The structure and activity of two cytochrome P450c21 proteins encoded in the ovine adrenal cortex.

The steroidogenic enzyme cytochrome P450c21 (CYP21A1) is synthesized in the adrenal cortex and is essential for cortisol and aldosterone production. We have studied the structure and activity of ovine P450c21 proteins by analysis and expression of the corresponding cDNAs. Two P450c21 mRNAs (2.2 and 1.7 kilobases) were detected in ovine adrenal RNA and corresponded to two types of P450c21 cDNA clones that differed in their 3' region. One clone encoded a protein similar in structure to bovine, murine, and human P450c21 proteins. The other clone contained a 3' deletion of about 500 nucleotides and encoded a P450c21 protein that was truncated by 18 residues at the carboxyl terminus. The boundaries of this deletion suggested that an additional splicing event was responsible for the shortened mRNA sequence. Detailed Southern analysis of ovine genomic DNA indicates that the two mRNAs are derived from one gene even though two P450c21 genes are present in the ovine genome. The activities of the two P450c21 proteins were determined by expressing the respective cDNA clones in COS cells. The complete P450c21 protein was an efficient catalyst of 21-hydroxylation reactions, whereas no 21-hydroxylation activity was detected in cells containing the P450c21 protein with the carboxyl-terminal deletion.     

Arctic shrub colonization lagged peak postglacial warmth: Molecular evidence in lake sediment from Arctic Canada

Arctic shrubification is an observable consequence of climate change, already resulting in ecological shifts and global‐scale climate feedbacks including changes in land surface albedo and enhanced evapotranspiration. However, the rate at which shrubs can colonize previously glaciated terrain in a warming world is largely unknown. Reconstructions of past vegetation dynamics in conjunction with climate records can provide critical insights into shrubification rates and controls on plant migration, but paleoenvironmental reconstructions based on pollen may be biased by the influx of exotic pollen to tundra settings. Here, we reconstruct past plant communities using sedimentary ancient DNA (sedaDNA), which has a more local source area than pollen. We additionally reconstruct past temperature variability using bacterial cell membrane lipids (branched glycerol dialkyl glycerol tetraethers) and an aquatic productivity indicator (biogenic silica) to evaluate the relative timing of postglacial ecological and climate changes at a lake on southern Baffin Island, Arctic Canada. The sedaDNA record tightly constrains the colonization of dwarf birch (Betula, a thermophilous shrub) to 5.9 ± 0.1 ka, ~3 ka after local deglaciation as determined by cosmogenic ¹⁰Be moraine dating and >2 ka later than Betula pollen is recorded in nearby lake sediment. We then assess the paleovegetation history within the context of summer temperature and find that paleotemperatures were highest prior to 6.3 ka, followed by cooling in the centuries preceding Betula establishment. Together, these molecular proxies reveal that Betula colonization lagged peak summer temperatures, suggesting that inefficient dispersal, rather than climate, may have limited Arctic shrub migration in this region. In addition, these data suggest that pollen‐based climate reconstructions from high latitudes, which rely heavily on the presence and abundance of pollen from thermophilous taxa like Betula, can be compromised by both exotic pollen fluxes and vegetation migration lags.     

Site-specific mutations in human ferredoxin that affect binding to ferredoxin reductase and cytochrome P450scc

Ferredoxins found in animal mitochondria function in electron transfer from NADPH-dependent ferredoxin reductase (Fd-reductase) to cytochrome P450 enzymes. To identify residues involved in binding of human ferredoxin to its electron transfer partners, neutral amino acids were introduced in a highly conserved acidic region (positions 68-86) by site-directed mutagenesis of the cDNA. Mutant ferredoxins were produced in Escherichia coli, and separate assays were used to determine the effect of substitutions on the capacity of each mutant to bind to Fd-reductase and cytochrome P450scc and to participate in the cholesterol side chain cleavage reaction. Replacements at several positions (mutants D68A, E74Q, and D86A) did not significantly affect activity, suggesting that acidic residues at these positions are not required for binding or electron transfer interactions. In contrast, substitutions at positions 76 and 79 (D76N and D79A) caused dramatic decreases in activity and in the affinity of ferredoxin for both Fd-reductase and P450scc; this suggests that the binding sites on ferredoxin for its redox partners overlap. Other substitutions (mutants D72A, D72N, E73A, E73Q, and D79N), however, caused differential effects on binding to Fd-reductase and P450scc, suggesting that the interaction sites are not identical. We propose a model in which Fd-reductase and P450scc share a requirement for ferredoxin residues Asp-76 and Asp-79 but have other determinants that differ and play an important role in binding. This model is consistent with the hypothesis that ferredoxin functions as a mobile shuttle in steroidogenic electron transfer, and it is considered unlikely that a functional ternary complex is formed.     

A modified and automated version of the 'Fluorimetric Detection of Alkaline DNA Unwinding' method to quantify formation and repair of DNA strand breaks

Background  

Formation and repair of DNA single-strand breaks are important parameters in the assessment of DNA damage and repair occurring in live cells. The 'Fluorimetric Detection of Alkaline DNA Unwinding (FADU)' method [Birnboim HC, Jevcak JJ. Cancer Res (1981) 41:1889–1892] is a sensitive procedure to quantify DNA strand breaks, yet it is very tedious to perform.     

The effects of intracerebroventricular administration of biologically active peptides in conscious sheep

The present study records the physiological effects of 24-hour intracerebroventricular infusion of a variety of biologically active peptides in conscious sheep. A number of peptides including AVP and TRH produced increases in mean arterial pressure, heart rate and body temperature. There was an overall positive correlation between peptide-induced changes in body temperature and changes in either mean arterial pressure or heart rate. TRH and β-endorphin had marked effects on behaviour and several peptides reduced food and water intake. Several peptides increased urinary sodium excretion, however, few peptides changed plasma electrolyte concentrations. TRH produced small effects on plasma ACTH and plasma glucose concentrations. The peptides in this study produced physiological changes which were probably mediated by their actions on the central nervous system.     

Linkage between the Colton blood group locus and ASSP11 on chromosome 7

In an attempt to assign the Colton blood group locus (CO) we have successfully revisited chromosome 7. CO is linked to the argininosuccinate synthetase pseudogene 11 locus (ASSP11) with z = 5.79 at theta = 0.07 for combined paternal and maternal meioses. We propose a 7p position for CO.