Materials of Conference of Young Scientists Dedicated to the 35th Anniversary of Institute of Molecular Biology and Genetics of the National Academy of Sciences of Ukraine
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Інститут молекулярної біології і генетики НАН України
2008
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nasplib_isofts_kiev_ua-123456789-1577382025-02-23T17:56:45Z Materials of Conference of Young Scientists Dedicated to the 35th Anniversary of Institute of Molecular Biology and Genetics of the National Academy of Sciences of Ukraine Материалы конференции молодых ученых посвященной 35-летию Института молекулярной биологии и генетики Национальной академии наук Украины Матеріали конференції молодих вчених присвяченій 35-річчю Інституту молекулярної біології і генетики Національної академії наук України Хроніка та інформація 2008 Article Материалы конференции молодых ученых посвященной 35-летию Института молекулярной биологии и генетики Национальной академии наук Украины // Біополімери і клітина. — 2008. — Т. 24, № 5. — С. 349-367. — англ. 0233-7657 https://nasplib.isofts.kiev.ua/handle/123456789/157738 uk Біополімери і клітина application/pdf Інститут молекулярної біології і генетики НАН України |
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Хроніка та інформація Хроніка та інформація Materials of Conference of Young Scientists Dedicated to the 35th Anniversary of Institute of Molecular Biology and Genetics of the National Academy of Sciences of Ukraine Біополімери і клітина |
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Materials of Conference of Young Scientists Dedicated to the 35th Anniversary of Institute of Molecular Biology and Genetics of the National Academy of Sciences of Ukraine |
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Materials of Conference of Young Scientists Dedicated to the 35th Anniversary of Institute of Molecular Biology and Genetics of the National Academy of Sciences of Ukraine |
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Materials of Conference of Young Scientists Dedicated to the 35th Anniversary of Institute of Molecular Biology and Genetics of the National Academy of Sciences of Ukraine |
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Materials of Conference of Young Scientists Dedicated to the 35th Anniversary of Institute of Molecular Biology and Genetics of the National Academy of Sciences of Ukraine |
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Materials of Conference of Young Scientists Dedicated to the 35th Anniversary of Institute of Molecular Biology and Genetics of the National Academy of Sciences of Ukraine |
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materials of conference of young scientists dedicated to the 35th anniversary of institute of molecular biology and genetics of the national academy of sciences of ukraine |
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Інститут молекулярної біології і генетики НАН України |
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2008 |
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Хроніка та інформація |
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Материалы конференции молодых ученых посвященной 35-летию Института молекулярной биологии и генетики Национальной академии наук Украины // Біополімери і клітина. — 2008. — Т. 24, № 5. — С. 349-367. — англ. |
| series |
Біополімери і клітина |
| first_indexed |
2025-11-24T04:45:01Z |
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2025-11-24T04:45:01Z |
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1849645598131617792 |
| fulltext |
349
CONFERENCE OF YOUNG SCIENTISTS
N A T I O N A L A C A D E M Y O F S C I E N C E S O F U K R A I N E
Conference of Young Scientists
Dedicated to the 35th Anniversary of
Institute of Molecular Biology and Genetics
of NAS of Ukraine
Collection of Theses
C O N T E N T S
BOYARSHIN K. S., YAREMCHUK A. D., TUKALO Ì. À. Study on the structural basis of the prokaryote-type prolyl-tRNA synthetase from
Enterococcus faecalis editing activity by the methods of site directed mutagenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 351
CHEKANOV M. A., SYNYUGIN A. R., LUKASHOV S. S., YARMOLUK S. M. Synthesis of 2-phenylisothiazolidin-3-one 1,1-dioxides as
inhibitors of human protein kinase CK2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 352
FESAY O. A. Aassociation between cag repeats number in the exon 1 of androgen receptor gene and impaired spermatogenesis . . . . . . . . . . . 353
GORBENKO O. M., OVCHARENKO G. V., VOLKOVA D. D., MAYILO D. S., FILONENKO V. V., GOUT I. T. Production and
characterization of monoclonal antibody specific to Fibroblast Growth Factor Receptor 1 (FGFR1) . . . . . . . . . . . . . . . . . . . . . . . . . . 354
GRYSHKOVA V. S., LITUEV D. S., FILONENKO V. V., KIYAMOVA R. G. The model for the investigation of novel ovarian cancer marker –
sodium-dependent cotransporter NaPi2b . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 355
KOTEY I. M., BALANDA A. O., LUKASHOV S. S., YARMOLUK S. M. Thienopyrimidinone derivatives as a new class of protein kinase CK2
inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 356
LIVSHYTS G. B., PODLESNAJA S. S., KRAVCHENKO S. A. FSH receptor and INHa1 genes association analysis with diminished ovarian
reserve in women . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 357
MARTSENYUK O., HUPPERTZ B., SIWETZ M., MISLANOVÀ C., OBOLENSKAYA M. Cytotoxicity of homocysteine and its transsulfuration
pathway in human placenta . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 358
NOVOKHATSKA O., TSYBA L., SKRYPKINA I., NIKOLAIENKO O., DERGAI O., RYNDITCH A. ITSN adaptor protein family:specificity of
partners interaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 359
SLONCHAK A., MARTSENYUK O., WIDLAK P., RZESZOWSKA-WOLNY J., OBOLENSKAYA M. Interactions of human glutathione
S-transferase P1 promoter with placental nuclear proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 360
SOLDATKIN Î. Î. Optimization of bioselective elements operation of multibiosensor for toxins detection . . . . . . . . . . . . . . . . . . . . . . 361
SOLOVYOV O. O., LIVSHITS L. A. Application of Real-Time PCR for detection of mutations . . . . . . . . . . . . . . . . . . . . . . . . . . . 362
SYNYUGIN A. R., CHEKANOV M. A., LUKASHOV S. S., YARMOLUK S. M. Design of protein kinase CK2 inhibitors based on the p-quinone
arylsulfonelimides scaffold . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 363
TOKOVENKO B. T., GOLDA R. Ya., PROTAS O. M., OBOLENSKAYA M. Yu. COTRASIF: genomics tool for systems biology . . . . . . . . 364
TWARDOVSKA M. O., KONVALIUK I. I., STRASHNIUK N. M., BUBLYK O. M., MEL’NYK V. M. Interspecies polymorphism of gentiana l
genus. Members: the results of RAPD-analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 365
VEREMIEVA M., NEGRUTSKII B. Investigation of the expression of multisubunit translation elongation factor 1 in human carcinomas . . . . . 366
VOLKOVA K. D., KOVALSKA V. B., SUBRAMANIAM V., YARMOLUK S. M. Characterization of complexes of T-284 and SH-516 cyanine
dyes with alpha-synuclein fibrils . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 367
350
ISSN 0233-7657. Á³îïîë³ìåðè ³ êë³òèíà. 2008. Ò. 24. ¹ 5
351
CONFERENCE OF YOUNG SCIENTISTS
Study on the structural basis of the prokaryote-type
prolyl-tRNA synthetase from Enterococcus faecalis
editing activity by the methods of site directed
mutagenesis
K. S. Boyarshin, A. D. Yaremchuk, Ì. À. Tukalo
Institute of Molecular Biology and Genetics of National Academy of Science of Ukraine
kboyarshin@mail.ru
An insertion domain of the most of prokaryotic
prolyl-tRNA synthetases (ProRS) exhibits
post-transfer editing activity. The mechanism of
tRNA-dependent editing by ProRS has to be defined.
The present work is aimed to study the structure of
the active site of enterobacteria E. faecalis ProRS
(ProRSEF) editing domain. On the grounds of the
putative structure of the editing domain active center,
based on the ProRSEF crystal structure and computer
modeling data [1], following amino acids positions
have been chosen for the site directed mutagenesis
(alanine scanning): E218, T257, K279, G331, S332,
G334, H366. In the ProRSEF gene, cloned previously,
mutations have been obtained by QuickChange®
method (Stratagene). Mutant genes have been checked
by sequencing. Mutant proteins were overexpressed in
Escherichia coli, BL-21 Star strain. The purification
procedure included graded salting-out, chroma-
tography on DEAE-sepharose and chromatography on
Toyopearl HW-60. The purity of the proteins was
checked by OD
260/280
ratio and by electrophoresis in
PAAG, and in all cases was over 95 %. The
aminoacylation activity was determined for all
ProRSEF mutants. To check editing activity of the
ProRSEF mutant forms by alanyl-tRNA hydrolysis, a
chimeric tRNA, cognate for both prolyl- and
alanyl-tRNA synthetases, was created. Determination
of the mutant forms editing activity allowed to reveal
next amino acid residues, important for the editing
process: K279, G331, H366. The comparison of these
data with structural data enabled us to suppose that
K279 is involved in substrate positioning in the editing
active center, G331 takes part in putative catalytic
water molecule binding and activating, and H366
maintains G331 in the right conformation. The
existence of a bound water molecule in the editing
active center was predicted by computer modeling.
In summary, seven mutant forms of ProRSEF with
mutations in the editing domain have been obtained,
and their editing activity has been compared with the
wild type of ProRSEF. Three amino acid residues,
important for the editing activity, K279, G331 and
H366, have been revealed. The hypothesis is proposed,
that the water molecule, hydrolyzing alanyl-tRNAPro, is
positioned in the active center of the ProRS editing
domain.
1. Crepin T., Yaremchuk A., Tukalo M., Cusack S.
(2006) Structure, 14, 1511– 1525.
352
COLLECTION OF THESES
Synthesis of 2-phenylisothiazolidin-3-one 1,1-dioxides as
inhibitors of human protein kinase CK2
M. A. Chekanov, A. R. Synyugin, S. S. Lukashov, S. M. Yarmoluk
Institute of Molecular Biology and Genetics of National Academy of Science of Ukraine
chekanov-maxx@ukr.net
Protein kinase CK2 (Casein Kinase 2) is an
extremely pleiotropic Ser/Thr kinase with high
constitutive activity. The observation of CK2
deregulations in various pathological processes
suggests that CK2 inhibitors have a therapeutic
value, particularly as anti-neoplastic and antiviral
drugs.
We have developed series of 2-phenyliso-
thiazolidin-3-one 1,1-dioxides as new class of
compounds with high CK2 inhibitory activity. This
class of inhibitors was identified by high-throughput
docking of the virtual library of compounds in the
ATP-binding site of human CK2. Synthesis of the
compound series was carried out using combinatorial
synthesis techniques, such as parallel synthesis in
wheaton sample vials with product isolation
bycentrifugation. Series of 55 compounds were tested
in vitro for CK2 inhibitory activity. It was found that
N-(3-acetylphenyl)-2-chloro-4-(4-methyl-1,1-dioxido-
3-oxoisothiazolidin-2-yl)benzamide has IC
50
value of
20 mM. To perform further structure optimization,
another 7 compounds of this class were synthesized.
Five of them showed IC
50
value less than 20 mM. The
most active compound is 4-{[2-chloro-4-(1,1-dioxi-
do-3-oxoisothiazolidin-2-yl)benzoyl]amino}-2-hydro-
xybenzoic acid with IC
50
value of 3 mM.
Thus, we suppose that the represented compounds
are promising class of novel CK2 inhibitors. To obtain
more active compounds of this class, we plan to
continue QSAR modeling and structure optimization of
the 2-phenylisothiazolidin-3-one 1,1-dioxide scaffold.
353
CONFERENCE OF YOUNG SCIENTISTS
Association between CAG repeats number
in the exon 1 of androgen receptor gene
and impaired spermatogenesis
O. A. Fesai
Institute of Molecular Biology and Genetics of National Academy of Science of Ukraine
olga_fesay@ukr.net
Androgens are required for male sex determination,
development and spermatogenesis. The androgen
activity is mediated by an androgen receptor. The
androgen receptor (AR) gene is located on
chromosome Xq11-12. The AR gene has a repetitive
DNA sequence in the exon 1 that encodes a
polyglutamine tract. Within the normal polymorphic
range this (CAG)
n
tract length is inversely related to
the transcriptional activity of the AR gene.
The aim of our research was to study the association
between CAG repeats number in the exon 1 of AR gene
and impaired spermatogenesis.
DNA isolated from blood samples of 158 infertile
males (with azoospermia and oligozoospermia) was
amplified by PCR targeting the AR (CAG)
n
tract. DNA
isolated from blood samples of 124 fertile males served
as a control. A fragment analysis of Cy5-labeled PCR
products on an automated DNA analyzer «A. L. F.-
express» was used to calculate the CAG repeats
number. To determine correct number of the CAG re-
peats three different alleles were sequenced using ABI
3130 Genetic Analyzer. The nomenclature of alleles in
our study corresponds to the CAG repeats number.
The CAG repeats number of AR gene in the infertile
male group was more widely distributed than in control
croup. There was a significant difference in CAG
alleles in the infertile males versus controls (p = 0.023).
Short alleles contained 7 CAG repeats were detected
only in males with azoospermia. Long alleles contained
32 and 33 CAG repeats were detected only in males
with oligozoospermia. These alleles were not found in
control group. Severely oligozoospermic males had
longer CAG repeat tract than azoospermic males. The
results of our study allow to suppose that short CAG
repeats can lead to disease of androgen-dependent
tissues. On the other hand, males with longer alleles of
AR gene within the normal range of CAG repeats may
have decreased AR function that results in reduced
spermatogenesis.
Thus, the molecular-genetic analysis of the CAG
repeats number in AR gene as well as genetic
counseling are very important for patients with male
infertility, especially if they are included in an assisted
reproductive technologies program.
354
COLLECTION OF THESES
Production and characterization of monoclonal antibody
specific to Fibroblast Growth Factor Receptor 1
(FGFR1)
O. M. Gorbenko1, G. V. Ovcharenko1, D. D. Volkova1, D. S. Mayilo1,2,
V. V. Filonenko1, I. T. Gout1,3
1Institute of Molecular Biology and Genetics of National Academy of Science of Ukraine
2Taras Shevchenko National University of Kyiv
3University College London, Gower Street, London, WC1E 6BT, UK
termite@ukr.net
Fibroblast growth factor receptor 1 is a
transmembrane receptor activated by FGF-1 and by
related FGF-2. It belongs to the large class of
tyrosine kinase receptors.
In this study, we report the production and
characterization of monoclonal antibody against FGF-1
receptor. The antigen for mouse immunization has been
chosen by Bcepred software. Chosen sequence
encoding loop II-III for FGFR1 extra cellular domain
was cloned into pGEX4T1 and pET42a vectors and
expressed in bacteria. Recombinant proteins were
purified by electroelution from gel and by NiNTA
affinity chromatography. GST-loopII-IIIwas used as
antigen for mouse immunization whereas
His-GST-loopII-III was used in primary hybridoma
screening by ELISA and Western blot. Primary
screening allowed us to select 14 positive clones, which
have been checked for their crossreactivity with
FGFR3wt. 10 clones showed no crossreactivity.
Among them there are two hybridoma clones, which
could specifically recognize endogenous FGFR1 in
Western blot of NIH3T3L1 cell lysates. The selected
positive clones were subcloned twice using a limiting
dilution method.
355
CONFERENCE OF YOUNG SCIENTISTS
The model for the investigation of novel ovarian cancer
marker – sodium-dependent cotransporter NaPi2b
V. S. Gryshkova, D. S. Lituev, V. V. Filonenko, R. G. Kiyamova
Institute of Molecular Biology and Genetics of National Academy of Science of Ukraine
vitalinochka@ukr.net
Ovarian cancer is the most common gynecologic
cancer that is usually far advanced before it is
diagnosed and thus patients have a poor prognosis
and survival rate. Identification and characterization
of novel ovarian cancer markers is important for
understanding the molecular mechanisms of
malignant transformation and for the development of
novel diagnostic and immunotherapeutic approaches
in gynecologic oncology.
Recently, we have identified the sodium-dependent
phosphate transport protein 2b (NaPi2b) as a new
ovarian cancer antigen based on the immunoscreening
of ovarian cancer cell line (OVCAR3) cDNA library
with MX35 monoclonal antibodies obtained at Ludwig
Institute for Cancer Research by mice immunization
with ovarian carcinoma cells.
The human protein, NaPi2b encoded by SLC34A2
gene is involved in the homeostasis of inorganic
phosphate. NaÐi2b is a membrane protein with the NH
2
-
and COOH-termini located on the cytoplasmic side of
the membrane, 8 transmembrane domains and a large
extra cellular loop (188–361 aa). Previously, we have
generated monoclonal antibodies (L2/20) against the
large extra cellular loop (188–361 aa) of NaÐi2b and
determined a region of the transporter molecule
thatincludes the epitope for these antibodies (311–340
aa) which happened to be the same as for the MX35
antibodies.
The present study was aimed to create a model for
the investigation of functional activity of a new marker
of ovarian cancer – sodium-dependent phosphate
transporter NaÐi2b, in malignant cells. The stable cell
lines expressing wild type and mutant forms of NaPi2b
were created by stable transfection of HEK293 cells.
We have chosen the mutations of NaPi2b that
according to the data base search could be potentially
associated with ovarian cancer. There are deletion of 6
aa in C-terminus and point mutation T330V in the extra
cellular loop of NaPi2b. The expression of NaPi2b by
stable cell lines was confirmed by Western-blot
analysis using tag-specific and anti-NaPi2b antibodies.
In addition we have shown that amino acid
substitution T330V resulted in loss of NaPi2b
recognition by both antibodies in Western-blot analysis
that could be explained by destruction of the epitope for
anti-NaPi2b antibody. The experimental system
described here will have strong implications for the
further investigation of the NaPi2b function in normal
and pathological states.
356
COLLECTION OF THESES
Thienopyrimidinone derivatives as a new class of protein
kinase CK2 inhibitors
I. M. Kotey, A. O. Balanda, S. S. Lukashov, S. M. Yarmoluk
Institute of Molecular Biology and Genetics of National Academy of Science of Ukraine
im_kotey@ukr.net
Signal transduction by phosphorylation of proteins is
an important mechanism that is responsible for the
most of physiological and pathological processes in
the cell. Protein phosphorylation is realized by
protein kinases – enzymes, which regulate such
essential events as cell growth and proliferation.
Enhanced activity of protein kinases is often
associated with a number of disorders, including
carcinogenesis, inflammation and viral infections.
Therefore, the creation of protein kinase inhibitors is
of significant value in modern drug discovery
industry.
Here we represent a novel class of inhibitors of
protein kinase CK2 – an enzyme playing, in particular,
crucial role in the suppression of cell apoptosis that is
one of the causes of tumor development. At the first
stage, we performed docking of virtual compound
library (20,000 compounds in total) using crystal
structure of the ATP-binding site of CK2. 100
thienopyrimidinone derivatives selected by docking
engine were synthesized. The synthesis was carried out
in the following way. 2-Amino-3-carbethoxy-
thiophenes were obtained by the Gewald reaction.
Their thermal condensation in formamide gave
3-H-thienopyrimid-4-ones that were further alkylated
in 3-position of pyrimidine moiety. Most of the
incorporated side chains contained carbalkoxy-groups
that were hydrolyzed into carboxylic acids.
At the next stage, the set of synthesized compounds
was tested in vitro towards CK2. The testing results
revealed that about one-third of the investigated
compounds displayed ²Ñ
50
values from 15 to 30 ìÌ.
Further structural optimization of the thienopyri-
midinone scaffold allowed toobtain more active deriva-
tive with ²Ñ
50
of 2.5 ìÌ.
Hence, using docking technique and organic
synthesis methodologies, a new class of CK2 inhibitors
was identified. These inhibitors may be of interest for
further structural optimization and evaluation of
biological activity.
357
CONFERENCE OF YOUNG SCIENTISTS
FSH receptor and INHa1 genes association analysis with
diminished ovarian reserve in women
G. B. Livshyts, S. S. Podlesnaja, S. A. Kravchenko
Institute of Molecular Biology and Genetics of National Academy of Science of Ukraine
livshits@imbg.org.ua
Objectives: Premature ovarian failure (POF) is a
secondary gonadotrophic amenorrhoea affecting 1–
3 % of females. FSH (follicle stimulation hormone)
and its receptor (FSHR) play a major role in the
development of follicles and regulation of
steriogenesis in the ovary. Mutations in the FSHR
might theoretically lead to an impaired signal
transduction and thereby to a diminished ovarian
reserve. Genes encoding the three inhibin subunits
can be proposed as candidates for POF due to its role
in the negative feedback control of FSH. We
investigate Asn680Ser and Thr307Ala transitions in
FSHR gene and Ala257Thr transition in INHa1 gene
as markers for diminished ovarian reserve in women:
with clinical POF diagnosis and women with poor
response (less than 4 oocytes after standard protocol
of FSH stimulation) – «poor responders».
Methods: The FSHR variants Asn680Ser and
Thr307Ala, and Ala257Thr transition in INHa1 gene
were analyzed by PCR and RFLP. Statistical analysis
was performed by c2 test, Fisher’s exact test, like-
lihood-ratio test and Expectation-Maximization
algorithm.
Results: The frequency of Ala307-Ser680 (AS)
allele of FSHR gene was significantly higher both in
POF group and in «poor responders» group comparing
to control group. The carriers of INHa1 gene Ala257Thr
transition predominated in the «poor responders» group.
Quantity of oocytes after stimulation ovulation in women
with INHa1 gene Ala257Thr transition was significantly
decreased in comparison to patients without such mutation.
Our data shows the prevalence of FSHR gene AS
allele in both patients groups: group of POF patients
(45.7 %) and «poor responders» group (52.8 %),
comparing to control group (35.1 %).
Conclusions: Our data about FSHR and INHa1
genotype association with ovarian reserve and response
to FSH represent that the best stimulation protocols can
based on the individual’s genetic profile in order to reduce
side-effects and costs and improve the delineation
stimulation protocols.
358
COLLECTION OF THESES
Cytotoxicity of homocysteine and its transsulfuration
pathway in human placenta
O. Martsenyuk1, B. Huppertz2, M. Siwetz2, C. Mislanovà3, M. Obolenskaya1
1Institute of Molecular Biology and Genetics, NAS of Ukraine
2Department of Cell Biology, Histology and Embryology, Medical University of Graz, 8010 Graz, Austria
3Slovak Medical University, 833 03 Bratislava, Slovak Republic
o.p.martsenyuk@imbg.org.ua
Background: Elevated level of homocysteine (Hcy)
increases the risk of developmental defects and
placenta-mediated diseases, such as preeclampsia,
spontaneous abortion, etc. The problem is whether
placenta has some metabolic resources to withstand
the Hcy loading. Our previous data have revealed the
unexpectedly high cysteine/glutathione (Cys/GSH)
ratio = 3:1 in human placenta contrasting with 1:6 in
maternal blood and 3:2 in embryonic liver. These
data together with relatively high correlation
between Hcy and Cys content (k
s
= 0.5, p < 0.001)
served as a basis to advance a hypothesis that
transsulfuration pathway via cystathionine-b-syn-
thase (CBS) is active in human placenta.
Here we aimed to analyze the influence of increased
concentration of Hcy supplemented with folic acid or
not on the proliferation in the villous explants and to
examine the presence of CBS in human placenta.
Methods: Placental explants (10–25 mg of villous
tissues) were obtained from normal term placentas
(38–40 weeks) and from abortive material (6–11
weeks). Explants were cultivated in DMEM/F12 in the
presence of 20, 40, 80 µM Hcy with or without 20 nM
folic acid. Cultures were run for 48 h at 37 °C and 5 %
CO
2
, 20 % oxygen. After cultivation the explants were
embedded in paraffin and immunohisto chemically
(IHC) stained for Ki-67, and cytokeratin 18-neo-
epitope (M30). Proliferation index was calculated as
the number of Ki-67 positive nuclei in cytotrophoblast
cells per 100 µm of the villous circumference. The
presence of CBS was examined by Western blot and
IHC analyses with corresponding antibodies.
Results: Proliferation index in the first trimester
explants is higher than in the term explants (3,8 vs. 0,6).
The cultivation with increasing concentrations of Hcy
(20, 40 and 80 µM) is followed by gradual decrease of
proliferation index (0.53, 0.48 and 0.4 in the term
explants and 3.3, 2.7 and 2.0 in the first trimester
explants). Higher concentrations of Hcy retard the
nuclei transition from cyto- to syncytiotrophoblast and
provoke apoptotic processes (higher M30 staining).
The addition of folic acid at the background of 40 and
80 µM Hcy slightly increases proliferation index till
2.96 and 2.20 in the first trimester explants and till 0.50
at 40 µM Hcy in the term placenta explants. The CBS
was detected in the first trimester and term placental
explants.
Conclusions: The increased level of Hcy decreases
the proliferation index in placenta explants, induces the
tissue destruction and apoptosis. The additional folic
acid slightly reverses the cytotoxic effect of Hcy. We
suppose that the elevated level of Hcy induces the
disbalance of methylation processes and DNA
synthesis. We expect that Hcy may be trans-sulfurated
in human placenta. A role of this pathway in the
placental physiology merits further investigation.
The investigations were supported by grant of
ÌÎÍ N 28-2008, INTAS YSF 06-1000014-5961.
359
CONFERENCE OF YOUNG SCIENTISTS
ITSN adaptor protein family: specificity of partners
interaction
O. Novokhatska, L. Tsyba, I. Skrypkina, O. Nikolaienko, O. Dergai, A. Rynditch
Institute of Molecular Biology and Genetics of National Academy of Science of Ukraine
olga.novokhatska@gmail.com
Human intersectins (ITSN 1 and 2) are members of a
conserved family of adaptor proteins encoded by two
paralogous genes. ITSN1 is known to participate in
multiple cellular processes including endocytosis,
mitogenic signaling, actin cytoskeleton rearrange-
ments and apoptosis while the function of ITSN2 is
to be elucidated. In this work we intended to examine
interaction of ITSN2 SH3 domains with protein part-
ners that could be common to ITSN1 or different. For
this purpose cDNAs encoding ITSN2 SH3 domains
(SH3A–E) were cloned into pGEX4T3 vector, prote-
ins were expressed, purified in E. coli system and us-
ed for in vitro analysis. Pull-down experiments were
carried out using mouse brain lysates (for dynamin 1
and SOS1 proteins) or lysates of MCF7 cells over-
expressing adaptor protein CIN85/Ruk and ubiquitin
ligase c-CBL.
Using GST pull-down assays we showed the inter-
action of ITSN2 SH3 domains with dynamin 1, mole-
cule thought to drive the endocytosis late events by in-
teracting with multiple endocytic proteins and phos-
pholipids. Dynamin 1 strongly bound to SH3 A, C and
with highest affinity to SH3E domain but not to SH3 B
and D. No significant difference was observed compa-
ring ITSN 1 and 2 SH3 domains interaction with dyna-
min 1. The ability of multiple SH3 domains in ITSN2 to
bind dynamin 1 suggests that ITSN2 can cluster several
dynamin molecules during endocytosis.
The interaction of ITSN1 SH3A domain with Ras
exchange factor SOS1 was reported. It is also known
that expression of ITSN1 is detected in both prolifera-
ting and differentiating neurons, while ITSN2 is mainly
expressed in the latter. Given that we analyzed ITSN2
participation in signal transduction mechanisms thro-
ugh SOS1 binding. We showed that ITSN2 SH3 A, C
and E domains interact with SOS1 but the affinity is
less when compared to that of ITSN1 SH3A domain.
The results obtained indicate the role for ITSN2 in
linking endocytosis and signal transduction pathways.
Considerable differences were observed while
examining ITSN2 interactions with ubiquitin ligase
c-CBL and adaptor protein CIN85/Ruk. Comparing to
ITSN1 only two of ITSN2 SH3 domains (SH3 C and E)
were involved in binding to c-CBL in vitro. It is worth-
while to mention that SH3A domain is the most diver-
gent one when ITSN 1 and 2 are compared which might
impose differences in its ability of binding partners.
The results of pull-down assays showed that only
SH3A domain of ITSN2 binds to CIN85/Ruk with low
affinity. This allowed us to suggest that interaction of
ITSN2 with CIN85/Ruk does not occur in vivo since
there is an intramolecular interaction of CIN85/Ruk
own SH3A domain with its own proline-rich region.
Using in silico prediction we identified new ITSN
interacting partner semaphorin 6A (Sema6A) implica-
ted in retrograde signaling and cytoskeletal rearrange-
ments during neuro- and organogenesis. The interacti-
on of Sema6A with ITSN is mediated by SH3A domain
of ITSN2 and SH3 A, C and E domains of ITSN1.
In this study we first demonstrated in vitro interac-
tion of ITSN2 with endocytic GTPase dynamin 1, gua-
nine nucleotide exchange factor for Ras SOS1, ubiqui-
tin ligase c-CBL, adaptor protein CIN85/Ruk, new in-
tersectins interactor Sema6A and showed differences
in binding properties of ITSN 1 and 2 with these
proteins.
This work was supported by INTAS Ref. Nr 05-
1000004-7762 and grant of NASU for young
scientists.
360
COLLECTION OF THESES
Interactions of human glutathione S-transferase P1
promoter with placental nuclear proteins
A. Slonchak, O. Martsenyuk, P. Widlak, J. Rzeszowska-Wolny, M. Obolenskaya
Institute of Molecular Biology and Genetics of National Academy of Science of Ukraine
elephass@gmail.com
Background: GSTP1 is a phase II detoxification
enzyme, which participates also in intracellular
signal transduction, molecular transport and NO
storage. Down-regulation of GSTP1 expression in
human placenta is associated with pregnancy
disorders. The goal of this work was to recognize
how GSTP1 promoter realizes its regulatory function
in human placenta. We investigated the level of
promoter methylation and the interactions of its
binding sites with placental nuclear proteins.
Materials and methods: DNA from placenta was
purified, treated with bisulphate and the level of
methylation was assessed by methylation-specific
PCR. The DNA-microarray database was screened for
the placental expression of transcription factors, known
to regulate this gene in tumors. The region of human
GSTP1 promoter was PCR-amplified and cloned. After
excision from plasmid it was end-labeled with
g-32P-ATP and used in competitive EMSA with
placental nuclear extracts. Synthetic oligonucleotides
corresponding to ARE, iARE, NF-kB-like, NF-kB,
CRE and GATA-binding sites were annealed,
end-labeled with g-32P-ATP and used in EMSA with
placental nuclear extracts and competitive
oligonucleotides corresponding to the consensus
sequences for AP-1, Mafs, ERb, RARa, NF-kB,
CREB and GATA transcription factors.
Results: The computer analysis revealed the
specific pattern of transcription factors expression in
human placenta, represented by highly expressed
GATA2, GATA3, Fos-B, Nrf3 and MafK, and
moderately expressed c-Fos, Juns, Mafs, ERb, RARa
and NF-kB. Competitive EMSA with promoter
fragment provided the evidence that ARE and
NF-kB-like sites are involved in the interactions with
placental nuclear proteins. Using EMSA with synthetic
oligonucleotides we revealed that ERb and Maf
proteins, but not AP-1 and RAR, interact with GSTP1
ARE and inner ARE elements, NF-kB factor binds to
NF-kB and NF-kB-like elements, and GATA proteins
to GATA element.
Conclusions: Binding of ERb, Maf, NF-kB and
GATA proteins to the corresponding binding sites of
promoter, but not CpG methylation is responsible for
the regulation of GSTP1 transcription in human
placenta. We suppose that GSTP1 in human placenta is
a responsive gene for estrogens and intracellular redox
state.
361
CONFERENCE OF YOUNG SCIENTISTS
Optimization of bioselective elements operation of
multibiosensor for toxins detection
Î. Î. Soldatkin
Institute of Molecular Biology and Genetics of National Academy of Science of Ukraine
alex_sold@yahoo.com
The investigation presents the development of
highly sensitive and selective multibiosensor based
both on a number of immobilized enzymes as
bioselective elements and the matrix of ion-selective
field effect transistors as transducers of biochemical
signal into the electric one. To develop bioselective
elements of multibiosensor, such enzymes as
acetylcholinesterase, butyrylcholin esterase, urease,
glucose oxidase, and three-enzyme system
(invertase, mutarotase, glucose oxidase) were used.
Obtained bioselective elements were shown to
demonstrate high sensitivity to corresponding
substrates in direct enzymatic analysis, which lasted
10 min. Dynamic range of substrate
determination (0.1 mM–1.5–10 mM) was shown to
depend on enzymatic system and to differ
specifically in upper threshold. Current work
presents the investigation on the dependence of
multibiosensor response to pH, ionic strength and
buffer capacity of the solution; optimal conditions
for simultaneous operation of all bioselective
elements of the multibiosensor were selected; the
data on cross-influence of substrate of all enzymes
used were obtained. The developed multi-analyzer
was shown to demonstrate sufficient signal
reproducibility. Therefore multibiosensor turned out
to be suitable for real sample analysis.
362
COLLECTION OF THESES
Application of Real-Time PCR for detection of
mutations
O. O. Solovyov, L. A. Livshits
Institute of Molecular Biology and Genetics of National Academy of Science of Ukraine
fancyflight@yardex.ru
The traditional methods for detection of mutations
such as restriction analysis, SSCP, DGGE, direct
sequencing are labour-intensive and time-con-
suming. The use of Real-Time PCR techniques can
essentially shorten the time of analysis and avoid
contamination of probes that is very important for
molecular-genetic diagnostics. Our aim was to
develop a closed-tube system for the scanning of
mutations using Real-Time PCR techniques: melting
analysis of amplicons and allele-specific Real-Time
PCR. We studied the 3 bp deletion dF508 in exon 10
of CFTR gene using melting analysis. We designed
specific oligonucleotide primers and optimized the
PCR conditions for obtaining amplification product
of 44 bp for the DNA sample without deletion and 41
bp for the sample with dF508. It was shown that
SYBR Green I dye is stabilizing the DNA duplex,
thereby increasing the melting temperature of the
complex. SYBR Green I dye is supplied with the
concentration 10000 .́ We determined that the
optimal dye concentration was 5 .́ The difference in
the T
m
between the normal sample
and the sample with dF508 was 0.9–1 îC. The
samples were checked by heteroduplex analysis to
confirm the presence of dF508. We have also studied
deletions/insertions in genes BRCA1/2 and CHEK2 –
the susceptibility genes for hereditary breast cancer:
BRCA1 5382insC, BRCA1 185delAG, BRCA1
4153delA, BRCA2 6174delT, CHEK2 1100delC
using the allele-specific Real-Time PCR. This
method allows discriminating heterozygous carriers
of mutations using two pairs of primers, one of which
is homologous to normal DNA sample and the other
one – to the DNA with mutation. We designed spe-
cific oligonucleotide primers and optimized the PCR
conditions for amplification with SYBR Green I. We
used samples with abovementioned mutations as
positive controls in our study. Thus, we believe that
the developed assays for detection of mutations
using melting analysis and allele-specific Real-Time
PCR in combination with direct sequencing can be
used for molecular-genetic diagnostics and scre-
ening programmes.
363
CONFERENCE OF YOUNG SCIENTISTS
Design of protein kinase CK2 inhibitors based on the
p-quinone arylsulfonelimides scaffold
A. R. Synyugin, M. A. Chekanov, S. S. Lukashov, S. M. Yarmoluk
Institute of Molecular Biology and Genetics of National Academy of Science of Ukraine
drumms@mail.ru
During recent decade the inhibitors of protein
kinases draw great attention as anticancer and
antiviral drug candidates. The protein kinase CK2
(casein kinase 2, CK2) is known to show enhanced
activity in wide range of tumors, inhibit cell
apoptosis and is used by many viruses for their own
protein phosphorylation. Therefore, the inhibitors of
CK2 are considered prospective for pharmacological
application.
We developed a novel class of CK2 protein kinase
inhibitors – halogen derivatives of p-quinone
arylsulfonelimides. Flexible docking in ATP-binding
site of CK2 was used for selection of 50 compounds
from virtual library of about 1000 p-quinone
arylsulfonelimide derivatives. About 30 compounds
from selected ones were synthesized and tested in vitro
against CK2. Some of them showed high inhibitory
activity (²Ñ
50
from 0.5 to 20 mM).
Starting p-arylsulfonamidophenols were prepared
via reaction of arylsulfochlorides with p-aminophenol
derivatives in presence of triethylamine or sodium
hydrocarbonate. Oxidation of p-arylsulfonamidophe-
nols to p-quinone arylsulfonelimides was carried out
using potassium bichromate in 20 % sulphuric acid or
lead tetraacetate. Halogenation of quinoneimine
moiety was conducted by action of corresponding
hydrogen halogenides. This reaction was accompanied
by reduction of quinoneimide ring into amidophenole
that was oxidated again under the same reaction
conditions. The resulted halogen derivatives of
p-quinone arylsulfonelimides were tested in vitro using
g-labeled ATP.
The halogen derivatives of p-quinone
arylsulfonelimides with two or more halogen atoms
substituted in quinoneimide moiety showed higher
inhibitory activity. Substituents in arylsulfonyl moiety
have lesser influence on the inhibitory activity. Further
optimization of this scaffold will be performed.
364
COLLECTION OF THESES
COTRASIF: genomics tool for systems biology
B. T. Tokovenko, R. Ya. Golda, O. M. Protas, M. Yu. Obolenskaya
Institute of Molecular Biology and Genetics of National Academy of Science of Ukraine
b.t.tokovenko@imbg.org.ua
Summary: A new tool has been developed
(COTRASIF, conservation-aided transcription
factor binding site finder) for the genome-wide
identification of the putative transcription factor
binding sites (TFBS) in eukaryotic gene promoters.
Motivation and aim: Promoter analysis and TFBS
identification are essential for the understanding of
gene regulatory networks. Increasing specificity of the
TFBS prediction in eukaryotic gene promoters is a
challenging task for bioinformatics.
Based on our previous research, we observed better
specificity of the TFBS search when comparing
promoters of orthologous genes of the evolutionary
close species (e. g. rat and mouse).
Our aim was to develop an easy-to-use web-tool for
genome-wide identification of putative TFBS with
enhanced results quality.
Methods and algorithms: COTRASIF is built upon
the semi-automatic importer of promoters from the
Ensembl genome annotation database. Currently
COTRASIF has 11 genomes available (including the
human, rat, and mouse genomes).
Promoters are defined as 800 bp upstream from
transcription start site, plus the 5' UTR coding se-
quence. For the initial TFBS search, either classical
position-weight matrix (PWM) approach or the
recently developed HMM-based (hidden Markov
models) search method were used, For PWM method,
frequency matrices are needed as input; for HMM – a
list of at least 3 known sequences of the TFBS, plus an
optional position frequency matrix.
Initial search results can be further analyzed using
the built-in gene orthology filter. The orthology
information is automatically obtained from the
Ensembl Compara genome alignments database. If the
putative TFBS is present in the promoters of the genes
of both orthologous genes being analyzed, then it has
higher probability of being functional.
Results: We developed a web-accessible tool
(conservation-aided transcription factor binding site
finder, COTRASIF) for the genome-wide conser-
vation-aided TFBS search.
Further development includes: addition of new
genomes; integration of the Gene Ontology category
enrichment functional analysis (hypergeometric and
Bayesian); more formats of results output; specialized
web-API (application programming interface) for
enabling easy use of COTRASIF by other tools.
Availability: COTRASIF is freely available at
http://biomed.org.ua/COTRASIF/.
365
CONFERENCE OF YOUNG SCIENTISTS
Interspecies polymorphism of gentiana l genus.
Members: the results of RAPD-analysis
M. O. Twardovska1, I. I. Konvaliuk 1, N. M. Strashniuk2, O. M. Bublyk1,
V. M. Mel’nyk1
1Institute of Molecular Biology and Genetics NAS of Ukraine
2Ternopil National Volodymyr Hnatiuk Pedagogical University
twardovska 06@mail.ru
Gentiana L. – the biggest genus of the family
Gentianaceae Juss. (~ 400 species) which is pre-
sented by 10 species in Ukraine. The genus scope and
classification are treated differently by various
authors due to both insufficient characterizations of
Gentiana species and problems with the genus
boundaries determination. As many problems
concerning the systematics and evolution of
Gentiana L. genus are still unsolved the genetic and
molecular analysis of gentians is of special
importance.
The aim of the work was to study gentians
interspecies variability by means of the RAPD-PCR
technique.
To perform a genetic analysis of the intact plants
G. lutea, G. punctata, G. acaulis, G. asclepiadea,
G. cruciata, G. pneumonanthe and G. verna 27
decamer RAPD-primers were used. Following
preliminary screening there were chosen 19 primers,
which generated unique amplification 1–14 fragments
for each object within the range of 2.7–0.3 kb. Overall,
it was taken into account 460 fragments. Polymorphism
of RAPD-spectra is proved by presence or absence of
individual amplicons and varying intensity of
fluorescence for some of the homologous fragments.
The results of the RAPD-analysis revealed the
gentians interspecies polymorphism. We failed to
disclose any amplicon shared by all the objects
involved. The only fragment of 720 bp being amplified
with À01 primer was shared by each species except for
G. verna. G. punctata showed much higher number of
this amplicon as compared with the other studied
species.
On the whole, RAPD-analysis demonstrated some
peculiarities of G. verna which according to the
electrophoretic profiles of amplification products
differs substantially from the rest of species. The
nearest to G. lutea appeared to be G. punctata species,
to G. asclepiadea – G. pneumonanthe. According to Ho
and Liu classification (1990) these pairs of species are
included into Gent³ana ànd Pneumonanthe sections,
respectively.
Thus, genetic investigations of G. lutea, G. pun-
ctata, G. acaulis, G. asclepiadea, G. cruciata, G. pneu-
monanthe and G. verna, performed by the RAPD-PCR
technique, revealed the interspecies polymorphism for
these gentians G. lutea and G. punctata, as well as
G. asclepiadea and G. pneumonanthe are the most
similar judging from their electrophoretic profiles.
These results are in accordance with the literature data
(Ho, Liu, 1990; Yuan, 1996) relative to the systematic
and evolution of these species within the Gent³ana L.
genus.
366
COLLECTION OF THESES
Investigation of the expression of multisubunit
translation elongation factor 1 in human carcinomas
M. Veremieva, B. Negrutskii
Institute of Molecular Biology and Genetics of National Academy of Science of Ukraine
vermarina@list.ru
Translation elongation factor 1 (eEF1) regulates the
specific interaction of aminoacyl-tRNA with the
ribosome during the elongation phase of protein
biosynthesis. It is comprised of four subunits: eEF1A
(eEF1A1 and eEF1A2 isoforms), eEF1Ba, eEF1Bb
and eEF1Bg.
Previously, eEF1 was demonstrated to be related to
carcinogenesis. The eEF1A1 mRNA is overexpressed
in malignant tissues of pancreas, colon, breast, lung and
oesophagus. The eEF1A2 mRNA is overexpressed in
ovary and breast cancer. The overexpression of the
eEF1Bb and eEF1Bg mRNAs was also found in certain
types of human cancer. No data exist concerning the
expression of different eEF1 subunits at the protein
level.
In our studies, the expression of different subunits
of eEF1 in samples of cardioesophageal carcinoma
(CEC), renal cell carcinoma (RCC) and non-small cell
lung carcinoma (NSCLC) was studied at the level of
protein. Since the only available commercial antibodies
for eEF1 were anti-eEF1A1/2 antibodies, we obtained
the polyclonal antibodies for eEF1Ba, eEF1Bb and
eEF1Bg, as well as antipeptide polyclonal antibodies
specific exclusively for eEF1A2. Cytosolic proteins
were extracted from the tissues and examined by
Western blot analysis.
Our preliminary data show 1.5 to 2-fold increase in
the eEF1A expression in CEC and NSCLC.
Importantly, no corresponding changes in the amount
of eEF1Ba have been observed.
CONFERENCE OF YOUNG SCIENTISTS
367
Characterization of complexes of T-284 and SH-516
cyanine dyes with alpha-synuclein fibrils
K. D. Volkova1, V. B. Kovalska1, V. Subramaniam2, S. M. Yarmoluk1
1Institute of Molecular Biology and Genetics, NAS of Ukraine
2MESA + Institute for Nanotechnology, University of Twente, The Netherlands
volkova_katya@ukr.net
Parkinson’s disease and other related disorders are
characterized by the accumulation of fibrillar
aggregates of a-synuclein (ASN) inside brain cells.
Recently we have firstly proposed fluorescent
cyanine dyes for specific detection of amyloid
formations. It was shown that benzothiazole mono-
(T-284) and trimethinecyanines (SH-516) bind
fibrillar ASN with significant enhancement of the
emission intensity [1].
This study aimed to characterize dye/ASN fibril
complexes by means of fluorescence lifetime analysis
and atomic force microscopy. The ability of T-284 and
SH-516 dyes to selectively recognize amyloid proteins
of various amino acid compositions and to monitor the
kinetics of proteins fibrillogenesis process was also,
studied. Some prospects towards practical application
of these dyes were revealed.
Total lifetime value for the free Ò-284 dye in
aqueous solution was about 0.25 ns, while in the
presence of fibrillar ASN it increased almost in order of
magnitude up to 2.3 ns. Upon interaction with ag-
gregated ASN in fluorescence decay of T-284 the
additional slow component appeared, which may be
related to protein-embedded dye molecules.
Studies on the dyes selectivity were carried out on
various fibrillar proteins, namely insulin, lysozyme,
wild-type ASN and the known Parkinson
disease-related ASN mutants A30P and A53T.
It was found, that both dyes exhibit fluorescence
response in the presence of fibrillar proteins species,
comparable to that of the classic amyloid stain
Thioflavin T. T-284 appeared to be somewhat more
specific to fibrillar wild-type ASN and A53T mutant.
On the other hand, for trimethine SH-516 the least
protein-to-protein variability was found (with the
exception for lysozyme).
Both dyes appeared to have ability to follow the
step-by-step transition of monomeric wild type ASN,
A30P and A53T proteins into fibrils, demonstrating
good results reproducibility, much better than it was
observed for dye Thioflavin T. The presence of fibrils
was confirmed with AFM. It was shown that
quantification of the flibrillar ASN is possible in the
range from 1 to about 30 mg/ml using studied cyanine
dyes.
Trimethinecyanine SH-516 appeared to be able to
penetrate inside neuroblastoma SH-SY5Y cells, which
contain aggregated ASN, and selectively increase the
fluorescence intensity.
1. Volkova K. D., Kovalska V. B., Balanda A. O.,
Losytskyy M. Yu., Golub A. G., Vermeij R. J.,
Subramaniam V., Tolmachev O. I., Yarmoluk S. M.
(2008). Specific fluorescent detection of fibrillar
alpha-synuclein using mono- and trimethine cyanine
dyes. Bioorg. Med. Chem. 16(3), 1452–1459.
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