Department of Biophysics and Radiation Biology

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.

Endocannabinoid CB1 receptor (CB1R) Single photon emission computed tomography (SPECT) Molecular imaging biomarker [ 125 I]SD7015 Knock-out CB1R-/-mouse Multiplexed multipinhole dedicated small animal SPECT/CT system a b s t r a c t We aimed to evaluate the novel high-affinity and relatively lipophilic CB 1 receptor (CB 1 R) antagonist radioligand [ 125 I]SD7015 for SPECT imaging of CB 1 Rs in vivo using the multiplexed multipinhole dedicated small animal SPECT/CT system, NanoSPECT/CT PLUS (Mediso, Budapest, Hungary), in knock-out CB 1 receptor knock-out (CB 1 R-/-) and wildtype mice. In order to exclude possible differences in cerebral blood flow between the two types of animals, HMPAO SPECT scans were performed, whereas in order to confirm the brain uptake differences of the radioligand between knock-out mice and wildtype mice, in vivo scans were complemented with ex vivo autoradiographic measurements using the brains of the same animals. With SPECT/CT imaging, we measured the brain uptake of radioactivity, using %SUV (% standardised uptake values) in CB 1 R-/-mice (n = 3) and C57BL6 wildtype mice (n = 7) under urethane anaesthesia after injecting [ 125 I]SD7015 intravenously or intraperitoneally. The Brookhaven Laboratory mouse MRI atlas was fused to the SPECT/CT images by using a combination of rigid and non-rigid algorithms in the Mediso Fusion TM (Mediso, Budapest, Hungary) and VivoQuant (inviCRO, Boston, MA, USA) softwares. Phosphor imager plate autoradiography (ARG) was performed on 4 m-thin cryostat sections of the excised brains. %SUV was 8.6 ± 3.6 (average ± SD) in CB 1 R-/-mice and 22.1 ± 12.4 in wildtype mice between 2 and 4 h after injection (p < 0.05). ARG of identically taken sections from wildtype mouse brain showed moderate radioactivity uptake when compared with the in vivo images, with a clear difference between grey matter and white matter, whereas ARG in CB 1 R(-/-) mice showed practically no radioactivity uptake. [ 125 I]SD7015 enters the mouse brain in sufficient amount to enable SPECT imaging. Brain radioactivity distribution largely coincides with that of the known CB 1 R expression pattern in rodent brain. We conclude that [ 125 I]SD7015 should be a useful SPECT radioligand for studying brain CB 1 R in mouse and rat disease models.

Objectives: In the early recognition of portal vein ligation (PVL) induced tumor progression, positron emission tomography and magnetic resonance imaging (PET/MRI) could improve diagnostic accuracy of conventionally used methods. It is unknown how PVL affects metabolic patterns of tumor free hepatic tissues. The aim of this preliminary study is to evaluate the effect of PVL on glucose metabolism, using PET/MRI imaging in healthy rat liver.

Methods. Arthritis was induced in PACAP ؊/؊ and wild-type (PACAP ؉/؉ ) mice by K/BxN serum transfer. General features of the disease were investigated by semiquantitative scoring, plethysmometry, and histopathologic analysis. Mechano-and thermonociceptive thresholds and motor functions were also evaluated. Metabolic activity was assessed by positron emission tomography. Bone morphology was measured by in vivo micro-computed tomography, myeloperoxidase activity and superoxide production by bioluminescence imaging with luminol and lucigenin, respectively, and vascular permeability by fluorescent indocyanine green dye study.

This study aimed at investigating some respects of binding and interaction between water-soluble drugs and liposomal carrier systems depending on their size and lamellarity. As model substance, ciprofloxacin hydrochloride (CPFX) was incorporated into giant unilamellar vesicles (GUVs) to study their CPFX encapsulation/binding capacity. To characterize molecular interactions of various CPFX microspecies with lipid bilayer, zeta potential and electron paramagnetic resonance (EPR) spectroscopy measurements were performed. The increase of the zeta potential at pH 5.4 but no change at pH 7.2 was interpreted in terms of the CPFX microspecies' distribution at the two pH values. EPR observations showed an increased fluidity because of CPFX binding to GUVs. We worked out and applied a three-compartment dialysis model to separately determine the rate of drug diffusion through the liposomal membrane. Equilibrium dialysis showed (a) different permeation of CPFX through the membranes of GUVs and multilamellar vesicles (MLVs), with characteristic half-lives of 54.4 and 18.1 h, respectively; and (b) increased retention of CPFX in case of GUVs with released amounts of 70% compared with about 97% in case of MLVs. Our results may provide further details for efficient design of liposomal formulations incorporating water-soluble drugs.

The rheological properties of living cells strongly depend on their cytoskeletal structures, which are composed of polymer networks and responsible for fundamental cellular functions. In particular, the actin network plays a major role in determining the rheological properties of living cells. In order to elucidate how the rheological properties of individual cells are affected by actin filamentous structures, we measured the number distribution of complex shear modulus of single cells, which were treated by actin modification drugs and cultured on microarray substrates, by atomic force microscopy. A force modulation mode experiment was employed to measure the complex shear modulus of single cells in a frequency range of 2-200Hz. When the cells were treated with actin-stabilizing drug, jasplakinolide, and actin-disrupting drug, cytochalasin D (CD), the storage modulus G' and loss modulus G'' increased and decreased, respectively. The changes in G'' were smaller comparing to those in G'. The moduli exhibited a weak power-law dependence on frequency [1], whereas the increasing and decreasing of G' and G'' were accompanied by a decreasing and increasing power-law exponent respectively. Furthermore, their corresponding logarithmic standard deviation s showed a slight change in the case of jasplakinolide treatment whereas it became small and attained a constant value at higher frequencies in CD treatment [2]. The results indicated that individual differences of cell rheology enhanced as actin cytoskeletal structures were stabilized. Furthermore, it was implied that the observed frequency dependence of s was attributed to a frequency susceptibility of actin filaments.

Application of porphyrins as photosensitizers is based on their light-triggered generation of reactive oxygen species (ROS) that may cause oxidative tissue damage and ultimately kill cells. Cellular membranes are the action grounds of many sensitizers due to their hydrophobic or amphiphilic character as well as the location of many of the targets attacked by ROS. Hence, the binding ability and location of porphyrins in liposomes as simple models of cellular membranes are of outstanding interest. Here we compare mesoporphyrin IX dimethyl ester (MPE) and its nonesterified form, mesoporphyrin IX dihydrochloride (MPCl). Monocomponent small unilamellar vesicles formed of various saturated phosphatidylcholines with incorporated mesoporphyrins were investigated. We determined the binding parameters and the inhomogeneous distribution functions (IDFs) by different fluorescence techniques. We found in general that the binding ability of MPE is considerably greater than that of MPCl. In the case of MPCl, the IDFs suggest that only one of the two binding site types identified earlier for MPE ("site II") exists; the other one ("site I") vanishes while a new one appears ("site III"). We can confirm that "site I" is located between the two lipid layers, "site II" is situated between the hydrocarbon chains, while the location of the novel "site III" is along the outer part of the hydrocarbon chains partially inserted between the lipid head groups.

Binding of photosensitizers to target cells is a crucial step during the photodynamic effect. Sensitizer distribution is a good indication of whether the chemical is a good candidate for perturbing cell membrane integrity. Hence, the photophysical properties of porphyrinoid sensitizers in microheterogeneous systems such as liposomes are of outstanding interest. Here we present a site-selective fluorescence study of liposome systems. Monocomponent, small unilamellar vesicles formed of different phosphatidylcholines with incorporated mesoporphyrin were investigated. The size distribution of liposomes was measured by dynamic light scattering after each step of the experiment. On the basis of fluorescence line narrowing spectra of mesoporphyrin, the inhomogeneous distribution function was determined in order to characterize the photosensitizer location. The dual character of the functions revealed two different locations. Decomposition of the inhomogeneous distribution functions into Gaussians and the analysis of the fit results suggest that one of the locations for mesoporphyrin is between the two lipid layers, and the other one is between the hydrocarbon chains of the lipid molecules.

Aim: To carry out a systematic study on the effect of different storage conditions on the number as well as the physical and functional properties of antibacterial extracellular vesicles (EVs) derived from human neutrophilic granulocytes. Methods: Production of EVs with antibacterial properties was initiated by opsonized Zymosan A particles. The number of released fluorescent EVs was determined by flow cytometry following careful calibration. Physical properties and size of EVs were investigated by flow cytometry, dynamic light scattering and electron microscopy. Functional properties of EVs were tested by bacterial survival assay. Results: Storage at '208C or '48C resulted in a significant decrease of EV number and antibacterial effect after 1 day. Storage at (208C did not influence the EV number up to 28 days, but induced a shift in EV size and almost complete loss of antibacterial function by 28 days. Storage at (808C had no significant effect either on EV number or size and allowed partial preservation of the antibacterial function up to 28 days. Snap-freezing did not improve the results, whereas the widely used cryoprotectants induced EV lysis. Conclusion: Storage significantly alters both the physical and functional properties of EVs even if the number of EVs stays constant. If storage is needed, EVs should be kept at (808C, preferably not longer than 7 days. For functional tests, freshly prepared EVs are recommended.