Effects of oxygen inhalation on the antioxidant capacity of lungs, livers, and brains in normal and vitamin E-deficient rats at various ages.

The effects of oxygen inhalation for 48 h on the antioxidant capacity of lungs, livers, and brains in normal and vitamin E-deficient rats at various ages were examined. The activity levels of catalase, glutathione peroxidase, and superoxide dismutase, and the level of vitamin E in tissue homogenates were assayed as the indices of antioxidant capacity. Oxygen inhalation mostly decreased antioxidant enzyme activity in lungs. In particular, the catalase activity was much decreased. The glutathione peroxidase activity tended to be decreased. The superoxide dismutase activity was decreased in 32-month-old rats. Vitamin E deficiency did not augment oxidative damage due to oxygen inhalation. There appears to be no age effect on the oxygen-induced decrease in the antioxidant enzyme activities of lungs, except the superoxide dismutase activity in very old rats. Oxygen inhalation had some effects on the antioxidant capacity of livers and brains. For example, oxygen inhalation decreased the vitamin E concentration of livers in 32-month-old, normal rats. These results suggest that the antioxidant capacity of lungs is directly damaged by oxygen inhalation and that the antioxidant capacity of livers and brains is indirectly affected through lung damage. Antioxidant capacity may be maintained without large variation during young and middle ages, but its redundancy for emergency use may be diminished in old age.

Induction of labour using prostaglandin E2 gel: the effect of changing the time of first insertion.

This study forms part of an on-going audit into induction of labour using prostaglandin E2 vaginal gel at a district general hospital. It looks at the effect of changing the time of first insertion of the gel from 2200 h to 1400 h in 80 primiparous women between 37 and 42 completed weeks' gestation, booked for induction of labour. Significant reductions in total cost of admission and length of hospital stay were associated with induction at 1400 h, and a trend to shorter time from induction to rupture of membranes and lower Caesarean section rate was also noted. Larger studies are needed.

Age-related alterations in antioxidant capacity and lipid peroxidation in brain, liver, and lung homogenates of normal and vitamin E-deficient rats.

Age-related alterations in both antioxidant capacity and lipid peroxidation in the cerebrum, lung and liver homogenates of normal and vitamin E-deficient rats were investigated. The antioxidant capacity, which includes superoxide dismutase, catalase and glutathione peroxidase activities and vitamin E (alpha-tocopherol) concentration, was relatively stable throughout the lifespan. It was observed, however, that catalase and glutathione peroxidase activities in livers of old rats decreased and that vitamin E concentration in lung and liver increased with age. In vitamin E-deficient animals, catalase activity in liver increased and glutathione peroxidase activity in liver and lung decreased. Lipid peroxidation was monitored by use of three different indices, i.e. the thiobarbituric acid (TBA) value, oxygen absorption and conjugated-diene formation. In the absence of any initiator, neither oxygen absorption into tissue homogenates nor conjugated-diene formation in lipid extracts from the homogenates occurred. The TBA value of each cerebrum homogenate incubated under air or an oxygen atmosphere was larger than that of the corresponding unincubated cerebrum homogenate. From comparison between the TBA value and oxygen absorption, this increase in the TBA value was suggested to be due to some reactions other than lipid peroxidation. Although tissue homogenates examined contained TBA-reacting materials, no lipid peroxidation seems to arise during incubation of them. No age-related alterations in the TBA value and oxygen absorption in rat tissue homogenates were observed. Vitamin E deficiency had no effect on the TBA values of cerebrum and lung homogenates, while it seemed to increase the TBA values of liver homogenates. Vitamin E deficiency had no effect on oxygen absorption in these tissue homogenates. The induction period of initiator-induced conjugated-diene formation in lipid extracts from liver and lung homogenates from normal and vitamin E-deficient rats tended to be extended with age. Vitamin E deficiency decreased the induction period of initiator-induced conjugated-diene formation. As a result, the length of the induction period was found to be proportional to vitamin E concentration in lipid extracts. The overall antioxidant capacity of rat tissues appears to be maintained without large variation during ageing. Decreases in the capacity of some antioxidant factors may be compensated by increases in the capacity of other factors.

Effects of 2,2'-azobis (2-amidinopropane) hydrochloride in vivo and protection by vitamin E.

2,2'-Azobis (2-amidinopropane) hydrochloride (AAPH), a compound that decomposes spontaneously to generate free radicals, was administered intraperitoneally to rats. High doses (greater than or equal to 70 mg/kg) were always fatal within a few hours. At nonlethal levels, AAPH was found to be absorbed into the circulation where it remained with a half-life of about 30 min. Lipid peroxidation was observed to occur in the liver and, to a much smaller extent, the kidney and heart of treated rats; levels of thiobarbituric acid-reactive substances were unchanged in the lung and brain, and significantly reduced in the plasma. Serum lipid levels were also lower in the AAPH-treated rats. Orally administered vitamin E, but not its water soluble analog, prevented the accumulation of TBA-reactive substances in the livers of AAPH-treated rats in a dose-dependent manner, but had no effect on mortality or the changes in serum lipid levels. The data suggest that intraperitoneally administered AAPH is absorbed into the circulation and can induce lipid peroxidation in vivo, but that toxicity may also arise through nonradical mechanisms. Furthermore, the free radical toxicity of AAPH in vivo may not be so general as previously suggested.

Comparative clinical trial of fenticonazole ovule (600 mg) versus clotrimazole vaginal tablet (500 mg) in the treatment of symptomatic vaginal candidiasis.

A multi-centre, randomized, single-blind, parallel-group clinical trial was undertaken in 50 patients (26 fenticonazole, 24 clotrimazole) with symptomatic vaginal candidiasis to compare the antifungal efficacy and tolerability of single-dose intra-vaginal treatment with a fenticonazole ovule (600 mg) or a clotrimazole vaginal tablet (500 mg). Assessment was by laboratory mycological investigation and symptomatic assessments for a period of 3 weeks from the day of treatment. Of the 50 patients, 43 (23 fenticonazole, 20 clotrimazole) returned for assessment 1 week after drug administration and 32 (17 fenticonazole, 15 clotrimazole) were re-assessed 3 weeks after drug administration. Both treatments resulted in very similar and highly significant improvements in symptoms, associated with disappearance of detectable Candida in approximately 70% of patients. There were no significant differences between treatments and no appreciable incidence of relapse during the 3-week period of observation. At the end of this period, 10 (59%) of 17 fenticonazole patients were totally disease-free, as compared with 10 (67%) of 15 patients after clotrimazole treatment. The cure rate observed was somewhat less than that previously seen when intra-vaginal cream formulations of the same two drugs were given on a multiple-dose basis. Both drugs were very well tolerated, with no reports of appreciable local or systemic adverse reactions to either drug.

The inactivation of alpha-1-proteinase inhibitor by gas-phase cigarette smoke: protection by antioxidants and reducing species.

The abilities of a number of compounds of biological interest to protect alpha-1-proteinase inhibitor (alPI) against the loss of elastase inhibitory capacity (EIC) resulting from exposure to gas-phase cigarette smoke have been tested. We have identified several species that protect AlPI. Amino acids prevent the loss of EIC in a manner that correlates with their pK alpha-values; only the unprotonated amine provides protection. Catalase partially prevents the loss of EIC, suggesting that hydrogen peroxide produced from the reduction of oxygen in cigarette smoke extracts is responsible for at least some of the smoke-induced inactivation. The best protection against smoke-induced loss of EIC was provided by two biologically important antioxidant species: glutathione and ascorbic acid. Both species provide almost complete protection to alPI under the experimental conditions used. The nature of species that protect AlPI against the inactivation caused by exposure to gas-phase smoke provides clues upon which speculations about the mechanism of this inactivation may be based. The identification of protective species could lead to the development of compounds that smokers could take (for example, vitamin C) that would protect their lung tissue against the oxidative damage caused by cigarette smoke.

Human alpha-1-proteinase inhibitor is inactivated by exposure to sidestream cigarette smoke.

Direct exposure of human alpha-1-proteinase inhibitor (alpha 1PI) to sidestream cigarette smoke causes an initial, rapid loss of elastase inhibitory capacity followed by a slow, gradual loss of activity as the protein is incubated in the smoke-exposed aqueous buffer solution. The exposure used here gives a total of 40% inactivation using one cigarette. This biphasic pattern of inactivation is similar to the inactivation seen following the direct exposure of alpha 1PI to mainstream smoke. We suggest that although exposures to sidestream smoke by nonsmokers are generally lower than the exposures to mainstream smoke experienced by smokers, sidestream smoke has the potential to produce similar types of damage as mainstream smoke, including emphysema-like damage, and should not be regarded as innocuous.

Mechanisms of cigarette smoke toxicity: the inactivation of human alpha-1-proteinase inhibitor by nitric oxide/isoprene mixtures in air.

A mixture of nitric oxide (NO) and isoprene in air has been studied as a model for gas-phase cigarette smoke. We have shown that this model system duplicates many of the properties of cigarette smoke including the inactivation of human alpha-1-proteinase inhibitor (a1PI). In this study, buffered solutions of a1PI were exposed to puffs of air containing 300 ppm NO and 400 ppm isoprene. Bubbling of the NO/air/isoprene gas stream directly through buffered protein solutions causes a1PI to undergo a fast loss of inhibitory capacity. This fast inactivation is not observed when a1PI is exposed to aqueous extracts of the NO/air/isoprene mixture. Both direct exposure and exposure to aqueous extracts, however, cause a1PI to undergo a slow loss of activity that continues for several days as the protein is incubated in the buffer solutions. Gas-phase cigarette smoke has already been shown to cause this same two-phase inactivation of a1PI. The inactivation of a1PI by the model system is dependent on the presence of oxygen in the gas stream, suggesting that the oxidation of nitric oxide to nitrogen dioxide in air is involved in the formation of the inactivating species. The nature of these species remains to be determined; however, small alkoxyl or peroxyl radicals (such as are spin-trappable from gas-phase smoke as well as from the NO/air/isoprene system) do not appear to inactivate a1PI. One possibility is that the inactivating species are metastable compounds formed by radical processes in the gas phase of both cigarette smoke and our model system. Our data suggest that one possible class of species is peroxynitrates.

Inactivation of human alpha 1-proteinase inhibitor by cigarette smoke: effect of smoke phase and buffer.

In order to resolve a discrepancy in the literature, we have examined the in vitro inactivation of human alpha 1-proteinase inhibitor by direct exposures either to whole cigarette smoke or to filtered (i.e., gas-phase) smoke. Wyss and coworkers (2) reported that whole smoke does not inactivate the protein, whereas we reported that gas-phase smoke does. We now find that direct exposure to gas-phase cigarette smoke causes a slightly greater inactivation of the protein than does direct exposure to whole cigarette smoke, confirming our earlier suggestion that whole smoke is less oxidizing than is gas-phase smoke. This difference, however, does not explain the dramatic difference between our previous findings and those of Wyss and coworkers (2). The explanation for the discrepancy lies in the nature of the buffers used. Wyss and coworkers used Tris buffer and the use of Tris quenches the inactivation process almost completely. Our experiments used phosphate buffer. We suggest that Tris is an unsuitable buffer for use in experiments that probe the effects of cigarette smoke.

Direct solid-phase enzyme immunoassay of progesterone in saliva.

We have developed a rapid enzyme immunoassay for progesterone in saliva. This solid-phase assay is carried out on microtitre plates with no extraction or centrifugation steps. The detection limit of the assay is 200 fg per well (3.2 pmol/L). Intra- and interassay coefficients of variation for low, medium, and high concentrations of progesterone were 7.5, 16.0; 9.1, 8.3; and 8.7, 6.7%, respectively. Correlation between total plasma progesterone (assayed by enzyme immunoassay with extraction) and salivary progesterone concentrations was good (r = 0.848, p less than 0.001, n = 56). We found the assay useful for monitoring ovarian function. The analytical procedure is convenient, and one person can assay more than 200 saliva samples per working day. The turnaround time for 36 samples is 2 h, including 1.5 h of incubation time, when previously coated plates are used. We conclude that such assays are very suitable for measuring progesterone in serial saliva samples and could become the preferred method for monitoring ovarian function.

Inactivation of human alpha-1-proteinase inhibitor by gas-phase cigarette smoke.

Direct exposure of human alpha-1-proteinase inhibitor to the gas-phase smoke from one cigarette results in an initial rapid loss of elastase inhibitory capacity, the amount of which is dependent upon the age of the smoke. This short-term inactivation is not seen when the protein is exposed to aqueous extracts of cigarette smoke (as had been done in the past). Both exposure regimens result in a slow inactivation occurring over several days. We suggest that the short-term inactivation may be due to a peroxynitrate (or a similar reactive species) that is formed from radicals in the gas phase but is unstable in aqueous solution.

Template-independent poly(A) x poly(U) synthesizing activity of different forms of Bacillus subtilis RNA polymerase.

Several, but not all, forms of bacillus subtilis RNA polymerase found in vegetative and sporulating cells can synthesize poly(A) x poly(U) in vitro. The vegetative delta-containing form of RNA polymerase (E delta) has little or no poly(A) x poly(U)-synthesizing activity, whereas RNA polymerase core (E) and sigma-containing core (E delta) both have significant activity. When purified vegetative delta factor was added to core, the core synthetic activity was reduced essentially to that of the vegetative enzyme E delta. When E sigma enzymes from vegetative and sporulating cells were compared for their salt sensitivity, it was found that the sporulation enzyme E sigma retained much more of its activity at 0.1 M KCl than the vegetative enzyme E sigma. Furthermore, when sporulation enzyme E delta 1 was compared with vegetative enzyme E sigma, it was found that the activity of the E sigma 1 form was much more resistant to high KCl concentrations than that of the vegetative E sigma form. These differences in enzyme activity, as affected by salt concentrations, suggest that the conformations of the sporulation E sigma and E delta 1 enzymes are different from that found in vegetative E sigma enzyme. These differences in conformation may be involved in selective gene expression during sporularion.

A mutation to 5-methyltryptophan dependence in the tryptophan (trp) operon of Salmonella typhimurium. II. Studies of 5-methyltryptophan-dependent mutants and their revertants.

Mutants of S. typhimurium with a defect in the first structural gene of the trp operon can utilize anthranilic acid (AA) as a growth factor. Among a group of 5-methyltryptophan (MT) resistant derivatives of trpA mutants we encountered several with a novel phenotype: they actually grew better in the presence of MT than in its absence. Normally MT inhibits growth of S. typhimurium at the concentration we employed due to its ability to act as co-repressor of the trp operon and as a feedback inhibitor of anthranilate synthetase (AS) the first enzyme for tryptophan biosynthesis. Mutations to MT-dependence were only found in strains carrying extremely polar trpA mutations. In all cases analyzed, mutations causing MT-dependence mapped at the extreme operator distal end of trpA. The mutation trpA515 responsible for MT-dependence in strain SO61 (genotype trpA49trpA515) was recombined away from the polar mutation. The strain thus obtained, SO495 was totally dependent on MT for growth on AA supplement. Strain SO495 lacks AS and under repressing growth conditions synthesizes the trp enzymes constitutively at 2--3 times the basal level. Under derepression, while the levels of the distal enzymes, as represented by tryptophan synthetase--beta subunit (TSbeta), did not increase there was a marked drop in the activity of anthranilate-PRPP phosphoribosyltransferase, (PRT) the enzyme catalyzing the second step of tryptophan biosynthesis. trpA515 was found to revert to prototrophy at a low frequency (about 10(-8)) which was not increased by chemical mutagens or ultraviolet radiation. In contrast, it was found to revert to MT-independence (growth on AA in the absence of MT) at a fairly high spontaneous frequency (about 10(-6)) and this frequency could be increased approximately tenfold by mutagens causing base substitutions or deletions but not by frameshift mutagens. About one hundred MT-independent revertants of trpA515 were mapped and found to fall into three general classes: (A) mutations at or near the trpA515 site (B) secondary mutations located upstream from trpA515, (C) deletions of various sizes. Based on a detailed genetic and physiological study of twelve representative MT-independent revertants, it appears that trpA515 may be caused by the insertion of a piece of DNA with some of the properties described for the IS elements found in Escherichia coli. The trpA515 insertion should contain (in this order), a transcription terminator, a low efficiency promoter and, probably, a translation start signal.