Inhibition of exogenous NADH oxidation in plant mitochondria by chlorotetracycline in the presence of calcium ions

Chlorotetracycline inhibits the uncoupled oxidation of exogenous NADH by Jerusalem artichoke (Helianthus tuberosus L.) mitochondria extensively (over 80%) and rapidly (inhibition complete in 10 s) in the presence of added Ca²⁺. Half-maximal inhibition is observed at 15 μM chlorotetracycline in the presence of 2 mM Ca²⁺. The oxidation of succinate is only affected marginally by chlorotetracycline plus Ca²⁺. The inhibition of NADH oxidation and the fluorescence of CTC are well correlated. Mn²⁺ is the only other cation which shows an (increased) inhibition in the presence of chlorotetracycline. The inhibition by Ca²⁺ and chlorotetracycline disappears at acid pH, and the pH optimum in their presence is 6.4. The inhibition caused by other lipid-soluble Ca²⁺-chelators is not reversible or is enhanced by the addition of excess Ca²⁺. In contrast, inhibition caused by relatively water-soluble chelators is completely reversed by added Ca²⁺. It is suggested that a neutral 1:2 complex is formed between Ca²⁺ and chlorotetracycline which can substitute for Ca²⁺ bound at sites in the lipophilic phase of the inner mitochondrial membrane, which are essential for the activity of the external NADH dehydrogenase.     

The relationship between sister-chromatid exchange and perturbations in DNA replication in mutant EM9 and normal CHO cells

The majority of the high (12-fold elevated) baseline sister-chromatid exchanges (SCEs) that occur in the CHO mutant line EM9 appear to be a consequence of incorporated BrdUrd, and they arise during replication of DNA containing BrdUrd in a template strand. In normal CHO cells the alkaline elution patterns of DNA newly replicated on a BrdUrd-containing template are significantly altered compared with those seen during the replication on an unsubstituted template. The nascent DNA synthesized on such an altered template is delayed in reaching mature size, possibly because replication forks are temporarily blocked at sites occurring randomly along the template. Transient blockage of replication forks may be a prerequisite for SCE. The delay in replication on BrdUrd-substituted templates was greater in EM9 cells than in parental AA8 cells and was also greater in AA8 cells treated with benzamide, an inhibitor of poly(ADPR) polymerase, than in untreated AA8 cells. Under these conditions, treatment with benzamide also produced a 7-fold increase in SCEs in AA8. An EM9-derived revertant line that has a low baseline SCE frequency showed less delay in replication on BrdUrd-substituted templates than did EM9. However, under conditions where the template strand contained CldUrd, which was shown to produce 4-fold more SCEs than BrdUrd in AA8 cells, the replication delay in AA8 was not any greater in the CldUrd-substituted cells. Thus, other factors besides the delay appear to be involved in the production of SCEs by the template lesions resulting from incorporation of the halogen-substituted pyrimidine molecules.     

Mutations resistant to bromodeoxyuridine in mouse lymphoma cells selected by repeated exposure to EMS characteristics of phenotypic instability and reversion to HAT resistance by 5-azacytidine

Mutations induced by repeated EMS treatments were investigated by using mouse L5178Y cells. The frequency of TG^r mutations increased linearly with the number of EMS treatments whereas the yield of BrdUrd^r mutations showed a curvilinear dose-response curve. The BrdUrd^r frequency was roughly proportional to the square of the TG^r frequency and the results were compatible with the hypothesis that BrdUrd^r cells were induced by two mutational events within a cell. Most of the BrdUrd^r colonies isolated after 6 EMS treatments, however, were unstable. When BrdUrd^r colonies that had arisen in BrdUrd medium after 2 weeks' incubation were isolated in normal medium, the descendant cells showed a nearly normal level of thymidine incorporation and low plating efficiencies of about 1% in BrdUrd medium. In contrast, after isolation of the same colonies in BrdUrd medium, a low level of thymidine incorporation and high plating efficiencies in BrdUrd medium were observed in the descendant cells.

Reverse selection from BrdUrd^r to HAT^r was accomplished with frequencies of 10⁻⁶−10⁻³ for the descendants grown in BrdUrd medium, and AzaCyd treatment drastically increased the reversion frequency to nearly 10⁻¹. Further re-revertants from HAT^r to BrdUrd^r were also found with frequencies of 10⁻³−10⁻² without treatment. These results indicate that the initial BrdUrd^r cells did not result from inactivation of the thymidine-kinase gene but that the mode of gene expression was altered in some way.     

Cross-resistance studies with V79 Chinese hamster cells adapted to the mutagenic or clastogenic effect of N-methyl-N′-nitro-N-nitrosoguanidine

When V79 cells are exposed to a single low dose of MNNG or MNU they acquire resistance to the mutagenic or to the clastogenic effect of the agents. Here the effect of MNNG pretreatment on mutagenesis (6-thioguanine resistance) and aberration formation in cells challenged with various mutagens/clastogens is reported. MNNG-adapted cells were resistant to the mutagenic effects of MNU and, to a lower extent, of EMS. No mutagenic adaptation was observed when MNNG-pretreated cells were challenged with MMS, ENU, MMC or UV.

Cells pretreated with a dose of MNNG which makes them resistant to the clastogenic effect of this compound were also resistant to the clastogenic activity of other methylating agents (MNU, MMS), but not so with respect to ethylating agents (EMS, ENU). Cycloheximide abolished the aberration-reducing effect of pretreatment. However, when given before the challenge dose of MNNG, MNU or MMS, it drastically enhanced the aberration frequency in both pretreated and non-pretreated cells. No significant enhancement of aberration frequency by cycloheximide was found for ethylating agents.

The results indicate that clastogenic adaptation is due to inducible cellular functions. It is concluded that mutagenic and clastogenic adaptation are probably caused by different adaptive repair pathways.     

Oxo fatty acid esters from Cryptocoryne spiralis rhizomes

Two new compounds, isolated from the rhizomes of Cryptocoryne spiralis, have been characterized as ethyl 14-oxotetracosanoate and 15-oxoeicosanyl 14-oxoheptadecanoate by spectral data and chemical studies. Hentriacontane and sitosterol have also been isolated and identified.     

Fibre length and gibberellins A1 and A20 are decreased in Eucalyptus globules by acylcyclohexanedione injected into the stem

Trinexapacethyl (TriEt), an acylcyclohexanedionetype inhibitor of gibberellin (GA) biosynthesis, was applied to 3-year-old Eucalyptus globules saplings by localised injection near the base of each stem. The objective was to alter cambial region GA levels and to study the effects on secondary xylem fibre development. Seven weeks later wood samples, with bark and cambial region intact, were removed 10 and 30 cm above the point of injection. Fusiform cambial cell dimensions were compared with those of fibre-tracheids in the most recently formed 100 um of secondary xylem. Increasing TriEt applications from 5 to 5 000 mg active ingredient significantly reduced average fibre length, and to a lesser extent average fusiform cambial cell length. Also reduced was the number of cells in the cambial zone and the number of differentiating fibres with primary walls. However, no trends were evident for changes in fibre diameter, the proportion of vessel elements or the ratio of cambial ray cells to fusiform cambial cells. Two gibberellins (GA₁ and GA₂₀), indole-3-acetic acid (IAA) and abscisic acid (ABA) were quantified in cambial region tissues by gas chromatographymass spectrometry using stable isotope labelled internal standards. Increasing TriEt application reduced both GA₁ and GA₂₀ levels. Effects on IAA and ABA were not significant, although their levels tended to be lower at the highest TriEt application rate. The elongation of secondary xylem fibres was positively correlated with higher levels of endogenous GA₁ (r_s= 0.74, P < 0.01) and GA₂₀ (r_s= 0.72, P < 0.01). These results support a causal role for GA₁ in cambial cell division. They are also consistent with the hypothesis that the elongation of differentiating secondary xylem fibres in woody an–giosperms is dependent on GA₁ levels in the cambial region.     

Thin-layer chromatography and high-performance liquid chromatography for the assay of fatty acid compositions of individual phospholipids in platelets from non-insulin-dependent diabetes mellitus patients: effect of eicosapentaenoic acid ethyl ester administration

Eight major phospholipids were separated by a TLC method with a one-dimensional developing system without any pretreatment of the plate and the fatty acids incorporated into each phospholipid class were analysed by an improved HPLC method with a simple elution system, which has advantages with respect to resolution and analysis time. The fatty acid compositions of individual phospholipids in platelets were investigated following administration of ethyl cis-5,8,11,14,17-eicosapentaenoate for more than 13 weeks to patients with non-insulin-dependent diabetes mellitus. The cis-5,8,11,14,17-eicosapentaenoic acid compositions of all phospholipid classes were significantly increased with decreasing platelet aggregation rates after the administration. These results suggested that the present method provides the complete separation of individual phospholipids in sufficient amounts to allow fatty acid analysis on the isolated phospholipid moieties.     

In vitro anti- and pro-oxidative effects of natural polyphenols

The anti- and pro-oxidative effects of phenolic compounds and antioxidants were studied in two different in vitro model systems utilizing ethyl linoleate and 2′-deoxyguanosine (2′-dG) as oxidative substrates, and a Fenton reaction (H₂O₂, Fe²⁺) to initiate oxidation. Oxidation of the biomolecules in both model systems exhibited dose dependency. In the 2′-dG assay, oxidation was closely related to H₂O₂ generation, which occurred during autoxidation of the phenolics. Hydroxylating activity was greatly enhanced by Mn²⁺ and Cu²⁺, but not by Zn²⁺ or Co²⁺. Ethyl linoleate peroxidation was inhibited by low concentrations of catechol, quercitin, and instant coffee. However, peroxidation was promoted by high concentrations of the same compounds, probably by recycling of chelated inactive Fe³⁺ to the active Fe²⁺ state.