A role for the transient increase of cytoplasmic free calcium in cell rescue after photodynamic treatment.

Chinese hamster ovary (CHO) cells and T24 human bladder transitional carcinoma cells were treated with the photosensitizers aluminum phthalocyanine (AlPc) and hematoporphyrin derivative (HPD), respectively. Exposure of both sensitized cell lines to red light caused an immediate increase of cytoplasmic free calcium, [Ca2+]i, reaching a peak within 5-15 min after exposure and then returning to basal level (approximately 200 nM). The level of the peak [Ca2+]i depended on the light fluence, reaching a maximum of 800-1000 nM at light doses that kill about 90% of the cells. Loading the cells with the intracellular calcium chelators quin2 or BAPTA prior to light exposure enhanced cell killing. This indicates that increased [Ca2+]i after photodynamic therapy (PDT) contributed to survivability of the treated cells by triggering a cellular rescue response. The results of experiments with calcium-free buffer and calcium chelators indicate that both in CHO cells treated with AlPc and with HPD-PDT of T24 cells extracellular Ca2+ influx is mainly responsible for elevated [Ca2+]i. PDT is unique in triggering a cell rescue process via elevated [Ca2+]i. Other cytotoxic agents, e.g., H2O2, produce sustained increase of [Ca2+]i that is involved in the pathological processes leading to cell death.     

Protein damage, induced by small amounts of photodynamically generated singlet oxygen or hydroxyl radicals

The influence of limited oxidation of glyceraldehyde-3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate:NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12), alcohol dehydrogenase (alcohol:NAD+ oxidoreductase, EC 1.1.1.1) and myoglobin by singlet oxygen and by hydroxyl radicals was investigated. The intrinsic fluorescence of glyceraldehyde-3-phosphate dehydrogenase and alcohol dehydrogenase decreased rapidly during oxidation, indicating a conformational change of the protein molecules. The free energy of isothermal unfolding in urea solutions was increased by singlet oxygen, but decreased by hydroxyl radical attack. The velocity of refolding of the denatured protein after dilution of the denaturant was increased by exposure to either singlet oxygen or hydroxyl radicals, with one exception: the velocity of refolding of myoglobin, oxidized by singlet oxygen, was strongly decreased. Hydroxyl radicals produced covalently crosslinked protein aggregates and some fragmentation, whereas singlet oxygen produced only crosslinked aggregates with glyceraldehyde-3-phosphate dehydrogenase and alcohol dehydrogenase. All oxidized proteins were more susceptible to proteolysis by elastase and proteinase K, as compared to the undamaged proteins. Singlet oxygen-induced crosslinked aggregates were degraded very rapidly by elastase. Hydroxyl radical-induced aggregates of glyceraldehyde-3-phosphate dehydrogenase were also degraded very rapidly by this enzyme, but hydroxyl radical-induced aggregates of alcohol dehydrogenase were resistent to enzymatic degradation. The results indicate that limited protein oxidation may have a pronounced influence on several properties of the protein. The effects vary, however, with varying proteins and with the oxidizing species.     

Safeguarding marine life: conservation of biodiversity and ecosystems

Reviews in Fish Biology and Fisheries - Marine ecosystems and their associated biodiversity sustain life on Earth and hold intrinsic value. Critical marine ecosystem services include maintenance of...     

Anomalous properties of water in macromolecular gels.

Low molecular weight solutes often exhibit elution characteristics on gel filtration columns which deviate from ideal behaviour. In many previous studies this anomalous behaviour was attributed to the existence of extremely narrow pores in the gel, inaccessible even to very small solute molecules, to explain Kd values lower than unity. Kd values of small solutes higher than unity were usually ascribed to adsorption of the solute to the gel matrix. In the present paper several observations are presented that contradict these suggestions. Experimental evidence indicates that with small solute molecules Kd values differing from unity can be fully explained by the anomalous properties of vicinal water layers at the gel matrix-water interface.     

Effect of xylose incubation on the glucose transport system in Saccharomyces cerevisiae

Incubation of Saccharomyces cerevisiae with xylose and ethanol for 16 hours leads to a decrease of hexokinase (and glucokinase) activity in the cells. It does not alter the levels of polyphosphate, orthophosphate and ATP. The transport of the glucose derivative 2-deoxy-D-glucose, a sugar that can be phosphorylated, is inhibited after this treatment, whereas transport of 6-deoxy-D-glucose, which has a blocked phosphorylation site, is not inhibited. Even though, both deoxyglucoses use the same transport system. The decrease in initial velocity of 2-deoxy-D-glucose transport is most pronounced under anaerobic conditions. Incubation of the cells with antimycin A, a treatment which has a similar effect as anaerobiosis, shows, that the inhibition of the transport of 2-deoxy-D-glucose is presumably the result of an increase in the Km of the carrier transport. Transport of glucose is probably regulated by kinase enzymes.     

Photodynamic treatment of yeast cells with the dye toluidine blue: all-or-none loss of plasma membrane barrier properties.

Photodynamic treatment of Kluyveromyces marxianus with the sensitizer Toluidine blue leads to the loss of colony forming capacity. In this paper, the influence of this treatment on the barrier properties of the plasma membrane has been studied. Photodynamic treatment with the dye Toluidine blue resulted in efflux of potassium ions and E260-absorbing material. Moreover, cells became stainable with erythrosine. It is concluded that the permeability change induced by photodynamic treatment proceeds in an all-or-none fashion. Treatment of this yeast strain, with the dye and light, also induced a diminution of the cell volume. This process is most likely not coupled to the cellular potassium content, but rather to the integrity of the vacuole. These data suggest that the vacuole has an important function in the maintenance of cell volume. Finally, it was observed that the loss of cell viability was not induced by the all-or-none loss of barrier properties.     

The influence of ozone on human red blood cells. Comparison with other mechanisms of oxidative stress

Exposure of red blood cells to ozone resulted in K+ leakage, lipid peroxidation and inhibition of some membrane-associated enzymes. On the other hand, carrier-mediated transport of glucose, leucine, sulfate and glycerol and the nonspecific permeation of glycerol, L-glucose and erythritol were not affected by ozone. The cellular level of reduced glutathione declined, whereas the ATP content of the cells was quite insensitive to ozone exposure. It was shown that, most probably, lipid peroxidation and K+ leakage are not causally related. Further, K+ leakage did not reflect gradual, progressive loss of K+ from all cells simultaneously, but occurred in an all-or-none fashion. Finally, ozone-induced damage was compared to damage induced by H2O2, t-butyl hydroperoxide and photosensitizers plus light. It appeared that the pathways leading to membrane deterioration are quite dissimilar in these various forms of oxidative stress.     

Transport and transport-associated phosphorylation of galactose in Saccharomyces cerevisiae

The galactose transport mechanism in three strains of Saccharomyces cerevisiae was investigated in some detail, both in glucose-grown cells and in galactose-induced cells.