L14.1 Session 14: Clinical Biochemistry Lectures L14.2

Transkrypt

Session 14: Clinical Biochemistry
Lectures
L14.1
L14.2
Systemic connective tissue diseases:
pathophysiology and laboratory diagnosis
Molecular mechanisms of the damaging
effect of glucose on tissues
Grażyna Odrowąż-Sypniewska
Bogdan Solnica
Collegium Medicum NC University, Department of Laboratory
Medicine, Toruń, Poland
Jagiellonian University Medical College, Department of Diagnostics,
Kraków, Poland
e-mail: Grażyna Odrowąż Sypniewska <[email protected]>
e-mail: Bogdan Solnica <[email protected]>
The cause and pathophysiology of immunologic rheumatic
inflammatory diseases is poorly understood however, genetic, hormonal, environmental and immunologic factors
seem to play an important role. These diseases feature autoantibodies and the antigenic reactivity profile of each
may be helpful in establishing the diagnosis. It is suggested
that autoantibodies are pathogenic in these ilnesses. For
unclear reasons, in immunologic rheumatic inflammatory
diseases the organ system damage involves joints, skin,
muscle and exocrine glands. Better understanding a role
for immunologically mediated inflammation in producing
tissue damage by identification of key players will help in
developing highly efficacious therapeutic agents that can be
used for treatment.
Recognition of the autoantibody profile facilitates disease
classification whereas measurement of unspecific serum
inflammatory markers helps to evaluate severity of the clinical disease and the effect of treatment. Among rheumatic
diseases, classic connective tissue diseases such as rheumatoid arthritis (joint inflammation and destruction), juvenile idiopathic arthritis (inflammatory articular disorders
presenting as polyarthritis or oligoarthritis), seronegative
spondyloarthritis (inflammatory symptoms affecting the
spine, the peripheral joints, tendon or ligament insertions),
systemic lupus erythematosus (multisystemic autoimmune
disease affecting the joints, skin, heart, lungs, central nervous system, kidneys and hematopoietic system), Sjogren’s
syndrome (autoimmune disorder affecting salivary and lacrimal glands), antiphospholipid syndrome (a prothrombotic disorder), scleroderma (characterized by excess collagen
deposition in the skin and visceral tissues with vascular abnormalities) and polymyositis/dermatomyositis (idiopathic
inflammatory myopathies) will be discussed. Clinical and
immunological features and the role of traditional and novel serological (anti-cyclic citrullinated peptide autoantibodies, anti-mutated citrullinated vimentin autoantibodies) and
other laboratory tests (hsC-reactive protein, serum amyloid
A, matrix metalloproteinase-3, cartilage oligomeric matrix
protein, C-terminal telopeptides of collagen type II) in the
diagnosis and monitoring of connective tissue diseases will
be presented.
Glucose is the intensively metabolized substrate in human body. It is the main source of energy and is used in
synthesis of glycoprotein polysaccharide components and
a number of other substances including glucuronate, glycans, some organic acids and polyols. Glucose physiologically reaches relatively high concentrations around 5 – 6
mM in both extracellular and intracellular fluid. Diabetes
is a group of metabolic diseases characterized by sustained
hyperglycemia. Elevated blood glucose concentrations occurring in the natural course of the disease and in poorly
controlled patients causes several mechanisms leading to
tissue damage and development of chronic diabetes complications. Hyperglycemia-related tissue damage involves
various processes such as: oxidative stress, increased generation of advanced glycation end-products, increased glucose flux through the polyol and hexosamine pathways and
protein kinase C activation. The common factor triggering
these mechanisms is hyperglycemia-induced mitochondrial
overproduction of the superoxide anion radical. All these
processes are interlinked on several levels and cause not
only direct cell and tissue damage but also activate signaling pathways leading to increased synthesis and releasing
of inflammatory, vasoactive and procoagulatory mediators,
and several extracellular matrix components. These mechanisms cause increased oxidative stress and inflammatory
response, vasoconstriction, hypercoagulability, extracellular
matrix expansion and other processes contributing to microvascular complcations and accelerated atherosclerosis in
diabetes. In clinical practice, the degree of hyperglycemia
trigering all these damaging mechanisms is assessed by
glycemia monitoring and HbA1c assays. Because the treatment interfering with one or more of interlinked mechanisms can not be effective, hyperglycemia is the main target
for therapies preventing complications of diabetes nd the
available biomarkers of oxidative stress, inflammation or
hypercoagulability are not used for the routine evaluation
of patients with diabetes.
45th Annual Meeting of the Polish Biochemical Society
L14.3
Oral Presentations
Acetyl-CoA: to be or not to be for neuronal
and glial cells in encephalopathic brain
O14.1
Andrzej Szutowicz, Hanna Bielarczyk, Agnieszka
Jankowska-Kulawy, Anna Ronowska
Department of Laboratory Medicine, Medical University of Gdańsk,
Gdańsk, Poland
e-mail: Andrzej Szutowicz <[email protected]>
Early inhibition of brain energy metabolism is a characteristic feature of several encephalopathies. Several pathogens, are considered as important neurodegenerative signals. However, mechanisms of preferential loss of central
cholinergic neurons in several brain pathologies remain
unknown. We demonstrate that neuronal acetyl-CoA
metabolic pathways, may be a primary target for neurotoxicity. Several compounds, such as aluminum, zinc, NO
excess, interleukin 1b, hypoglycemia and thiamine deficit
decreased viability and transmitter functions of cholinergic cells both in cellular and whole brain models of neurodegeneration. They decreased mitochondrial acetyl-CoA
more significantly in differentiated than in nondifferentiated cholinergic neurons causing proportional loss of their
viability. Significant inverse correlation was found between
pyruvate dehydrogenase activity, mitochondrial acetyl-CoA
and the rate of cholinergic cell death. Neurotoxins also
decreased, and neuroprotectants restored acetyl-CoA level
in cytoplasmic compartment, resulting in respective shifts
in ACh content and its quantal release. Significant direct
correlation existed between cytoplasmic acetyl-CoA and
cholinergic marker levels in these conditions. Neurotoxic
signals were less harmful for resting microglial or astroglial
than for neuronal cells. However, the stimulation of neuroglia by lipopolysacharide markedly increased their cytotoxic activity along with decrease their acetyl-CoA stores.
On the other hand lipopolysacharide had no direct effect
of cholinergic neuronal cells. Presented data indicate that
in encephalopathic brains the cholinergic neurons viability
and their transmitter functions are affected by alterations
of two functionally different intracellular pools of acetylCoA.
Acknowledgements:
Supported by MNiSW and GUMed projects NN401233333 and St-57.
191
Activity of selected antioxidant
enzymes and degradation of lipids in
the pathogenesis of osteoarthritis
Agnieszka Fitowska1, Beata Hapeta1, Michał Dobrakowski1,
Aleksandra Kasperczyk1, Tomasz Stołtny2, Bogdan Koczy2,
Alina Ostałowska1, Ewa Birkner1, Sławomir Kasperczyk1
1Medical University of Silesia in Katowice, Department of Biochemistry
in Zabrze, Poland; 2 District Orthopaedic Hospital, Department of
Orthopaedics, Piekary Slaskie, Polnad
e-mail: Agnieszka Fitowska <[email protected]>
Introduction: Osteoarthritis is a chronic degenerative
process of multifactor etiology, still largely unexplained.
Recent studies suggest that the advancement of the fundamental processes of disease pathogenesis - the degradation
of articular cartilage and the formation of inflammatory
infiltration within the synovial membrane - correlates to
the amount of released mediators of inflammation and the
resulting reactive oxygen species. In determining the activity of antioxidant enzymes and concentration of lipid peroxidation products in synovial membrane can be indirectly
inferred about its oxidation-reduction status.
Aim: The aim of this study was to compare the activity of
selected antioxidant enzymes, concentrations of the thiol
groups, malondialdehyde, lipid peroxides and lipofuscin in
the physiological synovial membranes and damaged by the
degenerative process.
Material and Methods: The material studied fragments
of the synovial membrane were taken during surgery of the
hip arthroplasty 48 male patients with osteoarthritis (osteoarthritis, OA) and 19 men after traumatic hip fracture who
were the control group. In the supernatants determined activity of superoxide dismutase (SOD) by Oyanagui, catalase (CAT) by Johansson and Borg, glutathione peroxidase
(GPx) according to Paglia, and the protein concentration
by Lowry, thiol groups according to Koster, lipid peroxides
by Södergrena, malondialdehyde (MDA) by Okawy and
lipofuscin according to Tsuchida.
Results: SOD activity was significantly higher in the study
group (OA) than in controls, while GPx and thiol levels
were significantly lower. In terms of catalase activity and
concentrations of protein, lipid peroxides, MDA and lipofuscyny the two groups did not differ significantly.
Conclusions: The results can be concluded that in the
course of degenerative disease comes to the modification
of the antioxidant, but this process appears to have no effect on the intensity of the degradation of lipids.
Abstracts
192
O14.2
O14.3
The influence of gonadoliberin’ analogs on
GnRH receptor’ expression in rat organs
Do the changes in the concentration
of growth hormone and ovarian
hormones caused by administration of
GnRH analogs modify the expression
of hormone-dependent CYP3A
isoforms and pregnane X receptor?
Karolina Kociszewska1, Aleksandra Suszka-Świtek2,
Piotr Czekaj2, Danuta Plewka2, Katarzyna WronaBogus2, Aleksandra Bryzek2, Danuta Kozłowska-Rup2,
Karolina Smorzyk1, Ryszard Wiaderkiewicz2
1Students’ Scientific Society, 2Department of Histology, Medical
University of Silesia, Katowice, Poland
e-mail: Karolina Kociszewska <[email protected]>
Gonadoliberin receptor (GnRHR) constitutes the target
for GnRH-superior decapeptide of hypothalamic-pituitary-gonadal axis and for GnRH analogs receptor’ agonists
and antagonists, which are currently used in oncology and
gynecology. Desensitization of the pituitary gland is the
effect of their work, however mechanism of their action
differs. It is sugessted that extrapituitary GnRHRs participate also in the creation of local axes. In the ovary GnRHR
could take part in changes in the course of the cycle and
evoke the ovulation preceeding pituitary stimulation
The aim of the study was to evaluate the influence of a
long-term GnRH analogs administration on GnRHR
mRNA and protein expression in the pituitary gland and
ovary of SPD mature rat females. In the course of 1-,2and 3-month administration of a low-dose (6ug/kg of b.w.)
of GnRHR agonist (dalarelin) and antagonist (cetrorelix)
and after 1,2 and 4 weeks after the end of their application,
the expression of the GnRHR was evaluated immunohistochemically and by RT-PCR.
After both cetrorelix and dalarelin administration the
number of cells with GnRHR expression in the anterior
pituitary and pituitary pars intermedia decreased. Changes
on the mRNA level confirmed changes of GnRHR protein
expression. In the ovary, the number of corpora lutea containing GnRHR-positive cells considerably increased after
dalarelin administration, however receptor expression kept
on constant, moderate level. It was in contrast to cetrorelix-treated rats where decreasing GnRHR expression was
found in granular and thecal cells of both maturing and
growing follicles. All these changes were reversible.
It was concluded, that GnRHR antagonist causes partial
desensitization of both hypothalamic-pituitary-ovarian and
local axis, in the opposite to agonist, which inhibits only the
main axis, permitting further function of ovarian axis. Desensitization caused by antagonist appears earlier comparing to agonist. Desensitization is reversible, however time
of return to control values varies. Results indicate on probable interaction among GnRH, GnRHR on corticotrophes
and ACTH secretion in the pituitary pars intermedia, what
probably modifies either gondotropins’ secretion and hypothalamic-pituitary-adrenal axis’ function. Outcomes give
bases to creation a new analogs’ generation, which could
hit only local axis. In connection of this, they could be deprived of many undesirable effects resulting from the main
axis inhibition.
Rafał Skowronek1, Piotr Czekaj2, Aleksandra SuszkaŚwitek2, Danuta Kozłowska-Rup2, Aleksandra
Bryzek2, Danuta Plewka2, Ewa Czech2, Wojciech
Maruszczyk1, Anna Wiaderkiewicz2
1Students’ Scientific Society; 2Department of Histology, Medical
University of Silesia, Katowice, Poland
e-mail: Rafał Skowronek <[email protected]>
The expression of some drug-metabolizing enzymes, including cytochromes P450 (CYP), is sexually specific. The
major regulators of sexual dimorphism of CYP expression
are growth hormone (GH) and sex hormones. Temporal
GH secretion is pulsatile in males and continuous in females. Therapy with analogs of GnRH (GnRH-a) results
in repression of hypothalamic-pituitary-gonadal axis. Thus,
the changes in levels of gonadotropins, sex hormones and
— indirectly — GH, can modify the metabolism of drugs
and toxins.
The aim of the study was to indicate, whether the blood
changes of ovarian hormones and GH caused by a longterm administration of a new GnRH receptor’ agonist
— dalarelin and antagonist — cetrorelix influence the
expression of hormone-dependent CYP3A cytochromes
regulated by pregnane X receptor (PXR) within the liver.
SPD adult female rats were administered i. p. with dalarelin
or cetrorelix (6 μg/kg of b.w.). Blood was taken after 2 and
3 months of drug administration and 1 and 2 weeks after
its finishing. The concentration of GH was measured with
ELISA. The concentration of estradiol, testosterone and
progesterone was measured using RIA method. Expression
of CYP3A1, CYP3A2, CYP3A9 and PXR within the liver
acinus was analyzed immunohistochemically. Appropriate
mRNAs (RT-PCR) and proteins (Western blotting) were
identified and quantified by densitometry.
During the period of drug administration, dalarelin only
slightly changed the level of GH, whereas cetrorelix increased the level of GH especially in the light phase of
the 24-hour secretory profile. We didn’t observe significant
changes in the total levels of ovarian hormones. Dalarelin and cetrorelix, in a different way changed the expression, but not the localization of CYP3A isoforms within
the liver. Changes of GH concentration caused by dalarelin
correlated with the increase of ‘male’ CYP3A2/ ‘female’
CYP3A9 expression pattern, while changes caused by cetrorelix were correlated with the enhancement of ‘female’
CYP3A9 expression. These changes were poorly correlated
with PXR expression.
The long-term administration of GnRH-a (especially cetrorelix) causes significant changes in the blood concentration of GH and hepatic CYP3A expressions. Effects
of the therapy with GnRH-a can be potentially associated
with growth disturbances in young individuals and toxic effects caused by modified CYP3A-dependent metabolism
of drugs in females.
O14.4
Posters
Influence of monocyte chemoattractant
protein 1 on adventitia cell migration
P14.1
Joanna Soin1, Małgorzata Dutkiewicz1,
Piotr Religa2,3, Katarzyna Koziak1
1Department of General and Nutritional Biochemistry, The Warsaw
University of Medicine, Warsaw, Poland; 2Karolinska Hospital, Karolinska
Institutet, Stockholm, Sweden; 3Department of Internal Medicine, The
Warsaw University of Medicine, Warsaw, Poland
193
Maternal and newborn total plasma and red
blood cells homocysteine and birth defects
Andrew R. Plotsky1, Aleksander V. Naumov1, Elena A. Sergey2
1Grodno State Medical University, Grodno, Belarus; 2Grodno Regional
Perinatal Center, Grodno, Belarus
e-mail: Joanna Soin <[email protected]>
e-mail: Plotsky Andrew <[email protected]>
The major cause of transplanted organs dysfunction is vasculopathy characterized by vessel inflammation and intimal
hyperplasia due to the migration of smooth muscle cells
(SMCs) for example. Possible sources of these cells can be
stem cells, progenitor cells, or other cells after growth factors transformation.
Our experiments indicated that in neointima formation
SMCs and adventitia cells are involved [1].
We would like to establish what agents contribute to adventitia cell migration into the vessel intima.
We performed the migration tests (Boyden chamber
method) with SMCs and adventitia cells tissue cultures to
estimate the influence of the different chemoattractans.
Monocyte chemoattractant protein 1 (MCP-1) caused the
significant increase in both SMCs and adventitia cell migration.
Background: Mild hyperhomocysteinemia has been associated with birth defects and other adverse outcomes of
pregnancy — abruption of placenta, preeclampsia, unexplained fetal loss. Most of researches are devoted studying
of maternal plasma homocysteine. Data on level of total
homocysteine (tHcy) in the umbilical cord in a cases of
fetal malformations are not sufficient and controversial.
Objective: We aimed to assess the level of total homocysteine both in maternal blood and blood from umbilical
cord on normal pregnancy and in cases of congenital birth
defects.
Methods: We studied 15 women with birth defects offspring — anomalies of central nervous system, cardiac defects and clefts, and 17 women with a healthy children as
control. Fetal malformations were diagnosed at antenatal
period and confirmed after birth. Time of delivery was 38 40 weeks. Venous maternal blood and placental blood from
umbilical cord were taken immediately after delivery of babies. Total homocysteine was determined by high-performance liquid chromatography (HPLC) with fluorescent detection. Nonparametric statistical analysis was performed.
Results: There were no differences between delivering babies on bodyweight, Apgar score. Maternal and newborn
plasma total homocysteine concentrations according case
and control status did not differ (p > 0.05). Newborn concentrations were lower than maternal values. Maternal red
blood cells total homocysteine were significantly higher in
the study group as compared with controls — 1.95 (1.36–
2.06) mcM/L and 1.25 (0.96–1.52) mcM/L respectively (р
= 0.043). We found no differences in the level of total homocysteine in newborn red blood cells (p > 0.05).
Conclusions: We found no differences in level of tHcy in
the blood plasma both mothers and newborns, and it’s at
some extend contradicts the published data. This is probably due to the timing of study, namely, at term pregnancy.
From the other side we found significantly increased level
of tHcy in red blood cells of mothers with affected offspring. Probably high level of tHcy in cells leads to dramatic events during embryogenesis due to accumulations of
S-adenosyl homocysteine and inhibitions of methyltransferase reactions.
Reference:
Religa P et al. (2009) PlosOne 4: e4187 .
194
Abstracts
P14.2
P14.3
Cytotoxic impact of phenolic compounds
derived from four Lamiaceae species
on human breast cancer cells
Thiol levels, protein carbonylation
and anaerobic sulfur metabolism in
erythrocytes of peritoneal dialysis
and predialysis patients
Izabela Berdowska1, Bogdan Zieliński1,
Julita Kulbacka1, Izabela Fecka2
1 Wrocław Medical University, Department of Medical Biochemistry,
Poland, 2 Department of Pharmacognosy, Wrocław, Poland
e-mail: Izabela Berdowska <[email protected]>
In search for the phytochemicals exerting anticancer properties, phenolic extracts and purified compounds from Wild
Thyme — Thymus serpyllum, Common Thyme — Thymus
vulgaris, Marjoram -— Majorana hortensis, and Peppermint —
Mentha piperita were tested on adriamycin-resistant human
breast cancer cell line (MCF-7/Adr), and compared with
their effect on wild-type line (MCF-7/wt). The four extracts as well as flavonoid glycosides (eriodictiol-7-O-rutinoside, luteolin-7-O-glucuronide, luteolin-7-O-rutinoside),
arbutin, caffeic acid, and rosmarinic acid were incubated
with the two cell lines in 96-well plates for 48 hours, and
tested with the MTT assay. The general cytotoxicity pattern
was similar for both MCF-7 cell lines. The comparison of
phenolic extracts exhibited the highest cytotoxicity in the
case of Marjoram, and this effect was more pronounced
in MCF-7/Adr, whereas Peppermint extract showed the
lowest cytotoxicity; especially against MCF-7/wt cells.
Similarly, in the previous studies Peppermint extract had
exhibited antiapoptotic, and Marjoram the strongest proapoptotic activity against human leukaemia cells, in comparison with the remaining two extracts.The evaluation of
phenolic compounds’ impact showed rosmarinic acid to be
the most, and arbutin the least toxic (with similar EC50 values in both cell lines). Caffeic acid, eriodictiol-7-O-rutinoside, and luteolin-7-O-glucuronide showed higher toxicity
against MCF-7/wt as compared with adriamycin-resistant
cell line. The obtained results demonstrated most of the
tested purified phytochemicals to be more toxic against
non-resistant cells, and all four phenolic extracts proved to
be more toxic against resistant breast cancer cell line.
Anna Bilska, Małgorzata Iciek, Danuta KowalczykPachel, Maria Sokołowska-Jeżewicz, Lidia Włodek
The Chair of Medical Biochemistry, Jagiellonian University, Medical
College, Cracow, Poland
e-mail: Anna Bilska <[email protected]>
Erythrocytes of chronic kidney disease (CKD) patients
in predialysis period (ND) contained decreased levels of
sulfane sulfur, non protein thiols (NPSH) and total thiols
(TSH). On the other hand, in erythrocytes of end-stage
renal failure (ESRF) patients treated with continuous ambulatory peritoneal dialysis (CAPD), sulfane sulfur, NPSH
and TSH level remained at the level observed in healthy
controls. These changes indicate a disturbed thiol balance
and anaerobic cysteine metabolism in ND CKD patients,
whereas CAPD patients did not show these disorders.
γ-Cystathionase activity was equally elevated in ND and
CAPD patients, which means that CKD pathology is accompanied by an increased expression of this enzymatic
activity in erythrocytes. Erythrocytic rhodanese activity was
unchanged and stayed at the control level in both groups.
Protein carbonylation rate was equally enhanced in both
patient groups which indicated acceleration of oxidative
processes and inability of CAPD to correct these changes
in erythrocytes.
195
P14.4
P14.5
Determination and quantification of
cystathionine in various regions of human
brain using the RP-HPLC method
Concentration of serum prostaglandin E2
in squamous cell carcinoma of esophagus
Patrycja Bronowicka-Adamska, Halina Jurkowska,
Maria Wróbel, Jacek Zagajewski
Chair of Medical Biochemistry, Jagiellonian University Medical College,
Kraków, Poland
e-mail: Patrycja Bronowicka Adamska <[email protected]>
Cystathionine is an important intermediate of the transsulfuration pathway in mammalian tissues. The level of cystathionine reflects the activity of cystathionine β-synthase
(CBS, EC 4.2.1.22), the enzyme responsible for biosynthesis of cystathionine from serine and homocysteine, and
cystathionine γ-lyase (CSE, EC 4.4.1.1), which degrades
cystathionine to cysteine, α-ketobutyrate, and ammonium
ions. Using the RP-HPLC method, we investigated the level of cystathionine, cysteine, reduced (GSH) and oxidized
(GSSG) glutathione in the various regions (cerebellum,
hypothalamus, thalamus, subcortical nuclei, hippocampus,
frontal and parietal cortex) of post-mortem collected human brain. The greatest level of cystathionine was found
in the thalamus and it was about ten times higher than
in the cerebellum. In the thalamus, the high level of cystathionine was correlated with no detectable CSE activity.
The thalamus and hippocampus showed the highest level
of reduced glutathione (GSH) and the highest content of
cysteine was detected in the thalamus, hypothalamus and
subcortical nuclei in comparison to other regions of human brain. In thalamus homogenates, the activity of CBS
was examined in the presence of 8 mM homoserine as the
CBS substrate and in the presence of 0.15 mM DL-propargylglycine (PPE) as the CSE inhibitor. After 15 minutes
of incubation with homoserine, we observed an increase in
the level of cystathionine, cysteine and glutathione and lack
of α-ketobutyrate. The difference in the cystathionine level
between the homogenates with and without PPE was used
to estimate the activity of CBS in the tissue homogenates.
The thus determined activity of CBS in the homogenate
of thalamus was 4.13 pmol/mg·min–1. The relatively high
CBS activity and lack of CST activity confirms the main
role of CBS in hydrogen sulfide generation in the brain.
Dorota Diakowska1, Krystyna Markocka-Mączka1, Krzysztof
Grabowski1, Andrzej Lewandowski1, Małgorzata KrzystekKorpacka2, Małgorzata Matusiewicz2, Witold Diakowski3
1Department of Gastrointestinal and General Surgery, Silesian Piasts
University of Medicine, Wroclaw, Poland; 2Department of Medical
Biochemistry, Silesian Piasts University of Medicine, Wroclaw, Poland;
3Faculty of Biotechnology, Wroclaw University, Wroclaw, Poland
e-mail: Dorota Diakowska <[email protected]>
Background: Prostaglandins play a critical role in tumor
growth and development. Prostaglandin E2 (PGE2) expression is associated with cancer cachexia and tumor angiogenesis. Serum levels of PGE2 (sPGE2) are elevated in
patients with head and neck squamous cell carcinoma and
esophageal carcinoma. However, the relationship between
concentrations of sPGE2 and stages of cancer progression
in esophageal squamous cell carcinoma (ESCC) is unclear.
The aim of this study was to investigate the prognostic relevance of sPGE2 with tumor invasion and its connection
with proangiogenic vascular endothelial growth factor-A
(VEGF-A).Methods. We analyzed the sPGE2 levels in 67
patients with esophageal squamous cell carcinoma in different stages, and compared the results with serum levels
of 32 healthy donors. We also determined serum VEGFA (sVEGF-A) levels in patients and control groups. Immunoenzymatic ELISA tests were used to measurement
of sPGE2 and sVEGF-A concentrations. Results. We
observed a significantly higher concentrations of sPGE2
in ESCC patients than in control (p < 0.001), especially in
stage II (p < 0.001). Concentrations of sPGE2 decreased
with increasing depth of tumor invasion and sPGE2 levels
were significantly higher in patients with T2 tumor than determined in patients with T4 tumor (p < 0.05). The sPGE2
firmly correlated with sVEGF (r=0.76, p < 0.001). Conclusion. Elevated level of sPGE2 in early stages of tumor may
raise hopes for using of this compound as marker of cancer presence in ESCC. It’s correlation with sVEGF-A suggests that PGE2 production can promote esophageal squamous cell carcinoma cell proliferation and angiogenesis.
196
Abstracts
P14.6
P14.7
Study on the structure and function of
the LA loop — a regulatory element of
the HtrA protease from Escherichia coli
Level of a serotonin of plasma
at pregnant women.
Donata Figaj1, Artur Giełdoń2, Barbara
Lipińska1, Joanna Skórko-Glonek1
1University
of Gdańsk, Department of Biochemistry, Kładki 24, 80822 Gdańsk, Poland; 2University of Gdańsk, Faculty of Chemistry,
Sobieskiego 18, 80-952 Gdańsk, Poland
e-mail: Donata Figaj <[email protected]>
HtrA (DegP) from Escherichia coli is a periplasmic protein
that belongs to a big family of serine proteases present in
most of studied organisms. It’s main function is protection
of bacterial cells from the effects of the stressful conditions (e.g. heat shock, oxidative or reducing stress). It is an
important virulence factor of pathogenic E. coli strains.
HtrA recognizes and binds incorrectly folded proteins that
arise in the cell subjected to a stress and prevents their accumulation and aggregation in the extracytoplasmic space.
HtrA shows two activities, proteolytic and chaperone, and
both of them are required for full protection of the cell.
The proteolytic activity of HtrA is strongly dependent on
temperature. At temperatures below 30oC this protein hydrolyzes substrates at very low rate and acts mainly as a
chaperone. At higher temperatures the proteolytic activity
dominates. The low proteolytic activity of HtrA at temperatures below 30oC can be explained basing on the crystal structure of this protein. Proteolytically inactive HtrA
exists in form of hexamer, where catalytic centers are in
the improper configuration for the catalysis. Moreover the
access to the active sites is blocked by the regulatory loops
L1, L2 and LA. During the activation process the HtrA
hexamers undergo rearrangement into 12- or 24-mers. As
the result of this process the LA loop stops interacting with
the L1 and L2 loops. This enables the L1 loop to position
Ser 210 in the right orientation to interact with His 105 and
Asp135 that form together the active center. The structural
changes can be induced thermally or allosterically.
The aim of this study is to characterize the LA loop: it’s
structure and role in the regulation of proteolytic activity
of HtrA. As the LA segment has not been traced by crystallography, we are building a theoretical model of LA and
confirming its reliability in an experimental way. The amino
acid residues involved in potentially important interactions
(according to the model) has been replaced with residues
of different character. We obtained the following HtrA
variants: HtrAE59V, HtrAP43G, HtrAQ47L, HtrAQ64A
and HtrAR207L. The introduced mutations influence the
proteolytic activity of HtrA. For example mutation E59V
stimulates whereas mutation R207L decreases the rate of
substrate cleavage.
Victoria V. Furs1, Alexandr V. Naumov2,
Yevgeny M. Doroshenko2
Grodno State Medical University 1Obstetrics and Gynecology
Department, 2Biochemistry Department, Grodno, Belarus
e-mail: Victoria Furs <[email protected]>
Background: The placenta has no nerves. This fact does
a placenta unique in respect of mechanisms of regulation
and integration of functions carried out by it, a particular interest cause local humoral factors. Placenta functions
are regulated producing in it biologically active mediators
which now it is verified more than 100. One of the basic
groups is biogene amines (serotonin, melatonin, etc.).
Serotonin is a monoamine neurotransmitter. Serotonin is
synthesized from the amino acid tryptophan. Hyperserotoninemia has been associated with pregnancy complications (pre-eclampsia, spontaneus abortion). The serotonin
spastic stricture of arterioles causes activation of thrombocytes that leads still to larger emission of a serotonin on
periphery. Such role of a serotonin proves to be true rising
of concentration of a metabolite of a serotonin 5-hydroxyindoleacetic acid in urine at patients with pre-eclampsia.
Objective: To measure the concentration of plasma total
serotonin in pregnant women during the various periods
of pregnancy.
Methods: Blood samples were collected into tubes containing heparin, immediately chilled in the ice and centrifuged. Samples prepared at the day of analyses. Serotonin
was determined by high-performance liquid chromatography (HPLC) with fluorescence detection in healthy pregnant women (n = 20) in 25–28 weeks and in 38–40 weeks
(n=20) of pregnancy.
Results: Plasma serotonin was significantly higher in pregnant women in later terms of pregnancy (p < 0.05). In the
first group (25–28 weeks) plasma serotonin was less unlike
women on the day before labours at which the highest level
serotonin was marked.
Conclusion: We have studied serotonin level on different
durations of gestation and have shown augmentation, a
plasma serotonin to proportionally duration of gestation.
Us the further studying of levels of biogenic amines at
pregnant women with normal and complicated pregnancy
and the role of a serotonin for fuller understanding in pregnancy is supposed.
Reference:
Ajmalazian EK et al. (2003) J Obst Female Illnesses 4: 7–9.
Khong TJ et al. (1986) J Obst Gynaecol 93: 1049–1059.
Sirotkin AV et al. (1997) J Endocrinol 154: 1–5.
Pijnenborg K et al. ( 1988) Reprod Nutr Dev 28: 1581–1586.
P14.8
P14.9
Effect of prosequence removal
on the human thyroid peroxidase
structure and function
Hypercoagulability is the most prevalent
for patients undergoing total hip
replacement after femoral neck fracture
Marlena Godlewska1, Wanda Krasuska1, Monika Góra2
Tatiyana Grinevich
1Medical
Grodno State Medical University, Grodno, Belarus.
Centre of Postgraduate Education, Department of
Biochemistry and Molecular Biology, Warsaw, Poland; 2Institute of
Biochemistry and Biophysics PAS, Department of Genetics, Warsaw,
Poland
e-mail: Marlena Godlewska <[email protected]>
Human thyroid peroxidase (hTPO) plays a key role in
thyroid hormone synthesis and is also a main autoantigen in autoimmune thyroid diseases. After synthesis this
membrane-associated protein undergoes posttranslational
modifications such as glycosylation, heme fixation and proteolysis in the N-terminal part. N-sequencing showed that
the N-terminal part of mature TPO begins at T109 both
in human thyroid and in mammalian CHO cells transfected with hTPO cDNA. It has been already shown that in
myeloperoxidase, a protein highly homologous to hTPO,
the prosequence may play an important role in peroxidase
activity, proper folding and transport. To explain the role
of the prosequence in hTPO structure and function we
generated two N-terminally truncated hTPO variants starting at residues F76 and T109. Both constructs were stably
expressed in CHO cells. Using immunodetection and densitometric analysis we showed that prosequence removal
does not change the expression level of hTPO variants in
comparison with wild type hTPO. Furthermore, we tested
by flow cytometry the reactivity of truncated TPO forms
incorporated into the membrane of living CHO cells with
anti-TPO antibodies. This experiment confirmed that the
absence of the prosequence has no impact on TPO transport to the membrane of CHO cells. Additionally, we have
found in the present study that the three-dimensional structure of the protein is not considerably affected by the lack
of the N-terminal part. These conclusions were based on
the results obtained by ELISA and flow cytometry analysis,
where interactions between truncated hTPO and anti-TPO
antibodies recognizing conformational epitopes were examined. In summary, the data presented here provide evidence that the presence of the prosequence is not crucial
for the proper folding and transport of hTPO produced
in CHO cells.
Acknowledgements:
This work was supported by CMKP 501-1-1-25-04/09 grant.
197
e-mail: Tatiyana Grinevich <[email protected]>
The most progressive and radical treatment method of
femoral neck fractures for elderly and old patients is a total
hip replacement. Hypercoagulability and as consequence
venous thromboembolism (VTE) after total hip replacement is a major source of morbidity and mortality in traumatology. Standard tests, such as a prothrombin time (PT)
with its derivatives (international normalized ratio (INR))
and activated partial thromboplastin time (APTT), possess minimal information for diagnosis of hypercoagulation. For this purpose we suggest to use a new method
named rotation thromboelastometry (ROTEM) (Pentapharm, Germany) [1]. Reagent-supported thromboelastometry with the rotation thromboelastography (ROTEM) is a
whole blood assay that evaluates the visco-elastic properties during blood clot formation and clot lysis.
Objectives: This study was performed to determine the
course of coagulation for patients undergoing total hip replacement after femoral neck fracture.
Method: The effect of surgery for femoral neck fracture
on whole blood coagulation and the relationship of altered
coagulation to deep venous thrombosis were investigated
for 17 patients with mean age 60.6+/-18.6 years. All patients undergoing total hip replacement received anticoagulant agents during their hospitalizations (average stay, 10 to
11 days). At admission, standard coagulation assays were
performed and ROTEM parameters such as for the clotting time (CT), clot formation time (CFT), maximum clot
firmness (MCF), alpha-angle (ALP) with two activated tests
(INTEM, EXTEM).
Results: Trauma brings on significant modifications of
coagulation. There was a high value of the maximum
clot firmness (MCF) in both EXTEM (MCF = 66 mm,
р < 0.0003) and INTEM (MCF = 65.2 mm, р < 0.0002)
tests and alpha-angle (ALP) in both EXTEM (ALP = 73,
р < 0.002) and INTEM (ALP = 77, р < 0.002) tests which
leads to a condition of a hypercoagulation for the surveyed
patients.
Conclusion: The activation of coagulation, as monitored by rotation thromboelastometry, is predominant in
total hip arthroplasty. The findings suggest that to reduce
thromboembolic outcomes, patients undergoing total hip
replacement need earlier, more intensive and prolonged
prophylaxis.
References:
Rugeri L, Levrat A, David JS et al. (2007) J Thromb Haemost 5: 289–295.
Abstracts
198
P14.10
P14.11
The role of the disulphide bond in the
stabilization of the structure of the
LA loop, a regulatory element of the
HtrA protease from Escherichia coli
Whole blood aggregometry is more
effective method to test inhibitory
effect of extract from Aronia
melanocarpa berries on platelets than
light transmission aggregometry
Tomasz Koper1, Anna Sobiecka-Szkatuła1, Artur
Giełdoń2, Agnieszka Polit3, Katarzyna Guzow2,
Wiesław Wiczk2, Aleksander A. Kubicki4, Piotr Bojarski4,
Barbara Lipińska1, Joanna Skórko-Glonek1
1University
of Gdańsk, Department of Biochemistry, Kładki 24, 80822 Gdańsk, Poland; 2University of Gdańsk, Faculty of Chemistry,
Sobieskiego 18, 80-952 Gdańsk, Poland; 3Jagiellonian University,
Department of Biochemistry, Biophysics and Biotechnology,
Gronostajowa 7, 30-387 Kraków, Poland; 4University of Gdańsk, Institute
of Experimental Physics, Wita Stwosza 57, 80-952 Gdańsk, Poland
e-mail: Tomasz Koper <[email protected]>
HtrA (DegP), a periplasmic heat shock protein from Escherichia coli, is a model protein of the HtrA family, the evolutionarily conserved serine proteases, widely distributed
among living organisms. HtrA from E. coli is a key proteolytic factor of the periplasmic quality control system. It
degrades irreversibly damaged polypeptides which appear
under stress conditions (e.g. heat shock, oxidative stress, reducing stress). It is indispensable for the survival of E. coli
cells under heat shock conditions. Moreover, prokaryotic
HtrA homologs are responsible for a virulence of a wide
range of pathogenic bacteria. Proteolytic activity of HtrA
is strongly dependent on temperature. At temperatures
below 30ºC the activity is very low and it is believed that
HtrA acts then mainly as a chaperone. The activity gradually increases along with the rise of temperature, reaching
its maximum at the range of 45–50ºC.
Under reducing conditions at low temperatures (20–30ºC)
the proteolytic activity of HtrA in vivo is much higher when
compared to the activity under physiological conditions.
HtrA contains a single disulphide bridge connecting Cys57
and Cys69, located within the LA loop. The LA loop plays
a regulatory function - in the inactive conformation it covers the entrance to the active site. The redox state of this
structure may influence the activity of HtrA. We expect
that the reduced form of LA (lacking the S-S bond) is more
flexible and therefore its inhibitory effect on active center
is weaker. To confirm this hypothesis we constructed two
htrA genes, coding for two HtrA variants, each containing
a single Trp residue placed within the LA loop. One of the
variants lacks the disulphide bridge due to the substitution
of two cysteines with alanines. The Trp indole ring serves
as an internal probe for monitoring the conformational
changes of the loop. The relative exposition of the Trp residues to the solvent was examined by quenching its steadystate fluorescence by acrylamide and fluorescence lifetime
measurements at the temperature range of 20–45ºC. The
Stern-Volmer constants, fluorescence lifetimes and bimolecular quenching constants were calculated.
The obtained results indicate that at the low temperatures
the LA loop from the protein lacking S-S bond is more
exposed to the solvent than the one from the protein
which keeps it. It suggests that disulphide bridge plays an
important role in the stabilization of the structure of the
LA loop and its reduction probably facilitates adopting an
active conformation.
Anna Kosiorek1, Kamila Kostrzewska1, Beata
Olas2, Cezary Watala1, Jacek Golanski1
1Department of Haemostasis and Haemostatic Disorders, Medical
University of Lodz, Lodz, Poland; 2Department of General Biochemistry,
University of Lodz, Lodz, Poland
e-mail: Anna Kosiorek <[email protected]>
The platelet activation plays an important role in normal
hemostasis but it is also associated with the occurrence of
cardiovascular diseases. The platelets response to various
agonists can be monitored on the basis of shape change,
lag phase and primary and secondary aggregation. There
are many natural compounds which have been shown to
decrease platelet activation. For example, the plant extracts
where polyphenols are present at high concentrations have
anti-platelet action. Extract from Aronia melanocarpa
fruits (AM) is a complex mixture of polyphenols that have
been shown previously to reduce a platelet aggregation.
The aim of the study was to compare the usefulness two
techniques commonly used for measuring platelet aggregation light transmission aggregometry (LTA) and impedance
whole blood aggregometry (WBA) to assess the inhibitory
effect of AM extract (concentration 25, 50, 100, 200 μg/
ml).
The extract from Aronia melanocarpa berries (Aronox)
was supplied by Agropharm SA. The effects of AM extract
on platelet aggregation was tested in the group of 20 volunteers. In LTA and WBA PRP and whole blood, respectively were preincubated with AM extract for 10 minutes
and then platelet aggregation was stimulated with ADP (5
μM).
Using WBA, we observed inhibitory effect of extract
from Aronia melanocarpa in concentration of 50 μg/ml
(p=0.0407), 100 μg/ml (p=0.0137), 200 μg/ml (p < 0.0001).
Using LTA we detected inhibitory effect studied extract
only in concentration of 200 μg/ml (p=0.0255).
We conclude that WBA technique allows the detection of
the inhibitory effect of extract from Aronia melanocarpa
berries in lower concentration then LTA method.
Acknowledgements:
This study was supported by the project N405 065034 of the Polish State
Committee for Scientific Research.
P14.12
P14.13
The transferase activity and properties of
neutral alpha glucosidase from the blood
serum of the carp (Cyprinus carpio L.)
The most important and effective
endogenous antioxidant glutathione
in children with glomerulonephritis
Julia Kotońska-Feiga
Wroclaw University of Environmental and Life Sciences, Institute of
Biology, Wroclaw, Poland
e-mail: Julia Kotońska <[email protected]>
The activity of neutral alpha glucosidase has been found in
a blood serum of several species of mammals. In human
blood serum its activity is very low, but in pig serum is three
orders higher. Neutral alpha glucosidase has two kinds of
activity: hydrolytic and transferase. Similarly is in a fishes.
In serum of carp, both activities are well determinable, and
their levels are of the range observed in pig. Activities of
this enzyme in carp show considerable seasonal variability.
It is connected with metabolic changes intensity in moderate climate fish.
The synthetic N-glycosides of mono and disaccharides
were used in assay of transferase activity. The products of
transglycosylation were than separated and identified by the
HPLC, and determined by measure of absorbance at 365
nm. The carp blood serum enzyme, which was used for the
tansferase assay, was seventy fold purified by the DEAE
cellulose ion exchange chromatography.
The pH 7.0 appeared to be the optimal of transferase activity, while pH 6.8 was the best for the hydrolytic (maltase)
activity of the carp blood serum enzyme. The enzyme is
active at relatively wide range at temperatures, depending
on the access to substrate and the time exposition. Ten
minutes exposure of the protein enzyme on 60oC did not
change its activity. The substrate specification toward to pnitropenyl-N-maltoside and p-nitrophenyl-N-maltotrioside
(both donors and acceptors from transferred glucose) are
very similar, Km = 11.8 and 12 mM respectively. The equatorial position of the hydroxyl group at fourth carbon of
sugar, necessary for the acceptor properties of p-nitrophenyl-N-glycosides derived some pentoses and hexoses.
199
Alexander V. Naumov, Elena A. Konuch
Grodno State Medical University, Grodno, Belarus
e-mail: Konuch lena <[email protected]>
Glutathione (GSH) is the major endogenous antioxidant
produced by the cell. GSH participates directly in the neutralization of free radicals, reactive oxygen compounds,
maintains exogenous antioxidants such as vitamins C and
E in their active forms. In addition, GSH plays a role in
the detoxification of many xenobiotics. Glutathione is an
essential component of the human immune response. It
takes up and gives off hydrogen and is important in cellular
respiration. A deficiency of glutathione can cause hemolysis and oxidative stress.
The aim of our study was to evaluate plasma GSH levels
and their determining factors in children with acute and
chronic glomerulonephritis. The baseline levels of plasma
GSH, serum and urine protein, creatinine, blood pressure (BP), levels of erythrocyturia were measured after an
overnight fast. Fasting GSH levels in plasma samples were
measured by high-performance liquid chromatography
with fluorescence detection.
18 children and adolescents with a mean age of 13.94±3.49
years (range 6–17 years) with acute glomerulonephritis and
11 children and adolescents with a mean age 13.45±3.80
years (range 4–17 years) with chronic glomerulonephritis
were included in the study. In accordance to our previous
communication the normal range for plasma GSH in adolescents is 3.71±0.48 μM/L (before) and 2.71±0.21 μM/L
after 10 days of appropriate vitamin (B9, B12) diet. The
mean plasma GSH in the patients with acute glomerulonephritis was 6.16±2.6 μmol/L and 6.29±2.37 μmol/L in the
patients with chronic glomerulonephritis.
The GSH levels of both groups of patients were significantly correlated with period of glomerulonephritis
(r=0.46, P=0.007), the level of serum albumen (r=0.72,
P=0.0001) and calcium (r=0.54, P=0.003), urine creatinin
level (r=0.41, P=0.04) and systolic BP (r=0.56, P=0.001).
We also find negative correlations between GSH level and
the level of ESR (r=–0.35, P=0.04), minute dieresis (r=
–0.62, P=0.001) and oedema at first days of the disease (r=
–0.51, P=0.016).
Glutathione is the major endogenous antioxidant produced
by the cell. So the regulation of GSH metabolism may provide some effect in treatment and prognosis of glomerulonephritis, minimising the risk of chronic disease and promoting longevity.
Abstracts
200
P14.14
P14.15
Changes in expression of human serine
proteases HtrA1, HtrA2, HtrA3 genes in
benign and malignant thyroid lesions
Growth rate of Desulfovibrio
desulfuricans bacteria in the presence
of various body iron sources
Dorota Żurawa-Janicka1, Jarosław Kobiela2,Tomasz
Stefaniak2, Joanna Skórko-Glonek1, Andrzej
Łachiński2, Barbara Lipińska1
Marzena Jaworska Kik1, Jolanta Lodowska2, Daniel
Wolny1, Zofia Dzierżewicz1, Ludmiła Węglarz2
1Department
2Department
of Biochemistry, University of Gdansk, Gdansk, Poland;
of General, Endocrine and Transplant Surgery, Medical
Universityof Gdansk, Gdansk, Poland
e-mail: Barbara Lipińska <[email protected]>
Human HtrA 1/2/3 proteins are serine proteases involved
in essential physiological processes, such as maintenance
of mitochondrial homeostasis, apoptosis and cell signaling.
Disturbances in their function contribute to the development of several diseases including cancer. It is believed
that HtrA1 functions as a tumor suppressor, promoting cell
death. HtrA2 plays a pivotal role in the inductionof apoptotic pathways by activation of caspases and via its proteolytic activity. Function of HtrA3 is still unclear. However,
the HtrA3 level changes drastically in some neoplastic tissues.
The aim of the study was to find out whether the HtrA
proteins can serve as molecular markers in thyroid cancer. To achieve this goal we assayed expression of HtrA1,
HtrA2 and HtrA3 genes in tissues of benign and malignant
thyroid lesions and tissues of control groups. We also analyzed levels of HtrA proteins indifferent histological types
of thyroid cancer, follicular thyroid carcinoma (FTC) and
papillary thyroid carcinoma (PTC). We used Western blotting technique to estimate protein levels of HtrA1, HtrA2
and, for the first time, both isoforms of HtrA3, HtrA3-S
(short) and HtrA3-L (long).
We found that the HtrA1 protein level was increased in
FTC compared to control tissues while it was unchanged in
PTC and in benign lesions. The level of HtrA2 was higher
in a group of thyroid cancer compared to normal tissues.
These results indicate the implication of HtrA1 and HtrA2
in the development of thyroid cancer and suggest that
HtrA1 could be use as a diagnostic marker in separation of
FTC from follicular thyroid adenoma.
We also found that the HtrA3-S level was significantly
higher in PTC compared to control tissues while it was unchanged in FTC. Interestingly, the HtrA3-L expression was
higher in normal tissues from patients with thyroid cancer
compared to normal tissues from patients with benign lesions. Our results indicate that both isoforms of HtrA3 are
involved in the development of thyroid cancer. Taking into
account our results and a fact that PTC is characterized by
more aggressive phenotype it ispossible that the HtrA3-S
expression may be correlated with malignancy of thyroid
cancer. It is also possible that a higher level of HtrA3-L in
normal thyroid tissues could be correlated with the risk of
development of malignant thyroid lesion. 1Department
of Biopharmacy, 2Department of Biochemistry, Faculty of
Pharmacy, Medical University of Silesia, Katowice, Poland
e-mail: Jolanta Lodowska <[email protected]>
One of the factors regulating the ability of pathogenic
bacteria to colonize a host organism is an access to iron
ions that are crucial participants of many physiological
functions including cell division. The uptake of these ions
from an internal environment and metabolic reserves of
the infected macroorganism determines pathogenicity of
bacteria. Staphylococci as facultative anaerobes absorb iron
from heme ferroproteins (hemoglobin, myoglobin), nonheme ferroproteins (ferritin, hemosiderin) and also from a
bovine transferrin and lactoferrin [1], whereas enterococci
utilize iron from transferrin and lactoferrin mainly [2]. Haemophilus influenze type B, notably pathogenic for humans,
takes iron from both bovine and human transferrin but
not from lactoferrin [3]. It is unknown which of the body
iron sources are used by Desulfovibrio desulfuricans bacteria.
Though these bacteria are constituents of natural microflora of a human gastrointestinal tract, they are suspected
to be a pathogenic factor of some disorders of intestine
such as Crohn’s disease and ulcerative colitis.
The aim of study was to evaluate D. desulfuricans growth rate
on media containing various body iron sources.
Standard soil strain La 2226 (DMS 642) from Swiss National Type Cultures Collection in Lausanne and six wild
intestinal strains DV/A, DV/B, DV/C, DV/H, DV/I,
DV/I/1of D. desulfuricans were used in the experiment.
Bacteria were cultured on Postgate’s pyruvate medium containing 0.0015 g/dm3 of the proper iron source (human hemoglobin and transferrin, bovine hemoglobin, transferrin,
lactoferrin and hemin, equine myoglobin and cytochrome
c) in an anaerobic condition (80% N2, 10% CO2 and 10%
H2). The bacterial growth rate was evaluated by quantification of colonies after 48h of the culture.
Intrastrain differences in the growth of D. desulfuricans on
every of the used iron sources were observed. La2226, cultured on the medium containing both human and bovine
transferrin, was the most slowly proliferating strain. The
most intensive rate of growth was observed for DV/I and
DV/I/1 strains cultured on the medium suplemented with
equine myoglobin and cytochrome c, and bovine lactoferrin. Human and bovine hemoglobine stimulated DV/H
strain growth the most.
References:
Lisiecki P et al. (1996) Med Dosw Mikrobiol 48: 5–13.
Sobiś-Glinkowska M et al. (2001) Med Dosw Mikrobiol 53: 9–15.
Morton DJ et al. (1990) J Gen Microbiol 136: 927–933.
P14.16
P14.17
Midkine as a potential differential
marker for sepsis
Relative changes in lipopolysaccharidebinding protein (LBP) predict lethal
outcome in septic patients.
201
Małgorzata Korpacka1, Magdalena Mierzchała1,
Iwona Misa1, Dorota Diakowska2, Grażyna
Durek3, Małgorzata Matusiewicz1
Magdalena Mierzchała1, Małgorzata
Korpacka1, Grażyna Durek2
1Dept. of Medical Biochemistry, 2Dept. of Gastrointestinal and General
Surgery, 3Dept. of Anesthesiology and Intensive Care, Wroclaw Medical
University, Wroclaw, Poland
1Wroclaw Medical University, Department of Medical Biochemistry,
Poland, 2 Wroclaw Medical University, Department of Anaesthesiology
and Intensive Care, Poland
e-mail: Malgorzata Matusiewicz <[email protected]>
Severe sepsis is a leading cause of death in the intensive
care units (ICU). The disease is complex with variable and
unspecific symptoms, for which biomarkers are sought after to facilitate differential diagnosis, severity evaluation,
and outcome prediction. Midkine is a proinflammatory and
angiogenic cytokine, the value of which as a biomarker has
already been demonstrated in several inflammation-based
diseases. Midkine was measured by ELISA in 38 septic patients at ICU admission and on subsequent days (1–5) and
compared with 88 patients with active IBD (inflammation
without infection) and 99 healthy controls. Cytokine was
elevated in sepsis as compared to IBD and healthy subjects
(1379 vs 538 and 89 ng/L, p<0.001). Using 500 ng/L as a
threshold, 73.7% of septic patients had elevated midkine
as compared to 38.6% of active IBD patients and none
of healthy subjects (p<0.001). Over one-fourth of septic patients but none with active IBD had midkine levels
>2500 ng/L. Midkine was significantly higher in septic
shock than severe sepsis on the 5th day (4916 vs. 712 ng/L,
p=0.017). Moreover, its values differed over the examined
days (p<0.001) - gradually decreasing in severe sepsis and
persisting high in septic shock. No significant differences
in midkine levels were observed between sepsis survivors
and non-survivors (p=0.996) but midkine tended to remain
high throughout the examined days in non-survivors and
decline in survivors (p=0.225). Time course of midkine
tended to be affected also by infection site: patients with
abdominal infection had lower at admission but relatively stable cytokine levels, whereas those with pulmonary/
other infections had higher midkine concentrations, which,
however, decreased during first five days to become lower on the 5th day. Midkine did not correlate with clinical
scores APACHE II and SOFA, with CRP or procalcitonin
but corresponded with WBC (r=0.42, P=0.015), lipopolysaccharide-binding protein (r=0.47, P=0.005), and IL-6
(r=0.36, P=0.045).
Concluding, sepsis is associated with elevation in midkine
levels almost ten times higher than found in inflammation
without infection, hence, midkine owns a potential as a differential marker.
e-mail: Magdalena Mierzchała <[email protected]>
A number of biomarkers has been evaluated as possible
diagnostic tools facilitating evaluation and management of
septic patients, LBP among others, but results were conflicting. We hypothesized that changes in LBP expressed
as chain indices rather than LBP absolute values measured
at its maximum might serve as biomarkers in sepsis. LBP
was measured (ELISA) daily in 40 septic patients and compared to 20 healthy subjects. Chain indices were calculated
to express relative changes in LBP levels across days examined (1-5). LBP in sepsis was significantly higher than in
controls (47.1 vs. 11.5 mg/l, p<0.0001); being the highest
at admission and declining gradually during following days.
At admission, LBP was higher in survivors than non-survivors (50 vs. 40 mg/l) but decreased to become significantly
lower at 5th day whereas LBP in non-survivors remained
high (25 vs. 51 mg/l, respectively). Average rate of relative changes in LBP based on chain indices and calculated
for survivors and non-survivors was 0.808 (interpreted as
decrease by 19.2%) and 1.055 (increase by 5.5%), p=0.005,
respectively. LBP as outcome predictor (ROC analysis) had
fair accuracy when expressed in terms of its relative changes (AUC=0.81, p=0.0001) as compared to none when
measured at admission (AUC=0.5, p=0.979). With cut-off
value >0.899 (decrease >10.1%) average rate of relative
changes as predictor of poor prognosis had 83% sensitivity and 73% specificity as compared to 67% sensitivity
and 50% specificity of LBP’s admission level. At admission, LBP did not correlate with the disease clinical severity
scores APACHE II and SOFA and with other inflammatory indices except for WBC (r=0.37, P=0.020), whereas
average rate of relative changes in LBP based on chain
indices correlated with APACHE II (r=0.47, P=0.003),
SOFA (r=0.49, P=0.002), CRP (r=0.63, P=0.0001), and
procalcitonin (r=0.36, P=0.028). Concluding, LBP evaluated at its maximum poorly reflects the disease clinical
severity, intensity of inflammatory response and had no
predictive power. In turn, average rate of relative changes
in LBP concentrations based on chain indices corresponds
with sepsis severity and has fair accuracy as a predictor of
lethal outcome.
Abstracts
202
P14.18
P14.19
Homocysteine blood levels of
patients with eczema
Circadian concentrations of DHEA and bone
status in obese postmenopausal women
A. V. Naumov1, Y. M. Doroshenko1, A. I. Novoseletskaya2
1Grodno
2Belarusian
State Medical University, Grodno, Belarus;
Medical Academy of Post-Graduate Education, Department of
Dermatovenerology, Belarus
e-mail: Aliautsina Novasialetskaya <[email protected]>
The purpose of work: to reveal in dynamics changes in
homocysteine (Hcy) serum levels of patients with eczema
during standard therapy.
Materials and methods: 17 patients, suffering from various forms of eczema, were included in research. Average
age 47.8±12.0 years.
The diagnosis of eczema was established by detection of
morphological elements typical for this disease.
The assay of tHcy in blood serum of patients by using
high-performance liquid chromatography (HPLC) with
pre-column derivatization and fluorescence detection was
performed. Obtained data were processed with use of operational system Statistica 6.0.
Results: There were 13 men and 4 women in group of the
surveyed patients. There were 6 patients in the age younger
40 years, and older 40 years (11 patients). There were 13
patients with the small area of damage (less than 25 % of a
body surface) and 4 persons with the area more than 25 %.
Before treatment in the general group of patients (n=17)
Hcy level was 9.1±1.84 mcM/L, and on the 10-th day of
treatment — 7.33±0.66 mcM/L. No significant changes
were revealed statistically (p=0,72). In comparison to Hcy
levels of patients with eczema and healthy people (5,3
mcM/L [2] or 6.85 mcM/L [1]) hyperhomocysteinemia
was observed (HHcy). Unfortunately, in this work the control group has not been picked up. Hcy levels changed variously during treatment: in 5 patients Hcy levels were not
changed, in 6 – decreased to norm, and in 6 – increased.
Conclusions: 1. Hyperhomocysteinemia was observed in
patients with eczema. 2. This research confirmed the absence of statistically significant changes in Hcy serum level
in patients with eczema during 10-days of standard therapy
due to various patients. 3. Three variants of homocysteine
changes during therapy confirm availability of possible different molecule etiology mechanism of eczema.
References:
1. Navumau AV, Zolotukhin MM, Plotski AR (2006) Acta Biochim Pol 53:
Suppl 1, 195.
2. Predko VA, Spas VV, Yakubtsevich RE, Naumov AV, Lazovskaia MV
(2009) Acta Biochim Pol 56: Suppl 3, 206.
Zofia Ostrowska1, Beata Kos-Kudła2, Bożena Szapska1, Kinga
Wołkowska-Pokrywa1, Bogdan Marek2, Dariusz Kajdaniuk2
1Division
of Clinical Biochemistry, Zabrze, 2 Department of
Pathophysiology and Endocrinology, Zabrze, Poland; Medical University
of Silesia in Katowice, Poland
e-mail: Zofia Ostrowska <[email protected]>
Background: Postmenopausal increase of adipose tissue
mass is associated with elevation of circulating androgens,
especially adrenal androgens. A relationship has been found
between body mass, BMI and DHEAS as well as bone turnover in postmenopausal women. It has also been suggested
that the effect could be mediated by the RANKL/RANK/
OPG system. The purpose of our investigations was: 1)
to determine whether a relationship exists in postmenopausal subjects between circadian DHEA concentrations,
BMD and bone metabolism as assessed based on serum
OC and CTx; 2) to establish whether OPG and its ligand,
sRANKL, might be engaged in this relationship. Patients
and methods: BMD was measured in 35 postmenopausal
subjects (20 obese and 15 healthy women) using DXA images of lumbar spine (L2-L4) obtained with Lunar DPX,
USA. Blood was sampled at 3-hour intervals for 24 hour
(starting at 08:00). Serum was obtained by centrifugation
and assayed for DHEA, OC, CTx, OPG and sRANKL
concentrations by ELISA. Results: Obese women showed
a significant increase in body mass as well as in BMI, WHR
and BMD compared to the control. Their mean circadian
concentrations of OC, CTx, OPG and sRANKL, and circadian rhythm amplitudes of CTx, OPG and sRANKL
were markedly decreased; a shift was also observed in
sRANKL rhythm acrophase. Mean circadian DHEA and
its circadian amplitude were significantly increased. A positive correlation was revealed between BMD, DHEA concentration, and OPG/sRANKL ratio whereas a negative
correlation was found between BMD and sRANKL. Circadian DHEA concentrations correlated negatively with OC,
CTx and sRANKL, and positively with OPG. A negative
correlation was seen between circadian OC and sRANKL
as well as between OPG and sRANKL. Conclusions: 1)
Postmenopausal obesity alters circadian DHEA secretion;
these alterations are associated with BMD cxincrease, bone
metabolism decrease, and the suppression of circadian
OPG and sRANKL oscillations; 2) Increased DHEA secretion observed in obese postmenopausal subjects might
exert a beneficial affect on bone tissue, probably via a shift
in the OPG/RANKL ratio towards OPG.
P14.20
P14.21
Direct effects of selected „appetite
controlling” peptides on lipogenesis and
glucose uptake in isolated rat adipocytes
Alteration in plasma fibronectin
domain concentration associated
with uveitis in children
Ewa Pruszyńska-Oszmałek, Przemysław Kaczmarek,
Dawid Szczepankiewicz, Maciej Sassek, Marek Skrzypski,
Iwona Hertig, Paweł Maćkowiak, Krzysztof W. Nowak
Magdalena Przybysz1, Bożena Polańska2
Department of Animal Physiology and Biochemistry, University of Life
Sciences, Wołyńska 35, 60-637 Poznań, Poland
e-mail: Ewa Pruszyńska Oszmałek <[email protected]>
The worldwide obesity epidemic has prompted renewed
interest in understanding the mechanism of energy homeostasis. Neural and endocrine signals from peripheral tissue
to brain are thought play a vital role in the short-term regulation of appetite. Several peripheral hormones (expressed
mainly in gut, pancreas or adipose tissue) including obestatin and neuromedin U (NmU) act as satiety signals, whilst
orexin (OXA) and ghrelin act as a hunger hormone by
increasing appetite. Despite of the vital, central action of
these peptides a direct modulation of energy metabolism
processes could be possible as well. Modulation of peripheral hormones/neuropeptides could provides an effective
long-term therapy for the treatment of obesity.
The aim of present study was to determined the direct
effect of selected peripheral hormones/neuropeptides
on lipid and carbohydrates metabolism in isolated rat adipocytes.
Adipocytes were isolated from epididymal fat pad of Wistar rats. Basal and stimulated lipogenesis and glucose uptake
were assayed after incubation with different doses of tested
peptides: neuropeptide U and orexin A (1 nM, 10 nM and
100 nM). Lipogenensis was studied as the incorporation of
[U-14C] glucose into lipids. Glucose transport was studied
as amount of 2-deoksy-D-[-1-3H]glucose transported to
adipocytes.
We proved that all tested peptides significantly affects fat
tissue metabolism in distinct manner. Orexin A elevated
lipogenesis and glucose uptake. The opposite effect was
observed for NmU. In general the prominent and rational
link between the physiological features of central effect
generated by particular peptide and observed direct effort
to adipocytes metabolic function could be figure out.
203
1Department of Chemistry and Immunochemistry, 23rd Department
and Clinic of Pediatrics, Immunology and Rheumatology of
Developmental Age, Wroclaw University of Medicine, Wroclaw, Poland
e-mail: Magda Przybysz <[email protected]>
Background: Fibronectin (FN) is a large glycoprotein
found in many tissues, on cell surfaces and in body fluids.
Because of its multidomain structure and ability to interact with many ligands including immune complexes, FN
is involved in inflammatory processes and plays a role in
wound healing and tissue repair. The aim of this study was
to determine molecular FN status in plasma of patients
suffering from uveitis in comparison to some other inflammatory diseases.
Methods: Blood plasmas were obtained from children
with uveitis and other autoimmune as well as non-autoimmune diseases. The control group consists of plasmas derived from healthy children.
The concentrations of FN domain were performed by
ELISA with 5 monoclonal anti-FN antibodies (“TaKaRa”,
Japan) recognizing cell-binding (CBDFN), collagen-binding (CollagenFN), fibrin-binding (FibrinFN), C-terminal
(CtFN) and N-terminal (NtFN) domains. Molecular forms
of FN were detected by Western blot analysis with monoclonal antibody specific to cell-binding FN domain.
Results: Concentration of CBDFN was significantly lower, while NtFN was significantly higher in the plasmas from
uveitis than in plasmas other autoimmune diseases. Moreover concentration of CBDFN was significantly lower in
uveitis than in control group. Plasma FN imunobloting pattern did not show any differences associated with patient’s
disease state.
Conclusion: Results show that uveitis could be differentiated from other autoimmune diseases as well as from
healthy individuals by determination of plasma CBDFN
and NtFN concentrations. Our findings would suggest
that FN is engaged in inflammatory processes occurring
in uveitis.
Abstracts
204
P14.22
P14.23
The construction of plasmid vector with
angiopoeitin-1 and its ectopic expression
in human umbilical vein cells (HUVECs)
Adipokines secretion in diabetes Wistar rats
Agata Radwańska1, Alicja Witek 2, Dagmara
Baczyńska1,2, Tadeusz Dobosz1,2
1Regional
Specialist Hospital in Wrocław, Research and Development
Center, Wrocław, Poland; 2Laboratory of Molecular Techniques,
Department of Forensic Medicine, Medical University, Wrocław, Poland
e-mail: Agata Radwańska <[email protected]>
Gene therapy is the one of the alternative methods of
medical treatment, particularly in a case when the traditional techniques are not satisfactory. In previous investigation, our group have applied VEGF gene in a therapy of
patients with critical limb ischemia.
The aim of this study was the construction of plasmid vector with cDNA of human angiopoeitin 1 gene (Ang-1). As
a source for RNA isolation we used a heart tissue derived
from the patient died of ischemic heart disease. Afterwards
the Reverse Transcription and PCR reactions were done.
Obtained DNA was cloned to pcDNA3 vector, allowing
for gene expression under a control of strong CMV promoter.
Human umbilical vein cell line (ATCC) was transfected
transiently with constructed plasmid and Fugene 6 Transfection Reagent. As positive control we used earlier characterized plasmid vector with VEGF cDNA. Analysis of
Ang-1 mRNA level was done 24 h and 48 h after transfections by Real-Time PCR method. The efficiency of Ang1 expression after transfection with pcDNA3-Ang1 was
above 30 times higher than mock transfected control cells.
Our results confirmed that prepared plasmid construct can
be efficiently expressed in the cells. The influence of Ang-1
expression on migration and proliferation capabilities of
the cells is under investigation.
Acknwledgements:
This work was supported by European Foundation for Regional Development and Polish Government PO IG 2007-2013 within the project
“Wrovasc- Integrated Centre of Heart-Vascular Medicine”.
Maciej Sassek, Ewa Pruszyńska-Oszmałek, Dawid
Szczepankiewicz, Marek Skrzypski, Iwona Hertig,
Przemysław Kaczmarek, Paweł Maćkowiak
University of Life Sciences, Department of Animal Physiology and
Biochemistry, Wolynska 35, 60-637 Poznan, Poland
e-mail: Maciej Sassek <[email protected]>
Our modern understanding of adipocytes is tightly connected with adipokines. These compounds are produced by
adipocytes and regulate a few areas of our body functioning. Present we know several dozen of various adipokines
but the most popular are: leptin, adiponectin and resistin.
The aim of this study was to determine serum concentrations three mentioned above adipokines in two induced
types of diabetes. Type I diabetes mellitus (DM) was induced in 10-weeks old Wistar rats by streptozotocin (STZ)
injection in dose 35 mg/100 g body weight. Type II DM
was induced in 2-days old Wistar rats by STZ in dose 80
mg/1 kg body weight. In both cases STZ was prepared in
0,05 mol/l citrate buffer, pH = 4.0. Control and diabetes
12-week old rats were decapitated and serum samples were
collected (clot was separated by centrifugation). Resistin
and leptin serum concentrations were determined by RIA
kits (Millipore), adiponectin serum concentration was determined by ELISA kit (Invitrogen).
We observed statistical differences of individual adipokines secretion in compare to control groups. Resistin serum concentration was increased in both types of diabetes. Opposite result was obtained for adiponectin. In both
types of diabetes serum concentration of this protein was
diminished. Leptin exhibited two different trends. In type I
DM its concentration was reduced but in type II DM concentration was raised.
P14.24
P14.25
Gene expression profiling of PBMC
from children with birch-apple
syndrome and allergic asthma
Influence of antiasthmatic drugs
concentration on bronchial
epithelial cell line proliferation
Wieslawa Woronowicz1, Beata Cudowska2, Maciej
Kaczmarski2, Aleksandra Szczepankiewicz3, Natalia
Schöneich3, Anna Breborowicz3, Luiza Handschuh1,4,
Agnieszka Ciesielska1, Marek Figlerowicz1, Michal M. Sikorski1
Aleksandra Szczepankiewicz1, Maria Wołuń-Cholewa2,
Wanda Butowska2, Paulina Sobkowiak1, Zdzisława
Kycler1, Jerzy Warchoł2, Anna Bręborowicz1
1Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznan,
Poland; 2III Department of Pediatrics, Gastroenterology and Allergology,
Medical University of Bialystok, Bialystok, Poland; 3Department of
Pulmonology, Alergy and Clinical Immunology, Poznan University
of Medical Sciences, Poznan, Poland; 4Department of Hematology,
Poznan University of Medical Sciences, Poznan, Poland
e-mail: Michał Sikorski <[email protected]>
The birch-apple syndrome, an example of cross-reactive
allergy, belongs to the type I of IgE-mediated hypersensitivity. The birch pollen protein Bet v 1 acts as a sensitizer
or primary allergen and is able to trigger the immune system to produce IgE antibodies. The homologous Mal d 1
protein from apple is in turn an incomplete allergen which
triggers mast cells loaded with anti-Bet v 1 IgE and elicits
allergic symptoms. Asthma is the most common chronic
disease of childhood which usually induces IgE-dependent
hypersensitivity in response to environmental triggers. The
aim of our study was to identify the genes that are up- or
down-regulated in birch apple syndrome and those that can
differentiate between allergic and non-allergic asthma in
peripheral blood mononuclear cells (PBMC) from pediatric
patients using microarray technology. Microarrays containing oligonucleotide probes (50-nucleotide-long) for 183
genes (involved in IgE-dependent allergy and asthma) were
designed and printed onto epoxide slides. Total RNA was
isolated from the whole PBMC fractions collected from
children with birch-apple syndrome, asthma and non-allergy children. RNA was reversibly transcribed to cDNA and
amplified with PCR. The products were used as the templates to obtain ssDNAs labeled with Alexa 555 and Alexa
647 dyes. Labeled samples were hybridized with the arrays
(overnight hybridization: 50–45–40oC). Scanned images
were analyzed with GenePixPro 6.0. R Bioconductor packages were used for normalization the data and differential
expression analysis. For validation of the microarray data
real-time PCR was used. Microarray data analysis revealed
that there are only a few genes that can potentially differentiate between non-allergic children and patients with
birch-apple syndrome and asthma. The best candidate was
chemokine gene CCL1. However, the results of real-time
PCR experiment did not confirm significant differences
in expression levels of this gene in two analyzed groups.
Presumably, the differences in gene expression profiles are
rather subtle or concern genes which were not included in
our analysis. In allergic asthmatic patients, when compared
to asthmatic patients without allergic background the expression of 4 genes involved in IgE-dependent reactions
was substiantially up-regulated. Although the up-regulated
expression of those genes was confirmed by QPCR we
did not observed significant differences between analyzed
groups, with the exception of CSF1 gene.
205
1Laboratory of Molecular and Cell Biology, 2Department of Pediatric
Pulmonology, Allergy and Clinical Immunology, 3Department of Cell
Biology, Poznan University of Medical Sciences, Poznan, Poland
e-mail: Aleksandra Szczepankiewicz <[email protected]>
Background: Bronchial epithelial cell lines enable analysis
of influence of different factors, such as exposure to inhaled antiasthmatic agents, on their morphology and function in vitro. We aimed to investigate if chronic exposure to
different concentrations of therapeutic agents used in the
inhalation therapy of asthma influence bronchial epithelial
cells growth and morphology.
Methods: Bronchial epithelial cell line 16HBE14o- was
used. We analyzed morphological changes of cells treated
with different drug concentrations: budesonide, fluticasone
propionate, salbutamol and ipratropium bromide. Observations were performed every 24 hours for 4 days. Cytotoxicity of different drug concentrations was analyzed by
fluorescent staining and XTT viability test.
Results: Positive correlation between drug concentration
and growth inhibition was observed. The only exception
was ipratropium bromide which was toxic at all studied
concentrations. Steroids at the two highest concentrations
led to significant growth inhibition with fluticasone propionate being more potent inhibitor than budesonide. Incubation with salbutamol also demonstrated inhibitory influence on cell proliferation at the two highest concentrations.
Ipratropium bromide was toxic for the bronchial cells at
all concentrations leading to significant growth inhibition.
Conclusions: Our observation that lower drugs concentrations are less harmful for the cells could be considered
when planning antiasthmatic treatment schedule.
Abstracts
206
P14.26
P14.27
Distribution of Neuromedin U,
NmUR1 and NmUR2 in rodent
adipose tissue — verification of fat
NmU receptor functionality
Effectiveness of solubleTNFα receptor
I encoded on plasmid vector in
experimental antifibrotic therapy
Przemyslaw Kaczmarek, Ewa PruszynskaOszmalek, Maciej Sassek, Marek Skrzypski, Dawid
Szczepankiewicz, Krzysztof W. Nowak
Poznan Life Sciences University,Department of Animal Physiology and
Biochemistry, Wolynska 35, 60-637 Poznan, Poland
Małgorzata Przybyszewska1, Joanna Miłoszewska1, Sylwia
Rzońca1, Halina Trembacz1, Kazimiera Pyśniak2, Agnieszka
Walewska3, Agnieszka Kotlarz1, Maciej Małecki1
Maria Sklodowska-Curie Memorial Institute of Oncology and Cancer
Centre, 1Cell Biology Department,2Genetic and Animal Breeding
3Medical Physics Department, Warsaw, Poland
e-mail: Dawid Szczepankiewicz <[email protected]>
e-mail: Halina Trembacz <[email protected]>
Neuromedin U (NmU) is a novel multifunctional neuropeptide distributed with the highest level in the gastrointestinal tract (GI) and pituitary and acting via two G-protein coupled receptors NmUR1 and NmUR2. The former
is predominantly expressed in GI tract whereas the latter
is mainly located within CNS. Current evidence suggests
a role of NmU in pain, regulation of feeding and energy
homeostasis, stress, cancer and immune mediated inflammatory disease. It is clear that NmUR2 mediates the central
effects of NmU and therefore NmUR2 receptor has been
proposed to be a potential target for developing new therapeutics for treatment of eating disorders, obesity and stress
related disorders. However, the peripheral action of NmU
via activation of NmUR1 in several tissues directly engaged
in energy metabolism might be also relatively promising.
Recently, we investigated the role of NmU in insulin secretion. We indicated involvement of NmU in regulating the
pancreatic branch of adipo-insular axis [1]. More recently,
we postulated somatostatin participation in NmU generated suppression of insulin release [2].
Here we decide to examine the possible, direct effect of
NmU on metabolism and endocrine function of adipose
tissue At the beginning, the NmU, NmUR1 and NmUR2
expression pattern in different fat depot (subcutaneous,
epidydymal, mesenteric and perirenal adipose tissue) was
assayed using real time PCR, western blotting and immunohistochemistry. Specific expression of NmU and both
receptors in distinct manner, depending on adipose origin was clearly demonstrated. Interestingly, the NmUR2
expression seems to beremarkable high in comparison to
remaining peripheral tissues i.e. pancreas. In addition, different expression profile was elucidated in differentiated
mouse 3T3L1 cells and NmUR1/NmUR2 ratio varies during adipogenesis. In order to confirm the full functionality
of NmU signal transduction apparatus in cultured 3T3L1
cells, Ca2+ flux (FLIPR assay) and cytoplasmatic cAMP
concetration was carried out.
TNFα, a proinflammatory cytokine, plays a significant role
in fibroblast and myofibroblast proliferation and recruitment in the course of fibroproliferative disorders of lung.
The increasing TNFα protein level induces TGFβ expression and ECM deposition. Prevention or moderation of
chronic lung radiation injury by TNFα activity neutralization would have important clinical implications. Pathological inflammatory and fibrotic changes could be attenuated
by an inhibition of TNFα synthesis or activity. Recently, a
TNFα antagonists derived from the extracellular domain
of the soluble TNF receptors I and II have been developed
for clinical application. Local or systemic gene transduction
may be a promising way to enhance the levels of soluble
TNFα receptors. The aim of our study was to develop an
experimental antifibrotic gene therapy, using pcDNA3.1
plasmid vectors encoding sTNFR-I, the mouse soluble
TNFα receptor I. In an in vitro study on TNF-sensitive
mouse sarcoma L1 cell line, we confirmed the ability of
psTNFR-I product to neutralize TNFα. In vivo gene delivery was conducted by intramuscular injection psTNFRI plasmid complexed with PEI-25kDa. Measurements
of post-transfection sTNFR-I plasma levels by ELISA
showed that in vivo gene transfer was effective. The cationic
polymer, PEI, was found to enhance transfection efficiency
in vivo. psTNFR-I/PEI mix (in 1:3 ratio) caused no toxicity
in the transfected mice. C57Bl/6J mice were exposed on a
single dose of 20Gy on the thorax. The animals received
psTNFR-I/PEI or control pcDNA/PEI injection 3 days
before irradiation and 10x plasmid injection, once a week,
during 2.5 months following irradiation. The psTNFR-Itreated animals developed lethal fibrotic syndrome and died
7–8 weeks later than control mice. The data suggest, that
prolonged, systemic administration of soluble TNFR-I by
nonviral, intramuscular gene transduction in post-radiation
inflammatory phase prolonged life-time and attenuated the
symptoms of lung fibrosis of mice, and could be a simple
and safe method to partially TNFα neutralization and prevention of radiation-induced lung injury.
References:
1. Kaczmarek et al. (2006) IntJourn Mol Med 18: 951–995.
2. Kaczmarek et al. (2009) Pancreas 38: 951–995, 208–212.
Acknowledgements:
The study was supported by MSRIT grant No N N401 062435.
Acknowledgements:
Supported by MNiSW grant N401 077 32/1925.
P14.28
P14.29
Homocysteine level as a prognostic
indicator in clinical practice
Taq1B polymorphism of the CETP gene
is a determinant of anthropometric
and biochemical parameters of the
response to dietary components
Anna Volkovich, Alexander Buben, Alexander Naumov
Grodno State Medical University, Grodno, Belarus
e-mail: Anna Volkovich <[email protected]>
Homocysteine is a sulfurcontaining amino acid which is a
product of methionine metabolism in organism.
Now, after carrying out of numerous researches, there is
proved close connection between increasing homocysteine
(Hcy) level and development of some diseases. So, the
blood plasma homocysteine level has important prognostic meaning in a cardiovascular pathology: it is established,
that Hcy leads to endothelial cells apoptosis, the activation
of thrombocytes aggregation, to low density lipoproteins
oxidation, nitrogen oxides bioavailability decrease, that, in
turn, increases the risk of cardiovascular diseases. Besides
of it, hyperhomocysteinemia can be the reason of some
neurodegeneratives diseases, pregnancy complications, defects in fetation [1].
The high importance of development of homocysteine
concentration methods for its definition in biological liquids of human body is clear now. According to literary data
the maintenance of the tHcy in blood plasma of healthy
person varies in a range between 5–15 μmol/l. Concentration of 15–30 μmol/l corresponds the moderate, 30–100
μmol/l — average, above 100 μmol/l — heavy hyperhomocysteinemia [3].
However thanks to new HPLC methods, including used in
our laboratory [2], defined Hcy level (~3000 analysis) does
not exceed 7 μmol/l in healthy patients blood plasma, and
only in pathologicall cases increases up to 15 μmol/l [4],
[2]. The usage of new modifications for Hcy identification
methods gives the bases to reconsider a scale of Hcy levels
in clinical practice.
References:
1. Stanger O, Herrmann W, Pietrzik K, Fowler B, Geisel J, Dierkes J,
Weger M (2004) Z Kardiol 93: 439–453.
2. Navumau AV, Zolotukhin MM, Plotski AR (2006) Acta Biochem Poln 53:
Suppl 1, 195.
3. Kang SS, Wong PW, Malinow MR (1992) Annu Rev Nutr 12: 279–298.
4. Navumav AV, Matveenko PA, Doroshenko EM, Snezhitskiy VA (2009)
Acta Biochem Polon 56: Suppl 3, 198.
207
Teresa Wesołowska, Kornel Chełstowski,
Grażyna Adler, Hanna Bukowska
Department of Laboratory Diagnostics and Molecular Medicine,
Pomeranian Medical University, Szczecin, Poland
e-mail: Teresa Wesolowska <[email protected]>
The Taq1B cholesteryl ester transfer (CETP) gene polymorphism (B1B2) is a determinant of HDL-C in nondiabetic population. This gene effect appears to be modified by
environmental factors. A population sample of 87 healthy
youth (age range: boys 14–29 yrs and girls 15–28 yrs) was
enrolled in this study.We studied the effect of Taq1B polymorphism of the cholesteryl ester transfer protein (CETP)
gene on anthropometric (body mass, adipose tissue mass,
water volume) and biochemical parameters (serum lipoproteins, phospholipids, cholesteryl esters) in response to
components of a common diet. The type of foods and
frequency of their consumption was disclosed basing on
information provided by the participants concerning the
diet during the last three days prior to blood sampling and
body measurements. Intake of solid fats (butter), chocolate, sparkling soft drinks, tea, vegetables, and fruits exerted
a significant effect - albeit varied in B1B1 and B2B2 homozygotes - on high and low density lipoproteins, lecithin,
cholesteryl oleate and linoleate, body mass index, and body
water volume. The highest body mass index was revealed in
B2B2 homozygotes whose daily diet always contained more
than 1500 ml of fluids (p < 0.0043), chocolate (p < 0009),
and vegetables. Everyday consumption of chocolate by
B2B2 homozygotes was associated with lowest levels of
lecithin and highest levels of lysolecithin (p < 0.047). B1B1
homozygotes of the CETP gene preferred sparkling soft
drinks (p < 0.018) and solid fats (p < 0.0004).
Differences in the serum lipid response of B2B2 homozygotes to dietary components may likewise be due to limited
activity of CETP and to increased transport catalyzed by
the lecithin-cholesterol acyltransferase
Abstracts
208
P14.30
P14.31
Dietary fat and leptin to adiponectin ratio
in patient with metabolic syndrome
Melatonine as a regulator of red
bloos cells membranes
Dominika Wnęk, Małgorzata Malczewska-Malec, Beata
Kieć-Wilk, Anna Zdzienicka, Małgorzata Kwaśniak
Victoria M. Naidzina, Ilyia B. Zavodnik, Aliaksandr V. Navumav
Department of Clinical Biochemistry, Jagiellonian University Medical
College, Cracow, Poland
e-mail: Dominika Wnęk <[email protected]>
Background: The dominant underlying risk factors for
metabolic syndrome appear to be abdominal obesity and
insulin resistance mainly in skeletal muscle, adipose tissue
and liver. Leptin and adiponectin are released from the adipose tissue however each in the opposite relation to the fat
mass and exert the opposite activity in relation to development of consequences of obesity. The increase of the leptin to adiponectin ratio (lep/adip) is considered as indicator
of metabolic syndrome complications (Malczewska-Malec
2009). Aim of the study: was to estimate if the dietary
fatty acids: saturated fatty acids (SFA), monounsaturated
fatty acids (MUFA), polyunsaturated fatty acids (PUFA)
may changes lep/adip ratio in patient with metabolic
syndrome. Methods: 43 polish patients (35–70 yrs) with
metabolic syndrome were assigned to the 3 months dietary
intervention. Subjects were randomised to 4 diet groups:
group A (37 % of fat, high SFA diet), group B (37 % of
fat, high MUFA diet), group C (28 % of fat, high carbohydrate complex), group D (28 % of fat, high carbohydrate
complex, enriched with n-3 PUFA).Blood leptin and adiponectin concentration were determined in all patients before and after 3 months dietary intervention. Results: We
have not observed any significant changes in the lep/adip
ratio before and after diet intervention (Table). However
we observed a moderate, unwanted increase of this ratio in
the B and C groups, when decrease of this ratio was found
in the PUFA diet D group.
Table. Mean value of lep/adip ratio before and after dietary intervention according to the groups.
diet before A 5.17 ±1.9 B 8.10 ±7.16 C 7.52 ±7.93 D 8.51 ±6.13 after
5.02 ±3.23
11.69 ±10.29
9.53 ±11.89
7.68 ±3.60
Conclusion: Supplementation of n-3 PUFA in diet seems
to improve the lep/adip ratio in patients with metabolic
syndrome. More clinical data are required.
Acknowledgements:
Supported by: FW7 EU LIPGENE IP project: Lipgene- Diet, genomic
and the metabolic syndrome: an integrated nutrition agro-food, social an
economic analysis (Contract FOOD-CT-2003-505944).
Grodno State Medical University, Grodno, Belarus. Grodno State
University, Department of Biochemistry, Grodno, Belarus
e-mail: viktoria zalesskaia <[email protected]>
Melatonine (N-acetyl-5-methoxytryptamine) a tryptophan
derivative, are potent endogenously produced antioxidant
becouse for its capacity to act as effective free radical scavenger.
Experimental data revealed that melatonine is a liposoluble
molecule, interacting with membranes of blood cells, raises
their osmotic stability. In the in vitro experiments preliminary addition of melatonine to suspension of red blood
cells in concentration of either 0.1 or 0.4 mmol markedly shifts standard curves of red blood cells lysis towards
smaller concentration of osmolyte.
Hence, melatonine while possessing the expressed capabilities as both direct and indirect antioxidant, directly reduces
osmotic fragility of red blood cells. It also interferes with
oxidative damage of red blood cells membranes which
probably takes place in the process of hemolysis.
Due to ability of melatonine to penetrate the cell membranes easily the cells can collect it, protecting red bloos
cells from haemolysis under oxidative stress.
P14.32
P14.33
Peripheral blood leukocytes
expression of ARs in GDM subjects
Effect of luteolin-7-O-glucuronide and
arbutin on staurosporine-induced
apoptosis in human leukaemia cell line
Andrzej Zieleniak1, Marzena Wójcik1, Iwona
Nadel2,3, Katarzyna Cypryk2,3
1Department of Structural Biology, 2Diabetology and Metabolic
Diseases Clinic, Medical University of Lodz, Łódź, Poland; 3Polish
Mother’s Memorial Hospital, Research Institute, Łódź, Poland
e-mail: Andrzej Zieleniak <[email protected]>
Adenosine receptors (ARs) are the G-protein-coupled
membrane molecules found in almost every kind of human
tissues. To date, four subtypes (A1, A2A, A2B, A3) of ARs
have been characterised, each with a distinctive expression
pattern. Adenosine as a natural agonist of these receptors
is known to play an important role in regulation of many
physiological processes, such as synaptic transmission,
sleep regulation, platelet aggregation, inflammation, glucose homeostasis or lipolysis. Adenosine signalling pathway may trigger different, even opposite metabolic effects,
depending on cell type and physiological state.
Many of the ARs mediated cell metabolism changes are
connected to energetic homeostasis. In particular, regulation of lipolysis and inflammation are important during
pathological conditions corresponding to metabolic syndrome. There are experimental data proving the expression
level changes of ARs may occur in some cell types in diabetic subjects (Sakowicz-Burkiewicz et al., 2006).
The purpose of our project was to verify a value of peripheral blood leukocytes as a relatively non-invasive source of
biological material to study ARs expression in Gestational
Diabetes Mellitus (GDM) diagnosed women.
We evaluated ARs expression level changes in GDM
subjects (n=70) in compare to non-GDM pregnant ones
(n=35) by a semi-quantitative PCR method followed by gel
densitometry. The levels of ARs mRNA were correlated
with biochemical and anthropological parameters.
We have observed statistically significant (p-value<0.001)
increase of A2A, A2B (of 39% and 77% respectively) and A3
(63%, p-value=0.037) mRNA levels in GDM group. There
has been detected a monotonic correlation (p-value<0.05)
of above mentioned receptors to HDL level (negative) and
oral glucose tolerance test (OGTT) after 120 min value
(positive).
In the light of these results, expression of leukocyte A2A,
A2B and A3 receptors rise significantly in GDM patients,
and a relation to some biochemical markers levels occurs. It
has been also shown, that peripheral blood leukocytes seem
to be promising biological material useful in ARs expression study in context of diabetes.
References:
Sakowicz-Burkiewicz M et al. (2006) Immunology 118: 402–412.
Acknowledgments:
The project was co-financed by The European Social Fund and the national budget within the framework of Measure 2.6 of the Integrated
Regional Operational Programme: “Stypendia wspierające innowacyjne
badania doktorantów” Project.
209
Bogdan Zieliński1, Izabela Berdowska1, Jolanta
Saczko1, Ewa Seweryn1, Izabela Fecka2
1Wrocław Medical University, Department of Medical Biochemistry,
Chalubińskiego 10, 50-368 Wrocław, Poland; 2 Wrocław Medical
University, Department of Pharmacognosy, Nankiera 1, 51-140
Wrocław, Poland
e-mail: Bogdan Zielinski <[email protected]>
Polyphenols including: luteolin-7-O-glucuronide and arbutin, are natural components in our diet exhibiting beneficial
properties such as antioxidant, anti-inflammatory, and anticarcinogenic. Another aspect is their mitochondrial toxicity and potential apoptosis-inducing properties. Luteolin7-O-glucuronide was confirmed among the others in the
genus of Thymus, Salvia and Lycopus. It reveals antioxidant,
antiallergic and antigonadotropic effects. Arbutin shows
urinary anti-infective and skin bleaching properties (inhibitor of melanin synthesis). The purpose of this study was
the evaluation of these two compounds’ effect on human
leukaemia cells subjected to staurosporin-induced apoptosis. Jurkat T cells were incubated with each of the compounds in 24-well plates for 17 h, and subsequently with
staurosporine for 3 h. For comparison, the cells were incubated with the same concentrations of the chemicals for 20
h without staurosporine. The status of Jurkat cells after the
experiment was assessed using MTT assay and caspase-3
colorimetric assay. Neither of the substances proved to
be cytotoxic against Jurkat T cells within the 30–300 nM
range, when tested with MTT assay. Arbutin alone tended
to non-significantly stimulate caspase-3 activity, but this
effect was more clear in the samples induced with staurosporine. Luteolin-7-O-glucuronide demonstrated reversed effect decreasing caspase-3 activiy, which was more
clear and statistically significant in staurosporine-simulated
cells. Similarly, as demonstrated in the previous studies,
preincubation of Jurkat cells with luteolin-7-O-rutinoside,
eriodictiol-7-O-rutinoside, and rosmarinic acid prior to apoptosis induction resulted in decrease in caspase-3 activity, whereas preincubation with caffeic acids stimulated the
enzymatic activity. These preliminary results suggest the
modulatory properties of the two compounds with regard
to apoptosis in Jurkat cells.

L14.1 Session 14: Clinical Biochemistry Lectures L14.2

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