4. New Analytical and Diagnostic Approaches
4.1 Bioinformatics and Proteomics
S4.1-1
Strategies tocovermicrobial proteomes
P. R. Jungblut
Core Facility Protein Analysis, Max Planck Institute for Infection
Biology, Berlin, Germany. E-mail: jungblut@mpiib-berlin.mpg.de
The aim of our studies is to get access to as much as possible of
the protein species present in the proteome under analysis.
Already at the low complexity level of proteasomes it becomes
clear that proteins are massively modified. There are about 70
spots representing different protein forms from which only some
of them were identified at the protein species level. The mycobac-
terial proteome of a cell extract of late growth phase state
revealed by 2-DE/MS 1800 spots with 379 different proteins iden-
tified (10% of the predicted genes). Isotope coded affinity tag
(ICAT)-LC/MS resulted in 619 (15% of predicted genes) identi-
fied proteins. Because of the overlap of 158 proteins showing the
high complementarity of 2-DE/MS and ICAT-LC/MS the prote-
ome is covered at present by 21%. For Helicobacter pylori we
revealed with a pre-fractionation strategy about 520 different
proteins (33% of predicted proteome). One of the proteins deter-
mined as vaccine candidate was successful as vaccine in animal
models and is now in a clinical study. A proteome 2-DE database
was established (http://www.mpiib-berlin.mpg.de/2D-PAGE),
which contains data of more than 10 different bacterial species.
Proteomics does not end with the identification of a protein. At
present identification at the protein species level, which needs
100% sequence coverage is very time-consuming and affords fur-
ther technology development. Today, the enormous information
content of a 2-DE gel is only accessible to a small extent.
S4.1-2
Targeted peptide-centric protemics: a versatile
tool for quantitative proteomics
J. Vandekerckhove, L. Martens, J. Pinxteren, P. Van Damme,
X. Hanoulle, B. Ghesquie
`
re, A. Staes, J. Van Damme,
E. Timmerman, M. Goethals, H. Demol, K. Hugelier and
K. Gevaert
Department of Biochemistry and Medical Protein Research, VIB
and Ghent University, Ghent, Belgium.
E-mail: joel.vandekerckhove@ugent.be
We have developed a peptide-centric proteome method, based on
the principle of diagonal chromatography. The procedure has
been called COmbined FRActional DIagonal Chromatography
(COFRADIC) and can sort for any kind of peptide or subset of
peptides, provided it can be selectively altered by chemical or
enzymatic reaction. We can sort for either methionine or cysteine
peptides. In addition, the protein N-terminal peptides can be iso-
lated from highly complex mixtures, such as tryptic digests of
total cell or tissue lysates. The latter approach holds interesting
possibilities: (a) the complexity of a proteome is reduced to a one
protein-one peptide ratio, allowing high coverage proteomics of
complex samples; (b) the N-termini of proteins each harbor
highly characteristic sequences opening the possibility to analyze,
two proteomes at the same time such as is the case in xeno-pro-
teomics; and (c) protein cleavage or processing yields novel
N-termini which can be specifically isolated allowing global ana-
lysis of protein degradation events.
Examples are shown of: (a) high-coverage proteomics of human
multipotent progenitor cells; (b) mouse plasma proteomics after
human hepatocyte grafting; (c) global analysis of apoptotic cells;
(d) glycoproteomics in human interstitial fluids; and (e) analysis
of oxidative stress in CSF.
We have further adapted our COFRADIC procedure to identify
different types of ATP-binding to proteins in a total cellular con-
text.
S4.1-3
Learning from evolution to predict protein
structure and function
A. Tramontano, D. Cozzetto, A. Giorgetti, D. Raimondo
Department of Biochemical Sciences, University ‘La Sapienza’,
Rome, Italy. E-mail: anna.tramontano@uniroma1.it
Since decades, the problem of deciphering the code that relates
the amino acid sequence of a protein and its native three-dimen-
sional structure has been the subject of innumerable investiga-
tions and, in spite of the many frustrations caused by its
elusiveness, the interest in the problem is not fading away, on the
contrary. What stands in our way, notwithstanding all these
efforts, is the complexity of protein structures. In a protein struc-
ture thousands of atoms are held together by weak forces and
give rise to a conformation which is only marginally stable. It is
very unlikely that we can use the laws of physics to compute the
native functional structure of a protein in the foreseeable future.
However, we have at our disposal the experimentally solved
structures of a reasonable number of proteins, a few thousands
as of today.
Among them, there are many examples of proteins that descend
from a common ancestral protein. We know that these proteins
are functional, or at least not deleterious, as they have been
accepted in the population, therefore they are expected to have a
stable native structure. Because of the requirement that the evo-
lutionary changes preserve function, we also expect their struc-
ture to be similar. This has allowed us to devise methods to
construct models of the structure of their evolutionary neigh-
bours.
I will describe how we can evaluate and estimate the quality of a
protein model and analyze how this impacts on its usefulness.
Abstracts
24
4.2 Clinical Proteomics
S4.2-1
Proteomic strategiesto the analysis of breast
apocrine carcinomas
J. E. Celis, I. Gromova, P. Gromov, J. M. A. Moreira,
T. Cabezo
´
n, E. Friis and F. Rank
Danish Centre for Translational Breast Cancer Research, Copen-
hagen, Denmark. E-mail: jec@cancer.dk
Breast apocrine carcinomas represent about 0.5% of all invasive
breast cancers, and despite their distinct morphological features,
there are at present no standard molecular criteria available for
their diagnosis. Recent proteome expression profiling studies of
breast apocrine macrocysts, normal breast tissue and breast
tumours have identified specific apocrine biomarkers (15-PGDH
and HMG-CoA reductase) present in early and advanced apocrine
lesions. These biomarkers in combination with proteins found to
be characteristically upregulated in pure apocrine carcinomas
(psoriasin, S100A9, p53) provide a protein signature distinctive
for benign apocrine metaplasias. These studies have also presented
compelling evidence for a direct link, through the expression of
15-PGDH, between early apocrine lesions and pure apocrine carci-
nomas. Moreover, specific antibodies against components of the
signature have identified precursor lesions in the linear progression
to apocrine carcinoma. Finally, the identification of proteins that
characterize the early stages of mammary apocrine differentiation
such as 15-PGDH, HMG-CoA reductase and COX-2, has opened
a window of opportunity for chemoprevention.
S4.2-2
Uniprot and beyond: integrated database
infrastructure for the life sciences
R. Apweiler
EMBL-EBI, Sequence Database Group, Wellcome Trust Genome
Campus, Hinxton, UK. E-mail: apweiler@ebi.ac.uk
The increasing sophistication of electronic mechanisms to store,
manipulate and communicate information has transformed the
way we work. In particular, the opportunities and pitfalls for sci-
ence opened up by information technology are profound.
Nowhere has this been more apparent than in molecular biology.
It is possible to collect, analyse, communicate and share huge
amounts of information rapidly and accurately. Molecular bio-
logy, driven by the need to deal with large volumes of informa-
tion, was quick to build large collections of shared scientific
information. Nowadays, access to and skill in exploiting elec-
tronic information repositories is crucial to biological research. It
is on such resources (especially the protein sequence databases),
their history, their usage, their advantages, their pitfalls and how
scientific progress leads to new challenges for bioinformatics and
the development of resources for biological information that my
talk will concentrate.
S4.2-3
Nanobiotechnology in clinical proteomics
A. I. Archakov and Y. D. Ivanov
Institute of Biomedical Chemistry RAMS, Pogodinskaya, Moscow,
Russia. E-mail: archak @ibmc.msk.ru
A certain concentration barrier exists in proteomics: the protein
molecules residing in the concentration range below 10
)12
M can-
not be identified. The atomic force microscopy (AFM) nanotech-
nological method appears to be one of the most perspective
diagnostic tools and enables not to measure protein concentration
but merely to count single protein molecules and their complexes.
Biospecific fishing method enables to extend the concentration
limits of proteomics, as it enables to fetch the protein from the
solution and therefore to increase its concentration on the surface
of the biochip with immobilized partner proteins. Based on com-
bination of AFM and biospecific fishing method we have created
the AFM biochips for registration of hepatitis B and C and ovar-
ian cancer biomarkers in patient serum. The described approach
attains the concentrations of about 10
)15
M; however, to reveal
the lower concentrations (e.g. 10
)18
M) it is necessary to convert
the reversible complex formation into irreversible process.
Several other nanotechnological approaches can be useful for
proteomic research, among them are nanowire electrical detectors
and optical biosensors. The optical biosensor method enables to
register in real time the intermolecular interaction reactions
(including Ag/Ab) without labels and calculate the kinetic rate
constants and affinities for the complexes. Optical principle of
detection can be easily implemented in the widely available CD-
ROM readers, which convert the biospecific Ag/Ab binding
directly into the informational signal. Nanowire electrical detec-
tor is designed as nanowire arrays modified with antibodies.
These detectors show discrete conductance changes with biospe-
cific binding of viruses to nanowire.
4.3 DNA and Protein Microarrays
S4.3-1
Identification and analysis of signaling
networks regulating the development of the
mammalian cerebral cortex
G. Eichele
Max Planck Institute for Experimental Endocrinology, Hannover,
Germany
Determining the spatiotemporal distribution of gene products at
cellular resolution on a transcriptome-wide scale is crucial for
functional genomics but presents a daunting task. To tackle this
challenge, we have developed a platform that consists of high-
throughput detection of gene products and a web database – gene-
paint.org – from which annotated gene expression patterns can be
retrieved and viewed. This platform was used to construct a mid-
gestation mouse gene expression atlas containing a balanced repre-
sentation of 10% of the transcriptome. As proof of concept, we
analyze the Small eye mouse mutant that lacks the transcription
factor Pax6, indispensable for the proper development of many
organs including the cerebral cortex. From genepaint.org we
retrieved 300 genes regionally expressed in developing cortex, 87
of which we then found to be differentially expressed in Small eye
cortex, mostly in proliferative zones and mediating cell division,
migration, and fate determination, processes known to be abnor-
mal in Small eye. Band-shift assays suggest that many of the dif-
ferentially expressed genes are direct Pax6 transcriptional targets.
We discuss the emerging regulatory network of genetic cascades
that contribute to mammalian brain development.
Abstracts
25
S4.3-2
High-density protein and antibody arrays
D. J. Cahill
Conway Institute, University College Dublin, Dublin, Ireland.
E-mail: dolores.cahill@ucd.ie
The ability to systematically interrogate thousands of human pro-
teins simultaneously will facilitate large-scale biological approa-
ches. We have generated a set of over 10 000 different human
recombinant proteins. Results of screening antibodies and pep-
tides against these thousands of proteins, including validation of
the results obtained, will be shown. Recent results of profiling
the antibody repertoire in sera from subjects with autoimmune
disease versus controls will also be described. Recent improve-
ments to optimize the protein and antibody chip format will be
presented.
S4.3-3
Microarray-based comparative genomic
hybridization (array-CGH)
H. Fiegler
The Wellcome Trust Sanger Institute, The Wellcome Trust
Genome Campus, Hinxton, Cambridge, UK.
E-mail: heike@sanger.ac.uk
Comparative genomic hybridization has become a widespread
method for a genome-wide analysis of DNA copy number
changes in tumours. However, the detection of amplifications
and deletions is limited when performing CGH to metaphase
chromosomes. Replacing metaphase chromosomes with mapped
sequences arrayed onto glass slides overcomes these limitations.
As a result, resolution is drastically improved and limited only
by the insert size and the density of the mapped sequences used.
We have initially constructed an array with a resolution of
approximately 1 Mb across the human genome. We have now
increased this resolution by using overlapping tiling path clones
for array construction.
Array-based CGH (array-CGH) has initially been used for the
analysis of copy number changes in tumours. We have also
applied this technology to detect microduplications and microde-
letions in patients with constitutional rearrangements. In an ini-
tial study using the 1 Mb array we detected copy number
changes in 24% of the patients studied. However, five of the 12
copy number changes identified were found to be inherited from
one of the phenotypically normal parents. This suggested that
these imbalances might not be linked to the phenotype of the
patient but represent copy number variations in the normal pop-
ulation. In a newly established research programme we are now
screening more than 1000 normal individuals for copy number
variations using the whole genome tiling path array.
4.4 Pharmacogenomics and Toxicogenomics
S4.4-1
Pharmacogenomics and pharmacoproteomics
in cardiovascular diseases and risks
G. Siest
Universite
´
Inserm U.525 Equipe IV, Henri Poincare
´
Nancy 1,
Nancy, France. E-mail: gerard.siest@pharma.uhp-nancy.fr
We have recently proposed a five-step pharmacogenomic
approach for a new cardiovascular drug. After making a sum-
mary of the most well-known genes involved in drug metabolism
(in particular the new transporter ones) we will take as example
of drugs the statins. These drugs are responding differently in
human in function on multiple polymorphisms and we will give
examples with common ApoE polymorphisms influencing the
hypolipaemic effects of statins. These drugs have also pleiotropic
effects and are decreasing inflammation including some plasma
markers. This is an example of the need to separate the clinical
diseases phenotypes and to propose specific metabolic pathways.
However, given the current existing pharmacogenomic tools and
the development of the pharmacoproteomics field, a complete
strategy needs the integration of gene products, including some
peptides measured in laboratory medicine. These biomarkers
could be the metabolites resulting from drug-metabolising
enzymes activities, clusters of peptides related to an inflammatory
process, specific proteins or the metabolites resulting from envi-
ronmental effects. With the extraordinary amount of information
which could now be given by the proteins separated from plasma
by proteomic devices, we now have to add all these data to the
genomic and transcriptomic ones. Biological systems are then the
only way to put all this information in a comprehensive and clin-
ically usable set of data.
S4.4-2
Pharmacogenomics: towards individualized
drug therapy
M. Eichelbaum
Dr Margarete-Fischer-Bosch-Institut, fu
¨
r Klinische Pharmakologie,
Stuttgart, Germany. E-mail: michel.eichelbaum@ikp-stuttgart.de
With the complete sequence of the human genome available,
individualized medicine may soon become reality. Genomic infor-
mation may allow more accurate prediction of an individual’s
drug response and selection of the appropriate drug dosage to
achieve the optimal therapeutic response, avoid therapeutic fail-
ure and minimize side effects and toxicity.
Although many genes encoding proteins involved in the metabo-
lism, transport and mechanism of action of medications are
known to exhibit polymorphism in humans, use of this knowledge
in routine clinical practice is limited. Excepting a few examples of
drug-metabolizing enzymes, the contribution of genetic polymor-
phisms to individual differences in drug effects and toxicity is not
well understood. Moreover, most studies have focused on the
effect of a single polymorphism on drug response. This approach
neglects the fact that drug response phenotype, like most disease
phenotypes, is a complex polygenic trait also determined by non-
genetic factors (three). The extent to which genetic factors con-
tribute to the drug response/toxicity phenotype depends on the
extent of the candidate gene’s influence on drug disposition and
effects. Even if a gene has a large effect on a drug’s pharmacoki-
netics or pharmacodynamics, the presence of, a single nucleotide
polymorphism (SNP) in that gene will not provide an unequivocal
answer but, rather, will indicate the likelihood that an individual
patient will show an altered drug response.
Abstracts
26
S4.4-3
Ecogenetics and pharmacogenetics
of coronaropathies
P. F. Pignatti
Department of Mother and Child, Biology and Genetics, University
of Verona, Verona, Italy. E-mail: pierfranco.pignatti@univr.it
A few examples from our work on the interactions between gen-
etic and environmental factors to determine a modification of (a)
disease risk or (b) response to treatment will be discussed.
Given the interactions between MTHFR gene and pholatemia in
determining hyperomocysteinemia (Girelli et al. 1998), threshold
values of pholatemia relative to genotype will be indicated (Gi-
relli et al. 2003).
A correlation between APOCIII genotypes and coronaropathies
(Olivieri et al. 2002) or metabolic syndrome (Olivieri et al. 2003)
will be the second example, including the response to dietary
PUFA (Olivieri et al. 2005).
The third ecogenetic example will be the correlation between
PON genotypes and miocardial infarction risk in coronaropatics
relative to smoking habit (Martinelli et al. 2004), or metabolic
syndrome (Martinelli et al. 2005).
Pharmacogenetic response to clopidogrel platelet antiaggregating
therapy in patients who underwent coronary stenting, and the
role of several genes involved in the drug’s mechanism of action
or metabolism will last be analyzed: GPIIIa (Angiolillo et al.
2004a), GPIa (Angiolillo et al. 2004b, 2005a), and P2Y12 recep-
tor (Angiolillo et al. 2005b), or CYP3A4 (Angiolillo et al. sub-
mitted), respectively.
4.5 Glycobiology and Glycomics
S4.5-1
Carbohydrate-based vaccines
J. F. G. Vliegenthart
Department Bioorganic Chemistry, Bijvoet Center, Utrecht
University, Utrecht, the Netherlands.
E-mail: j.f.g.vliegenthart@chem.uu.nl
In recent decades, significant progress has been made in the struc-
ture determination and chemo (enzymatic) synthesis of glycan
chains that are part of cell surface molecules. This development
has rendered feasible the preparation of new generations of car-
bohydrate-based vaccines. They are urgently needed to prevent
bacterial infections, because a gradual increase in the resistance to
antibiotics has been observed leading to a serious health thread.
Recently, the application of a human vaccine against Hemophilus
influenzae type B has found approval. This vaccine consists of a
fully synthetic glycan that has been coupled to a suitable carrier
protein. Obviously, the problems arising from an infection by
(tropical) parasites has focused the attention on their cell surfaces
glycans, which are being explored as immuno-epitopes for prepar-
ing glycoconjugate vaccines. Also the possible application is
investigated of vaccines directed against cancer-related cell surface
epitopes. These vaccines are primarily aimed to prevent the recur-
rence of cancer after treatment and/or to avoid the formation of
metastasis. The development in this area will be discussed.
S4.5-2
Sialic acid-binding proteins, siglecs, as
modulators of immune responses
P. R. Crocker
Cell Biology and Immunology, University of Dundee, Dundee, UK.
E-mail: p.r.crocker@dundee.ac.uk
Siglecs are transmembrane sialic acid-binding receptors of the
immunoglobulin superfamily expressed in the haemopoietic,
immune and nervous systems where they play roles in cellular
adhesion and signalling. Sialoadhesin (Sn) is a prototypic siglec
that mediates adhesive functions of macrophages. We have
recently generated Sn-deficient mice, which exhibit subtle differ-
ences in subsets of T and B cells, but no defect in steady-state
haemopoiesis. Immune responses are significantly altered in
Sn-deficient mice indicating that this receptor is required for opti-
mal responses of T and B cell to foreign antigens. The CD33-
related siglecs are a distinct subset expressed predominantly by
cells of the innate immune system. They typically contain two
conserved tyrosine-based signalling motifs, comprising a mem-
brane proximal immune receptor tyrosine-based inhibitory motif
(ITIM) and an ITIM-like motif that can recruit and activate the
tyrosine phosphatases, SHP-1 and SHP-2. We have studied the
inhibitory signalling functions of CD33-related siglecs and shown
that even in the absence of tyrosine phosphorylation, Siglec-5 is
able to deliver inhibitory signalling functions. Together with
other published data, these findings suggest that glycan-depend-
ent interactions of siglecs leads to modulation of leucocyte activa-
tion and may play an important role in preventing inappropriate
tissue damage during immune and inflammatory responses.
S4.5-3
Oligosaccharide microarrays to unravel
carbohydrate recognition in the immune
system
T. Feizi
The Glycosciences Laboratory, Imperial College London,
Northwick Park and St Mark’s Hospital Campus, Harrow, UK.
E-mail: t.feizi@imperial.ac.uk
The diverse oligosaccharides of glycoproteins, glycolipids, prote-
oglycans and polysaccharides are potentially a vast source of
information, harbouring ‘glyco codes’ that are waiting to be deci-
phered in various contexts of biological and medical importance.
Towards developing a knowledge base of biological systems that
operate through oligosaccharide recognition, we have developed
a carbohydrate microarray platform. The approach is based on
the neoglycolipid technology that we introduced in 1985 to
address the need for a microscale technology to present oligosac-
charides in a clustered form for studies of carbohydrate–protein
interactions. This is a powerful means of generating probes: from
N- and O-glycans, glycolipids, glycosaminoglycans, polysaccha-
rides and diverse chemically synthesized oligosaccharides. A key
development has been to combine carbohydrate–protein interac-
tion studies with mass spectrometry in situ to determine mono-
saccharide composition, sequence and branching pattern, as well
exotic substituents at low picomole levels. Our state-of-the-art
microarray system, with almost 300 sequence-defined oligosac-
charide probes, is continually expanding towards thousands, and
has provision for generating ‘designer’ microarrays from desired
sources. I shall describe ligand assignments to classic receptors of
(C-type lectins and siglecs) and a newly described receptor Dec-
tin-1 of the immune system (Nat Biotechnol 2002; 20: 1011; J Biol
Chem, ahead of print, December 2005).
Abstracts
27
4.6 New Proteomic Assays for Cancer Biomarkers
S4.6-1
Clinical utility of biomarkers in cancer
M. Mu
¨
ller
Institute of Laboratory Diagnostics, Kaiser Franz Joseph Hospital,
Vienna, Austria. E-mail: mathias.mueller@wienkav.at
Biomarkers in cancer (approximately 25% of the mortality rate
in the population) are characteristic for the malignant phenotype
of a tumor cell. The tumor markers belong to various groups of
substances: enzymes, hormones, oncofetal antigens, carbohydrate
markers, blood group carbohydrates, proteins, and genetic mark-
ers. The concentration of the circulating marker depends upon its
expression, its production, its release, its catabolism, its clearance
as well as upon the blood supply of the tumor. They can be iden-
tified in intact cells by immuno-histopathology, flowcytometry,
cancer genetics, and proteomics technologies. In blood they are
commonly measured by immunochemical techniques.
In addition to established markers for malignancy several new
biomarkers based on genomics, proteomics, and bioinformatics
research are now evolving. There are promising results on cal-
reticulin for bladder cancer, on mammaglobin, on autoantibod-
ies, on the combination of the tumor-suppressor protein p53,
the growth factor C-erbB2, and the cell adhesion protein
E-cadherin for breast cancer and on surface proteins for colon
carcinoma.
The ideal tumour marker for differentiation between normal and
neoplastic conditions offers a high degree of organ specificity and
at the same time correlates with the prognosis and stage of the
malignancy. These goals have not yet been reached in most cases.
However, multivariate combination of markers results in signifi-
cant improvements of the diagnostic validities.
S4.6-2
Endogenous proteinase inhibitors as future
targets in the treatment of cancer
A. S. Schroll
1
, U. Lademann
1
, I. J. Christensen
2
, J. Foekens
3
,
H. J. Nielsen
2
,S.Wu
¨
rtz
1
, L. Davidsen
1
and N. Bru
¨
nner
1
1
Veterinary Pathobiology, KVL, Frederiksberg, Denmark,
2
Department of Surgical Gastroenterology, Hvidovre Hospital,
Copenhagen, Denmark,
3
Rotterdam University, Rotterdam, the
Netherlands. E-mail: nbr@kvl.dk
Proteinases and their natural occurring inhibitors are involved in
cancer progression. As such, they may be used as biomarkers or
represent targets for new treatment interventions. We have devel-
oped and validated immunoassays for the detection and quantita-
tion of Tissue Inhibitor of Metalloproteinases-1 (TIMP-1) in
plasma and tumor tissue. Analyses of plasma samples collected
retrospectively have shown that patients with colorectal cancer
(CRC) have elevated plasma TIMP-1 levels as compared to non-
CRC individuals. We could show that plasma TIMP-1 was eleva-
ted in early as well as late stage disease and that plasma TIMP-1
detected the disease with high sensitivity and high specificity
(Holten-Andersen et al. Clin Cancer Res 2002). Based on these
results, we have now performed a large prospective study to val-
idate TIMP-1 as a marker for early detection of CRC. The study
includes plasma samples from 5330 individuals who have under-
gone endoscopy. At the same time, we have shown in experimen-
tal models that TIMP-1 protects against chemotherapy-induced
apoptosis. By inhibiting TIMP-1 expression, cancer cells become
significantly more sensitive to chemotherapy. In support of an
apoptosis-inhibiting function of TIMP-1 in cancer, we have
shown that in patients with metastatic breast cancer and high
tumor tissue TIMP-1 levels, the objective response to conven-
tional chemotherapy is 0% as opposed to 45% in patients with
low TIMP-1 tumor tissue levels.
S4.6-3
SH2-profiling: a new approach for the global
characterization of the tyrosine
phosphorylation state
K. Dierck
1
, K. Machida
2
, B. J. Mayer
2
and P. Nollau
1
1
Department of Clinical Chemistry, Center of Clinical Pathology,
University Medical Center Hamburg-Eppendorf, Hamburg,
Germany,
2
Department of Genetics and Developmental Biology,
Raymond and Beverly Sackler Laboratory of Genetics and
Molecular Medicine, University of Connecticut Health Center,
Farmington, CT, USA. E-mail: nollau@uke.uni-hamburg.de
Deciphering complex signaling networks is of great importance
for the understanding of many important biologic functions such
as cell proliferation, differentiation or apoptosis. Dysregulation
of signal transduction plays a critical role in the pathogenesis of
many malignant and other diseases. In this context, the phos-
phorylation of tyrosine residues is a key event and it is of great
interest to gain detailed insights into the state and regulation of
the phosphoproteome. Current techniques for the comprehensive
characterization of the cellular state of tyrosine phosphorylation
are limited. Applying src-homology regions 2 (SH2)-domains, we
developed a novel strategy termed SH2-profiling for the analysis
of phosphotyrosine-dependent signaling networks. SH2-domains
are modular-binding domains present in many important signa-
ling proteins specifically recognizing tyrosine phosphorylated pro-
teins and thus serving as ideal tools for the comprehensive
analysis of the tyrosine phosphorylation state. Our current
SH2-profiling data demonstrate that tumor- and patient-specific
phosphorylation profiles are detectable with high sensitivity and
specificity in small amounts of complex protein mixtures allowing
to uncover aberrant signaling pathways with the potential to clas-
sify tumors and to develop rational therapeutic concepts based
on differential signaling states.
Abstracts
28
4.7 Imaging and Noninvasive Techniques: Fluorescence
Techniques
S4.7-1
Quantum dot ligands reveal a new mechanism
for retrograde transport of activated growth
factor receptor
T. M. Jovin, D. S. Lidke and D. J. Arndt-Jovin
Department of Molecular Biology, Max Planck Inst. f. Biophys.
Chem., Goettingen, Germany. E-mail: tjovin@gwdg.de
Biotinylated epidermal growth factor (EGF) attached to strept-
avidin-conjugated Quantum Dots (QDs) binds to its cell surface
receptor (EGFR, ErbB1, HER1). Wide field and laser scanning
microscopy have revealed the ligand–receptor complexes diffuse
freely on the cell surface, including filopodia, cellular extensions
with an F-actin bundle core. Interaction of at least two com-
plexes leads to activation of the receptor tyrosine kinase and
initiation of retrograde transport from the filopodia to the cell
body. According to a FRET assay based on QDs as donors,
endocytosis of the complexes occurs at the base of the filopodia.
We have measured the rates of free diffusion and retrograde
transport as a function of cell type, temperature, presence of var-
ious inhibitors, and mutations or manipulation of the expression
levels of putative protein components of the transport mechan-
ism. Diffusion constants and transport rates were determined
with single molecule sensitivity by tracking individual QD–recep-
tor complexes, exploiting the high sensitivity provided by an elec-
tron multiplying CCD camera. The QDs, the associated receptors
(visualized as a fusion protein with eGFP), and F-actin are trans-
ported with the same rate on filopodia, implying a coupling of
receptor movement to treadmilling of the F-actin bundles. We
conclude that filopodia act as non-linear biosensors for growth
factors located far from the cell body (Lidke et al. J Cell Biol
2005; 170: 619–626).
S4.7-2
Functional optical imaging of the neocortex in
action
A. Grinvald
Department of Neurobiology, The Weizmann Institute of Science,
Rehovot, Israel, E-mail: amiram.grinvald@weizmann.ac.il
During the last few decades, neuroscientists have benefited from
the emergence of many powerful functional imaging techniques
that cover broad spatial and temporal scales. We can now image
single molecules controlling cell differentiation, growth and
death; single cells and their neurites processing electrical inputs
and sending outputs; neuronal circuits performing neural compu-
tations in vitro; and the intact brain. At present, imaging based
on voltage-sensitive dyes (VSDI) offers the highest spatial and
temporal resolution for imaging neocortical functions in the liv-
ing brain, and has paved the way for a new era in the functional
imaging of cortical dynamics. It has facilitated the exploration of
fundamental mechanisms that underlie neocortical development,
function and plasticity at the fundamental level of the cortical
column. To follow neuronal computations at this fundamental
level requires a spatial resolution of at least 200 lm. Further-
more, because communication occurs over milliseconds, an ima-
ging approach with millisecond time resolution is required.
Without an imaging technique that can reveal the spatio-tem-
poral patterns of the subthreshold input, perception and other
higher brain functions cannot be fully understood. Finally, to
investigate higher brain functions, it is essential to be able to
image the neocortical function of behaving mammals, partic-
ularly monkeys. In the present study, we review the technique of
VSDI and describe some of its applications.
S4.7-3
Visualization and sensing of intermolecular
interactions with two-color fluorescent probes
A. Demchenko
Laboratory of Spectroscopy, Research Institute for Genetic
Engineering and Biotechnology, Gebze-Kocaeli, Turkey. E-mail:
dem@gmbae.tubitak.gov.tr
The key issue in biosensing is the transformation of response to
sensor–analyte interaction into measurable signal. We developed
a new generic technology for fluorescence response that may find
a broad range of applications – from homogeneous assays in
solutions to microarrays, microfluidic devices, nanosensors, and
whole-cell imaging. In sensing event, the novel functional dyes
change their color due to interplay of intensities between blue-
green and orange-red well-separated emission bands. Due to two-
channel ratiometric detection the signal is self-calibrating and
allowing easy quantitative assays. These dyes belong to the fam-
ily of 3-hydroxychromones (3HCs) exhibiting an excited-state
intramolecular proton transfer (ESIPT) reaction. By proper
chemical substitutions we endowed 3HCs the ability to
respond in a wavelength-ratiometric manner to all major types of
non-covalent interactions that can be used in sensing – to polar-
ity, hydrogen bonding ability and to electrostatic fields. Their
derivatives for covalent labeling of proteins and peptides were
synthesized. We present the data showing how with incorpor-
ation of new dyes the biomembrane modes such as micelles and
phospholipid vesicles can be transformed into nanosensor
devices. In cellular research a fast response to electrostatic mem-
brane potential can be achieved and the membrane changes
occurring on apoptosis detected.
Abstracts
29
4.8 Imaging and Noninvasive Techniques: MRI
S4.8-1
Use of intracortical recordings and fMRI to
study mass action
N. K. Logothetis
Department of Physiology of Cognitive Processes,
Max-Planck-Institute for Biological Cybernetics, Tuebingen,
Germany. E-mail: conchy.moya@tuebingen.mpg.de
The combination of fMRI with electrophysiology, histology, and
neurochemistry promises great insights into a level of neural
organization that could have never been studied with either tech-
nique alone. My talk will focus on (a) spatially resolved fMRI/
MRSI; (b) the study of in vivo connectivity using fMRI and elec-
trical microstimulation, or manganese-enhanced MRI; (c) com-
bined physiology and MRI for examining the electrical activity
occurring during increases and decreases of BOLD activation;
and (d) molecular imaging based on smart agents. MRSI optimi-
zation enabled sufficiently high spectral dispersion and spatiotem-
poral resolution to obtain isolated glutamate maps in the primate
brain. Ongoing research attempts the differentiation of brain
structures in the millimeter range and/or detection of small con-
centration differences in the same structure (activated versus non-
activated cortex). Finally, I will report our first attempt to
develop chelates for Gd and endow them with the appropriate
coordinating groups that reversibly block some of the Gd’s free
coordination sites with changes in pH or [Ca++].
S4.8-2
Functional and molecular assessment of
breast cancer using high field MRI and MRS
M. Garwood
Radiology/CMRR, University of Minnesota, Minneapolis, MN,
USA. E-mail: gar@cmrr.umn.edu
The techniques of magnetic resonance spectroscopy (MRS) and
imaging (MRI) can be used to assess molecular, functional, and
anatomical features of breast lesions in patients non-invasively.
MRI scanning of the breast following intravenous injection of a
contrast-enhancing agent such as Gd-DTPA is useful for deter-
mining the presence or absence of a breast lesion, and provides
information about lesion morphology, blood flow, and vascular
properties. In the same examination, in vivo proton (1H) MRS
can be used to obtain molecular information about the lesion(s).
Resonances from choline-containing compounds are commonly
observed in 1H spectra of malignant lesions, but not in benign or
normal tissues. The primary constituents are those with a tri-
methylamine moiety, including free choline, phosphocholine, and
glycerophosphocholine. Used in combination, these MR tech-
niques are capable of providing information to make an accurate
non-invasive diagnosis and to identify a clinical response early in
the course of cancer therapy. Current methods and technical
issues in performing MRS and MRI of breast at high magnetic
field (4 Tesla) will also be discussed.
S4.8-3
MRI-guided gene therapy
E. Atalar
1
and X. Yang
2
1
Electrical and Electronics Engineering, Bilkent University,
Ankara, Turkey,
2
Department of Radiology, Johns Hopkins
University, Baltimore, MD, USA. E-mail: ergin@ee.bilkent.edu.tr
Gene therapy is designed to target the causes rather than the
symptoms of diseases. Our current knowledge about the biodis-
tribution and pharmacokinetics of gene therapy relies mainly on
results of in vitro laboratory examinations of tissues obtained at
biopsy or autopsy. In many cases, actual distribution of gene
materials is unknown. Thus, it is essential to develop non-inva-
sive methods to monitor and guide gene therapy.
To date, different imaging modalities, such as ultrasound, nuclear
medicine, and optical imaging, have been applied in monitoring
of gene therapy. Magnetic resonance imaging (MRI) has several
prominent advantages comparing to other imaging techniques,
including the abilities to generate high-contrast and high-resolu-
tion images, to obtain multiple diagnostic evaluations of organ
functions and morphology, and to provide multiple image planes
with no risk of ionizing radiation. Recent efforts have focused on
using MR technology to monitor gene delivery, to enhance gene
transfection/transduction, and to track gene expression. This
review summarizes the current status of MRI-guided gene ther-
apy.
Abstracts
30
. Diagnostic Approaches
4.1 Bioinformatics and Proteomics
S4.1-1
Strategies to cover microbial proteomes
P. R. Jungblut
Core Facility Protein Analysis, Max. also determined by non-
genetic factors (three). The extent to which genetic factors con-
tribute to the drug response/toxicity phenotype depends on the
extent