Lipids and carbohydrates biochemistry

Session I. Lipids and Carbohydrates Biochemistry
Lectures

L9.2

L9.1

The role of long-chain fatty acids in the
pathogenesis of the Metabolic Syndrome

Lipid compounds of the umbilical cord artery
Lech Romanowicz, Edward Bańkowski
Department of Medical Biochemistry, Medical University of
Białystok, Białystok, Poland
e-mail: Edward Bańkowski <edward12@umwb.edu.pl>
The lipid composition of arterial walls changes during development, ageing and pathological processes.
Preeclampsia is the most common pregnancy-associated
pathological syndrome. It is accompanied by significant
remodelling of extracellular matrix both in the umbilical cord vessels and in the surrounding Wharton’s jelly.
The lipids of the umbilical cord were not studied till now,
therefore it was decided to evaluate the specific features of
lipid composition of the umbilical cord artery (UCA) and
its alteration in preeclampsia. Thin layer chromatography
and high-performance liquid chromatography were employed for these analyses. It was found that UCA wall,
as most human tissues, contains free fatty acids, mono-,
di- and triacylglycerols, free cholesterol and its esters.
The characteristic feature of UCA wall is the presence of
high amount of long chain polyunsaturated fatty acids
(PUFA), including eicosapentaenoic acid (C20:5) and docosahexaenoic acid (C22:6), which are rather minor lipid
components of most human tissues. They exist both in a
free form and in a form of acylglycerols and cholesterol
esters. Preeclampsia is associated with a deep decrease in
most free fatty acids and acylglycerols. The total amount
of long chain PUFA: C18:2, C:18:3, C20:4, C20:5 and C22:6
in these lipid fractions decreased by half. In the same time
an increase in free cholesterol and its esters was observed.
The role of these phenomena in pathobiology of UCA and
foetal circulation is discussed. The shortage of PUFA may
reduce prostaglandin synthesis in arterial wall and impair blood flow in foetal vascular system.

Agnieszka Dobrzyń
Laboratory of Cell Signaling and Metabolic Disorders, Nencki
Institute of Experimental Biology PAS, Warszawa, Poland
e-mail: Agnieszka Dobrzyń <adobrzyn@nencki.gov.pl>
The incidents of obesity has increased dramatically in
recent years, making it one of the most pressing public
health concerns worldwide. Obesity is commonly associated with comorbid conditions, most notably insulin resistance, diabetes, heart disease, and hypertension, and
the coexistence of these diseases has been termed the Metabolic Syndrome. The precise etiology of the many abnormalities that occur in the obese people is still unknown;
however an increasing body of evidence indicates that
several manifestations of the Metabolic Syndrome and
type 2 diabetes mellitus are associated with alterations
in intracellular lipid metabolism. Obese humans and animals not only accumulate significant amounts of triglyceride in adipose tissue, but also in liver, muscle and other
peripheral tissues. Storage of even modest caloric surplus in lean tissues leads to lipid-induced dysfunction in
those tissues. If non-adipose tissues are exposed to excess
of long-chain fatty acids, unless leptin action increases
their oxidation sufficiently, unoxidized fatty acids enter
damaging pathways of nonoxidative metabolism, such
as ceramide synthesis. Free fatty acids and ceramides are
probably the most toxic lipids and are a cause of lipoapotosis of pancreatic β cells and myocardiocytes leading
to diabetes and lipotoxic cardiomyopathy. In the skeletal
muscle, the resulting functional impairment causes insulin resistance. These facts demonstrate that reduction
in intracellular fatty acid accumulation in non-adipose
tissue may be a very promising therapeutic strategy in
the prevention of diabetes and other components of the
Metabolic Syndrome.

43rd Annual Meeting of the Polish Biochemical Society and the 10th Conference of Cell Biology
Vol. 55

L9.3

L9.4

Nitric oxide and cPLA2 regulation in
Parkinson and Alzheimer diseases

205

Role of fatty acids and carnitine in the heart

Małgorzata Chalimoniuk1, Anna Stolecka1,2,
Jozef Langfort2, 3, Joanna B. Strosznajder1
1Department

of Cellular Signaling, Medical Research
Centre PAS, Warszawa, Poland; 2Department of Physiology,
Academy of Physical Education, Katowice, Poland;
3Department of Experimental Pharmacology, Medical
Research Centre PAS, Warszawa, Poland
e-mail: Malgorzata Chalimoniuk <mchalim@yahoo.com>
Cytosolic phospholipase A2 (cPLA2) appears to play an
important role in the central nervous system. It belongs
to a family of enzymes which catalyze the hydrolysis of
the sn-2 position of glycerophospholipids, generating free
fatty acids and lysophospholipids. The products of these
reactions may exist as second messenger themselves, or
be metabolized to eicosanoids and lysophospholipids. In
Parkinson’s and Alzheimer’s diseases, many studies have
observed increased expression and activity of cPLA2, accompanied with increased release of AA, induction of
COX-2 enzyme and higher production of prostaglandins.
We investigated if the cGMP/cCGP-dependent protein kinase (PKG) signaling pathway was involved in cPLA2 activation in Parkinson and Alzheimer diseases. We found
increased levels of total and phosphorylated cPLA2 and
increased AA release in the nigrostriatal system of MPTPinduced parkinsonism mice and in experimental model
of AD [in PC12 cells with the Swedish double mutation
in amyloid beta precursor protein (APPsw) and in transfected with human APP (APPwt)]. We used cPLA2-specific inhibitors and Ca2+-independent PLA2 (iPLA2), and
we found that cPLA2 released more AA after stimulation with MPTP/MPP+ than iPLA2 and that there was a
time-dependent delay of AA release by iPLA2 compared
to cPLA2. Moreover, our results indicated that the direct
relationship between Ab concentration, NOS activity and
cPLA2 level and AA release in experimental model of AD.
The PKG inhibitor KT5823 decreased MPTP-induced AA
release in the nigrostriatal pathway. KT5823, in addition
to PKC and ERK1/2 inhibitors, decreased cPLA2 activity
as well as total and phosphorlyated cPLA2 protein levels
in the midbrain and striatum of MPTP-induced parkinsonism mice.. Inhibition occurred within 30 minutes and
persisted for up to 24 hours. Similar results were also observed in MPP+-treated PC12 cells. Dual treatment with
PKG and PKC inhibitors had the same effect on cPLA2 activity and protein levels. PKG is involved in the enhancement of cPLA2 phosphorylation at Serine-505 and in AA
release in PC12 cells exposed to MPP+. In PC12 cells, inhibitors of cPLA2 and PKG increased viability and prevented MPP+-induced apoptosis. Our results indicate that
the nNOS/cGMP/PKG pathway stimulates cPLA2 phosphorylation at Ser-505 by activation of PKC or ERK1/2.
Our results also suggest that upregulation of the nNOS/
cGMP pathway observed in experimental models of PD
and AD may mediate neuron degeneration and death
through activation of cPLA2.

Ryszard T. Smolenski
Department of Biochemistry, Medical University of Gdansk,
Gdańsk, Poland
e-mail: Ryszard Smolenski <r.smolenski@ic.ac.uk>
Fatty acids are predominant energy substrates for cardiomyocytes in the healthy heart, but it is now well appreciated that it is also dangerous fuel for cardiac metabolism,
particularly in the ischemic or failing heart. Both basic
research and clinical evidence highlight deleterious effects of excess of fatty acids on cardiac cell viability and
function. Cardiomyocytes maintain one of the highest
levels of fatty acid binding protein among all cells in the
body that indicates importance of sequestration of free
fatty acid molecules inside the cell. Number of mechanisms could contribute to impairment of cardiomyocyte
function by fatty acids. These could be a consequence of
biophysical or chemical properties of fatty acids such as
detergent effect or specific effects on enzymes or membrane transport. There is strong clinical evidence that correlates deterioration of heart function in patents suffering
from heart failure with elevated blood free fatty acid concentration. Reduction of fatty acid entry into cardiac cells
and replacement of its metabolic role with carbohydrate
substrates is therefore an important target for treatment
of heart failure and ischemic heart disease. Carnitine is
vital for cardiomyocyte fatty acid transport across mitochondrial inner membrane but its role under pathological
conditions extends into additional pathway to sequester
fatty acids inside cardiac cells. Carnitine supplementation
is therefore believed to provide therapeutic benefits in
heart disease. Our studies on carnitine metabolism in the
failing heart provided further support for this concept.
First, contrary to common view that carnitine is supplied
exogenously to cardiac cells we have shown that key
enzyme of this pathway arginine:glycine amidinotransferase (AGAT) is present and active in the heart. Furthermore, we have shown that expression and activity of
AGAT is increased several times in heart failure, possibly
indicating activation of compensatory mechanism under
conditions of increased demand for carnitine. Optimizing
fatty acid metabolism in the heart seems to be important
in heart failure and ischemic heart disease and this pathway could be target for new effective drugs.

The Congress of Biochemistry and Cell Biology — Abstracts
206

L9.5

L9.6

Regulation of the activity of glycolytic
enzymes upon their binding to lipid
and membrane structures

2008

Surface plasmon resonance (SPR) —
applications in carbohydrate analysis

Jan Gutowicz
Department of Physical Chemistry of Microorganisms,
Institute of Genetics and Microbiology, University of
Wroclaw, Poland
e-mail: Jan Gutowicz <gutowicz@microb.uni.wroc.pl>
Interaction between most of glycolytic enzymes and intracellular membranous or protein insoluble structures
in vitro as well as their co-localization in situ have been
evidenced in a wide range of plant, animal and microbial cells. In this report, the representative recent studies
(own studies of the author and his coworkers included)
on the interaction, its mechanism, and its consequences
for the molecular and functional properties of some glycolytic enzymes are reviewed and discussed. The interaction results in reversible binding of the enzymes to the
structures. Since in almost all studied cases the binding
is highly sensitive for the change of environmental conditions like pH and ionic strength influencing ionic bonds
the multi-elelectrostatic interaction between the molecules of the enzymes and the charged surface of the cellular substructure binding sites has been postulated to be
the initial and main forces in the binding mechanism. The
implications of the association with negatively charged
surface for the activity have been studied using phospholipid bilayer model structures. The electrostatic interaction
mechanism has been supported by the studies. In many
of the studied enzymes more specific substances like metabolites, substrates, cofactors, some drugs etc. have been
shown to alter the binding. The binding of the enzymes
to both: natural membrane or phospholipid bilayer structures induces modification of their catalytic properties
and the effect differs among the izozyme forms. For aldolase, glyceraldehyde-3-phosphate dehydrogenase, pyruvate kinase and lactate dehydrogenase the contribution
of conformational changes in the activity modification
upon the binding with anionic phospholipid bilayers has
been evidenced. All these findings support the concept
that the equilibrium between bound and unbound forms
of the enzymes in vivo is sensitive for metabolic state of a
cell. The role of the capacities of the enzymes for such dynamic association is discussed in terms of the regulation
of the enzyme activity and of the pathway.

Hubert Krotkiewski1, Bożena Krotkiewska2
1Ludwik

Hirszfeld Institute of Immunology & Experimental
Therapy, PAS, Wrocław, Poland; 2Department of
Biochemistry, Medical University, Wrocław, Poland
e-mail: Hubert Krotkiewski <krotkiew@iitd.pan.wroc.pl>
BIAcore optical biosensor, constructed in 1990 (Biacore
AB, Uppsala, Sweden) was designed to measure, in a real
time, an interaction between two substances (biological
interaction analysis, BIA), for example antigen and antibody, lectin and glycoprotein, etc. As a detection method
in the instrument the surface plasmon resonance (SPR)
technique was used; it is an optical phenomenon which
arises during the total inner light reflection when light
illuminates thin conducting films under specific conditions, originally discovered by Turbadar (1959). During
BIAcore operation the following methodology is used:
one of two interacting substances, a ligand, is covalently
bound to a working channel of a sensor chip; the second
interacting substance, an analyte, is introduced into the
working channel as a solution of known concentration,
under controlled flow rate in the range of 1–100 µl/min. A
detector, based on the surface plasmon resonance, detects
the mass increase in the channel, which is a direct consequence of the ligand-analyte complex formation; a signal
generated by the detector is given in the relative resonance units (RU); it is proportional to an increase of mass
in the working channel (1 RU = 1 pg/mm2 for protein). In
the course of investigating the carbohydrate structures,
using BIAcore, the following sets of experiments were
performed: analysis of the level of desialylation of human glycophorin A (use of Psathyrella velutina, Triticum
vulgaris and Sambucus nigra lectins), analysis of reaction
of Triticum vulgaris (wheat germ agglutinin, WGA) with
glycophorin A, isolated from erythrocytes of blood group
A, B and O (looking for fine differences in glycophorin
glycosylation), analysis of the level of agalactosylation of
IgG, isolated from the patients with rheumatoid arthritis (RA) before and after pharmacological treatment, using Ricinus communis and Griffonia simplicifolia II lectins.
The method with BIAcore use showed up as a procedure
sensitive, without any need of chemical derivatization of
the reagents and repeatable. In all experiments the lectins
served as the ligands and the proteins were used as the
analytes.
Reference:
Turbadar T (1959) Proc Phys Soc (London) 73: 40–44.

Vol. 55

L9.7

Oral Presentations

The role of hexokinase in sugar sensing
and signaling in plant cells

207

O9.1

Iwona Ciereszko
Institute of Biology, University of Białystok, Białystok, Poland
e-mail: Iwona Ciereszko <icier@uwb.edu.pl>
Sugars affect germination, plant growth and metabolic
processes via the expression of numerous genes. Glucose,
sucrose and other sugars might serve as elicitors of sugar
signaling. Plants developed mechanisms of sensing and
transduction of sugar signals. Hexokinase (EC 2.7.1.1),
sugar transporters and specific sugar receptors have
been proposed as components of sugar sensing machinery. However, only hexokinase1 (HXK1) is the relatively
well characterized sugar receptor until today. HXK1
play a dual role in glucose signaling and hexose phosphorylation (enzyme of the glycolytic pathway), both in
plants and other organisms (yeast, animals). Genetic and
biochemical analyses of Arabidopsis mutants, gin2, have
shown that diverse glucose responses can be mediated
by the HXK1 mutations with little or no catalytic activity
and provided evidence for uncoupling of glucose-signaling functions from metabolic activities. Hexokinases are
encoded by a relatively large gene family (e.g. 6 genes
in Arabidopsis or 10 genes in rice). Different subcellular
localizations were determined for HXK protein family
members. HXK proteins can be soluble in the cytosol or
associated with organelles like mitochondrium, chloroplast, Golgi and nucleus. Recent studies have provided
evidence for the nuclear localization of hexokinase1 and
showed that this nuclear HXK1 forms a signaling complex with VHA-B1 (vacuolar H+-ATPase B1) and RPT5B
(19S regulatory particle of proteasome). Such complex
directly modulates gene transcription, independently
of glucose metabolism. The role of actin cytoskeleton in
HXK-dependent glucose signaling during plant growth
has recently also been suggested.

Raffinose family of oligosaccharides and
galactosyl cyclitols in legume seeds
Lesław Lahuta, Ryszard Górecki
University of Warmia and Mazury, Department of Plant
Physiology and Biotechnology, Olsztyn, Poland
e-mail: Lesław Lahuta <lahuta@uwm.edu.pl>
One of the major reasons for limited consumption of grain
legume seeds is the presence of relatively high quantities
of the raffinose family of oligosaccharides (RFOs) that result in flatulence. Therefore, legume plant breeders try to
reduce or eliminate the flatulence-producing oligosaccharides from seeds. RFOs are ubiquitous in legume seeds
but their compositions vary among species. The seeds of
some species contain also α-D-galactosides of myo-inositol isomers or methylated ethers (galactosyl cyclitols). The
presence of galactosyl cyclitols in seeds is correlated to the
presence of appropriate cyclitols in vegetative tissues. Accumulation of both types of oligosaccharides starts during
seed filling and accelerates during seed maturation drying. Changes in the activity of enzymes of the RFO pathway throughout seed development and maturation and
catalytic properties of purified or recombinant enzymes
indicate that in the biosynthesis of RFOs and galactosyl
cyclitols the same set of enzymes is engaged. Therefore, it
is possible to improve nutritional quality of legume seeds
by the replacement of RFOs by defined α-D-galactosides
of cyclitols (with lower flatulence potential and heath promoting property). Before this effort proceeds to far, seed
physiologists need to determine the physiological effects
of such modification of oligosaccharides in seeds. Feeding
free cyclitols into isolated developing seeds or via the cut
stem explants was introduced as an experimental model
to obtain seeds of Pisum sativum L., Phaseolus vulgaris L.,
Lathyrus odorathus L. and Vicia sp. with modified α-D-galactosides composition. The amount and composition of
newly formed galactosyl cyclitols was closely related to
genotype, seed developmental stage and cyclitols type
and their concentration used in experiments. The formation of galactosyl cyclitols inhibited the accumulation of
higher homologues of raffinose (especially verbascose)
and decreased the total content of RFOs in seeds. The accumulation of new cyclitols and galactosyl cyclitols does
not affect seed physiological quality (germinability and
desiccation tolerance).
Acknowledgments:
This work was partially supported by grant N303 125 32/4015
obtained from Ministry of Sciences and Education in Poland.

208

O9.2

O9.3

N-oligosaccharides of α3β1 and αvβ3 integrins in
metastatic melanoma WM9 and WM239 cell lines

Underappreciated catabolism of
glycoconjugates in alcohol abuse

Małgorzata Przybyło1, Marcelina E. Kremser1,
Dorota Hoja-Łukowicz1, Ewa Pocheć1,
Angela Amoresano2, Andrea Carpentieri2,
Monika Bubka1, Anna Lityńska1

2008

Napoleon Waszkiewicz1, Sławomir D.
Szajda2, Anna Jankowska3, Agata Szulc1,
Beata Konarzewska1, Krzysztof Zwierz2

1Department

of Glycoconjugate Biochemistry, Institute
of Zoology, Jagiellonian University, Kraków, Poland;
2Dipartimento di Chimica e Biochimica, Complesso
Universitario Monte S’Angelo, Napoli, Italy
e-mail: Małgorzata Przybyło <malgorzata.przybylo@
uj.edu.pl>
It is well documented that glycan synthesis is altered in
some pathological processes, including cancer. The most
frequently observed alterations during tumourigenesis
are extensive expression of β1,6-branched tri- and tetraantennary complex type N-glycans, the presence of poly-Nacetyllactosamine structures, and high sialylation of cellsurface glycoproteins. Carbohydrates have been shown to
interact with proteins during recognition events, so any
changes in the structure of the sugar component may influence the glycoprotein’s ligand binding ability. Integrins,
a large family of cell membrane glycosylated receptors, are
involved in important processes such as cell-cell and cellECM adhesion. Their altered expression is associated with
tissue invasion and metastasis in many types of cancer. In
melanoma, α3β1 and αvβ3 integrins are recognised as specific markers of tumour progression, and αvβ3 in particular
is used as a marker to distinguish the radial growth phase
(RGP) from the vertical growth phase (VGP). The aim of
this study was to analyse glycan pools of α3β1 integrin purified from human melanoma cell line WM239 (skin metastasis), with the use of MALDI MS, and to compare them
with previously analysed glycosylation patterns for integrin α3β1 from WM9 cells and αvβ3 from both cell lines.
Integrin α3β1 was purified from cell extracts by affinity
chromatography. The collected material was separated
by SDS/PAGE under non-reducing condition, and protein bands corresponding to a3 and b1 subunits were excised from PVDF sheet. After protein alkylation, in situ
digestion, sugar extraction and microcolumn clean-up of
sugars, MALDI MS was performed and oligosaccharides
were detected as [M + Na+] or [M + H+] ions in the positive
ion mass spectrum.
Integrins α3β1 and αvβ3 purified from two melanoma cell
lines derived from particular metastasis sites differ in
their glycosylation profiles. Both subunits of α3β1 integrin
in WM239 cells showed less diverse glycan types than did
WM9 cells. Our previous results concerning αvβ3 integrin
glycosylation have shown reverse correlation: the glycan
pool from WM239 cells was much more diverse than in
WM9 cells. Nevertheless, both integrins showed the presence of tumour-associated glycans regardless of the differences in glycosylation profiles between these integrins
in the two cell lines.
Acknowledgements:
This work was supported by the Polish State Committee for Scientific Research (PB/0939/P05/2004/26).

1Department

of Psychiatry, 2Department of Pharmaceutical
Biochemistry, 3Department of Paedodontics, Medical
University of Białystok, Białystok, Poland;
e-mail: Napoleon Waszkiewicz <napwas@wp.pl>
Glycoproteins, glycolipids, and proteoglycans, are referred to as glycoconjugates or complex carbohydrates [1,
4]. Glycoconjugates are important for maintaining cellular
and extracellular homeostasis. The biological roles of the
oligosaccharide units of individual classes of glycoconjugates include: maintaining conformation and stability of
proteins, being target for microorganisms and masking of
such a target structures, control of the half-life of proteins
and cells, modulation of protein functions, mediating of
ligands binding, as well as cell-matrix and cell-cell interactions. Alcohol abuse is a widespread social and medical problem in a large section of populations all over the
world [2, 3]. Ethanol and its metabolites induce a variety
of pathogenic reactions, affecting almost every organ of
human body, disrupting also developmental processes
(e.g. alcoholic liver disease and fetal alcohol syndrome).
Ethanol, acetaldehyde, reactive oxygen species, and other
metabolites of alcohol (e.g. fatty acid ethyl esters), participate in destabilization of processes of glycoconjugate
metabolism [2–4]. As the majority of changes in the cellular metabolism of glycoconjugates are ascribed to the
alcohol-induced alterations in synthesis, transport, glycosylation and secretion of glycoconjugates [2, 3], the degradative and elimination processes, of which the former
occurs also in extracellular matrix, seem to be underappreciated. We therefore reviewed all of these aforementioned crucial areas of the glycoconjugate metabolism
and went into conclusion that further research, based on
all metabolic steps of the glycoconjugate metabolism, including important catabolic process, need to bridge the
transdisciplinary research in enhancing our understanding of alcohol- and glycoconjugate-related diseases.
References:
1. Varki A (1993) Glycobiology 3: 97–130.
2. Tomas M et al. (2002) J Neurochem 83: 601–612.
3. Lands WE (1993) Glycobiology 3: 415–416.
4. Waszkiewicz N et al. (2008) Alcohol Alcohol doi: 10.1093/alcalc/
agn027.

Vol. 55

Posters

P9.2

P9.1

209

Evaluation of the influence of age on the
activity of salivary lysosomal exoglycosidase
in diabetes type 2

Changes in mammalian erythrocyte
membrane permeability induced by
amphotericin B, nystatin and verapamil
Agnieszka Knopik-Skrocka, Józef Bielawski,
Joanna Grochowska, Marta Mot
Institute of Experimental Biology, Department of Cell Biology,
Adam Mickiewicz University of Poznań, Poland;
e-mail: Agnieszka Knopik-Skrocka <askro@amu.edu.pl>
Erythrocyte membrane lipids (cholesterol, phospholipids) are very important targets of some hemolytic agents.
The membrane activity of amphotericin B (AmB) and nystatin (Nys), belonging to antimicrobial polyene antibiotics, is the effect of sterol-antibiotic complex formation [1,
2]. Similar to AmB and Nys, verapamil (Ver) known as a
calcium channel blocker and P-gp inhibitor shows a high
affinity to membrane lipids, mainly to anionic phospholipids [3]. The hemolytic activity of AmB and Nys is highly
dependent on red blood cell (RBC) membrane specie`s
and specimen’s features [4–6]. Its modification is caused
by some changes in incubation conditions [2, 3]. In the
present study, it is of interest how the changes in chemical
composition and temperature of incubation medium influence the hemolysis induced by the polyenes in chosen
mammalian erythrocytes. Ver interacts with RBC’s membranes inducing cell shape changes [7]. However, the kinetics of Ver-induced hemolysis and the effect of species
features of RBC’s membranes are not known in detailes.
In standard conditions (isoosmotic KCl, 37oC), the kinetics of
hemolysis induced by Ver, measured with absorption spectrophotometric method (l =590 nm), was very similar to that
of AmB and Nys. However, the resistance (C50) of the mammalian erythrocytes to Ver was about 3 orders of magnitude
higher than to AmB and 2 orders of magnitude higher than
to Nys. No statistic difference was found between C50 calculated for human and pig erythrocytes treated with Ver.
The kinetics of AmB or Nys-induced hemolysis was modified when KCl was replaced by other isoosmotic media.
However, smaller changes in C50 values were found for
Nys. For both antibiotics, the effect of cations and anions
tested is dependent on specie’s features of erythrocyte
membrane.
With the increase of temperature from 15oC to 37oC, the
rates of hemolysis induced by the polyenes in human as
well as in pig RBCs do not increase markedly. The results
for AmB and Nys are similar. The values of the ratios C50
37oC/C50 15oC, calculated from the rates of hemolysis,
were near 1.0.
References:
1. Knopik-Skrocka A, Bielawski J (2002) Cell Mol Biol Lett 7: 31–
48.
2. Knopik-Skrocka A et al. (2003) Cell Mol Biol Lett 8: 439–454.
3. Knopik-Skrocka A, Buldańczyk A (2004) Biol Lett 41: 27–39.
4. Speelmans G et al. (1995) Biochim Biophys Acta 1238: 137–146.
5. Deuticke B (1968) Biochim Biophys Acta 163: 494–500.
6. Coutinho A, Prieto M (1995) J Biophys 69: 2541–2557.
7. Charbonneau C et al. (2001) Biophys Chem 91: 125–133.

Slawomir D. Szajda1, Malgorzata Knas1,
Katarzyna Knas-Karaszewska2, Jakub
Karaszewski3, Malgorzata BorzymKluczyk1, Anna Stypulkowska1, Wiesław
Zarzycki4, Krzysztof Zwierz1
1Department

of Pharmaceutical Biochemistry, 3Department
of Maxillofacial Surgery, 4Department of Endocrinology,
Diabetology and Internal Diseases, Medical University of
Białystok, Poland; 2Unpublic Institution of Health Care
“Stomatology Dr. Knas”, Białystok, Poland
e-mail: Slawomir Szajda <spoak@umwb.edu.pl>
The diabetes (DM — diabetes mellitus) is a systemic disease which has an influence on glucose level in the blood.
Type 2 diabetes mellitus is a disease that affects a rapidly
increasing number of patients. Most patients with Type 2
diabetes will develop vascular complications. This may
be microvascular disease, such as nephropathy, retinopathy or polyneuropathy, and also macrovascular disease,
such as coronary heart disease, stroke or peripheral artery
disease. β-galactosidase is one of the lysosomal exoclycosidases which catalyzes removal of galactose residues
from the non-reducing end of oligosaccharide chains of
glycoconjugates. β-galactosidase also degrades lactose to
galactose and glucose in small intestine. The aim of the
study was evaluation of the influence of age on salivary
lysosomal exoglycosidase (β-galactosidase) in diabetes
type 2.
Material and methods: 3–5 ml of unstimulated saliva was
obtained one to three hours after last meal from the patients with diabetes type 2. 40 patients were divided into
two groups: I — patients to 65 years old and II — patients
after 65 years old. Unstimulated saliva was collected, by
spitting method, into polyethylene tubes, kept on ice. βgalactosidase activity was determined by Chatteriee et al
method in Zwierz et al. modyfication.
Results: We showed significant decrease in activity (pKat/
kg protein) of β-galactosidase (p=0.008126) in group II
(patients after 65 years old with type 2 diabetes) in comparison to group I (patients to 65 years old with type 2
diabetes).
Conclusion: Age has influence on enzymatic activity of
saliva in patients with type 2 diabetes.

210

2008

P9.3

P9.4

Evaluation of the influence of diabetes type 1 on
the activity of salivary lysosomal exoglycosidases

Evaluation of the influence of diabetes type 1
on exoglycosidases activity in human saliva

Krzysztof Zwierz1, Malgorzata Borzym-Kluczyk1,
Malgorzata Knas1, Katarzyna Knas-Karaszewska2,
Jakub Karaszewski3, Slawomir D. Szajda1,
Anna Stypulkowska1, Wiesław Zarzycki4

Malgorzata Knas1, Katarzyna Knas-Karaszewska2,
Jakub Karaszewski3, Malgorzata Borzym-Kluczyk1,
Slawomir D. Szajda1, Anna Stypulkowska1,
Wiesław Zarzycki4, Krzysztof Zwierz1

1Department

Białystok, Białystok, Poland; 2Unpublic Institution of Health
Care “Stomatology Dr. Knas”, Białystok, Poland
e-mail: Krzysztof Zwierz <kzwie@umwb.edu.pl>

1Department

Type 1 diabetes is usually diagnosed in children and
young adults, and was previously known as juvenile diabetes. In type 1 diabetes, the body does not produce insulin. Insulin is a hormone that is needed to convert sugar
(glucose), starches and other food into energy needed
for daily life.Glucuronidase is located in lysosomes and
plays an important role in recycling cellular components
by cleaving glucuronide moieties from proteins. Glucuronidase exhibits both endo-glycosidase and exo-glycosidase activities, meaning that it can cleave monosaccharides from the middle of a chain or from the end. One of
diagnosing method of periodontal disease in a patients is
detecting elevated concentrations of β-glucuronidase in
saliva. The aim of the study was evaluation of the influence of diabetes type 1 on the activity of salivary lysosomal exoglycosidase — β-glucuronidase.
Material and methods: 3–5 ml of unstimulated saliva was
obtained one to three hours after last meal from the patients with diabetes type 1. Unstimulated saliva was collected, by spitting method, into polyethylene tubes, kept
on ice. β-glucuronidase activity was determined by Chatteriee et al. method in Marciniak et al. modyfication.
Results: We showed significant increase in concentration
of activity (nmol/ml/min) β-glucuronidase activity (p=
0.000000) in the saliva in type 1 diabetic patients in comparison to control group.
Conclusion: The significant increase of the activity of
β-glucuronidase probably is connected with increase in
catabolism of glycoconjugates mostly related to inflammatory state of tissues.

Diabetes mellitus is a syndrome characterized by disordered metabolism and abnormally high blood sugar (hyperglycaemia) resulting from low levels of the hormone
insulin with or without abnormal resistance to insulin’s
effects. Diabetes type 1 is usually due to autoimmune
destruction of the pancreatic beta cells. N-acetyl-β-hexosaminidase and β-galactosidase are lysosomal exoglycosidases that degrade oligosaccharide chains of glycoconjugates, such as glycoproteins and glycolipids that
constitute cell membranes, and glycosaminoglycan chains
of proteoglycans that constitute extracellular matrix. Nacetyl-β-hexosaminidase releases N-acetylglucosamine
and N-acetylgalactosamine, and β-galactosidase releases galactose from non-reducing end of oligosaccharide
chains of lycoconjugates. Changes in the levels of these
enzymes could be associated with breakdown of the periodontal ligament or the oral mucosa.The aim of the study
was evaluation of the influence of diabetes type 1 on the
activity of salivary lysosomal exoglycosidases.
Material and methods: 3–5 ml of unstimulated saliva
was obtained one to three hours after last meal from the
patients with diabetes type 1. Unstimulated saliva was
collected, by spitting method, into polyethylene tubes,
kept on ice. Lysosomal exoglycosidases (N-acetyl-β-Dhexosaminidase and β-galactosidase) activity was determined by Chatteriee et al. method in Zwierz et al. modyfication.
Results: We showed significant increase in concentration
of activity (nmol/ml/min) N-acetyl-β-D-hexosaminidase
(p=0.000000) and β-galactosidase (p=0.000000) in the saliva in type 1 diabetic patients in comparison to control
group.
Conclusion: The significant increase of the activity of
lysosomal exoglycosidases probably is connected with
increase in catabolism of glycoconjugates mostly related
to inflammatory state of tissues.

Białystok, Białystok, Poland; 2Unpublic Institution of Health
Care “Stomatology Dr. Knas”, Białystok, Poland
e-mail: Małgorzata Knaś <knass@umwb.edu.pl>

Vol. 55

P9.5

P9.6

Activity of a-fucosidase (a-Fuc) in
rats liver after 5 days of hypoxia

Enzymes of glycogen catabolism from
Contracaecum rudolphii (Nematoda)

Danuta Dudzik1, Małgorzata Knaś1,
Róża Wisniewska2, Małgorzata BorzymKluczyk1, Krzysztof Zwierz1

211

Krystyna Żółtowska1, Elżbieta
Łopieńska-Biernat1, Jerzy Rokicki2

1Department

2Department

of Pharmaceutical Biochemistry,
of Pharmacology, Medical University of Białystok, Białystok,
Poland
e-mail: Danuta Dudzik <danutadu@op.pl>
Background: Hypoxia is defined as a reduction in the
amount of oxygen passing into the blood and it is a state
of oxygen deficiency in the blood which is sufficient to
cause an impairment of organism function. Hypoxia is
caused by the reduction in partial pressure of oxygen, inadequate oxygen transport, or the inability of the tissues
to use oxygen. α-fucosidase is one of the lysosomal exoglycosidases which catalyzes removal of fucose residues
from the non-reducing ends of oligosaccharide chains of
glycoconjugates. The aim of our work was determination
of α-fucosidase activity in rats liver after 5 days of hypoxia.
Materials and Methods: Hypoxia was induced by five
days of anoxemia resulting from putting laboratory rats
into gas mixture (2% oxygen and 98% nitrogen) with stable flow, and without pressure changes until first apnoea
incident. Activity of α-fucosidase (pKat/kg of protein) in
rats liver was determined by Chatteriee et al. method in
the modification of Zwierz et al.
Results: The mean activity of α-fucosidase in homogenates of rats liver were: in control group – 0.633 nKat/kg
of protein and – 1.88 nKat/kg of proiein, after 5 days of
hypoxia. We observed significant increase in the activity of α-fucosidase in liver tissue after 5 days hypoxia, in
comparison to the control group (p=0.000000).
Conclusions: It may be suggested that hypoxia causes
increase in degradation of glycoconjugates containing fucose in liver tissue.

1Department

of Biochemistry, Faculty of Biology, University
of Warmia and Mazury, Olsztyn, Poland, 2Invertebrate
Zoology Division, Gdańsk University, Gdańsk, Poland
e-mail: Krystyna Żółtowska <k.zoltowska@uwm.edu.pl>
The nematode Contracaecum rudolphii is a cosmopolitan
parasite of piscevorous birds as cormorants, pelicans and
see ducks. It can contribute to mortality in birds and causes economical losses to the fish industry. In the last decade
this species is starting to close its development cycle also
in Poland. Biochemistry of this parasite is particularly interesting because of the complexity of its life cycle. Only
little information was found on the metabolism of C. rudolphii. Sugars are an important energetic source for parasitic nematodes. This study was aimed at carbohydrates
and the enzymes of glycogen catabolism from C. rudolphii.
Larvae third (L3) and fourth (L4) stage and the adult male
and female C. rudolphii were isolated from stomachs (n =
10) of cormorants (Phalacrocorax carbo) from lake Bełdany
(north-eastern Poland). In the extracts from them the contents of total sugars, glycogen, trehalose and glucose were
estimated. The activities of glycogen phosphorylase and
enzymes hydrolyzing glycogen as α-amylase, glucoamylase and disaccharidases: maltase, lactase and saccharose
were studied. The concentration of total sugars was 6 - 8%
of fresh matter. Female parasites accumulated significantly more sugars than male. The concentrations of studied
sugars may be put in order: glycogen, glucose, trehalose.
Glycogen was a main sugar of this parasite. Its content
was higher in adult nematodes than in their larvae. Differently trehalose was more in larvae than in adult. The
activity of glycogen phosphorylase was especially high
in L3 (2.89 μmol/mg). Both amylases had high activities,
and they were almost the same in all parasite stages. The
disaccharidases activities were higher in larvae than in
adults. Among the disaccharidases activities maltase was
the most active (2.67 μmol/mg in L3). Our results indicate
that at C. rudolphii age dependent differences in carbohydrate catabolism were appearing.

212

2008

P9.7

P9.8

The properties of trehalose 6-phosphate
synthase from the third larval stage of
Anisakis simplex (Nematoda, Anisakidae)

Increase of 11-beta-hydroxysteroid
dehydrogenase type I mRNA level in rat white
adipose tissue after starvation-refeeding

Elżbieta Łopieńska-Biernat1, Marta
Czubak1, Jerzy Rokicki2

Tomasz Śledziński

1Department

of Biochemistry, Faculty of Biology, University
of Warmia and Mazury, Olsztyn, Poland; 2Invertebrate
Zoology Division, University of Gdansk, Gdynia, Poland
e-mail: Elżbieta Łopieńska-Biernat <ela.lopienska@uwm.
edu.pl>
Anisakis simplex is a parasite gastrointestinal nematode
with a complex life cycle; the definitive hosts — marine
mammals – as well as in humans. As anisakiasis is a serious condition that be fatal to a patient and may cause
allergic reaction in patients sensitive to A. simplex allergens, it necessary to intensify research on biochemisty of
the parasite’s larvae. For example, little is known about
its carbohydrates, in particular — synthesis of trehalose.
This sugar is of special importance for parasites owing to
its physical and chemical properties. Besides the function
of energy reserve, it fulfills a protective role under stress
conditions. There is non information available on synthesis of trehalose in the third larval stage of A. simplex that’s
why in the present research we decided to mark determine the properties of enzyme participating in synthesis
of trehalose of A. simplex. Activity of TPS (EC 2.4.1.15),
was determined using the method by Giaever et al. (1988).
The end of the product of reaction – trehalose was determined using HPLC. Protein content according to Bradford (1976).The optimum pH was 7.0. TPS showed optimal activity at 55oC. The activity of enzyme decreased
rapidly at a temperature higher than 60oC. The activity of
TPS was found to be unaffected by the 15-min. preincubation at temperatures up to 50oC. Temperatures higher
than 65oC resulted in inactivation of enzyme. Trehalose
showed an activator effect on the enzyme. At 300 mmol
trehalose increased 24-fold of TPS activity. Fructose and
sorbitol exhibited an activity effect at 100 mmol 2-fold
and 3-fold respectively, higher concentrations up at 400
mmol had inhibitory effect on the enzyme. Glucose and
proline had an inhibiting effect on the enzyme at 100 and
200 mmol concentration. Na+ had an activator effect on he
enzyme, maximal effect was observed at 50 mmol, where
it increased the enzyme activity by about 5-fold, higher
concentration of NaCl to 400 mmol inhibited almost 27%
control activity. K+ showed no significant effect on the
enzyme. Mg2+ had an activator effect at higher concentrations (20, 25 mmol) about 30%. Co2+ and Cu2+ had an
inhibitory effect with a maximum at 5 mmol.
Reference:
Bradford J (1976) Anal Biochem. 72: 248-254.
Giaever H M et al. (1998) J Bacteriol 170: 2841-49.

Acknowledgements:
The study was supported by the Polish Ministry of Science and
Higher Education grant No. P04C02428.

Department of Pharmaceutical Biochemistry, Medical
University of Gdansk, Gdańsk, Poland
e-mail: Tomasz Śledziński <tsledz@amg.gda.pl>
11-beta-hydroxysteroid dehydrogenase type I (11βHSD1)
catalyses conversion of less active cortison (11-dehydrocorticosterone in rodents) to more active cortisol (corticosterone in rodents). 11βHSD1 gene is expressed in liver,
white adipose tissue (WAT) and other tissues. Cortisol
acts on adipose tissue promoting adipocytes differentiation. Mice overexpressing 11βHSD1 gene display enhanced adipocytes differentiation measured by increased
fat cell size, and consequently higher adipose tissue mass.
Increased activity of 11βHSD1 has been postulated to play
important role in pathogenesis of obesity. Another important factor for the development of obesity is increased
fatty acid biosynthesis. It has been shown that fastingrefeeding leads to increased lipogenesis. The aim of the
study was to investigate the effect of fasting-refeeding
on 11βHSD1 gene expression in WAT of rats. 11βHSD1
and lipogenic enzymes genes mRNA level were analysed
by real-time PCR in perirenal WAT of 2 months old male
rats fasted for 3 days and then fed for another 3 days ad
libitum. The results were compared to results obtained
in control rats. As expected fasting-refeeding resulted
in increase of lipogenic enzymes genes expression (fatty
acid synthase and malic enzyme) in WAT of rats. The new
finding of this study is that fasting-refeeding leads also to
increase of 11βHSD1 gene expression in WAT of rats.

Vol. 55

P9.9

P9.10

Synthesis, uptake and biotransformation of
[3H]-pantethine in brain structures after its
intracerebroventricular administration

Analysis of expression and tissue localization
of phosphoglucan water dikinase (PWD)
gene from Solanum tuberosum L.

Valery Hurynovich1, Inna Katkovskaya1,
Gennady Badun2, Zinaida Tyasto2, Natalya
Gulyaeva3, Andrey Moiseenok1

213

Sławomir Orzechowski1, Joanna Simińska1,
Agnieszka Grabowska1, Mirosław Sobczak2

1Institute

of Pharmacology and Biochemistry NAS, Grodno,
Belarus; 2M.V. Lomonosov Moscow State University, Moscow,
Russian Federation; 3Institute for Higher Nervous Activity
and Neurophysiology, RAS, Moscow, Russian Federation
e-mail: Valery Hurynovich <vgurinovich@rambler.ru>
To obtain the labeled preparation, we used tritium thermal activation and D-pantethine substance (Sigma, USA).
The biotransformation of [3H]-PT was studied after its
cerebroventricular administration (4 μl, uni- or bilaterally,
with specific radioactivity of 1.2 mCi/ml (1.6 Ci/mmol)) to
Wistar male rats. After dithiothreitol treatment (10 mM),
tissue perchlorate extracts were assayed for radionuclides
using HPLC in the regimen of isocratic elution on a 10 μm,
250×4 mm μBondapak C18 column with a mobile phase of
50 mM potassium phosphate buffer-methanol in the ratio
of 91.5:8.5 (v/v). [3H]PT uptake by brain structures was the
following: hippocamp > large hemisphere cortex > frontal
cortex > brain stem > cerebellum. Maximum level of [3H]PT and its metabolites was noted in the hippocamp and
large hemisphere cortex after 10–20 min when the radionuclide content 5 to 10-fold exceeded the levels of labeled
products in other brain structures. No differences were
found in [3H]-PT biotransformation after its uni- and bilateral administration. The samples assayed showed PT
as pantetheine (PN). After 10–20 min, the hippocamp
and the large hemisphere cortex demonstrated formation
of the following metabolites: phosphopantothenic acid
(PPA) (6–10%), pantothenic acid (PA) (2–9%), phosphoPN (PPN) (52–57%), PN (26–31%). After 1 h following the
administration of the preparation, the PPA and PA fractions were found to increase up to 23–30% while the PPN
and PN fractions – to decrease (32–45% and 5–9%, respectively). Since that time [3H]-CoA fraction was seen in the
hippocamp (trace amounts) and the large hemisphere
cortex (1–3% after 1-10 h, and 11% after 24 h). After 3–24 h
following the [3H]PT intracerebroventricular administration the amount of the labeled PPA and PA constituted
the bulk of PA labeled metabolites, whereas the amount
of PPN and PN fractions decreased down to 6–17 and 5–
11%, respectively. [3H]PT biotransformation in the frontal
cortex, brain stem and cerebellum was distinguished by
a rapid increase in the PPA fraction (27–40%), CoA (4%)
being detected in the frontal cortex at this period. The results obtained indicate a possibility of direct PT transport
(in the form of PN) into the CNS structures, biotransformation to phosphopantetheine and CoA, PN hydrolysis
by pantetheine kinase from brain (Vecsei L, 1990) to give
PA and its phosphorylation leading to considerable accumulation of phosphopantothenic acid.

1Department

of Biochemistry, 2Department of Botany, Faculty
of Agriculture and Biology, Warsaw University of Life
Sciences, Warszawa, Poland
e-mail: Sławomir Orzechowski <slawomir_
orzechowski@sggw.pl>
Starch is the most abundant storage carbohydrate produced in plants. All transient starch granules synthesised
during the day undergo the breakdown during the night,
providing sugars that become included in the metabolism
of whole plant. Key meaning in the starch degradation is
attributed to α-amylase, product of its activity β-maltose
is transported to the cytosol where it is subjected farther
conversions. There are same important elements affecting rate of starch decomposition: day durnal cycle, starch
phosphorylation and posttranslational regulation of enzyme activities. We isolated from Solanum tuberosum L.
tubers and partly described expression pattern of a novel
protein implicated in starch metabolism — phosphoglucan, water dikinase (PWD, EC 2.7.9.5). We cloned a part
of PWD sequence from potato. The cDNA sequence designated as StPWD1 was submitted to the National Center
for Biotechnology Information (accession no. EL595870).
On the basis of this sequence we prepared specific primer
pairs to localize expression of PWD gene in different potato tissues and organs using in-situ RT-PCR methods.
Expression of PWD takes place both in heterotrophic and
autotrophic tissues at potato. This is in agreement with
our previous observations and implies a possible involvement of PWD in storage and transient starch metabolism.
We also observed higher levels of PWD expression at the
end of light period than during the rest of the day. Such
diurnal changes in transcript abundancy are typical for
genes involved in starch degradation in chloroplasts. Apparently, PWD plays a regulatory role in starch degradation in potato.
Acknowledgements:
This project was supported by Ministry of Science Grant no.
N302061134.

214

2008

P9.11

P9.12

The effect of JA-Me on the degradation of α-Dgalactosides in germinating yellow lupin seeds

The influence of trensgenic BT corn pollen
on chosen carbohydrates and protein level in
worker honeybees (Apis mellifera carnica)

Bartosz Nitkiewicz1, Lesław B. Lahuta2,
Kazimierz Zalewski1
1Department

of Biochemistry, 2Department of Plant
Physiology an Biotechnology, University of Warmia and
Mazury, Olsztyn, Poland
e-mail: Bartosz Nitkiewicz <b.nitkiewicz@uwm.edu.pl>
The aim of the study was the determination of the effect
of jasmonate metyl-ester (JA-Me) at different concentrations (10–3–10–6 M) on the degradation of α-D-galactosides during 48 h of yellow lupin (Lupinus luteus L. cv.
Polo) seeds germination. The composition and amounts
of soluble carbohydrates were analyzed by gas chromatography method in axes and cotyledons (separately) after 0, 24 and 48 h of seeds germination.
In dry seeds raffinose family of oligosaccharides (RFOs)
were the main fraction of soluble carbohydrates. Seeds
contained also α-D-galactosides of D-pinitol (galactosyl
pinitols) and α-D-galactosides of D-chiro-inositol (fagopyritols), but at several fold lower concentration than that
of RFOs. During germination the degradation of RFOs
(and other galactosides) started earlier in axis, than in
cotyledons. The level of sucrose, fructose, glucose and
free cyclitols in axis increased according to hydrolysis of
oligosaccharides.
In axis tissues JA-Me delayed degradation of RFOs, regardless of its concentration. The rate of hydrolysis of
RFOs decreased according to increasing concentration of
JA-Me, as expected. However, during the first 24 hours of
germination JA-Me at 10–5 and 10–6 M induced disappearance of RFOs in cotyledons tissues, opposite to axis. Thus
it can be concluded, that the effect of JA-Me on the hydrolysis of α-D-galactosides in germinating yellow lupin
seeds is associated with changes in the susceptibility of
both axis and cotyledons tissues to this growth regulator.

Marek Farjan1, Zbigniew Lipiński1, Krystyna
Żółtowska1, Benedikt Polaczek23
1Division

of Biochemistry, Faculty of Biology, University of
Warmia and Mazury, Olsztyn, Poland; 2Institute of Zoology,
Free University of Berlin, Berlin, Germany
e-mail: Marek Farjan <marek.farjan@wp.pl>
Honeybee is of paramount importance as a pollinator
in the natural environment and agriculture. A recent
increase of the genetically modified crops areas require
researches about their thread to the beneficial insects.
Carbohydrate metabolism is an essential marker of the
physiological condition of insects. We have examined
how GM corn pollen affects the levels of protein content
as well as maltose and trehalose – sugars crucial in insects
biochemistry. We have chosen the pollen of Limagrain LG
22.43 (without genetic modifications, experimental field
near Olsztyn, Poland) and MON863 x MON810 corn (experimental field near Berlin, Germany), which is a hybrid
of two lines containing cry3Bb1 and cry1Ab genes, from
bt genes family, coding Bt crystal proteins, thus making
plants resistant to coleopteran and lepidopteran pests. In
order to assess potential impact of transgenic insect-resistant (MON 863 x MON 810) Bt corn-pollen consumption on hive honeybees, 2-, 3-, 4- and 5-day old workers
were chosen. The bees were placed in queen shipment
cages and fed during 5 days with a honey breed - mixture of honey and sugar (control), honey with non-transgenic Limagrain corn pollen (gr I) and honey with Bt corn
pollen (gr II). The protein content, trehalose and maltose
levels were estimated. The protein content was measured
by Bradford method. HPLC was used to determine trehalose and maltose content. There were no significant differences in protein content tested among the groups of
bees fed all of the tree diets (example for 7 days-old bees:
control — 2.97±0.49, gr I — 2.68±0.57, gr II — 2.63±0.40 mg
protein/g tissue). The sugars contents were singnificantly
higher in the control (0.889±0.027 and 0.243±0.075 mg/g
tissue) than in both of corn pollen consuming groups
(gr I 0.483±0.001 and 0.176±0.031 mg/g tissue, and gr II
0.459±0.145 and 0.147±0.039 mg/g tissue) respectively for
trehalose and maltose. Our data indicate that the transgenic pollen has no adverse impact on studied markers
of young hive honeybees under our experimental conditions.

Vol. 55

P9.13

P9.14

Purification and characterization of
trehalose-6-phosphate synthase from
muscle of Ascaris suum (Nematoda)

215

The effect of membrane cholesterol depletion
on hemolytic activity of bile salts

Małgorzata Dmitryjuk, Marta Dopieralska,
Elżbieta Łopieńska-Biernat, Marek Farjan
Department of Biochemistry, Faculty of Biology, University of
Warmia and Mazury, Olsztyn, Poland
e-mail: Małgorzata Dmitryjuk <m.dmit@uwm.edu.pl>
The synthesis of trehalose in nematodes is catalysed by
the action of two enzymes: trehalose-6 phosphate synthase (TPS; EC2.4.1.15) that catalyzes the condensation
reaction of UDP-glucose and glucose-6-phosphate to give
intermediate trehalose-6-phosphate (T6P), and trehalose6-phosphate phosphatase (TPP; EC3.1.3.12) that convert
T6P to free trehalose and Pi. The properties of TPS in nematodes was only researched in Aphelenchus avenae (Loomis
et al. 1980). The first information concerning the pathway
of trehalose synthesis in reproductive tissues and muscle
of A. suum presented Feist et al. (1965).
The goal of these researches was purification of TPS from
muscles of parasitic nematode A. suum and investigating its properties such as optimum pH and temperature,
thermal stability, isoelectric point and molecular weight,
Km, Vmax, substrate specificity and influence of potential
activators and inhibitors on activity of the enzyme.
Activity of TPS was determined by Giaever et al. (1988).
The end product of reactions — trehalose was determined
using HPLC. Protein concentration was determined by
the method of Bradford (1976). Absorbance at 280 nm
was used for monitoring protein in the column eluate. To
determine the optimum pH was used 0.1 mol acetic acidammonia buffer in range of pH 3.0–8.6. Enzyme optimal
temperature and thermal stability was studied measuring
TPS activity at 20–90oC.
Trehalose-6-phosphate synthase was isolated from muscle of A. suum 265-fold by fractionating with ammonium
sulfate, column chromatography on DEAE-cellulose and
gel filtration on sepharose 6B. TPS was a monomer with
molecular mass on native and on SDS/PAGE of 66 kDa.
The enzyme has the optimum pH 3.8, pI 5.4, Km of 6.6 ×
10–4 mol and Vmax = 3.5 nmol × min–1 × mg–1 for UDPG
and Km of 1.8 × 10–3 mol and Vmax = 6.2 nmol × min–1 ×
mg–1 for G6P. Basides glucose-6-phosphate, it is use fructose-6-phosphate as acceptor of glucose and it does not
act on glucose and fructose. TPS from A. suum muscles
was activated by 10 mmol MgCl2, NaCl or CaCl2 and inhibited by EDTA, KCl, FeCl3 and ZnCl2.
References:
Bradford MM (1976) Anal Biochem 72: 248–254.
Feist CF et al. (1965) J Parasitol 51: 76–78.
Giaever HM et al. (1988) J Bacteriol 170: 2841–2849.
Loomis SH et al. (1980) J Exp Zoology 211: 311–320.

Acknowledgements:
This work was supported by Grant: 2PO4C 124 29, from 2005
to 2008.

Lucyna Mrówczyńska, Katarzyna
Wawrzyniak, Józef Bielawski
Department of Cell Biology, Institute of Experimental Biology,
Poznań, Poland
e-mail: Lucyna Mrówczyńska <lumro@amu.edu.pl>
Sterols are essential membrane components of eukaryotic
cells and are important for membrane organization and
function. Cholesterol is the most representative sterol
present in higher eukaryotes. The membrane fluidity
(cholesterol/phospholipid ratio) of a cell determines the
extent to which it is susceptible to compounds with detergent properties. The present study was undertaken to examine the effect of membrane cholesterol content on the
kinetics of hemolysis induced by unconiugated bile salts
differ in number of hydroxyl groups and hydrophobicity
index (HIx) – two hydrophobic (monohydroxy litocholic LChol and dihydroxy deoxycholic — NaDChol) and one
hydrophilic (trihydroxy cholic — NaChol). Using metylbeta-cyclodextrin we directly get the depletion of ~33%
cholesterol from the erythrocyte membrane. Digitonin
known to from complexes with cholesterol in membranes
was used as positive control in our study. The kinetics of
hydrophobic and hydrophilic bile salts-induced hemolysis of erythrocytes treated with methyl-β-cyclodextrin
appeared to be similar to observe for the control cells and
indicated the colloid osmotic type. Depletion of membrane cholesterol increased bile salts-induced hemolysis
rate in the concentration under and above their CMC in
the order monohydroxy < dihydroksy < trihydroxy. No
significant decrease (p>0.05) the erythrocyte resistance
to deoxycholic and cholic bile salts was observed after
methyl-beta-cyclodextrin treatment. Cholesterol depletion did not influence the hemolytic activity of the most
hydrophobic lithocholic acid. To compare, depletion of
membrane cholesterol by treating with methyl-beta-cyclodextrin suppressed digitonin-induced hemolysis and
significantly increased the erythrocyte resistance to its action (p<0.05). Cholesterol depletion had no effect on the
erythrocyte shape. The obtain results showed that the
partial depletion of plasma membrane cholesterol is not
essential for the significant decrease of erythrocyte resistance against unconiugated hydrophobic and hydrophilic
bile salts. We conclude that bile salts are able to interact
with membrane differently depending on their physicochemical properties.

216

2008

P9.15

P9.16

Influence of JA-Me in biosynthesis of
phospholipids in germinating yellow
lupin (Lupinus luteus) seeds

Sex-related differences in lipogenic enzymes and
SREBP-1 genes expression in rat subcutaneous
adipose tissue by progesterone treatment

Kazimierz Zalewski, Bartosz Nitkiewicz

Ewa Stelmanska1, Elzbieta Goyke1,
Julian Swierczynski1

Department of Biochemistry, University of Warmia and
Mazury, Olsztyn, Poland
e-mail: Kazimierz Zalewski <k.zalewski@uwm.edu.pl>
The study presents jasmonic acid methyl ester influence
on biosynthesis and composition of phospholipid compounds in germinating Lupinus luteus var. Polo seeds.
Fully matured lupin seeds chosen to experiment acquired form seeds production central (OLZNAS-CH Sp.
z o. o.). Seeds were germinating for 48 h in 21oC in darkness. Phospholipid (TLC) isolation and separation was
conducted according to Nichols et al. (1965). Phosphorus
content was analyzed according to Ames (1966). 3H-sodium acetate (Polatom, Świerk) was used as a precursor
to lipids synthesis. Samples radioactivity was indicated
by scintilation counter LS-1801 (Beckman). Growth regulator was applied in four concentrations 10–3 M, 10–4 M,
10–5 M, 10–6 M. Seeds germinating in distilled water were
used as control sample.
Intensivity of different phospholipid synthesis varied and
depended mainly on JA-Me concentration applied.
Table 1. 3H-sodium acetate (185 kBq/cm3) incorporation to phospholipids of germinating lupin seeds (48 h, 21°C) (cpm/100 embryonic axes)
Phospholipid combination

PS

PI

PC

PG

PE

PA

H20

14708

15151

4131

16102

38210

11449

JA-Me 10-3 M

2380

8137

7396

21341

49280

2303

JA-Me 10-4 M

15658

13521

2003

15756

31731

4017

JA-Me 10-5 M

17517

10040

2063

12867

26548

2906

JA-Me 10-6 M

15790

11434

2923

22816

6443

3402

Table 2. 3H-sodium acetate (185 kBq/cm3) incorporation to phospholipids of germinating lupin seeds (48 h, 21°C) (cpm/100 cotyledons)
Phospholipid combination

PS

PI

PC

PG

PE

PA

H20

19875

23266

73435

27034

34478

11898

JA-Me 10-3 M

6768

25722

11568

39531

53723

11606

JA-Me 10-4 M

11383

19973

5264

59608

38199

15931

JA-Me 10-5 M

14538

18373

10660

32391

26168

21746

JA-Me 10-6 M

14488

17862

21697

20218

43002

11272

1Department

of Biochemistry, Medical University of Gdansk,
Gdańsk, Poland
e-mail: Ewa Stelmanska <bori@amg.gda.pl>
Steroid hormones play an important role in the regulation of lipogenesis, however the effect of progesterone on
lipogenesis is not clear.
The aim of present study was to investigate the influence
of pharmacological doses of progesterone on the lipogenic enzymes genes expression in subcutaneous adipose tissue of female and male Wistar rats.
Two-month old rats received a single dose (100 mg) of
progesterone as a subcutaneous implant. After 1 month
of such treatment rats were killed, blood and adipose tissues were collected. Fatty acid synthase, ATP citrate-lyase,
malic enzyme, and SREBP-1 genes expression in adipose
tissue was measured.
Serum progesterone concentration in progesterone-treated rats was higher than in control animals (76.8 ng/ml versus 31.3 ng/ml in female and 29.5 ng/ml versus 1.7 ng/ml
in male). Progesterone treated female rats gained weight
more rapidly than the control animals. In contrast no effect of progesterone on body weight gain in male rats was
found.
Lipogenic enzymes genes expression (measured as mRNA
level and enzyme activity) was significantly higher in
adipose tissue of female rats treated with progesterone
as compared with the control rats. Moreover, SREBP-1c
mRNA level in this group of rats was also significantly
higher. No significant differences in lipogenic enzyme
genes expression in subcutaneous adipose tissue of male
rats treated with progesterone as compared with the control rats were observed.
In the present study we demonstrated sex-related differences in lipogenic enzymes genes expression in subcutaneous adipose tissue by progesterone treatment. Moreover, our data suggested that SREBP-1c plays important
role in progesterone-induced stimulation of lipogenic
enzyme genes expression in female rats.

Vol. 55

P9.17

P9.18

Effect of platelet-activating factor (PAF) on
motility, capacitation and acrosome reaction
of frozen-thawed boar spermatozoa

217

Regulation of hormone-sensitive
lipase/cholesterol esterase (LIPE) gene
expression by transcription factor C/
EBPα in the adrenal cortical cells

Władysław Kordan, Marek
Lecewicz, Jakub Tobolski
Department of Animal Biochemistry and Biotechnology,
University of Warmia and Mazury in Olsztyn, OlsztynKortowo, Poland
e-mail: Władysław Kordan <wladyslaw.kordan@uwm.
edu.pl>
Fertility in mammals involves a series of specific receptor-ligand interactions between different substances during sperm-oocyte fusion. Capacitation and subsequent
acrosome reaction are important events in the process
of sperm-egg fertilization. It has been demonstrated that
platelet activating factor (1-O-alkyl-2-acetyl-sn-glycero3phosphorylcholine, PAF) can influence the fertilization
processes by modulating motility, capacitation and the
acrosome reaction in spermatozoa. However, there is no
information in the literature regarding the effect of PAF
on sperm function following semen cryopreservation.The
aims of this study were to 1) analyze sperm motility and
viability and 2) determine if frozen-thawed boar spermatozoa could undergo capacitation and acrosome reaction
following the supplementation of different concentrations
of PAF to the thawing medium.Ejaculates, collected from
four Large White race boars, were frozen using the Kortowo freezing protocol (Strzeżek et al., 1985). PAF used
at concentrations ranging from 1 × 10–8 to 1 × 10–5M was
supplemented to semen samples prior to and after freezing-thawing. Motility parameters were assessed using
a CASA system. Sperm viability was assessed using the
Live/DeadÒ Sperm viability kit (Molecular Probes). The
antibiotic chlorotetracycline was used to monitor sperm
capacitation status (Saling & Storey, 1979), whereas the
Giemsa staining method was used to assess the sperm
acrosomal status (Watson, 1975). It was confirmed that
the supplementation of PAF at concentrations ranging
from 1 × 10–5 to 1 × 10–6M had a beneficial effect on postthaw sperm quality characteristics. This phenomenon was
manifested in enhanced motility and survivability of the
frozen-thawed spermatozoa. Furthermore, PAF used at a
concentration of 1 × 10–5M caused a marked increase in
the percentage of membrane-intact frozen-thawed spermatozoa. In addition, the supplementation of 1 × 10–5M
PAF to the thawing medium seemed to induce capacitation and acrosome reaction in frozen-thawed spermatozoa. It appeared that PAF used at lower concentrations
did not have any significant effect on capacitation and
the acrosomal status of frozen-thawed boar spermatozoa.
The findings of this study emphasized the role of PAF in
mammalian reproductive processes.
References:
Saling, Storey (1979) J Cell Biol 83: 544–555.
Strzeżek J et al. (1985) Med Wet 6: 349–353.
Watson PF (1975) Vet Rec 97: 12–15.

Acknowledgements:
This study was supported by funds from UWM in Olsztyn
(0103.0206)

Maciej Czajkowski, Katarzyna Kulcenty,
Marcin Hołysz, Wiesław H. Trzeciak
Department of Biochemistry and Molecular Biology,
University of Medical Sciences in Poznań, Poznań, Poland
e-mail: Marcin Hołysz <mholysz@ump.edu.pl>
Experiments on human and animal cell lines revealed that
the steroidogenic process is regulated by corticotropin
(ACTH). This hormone activates protein kinase A (PKA),
which phosphorylates and thereby activates hormonesensitive lipase/cholesterol esterase (HSL) responsible for
cholesterol supply for steroid hormone synthesis.
We demonstrated that in the adrenal cortical cells Y-1
expression of LIPE gene, encoding hormone-sensitive
lipase/cholesterol esterase, was induced by activation of
the PKA signaling pathway. The analysis of the LIPE promoter sequence indicated that a number of transcription
factors might bind to this region. One of them is C/EBPα,
which binds to the proximal region of the promoter, between –46 and –59 bp. The overexpression of C/EBPα in
these cells increased transcriptional activity of the LIPE
promoter. The expression of the C/EBPα gene in these
cells was confirmed by detection of the C/EBPα transcript
by real-time qPCR and the presence of the C/EBPα protein by Western blotting.
Transfection of Y-1 cells with the vector containing the
LIPE promoter fragments, and co-transfection with the
vector overexpressing C/EBPα confirmed that LIPE transcription was stimulated by C/EBPα and probably its coregulators GATA-2 and GATA-3, which bind to the proximal region of the promoter between –30 and –60 bp.
To confirm that protein kinase A and C/EBPα took part in
the stimulation of LIPE expression, the cells were transfected with the vector overexpressing C/EBPα, along with
the vector containing the LIPE promoter fragments and
simultaneously were incubated with the activator of adenylyl cyclase – forskolin.
It is postulated that the cAMP-mediated activation of LIPE
expression is independent of the stimulatory effect of C/
EBPα. However, the possibility that C/EBPα and GATA
proteins may be phosphorylated by protein kinase A can
not be excluded.

218

2008

P9.19

P9.20

Regulation of hormone-sensitive
lipase/cholesterol esterase (LIPE) gene
expression by steroidogenic factor 1
(SF-1) in the adrenal cortical cells

The content of protein as well as fat, free fatty
acids and phospholipids in cotyledons of
developing seeds of Andean lupine (Lupinus
mutabilis Sweet) growing in vitro in various
conditions of carbon and nitrogen nutrition

Katarzyna Kulcenty, Maciej Czajkowski,
Marcin Hołysz, Wiesław H. Trzeciak
Department of Biochemistry and Molecular Biology,
University of Medical Sciences in Poznań, Poznań, Poland
e-mail: Katarzyna Kulcenty <mholysz@ump.edu.pl>
Synthesis of steroid hormones in the adrenal cortex is regulated by corticotropin (ACTH). This hormone activates
adenylyl cyclase, which results in increased synthesis of
cAMP and activation of protein kinase A (PKA).
A common transcription factor controlling gene expression via protein kinase A in the adrenal cortex is steroidogenic factor 1 (SF-1) also known as adrenal binding
protein-4 (Ad4BP). It regulates the expression of several
genes encoding cytochromes involved in steroid hormone synthesis as well as cholesterol transporters and the
ACTH receptor. An important step in steroid hormone
synthesis is free cholesterol supply by hormone-sensitive
lipase/cholesterol esterase (HSL) which hydrolyses cholesterol esters stored in the lipid droplets. It is not known,
however, whether SF-1 is involved in the expression of
the LIPE gene, encoding HSL. The aim of our investigations was to elucidate the mechanism of regulation of
LIPE gene expression by ACTH and to determine if SF-1
plays a role in this process.
The investigations were conducted in adrenocortical Y-1
cells in culture. Specific mRNA levels were determines by
real time PCR and the activities of SF-1 and LIPE promoters were estimated by means of luciferase reporter gene
expression. To investigate the role of SF-1 in the regulation of LIPE expression, the cells were transfected with
plasmids harbouring -343, -1150 and -2150 bp fragments
of the LIPE promoter fused to the firefly luciferase reporter gene and the transfection efficiency was normalised to
the Renilla luciferase.
It was evidenced that overexpression of SF-1 significantly
increased transcriptional activity of the LIPE promoter
fragments of -1150 and -2150 bp, but not -343 bp. The
analysis of LIPE promoter sequence revealed SF-1 binding sites at around -1400 bp. Electrophoretic mobility
shift assay (EMSA) using specific probe confirmed that
the interactions between SF-1 and the identified sequence
of the LIPE promoter were direct. To reveal the importance of SF-1 in PKA-dependent LIPE gene expression,
the SF-1 gene, was silenced using specific siRNA leading
to a significant reduction in transcriptional activity of the
LIPE gene. It was concluded that in the adrenocortical
cells SF-1 plays an essential role in the protein kinase A
-mediated regulation of LIPE gene transcription.

Sławomir Borek1, Stanisława Pukacka2,
Krzysztof Michalski3, Lech Ratajczak1
1Department

of Plant Physiology, Faculty of Biology,
Adam Mickiewicz University, Poznań, Poland; 2Institute
of Dendrology PAS, Kórnik, Poland; 3Plant Breeding and
Acclimatization Institute, Poznań, Poland
e-mail: Lech Ratajczak <borek@main.amu.edu.pl>
Accumulation of storage compounds in developing
seeds is very important for the following germination. It
is also important to human, for example as a source of
high quality oil or animal fodder. Could the protein seeds
of lupines be a good alternative to widely used soy? We
know that the quantity and quality of storage compounds
in seeds closely depend on cultivation conditions.
In lupine seeds, five (I-V) developmental stages can be distinguished upon morphological and anatomical features.
Total lipid level in whole developing seeds obtained from
field cultivation in 2007 year was determined. Lipid content
was higher in consecutive developmental stages. The maximum level was achieved in stage V and was about 12% of
fresh weight. In stage III endosperm disappears and lipid
content is about 50% of maximum level. In this stage cotyledons were isolated and cultivated in vitro for 96 hours on the
Heller’s medium with 60 mM sucrose or without the sugar.
The medium was additionally enriched in 35 mM asparagine (Asn) or 35 mM NaNO3. Light conditions were 75 mM
light quantum × m–2 × s–1. Soluble protein, total lipid content, free fatty acids and phospholipids were determined.
Protein content in cotyledons growing on medium without
sucrose, Asn and NaNO3 was the same as in cotyledons
used for the preparation of in vitro culture. Addition of sucrose to the medium caused an increase in protein content
and addition of Asn (with and without sucrose) resulted in
even greater increase in protein content. Similar effect was
caused by NaNO3, but it was weaker than in the case of Asn.
Addition of sucrose increased lipid level as well; however,
Asn inhibited this process. This effect was more intensive
in cotyledons growing on medium without the sugar. Phosphatidylinositol, phosphatidylserine, phosphatidylcholine,
phosphatidylglycerol and phosphatidylethanolamine were
detected. The highest concentration was found for phosphatidylcholine. The content of almost all phospholipids
was smaller in cotyledons growing on the medium with
Asn (with and without sucrose). Palmitic, stearic, oleic, linoleic, linolenoic and eicozic acid were determined. The highest concentration was found for linoleic and oleic acids. No
erucic acid was found. Our results show that higher accumulation of protein and lipid in developing cotyledons can
be achieved by good sucrose supply. However, in Andean
lupine seeds there is negative correlation between protein
and lipid accumulation.
Acknowledgements:
This work was supported by grant no. 2 P06A 004 29 from science fundings in years 2005–2008.

Vol. 55

P9.21

P9.22

Sucrose regulates activity of acyl-CoA oxidase
and PEP carboxykinase in germinating seeds
of yellow lupine (Lupinus luteus L.), white
lupine (Lupinus albus L.), and Andean lupine
(Lupinus mutabilis Sweet) growing in vitro

219

Characterisation of glycoforms
of ascitic fluids by lectins

Sławomir Borek, Lech Ratajczak
Department of Plant Physiology, Faculty of Biology, Adam
Mickiewicz University, Poznań, Poland
e-mail: Sławomir Borek <borek@main.amu.edu.pl>
Storage lipids, accumulated in developing seeds, are
converted into glucose and sucrose during germination.
Through catabolic and anabolic repression these sugars
can regulate expression of hundreds of genes. We know
that sugars repress degradation of storage carbohydrates,
proteins and lipids by catabolic repression. Regulatory
role of sugars is studied almost only in plants which
seeds are rich in starch or lipids. Protein seeds are not
investigated in those experiments.
Activity of acyl-CoA oxidase (β-oxidation) and PEP carboxykinase (gluconeogenesis) was assayed in isolated
embryo axes, excised cotyledons as well as in seedlings
axes and cotyledons growing in vitro for 96 hours in darkness on Heller’s medium with 60 mM sucrose or without
the sugar. We used seeds of three lupine species which
differ significantly in storage lipid content. Yellow lupine
contains ca. 6% of lipid in dry mass, white lupine 7–14%
and Andean lupine up to 20%.
Both enzymes were more active (calculated per gram
fresh weight) in excised and seedling cotyledons than
in embryo axes. Acyl-CoA oxidase in organs of yellow
lupine was more active in sugar deficiency, whereas in
white and Andean lupine the enzyme was more active in
organs growing on medium supplemented with sucrose.
PEP carboxykinase was more active in organs fed with
sucrose in each of the three lupine species.
The results are very difficult to interpret. The increase in
acyl-CoA oxidase activity in sugar-deficient conditions
can be explained upon catabolic repression, but only in
yellow lupine. It is striking that the activity of acyl-CoA
oxidase in white and Andean lupine as well as activity of
PEP carboxykinase in each of the three lupine species is
higher in organs growing on medium with sucrose. Probably this caused by specific features of protein seeds, in
which nitrogen metabolism is complex and there is intensive degradation of storage lipids in conditions of appropriate supply of tissues in basic respiratory substrate.
This issue, however, requires further investigations with
the use of molecular biology techniques.
Acknowledgements:
This work was supported by grant no. 2 P06A 004 29 from science fundings in years 2005-2008.

Iwona Radziejewska1, Małgorzata
Borzym-Kluczyk2, Joanna Wosek1
1Department

of Medical Chemistry, 2Department of
Pharmaceutical Biochemistry, Medical University of
Białystok, Białystok, Poland
e-mail: Iwona Radziejewska <iwona@umwb.edu.pl>
Ascitic fluid is a pathologic fluid which is accumulated
within the abdominal cavity. It is observed especially in
patients with cirrhosis, other severe liver diseases and
many cancers. Malignant ascites represent about 10% of
all cases. They are a manifestation of advanced malignant disease that is associated with a poor diagnosis and
significant morbidity. The exact mechanisms of ascites
formation are poorly understood. It is suggested that
lymphatic obstruction, immune modulators, vascular
permeability factors and metalloproteinases are involved
in these mechanisms. Because of a big variability of the
ascitic fluids, they have not been well characterized so far.
One of their components is a soluble form of MUC 1 mucin, highly O-glycosylated glycoprotein, carrier of many
various glycoform structures. It is known that in cancers
glycosylation of MUC 1 is altered, oligosaccharides are
reduced in chain lengths, the protein core is more densely glycosylated, very often a relatively higher amount of
sialic acid can be observed. The main goal of our study
was characterization of glycoform structures of ascitic fluids taken from patients with different clinical events. To
perform this ELISA tests with biotinylated lectins where
used. Results could be considered as useful in discrimination between benign and malignant diseases.

220

2008

P9.23

P9.24

Expression of MUC1 mucin in human
umbilical vein endothelial cells (HUVECs)

The conversion of cholesterol into derivatives
by the enzyme(s) located in the lysosomal
membranes of human placenta

Joanna Wosek1, Halina Porowska1, Krzysztof
Wnuczko2, Marek Szczepański2
1Department

of Medical Chemistry, 2Department of
Neonatology, Medical University of Białystok, Białystok,
Poland
e-mail: Joanna Wosek <wosekk@wp.pl>
Mucins are high-molecular-weight O-glycosylated proteins (50–80% of their mass is due to O-linked carbohydrate chains) that participate in the protection, lubrication, and acid resistance of the epithelial surface. Each
organ or tissue exhibits specific pattern of MUC gene
expression. MUC1 mucin is a type I transmembrane
glycoprotein, which level increases with malignant transformation. MUC1 is normally expressed at the apical
borders of glandular epithelial cells, by contrast, the polarization of MUC1 expression is lost in carcinoma cells.
Proposed functions for MUC1 include modulation of cell
adhesion, signal transduction, lubrication and hydration
of epithelial surfaces, and protection of epithelial surfaces
against infection. Although expression of transmembrane
mucins was originally thought to be restricted to epithelial tissues, in some recent studies MUC1 and MUC4 was
detected in several types of endothelial cells. This fact
directed us to search of MUC1 mucin on the surface of
human umbilical vein endothelial cells (HUVECs). The
expression of MUC1 mucin in HUVE cells was examined
by the method of Western blotting, ELISA and flow cytometry. The influence of TNF-α and interferon-gamma
on the expression and shedding of MUC1 as well as on
cell adhesion to ECM proteins was also tested. Our experiments confirmed the expression of MUC1 on the
surface of HUVECs, which was increased after treatment
with cytokines, like as α2β1 and α5β1 integrins expression
in the cells treated with TNF-α. Culture media contain
MUC1 glycoprotein in greater quantity than lysates, and
molecular weight of this protein was higher in medium
than in lysate. Shedding of MUC1 to medium was increased after incubation with cytokines. TNF-α treatment
caused a decrease in sialic acid level and T antigen level
was not changed. The adhesion to fibronectin was slightly increased in cells treated with TNF-α, but adhesion to
collagen type I was not changed. Vascular endothelial
cells may play an important role in angiogenesis, a critical process in wound-healing, inflammation, embriogenesis cancer and development. Expression of adhesion
and anti-adhesion molecules plays an important role in
the interaction of tumor cells with vascular endothelial
cells during tumor invasion and metastasis. The presence
of transmembrane mucin MUC1 in HUVECs may have
implication in interactions with different type of cells in
physiological and pathological processes.

Katarzyna Roszek, Wioletta Werner,
Michał Komoszyński
Biochemistry Department, Institute of General and Molecular
Biology, Nicolaus Copernicus University, Toruń, Poland
e-mail: Katarzyna Roszek <kroszek@umk.pl>
It is well known that cholesterol plays a crucial role in
maintaining the proper fluidity of the membranes. We
adapted the enzymatic CHOD/PAP method of cholesterol quantification [1] to the examination of cholesterol
metabolism in the lysosomal membranes. Our data indicate that in the lysosomal membranes of human placenta
there is an enzyme(s) converting cholesterol into its derivatives. Addition of 5 mM cholesterol to the lysosomal
membranes and incubation for 10 to 120 minutes effected in the linear decrease in cholesterol concentration. It
depended on the time of incubation and the amount of
membranes used. These data suggest the enzymatic conversion of cholesterol into its derivatives. Optimum pH
for the examined reaction was 5.0. The decrease in cholesterol concentration was also catalyzed by the partially
purified preparation of cholesterol sulphate sulphohydrolase [2], what excludes the non-enzymatic interactions
between lysosomal membranes and cholesterol. Besides,
cholesterol sulphate sulphohydrolase may be a part of
the multienzymatic complex involved in regulation of
cholesterol content in the lysosomal membrane.Our studies with HPLC method were focused on the qualitative
analyses of emerging cholesterol derivatives. The results
revealed that a steroid derivative with more hydrophilic
properties appeared simultaneously with the decrease in
cholesterol concentration. Currently, our research aims
at the identification of this compound. Concluding remarks: 1/ The concentration of membranous cholesterol
is precisely controlled since it influences the stability of
the membranes. 2/ Enzymatic conversion of cholesterol
may be the simple way for the regulation of cholesterol
content and lysosomal membranes fluidity.
References:
1. Richmond W (1973) Clin Chem 19: 1350–1356.
2. Roszek K, Gniot-Szulżycka J (2008) J Steroid Biochem Mol Biol
(in press).

Vol. 55

221

P9.25

P9.26

Dose-dependent effect of catechin on Pyruvate
Dehydrogenase Kinase (PDK) activity in rats

Does obestatin play a functional role in
metabolism of white adipocytes?

Małgorzata Tyszka-Czochara1, Joanna
Gdula-Argasińska1, Sylwia BobisWozowicz2, Ewa Leśko2, Beata Bystrowska3,
Marcin Majka2, Jerzy Jaskiewicz1

Dawid Szczepankiewicz, Ewa PruszyńskaOszmałek, Maciej Sassek, Marek Skrzypski,
Iwona Hertig, Agnieszka Wojtkowiak, Wojciech
Szlachcic, Paweł Maćkowiak, Krzysztof W. Nowak

1Department

Department of Animal Physiology and Biochemistry,
University of Life Sciences, Poznań, Poland
e-mail: Dawid Szczepankiewicz <dawidsz@up.poznan.pl>

of Analitycal Biochemistry, 2Department of
Transplantology, 3Department of Toxicology, Faculty of
Pharmacy, Collegium Medicum, Jagiellonian University,
Kraków, Poland
e-mail: Malgorzata Tyszka-Czochara <mtyszka@poczta.
fm>
Catechin is a polyphenolic compound found in a human
diet, especially in beverages such as tea. Current interest of
biological activities of polyphenolic compounds is due to
their antioxidant properties. Anti-hepatotoxic action of Catechin has been reported. Additionally, it was suggested that
Catechin may influence carbohydrate and lipid metabolism
but the molecular mechanism of influence on metabolic
pathways is still unclear. It was demonstrated in previous
work that Catechin affects both Pyruvate Dehydrogenase
Kinase (PDK) activity and PDK isoenzymes protein amount
in rats hepatocyte culture but the range and features of the
influence in vivo has not been studied yet.
PDK phosphorylates and causes inactivation of Pyruvate
Dehydrogenase Complex (PDH). The irreversible reaction
of decarboxylation of Pyruvate to AcetylCoA catalysed by
PDH links glycolysis with Citric Acid Cycle and, indirectly, with Fatty Acids synthesis in a liver. Precisely balanced
carbohydrate and lipid metabolism is critical to the whole
body homeostasis. Therefore PDH Complex is one of the
key enzymes which influences glucose as well as lipid metabolism. On the other hand PDK is a subject to regulation
by dietary factors and xenobiotics. Therefore it is of interest
to find out if Catechin influences PDK activity in vivo.
Aim of the study: This study investigated the dose-dependent effect of Catechin on PDK in rats in order to find
out how a diet enriched with established doses of polyphenolic compound influences PDK activity in a rat liver.
Diet and animals: Wistar male rats were fed ad libidum
for 14 days with a chow diet and each day were administred Catechin at several doses ranging from 0.5 mg/kg
body weight to 200 mg/kg body weight. Control rats were
administred dissolvent only. After two weeks of experiment rats were sacrificed and livers were collected.
PDK activity assay: PDK activity was measured by Arylamine Acetyltransferase coupled assay. The activity of
PDK was expressed as first order rate constant of inactivaction of the PDH complex by the PDK over period of
incubation of probe with ATP.
Results: It was established that Catechin caused a significant decrease in PDK activity after administration of 2.5
mg of phenolic acid/kg body weight as well as 1 mg/kg
body weight when compared to the control group. The
dose of polyphenol 2.5 mg/kg body weight was the most
effective in the reduction of PDK activity. In contrast, a
higher dose of Catechin, 200 mg/kg body weight/day, increased PDK activity.

Ghrelin and obestatin are encoded by the preproghrelin gene and originate from posttranslational processing
of the preproghrelin peptide. Obestatin like a ghrelin is
present in the stomach, but in contrast to ghrelin, obestatin inhibits appetite. Preliminary, it was found that obestatin acts by GPR39 receptor. However, it was presented
that obestatin may acts by other type of receptor like glukagon-like peptide 1 receptor (GLP-1R).
In our study, we analyzed obestatin action on lipolysis
and lipogenesis in isolated rat adipocytes. We also studied changes of expression of putative obestatin receptors: GPR39 receptor and GLP-1 receptor in adipocytes.
Moreover, we investigated expression of ghrelin receptor
(GHSR-1a).
Adipocytes were isolated from epididymal fat pad of
Wistar rats according to the method of Rodbell (1964). Isolated adipocytes (106 cells/ml) were incubated with KBRHEPES buffer supplemented with obestatin at concentration 1, 10 and 100 nM, in the absence (basal) or presence
of adrenalin. Incubations were carried out at 37oC for 120
min. The lipolysis was determined by measuring the level
of released glycerol in incubation medium.
Lipogenensis was studied as the incorporation of [U-14C]
glucose into lipids. Adipocytes (106 cells/ml) in the same
medium as described above without (basal) or with insulin, in the additional presence of [U-14C] glucose. Fraction
of lipids was added to scintillation liquid for β-counting.
Total RNA was isolated from adipocytes using TriPure reagent (Roche). Reverse transcription was undertaken using ImProm-II Reverse Transcription System (Promega).
The cDNA was amplified by multiplex PCR using primers and universal probes (Roche) for GHSR-1a, GPR39
and GLP-1R and internal standard GAPDH. Analysis
was made using LightCycler Software Version 4.5 and
was based on the relative quantification method with efficiency correction.
Our results for the first time present that obestatin influences on lipolysis and lipogenesis. In our study, we found
that glycerol concentration was significantly higher in
comparison to the control with higher concentration of
obestatin in adrenalin-stimulated adipocytes. We also
observed that obestatin significantly inhibited lipogenesis in both basal and insulin-stimulated adipocytes. In
addition, we presented expression of GHSR-1a. We also
found, that GPR39 and GLP-1R expression was present in
adipocytes. Moreover, we observed that GPR39 had significantly higher expression after obestatin stimulation in
comparison to the control.

222

2008

P9.27

P9.28

Nuclear hormone receptors LXR and PPAR in
the regulation of glucose and lipid homeostasis

The role of peroxisome proliferator-activated
receptors (PPARs) in the regulation of adipokine
gene expression in isolated mature adipocytes

Zdzislaw Kochan
Gdańsk, Poland
e-mail: Zdzislaw Kochan <kochanz@amg.gda.pl>
The liver X receptors (LXRα and β) and peroxisome
proliferator-activated receptors (PPARα, β/δ, and γ) are
members of the nuclear hormone receptor family, a large
family of ligand-dependent transcription factors that coordinate gene expression in response to hormonal and
metabolic signals. The binding of ligand to these receptors usually induces the release of corepressors and the
recruitment of coactivators, thus triggering the transcription of target genes. Downstream targets of LXR include
genes involved in the regulation of cholesterol homeostasis: 7α-hydroxylase (CYP7A1), the lipid scavenger
receptors (SR-B1 and FAT/CD36), and members of the
ABC family of membrane transporters (ABCA1, ABCG1,
ABCG5 and ABCG8). Moreover, LXRs and PPARs activate the transcription of genes that affect uptake of free
fatty acids–lipoprotein lipase (LPL) and fatty acid transport protein (FATP); stimulate de novo lipogenesis and
promote lipid storage in the form of triacylglycerols fatty acid synthase (FAS), acetyl-CoA carboxylase (ACC)
and stearoyl-CoA desaturase (SCD1). Among direct targets of these nuclear receptors is also a major regulator of
lipogenesis–sterol regulatory element binding protein 1c
(SREBP-1c). In adipose tissue, PPARγ induces the expression of genes which promote recycling of intracellular
fatty acids and glycerol–glycerol kinase (GyK) and phosphoenolpyruvate carboxykinase (PEPCK). Both LXRs
and PPARs appear to serve as lipid sensors, as they are
activated by oxidized cholesterol and naturally occurring
fatty acids or fatty acid derivatives, respectively. Moreover, it has recently been demonstrated that LXR may bind
glucose, thus integrating glucose sensing and regulation
of lipid metabolism. Considering their role in the regulation of glucose and lipid homeostasis, LXRs and PPARs
may represent promising therapeutic targets for many
common disorders, including obesity, type 2 diabetes,
hyperlipidaemia, and atherosclerosis.

Joanna Karbowska
Gdańsk, Poland
e-mail: Joanna Karbowska <jonka@amg.gda.pl>
Nuclear receptors from the PPAR family are ligand-activated transcription factors involved in the regulation
of gene expression. So far, three isoforms of PPARs have
been described: PPARα, which is most abundant in the
liver, kidney and heart; the ubiquitously expressed
PPARβ/δ; and PPARγ, which is highly expressed in fat
cells. PPARγ is adipocyte predominant transcription factor and ultimate effector of adipogenesis. During adipocyte differentiation, the genes encoding adiponectin,
resistin and leptin are induced; thus to examine the sole
effect of PPAR activation on these adipokines we measured gene expression and protein release of adiponectin,
resistin and leptin in isolated mature fat cells incubated
with agonists for PPARα, PPARβ/δ and PPARγ.
Mature adipocytes were prepared from epididymal fat
pads of male Wistar rats by collagenase digestion. Freshly
isolated cells were incubated in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 25 mM glucose and 10% fetal bovine serum and treated with PPAR
agonists for 24 h at 37°C, 5% CO2. Total RNA was then
extracted from adipocytes and the mRNA expression of
adiponectin, resistin and leptin was quantified by realtime RT-PCR.
Leptin gene expression in mature adipocytes was downregulated by PPARγ ligands. In contrast, PPARγ ligands
induced gene expression of resistin in these cells. Moreover, this effect was more pronounced when dual agonists
of PPARα and PPARγ were used, confirming the role of
PPARα in the regulation of resistin transcription. Unexpectedly, agonists of PPARγ had no effect on adiponectin
gene expression in isolated rat fat cells.
These results indicate that ligand-dependent activation of
PPARs modulates the endocrine functions of isolated mature adipocytes in a different manner than in adipocyte
cell lines.

Vol. 55

223

P9.29

P9.30

Effect of food restriction on glycogen
synthase activity in rat heart

Effect of orexin A on lipid metabolism
in isolated rat’s white adipocytes

Ewa Kossowska, Dorota Kazimierska,
Paulina Pawłowska

Tatiana Wojciechowicz, Marek Skrzypski, Ewa
Pruszyńska-Oszmałek, Dawid Szczepankiewicz,
Przemysław Kaczmarek, Maciej Sassek,
Emilia Kotowska, Krzysztof W. Nowak

Gdańsk, Poland
e-mail: Ewa Kossowska <ewakos@amg.gda.pl>
Myocardial glycogen may be considered as a depot form
of glucose. It is generally belived to be synthesized by
myocardiocytes under conditions of rich glucose supply.
Cardioprotective effect of glycogen have been proposed
as a protective mechanisms against ischemic heart stress.
Glycogen is directly synthesized from UDPG by glycogen synthase-enzyme existing in two forms: “I”(active),
and “D”(less active, requiring G-6-P for its further activation). During fasting the amount of myocardial glycogen
increas. Our previous studies demonstrated an increase
in myocardial glycogen content in rats kept 30 days at the
restricted diet (50% of “ad libitum” diet) in comparison
with rats at the unrestricted diet (control group). After
subsequent two days of refeeding the content of the myocardial glycogen in the investigated group of rats was increased. The aim of the present study was to investigate
how the restriction diet alone as well as in combination
with the subsequent refeeding can modify glycogen synthase activity in the hearts of rats. The experimental results showed stimulation of both forms of glycogen synthase by the restricted diet. In cardiomyocytes of the rats
refeeded after the previous limited fasting, the activity
of the enzyme was higher than in not refeeded rats. The
observed increase of glycogen amount in cardiomyocytes
of rats kept at described experimental conditions seems
probably be a result of glycogen synthase induction.

Department of Animal Physiology and Biochemistry,
University of Life Sciences, Poznań, Poland
e-mail: Tatiana Wojciechowicz <tatianawojciechowicz@
wp.pl>
Orexin-A and orexin-B (hypocretins), hypothalmic neuropeptides, were identified in neural cells of the lateral
hypothalmic area (LHA). Both peptides are coded by the
same gene (HCRT). They are widely distributed throughout the body, interacting with two G protein-coupled
receptors (GPCR), orexin receptor-1 (OX1R) and orexin
receptor-2 (OX2R). Orexins play a predominant role in
feeding and sleep behavior. Orexins and their receptors
were also found in the peripheral tissues including white
adipose tissue. In our study we focused on effects of orexin A on lipid metabolism (lipolysis and lipogenesis) in
isolated rat’s adipocytes.
Adipocytes were isolated from epididymal fat pad of
Wistar rats according to the method of Rodbell (1964).
Isolated adipocytes (106 cells/ml) were incubated with
KBR-HEPES buffer supplemented with orexin A at concentration 1, 10 and 100 nM, in the absence (basal) or presence of epinephrine. Incubations were carried out at 37oC
for 120 min. After incubation media were explanted and
stored at –80oC. The lipolysis was determined by measuring the level of released glycerol in incubation medium
using a commercially available colorimetric kit.
Lipogenensis was studied as the incorporation of [U-14C]
glucose into lipids. Adipocytes (106cells/ml) were placed
in the medium supplemented with orexin A at concentration 1, 10 and 100 nM and without (basal) or with insulin,
in the additional presence of [U-14C] glucose. Fractions of
lipids were extracted using Dole’s solution and added to
scintillation liquid for β-counting.
Our results indicate that orexin A influences on lipolysis
and lipogenesis. In our study, we found that glycerol concentration was significantly lower in group treated with
orexin A when compared to the control. This effect was
observed both, in basal and epinephrine-stimulated lipolysis. We also observed that orexin A stimulated lipogenesis in both basal and insulin-stimulated adipocytes.

224

P9.31
Lipids and insoluble carbohydrates in
thallus cells of the Antarctic lichens
Irena Giełwanowska1,2, Ewa Gojło1,
Ryszard Górecki1, Maria Olech2,3
1Department

of Plant Physiology and Biotechnology,
University of Warmia and Mazury, Olsztyn, Poland;
2Department of Antarctic Biology, PAS, Warszawa, Poland;
3Zdzisław Czeppe Department of Polar Research and
Documentation, Institute of Botany, Jagiellonian University,
Kraków, Poland
e-mail: Irena Giełwanowska <i.gielwanowska@uwm.
edu.pl>
Antarctic lichens are organisms adapted to life under the
conditions of extremely low temperatures, dehydration
and solar radiation. They carry out the process of photosynthesis with the minimum of light and at a temperature
below 0oC, or even below the temperature of ice nucleation in the cellular fluids of the thallus. Lichens stimulate
ice deposition in intercellular spaces, just like vascular
plants. They transform freezing loosely bound water into
tightly bound water, which does not freeze, in order to
avoid freezing of the intracellular water.
We studied the ultrastructure of cell walls and protoplasts of cells in lichen thalli and the location of insoluble polysaccharides and other metabolites in six species
of Antarctic lichens. These are Bryoria forsteri, Caloplaca
regalis, Cetraria aculeata, Ramalina terebrata, Sphaerophorus
globosus and Usnea antarctica.
The lichens were collected in the vicinity of the Polish H.
Arctowski Antarctic Station on King George Island (South
Shetland Islands) during Arctic summer (2006/2007) at
two times: during a sunny warm (about 9–11oC) and
cloudy cool (–1–0oC) day. Several-millimetre thallus fragments were fixed in Carnoy’s fixer (for the PAS reaction
to insoluble polysaccharides) and in 3.5% glutaraldehyde
in 0.5M phosphate buffer at pH 7.0–7.2 for the anatomical (in light) and ultrastructural (in transmission electron
microscope) observations.
In all the studied lichen species, both in the thalli collected on a sunny and cloudy day, algal cells had a similar
protoplast structure. The occurrence of a wide space between the cell wall and the protoplast, as well as a very
tight arrangement of all cell organelles, was their most
characteristic feature. PAS positive starch grains, areas
of cytoplasm with lipid grains and huge drops of dense
osmophilic material were visible in the central part of the
photobionts. This material periodically accumulated on
the protoplast periphery, in the area close to the wall. The
same material filled intercellular spaces in thalli and occurred inside the mycobionts.

2008

Lipids and carbohydrates biochemistry

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