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The Na(+)/H(+) antiport activity encoded by the seven-gene mrp operons of Bacillus subtilis and alkaliphilic Bacillus pseudofirmus OF4 were cloned into a low copy plasmid, were expressed in several Escherichia coli mutant strains and compared side-by-side with similarly cloned nhaA, a major secondary antiporter from E. coli. All three antiporter systems exhibited electron donor-dependent antiport in a fluorescence-based vesicle assay, with NhaA being the most active. In whole cells of the same antiporter-deficient strain from which the vesicles were made, E. coli KNabc, Mrp-mediated Na(+) exclusion was significantly more protonophore-resistant than that conferred by NhaA. The Mrp systems were also more efficacious than NhaA: in supporting anaerobic Na(+) resistance in wild type and a terminal oxidase mutant strain of E. coli (SBS2115); and in increasing non-fermentative growth of an NADH dehydrogenase-minus E. coli mutant (ANN0222). The results suggest the possibility that the Mrp systems may have both secondary and primary energization capacities.

Oligonucleotide-directed triplex formation attracts much attention due to its potential usefulness in diagnostic and biotechnological applications. Among other aspects, the research embraces numerous studies probing the influence of intercalating ligands on triplex stability. The effect of the intercalator on triplex formation and stability is known to depend on nucleotide sequence, type of intercalator and solution conditions. The present work is aimed at determining the average number of intercalated ethidium bromide (EtBr) and acridine orange (AO) molecules leading to the most effective stabilization of triplexes. First, fluorescing complexes of intramolecular parallel (recombinant) triplex 5'-d(CATGCTAACT)-L-d(AGTTAGCATG)-L-d(CATGCTAACT)-3' (parARB) and classical antiparallel 5'-(dA)10-L-(dT)10-L-(dT)10-3'(antiATT) (L = -pO(CH2CH2O)3p-) with EtBr and AO were characterized, binding constants were obtained and compared to those for homologous DNA duplexes. Then the total EtBr and AO concentrations corresponding to an average of one, two or three intercalated molecules per oligonucleotide were estimated. Thermal denaturation of parARB and antiATT complexes with an average of one, two or three bound molecules was carried out, thermodynamic parameters of the triplex-to-duplex and duplex-to-open-strand transitions were evaluated using a three-state model. The ability of EtBr and AO to stabilize or destabilize both parallel (recombinant) and classical antiparallel triplexes was found to depend strongly on the concentration of bound intercalator. The triplexes were shown to be stabilized by intercalation of the first and second EtBr or AO molecules, while binding of the third intercalator molecule to 10 nucleotide long triplex resulted in significant triplex destabilization.

The fluorescent dye 10-N-nonyl acridine orange (NAO), known as specifically associated with mitochondria, has been reported to have a cytotoxic effect when high doses were applied to cells. Presently, the biochemical basis of its toxicity was investigated on isolated rat liver mitochondria. At low concentrations, NAO strongly inhibited state 3 respiration and ATP synthesis. At high concentrations, electron transport, ATP hydrolysis, Pi-transport and adenine nucleotide activities were also decreased. All these inhibitions can be explained by probe-cardiolipin interactions which could induce the collapse of energy conversion and/or the modification of membrane fluidity.

To determine whether protein kinase C plays a role in the actions of insulin and growth hormone in rat adipocytes, we tested the effect of acridine orange, a potent inhibitor of kinase C, on the lipogenic activity of both hormones. This compound completely inhibited the effects of insulin, growth hormone and phorbol ester 12-myristate 13-acetate, whereas 9-acridine carboxylic acid, an analog of acridine orange which does not inhibit kinase C, had no effect. Acridine orange did not act through inhibition of hormone binding. These data are consistent with the involvement of kinase C in the action of insulin and growth hormone on lipogenesis in rat fat cells.

The storage disaccharide, trehalose, is accumulated in yeast during a temperature shift from 30 to 45 degrees C. The response peaks at 90 min and is transient since levels of trehalose decline rapidly in cells returned to 30 degrees C. Storage of trehalose is inhibited when cells are incubated in the presence of acridine orange or ethidium bromide prior to and during temperature shift, suggesting a requirement for de novo RNA synthesis. Accumulation of trehalose occurs when cells are exposed to either ethanol, copper sulphate or hydrogen peroxide at 30 degrees C, indicating that the phenomenon may be a general response to physiological stress. Parallels are drawn between the trehalose accumulation response and the heat shock response in yeast.