Sponsored Oral presentation SO14 Posters P14.1 Session 14: Varia

Sponsored Oral presentation SO14 Posters P14.1 Session 14: Varia

Session 14: Varia
Sponsored Oral presentation
Posters
SO14
P14.1
LCMS-QIT-TOF: High-speed liquid
chromatograph mass spectrometer
for structural analysis
Antimicrobial peptides of Galleria
mellonella degraded in vitro and in vivo
by Pseudomonas aeruginosa protease B
Marek Szklarczyk1*, Marcus
Mreyen2, Alan Barnes2
Mariola Andrejko*, Magdalena MizerskaDudka, Teresa Jakubowicz
1SHIM-POL,
Warszawa, Poland, 2Shimadzu Europe GmbH,
Duisburg, Germany
*e-mail: Marek Szklarczyk < [email protected]>
Department of Invertebrate Immunology, Maria CurieSklodowska University, Lublin, Poland
*e-mail: Mariola Andrejko < [email protected]>
There are several types of LC/MS instruments used in the
different laboratories. The single quadrupole and triple
quadrupole instrumets are primarily used for quantitative analysis, while the ion trap, LC-TOF and Qq-TOF instruments are basicly used for qualitative analysis. Triple
quadrupoles, the excellent instruments for quantitative
analysis, unfortunately falter in terms of mass accuracy
and resolution. Ion traps are excelled in structural analysis
due their ability to perform MSn analysis, however, their
mass accuracy and resolution, like for a triple quadrupole
MS is strongly limited. The Qq-TOF instruments features
excellent resolution and mass accuracy, but because their
construction they do not have ability to perform higher
stage analysis than MS3 what makes limited their application in structural analysis.
Shimadzu developed a new type of hybrid mass spectrometer, the LCMS-QIT-TOF, which possesses both the
MSn ability of an ion trap, and the excellent resolution
and mass accuracy of a TOF analyzer. This hybrid instruments opens new doors to the prediction of elemental
composition and structural analysis. The special construction of ion trap, four parts, greatly increases accurate
MSn fragmentation. The so call Quadrupole (four parts)
Ion Trap (QIT) is in the unique linear arrangement with
TOF analyser what increases speed of analysis and allows
to avoid trap saturation. It allows to get qualitative information within a limited HPLC peak elution time.
In this presentation we will present the unique construction of the LCMS-QIT-TOF instrument pointing the innovative octopole lens construction. The several chemical
and biochemical applications will be presented, e.g. for
flavonoids and proteins. The application of LCMS-QIT_
TOF instrument for analysis of small and large molecules
will be shown. The step by step multi stage, MSn, analysis
will be explained.
The universality of new instrument makes possible its application in qualitative analysis, structural and metabolite
ID studies, impurity analysis, proteomics and biomarker
studies.
Insects defense mechanisms are based on cellular and humoral innate immune response systems. One of the most
important mechanisms of humoral defense is induction
of antimicrobial peptides and proteins in response to infection with bacteria or inoculation of bacteria products
(lipopolisaccharide-LPS, peptidoglycan) into the body
cavity. Cecropins, insect defensins, glicine-rich and proline-rich peptides, lysozyme, hemolin and others have
been identified among inducible peptides/proteins in
challenged insects.
It is known that P. aeruginosa is an opportunistic human
pathogen responsible for many types of infectious diseases. Bacteria secrete several extracellular proteolytic
enzymes that have been implicated as virulence factors.
Among them there are: protease IV, alkaline protease
(aeruginolysin), two elastases, namely Las A (staphylolysin) and Las B (pseudolysin).
It is known that the insect-pathogenic strain P. aeruginosa
impaired cellular defenses of the greater wax moth G. mellonella larvae by hemocyte breakdown. It was also demonstrated that P. aeruginosa serine protease IV degraded in
vitro and in vivo apolipophorin-III (apolp-III) from hemolymph of G. mellonella larvae, while lysozyme appeared to
be resistant for degradation.
In this study we tested the expression level and activity
of peptides in hemolymph of G. mellonella larvae infected
with P. aeruginosa in different periods of time. Significant
increase of peptides activity 18 h after infection was detectable while after 30 h postinjection time only trace of
activity was noted. Decrease of antimicrobial activity was
correlated with significant decrease of peptides of Mr 4–6
kDa revealed by Tris- tricine SDS/PAGE.
In the following experiments we demonstrated that antimicrobial peptides in hemolymph of G. mellonella was degraded in vitro by P. aeruginosa metalloprotease - elastase
B. When immune hemolymph preincubated with enzyme
fraction was separated by Tris-tricine SDS/PAGE, peptide
bands with molecular mass 4–5 kDa were not observed
in comparison to control immune hemolymph. Peptides
degradation was inhibited by 1 mM EDTA, additionally
demonstrating that digestion was catalysed by metallo-
42nd Meeting of the Polish Biochemical Society
Vol. 54 protease. By using bioautography it was also demonstrated that in elastase B treated hemolymph samples antimicrobial activity was absent.
The experimental evidence reported here indicates that
peptides proteolytic degradation in G. mellonella hemolymph may be caused by P. aeruginosa elastase B. This protease seems to participate in the virulence against insect
immune response.
199
P14.2
The participation of ClpB80 and
ClpB95 proteins in thermotolerance and
removal of the heat-aggregated proteins
from Escherichia coli ΔclpB cells
Izabela Guenther*, Andrzej Taranta, Marta
Cichorska, Sabina Kędzierska-Mieszkowska
Department of Biochemistry, University of Gdańsk, Gdańsk,
Poland
*e-mail: Izabela Guenther < [email protected].
pl>
ClpB from Escherichia coli is an ATP-dependent ring-forming chaperone that mediates the resolubilization of aggregated proteins in cooperation with the DnaK chaperone
system. ClpB belongs to the Hsp100/Clp superfamily
of AAA+ proteins and is composed of two ATP-binding
AAA+ modules, the middle domain which forms a coiledcoil structure and the N-teminal and C-terminal domains.
Two isoforms of ClpB are produced in vivo: the full-length
ClpB95 and the ClpB80, which does not contain the N-terminal domain. The physiological function of the different
isoforms of ClpB has not been explained yet. We examined the ability of the two different-sized ClpB homologues to confer thermotolerance and remove heat-aggregated proteins from Escherichia coli cells (the S fraction).
For this purpose we constructed series of plasmids allowing production of the ClpB95/ClpB80 pair, ClpB95 alone,
or ClpB80 alone. We found that in the ΔclpB mutant cells
expressing ClpB95/ClpB80, the S fraction disappeared 20
min after transfer of the culture from 45°C to 37° C. In
contrast to this, in the clpB mutant overexpressing ClpB95
or ClpB80 alone elimination of the S fraction was retarded
— nearly 50% of the aggregated proteins remained stable
20 min after heat shock. These results indicate that cooperation of both isoforms of ClpB is required to protect
Escherichia coli from thermal killing and to efficiently remove protein aggregates after heat shock.
Abstracts
200
P14.3
P14.4
Cytotoxicity of 3-(2-benzoxazol5-yl)alanine derivatives
Comparison of the influence of selected
polysiloxane derivatives on the growth
of Chlorella vulgaris algal cultures
Ewa Mulkiewicz, Katarzyna
Guzow*, Wiesław Wiczk
Faculty of Chemistry, University of Gdańsk, Gdańsk, Poland
*e-mail: Katarzyna Guzow < [email protected]>
3-(2-benzoxazol-5-yl)alanine derivatives are a group of
unnatural amino acids which can be used as fluorescent
probes [1, 2]. Moreover, some of them are active against
Bacillus subtilis, Pichia pastoris, Candida albicans, Aspergillus niger [3]. Because of that, cytotoxicity of those compounds was studied using in vitro methods. Tests were
performed for 40 derivatives using tumour cell lines (rat
glial cells C6 and mouse fibroblasts A9) as well as normal cell line (embryonic kidney cells Hek 293). WST-1
test was used to determine concentrations of the compounds causing 50% reduction of the cells viability compared with control values (EC50) [4,5]. It was found that
only 7 compounds among all studied are not toxic to
the tumour rat glial cells (3-[2-(2-naphthyl)benzoxazol5-yl]alanine, 3-[2-(4-biphenyl)benzoxazol-5-yl]alanine,
3-[2-(4-methylphenyl)benzoxazol-5-yl]alanine, 3-[2-(2benzofuryl)benzoxazol-5-yl]alanine, 3-[2-(2,4-dihydrox
yphenyl)benzoxazol-5-yl]alanine methyl ester, 3-[2-(4carboxyphenyl)benzoxazol-5-yl]alanine methyl ester,
3-[2-(2-imidazolyl)benzoxazol-5-yl]alanine). In the case
of Hek293 cell line, only one compound among studied
was not toxic — 3-[2-(4-phenylboronic acid)benzoxazol5-yl]alanine methyl ester. For all cell lines studied, similar
structure-activity relationship was observed. However, the
mouse fibroblasts A9 were more sensitive to the presence
of the compounds studied than rat glial cells C6. Also, in
some cases of the compounds studied the hormetic effect
was observed. Most of compounds studied was less toxic
to the normal cell line in comparison with tumour cell
lines, except for 3-[2-(8-quinolinyl)benzoxazol-5-yl]alanie
methyl ester and 3-[2-(2,5-dimethoxyphenyl)benzoxazol5-yl]alanine methyl ester which were more toxic.
References:
1. Guzow K, Szabelski M, Karolczak J, Wiczk W (2005) J Photochem Photobiol A: Chem 170: 215.
2. Szabelski M, Rogiewicz M, Wiczk W (2005) Anal Biochem 342:
20.
3. Guzow K, Obuchowski M, Wiczk W (2006) Acta Biochim Polon
53 (suppl): 184.
4. Ranke L, Molter K, Stock F, Bottin-Weber U, Poczobutt J, Hoffmann J, Ondruschka B, Filser J, Jastorff B (2004) Ecotoxicol Environ Saf 58: 396.
5. Van Ewijk PH, Hoekstra JA (1993) Ecotoxicol Environ Saf 25:
25.
Acknowledgement:
This work was financially supported by the Polish Ministry of
Science and Higher Education under grants BW 8000-5-0287-6
and BW 8000-5-0049-7.
2007
Grażyna Janikowska1*, Urszula Mizerska2,
Witold Fortuniak2, Julian Chojnowski2
1Division
of Analytical Chemistry, Medical University
of Silesia, Katowice, Poland, 2Center of Molecular and
Macromolecular Studies PAS, Łódź 90-363 Poland
*e-mail: Grażyna Janikowska < gjanikowska@slam.
katowice.pl>
Chlorella algae are recommended by OECD (Organization
of Economic Cooperation Development) and other world
agencies (EPA, ISO) to investigate influence of chemical
matters on aquatic environment, as also estimation of
cleanness of waters [1]. Algal tests permit to rate influence
on examined chemicals in aquatic environment, one from
links of trophic chain. Serve to estimations of biological
activity of chemical substances. It is known that quarternary amonium salts are acting on biological microorganisms such as bacteria or fungi and the cytotoxic activity
were demonstrated [2]. The aim of present work was comparison of the influence of selected siloxane copolymers
with quarternary ammonium groups on growth of alga
Chlorella vulgaris cultures. Cultures of Chlorella vulgaris Beijerinck 1890 strain A-8 carried out in 300 ml Erlenmayer
flasks with bacteriological corks, in liquid medium KühlLorenzen modified by Borns and others [3], in conditions
of continuous lighting with mercury-glow lamp (4800 lx)
and continuous mixings with magnetical stirrers. Both
cultures, control cultures and cultures with addition of
suitable quantities investigated polysiloxane derivatives,
such as alpha, omega – trimethyl polysiloxy-{chloric[3(N-n-octyl-N,N-dimethyl ammonium) propyl}methyl
siloxane and alpha, omega-trimethyl polysiloxy-{{chloric-[3(N-n-octyl-N,N-dimethyl ammonium) propyl] ethyl
siloxane}-co-(3-chlorpropyl) methyl siloxane}. The initial
density of cultures about 300 thousands cells on 1 ml of
culture were used in investigations. Observed changes in
biological activity of investigated polysiloxanes we can
see in growth of Chlorella vulgaris alga cells. The investigated copolymers in heterogeneous manner acts on alga
cells. Ascertained changes show univocally that upper
named polysiloxanes disturb equilibrium in proliferation
and apoptosis process of cells in relation to control cultures. The algicidal effect of these derivatives are different
and decide about their quantities, chemical construction
and time of acting on algas.
References:
1. OECD Guidelines for the testing of chemicals, Paris 1993.
2. Hoogerheide J (1944) J Bacteriol 49: 277.
3. Borns E, Böhm H, Schultze D (1973) Wiss Hefte d Päd Inst Köthen
2: 55.
42nd Meeting of the Polish Biochemical Society
Vol. 54 P14.5
P14.6
Humoral immune response of Galleria mellonella
larvae after infection with entomopathogenic
strain Bacillus thuringiensis subsp. kurstaki
Thermolysin – an inductor of Galleria
mellonella immune response
Kowalski*,
Patryk
Teresa
Jakubowicz, Iwona Wojda
Department of Invertebrate Immunology, Maria CurieSklodowska University, Lublin, Poland
*e-mail: Patryk Kowalski < [email protected].
lublin.pl>
Bacillus thuringiensis is a Gram-positive, spore forming
bacteria, that produces a parasporal crystals during the
stationary phase of growth. It is a species of outstanding
scientific interest due to its leading role in microbial pesticides industry. The main components of its parasporal inclusions: delta-endotoxins represent the most successful
use of insect biological control agent to date. Bacillus thuringiensis discovered to science in 1911 is already a useful
alternative to synthetic chemical pesticide application in
agriculture. It is also a key source of genes for transgenic
expression to provide pest resistance in plants. Production improvement and engineering of new pore-forming
toxins are the main scopes of Bacillus thuringiensis industry science.
We investigated the other side of this insect-pesticide battle, examining model organism Galleria mellonella, which
is a honey bee hive pest. In our study we observed the
humoral immune-response of this lepidopteran to Bacillus thuringiensis subsp. kurstaki. Bacteria kills wax moth
efficiently in dosage-dependent manner. We studied the
appearance of antimicrobial activity in hemolymph from
immune-stimulated larvae. Both antibacterial and antifungal activity increased within 7 hours after infection
and decreased afterwards, resulting in animal death. We
showed that infection triggers MAP kinases pathways.
Activation of MAP-kinases routes, measured as double
phosphorylation of ERK, p38 and JNK MAP kinases in
fat body cells at the beginning of immune response was
observed. This may point at their importance in immune
process since the fat body plays the main role in humoral
response.
201
Magdalena Mizerska-Dudka*, Mariola
Andrejko, Teresa Jakubowicz
Department of Invertebrate Immunology, Maria CurieSklodowska University, Lublin 20-033 Poland
*e-mail: Magdalena Mizerska-Dudka < mariola.
[email protected]>
Insects have developed unique, innate immmune system.
They are able to synthesize a large number of antimicrobial peptides, as a response to invading microorganisms.
It is well documented that insects sense foreign entities
by cell wall components common in bacteria or fungi,
but absent from animals. Recent studies on Galleria mellonella immune system revealed that induction of innate
immunity in G. mellonella does not necessary require recognition of microbial cell wall components. The presence
of metalloprotease activity (thermolysin-like activity) in
hemolymph alone is sufficient to elicit innate immune
response in G. mellonella. Many entomopathogenic bacteria, fungi or viruses produce metalloproteases as virulence factors. Secretion of these enzymes into hemolymph
during pathogenesis leads to degradation of extracellular matrix protein – collagen IV and releasing peptides
( < 3 kDa) with strong immune stimulatory activity. These
peptide fragments activate a signaling pathway leading
to expression of immune proteins such as lysozyme and
inducible gallerimycin or/and other antimicrobial peptides and IMPI (Insect Metalloproteases Inhibitors).
Our results demonstrated that injection of thermolysin
at different, sublethal concentrations (0.1; 0.2; 0.3; 0.4; 0.5
µg/larva) into hemocel of G. mellonella larvae elicited immune response. We observed that lysozyme activity estimated 24 h after thermolysin injection increased significantly depending on thermolysin dose. Our studies also
revealed that thermolysin injection induced antimicrobial
activity against Gram-negative E.coli D31 depending on
thermolysin concentration used. Similar results were
observed after immunization G. mellonella larvae with
heat-killed cells of entomopathogenic strain Pseudomonas
aeruginosa, ATCC 27853. Using tricine SDS/PAGE electrophoresis we observed 2 major polypeptide bands below
6.5 kDa after immunization with both thermolysin and
microbial elicitors of humoral immune response. These
bands present inducible antimicrobial peptides G. mellonella larvae. Injection of thermolysin in sublethal doses
induced also thermolysin-inhibitory activity. It is known
that immunization G. mellonella larvae with heat-killed
cells P. aeruginosa developed protective immunity against
subsequent infection with viable cells entomopathogenic
strain P. aeruginosa, ATCC 27853. In our experiment we
achieved about 46% survival rate for immunized G. mellonella larvae within 48 h after infection with Pseudomonas
aeruginosa. Whereas immune response induced by sublethal doses of thermolysin elicited smaller survival rate,
ranging from 15 to 20%, depending on thermolysin dose.
Abstracts
202
P14.7
P14.8
Protein kinase A activity in haemocytes
of Galleria mellonella larvae upon
immune challenge conditions
Analysis of biofilm formation by
natural isolates of Bacillus spp.
Małgorzata Cytryńska, Agnieszka ZdybickaBarabas*, Teresa Jakubowicz
Department of Invertebrate Immunology, Maria CurieSklodowska University, Lublin, Poland
*e-mail: Agnieszka Zdybicka-Barabas < barabas@biotop.
umcs.lublin.pl>
Protein kinase A (PKA) activity was detected in haemocytes of the greater wax moth, Galleria mellonella larvae
using kemptide, a specific peptide substrate. The enzyme
was activated in vitro by 1 μM concentration of cAMP,
its analogs 8-Br-cAMP, 8-Chl-cAMP, as well as by BzcMP.
Cyclic GMP was much less effective in haemocyte PKA
activation. Incubation of G. mellonella haemocytes in vitro in the presence of a cell-permeable, specific PKA inhibitor, Rp-8-Br-cAMPS, induced changes in haemocyte
morphology resembling those caused by live bacteria.
Immune challenge of G. mellonella larvae with bacteria
led to changes in haemocyte PKA activity. Gram-positive
Micrococcus luteus was a better inducer of PKA activity
than Gram-negative Escherichia coli. The kinetics of activity changes was dependent on the used bacteria and considerably differed from that observed in water-treated insects. Four potential PKA substrates of 155 kDa, 44 kDa,
40 kDa and 22 kDa were detected in haemocytes of naive larvae by phospho-motif antibodies recognizing PKA
phosphorylation consensus site. The modification level of
40 kDa protein changed in haemocytes of water- as well
as both bacteria-treated G. mellonella larvae, whereas that
of 155 kDa protein changed only after E. coli injection.
Additionally, in the haemocytes of bacteria-challenged
insects a transient phosphorylation of 36 kDa protein
was detected.
2007
Mariusz Bikowski*, Michał P. Obuchowski
Medical University of Gdańsk, Gdańsk, Poland
*e-mail: Mariusz Bikowski < [email protected]>
The endospore-forming rhizobacteria from the Bacillus
genus living in association with plant roots are universal
symbionts of higher plants, which enhance the adaptive
potential of their hosts. These Plant Growth-Promoting
Rhizobacteria (PGPR) have numerous traits which allow
them to act as biocontrol agents. One of them is suppression of diseases caused by phytopathogens thanks to
produced wide range of antimicrobial molecules. Bacillus
subtilis is the most outstanding representative with regards to its ability to excrete a variety of antibacterial and
antifungal compounds among which are lipopeptides of
the surfactin, iturin and fengycin families.
Samples of various plant roots (n = 86) were collected from
fields in neighbourhood of Gdansk, Poland, and bacteria
from its rhizosphere were isolated. Method of isolation
was based on resistance of Bacillus endospores to high
temperature. In our search for biological agents to control plant pathogens, we obtained 620 bacterial isolates.
To confirm their preliminary belonging to Bacillus genus,
gram staining, colony morphology, a catalase reaction
and nitrate reduction test were assayed.
Moreover, isolates were analyzed according to their ability to biofilm formation. Measurement of biofilm mass
was adapted from the protocol published by O’Toole
and Kolter (1998) and modified in our laboratory. Studies
investigated growth and biofilm production of isolated
strains in minimal defined medium (Belitzky medium)
with glucose as a sole carbon source, and test was assayed by the ability of cells to pellicle formation in air/liquid interfaces of 96-well plates made of polystyrene. The
biofilm was detected by staining with crystal violet. The
procedure was performed three times for all strains, and
the averages and standard deviations were calculated for
all repetitions of the experiment.
The domesticated strain Bacillus subtilis 168 and natural
wild-type Bacillus subtilis 3610 formed a biofilm in amount
of crystal violet units (CV) 0.95 and 1.80, respectively. Results appeared to detect differences in biofilm-forming
ability among investigated strains. Biofilm production at
24 h of incubation varied greatly between different natural isolates, ranging from CV of 0.1 to 2.4.
42nd Meeting of the Polish Biochemical Society
Vol. 54 P14.9
Inactivation of pathogenic bacteria in fish
products using high pressure processing (HPP)
Bożena Windyga1,2, Monika FonbergBroczek2*, Halina Scieżyńska1, Anna
Grochowska1, Krystyna Górecka1, Kamila
Pawłowska1, Jacek A. Szczawiński3
1Department
of Food Research and Consumer Articles,
National Institute of Hygiene, Warszawa, Poland, 2Institute
of High Pressure Physics PAS, Warszawa, Poland,
3Department of Food Hygiene and Public Health Warsaw
Agricultural University, Warszawa, Poland
*e-mail: Monika Fonberg-Broczek < mfonberg@
unipress.waw.pl>
In the last decade of 20th century, high pressure science
and technique have found application in new areas: biology, biochemistry and production of novel foods. In Poland, the research on high pressure processing (HPP) of
foods has been initiated in 1993 in the Institute of High
Pressure Physics, Polish Academy of Sciences in cooperation with National Institute of Hygiene.
Effect of HPP on food and microbial contamination of
food depends not only on pressure level, but also on time
of exposure, temperature, pH and chemical composition
of processed products. Vegetative forms of microorganisms are more susceptible to high pressure than microbial
spores which may be destroyed only in the stage of germination.
High pressure pasteurization is an athermic technology
of microbiological decontamination. While destructing
microbiological agents it does not brake covalent bonds,
neither primary structure of peptides. The influence of
high pressure on cell systems includes changes in molar
volume of elements. High pressure processed foods have
longer shelf life without the risk of psychrophylic pathogenic bacteria growth.
The aim of the study was to define the impact of high
pressure HPP on pathogenic bacteria including Listeria
monocytogenes and Staphylococcus aureus in smoked vacuum packed fish – mackerel. Samples were subjected to
various pressure levels: 200, 300, 400 and 500 MPa in a
special high pressure chamber. The HPP was conducted
in temperatures of 2°C or 40°C and the time of exposure
was 5, 10 or 15 minutes. Number of bacteria was estimated after 2–4 hours following HPP and compared to
control samples not subjected to HPP.
For each of the studied bacteria 42 HPP cycles were performed. The following results were obtained:
Listeria monocytogens was more susceptible to HPP than
Staphylococcus aureus irrespective of temperature,
Listeria monocytogenes was completely inactivated at
pressure level of 500 MPa in all samples of smoked fish
irrespective of the time of HPP exposure,
HPP is more effective against Stapylococcus aureus at the
temperature of 2°C as compared to 40°C, and Listeria
monocytogenes is more susceptible to HPP at 40°C.
Pressure of 200 MPa does not have a significant effect on
the number of Staphylococcus aureus bacteria.
203
Acknowledgments:
The work was performed within a research project MNiI PO6T
006.29, 2005-2008.
References:
Food and Agriculture Organization (1999) Report of the FAO expert consultation on the trade impact of Listeria in fish products.
Rome: FAO.FAO Fisheries Report nr 604, 34.
Arabas J, Szczepek J, Dmowski L, Heinz V, Fonberg-Broczek M
(1999) New technique for kinetic studies of pressure-temperature induced changes of biological materials. Adv High Pressure
Biosci Biotechnol (Ludwig H, ed) pp 537–540, Springer-Verlag.
Abstracts
204
P14.10
Inactivation of yveS-yvfF genes from eps
operon lead to inhibition of multicellular
behaviours such as: biofilm formation
and swarming in Bacillus subtilis
Krzysztofa Nagórska1*, Adam Ostrowski2,
Krzysztof Hinc2, Michał P. Obuchowski2
1Deptartment
of Molecular Biology, University of Gdansk,
Gdańsk, Poland, 2Medical University of Gdańsk, Gdańsk,
Poland
*e-mail: Krzysztofa Nagórska < [email protected]>
B. subtilis — prevalent inhabitant of soil, belonging to
aerobic, spore-forming genus Bacillus, establish symbiotic
relationship with the plants by forming robust biofilms
on the roots surface. Biofilms, prevalent form of existence of microorganisms in every ecosystems, are viewed
as highly-structured communities of bacteria embedded
in an extracellular matrix. In laboratory conditions the
most common form of this structure formed by B. subtilis are pellicles — floating biofilms formed at air-liquid
interface. In case of B. subtilis, eps operon employing 15
genes, seems to participate in exopolysaccharide (EPS)
synthesis, which is the main component of mentioned
matrix. However, the function of particular genes remain
unknown. Searching for influence of proteins encoded by
those genes on multicellular behaviours, we performed
series of insertional mutations in distal part of the eps
operon including eight genes (yveS, yveT, yvfA, yvfB,
yvfC, yvfD, yvfE and yvfF). Obtained results, with usage
crystal violet staining, showed drastic decrease in the
amount of produce EPS. Furthermore, mutant strains,
excluding the last gene of the eps operon, was unable to
develop biofilm community (yveS and yveT mutants) or
produced only fragile peaces of pellicles accumulating in
the bottom of the cultivation flask. Consequently, the architecture of the colony on a solid agar medium of mutant
strains were considerable different from colony formed
by parental strain 168. Confocal microscopy (CLSM) as
well, confirmed significant changes in three-dimensional
structures of those fragments of pellicles in comparison
to mature biofilms form by wild strain at the air/liquid
interface. The main differences is irregular, chaotic structure due to the lack of bundles, defined as long chains of
morphological changed cells. What is interesting, IPTGdependent induction of pspac promoter driving the expression of every gene in constructed fusions, led to restore biofilm formation ability only in case of yveS and
yveT genes.
Relying on coordinated movement, generating by successive waves of moving cells on solid media- swarming is
believed to share some features with biofilm formation
process. Since, exopolysaccharide was proved to posses
properties facilitating active movement in case of Gramnegative bacteria, we decided to check if this is the issue
in case of B. subtilis. The first set of experiments showed
that, strains harboring mutations in group of yveS-yvfF
genes, displayed a completely nonswarming phenotype
in 168 background on Luria-Bertani solid medium. However, introduction gene encoded surfactin, crucial for
2007
swarming on fully defined media, caused restoration of
swarming ability in mutant defective in EPS synthesis. It
was found, that the colonizing behaviour and the pellicle
formation of Bacillus subtilis depend on producing EPS.
This is another step in confirming hypothesis that biofilm and swarming can have overlapping control mechanisms.
42nd Meeting of the Polish Biochemical Society
Vol. 54 P14.11
P14.12
Effects of phage T4 on NF-κB activity
in human mononuclear cells
Cloning of the human IFN-β synthetic
sequence and analysis of expression
level in two types of vectors
Małgorzata Mitkiewicz*, Jakub Siednienko,
Ewa Kurowska, Beata Weber-Dąbrowska,
Andrzej Górski, Wojciech A. Gorczyca
Institute of Immunology and Experimental Therapy PAS,
Wrocław 53-114 Poland
*e-mail: Małgorzata Mitkiewicz < [email protected].
pan.wroc.pl>
The natural “killers” of bacteria termed bacteriophages
(phages) are known for a long time. They again attracted increased attention quite recently as a potential tool
in therapy of infections not susceptible to antibiotics.
However, to ensure that such an approach is safe, it is
important to examine whether or not phages influence an
activity of human cells. Of particular interest are immune
cells which rapidly change activities at inflammation and
release variety of mediators. Since a key role in the expression most of them plays transcriptional factor NF-κB
(Nuclear Factor kappaB), any changes of its activity are
important indicators of initiated cellular response. Therefore, the aim of our studies was to determine whether
bacteriophages T4 are able to affect an activity of NF-κB
in human peripheral blood mononuclear cells (PBMC) as
well as monocytic cell lines U937 and THP-1. We have
also examined an effect of phages T4 on the NF-κB activity induced in these cells by virus HSV-1. Using electromobility shift assay (EMSA), we show that phages T4
did not change NF-κB activity, while it was significantly
elevated by HSV-1 in examined cells. Moreover, cells first
treated with the phages and then with HSV-1 show decreased NF-κB activity in comparison with cells treated
exclusively with HSV-1. These results indicate that phages T4 not only do not activate NF-κB but also may inhibit
in yet unrecognized way its activation caused by other
viruses.
Acknowledgements:
Supported by the grant No. 2PO5B04727 from the Ministry of
Education and Science, Poland.
205
Jolanta Kuthan-Styczeń*, Luiza Chojnacka,
Grażyna Płucienniczak, Andrzej Płucienniczak
Institute of Biotechnology and Antibiotics, Warszawa, Poland
*e-mail: Jolanta Kuthan-Styczeń < [email protected]>
The synthetic human IFN-β structural gene was fused to
synthetic ubiquitin sequence and adapted to the E. coli
codon usage. We compared the stability of the same IFNβ sequence in two types of vectors: pT7RS with T7 promoter and pDB with deo P1P2 promoter. E. coli laboratory strains: BL21 DE3, NM522 and DH5α were used in
transformation procedure. The stability was confirmed
for over 40 generations in the E. coli BL21DE3 strain
which was transformed with pT7RSIFNβ vector. The results were demonstrated by comparing two parameters:
the level of recombinant protein expression and the ratio
of antibiotics resistant clones in general population. Identification of expression level was analysed by SDS/PAGE.
With the use of the plasmid/host combination we could
directly increase the ratio on expressions level.
Abstracts
206
P14.13
Inhibition of proteasome activity interferes
with laccase production in tunicamycintreated cultures of Trametes versicolor
Magdalena Staszczak*, Joanna Sajewicz
Department of Biochemistry, Maria Curie-Sklodowska
University, Lublin, Poland
*e-mail: Magdalena Staszczak < [email protected].
lublin.pl>
Trametes versicolor belongs to white rot fungi which are
the only organisms able to degrade lignin efficiently.
Lignin degradation caused by these Basidiomycetes is accomplished by the action of secreted enzymes, the best
characterized of which are laccases, lignin peroxidases,
and manganese peroxidases. Lignin-modifying enzymes
are mainly produced during secondary metabolism triggered in the white rot fungi by nutrient deprivation.
Ligninolytic enzymes have been investigated mainly
because of their ecological significance and industrial
applications for pulping and bleaching as well as for bioremediation. Our previous studies have demonstrated
that ubiquitin/proteasome-mediated proteolytic pathway
is involved in the regulation of ligninolytic activities in
the wood-decaying fungus T. versicolor upon nitrogen
and carbon starvation (Staszczak (2002) Enzyme Microb
Technol 30: 537–541). This highly selective non-lysosomal
pathway requires ATP and the 26S proteasome, a large
~2.5 MDa multisubunit complex which degrades protein
targets marked by a covalently attached polyubiquitin
chain. The 26S proteasome plays a major regulatory role
in eukaryotic cells; it degrades many important proteins
involved in cell cycle control, signaling pathway, and in
general metabolism, including transcription factors and
key metabolic enzymes. Proteasemes are implicated in
stress response by removing damaged, denatured, or
misfolded proteins. Our recent studies have indicated
that proteasomal degradation of intracellular proteins
is involved in the regulation of T. versicolor laccase in
response to cadmium (Staszczak M, Jarosz-Wilkołazka
A (2005) Biochimie 87: 755–762). Laccase (benzenediol:
oxygen oxidoreductase; EC 1.10.3.2), a major ligninolytic
enzyme of this fungus, is a glycosylated protein. Secretion of glycoproteins can be influenced by tunicamycin,
an endoplasmic reticulum (ER) stress inducer. Tunicamycin, a nucleoside antibiotic produced by Streptomyces lysosuperficus inhibits N-linked glycosylation and blocks the
formation of N-glycosidic protein-carbohydrate linkages.
Secretory proteins that ultimately fail to fold properly are
removed from the ER and degraded in the cytosol by the
26S proteasome. In the present study we tested whether the blocking of proteasome function would interfere
with laccase production in tunicamycin-treated cultures
of T. versicolor. These studies were performed using carbobenzoxy-Leu-Leu-leucinal (MG 132), one of the most
potent peptide aldehyde inhibitors of the proteasome,
added (to a final concentration of 40 µM) either alone or
in combination with tunicamycin (5 µg/ml) at the time
of transfer of seven-day old mycelia to nutrient-deprived
(carbon or nitrogen depleted) media or to nutrient-suffi-
2007
cient (trophophasic) media. Laccase activities (secreted as
well as intracellular) and isozyme patterns were assessed
in cultures treated with tunicamycin for 6 h or 24 h in the
absence or presence of MG 132.
42nd Meeting of the Polish Biochemical Society
Vol. 54 207
P14.14
P14.15
Search for proteins involved in phagosome
maturation in unicellular eukaryote
Changes in cellular localization of CacyBP/
SIP during differentiation of NB-2a cells
Emilia Wypych*, Liliana Surmacz, Magdalena
Osińska, Jolanta Wiejak, Henryk Bilski,
Kazimierz Krawczyk, Elzbieta Wyroba
Gabriela Schneider1*, Krzysztof Nieznanski1,
Ewa Kilanczyk1, Jacek Kuznicki1,2, Anna Filipek1
Nencki Institute of Experimental Biology PAS, Warszawa,
Poland
*e-mail: Emilia Wypych < [email protected]>
During phagocytosis cells internalize large particles that
are subsequently degraded in lysosomes. Digestion capability of phagosomes is acquired in the process of maturation that culminates in the fusion of phagosomes with
lysosomes leading to formation of the phagolysosomes.
In single celled eukaryote Paramecium we identified the
components indispensable for this process: homologues
of lysosomal membrane protein 2 (LAMP-2) and a7 subunit of the 26S proteasome — the interacting partner of
Rab7 (Dong et al. J Biol Chem 279: 21334) that was cloned
by us in this cell (Surmacz et al. Acta Biochim Polon 53:
149).
LAMP-2 cross-reacting polypeptide of ~106 kDa was
glycosylated as shown by Pro-Q Emerald fluorescent
staining and Western analysis of the same blot preceded
by PNGase F digestion. During phagocytosis LAMP-2
expression was 2.5-fold higher than in the control cells
when P2 protein fractions (100 000 × g) of equal load were
quantified by immunoblotting.
LAMP-2 homologue was localized by confocal microscopy and ultrastructural studies. It was detected in
lysosomes and in the phagolysosomes where it colocalized with Rab7 during internalization of latex beads. The
presence of proteasome-related polypeptides of ~22 and
~24 kDa was revealed by immunoblotting with the antibody against human a7 of 20S proteasome. By electron
microscopic analysis a7 subunit of 26S proteasome was
detected in the vicinity of phagosomal membrane in the
tiny vesicles. In some of them it colocalized with Rab7.
The proteasomal antigen was not observed in LAMP-2positive primary lysosomes. These results indicate that
LAMP-2, Rab7 and a7 subunit of 26S are localized to
phagolysosomal compartment and may be involved in
the process of phagosome maturation in Paramecium.
1Nencki
Institute of Experimental Biology PAS, Warszawa,
Poland, 2International Institute of Molecular and Cell
Biology, Warszawa, Poland
*e-mail: Gabriela Schneider < [email protected].
pl>
The CacyBP/SIP protein was originally discovered as a
S100A6 (calcyclin) target (Filipek & Kuźnicki, 1998) and
later as a Siah-1 interacting protein (Matsuzawa & Reed,
2001). CacyBP/SIP also interacts with some other members of the S100 family (Filipek et al., 2002a) and with the
Skp1 protein. The function of CacyBP/SIP is not clear at
present although there are some results published suggesting that it can be a component of the SCF ubiquitin
ligase (Matsuzawa & Reed, 2001) and that it might play
a role in cell differentiation (Au et al., 2006). In particular,
it has been shown that during differentiation of rat neonatal cardiomyocytes CacyBP/SIP is up-regulated. Since
CacyBP/SIP is highly expressed in neuroblastoma NB2a
cells (Filipek et al., 2002b), in this work we examined the
influence of CacyBP/SIP on differentiation of these cells.
We found that in differentiated cells, the level of CacyBP/
SIP, established by western blot, was higher than in undifferentiated ones. Immunofluorescence experiment
showed that CacyBP/SIP staining becomes very intensive
in the cellular processes formed upon cell differentiation.
This suggests that CacyBP/SIP plays a role in differentiation of neuroblastoma NB2a cells and points to a possible
function of this protein in differentiation of brain neurons
under physiological and pathological conditions. The
role of CacyBP/SIP in a signaling pathway involved in
the differentiation process is under investigation in our
laboratory.
Acknowledgements:
This work was supported by the State Committee of Scientific
Research grant to A.F (2 P04A 01030) and statutory funds from
the Nencki Institute of Experimental Biology. G. Schneider is a
recipient of a scholarship from the President of the Polish Academy of Sciences, and E. Kilanczyk and J. Kuznicki are recipients
of a stipend from the Foundation for Polish Science.
References:
Au KW, Kou CY, Woo AY, Chim SS, Fung KP, Cheng CH, Waye
MM, Tsui SK (2006) J Cell Biochem 98: 555–566.
Filipek A, Kuznicki J (1998) J Neurochem 70: 1793–1798.
Filipek A, Jastrzebska B, Nowotny M, Kuznicki J (2002b) J Biol
Chem 277: 21103–21109.
Filipek A, Jastrzebska B, Nowotny M, Kwiatkowska K, Hetman
M, Surmacz L, Wyroba E, Kuznicki J (2002a) J Biol Chem 277:
28848–28852.
Matsuzawa S, Reed J (2001) Mol Cell 7: 915–926.
Abstracts
208
2007
P14.16
P14.17
Isolation and preliminary characteristics
of b-N-acetylglucosaminidase of Rainbow
trout (Oncorhynchus mykiss) milt
The effect of arsenic trioxide on F-actin
reorganization in nuclei isolated from HL-60 cells
Beata
Sarosiek*,
Beata Cejko, Jan Glogowski
Institute of Animal Reproduction and Food Research PAS,
Olsztyn, Poland
*e-mail: Beata Sarosiek < [email protected]>
The role of b-N-acetylglucosaminidase (b-NAGase)
which existed in several species sperm acrosome, was
investigated by many years. This enzyme, together with
acrosine play an important role in mammals, amphibians
and ascidians toward the sperm penetration of oocyte.
Teleosts fish possess spermatozoa that lack an acrosome
and our earlier experiments revealed that did not contain
arylsulfatase, one of acrosomal enzymes. Interestingly
we found the b-NAGase activity in Rainbow trout milt
plasma and spermatozoa. The characteristics of this enzyme might revealed the role of b-NAGase in not-acrosomal sperm.
Milt obtained from Rainbow trout was centrifuged to obtain milt plasma and spermatozoa, and its was used to isolation b-NAGase from each sources separately. After ion
exchange chromatography (Q Sepharose) of milt plasma
we obtained 2 protein peaks possessing b-NAGase activity in milt plasma, and one protein peak in sperm extracts.
This step of purification enabled us to obtain about 5-7
fold purification. But after gel filtration (Superdex 200) of
Rainbow trout milt plasma peaks I and II we obtained
165 and 242 fold purification respectively. The molecular
mass established during gel filtration was 127 kDa for
Rainbow trout spermatozoal b-NAGase, and 74 kDa for
each forms from milt plasma. SDS/PAGE electrophoresis
revealed that after Rainbow trout sperm extract chromatography we observed 3 protein bands. The electrophoresis of peaks of Rainbow trout milt plasma revealed 2 and
6 bands in the 1st and 2nd peaks respectively.
The kinetic parameters were determined for not-purified enzymes: milt plasma and spermatozoa extracts. The
optimum pH for Rainbow trout milt plasma and sperm
extract b-NAGase was at the range 4.4–4.8. We also calculated the Km value, it was 7.79 × 10-4M for Rainbow trout
milt plasma b-NAGase and 5.80 × 10-4M for Rainbow
trout sperm extract b-NAGase. The incubation at 56oC by
20 minutes of all sources of enzymes revealed that at this
temperature 100% of b-NAGase activity was observed in
spermatozoa extracts, but in Rainbow trout milt plasma
only 2% of b-NAGase activity was observed. Form of enzyme observed in spermatozoa extracts was thermal stable.
The preliminary results indicated that in Rainbow trout
milt might exist two or three forms of b-NAGase, however further study (hydrophobic chromatography and/or
Concavalin A Sepharose) are necessary for obtaining a
detailed characteristics of all those enzymes forms.
Acknowledgements:
This study is supported by Ministry of Science and Higher Education Grant No N 308 018 31.
Magdalena Izdebska1, Maciej
Ostrowski2*, Alina Grzanka1
1Department
of Histology and Embriology, Collegium
Medicum, 2Department of Biochemistry, Nicolaus Copernicus
University, Bydgoszcz, Poland
*e-mail: Maciej Ostrowski < [email protected]>
Actin is one of the major constituents of the cytoskeleton
in eukaryotic cells. A large number of cellular processes,
including cytokinesis, endocytosis and chemotaxis are
mediated by polymerization of actin filaments. Actin as
a cytoskeletal protein is believed to be connected with
chromatin reorganization in the cell nucleus as well as
with apoptosis. Previous studies showed actin association
with RNA polymerases and suggest significance of actin
in transcription. The main purpose of this study is the
evaluation of fibrilar actin (F-actin) reorganization in nuclei isolated from the human leukemia cell line HL‑60, after induction of apoptosis, using arsenic trioxide (As2O3).
The cells were treated with three different doses of cytostatic drug during 24 hours. Nuclei were isolated and
purified by centrifugation in gradient of glycerol. Purity
and integrity of isolated nuclei were determined spectrophotometrically and with the use of transmission electron microscopy (TEM). Changes in F-actin organization
were qualitative performed by fluorescence microscopy
with the use of phallacidin — BODIPY and semi-quantitative measured by flow cytometry assay. Morphological
features of apoptosis were observed in light and fluorescence microscope.
42nd Meeting of the Polish Biochemical Society
Vol. 54 209
P14.18
P14.19
Noradrenergic neurons lesion as neonates and
central histaminergic system activity in adult rats
Glucose-depleted medium reduces the collagen
content of human skin fibroblast cultures
Przemysław Nowak1, Ryszard Szkilnik1*,
Adam Kwieciński1, Krystyna ŻwirskaKorczala2, Jerzy Jochem2, Łukasz Noras2,
Richard M. Kostrzewa3, Ryszard Brus1
Marzanna Cechowska-Pasko1*, Jerzy
Pałka2, Edward Bańkowski1
1Department
of Pharmacology, 2Department of Physiology,
Medical University of Silesia, Zabrze, Poland,3Quillen
College of Medicine, East Tennessee State University, Johnson
City, United States
*e-mail: Ryszard Szkilnik < [email protected]>
DSP-4 [N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine]
is a neurotoxin which induces acute and selective degeneration of both central and peripheral noradrenergic
nerve terminals in mammals. In rats, DSP-4 crosses the
blood-brain barrier and produce long-lasting degeneration of the central noradrenergic neurons. Previously we
showed that DSP-4 injected on the day 1st and 3rd of life
of rats dramatically decreased noradrenaline (NA) level
in the brain and modified function of the central serotoninergic, dopaminergic and GABA-ergic neurotransmitter systems in adult animals. The aim of this study was to
examine effect of DSP-4 applied to newborn rats on the
central histaminergic system in adult rats examined by
biochemical and behavioral methods.
Newborn Wistar rats were injected with DSP-4 at 50.0
mg/kg sc twice, on the day 1st and 3rd of postnatal life.
Control newborn rats were injected with saline. When
rats attained age of 8 weeks the level of NA was assayed
in different parts of the brain (HPLC/ED). Beside the content of histamine (H) the brain was estimated by immunoenzymatic method. Then in a separate group of adult
male rats with neonatally lesioned central noradrenergic
system and control, several behavioral tests were performed. For this study three histamine receptor antagonists were used such as: S(+)chlorpheniramine (for H1),
cimetidine (for H2) and thioperamide (for H3). It was
shown that neonatal lesion of the central noradrenergic
neurons significantly decreased H level in the frontal
cortex, and changed the central histamine receptors reactivity to antagonists (mainly H2 and H3) examined by
behavioral methods in adult rats.
Acknowledgements:
This study was supported by Medical University of Silesia
(2007).
1Department
of Medical Biochemistry, 2Department of
Medicinal Chemistry Medical Academy of Bialystok,
Białystok, Poland
*e-mail: Marzanna Cechowska-Pasko < mapasko@tlen.
pl>
Glucose deprivation appeared to be a factor which induces ORP150 expression in the human skin fibroblasts
cultures. Whereas glucose deprivation resulted in a slight
(statistically insignificant) decrease of protein content in
these cultures, a marked decrease of collagen content was
observed, resulting in a distinct reduction of hydroxyproline : protein ratio. Furthermore, the appearance of ORP150
in glucose deprived cultures coexisted with an increase of
gelatinolytic activity and slight reduction in the expression of IGF-I receptor. Since IGF-I is a main stimulator of
collagen synthesis the reduction in the expression of IGFI receptor may result in a decrease of collagen synthesis.
It is suggested that ORP-150 is a chaperon, which protects
intracellular proteins against proteolytic effects exerted
by hypoxia or glucose shortage. Since the total amount of
protein in fibroblast cultures did not change much it appears that collagen (in contrast to other proteins) was not
efficiently protected. The decrease in collagen synthesis
and the enhancement of collagen degradation by gelatinases may result in distinct reduction of collagen content
in glucose-deprived fibroblast cultures.
Abstracts
210
2007
P14.20
P14.21
Effect of sodium azide and a protease
inhibitor cocktail on the stability of
statherin in whole human saliva
Interaction of melanin with p-aminobenzoic acid
Elżbieta Kamysz1, Dominika Jackiewicz
Barańska1*, Leszek Łobocki1,
Zbigniew W. Maćkiewicz1, Barbara
Kochańska2, Jolanta Ochocińska2
1Faculty
of Chemistry, University of Gdańsk, Gdańsk, Poland,
University of Gdansk, Gdańsk, Poland
*e-mail: Dominika Jackiewicz Barańska < dominika.
[email protected]>
2Medical
Statherin is a 43-amino acid residue miniprotein produced by salivary glands. Saliva contains various enzymes which might be able to decompose this peptide.
However, not only endogenous enzymes are responsible
for a more or less specific turnover of statherin in saliva.
Micro-organisms are also potent degradation agents of
salivary components.
The purpose of this study was to investigate the stability
of statherin in saliva in the presence of a protease inhibitor cocktail and sodium azide.
Samples of whole non-stimulated saliva were collected
from four volunteers between 9.00 and 10 a.m. Two hours
prior to saliva collection they were refrained from eating,
drinking, smoking, and oral hygiene. Saliva from each
individual was collected over a 5 min period by spitting
into chilled disposable polypropylene tubes. The samples
of saliva were mixed and divided into three sub-samples
labeled A, B and C. A 1% sodium azide solution was added to sample A in proportion 1 : 1. The protease inhibitor
cocktail was added to sample B in proportion 3:1. Sample
C was a control. The presence of statherin was determined
by the matrix-assisted laser desorption/ionization-timeof-flight mass spectrometry (MALDI-TOF MS) technique.
The analyses were performed directly after preparation
of the samples and later every 24 hours. The samples of
saliva were stored at room temperature (RT). The native
statherin has been found to be decomposed in all the samples after 24 hours at RT. Then the synthetic statherin was
dissolved in a 10 mM TRIS-HCl buffer of pH 8.2 (concentration 0.5 mg/ml) and was added to samples A, B and C
to concentration of 35 μg/ml. In all samples the statherin
was decomposed within 48 hours.
Our findings show that the addition of sodium azide, a
potent metabolic poison preventing microbial contamination, to whole human saliva did not protect the saliva
samples against decomposition of both the native and
the synthetic statherin (sample A). A similar situation
was noticed in sample B containing the protease inhibitor
cocktail. Salivary specific enzymes were inactivated at the
beginning of the experiment, but also the native and synthetic statherins were decomposed in that sample.
In this presentation, difficulties encountered in maintaining the stability of the native and synthetic statherins in
the three samples will be discussed.
Acknowledgements:
This work was partially supported by the University of Gdańsk
(BW 8000-5-0380-7) and the Medical University of Gdańsk ( ST31) grants.
Janina M. Trzcionka*, Ewa Z.
Buszman, Paweł Kaczmarczyk
Department of Chemistry and Drug Analysis, Medical
University of Silesia, Sosnowiec, Poland
*e-mail: Janina M. Trzcionka < ezarzycka@slam.
katowice.pl>
Photodermatoses are a group of dermatological diseases
caused by hypersensitivity to UV band of the sunlight.
Hypersensitivity of skin to UV light may manifest itself
as phototoxic or photoallergic reaction. Phototoxic reactions may be caused by a variety of drugs such as sulfonamides, tetracyclines, phenothiazines, furocumarines,
salicylates, thiazides and others.
Melanins fulfill important physiological functions by
absorbing UV radiation and protecting DNA from the
UV radiation related damage. These biopolymers bind
some of the chemical compounds used as drugs and
thus affect both their metabolism and therapeutic effect.
Drugs binding to melanin by electrostatic interactions
do not cause toxic side effects while in the case of drugs
participating in free-radical type reactions the toxicity
increases.
Sulfonamides are the derivatives of 4-aminobenzenosulfonic acid. Anti-bacterial properties of these compounds depend on the presence of an amino group in
the para position in relation to the sulfonamide group.
Sulfonamides are anti-metabolites of folic acid which is
necessary for the synthesis of purine nucleotides. The
inhibition of biosynthesis of folic acid is related to structural similarity of sulfonamides and p-aminobenzoic
acid (PABA).
The objective of this study was to evaluate the binding
of PABA, known as skin photosensitizer, with melanin.
The study was conducted in vitro using a model eumelanin synthesized from l-DOPA. Kinetics of drugs-melanin
complexes formation as well as binding parameters were
determined. Binding of p-aminobenzoic acid to melanin
was studied as follows: 5 mg of melanin were placed in
plastic test-tubes, where drug solutions were added to a
final volume of 5 ml. The initial concentration of PABA
ranged from 2 × 10–5M to 1.5 × 10–4M. All samples were incubated for 1, 3, 6, 15, 24 and 48 h at room temperature.
The suspensions were filtered after incubation. The concentration of unbound drug in each filtrate with respect
to the control sample was determined spectrophotometrically, using wavelenght of 265 nm. All spectophotometric
measurements were made using the UV-VIS spectrophotometer JASCO model V-530.
It has been demonstrated that p-aminobenzoic acid forms
complexes with model melanin in vitro. The amount of
PABA bound to melanin increases with the increasing of
initial drug concentration and the incubation time. An
analysis of PABA binding to melanin by the use of Scatchard plots has shown that only one class of binding sites
parcitipates in PABA-melanin complexes formation with
the association constant K = 6.54 × 103 M–1.
42nd Meeting of the Polish Biochemical Society
Vol. 54 The ability of p-aminobenzoic acid to form complexes
with melanin in vitro may be one of the reasons of phototoxic effects of this drug in vivo as a result of its accumulation in the skin melanin.
Acnowledgements:
This work was financially supported by the Medical University
of Silesia.
211
P14.22
Kinetic changes in activity of HR-peroxidase,
induced by low doses of phenol
Elżbieta Malarczyk*, Janina Kochmańska-Rdest
Department of Biochemistry, Maria Curie-Sklodowska
University, Lublin, Poland
*e-mail: Elżbieta Malarczyk < [email protected].
lublin.pl>
The strong experimental evidences proved that HR-peroxidase is very sensitive detector of subtle changes in
concentration of phenol, used as cofactor of peroxidaseoxidase reaction. The effects of low doses of phenol, prepared by successive dynamic dissolution in water or in
75% ethanol, on HRP activity were tested in systematic
manner with colorimetric as well as luminometric methods. The sinusoidal shape of these relations showed the
concrete phenol dilutions, which activated HRP (maximal points) and others with inhibiting doings (minimal
points). These results were dependent on kind of dilutor.
The dilutions with maximal values showed the higher
relativity to hydrogen peroxide, whereas dilutions with
minimal activity, corresponded mainly with oxidase type
of reaction. Also the scan-kinetics revealed the distinct
differences in shapes of UV-Vis spectra for two tested
groups of phenol dilutions, which diverse also in Km values, lower for maximal and higher for minimal dilutions.
The conception to recognize of HRP as a detector of dissolution of phenols is proposed.
Abstracts
212
P14.23
The comparison of free and melaninbound lipofuscin isolated from pig iris
Jolanta Lodowska1*, Ewa Chodurek2,
Ludmiła Węglarz1, Daniel Wolny2, Sławomir
Kurkiewicz3, Leszek Krzyżanowski4, Natalia
Sakina5, Barbara Bilińska4, Mikhail Ostrovsky5,
Agnieszka Petela6, Dariusz Dobrowolski7
1Department
of Biochemistry, 2Department of Biopharmacy,
of Instrumental Analysis, 4Department
of Physical Pharmacy, Medical University of Silesia,
Sosnowiec, Poland,Poland, 5Institute of Biochemical
Physics RAS, Moscow, Russian Federation, 6Department
of Medical Physics, University of Silesia, Katowice, Poland,
7Ophthalmology Department, District Railway Hospital,
Katowice, Poland
*e-mail: Jolanta Lodowska < [email protected].
pl>
3Department
Lipofuscin is a protein-lipid aggregate, which originates
from incompletely degraded cell components, as a result
of lysosomes dysfunction. This pigment, recognized as a
marker of ageing can induce phototoxic reactions. Melanin, synthesized in the iris epithelium before birth and
in stroma during the first weeks after birth has photo
protective activity [1]. Structural modifications which occur in this biopolymer with ageing lead to the loss of its
antioxidative properties or even to the toxic effects. Partially degraded melanin is probably bound to lipofuscin
to form granulation called melanolipofuscin.
The aim of this study was to determine the chemical composition of free and melanine bound lipofuscin, isolated
from pig iris.
This aim was realized by isolation of lipofuscin and
melanolipofuscin from pig iris cells homogenate [2], followed by thermolysis at 770oC and the analysis of obtained products by GC/MS.
In the pyrolytic profile of free and melanin-bound lipofuscin, the products of thermal degradation of melanins
and proteins, such as derivatives of benzene, phenol, pyridine, pyrrole, indole and nitryls, have been identified.
Aliphatic carbohydrates and derivatives of fatty acids
have also been identified. The quantities of the products
derived from melanins and proteins were much greater
in melanolipofuscins compared to lipofuscin, where fatty
acid derivatives were predominant compounds (44.6%).
There was significant quantitative differentiation of
pyrrole derivatives obtained from both lipofuscins and
melanolipofuscins. These derivatives might have been
products of thermolysis not only melanins but also of
prolyl residues of lipofuscin protein component. Significant amounts of aliphatic carbohydrates detected during
thermal degradation of biopolymers could probably result from decarboxylation of fatty acids or from melanin
thermolysis as the alkyl radicals were generated from
these compounds at high temperature and atmosphere of
neutral gas.
References:
1. Kałużny BJ, Kałużny JJ (2002) Okulistyka 2: 9–14.
2007
2. Boulton M, Docchio F, Dayhaw-Barker P, Ramponi R, Cubeddu R (1990) Vision Res 30: 1291–1303.