Binding sites for atrial natriuretic factor (ANF) in kidneys and adrenal glands of spontaneously hypertensive (SHR) rats

Binding sites for atrial natriuretic factor (ANF) were studied in kidneys and adrenal glands of 17 week old male spontaneously hypertensive (SHR) and Wistar-Kyoto (WKY) normotensive rats by quantitative autoradiography using 125I-ANF-28. In kidney, 125I-ANF-28 binding sites were found in high concentrations in glomeruli and in much lower concentrations in the renal papilla. In adrenal gland, 125I-ANF-28 binding sites were highly localized to the zona glomerulosa and were of moderate density in the inner cortical regions. ANF binding sites did not occur in the adrenal medulla. The maximum binding capacity (Bmax) of 125I-ANF-28 was reduced by 50% in the kidney glomeruli of SHRs compared to WKY controls. In contrast, the affinity constant (Ka) for 125I-ANF-28 was elevated by 100% in kidney glomeruli of SHRs. There were no significant strain differences in values for Bmax or Ka for 125I-ANF-28 binding in the adrenal zona glomerulosa. These findings suggest that the natriuretic and diuretic actions of ANF within kidney glomeruli may be compromised in adult SHR rats and these alterations may contribute to the development and maintenance of hypertension in rats of this strain.     

Molecular analysis of high-copy insertion sites in maize

High-copy transposon mutagenesis is an effective tool for creating gene disruptions in maize. In order to molecularly define transposon-induced disruptions on a genome-wide scale, we optimized TAIL-PCR to amplify genomic DNA flanking maize Robertson’s Mutator insertions. Sample sequencing from 43 Mutator stocks and the W22 inbred line identified 676 non-redundant insertions, and only a small fraction of the flanking sequences showed significant similarity to maize repetitive sequences. We further designed and tested 79 arbitrary primers to identify 12 primers that amplify all Mutator insertions within a DNA sample at 3.1-fold redundancy. Importantly, the products are of sufficient size to use as substrates or probes for hybridization-based identification of gene disruptions. Our adaptation simplifies previously published TAIL-PCR protocols and should be transferable to other high-copy insertional mutagens.     

A cryptic invasion within an invasion and widespread introgression in the European water frog complex: consequences of uncontrolled commercial trade and weak international legislation

In Western Europe, many pond owners introduce amphibians for ornamental purposes. Although indigenous amphibians are legally protected in most European countries, retailers are circumventing national and international legislation by selling exotic nonprotected sibling species. We investigated to what extent non‐native species of the European water frog complex (genus Pelophylax) have become established in Belgium, using morphological, mitochondrial and nuclear genetic markers. A survey of 87 sampling sites showed the presence of non‐native water frogs at 47 locations, mostly Marsh frogs (Pelophylax ridibundus). Surprisingly, at least 19% of all these locations also harboured individuals with mitochondrial haplotypes characteristic of Anatolian water frogs (Pelophylax cf. bedriagae). Nuclear genotyping indicated widespread hybridization and introgression between P. ridibundus and P. cf. bedriagae. In addition, water frogs of Turkish origin obtained through a licensed retailer, also contained P. ridibundus and P. cf. bedriagae, with identical haplotypes to the wild Belgian populations. Although P. ridibundus might have invaded Belgium by natural range expansion from neighbouring countries, our results suggest that its invasion was at least partly enhanced by commercial trade, with origins as far as the Middle East. Also the invasion and rapid spread of Anatolian lineages, masked by their high morphological similarity to P. ridibundus, is likely the result of unregulated commercial trade. We expect that Anatolian frogs will further invade the exotic as well as the native range of P. ridibundus and other Pelophylax species elsewhere in Western and Central Europe, with risks of large‐scale hybridization and introgression.     

Chromatographic purification of the chloroplast ATP synthase (CF0-CF1) and the role of CF0 subunit IV in proton conduction

Chromatographic procedures were developed to purify chloroplast ATP synthase (CF0-CF1) in large amounts and to resolve subunits from this enzyme. The ATP synthase thus obtained has high ATP-Pi exchange and Mg2(+)-ATPase activities upon incorporation into asolectin liposomes. The purity of this preparation was about 95%. By modifications of this chromatographic procedure, we purified subunit IV-deficient CF0-CF1, subunit IV-deficient CF0, and subunit IV. Both ATP-Pi exchange and Mg2(+)-ATPase activities were impaired by depletion of subunit IV from CF0-CF1. Partial restoration of these activities was obtained by reconstituting subunit IV-deficient CF0-CF1 with subunit IV. The impairment of these activities was likely caused by a loss in proton conductivity of CF0 upon removal of subunit IV. The dicyclohexylcarbodiimide-sensitive Mg2(+)-ATPase of subunit IV-deficient CF0-CF1 was not as sensitive to the depletion of subunit IV as ATP-Pi exchange. Nearly 90% of subunit IV could be removed, but Mg2(+)-ATPase activity was inhibited by only 40-60%. Thus subunit IV of CF0-CF1 may not participate directly in proton transfer but may have a role in organizing and/or stabilizing CF0 structure.     

Mitogenesis induced by calcium-containing crystals. Role of intracellular dissolution

Hydroxyapatite, like other calcium-containing crystals previously studied by us, is mitogenic for cultured human fibroblasts. This mitogenic effect is not a result of increased ambient calcium concentration due to extracellular crystal dissolution. Synthetic crystals labelled uniformly with calcium 45 (45Ca) undergo endocytosis when incubated with cells and are solubilized. Such solubilization is inhibited by chloroquine or ammonium chloride in concentrations that significantly block the mitogenic effect of crystals but not that of serum. The data suggest that mitogenesis and intracellular crystal dissolution are related phenomena.     

Ethylene dibromide transformation under methanogenic conditions

Ethylene dibromide present at a low concentration (less than 100 micrograms/liter) was transformed by reductive dehalogenation under methanogenic conditions in batch bacterial cultures and in a continuous-flow, methanogenic, fixed-film, laboratory-scale column.     

Measurement of proton-linked transport activities in pyranine loaded chloroplast inner envelope vesicles

A method has been devised for loading chloroplast inner envelope vesicles prepared from pea (Pisum sativum L. var Progress No. 9) or spinach (Spinacia oleracea L.) with 8-hydroxypyrene-1,3,6-trisulfonate (pyranine), a membrane impermeant, fluorescent pH indicator. Two known proton-linked transport activities of the inner envelope, glycolate/H⁺ co-transport and phosphate/phosphoglycerate exchange have been shown to cause quenching of the internal pyranine fluorescence. This represents the first demonstration that these vesicles are sealed and competent for transport measurements. The technique, as it now stands, is essentially qualitative. It does, however, offer advantages over transport measurements with intact chloroplasts, for example compatibility with rapid mixing techniques and accessibility of the transport proteins to antibodies.     

Effects of toxicity, aeration, and reductant supply on trichloroethylene transformation by a mixed methanotrophic culture.

The trichloroethylene (TCE) transformation rate and capacity of a mixed methanotrophic culture at room temperature were measured to determine the effects of time without methane (resting), use of an alternative energy source (formate), aeration, and toxicity of TCE and its transformation products. The initial specific TCE transformation rate of resting cells was 0.6 mg of TCE per mg of cells per day, and they had a finite TCE transformation capacity of 0.036 mg of TCE per mg of cells. Formate addition resulted in increased initial specific TCE transformation rates (2.1 mg/mg of cells per day) and elevated transformation capacity (0.073 mg of TCE per mg of cells). Significant declines in methane conversion rates following exposure to TCE were observed for both resting and formate-fed cells, suggesting toxic effects caused by TCE or its transformation products. TCE transformation and methane consumption rates of resting cells decreased with time much more rapidly when cells were shaken and aerated than when they remained dormant, suggesting that the transformation ability of methanotrophs is best preserved by storage under anoxic conditions.     

Nitrite Transport in Chloroplast Inner Envelope Vesicles (I. Direct Measurement of Proton-Linked Transport)

Chloroplast inner envelope membrane vesicles that are loaded with the pH-sensitive fluorophore, pyranine, show rapid internal acidification when nitrite is added. Acidification is dependent upon [delta]pH, with the inside of vesicles being alkaline with respect to the outside. The rate of vesicle acidification was directly proportional to the concentration of nitrite that was added and the imposed pH difference across the membrane. In contrast, added nitrate had no effect on vesicle acidification. Nitrite also caused acidification of asolectin vesicles. The extent of vesicle acidification is dependent on the internal volume of vesicles. Inner envelope and asolectin vesicles that were prepared by extrusion were approximately the same size, allowing them to be compared when the final extent of acidification, measured after the pH gradient had collapsed, was similar. The rate of nitrite-dependent acidification was similar in these two preparations at any single nitrite concentration. These results indicate that nitrite movement occurs by rapid diffusion across membranes as nitrous acid, and this movement is dependent on a proton gradient across the lipid bilayer. Under conditions approximating those in vivo, the rate of diffusion of nitrous acid far exceeds that of nitrite reduction within chloroplasts.