A method for flow cytometric cell cycle analysis of normal and psoriatic human epidermis based on a detergent/citric acid technique for suspension of nuclei

A new method is described for flow cytometric cell cycle analysis of normal and psoriatic human epidermis, based on non-enzymatic tissue disaggregation. The epidermis was isolated by treatment with acetic acid and stored by freezing. After thawing, the epidermis was disintegrated into a nuclear suspension by 3 steps: incubation with dithiotreitol, whirling in a buffer (pH 7.4) with the non-ionic detergent Nonidet P40, EGTA, RNase and spermine, and whirling after addition of citric acid to a final concentration of 1% (pH 2.4). The suspension was stained with propidium iodide and filtered before flow cytometry. The yield of suspended nuclei was approximately 70% of the original number of cells in the tissue. The detergent/citric acid method was found to be preferable to an ultrasonication method previously used on human epidermis. All cell cycle and cell maturation stages were represented in the detergent/citric acid suspension, in contrast to the selection of immature G1, S and G2 stages with enzymatic methods. In the analysis of psoriatic epidermis inadequately matured (parakeratotic) cells were present in the suspension and had to be discriminated by gating on light scattering intensity, as they were not susceptible to lysis and did not stain properly. The fraction of S phase nuclei was on average 1.9% in normal and 7.7% in psoriatic epidermis, thus confirming the results of other investigators using enzymes. The presence of mitotic figures in the suspension was demonstrated by flow sorting. In this way the mitotic fraction was estimated to 0.06% in normal and 0.22% in psoriatic epidermis, confirming histological data of other investigators.     

The catalytic activities of DNA topoisomerase II are most closely associated with the DNA cleavage/religation steps.

DNA topoisomerase II regulates the three-dimensional organisation of DNA and is the principal target of many important anticancer and antimicrobial agents. These drugs usually act on the DNA cleavage/religation steps of the catalytic cycle resulting in accumulation of covalent DNA-topoisomerase II complexes. We have studied the different steps of the catalytic cycle as a function of salt concentration, which is a classical way to evaluate the biochemical properties of proteins. The results show that the catalytic activity of topoisomerase II follows a bell-shaped curve with optimum between 100 and 225 mM KCl. No straight-forward correlation exists between DNA binding and catalytic activity. The highest levels of drug-induced covalent DNA-topoisomerase II complexes are observed between 100 and 150 mM KCl. Remarkably, at salt concentrations between 150 mM and 225 mM KCl, topoisomerase II is converted into a drug-resistant form with greatly reduced levels of drug-induced DNA-topoisomerase II complexes. This is due to efficient religation rather than to absence of DNA cleavage as witnessed by relaxation of the supercoiled DNA substrate. In the absence of DNA, ATP hydrolysis is strongest at low salt concentrations. Unexpectedly, the addition of DNA stimulates ATP hydrolysis at 100 and 150 mM KCl, but has little or no effect below 100 mM KCl in spite of strong non-covalent DNA binding at these salt concentrations. Therefore, DNA-stimulated ATP hydrolysis appears to be associated with covalent rather than non-covalent binding of DNA to topoisomerase II. Taken together, the results suggest that it is the DNA cleavage/religation steps that are most closely associated with the catalytic activities of topoisomerase II providing a unifying theme for the biological and pharmacological modulation of this enzyme.     

Assessing Greater Sage-Grouse Selection of Brood-Rearing Habitat Using Remotely-Sensed Imagery: Can Readily Available High-Resolution Imagery Be Used to Identify Brood-Rearing Habitat Across a Broad Landscape?

Greater sage-grouse populations have decreased steadily since European settlement in western North America. Reduced availability of brood-rearing habitat has been identified as a limiting factor for many populations. We used radio-telemetry to acquire locations of sage-grouse broods from 1998 to 2012 in Strawberry Valley, Utah. Using these locations and remotely-sensed NAIP (National Agricultural Imagery Program) imagery, we 1) determined which characteristics of brood-rearing habitat could be used in widely available, high resolution imagery 2) assessed the spatial extent at which sage-grouse selected brood-rearing habitat, and 3) created a predictive habitat model to identify areas of preferred brood-rearing habitat. We used AIC model selection to evaluate support for a list of variables derived from remotely-sensed imagery. We examined the relationship of these explanatory variables at three spatial extents (45, 200, and 795 meter radii). Our top model included 10 variables (percent shrub, percent grass, percent tree, percent paved road, percent riparian, meters of sage/tree edge, meters of riparian/tree edge, distance to tree, distance to transmission lines, and distance to permanent structures). Variables from each spatial extent were represented in our top model with the majority being associated with the larger (795 meter) spatial extent. When applied to our study area, our top model predicted 75% of naïve brood locations suggesting reasonable success using this method and widely available NAIP imagery. We encourage application of our methodology to other sage-grouse populations and species of conservation concern.     

Seasonal and annual variability in the phytoplankton community of the Raunefjord,west coast of Norway from 2001–2006

Changes in phytoplankton community composition potentially affect the entire marine food web. Because of seasonal cycles and inter-annual variations in species composition, long-term monitoring, covering many sequential years, is required to establish a baseline study and to reveal long-term trends. The current study describes the phytoplankton biomass variations and species composition in relation to hydrographic and meteorological conditions in the Raunefjord, western Norway, over a 6-year period from 2001 to 2006. The extent of inflow or upwelling in the fjord varied from year to year and resulted in pronounced differences in water column stability. The annual phytoplankton community succession showed some repeated seasonal patterns, but also high variability between years. Two to four diatom blooms were observed per year, and the spring blooms occurring before water column stratification in March were dominated by Skeletonema marinoi and Chaetoceros socialis, and other Chaetoceros and Thalassiosira spp. Blooms of the haptophytes Phaeocystis pouchetii and Emiliania huxleyi were irregular and in some years totally absent. Although E. huxleyi was present all year round it appeared in bloom concentrations only in 2003, when the summer was warm and the water column characterized by high surface temperatures and pronounced stratification. The annual average abundance of both diatoms and flagellates increased during the six years. Despite the high variation from year to year, our investigation provides valuable knowledge about annual phytoplankton community patterns in the region, and can be used as a reference to detect possible future changes.     

Mutagenic activity of nine N,N-disubstituted hydrazines in the Salmonella/mammalian microsome assay.

The mutagenic activity of N,N-dimethyl-, N,N-diethyl-, N,N-dibutyl-, N,N-diisobutyl-, N,N-di(p-tolyl)-, N-ethyl-N-phenyl-, N,N-dibenzyl-, N,N-diphenyl- and N,N-diisopropylhydrazine was examined in the Salmonella/mammalian microsome assay using the strains TA1535, TA1537, TA97, TA98, TA100, TA102 and TA1530. All nine hydrazines were mutagenic in at least one tester strain, although of borderline significance for some of the compounds. The mutagenic potencies of the hydrazines varied 2-3 orders of magnitude, from very weak to moderate mutagenic activity. In general, the addition of S9 resulted in a lowering of the mutagenic activity and a lowering of the toxic properties of the hydrazines. The test results were relatively difficult to evaluate due to toxic effects of many of the test compounds on the test bacteria which may have resulted in an underestimation of the mutagenic potencies of some of the compounds. The pattern of mutagenic activity of the hydrazines in the different tester strains indicates that more than one mechanism of action may be involved in the mutagenicity.     

Isolation of isoflavones from soy-based fermentations of the erythromycin-producing bacterium Saccharopolyspora erythraea

A search for an abundant and economical source of isoflavones, particularly genistein, led to the discovery that the erythromycin-producing organism Saccharopolyspora erythraea also produces this promising new cancer-prevention agent. Erythromycin fermentation is a large-scale, soybean-based process used world-wide for the commercial production of this medically important antibiotic. Results from this study indicate that genistin (the glucoside form of genistein), which is added to the fermentation in the soybean media, was converted to genistein through the action of a β-glucosidase produced by the organism. Genistein was co-extracted with erythromycin from the fermentation broth, then separated from erythromycin during the second step of the purification process for the production of erythromycin. Received 10 September 1996 / Received revision: 22 November 1996 / Accepted: 7 December 1996     

Selenocysteine confers the biochemical properties characteristic of the type I iodothyronine deiodinase.

The conversion of thyroxine to 3,5,3'-triiodothyronine (T3) is the first step in thyroid hormone action, and the Type I iodothyronine deiodinase supplies most of this extrathyroidal T3 in the rat. We found that the cDNA coding for this enzyme contains an in-frame UGA encoding the rare amino acid selenocysteine. Using site-directed mutagenesis, we have converted selenocysteine to cysteine and expressed the wild-type and cysteine mutant enzymes in JEG-3 cells by transient transfection. The kinetic properties of the transiently expressed wild-type enzyme are nearly identical to those reported for rat liver Type I deiodinase. Substitution of sulfur for selenium causes a 10-fold increase in the Km of the enzyme for the favored substrate 3,3',5'-triiodothyronine (rT3), a 100-fold decrease in the sensitivity of rT3 deiodination to competitive inhibition by gold and a 300-fold increase in the apparent Ki for uncompetitive inhibition by 6-n-propylthiouracil. These results demonstrate that selenium is responsible for the biochemical properties which characterize Type I iodothyronine monodeiodination.