Single strand breaks and mutagenesis in yeast induced by photodynamic treatment with chloroaluminum phthalocyanine.

Photodynamic treatment of the yeast Kluyveromyces marxianus with the sensitizer aluminum phthalocyanine results in loss of clonogenicity. In this paper the effect of this treatment on DNA of this yeast was investigated by searching for single strand breaks and forward mutations. Using the alkaline step elution technique it was found that illumination of the yeast in the presence of aluminum phthalocyanine resulted in an increase in single strand breaks. These could, partially, be repaired by post-incubating illuminated cells in growth medium. At comparable survival levels, photodynamic treatment with aluminum phthalocyanine induced fewer single strand breaks than X-ray treatment. By using a medium containing 5-fluoroorotic acid, mutants in the uracil biosynthetic pathway were selected. Photodynamic treatment resulted in a light dose dependent increase of the mutation frequency. The observed mutagenicity of photodynamic treatment of the yeast with phthalocyanine was lower than the mutagenicity of UVC and X-ray treatment at equal colony forming capacity, indicating that photodynamic treatment is the least mutagenic of those treatments. It is concluded that photodynamic treatment of K. marxianus results in DNA damage. Saccharomyces cerevisiae rad14 and rad52 mutants were used to determine the effect of the nucleotide excision repair and recombinational repair pathways, respectively, on survival after photodynamic treatment. Our data indicate that DNA damage is not the main determinant for cell killing by photodynamic treatment and that the type of damage induced is apparently not subject to RAD14- or RAD52 controlled repair.

Intracellular damage in yeast cells caused by photodynamic treatment with toluidine blue.

The positively charged photosensitizer toluidine blue (TB) can induce loss of clonogenicity in Kluyveromyces marxianus. Previous studies have revealed that, as a consequence of the localization of this dye at the cell surface, photodynamic action results in extensive damage at the level of the plasma membrane. In this paper, a study is reported on the effect of photodynamic treatment with TB on intracellular enzymes. It is shown that treatment with TB and light resulted in the inhibition of alcohol dehydrogenase, cytochrome c oxidase, glyceraldehyde-3-phosphate dehydrogenase and hexokinase. Photodynamic treatment also lowered the ATP levels. The ATP levels could be partially restored in the presence of glucose but not with ethanol. Toluidine blue binding experiments revealed that photodynamic treatment caused a rapid increase in the amount of cell-associated dye. Moreover, it also appeared that this treatment decreased the binding of TB to the cell surface. It is concluded that TB enters the cell during the first minutes of illumination, whereafter intracellular enzymes are inactivated. The data indicate that photodynamic damage of intracellular sites contributes to the loss of viability.

Inhibition of transport systems in yeast by photodynamic treatment with toluidine blue.

Photodynamic treatment of yeast with the sensitizer Toluidine blue results in loss of cell viability. In previous investigations it was suggested that plasma membrane damage might be responsible for the loss of colony forming capacity. In this context the influence of photodynamic treatment on transmembrane transport systems was studied. It appeared that the uptake of the sugars glucose, lactose and galactose, the amino acids arginine, phenylalanine, glycine and aspartic acid and of the inorganic compound phosphate was inhibited by photodynamic treatment. The different elements of the energy providing system necessary for active transport, viz. the plasma membrane ATPase and the protonmotive force, were not significantly affected by Toluidine blue and light, indicating that inhibition of transport is not caused by a reduction of the membrane potential or the transmembrane pH gradient. These observations suggest that the transport carriers themselves were damaged by treatment with Toluidine blue and light. This could be confirmed in experiments, in which the lactose and galactose transport proteins of treated and untreated cells were reconstituted in plasma membrane vesicles. It appeared that the carriers, obtained from photodynamically treated Kluyveromyces marxianus cell, had lost their transport capacity.

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.

Regulation of sugar transport systems of Kluyveromyces marxianus: the role of carbohydrates and their catabolism.

In Kluyveromyces marxianus grown on a glucose-containing synthetic medium four different sugar transporters have been identified. In cells, harvested during the exponential phase, only the constitutive glucose/fructose carrier, probed with 6-deoxy-D-glucose or sorbose, appeared to be active. In cells from the stationary phase three proton symporters can be active, recognizing 6-deoxyglucose (a glucose/galactose carrier), sorbose (a fructose carrier) and galactosides (lactose carrier), respectively. These symporters appeared to be sensitive to catabolite inactivation. This process is induced by incubating cells in the presence of glucose, fructose or mannose. Catabolite inactivation was not influenced by the inhibitor of protein synthesis, anisomycin. Derepression of the proton/sorbose and the proton/galactoside symporters proceeded readily when cells were incubated in a medium without glucose. Activation of the proton/galactose symporter needed, in addition, the presence of specific molecules (inducers) in the medium. The activation of each of these active transport systems was inhibited by anisomycin, showing the involvement of protein synthesis.

The role of ATP in the control of H+-galactoside symport in the yeast Kluyveromyces marxianus.

Transport of methyl beta-D-thiogalactoside and p-nitrophenyl beta-D-galactoside is shown to proceed through the H+-lactose symporter of Kluyveromyces marxianus. Uptake of these compounds is strongly reduced under anaerobic conditions or aerobically in the presence of antimycin. It is shown that antimycin treatment affects p-nitrophenyl beta-D-galactoside uptake in a similar way as it affects the cellular amount of ATP, suggesting regulation of p-nitrophenyl beta-D-galactoside transport by ATP. Also, manipulation of cellular ATP by antimycin treatment followed by glucose incubation, or by aerobic incubation of cells with 2-deoxy-D-glucose, showed a similar dependence of galactoside uptake on the ATP level. Transport of the lipophilic cation tetraphenylphosphonium is affected by ATP variations in a similar way as galactoside influx. It is concluded that ATP regulates H+-galactoside symport by its influence on charge translocation. It is discussed that a membrane ATPase probably plays a central role in the control of the activity of H+-sugar symport.

Alternative-substrate inhibition of L-lactate transport via the monocarboxylate-specific carrier system in human erythrocytes.

The L-lactate/proton symport system of the red blood cell membrane was studied under conditions of alternative-substrate inhibition by glycolate. At constant pH of the medium glycolate caused competitive inhibition of L-lactate transport. In Lineweaver-Burk plots of 1/v against 1/[H], on the other hand, glycolate caused an uncompetitive inhibition. These observations indicate, that the monocarboxylate carrier exhibits ordered substrate binding, with the proton binding first.

Kinetic analysis of L-lactate transport in human erythrocytes via the monocarboxylate-specific carrier system.

Three parallel pathways of L-lactate transport across the membrane of human red blood cells can be discriminated: (a) by nonionic diffusion; (b) via the band 3 anion exchange protein; and (c) via a specific monocarboxylate carrier system. Influx of lactate via the latter system leads to alkalinization of the medium, suggesting lactate-proton symport. Kinetic analysis of initial lactate influx via the monocarboxylate carrier indicates a symport system with ordered binding of the two ligands, in the sense that a proton binds first to the translocator, followed by lactate binding to the protonated carrier. The influence of varying trans-pH under conditions of net (zero-trans) flux with constant cis-pH indicates that the monocarboxylate translocator should be considered as a mobile carrier, with the ligand-binding sites exposed alternatively to the outside and the inside of the membrane.

Studies on erythrocyte membranes of patients with Huntington's disease.

Several red cell membrane properties and activities of membrane-bound enzymes were investigated in blood samples of patients with Huntington's disease. (Na(+)+K(+)) ATPase activity and cell deformability appeared to be normal, in contradiction to preceding reports from other laboratories. With other techniques sensitive to relatively small changes in membrane structure, no abnormalities were found in Huntington's disease red cell membranes. These investigations do not support the concept that a generalised membrane abnormality is present in Huntington's disease.

Hypertonic cryohemolysis of human red blood cells.

Hypertonic cryohemolysis of human erythrocytes is caused by incubation of the cells in hypertonic medium at a temperature of 20--50 degrees C (stage 1), with subsequent cooling to 0 degrees C (stage 2). In 0.86 M sucrose hemolysis increases, with increasing stage 1 temperature, whereas in 1 M NaCl cryohemolysis has a temperature optimum at a stage 1 temperature of about 30 degrees C. Cryohmeolysis is inhibited by preceding ATP depletion of the cells and by preincubation of the cells in hypertonic medium at 0 degrees C. In general, anesthetics inhibit cryohemolysis strongly. Only in 1 M NaCl at stage 1 temperatures in the range of 40--50 degrees C is cryohemolysis stimulated by these drugs, if present during the entire incubation period. This effect is abolished, however, when the anesthetic is added after prior incubation of the cells at 40--50 degrees C in 1 M NaCl. Ghost-bound ANS fluorescence indicates complicated conformation changes in the membrane structure during the various experimental stages leading to cryohemolysis. Some of the experimental results can be considered as examples of molecular hysteresis, thus indicating several different metastable structures of the membrane, under various experimental conditions. The described results support the working hypothesis of Green and Jung that the experimental procedure results in membrane protein damage, preventing normal adaptation of the membrane during cooling.