Active transport of L-sorbose and 2-deoxy-D-galactose in Saccharomyces fragilis.

Sorbose and 2-deoxy-D-galactose are taken up in Saccharomyces fragilis by an active transport mechanism, as indicated by the energy requirement of the process and the accumulation of free sugar against the concentration gradient. There are no indications for transport-associated phosphorylation as mechanism of energy coupling with these two sugars. The measured sugar-proton cotransport and the influx inhibition by uncouplers suggest a chemiosmotic coupling mechanism. Thus there are at least two different active transport mechanisms operative in Saccharomyces fragilis: transport-associated phosphorylation in the case of 2-deoxy-D-glucose and chemiosmotic coupling in the case of sorbose and 2-deoxy-D-galactose. The differences between the two mechanisms are discussed. Uncouplers do not stimulate downhill sorbose transport in energy-depleted cells and evoke an almost complete inhibition of efflux and of exchange transport. The differences between this sugar-proton cotransport system and similar systems in bacteria and Chlorella are discussed.     

Growth characteristics and ion contents of non-selected and salt-selected callus lines of highbush blueberry (Vacdnium corymbosum) cultivars Blue Crop and Denise Blue

Non-selected and sodium chloride selected callus lines of Vacdnium corymbosum L.cv Blue Crop and cv. Denise Blue were grown on media supplemented with 0–100 mM NaCl. For both cultivars, fresh weight and dry weight yields were greater in selected lines on all levels of NaCl. Selected lines of Blue Crop displayed better growth than selected lines of Denise Blue at most concentrations of NaCl. Internal Na⁺ and Cl^– concentrations in selected and non-selected lines of both cultivars increased as external concentration was raised. However, selected lines of Blue Crop and Denise Blue accumulated more Na⁺ and Cl^– than non-selected lines. Selected lines of both cultivars maintained higher levels of K⁺ than non-selected lines on all external NaCl levels. Selected lines of Blue Crop had higher levels of Na⁺ and Cl^– than that of Denise Blue. The results suggest Na⁺ and Cl^– accumulation could be a mechanism allowing better growth in selected lines at moderate salinity levels (50–75 mM NaCl).     

The binding of zinc,but not cadmium,by phytic acid in roots of crop plants

Plant species adapted to soils enriched with heavy metals often accumulate these metals in their above or below ground organs. In this study, electron probe microanalysis of fractured, quench-frozen root specimens of common crop species shows that an appreciable quantity of Zn can be bound as Zn phytate (myo-inositolkis-hexaphosphate) within small vacuoles of cells in the root elongation zone of lucerne, soybean, lupins, tomato, rapeseed, cabbage, radish, maize and wheat exposed to high levels of Zn (80–300 M). Globular deposits of Zn phytate are most frequently observed in the endodermis of dicotyledonous species and in the pericycle of monocotyledonous species, but may also occur in the stele and inner cortex after prolonged exposure to toxic levels of Zn. The deposits could not be found in Zn-treated sunflower, field peas and Italian ryegrass. In three crop species, lucerne, soybean and maize, Zn-induced phytate globules were frequent, but exposure of roots to 30 M Cd did not induce the formation of Cd-containing globular deposits as observed inLemna minor (Van Steveninck et al., 1990a, 1992). Simultaneous Zn and Cd treatment induced the formation of Zn phytate globules as effectively as Zn alone, and Cd was not detected in the deposits.     

Detection of a yeast polyphosphate fraction localized outside the plasma membrane by the method of phosphorus-31 nuclear magnetic resonance

Non-penetrating cations, like UO2+(2) and Eu3+, are bound to the outside of yeast cells in a reversible fashion. Binding of these ions was attended with a decrease of the 31P NMR polyphosphate signal. Subsequent addition of EDTA to the suspension restored the original spectrum. These experiments confirm the localization of a polyphosphate fraction outside the plasma membrane of yeast.     

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.     

Site-specific and bulk-phase generation of hydroxyl radicals in the presence of cupric ions and thiol compounds.

Addition of a thiol compound to a solution containing Cu2+ and H2O2 resulted in the generation of hydroxyl radicals (OH.). These radicals were able to oxidize salicylic acid and tryptamine in a reaction that was strongly inhibited by the OH.-scavenger mannitol. Covalent coupling of the thiol compound to tryptamine did not significantly influence the degradation of the indole moiety subsequent to addition of H2O2 and Cu2+. The inhibiting effect of mannitol, however, was strongly reduced, indicating that the scavenger could not interfere with site-specific reactions of OH..     

The influence of porphyrins on iron-catalysed generation of hydroxyl radicals.

Uroporphyrin I, haematoporphyrin and haematoporphyrin derivative had no effect on O2-. generation during oxidation of hypoxanthine by xanthine oxidase and on the formation of hydroxyl radicals (OH.) in the hypoxanthine/xanthine oxidase/Fe3+-EDTA/deoxyribose system. On the other hand, these porphyrins strongly inhibited O2-. formation in a horseradish peroxidase/H2O2/NADPH mixture, whereas they augmented OH. generation in this system after addition of Fe3+-EDTA. Experimental evidence suggests that these observations should be ascribed to the formation of a porphyrin anion radical in the horseradish peroxidase/NADPH system. The formation of this anion radical was confirmed by e.s.r. spectroscopy. This radical is apparently unable to reduce cytochrome c, but it can replace O2-. in the OH.-generating Haber-Weiss reaction.     

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.