The cellular internalization of liposome encapsulated protoporphyrin IX by HeLa cells.

The proper lipid composition of liposomes designed to carry drugs determines their surface properties ensuring their accumulation within selected tissue. The electrostatic potential and surface topology of liposomes affect the internalization by single cells. The high-resolution imaging of cancer cells and the distribution of protoporphyrin-loaded liposomes within the cytoplasm and its dependence on the liposome surface properties are presented. In the paper, HeLa cells were used to investigate the uptake of porphyrin-loaded liposomes and liposomes alone by means of confocal and differential interference contrast microscopies. The effect of liposomes surface electrostatic potential and surface topology on their intracellular distribution was evaluated. The time evolution of the intracellular distribution of liposomes labelled with Rhodamine-PE was examined on HeLa cells. These studies allow for the identification of the liposome lipid composition so the efficient delivery of the active substance to cancer cells will be achieved. The obtained results showed that neutral PC-liposomes are the most efficiently internalized by HeLa cells. Moreover, results showed that properties of liposomes affect not only the internalization efficiency of the photosensitizer but also its distribution within the cells, as revealed by colocalization measurements.

Analysis of spatial correlations between patterns of DNA damage response and DNA replication in nuclei of cells subjected to replication stress or oxidative damage.

Sites of DNA replication (EdU incorporation) and DNA damage signaling (γH2AX) induced by camptothecin (Cpt) or hydrogen peroxide (H2O2) form characteristic patterns of foci in cell nuclei. The overlap between these patterns is a function of the number of DNA double strand breaks (DSBs) formed in replication sites. The goal of this study was to optimize a method of quantitative assessment of a degree of correlation between these two patterns. Such a correlation can be used to estimate a probability of inducing damage in sections of replicating DNA. The damage and replication foci are imaged in 3D with confocal microscopy and their respective positions within nuclei are determined with adaptive image segmentation. Using correlation functions spatial proximity of the resultant point patterns is quantified over the range of distances in cells in early-, mid- and late S-phase. As the numbers (and nuclear densities) of γH2AX and replication foci differ significantly in the subsequent substages of S phase, the detected association values were corrected for the expected random overlap between both classes of foci. Thus, the probability of their nonrandom association was estimated. Moreover, self association (clustering) of DNA replication sites in different stages of S-phase of the cell cycle was detected and accounted for. While the analysis revealed a strong correlation between the γH2AX foci and the sites of DNA replication in cells treated with Cpt, only a low correlation was apparent in cells exposed to H2O2. © 2013 International Society for Advancement of Cytometry.

Light-induced isomerization of the LHCII-bound xanthophyll neoxanthin: possible implications for photoprotection in plants.

Light-harvesting pigment-protein complex of Photosystem II (LHCII) is the largest photosynthetic antenna complex of plants and the most abundant membrane protein in the biosphere. Plant fitness and productivity depend directly on a balance between excitations in the photosynthetic apparatus, generated by captured light quanta, and the rate of photochemical processes. Excess excitation energy leads to oxidative damage of the photosynthetic apparatus and entire organism and therefore the balance between the excitation density and photosynthesis requires precise and efficient regulation, operating also at the level of antenna complexes. We show that illumination of the isolated LHCII leads to isomerization of the protein-bound neoxanthin from conformation 9'-cis to 9',13- and 9',13'-dicis forms. At the same time light-driven excitation quenching is observed, manifested by a decrease in chlorophyll a fluorescence intensity and shortened fluorescence lifetimes. Both processes, the neoxanthin isomerization and the chlorophyll excitation quenching, are reversible in dim light. The results of the 77K florescence measurements of LHCII show that illumination is associated with appearance of the low-energy states, which can serve as energy traps in the pigment-protein complex subjected to excess excitation. Possible sequence of the molecular events is proposed, leading to a protective excess excitation energy quenching: neoxanthin photo-isomerization→formation of LHCII supramolecular structures which potentiate creation of energy traps→excitation quenching.

Interaction of human peripheral blood monocytes with apoptotic polymorphonuclear cells.

Macrophages have the potential to recognize apoptotic neutrophils and phagocytose them while the same function for monocytes is uncertain. In fact, early findings indicated that monocytes started to phagocytose neutrophils on the third day of differentiation to macrophages. Here we show, using flow cytometry and confocal microscopy, that peripheral blood monocytes phagocytose apoptotic but not freshly isolated granulocytes. Recognition of apoptotic cells is predominantly connected with CD16(+) monocytes (CD14(high) CD16(+) and CD14(dim) CD16(+)) and requires CD36. Clearance of apoptotic polymorphonuclear leucocytes appears to be independent of the CD14 mechanism. Uptake of apoptotic Jurkat T cells by monocytes is CD14 and CD36 dependent. Liposomes containing phosphatidyl-l-serine reduce binding of apoptotic polymorphonuclear leucocytes. Lipopolysaccharide-activated subpopulations of monocytes while in contact with apoptotic cells produce more anti-inflammatory cytokine interleukin-10 whereas the production of pro-inflammatory cytokines, tumour necrosis factor-alpha and interleukin-1beta is reduced.