The distribution of Ca 2+ in intact cells was monitored with fluorescent probes: fura-2 for cytosolic [Ca 2+ ] and rhod-2 for mitochondrial [Ca 2+ ]. It was found that in neoplastic cells, such as Ehrlich ascites tumour and Zajdela hepatoma, but not in non-malignant cells, such as fibroblasts, glucose and deoxyglucose elicited release of Ca 2+ from endoplasmic reticulum stores and an increase in Ca 2+ concentration in the cytosol. Parallel to this, a decrease in the rate of Ca 2+ extrusion from the cell and an enhanced uptake of Ca 2+ by mitochondria were observed. The increase in mitochondrial [Ca 2+ ] was accompanied by an increase in the mitochondrial membrane potential and the reduction state of nicotinamide nucleotides. F 1 F o -ATPase in submitochondrial particles of Zajdela hepatoma was strongly inhibited in the presence of micromolar Ca 2+ concentrations, whereas this activity in submitochondrial particles from rat liver appeared to be less sensitive to Ca 2+ . Indications of glycosylation of Ehrlich ascites tumour cell proteins were also obtained. These data strengthen the proposal [Bogucka, K., Teplova, V.V., Wojtczak, L. and Evtodienko, Y. V. (1995) Biochim. Biophys. Acta 1228, 261±266] that the Crabtree effect is produced by mobilization of cell calcium, which is subsequently taken up by mitochondria and inhibits F 1 F o -ATP synthase.Keywords: Calcium; Crabtree effect; Ehrlich ascites tumour; mitochondrial membrane potential; Zajdela hepatoma.Many kinds of tumour exhibit an unusual reaction towards glucose: addition of millimolar concentrations of glucose to the culture medium results in a decrease in the cell respiration rate. This behaviour, known by the name of its discoverer as the Crabtree effect [1], has also been observed in some non-malignant cells and tissues, such as spermatozoa [2,3], proliferating thymocytes [4], intestinal mucosa [5] and mammalian embryos at their very early developmental stage [6]. Common features of all these tissues, both malignant and normal, are a high proliferation rate and/or a high glycolytic rate. Although the Crabtree effect has been known for seven decades (for an early review see [7]), its molecular mechanism is not fully understood. First attempts to explain it pointed to the competition between respiration and glycolysis for precursors of ATP production, i.e. ADP [8,9] and inorganic phosphate [10±12]. However, this explanation is not satisfactory because deoxyglucose, which is phosphorylated in the cytoplasm but not further metabolized and therefore does not generate ATP, produced even stronger inhibition of respiration than did glucose [13] and phosphate transport into the cell has been shown to be fast enough [13].Other possible explanations of the mechanisms of the Crabtree effect were a shift in intracellular pH [7], a change in permeability properties of the inner mitochondrial membrane [14], a specific isoenzyme pattern of the glycolytic pathway and regulatory behaviour of key enzymes of this pathway [15], and specific topography of enzymes in rap...

Isothiocyanates (ITCs) derived from cruciferous plants reveal antibacterial activity, although detailed mechanism is not fully elucidated. Recently it has been reported that ITCs induce the stringent response in Escherichia coli strains. The aim of this work was to determine whether two isothiocyanates, sulforaphane (SFN) and phenethyl isothiocyanate (PEITC), similarly as in E. coli induce stringent response in Bacillus subtilis, model Gram(+) bacterium, and test their potency against a panel of clinical isolates belonging to Gram(+) or Gram(−) groups. Minimal inhibitory concentrations were determined as well as effect of ITCs on membranes integrity, synthesis of DNA, RNA and stringent response alarmones was assessed. SFN and PEITC are effective against B. subtilis and bacterial isolates, namely E. coli, K. pneumonia, S. aureus, S. epidermidis and E. faecalis. Interestingly, in B. subtilis and E. faecalis the inhibition of growth and nucleic acids synthesis is independent of ppGpp accumulation. In bacteria, which do not induce the stringent response in the presence of ITCs, membrane integrity disruption is observed. Thus, ITCs are effective against different pathogenic bacteria and act by at least two mechanisms depending on bacteria species.

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Phosphorylation of ADP and hydrolysis of ATP by isolated mitochondria from Ehrlich ascites tumour cells is greatly reduced when the mitochondria have been preloaded with Ca2+ (50 nmol/mg protein or more). Translocation of ADP is diminished in Ca(2+)-loaded mitochondria. However, ATPase in toluene-permeabilized mitochondria and in inside-out submitochondrial particles is also strongly inhibited by micromolar concentrations of Ca2+, indicating that, independently of adenine nucleotide transport, F1Fo-ATPase is also affected. ATP hydrolysis by submitochondrial particles depleted of the inhibitory subunit of F1Fo-ATPase (the Pullman-Monroy protein inhibitor) is insensitive to Ca2+; however, this sensitivity is restored when the particles are supplemented with the inhibitory subunit isolated from beef heart mitochondria. In view of the previous observations that glucose elicits in Ehrlich ascites tumour cells an increase of cytoplasmic free Ca2+ (Teplova, V.V., Bogucka, K., Czyz, A., Evtodienko, Yu.V., Duszyński, J. and Wojtczak, L. (1993) Biochem. Biophys. Res. Commun. 196, 1148-1154) and that this calcium is then taken up by mitochondria, resulting in a strong inhibition of coupled respiration (Evtodienko, Yu.V., Teplova, V.V., Duszyński, J., Bogucka, K. and Wojtczak, L. (1994) Cell Calcium 15, 439-446), the present results are discussed in terms of the mechanism of the Crabtree effect in tumour cells.

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