Báo cáo khoa học:Invited Lectures docx

We have evidence that DNA is intro-duced into the cell through the positively charged pore formed by the PilQ complex and that other DNA binding components processDNA inside the periplas

Invited Lectures

A1-L5

Mitotic chromosome condensation and segration

C D’Ambrosio1, Y Katou2, K Shirahige2, F Uhlmann1

Furthermore, condensin is required during anaphase to promote sister chromatid resolution In the absence of condensin, anaphase ges and segregation defects are observed How condensin promotes sister chromatid resolution is unknown We have used the buddingyeast rDNA as a model locus, whose segregation depends on condensin during anaphase We show that anaphase bridges in a condensinmutant are resolved by ectopic expression of a foreign (Chlorella virus) but not endogenous yeast topoisomerase II (topo II) This sug-gests that catenation prevents sister rDNA segregation, and that yeast topo II is ineffective in decatenating the rDNA, and maybe otherchromosomal regions, in the absence of condensin

brid-B5-L1

The Meningococcal Transformation Machine

T Tønjum

Centre for Molecular Biology and Neuroscience, University of Oslo, Oslo, NORWAY

Neisseria meningitidisis a leading cause of bacterial meningitis and septicaemia worldwide This bacterium is constitutively competentfor transformation throughout its entire life-cycle Transformation in neisserial species is particularly important for genetic exchange anddiversity and is coupled to the expression of type IV pili Meningococcal type IV pili are present on the cell surface as bundled filamen-tous appendages and are assembled, extruded and retracted by pilus biogenesis components These proteins are homologous to type IIsecretion components in Gram-negative species

The binding and uptake of transforming DNA into the meningococcal cell can be divided into four stages: entry through an outer brane pore, transit of the periplasm, transport across the inner membrane and genome integration We propose that the early stage ofmeningococcal transformation is coupled to pilus retraction and that transforming DNA is non-specifically attached to retracting pili.Previously, we have shown that pili directly interact with the secretin PilQ in the outer membrane We have evidence that DNA is intro-duced into the cell through the positively charged pore formed by the PilQ complex and that other DNA binding components processDNA inside the periplasm and inner membrane

mem-To address the multi-step nature of DNA binding and uptake during transformation, we have identified DNA binding components inmeningococcal cellular fractions By using a combination of molecular and imaging approaches, DNA binding candidates are beingassessed for their structure-function relationships to define how they act and interact, with each other and with DNA The goal is todefine how these DNA binding components provide dynamic multi-site targeting and entry of DNA

Global Mapping of the Yeast Genetic Interaction Network

C Boone, III

University of Toronto, Toronto, ON, CANADA

Synthetic Genetic Array (SGA) analysis automates yeast genetics, enabling a number of different large-scale/systematic studies In ular, we are attempting to generate the complete synthetic lethal genetic interaction map for yeast cells This map can be used to definecomplexes and pathways in the cell, but perhaps more importantly, it adds functional information to the protein-protein interactionmap, identifying complexes and pathways that buffer one another and somehow work together as backup systems One of our majorchallenges has been to generate a quantitative model for scoring genetic interactions based upon plate images and the predicted fitness

partic-of the double mutant relative to each single mutant Another challenge has been to develop robotic platforms for our own put analysis and individual users in other labs I will present the results of our latest network compilation and describe our plans fortackling the yeast genome In addition, I will describe how the yeast genetic interaction network can be used to interpret chemical-geneticinteractions and link bioactive compounds to their target pathways

high-through-E2-L2

Multi-factorial disease and robustness: Where Systems Biology makes a difference

H V Westerhoff

Manchester Center for Integrative Systems Biology and Netherlands Institute for Systems Biology, Manchester, UNITED KINGDOM

As we now know what Systems Biology is, it may be worthwhile to examine the difference it will make In particular it may be useful toexamine where such a difference is needed and how it may be brought about by those who are interested

There are a number of diseases that can be related to a single faulty gene product and or a single invading microorganism Quite a few

of these ‘monofactorial’ diseases can be cured, or structural molecular biology has defined a strategy to identify or design the ponding drugs However, there is a more substantial number of diseases for which a vast amount of biomedical research has been moun-ted and that cannot be cured The research has led to substantial scientific success and biochemical understanding, but to little if anyprogress in defining cures The examples are on the long list of ‘multifactorial diseases’ and include type-2 diabetes, obesity, heart dis-ease, cancer and arthritis

corres-The approach that has been successful for monofactorial diseases is not optimal vis-a`-vis these diseases Finding cures for these diseasesmay require a shift in strategy These diseases are ‘network’ or ‘Systems Biology diseases’ and to manage them one needs to addressfaults in the network rather than in just a molecule

I shall discuss some of the ways the Manchester-Amsterdam Systems Biology axis tries to devise such network-based strategies Theseinclude differential network based drug design against Trypanosoma brucei, modular kinetics of a connection between type-2 diabetesand obesity, and new paradigms for searching new anti-tumor drugs Fragility and robustness are Leitmotive, but differentially so

Insight into alternative-splicing mechanisms with the solution structures of several RRM-RNA complexes

F Allain

Institute of Molecular Biology and Biophysics, Zu¨rich, SWITZERLAND

Alternative-splicing is probably the most effective post-transcriptional gene regulatory event, as more than 60% of the human genes arealternatively spliced Defects in alternative-splicing are the cause of many genetic diseases We recently determined the structure of sev-eral important human alternative-splicing factors in complex with RNA, namely the Poly-pyrimidine Tract Binding Protein (PTB1),Fox-12, SRp203 and of a potential splicing factor RBMY4 The structures of PTB and Fox-1 showed that these alternative-splicing fac-tors bind RNA sequence-specifically and might control the fate of an alternative-exon in remodeling the RNA The structures of SRp20and of RBMY in complex with RNA revealed unusual RNA recognition modes SRp20 binds RNA in a semi-sequence specific manner3and the human RBMY binds a stem-loop RNA whereas its mouse homolog doesn’t4 Implications of these structural findings for under-standing the mechanism of action of these alternative-splicing factors will be discussed

References:

1 Oberstrass, F.C et al Structure of PTB bound to RNA: specific binding and implications for splicing regulation Science 309, 2054–7(2005)

2 Auweter, S.D et al Molecular basis of RNA recognition by the human alternative splicing factor Fox-1 Embo J 25, 163–73 (2006)

3 Hargous, Y et al Molecular basis of RNA recognition and TAP binding by the SR proteins SRp20 and 9G8 Embo J 25, 5126–37(2006)

4 Skrisovska, L et al The testis-specific human protein RBMY recognizes RNA through a novel mode of interaction EMBO Rep 8,372–79 (2007)

A1-13

Characterization of a subtelomeric satellite DNA

in the mollusc Donax trunculus

V Petrovic1, C Pe´rez-Garcı´a2, J J Pasantes2, E Prats3, M Plohl1

1Rudjer Boskovic Institute, Zagreb, CROATIA,2University of Vigo,

Vigo, SPAIN,3Institut de Biologia Molecular de Barcelona – CSIC,

Barcelona, SPAIN

Satellite DNAs are non-coding, tandemly repeated DNA sequences

that comprise long arrays in the genome and are usually located

within heterochromatic regions of chromosomes Among marine

invertebrates, satellite DNAs have so far been studied in detail

only in a couple of taxonomic groups We detected a novel satellite

DNA present in the genome of the bivalve mollusc Donax trunculus

The monomer repeat length of this satellite is 169 bp, while the

sequence analysis reveals high sequence conservation maintained

throughout the entire monomer length In contrast to the other

satellites detected previously in the genome of D trunculus

(Petrovic and Plohl, Gene 2005, 362:37, and references therein),

this is a GC rich satellite that has also been shown to exhibit CpG

site methylation In addition, this is the most abundant (5 %)

among all detected satellites Fluorescence in situ hybridization

revealed that this satellite is located in subtelomeric regions on

more than half of D trunculus chromosome pairs Its location

on a subset of chromosomes and homogeneity of randomly cloned

variants indicate different homogenization mechanisms acting

between chromosomes, and/or different evolutionary history of

chromosomes in the D trunculus genome

A1-14

The evolutionary role of the LCR16A element in

Mendelian disorders

O Symmons, H de Boussac, A Va´radi, T Aranyi

Institute of Enzymology, Hungarian Academy of Sciences,

Budapest, HUNGARY

With the completion of the human genome, a major challenge is to

determine correlation between genome evolution and phenotypic

changes The best studied human phenotypes are Mendelian

disor-ders Two such examples are Polycystic Kidney Disease (PKD)

and Pseudoxanthoma Elasticum (PXE) caused by PKD1 and

ABCC6, respectively Both genes were mapped to the short arm of

Chr 16 However, their study is complicated by the presence of

multiple pseudogenes of high degree of similarity and also located

on Chr 16 Our aim was to characterize these pseudogenes and

understand the mechanism and potential impact of their

duplica-tion We found that every pseudogene is situated in close proximity

to an LCR16a element, which is a very proliferative duplicon on

Chr 16 LCR16a was proposed to be the driving force for the

for-mation of intrachromosomal duplication blocks We analyzed the

phylogenetic relationship of the human LCR16a elements, which

form three distinct clusters The duplicated copies of the ABCC6

and PKD1 genes are all associated with the same cluster We

dem-onstrated that some of the duplication events were hominin-specific

and showed that this can lead to hominin-specific chimeric

tran-scripts We have also investigated the possible consequences of the

presence of pseudogene sequences in the aetiology of PXE and

PKD Our results demonstrate that non-allelic homologous

recom-bination has occurred and is manifested in these regions and gene

conversion is still an on-going process

A2-27 The BAH domain of Sir3 is the primary nucleosome binding domain in the SIR silencing complex

M Onishi1, G Liou1,2, D Moazed1

1

Harvard Medical School, Boston, MA,2Division of Molecular andGenomic Medicine, National Health Research Institutes, Miao-LiCounty, TAIWAN

In Saccharomyces cerevisiae, Silent information regulator (Sir) teins are required for regional gene silencing at the silent matingtype cassettes and telomeres The initiation of transcriptional silen-cing at these domains is proposed to involve the recruitment of theSir complex, composed of Sir2, Sir3, and Sir4, by DNA-bindingproteins This recruitment is followed by iterative cycles of NAD-dependent deacetylation, production of O-acetyl-ADP-ribose, andbinding of Sir3 and Sir4 to the deacetylated nucleosomes, andleads to the creation of extended silent chromatin domains Thesesilenced regions have histones that are hypoacetylated and hypo-methylated, while disruption of enzymes that acetylate and methy-late histones leads to mislocalization of Sir3 Thus, histonemodifications play a crucial role in the assembly of silent chroma-tin However, while Sir3 has been shown to have histone and nu-cleosome binding properties in vitro, specific binding of Sir3 tonucleosomes, as it occurs in vivo, has yet to be observed We showthat the Bromo-adjacent-homology (BAH) domain of Sir3 is neces-sary for nucleosome binding and that this binding is regulated byhistone acetylation and methylation These results suggest that theBAH domain, found in many eukaryotic chromatin proteins, binds

pro-to specifically modified nucleosomes

A2-28 Post synthetic acetylation of HMGB1 protein stimulates DNA end-joining

I Ugrinova1, E Mitkova1, C Moskalenko2, I Pashev1,

E A Pasheva1

1

Institut Molecular biology,Bulgarian.Academy of Sciences, Sofia,BULGARIA, 2Laboratoire Joliot-Curie, Ecole Normale Superieure,Lyon, FRANCE

The ability of HMGB1 protein to recognize bent DNA and toinduce bending in linear duplex DNA defines HMGB1 as an archi-tectural factor We already demonstrated that the binding affinity

of the protein to various bent DNA structures is enhanced upon invivo acetylation at Lys 2 Here we investigate how this modifica-tion of HMGB1 affects its ability to bend DNA We report thatthe modified protein cannot bend short DNA fragments but,instead, it stimulates joining of the same fragments via their ends.The same properties demonstrates in vivo acetylated HMGB1 lack-ing its acidic tail Further in vitro acetylation with acetyltransferaseCBP of the truncated protein at Lys81 (possible upon tail removalonly), restores the protein’s bending ability, while the stimulation

of DNA end-joining is strongly reduced We conclude, therefore,that the ability of HMGB1 to bend DNA or to stimulate end-join-ing is modulated in vitro by acetylation In an attempt to explainthe properties of in vivo acetylated HMGB1, its complexes withDNA have been analysed by both protein-DNA cross linking andAFM (atomic force microscopy) Unlike the parental protein,bound mainly within the internal sequences, the acetylatedHMGB1 binds preferentially to DNA ends We propose that theloading of acetylated protein on DNA ends accounts for both thefailure to bend DNA and the stimulation of DNA end-joining

Analysis of restriction fragments distribution in

genomic fungal DNAs.

V N Tomilov1, D A Gonchar1, M A Abdurashitov1,

A Schueffler2, H Anke2, S K Degtyarev1

1SibEnzyme Ltd., Novosibirsk, RUSSIAN FEDERATION,2IBWF,

Kaiserslautern, GERMANY

Theoretical diagrams of fungal chromosomal DNA (Magnaporthe

grisea, Mycosphaerella graminicola and Schizosaccharomyces

po-mbe) cleavage at more than 125 different 4 - 8 nucleotides

sequences have been simulated based on recently determined DNA

primary structures All chosen DNA sequences are the recognition

sites of restriction enzymes In all diagrams obtained we have

selec-ted the fragments presenselec-ted in peak’s quantities Each peak has

been characterized by multiplication of the number of fragments

by their length In the analysis we have considered peaks with a

value of 0.15% or higher compared to the length of the

corres-ponding chromosomes Calculated Schizosaccharomyces pombe

DNA digestions did not contain such peaks Theoretical cleavage

of Magnaporthe grisea and Mycosphaerella graminicola DNAs at

the appropriate recognition sites of restriction enzymes resulted in

more than 10 such peaks A more detailed study has shown that

the presence of peaks in DNA digestions depends on genome

com-plexity Hydrolysis of the fungal DNA with several restriction

enzymes has confirmed the presence of the predicted fragments

A3-12

Nitric oxide levels and eNOS gene

polymorphism in patients with coronary artery

disease documented by angiography

G Yilmaz1, I Mehmetoglu1, S Kurban1, H Aacar2

1

Meram Faculty of Medicine, Department of Biochemistry,

Univer-sity of Selcuk, Konya, TURKEY, 2Meram Faculty of Medicine,

Department of Medical Genetics, University of Selcuk, Konya,

TURKEY

The aim of this study was to investigate 4a/4b VNTR

polymorph-ism in intron 4, the G10-T polymorphpolymorph-ism in intron 23 and plasma

NO levels in patients with coronary artery disease (CAD)

docu-mented by angiorgaphy and healty controls

The study was performed on 106 patients (30 F, 76 M) aged 40–70

years and 89 healty controls (40 males, 49 females) aged 41–73

years All patients had more than 50% stenosis in at least one

cor-onary artery documented by angiography

Blood samples were obtained from all subjects after fasting NO

was measured as NOx by Griess reaction on EDTA samples and

eNOS gene polymorphism was investigated by PCR-RFLP

tech-nique

NO levels of the patients and the controls were 47,78 ± 27 lmol/

L and 41,44 ± 25,9 lmol/L respectively the difference of which

was significant

There was no significant difference between allelic frequencies of

eNOS gene intron 4 a/b VNTR and intron 23 polymorphisms of

the groups

Therefore, our results show that eNOS gene intron 4 a/b VNTR

and intron 23 polymorphisms are not independent risk faktors for

CAD in a central area (Konya) of TURKEY

A4-11 The role of miRNAs in DNA replication and damage repair

E Zlotorynski, J van Duijse, R AgamiThe Netherlands Cancer Institute, Amsterdam, THE NETHER-LANDS

The miRNA genes encode short RNAs, which suppress the sion of mRNAs bearing complementary target sequences Each mi-RNA is predicted to target up to hundreds of genes, but thefunction of only a handful miRNAs has been determined, under-ling the need for a systematic screening approach to identify theirroles in development and disease Primary human cells grown withlow levels of DNA replication inhibitors undergo irreversiblegrowth arrest due to the accumulation of DNA damage In order

expres-to identify miRNAs that can modulate DNA replication and DNAdamage repair, we performed a screen for miRNAs that can affectreplication inhibition-induced growth arrest Human primary cellswere transduced with a human miRNA expression library, andgrown for 3 weeks with low levels of the replication inhibitorsAphidicolin, Hydroxyurea, Etoposide or Doxorubicin The relativeabundance of miRNA expressing cells was then measured byhybridization of the DNA of the different cell populations on amiRNA micro-array We subsequently validated the screen resultsfor each miRNA individually Several miRNAs were found tooverride the replication inhibition-induced arrest, whereas otherswere found to enhance the effect of replication inhibition We arecurrently studying the mechanism of action of these miRNAs, inorder to understand their role in DNA replication and DNA dam-age repair

A4-12 Identification of novel regulators of microRNA function

M Kedde, R AgamiNetherlands Cancer Institute, Amsterdam,THE NETHERLANDS

The 3¢-untranslated regions (UTRs) of many mRNAs are subject

to posttrancriptional regulation by microRNAs (miRNAs), whichfunction as repressors of their target mRNAs Little is knownabout the regulation of miRNA targeting and the reversibility ofthe process Here I will present evidence that certain RNA bindingproteins can repress miRNA activity both in vitro and in vivo

Role of miRNAs in replicative senescence of

endothelial cells

K Fortschegger, M Wieser, H Katinger, R Voglauer, J Grillari

University of Natural Resources and Applied Life Sciences, Vienna,

AUSTRIA

In vitroreplicative senescence of human umbilical vein endothelial

cells (HUVECs) is widely used as a model system for aging

Indeed, senescent cells can be detected in atherosclerotic plaques

and might contribute to aging per se and the development of

car-diovascular disease We aim to elucidate to what extent the

expres-sion profile of micro-RNAs (miRNAs) is changed throughout in

vitro senescence and whether these changes are responsible for

observed differences in protein expression patterns

Early passage HUVECs of five donors are compared with their

senescent counterparts regarding miRNA expression profiles

LNA-microarray analysis reveals consistent differential expression

of at least 12 miRNAs, most of them upregulated in senescent

cells Differential regulation is confirmed by Northern blot analysis

or quantitative realtime PCR Selected miRNAs will be

investi-gated regarding their potential targets and their influence on

cul-tured cells when overexpressed or blocked Furthermore cloning

and sequencing of the miRNA-pool will help to identify

HUVEC-specific small regulatory RNA species

Some of the miRNAs which we found upregulated in senescent

cells have growth factor receptor mRNAs as their putative targets

The suppressed expression of such proteins would perfectly agree

with the observed growth arrest of senescent cells Future

experi-ments will predominantly address this topic

Acknowledgement: Supported by the GEN-AU II Pilot Program

A5-44

Efficient initiation of k DNA replication requires

transcription and interaction

between RNA polymerase and the kO

replication protein

A Szambowska, G Wegrzyn, M Glinkowska

University of Gdansk, Gdansk, POLAND

Initiation of k DNA replication in vivo and in crude in vitro system

is strongly dependent on transcription initiated at the k pR

promo-ter This transcription event is indispensable for transcriptional

activation of orik and expression of the kO and kP genes

Forma-tion of the k replicaForma-tion initiaForma-tion complex requires both kO and

kP initiators and many E coli proteins; including DnaB, primase,

SSB, DNA polymerase III holoenzyme as well as DnaK, DnaJ

and GrpE chaperones

In this study, we investigated in more detail the role of

transcrip-tion and RNA polymerase in initiatranscrip-tion of replicatranscrip-tion at orik

Using gel mobility shift assay, we detected stimulation of kO

bind-ing to iterones and its self-assembly into a nucleosome-like

struc-ture by RNA polymerase and transcription In addition, we

observed enhanced dimmer formation by kO protein in the

pres-ence of both RNA polymerase and transcription Stimulation of

O-some formation by trancription was also confirmed by gel

filtra-tion These experiments revealed, however, that transcription

caused no significant disassembly of nucleoprotein structure

formed by kO Glutaraldehyde - crosslinking experiments revealed

also a possible direct interaction between kO protein and b

-sub-unit of the E coli RNA polymerase We obtained additional

evidence supporting this hypotesis using in vitro replication

experi-ments, in which a plasmid containing phi10 promoter of

bacterio-phage T7, instead of the k pRpromoter, was used as a template

Interestingly, such a plasmid did not replicate in vitro in the

presence of T7 RNA polymerase Using in vitro permanganate

footprinting method we detected also that RNA polymerase and

trancription process enhance orik unwinding

A5-45 External cell control PCR: a new strategy for qPCR normalization

A Bors1, P Ribiczey1, G Ko¨blo¨s2, Z Ujfaludi3, A Va´radi2,

T Kova´cs1, T Aranyi2

1National Medical Centre, Institute of Haematology and logy, Budapest, HUNGARY, 2Institute of Enzymology, HungarianAcademy of Sciences, Budapest, HUNGARY,3University of Szeged,Faculty of Sciences, Dept of Biochemistry and Molecular Biology,Szeged, HUNGARY

Immuno-Quantitative RT-PCR (qRT-PCR) is a widely used method todetermine relative gene expression levels Quantification of theobserved expression levels becomes reliable after normalization tothe expression of an internal standard gene However, the expres-sion of commonly used internal standard genes is often unstable,which may compromise quantification To overcome the drawback

of unstable internal standards, we developed a new method, calledexternal cell control PCR (eccPCR) This method is based on theaddition of control cells to the studied cells before RNA extractionand qRT-PCR Only the control cells express the reference gene,while only the studied cells express the gene of interest Here wepresent the validation of the method in various model systemsincluding both adherent and non-adherent cells and either mam-malian or Drosophila external control cells

A5-46 Role of Arabidopsis SR protein atRSp31 in plant growth and development.

M Maronova1, M Kalyna1, C G Simpson2, J W Brown2,

Our aim was to characterize the function of plant specific SR tein atRSp31 in plant growth and development using plants over-expressing atRSp31 and atRSp31 mutant plants A phenotypicstudy revealed that increased levels of atRSp31 negatively affectedplants upon oxidative stress, salt, abscisic acid and sugar treat-ments Loss of function mutation showed an improved response tothese stress conditions

pro-Interestingly, plants overexpressing atRSp31 showed late floweringphenotype and early senescence

Corroborating these data, microarray analysis of overall expressionchange in atRSp31 overexpressing plants pointed out genesinvolved in response to diverse biotic and abiotic stimuli, genesparticipating in ROS metabolism, carbohydrate metabolism, agingand senescence Moreover, several other SR proteins appeared to

be affected

Further, to monitor the influence of atRSp31 on the known native splicing events, we used a high throughput RT-PCR screen.The most prominent changes were observed in the splicing patterns

alter-of the SR protein atSRp30, the floral repressor MAF2 (AGL31),the two DNA repair proteins RAD1 and RAD23, and severalunknown proteins

Our results suggest that atRSp31 is a negative regulator of stressresponse It participates in response to wounding, oxidative stress,abscisic acid and salt stress It is involved in flowering control andaging process

Properties of the Escherichia coli DnaA protein

required for transcriptional activation of

bacteriophage k pRpromoter

M Glinkowska1, A Szambowska1, M S Thomas2, G We˛grzyn1

1University of Gdan´sk, Gdansk, POLAND,2University of Sheffield,

Sheffield, UNITED KINGDOM

DnaA protein is a bacterial replication initiator It recognizes oriC

and governs subsequent assembly of the replication complex

DnaA was also demonstrated to act as a transcription factor

Apart from influencing mRNA synthesis of several bacterial genes,

it also regulates transcription starting from phage lambda pR

pro-moter, through a mechanism that requires DnaA binding to two

non-canonical DnaA-boxes situated downstream of transcription

start site

DnaA belongs to the AAA+ superfamily of ATP-ases For its

function as a replication initiator, it requires formation of

higher-order nucleoprotein complexes and self-oligomerization Domain I

and III of DnaA contain specific sites necessary for interaction of

DnaA monomers

In this study we investigated the role of ATP binding by DnaA

and domain I-mediated oligomerization in the stimulation of

tran-scription starting from the pRpromoter We present an evidence

that DnaA devoid of its first domain is both able to bind to the pR

region and stimulate activity of this promoter in vitro We also

demonstrate that DnaA mutants, incapable of binding nucleotides

or hydrolyzing ATP, are still active in transcriptional activation of

the pRpromoter both in vivo

The role of distinct DnaA domains in stimulating transcription

from pRwill be discussed and a mechanism for transcription

acti-vation by DnaA will be proposed

A5-48

NMR investigation on 15.5K associated

complexes

P Li, D Raghunathan, S Liu, S Nottrott, O Dybkov,

R Lu¨hrmann, M C Wahl, T Carlomagno

Max-Planck-Institute for Biophysical Chemistry, Goettingen,

GERMANY

15.5K is one of the U4 specific proteins in the spliceosome Its

binding to the 5’ stem-loop of U4 snRNA (U4 5’-SL) nucleates

bindings of other U4/U6 specific proteins, namely hPrp31, which

contains Nop domain, and 20K/60K/90K protein complex The

length of stem II of U4 5’-SL is found to be crucial for the binding

of hPrp31 Binding of 15.5K is also require prior to the association

of other Nop domain containing core proteins of Box C/D

snoR-NAs in the nucleoli (Lukowiak, 2000; Watkins, 2002) We use

NMR spectroscopy methods to study the specificity required in

these binding events Unlabeled full-length hPrp31 was titrated to

[15N,2D,1HN]15.5K bound to unlabeled U4 5’-SL resulting a ca

80KDa tri-complex Saturation transfer experiment on the

tri-com-plex revealed direct protein-protein contacts on the a2 helix and

part of the a3 helix of 15.5K hPrp31(aa.78–333) construct yield

the same binding surface, suggesting that Nop domain is sufficient

for binding to the primary RNP and the C-terminus of hPrp31 is

not involve in protein-protein interaction From the binding

sur-face, we proposed models of the tri-complex with HADDOCK

program The crystal structure of the hPrp31(aa.78–333)-15.5K-U4

5’-SL complex and the HADDOCK model is consistent with the

each other We have shown that the Nop domain is a genuine

RNP binding module exhibiting RNA and protein binding

surfa-ces The discrimination between the RNAs rises from structural

rather than sequence differences From the structure as well as our

model, it is seen that the elongation of stem II results steric

hindrance, which abolishes hPrp31 binding Currently the

com-plexes of 15.5K with U3 and U14 Box C/D snoRNAs are also

been studied

A5-49 Cellular localization of the proto-oncoprotein TAF15

M Marko, A Vlassis, A Guialis, M LeichterNational Hellenic Research Foundation, Athens, GREECE.TAF15 is the least studied member of the TET protein family(TLS/FUS, EWS and TAF15) Chromosomal translocations fusingTET genes with genes of specific transcription factors can lead tothe expression of oncogenic fusion proteins

While the oncogenic chimeric proteins are extensively studied,much less is known about their normal counterparts To under-stand how TAF15 is regulated on the spatial level we performed

an analysis of the TAF15 domain structure with respect to nuclearand subnuclear localization Nuclear localization signals were iden-tified by expression of TAF15 and TAF15 deletion mutants fused

to EGFP The experiments revealed that the very C-terminus ofTAF15, as for EWS, functions as a nuclear retention signal TheN-terminus of the protein localizes to the cytoplasm and nucleus.When transcription is inhibited TAF15 localizes to nucleolar caps.The N- and C-terminal parts share, independently of each other,this property with the full length protein

Biochemical and in situ fractionation experiments show that a stantial fraction of TAF15 is associated with the nuclear matrix, aproperty which can be mainly ascribed to the TAF15 N-terminus.Immunoprecipitation assays identified two scaffold attachment fac-tors SAF-A/hnRNPU and SAF-B as binding partners Both SAF-

sub-B and SAF-A can associate directly with the TAF15 N-terminus invitro Together our findings indicate that nuclear and subnuclearlocalization of TAF15 is mediated by multiple domains of the pro-tein and by different binding partners

A5-50 The specific transcription factor TAF15 can associate with a subset of the spliceosomal U1 snRNP complex

M Leichter1, M Marko1, M Patrinou-Georgoula1, L Tora2,

A Guialis1

1National Hellenic Research Foundation, Athens, GREECE,

2IGBMC/CNRS, Strasbourg, FRANCE

Nuclear proteins able to bind RNA/ssDNA comprise an emergingclass of multi-functional proteins with an anticipated role in coup-ling transcriptional and post-transcriptional events This is the case

of the highly homologous specific transcription factors of the TETfamily: TLS/FUS, EWS and TAF15 (TAFII68) We have beeninterested in ascribing structural/functional properties to the leaststudied member, TAF15 Anti-TAF15 immunoprecipitation assays(IPs) on HeLa nuclear fractions, prepared at higher than physiolo-gical salt-conditions, revealed the association of TAF15 with aminor fraction of the spliceosomal U1 snRNP, as shown by thedetection in the immune pellet of both U1 snRNA and U1-70kand Sm proteins Moreover, the presence of TAF15 in IPs of anti-U1-70k (as well as anti-U1 RNP autoAbs) was also evident Pull-down assays with recombinant TAF15 and U1 snRNP-specificproteins were applied to identify components directly involved inthis association This revealed interactions between the N-terminaldomain of TAF15 and U1C (and to a lesser degree U1A) proteins,while U1snRNA was not able to directly interact with TAF15 invitro The ability of TAF15 to directly contact RNA Pol II tran-scripts of a high turn over rate was shown in in vivo UV cross-linking studies In addition the association of TAF15 with premRNA was demonstrated by in vitro splicing assays All thesefindings suggest the existence of a functionally discrete U1 snRNPsubset and provide support to the current view on an extensiveinvolvement of U1 snRNP components in the early steps ofcoordinated gene expression and the involvement of TAF15 incoupling these processes

Investigation of the structure and zinc-binding

properties of the RING finger domain from the

human splicing-associated protein RBBP6

M A Kappo1, T Mulaudzi1, R A Atkinson2, E AB3,

R Boelens3, D J G Rees1, D J R Pugh1

1University of the Western Cape, Bellville, SOUTH AFRICA,

2

IGBMC, Strasbourg, FRANCE, 3University of Utrecht, Utrecht,

THE NETHERLANDS

RBBP6 is a 250 kDa human protein known to interact with both

p53 and pRb tumour-suppressor proteins as well as being involved

in mRNA processing and transcriptional regulation RBBP6

con-tains a RING finger domain and an N-terminal ubiquitin-like

domain, suggesting a role in ubiquitylation Recently RBBP6, in

particular the RING finger domain, was shown to be required for

the ubiquitylation of p53 by Hdm2, the human homologue of

Mdm2, with knock-down of RBBP6 leading to accumulation of

p53, concomitant wide-spread apoptosis and embryonic lethality,

prompting speculation that RBBP6 may function as a scaffold for

the interaction of p53 with Hdm2

As a first step towards investigating the interaction of RBBP6 with

p53 and Hdm2, we have expressed the RING finger domain from

human RBBP6 as a GST-fusion and determined its solution

struc-ture using NMR In addition to the conserved Cysteines typical of

RING fingers, the domain also shares the conserved hydrophobic

residues characteristic of U-box proteins, which are able to fold in

the absence of bound zinc, raising the question of whether the

domain requires zinc in order to fold Using NMR we have shown

that zinc is required for the domain to fold Addition of Cd-EDTA

leads to changes in the 15N-HSQC spectrum of the domain

consis-tent with the replacement of zinc ions by cadmium ions Further

refinement is being undertaken in order to determine the identities

of the residues involved in coordination of the zinc ions Attempts

to identify the coordinating residues directly using 113Cd-1H

HSQC spectra are also ongoing

A5-52

NMR and SAXS studies of the ribonuclease

RegB / ribosomal protein S1 system.

P Aliprandi1, P Salah1, P Giraud1, F Mareuil1, J Perez2,

M Uzan3, C Sizun1, F Bontems1

1Institut de Chimie des Substances Naturelles, Gif-sur-Yvette,

FRANCE,2Synchrotron SOLEIL, Gif-sur-Yvette, FRANCE,3

Insti-tut Jacques Monod, CNRS et Universite´ Paris VI et VII, Paris,

FRANCE

S1 is the largest protein (61 kDa) of the Escherichia coli ribosome

It is strictly required to the correct recognition of the translation

initiation codon by the 30S subunit on the messenger RNA when

the Shine-Dalgarno region is degenerated S1 is also used by

sev-eral bacteriophages at their own benefit In particular, it increases

the reaction rate of the T4 endoribonuclease RegB, which

inacti-vates some of the phage early mRNAs, when their translation is

no more required, by cleaving them in the middle of their

Shine-Dalgarno sequence

We are interested in analyzing and comparing S1 mode of actions

in both functions We studied by NMR the structure of RegB and

demonstrated that it belongs to a family of ribonucleases acting on

translated mRNA in the ribosome We also showed that the same

region of S1 is responsible for the recognition of the initiation

codon and for RegB activation We analyzed by NMR and SAXS

the structural and functional properties of this region We now

have undertaken to study the ternary complex formed by RegB, S1

and some mRNA in solution and in the ribosome

A5-53 Cytoplasmic recycling of 60S pre-ribosomal factors depends on the AAA-protein Drg1

L Kappel1, G Zisser1, B Pertschy2, M Tengg1, E Liebminger1,

B Nobis1, G Ho¨genauer1, H Bergler1

1Karl-Franzens-Universita¨t Graz Institut fu¨r MolekulareBiowissenschaften, Graz, AUSTRIA,2Biochemie-ZentrumHeidelberg, Heidelberg, GERMANY

Allelic forms of DRG1/AFG2 confer resistance to the drug zaborine, an inhibitor of ribosome biogenesis in yeast We showhere that the AAA-ATPase Drg1 is essential for 60S maturationand associates with 60S precursor particles in the cytoplasm.Functional inactivation of Drg1 leads to an increased cytoplas-mic localization of shuttling pre-60S maturation factors likeRlp24, Arx1 and Tif6 Surprisingly, Nog1, a nuclear pre-60S fac-tor, was also relocalized to the cytoplasm under these condi-tions, suggesting that it is a previously unsuspected shuttlingpre-ribosomal factor that is exported with the precursor particlesand very rapidly reimported Proteins that became cytoplasmicunder drg1 mutant conditions were blocked on pre-60S particles

dia-at a step thdia-at precedes the associdia-ation of Rei1, a ldia-ater actingpre-ribosomal factor We conclude that functional Drg1 isrequired for the release of shuttling proteins from the pre-60Sparticles at a maturation step that closely follows the nuclearexport of particles, thus defining a novel step in eukaryotic ribo-some maturation

B1-46 Mitochondrial competence: The DNA import mechanism

N Ibrahim1, F Weber-Lotfi1, M Koulintchenko1,2, Y nov3, H Handa4, R Lightowlers2, A Dietrich1

Konstanti-1IBMP-CNRS, Strasbourg Cedex, FRANCE, 2Newcastle sity, Newcastle Upon Tyne, UNITED KINGDOM, 3SIFIBR-RAS,Irkutsk, RUSSIAN FEDERATION,4NIAS, Tsukuba, JAPAN.There are considerable gaps in the understanding of the mitoch-ondrial genetic systems and sterility- or disease-related mutations

Univer-in the mitochondrial DNA cannot be complemented This ismainly due to the fact that conventional transformation of mito-chondria has been unsuccessful for plants and mammals How-

mitochondria have a natural potential to incorporate, repair andexpress foreign DNA To understand and optimize the process,

we studied the import mechanism through biochemical and siological approaches The voltage-dependent anion channel(VDAC) was identified as the putative translocator through theouter membrane In the case of plant mitochondria, DNA importseems to follow nucleotide transport pathways through the innermembrane and to be concomitant with phosphate uptake Todirectly identify the import complex, we designed DNA substratesdestined to get stuck in the membranes during translocation.Concerning the in vivo relevance of the process, we hypothesizethat it is the basis for paternal transmission of an 11.6 kb mit-ochondrial plasmid in Brassica napus The idea is supported byDNA import experiments with isolated Brassicacea mitochondria.From all these results, we expect to progress towards mitochond-rial transformation in vivo

VAMP2 affects the U-type inactivation of Kv2.1

through interaction with N-terminus of the

channel

D Chikvashvili, A Lvov, D Greizer, I Lotan

Tel-Aviv University, Tel-Aviv, ISRAEL

We have previously demonstrated that physical and functional

interactions of Kv2.1 with syntaxin 1A or the binary t-SNARE

complex were mediated by the C-terminus of the channel

Recently, we focused on VAMP2, the vesicle associated partner of

Syx and SNAP-25 in the ternary SNARE complex Two-electrode

voltage clamp analysis in oocytes showed that VAMP2 has an

effect on the steady-state and kinetic parameters of the U-shaped

inactivation of Kv2.1 Using co-immunoprecipitation from plasma

membranes of Xenopus oocytes combined with in vitro binding

assay with recombinant proteins, we demonstrated that VAMP2

interacts physically with Kv2.1 Notably, although VAMP2 can

bind both the C- and N-termini of Kv2.1, as determinated by in

vi-trobinding analysis, we present evidence that the interaction with

the N-terminus is important for its functional effect on

inactiva-tion Firstly, effects of VAMP2 on Kv2.1 and Kv2.2 channels,

which share high degree of their sequences homology at the

N-ter-minus and the transmembrane region, are similar, reflecting

VAMP2’s ability to bind these channels at their N-termini

Second-arily, deletions at the C-terminus of Kv2.1 do not abolish the

effects of VAMP2 on the channel inactivation Finally, injection of

recombinant protein corresponding to the N-terminus reverses the

effect of VAMP2 on the inactivation of Kv2.1 Taken together our

results show that VAMP2 functions as a regulatory protein that

modulates the U-shaped inactivation of Kv2.1 through interaction

with the N-terminus of the channel

B1-48

Enzymatic properties and subcellular

localization of Arabidopsis

beta-N-acetylhexosaminidases

E Liebminger1, J S Bondili2, J Schoberer1, F Altmann2,

J Glo¨ssl1, H Steinkellner1, L Mach1, R Strasser1

1

Institute of Applied Genetics and Cell Biology, Vienna,

AUSTRIA,2Departement of Chemistry, Vienna, AUSTRIA

Plant glycoproteins contain substantial amounts of N-glycans

lack-ing terminal GlcNAc residues at their non-reduclack-ing ends It has

been proposed that this is due to the action of b-hexosaminidases

during late stages of N-glycan processing or in the course of

N-glycan turnover We have now cloned the three putative

b-hexosaminidase sequences from Arabidopsis thaliana When

heterologously expressed as soluble forms in insect cells, the

enzymes (termed HEXO1-3) could all hydrolyze substrates like

pNP-GlcNAc, pNP-GalNAc, chitotriose and chitobiose, albeit to a

varying extent With N-glycan substrates, HEXO1 displayed a

more than 1000 times higher specific activity than HEXO2 and

HEXO3 Subcellular localization studies with HEXO-fluorescent

protein fusions transiently expressed in Nicotiana benthamiana

plants showed that HEXO1 is a vacuolar protein In contrast,

HEXO2 and HEXO3 are mainly located at the plasma membrane

Analysis of HEXO expression in leaves revealed that HEXO1 is

present in the soluble protein fraction, while HEXO3 is mainly

membrane-bound and HEXO2 was not detected at all These

results indicate that HEXO1 participates in N-glycan trimming in

the vacuole, whereas HEXO3 could be responsible for the

process-ing of N-glycans present on secretory glycoproteins

B2-20 2D-PAGE versus shotgun proteomics:

Secretome analysis of immature and differently stimulated human dendritic cells

N C Gundacker, V J Haudek, A Slany, H Wimmer, E Bayer,

V Bochkov, J StoecklMedical University of Vienna, Vienna, AUSTRIA

Dendritic cells (DC), the most potent and specialised senting cells, play a key role in the regulation of adaptive immu-nity The secretome contains the most directly acting proteinsaffecting other cells Immature human DCs were functionally acti-vated with lipopolysaccharide (LPS), lipid oxidation productsderived from 1-palmitoyl-2-arachidoyl-sn-glycerol-3-phosphorylch-olin (OxPAPC) and human rhinovirus (HRV) During treatment,cells were metabolically labeled with 35S-methionine/cysteine In2D-gels, secreted proteins were specifically detected by autoradiog-raphy and accessible to accurate quantification Protein identifica-tion, however, was more successful by shotgun analysis usingnano-flow HPLC peptide separation combined with a electrosprayion trap mass spectrometer Here, a semi-quantitative assessmentwas performed with the spectral count method By means of ahome-made database, contaminants from residual fetal calf serumand cytoplasmic proteins were subtracted As a result, 148 differentsecreted proteins were identified Secretion was induced by LPSand HRV, but rather repressed by OxPAPC 18 proteins wereexclusively found secreted by LPS-treated, 6 by OxPAPC-stimula-ted and 16 by HRV-infected DCs The present approach proved to

antigen-pre-be very powerful for the analysis of this meaningful protein tion

frac-B3-20 Characterising the multitude of chloroplast protein import receptors in Arabidopsis thaliana

M Gutensohn, B HustMartin-Luther-University Halle-Wittenberg, Halle/Saale,GERMANY

The majority of chloroplast proteins are encoded in the nucleusand have to be imported into the organelle after their synthesis inthe cytosol as precursor proteins The transport of precursorsacross the two chloroplast envelope membranes is mediated by theinteraction with two import machineries, the Toc and Tic complex.The core of the Toc complex consists of two receptor proteins,Toc34 and Toc159, involved in initial binding of precursor proteins

at the chloroplast surface and a translocation pore, Toc75 In bidopsis two homologs of Toc34 (atToc33, atToc34) and fourhomologs of Toc159 (atToc159, atToc132, atToc120, atToc90),however, only one ortholog of Toc75 (atToc75-III), have beenidentified For the functional characterisation we have isolatedknockout mutants for all six Arabidopsis Toc receptors as well asfor the translocation pore The atToc75-III knockout mutant has

Ara-an embryo lethal phenotype with Ara-an extremely early arrest ofdevelopment In contrast, the Toc receptor mutants show remark-ably different phenotypes suggesting more specialized functions forthese import receptors Detailed characterisation of the Toc recep-tor mutants, including proteome and expression analyses, as well

as in vitro studies demonstrated that each of these Toc receptorspreferentially interacts with different groups of precursor proteins.The composition of Toc complexes in Arabidopsis has been ana-lysed by biochemical approaches using specific antibodies as well

as by a genetic approach using a series of Toc receptor doublemutants

The Pex3p-Pex19p interactions modulated by

PMP cargo proteins

C P Grou1,2, M P Pinto1,2, I S Alencastre1, M E Oliveira1,2,

C Sa´-Miranda1, M Fransen3, J E Azevedo1,2

1IBMC, Porto, PORTUGAL,2ICBAS, Porto, PORTUGAL,

3Katholieke Universiteit Leuven, Leuven, BELGIUM

Biogenesis of the mammalian peroxisomal membrane seems to

require the action of only three peroxins: Pex3p, Pex16p, two

intrinsic membrane proteins of the peroxisome, and Pex19p, a

pro-tein that displays a dual subcellular distribution (cytosolic and

per-oxisomal) Pex3p is believed to be the docking protein for

Pex19p-PMP complexes at the peroxisomal membrane We have analyzed

the Pex3p-Pex19p interaction by Native-PAGE Our data indicate

that the Pex3p-binding affinity of Pex19p is highly increased when

this peroxin is pre-loaded with a PMP These findings suggest the

existence of a cargo-induced peroxisomal targeting for Pex19p

Supported by: Fundac¸a˜ o para a Cieˆncia e Tecnologia Grant

POCTI2010 and FEDER Funds, Portugal, European Union VI

Framework program Grant LSHG-CT-2004-512018, Peroxisomes

in Health and Disease, the Flemish Government (Geconcerteerde

Onderzoeksacties Grant GOA/2004/08) and Fonds voor

Wet-enschappelijk Onderzoek Vlaanderen (Onderzoeksproject Grant

G.0237.04)

B3-22

The role of Pex19p in the biogenesis of

peroxisomal membrane proteins

M P Pinto1,2, C P Grou1,2, I S Alencastre1,2,2,

M E Oliveira1,2, C Sa´-Miranda1, M Fransen3, J E Azevedo1,2

1

IBMC, Porto, PORTUGAL,2ICBAS, Porto, PORTUGAL,

3

Katholieke Universiteit Leuven, Leuven, BELGIUM

Only three proteins, Pex3p, Pex16p and Pex19p, were found to be

involved in the biogenesis of the mammalian peroxisomal

mem-brane Pex3p and Pex16p are two intrinsic membrane proteins of

the peroxisome, while Pex19p localizes both to cytosol and to the

organelle The ability of Pex19p to interact with most peroxisomal

membrane proteins (PMPs) led to the proposal that this peroxin

functions as an import receptor or as a chaperone in the assembly/

disassembly of membrane protein complexes Using an in vitro

import system, we show that insertion of a reporter protein into

the peroxisomal membrane is Pex19p-dependent and does not

require ATP/GTP hydrolysis By programming the system with

recombinant versions of Pex19p, we demonstrate that Pex19p

forms a complex with its cargo PMP before the peroxisomal

dock-ing/insertion steps

Supported by: Fundac¸a˜ o para a Cieˆncia e Tecnologia Grant

POCTI2010 and FEDER Funds, Portugal, European Union VI

Framework program Grant LSHG-CT-2004-512018, Peroxisomes

in Health and Disease, the Flemish Government (Geconcerteerde

Onderzoeksacties Grant GOA/2004/08) and Fonds voor

Wet-enschappelijk Onderzoek Vlaanderen (Onderzoeksproject Grant

G.0237.04)

B3-23 Mdm38p and its role in mitochondrial dynamics

K Nowikovsky1, R J Schweyen1, S Reipert2, R J Devenish3

1MFPL, Department of Genetics, Wien, AUSTRIA, 2MFPL,Department of Molecular Cell Biology, Wien, AUSTRIA, 3Depart-ment of Biochemistry and Molecular Biology, and the ARC Centre

of Excellence in Structural and Functional Microbial Genomics,Monash University, Victoria, AUSTRALIA

Yeast MDM38/YOL027c and its human homologue LETM1encode an integral protein of the inner mitochondrial membrane

As previously reported, LETM1 is the candidate gene for seizures

in the Wolf-Hirschorn-syndrom (Zollino et al., 2003) Loss of theMDM38 gene product in yeast mitochondria results in a variety

of phenotypic effects including reduced content of respiratorychain complexes, altered mitochondrial morphology and loss ofmitochondrial K+/H+ exchange activity resulting in osmoticswelling By use of doxycycline-regulated shut-off of MDM38gene expression we showed that loss of K+/H+ exchange activ-ity and mitochondrial swelling are early events, associated with areduction in membrane potential and fragmentation of the mit-ochondrial reticulum The use of a novel fluorescent biosensordirected to the mitochondrial matrix revealed that the loss ofK+/H+ exchange activity was immediately followed by morpho-logical changes of mitochondria and vacuoles, the close associ-ation of these organelles and finally uptake of mitochondrialmaterial by vacuoles Nigericin, a K+/H+ ionophore, fully pre-vented these effects of Mdm38p depletion We conclude thatosmotic swelling of mitochondria triggers selective mitochondrialautophagy, or mitophagy Furthermore, we provide evidence thatfragmentation of the mitochondrial network is a prerequisite formitochondrial degradation

B3-24 Biogenesis of b-barrel proteins in the outer membrane of human mitochondria

V Kozjak-Pavlovic, K Ross, N Benlasfer, S Kimmig,

A Karlas, T RudelMax Planck Institute for Infection Biology, Berlin, GERMANY.Voltage-dependent anion-selective channel (VDAC), also known asmitochondrial porin, is one of the few proteins in the outer mit-ochondrial membrane predicted to have a ß-barrel topology Fromstudies in fungi, it is known that VDAC, similar to other ß-barrelproteins, requires the translocase of the outer mitochondrial mem-brane (TOM), as well as the sorting and assembly machinery(SAM) for its translocation and proper integration into the outermembrane of mitochondria We studied the biogenesis of VDAC

in human mitochondria by depleting the components of the ochondrial import machinery by using RNA interference We gen-erated stably transduced cell lines that showed inducibleknockdown of Tom40, Sam50, Tom70 and metaxin 2 Using thesecell lines, we were able to show that besides the TOM complexand Sam50 (Tom55/Omp85), so far the only known component ofthe human SAM complex, metaxins were also necessary for VDACbiogenesis Metaxin 2-depleted mitochondria had reduced levels ofboth metaxin 1 and metaxin 2, and were deficient in the importand assembly of VDAC and Tom40, another ß-barrel precursor,but not in the import of three matrix-targeted precursors We alsoobserved a reduction in the amounts of metaxin 1 and metaxin 2

mit-in Sam50-depleted mitochondria, implymit-ing a connection betweenthese three proteins However, we found metaxin 1 and metaxin 2

in a high-molecular-weight complex distinct from the taining complex We propose that in human mitochondria Sam50and metaxins act sequentially to promote integration of ß-barrelproteins into the outer mitochondrial membrane

On the mechanism of peroxisomal protein

translocation: functional implications of two

missense mutations in Pex5p

A F Carvalho1,2, C P Grou1,2, M P Pinto1,2,

I S Alencastre1,2, J Costa-Rodrigues1, M Fransen3,

C Sa´-Miranda1, J E Azevedo1,2

1

IBMC, Porto, PORTUGAL,2ICBAS, Porto, PORTUGAL,

3

Katholieke Universiteit Leuven, Leuven, BELGIUM

The import of proteins into the peroxisome matrix is an essential

step in peroxisome biogenesis Most newly synthesized peroxisomal

proteins are targeted to the organelle by Pex5p Pex5p interacts

with these proteins in the cytosol, transports them to the

peroxi-somal docking/translocation machinery, promotes their

transloca-tion across the membrane, and is recycled back to the cytosol to

catalyze further rounds of transportation Here, we describe the

mechanistic implications of two single-amino acid substitutions in

Pex5p The first mutation, Cys11Ser, blocks the recycling of Pex5p

back into the cytosol at the step in which stage 2 Pex5p is

conver-ted into stage 3 Pex5p The mutation Asn526Lys, described in a

child with NALD and shown to abolish the PTS1-binding capacity

of Pex5p, results in a protein exhibiting import capacity Protease

assays suggest that the Asn526Lys mutation causes conformational

alterations at the N-terminal half of Pex5p mimicking the ones

induced by binding of a PTS1-containing peptide to the normal

peroxin

Supported by: FCT (POCTI program) and FEDER funds, and by

European Union VI Framework program Grant

LSHG-CT-2004-512018, Peroxisomes in Health and Disease

B4-44

PDZ-domain interactions control the surface

stability and degradation of the EAAC1

glutamate transporter in MDCK cells

A D’Amico1, A Soragna1, N Panzeri1, E Di Cairano1,

N Anzai2, F Sacchi1, C Perego1

1

University of Milano, Milano, ITALY, 2Kyorin University School

of Medicine, Tokyo, JAPAN

The glutamate transporter EAAC1/EAAT3 mediates the uptake of

glutamate from the synaptic cleft as well as the absorption of

di-carboxylic amino acids in epithelial cells EAAC1 cell surface

dis-tribution is dynamically regulated by membrane trafficking, but

the precise molecular mechanisms underlying this event are far

from being fully elucidated

Here, we identified a conserved consensus sequence (-S-Q-F) for

interaction with class I PDZ domains in the C-terminus of EAAC1

and investigated the role of this motif in the transporter

localiza-tion, in MDCK cells

Confocal microscopy and cell surface biotinylation experiments

demonstrated that removal or manipulations of the

PDZ-interact-ing sequence affected the cell surface expression of the transporter

without altering its apical targeting or substrate affinity The

decreased cell surface expression was caused by a greater

internal-ization rate Furthermore, the internalized truncated transporter

did not accumulated in the recycling compartment, but it was

tar-geted to lysosomes and degraded

We propose that PDZ mediated interactions control EAAC1 cell

surface expression through inhibition of basal transporter

endocy-tosis and regulation of its degradation

Future directions will aim at identifying the EAAC1 PDZ

interact-ing proteins, in epithelia and neurons as well as the precise

transporter expression

B4-45 Insulin increases jejunal glucose absorption and reduces jejunal Na+-K+ATPase activity via PI3 kinase, PKC, p38 and ERK.

S I Kreydiyyeh, Sr., M F SerhanAmerican University of Beirut, Beirut, LEBANON

This work was conducted to study the effect and mechanism ofaction of insulin on glucose uptake by rat jejunum and culturedCaco-2 cell Rats were injected intraperitoneally with insulin and

30 min later, a segment of the jejunum was perfused with labeled3-O-methyl-D-glucose [14C] (3OMG) Insulin increased 3OMGabsorption and decreased its retention in jejunal cells and their

Na+-K+ATPase activity The uptake of 3OMG by Caco-2 cellswas also investigated and calculated by measuring its retentioninside the cells and its disappearance from the incubation medium.Phloretin and phoridzin were used as respective inhibitors of sero-sal and mucosal transport Insulin increased significantly mucosaland serosal transport and reduced the activity and protein expres-sion of the Na+-K+ pump The effect of the hormone on the

Na+-K+ pump was pursued further and studied in presence ofinhibitors of different second messengers known to be involved ininsulin signaling The hormone was found to inhibit the pump via

a mechanism involving P-I-3 kinase, PKC, p38 and ERK PKCwas found to lie downstream of both p38 and ERK It was conclu-ded that the increase in intestinal absorption of glucose is due to

an increase in the number of transporters and not to an increase inthe sodium gradient established by the pump The observeddecrease in the activity of the pump may be considered as atype of negative feedback to counterbalance the increase intransporters

B4-46 Structural study of a potential therapeutic target: the antibiotics efflux pump

MexXY-OprM from Pseudomonas aeruginosa

G Phan, I Broutin, M Lascombe, P Benas, A DucruixLaboratoire de Cristallographie et RMN Biologiques, UMR 8015CNRS-Paris Descartes, Paris, FRANCE

The Gram-negative bacteria Pseudomonas aeruginosa is a ously opportunistic human pathogen mainly implied in hospitalinfection This bacterium preferentially infects immuno-depressedindividuals related to AIDS, diabetes mellitus or severe burns It isalso the predominant cause of nosocomial pneumonia and lunginfection in patients with cystic fibrosis It has been largely shownthat this organism exhibits resistance to multiple antimicrobialagents, partly because of a membrane efflux system from the RNDfamily (Resistance Nodulation cell Division), called Mex (Multi-drug Efflux pump) This efflux pump displaces drug vectoriallyfrom the bacterium using proton electrochemical force Facing theantibiotic resistance, inhibition of bacterial efflux mechanism seems

notori-to be a promising therapeutic strategy

In 2000, the genome sequence of Pseudomonas aeruginosa reveals theexistence of 12 potential efflux pumps, 9 of which have been experi-mentally characterized to date 2 of them are the most prevalentefflux systems in clinical strains and confer significant resistance toantibiotics, namely MexAB-OprM and MexXY-OprM

Our researches focus on the structurally uncharacterized OprM efflux pump and its specificity toward aminoglycoside anti-biotics We aim at understanding how those membrane proteinsassemble to form a functional pump, how allostery mechanism,substrate specificity interaction and channel opening may occurduring drug transport

MexXY-At present, a large production of OprM allowed us to solve itscrystallographic structure at 2.4 A˚ resolution Besides, crystalliza-tion of MexX and purification of MexY are in progress

Reversible dissociation of V-ATPases: fact or

artefact?

A Albertmelcher, O Vitavska, M Huss, H Wieczorek

University of Osnabru¨ck, Osnabru¨ck, GERMANY

Primary active proton transport by eukaryotic V-ATPases is

regu-lated via the reversible disassembly of the V1VOholoenzyme into

its peripheral, catalytic V1 complex and its membrane bound,

proton translocating VO complex This nutrient dependent

phenomenon had been first detected in the midgut epithelium of

non-feeding, molting tobacco hornworms (Manduca sexta), and in

glucose deficient yeast cells (Saccharomyces cerevisiae)

Immunocyt-ochemical investigation of non-feeding starving instead of molting

tobaccco hornworms revealed that subunits of the V1complex are,

as expected, found in the cytoplasm, but that an astonishingly high

amount appeared to be membrane bound To clarify this

surpri-sing finding we investigated the reversible disassembly under

approximately physiological conditions in living yeast cells, taking

advantage of the existence of yeast strains with V-ATPase subunits

fused to green fluorescent protein By using yeast cells with

fluores-cent subunits of the V-ATPase, we found that only the V1subunit

C is released after displacement of extracellular glucose with

galac-tose, whereas the other V1 subunits remain at or near the

mem-brane Neither disassembly nor reassembly of subunit C depend on

the actin cytoskeleton, whereas disassembly but not reassembly

depends on functional microtubules Results from overlay blots

and pull-down assays support the assumption that subunit C

inter-acts directly with microtubules without involvement of linker

proteins

B4-48

ATP-binding to NBD1 of multidrug resistance

protein ABCC1 probed by NMR

C Sizun1, O Ramaen1, J M Girard1, O Pamlard1,

F Bontems2, E Jacquet1, J Y Lallemand1

1

ICSN-CNRS, Gif-sur-Yvette, FRANCE,2ICSN-CNRS,

Palaiseau, FRANCE

Multidrug Resistance Protein ABCC1 belongs to the ATP-binding

cassette (ABC) superfamily of membrane transport proteins It

contributes to chemotherapy failure by conferring cell resistance to

a wide range of chemotherapeutic agents The ABC transporter

topology consists of two cytosolic nucleotide binding domains

(NBD) and two transmembrane domains (TMD), arranged as

TMD-NBD dimers Whereas the TMDs are implicated in substrate

recognition/transport, the highly conserved NBDs are responsible

for ATP binding/hydrolysis NBD dimerization is considered as a

prerequisite to ABC ATPase activity Despite plenty of

biochemi-cal and structural data for prokaryotic/eukaryotic ABC

transport-ers, the exact mechanisms of the catalytic cycle and the mechanical

TMD/NBD coupling remain unclear Human ABCC1-NBD1

par-ticipates in a degenerate ATP-binding site and is deficient in

AT-Pase activity as an isolated domain and in the full-length

transporter The unusual conformation of the catalytic site

observed in the X-ray crystal structure of MgATP-bound

ABCC1-NBD1 gave structural clues about the lack of ATPase activity

Here we use NMR to gain further insight into nucleotide binding

and subsequent structural and dynamic changes Using the

back-bone assignment of NBD1, we carried out chemical shift titration

experiments at various nucleotide concentrations and temperatures

This allowed not only to map the canonical ATP-binding residues,

but also residues located far from the nucleotide binding pocket

The identified regions are assumed to be involved in TMD/NBD

communication according to prokaryotic models Comparison

between APO and MgATP-bound NBD1 suggests a hinge rotation

in the Q-loop

C1-66 X-ray structures of the 2:1 complex between prolactin and its receptor

I Broutin1, J B Jomain2, E Tallet2, J Van Agthoven1,

V Goffin2, A Ducruix1

1Laboratoire de cristallographie et RMN biologiques, CNRSUMR8015, Paris, FRANCE, 2Centre de Recherche Croissance etsignalisation, Inserm U845, Paris, FRANCE

Prolactin is a polypeptide hormone whose major biological actionsare related to normal lactation and reproduction With the growthhormone (GH) and the placental lactogen (PL) it forms a peptidichormone family They all interact with receptors which belong tothe cytokine family hGH activates both hGHR and hPRLR, hPLdoes not interact with the hGHR, but activates solely the hPRLR,similar to hPRL As PRLR was recently evidenced to be involved

in the etiology and progression of breast and prostate tumors,there is a real need to develop new therapeutic strategies to blockthe activation of this receptor However, further development oflead compounds remains difficult due to the lack of key informa-tion on the molecular and cellular biology of the PRLR

The ligand binds in a two-step process, forming a homodimer sisting of one molecule of PRL and two molecules of receptor.When these partners are mixed in vitro, only 1:1 complexes areformed

con-Different structures already exist for GH/GHR and PL/PRLR as2:1 or 1:1 complexes, but none involving PRL with PRLR Anaffinity-matured hPRL variant containing a minimum of mutationswas constructed in order to stabilized the 2:1 complex Here wepresent two X-ray structures of this complex at 3.8A˚ resolutioncrystallized in two different space groups, which reveal the details

of the interactions of site2

of our study was to examine the role of different activated G tein alpha subunits in actin cytoskeleton reorganization For thepurpose of the study autofluorescently-tagged b-actin (pEYFP-actin) was co-expressed together with receptor constructs (e.g neu-rokinin type 1 receptor (NK1-R) and b2-adrenergic receptor (b2-AR)) or constitutively active mutants of Gaq, Ga11, Ga12, Ga13and Gas in the HEK 293 cells Evaluation of the autofluorescent-ly-labeled actin filaments was performed with the use of confocalmicroscope Activation of Rho A and inositol phosphate accumu-lation (IP) were also monitored The acquired data shows that theactivation of Gaq/11-coupled NK1-R resulted in actin cytoskeletonrearrangement, stress fiber formation, IP accumulation and Rho Aactivation Comparable changes in actin cytoskeleton assemblywere also observed after the expression of constitutively activemutants of Gaq, Ga11, Ga12 and Ga13 On the contrary the acti-vation of Gas did not induce any apparent changes in actin cyto-skeleton organization

Essential role of IP3R2 in NFAT transcriptional

activity in HEK293 cells

A Z Caron, G Guillemette

University of Sherbrooke, Sherbrooke, PQ, CANADA

Ca2+is a ubiquitous intracellular messenger involved in many

cel-lular processes including fertilization, mitosis, neuronal

transmis-sion, contraction of muscles, gene transcription, and cell death

Inositol 1,4,5-trisphosphate (IP3) releases Ca2+ from intracellular

stores by activating three subtypes of receptor-channel (IP3R1,

IP3R2, and IP3R3) that share basic properties but differ in terms

of regulation However, it is not known how the different subtypes

of IP3R influence the Ca2+ signalling patterns and contribute to

the specific cellular responses Here, we show that a weak

stimula-tion with carbachol (CCh), a muscarinic receptor agonist, evoked

typical intracellular Ca2+ oscillations in HEK 293 cells that

express the three subtypes of IP3R The knockdown of IP3R2

abol-ished the Ca2+ oscillatory response whereas the knockdown of

IP3R1 or IP3R3 caused minor change in Ca2+ signalling The

knockdown of IP3R3 slightly increased the duration of the

oscilla-tory process, suggesting an anti-oscillaoscilla-tory role Interestingly the

knockdown of IP3R2 importantly reduced the activation of the

nuclear factor of activated T cells (NFAT) known to be regulated

by Ca2+ This reduction of NFAT activation was still observable

when IP3R2-KO cells were stimulated with a maximal dose of

CCh These results illustrate different modes of Ca2+ signalling

associated with the different subtypes of IP3R and they suggest

that IP3R2 is essential for the activation of NFAT in HEK 293

cells Supported by CIHR and HSFC

1Institute for Cancer Research, Vienna, AUSTRIA, 2Otto Wagner

Spital, Vienna, AUSTRIA

Self sufficiency of growth signals and desensitized signal

transduc-tion systems are hallmarks of human cancer One group of

pro-teins shown to be deregulated in malignant tumors of diverse

tissues is the family of Sprouty proteins which inhibit

RTK-medi-ated signaling The purpose of this study was to characterize the

mechanisms involved in Sprouty expression in normal and tumor

cells

The first series of experiments were designed to explore the precise

expression patterns of Sprouty proteins during cell cycle Sprouty4

expression fluctuated throughout the cell cycle and its expression

differed markedly compared to the cell cycle-specific expression

pattern of Sprouty2 and Sprouty1 By analyzing mRNA and

pro-tein levels we studied to which extent different regulation

mecha-nisms influence the expression levels of Sprouty4 Analysis of

changes in Sprouty expression after stimulation of serum arrested

cells using selected growth factors showed that activation of RTK

mediated signaling contributes to induce Sprouty4 protein

expres-sion Correspondingly expression of constitutive active Ras

induced Sprouty4 expression in logarithmically growing cells

Additionally we studied the influence of hypoxia on Sprouty4

expression, since initial experiments revealed that Sprouty4 is

highly expressed in endothelial cells

These results suggest that Sprouty4 protein is tightly and

specific-ally regulated by multiple mechanisms

C1-70 Osteoprotegerin and RANKL levels in multiple sclerosis

S Kurban1, Z Akpinar2, I Mehmetoglu1

1Meram Faculty of Medicine, Department of Biochemistry, sity of Selcuk, Konya, TURKEY, 2Meram Faculty of Medicine,Department of Neurology, University of Selcuk, Konya, TURKEY.Multiple sclerosis (MS) is a progressive and inflammatory autoim-mune disorder associated with osteoporosis The underlying patho-physiology of the bone disease in MS is uncertain

Univer-The purpose of this study was to examine whether the

osteoproteger-in (OPG)/receptor activator of NF- B ligand (RANKL) system isinvolved in the pathogenesis of the MS associated osteoporosis.For this purpose, OPG, RANKL, osteocalcin (OC) and parathor-mon (PTH) levels of fifteen healthy individuals (10 F, 5 M) andthirteen age-matched patients (10 F, 2 M) with MS were measured.Serum OPG and RANKL levels were higher in MS patients than

in healthy controls (p<0.01 and p<0.05 respectively) But OCand PTH levels of the groups were not different

RANKL is produced by activated T cells and directly induce oclastogenesis Therefore, our findings of increased RANKL levels

oste-in patients with MS can provide an explanation for bone loss ciated with the disease that involve the immune system

asso-OPG is also known as osteoclastogenesis inhibitory factor whichacts as a soluble decoy receptor for RANKL It inhibits osteoclastmaturation and osteoclast activation Therefore, our findings sug-gest that the effect of RANKL can be counterbalanced by OPG in

MS patients

On the other hand, the elevated OPG level without OC elevationindicates that the excessive amount of OPG may partly be derivedfrom other cells than osteoblast in MS

In conclusion, we showed that OPG and RANKL may play a role

in the osteoporosis of MS patients and follow up studies may vide further information on the association between MS relatedosteoporosis and OPG/RANKL system

pro-C1-71 Phosphorylation of Erp1 by p90rsk is required for cytostatic factor arrest in Xenopus laevis

T Nishiyama, T Kishimoto, K OhsumiTokyo Institute of Technology, Midori-ku, Yokohama, JAPAN.Until fertilization, the meiotic cell cycle of vertebrate eggs is arres-ted at metaphase of meiosis II by a cytoplasmic activity termedcytostatic factor (CSF), which causes inhibition of the anaphase-promoting complex/cyclosome (APC/C), a ubiquitin ligase that tar-gets M-metaphase cyclins for degradation Recent studies indicatethat Erp1/Emi2, an inhibitor protein for the APC/C, has an essen-tial role in establishing and maintaining CSF arrest, but its rela-tionship to Mos, a MAPK kinase kinase that also has an essentialrole in establishing CSF arrest through activation of p90Rsk, isunclear We investigated whether the Mos-MAPK pathway isinvolved in Erp1 regulation, using Xenopus oocytes and their cell-free extracts Our results indicate that Erp1 is a substrate ofp90Rsk, and that Mos-dependent phosphorylation of Erp1 byp90Rsk at Thr 336, Ser 342 and Ser 344 is essential for establishingCSF arrest and stabilizing Erp1 Mos-dependent phosphorylation

of Erp1 enhances, but does not generate, the activity of Erp1 thatmaintains metaphase arrest Our results also suggest that Erp1inhibits cyclin B degradation by binding the APC/C at its C-ter-minal destruction box, and this binding is also enhanced by theMos-dependent phosphorylation Thus, Mos and Erp1 collaborat-ively establish and maintain metaphase II arrest in Xenopus eggs.The link between Mos and Erp1 provides a novel molecularexplanation for the integral mechanism of CSF arrest in unferti-lized vertebrate eggs

Diet affects the asymmetrical, thyroid hormone

induced metamorphosis in flatfish

M Moren1, A M Schreiber2

1NIFES (National Institution of Nutrition and Seafood Research),

Bergen, NORWAY, 2Carnegie Institution of Washington,

Depart-ment of Embryology, 3520 San Martin Drive, Baltimore, MD, USA

Flatfish species are the most asymmetrically-shaped group of

verte-brates They are not asymmetrical when they hatch After a period

of growth thyroid hormones (TH) transformation from

symmetri-cal larvae to asymmetrisymmetri-cal juvenile begins: one eye moves to the

same side at the other eye including remodeling of cranial bones

By staining the calcified bone in vivo with calcein, we can follow

the alteration of the frontale processes during metamorphosis If

TH is inhibited, no asymmetry appears (Schreiber, 2006) Pictures

of asymmetrical development will be shown Several genes are

directly regulated by TH during metamorphosis (Schreiber et al.,

in prep.) We have found that FABP6 seems to be down-regulated

during metamorphosis The distribution of this protein will be

shown for Atlantic halibut and southern flounder The migration

of the eye can be arrested in hatchery-produced Atlantic halibut

The causatives are still unknown, but the development is successful

when larvae are fed their natural diet (Hamre et al., 2005) Hence,

the metamorphosis is influenced by diet Hamre et al (2007) have

shown that several of the nutrients, that may affect the

develop-ment, are absent or in very low levels in commercial diets Some

will be presented PGE2, a derivative of arachidonic acid, is

essen-tial for osteoblast activity (Watkins et al., 2001) PGE2

up-regu-lates Cbfa1 and osterix which is required for the differentiation

and activation of osteoblasts (Zhang et al., 2002) We will present

results from in situ hybridization of osterix in both species

C1-73

Acute-phase and developmentally related role

of STAT3 and NFkB in alpha2-macroglobulin

expression

J Arambasic, A Uskokovic, S Dinic, M Mihailovic,

M Vidakovic, N Grdovic, I Grigorov, S Ivanovic-Matic,

D Bogojevic, G Poznanovic

Institute for Biological Research, Belgrade, SERBIA

Alpha2-Macroglobulin (MG) is acute phase (AP) protein highly

synthesized in fetal but low in the adult rat liver in basal state AP

induced MG synthesis in fetals and adults is regulated at the

tran-scriptional level STAT3-NFkB role in MG gene expression in fetal

and adult rats, under basal and turpentine-induced AP response

was examined Combined techniques of DNA affinity

chromatog-raphy and immunoblot analysis revealed that AP-induced

tran-scriptional activity of MG gene in adults was connected with

STAT3 and NFkB presence on the MG gene promoter suggesting

their positive regulatory role Low level of NFkB DNA binding in

control fetuses and its unchanged affinity during the AP response,

point its role in constitutive rather than in AP-inducible MG

regu-lation In basal state of fetuses, 91 and 86 kD STAT3 isoforms are

involved in MG gene transcription, while only 91 kD isoform

retained this ability during AP response Our results suggest that

transcriptional regulation of MG gene expression in vivo is driven

by the AP and developmentally related mechanisms which partly

rely on the interplay of STAT3 and NFkB

C1-74 c-JUN is activated by the hedgehog pathway transcription factor GLI2

S Laner-Plamberger, T Eichberger, A Kaser, F Aberger,

A FrischaufUniversity of Salzburg, Salzburg, AUSTRIA

Constitutive activation of the Hh signaling pathway is known tolead to tumourigenesis in skin (basal cell carcinoma, BCC) throughthe enhanced activity of its main mediators: the zinc finger tran-scription factors GLI1and GLI2 Therefore, GLI target gene iden-tification will lead to a more comprehensive understanding of themolecular mechanism of tumour development and maintenance.Global expression analysis in inducible HaCaT keratinocytesrevealed 450 genes differentially expressed in response to expres-sion of GLI1 or GLI2 (Eichberger et al., 2006) Among those theleucine zipper transcription factor and activator protein 1 (AP1)family member c-JUN is particularly interesting because of itsinvolvement in keratinocyte proliferation Here we show byRTPCR that c-JUN mRNA levels rise about 20 fold upon GLI2but not GLI1 induction The c-JUN upstream regulatory regioncontains a cluster of five potential GLI binding sites and is activa-ted by GLI2 in a luciferase reporter assay In vivo binding ofGLI2 to the region was shown by Chromatin immunoprecipitation(ChIP) Mutation analysis of each potential binding site identified

a single binding site as necessary for activation We therefore clude that c-JUN transcription can be directly activated by GLI2.This will permit us to investigate a possible role of c-JUN in ourinducible HaCaT cell model of basal cell carcinoma

con-C1-75 Analysis of MAPK signaling by phosphoproteomics and chemical genetics

S Morandell1, K Grosstessner-Hain2, I Feuerstein3,

T Lindhorst4, K Mechtler2, G K Bonn3, L A Huber1 1

Medical University, Innsbruck, AUSTRIA,2Institute for MolecularPathology, Vienna, AUSTRIA,3Leopold Franzens University, Inns-bruck, AUSTRIA,4Ugichem GmbH, Innsbruck, AUSTRIA.The MAPK signaling pathway is a key player in the regulation ofvarious cellular processes One essential task for phosphoproteo-mics analyses of signal transduction is the development of strat-egies for the identification of low abundant signaling molecules incombination with the detection of post translational modifications

We established a sensitive and specific approach for the tion of direct kinase substrates by combining sample prefractiona-tion, reduction of background phosphorylation and ChemicalGenetics Protein extracts from genetically modified mouse celllines are used for assays with mutated kinases The kinases useATP-analogs that are not bound by wild type kinases to increasethe specificity of kinase reactions Phosphorylated proteins are ana-lyzed with a chromatography based IMAC approach, optimizedfor complex protein mixtures, in combination with mass spectrom-etry Peptides from two samples are labeled differentially for relat-ive quantification The established screening platform was usedsuccessfully for the identification of direct Mek1 substrates Forthe future we plan to use this approach to reveal substrate phos-phorylation in the late endosomal MAPK signaling complex.Work is supported by the Austrian Genome Program (GEN-AU),Vienna, Austria and by the Special research Program ‘‘Cell Prolif-eration and Cell Death in Tumors’’ (SFB021, Austrian ScienceFund)

Endothelial nitric oxide synthase promotes

neonatal cardiomyocyte proliferation by

inhibiting TIMP-3 expression

L Hammoud1, X Lu2, F Xiang1, F Brunner3, K Leco1,

Q Feng1,2

1University of Western Ontario, London, ON, CANADA, 2Lawson

Health Research Institute, London, ON, CANADA,3University of

Graz, Graz, AUSTRIA

Objective: We have recently demonstrated that endothelial nitric

oxide synthase (eNOS) promotes cardiomyocyte proliferation

However, mechanisms by which eNOS regulates cardiomyocyte

proliferation are not fully understood The goal of the present

study was to investigate the role of tissue inhibitor of

metallopro-teinase-3 (TIMP-3) in eNOS-mediated cardiomyocyte

prolifer-ation

Methods and Results: Experiments were conducted in cultured

neonatal mouse cardiomyocytes TIMP-3 expression was

signifi-cantly decreased in wild-type (WT) cardiomyocytes treated with

an adenoviral eNOS (Ad-eNOS) Furthermore, TIMP-3 levels

were significantly decreased in cardiomyocytes derived from

eNOS transgenic mice Inversely, TIMP-3 transcript levels were

significantly elevated in eNOS-/- cardiomyocytes The inhibitory

effect of NO on TIMP-3 expression was dependent on

S-nitrosy-lation of c-jun, a subunit of AP-1 Cell proliferation was

increased in TIMP-3-/-cardiomyocytes while recombinant TIMP-3

decreased proliferation in both TIMP-3-/- and WT

cardiomyo-cytes Furthermore, the decline in proliferation observed in

eNOS-/- cardiomyocytes was abrogated when TIMP-3 expression

was blocked by an anti-TIMP-3 antibody In vivo cardiomyocyte

proliferation was assessed by Ki67 immunostaining on postnatal

day 1 hearts Ki67 positive cardiomyocytes was decreased in

eNOS-/-, but increased in eNOS-Tg and TIMP-3-/- hearts

com-pared to WT controls

Conclusions: Our study suggests that eNOS promotes neonatal

cardiomyocyte proliferation by inhibiting TIMP-3 expression

C1-77

Effects of estrogen and conjugated estrogen on

adipogenesis in human mesenchymal stem

cells

C Linehan, L M Oconnor

National University of Ireland Galway, Galway, IRELAND

Obesity is a prevalent and growing health hazard in industrial

countries It is a risk factor for non-insulin-dependent diabetes,

car-diovascular disease, osteoarthritis, some cancers and multiple

reproductive and metabolic disorders There is an urgent need to

understand the process of adipocyte growth and differentiation in

order to address this growing epidemic

Objective: This project seeks to provide quantitative and

qualitat-ive evidence of a role for sex-steroid hormones by both genomic

and non-genomic signalling systems in the process of adipogenesis

Materials and Methods: MSCs were induced to differentiate in the

presence of varying concentrations of estrogen and estrogen

conju-gated to BSA Results: We evaluated differences in the outcome

by monitoring (i) the time taken for each test to differentiation by

comparison with both positive and negative controls (ii) the level

of differentiation (as measured by triacylglycerol assay) the level

of PPAR (by real time-PCR) in each assay on a daily basis We

demonstrated that significant differences exist between adipocyte

differentiation in the presence of estrogen and conjugated with

respect to the level of differentiation achieved and the signalling

mechanisms through which the processes operate

C2-151 Substituted 9-arylideneanthrones as potential agents for leukemia therapy; mode of action

E Levy-dahary1, Z Rappoport2, D Arad3, O Shpilberg4,

I Levy5, I Nathan1

1Ben- Gurion University of the Negev & Soroka University Medical ter, Beer- Sheva, ISRAEL,2The Hebrew University of Jerusalem, Jeru-salem, ISRAEL,3NLC Pharma, Tel-Aviv, ISRAEL,4Medical CenterRabin, Campus Belinson, Petach Tikva, ISRAEL,5Institute of Hematol-ogy Soroka University Medical Center, Beer- Sheva, ISRAEL

Cen-In a search for novel derivatives, which can serve as antileukemicagents we have screened and tested a large series of vinylic com-pounds on human leukemic cell lines: HL-60 promyelocytic leuke-

monocytic leukemia The ring-substituted 9-benzylideneanthroneshad the strongest antileukemic effect with LC50< 0.1 lg/ml buthad no effect on lymphocytes from healthy donors under thesame experimental conditions These substituted 9-arylidenean-thrones killed the leukemic cells by induction of apoptosis asmeasured by acridine orange/ ethidium bromide staining andphosphatidyl serine exposure using annexin V-FITC These com-pounds also caused G1 arrest in CCRF-CEM cell line The signa-ling pathway leading to cell death were studied PKC-e activationwas manifested by translocation of the enzyme from the cytosol

to the membrane, as assessed by western blotting In addition,the production of reactive oxygen species (ROS) was observed asdetermined by the fluorescent probes specific to H2O2 and O2-

DCFH-DA and hydroethidine, respectively The lipophilic oxidant vitamin E was able to abolish the ROS generation andthe apoptotic activity of these 9-arylidene anthrones Primary cellsobtained from CLL patients underwent apoptosis in response totreatment with 9-arylideneanthrones The results imply the antile-ukemic mechanism of these arylideneanthrones and their potential

anti-as antileukemic agents

C2-152 Involvement of 14-3-3sigma in antitumor effects

of cis-4-hydroxy-L-proline (CHP) in human epithelial tumor cell lines

U Olszewski1, A Ellinger2, R Zeillinger1, E Ulsperger1,

The role of NUR77 protein in apoptosis of

normal thymocytes and malignant lymphoid

cells

A Rapak, I Stasik, E Ziolo, L Strzadala

Institute of Immunology and Experimental Therapy, Wroclaw,

POLAND

Nur77 (TR3, NGFI-B) is an orphan nuclear receptor affecting

sev-eral important intracellular processes like apoptosis, differentation

and proliferation Nur77 may act as a transcription factor or as an

adaptor protein on mitochondrial membrane facilitating the release

of cytochrome c to the cytosol Nur77 is involved in negative

selec-tion of thymocytes but the exact mechanism of its acselec-tion is

unknow

We showed that induction of Nur77 by ionomycin and its ability

to bind DNA is not sufficient to initiate the apoptotic process in

lymphoma cell line, but apoptosis was restored by treatment with

immunosupressant FK506 We found that Nur77 is present on

mitochondria both in thymocytes undergoing apoptosis and in

lymphoma cells resistant to apoptosis after treatment with

ionomy-cin However cytochrome c release and apoptosis in thymic

lymphomas is abolished despite Nur77 mitochondrial targeting,

but can be restored by FK506 The resistance of lymphoma cells

to calcium mediated apoptosis is associated with cytochrome c

release and is FK506 sensitive

C2-154

Role of PKN isoforms in tumour cell migration/

invasion

S Lachmann1, A Jevons1, P J Parker1,2

1Protein Phosphorylation Laboratory, London Research Institute, 44

Lincoln’s Inn Fields, London, UNITED KINGDOM, 2Division of

Cancer Studies, Kings College School of Medicine, Guy’s Hospital,

London, UNITED KINGDOM

The mammalian Protein Kinase N (PKN) family of

Serine/Threon-ine kinases comprises three isoforms While the catalytic domains

of the PKNs are highly related to those of the PKC family, the

regulatory domains are distinct They contain stretches of a leucine

zipper like sequence which can bind Rho family GTPases Since

small GTPases are major regulators of the cellular cytoskeleton

and hence important for processes such as cell migration and

inva-sion we focused on the potential role(s) of specific PKN isoforms

therein It has been reported that PKN3 is required for prostate

tumour cell invasion The lack of influence of PKN1 and PKN2 in

this model might reflect very different roles for these proteins or

simply tissue specific isoform expression To investigate this, we

chose two tumour model systems to study the roles of PKN1 and

PKN2: a breast tumour model in which all three isoforms are

expressed and a bladder tumour model in which PKN2 is the

pre-valent isoform Interestingly, siRNA mediated depletion of PKN2

alone in bladder tumour cells is sufficient to inhibit cell migration

However, in breast tumour cells depletion of all three isoforms is

required to observe a similar effect PKN1/2 are also required for

efficient invasion in both cell types Currently we are addressing

the question whether the PKN isoforms act redundantly or

whe-ther they signal through parallel pathways

C2-155 Downregulation of angiogenesis in vivo by DNAzymes to b1 mRNA integrin subunit

I Sacewicz, M Wiktorska, T Wysocki, C S Cierniewski,

J NiewiarowskaMedical University of Lodz, Department of Molecular and MedicalBiophysics, Lodz, POLAND

Angiogenesis, formation of new vessels from preexisting ones,occurs both under physiological (wound healing, reproducingcycle) and pathological conditions (retinopathy, cancer) Adhesion,migration and proliferation of endothelial cells to extracellularmatrix proteins as well as invasiveness and cellular survival of can-cer cells play important roles in biological processes such as angio-genesis, tumor growth and metastasis These cellular events aremediated by integrins, particularly by members of the b1 sub-family Recently, we showed that gene-inactivating agent - metylat-

ed derivative of DNAzyme to b1 mRNA, can be used todownregulate neovascularization process in mouse models Angio-genesis was examined using a bFGF supplemented Matrigel plugs

in BALB/c mice Local administration of the DNAzyme every ond day markedly reduced revascularization of Matrigel plugs InHuman Tumor Xenograft Models (PC-3 and CX 1.1 cell lines)where BALB/cA nude (nu-/-)-B6.Cg - Foxn1nuathymic mice wereused DNAzyme decreased tumor volume and kinetic of its growth

sec-In this report we provide evidences showing that the active form

of mouse DNAzyme to b1 integrin subunit decreased intensity ofneovascularisation process, evaluated by measurement of haemo-globin concentration as well as PECAM-1 immunohistochemicalstaining In conclusion, these DNAzymes might ultimately provide

a therapeutic means to inhibit pathological angiogenesis and stasis of carcinoma cells

meta-C2-156 Evaluation of para-phenylendimaleimide in tumour cell death

J Garcı´a1, L Cedillo1, J G Trujillo2

by PMA and ionomycin served as the control, resulting in a GSHconcentration of 8.76 nM/106 cells The time and concentrationdependent cytotoxic effect of the tested compound was evaluated

by Mosman method and was compared mole to mole with thesame effect produced by the diethyl maleate (DEM) The maxi-mum cytotoxic effect was 70% for SiHa cells at 21hours and 80%for L-5178Y cells at 8 hours, in each case with 10 millimoles ofp-PDI, showing no significant difference with DEM The controlcells did not show any cytotoxic effect from either p-FDI or DEM

at any concentration or time The percentages of early apoptosisdetected at 2 hours by using a flow cytometry with Anexin V-PEand 7-AAD were: 8.22 for SiHa, 3.03 for L-5178Y and 3.05 forthe splenic cells The necrotic populations were 49.24, 15.45, 9.42respectively Finally the late apoptosis was observed by the TUN-NEL technique through microscopic analysis In conclusion, p-PDIreacts selectively but not enzymatically with the thiol group andforms the 1,4 Michael aduct

Antitumor effects of oncolytic recombinant

adenoviral vectors containing L- plastin

promoter in hepatocellular carcinoma cells

I Chung1, S Kim2, K Jung2, A B Deisseroth2

1College of Pharmacy, Duksung Women¢s University, Seoul,

Diego, CA, USA

The replication competent adenoviral vectors, AdLPE1A and

Ad-LPCDIRESE1A were generated and reported previously to have

cytotoxic effects in some cell lines AdLPE1A vector contains

tumor-specific L-plastin promoter (LP) to regulate the expression

of E1A gene, which is essential for viral replication

In AdLPCDIRESE1A vector, the expression of cytosine deaminse

(CD) and E1A genes are under the control of LP

CD enzyme can deaminate the nontoxic prodrug 5-fluorocytosine

(5-FC) to the toxic 5-fluorouracil (5-FU)

We have conducted in vitro preclinical study to evaluate

effective-ness of these vectors on human hepatic carcinoma which is the

most common internal malignancy in Korea

The cytotoxic effect was evaluated previously in HepG2 cells which

had showed the best LacZ expression by infection of AdLPLacZ

among liver carcinoma cell lines tested in previous study The

effic-acy of cytotoxicity was measured by MTT test, cell counting and

photographs We infected HepG2 cells with various MOI of

vec-tors alone or concurrent with 5-FC Additive cytotoxic effects were

observed by the coadministration of 5-FC with the vector

contain-ing CD The replication competent AdLPE1A and

AdLPCDIR-ESE1A vectors were directly cytotoxic to HepG2 cells We also

observed the additive cytotoxic effects when AdLPCDIRESE1A

vector had been coadministrated with 5-FC

The results from present study suggest that the use of AdLPE1A

and AdLPCDIRESE1A/5FC system may be value in treatment of

liver cancer Further in vivo animal studies are needed for clinical

trial

C2-158

Protein phosphatase M1B; A novel Gankyrin

binding protein

S Kongkham, R Layfield, S Dawson

School of Biomedical Sciences,Medical School, University of

Nottingham, Nottingham, UNITED KINGDOM

Gankyrin, whose name derives from gann meaning cancer in

Jap-anese, has been found overexpressed in hepatocellular carcinoma

Many studies show that gankyrin acts as an oncoprotein which

promotes cell proliferation Its function was investigated and

revealed that gankyrin causes the degradation of both the pRB

and p53 tumour suppressor proteins; this may be mediated via the

interaction of gankyrin with Mdm2 However, other possible

gank-yrin associated proteins involved in tumorigenesis remain to be

clarified Here we investigated the properties of a new gankyrin

binding protein and its effect in combination with gankyrin on the

NF-jB pathway induced by TNF-a

This study detected new gankyrin binding proteins using stable

overexpression of FLAG-tagged gankyrin in HEK293 cell lines

and anti-Flag M2 affinity agarose to precipitate gankyrin binding

proteins Mass spectrometry/QTOF and Mascot database searches

identified a 40 kDa band as protein phosphatase M1B (PPM1B)

The confirmation of this protein as PPM1B was achieved with

western blotting using an anti-PP1B antibody Moreover, GST

pull-down and coimmunoprecipitation assays confirmed the

phys-ical interaction between PPM1B and gankyrin NF-jB reporter

assays reveal that gankyrin activates the NF-jB pathway, whereas

PPM1B down-regulates this pathway

C2-159 The leader region of Laminin B1 mRNA confers cap-independent translation

M PetzMedical University of Vienna, Vienna, AUSTRIA

Translation initiation of eukaryotic mRNAs generally occurs bycap-dependent ribosome scanning However, certain mRNAs con-tain internal ribosome entry sites (IRES) allowing cap-independenttranslation Several of these IRES-competent transcripts and theircorresponding proteins are involved in tumourigenesis This studyfocused on IRES driven translation control during the epithelial tomesenchymal transition (EMT) of hepatocytes that reflects crucialaspects of carcinoma progression Expression profiling of EMTrevealed Laminin B1 (LamB1) to be translationally upregulated.The 5’-untranslated region (UTR) of LamB1 was potent to directIRES-dependent mRNA utilization of a bicistronic reporter con-struct Stringent assays for cryptic promoter and splice sitesshowed no aberrantly expressed transcripts, suggesting that thereporter activity provided by the leader region of LamB1 mRNAexclusively depends on IRES In accordance, LamB1 expressionincreased upon negative interference with cap-dependent transla-tion by expression of human rhinovirus 2A protease or heat shock

of cells Finally, the enhanced expression of LamB1 during EMTcorrelated with an elevated IRES activity Together, these dataprovide first evidence that the 5’-UTR of LamB1 contains a bonafide IRES that directs translational upregulation of LamB1 duringstress conditions and neoplastic progression of hepatocytes

C2-160 Cross-talks of Raf/MEK/ERK, TRAIL/Apo2 and mevalonate cascades modulate

sup-of prostate carcinoma cell lines with different ness phenotype to c-Tocotrienol was initially analyzed in relation

androgen-responsive-to co-treatment with (a) Lovastatin, (b) Z-VADfmk, a pan-caspaseinhibitor, (c) Geranylgeraniol (GGO), a mevalonate-pathwaymodulator and (d) TNF-related apoptosis inducing ligand(TRAIL) Sensitization of both cell lines to c-Tocotrienol aftermodulation of the mevalonate pathway by Lovastatin was not res-cued but further enhanced by both pan-caspase inhibition withZ-VAD and mevalonate modulation with GGO Immunoblot ana-lysis disclosed augmentation of cleaved effector caspases and con-current modulation of the mitochondrial apoptotic cascades withupregulation of BAX in a c- Tocotrienol - dose dependent manner.Lovastatin sensitized both cell lines to c- Tocotrienol-induced inhi-bition of G-protein prenylation and activation of the TRAIL/Apo2 apoptotic cascade as shown by RAP1A and Death Receptorlevels, respectively This effect was enhanced by pan-caspase inhibi-tion while a low-level ERK-phosphorylation induced by c- Tocot-rienol in the androgen unresponsive cells was reversed byLovastatin

Conclusively, cross-talks between Raf/MEK/ERK, TRAIL/Apo2and mevalonate cascades play a key role in the synergism betweenc-Tocotrienol and Lovastatin independently of effector caspaseactivation Signalling molecules such as phospho-ERK, Death Re-ceptors and small G-proteins can serve as intermediate markers ofresponse

Characterization of novel blood and lymphatic

endothelial antigens by antibody phage display

T M Keller, B Hantusch, R Kalt, A Davidovits,

D Kerjaschki

Medical University of Vienna, Vienna, AUSTRIA

The microvascular system is a specialized compartment

constitu-ting a network of vessels that are lined by endothelial cells While

it is the task of blood vessels to provide all regions of the organsim

with fluid, nutrients and necessary cells, the lymphatic vessels not

only accomplish their evacuation but concomitantly assume

important immunologic tasks We have chosen to use an scFv

anti-body phage library to screen the membrane proteins of cultured

human lymphatic versus blood endothelial cells Screening of a

scFv-phage library was performed on human LECs and BECs

in a series of seven consecutive panning rounds The quality of

the enrichment was stringently controlled by comparison with

unspecific WT-phage enrichment and DNA-digests of phagemid

amplificates Then, single clones were tested for presence of

an integer scFv insert and the DNA sequences were determined

The amplified phage clones are actually stringently tested in

cell based assays for binding towards LECs versus BECs with

high affinity and specificity Specific antibody reactivities will be

confirmed in classical immunodetection assays on endothelial

cells as well as tissue sections Novel specific antibodies may serve

as diagnostic and therapeutic tools in inflammatory as well as

malignant disease Moreover, the retrieval of such antibodies

may identify therapeutic drug targets for inflammation and/or

cancer therapy

C2-162

Distinct actions of thrombin on growth and

survival of endothelial cells

N E Tsopanoglou, P Zania, C S Flordellis, M E Maragoudakis

University of Patras, Medical School, Department of Pharmacology,

Rio-Patras, GREECE

Many of the effects of thrombin in vascular cells can contribute to

activation of angiogenesis by thrombin The most prominent effect

is considered to be a direct promotion of endothelial cell

prolifer-ation In this report, however, we showed that thrombin is a poor

growth factor for human endothelial cells and its limited mitogenic

effect is mediated indirectly by the release of heparin

binding-epi-dermal growth factor, subsequent to protease-activated receptor-1

(PAR-1) activation On the other hand, it was demonstrated that

thrombin is a potent anti-apoptotic factor for endothelial cells,

pointing to a novel role of thrombin in vascular protection

Analy-sis by Annexin V/propidium iodide double staining revealed that

thrombin, specifically, promoted survival of serum-starved

endot-helial cells in a concentration-dependent manner This effect of

thrombin was largely independent of its catalytic activity and was

mediated through interaction with amb3 integrin, while the

involve-ment of PAR-1 was limited These results provide new insights in

understanding the role of thrombin in vascular development Both

angiogenic and anti-apoptotic actions of thrombin may contribute

to the formation of functional blood vessels and this may be a

basis for developing new agents for application in

angiogenesis-dependent diseases

This work was supported by the European Social Fund

Opera-tional Program for EducaOpera-tional and VocaOpera-tional Training II,

Pro-gram Pythagoras II

C2-163 Angiotensin II triggers apoptosis in cardiac fibroblast but not in myofibroblast

overexpressing the type 1 receptor

G A Diaz-Araya1, R Vivar Sanchez1, C Soto1, M Copaja1,

F Mateluna1, P Aranguiz1, J P Mun˜oz1, M Chiong1,

W G Thomas2, S Lavandero1 1

University of Chile, Santiago de Chile, CHILE,2MolecularEndocrinology, Baker Medical Research Institute, Melbourne,AUSTRALIA

The function of angiotensin II receptor type 1 and type 2 (AT1Rand AT2R) on cardiac fibroblasts and myofibroblast viability inconditions where they are up-regulated is unknown Using adeno-virus we ectopically expressed the AT1R and AT2R in cultured ratcardiac fibroblasts and myofibroblast and investigated the effect oncell viability Angiotensin II 100 nM, decreased the cell viabilityonly in cardiac fibroblasts expressing AT1R In cardiac fibroblastexpressing the AT2R or myofibroblast expressing the AT1R orAT2R, no effect of Ang II 100 nM on cell viability was observed.Cell viability was linked to an early decrease (starting at 6 h) inmitochondrial membrane potential (wMMP) in cardiac fibroblastsexpressing AT1R Cardiac fibroblast apoptosis, assessed by Propi-dium Iodine (PI), was detected 18 h after Ang II Both wMMPand apoptosis were blocked by Losartan 10lM, U73122 1lM, andGo¨ 68074 100nM No differences in expression levels of bcl-2 andbax protein between cardiac fibroblast and myofibroblast wereobserved However, Ang II only reduces the ratio bcl-2/bax in car-diac fibroblast We conclude that Ang II trigger apoptosis in car-diac fibroblasts expressing AT1R by a PLC-PKC dependentsignalling pathway by an early decrease in wMMP

C2-164 Requirement of glycosaminoglycans in death receptor-induced apoptosis

W Chien, Y Wu, M LaiInstitude of Molecular Biology, Acadimia Sinica, Taiwan, ROC,Taipei, TAIWAN

Proteoglycans are cell surface molecules that regulate several gical functions through their glycosaminoglycan (GAG) chains.Death receptors initiate apoptosis by assembly of protein complex

biolo-at cell membrane Whether glycosaminoglycans may regulbiolo-ate debiolo-athreceptor-induced apoptosis remains unclear We found that glycos-aminoglycans modulate cell death triggered by Fas and TRAIL.Exogenous addition of heparin and dermatan sulfate effectivelysuppressed death receptor-induced apoptosis in Jurkat cells Thepotential function of glycosaminoglycans in Fas- and TRAIL-trig-gered cell death was further elucidated using Sog9 cells, lackingkey enzyme to synthesize glycosaminoglycans, and the parental Lcells Fas induced significant amount of apoptosis in wild-type Lcells, but not in Sog9 cells, suggesting a requirement of glycosami-noglycans in Fas-mediated cell death To investigate the possiblecross-coupling between glycosaminoglycans and death receptor, wechose to knock down syndecan-2, the major transmembrane prote-oglycan in Jurkat cells Downregulation of syndecan-2 by specificsiRNA inhibited Fas- and TRAIL-induced apoptosis in Jurkatcells The reduction in cell death was accompanied with a dimin-ished caspase-8 activation, suggesting a blockage at death receptorinitiation stage when syndecan-2 was downregulated The cell sur-face levels of DR5 were not affected by syndecan-2 knockdown,while Fas expression on cell membrane was instead elevated Ourresults suggest an essential role of proteoglycans in death receptor-mediated apoptosis

Cytoplasmic localization of survivin is

implicated in lymphnode metastasis of

colorectal cancer patients

F Shimamoto1, H Tuncel2, G Qi3, E Aoki4, T Takata3,

M Tatsuka5

1Prefecture University of Hiroshima, Hiroshima, JAPAN, 2Istanbul

University, Istanbul, TURKEY,3Graduate School of Biomedical

Sci-ence, Hiroshima University, Hiroshima, JAPAN,4Hiroshima

Univer-sity, Hiroshima, JAPAN,5Research Institute of Radiation Biology,

Hiroshima University, Hiroshima, JAPAN

Overexpression of survivin, a member of the inhibitor of apoptosis

protein (IAP) family, is commonly detected in colorectal cancers

but not in normal colonic mucosa The IAP function of survivin is

believed to be critical for cancer progression Survivin is also

known to be a component of chromosome passenger protein

(CPP) complex essential for chromosome segregation and

cytokin-esis Recently, nuclear export signal of survivin is reported to be

important to the CPP function Here, we show that cytoplasmic

localization of survivin is implicated in lymphnode metastasis of

colorectal cancer patients Immunohistochemical analyses reveal

that increased expression levels of cytoplasmic survivin are found

in lymphnode-metastasized tumors of colorectal cancer patients

On the other hand, another CPP protein, Aurora-B, is mainly

found in the nucleus of both cancerous and normal cells Although

Aurora-B is also overexpressed in colorectal cancers, the expression

levels of Aurora-B are not indistinguishable between primary

tum-ors and lymphonode-metastasized tumtum-ors These results indicate

that survivin has a crucial role in colonic lymphnode metastasis,

suggesting that survivin is a potential target for anti-metastatic

agents

C2-166

Immunotoxin containing Pseudomonas

Exotoxin A induces cell death in a malignant

melanoma cell line

K Risberg, Ø Fodstad, Y Andersson

Dept Tumor Biology, Rikshospitalet-Radiumhospitalet Medical

Cen-ter, Oslo, NORWAY

Metastatic malignant melanomas are generally drug resistant and

have a very poor prognosis Hence, there is an obvious need for

new therapies, preferably approaches that have different

mecha-nisms of action

Immunotoxins are compounds that are being developed for cancer

therapy Immunotoxins consist of a toxin which is linked to an

antibody which recognizes cancer specific antigens Once

internal-ized, the immunotoxin induces cell death through both inhibition

of protein synthesis and induction of apoptosis

We have chemically conjugated Pseudomonas Exotoxin A (PE) to

the antibody 9.2.27 which recognizes the surface molecule

HMW-MAA expressed on most malignant melanomas and melanoma cell

lines The 9.2.27PE immunotoxin (IT) induced inhibition of

pro-tein synthesis followed by decreased cell viability in the malignant

melanoma cell line FEMX Hyperpolarization of the mitochondrial

membrane was observed suggesting that cytochrome c, a molecule

that triggers activation of caspase-3, was retained within the

mito-chondria The caspase-3 activation is therefore not induced via the

apoptotic intrinsic pathway, but through another mechanism in the

FEMX cells Chromatin condensation, an early apoptotic effect,

was observed in the IT treated cells using the EM technique

How-ever, DNA fragmentation, a typical feature of apoptosis, was not

A to enhance the transforming activity of oncogenic H-Ras tionally we investigated the influence of Aurora A of the trans-forming capacity of H-RasV12 in combination with E1A, where

Addi-we observed that Aurora A inhibited transformation

Accordingly we found that Aurora A overexpression has an tory influence on cell proliferation of normal fibroblasts Furtherinvestigations revealed that serum deprived cells enter S-phase sig-nificantly later in cells overexpressing Aurora A as compared tomock treated cells Since an interplay of Aurora A with p53 hasbeen reported, we postulated that this interaction is responsible forthe inhibitory function of Aurora A at the G1/S transition of thecell cycle Therefore we tested the influence of Aurora A overex-pression on the cell cycle of a p53 mutated cell line In these cellsoverexpression of Aurora A had no influence on the duration ofthe cell cycle Based on these results we further tested the influence

inhibi-of Aurora A kinase overexpression in UV treated cells in the ence or absence of p53 activity

pres-Taken together the presented results identified the existence of anew Aurora A role at the G1/S transition

Periodontal disease is one of the most common infectious diseases

of humans, yet its osteoclastogenic pathways are not fully ted We have developed a murine model in which alveolar boneloss is induced by oral infection with Porphyromonas gingivalis, agram-negative periodontopathic bacterium from humans In thepresent study B cell normal BALB-J mice lost alveolar bone 6weeks after oral infection, while delta heavy chain knockoutBALB-Igh-5-/-Jmice showed no bone loss These mice lack IgD ontheir surfaces and have delayed affinity maturation The presence

elucida-of B cells was confirmed by flow cytometry, and decreased specificantibody in the IgD knockouts was shown by ELISA, corrobor-ating our previous finding that specific antibody does not preventbone loss IgD is known to be an effective B cell antigen presenter

to, and activator of, CD4+helper T cells After P gingivalis tion the percent of total CD4+T cells and the percent of CD69+

infec-activated CD4+T cells went up in the normal mice but not in theIgD knockout mice Activated CD4+T cells or B cells can directlystimulate osteoclasts through RANKL and we have previouslyshown that CD4+T cell deficient mice do not lose alveolar boneafter oral infection Here, we demonstrate that B cell IgD activa-tion of CD4+ T cells is an additional pathway to alveolar boneresorption Supported by U.S Public Health Service grants R01DE10728 to PB and R01 DK56597 to DR