Oral presentations O4.1 O4.2 Session 4: Biochemistry of Lipids

Session 4: Biochemistry of Lipids
Oral presentations
O4.2
O4.1
Metabolic effects of conjugated linoleic acid
Occurrence of trans fatty acid isomers and
CLA in selected Polish food products.
Mieczysław Obiedziński*
Division of Food Biotechnology and Microbiology, Warsaw
Agricultural University, Warszawa, Poland
*e-mail: Mieczysław Obiedziński < mieczyslaw_
[email protected]>
Fatty acids containing trans unsaturated double bonds
occur in nature, but the most common dietary source are
“man-treated” fats. Because of the possible link between
trans-fatty acids consumption and human disease-mainly
cardiovascular disease and cancer, the health effects of
trans-FA have been studied for over the last fifty
years. Trans fatty acids are unsaturated fatty acids with
at least a double trans configuration, resulting in a more
firm molecule close to a saturated fatty acid. These appear
in dairy fat because of ruminal activity, and in hydrogenated oils; margarines, shortenings and baked goods contain relatively high levels of trans fatty acids. Conjugated
linoleic acids (CLA) are a group of linoleic (18:2)-derived
isomers with conjugated double bonds, mostly at carbon
atoms 9 and 11 or 10 and 12, with all possible cis and trans
combinations. CLA occur naturally in several foods but
they are found in highest concentrations in milk fat and
body fat of ruminants the predominant fatty acid isomer
is the 18:2 9c,11t. CLA are also present in plant oils and
partially hydrogenated oils but in very low amounts.
In this paper the results of the studies on trans fatty acids
in different food products determined by high resolution
gas chromatography combined with mass spectrometry
are presented and reviewed.
Keywords: fatty acids with trans configuration, conjugated linoleic acid, trans FA analysis
Elżbieta Bartnikowska*
Faculty of Human Nutrition and Consumer Sciences, Warsaw
Agricultural University, Warszawa, Poland
*e-mail: Elżbieta Bartnikowska < elzbieta_
[email protected]>
The term „conjugated linoleic acid” (CLA) refers to a
mixture of positional and geometric isomers of octadecadienoic acid with conjugated bonds. Results from experiments in animals indicate that this compound prevents
and counteracts chemically induced carcinogenesis,
prevents lipid metabolism and atherosclerosis induced
via alimentary tract, improves impaired glucose metabolism, exerts protective effects on bone health as well as
reduces body fat and increases lean body mass. Detailed
mechanism(s) remain to be explained.
In this paper the results of the researches on metabolic
effects of CLA are reviewed.
Keywords: monounsaturated fatty acids with trans configuration, conjugated linoleic acid, vaccenic acid, metabolic effects of CLA.
Abstracts
34
2007
O4.3
O4.4
Effect of food restriction on lipid
metabolism in adipose tissue
Molecular mechanism of lipid-induced insulin
resistance — the role of stearoyl-CoA desaturase
Julian Swierczynski*
Agnieszka Dobrzyń*
Medical University of Gdańsk, Gdańsk, Poland
*e-mail: Julian Swierczynski < [email protected]>
Laboratory of Cell Signaling and Metabolic Disorders, Nencki
Institute of Experimental Biology, Warszawa, Poland
*e-mail: Agnieszka Dobrzyń < [email protected]>
Food restriction is an intervention that may provide a
method of body weight control to prevent and/or to treat
of obesity — a major risk factor for diabetes, hypertension and atherosclerosis. However, its effectiveness and
health benefits is still controversial. Usually some patients after food restriction gain body weight even over
the value before treatment. The molecular mechanism(s)
contributing to weight regain after food restriction is not
clear. Our studies have shown that food restriction induces in rats a substantial decrease in: a) body and fat
mass; b) serum triacylglycerol concentration; c) serum
concentrations of factors controlling food intake and energy expenditure (leptin and NPY) and increase in serum
adiponectin concentration. Moreover, food restriction increases in GLUT 4 mRNA level, lipogenic enzymes (fatty
acid synthase, acetyl-CoA carboxylase, ATP-citrate lyase
and malic enzyme) genes expression and measured in
vivo fatty acids synthesis in adipose tissue. The increase
in lipogenic activity of adipose tissue was associated with
the increase of SREBP-1 gene expression (measured at the
level of RNA and protein) in epididymal and perirenal
white adipose tissue. Thus, food restriction increases the
mature (nuclear) form of SREBP-1c level which in turn as
transcription factor activates lipogenic enzymes genes expression and consequently fatty acid and triacylglycerol
production. Moreover, the increase of NPY mRNA abundance in hypothalamus of rats maintained on restricted
diet was observed. The results obtained suggest that food
restriction could display beneficial effects including: decrease in body and fat mass, decrease in serum triacylglycerol concentration and increase concentration of serum
adiponectin, an important anti-atherogenic, anti-diabetic
and anti-inflammatory adipokine. The reduction of fat
mass, serum triacylglycerol concentration and increase
in serum adiponectin concentration might be an essential
preventive factors for cardiovascular disease. However,
the increase in lipogenic activity of adipose tissue, decrease in serum leptin concentration and increase of NPY
mRNA level in hypothalamus could be rather detrimental to dietary restricted patients because may contribute
to weight gain after food restriction.
Acknowledgements:
Project partly supported by Polish Ministry of Science and Higher Education, grant No. N401 009 31/0150.
While diabetes has traditionally been thought of as a
disorder of carbohydrate metabolism, more recent work
has indicated that diabetes is also a disease of lipid metabolism. Increased deposition of lipid in tissues other
than white adipose leads to insulin resistance and the
development of diabetes. One of the proposed reasons is
that excess lipids, particularly lipids that are deposited
in insulin-sensitive cell types other than adipocytes, can
inhibit insulin signaling. The precise identity of the lipid factor responsible is not known, although free fatty
acids, fatty acyl-CoA, diacylglycerol and ceramide are
likely suspects. By activating protein kinase C, the lipid
molecules seem to reduce the activity of insulin receptor
substrate 1 (IRS-1), a key component of the insulin signaling pathway. Altered lipid metabolism in skeletal muscle as seen in the insulin-resistant states largely depend
on the aberrant expression of genes encoding metabolic
key enzymes. Consequently, regulators of tissue specific
metabolic pathways are attractive candidates for novel
therapeutic intervention strategies, but are still mostly
unexplored. In recent years, several candidate genes have
been proposed as therapeutic targets. Stearoyl-CoA desaturase1 (SCD1) is of special significance, because SCD1
is the major gene target of leptin, the central mediator
in an endocrine circuit regulating energy homeostasis.
SCD has been established by my previous studies as a
new critical regulatory switch in the control of metabolic
pathways and the maintenance of body weight. Using
stearoyl-CoA desaturase null mouse model we have demonstrated that SCD plays a major role in the regulation
of lipid and carbohydrate metabolism as well as in the
development of obesity and diabetes. By aberrantly affecting expression of key metabolic enzymes in the liver,
the SCD1 deficiency promotes fatty acid oxidation and
thus represents a critical molecular checkpoint for maintenance of hepatic lipid homeostasis. We have shown that
mice with a targeted disruption of the SCD1 gene have
improved glucose tolerance compared to wild-type mice,
despite lower fasting plasma insulin levels. Our studies
established that in skeletal muscle and in brown adipose
tissue, basal tyrosine phosphorylation of the IRS1 and
IRS2, the association of both IRS1 and IRS2 with the ap85
subunit of phosphatidyl-inositol 3-kinase, the phosphorylation of Akt and membrane GLUT4 translocation are all
elevated in SCD1–/– mice compared with wild-type mice.
The precise mechanism of SCD action on insulin signaling
remains to be established, however, our findings on SCD
point to a very promising novel strategy for the treatment
of obesity and insulin resistance.
42nd Meeting of the Polish Biochemical Society
Vol. 54 35
O4.5
O4.6
Stearoyl-CoA desaturase 1 deficiency
decreases fatty acid oxidation in the heart
Biosynthesis, transformations and metabolic
functions of arachidonic acid derivatives in
macrophages. Role in the atherosclerotic process
Paweł Dobrzyń1*, Agnieszka
Dobrzyń1, James M. Ntambi2
1Laboratory
of Cell Signaling and Metabolic Disorders,
Nencki Institute of Experimental Biology, Warszawa, Poland,
2Department of Biochemistry, University of WisconsinMadison, Madison, United States
*e-mail: Paweł Dobrzyń < [email protected]>
Stearoyl-CoA desaturase (SCD) is a regulatory enzyme
in lipogenesis, catalyzing the rate-limiting step in the
overall de novo synthesis of monounsaturated fatty acids mainly oleate and palmitoleate from stearoyl- and
palmitoyl-CoA, respectively. SCD1 deficiency activates
metabolic pathways that promote fatty acid β-oxidation
and decrease lipogenesis in liver and skeletal muscles.
SCD1–/– mice have increased energy expenditure, reduced
body adiposity, increased insulin sensitivity and are resistant to diet-induced obesity and liver steatosis. In the
present study, using the SCD1–/– mouse model, we have
shown that SCD1 deficiency causes decrease in fatty acids
transport and oxidation in the heart. The concentrations
of free fatty acids (FFA) and triglycerides (TG) in blood
plasma were significantly lower in SCD1–/– mice compared to wild-type controls. To estimate the rate of the
membrane fatty acid transport into the heart of SCD1–/–
mice we measured the level of both fatty acid translocase
(FAT)/CD36 and fatty acid transport protein (FATP) in
isolated cardiomyocyte membranes by Western blotting.
The contents of the both proteins were significantly lower
in the heart of SCD1–/– mice compared to wild-type mice.
Decreased rate of membrane transport of fatty acids into
the heart was associated with reduced intracellular lipid
levels. The contents of FFA and TG were decreased by
30.6 and 41.9 %, respectively, in the heart of SCD1–/– mice
in comparison to wild type mice. The rate of fatty acid
oxidation is controlled by the rate of their transfer into the
mitochondria through carnitine palmitoyltransferase 1
(CPT1). Both CPT1 protein level and activity were significantly decreased in the heart of SCD1–/– mice in comparison to wild type controls. Consequently, also the rate of
palmitoyl-CoA oxidation in the heart of SCD1–/– mice was
37.1% lower compared to wild-type mice. Because FFA
and TG contents in both blood plasma and cardiomyocytes are significantly reduced in SCD1–/– mice, it is concluded that SCD1 deficiency, by increasing whole body
energy expenditure and reducing lipogenesis, decreases
fatty acids availability for utilization by the heart.
Ewa Stachowska*
Department of Biochemistry and Medical Chemistry,
Pomeranian Medical University, Szczecin, Poland
*e-mail: Ewa Stachowska < [email protected]>
Eicosanoids are lipid derivatives of three polyunsaturated 20-carbon fatty acids: dihomogammalinolenic acid
(C20:3 n–6), arachidonic acid (C20:4 n–6) and eicosapentaenoic acid (C20:5 n–3). The most common of them is
arachidonic acid, the main precursor of eicosanoids in
macrophages. In the course of eicosanoid synthesis, arachidonic acid released from membrane phospholipids is
transformed via two pathways – by cyclooxygenases to
cyclic compounds, i.e. prostaglandins, prostacyclins and
thromboxanes, and by lipoxygenases to non-cyclic compounds — leukotrienes and hydroperoxy derivatives of
arachidonic acid and linoleic acid. Two main classes of
phospholipases A2 (which are subdivided in isoforms
within one class) are found in macrophages: cytoplasmatic phospholipase A2 and secretory phospholipase A2.
Both classes participate in regulation of physiological
and pathological processes, e.g. in release of pro-inflammatory mediators and stimulation of inflammatory processes. Lipoxygenases constitute a heterogeneous family of non-heme enzymes oxygenating polyunsaturated
fatty acids. This family of enzymes includes a number of
isoforms, including 15-lipoxygenases present in macrophages and 5-lipoxygenase. The role of lipoxygenases is
to synthesise hydroperoxides of fatty acids, which play
signalling functions in the body: 15S- and 12S-HpETE
and 13S-and 9S-HpODE. HpODE and HpETE are labile
compounds which undergo rapid reduction to hydroxyl
derivatives. 5-LO is a cytoplasmatic enzyme which initiates transformation of arachidonic acid to leukotriene
A4 and its derivatives. Arachidonic acid (released from
nuclear membrane by cPLA2) is transformed by 5-LO to
5-hydroperoxyeicosatetraenoic acid (5-HpETE), which
may be converted to hydroxyeicosatetraenoic acid (5HETE) with participation of glutathione peroxidase or to
leukotriene A4 (LTA4). LTA4 may then be metabolised via
two pathways: by cytoplasmatic hydrolase to leukotriene
B4 or by LTC4 synthase to cysteinyl leukotrienes (LTC4,
LTD4, LTE4). Two cyclooxygenase isoforms are present in
macrophages: cyclooxygenase-1 (COX-1) and cyclooxygenase- 2 (COX-2). COX-1 is a constitutive enzyme, COX-2
is potently stimulated by cytokines, growth factors and
free radicals. Both isoforms of cyclooxygenases may be a
source of prostaglandins in macrophages. Induction (and
activity) of COX-2 increases significantly in the course of
monocyte differentiation. A number of factors involved
in this process were observed: AP2, STAT-1, STAT-3, IL-6.
The best known of them is the NFκB transcription factor
which is additionally responsible for induction of secretory phospholipase A2. Expression of enzymes metabolising arachidonic acid (sPLA2, 15-LO, COX-2 5-LO) was
shown to significantly increase in advanced atheroscle-
Abstracts
36
rotic lesions. The pro-atherogenic activity of these enzymes is related to LDL oxygenation, synthesis of pro-inflammatory leukotrienes and prostanoids, and regulation
of activity of matrix metalloproteinases.
2007
O4.7
HETEs changes during kidney
allograft reperfusion
Barbara B. Dołęgowska1*, Wojciech
Blogowski1, Leszek Domański2, Krzysztof
Safranow1, Maria Olszewska1, Katarzyna
Jakubowska1, Dariusz Chlubek1
1Department
of Biochemistry and Medical Chemistry,
of Nephrology, Transplantology and Internal
Medicine, Pomeranian Medical University, Szczecin, Poland
*e-mail: Barbara B. Dołęgowska < [email protected].
szczecin.pl>
2Department
Introduction: Metabolites of arachidonic acid (AA), eicosanoids, are known to play a significant role in the regulation of renal homeostasis, and are intensively examined
in various kidney diseases. However, almost no studies
address lipoxygenase (LOX) pathway of AA metabolism
during renal transplantation in humans. LOX metabolites
(hydroxyeicosatetraenoic acids — HETEs, lipoxins and
leukotriens) may significantly contribute to graft protection and, paradoxically, to graft rejection.
The aim: of this study was to determine, whether succesful renal transplantation, defined by immediate activation
of graft function, is accompanied by significant changes
in lipoxygenase pathway of AA metabolism.
Materials and methods: 16 renal recipients were included in the study. Blood was taken intraoperatively directly
before and in the 1, 3, 5 minute of graft reperfusion. The
concentrations of 5-, 12- and 15-HETE were measured using liquid–chromatography (RP–HPLC).
Results: In the first minute of reperfusion the highest concentration of 12-HETE was stated and within the next 5
minutes of reperfusion a significant constant decrease in
the concentration of this eicosanoid was observed. Moreover, the highest level of 12-HETE was accompanied by
significant decrease in the concentrations of 5- and 15HETE. The concentrations of 5- and 15-HETE in the third
minute of reperfusion increased, and in the fifth minute
significantly decreased.
Conclusion: To our knowledge we are first to report, that
successful human renal transplantations are accompanied
by significant changes in the lipoxygenase pathway of
arachidonic acid metabolism. We hypothesize, that precise analysis of HETE changes might be used as a novel
clinical marker of severity of I/R injury.
42nd Meeting of the Polish Biochemical Society
Vol. 54 37
Posters
P4.2
P4.1
Interaction of aIbI and aIIbII spectrins and their
truncated mutants with phospholipid monolayers
Effect of biotic and abiotic stress factors on the
content of isoprenoids in the tobacco leaves
Bajda1*,
Agnieszka
Dorota KonopkaPostupolska1, Agnieszka Sirko1, Malgorzata
Lewandowska1, Ewa Skorzynska-Polit2,
Maria Drazkiwicz2, Ewa Swiezewska1
1Institute
of Biochemistry and Biophysics PAS, Warszawa,
Poland, 2Maria Curie-Sklodowska University, Lublin, Poland
*e-mail: Agnieszka Bajda < [email protected]>
The biological role of free polyisoprenoid alcohols is
uncertain but biophysical experiments have shown that
isoprenoids increase the permeability and fluidity of the
model membranes. In contrast to polyisoprenoid alcohols
the biological role of prenylated quinones (plastoquinonePQ and ubiquinone) is well established. Both are components of the electron transport machinery. Additionally
both of them are postulated to function as antioxidants.
There is a little information describing role of isoprenoids
in the plant response to stress conditions (1) however the
increase of biosynthesis of quinones under oxidative stress
was observed (2). We tried to explain whether polyisoprenoids alcohols and PQ participate in defence mechanisms
activated in plants upon various stress conditions. Viral
(TMV-Tobacco Mosaic Virus) infection of tobacco plants
was used as a model of biotic stress. In our experiments
Nicotiana tabacum cv Samsun NN (resistant to TMV) was
applied. The increase of solanesol (Sol), dolichol (Dol)
and plastoquinone (PQ) content in TMV-infected leayes
suggested the possible involvement of analyzed isoprenoids in plant response to viral infection. In parallel we
analyzed activity of selected antioxidant enzymes, lipoxygenase (LOX) and lipid peroxidation in infected leaves.
The increase of antioxidant enzyme activities and lipid
peroxidation was observed directly after viral infection
in contrast to LOX activity. It is widely accepted that SA
plays central role in biotic stress signaling upon pathogen
infection. We checked if SA is an inducer of isoprenoid
accumulation. Tobacco leaves was sprayed with SA but
the content of PQ, solanesol and dolichols remained unchanged. Similarly, after wounding, the changes of content of analyzed isoprenoids were not observed.
We also investigated the effect of sulphur starvation on
the accumulation of isoprenoids in tobacco leaves (LA
Burley 21), (3). The significant increase of PQ accompanied by a moderate increase of Sol content was observed.
In contrast, Dol content was decreased.
The increase of PQ content observed upon TMV infection
and sulphur starvation suggests its plausible role in plant
defence mechanism, most probably as an element of the
antioxidant system. Parallel increase of Sol level probably
reflects the biosynthetic relationship between solanesyl
diphospate (the precursor of PQ side chain) and PQ. Doubled amount of dolichols observed in infected tobacco
leaves also suggests their role in plant.
References:
1. Bajda A et al. (2005) Acta Biochim Polon 52: 233–241.
2. Wanke M et al. (2000) Biochim Biophys Acta 1463: 188–194.
3. Wawrzynska A et al. (2005) J Exp Bot Jun 56: 1575-1590.
Witold Diakowski*, Anita HryniewiczJankowska, Ewa Bok, Anna Chorzalska,
Ewa Plażuk, Aleksander F. Sikorski
Faculty of Biotechnology, Wrocław University, Wrocław,
Poland
*e-mail: Witold Diakowski < [email protected].
pl>
Spectrin is the protein of membrane skeleton of red blood
cell which forms elongated tetramer made up of 2 aIbI
heterodimers linked head-to-head, and attached at their
distal ends to junctional complexes. This protein, linked
to the membrane through interactions with transmembrane proteins, can also bind to the aminophospholipids
of the inner membrane leaflet, as has been established
and described by many authors, and this interaction
can play an important role in stabilizing the erythrocyte
membrane. Main binding site engaged in this interaction
was identified as fragment of β-spectrin ankyrin binding
domain. Spectrin consists largely of series of homologous
repeating 106 amino acid units, each of them form triple
helical coiled-coil bundle with the helices marked as A, B,
and C, of which A and C are parallel and B is antiparallel.
At room temperature spectrins and their mutants show
45–75% of a helix, as measured by circular dichroism
spectra, and seem to be highly folded.
We attempted to answer the question; how termal unfolding and exposing of hydrophobic amino acids residues
affect spectrin-lipids interaction?
We report the results of monolayer experiments carried on PE/PC 6/4 phospholipid mixtures at pH 7.5, that
are optimal for spectrin-phospholipid interaction. The
changes in monolayer surface pressure after adding native folded and heat-denaturated (70oC, 30 min) protein
to the subphase were tested. We used spectrin and fodrin
b-subunit ankyrin binding domain and a series of their
truncated mutants.
We have observed an increase in the surface pressure of
the monolayer for all used non-denaturated proteins, that
indicate the penetration of monolayer by spectrins and
their mutants. In contrast, heat-treated proteins induce a
much smaller increase of surface pressure than observed
in case of native proteins or even a decrease in surface
pressure was observed. It seems that unfolding of spectrins and their β-spectrin ankyrin binding domain fragments markedly decreases the ability of studied proteins
to penetrate the phospholipid monolayer. Therefore these
results indicate that the ability to penetrate phospholipid
monolayer by spectrins is a property of native proteins
not a result of unfolding and denaturation.
Acknowledgements:
Supported by KBN grant no. 2P04A 021 27.
Abstracts
38
P4.3
The degree of substitution of Desulfovibrio
desulfuricans lipopolysaccharides and
their lipid A component by phosphates
Jolanta Lodowska1, Marzena Jaworska-Kik2, Daniel
Wolny2, Zofia Dzierżewicz2*, Ludmiła Węglarz1
1Department
of Biochemistry, 2Department of Biopharmacy,
Medical University of Silesia, Sosnowiec, Poland
*e-mail: Zofia Dzierżewicz < [email protected]>
Bacteria of the Desulfovibrio desulfuricans species are
Gram-negative, anaerobic, sulphate-reducing rods that
settle various ecosystems including human digestive
track. These bacteria are suggested to be involved in the
pathogenesis of some colon disorders such as ulcerative
colitis or Crohn disease. Endotoxin (lipopolysaccharide,
LPS) is the integral component of bacterium cell outer
membrane and a factor responsible for the virulence of
D. desulfuricans bacteria. The biological activity of LPS is
determined by its chemical structure, including the degree of lipid A saccharide hydroxyl groups substitution
by phosphates. The less lipid A is substituted by phosphates the less active it is.
The aim of the study was to establish the degree of substitution of endotoxin and lipid A, derived from D. desulfuricans, by phosphates. The standard soil strain La2226
and the wild strains (DV/A, DV/B, DV/H, DV/I, DV/I/1)
of D. desulfovibrio have been used. The strains have been
cultured for 10 days on pyruvate Postgate medium (30ºC,
pH 7.5) at anaerobic conditions (80% N2, 10% H2, 10%
CO2) (3). LPS was isolated from a biomass by water-phenol extraction according to the Westphal et al. method (4).
The quantity of phosphorus in LPS and in lipid A, liberated from LPS by mild acid hydrolysis, was established
by the Bartlett method (1).
The results of spectrophotometrical analysis showed the
interstrain differences in phosphorus content in both LPS
and lipid A of D. desulfovibrio. This could result from not
only different level of LPS and lipid A phosphorylation
but also from variations in the average molecular mass
of these heteropolymers. LPS extracted from a particular
bacterial strains is not structurally homogenous and rather, it consists of a group of molecules differing in molecular mass, structural details and physicochemical properties. Smaller quantities of phosphorus in LPS suggest the
greater quantitative contribution of sugar component to
endotoxin structure. Previous studies by Dzierżewicz et
al. (2) revealed macromolecular differentiation of endotoxins from DV/A and La2226 isolates indicating that the
intestinal wild strain is characteristic of a greater content
of LPS molecules containing more complex polysaccharide fragment and therefore of the greater molecular mass
than the soil strain. The interesting finding of our study
was also related to the different phosphorus quantity in
LPS derived from DV/I and DV/I/1 strains isolated from
the feces and biopsy specimen, respectively, of the same
patient.
References:
1. Bartlett G (1959) J Biol Chem 234: 466–468.
2007
2. Dzierżewicz Z, Orchel A, Komarska-Szostak A, Wawszczyk J,
Węglarz L, Szczerba J, Wilczok T (2005) Ann Acad Med Siles 59:
9–16.
3. Postgate JR (1984) The sulphate-reducing bacteria. Cambridge
University Press. Cambrige.
4. Westphal O, Luderitz O, Bister F (1952) Z Naturforsch 78: 148–
155.
42nd Meeting of the Polish Biochemical Society
Vol. 54 P4.4
39
P4.5
Basis of oleanolic acid antibacterial effect
Grudniak1*,
Kurek1,
Anna M.
Anna
Anna
Klicka1, Łukasz Samluk1, Krystyna I. Wolska1,
Ewa Wiktorowska2, Wirginia Janiszowska2
1Institute
of Microbiology, 2Institute of Biochemistry, Faculty
of Biology, University of Warsaw, Warszawa, Poland
*e-mail: Anna M. Grudniak < [email protected]>
Pentacyclic triterpenoids extracted from a variety of
plants have many biological activities which made them
useful in medicine and pharmacy. Their antifungal and
antiviral effect are well documented. In contrary the data
concerning their antibacterial activity are scarce and no
cellular target of their action is identified so far. We decided to study the effect of oleanolic acid (OL) obtained
from marigold (Calendula officinalis L.) on gram-positive
and gram-negative bacteria including two pathogenic
species – Listeria monocytogenes and Yersinia enterocolitica.
We showed that OL preferentially inhibits the growth and
survival of gram-positive bacteria. In sublethal concentration OL influences the shape of Escherichia coli and Bacillus
megaterium in different way. Escherichia coli cells become
longer and B. megaterium much shorter in the presence
of this compound. Penicillin bounding proteins, PBPs,
which are involved in the bacterial shape determination
are not the target of OL activity. OL is also the very potent
inhibitor of bacterial sporulation. OL preferentially binds
to cell envelope fraction of E. coli but in B. megaterium and
L. monocytogenes it was equally distributed between cytoplasm and envelope fractions. We demonstrated that OL
enhances lysis of E. coli and L. monocytogenes induced by
Triton X and the lysis of B. megaterium induced by Triton
X and lysosyme. This compound inhibits lysis in vitro of
murein isolated from all tested gram-positive and gramnegative bacteria. OL does not induce stress response as
concluded on the basis of its inability to influence the cellular level of DnaK chaperone. Our results suggest that
bacterial envelopes can be the potential target of OL activity however the precise mode of its action is not known
yet.
Acknowledgements:
This work was supported by founds from the University of Warsaw No BW-1720/22.
Arachidonic acid contained in triacylglycerol
is a potential factor determining phagocytic
function of human monocytes/macrophages
Izabela Gutowska1*, Ewa Stachowska1*,
Magdalena Baśkiewicz-Masiuk2, Jacek
Kijowski3, Violetta E. Dziedziejko1, Bogusław
B. Machaliński2, Dariusz Chlubek1
1Department
of Biochemistry and Medical Chemistry,
of General Pathology, Pomeranian
Medical University, Szczecin, Poland, 3Department of
Transplantation, Jagiellonian University Medical College,
Kraków, Poland
*e-mail: Izabela Gutowska < [email protected]>
*e-mail: Ewa Stachowska < [email protected]>
2Department
Peripheral blood monocytes are populations of cells of high
importance for resistance of the body. When migrating to
inflammation sites, they constitute populations cells able to
phagocyte and to present an antigen to other immune cells.
The artificial neural network analysis is a method of data
analysis which is to emulate the brain’s way of working.
Neural networks are easier in use than traditional statistical methods, since they construct models needed by the
user themselves.
Methods: Monocytes were isolated from peripheral blood
of 31 healthy donors by the lymphocyte separation media density gradient and then cultured for 7 days in the
RPMI medium with 10% autologous serum. Phagocytosis was determined using the PHAGOTEST kit, and then
analysed by flow cytometry (FACSCalibur, Becton Dickinson). The percentage of monocytes/macrophages engaging
in phagocytosis and the percentage of cells presented the
CD 68 antigen were determined. The fatty acids content in
blood was measured by gas chromatography (Perkin Elmer 8500). The cholesterol fractions and the triacylglycerol
(TG) content were measured in the fasting plasma with
commercial test kits (bio-Merieux, France). Phosphorylation of the ERK1/2 kinase and PPAR-γ2 was measured by
the western blot method. The neural networks were used
as a calculation method with the SNN (Statistica Neural
Networks) STATSOFT software package.
Results: The content of arachidonic acid (AA) in the blood
was a factor strongly determining monocyte/macrophage
phagocytosis. At the same time, this feature was also
strongly determined by plasma concentration of triacylglycerols and HDL-cholesterol. Importantly, linoleic acid
(arachidonic acid precursor) changed monocyte/macrophage phagocytosis only to a small extent. Different intensities of ERK1/2 kinase phosphorylation were noted in
the environments of arachidonic acid and linoleic acid.
Conclusion: Monocyte/macrophage phagocytosis is a
phenomenon dependent on many external factors. The
intensity of this process is associated with PPAR-γ activation. Phosphorylation is one of the mechanisms of activation/inactivation of PPAR-γ in macrophages. It seems that
arachidonic acid, while inhibiting ERK1/2 phosphorylation, may activate PPAR-γ, causing intensified monocyte/
macrophage phagocytosis.
Abstracts
40
P4.6
Biotransformation of 4’-phosphopantothenic
acid in brain structures after its
intracerebroventricular administration
Valery A. Hurynovich1*, Inna N.
Evkovich1, Gennady A. Badun2, Natalya
V. Gulyaeva3, Andrey G. Moiseenok1
1Institute
of Pharmacology and Biochemistry NASB, Grodno,
Belarus, 2M.V. Lomonosov Moscow State University, ,
Moscow, Russian Federation, 3Institute for Higher Nervous
Activity and Neurophysiology, RAS, Moscow, Russian
Federation
*e-mail: Valery A. Hurynovich < vgurinovich@rambler.
ru>
The occurrence of coenzyme A (CoA) in the amount of
42–88 nmol/g wet tissue in the large hemispheres (Deutsch, 2002, 2003) suggests an in situ system of coenzyme
biosynthesis from the vitamin precursor, L-cysteine, and
ATP in which production and biotransformation of 4’phosphopantothenic acid (PPA) play a key role. The biotransformation of [3H]PPA, obtained by thermal activation of tritium with the specific activity of 9.8 GBq/mmol,
was studied after its cerebroventricular administration
(0.6 μCi, uni-or bilaterally) to Wistar male rats. After 1020 min following the administration, cerebrospinal fluid
showed unchanged PPA. In total, brain structures retained up to 39–42% of the radionuclide (10–20 min), and
after 6 h, the radionuclide content was decreased down
to 17%, which was not accompanied by a significant radioactivity release into the blood circulation. [3H]PPA
biotransformation was studied by HPLC of tissue perchlorate extracts in the regimen of isocratic elution on a
column filled with reverse-phase sorbent (Separon SGX
C18). A preparation synthesized at our laboratory was
used as standard. The radionuclide distribution among
brain structures was investigated using HPLC of tissue
perchlorate extracts. Twenty minutes after the radionuclide administration, the structures with high [3H]PPA
uptake (large hemisphere cortex, brain stem) showed the
following metabolites: PPA, 20 %, pantothenic acid (PA),
58%, phosphopantetheine (PP-SH), 18%, and CoA, 3%.
Within the subsequent periods (3–6 h) the PPA fraction
was reduced, whereas the PA fraction was increased up
to 64–77% and the PP-SH and CoA fractions remained
at the initial levels. In other structures (cerebellum, hyppocampus, frontal cortex), the radionuclide distribution
was the same, with 41 and 28% of PPA being revealed in
the hyppocampus after 10 and 20 min, respectively. The
PPA level in the large hemisphere cortex was somewhat
lower: 37 and 20%, respectively. A maximally decreased
PPA fraction was found in the large hemisphere cortex
and cerebellum, whereas a maximally increased PP-SH
fraction was detected in the brain stem and cerebellum
6h after the administration. At that period the CoA fraction accumulated from 1.5 to 5% of tissue radioactivity.
The results suggest that [3H]PPA absorption by brain
structures was accompanied by rapid dephosphorylation
of the compound with concomitant (subsequent) phos-
2007
phorylation in a pantothenate kinase reaction and further
use by CoA biosynthetic enzymes and/or PPA absorption
by brain structures and its rapid utilization by a direct
conjugation with cysteine in a phosphopantothenoyl-Lcysteine synthetase reaction. Our findings confirm a key
role of PPA or its metabolism to PP-SH in CoA system
stabilization in neurostructures.
42nd Meeting of the Polish Biochemical Society
Vol. 54 P4.7
P4.8
Cholesterol biosynthesis feedback inhibition
by a cholesterol enriched diet in the course
of experimental chronic renal failure (CRF)
Characterization of the phospholipids
from Legionella bozemanii
Chmielewski2,
Sucajtys-Szulc2,
Michał
Elżbieta
Ewa Kossowska1*, Julian Świerczyński1,
Bolesław Rutkowski2, Wojciech Bogusławski3
1Department
2Department
of Biochemistry,
of Nephrology,
Transplantology and Internal Medicine, 3Department of Social
and Clinical Gerontology. Medical University of Gdańsk,
Gdańsk, Poland
*e-mail: Ewa Kossowska < [email protected]>
Hypercholesterolemia in the course of chronic renal failure (CRF) is a significant risk factor for cardiovascular
complications. It has been found that experimental CRF
is associated with increased liver SREBP-2 mRNA level
as well as in mature SREBP-2 protein abundance. This is
probably the cause for increase in HMG-CoA reductase
gene expression and, consequently, for increase in liver
cholesterol synthesis and hypercholesterolemia in CRF
rats. Surprisingly, enhanced liver cholesterologenesis in
CRF rats occured even though cholesterol concentration
in plasma and hepatocytes is increased, pointing to the
hypothesis, that the physiological feedback inhibition of
cholesterol synthesis may be disturbed in CRF rats. Therefore it was interesting to know if the dietary cholesterol
exerts its inhibitory effect on the liver cholesterologenesis
in the CRF rats. Control and CRF rats (achieved by 5/6
nephrectomy model) were kept on the normal or 1% cholesterol rich diet. After three days of cholesterol rich diet
feeding, animals were killed under light ether anesthesia.
It has been found that cholesterologenesis in the course
of CRF, is inhibited by exogenous alimentary cholesterol.
HMG-CoA reductase activity and HMG-CoA reductase
mRNA level decreased significantly in the liver of control
and CRF animals keep on the cholesterol enriched diet.
The changes in cholesterol synthesis, HMG-CoA activity
and HMG-CoA mRNA abundance were strictly associated with changes in SREBP-2 mRNA and mature SREBP
level. Obtained results suggest that cholesterol enriched
diet exerts similar effect on cholesterol synthesis in control and CRF rats.
41
Marta Palusińska-Szysz1*, Rafał Kalityński2,
Andrzej L. Dawidowicz2, Ryszard Russa1,
Monika Bodys1, Wincenty J. Drożański1
1Department
of General Microbiology, 2Department of
Chromatographic Methods, Maria Curie-Skłodowska
University, Lublin, Poland
*e-mail: Marta Palusińska-Szysz < martasz@biotop.
umcs.lublin.pl>
The genus Legionella includes waterborne gram-negative
bacilli that occasionally cause pneumonia in humans.
The number of species classified in the genus Legionella is
steadily increasing and presently includes at least 52 species with 70 associated serogroups. Legionella pneumophila
— the first Legionella species defined is still the dominating species among clinical isolates, but a high ratio of the
recently defined species was found to be also associated
with clinical cases. Among the most frequently occurring
isolates is L. bozemanii serogroup 1.
The aim of this study was to investigate the chemical
composition of lipids and fatty acids profile from the bacteria grown on artificial medium BCYE. The lipids and
phospholipids were extracted with chloroform/methanol and separated into nonpolar and polar fraction by
silicic acid column chromatography. The phospholipids
composition was determined by two-dimensional thinlayer chromatography. Phospholipids were analyzed using high performance liquid chromatography coupled
on-line with mass spectrometry (HPLC/ESI-MS) underpositive ionization mode. Identification of the individual
phospholipid molecular species was based on the m/z
ratio of their pseudomolecular ions, mostly sodium ion
adducts and head group-specific up-front fragmentation
products. Cardiolipin (CL), phosphatidylglycerol (PG),
phosphatidylethanolamine (PE), phosphatidylcholine
(PC) and two methylated derivatives of PE, i.e. phosphatidyl-N,N-dimethylethanolamine (DMPE) and phosphatidyl-N-monomethylethanolamine (MMPE), were
found to make up the phospholipids of the analyzed bacteria. Characteristic feature of L. bozemanii phospholipids
was the presence of branched iso and anteiso fatty acids as
well as cis-9-10-methylenehexadecanoic acid. The possible taxonomic implication of these data are discussed.
Abstracts
42
2007
P4.9
P4.10
An attempt to evaluate the effect of
selected lipids on fluoride and calcium
content in the skull bones and antlers
of roe deer (Capreolus capreolus L.)
Sphingomyelins and ceramides of umbilical cord
tissues and their alterations in preeclampsia
Sylwia Piotrowska*, Zygmunt
Machoy, Dariusz Chlubek
Department of Biochemistry and Medical Chemistry,
Pomeranian Medical University, Szczecin, Poland
*e-mail: Sylwia Piotrowska < [email protected]>
Antlers are an osseous structure characteristic of the Cervidae family. Roe deer antlers are composed of 44% organic and 56% inorganic compounds. The purpose of this
study is to evaluate the effect of total lipid content and five
fatty acids — linoleic (C 18:2Δ9.12), oleic (C 18:1Δ9), palmitic (16:0), stearic (18:0) and eicosadienic (C 20:2Δ11.14)
— on calcium (Ca) and fluoride (F) content in the skulls
and antlers of 2 groups of roe deer (Capreolus capreolus
L.). The first group comprises young deer (1–2 years) and
the second comprises old deer (4–8 years). The obtained
lipid extracts were used to measure total lipids using the
spectrophotometric method, and the fatty acids were determined quantitatively using the gas chromatography
technique with heptadecanoic acid (C 17:0) as the internal standard. Fluoride content was determined using the
ion‑selective electrode method and calcium content was
determined by atomic spectrometry absorption (ASA).
The results were analysed statistically with STATISTICA
6.1 software using the Wilcoxon ranked pair test and the
Mann-Whitney U‑test, and calculating the Spearman’s
rank correlations. The summary of the study shows that
subtle changes in the ratio of linoleic, oleic, palmitic and
stearic acids have an effect on calcium and fluoride accumulation in the antlers and bones of young and old roe
deer (Capreolus capreolus L.). They regulate the mineralisation process, the regeneration process, and the shedding
and involution of antlers. In young animals, the calcification of antlers is stimulated by oleic acid and inhibited
by palmitic acid. In old animals, oleic, palmitic, linoleic
and stearic acids stimulate the antler involution process. There are no statistically significant changes in the
content of fatty acids (linoleic, oleic, palmitic, stearic and
eicosadienic), total lipids, or calcium and fluorides in the
antlers or skull bones of roe‑deer (Capreolus capreolus L.)
over the course of their lives. Only eicosadienic acid fails
to correlate with the parameters tested, suggesting that it
has no effect on the hard tissue metabolism of roe deer.
Lech Romanowicz*, Edward Bańkowski
Department of Medical Biochemistry, Medical Academy of
Bialystok, Białystok, Poland
*e-mail: Lech Romanowicz < [email protected]>
Preeclampsia is accompanied by an extensive remodeling
of the extracellular matrix of the umbilical cord. First of
all it is accompanied by an increase of collagen content
in the umbilical cord arteries and in Wharton’s jelly but
a decrease in umbilical cord vein. Furthermore preeclampsia distinctly reduces proteolytic and gelatinolytic
activity, especially after activation with various agents. It
is of interest if preeclampsia make any influence on cells
from umbilical cord tissues. So we decided to determine
sphingomyelins and ceramides content in umbilical cord
tissues. Studies were performed on the umbilical cord arteries, vein and Wharton’s jelly taken from 10 newborns
delivered by healthy mothers and 10 newborns delivered
by mothers with preeclampsia. Sphingomyelins and ceramides were isolated by Thin Layer Chromatography,
fatty acids were liberated by basic hydrolysis and analyzed by HPLC of their p-bromophenacyl derivatives using detection on 254 nm. It was found that preeclampsia
was associated with an increase in sphingomyelin content
in umbilical cord arteries and a decrease in umbilical cord
vein. Preeclampsia evoked reduction in ceramides content in umbilical cord vein too. Saturated fatty acids were
the main group of fatty acids incorporated to sphingomyelins and ceramides from all control and preeclamptic
umbilical cord tissues. The estimation of sphingomyelins
and ceramides relationship allowed to find that preeclampsia caused significant increase in relative contribution of sphingomyelins from umbilical cord arteries and
vein. Such a remodeling of plasma membrane could
change metabolism of cells from umbilical cord tissues of
newborns delivered by mothers with preeclampsia.
42nd Meeting of the Polish Biochemical Society
Vol. 54 P4.11
P4.12
Vascular cell adhesion molecule-1 as a risk factor
of atherosclerosis in diabetes mellitus patients
Application of the enzymatic assay of
cholesterol to the examination of cholesterol
metabolism in the lysosomal membranes
Ewa Romuk*, Jolanta Jagosz, Bronisława SkrzepPoloczek, Krzysztof Strojek, Ewa Birkner
Department of Biochemistry, Medical University of Silesia,
Zabrze, Poland
*e-mail: Ewa Romuk < [email protected]>
Background: Diabetes mellitus (DM) is the chronic disease that is associated with an increased risk of premature
atherosclerosis. The “response to injury” hypothesis postulates that the initial step of atherogenesis is represented
by endothelial dysfunction. The first sign of disease activity is an upregulation of adhesion molecules such as
vascular cell adhesion molecule VCAM-1. The aim of our
study was to evaluate the concentration of VCAM-1 in
patients with diabetes mellitus.
Materials and methods: We have studied 20 patients with
type I DM, 20 patients with type II DM and 20 healthy
persons without DM. Total cholesterol, HDL -cholesterol,
triglicerydes we have measured with the use of standard
kits (Alpha Diagnostic, Poland), LDL-cholesterol was
measured directly with the biomerieux kit (bioMerieux,
France). Serum VCAM-1 concentration was measured by
ELISA kit (Biomedica, Germany).
Results: Concentration of VCAM-1 in DM II was statistically significant higher than in type I DM (918.55 ± 280.23
ng/ml vs. 759.14 ± 105.45 ng/ml, p < 0.05). VCAM-1 concentration in type I DM and type II DM was statistically significant higher tan in control group (759.14 ± 105.45 ng/ml
vs. 595.61 ± 198.23 ng/ml, p < 0.05 and 918.55 ± 280.23 ng/ml
vs. 595.61 ± 198.23 ng/ml, p < 0.05). There were no changes
in lipids parameters.
Conclusion: The present study demonstrated that in diabetes mellitus patients endothelial function is abnormal.
Increased VCAM-1 concentration suggests that this molecule plays an important role in the initiation of atherosclerosis.
43
Katarzyna Roszek*, Edyta Kuczkowska,
Justyna Waloch, Michał A. Komoszyński
Department of Biochemistry, University of Mikołaj Kopernik
Toruń, Poland
*e-mail: Katarzyna Roszek < [email protected]>
Steroid sulphohydrolase (EC 3.1.6.2) has received in recent years the considerable attention due to its involvement in pathogenesis of diseases like X-linked ichthyosis
or breast cancer. Steroid sulphohydrolase from human
placenta lysosomal membranes is highly specific to cholesterol sulphate as a substrate and acts optimally at pH
3.4. The cholesterol sulphate sulphohydrolase (CHS-ase)
is capable of hydrolysing the exogenous cholesterol sulphate as well as the cholesterol sulphate present in lysosomal membranes. Free cholesterol and sulphate ion are
products of the enzymatic reaction catalyzed by this enzyme.
Up to now the determination of CHS-ase activity was
based on the loss of cholesterol sulphate assayed by the
method of Roy [1]. We adapted the enzymatic CHOD/
PAP method [2] of cholesterol quantification to the determination of CHS-ase activity. Application of this method
in crude lysosomal fraction required the addition of 30
mM sodium cholate for the effective solubilization of lysosomal membranes and free cholesterol.
The amount of cholesterol released in the enzymatic reaction catalyzed by CHS-ase in the lysosomal membranes
rised during 60 minutes of incubation to 8.95 ± 1.2 μmoles/
ml and unexpectedly, after next 60 minutes decreased to
4.05 ± 0.03 μmoles/ml. Addition of exogenous cholesterol
(5 μmoles/ml) to the lysosomal membranes and incubation under the above conditions also effected in the linear
decrease of cholesterol concentration depending on the
amount of membranes used.
The above changes suggest the conversion of cholesterol
to other derivatives. Our current studies are focused on
the identification of these derivatives.
Conclusions: 1) The application of enzymatic CHOD/PAP
method of cholesterol quantification allowed us to find
that cholesterol produced by CHS-ase in the lysosomal
membranes is subsequently converted to other derivatives. 2) The concentration of membranous cholesterol
must be precisely controlled since it influences the stability of the membranes. Cholesterol sulphate is then the
reserve pool of the inactive steroid for the production of
cholesterol as well as its derivatives. 3) We presume that
CHS-ase present in lysosomal membranes from human
placenta may be a part of the multienzymatic complex
involved in regulation of cholesterol and its derivatives
content in these membranes.
References:
Roy A. (1956) The enzymic synthesis of steroid sulphates. Biochem J 63: 294–300.
Richmond W (1973) Preparation and properties of a bacterial cholesterol oxidase from Nocardia sp. and its application to enzyme
assay of total cholesterol in serum. Clin Chem 19: 1350–1356.
Abstracts
44
P4.13
Influence of conjugated linoleic acid
diens (CLA) on the glutathione peroxidase
and catalase activity in macrophages
Marta Rybicka*
Department of Biochemistry and Medical Chemistry,
Pomeranian Medical University, Szczecin, Poland
*e-mail: Marta Rybicka < [email protected]>
Conjugated linoleic acid diens is a term used to describe
positional and geometric isomers of linoleic acid with the
presence of conjugated double bonds. The dietary sources
of CLAs are above all animal fats, mainly the fats of meat
(mainly beef) and milk products. CLAs are also formed
during thermal processing of meat. The positive influence
of CLAs on the reduction of the total body fat mass was
used by the pharmaceutical industry for the production
of products supporting weight reduction based on CLAs.
Commercial sources of CLA predominantly contain cis-9,
trans-11 (~40%) and trans-10, cis-12 (~40%) CLA isomers.
Reactive oxygen species are toxic products formed as
a result of reduction of molecular oxygen in the cell. In
monocytes, ROS sources are the mitochondrial electron
transport chain, cyclooxygenases, lipooxygenases, cytochrome P-450 and NAD(P) oxidase. There are factors
which protect cells from excessive oxidation: chemical
antioxidants, and also enzymes – e.g. catalase (Cat), glutathione peroxidase (GPx), soperoxide dismutase SOD.
In macrophages (cells crucial for atherosclerotic process)
cultured with CLA the activation of ROS generation, free
radical peroxidation of arachidonic acid to isoprostanes
and activation of apoptosis process was observed.
The aim of the study was to estimate the influence of main
CLA isomers on the macrophage catalase and glutathione
peroxidase activity.
THP-1 monocytes were treated with 100 nM PMA for
24 hr, then the adherent macrophages were washed
three times with phosphate-buffered saline (PBS) and incubated with fatty acids for 48 hr at 37oC. Fatty acids were
added as 4 mM stock solution dissolved in 1 mM fatty
acid free FBS. Isomers of CLA (cis-9, trans-11 & trans-10,
cis-12) were used at final concentrations of 30 μM. The
viability of cells was tested by trypan blue exclusion. After incubation cells were sonicated to homogenous suspension. In the suspension the catalase and glutathione
peroxidase activity was estimated (by the spectrophotometric method). The protein concentration was measured
by the Bradford method. For related samples significance
was first checked with Friedman ANOVA, then significant results were subjected to the Wilcoxon matched-pair
test. P < 0.05 was considered significant.
In macrophages obtained from THP-1 and incubated with
fatty acids the activity of Cat and GPx were decreased
after incubation with CLA (p < 0.05). A significant fall in
Cat activity 52% as compared to control was noted for
trans-10, cis-12 isomer, and 47 % for cis-9, trans-11 (both
p < 0.05).
A significant reduction in GPx activity — 49% (p < 0.05)
was also noted for trans-10, cis-12 CLA, and 57 % (p < 0.05) for cis-9, trans-11 CLA as compared to control.
2007
CLAs may act as an inhibitor of catalase and glutathione peroxidase activity in macrophages, and can lead to
impairment of their defence ability against prooxidative
factors.
42nd Meeting of the Polish Biochemical Society
Vol. 54 P4.14
Changes in polyisoprenoid alcohols
accumulation upon abiotic stress in plants
Karolina J. Skorupinska-Tudek1*, Julita
Sternik1, Agnieszka Bajda1, Grazyna
Klobus2, Ewa Swiezewska1
1Institute
of Biochemistry and Biophysics PAS, Warszawa,
Poland, 2Wrocław University, Wrocław, Poland
*e-mail: Karolina J. Skorupinska-Tudek < karolina@ibb.
waw.pl>
Plants have created many well characterized defense
mechanisms against environmental stress however new
compounds involved in these processes are still discovered [1].
Polyisoprenoid alcohols (dolichols and polyprenols) are
linear five-carbon unit polymers occurring in almost all
living cells constituting families of prenologues.
In this study the effect of salinity, heavy metals, lower
temperature and phosphate and nitrogen starvation on
polyisoprenoid alcohol accumulation were tested.
In vitro root cultures of Cucumis sativus and Coluria geoides
were applied as models. Dolichols were extracted and analyzed as described previously [2]. Roots of both plants
accumulate a mixture of dolichols from Dol-15 to 22, with
Dol-16 or Dol-17 dominating for Coluria geoides and Cucumis sativus, respectively.
Dolichol content was increased upon all abiotic stress conditions tested. In the presence of NaCl (60–100 mM) dolichol content was doubled in comparison to the control
while when the medium was supplemented with CdCl2
(80 μM) the content of dolichol reached approx. 130% of
the control. Prolonged cultivation of Coluria roots at 7°C
resulted in significant elevation (approx. 200% of control)
of the dolichol content.
On the contrary to the above mentioned stress conditions
starvation resulted in the decreased dolichol accumulation (approx. 75% and 60% level of the control for phosphate and nitrogen deficiency, respectively). Estimation
of dolichol content in membrane fractions of plant cells
is in progress.
References:
1. Mittler R (2006) Trends Plant Sci 11: 15.
2. Skorupinska-Tudek K et al. (2003) Lipids 38: 981.
45