Stability and sequence-specific DNA binding of activation-labile mutants of the human glucocorticoid receptor

The stability and DNA-binding properties of activation-labile (act1) human glucocorticoid receptors (hGRs) from the glucocorticoid-resistant mutant 3R7.6TG.4 were investigated. These receptors are able to bind reversibly associating ligands with normal affinity and specificity, but become unstable during attempted activation to the DNA binding form [Harmon et al. (1984) J. Steroid Biochem. 21, 227-236]. Affinity labeling and immunochemical analysis demonstrated that act1 receptors are not preferentially proteolyzed during attempted activation. In addition, analysis of binding to calf thymus DNA showed that after loss of ligand, act1 receptors retain the ability to bind to DNA nonspecifically. A 370 bp MMTV promoter fragment containing multiple GREs and an upstream 342 bp fragment lacking GRE sequences were used to assess the binding of act1 hGR to specific DNA sequences. Immunoadsorption of hGR-DNA complexes after incubation with 32P-end-labeled fragments showed that both normal and act1 hGR bound selectively to the GRE-containing fragment in an activation-dependent manner. Binding of both normal and act1 hGRs could be blocked with a synthetic oligonucleotide containing a perfect palindromic GRE, but not with an oligonucleotide in which the GRE was replaced by an ERE. Analogous results were obtained for normal and act1 hGR activated in the absence of ligand, or after incubation with the glucocorticoid antagonist RU 38486. These results suggest that sequence-specific binding of the hGR does not require the presence of bound ligand and suggest a role for the ligand in trans-activation of hormonally responsive genes.     

A major locus for resistance to <Emphasis Type="Italic">Botryosphaeria dothidea</Emphasis> in <Emphasis Type="Italic">Prunus</Emphasis>

Species in the fungal family Botryosphaeriaceae are significant pathogens of peach. The climatic conditions in the Southeastern USA are conducive to the development of peach fungal gummosis (PFG) with an estimated yield reduction of up to 40% in severe cases. Genotypes with resistance to this PFG were identified in interspecific crosses and segregating backcross populations generated using Kansu peach (Prunus kansuensis Rehder), almond [Prunus dulcis (Mill.) D.A. Webb], and peach [Prunus persica (L.) Batsch]. Hybrids were evaluated for four consecutive years in field conditions. Data generated was validated in different environments using clonal replicates of the hybrids. The F₁ and BC₁F₁ segregation population data suggest a dominant allele for PFG resistance originating from almond. Segregation and mapping analysis located the PFG resistance locus on a chimeric linkage groups 6–8 near the leaf color locus. The molecular markers identified will facilitate marker-assisted selection (MAS) and introgression of this resistance trait into commercial peach germplasm.     

Water balance of conifer logs in early stages of decomposition

Seasonal and long-term changes in the water balance of conifer logs during the first 8 years of decomposition were studied in an old-growth Pseudotsuga/Tsuga forest in the Oregon Cascade Mountains. Measurements were made of the moisture content of outer bark, inner bark, sapwood, and heartwood and of the flow of water into and out of logs of four species (Abies amabilis, Pseudotsuga menziesii, Thuja plicata, and Tsuga heterophylla). After the logs had decomposed from 1 to 2 years, 38–47% of the canopy throughfall landing upon them ran off the surface, 29–34% leached from the bottom, and 21–30% was absorbed and evaporated. After 8 years of decomposition, water entering and then leaching from logs increased 1.3 times while runoff decreased a similar amount. The proportion of water stored by and evaporated from logs in this study indicates that in old growth forests they may intercept 2–5% of the canopy throughfall to the forest floor and that, even in early stages of decomposition, they may affect the hydrological cycle of Pacific Northwest old-growth forests.This is paper 2945 of the Forest Research Laboratory, Oregon State University, Corvallis     

Hormone-independent repression of AP-1-inducible collagenase promoter activity by glucocorticoid receptors.

The role of the ligand in glucocorticoid receptor-mediated transactivation and transrepression of gene expression was investigated. Half-maximal transactivation of a mouse mammary tumor virus-chloramphenicol acetyltransferase reporter gene in transfected cells expressing the human glucocorticoid receptor mutant GRL753F, from which the rate of ligand dissociation is four to five times higher than the rate of dissociation from normal receptors, required a 200- to 300-fold-higher concentration of dexamethasone than was required in cells expressing the normal receptor. Immunocytochemical analysis demonstrated that this difference was not the result of a failure of the mutant receptor to accumulate in the nucleus after steroid treatment. In contrast, in cells cotransfected with a reporter gene containing the AP-1-inducible collagenase gene promoter, the concentration of dexamethasone required for 50% transrepression was the same for mutant and normal receptors. Efficient receptor-mediated transrepression was also observed with the double mutant GRL753F/C421Y, in which the first cysteine residue of the proximal zinc finger has been replaced by tyrosine, indicating that neither retention of the ligand nor direct binding of the receptor to DNA is required. RU38486 behaved as a full agonist with respect to transrepression. In addition, receptor-dependent transrepression, but not transactivation, was observed in transfected cells after heat shock in the absence of the ligand. Taken together, these results suggest that unlike transactivation, transrepression of AP-1 activity by the nuclear glucocorticoid receptor is ligand independent.     

Site- and branch-specific sialylation of recombinant human interferon-gamma in Chinese hamster ovary cell culture

Since sialic acid content is known to be a critical determinant of the biological properties of glycoproteins, it is essential to characterize and monitor sialylation patterns of recombinant glycoproteins intended for therapeutic use. This study reports site- and branch-specific differences in sialylation of human interferon-gamma (IFN-gamma) derived from Chinese hamster ovary (CHO) cell culture. Sialylation profiles were quantitated by reversed-phase HPLC separations of the site-specific pools of tryptic glycopeptides representing IFN-gamma's two potential N-linked glycosylation sites (i.e., Asn(25) and Asn(97)). Although sialylation at each glycosylation site was found to be incomplete, glycans of Asn(25) were more heavily sialylated than those of Asn(97). Furthermore, Man(alpha1-3) arms of the predominant complex biantennary structures were more favorably sialylated than Man(alpha1-6) branches at each glycosylation site. When the sialylation profile was analyzed throughout a suspension batch culture, sialic acid content at each site and branch was found to be relatively constant until a steady decrease in sialylation was observed coincident with loss of cell viability. The introduction of a competitive inhibitor of sialidase into the culture supernatant prevented the loss of sialic acid after the onset of cell death but did not affect sialylation prior to cell death. This finding indicated that incomplete sialylation prior to loss of cell viability could be attributed to incomplete intracellular sialylation while the reduction in sialylation following loss of cell viability was due to extracellular sialidase activity resulting from cell lysis. Thus, both intracellular and extracellular processes defined the sialic acid content of the final product. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 390-398, 1977.     

The Release and Detection of Endotoxin from Liposomes

Incorporation of lipopolysaccharide (LPS) into liposomes dramatically reduces its ability to coagulateLimulusamebocyte lysate (LAL). The coagulation of LAL is commonly used to signal the presence of endotoxinin vitro.This study demonstrates a simple method to release masked endotoxin from liposomal dispersions using moderate amounts of detergent to form mixed micelles containing lipid, detergent, and LPS. Several parameters were found to affect the degree of liposome solubilization and/or the sensitivity of the LAL assay. These included detergent type and concentration, temperature for solubilization, lipid composition, liposome morphology, and time for test incubation. The nonionic detergent polyoxyethylene 10 lauryl ether (C₁₂E₁₀) proved to be unique in its ability to solubilize liposomes and minimally interfere with endotoxin detection. The LAL endotoxin detection limit for samples dispersed in C₁₂E₁₀varied with the phospholipid component; the sensitivity decreased in the order DSPC > DPPC = EPC DMPC. Cholesterol lowered the solubility limit of the liposomes, but did not appear to affect the LAL assay sensitivity once the liposomes were completely solubilized. The presence of negatively charged phospholipids, DSPG and Pops, also lowered the solubility limit. Pops, but not DSPG, at 10 mol% further decreased the LAL endotoxin detection limit. This detergent-solubilization method should be useful in liposomal LPS immunological studies or in other situations where accurate determination of endotoxin concentration is important.     

Colonization of a novel depauperate habitat leads to trophic niche shifts in three desert lizard species

In a novel, depauperate ecosystem, colonizing species may experience changes in their trophic niche as a result of a new resource base and fewer competitors and predators. To examine trophic niche shifts of recent colonists, we focused on three ecologically and phylogenetically divergent lizard species that inhabit both the geologically distinctive depauperate habitat of White Sands and the surrounding Chihuahuan ‘dark soil’ desert in New Mexico. In White Sands the three species comprise the entire lizard community, whereas in the dark soils habitat, they constitute less than half of the lizard community abundance. As a result, we hypothesized that the three focal species would collectively represent a greater variety of trophic positions in the White Sands habitat than in the dark soils habitat. We hypothesized that the extent of shifts in each species’ trophic position would parallel diet and ecomorphology differences between habitats. To test these hypotheses, we combined analysis of lizard stomach contents with carbon and nitrogen stable isotopes in the context of previously published ecomorphology measurements. Stable isotope data indicated that as predicted, species were more different from one another in White Sands than in dark soils, suggesting community‐wide ecological release. Overall, all species were lower on the White Sands food chain; however, only one species decreased trophic level significantly, one increased trophic level variance, and one did not change significantly. Furthermore, stomach content data paralleled both stable isotope and ecomorphological data, showing different degrees of dietary overlap between habitats, depending on the species. That species’ differences in trophic ecology also correspond with ecomorphological differences suggests that these factors are either causally linked or collectively responding to similar ecological pressures, such as competition. By examining diet, trophic position, and ecomorphology of three colonist species, we demonstrate both species‐specific and community‐wide trophic differences in adjacent, but distinct habitats.     

Associations of Circulating Oxidized LDL and Conventional Biomarkers of Cardiovascular Disease in a Cross-Sectional Study of the Navajo Population

The prevalences of cardiovascular disease (CVD) and type 2 diabetes (T2D) have increased among the Navajo Native American community in recent decades. Oxidized low-density lipoprotein (oxLDL) is a novel CVD biomarker that has never been assessed in the Navajo population. We examined the relationship of oxLDL to conventional CVD and T2D risk factors and biomarkers in a cross-sectional population of Navajo participants. This cross-sectional study included 252 participants from 20 Navajo communities from the Diné Network for Environmental Health Project. Plasma samples were tested for oxLDL levels by a sandwich enzyme-linked immunosorbent assay. Univariate and multivariate analyses were used to determine the relationship of oxLDL and oxidized- to non-oxidized lipoprotein ratios to glycated hemoglobin (HbA1c), C-reactive protein (CRP), interleukin 6 (IL6) and demographic and health variables. Type 2 diabetes, hypertension and obesity are very prevalent in this Navajo population. HbA1c, CRP, body mass index (BMI), high-density lipoprotein, and triglycerides were at levels that may increase risk for CVD and T2D. Median oxLDL level was 47 (36.8–57) U/L. Correlational analysis showed that although oxLDL alone was not associated with HbA1c, oxLDL/HDL, oxLDL/LDL and CRP were significantly associated with HbA1c and glucose. OxLDL, oxLDL/HDL and oxLDL/LDL were significantly associated with CRP. Multivariate analysis showed that triglycerides were a common and strong predictor of oxLDL, oxLDL/HDL and oxLDL/LDL. OxLDL was trended with HbA1c and glucose but did not reach significance, however, HbA1c was an independent predictor of OxLDL/HDL. CRP trended with oxLDL/HDL and was a weak predictor of oxLDL/LDL. This Navajo subset appears to have oxLDL levels comparable to subjects without evidence of CVD reported in other studies. The high prevalence of T2D, hypertension and obesity along with abnormal levels of other biomarkers including HbA1c indicate that the Navajo population has a worsening CVD risk profile.