Pál Gróf | Semmelweis University (original) (raw)

Papers by Pál Gróf

Fluoroquinolone antibacterial agents are among the drugs most commonly causing phototoxic side ef... more Fluoroquinolone antibacterial agents are among the drugs most commonly causing phototoxic side effects. The phototoxicity may be originated in formation of reactive oxygen species upon ultraviolet exposure. Researches aiming the liposomal encapsulation of fluoroquinolones, expecting an increase in their therapeutic index, enhance the importance of studies on physicochemical properties and photostability of liposomal preparations. We studied the photodegradation of ciprofloxacin,

Small unilamellar liposomes were made of dipalmitoyl-phosphatidylcholine and dioleoyl-phosphatidy... more Small unilamellar liposomes were made of dipalmitoyl-phosphatidylcholine and dioleoyl-phosphatidylcholine, and photosensitized by a symmetrically or an asymmetrically substituted glycosilated tetraphenyl-porphyrin derivative. As differential scanning calorimetry and electron paramagnetic resonance spectroscopy (EPR) revealed these porphyrin derivatives were localized in different depth within the lipid bilayer. Both porphyrin derivatives were able to induce photoreaction and consequent structural changes in the membrane. 5-, 12-, or 16-doxyl stearic acid labeled lipid bilayers were applied and the efficiency of photoinduced reaction was followed by the decay of their EPR signal amplitude. Light dose-dependent destruction of nitroxide radical proved to be dependent on the position of spin label. In this process the porphyrin localized in closer connection with the double bond of unsaturated fatty acid was more effective. EPR signal decay was also dependent on the unsaturated fatty acid content of the liposome and the oxygen saturation of the solvent.

Scientia pharmaceutica, 2013

Developments in nanotechnology and in the formulation of liposomal systems provide the opportunit... more Developments in nanotechnology and in the formulation of liposomal systems provide the opportunity for cosmetic dermatology to design novel delivery systems. Determination of their physico-chemical parameters has importance when developing a nano-delivery system. The present study highlights some technological aspects/characteristics of liposomes formulated from egg or soy lecithins for topical use. Alterations in the pH, viscosity, surface tension, and microscopic/macroscopic appearance of these vesicular systems were investigated. The chemical composition of the two types of lecithin was checked by mass spectrometry. Caffeine, as a model molecule, was encapsulated into multilamellar vesicles prepared from the two types of lecithin: then zeta potential, membrane fluidity, and encapsulation efficiency were compared. According to our observations, samples prepared from the two lecithins altered the pH in opposite directions: egg lecithin increased it while soy lecithin decreased it w...

The Journal of Physical Chemistry A, 2004

ABSTRACT The permanganate/oxalate reaction has been known for more than a century; however, its m... more ABSTRACT The permanganate/oxalate reaction has been known for more than a century; however, its mechanism is still subject to debate. The latest general publication by a French group established a model that involved 14 steps including 8 equilibria. The model was found to be able to simulate experimentally observed phenomena and to account for the bistability in a continually stirred tank reactor (CSTR). However, some earlier reported observations that we found in the literature seem to be inconsistent with this model. We performed electron paramagnetic resonance (EPR) measurements and stopped-flow studies with spectrophotometrical detection to shed light on these contradictions. We found that one of the key steps of the model describing the decomposition of Mn(VII) is not acceptable at least with the indicated rate constant. The only other step in which permanganate is involved is not capable of accounting for the autocatalytic nature. Our striking observation that the reaction still can be autocatalytic when applying a large stoichiometric excess of manganous ions points out that autocatalysis cannot be purely explained by a positive Mn2+ feedback loop. Thus, we propose that the surface-catalyzed formation of colloidal manganese dioxide from Mn(II) and Mn(VII) provides a second positive feedback loop in the reaction.

European Journal of Biochemistry, 1983

PLoS ONE, 2013

Optical waveguide lightmode spectroscopic (OWLS) techniques were probed for monitoring ion permea... more Optical waveguide lightmode spectroscopic (OWLS) techniques were probed for monitoring ion permeation through channels incorporated into artificial lipid environment. A novel sensor set-up was developed by depositing liposomes or cell-derived membrane fragments onto hydrophilic polytetrafluoroethylene (PTFE) membrane. The fibrous material of PTFE membrane could entrap lipoid vesicles and the water-filled pores provided environment for the hydrophilic domains of lipid-embedded proteins. The sensor surface was kept clean from the lipid holder PTFE membrane by a water-and ionpermeable polyethylene terephthalate (PET) mesh. The sensor set-up was tested with egg yolk lecithin liposomes containing gramicidin ion channels and with cell-derived membrane fragments enriched in GABA-gated anion channels. The method allowed monitoring the move of Na + and organic cations through gramicidin channels and detecting the Cl -channel functions of the (a 5 b 2 c 2 ) GABA A receptor in the presence or absence of GABA and the competitive GABA-blocker bicuculline.

Physiologia Plantarum, 2007

The reduction of dehydroascorbate (DHA) was investigated in plant mitochondria. Mitochondria isol... more The reduction of dehydroascorbate (DHA) was investigated in plant mitochondria. Mitochondria isolated from Bright Yellow-2 tobacco cells were incubated with 1 mM of DHA, and the ascorbate generation was followed by high-performance liquid chromatography. Mitochondria showed clear ability to reduce DHA and to maintain a significant level of ascorbate. Ascorbate generation could be stimulated by the respiratory substrate succinate. The complex I substrate malate and the complex I inhibitor rotenone had no effect on the ascorbate generation from DHA. Similarly, the complex III inhibitor antimycin A, the alternative oxidase inhibitor salicylhydroxamic acid, and the uncoupling agent 2,4-dinitrophenol were ineffective on mitochondrial ascorbate generation both in the absence and in the presence of succinate. However, the competitive succinate dehydrogenase inhibitor malonate almost completely abolished the succinate-dependent increase in ascorbate production. The complex IV inhibitor KCN strongly stimulated ascorbate accumulation. These results together suggest that the mitochondrial respiratory chain of plant cells -presumably complex II -plays important role in the regeneration of ascorbate from its oxidized form, DHA.

Photochemistry and Photobiology, 1999

The formation of cyclobutane pyrimidine dimers (CPD) and 8-oxo-7,8-dihydro-2'-deoxyguanosine was ... more The formation of cyclobutane pyrimidine dimers (CPD) and 8-oxo-7,8-dihydro-2'-deoxyguanosine was investigated in Chinese hamster ovary cells upon exposure to either UVC, UVB, UVA or simulated sunlight (SSL). Two cell lines were used, namely AT3-2 and UVL9, the latter being deficient in nucleotide excision repair and consequently UV sensitive. For all types of radiation, including UVA, CPD were found to be the predominant lesions quantitatively. At the biologically relevant doses used, UVC, UVB and SSL irradiation yielded 8-oxodGuo at a rather low level, whereas UVA radiation produced relatively higher amounts. The formation of CPD was 102 and 105 more effective upon UVC than UVB and UVA exposure. These yields of formation followed DNA absorption, even in the UVA range. The calculated relative spectral effectiveness in the production of the two lesions showed that emcient induction of 8-oxodGuo upon UVA irradiation was shifted toward longer wavelengths, in comparison with those for CPD formation, in agreement with a photosensitization mechanism. In addition, after exposure to SSL, about 19% and 20% of 8-oxodGuo were produced between 290-320 nm and 320-340 nm, respectively, whereas CPD were essentially (90%) induced in the UVB region. However, the ratio of CPD to 8-oxodGuo greatly dflered from one source of light to the other: it was over 100 for UVB but only a few units for UVA source. The extent of 8-oxodGuo and CPD was also compared to the lethality for the different types of radiation. The involvement of 8-oxodGuo in cell killing by solar UV radiation was clearly ruled out. In addition, our previously reported mutation tThis paper is dedicated to the memory of Professor Raymond Latarjet who has been one of the most distinguished directors of the Institut Curie. His scientific impact in the field of photo-and radiobiology has been outstanding. It is a great honor to contribute to this special issue compiled in his memory. spectra demonstrated that the contribution of 8-oxodGuo in the overall solar UV mutagenic process is very minor.

Journal of Photochemistry and Photobiology B: Biology, 2002

Sunlight is the most important environmental UV source, affecting not only human health but also ... more Sunlight is the most important environmental UV source, affecting not only human health but also the whole terrestrial ecosystem. The use of artificial sources is advantageous since it is independent of geographical location and seasonal variations, however, in some photobiological/photochemical studies the choice of a specific UV source in relation to the biological end-point studied is sometimes questionable. Furthermore, it is often difficult to compare the results obtained in different laboratories due to &amp;amp;amp;amp;amp;amp;amp;#39;slight&amp;amp;amp;amp;amp;amp;amp;#39; differences in the physical characteristics of the UV sources used. In an attempt to address these issues we calculated and compared the physical characteristics and the biological efficiency in UV-B and UV-A regions for two biological end-points (CPD and Fpg-sensitive sites formation) for frequently used UV-B, UV-A sources and solar light simulators (SLS). Our calculation shows that FS20 lamp is appropriate for studying the biological effects of UV-B radiation although differences in spectral characteristics of the associated filters may lead to at least 2-fold yields in CPD production. Furthermore, the use of a SLS with a Kodacel filter alone is inadequate for studying environmental UV effects. A metal-halide source with a Schott WG345 filter is appropriate for studies on biological effects due to UV-A region. Relative exposure duration was calculated to achieve equal amount of CPD or Fpg-sensitive sites, provided equal, total UV-(A+B) irradiance for the different UV sources.

Journal of Photochemistry and Photobiology B: Biology, 2005

The effect of the symmetry and polarity of the porphyrin molecules on their membrane localization... more The effect of the symmetry and polarity of the porphyrin molecules on their membrane localization and interaction with membrane lipids were investigated by electron paramagnetic resonance (EPR). For this purpose, two glycoconjugated tetraphenyl porphyrin derivatives were selected, respectively, symmetrically and asymmetrically substituted. Small unilamellar liposomes composed of dipalmitoylphosphatidylcholine (DPPC) and spin labeled stearic acids were prepared. The spin probe was located at the 5th or 7th or 12th or 16th position of the hydrocarbon chain in order to monitor various regions of the lipid bilayer. EPR spectra of porphyrin-free and porphyrin-bound liposomes were recorded at various temperatures below and above the phase transition temperature of DPPC.

Journal of Photochemistry and Photobiology B: Biology, 2004

The ultraviolet A (UVA) radiation component of sunlight (320-400 nm) has been shown to be a sourc... more The ultraviolet A (UVA) radiation component of sunlight (320-400 nm) has been shown to be a source of oxidative stress to cells via generation of reactive oxygen species. We report here some consequences of the UVA irradiation on cell membranes detected by electron paramagnetic resonance (EPR) spectroscopy. Paramagnetic nitroxide derivatives of stearic acid bearing the monitoring group at different depths in the hydrocarbon chain were incorporated into human fibroblasts membranes to analyze two main characteristics: kinetics of the nitroxide reduction and membrane fluidity. These two characteristics were compared for control and UVA-irradiated (0-250 kJ/m 2 ) cells. The term relative redox capacity (RRC) was introduced to characterize and to compare free radical reduction measured by EPR with some well-known viability/clonogenicity tests. Our results showed that UVA-irradiation produces a more rigid membrane structure, especially at higher doses. Furthermore, we found that trends agree in survival measured by neutral red (NR), trypan blue (TB), and clonogenic efficiency compared with RRC values measured by EPR for low and medium exposure doses. Above 100 kJ/m 2 , differences between these tests were observed. Antioxidant effect was modeled by a-tocopherolacetate treatment of the cells before UVA irradiation. While NR, TB and clonogenicity tests showed protection at the highest UVA doses (>100 kJ/m 2 ), results obtained with EPR measurements, both membrane fluidity and kinetics, or using MTT test did not exhibit this protective effect.

Journal of Biological Chemistry, 2000

In order to better understand the relative contribution of the different UV components of sunligh... more In order to better understand the relative contribution of the different UV components of sunlight to solar mutagenesis, the distribution of the bipyrimidine photolesions, cyclobutane pyrimidine dimers (CPD), (6 -4) photoproducts ((6 -4)PP), and their Dewar valence photoisomers (DewarPP) was examined in Chinese hamster ovary cells irradiated with UVC, UVB, or UVA radiation or simulated sunlight. The absolute amount of each type of photoproduct was measured by using a calibrated and sensitive immuno-dot-blot assay. As already established for UVC and UVB, we report the production of CPD by UVA radiation, at a yield in accordance with the DNA absorption spectrum. At biologically relevant doses, DewarPP were more efficiently produced by simulated solar light than by UVB (ratios of DewarPP to (6 -4)PP of 1:3 and 1:8, respectively), but were detected neither after UVA nor after UVC radiation. The comparative rates of formation for CPD, (6 -4)PP and DewarPP are 1:0.25 for UVC, 1:0.12:0.014 for UVB, and 1:0.18:0.06 for simulated sunlight. The repair rates of these photoproducts were also studied in nucleotide excision repair-proficient cells irradiated with UVB, UVA radiation, or simulated sunlight. Interestingly, DewarPP were eliminated slowly, inefficiently, and at the same rate as CPD. In contrast, removal of (6 -4) photoproducts was rapid and completed 24 h after exposure. Altogether, our results indicate that, in addition to CPD and (6 -4)PP, DewarPP may play a role in solar cytotoxicity and mutagenesis.

Food and Chemical Toxicology, 2013

Citrinin (CTN) is a toxic fungal metabolite that is a hazardous contaminant of foods and feeds. I... more Citrinin (CTN) is a toxic fungal metabolite that is a hazardous contaminant of foods and feeds. In the present study, its acute toxicity and effects on the plasma membrane of Schizosaccharomyces pombe were investigated. The minimum inhibitory concentration of CTN against the yeast cells proved to be 500 lM. Treatment with 0, 250, 500 or 1000 lM CTN for 60 min resulted in a 0%, 2%, 21% or 100% decrease, respectively, in the survival rate of the cell population. Treatment of cells with 0, 100, 500 or 1000 lM CTN for 20 min induced decrease in the phase-transition temperature of the 5-doxylstearic acid-labeled plasma membrane to 16.51, 16.04, 14.18 or 13.98°C, respectively as measured by electron paramagnetic resonance spectroscopy. This perturbation was accompanied by the efflux of essential K + from the cells. The existence of an interaction between CTN and glutathione was detected for the first time by spectrofluorometry. Our observations may suggest a direct interaction of CTN with the free sulfhydryl groups of the integral proteins of the plasma membrane, leading to dose-dependent membrane fluidization. The change in fluidity disturbed the ionic homeostasis, contributing to the death of the cells, which is a novel aspect of CTN cytotoxicity.

FEBS Letters, 1987

Vibrational Raman spectroscopy has been used to study the conformation of the ! 1 S form of acety... more Vibrational Raman spectroscopy has been used to study the conformation of the ! 1 S form of acetylcholinesterase from Torpedo californica. Secondary structure analysis by the method of Williams [(1983) J. Mol. Biol. 166, 581-603] shows 49% ~-helical structure, 23% fl-sheets, 11% turns and 15% undefined structure. Secondary structure estimates obtained for this enzyme by Raman spectroscopy and circular dichroism have been analyzed. Raman spectroscopy; Acetylcholinesterase; Secondary structure Published by Elsevier Science Publishers B.V. (Biomedical Division) 202 00145793/87/$3.50

Cytometry Part A, 2010

Effects of some detergents-most frequently used in membrane raft studies-on the polymerization pr... more Effects of some detergents-most frequently used in membrane raft studies-on the polymerization properties of actin were examined under in vitro and in vivo conditions, for protein and cellular investigations, respectively. Under in vitro conditions the polymerization rates were measured with pyrene-labeled actin. We found that polymerization rate depended on the detergent concentration by following either biphasic characteristics or only decreasing tendency. The strongest effects were observed at relatively low detergent concentrations. SDS-PAGE electrophoresis and dynamic light-scattering measurements provided further evidences for the size distribution of actin filaments formed under the influence of detergents. Comparing the polymerization rates measured in the presence of different detergents to those obtained with various magnesium and KCl concentrations showed that detergents may influence the actin polymerization at three levels by modifying: (i) the monomer-monomer interaction, (ii) the local ionic strength, and (iii) the affinity of actin for various cations. In vivo studies on NIH 3T3MDR1 cells using TRITC-phalloidin detected fast depolymerization of large extent around the critical micellar concentrations of the detergents. We concluded that microdomain insolubility observed in the presence of detergents is hardly to be the result of the stabilization of the submembrane actin cytoskeleton merely; rather inter-lipid and lipid-protein interactions are also involved within the detergent-resistant membranes. '

Chemistry and Physics of Lipids, 2007

Biophysical Journal, 2009

In this study, experiments were carried out in the conventional and saturation-transfer electron ... more In this study, experiments were carried out in the conventional and saturation-transfer electron paramagnetic resonance (EPR) time domains to explore the effect of mDia1-FH2 formin fragments on the dynamic and conformational properties of actin filaments. Conventional EPR measurements showed that addition of formin to actin filaments produced local conformational changes in the vicinity of Cys-374 by increasing the flexibility of the protein matrix in the environment of the label. The results indicated that it was the binding of formin to the barbed end that resulted in these conformational changes. The conventional EPR results obtained with actin labeled on the Lys-61 site showed that the binding of formins could only slightly affect the structure of the subdomain 2 of actin, reflecting the heterogeneity of the formin-induced conformational changes. Saturation transfer EPR measurements revealed that the binding of formins decreased the torsional flexibility of the actin filaments in the microsecond time range. We concluded that changes in the local and the global conformational fluctuations of the actin filaments are associated with the binding of formins to actin. The results on the two EPR time domains showed that the effects of formins on the substantially different types of motions were uncoupled.

Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology, 1995

Raman spectra of human butyrylcholinesterase (BuChE; E.C. 3.1.1.8) were analyzed in the native st... more Raman spectra of human butyrylcholinesterase (BuChE; E.C. 3.1.1.8) were analyzed in the native state and after conjugation with organophosphates (soman, DFP and paraoxon). The secondary structure of the native BuChE in Tris-HCl buffer (pH 7.5), determined from analysis of the amide I polypeptide vibration band, indicates 47% a-helices, 26% r-sheets, 16% turns and 12% undefined structure. We obtained the same values for paraoxon-phosphorylated BuChE, but 39% helical structure, 31% /3-sheets, 17% turns and 13% undefined structure for 'aged' DFP-BuChE conjugates and 36% helical structure, 34% /3-sheets, 20% turns and 10% undefined structure for 'aged' soman-BuChE conjugates. The ~ 10% decrease of a-helical structure observed upon phosphorylation by DFP and phosphonylation by soman, probably corresponds to the 'aging' process, which does not take place in the case of paraoxon. Considerable differences have been observed between native, paraoxon inhibited and 'aged' BuChE in aromatic ring vibrations, suggesting that the dealkylation of organophosphate conjugates modifies the environment or the interactions of aromatic amino-acid residues. In the aliphatic side chains an increase of the number of gauche configurations has been observed in 'aged' DFP-BuChE and soman-BuChE.

Biochimica et Biophysica Acta (BBA) - Biomembranes, 2004

Interaction of pore-forming toxins, syringopeptin22A (SP22A), syringomycin E (SRE) and syringotox... more Interaction of pore-forming toxins, syringopeptin22A (SP22A), syringomycin E (SRE) and syringotoxin (ST), with model membranes were investigated. Liposomes were prepared from saturated phospholipids (DPPC or DMPC) or from binary mixtures of DPPC with varying amount of DOPC or cholesterol. The effects of the three toxins on the molecular order and dynamics of the lipids were studied using electron paramagnetic resonance (EPR) techniques. SP22A was the most-, SRE less-, and ST the least effective to increase the ordering and to decrease the rotational correlation time of the lipid molecules. The effects were more pronounced: (a) on small unilamellar vesicles (SUVs) than on multilamellar vesicles (MUVs); (b) on pure DPPC than on DPPC -cholesterol or DPPC -DOPC mixtures. Fluidity changes, determined from EPR spectra at different concentrations of the toxin, suggested the shell structure of the lipid molecules in pore formation. EPR spectra observed at different depth of the hydrocarbon chain of the lipid molecules implied an active role of the lipid molecules in the architecture of the pores created in the presence of the three toxins. Temperature dependence of the fluidity of the SUVs treated with toxins has shown an abrupt and irreversible change in the molecular dynamics of the lipid molecules at a temperature close to the pretransition, depending on the toxin species and the lipid composition. Coalescence and aggregation of the SUVs were proposed as the origin of this irreversible change. D

Biochimica et Biophysica Acta (BBA) - General Subjects, 2015

Actin filament bundling proteins mediate numerous processes in cells such as the formation of cel... more Actin filament bundling proteins mediate numerous processes in cells such as the formation of cell membrane protrusions or cell adhesions and stress fiber based locomotion. Among them alpha-actinin and fascin are the most abundant ones. This work characterizes differences in molecular motions in actin filaments due to the binding of these two actin bundling proteins. We investigated how alpha-actinin and fascin binding modify the conformation of actin filaments by using conventional and saturation transfer EPR methods. The result characteristic for motions on the microsecond time scale showed that both actin bundling proteins made the bending and torsional twisting of the actin filaments slower. When nanosecond time scale molecular motions were described the two proteins were found to induce opposite changes in the actin filaments. The binding of one molecule of alpha-actinin or fascin modified the conformation of numerous actin protomers. As fascin and alpha-actinin participates in different cellular processes their binding can serve the proper tuning of the structure of actin by establishing the right conformation for the interactions with other actin binding proteins. Our observations are in correlation with the model where actin filaments fulfill their biological functions under the regulation by actin-binding proteins. Supporting the general model for the cellular regulation of the actin cytoskeleton we showed that two abundant actin bundling proteins, fascin and alpha-actinin, alter the conformation of actin filaments through long range allosteric interactions in two different ways providing the structural framework for the adaptation to specific biological functions.