Search

Restriction endonucleases (RE) have been used in cytogenetic studies to mimic the DNA double-strand break (dsb)-inducing action of radiation. In the experiments presented here, we have treated electroporated CHO cells with RE and have measured the resulting dsb using the filter elution technique under non-denaturing conditions (pH 9.6). PvuII, which generates blunt-ended dsb, gave rise to a significant number of measurable dsb. The frequency of the dsb induced by PvuII is shown to increase over a 3-12-h post-treatment incubation period, which implies that the RE is active in the cell for a considerable length of time. We postulate that the accumulation of dsb reflects a competition between enzymatic incision and repair of the DNA. The presence of araA, a known inhibitor of DNA synthesis, did not affect the frequency of PvuII-induced breaks indicating a lack of an inhibitory effect of araA on the repair of RE-induced dsb. Two RE which cause cohesive-ended dsb, namely BamHI and EcoRI, were found to be ineffective in giving rise to measurable dsb. Our interpretation of this is that for cohesive-ended dsb (caused by BamHI and EcoRI) the rate at which these breaks are rejoined matches or exceeds the rate of enzymatic incision and hence no dsb were observed. In the case of PvuII, the possibly slower rate of repair of blunt-ended termini would on this hypothesis result in the observed net accumulation of dsb.

Bradley and Kohn (1979) showed that the neutral filter elution technique detects DNA double-strand breaks (dsb), yet there is still some uncertainty regarding the interpretation of results obtained with this technique (viz. the curvilinear dose-response curve and the rapid repair kinetics). In this report we have investigated the effect of the nucleoside analogue 9-beta-D-arabinofuranosyladenine (ara A), a known inhibitor of dsb repair, on the DNA repair in X-irradiated Ehrlich ascites tumour cells as measured by this technique. We have compared the effect of ara A on repair in these cells with results previously obtained with the same cell line and using the neutral velocity sedimentation and DNA unwinding techniques (Blöcher, 1982; Bryant and Blöcher, 1982). Our results suggest that the lesions measured by the neutral elution technique are different from those measured by neutral velocity sedimentation or long-term repair measured by DNA unwinding.

Transient hypothermia was employed to extend the G2 phase of CHO K1 cells in order to facilitate study of the repair of X-ray induced chromatid and DNA damage. Thus G2 + 1/2M at 37 degrees C of 2.9 h was lengthened to 5.7 h at 33 degrees C and 7.3 h at 29 degrees C. While chromatid break kinetics remained essentially unaltered at 33 degrees C, at 29 degrees C there was an initial shoulder followed by a decrease in breaks similar to that at 37 and 33 degrees C. Although fewer exchanges were observed at 33 and 29 degrees C than at 37 degrees C, a similar kinetic involving a sharp initial rise followed by a plateau was observed at 33 and 29 degrees C, and, as far as could be judged, also at 37 degrees C. The failure of G2 prolongation to influence the rate of break disappearance was taken as evidence in support of the view that the disappearance of chromatid breaks represented a repair process rather than the decline of chromosomal radiosensitivity throughout this phase, though the possibility of a reduced sensitivity close to the G2/M border remained open. This hypothesis was supported by the mainly flat kinetics of exchanges. The data were taken as further evidence that chromatid rejoining and misjoining (exchanges) are essentially different processes. The rates of repair of DNA double-strand breaks as measured by neutral filter elution were similar at 37 and 33 degrees C, while there was evidence of inhibition at 29 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

We have measured the concentrations of carnitine and the activities of the carnitine acyltransferases in immature rat ovaries which had been stimulated to develop with pregnant mare gonadotropin. The concentration of carnitine increased from 700 nmol/g wet wt. to 1 mumol/g wet wt. of ovary after hormonal administration and the activities of the acyltransferases also rose. These increases corresponded with cell division. After replication had ceased, when the new cells were emerging and actively making steroid hormones, the concentration of carnitine and activities of the transferases fell.

Lipid granules in ovaries of immature rats were confined to the interstitial tissue, and comprised 70% cholesteryl esters and 20% triacylglycerols, the balance being phospholipid and free cholesterol. Following treatment with gonadotropin the interstitial granules disappeared as cholesteryl esters were hydrolysed, but reformed in the follicle as it developed, first in the theca, then in the outer granulosa and finally in the inner cells. The cholesteryl ester: triacylglycerol ratio fell during follicular growth, but in the corpus luteum the ratio in the widely distributed granules was 1:1. Esterified fatty acids in both cholesteryl esters and triacylglycerols became longer and more unsaturated as development progressed. The same progression of granules across the follicles was evident in ovaries of normal adult rats. We concluded that lipid granules in interstitial tissue supplied the substrates for synthesis of new cells in adjacent developing follicles, and those in corpora lutea were a prerequisite for steroidogenic competence.

Changes in the concentrations of total coenzyme A, acetyl CoA, free carnitine and acetylcarnitine were measured in ovaries from immature rats before and after superovulation with 50 I.U. pregnant mare's serum gonadotropin. In addition, the concentrations of total CoA and total acid-soluble carnitine were measured in liver, adrenal glands and skeletal muscle from the same rats. Ovarian concentrations of total CoA, free carnitine and acetylcarnitine increased 3-fold on gonadotropin stimulation, whereas there was no marked change in total CoA and acid-soluble carnitine concentrations in the other organs. In ovary, the ratio of free CoA to acetyl CoA was about 2:1 during the growth period of follicular development and during active steroidogenesis in the luteal phase, but less than 1 when replication stopped and ovulation occurred. These results show that during periods of high energy demand the ovary has a good capacity to accommodate fatty acid oxidation, and supports the evidence that fatty acids are the major source of reducing equivalents for steroidogenesis at these times.

Enzymic changes in primary cultures of granulosa cells over 9 days were measured and compared with changes occurring during follicular development in vivo. Characteristic of in vivo development of granulosa cells was a large increase in activities of the NADP+-dependent isocitrate, glucose-6-phosphate dehydrogenases and malic enzyme, and smaller increases in the activities of the NAD+-dependent lactate and malate dehydrogenases. In vitro, the NAD+-dependent dehydrogenases increased in activity, while the NADP+-dependent enzymes showed transient or no changes. Despite the uncharacteristic metabolism, granulosa cells in culture could synthesize steroids. Our results suggest that the cells in vitro and in vivo use different metabolic pathways to support syntheses dependent on reducing equivalents.

The severity of selenium dependent nutritional myopathy in weaner sheep was markedly increased by the ruminal infusion of soluble phosphate (6.0 g d-1). Liver selenium concentration was also significantly lower in the sheep infused with sodium phosphate than in sheep fed the same diet and infused with sodium chloride. There were no significant differences in the selenium concentrations of other tissues examined (kidney, heart, skeletal muscle, blood). Blood glutathione peroxidase (GSH.Px) activity in the sheep affected with nutritional myopathy was considerably higher than values generally accepted to indicate a selenium deficiency. The results indicated that there was an interaction in the animal between selenium and phosphate leading to lower liver selenium concentrations. Results obtained from sheep intraruminally dosed with 75[Se]selenate suggested that an interaction between selenium and phosphate could also occur in the gastrointestinal tract.

The DNA unwinding technique has been used to measure the induction and repair of DNA strand breaks by X-rays in the X-ray-sensitive (xrs 5) mutant and its parent CHO K1 line of Chinese hamster cells. Results show that frequency of induction of DNA strand breaks was the same for both cell lines. The repair of single-strand breaks was found to be slightly slower in xrs 5 over the first 20 min after X-ray exposure, but the level of repair of ssb reached after an incubation of 1h following X-ray exposure in xrs 5 was the same as in CHO K1. Our results also show that the rate of repair of DNA double-strand breaks in xrs 5 cells was clearly slower than that in CHO K1, supporting the conclusion of Kemp et al. (1984) who used the neutral elution technique, that xrs 5 is defective in the repair pathway of DNA double-strand breaks.

The production of endogenous acetate by the liver has been investigated in lactating ewes using animals with indwelling arterial, and portal and hepatic venous cannulae. The capacity of the liver to produce acetate from acetyl-CoA in vitro has also been examined using homogenates prepared from liver biopsy samples. Mean arterial, portal and hepatic venous blood acetate concentrations in four ewes at 4 weeks lactation were 0.40, 1.00 and 1.46 mM respectively. The mean exogenous and endogenous acetate production rates were 56 and 54 mmol/h respectively, giving a total of 110 MMOL/h. The mean portal-hepatic venous difference in free fatty acid concentration was 81 muM. Converting this uptake of free fatty acids by the liver (based on palmitate as a standard) to 2-carbon equivalents, the acetate produced accounted for 70% of the fatty acids taken up. The correlation coefficient (r2) between uptake of free fatty acids and production of acetate by the liver was 0.83 (P less than 0.01). Calculation of the net acetate production in vivo gave a mean value for the production of acetate of 0.75 nmol/min. Calculation of the in vitro enzymic capacity of the liver to produce acetate from acetyl-CoA gave a mean of 0.94 mmol/min. These results indicate that enzymic production of acetate from acetyl-CoA, via carnitine acetyltransferase and acetylcarnitine hydrolase (see Costa and Snoswell 1975a), can adequately account for the substantial production of acetate by the liver in lactating ewes.

Chloramphenicol, erythromycin, gentamicin, oxytetracycline, penethamate and procaine benzyl penicillin were administered parenterally to cattle and the concentrations of these antibiotics in plasma and tears were assayed microbiologically. Concentrations in plasma and tears were significantly correlated for all antibiotics tested but the concentration of antibiotic in tears and the tear flow rate were not correlated. Lipophilic drugs diffused into the tears in higher concentrations than did drugs which were not lipophilic. Concentrations of lipophilic but not hydrophilic antibiotics in tears could be predicted from the Henderson-Hasselbach equation. In cattle, it is possible through parenteral administration of chloramphenicol, erythromycin, gentamicin or oxytetracycline to achieve antibiotic concentrations in the tears which are bacteriostatic to Moraxella bovis, a primary aetiological agent of infectious bovine keratoconjunctivitis.