Quantitative electrophoretic transfer of DNA from polyacrylamide or agarose gels to nitrocellulose

A method for efficient electrophoretic transfer of DNA fragments from polyacrylamide gels to nitrocellulose sheets was developed. Hybridization to these fragments can be performed by standard techniques. The method is also applicable to agarose gels, allowing this transfer method to be used for DNA ranging from 40 to at least 23,000 bp.     

Nucleosome arrangement in alpha-satellite chromatin of African green monkey cells.

By analyzing the accessibility of restriction endonuclease sites in African green monkey alpha-satellite chromatin, we demonstrate the absence of a unique phase relationship between nucleosomes and alpha-satellite DNA. The data indicate a minimum of three different positions for nucleosome cores relative to the alpha-satellite sequence and suggest a random distribution in at least some regions. In addition, while we confirm published reports that staphylococcal nuclease cuts the alpha-satellite sequence in chromatin at a highly preferred site, two-dimensional gel electrophoresis of nuclear digests demonstrates that this site is preferentially cut by staphylococcal nuclease even when it is within the nucleosome core. These data indicate that staphylococcal nuclease is not useful for determining nucleosome positions on alpha-satellite DNA, and perhaps on other specific DNA sequences as well.     

Effects of 2.45-GHz microwave radiation on embryonic quail hearts

Although exposure to nonionizing electromagnetic radiation has been reported to cause a variety of systemic alterations during embryonic development, there are few reports of the induction of specific physiologic or morphologic changes in the myocardium. This study was designed to examine the effects of microwave radiation on cardiogenesis in Japanese quail embryos exposed during the first eight days of development to 2.45-GHz continuous-wave microwaves at power densities of 5 or 20 mW/cm². The specific absorption rates were 4.0 and 16.2 mW/g, respectively. The ambient temperature for each exposure was set to maintain the embryonated eggs at 37.5 °C. This did not preclude thermal gradients in the irradiated embryos since microwaves may not be uniformly absorbed. The test exposure levels did not induce changes in either the morphology of the embryonic heart or the ultrastructure of the myocardial cells. Analysis of the enzymatic activities of lactate dehydrogenase, glutamic oxaloacetic transaminase, and creatine phosphokinase failed to reveal any statistically significant differences between the nonexposed controls and those groups exposed to either 5 or 20 mW/cm². The data indicate that 2.45-GHz microwave radiation at 5 or 20 mW/cm² has no effect on the measured variables of the Japanese quail myocardium exposed during the first eight days of development.     

Characterization of the lipoidal derivatives of pregnenolone prepared by incubation of the steroid with adrenal mitochondria.

Using mass spectrometric, radioisotopic, chromatographic and chemical techniques, five fatty acid esters of 3 beta-hydroxy-5-pregnen-20-one (pregnenolone) have been identified as components of the lipoidal derivatives biosynthesized in vitro with bovine adrenal mitochondria. The five compounds are: pregnenolone arachidonate, pregnenolone linoleate, pregnenolone oleate, pregnenolone palmitate, and pregnenolone stearate. The distribution of the fatty acids among these five esters is different from the previously reported (Cmelik, S.H.W., and Ley, H. (1977) Comp. Biochem. Physiol. 56B, 267-270) fatty acid composition of these organelles.     

Patterns of ehtylene production in senescing leaves

Changes in the patterns of ethylene production, chlorophyll content, and respiration were studied in relation to the senescence of intact leaves and leaf discs. The primary leaves of pinto bean, which abscise readily during natural senescence, and tobacco and sugar beet leaves, which do not abscise, were used. A decrease in the rate of ethylene production and respiration, during the slow phase of chlorophyll degradation, was observed in leaf-blade discs cut from mature leaves and aged in the dark. During rapid chlorophyll loss both ethylene production and respiration increased and then decreased. These climacteric-like patterns were shown by leaf discs of all three species. Discs taken from leaves that had been senescing on the plant also showed a climacteric-like rise in ethylene production but not in respiration, which decreased continuously with leaf age. Climacteric-like patterns in the rise of ethylene and respiration for leaf discs were also shown by the petioles of both bean and tobacco leaves. This indicates that the rise of ethylene and respiration is characteristic of the general process of senescence in leaves and is not restricted to the abscission process. In contrast to the ethylene-forming systems in climacteric fruits and many flowers, the one in leaves declines sharply in the early stages of senescence. The subsequent rise of ethylene production appears to be associated with the rapid phase of chlorophyll breakdown, and may indicate the final stage of the senescence process during which ethylene could be actively involved in inducing leaf abscission.     

Ethylene as a regulator of senescence in tobacco leaf discs

The regulatory role of ethylene in leaf senescence was studied with excised tobacco leaf discs which were allowed to senesce in darkness. Exogenous ethylene, applied during the first 24 hours of senescence, enhanced chlorophyll loss without accelerating the climacteric-like pattern of rise in both ethylene and CO₂, which occurred in the advanced stage of leaf senescence. Rates of both ethylene and CO₂ evolution increased in the ethylene-treated leaf discs, especially during the first 3 days of senescence. The rhizobitoxine analog, aminoethoxy vinyl glycine, markedly inhibited ethylene production and reduced respiration and chlorophyll loss. Pretreatment of leaf discs with Ag⁺ or enrichment of the atmosphere with 5 to 10% CO₂ reduced chlorophyll loss, reduced rate of respiration, and delayed the climacteric-like rise in both ethylene and respiration. Ag⁺ was much more effective than CO₂ in retarding leaf senescence. Despite their senescence-retarding effect, Ag⁺ and CO₂, which are known to block ethylene action, stimulated ethylene production by the leaf discs during the first 3 days of the senescing period; Ag⁺ was more effective than CO₂. The results suggest that although ethylene production decreases prior to the climacteric-like rise during the later stages of senescence, endogenous ethylene plays a considerable role throughout the senescence process, presumably by interacting with other hormones participating in leaf senescence.     

Production and action of ethylene in senescing leaf discs: effect of indoleacetic Acid, kinetin, silver ion, and carbon dioxide

Supraoptimal concentrations of indoleacetic acid (IAA) stimulated ethylene production, which in turn appeared to oppose the senescence-retarding effect of IAA in tobacco leaf discs. Kinetin acted synergistically with IAA in stimulating ethylene production, but it inhibited senescence. Silver ion and CO(2), which are believed to block ethylene binding to its receptor sites, delayed senescence in terms of chlorophyll loss and stimulated ethylene production. Both effects of Ag(+) were considerably greater than those of CO(2). IAA, kinetin, CO(2), and Ag(+), combined, acted to increase ethylene production further. Although this combination increased ethylene production about 160-fold over that of the control, it inhibited senescence. Treatment with 25 mul/l of ethylene in the presence of IAA enhanced chlorophyll loss in leaf discs and inhibited by about 90% the conversion of l-[3,4-(14)C] methionine to (14)C(2)H(4) suggesting autoinhibition of ethylene production.The results suggest that ethylene biosynthesis in leaves is controlled by hormones, especially auxin, and possibly the rate of ethylene production depends, via a feedback control system, on the rates of ethylene binding at its receptor sites.     

Influence of Ethylene on Indole-3-acetic Acid Concentration in Etiolated Pea Epicotyl Tissue

Indole-3-acetic acid levels are diminished about 50% in 5- to 6-day-old epicotyls of etiolated pea (Pisum sativum L.) seedlings treated with 10 to 36μl/l ethylene for 18 to 24 hr.     

Influence of Plant Hormones on Ethylene Production in Apple, Tomato, and Avocado Slices during Maturation and Senescence

Ethylene production by tissue slices from preclimacteric, climacteric, and postclimacteric apples was significantly reduced by isopentenyl adenosine (IPA), and by mixtures of IPA and indoleacetic acid, and of IPA, indoleacetic acid, and gibberellic acid after 4 hours of incubation. Ethylene production by apple (Pyrus malus L.) slices in abscisic acid was increased in preclimacteric tissues, decreased in climacteric peak tissues, and little affected in postclimacteric tissues. Indoleacetic acid suppressed ethylene production in tissues from preclimacteric apples but stimulated ethylene production in late climacteric rise, climacteric, and postclimacteric tissue slices. Gibberellic acid had less influence in suppressing ethylene production in preclimacteric peak tissue, and little influenced the production in late climacteric rise, climacteric peak, and postclimacteric tissues. IPA also suppressed ethylene production in pre- and postclimacteric tissue of tomatoes (Lycopersicon esculentum) and avocados (Persea gratissima). If ethylene production in tissue slices of ripening fruits is an index of aging, then IPA would appear to retard aging in ripening fruit, just as other cytokinins appear to retard aging in senescent leaf tissue.     

Maternal and neonatal disposition of pethidine in childbirth--a study using quantitative gas chromatography-mass spectrometry

An assay for pethidine using gas chromatography-mass spectrometry with single ion monitoring has been developed for its analysis in the blood of neonates whose mothers received pethidine during childbirth. Concentrations were determined from the height of the peak at m/e 218 derived from pethidine compared with that at m/e 234 derived from the internal standard lignocaine. Results from 10 mothers and their babies show the time-related passage of pethidine across the placenta. The elimination half-life of the drug from neonatal blood was 22.7h compared with 3h in the adult.