increased steadily during
embryonic transcription from 24 to 48 hpf. PR transcripts were detectable only in ovulated oocytes
and at 24 and 48 hpf. At these stages, there was a slight increase of ERα mRNA that initially was
very low. mPRα and β mRNAs were expressed in ovulated oocytes and faintly persisted during the
first 4 hpf. There was no subsequent embryonic expression of these transcripts. The possible
involvement of maternal mRNAs for glucocorticoid and sex hormone receptors in the programming
of early zebrafish development is intriguing, since they mainly occur at stages in which gene
replication predominates over transcription.
REFERENCES
1) McCormick MI (1999) Experimental test of the effect of maternal hormones on larval quality of a coral reef fish.
Oecologia 118, 412-422.
2) Feist G et al (1990) Sex steroid profiles of coho salmon (Oncorhynchus kisutch) during early development and sexual
differentiation. Gen Comp Endocrinol 80, 299-313.
3) Shiraishi K et al (1999) Changes in expression of prolactin- and cortisol-receptor genes during early-life stages of
euryhaline tilapia (Oreochromis mossambicus) in fresh water and seawater. Zool Sci 16, 139-146.
4) Belvedere P et al (1999) Long-term effects on body growth induced by oocyte exposure to exogenous cortisol and
estradiol-17β in rainbow trout, in: Roubous, W., Wendelaar Bonga, S.E., Vaudry, H., De Loot, A. (Eds), Recent developments
in Comparative Endocrinology and Neurobiology. Shaker Publishing, Maastricht, 258-260.
29
THE INTERACTION OF APOMYOGLOBIN WITH OLEIC ACID LEADS TO A PROTEIN
COMPLEX THAT DISPLAYS CELLULAR TOXICITY
Odra Pinato*, Barbara Spolaore, M. Canton, Patrizia Polverino de Laureto, Angelo Fontana
*Dipartimento di Scienze Farmaceutiche, CRIBI, Università degli Studi di Padova
ABSTRACT
Several studies have shown that fatty acids can induce the formation of toxic aggregates in some
amyloidogenic proteins involved in neurodegenerative diseases. In particular, it has been
demonstrated that oleic acid (OA) can induce the formation of oligomers in proteins such as tau (1),
superoxide dismutase (2) and amyloid β-peptide (3). Here, we have studied the interaction of OA
with horse heart apomyoglobin (apoMb), which is not related to any pathology. This single chain
and disulfide-free 153-residue protein is extensively used since decades as a model for studies of
protein structure, folding, misfolding and aggregation. The interaction of OA with apoMb (1:10
molar ratio) leads to the formation of an apoMb/OA complex given by protein oligomers, as
evidenced by protein cross-linking experiments with glutaraldehyde followed by SDS-PAGE.
Indeed, cross-linked oligomers of apoMb are formed in the presence of OA, while apoMb alone
does not form oligomers. Far-UV circular dichroism spectroscopy measurements indicated that
apoMb maintains a highly helical conformation in the presence of OA. We tested the toxicity of the
apoMb/OA complex on Jurkat cells and we have found that the protein complex causes cell death
by an apoptosis-like mechanism. Conversely, the protein is not active when tested alone and OA
shows a much reduced toxicity. Of interest, it has been demonstrated previously that OA can induce
cellular toxicity by an apoptosis-like mechanism (4). Our results suggest that the interaction of OA
with apoMb strongly enhance the water solubility of the otherwise insoluble fatty acid, thus leading
to a significant enhancement of its intrinsic apoptotic activity (3). We suggest that the apoMb/OA
complex acts as a delivery system of the toxic fatty acid to the cell. This mechanism could explain
also the previously reported toxicity of OA complexes with other proteins, including those with
human and bovine α-lactalbumin (LA) (6). We conclude that the toxic effect for tumor cells of the
LA/OA complex previously reported (6) is not protein-specific and we suggest that other proteins
can display similar toxic effects if combined in an OA complex.
REFERENCES
1) Kim YJ, Nakatomi R, Akagi T, Hashikawa T, Takahashi R (2005) Unsaturated fatty acids induce cytotoxic aggregate
formation of amyotrophic lateral sclerosis-linked superoxide dismutase 1 mutants. J. Biol. Chem. 280, 21515-21521.
2) Wilson DM, Binder LI (1997) Free fatty acids stimulate the polymerization of tau and amyloid beta peptides. In vitro
evidence for a common effector of pathogenesis in Alzheimer's disease. Am. J. Pathol. 150, 2181-2195.
3) Barghorn S et al (2005) Globular amyloid beta-peptide oligomer - a homogenous and stable neuropathological
protein in Alzheimer's disease. J. Neurochem. 95, 834-847.
4) Cury-Boaventura MF, Pompeia C, Curi R (2004) Comparative toxicity of oleic acid and linoleic acid on Jurkat cells.
Clin. Nutr. 23, 721–732.
5) Svensson M, et al (1999) Molecular characterization of alpha-lactalbumin folding variants that induce apoptosis in
tumor cells. J. Biol. Chem. 274, 6388-6396.
6) Mok KH et al (2007) HAMLET, protein folding, and tumor cell death. Biochem. Biophys. Res. Commun. 354, 1-7.
30
FEMALE PREFERENCES AND MULTIPLE MALE TRAITS IN THE PEACOCK BLENNY,
SALARIA PAVO.
Matteo Pizzolon*, Maria Berica Rasotto
ABSTRACT
Traditionally, studies of inter-sexual selection have focused primarily on single male sexual signals.
However, in many species, females base their mate choice on the simultaneous recognition of
multiple signals, rather than of one character only (1, 2). Why this occurs is debated and many
different hypotheses have been proposed, ranging from those that consider mate choice by multiple
cues as adaptive and increasing fitness (2) to those proposing it to be maladaptive and decreasing
fitness (3, 2).The aim of this project is to determine whether different male traits signal different
aspects of male quality and whether different quality traits co-vary or are traded off. Indeed, even if
male traits are usually considered correlated with male overall quality, often male attractiveness is
traded off against other male quality traits, such as fertility, parental care, or immune function.
The study species is the peacock blenny, Salaria pavo, a demersal spawner with male parental care
characterized by an evident sexual dimorphism, with males larger than females and exhibiting a
gaudy head crest and the first two anal fin rays modified in a pair of anal glands (4). These glands,
known to produce antimicrobial compounds, release a mucous secretion that males lay on egg
clutches while performing parental care (5). The research is based at the Hydrobiological Station in
Chioggia and the study area includes sites both inside and outside the Venetian Lagoon.
The analyses of field collected breeding males and their nests, relating the expression of secondary
sexual characters to mating success (expresses as eggs number x nest) indicated that anal glands and
head crest likely are under sexual selection pressure. These results were confirmed by preference
experiments where females were challenged with dummy males differing in anal gland or head crest
size. In addition I studied the anal gland development in relation to their possible role in parental
care efficiency. The anal gland secretion (AGS) has an inhibitory effect on the growth of both
Gram-negative and Gram-positive bacteria, including those causing the most severe fish diseases in
marine culture. The egg clutches cared by males deprived of anal glands have a significantly lower
survival than those cared by sham-operated males and non-viable eggs show clear signs of bacterial
infection. AGS, as well as its protein content, is proportional to gland size. These findings indicate
that i) antimicrobial production is a crucial component of male parental care and ii) female
preference for males with larger glands appears to be driven by the pursuit of direct fecundity
benefits (higher hatching success) related to male antimicrobial protection. The third year will be dedicated to study the relationships between the expression of head crest and
male quality, in terms of parental care effort (time spent inside nest, ventilation rate, etc.), immune
system quality (through injection of LPS antigen), and ejaculate quality (sperm number, viability
and velocity).
REFERENCES
1) Andersson M (1994) Sexual selection. Princeton University Press.
2) Candolin U (2003) The use of multiple cues in mate choice. Biol. Rev. 78, 575-595.
3) Møller AP, Pomiankowski A (1993) Why have birds got multiple sexual ornaments. Behav. Ecol. Sociobiol. 32, 167176.
4) Zander CD (1975) secondary sex characteristics of Blennioid fishes (Perciformes). Pubbl. Stn. Zool. Napoli. 39, 717–
727.
5) Giacomello E, Marchini D, Rasotto MB (2006) A male sexually dimorphic trait provides antimicrobials to eggs in
blenny fish. Biol. Lett. 2, 330-333.
31
BIO PhD & PostDoc Day ’09, Complesso Biologico A. Vallisneri”, Università di Padova
MICRORNAS PREDICTION ON GRAPE GENOME
Chiara Rigobello*, Alessandra Biliardi, Elisa Caniato, Andrea Telatin, Giorgio Valle
ABSTRACT
The recent sequencing of grape(1) (Vitis vinifera L.) brought out an huge amount of genomic data
and the obvious target of researchers is now to give sense to the produced information. The first
purpose is the identification and the functional annotation of protein coding genes throughout
specific predicting algorithms. Furthermore, some recent studies have underlined the role of non
coding regions in transcriptional regulation and control signalling. Thus, we have undertaken a
study on the 5’ and 3’UTRs and intronic regions of putative genes, in grape, to find specific motifs.
In particular, the study is focalized on microRNAs (miRNAs).
miRNAs are short (20-25 nt) non coding RNAs that can regulate the expression of transcripts, either
binding or degrading mRNA. In plants, it is supposed that mature miRNA binds the transcript in a
specific manner, in fact the “seed” (from nt 2 to nt 12) is very well conserved not only among
families, but also among species. The majority of these very short sequences has been discovered
through cloning and sequencing efforts, but nowadays, specific software are continuously
ameliorated to overcome these time consuming steps. The key role of these transcriptional
regulators is very well studied in mammals, so the large number of prediction tools is based on
animal miRNA characteristics. Only a few systems are available for plants and they often need a set
of pre-existing data(2,3). To face this issue, we have chosen five of the downloadable software,
according on particular parameters, and we have developed a multi-working approach for having a
much more faster method that gives a possible exclusive consensus for each miRNA gene. The
optimization of algorithms parameters was obtained through different steps, considering the better
result (maximum number of miRNAs found respect to the miRBase) for each genome. Once we got
the prediction, we calculated the sensibility and the precision (Positive Predictive Value) of each
algorithms, in order to balance their different features in our final prediction.
We tested the model on a group of miRNAs strictly related to Vitis vinifera ones and belonging to
plants whose genome has been already sequenced: Arabidopsis, Populus and rice. So, we have built
up three different sets of miRNAs, for each set excluding the known miRNA belonging to the
analyzed genome. Once we obtained all these evidences, we gave particular attention to some
distinctive properties of miRNAs(nucleotide content, sequence complexity, repeat elements).
Moreover, structural features of the potential precursor have been taken into account: hairpin and
hairpin-loop length, base pairing, number of bulges, gaps and mismatches.
With the incoming of innovative sequencing methods, different data are now available for grape
annotation: part of these data has been already used to improve annotation of the genome of Pinot
Noir, while we are going to validate our prediction on miRNAs through a library prepared
specifically on smallRNAs. Furthermore, data from Solexa sequencer have been analysed to remove
parts of adaptors necessary for the library preparation that could interfere with the alignment of the
reads. In addition, AT-rich regions, self-matches, annotated gene regions and repeats were removed.
REFERENCES
1) Olivier J et al (2007) The grapevine genome sequence suggests ancestral hexaploidization in major angiosperm
phyla. Nature 499, 463-468.
2) Zhang B et al (2006) Conservation and divergence of plant microRNA genes. The Plant Journal 46, 243-259.
3) Bentwich I (2005) Prediction and validation of microRNA and their targets. FEBS Lett 579, 5904-5910.
32
NOVEL ANCIENT MYOSINS IN MAMMALIAN STRIATED MUSCLES
Alberto C. Rossi*1,3, Cristina Mammucari1,3, Carla Argentini1,3, Carlo Reggiani2, Stefano
Schiaffino1,3
1. Dipartimento di Scienze Biomediche Sperimentali, Università degli Studi di Padova
2. Dipartimento di Anatomia e Fisiologia, Università degli Studi di Padova
*3. Venetian Institute of Molecular Medicine
Via Giuseppe Orus 2, 35129, 35131 Padova, Italy; Tel: +390497923234, [email protected]
ABSTRACT
We have examined the expression of two putative sarcomeric myosin heavy chain (MYH) genes,
MYH7b (also referred to as MYH14) and MYH15, identified in the mammalian genome and
potential orthologs of the genes coding for chick slow MYH 2 and ventricular MYH, respectively.
In rat and mouse, MYH7b transcripts are detected by RT-PCR in heart, extraocular, slow but not
fast skeletal muscles. In contrast, MYH15 transcripts are detected exclusively in rat and mouse
extraocular muscles. By immunofluorescence, MYH15 protein is specifically expressed in the
orbital layer of extraocular muscles and is not detected in other cranial muscles (tongue and
masseter) or in fast and slow leg muscles, except for the polar region of bag intrafusal fibers.
MYH15 is absent in embryonic skeletal muscle or heart and is first detected in extraocular muscles
at postnatal day 7. The MYH15 ortholog, chick ventricular MYH gene, is also expressed in most
fibers of the orbital layer of extraocular muscles of adult chicken, as well as in embryonic skeletal
muscle and heart but not in adult fast and slow skeletal muscles. In contrast, the Xenopus ortholog,
ventricular MYH gene is exclusively expressed in cardiac muscle. These findings point to striking
evolution of MYH15 function among vertebrate classes.
33
SMAD2 AND 3 TRANSCRIPTION FACTORS ARE CRITICAL FOR MYOSTATIN
SIGNALING AND COOPERATE WITH FOXO3 TO CONTROL MUSCLE MASS
Roberta Sartori*1,2,3, Giulia Milan1,2,3, Maria Patron1, Cristina Mammucari1,3, Bert Blaauw1, Reimar
Abraham1, Marco Sandri1,2,3
*1. Venetian Institute of Molecular Medicine, Padova, Italy. Email: [email protected]
2. Dulbecco Telethon Institute, Padova, Italy
3. Department of Biomedical Sciences, University of Padova, Italy.
Myostatin is an important negative modulator of muscle growth during myogenesis. However, the
role of the myostatin pathway in adulthood and the transcription factors involved in the signaling
are unclear. Using genetic tools we perturbed this pathway in adult myofibers, in vivo, to
characterize the downstream targets and their ability to activate an atrophy program. Mutants of
myostatin receptors are expressed specifically in skeletal muscles and the effects on fiber size and
on activation of atrogin-1 and MuRF1 promoters are monitored. Inhibition of the pathway induces
muscle growth although it does not block muscle loss during catabolic conditions. We further
characterize that the pathway is not sufficient to activate an atrophy program but requires AKT
inhibition and FoxO activation. In fact, FoxO inhibition either by RNAi or by expressing
constitutively active Akt completely prevents myostatin effects on muscle loss. Finally, we
determined that Smad2 and Smad3 are the transcription factors downstream of myostatin and that
Smads need to cooperate with FoxO3 to activate an atrophy program. These findings point to
myostatin inhibitors as good drugs to promote muscle growth during rehabilitation of patients but
useless to prevent activation of an atrophy program during disease.
REFERENCES
1) Cagnin S et al (2009) Reconstruction and functional analysis of altered molecular pathways in human atherosclerotic
arteries. BMC Genomics 10:13.
34
K+-SELECTIVE CHANNELS IN MITOCHONDRIA
Nicola Sassi*1, Umberto De Marchi1, Ildiko Szabò2, Mario Zoratti1,3
*1. Department of Biomedical Sciences, University of Padua
2. Department of Biology, University of Padova
3. CNR Institute of Neuroscience, Biomembranes Section, University of Padova
ABSTRACT
K -selective channels have been found to be present in the inner membrane of mitochondria. The
initial input for the project came with the discovery of the Shaker-family voltage-dependent Kv1.3
channel in the mitochondria of lymphocytes and lymphoma-derived (Jurkat) cells (Szabò et al.,
2005). Work published recently(Szabò et al., 2008) shows that it plays an important role in
apoptosis. Using a biochemical approach, we have now identified it also in the mitochondria of
MCF-7 (a breast cancer line) and PC3 (considered to be a prostate cancer line) cells.
We have also identified another potassium channel, the intermediate conductance Ca2+-activated
K+-selective channel KCa3.1 (IK), in electrophysiological recordings from the inner membrane of
mitochondria isolated from HCT116 (Human Colon Tumor 116) cells. Its presence has been
confirmed by Western blots. We are currently looking for the IK in the mitochondria of other cell
lines. It appears not to be present in the mitochondria of C-26 cells, a murine colon cancer line, and
of Caco-2, another human colon cancer line, even though the IK is present in the plasma membrane.
Both channels are predominantly expressed in the plasma membrane of cells, and thus constitute
examples of proteins with multiple cellular locations. Various K+ channels, including the ones just
mentioned, are upregulated in rapidly proliferating cells (e.g. lymphocytes) and in many cancer
types. The physiological role of the mitochondrial population remains to be clarified.
+
35
Indirizzo Biologia Evoluzionistica
CHARACTERISATION OF THE CARBONIC ANHYDRASE AND GLUTATHIONE
PEROXIDASE FROM ANTARCTIC TELEOST AND MANGROVE TELEOST
Giovanna Sattin*, Gianfranco Santovito, Mariano Beltramini, Arnaldo Cassini
ABSTRACT
Subject of the present research is the molecular characterisation of two key enzymes involved in
oxygen metabolism (glutathione peroxidase, GPX) (1) and osmoregulation and ion homeostasis
(carbonic anhydrase, CA) (2) in Antarctic and intertidal teleostei, in particular African mudskipper
species (Periophtalmus), that live in mangrove ecosystems.
GPX activity consists in the detoxification of hydrogen peroxide, and is one of the most important
ubiquitous antioxidant enzymes involved in antioxidant homeostatic control (3). For the ubiquitous
distribution, those proteins are well suited for molecular evolution studies also in consideration of
the fact that, for they multifunctional role, may be important for the adaptation of organisms to
peculiar environments (4).
CA is an ubiquitous enzyme and its highly conserved active site. It catalyzes the reversible
hydration of CO2 and plays a significant role in processes such as pH homeostasis, respiratory gas
exchange, photosynthesis, and ion transport (5), CA is one of the most important enzymes involved
in the osmoregulation in the gills (6).
The Antarctic and mangrove species have an interesting evolutionary history because they have
developed some adaptations that allow them to survive and to breed in waters where the
temperature, oxygen, ammonium and salt concentration deviate significantly from the average
recorded in the temperate waters (7,8). They represent paradigmatic cases for adaptation to different
temperatures and salinities in their environment (9).
Specimens of Trematomus bernacchii and T. eulepidotus, T. lepidorinus, three Antarctic teleosts,
have been sampled in the Ross Sea (Terra Nova Bay, 74°42’S, 164°7’E), and specimens of
Periophtalmus sobrinus, an air-breathing teleost, have been sampled in the mangrove of Gazi
(Kenya).
Total RNA was purified from gills and GPX and CA cDNAs have been characterised by a
combination of RT-PCR and 5’-3’RACE techniques. The obtained nucleotide sequences have been
compared with those of homologous genes already available in Genbank and have been used for
phylogenetic analysis.
REFERENCES
1) Herbette S, Roeckel-Drevet P, Drevet JR (2007) Seleno-independent glutathione peroxidises more than simple
antioxidant scavengers. FEBS J. 274, 2163-2180.
2) Esbaugh AJ, Tufts BL (2006) The structure and function of carbonic anhydrase isozymes in the respiratory system of
vertebrates. Respir Physiol Neurobiol. 154, 185-198.
3) Toppo S, Vanin S, Bosello V, Tosatto S (2008) Evolutionary and structural insights into the multifaceted glutathione
peroxidase (Gpx) superfamily. Antioxid Redox Signa. 10, 1501-1514.
4) Thompson JL, Thomas PM, Schuller KA (2006) Purification and properties of a glutathione peroxidase from
southern bluefin tuna (Thunnus maccoyii) liver. Comp Biochem Physiol B Biochem Mol Biol. 144, 86-93.
5) Henry PR (1996) Multiple roles of carbonic anhydrase in cellular transport and metabolism.
6) Henry PR, Swenson ER (2000) The distribution and physiological significance of carbonic anhydrase in vertebrate
gas exchange organs.
7) Georgalis T, Perry SF, Gilmour KM (2006) The role of branchial carbonic anhydrase in acid–base regulation in
rainbow trout (Oncorhynchus mykiss). J Exp Biol. 209, 518-530.
8) Nichols (2005) Chemical characterization of exopolysaccharides from antarctic marine bacteria.
9) Schmitz N, Verheyden A, Beeckman H, Kairo JG, Koedam N (2006) Influence of a salinity gradient on the vessel
characters of the mangrove species Rhizophora mucronata. Ann Bot (Lond) 98, 1321-1330.
36
OXIDATION OF THE MITOCHONDRIAL FUSION PROTEIN OPA1 IN THE HEART
Martina Semenzato*1, Christian Frezza2, Andrea Carpi1, Luca Scorrano2, Fabio Di Lisa1
*1. Dipartimento di Scienze Biomediche Sperimentali, Università degli Studi di Padova
Viale
G.
Colombo
3,
35131
Padova,
Italy;
Tel:
+390498276414
Email:
[email protected]
2. Dulbecco-Telethon Institute, Venetian Institute of Molecular Medicine, Via Orus 2, 35129
Padova, Italy
ABSTRACT
The dynamin-like GTPase Optic Atrophy 1 (OPA1) is required for mitochondrial fusion and
maintenance of normal cristae structure that is deranged during apoptosis. Since reactive oxygen
species (ROS) are produced by mitochondria, we aimed at investigating whether OPA1 is a target
of oxidative stress. Structural changes in OPA1 were assessed in samples from rat hearts perfused
under conditions of ischemia (30 min) and reperfusion (15 min) and of oxidative stress induced by
perfusion in the presence of 1 mM H2O2 for 15 min. Additional experiments were performed by
incubating isolated mitochondria in the presence of oxidizing solutions containing increasing
amounts of H2O2 (from 0,25 to 2 mM) and 0.2 mM Fe2SO4 to prompt massive ROS formation due
to the Fenton reaction. Both post-ischemic reperfusion and H2O2 perfusion induced OPA1 oxidation
as detected by the appearance of high molecular weight peptides due to the formation of disulfide
cross bridges. Oxidant-induced OPA1 oligomers that were also confirmed by Blue Native PAGE
besides being redox sensitive are of lower molecular mass than those involved in cristae
remodelling. Through homology modelling, we identified cysteines 853 and 874 as the amino
acidic residues that might be involved in disulfide crossbridges formation.
The aggregation of OPA1 correlated with mitochondrial fragmentation, as visualized by real time
confocal microscopy of a mitochondrially targeted red fluorescent protein expressed in HL-1
cardiomyoblasts exposed to oxidative stress, as well as with release of cytochrome c from
mitochondria.
In conclusion, these results provide the first evidence of OPA1 oxidation and suggest that the
covalent changes of OPA1 structure caused by oxidative stress might hamper its function
facilitating mitochondrial fragmentation and release of proapoptotic proteins.
REFERENCES
1) Di Lisa F, Canton M, Menabo R, Kaludercic N, Bernardi P (2007) Mitochondria and cardioprotection. Heart Fail.
Rev. 12, 249-260.
2) Cipolat S, Martins De Brito O, Dal Zilio B, Scorrano L (2004) OPA 1 requires mitofusins 1 to promote
mitochondrial fusion. Proc. Natl. Acad. Sci. U.S.A 101, 15927-15932.
3) Chen H, Chan DC (2005) Emerging functions of mammalian mitochondrial fusion and fission. Hum. Mol Genet. 14,
R283-R289.
4) Frezza C, Cipolat S, Martins de Brito O, Micaroni M, Beznoussenko GV, Rudka T, Bartoli D, Polishuck RS, Danial
NN, De SB, Scorrano L (2006) OPA 1 controls apoptotic cristae remodeling independently from mitochondrial fusion.
Cell 126, 177-189.
37
IDENTIFICATION OF MiRNAs CONTROLLING GROWTH FACTORS’ SIGNALING
PATHWAYS
Sandra Maria Soligo*, Luca Zacchigna, Graziano Martello, Masafumi Inui, Stefano Piccolo
*Dipartimento di Istologia, Microbiologia e Biotecnologie Mediche, Università degli Studi di
Padova
ABSTRACT
The Xenopus embryo offers an unsurpassed set of biological assays and experimental tools to
understand gene function and gene interaction in vivo. We found that Nodal/activin signalling is
controlled by the miR-15 family (1). Mir-15/16 is the first microRNA demonstrated to target the
TGF-beta signalling cascade. MiR-15/16 inhibits the translation of the Nodal type II receptor
ACVR2a. This was validated in multiple ways: i) overexpression of miR-15 inhibits the expression
of the endogenous ACVR2a and inhibits Phospho-Smad2 levels (immediate read-out of the receptor
activation); ii) inactivation of miR-15 leads to a enhancement of P-Smad2 levels; iii) the 3’UTR of
the ACVR2a gene, both in Xenopus and human cells contains functional miR-15 binding sites; iv)
the interaction between miR-15 and ACVR2a is specific as a mutation in the miR-15 3’UTR
binding sites or a mutant-mir-15 inactivate this interaction and the following biological effects.
More crucially, we demonstrated in vivo the biological effects of this new molecular interaction. In
gain-of-function assays, miR15/16 inhibits the development of the dorsal-most mesoderm, the
famous Spemann Organizer tissue. The localization of mir-15 is also extremely interesting. In
contrast to the known regulators of Nodal signalling, miR-15 and miR-16 are enriched on the
ventral side of the Xenopus embryo, thereby generating a dorsal bias in Acvr2a expression. Our
results offer a new mechanism by which miRNAs can specify cell lineage, that is, by partitioning a
growth factor receptor. In addition, we provide biological and biochemical evidence that expression
of miR-15 family members is under the negative control of Wnt/beta-catenin signalling.
We will also present work aimed at a better functional characterization of the role of 'human
microRNAs' in signalling pathways. Toward this goal, we are taking advantage of Reuven Agami’s
mirVec library, containing the collection of genomic fragment coding for almost all human
annotated microRNA (Rfam release 6, about 400 clones). We amplified these plasmids and
subcloned them all into the pCS2 expression vector. This step was required in order to be able to
synthesize RNA in vitro (i.e. the putative primary immature microRNA), as the original Agami’s
library was cloned into a retroviral vector.
We have also generated mRNA for about 200 independent clones and carried out a pilot study to
identify which of these microRNA can regulate Wnt TGFb and Notch signaling. To this end, we
injected each individual microRNA (10 ng) together with titrated doses of these ligands and the
reporter plasmid whose expression is activated by the corresponding pathways (2). Several primary
microRNAs were isolated as preliminal result. We are currently in the process of validating these
targets.
REFERENCES.
1) Martello G, Zacchigna L, Inui M, Montagner M, Adorno M, Mamidi A, Morsut L, Soligo S, Tran U, Dupont S,
Cordenonsi M, Wessely O, Piccolo S. (2007) MicroRNA control of Nodal signalling. Nature 449, 183-188.
2) Dupont S, Mamidi A, Cordenonsi M, Montagner M, Zacchigna L, Adorno M, Martello G, Stinchfield MJ, Soligo S,
Morsut L, Inui M, Moro S, Modena N, Argenton F, Newfeld SJ, Piccolo S. (2009) FAM/USP9x, a deubiquitinating
enzyme essential for TGFbeta signaling, controls Smad4 monoubiquitination. Cell. 9, 136, 123-135.
38
Scuola di Dottorato in Scienze delle Produzioni Vegetali
Indirizzo Agrobiotecnologie
TRANSCRIPTOME ANALYSIS OF RIPENING PEACH FRUITS TREATED WITH ETHYLENE,
AUXIN AND 1-MCP, AN ETHYLENE RECEPTOR INACTIVATING CHEMICAL.
Alice Tadiello*, Livio Trainotti, Giorgio Casadoro
ABSTRACT
The fruit acts as a protection for seeds during their development and facilitates their dispersal into the
environment with two different strategies: dry fruits at maturity scatter seeds by mechanical means,
while the seeds of fleshy fruit are dispersed by frugivorous animals. Fleshy fruit can be either
climacteric or non-climacteric, based on the fact that a peak in the rate of respiration and ethylene
production can be measured, or not, before the visible onset of the ripening process. Peaches are
climacteric fruits and their ripening is accompanied by dramatic changes in colour, tissue firmness,
flavour and aroma production. A complex network of signals is involved in the regulation of ripening
(1; 2) and, to such a purpose, ethylene has long been regarded as the main regulator of climacteric fruit
ripening. However, in past years it had been shown that, concomitant with ethylene production,
increases in the amount of auxin can also be measured in peach fruit mesocarp (3). In this work a
genomic approach with the µPEACH 1.0 microarray tool has been used in order to understand if such
an auxin increase is functional to an independent role played by the hormone during ripening. It has
thus been possible to show that auxin plays a role of its own during ripening of peaches. Many genes
involved in biosynthesis, transport and signalling (receptors, Auxin Response Factors and Aux/IAA) of
auxin had increased expression in the fruit mesocarp, thus strengthening the idea that this hormone is
actively involved in the ripening process. This study has also demonstrated the existence of an
important cross-talk between auxin and ethylene, with genes in the auxin domain regulated by ethylene
and genes in the ethylene domain regulated by auxin (4).
In order to better understand the role of hormones in the ripening of climacteric fruits, a large-scale
transcriptome analysis has been conducted also on peaches treated with 1-methylcyclopropene (1MCP) a chemical that is thought to inactivate the ethylene receptors. By using a recently developed non
destructive spectroscopic index (index of absorbance difference, IAD), which allows determining the
exact maturation and ripening stage of fruits (5), we could perform 1-MCP treatments on
homogeneously ripened fruit. The possibility of sorting out fruit at “pre-climacteric” (class 0) and
“onset of climacteric” (class 1) stages has yielded new findings on the regulation of this transition and
on 1-MCP action in peach. Moreover, this experimental system resulted useful to shed new light on the
regulation of ethylene synthesis and its cross-talk with auxin.
REFERENCES
1) Alba R, Payton P, Fei Z, McQuinn R, Debbie P, Martin GB, Tanksley SD, Giovannoni JJ (2005) Transcriptome and
selected metabolite analyses reveal multiple points of ethylene control during tomato fruit development. The Plant cell 17,
2954-2965.
2) Giovannoni JJ (2004) Genetic regulation of fruit development and ripening. The Plant Cell 16, S170-S180.
3) Miller AN, Walsh CS, Cohen JD (1987) Measurement of Indole-3-Acetic Acid in peach fruits (Prunus persica L. Batsch
cv Redhaven) during development. Plant Physiol 84, 491-494.
4) Trainotti L, Tadiello A, Casadoro G (2007). The involvement of auxin in the ripening of climacteric fruit comes of age.
The hormone plays a role of its own and has an intense interplay with ethylene in ripening peach. Journal of Experimental
Botany 58, 3299-3308.
5) Ziosi V, Noferini M, Fiori G, Tadiello A, Trainotti L, Casadoro G, Costa G (2008) A new index based on vis
spectroscopy to characterize the progression of ripening in peach fruit. Postharvest Biology and Technology 49, 319-329.
39
DETERMINATION OF FUNGAL ASSOCIATIONS OF THE ORCHID SPIRANTHES SPIRALIS
BY CULTURAL VS. DIRECT MOLECULAR METHODS
Alessandra Tondello1*, Barbara Baldan1, Andrea Squartini2
1
Dipartimento di Biologia, 2Dipartimento di Biotecnologie Agrarie, Università degli Studi di
Padova
*Viale G. Colombo 3, 35131 Padova, Italy; Tel: +390498276241; Email
[email protected]
ABSTRACT
Mycorrhizas are widespread intimate symbioses between members of fungal phyla and a vast
majority of plants. Orchid mycorrhiza is a category with important peculiarities as fungi often
sustain the life of these small-seeded plants during the delicate offspring stage and begin receiving
carbon only once the plant reaches its established form. The symbionts of some orchid species can
be routinely isolated, however isolation success in many orchids varies with season physiological
conditions. The symbionts of a number of orchids are difficult or yet impossible to isolate (1).
Spiranthes spiralis (L.) Chevall. is a submediterranean species; it has a remarkable phenology in
western Europe, where is the latest-blooming native species of orchid. Rosettes appear in summer,
stalks flower in September and the rosette die off in spring (2). We isolated mycorrhizal fungi from
plants, and in order to investigate their taxonomical identity, we extracted total DNA both from the
root tissues and from isolated fungi. The latter were cultured from inner root tissues upon surface
sterilization. Selective PCR amplification using ITS1-ITS4, ITS1F-ITS4 primers was carried out. In
parallel, using fluorescence and confocal microscopy on acridine orange-stained freehand sections
we demonstrate the presence a profuse cortical colonization by intracellular hyphal pelotons.
Electron microscopy examination of isolated fungi allowed to observe the details of this hyphal
ultra-structures.
REFERENCES
1) Lee Taylor D and McCormick MK (2008) Internal transcribed spacer primers and sequences for improved
characterization of basidiomycetous orchid mycorrhizas. New Phytologist 177, 1020-1033.
2) Willems JH and Dorland E (2000) Flowering frequency and plant performance and their relation to age in the
perennial orchid Spiranthes spiralis (L.) Chevall. Plant Biology 2, 344-349.
40
GAIN-OF-FUNCTION
OF
EXCITATORY
SYNAPTIC
TRANSMISSION
IN
MICROCULTURES OF CORTICAL NEURONS FROM KNOCK-IN MICE CARRYING THE
FAMILIAL HEMIPLEGIC MIGRAINE TYPE 1 S218L MUTATION
Dania Vecchia*1, Angelita Tottene1, Daniela Pietrobon1
1
ABSTRACT
Mutations in the CaV2.1alpha1 gene cause familial hemiplegic migraine type 1 (FHM1), a rare
subtype of migraine with aura. We have previously shown that knock-in (KI) mice with the R192Q
FHM1 mutation have an increased CaV2.1 Ca2+ current density in cerebellar and cortical neurons
and a facilitation of cortical spreading depression (1) (CSD), the phenomenon that underlies
migraine aura and may initiate migraine attacks. Recently we have demonstrated how gain-offunction of CaV2.1 Ca2+ channels causes the CSD susceptibility studying excitatory synaptic
transmission in microcultures of cortical neurons and in connected pairs of pyramidal cells and fastspiking interneurons in acute cortical slides of wild-type (WT) and R192Q KI mice (2). We found
that the R192Q mutation increases action potential (AP)-evoked Ca2+ influx and glutamate release
at cortical pyramidal cell synapses but does not alter inhibitory neurotransmission at fast-spiking
interneuron synapses. Using an in vitro model of CSD we showed a causative link between
glutamate release and CSD facilitation.
One of the goals of the research in the laboratory is the functional analysis of the novel S218L KI
mouse model, carrying a FHM1 mutation with a severe clinical phenotype of cerebral edema and
coma after minor head trauma and ataxia. It has been demonstrated: 1) an increased CaV2.1 Ca2+
current density in cerebellar granule cells in primary culture in KI mice, that was about twice as
large in homozygous as compared to heterozygous mice, revealing an allele-dose effect consistent
with the dominance of the mutation in FHM1. 2) a facilitation of CSD propagation in KI mice (3).
The gain-of-function of the CaV2.1 current and the facilitation of CSD propagation was larger in
S218L than R192Q homozygous mice, in correlation with the more severe clinical phenotype of
S218L (3).
To test the hypothesis that there is a correlation between enhanced synaptic glutamate release and
the clinical phenotype we studied excitatory synaptic transmission in microcultures of cortical
neurons from heterozygous S218L KI mice. We measured autaptic excitatory postsynaptic currents
(EPSCs) in response to action potentials elicited by brief depolarizing pulses on neurons in culture
for 8-14 days. The amplitude of the EPSC was larger in heterozygous S218L KI than WT mice, as a
consequence of an increased amplitude of the component inhibited by ω-AgaIVA and an increased
contribution of CaV2.1 channels in controlling release. The amplitudes of the ω-AgaIVA-insensitive
of the EPSC were similar in WT and in KI mice. Heterozygous S218L KI mice presented a
reduction of inhibition of excitatory neurotransmission by activation of GABAB receptors. The
increased of EPSC amplitude and the reduction of inhibition of excitatory neurotransmission by
activation of GABAB receptors were similar in heterozygous S218L KI and homozygous R192Q KI
mice. Our data show that the gain-of-function of excitatory synaptic transmission is similar in
heterozygous S218L and homozygous R192Q KI mice, in agreement with the more severe clinical
phenotype caused by S218L FHM1 mutation.
REFERENCES
1) van den Maagdenberg AM et al (2004) A Cacna1a knockin migraine mouse model with increased susceptibility to
cortical spreading depression. Neuron 41, 701-710.
2) Tottene AM et al (2009) Enhanced excitatory transmission at cortical synapses as the basis for facilitated spreading
depression in CaV2.1 knockin migraine mice. Neuron 61, 762-773.
3) van den Maagdenberg A et al (2009) A Cacna1aS218L knock-in mouse model for severe migraine with emiplegia,
ataxia, epilepsy and mild head trauma-induced brain edema. Submitted.
41
PHOSPHORYLATION OF THE N-TERMINAL DOMAIN OF p53 BY PROTEIN KINASE CK1
ISOFORMS DISCLOSES THE RELEVANCE OF SER-20 AND OF REMOTE DOCKING
SITE(S)
Andrea Venerando*, Oriano Marin, Giorgio Cozza, Lorenzo A. Pinna
Dipartimento di Chimica Biologica, Università degli Studi di Padova
ABSTRACT
The tumor suppressor p53 protein has been identified as key signal integrator molecule. Alterations
in its phosphorylation status can abolish its function resulting in uncontrolled cell growth (1). The
N-terminal transactivation domain of p53 doesn’t show evident specificity determinants for nonprimed phosphorylation by CK1. However, several CK1 isoforms have been reported to
phosphorylate this region in vitro and in cultured cells (2). These observations prompted us to assay
a series of peptides encompassing residues 1-28 of human p53 protein as substrates for different
CK1 isoforms. Phosphorylation assays with radiolabelled ATP show that only CK1 delta readily
phosphorylates p53 peptides, albeit with low efficiency (Km≈2mM). Mutational analysis of p53[128] peptides is consistent with a main role of serine 20 as a target of CK1. Phospho-aminoacid
analysis rules out any significant phosphorylation by CK1 of threonine 18 when serine 15 is not
previously chemically phosphorylated.
In the case of full length p53 protein, both CK1 alpha and delta are able to phosphorylate the
protein with Km in the sub-micromolar range. Although in this case the phosphorylation pattern is
more complex, a prominent contribution of serine 20 to overall phosphorylation of p53 is still
evident.
Kinetics suggest the existence of a remote docking site in the full length p53 that seems to be
isoform specific and increases the affinity for CK1 delta several orders of magnitude. A potential
docking site in the CK1 sequence could be located in the exposed basic loop between helices E and
F. This hypothesis is supported by the alignment of CK1 protein sequences and by in silico docking
model which underscores the importance of two out of three basic residues within this loop for the
interaction with corresponding binding box on p53.
REFERENCES
1) Vousden KH, Lu X (2002) Nat Rev Cancer 2, 594-604.
2) Bode AM, Dong Z (2004) Nat Rev Cancer 4, 793-805.
42
FAMILIAL ALZHEIMER’S DISEASE PS2 VARIANTS DECREASE ER CALCIUM
CONTENT: ROLE OF IP3 RECEPTORS AND PS2 CONFORMATION.
Enrico Zampese*, Lucia Brunello, Cristina Florean, Cristina Fasolato, Paola Pizzo
*Viale G. Colombo 3, 35131 Padova, Italy; Tel: +390498276067
ABSTRACT
Presenilins (PS1 and PS2) are components of the γ-secretase complex that is responsible of
β-amyloid peptide production through the final step of amyloid precursor protein (APP) processing.
They possess 9 transmembrane domains (TMDs) and their incorporation into γ-secretase complexes
requires a “presenilinase” cleavage between TMDs 6 and 7, thus leading to the formation of the
mature, dimeric active form, composed of N- and C-terminal fragments (NTF and CTF). PS and
APP mutations cause Familial Alzheimer’s Disease (FAD), the genetically inherited, early onset
and most aggressive form of Alzheimer’s Disease (AD). Calcium dysregulation has been reported
in association with PS FAD mutations, leading to the “Ca2+ overload hypothesis”: PSs have been
described as channel-forming proteins that allow the passive leakage of Ca2+ across the
endoplasmic reticulum (ER) membrane, decreasing its content and thus protecting cells against the
toxic effects of an excessive ER Ca2+ loading, an effect abolished by FAD mutations (1,2).
Our group previously demonstrated, by measuring ER Ca2+ concentration ([Ca2+]ER) and Ca2+
release from internal stores with different approaches, that PS mutations decrease rather than
increase the ER Ca2+ content (3). Recently, more evidence was obtained against the “Ca2+ overload
hypothesis”, showing that PS mutations, by sensitizing the IP3-receptor (IP3R) to its agonist,
increase the probability of Ca2+ release and favour a decreased ER Ca2+ content (4).
By using ER-targeted aequorin, we here show that the FAD mutant PS2-T122R dampens the ER
Ca2+ content, an effect that is only partially rescued by pharmacological block or down-regulation
of IP3Rs. Furthermore, pharmacological inhibition of ryanodine receptors (RyRs), gave similar
results; these data provide evidence that mechanisms other than IP3R sensitization lead to PS2induced [Ca2+]ER reduction.
We next investigated the structure of PS2 involved in this effect, by using ER-targeted aequorin and
different PS2 constructs aimed at mimicking its full length as well as dimeric form: our data provide
evidence that the full length form of PS2 is necessary to observe the [Ca2+]ER decrease, while the
dimeric form alone seems to be ineffective. This conclusion was further supported by data
demonstrating that a reduction in [Ca2+]ER could be obtained by treating the cells with a
“presenelinase” inhibitor or expressing ubiquilin-1, a protein reported to protect PS2 against
processing (5).
REFERENCES
1) Tu H et al (2006) Presenilins form ER Ca2+ leak channels, a function disrupted by familial Alzheimer's diseaselinked mutations. Cell 126, 981-993.
2) Nelson O et al (2007) Familial Alzheimer disease-linked mutations specifically disrupt Ca2+ leak function of
presenilin 1. J Clin Invest 117, 1230-1239.
3) Zatti G et al (2006) Presenilin mutations linked to familial Alzheimer's disease reduce endoplasmic reticulum and
Golgi apparatus calcium levels. Cell Calcium 39, 539-550.
4) Cheung KH et al (2008) Mechanism of Ca2+ disruption in Alzheimer's disease by presenilin regulation of InsP3
receptor channel gating. Neuron 58, 871-883.
5) Brunello L et al (2009) Presenilin-2 dampens intracellular Ca2+ stores by increasing Ca2+ leakage and reducing Ca2+
uptake. JCMM. Accepted.
43
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Alessia Angelin
Camilla Bean
Giorgia Beffagna
Clara Benna
Paola Berto
Cinzia Bertolin
Lucia Biasutto
Andrea Bisognin
Bert Blaauw
Francesco Boldrin
Stefano Cagnin
Andrea Carpi
Laura Cendron
Alex Costa
Paola Cusumano
Federica Dal Molin
Diego De Stefani
Sonia Fabris
Lara Fioravanzo
Susanna Franzoso
Erika Frare
Fabio Gasparini
Tiziano Gomiero
Valentina Lissandron
Carmen Losasso
Michela Magaraggia
Silvia Maistro
Sandro Malacrida
Diego Maruzzo
Ivan Micetic
Martina Milanetto
Enrico Moro
Roberto Moscatiello
Elisa Palumbo
Giovanna Pontarin
Samanta Raboni
Enrico Teardo
Isabella Tessari
Tania Tiepolo
Lucia Tombolan
Laura Varotto
Alessandro Vezzi
Nicola Vitulo
Barbara Zavan
THE CYCLOPHILIN INHIBITOR DEBIO 025 NORMALIZES MITOCHONDRIAL
FUNCTION, MUSCLE APOPTOSIS AND ULTRASTRUCTURAL DEFECTS IN COL6A1-/MYOPATHIC MICE
A. Angelin*1, T. Tiepolo2, E. Palma1, P. Sabatelli3, L. Merlini4, L. Nicolosi1, F. Finetti5, P.
Braghetta2, G. Vuagniaux6, J.-M. Dumont6, C.T. Baldari5, P. Bonaldo2 and P. Bernardi1
Departments of 1Biomedical Sciences/CNR Institute of Neuroscience and 2Histology, Microbioloy
and Biomedical Biotechnologies, University of Padova, Italy; 3IGM-CNR c/o Istituto Ortopedico
Rizzoli, Bologna, Italy; 4Section of Medical Genetics, University of Ferrara, and Laboratory of Cell
Biology, Istituto Ortopedico Rizzoli, Bologna, Italy; 5Department of Evolutionary Biology,
University of Siena, Italy; 6Debiopharm SA, Lausanne, Switzerland
* Viale G. Colombo 3, 35121 Padova, Italy; Tel: +390498276039; Email:[email protected]
ABSTRACT
Background and purpose. We have investigated the therapeutic effects of the selective cyclophilin
inhibitor D-MeAla3-EtVal4-cyclosporin (Debio 025) in myopathic Col6a1-/- mice, a model of
muscular dystrophies due to defects of collagen VI.
Experimental approach. We have studied calcineurin activity, T cell activation, propensity to open
of the permeability transition pore in mitochondria and skeletal muscle fibres, muscle ultrastructure
and apoptotic rates in Col6a1-/- mice before after treatment with Debio 025.
Key results. Debio 025 does not inhibit calcineurin, yet it desensitizes the mitochondrial
permeability transition pore in vivo. Treatment with Debio 025 prevents mitochondrial dysfunction
and normalizes the apoptotic rates and ultrastructural lesions of myopathic Col6a1-/- mice.
Conclusions and Implications. Desensitization of the mitochondrial permeability transition pore can
be achieved by selective inhibition of matrix cyclophilin D without inhibition of calcineurin,
resulting in an effective therapy of Col6a1-/- myopathic mice. These findings provide an important
proof of principle that collagen VI muscular dystrophies can be treated with Debio 025 and
represent an essential step toward a therapy of Ullrich Congenital Muscular Dystrophy and Bethlem
Myopathy because Debio 025 does not expose patients to the potentially harmful effects of
immunosuppression.
REFERENCES
Irwin WA, Bergamin N, Sabatelli P, Reggiani C, Megighian A, Merlini L, et al (2003) Mitochondrial dysfunction and
apoptosis in myopathic mice with collagen VI deficiency. Nat Genet 35, 267-271.
Angelin A, Tiepolo T, Sabatelli P, Grumati P, Bergamin N, Golfieri C, et al (2007) Mitochondrial dysfunction in the
pathogenesis of Ullrich congenital muscular dystrophy and prospective therapy with cyclosporins. Proc Natl Acad Sci
USA 104, 991-996.
Merlini L, Angelin A, Tiepolo T, Braghetta P, Sabatelli P, Zamparelli A, et al (2008) Cyclosporin A corrects
mitochondrial dysfunction and muscle apoptosis in patients with collagen VI myopathies. Proc Natl Acad Sci USA 105,
5225-5229.
Tiepolo T, Angelin A, Palma E, Sabatelli P, Merlini L, Nicolosi L, et al (2009) The cyclophilin inhibitor Debio 025
normalizes mitochondrial function, muscle apoptosis and ultrastructural defects in Col6a1-/- myopathic mice. Br J
Pharmacol In press.
45
EMERGING ROLE OF ANKRD2 PROTEIN IN THE NF-kB PATHWAY DURING IN VITRO
MUSCLE DIFFERENTIATION
Camilla Bean* and Gerolamo Lanfranchi
* Department of Biology, University of Padova, Viale G. Colombo 3, 35131 Padova, Italy; Tel:
+390498276368; Email: [email protected]
ABSTRACT
Ankrd2 is a member of the Muscle Ankyrin Repeat Protein family (MARPs), consisting of
sarcomere-associated proteins that can also localize in the nucleus. There are indications that
MARPs might function as shuttle proteins between the cytoplasm and nucleus, likely carrying
information to the genome, resulting in changes in the structure or function of the contractile
machinery. Recently, we have presented the first evidences that Ankrd2 protein may influence
myogenic differentiation. Examining both morphological and functional features of myocytes stably
overexpressing or silencing the Ankrd2 protein, we have shown that Ankrd2 gives an important
contribution to the coordination of proliferation and apoptosis during myogenic differentiation in
vitro, mainly through the p53 network (1). Over the past years, NF-kB protein, that shows striking
similarities with Ankrd2, has also been implicated by a series of studies, in skeletal muscle
differentiation. In particular, both proteins have multiple ankyrin repeats and phosphorylation sites.
Furthermore, Ankrd2 shares with I-kB (the NF-kB’s inhibitor protein) putative PEST motifs in
similar positions. The possibility that Ankrd2 could play a role in the NK-kB pathway is
particularly interesting since in the C2C12 myoblast cell line NF-kB functions as an inhibitor of
myogenic differentiation, and myoblasts lacking NF-kB activity displayed defects in cellular
proliferation and cell cycle exit upon differentiation. Likewise, Ankrd2 overexpression dramatically
affects myogenic differentiation (1). Then we have investigated whether Ankrd2 could play a role
in the NF-kB pathway. By using co-immunoprecipitation assay we find that Ankrd2 binds the p50
member of NF-kB family of transcription factors. Moreover, Ankrd2 co-localizes with p50 in the
nuclei of undifferentiated myoblasts, but after in vitro induction of myotubes formation, Ankrd2
moves to the cytoplasm. Finally, we show that Ankrd2 is regulated by the NF-kB pathway. In fact,
from chromatin immunoprecipitation experiments we show that in proliferating myoblasts the
transcriptional repressor p50 binds its box in the minimal promoter region of Ankrd2 gene and,
after differentiation induction, it is released from this recognition sequence. These findings suggest
that p50 contributes to confer spatial and temporal expression specificity to the Ankrd2 gene.
REFERENCES
1) Bean C, Facchinello N, Faulkner G, Lanfranchi G (2008) The effects of Ankrd2 alteration indicate its involvement in
cell cycle regulation during muscle differentiation. Biochim Biophys Acta – Mol Cell Biol 1783, 1023-1035.
46
IN VITRO AND IN VIVO EXPERIMENTAL MODELS FOR INVESTIGATING THE
MOLECULAR PATHOGENESIS OF ARRHYTHMOGENIC RIGHT VENTRICULAR
CARDIOMYOPATHY
Giorgia Beffagna*, Alessandra Lorenzon, Marzia De Bortoli, Paola Braghetta, Roberto Doliana,
Gessica Smaniotto, Giuseppe Lembo, Patrizia Sabatelli, Andrea Nava, Gaetano Thiene, Paolo
Bonaldo, Gian Antonio Danieli, Alessandra Rampazzo
Viale G. Colombo 3, 35131 Padova, Italy; Tel: +390498276214; Email :[email protected]
ABSTRACT
Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a heart muscle disease characterized
by a progressive myocardial atrophy followed by fibro-fatty replacement, involving mostly the right
ventricular myocardium1. Eight ARVC disease genes have been identified so far (RYR2, DSP, JUP,
PKP2, TGFbeta3, DSG2, DSC2 and TMEM43), four of them by our group. An impairment of
myocyte cell−to−cell adhesion has been postulated as a possible common pathogenetic pathway.
However, how the mutated desmosomal proteins result in a unique, predominantly right ventricular
cardiac phenotype is still unknown. Understanding the true mechanistic basis of ARVC could
enable both diagnosis and development of targeted therapies for those patients who have already
developed this life−threatening disease.
Mutation screening of PKP2, DSP, DSG2, DSC2 genes was performed in 110 ARVC index cases
diagnosed as affected on the basis of Task Force criteria. Mutations were detected in 52 cases
(47%)2. Of these, 16 (14.6%) carried a PKP2 mutation, 11 (10.0%) a DSP mutation, 8 (7.3%) a
DSG2 mutation, 3 (2.7%) a DSC2 mutation. Compound or double heterozygosity was identified in
14 probands (12.7%). This finding is particularly relevant to mutation screening strategy and to
genetic counselling.
In vitro functional studies have been performed to evaluate the pathogenic effects of DSC2
mutations p.E102K, p.I345T and p.A897KfsX3. Unlike wild-type DSC2 which was detected at cell
membrane, into cell−cell contact regions, the mutants DSC2 are predominantly localised in the
cytoplasm, with very scarce cell membrane localization3.
To investigate molecular pathogenesis of ARVC, cardiac-restricted DSG2 transgenic (Tg) mice
were generated using vectors containing the mouse alpha-myosin heavy chain promoter and fulllength wild-type and mutant DSG2 cDNA sequences. Transgenic founders for DSG2 mutations
G100R, N266S, K294E, Q558X and WT were obtained. Current experimental efforts are directed
to characterise the transgenic lines, as number of integrated copies and level of expression of the
different trasgenes. Preliminary evidences obtained by immunohystochemical analysis showed the
abnormal localisation of Q558X DSG2 protein into the cytoplasm and, on the contrary, the correct
localisation of N266S DSG2 protein into the desmosomal complexes. Phenotypic characterization
of transgenic lines will involve further histological and electrophysiological analyses.
REFERENCES
1) Nava A, et al (1989) Familial occurrence of right ventricular dysplasia: a study involving nine families. J Am Coll
Cardiol 12, 1222-1228.
2) Rampazzo A, et al (2008) Prevalence of desmosomal protein mutations and clinical features in a large cohort of
unrelated consecutive patients affected with arrhythmogenic right ventricular cardiomyopathy. Eur Heart J Suppl G.
3) Beffagna G, et al (2007) Missense mutations in Desmocollin-2 N-terminus, associated with arrhythmogenic right
ventricular cardiomyopathy, affect intracellular localisation of desmocollin-2 in vitro. BMC Medical Genetics 8, 65
47
FUNCTIONAL CHARACTERIZATION OF TIMELESS2 IN DROSOPHILA MELANOGASTER
Clara Benna*1, Corinna Wülbeck2, Silvia Bonaccorsi3, Maurizio Gatti3, Charlotte Förster2, Rodolfo
Costa1, Federica Sandrelli1
2. Universität Regensburg, Institut für Zoologie, Universitaetsstr. 31, 93040 Regensburg, Germany
3. Dipartimento di Genetica e Biologia Molecolare, Università di Roma “La Sapienza”, Roma, Italy
ABSTRACT
Drosophila melanogaster timeless2 (1) (tim2 or timeout) is the ortholog of mammalian Timeless (2)
(mTim), a member of an evolutionary conserved family of genes whose function is critical for
survival and may influence the circadian clock mechanism (3). We have characterized five tim2
PiggyBac insertional mutants (tim2-). Four out of five tim2- alleles cause lethality in homozygosity
and tim2-/tim2- mutants die few hours after puparium formation. In addition, cytological analysis of
tim2-/tim2- mutant larval brains revealed a high frequency of chromosome aberrations.
Constitutively induced tim2 RNAi resulted in severe heads defects leading to pupal death, although
at later developmental stages compared to tim2- mutant flies.
tim2 is located on the right arm of the third chromosome and spans 75 kb of genomic DNA. During
development, tim2 transcription leads to the production of a 4.5 kb major transcript and smaller
isoforms, expressed at very low levels, in each developmental stage tested. The expression level of
the main 4.5 kb tim2 isoform is very high in embryos and third instar larvae and low during pupal
life. In adults, tim2 is strongly expressed in both optic lobe and central brain.
To investigate the role of tim2 in the adult circadian clock, we induced, via dsRNAi, mRNA tim2
depletion in different areas of the Drosophila brain, and evaluated its effect on locomotor activity,
under different environmental regimes. We also analysed the locomotor behaviour of heterozygous
tim2-/+ adult flies. Our data indicate that the behavioural periodicity is not affected in either tim2
KD or tim2-/+ flies. In contrast, the responses to light-pulses, delivered at late night, seemed to be
altered in tim2-/+ and when tim2 depletion is triggered in a specific region of the optic lobe.
REFERENCES
1) Benna C, Scannapieco P, Piccin A, Sandrelli F, Zordan M, Rosato E, Kyriacou CP, Valle G, Costa R (2000) A
second timeless gene in Drosophila shares greater sequence similarity with mammalian tim. Curr. Biol. 10, R512-R513.
2) Gotter AL, Mangarano T, Weave DR, Kolakowsky LFJr, Possidente B, Sriram S, MacLaughlin DT, Reppert SM
(2000) A time-less function for mouse Timeless. Nature Neurosci. 3, 755-756.
3) Gotter AL (2006) A Timeless debate: resolving TIM's noncircadian roles with possible clock function. Neuroreport
17, 1229-1233.
48
HOMOLOGOUS AND HETEROLOGOUS EXPRESSION OF [FeFe]-HYDROGENASE FROM
CHLAMYDOMONAS REINHARDTII
Paola Berto*, Elisabetta Bergantino, Giorgio Giacometti, Paola Costantini
ABSTRACT
Hydrogenases, key enzymes in hydrogen metabolism in several microorganisms, are considered as
a possible future energy source. In particular, the ability of the green alga Chlamydomonas
reinhardtii to reduce protons and release hydrogen gas (H2) upon illumination by means of a
[FeFe]-hydrogenase represents a phenomenon of great scientific interest, because it holds the
promise of generating energy from nature’s most plentiful resources: solar energy and water.
However, the catalytic activity is strongly and irreversibly inhibited by the molecular oxygen
produced during photosynthesis; furthermore, the algal hydrogenase is expressed at very low levels
and only in conditions of strict anaerobiosis. This mutually exclusive nature of O2 and H2
photoproduction cannot be easily overcome and represents an important problem in the
development of H2 production biotechnology. The study of the structure-function relationship of
[FeFe] hydrogenases, which would help to clarify the molecular mechanisms underlying both H2
production and O2 sensitivity, requires the characterization of purified native and modified proteins.
The 3D structure of the [FeFe]-hydrogenase from C. reinhardtii has not been solved, mainly
because of the very low levels of protein which can be directly purified from the algae. I
overexpressed this enzyme in homologous and heterologous systems to obtain enough protein for
biochemical studies. The cyanobacterium Synechococcus PCC 7942, which holds a bidirectional
[NiFe]-hydrogenase is able to produce the [FeFe]-hydrogenase from C. pasteurianum in a
catalytically active form (1); so the [NiFe]-hydrogenase maturation pathway of cyanobacteria may
be flexible enough to allow the biosynthesis of functional Fe-only enzyme.
We expressed the
[FeFe]-hydrogenase from C. reinhardtii in the cyanobacterium
Synechocystis sp. PCC 6803. We obtained two constructs to stably
transform the cyanobacterium Synechocystis sp. PCC 6803, which
contains a bidirectional [NiFe]-hydrogenase with an active site very
similar to the one of Fe-only protein (2), and to enable the
cyanobacterium to express the C. reinhardtii [FeFe]-hydrogenase. The
recombinant strains expressing the algal [FeFe]-hydrogenase were able
to release H2 gas amounts 4 times higher than that of wild type strain.
These data open new perspectives about the indispensable presence of
HydE, HydF and HydG auxiliary proteins (3,4) to obtain a correctly
folded [FeFe]-hydrogenase.
REFERENCES
1) Asada Y, Koike Y, Schnackenberg J, Miyake M, Uemura I, Miyake J (2000) Heterologous expression of clostridial
hydrogenase in the cyanobacterium Synechococcus PCC 7942. Biochim. Biophys. Acta 1490, 269-278.
2) Armstrong FA (2004) Hydrogenases: active site puzzles and progress. Current Opinion in Chemical Biology 8, 133140.
3) Peters J, Szilagyi R, Naumov A, Douglas T (2006) A radical solution for the biosynthesis of the H-cluster of
hydrogenase. FEBS Letter 580, 363-367.
4) Posewitz MC, King PW, Smolinski SL, Zhang L, Seibert M, Ghirardi ML (2004). Discovery of two novel radical Sadenosylmethionine proteins required for the assembly of an active [Fe] hydrogenase. J .Biol. Chem. 279, 25711-25720.
49
IDENTIFICATION OF SUSCEPTIBILITY GENES IN FAMILIES WITH HIGH RECURRENCE
OF SCHIZOPHRENIA AND BIPOLAR DISORDER
Cinzia Bertolin*1, Giovanni Vazza1, Andrea Vettori1, Chiara Magri2, Sergio Barlati2, Sabina
Rampinelli3, Giulia Perini4, Pio Peruzzi5, Maria Luisa Mostacciuolo1
1. Department of Biology, Padua University
2. Department of Biomedical Sciences and Biotechnology, Brescia University
3. Mental Health Centre of Chioggia
4. Department of Neurology and Psychiatry, Padua
5. 1° Psychiatry Service, Padua Hospital
* Viale G. Colombo 3, 35131 Padova, Italy; Tel:+390498276214; Email: [email protected]
ABSTRACT
Schizophrenia (SZ) and Bipolar Disorder (BPD) are multifactorial disorders, also referred as
complex traits since many genes and environmental factors are recognized to contribute to their
predisposition. Several studies show that SZ and BPD are predominantly genetic disorders, with an
estimated hereditability of around 80%.
The main aim of this project is to identify loci and genes conferring the susceptibility to SZ and
BPD in a sample of families characterized by high recurrence of such psychiatric diseases, even in
the same sibship. All these families come from Chioggia, a little town in the Venetian lagoon.
Based on the history and the socio-cultural features, Chioggia’s population could be considered a
“closed community” that presumably derives from a small group of founders.
A first genome wide search, performed using microsatellite markers on a subset of 16 families,
identified a suggestive linkage peak on chromosome 15q26 (1) in a region where other groups
reported linkage signals for SZ and/or BPD. This highlights the potentiality of our sample and the
consistency of our methodological approach.
The first phase of the project was the improvement of the sample collection. In collaboration with
the Department of Mental Health of Chioggia, we collected new familial and sporadic cases as well
as unaffected relatives. Each new subject was clinically evaluated by the same psychiatrist and a
biological sample (blood or buccal swab) was collected from each one of them. Genealogical study
was carried out for each familial case; all family trees were then reconstructed and extended trough
the use of register office archives and church records of Chioggia. All these information, together
with clinical and anamnestic data were stored in a local database. Up to now we were able to collect
biological materials and clinical data for 197 subjects that belong to 66 pedigrees all originating
from Chioggia by at least 4 generations. 150 of these subjects, belonging to 31 pedigrees, have been
sent to BURLO-CBM SNP genotyping centre to be analyzed using the high-throughput Illumina
Infinium technology with the SNP arrays 370K. This analysis will allow us to perform genomewide high-resolution mapping as well as to identify and analyze the distribution of CNV (Copy
Number Variation) in our sample. This work is currently in progress and we expect to obtain the
first results soon.
In the meantime, in order to get phylogenetic information about maternal lineages of the collected
families, we characterized mitochondrial haplogroups and sequenced the hypervariable region 1
(HV1). Interestingly, on a total of 87 unrelated index cases analyzed, only 57 different maternal
lineages (defined by the same haplogroup and HVI haplotype) were identified suggesting that some
families may share the same mtDNA. To explore this hypothesis we sequenced the whole
mitochondrial genome of 31 selected unrelated index cases. The presence of the same mtDNA was
evidenced in 4 cases thus confirming a common female ancestor for all these families.
REFERENCES
1) Vazza G, Bertolin C, Scudellaro E, Vettori A, Boaretto F, Rampinelli S, De Sanctis G, Perini G, Peruzzi P,
Mostacciuolo ML (2007) Genome-wide scan supports the existence of a susceptibility locus for schizophrenia and
bipolar disorder on chromosome 15q26. Mol Psychiatry 12, 87-93.
50
DEVELOPING A PHARMACOLOGY OF PLANT POLYPHENOLS
Lucia Biasutto*1,2, Andrea Mattarei2, Alice Bradaschia1, Silvia Beltramello1, Nicola Sassi1, Ester
Marotta2, Spiridione Garbisa1, Cristina Paradisi2, Mario Zoratti1,3.
Università degli Studi di Padova, Dipartimenti di 1Scienze Biomediche Sperimentali e di 2Scienze
Chimiche, 3Istituto CNR di Neuroscienze.
ABSTRACT
Plant polyphenols are a vast family of natural compounds present in many foods and drinks. Many
of them have noteworthy biological properties, but their efficacy is severely hindered by their low
bioavailability. Because of inefficient absorption and of a rapid metabolism/degradation in the
intestinal and hepatic compartments, only small amounts of polyphenols are found in the
bloodstream, and mostly as metabolites.
Aiming at circumventing the obstacle posed by low bioavailability, we are developing “pro-drugs”
of polyphenols, resistant to metabolism during absorption and capable of regenerating the natural
compound thanks to the action of ubiquitous enzymes. Quercetin (3,3’,4’,5,7-pentahydroxyflavone)
and resveratrol (3,4’,5-trihydroxystilbene) are the model polyphenols used for our proof-ofprinciple project. We initially synthesised several carboxyester derivatives and studied their
transport/diffusion using supported monolayers of epithelial cells, ex vivo intestine segments and in
vivo pharmacokinetics. Carboxyester linkages, however, have proven to be too labile in a biological
context, and we are thus developing less hydrolysis-prone derivatives.
To increase the bioefficacy of polyphenols, an alternative to increasing their systemic levels could
be their specific accumulation at desired sites of action. Polyphenols are redox-active molecules,
and reactive oxygen species are involved in several pathologies. Mitochondria are major producers
of ROS and the site of key events for cell death. We have shown that quercetin can either inhibit or
induce the mitochondrial permeability transition, depending on whether its anti- or pro-oxidant
activity prevails.
We have thus synthesised derivatives of quercetin and resveratrol capable of accumulating into
mitochondria thanks to functionalisation with the triphenylphosphonium (TPP+) group. A first
investigation into the biological effects of these new compounds has been performed with isolated
mitochondria, showing their potential as co-inducers of the mitochondrial permeability transition, as
well as inhibitors of the respiratory chain and of mitochondrial ATP synthase.
Preliminary studies on cultured tumour and non-tumoural cells have shown that mitochondriotropic
compounds, as well as another construct produced during the project work, exhibit the typical
cytotoxic effects of chemotherapeutic agents.
51
A-MADMAN: ANNOTATION-BASED MICROARRAY DATA META-ANALYSIS TOOL
Andrea Bisognin*1, Alessandro Coppe1, Francesco Ferrari1, Davide Risso2, Marta Biasiolo1, Chiara
Romualdi1, Silvio Bicciato3, Stefania Bortoluzzi1
2. Dipartimento di Scienze Statistiche, Università degli Studi di Padova
3. Dipartimento di Scienze Biomediche Università di Modena e Reggio Emilia, Via G. Campi 287,
41100, Modena
ABSTRACT
Motivation:
Publicly available datasets of microarray gene expression signals represent an unprecedented
opportunity for extracting genomic relevant information and validating biological hypotheses.
However, the exploitation of this exceptionally rich mine of information is still hampered by the
lack of appropriate computational tools, which are able to overcome the critical issues raised by
meta-analysis.
Results:
We developed A-MADMAN, an open source web application which allows the retrieval,
annotation, organization and meta-analysis of gene expression datasets obtained from Gene
Expression Omnibus. A-MADMAN addresses and resolves several open issues in the meta-analysis
of gene expression data. In particular, A-MADMAN allows i) the batch retrieval from Gene
Expression Omnibus and the local organization of raw data files and of any related metainformation, ii) the re-annotation of samples to fix incomplete, or otherwise inadequate, metadata or
to create user-defined batches of data, iii) the integrative analysis of data obtained from different
Affymetrix platforms through custom chip definition files and meta-normalization.
The software, written in python, is based on the popular Django web framework and uses R as a
backend. A-MADMAN supports a collaborative working style for local or geographically dispersed
teams through LAN or Internet deployment options, but can be used also by a single researcher on
his Windows Personal Computer installing an all-in-one package that bundles all required
dependencies except R. The software comes with supporting material: program description,
installation instructions and step-by-step tutorial.
52
AKT ACTIVATION PREVENTS THE FORCE DROP INDUCED BY ECCENTRIC
CONTRACTIONS IN DYSTROPHIN-DEFICIENT SKELETAL MUSCLE
Bert Blaauw*1,2, Cristina Mammucari1,2, Luana Toniolo4, Lisa Agatea1,2, Reimar Abraham2, Marco
Sandri1,2,3, Carlo Reggiani4, Stefano Schiaffino1,2
*1. Department of Biomedical Sciences, CNR Institute of Neurosciences, University of Padova,
Padova, Italy; Email: [email protected]
2. Venetian Institute of Molecular Medicine (VIMM), Padova, Italy
3. Dulbecco Telethon Institute
4. Department of Human Anatomy and Physiology, University of Padova, Italy
ABSTRACT
Skeletal muscles of the mdx mouse, a model of Duchenne Muscular Dystrophy, show an excessive
reduction in the maximal tetanic force following eccentric contractions. This specific sign of the
susceptibility of dystrophin-deficient muscles to mechanical stress can be used as a quantitative test
to measure the efficacy of therapeutic interventions. Using inducible transgenesis in mice, we show
that when Akt activity is increased the force drop induced by eccentric contractions in mdx mice
becomes similar to that of wild type mice. This effect is not correlated with muscle hypertrophy and
is not blocked by rapamycin treatment. The force drop induced by eccentric contractions is similar
in skinned muscle fibers from mdx and Akt-mdx mice when stretch is applied directly to skinned
fibers. However, skinned fibers isolated from mdx muscles exposed to eccentric contractions in vivo
develop less isometric force than wild type fibers and this force depression is completely prevented
by Akt activation. These experiments indicate that the myofibrillar-cytoskeletal system of
dystrophin-deficient muscle is highly susceptible to a damage caused by eccentric contraction when
elongation is applied in vivo, and this damage can be prevented by Akt activation. Microarray and
PCR analyses indicate that Akt activation induces up-regulation of genes coding for proteins
associated with Z-disks and costameres, and for proteins with anti-oxidant or chaperone function.
The protein levels of utrophin and dysferlin are also increased by Akt activation.
53
TETRAHYMENA THERMOPHILA METALLOTHIONEINS: EFFECTIVE TOOLS FOR
RECOMBINANT PROTEIN EXPRESSION
Francesco Boldrin1*, Alessia Formigari1, Gianfranco Santovito1, Donna Cassidy-Hanley2, Theodore
G. Clark2 and Ester Piccinni1.
*1. Dipartimento di Biologia, Università degli Studi di Padova. Viale G. Colombo 3, 35131 Padova,
Italy; Tel: +390498276310 Email: [email protected]
2. Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell
University, Ithaca, NY, USA
ABSTRACT
The scientific community has recently taken into great consideration the use of Tetrahymena.
thermophila for the production of recombinant proteins for both biotechnological perspectives and
as research tools, since no system (i.e. bacteria, fungi, insect and vertebrate cell lines) is universally
suitable for the expression of foreign genes. T. thermophila appears to be an useful host to produce
membrane proteins from parasitic protozoa, such as Apicomplexa, because these parasites exhibit
the same codon usage as Tetrahymena. Moreover, like T. thermophila, a number of human
pathogens bear extremely AT-rich genomes, that are instable in conventional expression systems.
Therefore, T. thermophila has been proposed as experimental model system for functional and
structural characterization of proteins and production of vaccines.
The usefulness of T. thermophila as an experimental model organism has further increased with the
development of inducible expression vectors based on metallothionein (MTs) promoter genes. MTs
are a group of low molecular weight proteins that can bind group Ib and IIb transition metals,
forming tetra-metal-thiolate clusters with their numerous cysteine residues. One of the major
properties of MTs is their ability to be transcriptionally induced by heavy metals and other
physiological stressors. Generally, multiple isoforms encoded by separate genes are present in most
organisms, and different cells express distinct MT isoforms with varying levels of expression and
inducibility. To date, five different metallothionein genes have been identified in T. thermophila
whose promoters respond preferentially to cadmium or copper (namely MTT1, MTT2, MTT3, MTT4
and MTT5).
Here we demonstrate that the 5’-region of the MTT2 and MTT5 genes can act as efficient promoters
(once induced by copper and cadmium respectively) to drive the expression of heterologous genes
in the Tetrahymena system. In the latter case, a gene for a candidate vaccine antigen against
Ichthyophthirius multifiliis, a ubiquitous parasite of freshwater fish, was expressed at high levels in
transformed T. thermophila cell lines. Moreover, the recombinant protein was properly folded and
targeted to the plasma membrane in its correct three-dimensional conformation.
MTT2 promoter is highly regulatable simply by adding or depleting copper from the medium and
the strength of its induction increased with increasing concentrations of the metal, reaching levels
comparable to that achieved with the robust cadmium-inducible MTT1.
MTT5 promoter is inducible mainly by cadmium at levels two times higher than those obtained with
MTT1. Data from deletion/mutational analyses, performed on MTT5 5’-flanking region, show that a
region from –300 bp to –274 bp, named TtCdRE (Tetrahymena thermophila Cadmium-ResponseElement), is necessary to elicit high-level expression of the transgene following induction with
cadmium. This is the first report of a functional cis-acting element in a protozoan MT gene.
The data reported here indicated that MTT2 and MTT5 promoters may be attractive alternative to
the promoters usually employed for driving ectopic gene expression and could have a great impact
on biotechnological perspectives. Further deletion analyses and site-specific mutagenesis
experiments will be required to identify metal-responsive regulatory elements in both the
promoters.
54
RECONSTRUCTION AND FUNCTIONAL ANALYSIS OF ALTERED MOLECULAR
PATHWAYS IN HUMAN ATHEROSCLEROTIC ARTERIES
Stefano Cagnin*1,2, Michele Biscuola3, Cristina Patuzzo3, Elisabetta Trabetti3, Alessandra Pasquali3,
Paolo Laveder2, Giuseppe Faggian4, Mauro Iafrancesco4, Alessandro Mazzucco4, Pier Franco
Pignatti3, Gerolamo Lanfranchi1,2.
*1. Dipartimento di Biologia and 2. CRIBI, Università degli Studi di Padova
3. Department of Mother and Child, Biology and Genetics, Section of Biology and Genetics,
University of Verona, Verona, Italy
4. Division of Cardiac Surgery, University of Verona Medical School, Verona, Italy
ABSTRACT
Background: Atherosclerosis affects aorta, coronary, carotid, and iliac arteries most frequently than
any other body vessel. There may be common molecular pathways sustaining this process. Plaque
presence and diffusion is revealed by circulating factors that can mediate systemic reaction leading
to plaque rupture and thrombosis.
Results: We used DNA microarrays and meta-analysis to study how the presence of calcified plaque
modifies human coronary and carotid gene expression. We identified a series of potential human
atherogenic genes that are integrated in functional networks involved in atherosclerosis. Caveolae
and JAK/STAT pathways, and S100A9/S100A8 interacting proteins are certainly involved in the
development of vascular disease. We found that the system of caveolae is directly connected with
genes that respond to hormone receptors, and indirectly with the apoptosis pathway.
Cytokines, chemokines and growth factors released in the blood flux were investigated in parallel.
High levels of RANTES, IL-1ra, MIP-1alpha, MIP-1beta, IL-2, IL-4, IL-5, IL-6, IL-7, IL-17,
PDGF-BB, VEGF and IFN-gamma were found in plasma of atherosclerotic patients and might also
be integrated in the molecular networks underlying atherosclerotic modifications of these vessels.
Conclusions: The pattern of cytokine and S100A9/S100A8 up-regulation characterizes
atherosclerosis as a proinflammatory disorder. Activation of the JAK/STAT pathway is confirmed
by the up-regulation of IL-6, STAT1, ISGF3G and IL10RA genes in coronary and carotid plaques.
The functional network constructed in our research is an evidence of the central role of STAT
protein and the caveolae system to contribute to preserve the plaque. Moreover, Cav-1 is involved
in SMC differentiation and dyslipidemia confirming the importance of lipid homeostasis in the
atherosclerotic phenotype.
REFERENCES
1) Cagnin S, et al (2009) Reconstruction and functional analysis of altered molecular pathways in human atherosclerotic
arteries. BMC Genomics 10:13.
55
CARDIOPROTECTIVE EFFECTS OF ISCHEMIC PRECONDITIONING AND CYCLOPHILIN
D KNOCKOUT ARE ADDITIVE
Andrea Carpi*1, Roberta Menabò2, Emy Basso1, Paolo Bernardi1,2 and Fabio Di Lisa1,2
*1. Department of Biomedical Sciences, University of Padova
2. Insititute of Neuroscience, CNR
ABSTRACT
Ischemic preconditioning (IPC) is suggested to protect against ischemia/reperfusion (I/R) injury by
preventing opening of the mitochondrial permeability transition pore (PTP) upon post-ischemic
reperfusion (1,2). On the other hand, IPC protection has been reported to be abrogated by
cyclosporin A (CsA) that prevents PTP opening by binding to cyclophilin D (CypD) (3). However,
CsA effects other than PTP desensitization might explain the loss of protection. This study was
aimed at investigating the contribution of CypD, and indirectly of PTP, to IPC by exploiting a
genetic approach. Hearts isolated from CypD-/- mice (4) and wild type (WT) littermates were
perfused using the following protocols: (i) 40 min of global, no-flow ischemia (I), followed by 15
min of reperfusion (R); (ii) 3 cycles of 5 min I/5 min R (IPC) prior to the above mentioned I/R
protocol; (iii) 1 cycle of IPC. Release of lactate dehydrogenase (LDH) in the coronary effluent was
monitored during R to assess the loss of myocardial viability. The absence of CypD resulted in a
significant decrease of LDH release induced by post-ischemic reperfusion (23.9±3.7% vs 41±4.7%
of total LDH content in CypD-/- and WT mice, respectively). As expected the 3 cycle IPC protocol
resulted in a high degree of protection that however was significantly enhanced by CypD ablation
(17.2±2% vs 7.7±1.6% in WT and CypD-/-, respectively). The increase in IPC protection was
decreased when the less robust protocol of 1 cycle IPC was applied (19.9±12.1% vs 13.7±4.9 in WT
and CypD-/-, respectively) and appears to be rather specific. Indeed, IPC effects were not modified
by other protective strategies, such as treatment with mercaptopropionylglycine or deletion of
p66Shc that protect against I/R injury by preventing PTP opening (5). While arguing against the
requirement for PTP opening to elicit IPC protection, the present results indicate that the protection
afforded by CypD absence is additive with that of IPC suggesting that robust IPC might prevent
PTP opening at sites other than CypD. It is tempting to speculate that PTP opening is stimulated by
multiple pathways, and that the recent clinical evidence of cardioprotection by CsA (6,7) can be
improved by the compounds inhibiting the PTP without interfering with CypD.
REFERENCES
1) Di Lisa F, et al (2007) Mitochondria and cardioprotection. P.Heart Fail Rev. 12, 249-260.
2) Di Lisa F, Bernardi P (2006) Mitochondria and ischemia-reperfusion injury of the heart: fixing a hole. Cardiovasc
Res. 70, 191-199.
3) Hausenloy D, et al (2004) Transient mitochondrial permeability transition pore opening mediates preconditioninginduced protection. Circulation. 109, 1714-1717.
3) Basso E, et al (2005) Properties of the permeability transition pore in mitochondria devoid of Cyclophilin D. J Biol
Chem. 280, 18558-18561.
4) Migliaccio E, et al (1999) The p66shc adaptor protein controls oxidative stress response and life span in mammals
Nature. 402, 309-313.
5) Gomez L, et al (2009) Inhibition of mitochondrial permeability transition pore opening: translation to patients.
Cardiovasc Res. In Press.
6) Piot C, et al (2008) Effect of cyclosporine on reperfusion injury in acute myocardial infarction. N Engl J Med. 359,
473-481.
56
BIO PhD & PostDoc Day ’09 Complesso Biologico “A. Vallisneri”, Università di Padova
STRUCTURAL CHARACTERIZATION OF HIU- HYDROLASE, OHCU-DECARBOXYLASE
AND PUUE-ALLANTOINASE, THREE ENZYMES INVOLVED IN THE URIC ACID AND
ALLANTOIN DEGRADATION
Laura Cendron*1,2, Ileana Ramazzina3, Claudia Folli3, Rodolfo Berni3, Riccardo Percudani3,
Giuseppe Zanotti1,2
*1. Department of Biological Chemistry, University of Padova, Viale G. Colombo 3, 35131
Padova, Italy; Tel: +390498276286; Email: [email protected]
2. Venetian Institute of Molecular Medicine, VIMM, Padova
3. Department of Biochemistry and Molecular Biology, University of Parma, Parma, Italy
ABSTRACT
The three steps involved in the complete degradation of uric acid to S-allantoin have been
elucidated very recently by comparative analysis of different genomes. Inactivation of this pathway,
occurred during hominoid evolution, resulted in a high concentration of the antioxidant urate in the
blood and susceptibility to gout. Urate oxidase, the first enzyme of this pathway, had already been
characterized, whereas nothing was known about the other two. Furthermore, allantoin is one of the
most nitrogen-rich organic compounds. Some plants economise carbon by using this compound as a
nitrogen carrier. Conversely, soil animals eliminate the excess of nitrogen present in purine bases
mainly as uric acid and allantoin. In turn, microorganisms can utilise these compounds as nitrogen
sources, by degrading them to carbon dioxide, glyoxylate and ammonia. Allantoin, either deriving
from endogenous purine breakdown or imported through specific permeases, is hydrolysed by
allantoinase to allantoate. Since this step is required for the utilisation of the nitrogen present in the
allantoin molecule, no organism is expected to be able to use allantoin for growth without
possessing allantoinase activity. The already known allantoinase gene (DAL1), however, is missing
in many completely sequenced organisms able to use allantoin as a nitrogen source. In collaboration
with the group of R. Percudani, we demonstrated that an alternative allantoinase gene (puuE) can be
precisely identified by analyzing its logic relationship with three other genes of the pathway. The
novel allantoinase was previously annotated as polysaccharide deacetylase for its homology with
enzymes that catalyze hydrolytic reactions on chitin or peptidoglycan substrates. We carried out the
characterization by crystallographic techniques of the three enzymes that catalyze the conversion of
5-hydroxyisourate (HIU) to S-allantoin and further on, to allantoate: HIU-hydrolase, OHCUdecarboxylase and PuuE allantoinase. The proteins of interest have been purified, crystallized and
the structures solved either by molecular replacement or by using the anomalous signal of a Se-Met
enriched derivative. The active site has been explored by co-crystallization methods with substrate
analogs.
57
A CA2+-INCREASE IN THE PEROXISOME LUMEN ACTIVATES H2O2 SCAVENGING IN
PLANT LEAF CELLS IN VIVO.
Alex Costa, Ilaria Drago, Smrutisanjita Behera, Michela Zottini, Paola Pizzo, Julian I Schroeder,
Tullio Pozzan, Fiorella Lo Schiavo.
ABSTRACT
Oxidative stress is a major challenge for all cells living in an oxygen-based world and this is
particularly true for plant cells that produce massive amounts of diverse reactive oxygen species
(ROS). Among ROS, H2O2 can exert a dual role both as second messenger or cell death inducing
agent1,2,3. Peroxisomes are key players in cellular H2O2 homeostasis4 and evidence suggests that
Ca2+ plays a key role in modulating H2O2 levels: i) many stimuli induce both a cytoplasmic Ca2+
increase5,6 and H2O2 production5,7; ii) in vitro, Ca2+ and calmodulin (CaM) can bind to and activate
peroxisomal plant catalases8; iii) the opening of hyperpolarisation-activated Ca2+ channels requires
ROS9,10; iv) Ca2+ directly stimulates plasma membrane proteins involved in H2O2 production upon
biotic and abiotic stimuli2. However, no information is available on the participation of peroxisomes
in plant cell Ca2+ homeostasis or on the role of intraperoxisomal Ca2+ in plant physiology. By using
a novel genetically encoded Ca2+ indicator selectively targeted to the peroxisome lumen we here
demonstrate that peroxisome Ca2+ concentration, [Ca2+]p, rapidly equilibrates with the cytosolic
Ca2+ concentration, [Ca2+]c. Using the H2O2 sensor HyPer11 selectively targeted to cytosol or
peroxisomes, we also demonstrate that a rise in [Ca2+]p results in a major potentiation of H2O2
scavenging due to the activation of Arabidopsis CAT3. These data provide compelling evidence for
a major role for [Ca2+]p in ROS level regulation especially during the onset of senescence.
REFERENCES
1) Wang P, Song CP (2008) Guard-cell signalling for hydrogen peroxide and abscisic acid. New Phytol. 178, 703-718.
2) Apel K, Hirt H (2004) Reactive oxygen species: metabolism, oxidative stress, and signal transduction. Annu. Rev.
Plant Biol. 55, 373-399.
3) Alvarez ME, et al (1998) Reactive oxygen intermediates mediate a systemic signal network in the establishment of
plant immunity. Cell 92, 773-784.
4) Nyathi Y, Baker A (2006) Plant peroxisomes as a source of signalling molecules. Biochim. Biophys. Acta 1763,
1478-1495.
5) Leshem Y, Seri L, Levine A (2007) Induction of phosphatidylinositol 3-kinase-mediated endocytosis by salt stress
leads to intracellular production of reactive oxygen species and salt tolerance. Plant J. 51, 185-197.
6) Ranf S, et al (2008) Loss of the vacuolar cation channel, AtTPC1, does not impair Ca2+ signals induced by abiotic
and biotic stresses. Plant J. 53, 287-299.
7) Hetherington AM, Brownlee C (2004) The generation of Ca2+ signals in plants. Annu. Rev. Plant Biol. 55, 401-427.
8) Yang T, Poovaiah BW (2002) Hydrogen peroxide homeostasis: activation of plant catalase by calcium/calmodulin.
Proc. Natl Acad. Sci. U S A. 99, 4097-4102.
9) Demidchik V, Shabala SN, Davies JM (2007) Spatial variation in H2O2 response of Arabidopsis thaliana root
epidermal Ca2+ flux and plasma membrane Ca2+ channels. Plant J. 49, 377-386.
10) Pei ZM, et al (2000) Calcium channels activated by hydrogen peroxide mediate abscisic acid signalling in guard
cells. Nature 406, 731-734.
11) Belousov VV, et al (2006) Genetically encoded fluorescent indicator for intracellular hydrogen peroxide. Nat.
Methods 3, 281-286.
58
PDF-MODULATED VISUAL INPUTS AND CRYPTOCHROME DETERMINE EVENING
ACTIVITY IN DROSOPHILA
Paola Cusumano*1,2, Klarsfeld A. 2, Chélot E. 2, Picot M. 2, Richier B. 2, Rouyer F. 2
Italy; Tel: +39 049 82 76 228; Email: [email protected]
2. Institut de Neurobiologie Alfred Fessard, CNRS NGI UPR2216, Avenue de la Terrasse 91198,
Gif-sur-Yvette, France; Tel: +33 1 69823432
ABSTRACT
As many animal species, Drosophila melanogaster is crepuscular, showing most of its locomotor
activity at dawn and dusk. These activity rhythms are controlled by a circadian clock that is located
in the brain and relies on about 70 clock neurons in each hemisphere. Two distinct oscillators
control the bimodal activity profile in light-dark (LD) cycles (1,2). The lateral neurons that express
the neuropeptide Pigment dispersing Factor (PDF) control the morning anticipation (LN-MO:
Lateral Neurons Morning Oscillator) (2). The lateral neurons evening oscillator (LN-EO) is made of
four PDF-negative lateral neurons and plays a key role in promoting diurnal activity at dusk (2,3).
Synchronization of the behavioral clock to LD cycles relies on the blue-light photoreceptor
cryptochrome and the visual system (4). We show that the LN-EO autonomously synchronizes to
LD cycles through either the Cryptochrome (CRY) that it expresses or the visual system. When
CRY is not activated, flies depleted for the PDF neuropeptide (5) or the PDF receptor (6-8) display
reversed PER oscillations in the LN-EO and loose the evening activity. This shows that CRY or
PDF-modulated visual inputs phase PER cycling in the LN-EO to generate evening activity. Rescue
experiments indicate that the presence of evening activity rely on PDF secretion from the large
LNvs and PDF receptor function in the LN-EO. Surprisingly, this new PDF signaling pathway does
not require a functional clock in the large LNvs. The temporal control of Drosophila diurnal
behavior thus appears to result from a balance between CRY signaling in the evening LNs and a
clock-independent modulation of their visual inputs by PDF.
REFERENCES
1) Stoleru D et al (2004) Coupled oscillators control morning and evening locomotor behavior of Drosophila. Nature
431, 862-868.
2) Grima B et al (2004) Morning and evening peaks of activity rely on different clock neurons of the Drosophila brain.
Nature 431, 869-873.
3) Picot, M et al (2007) Light activates output from evening neurons and inhibits output from morning neurons in the
Drosophila circadian clock. PLoS Biol 5, e315.
4) Helfrich-Förster C et al (2001) The circadian clock of fruit flies is blind after elimination of all known
photoreceptors. Neuron 30, 249-261.
5) Renn SC et al (1999) A pdf neuropeptide gene mutation and ablation of PDF neurons each cause severe
abnormalities of behavioral circadian rhythms in Drosophila. Cell 99, 791-802.
6) Mertens I et al (2005) PDF Receptor Signaling in Drosophila Contributes to Both Circadian and Geotactic Behaviors.
Neuron 48, 213-219.
7) Lear BC et al (2005) A G Protein-Coupled Receptor, groom-of-PDF, Is Required for PDF Neuron Action in
Circadian Behavior. Neuron 48, 221-227.
8) Hyun S et al (2005) Drosophila GPCR Han Is a Receptor for the Circadian Clock Neuropeptide PDF. Neuron 48,
267-278.
59
IMAGING OF BACTERIAL TOXINS THAT INTERFERE WITH cAMP SIGNALING
PATHWAYS
Federica Dal Molin *, Irene Zornetta, Lucia Brandi, Morena Simonato, Fiorella Tonello, Cesare
Montecucco
Email:[email protected]
ABSTRACT
Many different microbial pathogens have developed virulence factors to alter cAMP intracellular
concentrations and cAMP-dependent processes. The high specificity of signals transduced by
cAMP is achieved by the tight compartmentation of the cellular machinery that controls this second
messenger (ACs, phosphodiesterases and anchoring proteins) (1). Hence, the alteration of cAMP
signaling pathways induced by pathogens depends on the localization that virulence factors can
reach inside the host cell. We studied Bacillus anthracis toxins, edema toxin (ET) and lethal toxin
(LT), composed of two enzymes, an adenylate cyclase (AC) named EF and a protease named LF,
and one common binding protein, named PA.
First, the intracellular localization of EF and LF was investigated. Two fluorescent chimeric
proteins, EF-EGFP and LF-EGFP, were produced; intoxicated cells were fixed to visualize the cell
binding, internalization along the endocytic pathway and translocation into the cytosol of the host
cell (2). It was found that both translocate from late endosomes around the nucleus, but EF-EGFP
still associates with the cytosolic side of those organelles, while LF-EGFP diffuses. A further aim is
to make swapped EF and LF, inverting the catalytic and the PA binding domain, to investigate the
structural features responsible for their different intracellular targeting.
Next, the increase of cAMP induced by ET was imaged by using cAMP-sensitive fluorescent
probes that allow a high temporal, spatial and quantitative resolution of cAMP dynamics in live
cells. We compared ET with CyaA, an AC from Bordetella pertussis, and with toxins that alter
cAMP via different mechanisms as pertussis toxin (PT) from Bordetella pertussis and cholera toxin
(CT) from Vibrio cholera (3,4). cAMP imaging revealed that in intoxicated cells ET creates a
gradient of cAMP decreasing from the perinuclear area to the plasma membrane, while CyaA
creates a gradient in the opposite direction, according to its localization at the cell membrane. This
is at variance with PT and CT that do not create cAMP gradients, but rather a cAMP increase
spreading throughout the cytosol. Nevertheless, they reach endogenous AC localized on the
cytosolic face of the plasma membrane, suggesting that a given localization of bacterial toxins
within the host cell does not necessarily determine a gradient of effects.
Further studies will deepen the interactions of ET-induced cAMP with the signaling pathways
mediated by cGMP, a second messenger that interweaves with cAMP in the regulation of cellular
processes and host immunity.
REFERENCES
1) Zaccolo M (2006) Phosphodiesterases and compartmentalized cAMP signalling in the heart. Eur J Cell Biol 85, 693697.
2) Puhar A, Montecucco C (2007) Where and how do anthrax toxins exit endosomes to intoxicate host cells? Trends
Microbiol 15, 477-482.
3) Dal Molin F et al (2006) Cell entry and camp imaging of anthrax edema toxin. EMBO J. 25, 5405-5413.
4) Dal Molin F et al (2008) cAMP imaging of cells treated with pertussis toxin, cholera toxin, and anthrax edema toxin.
Biochem. Biophys. Res.Commun 376, 429-433.
60
VOLTAGE-DEPENDENT ANION CHANNELS IN THE CONTROL OF APOPTOSIS AND
AUTOPHAGY
Diego De Stefani*, Cristina Mammucari, Roberta Siviero, Erika Zecchini, Rosario Rizzuto
Email:[email protected]
ABSTRACT
We investigated the genomic program controlled by PGC-1α (PPARγ-coactivator-1α), that
regulates mitochondrial number and oxidative metabolism at the transcriptional level. Its expression
is upregulated in various diseases and stress conditions (e.g. adaptive thermogenesis and fasting)
and coordinates the expression of a broad number of mitochondrial genes, including metabolic
enzymes, respiratory complexes and uncoupling proteins (1). We showed that the proteomic
changes cause a significant variation in mitochondrial responsiveness to cellular Ca2+ signals, and
thus in the efficacy of apoptotic stimuli for which Ca2+ acts as a fundamental sensitizing agent (2).
We have pursued this work by carrying out microarray analyses of mitochondrial signaling proteins
that are up- and down-regulated by PGC-1α. Among various interesting hits we have been obtained,
we focused on the voltage-dependent anion channel (VDAC) isoforms, that proved differentially
controlled by PGC-1α (VDAC1 is downregulated and VDAC2 and 3 are upregulated by PGC-1α).
As to the significance of this change, we could demonstrate that, while all isoforms are equally
effective in transferring Ca2+ ions across the outer mitochondrial membrane, they exhibit opposite
functions on cell survival in various stress conditions (3, 4). This could be due, at least in part, to a
VDAC1-selective coupling to the endoplasmic reticulum Ca2+ releasing channels (5), thus
suggesting the existence of a preferential signaling route transferring Ca2+-dependent apoptotic
stimuli from one organelle to the other. Moreover, VDAC2 appears essential for the induction of
macroautophagy, a process that has attracted major interest in the recent years, as it allows cells to
withstand nutrient deprivation and other stress conditions, before undergoing an eventually
morphologically distinct death event (type II cell death) (6). In particular, our data demonstrate that
VDAC2 physically interacts with the nutrient sensor mTOR (mammalian Target of Rapamycin)
impairing its activity. Apoptosis and autophagy share common regulatory molecules (e.g. Bcl-2)
and these data suggest that mitochondria may be the site where co-ordinated regulation of the two
processes occurs.
REFERENCES
1) Lin J, Handschin C, Spiegelman BM (2005) Metabolic control through the PGC-1 family of transcription
coactivators. Cell Metab 1, 361-370.
2) Bianchi K, Vandecasteele G, Carli C, Romagnoli A, Szabadkai G, Rizzuto R. (2006) Regulation of Ca2+ signalling
and Ca2+-mediated cell death by the transcriptional coactivator PGC-1alpha. Cell DeathDiffer13, 586-596.
3) Cheng EH, Sheiko TV, Fisher JK, Craigen WJ, Korsmeyer SJ. (2003) VDAC2 inhibits BAK activation and
mitochondrial apoptosis. Science 301, 513-517.
4) Rapizzi E, Pinton P, Szabadkai G, Wieckowski MR, Vandecasteele G, Baird G, et al. (2002) Recombinant
expression of the voltage-dependent anion channel enhances the transfer of Ca2+ microdomains to mitochondria. J Cell
Biol 159, 613-624.
5) Szabadkai G, Bianchi K, Varnai P, De Stefani D, Wieckowski MR, Cavagna D, et al (2006) Chaperone-mediated
coupling of endoplasmic reticulum and mitochondrial Ca2+ channels. J Cell Biol 175, 901-911.
6) Kroemer G, Levine B. (2008) Autophagic cell death: the story of a misnomer. Nat Rev Mol Cell Biol 9,1004-1010.
61
SIGNALLING AND REPAIRING DNA DAMAGE IN DIFFERENT PHASE OF CELL CYCLE
Sonia Fabris1,*, Maddalena Mognato1, Cristina Girardi1, Roberto Cherubini2 and Lucia Celotti1,2
Italy; Tel. +390498276286; E-mail: [email protected]
2. Laboratori Nazionali di Legnaro, I.N.F.N.
ABSTRACT
The genome is continually exposed to exogenous agent and endogenous processes that damage
DNA. The DNA double-strand break (DSB) represent the most severe damage for the genome
integrity and is the main lesion induced by ionizing radiation. If incorrectly repaired they can
provide the opportunity for inappropriate recombination with other sites in the genome leading to
tumorigenesis. There are two major pathways of DSB repair: non-homologous end joining (NHEJ)
and homologous recombination (HR). Both repair systems play an important role in mammalian
DSB repair, and the choice of which pathways to use depends on the cell cycle phase of the cell.
The aim of this work is to analyse the contribution of HR and NHEJ in DNA DSB repair of
proliferating and quiescent cells, after irradiation with γ rays and low-energy protons. We used a
human cell line of lung fibroblasts (CCD-34Lu) and determined the cellular distribution throughout
the cell cycle by FACS analysis. We studied the kinetics of induction and repair of DSB by in situ
immunofluorescence, following the formation and disappearance of ionizing radiation-induced foci
(IRIF) of γ-H2AX and 53BP1. RAD51 and Ku70 proteins have been selected as marker of HR and
NHEJ, respectively. To obtain information on single cells we analysed by confocal microscopy the
presence of 53BP1 and RAD51 foci in relation to an S/G2/M phase marker, CENP-F, a protein of
the nuclear matrix that gradually accumulates during the cell cycle until it reaches peak level in G2
and M phase cells and is rapidly degraded upon completion of mitosis.
In cycling fibroblasts we observed the maximum foci induction after 2 hours of irradiation with 0.5
Gy of protons (7.2 IRIF/cell) and after 24 hours DNA repair was almost completed (2.4 IRIF/cell).
By cytofluorimetric analysis we found a partial block in G2 phase after 6 hours of irradiation with a
decrease of S phase of a half in comparison to the control. Moreover, about 63% of S/G2/M phase
cells (CENP-F positive) showed RAD51 foci, which were presented also in 12% of CENP-F
negative cells. Western blot analysis showed that in proliferating cells, both irradiated and non
irradiated, have the same amount of Ku70 and RAD51 proteins. S/G2/M phase cells (CENP-F
positive) have a higher number of foci with respect to G1 cells and the IRIF number decreases
during the repair time faster in G1 cells in comparison with S/G2/M cells. In quiescent fibroblasts
foci of RAD51 were absent, in agreement with Western blot data, which instead showed the
presence of Ku70 in control and irradiated cells.
Our preliminary results demonstrate the constitutive expression of RAD51 and Ku70 in cycling
fibroblasts, regardless of radiation exposure and the absence of RAD51 in quiescent fibroblasts,
where NHEJ is the only DSB repair system working. Moreover, our data suggest that in cycling
fibroblasts the contribution of HR to DSB repair is higher than expected, since it occurred not only
after DNA replication as demonstrated by the presence of RAD51 foci in a part of CENP-F
negative cells. Strikingly, we observed that the number of IRIF decrease faster in G1 cells than in
S/G2/M phases, suggesting that the DSB rejoining is more efficient when NHEJ operates alone. On
the other hand the rejoining of DSB by NHEJ could result in deletion and genomic instability,
because of the error-prone nature of this repair pathway.
62
STUDY OF THE ROLE OF Aβ PEPTIDES ON ANGIOGENESIS RELATED TO ALZHEIMER
DISEASE
Lara Fioravanzo*, Marcella Folin
ABSTRACT
In the last years a growing amount of evidence suggests that Alzheimer’s disease (AD) is not only a
neurodegenerative disease but also a vascular pathology characterized by cerebral microvessel
alterations, in particular increased vascular density close to senile plaques (deposits of
proteinaceous material with the β-amyloid peptides, Aβ, as the major component) in comparison to
older non-demented people (5). The aim of the research was the attempt to understand some of the
pathogenic angiogenic mechanisms in order to develop clinic strategies for tissue regeneration. Our
in vivo studies on chick embryo ChorioAllantoic Membrane (CAM) and ex-vivo on aortic ring
demonstrated that Aβ1-40 and Aβ1-42 peptides increase the formation of capillary-like structures.
Diversely, in Rat Cerebral Endothelial Cell (RCECs) cultures they significantly decrease the
capillary-like structure formation. Our results confirm literature data (4; 1) where the effect of Aβ
peptides depends on the animal model and on where body zone cells come from. As no differences
were observed between the two isoform effects, only the Aβ1-42 peptide was assayed. Subsequent
studies pointed out no differences in RCEC VEGF-A release and mRNA transcription after 3, 24
and 48h of exposure to Aβ peptide. Moreover, diversely to in vivo results (6), in vitro Aβ1-42 did not
affect the VEGF-A release and mRNA expression on Rat Hippocampal Astrocytes (RHAs) at the
same times of treatment. Excluding that Aβ acts as an direct pro-angiogenic factor, RCECs were
cultured for 24 and 48h on 24 and 48h control and Aβ treated RHA culture medium (RHAm). Both
RHAm significantly increased the formation of capillary-like structures on RCECs, suggesting that
RHAs release pro-angiogenic cytokines. Thus the levels of IL-1β, IL-6 and TNF-α on 6, 12, 24 and
48h RHAm before and after the exposure to RCECs were determined. After 6 and 12h levels of all
the cytokines, in particular the levels of TNF-α, significantly increased in comparison to control on
Aβ1-42 treated RHAm. Literature data demonstrate that TNF-α increases expression and release of
other pro-angiogenic factors supporting its importance in neovascularisation (2). Moreover after
24h of exposure to both cell types, cell culture mediums significantly increased the levels of IL-6
while other cytokine levels did not vary. Finally cell viability results confirm literature data where
high concentrations of pro-inflammatory cytokines increase apoptosis, oxidative stress and downregulate eNOS bioactivity (3).
REFERENCES
1) Cantara S, Donnini S, Morbidelli L, Giachetti A, Schulz R, Memo M, Ziche M (2004) Physiological levels of
amyloid peptides stimulate the angiogenic response through FGF-2. FASEB J. 18, 1943-1945.
2) Hangai M, He S, Hoffmann S, Lim JI, Ryan SJ, Hinton DR (2006) Sequential induction of angiogenic growth factors
by TNF-alpha in choroidal endothelial cells. J Neuroimmunol. 171, 45-56.
3) Kofler S, Nickel T, Weis M, (2005) Role of cytokines in cardiovascular diseases: a focus on endothelial responses to
inflammation. Clin Sci 108, 205-213.
4) Paris D, Townsend K, Quadros A, Humphrey J, Sun J, Brem S, Wotoczek-Obadia M, DelleDonne A, Patel N,
Obregon DF, Crescentini R, Abdullah L, Coppola D, Rojiani AM, Crawford F, Sebti SM, Mullan M (2004) Inhibition
of angiogenesis by Abeta peptides. Angiogenesis 7, 75-85.
5) Vagnucci AH Jr, Li WW (2003) Alzheimer's disease and angiogenesis. Lancet 361, 605-608.
6) Zand L, Ryu JK, McLarnon JG (2005) Induction of angiogenesis in the beta-amyloid peptide-injected rat
hippocampus. Neuroreport. 16, 129-132.
63
A MUSCLE, TWO STATES
Susanna Franzoso*, Manuela Miuzzo, Elisa Bianchini, Giulia Pilia, Dorianna Sandonà, Romeo
Betto.
*Viale G. Colombo 3, 35131, Padova, Italy. Tel: +390498276026;
ABSTRACT
Our lab is dedicated to two main projects:
1) Processing, assembly and trafficking of sarcoglycan complex. Role of the ER quality-control and
ubiquitin-proteasome systems in the pathogenesis of sarcoglycanopathies.
Sarcoglycanopathies are progressive muscle-wasting disorders caused by genetic defects of four
glycoproteins known as α-, β-, γ-, and δ-sarcoglycan (SG), elements of a key transmembrane
complex of striate muscles. The proper assembly of the SG complex represents a critical issue of
sarcoglycanopathies, as several mutations perturb tetramer formation. Misfolded proteins are
generally discarded through a quality-control system that, however, could lead to the removal of
functional polypeptides. To investigate the ER processing of wild type and SG mutants, we
generated a heterologous cell system constitutively expressing three (βγδ-HEK) of the four SGs. In
these cells, sarcoglycan complex is formed only when also α-SG is present, whereas transfection
with disease-causing α-SG mutants determines the degradation of mutated proteins and the absence
of the complex in the cell membrane (1). This result closely reproduces the observation made in
muscle patients. Inhibition of proteasome reduced degradation of mutants and facilitated the
targeting of SG complex to the plasma membrane. Our efforts are mainly focused in the
identification of ER components involved in the recognition, retro-translocation and ubiquitination
of α-SG mutants. This knowledge will permit to selectively modulate ERAD system and prevent or
slow down degradation of α-SG mutants and facilitate SG complex membrane targeting.
2) Microgravity environment: effect on cultured adult muscle fibers and skeletal muscles.
Muscle atrophy is a physiological compensatory response to reduced use. Reduction of muscle mass
is due to enhanced protein degradation via the ubiquitin-proteasome and the autophagy-lysosome
systems. Our group is involved in the Italian Space Agency missions aimed at characterize the
signaling pathways involved in muscle atrophy during spaceflights, pathways that have never been
explored so far. In the unmanned Russian Foton-M3 mission (2007), we exposed to the
microgravity environment single muscle fibers from adult mice cultured in automated bioreactors.
Gene expression analyses demonstrated the dysregulation of genes involved in proteasomedependent degradation but not in autophagy-lysosome proteolysis (2). A second mission, scheduled
on August 2009, will study the long-term effects of spaceflight on skeletal muscle of mice
individually hosted in an automated rodent payload (Mice Drawer System, MDS) on board the
International Space Station. Since the MDS size is smaller than standard laboratory cages and may
affect overall mouse activity and muscle properties, we performed pre-flight tests on mice hosted
for 20, 60, and 100 days in MDS. Muscle morphology and gene expression analyses indicate that
mice well adapt to the prolonged housing in MDS (3).
REFERENCES
1) Gastaldello S, D’angelo S, Franzoso S, et al (2008) Inhibition of proteasome activity promotes the correct
localization of disease-causing alpha-sarcoglycan mutants in HEK-293 cells constitutively expressing β-, γ-, and δsarcoglycan. Am J Pathol. 173, 170-181.
2) Franzoso S, et al (2009) Cultured adult muscle fibers in the microgravity environment. The MYO experiment in the
Foton-M3 space flight mission. BAM 19, 65-76.
3) Betto R, Franzoso S, et al (2009) On ground preflight studies on skeletal muscles from mice kept in “Mice Drawer
System” (MDS) payload. BAM 19, 97-106.
64
BIOPHYSICAL AND BIOCHEMICAL METHODS TO UNRAVEL STRUCTURAL FEATURES
OF AMYLOIDOGENIC PROTEINS
Erica Frare
CRIBI Biotechnology Centre, University of Padua, Viale G. Colombo 3, 35121 Padua, Italy
Tel. +39-049-827-6157; E-mail: [email protected]
ABSTRACT
The formation of amyloid precipitates of otherwise soluble proteins characterise a number of
neurodegenerative disorders, including Alzheimer’s and Parkinson’s diseases. Nowadays the
analysis of the molecular features of protein amyloid aggregates is particularly timely, considering
that these diseases affect millions of individuals in modern world. However, the large molecular
weight, intrinsic heterogeneity and transient nature of protein amyloids make their study highly
challenging. We have used a combination of experimental techniques, such as circular dichroism,
fluorescence emission, Fourier-transform infrared (FT-IR) spectroscopy, electron microscopy,
limited proteolysis and mass spectrometry, in order to monitor the overall process of fibril
formation and to characterize distinct species on the aggregation pathway of a number of
amyloidogenic proteins. These studies were conducted on the SH3 domain of bovine phosphatidylinositol 3-kinase [1], hen and human lysozymes [2–4], bovine α-lactalbumin [5], human αsynuclein [6] and horse apomyoglobin (apoMb) [7]. Protein fragments have also been prepared in
order to study the aggregation propensity of different regions of a protein chain [2, 5–7].
The amyloid fibrils of lysozyme are formed by a protease-resistant core that was found to be the
most aggregation prone segment of the chain, suggesting that amyloid fibril formation initiates by
the partial unfolding of this region of the protein [2, 3]. The early oligomeric species of both SH3
domain and lysozyme were shown to be the most toxic protein species causing cell dysfunction and
death. Unlike the monomeric protein, oligomers have a misfolded structure that display enhanced
susceptibility to proteolysis, indicating a quite unfolded and flexible structure [1, 4]. These features
can explain their inappropriate interaction with cellular components, notably membranes, leading to
cellular toxicity.
Several protein fragments and truncated versions of proteins have been analysed for their
amyloidogenic behaviour, such as fragment 1–29 of apoMb [7]. Overall, these studies allowed us to
obtain important insights into the physicochemical determinants of fibril formation by proteins,
emphasizing that protein fragments obtained by limited proteolysis [8] are very useful for studying
protein aggregation. Indeed, an aspect that does not appear to be sufficiently emphasized in current
literature is that a large proportion of physiologically relevant amyloid precipitates are made up of
protein fragments derived from proteolysis of larger protein precursors.
REFERENCES
1) Polverino de Laureto P, Taddei N, Frare E, Capanni C, Costantini S, Zurdo J, Chiti F, Dobson CM, Fontana A (2003)
Protein aggregation and amyloid fibril formation by an SH3 domain probed by limited proteolysis. J. Mol. Biol. 334,
129-141.
2) Frare E, Polverino de Laureto P, Zurdo J, Dobson CM, Fontana A (2004) A highly amyloidogenic region of hen
lysozyme. J. Mol. Biol. 340, 1153-1165.
3) Frare E, Mossuto MF, Polverino de Laureto P, Dumoulin M, Dobson CM, Fontana A (2006) Identification of the
core structure of lysozyme amyloid fibrils by proteolysis. J. Mol. Biol. 361, 551-561.
4) Frare E, Mossuto MF, Polverino de Laureto P, Tolin S, Menzer L, Dumoulin M, Dobson CM, Fontana A (2009)
Characterization of oligomeric species on the aggregation pathway of human lysozyme. J. Mol. Biol. 387, 17-27.
5) Polverino de Laureto P, Frare E, Battaglia F, Mossuto MF, Uversky VN, Fontana A (2005) Protein dissection
enhances the amyloidogenic properties of α-lactalbumin. FEBS J. 272, 2176-2188.
6) Polverino de Laureto P, Tosatto L, Frare E, Marin O, Uversky VN, Fontana A (2006) Conformational properties of
the SDS-bound state of α-synuclein probed by limited proteolysis: Unexpected rigidity of the acidic C-terminal tail.
Biochemistry 45, 11523-11531.
7) Picotti P, De Franceschi G, Frare E, Spolaore B, Zambonin M, Chiti F, Polverino de Laureto P, Fontana A (2007)
Amyloid fibril formation and disaggregation of fragment 1-29 of apomyoglobin: Insights into the effect of pH on
protein fibrillogenesis. J. Mol. Biol. 367, 1237-1245.
8) Fontana A, Polverino de Laureto P, Spolaore B, Frare E, Picotti P, Zambonin M (2004) Probing protein structure by
limited proteolysis. Acta Biochim. Pol. 51, 299-321.
65
ANGIOGENIC-LIKE SPROUTING MECHANISM DURING NORMAL AND REGENERATIVE
DEVELOPMENT IN A COLONIAL ASCIDIAN
Fabio Gasparini*, Paolo Burighel, Lucia Manni, Giovanna Zaniolo
ABSTRACT
Tunicates are useful models for comparing differing developmental processes such as
embryogenesis, asexual reproduction, and regeneration, because they are the closest relatives to
vertebrates and are the only chordates to reproduce both sexually and asexually. Among them, the
ascidian Botryllus schlosseri forms colonies of numerous individuals embedded in the common
tunic, that is the peculiar extracellular matrix (ECM) of tunicates. In B. schlosseri colonies this
ECM holds also the colonial circulatory system (CCS): an anastomized network of vessels defined
by simple epithelia and connected to the open circulatory system of the zooids. Recently, we have
demonstrated that, during asexual propagation, new vessels form by means of a tubular sprouting
mechanism, resembling that occurring in other metazoans, particularly during vertebrate
angiogenesis. Moreover the immunohistology of CCS structures using antibodies against vertebrate
angiogenic factors (VEGF, FGF-2, EGF) and receptors (VEGFR-1, VEGFR-2, EGFR), have shown
that CCS sprouting occurs with their participation (1).
In B. schlosseri, the CCS possesses a remarkable regenerative potentiality, as shown by its ability to
reform tunic and peripheral vascular network. We have also studied the regeneration of
experimentally ablated CCS by analyzing the general dynamics of reorganization of vessels and
tunic, their ultrastructure, cell proliferation, and the immunohistology of regenerating structures (2).
Results show that the regenerative process of CCS occurs, similarly to normal growth, by the
sprouting mechanism, with participation of same angiogenic factors.
Statistical analyses on recent experiments are now indicating a significant increase of tunic and
vessels regeneration in groups of colonies injected with angiogenic factors (EGF, VEGF) with
respect to injected with PBS. This demonstrates that same factors have an angiogenic impact in B.
schlosseri and vertebrates circulatory systems.
Despite the different embryonic origin of their tissues (the former has an ectodermic origin, the
latter mesodermic), all these data indicate that an homologous morphodynamic mechanism,
controlled by homologous signal pathways, is involved during normal growth and regeneration of
both systems. As a conclusion 1) we can hypothesize that tubular sprouting had a parallel evolution
in these two structures, 2) our data support the idea that this morphogenetic mechanism was coopted during the evolution of various developmental processes in several taxa.
Sketch of sprouting in colonial circulatory system (CCS). a: common aspect of epithelium: the basement membrane (bm) faces the vessel lumen
(vl), the tunic (t) touches the apical surface of epithelium, and tight junctions (tj) are in apicolateral position. ec, epithelial cell; n, nuclei. b: In the
sprout apex, cells are thickened and form apical filopodia (arrows). c,d: During sprouting, a migrating epithelial cell (m-ec) protrudes toward the
tunic, but the basement membrane maintains its continuity. Tight junctions (arrowheads) of migrating cells shift from an apicolateral position
toward a basolateral position, while neighboring cells, which maintain the same junctions in the original apicolateral position, converge and touch
each other below the migrating cells. Modified from (1).
REFERENCES
1) Gasparini F, et al (2007) Tubular sprouting as a mode of vascular formation in a colonial ascidian (Tunicata). Dev
Dyn. 236, 719.
2) Gasparini F, et al (2008) Vascular regeneration and angiogenic-like sprouting mechanism in a compound ascidian is
similar to vertebrates. Evol Dev. 10, 591.
66
MULTICRITERA ANALYSIS OF AGROECOSYSTEMS
Tiziano Gomiero*, Maurizio G. Paoletti
ABSTRACT
Agriculture can be defined as the activity carried on by the human species aiming at managing the
natural environment in an attempt towards its domestication. The goal of this domestication is to
provide humankind with an adequate, controlled, and reliable source of food and fibre. To achieve
this goal, agriculture deals with the management of living systems at many scales while at the same
time affecting and being affected by the technical, social and cultural dimensions of human
development. Since thousand years agro-ecosystems are the environments within which human
species lives and evolved. Lately the need to take seriously in to account the long term
sustainability of agro-ecosystems became more and more pressing (1, 2, 3). Being on the interface
of human societies and ecosystems, agriculture is a dynamic, adaptive and evolving system. So in
order to face the challenge of sustainability, we have to adopt new ways of thinking where major
challenges are to take full account of (1): (*) The multi-functional nature of agriculture (that is not
only producing commodities, but also preserving the health of ecosystems, consumers and rural
communities), (*) The multi-scale nature of the complex network of relations among ecosystems
and socioeconomic systems, that requires considering simultaneously different but relevant
dynamics operating at different hierarchical levels.
My research deals with studying agro-ecosystems integrating their many different components by
adopting different perceptions/descriptions referring to different levels and scales: biophysical
perception (from the soil, and its fauna, to crops species), socioeconomic view, (from the farmer up
to that of the rural community, to local, regional and national socioeconomic systems), ecological
view, (from micro-ecosystems in the soil, to local agro-ecosystems, up to large ecological regions)
(1). Such an approch has been applied to different case studies (4, 5) and lately to new issues:
organic vs conventional farming (6) and agro-energy (7). By establishing relations between changes
in biophysical variables and economic variables, it is possible to describe with models the possible
effects – or better, the feasibility domains – in terms of values taken by a given indicator in relation
to another. This approach can also helps to combine ‘hard’ and ‘soft’ information when analysing
problems associated with the sustainability of agro-ecosystems.
REFERENCES
1) Gomiero T, Giampietro M, Mayumi K (2006) Facing complexity on agro-ecosystems: a new approach to farming
system analysis. International Journal of Agricultural Resources, Governance and Ecology, 5, 116-144.
2) Gomiero T, Paoletti MG, Pimentel D The concept of agriculture sustainability: a review. Critical Reviews in Plant
Science In preparation.
3) Gomiero T, Paoletti MG, Pimentel D Environmental impact of different agricultural management practices. Critical
Reviews in Plant Science In preparation.
4) Gomiero T, Giampietro M (2001) Multiple-Scale Integrated Analysis of farming systems: the Thuong Lo commune
(Vietnamese uplands) case study. Population and Environment, 22, 315-352.
5) Gomiero T, Giampietro M, Bukkens SM, Paoletti GM (1997) Biodiversity use and technical performance of
freshwater fish culture in different socio-economic context: China and Italy. Agriculture, Ecosystems and Environment
62, 169-185.
6) Gomiero T, Paoletti GM, Pimentel D (2008) Energy and environmental issues in organic and conventional
agriculture. Critical Review in Plant Science 27, 239-254.
7) Gomiero T, Paoletti MG, Pimentel D (2009) Biofuels: ethics and concern for the limits of human appropriation of
ecosystem services. Journal of Agriculture and Environmental Ethics In press.
67
UNIQUE CHARACTERISTICS OF CA2+ HOMEOSTASIS OF THE TRANS-GOLGI
COMPARTMENT
Valentina Lissandron*1, Paola Pizzo1,2 and Tullio Pozzan1,2,3
*1 Dipartimento di Scienze Biomediche Sperimentali, Università degli Studi di Padova
Viale G. Colombo 3, 35131 Padova, Italy; Tel: +390498276067;
2. CNR Institute of Neuroscience
3. Venetian Institute of Molecular Medicine, Padova
ABSTRACT
Taking advantage of a novel, FRET based, fluorescent Ca2+ indicator, selectively targeted to the
trans-Golgi lumen we here demonstrate that its Ca2+ homeostatic mechanisms are unique among
cellular organelles and distinct from those of the other Golgi sub-compartments: i) Ca2+ uptake
depends exclusively on the activity of the Secretory Pathway Ca2+ ATPase1 (SPCA1), while the
Sarco-Endoplasmic Reticulum Ca2+ ATPase (SERCA) is excluded; ii) no IP3-dependent Ca2+
release, but rather Ca2+ uptake is observed upon Ca2+ release from the ER and/or Ca2+ influx through
the plasma membrane; iii) fast and extensive Ca2+ release can be triggered by activation of
ryanodine receptors in cells endowed with robust expression of the latter channels (e.g. in neonatal
cardiac myocyte). We also demonstrate that the repertoire of Ca2+ handling mechanisms is
strikingly different in the different Golgi regions: SERCA and IP3 receptors predominate in the
compartment closer to the ER, while in an intermediate region the SERCA is still expressed but IP3
receptors are excluded.
68
THE HUMAN DNA TOPOISOMERASE I: STRUCTURAL STUDIES AND DRUG
RESISTANCE
Carmen Losasso*1, Erica Cretaio1, Mary-Ann Bjornsti2, Piero Benedetti1
Italy; Tel: +390498276289. E-Mail: [email protected];
2. Dept of Molecular Pharmacology, St. Jude Children’s Research Hospital, Memphis, TN 38104
ABSTRACT
Eukaryotic DNA topoisomerase IB (Top1p) catalyzes changes in DNA topology via the formation
of a covalent enzyme-DNA intermediate, which is reversibly stabilized by the anticancer agent
camptothecin (CPT). Crystallographic studies of a 70 Kd C-terminal fragment of human Top1p
bound to duplex DNA describe a monomeric protein clamp circumscribing the DNA helix.
Opposable ‘‘lip’’ domains form a salt bridge to complete Top1 protein clamping of duplex DNA.
The structures, which lack the N-terminal domain, comprise the conserved clamp, an extended
linker domain and the conserved C-terminal active site Tyr domain. Changes in DNA topology are
catalyzed by the formation of a transient phosphotyrosyl linkage between the active-site Tyr-723
and a single DNA strand. Substantial protein domain movements are required for DNA binding,
whereas the tight packing of DNA within the covalent Top1–DNA complex necessitates some DNA
distortion to allow rotation. To investigate the effects of Top1-clamp closure on enzyme catalysis,
molecular modeling was used to design a disulfide bond between residues Gly-365 and Ser-534, to
crosslink protein loops more proximal to the active-site tyrosine than the protein loops held by the
Lys-369–Glu-497 salt bridge. In reducing environments, Top1-Clamp was catalytically active.
However crosslinking the active-site proximal loops inhibited DNA rotation. Apparently, subtle
alterations in Top1 clamp flexibility impact enzyme catalysis in vitro. Yet, the catalytically active
Top1-Clamp was cytotoxic, even in the reducing environment of yeast cells. Remarkably, a shift in
redox potential in glr1- cells converted the catalytically inactive Top1Y723F mutant clamp into a
cellular toxin, which failed to induce an S-phase terminal phenotype. This cytotoxic mechanism is
distinct from that of camptothecin chemotherapeutics, which stabilize covalent Top1–DNA
complexes, and it suggests that the development of novel therapeutics that promote Top1-clamp
closure is possible (1, 4).
CPT bound to the covalent Top1p-DNA complex limits linker flexibility, allowing structural
determination of this domain. We previously reported that mutation of Ala653 to Pro in the linker,
increases the rate of enzyme catalyzed DNA religation, thereby rendering Top1A653Pp resistant to
CPT. Molecular dynamics studies suggested mutation-induced increases in linker flexibility alter
Top1p catalyzed DNA religation. To address the functional consequences of linker flexibility on
enzyme catalysis and drug sensitivity, we investigated the interactions of the A653P linker mutation
with a self-poisoning T718A mutation within the active site of Top1p. The A653P mutation
suppressed the lethal phenotype of Top1T718Ap in yeast, yet did not restore enzyme sensitivity to
CPT. However, the specific activity of the double mutant was decreased in vivo and in vitro,
consistent with a decrease in DNA binding. These findings support a model where changes in the
flexibility or orientation of the linker alter the geometry of the active site and thereby the kinetics of
DNA cleavage/religation catalyzed by Top1p (2, 3).
REFERENCES
1) Woo M, et al (2003) Locking the DNA topoisomerase I protein clamp inhibits DNA rotation and induces cell
lethality. Proc. Natl. Acad. Sci. USA 100, 13767-13772.
2) Losasso C, et al (2007) Alterations in Linker Flexibility Suppress DNA Topoisomerase I Mutant-induced Cell
Lethality. J. Biol. Chem. 282, 9855-9864.
3) Losasso C, et al (2008) A single mutation in the 729 residue modulates human DNA topoisomerase IB DNA binding
and drug resistance. Nucleic Acids Research 36, 5635-5644.
4) Palle K, et al (2008) Disulfide Cross-links Reveal Conserved Features of DNA Topoisomerase I Architecture and a
Role for the N Terminus in Clamp Closure. J. Biol. Chem. 283, 27767-2777.
69
PHOTOSENSITISED INACTIVATION OF MICROBIAL PATHOGENS: MEDICAL AND
ENVIRONMENTAL APPLICATIONS
Michela Magaraggia*, Clara Fabris, Marina Soncin, Giulio Jori
ABSTRACT
The increasing diffusion of infectious diseases represents a major challenge for human health
worldwide, especially as a consequence of the continuous emergence of antibiotic-resistant bacteria.
Thus, therapeutic alternatives based on different strategies are needed. Photodynamic therapy
(PDT) can be a useful approach, particularly for the treatment of localized infections. This therapy
is based on the combination of a molecule (namely photosensitiser), oxygen and light to produce
highly reactive oxygen species, which can induce damage to the microenvironment of the site
where it is generated (cell or tissue). It appears to be endowed with several favourable features for
the treatment of infections originated by microbial pathogens, including a broad spectrum of action,
the efficient inactivation of antibiotic-resistant strains, the low mutagenic potential, and the lack of
selection of photoresistant microbial cells (1). Therefore, intensive studies are being pursued in
order to define the scope and field of application of this approach.
Optimal cytocidal activity against a large variety of bacterial, fungal, and protozoan pathogens has
been found to be typical of photosensitizers that are positively charged at physiological pH values
(e.g., for the presence of quaternarized amino groups or the association with poly-lysine moieties)
and are characterized by a moderate hydrophobicity (n-octanol/water partition coefficient around
10). These photosensitizers in a micromolar concentration can induce a >4-5 log decrease in the
microbial population after incubation times as short as 30 minutes and irradiation under mild
experimental conditions, such as fluence-rates around 50 mW/cm2 and irradiation times shorter than
15 minutes.
In vivo studies show curative results in a) trout affected by Saprolegniosis-induced dermatitis (2)
and b) dogs affected by Gram(+)-induced dermatitis. Moreover, the photodynamic treatment can be
used to prevent the onset of infectious diseases in adult trout and trout eggs, as well as to optimize
the wellness and health of fish in aquaria with no detectable damage to plants or invertebrates.
PDT appears to represent an efficacious alternative modality for the treatment of localized
microbial infections through the in situ application of the photosensitizer followed by irradiation of
the photosensitizer-loaded infected area. Proposed clinical fields of interest of antimicrobial PDT
include the treatment of chronic ulcers, infected burns, acne vulgaris, and a variety of oral
infections.
REFERENCES
1) Jori G, Fabris C, Soncin M, Ferro S, Coppellotti O, Dei D, Fantetti L, Chiti G, Roncucci G. (2006) Photodynamic
therapy in the treatment of microbial infections: basic principles and perspective applications. Lasers Surg. Med. 38,
468-481.
2) Magaraggia M, Faccenda F, Gandolfi A, Jori G. (2006) Treatment of microbiologically polluted aquaculture waters
by a novel photochemical technique of potentially low environmental impact. J Environ Monit 8, 923-931.
70
MITOCHONDRIAL GENOME SEQUENCING OF PHOTOSYNTHETIC STRAMENOPILES
(CHROMALVEOLATA)
Silvia Maistro*1, Enrico Negrisolo2, Carlo Andreoli1
1. Dipartimento di Biologia, Università degli Studi di Padova
2. Dipartimento di Sanità Pubblica, Patologia Comparata e Igiene Veterinaria, Università degli
Studi di Padova, Agripolis
ABSTRACT
Over the last decade a number of complete mitochondrial genomes (mtDNAs) have been sequenced
and studied in order to elucidate the evolutionary relationships amongst various independent
eukaryotic lineages. These data allowed to better understand the evolution of included single genes
as well as genome size and structure (1). The bulk of complete mtDNA sequences currently
available is mainly from animal species, while only a few belong to Stramenopiles. This latter
group represents one of the major eukaryotic lineages. Stramenopilean taxa are characterized by
motile life cycle stage, in which the flagellate cells possess two differently-shaped flagella.
Heterotrophic stramenopiles, which are considered the most primitive forms, comprise bicosoecids
and oomycetes while photosynthetic organisms belong to several classes e.g. Bacillariophyceae,
Chrysophyceae, Phaeophyceae, Xanthophyceae (2). To date fifteen mitochondrial genomes have
been sequenced in Stramenopiles.
The Xanthophyceae is a class characterized by morphologically diverse taxa, ranging from
unicellular coccoid forms to multinucleate siphonous and multicellular filamentous species (3).
Previous studies on the class investigated the evolutionary history and phylogenetic relationships
amongst genera (4), families (5) and orders (6) adopting a multigenic approach that produced a new
classification for the whole class (6). To date no mtDNA genomes are known for Xanthophyceae.
Thus one of my post doc aims is focused to sequence the mitochondrial genomes of some taxa of
this clade. The new determined mtDNAs will allow to investigated several evolutionary issues
dealing with stramenopilean mtDNAs. Furthermore these sequences will be used to study the
phylogenetic position of Stramenopiles in the eukaryotic tree of life.
REFERENCES
1) Bullerwell, et al (2004) Evolution of the mitochondrial genome: protist connections to animals, fungi and plants.
Current Opinion in Microbiology 7, 528-534.
2) Outdot, et al (2001) The complete sequence of a brown algal mitochondrial genome, the Ectocarpale Pylaiella
littoralis (L.) Kjellm. Journal of Molecular Evolution 53, 80-88.
3) Van den Hoek, et al (1995) Algae. An Introduction to Phycology. Cambridge: Cambridge University Press.
4) Negrisolo, Maistro, et al (2004) Morphological convergence characterizes the evolution of Xanthophyceae
(Heterokontophyta): evidence from nuclear SSU rDNA and plastidial rbcL genes. Molecular Phylogenetics and
Evolution 33, 156-170.
5) Maistro, et al (2007) Molecular phylogeny and evolution of the order Tribonematales (Heterokonta, Xanthophyceae)
based on analysis of plastidial genes rbcL and psaA. Molecular Phylogenetics and Evolution 43, 407-417.
6) Maistro, et al (2009) Phylogeny and taxonomy of Xanthophyceae (Stramenopiles, Chromalveolata). Protist In press.
71
MOLECULAR GENETICS OF HUMAN RESISTANCE TO ENDURANCE AND HYPOBARIC
HYPOXIA
Sandro Malacrida*, Gian Antonio Danieli
* Dipartimento di Biologia, Università degli Studi di Padova, Viale G. Colombo 3, 35131 Padova,
ABSTRACT
Populations living at high altitudes show better endurance performances in hypoxic conditions than
acclimatized lowlanders. It has been suggested that genetic adaptation to hypoxia at population
level was achieved by natural selection along many generations, starting from resistance to
endurance, shared by all human ancestral populations. Selection acting on such genes should have
produced linkage disequilibrium between adaptive variants and specific alleles of flanking
polymorphic DNA markers. Since favourable alleles of genes involved in such biological
adaptation should be still very frequent in populations living in hypoxic environment, detection of
homozygosity for shared alleles of polymorphic DNA markers in unrelated individuals should
enable to identify short chromosomal segments harbouring genes involved in resistance to
endurance and to hypobaric hypoxia. Actually in 2007, by a genomic approach, we succeeded in
identifying human chromosomal segments which could include genes involved in adaptation to
hypobaric hypoxia and endurance in Sherpa porters. We performed a human genome scan using
998 polymorphic DNA markers in 7 unrelated Sherpa porters living in the Solu-Khumbu (Nepal)
area and we succeeded in detecting 8 DNA markers showing homozygosity for the same shared
allele. Analysis of additional DNA samples from two more Sherpa porters focused our attention on
three polymorphic DNA markers (D6S1697, D14S274, D17S1795) showing homozygosity for the
same shared allele in 8 out 9 tested individuals. Analysis of DNA samples from Sherpa and nonSherpa populations of Nepal proved HW equilibrium in both populations for markers D14S274 and
D17S1795, while an excess of heterozygotes was observed in the Sherpa population for marker
D6S1697. A significant difference in allele frequencies for D14S274 and D17S1795 between the
two populations was observed. These findings excluded the possibility that homozygosity for 3
specific loci in 8 unrelated individuals might be ascribed to inbreeding or recent genetic drift. We
therefore concluded that the chromosomal segments detected by such DNA markers might harbour
genes involved in adaptation to hypobaric hypoxia and endurance in Sherpas (1).
More recently we performed a new genome scan by GeneChip array (250,000 SNPs) on the same
DNA samples. Compared to STR maps, the GeneChip® Human Mapping 250K Array Set is
superior in uniform spacing and statistical power for detecting association to a given trait. Analysis
by IBDfinder program was used to identify IBD (Identical by Descent) regions. Such segments are
identified as stretches of chromosome devoid of heterozygous SNPs, and are scored to the number
of SNPs they contain (Cut-off value). In order to characterize true IBD regions IBD scores are
computed and adjusted. Very stringent Cut-off value in statistical analysis enables to identify over
20 candidate loci. Comparing genome scans results a few loci identified by GeneChip contains or
flanked loci identified by preliminary STRs genome scan. Differences in genome scan kits and
statistical analysis may explain differences in results obtained from the same sample.
REFERENCES
1) Malacrida S et al (2007) Association between human polymorphic DNA markers and hypoxia adaptation in Sherpa
detected by a preliminary genome scan. Ann Hum Genet 71, 630-638.
72
EVOLUTION AND DEVELOPMENT OF ARTHROPOD LIMBS
Diego Maruzzo
Dipartimento di Biologia,Università degli Studi di Padova
Via U. Bassi 58/B, 35131 Padova, Italy; Tel. +390498276238; E-mail: [email protected]
ABSTRACT
In the recent discussions on the evolution of animal form, arthropods play an important role not only as
useful models to understand general questions, such as the origin of evolutionary novelties or the
developmental basis of regional specialization, but also because their high morphological disparity has
eventually turned studies on specific arthropod features (like the evolution of arthropod trunk
segmentation or the evolution of arthropod limbs) into pivotal items of evolutionary developmental
biology. However, several aspects of the evolution and development of arthropod limbs are far from
clear; this is mainly because a) there is no consensus on arthropod phylogeny, b) the fine anatomy of
many limbs has not been studied, and c) still little data exists on comparative limb morphogenesis and
developmental genetics. Moreover, studying arthropod limb evolution is particularly complex because
developmental homologies should be evaluated in two ways: between similar limb types in different
taxa (special homology), and between different limb types within the same taxa (serial homology). In
my work, I try to understand arthropod limb evolution and development considering problems of both
special and serial homology, by comparing limb segmentation mechanism in few different limb types
and by studying the development and fine anatomy of some limb types within different arthropod
groups. Mechanisms of limb segmentation have been studied in different limbs of malacostracan
crustaceans with special focus on those limb parts that acquire new articles sequentially during
postembryonic development (1,2,3,4). A “growth zone” (a zone where new segmental elements are
produced) has been found and described for different limbs, and this is now object of more specific
studies of mine. The development and fine anatomy of some limb types is currently studied in
thecostracan and branchiopod crustaceans, two groups for which a relatively well-established
phylogeny (which is an essential background to reconstruct evolution) is available (5,6). This work, in
collaboration with J.T. Høeg and J. Olesen, has already produced two detailed descriptions of
previously poorly described limbs (7,8) and one broad comparative work (9).
REFERENCES
1) Maruzzo D, Bonato L, Brena C, Fusco G, Minelli A (2005) Appendage loss and regeneration in arthropods: a
comparative view. In Crustacea and Arthropod Relationships (Crustacean Issues, 16). Koenemann S. & Jenner R. eds. Pp.
215-245. Boca Raton: CRC Press.
2) Maruzzo D, Minelli A, Ronco M, Fusco G (2007) Growth and regeneration of the second antennae of Asellus aquaticus
(Isopoda) in the context of arthropod antennal segmentation. Journal of Crustacean Biology 27, 184-196.
3) Maruzzo D, Egredzija M, Minelli A, Fusco G (2008) Segmental pattern formation following amputation in the flagellum
of the second antennae of Asellus aquaticus (Crustacea, Isopoda). Italian Journal of Zoology 75, 225-231.
4) Maruzzo D The development of amphipod pleopods and the postembryonic patterning of crustacean limbs. In
preparation.
5) Pérez-Losada M, Høeg JT, Kolbasov GA, Crandall KA (2002) Reanalysis of the relationships among the Cirripedia and
the Ascothoracida and the phylogenetic position of the Facetotecta (Maxillopoda: Thecostraca) using 18S rDNA sequences.
Journal of Crustacean Biology 22, 661-669.
6) Richter S, Olesen J, Wheeler WC (2007) Phylogeny of Branchiopoda (Crustacea) based on a combined analysis of
morphological data and six molecular loci. Cladistics 23, 301-336.
7) Maruzzo D, Minelli A, Fusco G (2009) Segmental mismatch in crustacean appendages: the naupliar antennal exopod of
Artemia (Crustacea, Branchiopoda, Anostraca). Arthropod Structure & Development 38, 163-172.
8) Maruzzo D, Clare AS, Høeg JT Action of antennulary sensory setae during surface exploration in cyprids of Balanus
(Amphibalanus) amphitrite. In preparation.
9) Maruzzo D, Olesen J, Høeg JT The evolution of first antennae in branchiopod crustaceans. In preparation.
73
SOLUTION STRUCTURES AND ALLOSTERIC CONFORMATIONAL CHANGES OF 2×6MERIC AND 4×6-MERIC ARTHROPOD HEMOCYANINS
Ivan Mičetić*, Carmen Losasso, Pietro Benedetti, Luigi Bubacco, Paolo Di Muro, Mariano
Beltramini
ABSTRACT
Arthropod hemocyanins (Hcs) are large (from 450 kDa to over 2 MDa), extracellular, copper
containing oxygen transport proteins (1). They form oligomeric quaternary structures based on
different arrangements of a basic 6×75 kDa hexameric building block (2). In particular Carcinus
aestuarii and Squilla mantis Hcs present a 2×6-meric subunit arrangement while Upogebia pusilla
Hc has a more complex, 4×6-meric structure. The oxygen binding of Hcs is governed by
hierarchical interactions between different subunits of the native oligomers. In addition, allosteric
effectors modulate the oxygen binding properties. Biochemical studies have thoroughly described
the influence of protons and lactate on both affinity and cooperativity in the oxygen binding process
(3, 4). However, information on the structural changes that govern the allosteric behavior are still
lacking. Recent flash-photolysis and x-ray absorption spectroscopy (XAS) studies have shown that
the effect of protons mainly resides in changes of copper-imidazole complex geometry at the active
site level (5). No information is available on how such active site changes are propagated within the
oligomer, namely at an inter-subunit level. As far as the lactate effect is concerned, preliminary
XAS results show that there are no changes at the active site level, yet a marked influence on
affinity and cooperativity is observed.
In order to investigate structural changes that Hcs undergo in the presence of allosteric effectors
beyond the active site, the native structures of three 2×6 and 4×6-meric Hcs were studied by small
angle x-ray scattering (SAXS). The structure of native complexes was obtained by refining the
position and orientation of the crystallographic hexameric building block on the basis of recorded
SAXS data. The native structures of the two 2×6-meric Hcs show different inter-hexameric
arrangements. In the case of C. aestuarii Hc, the two hexameric building blocks are in contact
through four inter-hexameric subunits. S. mantis Hc shows a completely different topology: the two
hexameric building blocks are stacked one above the other with the three-fold symmetry axis of the
upper hexamer parallel to the same axis of the lower hexamer. In such a structure, five subunits
provide inter-hexameric contacts. For 4×6-meric U. pusilla Hc, a lower resolution model is
proposed giving insight to the symmetrical arrangement of the four hexameric building blocks. Due
to the complexity of the quaternary structure of this Hc, it was impossible to obtain a unique
structure based on SAXS data alone. Therefore, three best models are presented and are validated
against negative-stain electron microscopy projections of individual particles. Lactate-induced
conformational changes were observed in C. aestuarii Hc in the presence of increasing lactate
concentrations. In particular, the inter-hexameric distance decreases and the protein assumes a more
compact structure.
REFERENCES
1) Markl J, Decker H (1992) Molecular structure of the arthropod hemocyanins. Adv Comp Environ Physiol 13, 325376.
2) Volbeda A, Hol WG (1989) Crystal structure of hexameric haemocyanin from Panulirus interruptus refined at 3.2 Å
resolution. J Mol Biol 209, 249-279.
3) Dainese E, Di Muro P, Beltramini M, Salvato B, Decker H (1998) Subunits composition and allosteric control in
Carcinus aestuarii hemocyanin. Eur J Biochem 256, 350-358.
4) Weber E, Vinogradov N (2008) Nonvertebrate hemoglobins: functions and molecular adaptations. Physiol Rev 81,
569-628.
5) Hirota S, Kawahara T, Beltramini M, Di Muro P, Magliozzo R, Peisach J, Powers L, Tanaka N, Nagao S, Bubacco L
(2008) Molecular basis of the Bohr effect in arthropod hemocyanin. J Biol Chem 283, 31941-31948.
74
CHARACTERIZATION OF EXPRESSION AND FUNCTION OF NOVEL EMILIN GENES
IN MOUSE AND ZEBRAFISH
Martina Milanetto*1, Natascia Tiso1, Alvise Schiavinato2, Paola Braghetta2, Francesco Argenton1,
Paolo Bonaldo2
*1. Dipartimento di Istologia, Microbiologia e Biotecnologie Mediche, Università degli Studi di
Padova, Viale G. Colombo 3, 35131 Padova, Italy; Tel: +390498276099
ABSTRACT
Emilins are a family of glycoproteins of the extracellular matrix with common structural
organization and containing a characteristic N-terminal cysteine-rich domain. The prototype of this
family, Emilin-1, is found in human and murine organs in association with elastic fibers, and other
emilins were recently isolated in mammals.
To gain insight into these proteins in lower vertebrates, we investigated the expression of emilins in
the fish Danio rerio. Using sequence similarity tools, we identified eight members of this family in
zebrafish. Each emilin gene has two paralogs in zebrafish, showing conserved structure and
syntheny with the human ortholog. Whole-mount in situ hybridization (ISH) revealed that
expression of zebrafish emilin genes is regulated in a spatio-temporal manner during embryonic
development, with overlapping and site-specific patterns mostly including mesenchymal structures.
Expression of some emilin genes in peculiar areas, such as the central nervous system or the
posterior notochord, suggests that they may play a role in certain morphogenetic processes (1). The
most distinctive pattern is the one of emilin-3, the only component of the family lacking the Cterminal gC1q domain. While other emilin genes reveal a mesenchymal or cardiovascular
expression profile, the two zebrafish genes coding for emilin-3 are abundantly expressed at early
stages in notochord and floor plate, suggesting that emilin-3 may play key roles during early
development (1). Emilin-3 expression does not appear to be regulated by either Hedgehog, FGF or
Notch signaling, as indicated by ISH of embryos in which these molecular pathways were blocked
by drug treatments. Additionally, expression of the two Emilin-3 genes appears normal in embryos
mutant for Chordin (dino) or for BMP2 (swirl). We are currently investigating other relevant
pathways to check whether they regulate emilin-3 expression during early development. Moreover,
we are performing functional studies by microinjection of mRNA or morfolino oligonucleotides in
fertilized oocytes.
The studies in mice were concentrated on the gene coding for emilin-3. In the past years, targeted
Emilin3 gene inactivation in murine embryonic stem (ES) cells was already undertaken in our
laboratory. However, generation of Emilin3 knockout mice was unsuccessful and these previous
attempts revealed that, unlike other emilins, targeted inactivation of the Emilin3 gene is particularly
difficult. The frequency of homologous recombination of this gene is extremely low and
transmission of the inactivated allele to the F1 generation was not reached, probably also due to
karyotypic instability of targeted ES cell clones. Therefore, we carried out a new Emilin3 gene
targeting approach, performed by means of a large-scale experiment. We transfected R1 ES cells
with a Emilin3 targeting construct and, after double positive-negative selection, 1505 clones were
isolated; 995 of these individual cell clones were further expanded and investigated for identifying
clones in which a correct homologous recombination event had occurred. With such large number
of clones, molecular analysis by Southern blotting, a standard but time- and money-consuming
procedure, was not an easily affordable screening method for detecting rare homologous
recombinant clones. Therefore, we set up an optimal screening procedure by PCR. Six positive ES
clones were identified, and after additional karyotypic characterization four of them were used for
generation of chimeric mice by microinjection of ES cells into host blastocysts and implant in foster
females. Several chimeric mice were obtained from two independent clones, and they were bred to
obtain Emilin3 null mice which are currently under phenotypic analysis.
REFERENCES
1) Milanetto M, Tiso N, Braghetta P, Volpin D, Argenton F, Bonaldo P (2008) Emilin genes are duplicated and
75
dynamically expressed during zebrafish embryonic development.
Dev. Dyn. 237, 222-232.
ANALYSIS OF PANCREATIC BETA CELL DEVELOPMENT AND MAINTENANCE IN
ZEBRAFISH
Enrico Moro1, Lara Gnügge2, Paola Braghetta3, Marino Bortolussi1, Francesco Argenton1
2. Developmental Biology, Institute Biology 1, University of Freiburg, Hauptstrasse 1, D-79104
Freiburg, Germany
3. Department of Histology, Microbiology, and Medical Biotechnologies, I-35121 Padova, Italy
ABSTRACT
Most of pancreatic beta cell mass expansion during early postnatal stages and in adulthood is
considered to occur through a self-replication model, in which beta cells proliferate under both
physiological conditions and after partial pancreactomy (1, 2). Neogenesis, i.e. differentiation from
supposed ductal pancreatic progenitors, has been demonstrated in mice to act concurrently with beta
cell replication during pancreatic regeneration after a severe injury. (3). Both phenomena have been
investigated in higher vertebrates (mouse, rat and guinea pig) and different knock out models have
partially enabled to discover crucial mechanisms regulating beta cell mass homeostasis (4, 5).
The zebrafish model represents an attractive alternative tool to dissect pancreatic beta cell mass
dynamics during different life stages. In the present project, by combining time-lapse imaging of
live zebrafish larvae and immunostaining, we have performed a detailed reconstruction of the major
processes involved in fish beta cell mass genesis and maintenance. We have built a selective in vivo
system of proliferating beta cell ablation, based on the administration of the prodrug ganciclovir to
the transgenic line Tg(-1.2ins:TKGFP). Through the use of this conditional cell ablation tool, we
have demonstrated that in larval stages self-replication is the main mechanism of beta cell
expansion. The differentiation in vivo of pancreatic ductal cells into beta cells was also explored
through the analysis of the double transgenic line Tg(-3.5nkx2.2a:GFP)ia3/Tg(-1.2ins:DsRed)
during the first month of life. We have been able for the first time to trace the origin of new beta
cells from the duct epithelium in physiological conditions. Our results provide an extensive proof of
evidence that self replication and differentiation are not mutually exclusive in the early growth of
vertebrate beta cell mass (6). We, therefore, suggest the use of zebrafish as an in vivo platform to
analyse the pathways involved in beta cell differentiation and regeneration, which may be trivial in
the pathogenesis and management of diabetes.
REFERENCES
1) Dor Y, Brown J, Martinez OI, Melton DA (2004) Adult pancreatic beta-cells are formed by self-duplication rather
than stem-cell differentiation. Nature 429, 41-46.
2) Yesil, P.a.L., E. (2008) Islet dynamics: a glimpse at beta cell proliferation. Histol. Histopathol. 23, 883-895.
3) Inada A, Nienaber C, Katsuta H, Fujitani Y, Levine J, Morita R, Sharma A, Bonner-Weir S (2008) Carbonic
anhydrase II-positive pancreatic cells are progenitors for both endocrine and exocrine pancreas after birth. Proc Natl
Acad Sci U S A 105, 19915-19919.
4) Rane SG, Dubus P, Mettus RV, Galbreath EJ, Boden G, Reddy EP, Barbacid M (1999) Loss of Cdk4 expression
causes insulin-deficient diabetes and Cdk4 activation results in beta-islet cell hyperplasia. Nat. Genet. 22, 44-52.
5) Wang Z, Moro E, Kovacs K, Yu R, Melmed S (2003) Pituitary tumor transforming gene null male mice exhibit
impaired pancreatic beta cell proliferation and diabetes. Proc. Natl. Acad. Sci. U S A. 100, 3428-3432.
6) Moro E, Gnugge L, Braghetta P, Bortolussi M, Argenton F (2009) Analysis of beta cell proliferation dynamics in
zebrafish. Submitted.
76
BIO PhD & PostDoc Day ’09, complesso Biologico “A. Vallisneri”, Università di Padova
CALCIUM-MEDIATED PERCEPTION OF PLANT SYMBIOTIC SIGNALS BY NITROGENFIXING BACTERIA
Roberto Moscatiello*1, Andrea Squartini2, Paola Mariani1, Lorella Navazio1
*Viale G. Colombo 3, 35131 Padova, Italy, Tel: +390498276257;
2. Dipartimento di Biotecnologie Agrarie, Università degli Studi di Padova
ABSTRACT
Rhizobia are Gram-negative soil bacteria which can engage in a mutualistic association with
leguminous plants. During this interaction the bacteria invade the plant and highly specialized plant
organ are generated de novo on host roots (1). When living in symbiosis, rhizobia are able to fix
atmospheric nitrogen into forms usable by the plant. Nitrogen is the most often-limiting
macronutrient in many soils, and it is generally supplied as fertilizer. The rhizobium-legume
mutualistic association can reduce or eliminate nitrogen fertilizer requirements, resulting also in a
benefit to the environment (2). A successful symbiosis is the result of an elaborate developmental
program, regulated by the exchange of molecular signals between the two partners (3). During
growth in the rhizosphere of the host plant, rhizobia sense compounds such as flavonoids secreted
by the host root and respond by inducing bacterial nodulation (nod) genes, which are required for
the synthesis of rhizobial signal molecules of lipo-chitooligosaccharide nature, the Nod factors.
In host plants, intracellular Ca2+ changes are an integral part of the signalling mechanism. In
rhizobia it is not yet known whether Ca2+ can act as transducer of symbiotic signals. We introduced
a plasmid encoding the bioluminescent Ca2+ probe aequorin into Mesorhizobium loti and Rhizobium
leguminosarum bv. viciae to investigate whether a Ca2+ response is activated in rhizobia upon
perception of symbiotic signals. Host plant root exudates and specific flavonoids which induce
bacterial nod genes were found to trigger transient intracellular Ca2+ elevations. The Ca2+ responses
activated by these plant-to-bacteria signal molecules were abolished by the Ca2+ channel blocker
La3+ and the Ca2+ chelator EGTA. The Ca2+ dependence of nod gene expression strongly suggests
that the Ca2+ changes evoked by plant root exudates represent an essential prerequisite to convey
the plant symbiotic message into rhizobia. Moreover, no Ca2+ elevations were induced by non
specific flavonoids unable to trigger nod gene expression. These data indicate a newly described
early event in the molecular dialogue between plants and rhizobia and confirm that Ca2+ signalling
is operating in bacteria. The use of transgenic rhizobium strains expressing aequorin lends itself as a
promising novel approch for research in legume symbiosis (4).
REFERENCES
1) Oldroyd GED, and Downie JA (2008) Coordinating nodule morphogenesis with rhizobial infection in legumes.
Annual Review of Plant Biology 59, 519-546.
2) Garg N, Geetanjali (2007) Symbiotic nitrogen fixation in legume nodules: process and signaling. A review.
Agronomy for Sustainable Development 27, 59-68.
3) Cooper JE (2007) Early interactions between legumes and rhizobia: disclosing complexity in a molecular dialogue.
Journal of Applied Microbiology 103, 1355-1365.
4) Moscatiello R, Alberghini S, Squartini A, Mariani P, Navazio L (2009) Recombinant aequorin in Mesorhizobium
loti: a reporter to investigate how rhizobia sense plant symbiotic signals. Molecular Plant-Microbe Interactions
Submitted.
77
DNA REPLICATION DYNAMICS AT CHROMOSOMAL REGIONS UNDERGOING
GENOMIC INSTABILITY
Elisa Palumbo*, Laura Matricardi, Antonella Russo
ABSTRACT
The aim of the work is to investigate the replication dynamics at regions structurally prone to
undergo genomic instability. In this contest, we are analysing the replication pattern at the common
fragile site FRA6E (6q25.3-6q26), within the subregion where PARK2 is the most relevant gene
mapped, and at the region where the Frataxin gene (FXN) is located (9q13). The expansion of
trinucleotide repeat GAA/TTC within this locus by dynamic mutation is related to the onset of
Friedreich ataxia (FRDA). In both cases, the instability could be in part ascribed to deregulating
events occurring during the replication process (1-3).
1. The common fragile site FRA6E is a large region on the long arm of chromosome 6 (4). Common
fragile sites (CFSs) are expressed as breaks or gaps on metaphase chromosomes after partial
inhibition of the DNA replication process, mainly by treatment with aphidicolin (1,2,5). Up to date,
several theories aimed to explain the causes of CFSs instability suggest that the fragility may be
partially related to the incomplete replication of the region (1,2,6). A high resolution analysis of the
replication pattern of PARK2-FRA6E region has been performed by molecular combing, a single
molecule approach which provides a fine description of the structural and replication features of
chromosomal regions by means of microscope fluorescent analysis (7-8). The effect on the
replication progress of a relatively high dose of aphidicolin (APH 0.4 μM) was evaluated after 2 h
and 24 h at whole genome level. At 2 h a 10 fold slowing effect on the replication rate has been
observed with respect to the control (P < 0.001), with a partial rescue of fork progression after 24 h.
The replication dynamics at PARK2-FRA6E was analysed under replication stress (0.4 μM APH, 2
h) and control conditions with respect to an early replicating control region. The mean fork speed
and the inter-origin distance were determined and origins were mapped, addressing particular
attention to all deregulation events of the replication process. Differently from the control region,
the APH treatment at PARK2-FRA6E causes a rather strong deregulation effect on the replication,
leading possibly to the arrest of fork progression.
2. Three models have been proposed to explain the relation between DNA replication and repeat
instability, observed at loci involved in dynamic mutation. The repositioning of the replication
origins or an altered fork progression in the normal with respect to the expanded allele influence the
formation of the Okazaki initiation zone (3). In this frame, we are analysing the replication
dynamics at the normal allele at FXN locus by molecular combing, in order to define the density
and position of replication origins within the gene and in the flanking regions.
REFERENCES
1) Lukusa T, Fryns JP (2008) Human chromosome fragility. Biochim. Biophys. Acta 1779, 3-16.
2) Glover TW (2006) Common fragile sites. Cancer Letters 232, 4-12.
3) Cleary JD, Pearson CE (2005) Replication fork dynamics and dynamic mutations: the fork-shift model of repeat
instability. Trends Genet. 21, 272-280.
4) Russo A, et al (2006) Molecular characterisation of human common fragile site FRA6E, a large genomic region
spanning 9 Mb. In Trends in Genome Research, Clyde R. Williams (Ed.), Nova Science Publishers, Hauppauge, Inc.
5) Aguilera A, Gómez-González B (2008) Genome instability: a mechanistic view of its causes and consequences. Nat
Rev Genet. 9, 204-217.
6) Branzei D, Foiani M (2007) Interplay of replication checkpoints and repair proteins at stalled replication forks. DNA
Repair 6, 994-1003.
7) Caburet S, et al (2002) Combing the genome for genomic instability. Trends Biotechnol. 20, 344-350.
8) Herrick J, Bensimon A (2008) Global regulation of genome duplication in eukaryotes: an overview from the
epifluorescence microscope. Chromosoma 117, 243-260.
78
SUBCELLULAR
LOCALIZATION
AND
ACTIVITY
OF
p53R2-DEPENDENT
RIBONUCLEOTIDE REDUCTASE IN QUIESCENT HUMAN FIBROBLASTS
G. Pontarina, P. Ferraroa, C. Rampazzoa, P. Pizzob, A. Fijolekc, T. Pozzanb, L. Thelanderc, P.
Reicharda, V. Bianchia
Department of Biomedical Sciencesa and CNR Institute of Neurosciencesb , University of Padova,
35131 Padova, Italy; and Department of Medical Biochemistry and Biophysicsc, Umeå University,
SE-90187 Umeå, Sweden
E-mail: [email protected]
ABSTRACT
Background/hypothesis. Deoxyribonucleoside triphosphates (dNTPs) are the building blocks for DNA
synthesis. While nuclear DNA replication is a S-phase specific process exclusive of proliferating cells,
mitochondrial DNA (mtDNA) replication occurs allover the cell cycle also in post mitotic and differentiated
cells. dNTPs are produced by two pathways, de novo synthesis and salvage. In cycling cells the major supply
of dNTPs is the de novo reduction of ribonucleoside diphosphates to deoxyribonucleoside diphosphates by
ribonucleotide reductase (RNR). This enzyme consists of two proteins, R1 and R2, both necessary for
catalysis. The activity of the R1/R2 complex is strictly limited to S-phase due to R2 degradation by
proteolysis in late mitosis. Recently a p53 inducible RNR subunit, called p53R2, has been discovered which
can substitute for R2 outside S phase. The p53R2 protein is expressed at a 30 –fold lower level than R2 but it
is present overall the cell cycle. Which is its physiological function? p53R2 is induced after DNA damage
and it was proposed that the R1/p53R2 complex supplies dNTPs for DNA repair at the actual repair site after
translocation from the cytosol into the nucleus. However p53R2 is present in non cycling cells independently
of DNA damage. Our general hypothesis is that the main physiological function of p53R2/R1 complex is to
supply precursor for mtDNA in quiescent cells and the catalysis occurs in the cytosol independently of DNA
damage similar to the R1/R2 complex. In agreement with our hypothesis, genetic deficiency for p53R2 is
associated to a severe mtDNA depletion in differentiated tissues.
Investigation/methods. In proliferating and quiescent human fibroblasts we assessed the presence of the 3
different RNR subunits and measured the levels of the 4 dNTPs in the cytosol and mitochondria. We used
different procedures of cell fractionation and immunofluorescence in intact cells to establish the subcellular
localization of all 3 RNR subunits in the absence or presence of induced DNA damage. By kinetic isotope
flow experiments with 3H-cytidine we measured the in situ activity of RNR and with 3H-thymidine we
evaluated the relative contribution of de novo synthesis and salvage during proliferation and quiescence on
the basis of the specific radioactivity attained by the dTTP pool.
Results/conclusions. RNR activity requires the presence of R1 together with R2 or p53R2. In cycling
fibroblasts all 3 proteins are present. In quiescent cells R2 disappears, while R1 and p53R2 are still present,
p53R2 increasing 2-fold. By immunofluorescence with monoclonal and immunoaffinity purified antibodies
we demonstrated that independently of DNA damage, all 3 RNR subunits reside in the cytosol (1). The
dNTPs produced by the active enzyme diffuse into the nucleus or are transported into mitochondria. In
quiescent cells the disappearance of R2 leads to a 20-fold drop in pool sizes compared to cycling cells. The
dNTPs are produced by salvage and by the R1/p53R2 complex. Mitochondrial pool represents always 5% of
the total cellular dNTP pool, its composition reflecting the cytosolic one. The rate of de novo synthesis by
R1/p53R2 is about 40-fold lower than in cycling cells. However most deoxynucleotides are degraded to
deoxynucleosides and excreted into the culture medium (2). Our data support a primary function of p53R2
for mtDNA replication.
REFERENCES
1) Pontarin G, et al (2008) Ribonucleotide reduction is a cytosolic process in mammalian cells independently of DNA
damage. Proc Natl Acad Sci U S A 105, 17801-17806.
2) Pontarin G, et al (2007) p53R2-dependent ribonucleotide reduction provides deoxyribonucleotides in quiescent
human fibroblasts in the absence of induced DNA damage. J Biol Chem 282, 16820-16828.
79
LOCAL UNFOLDING IS REQUIRED FOR THE SITE-SPECIFIC PROTEIN MODIFICATION
BY TRANSGLUTAMINASE
Samanta Raboni*, Abhijeet Satwekar, Barbara Spolaore, Angelo Fontana
* CRIBI Biotechnology Centre, University of Padua, Viale G. Colombo 3, 35131 Padua
ABSTRACT
Transglutaminase (TGase) catalyzes an acyl transfer reaction between the γ-carboxyamide group of
a peptide-bound glutaminyl (Gln) residue (acyl donors) and a variety of primary amines (acyl
acceptors), including the amino group of lysine by formation of a thioacyl-intermediate with the
Cys residue in the active site. This reaction is similar to that catalysed by a Cys-protease as papain,
just in a reversed form, i.e., synthesis instead of hydrolysis of a peptide bond. The problem of
substrate specificity of TGase has been long investigated, but a clear-cut understanding of the
factors dictating substrate specificity of TGase has not yet been reached [1,2].
In our laboratory, we used microbial TGase to obtain site-specific modification at Gln residue(s) in
protein substrates. In the 153-residue polypeptide chain of apomyoglobin (apoMb) two sites of
modification were identified, despite the presence of many Gln residues in the sequence. The
primary site of modification is Gln91, located in a highly flexible and locally unfolded chain region.
It is interesting to note that the same chain regions modified by TGase suffer highly selective
limited proteolysis phenomena, implying a correlation between sites of TGase attack and/or limited
proteolysis and chain regions of enhanced flexibility.
Our studies indicate that TGase recognizes and interacts with Gln residues located in locally
unfolded or flexible regions of a protein substrate and, therefore, that enhanced chain flexibility
dictates the specificity of reactions on polypeptide substrates by TGases and proteases, as well as by
other enzymes involved in a number of post-translational modifications of proteins, such as
glycosylation and phosphorylation. Therefore, it is possible to predict the site(s) of TGase-mediated
modification of a protein on the basis of its structure and dynamics and, consequently, potential
effects on its physicochemical and functional properties.
Recently, we have successfully used TGase for the site-specific conjugation of the
poly(ethyleneglycol) polymer (PEG) to proteins of pharmaceutical interest, including human
growth hormone and granulocyte colony-stimulating factor. The novel enzymatic procedure offers
several advantages over the current chemical methods of protein conjugation using reactive
derivatives of PEG [3,4].
REFERENCES
1) Lorand L, Conrad SM (1984) Transglutaminases. Mol. Cell. Biochem. 58, 9-35.
2) Griffin R, Casadio R, Bergamini CM (2002) Transglutaminases: Nature’s biological glues Biochem. J. 368, 377-396.
3) Fontana A, Spolaore B, Mero A, Veronese FM (2008) Site-specific modification and PEGylation of pharmaceutical
proteins mediated by transglutaminases. Adv. Drug Deliv. Rev. 60, 13-28.
4) Mero A, Spolaore B, Veronese FM, Fontana A (2009) Transglutaminase-mediated PEGylation of proteins: Direct
identification of the sites of protein modification by mass spectrometry using a novel monodisperse PEG. Bioconjug.
Chem. 20, 384-389.
80
BIOCHEMICAL AND FUNCTIONAL CHARACTERIZATION OF CHLOROPLASTLOCATED PLANT GLUTAMATE RECEPTORS
Enrico Teardo*1, Elide Formentin1, Anna Segalla1, Manuela Zanetti1, Oriano Marin2, Giorgio Mario
Giacometti1, Fiorella Lo Schiavo1, Mario Zoratti3 and Ildikò Szabò1
1. Department of Biology; 2. CRIBI Biotechnology Center; 3. CNR Institute of Neuroscience and
Department of Biomedical Sciences, University of Padova, Italy.
ABSTRACT
In vertebrates, ionotropic glutamate receptors (iGluRs) are ligand-gated cation channels that
mediate the majority of the excitatory neurotransmission in the central nervous system. Mammalian
iGluRs are grouped into four subfamilies according to pharmacological properties and sequence
similarities: α-amino-3-hydroxy-5- methyl-4 isoxazole propionate (AMPA) receptors, kainate (KA)
receptors, N-methyl-D-aspartate (NMDA) receptors, and delta receptors.
In the model plant Arabidopsis thaliana, 20 genes encoding homologues of animal iGluRs have
been identified (1). Each protein displays a pore-forming loop, flanked by two conserved helices
(plus a third C-terminal helix), a glutamate-binding domain and an N-terminal region. The overall
structure is similar to that of animal glutamate receptors, but the ability of glutamate to regulate the
plant channel is still debated. According to phylogenetic analyses, the A. thaliana glutamate
receptor homologues can be subdivided into three separate subgroups (2). Studies with transgenic
plants suggested roles of members of the plant GLR family in Ca2+ fluxes (AtGLR2), coordination
of mitotic activity in the root apical meristem, regulation of abscisic acid biosynthesis and water
balance (AtGLR1.1), carbon/nitrogen sensing (AtGLR1.1) and resistance against fungal infection.
According to the endosymbiotic theory, cyanobacteria are considered to represent the precursors of
the present chloroplasts. Plant GluRs share a high homology with prokaryotic glutamate receptors:
the first to be identified and cloned is GluR0 from the cyanobacterium Synechocystis (2). The
putative products of the Atglr3.4 and Atglr3.5 genes display a possible targeting sequence for
chloroplast location and show a high degree of homology with cyanobacterial glutamate receptors.
In the present work, we provide multiple evidence for the chloroplast localization of two receptors,
GLR3.4 and GLR3.5 (splicing variant) in Arabidopsis cells and in spinach. By using specific
antibodies and confocal microscopy, we have localized these two iGluRs to the chloroplast in
Arabidopsis and to the inner envelope membrane in spinach. Purified inner envelope vesicles from
spinach display a cation-selective electrophysiological activity which is inhibited by DNQX, an
inhibitor of animal iGLRs known to act in plants as well. Furthermore, oxygen evolution
measurements suggest that chloroplast-located glutamate receptors may play a role in the regulation
of photosynthesis. These results identify for the first time glutamate receptors in chloroplasts and
their activity in native membranes at single channel level.
REFERENCES
1) Lam HM, Chiu J, Hsieh MH, Meisel L, Oliveira IC, Shin M, Coruzzi G (1998). Glutamate-receptor genes in plants.
Nature 396, 125-126.
2) Chiu J, DeSalle R, Lam HM, Meisel L, Coruzzi G (1999) Molecular evolution of glutamate receptors: a primitive
signaling mechanism that existed before plants and animals diverged. Mol. Biol. Evol. 16, 826-838.
81
CHIMERIC CONSTRUCTS FOR THE CONFORMATIONAL ANALYSIS AT SINGLE
MOLECULE LEVEL OF ALPHA-SYNUCLEIN AND ITS PATHOLOGICAL MUTANTS BY
ATOMIC FORCE MICROSCOPY
Isabella Tessari*1, Marco Brucale2, Massimo Sandal2, Bruno Samorì2, Luigi Bubacco*1
*Viale G. Colombo 3, 35131 Padova, Italy; Tel: +390498276340; E-mail: [email protected]
2. Dipartimento di Biochimica “G. Moruzzi”, Università degli Studi di Bologna
ABSTRACT
Human alpha-Synuclein (aSyn) is a natively unfolded protein whose aggregation into amyloid
fibrils is involved in the pathology of Parkinson disease (PD). The physiological function of the
protein is still poorly understood and the conformation adopted by aSyn in several putative
complexes with other proteins is still unclear. However, evidence is accumulating that aSyn in the
monomeric state, under in vitro physiological conditions, populates an ensemble of conformations
dynamically interchanging. On the other hand, also the aggregation process has been studied, with a
special interest for the early prefibrillar transient oligomers. The structure and dynamics of early
intermediates and of the determining factors leading to the aggregated states is still an unsolved
problems. Traditional bulk techniques can only provide ensemble-averaged information for
monomers and oligomers alike and cannot characterize the full complexity of the conformational
equilibria that trigger the aggregation process. We applied atomic force microscopy-based singlemolecule mechanical unfolding methodology (Single Molecule Force Spectroscopy - SMFS) to
study the conformational equilibrium of human wild-type and mutant aSyn. This methodology can
explore the full conformational space of a protein at single-molecule level, detecting even poorly
populated conformers and measuring their distribution in a variety of biologically important
conditions (1, 2).
In order to set up these experiments, some special chimeric polyproteins was designed and then
produced via molecular biology tools. To stretch an individual aSyn molecule by AFM, we need
handles to connect one end of the protein to the tip of the microscope and the other to the substrate.
To this aim, we followed the design proposed by J. Fernandez for the study of the random coiled
titin N2B segment (3), implementing the constructs with the insertion of the aSyn sequence in the
middle of the polyprotein. The overall structural pattern of these polyproteins is “M(n)-A-M(n)”,
where M is the flanking “marker” module, which is repeated in tandem (n) times, and A is the
central “analyte” module. The idea is that the flanking domains act both as "molecular handles"
(which help improving the success ratio and overall cleanness of the SMFS signal) but also as an
internal mechanical gauge (that generates a characteristic sawtooth shaped signal which allows the
unambiguous recognition of SMFS curves originating only from single molecules). For our first
works, the chosen flanking domain was the human titin I27 module, which was inserted in triplicate
on each side of a single aSyn module (wt or pathological mutated). However, this general strategy is
valid for various combinations of M and A and we are planning other constructs with a different
kind of molecular handles or with different permutations of the structural composition of
polyproteins (such as “M(n)-A(n)-M(n)”) in order to obtain new information about the structure and
the behaviour of aSyn especially in the aggregation process.
REFERENCES
1) Sandal M, et al (2008) Conformational equilibria in monomeric alpha-synuclein at the single-molecule level. PLoS
Biology 6, e6.
2) Brucale M, et al (2009) Pathogenic mutations shift the equilibria of alpha-synuclein single molecules towards
structured conformers. Chembiochem 10, 176-183.
3) Li H, et al (2002) Reverse engineering of the giant muscle protein titin. Nature 418, 998-1002.
82
PATHOGENIC MECHANISMS OF COLLAGEN VI MUSCULAR DYSTROPHIES
Tania Tiepolo1*, Paolo Grumati1, Anna Urciuolo1, Matilde Cescon1, Erika Rizzo1, Paola Braghetta1,
Alessia Angelin2, Elena Palma2, Emy Basso2, Patrizia Sabatelli3, Nadir M. Maraldi3, Luciano
Merlini4, Alessandra Ferlini4, Paolo Bernardi2, Paolo Bonaldo1
1
Department of Histology, Microbiology and Medical Biotechnologies, 2 Department of Biomedical
Sciences, University of Padova. 3 CNR-IGM, IOR, Bologna. 4 Department of Medical Genetics,
University of Ferrara.
* Tel: +390498276099; Email: [email protected]
ABSTRACT
Collagen VI is an extracellular matrix protein made of three genetically distinct chains and with a
broad distribution in several organs, including skeletal muscle. Mutations of COL6A1-A3 genes are
one of the most common causes of Bethlem Myopathy (BM) and Ullrich Congenital Muscular
Dystrophy (UCMD). Recently, we found that mutations of COL6A2 gene also cause another
inherited muscle syndrome (Autosomal Recessive Congenital Myosclerosis, ARCM), thus
expanding the spectrum of collagen VI disorders. ARCM is characterized by slender muscles with
firm 'woody' consistence and restricted movements of many joints due to severe muscle
contractures. In an Italian family affected by ARCM, we found that a homozygous nonsense
COL6A2 mutation leads to synthesis of a truncated α2(VI) chain, with impairment of collagen VI
assembly and abnormal microfilament organization (1). In a collaborative study, we identified three
novel genes (COL6A4-A6) coding for additional collagen VI subunits and showing tissue-regulated
expression, thus increasing the range of potential primary structures of collagen VI (2).
An excellent animal model of COL6 muscle disorders is represented by mice with targeted
disruption of the Col6a1 gene, previously generated in our laboratory. These mice show total lack
of collagen VI and display an early-onset myopathic syndrome affecting skeletal muscles and
characterized by spontaneous apoptosis and ultrastructural defects of mitochondria and
sarcoplasmic reticulum. Myofibers derived from Col6a1–/– mice have a latent mitochondrial
dysfunction caused by inappropriate opening of the permeability transition pore (PTP), an inner
membrane channel that plays a role in several forms of cell death and can be desensitized by
cyclosporin (Cs) A (3). Similarly to the animal model, patients affected by UCMD show an
increased rate of apoptosis in skeletal muscle in vivo, which is matched by a latent mitochondrial
dysfunction and spontaneous apoptosis as revealed in myoblast cultures derived from biopsies (4).
The results obtained in Col6a1–/– mice and in UCMD muscle cell cultures strongly indicate that
PTP desensitization by CsA might have a therapeutic potential for human collagen VI muscle
diseases (5,6). We carried out a pilot clinical trial with CsA in five patients affected by UCMD and
BM. The patients were given an oral formulation of CsA at 5 mg/kg/day for 1 month. The efficacy
of the CsA treatment, investigated by measuring different biological endpoints in muscle, was
demonstrated at different levels: normalization of mitochondrial dysfunction, decrease of apoptotic
rates, and increased muscle regeneration (7). These findings represent a significant step towards the
clinical goal of developing a therapy for collagen VI muscle diseases in humans.
In recent unpublished studies by our group in Col6a1 knockout mice we found that Col6a1-/- muscle
display marked changes in the activation status of some signal transducers involved in the control of
cell survival. Besides allowing the identification of the molecular pathways regulated by collagen
VI and how their orchestration results in the balanced survival of muscle cells, these studies may in
turn provide additional targets for therapeutic intervention.
REFERENCES
1) Merlini L, et al (2008) Neurology 71, 1245-1253.
2) Gara S, et al (2008) J Biol Chem 283, 10658-10670.
3) Irwin WA, et al (2003) Nature Genet. 35, 367-371.
4) Angelin A, et al. (2007) Proc Natl Acad Sci USA 104, 991-996.
5) Palma E, et al (2009) Hum Mol Genet In press.
6) Tiepolo T, et al (2009) Br J Pharmacol In press.
7) Merlini L, et al (2008) Proc Natl Acad Sci USA 105, 5225-5229.
83
ROLE OF INSULIN GROWTH FACTOR BINDING PROTEIN 2 IN RHABDOMYOSARCOMA
Lucia Tombolan*1, Cristiano De Pittà1, Angelica Zin2, Silvia Casara1, Angelo Rosolen2, Gerolamo
Lanfranchi1
*Viale G. Colombo 3, 35131 Padova, Italy; Tel: +390498276162; [email protected]
2. Clinica Pediatrica Oncoematologica, Ospedale di Padova.
ABSTRACT
Rhabdomyosarcoma (RMS) is the most frequent sarcoma of childhood with two major subtypes:
alveolar (ARMS) and embryonal (ERMS). ARMS are characterized, in about 75% of cases, by
specific chromosomal translocations that juxtaposed PAX3 or PAX7 to the FKHR gene.
We applied a gene expression profile approach to assess which biological differences exist between
PAX3-FKHR positive and negative ARMS. Our results suggest that PAX3-FKHR positive ARMS
possess a significantly different gene expression signature compared to PAX3-FKHR negative
tumors and that the expression level of a small set of genes can correctly classify the 2 ARMS
subtypes (1). Insulin Growth Factor Binding Protein-2 (IGFBP2) emerged as one of the most
interesting overexpressed genes in traslocation negative RMS as it is involved in several aspects of
growth and differentiation of tumors. This finding is confirmed by a meta-analysis study that we
have conducted on different microarray and SAGE datasets of RMS (2).
To better understand the role of IGFBP2 in RMS we have performed a set of functional studies.
To this purpose, we have studied the expression of IGFBP2 in 8 RMS cell lines, finding that
IGFBP2 mRNA and protein expression mirror the expression pattern of the corresponding RMS
patient samples analyzed by microarray. We have investigated the localization and distribution of
the protein in IGFBP2 overexpressing RMS cell lines, revealing that IGFBP2 is highly concentrated
in Golgi. IGFBP2 overexpression drammatically changes the Golgi morphology, since it appears
condensed in a juxtanuclear position and without its typical stack organization. This evidence was
confirmed also by electron microscopy. Moreover, after treatment with an inhibitor of protein
synthesis, like cycloheximide, we observed that the protein disappears from Golgi and is rapidly
secreted. This finding shows that IGFBP2 is accumulated in Golgi but is not clear if the abnormal
protein amount can influence the activity of the apparatus. It is known that IGFBP2 plays an
important role in several cancers, including pediatric solid tumors (3) and its expression is
correlated with the invasivness and higher tumorigenicity grade. Silencing of IGFBP2 in RMS cell
cultures was used as a tool to study its functional role and its relationships with cell growth and
invasivness. To determine the effects caused by the exogenously induced suppression of IGFBP2 on
RMS transcriptome, we have relied on the microarray technology. Some deregulated genes are
cyclin D1 and MCM2 involved in cell cycle progression, MMP2 that plays a role in invasivity and
CFL1 that is involved in the regulation of actin cytoskeleton and cell motility. Moreover, we have
observed a deregulation of some genes encoding for proteins of the secretory pathway and/or
protein transport. Subsequent FACS analysis and proliferation assay have confirmed that cells with
reduced levels of IGFBP2 have a decrease in proliferation rate and an evident G0/G1 phase arrest
which might be induced by dramatic reduction of cyclin D1 expression. RMS cell lines are very
useful model to study the function of this protein in RMS tumor biology. Nevertheless, such studies
are only a starting point to define the possible functional role of this protein in the clinical settings
or at least in tumors in vivo. To this purpose, we are assessing the IGFBP2 expression levels and
the morphological changes of the Golgi apparatus in tumor specimens as well as determining serum
levels of IGFBP2 in RMS patients that could be used as a prognostic parameter.
REFERENCES
1) De Pittà C, et al (2006) Gene expression profiling identifies potential relevant genes in alveolar rhabdomyosarcoma
pathogenesis and discriminates PAX3-FKHR positive and negative tumors. Int J Cancer 118, 2772-2781.
2) Romualdi C, et al (2006) Defining the gene expression signature of rhabdomyosarcoma by meta-analysis. BMC
Genomics 7, 287.
3) Firth SM, et al (2002) Cellular actions of the insulin-like growth factor binding proteins. Endocr Rev 23, 824-854.
84
ADVANCEMENTS IN THE FUNCTIONAL GENOMICS OF MYTILUS GALLOPROVINCIALIS
Laura Varotto*, Filippo Bernante, Umberto Rosani, Gerolamo Lanfranchi, Paola Venier
ABSTRACT
Marine mussels of the genus Mytilus are filter-feeding bivalves able to accumulate water
contaminants and commonly used in coastal water biomonitoring (1), yet their functional and
defence reactions are poorly characterized. Low competence for xenobiotic metabolism and
effective innate immunity could explain at least in part their resistance to toxic insults and potential
pathogens. Therefore, genomic approaches are needed to increase our knowledge on molecular and
cellular mechanisms involved in the mussel stress responses.
The first cDNA microarray of M. galloprovincialis, MytArray 1.0, has been defined at C.R.I.B.I. on
the basis of EST production from normal mussels, and it is currently tested with mussels exposed to
toxic and genotoxic stress in laboratory or in their natural environment (Venice lagoon, Italy) (2).
The resulting gene transcription patterns have revealed functional specificity of different mussel
tissues, specific subsets of transcripts significantly modulated by heavy metals or organic
contaminants and correct classification of mussels from fairly clean or contaminated lagoon sites (3,
4). Real-time PCR analysis and pollutant concentrations support the microarray data.
In the frame of the Integrated Project IMAQUANIM (FOOD-CT-2005-007103) aimed to define a
biotechnological platform for the European aquaculture, mussel EST sequencing and gene
expression profiling are progressing. In collaboration with A. Pallavicini (University of Trieste)
several new primary cDNA libraries and a final normalized library of high quality have been
prepared from mussels treated with bacterial antigens, biotoxins and chemical contaminants.
Following massive sequencing, the non redundant transcript tag collection has reached the size of
about 7k and the first annotated database of the Mediterranean mussel, Mytibase, has been
organized (5).
In the ESTs originated from haemocytes of immuno-stimulated mussels, we discovered a
remarkable abundance and molecular diversity of sequence identified as antimicrobial peptides.
These findings reveal the importance of such molecules in response to bacterial and viral pathogens,
and suggest ancient and specialized adaptation strategies against changeable non-self structures.
We are now applying advanced sequencing technologies to investigate in detail the variability of
these natural antibiotics. Moreover, a selection of immune-related transcripts have been used to
design oligonucleotide probes and arrange the first mussel Immunochip. Preliminary hybridization
experiments performed with RNAs amplified from immuno-stimulated mussels are revealing many
gene expression changes and, in general, a good performance of the new experimental tool. The in
house definition of a DNA microarray inclusive of the whole mussel transcript collection will
substantially expand the gene expression dataset of M. galloprovincialis and will provide new hints
on the physiological and genetic adaptation of mussels to the changing environment.
REFERENCES
1. Goldberg ED, Bertine KK (2000) Beyond the Mussel Watch new directions for monitoring marine pollution. Sci
Total Environ 247, 165-174.
2. Venier P, Pallavicini A, De Nardi B, Lanfranchi G (2003) Towards a catalogue of genes transcribed in multiple
tissues of Mytilus galloprovincialis. Gene 314, 29-40.
3. Venier P, De Pittà C, Pallavicini A, Marsano F, Varotto L, Romualdi C, Dondero F, Viarengo A, Lanfranchi G
(2006) Can gene expression trends reveal coastal water pollution? Mutat Res 602, 121-134.
4. Varotto L, Domeneghetti S, Rosani U, Pallavicini A, Bisol P, Lanfranchi G, Venier P. (2008) DNA microarray
analysis in M. galloprovincialis from the Venice lagoon. CORILA Research Programme 2004-2006 Vol VI. Ed. P.P.
Campostrini Venezia, pp. 269-277.
5. Venier P, De Pittà C, Bernante F, Varotto L, De Nardi B, Bovo G, Roch F, Novoa B, Figueras A, Pallavicini A,
Lanfranchi G (2009) MytiBase: a knowledgebase of mussel (M. galloprovincialis) transcribed sequences. BMC
Genomics 10, 72.
85
WORKING ON SEQUENCING DATA PRODUCTION AND ASSEMBLY
Alessandro Vezzi* Michela D’Angelo, Nicola Vitulo, Stefano Campanaro, Rosanna Zimbello,
Riccardo Schiavon, Davide Campagna, Alessandro Albiero, Chiara Rigobello, Giorgio Valle.
ABSTRACT
“Just over three years ago, it was announced that a first draft of the human genome sequence had
been completed. Gaps and errors remained, but the job of fixing those problems is now largely
done” (1). All of us knows the crucial contribution that a genome sequencing project gives in terms
of data production and subsequently how much it dramatically increases the genotypic/phenotypic
knowledge of the analyzed organism. But, to what extent do we have to trust the produced data?
Are the different genome sequencing strategies providing the same results with respect to accuracy
and assembly of the same genomic regions? I never worked in the human genome project but my
feelings are that: a) the public consortium (2) (clone-by-clone approach) performed better than the
Celera private company (3) (whole genome shotgun (WGS) approach); b) even now the human
genome is partly unfinished.
Starting from these few observations I will briefly point out some hints gained working on different
genome projects that our group carried out in the last years.
Surely the WGS approach is a fast and cheap methodology for getting the “complete” DNA
complement of an organism. It implies the random fragmentation of the genomic DNA, its cloning
in different types of vectors and then the sequencing of a sufficient number of clones in such a way
to obtain the entire sequence of the genome (4). As I said before, it is a fast approach, since it does
not required a physical map of the genome and, once the genomic libraries are built up, the
sequencing data are rapidly produced. But, the more the genome is complex in terms of type and
amount of repeat regions, the less the final sequence will be both accurate and reliable. The risks are
to create mis-assemblies, which affect any large scale analyses of the genome, and to completely
miss some part of the genome, possibly associated to diseases or other characters of the organism.
On the other hand, the clone-by-clone approach is much more expensive and considerably timeconsuming, since it implies the creation of an accurate physical map of the genome, the isolation of
specific clones to be sequenced (usually BAC clones), and finally the preparation and the
sequencing of shotgun plasmid libraries for each of the selected BACs (5). Since the approach
proceeds by extending the sequence from both ends of the so-called seed BACs (marker associated
BACs) it promptly allows to place each growing sequencing isle in its specific genomic location.
Nevertheless, since the BAC libraries are generally created by means of partial restriction digestion
of the genomic DNA, the process of extending from a BAC implies always a certain amount of
sequence redundancy. Moreover, depending on both BAC library quality and chance, it is possible
to reach a point where no other clones are found for extending some sequencing isles.
Thus, which is the best sequencing strategy to choose for a new genome project?
The answer is: “definitely the WGS!”. Even if the sequencing data are somehow less accurate, this
drawback is of minor importance with respect to their fast production and thus utilization. Also,
only the WGS approach is technically feasible with the emerging new sequencing technologies.
REFERENCES
1) Stein LD (2004) End of the beginning. Nature. 431, 915.
2) Lander SE, et al (2001) Initial sequencing and analysis of the human genome. Nature. 409, 860.
3) Venter JC, et al (2001) The sequence of the human genome. Science. 291, 1304.
4) Vezzi A, et al (2005) Life at depth: Photobacterium profundum genome sequence and expression analysis. Science.
307, 1459.
5) Mueller LA, et al (2009) A snapshot of the emerging tomato genome sequence: the Tomato Genome Sequencing
Consortium. The Plant Genome. In press.
86
INSERTION SEQUENCES DISTRIBUTION AND EVOLUTION IN PROKARYOTIC
GENOMES USING NETWORK APPROACH
Nicola Vitulo*, Riccardo Rosselli, Alessandro Vezzi, Giorgio Valle
ABSTRACT
Insertion Sequences (IS) are the simplest form of transposable elements, coding only the enzymes
allowing for their mobility. IS elements are transferred by all the classical mechanisms of horizontal
gene transfer (HGT). In association with other elements, they can mediate the transfer of genetic
information between genomes, as for example, genes for antibiotic resistance, virulence and new
metabolic pathway. Because of their effect, ISs are considered key elements for genome plasticity
and variability, providing important adaptive changes to genomes. ISs are found in most but not all
eubacterial and archeal genomes and many authors regard them as nearly ubiquitous. Many reports
indicate a high variability in the type and frequency of IS elements among bacteria (1).
ISs evolutionary history and occurrence among organisms is quite complex and a combination of
different events can describe the observed ISs distribution: vertical gene inheritance can explain the
co-presence of the same IS elements on the genome of phylogenetically correlated bacteria, while
horizontal gene transfer can be the reason for the occurrence of similar mobile elements on distantly
related organisms. Moreover gene loss and gene expansion events play an important role on IS
evolution. In this work we studied the ISs distribution among 765 fully sequenced bacteria, both
eubacterial and archeal genomes, applying a network approach for the data analysis.
At first we re-annotated and re-analyze the proteomes of the bacteria to detect as many as possible
IS elements. Using blast algorithm we performed a similarity search of the bacterial proteins against
ISfinder database (http://www-is.biotoul.fr/) (2). This database is a collection of about 2400 IS
protein from bacterial and archeal IS elements, grouped into 26 different family. Our analysis
identified 34362 proteins showing a significant similarity with an IS. To classify the IS elements,
we assigned each putative IS CDS to the family to which it shows the best BlastP hit in the ISfinder
database.
For each considered genome, a similarity measure was calculated as the number of ISs shared by
two organisms on the total number of unique ISs. The similarity score ranges from 0 if two
organisms do not share any insertion sequence to 100 if they share all the same ISs.
Using the similarity matrix, we produced a network in which each node represents an organism
connected to another one if the similarity value is higher of a defined cut-off. Using a cut-off of
34% we obtained a network with 447 nodes and 1212 edges that comprise 70% of organisms that
have at least one insertion sequence. Depending on the similarity value, it is possible to highlight
different aspects of the nature of the IS elements. As expected, for low similarity values, ISs seems
to be nearly ubiquitous, producing a high-interconnected network. Interestingly, for higher values,
the background noise decreases and groups of organisms phylogenetically related became clear.
These results suggest the existence of a “core” of IS elements characteristic for each phyla and often
for each taxonomic group, transferred with a vertical mechanisms and with a phylogenetic signal.
At the same time, similar ISs have been found in non-related organisms, highlighting the capability
to transpose from a genome to another. Further analyses are currently ongoing to understand any
possible correlation between ISs, co-transposed genes and groups of organisms that can have
similar habitat, lifestyle and metabolic pathways.
REFERENCES
1) Touchon M, Rocha EP (2007) Causes of insertion sequences abundance in prokaryiotic genomes. Mol. Biol. Evol.
24, 969-981.
2) Siguier P, Perochon J, Lestrade L, Mahillon J, Chandler M (2006) ISfinder: the reference centre for bacterial
insertion sequence. Nucleic Acid Research 34, D32-634.
87
HYALURONAN BASED SCAFFOLDS FOR TISSUE ENGINEERING
Barbara Zavan*
Dipartimento di Istologia, Microbiologia e Biotecnologie Mediche, Università degli Studi di Padova
ABSTRACT
Tissue engineering is a multidisciplinary field focused on in vitro reconstruction of mammalian
tissues. In native tissues, cells grow onto or into three-dimensional scaffolds made by the
extracellular matrix. In these natural conditions, cells proliferate, move in a three-dimensional
fashion, and maintain their phenotype. In order to allow a similar three-dimensional organization of
in vitro cultured cells, biocompatible scaffolds are needed. This need has provided immense
momentum in the research of “smart scaffolds” to use in cell culture. My post doc research
activities have been focused on tissue engineering by means of application of hyaluronan
derivatives, namely benzyl ester of hyaluronan (Hyaff® -Fidia Advanced Biopolymers- Abano T.
Italy). Hyaff® can be processed to obtain several types of devices such as tubes, membranes, nonwoven fabrics, gauzes, and sponges. All these scaffolds are highly biocompatible and when placed
inside the human body, do not elicit any adverse reactions and are resorbed by the host tissues.
Human hepatocytes (1), dermal fibroblasts and keratinocytes(2), chondrocytes(3), Schwann cells
(4), bone marrow derived mesenchymal stem cells (5) and adipose tissue derived mesenchymal
stem cells (6) have been successfully cultured in our lab. All these adult cell types have been able to
attach to the biomaterial fibers, proliferate, colonize the entire device maintaining they typical
phenotype. Moreover adult stem cells were able to commit in several tissues such as bone, cartilage,
fat.
The same scaffolds, in tube meshes, has been applied for vascular tissue engineering that has
emerged as a promising technology for the design of an ideal, responsive, living conduit with
properties similar to that of native tissue. During my post doc research, I’ve demonstrated that a
hyaluronan-based scaffold grafted in the abdominal rat aorta successfully guided the complete
regeneration of a well-functioning small diameter (2 mm) neoartery. When applied in a porcine
model (carotid artery) the tubular prosthesis (4 mm) has been able to develop into neovessel of
larger size: after 4 months biomaterial was almost completely degraded and replaced by a neoartery
segment composed of mature smooth muscle cells, collagen and elastin fibers organized in layers
and was completely covered on the luminal surface by endothelial cells. These studies confirm the
ability of these constructs to guide the development of a well-functioning neoartery, with the
remarkable additional attribute of facilitating the formation of organized layers of elastin fibers.
REFERENCES
1) Zavan B, et al, Abatangelo G, Cortivo R. (2005) Extracellular matrix-enriched polymeric scaffolds as a substrate for
epatocyte cultures: in vitro and in vivo studies. Biomaterials 26, 7038-7045.
2) Tonello C, Zavan B, et al (2005) In vitro reconstruction of an endothelialized skin substitute provided with a
microcapillary network using biopolymer scaffolds. FASEB J. 19,1546-1548.
3) Brun P, Zavan et al (2008) Characteristics of repair tissue in second-look and third-look biopsies from patients
treated with engineered cartilage: relationship to symptomatology and time after implantation. Arthritis Res Ther. 10,
R132.
4) Zavan B, et al (2008) New 3D hyaluronan-based scaffold for in vitro reconstruction of the rat sciatic nerve. Neurol
Res. 30, 190-196.
5) Zavan B, et al (2007) Osteogenic and chondrogenic differentiation: comparison of human and rat bone marrow
mesenchymal stem cells cultured into polymeric scaffolds. Eur J Histochem. 51, 1-8.
6) Aguiari P, Zavan B et al. (2008) High glucose induces adipogenic differentiation of muscle-derived stem cells. Proc
Natl Acad Sci U S A. 105, 1226-1231.
7) Lepidi S, Zavan B et al (2006) Hyaluronan biodegradable scaffold for small-caliber artery grafting: preliminary
results in an animal model. Eur J Vasc Endovasc Surg. 32, 411-417.
8) Lepidi S, Zavan B, et al (2005) In vivo regeneration of small-diameter (2 mm) arteries using a polymer scaffold.
FASEB J. 20, 103-105.
9) Zavan B, et al (2008) Neoarteries grown in vivo using a tissue-engineered hyaluronan-based scaffold. FASEB J. 22,
2853-2861.
88

La ricerca dei Dottorandi e dei Post Doc al Vallisneri

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