Estrogen induces a systemic growth factor through an estrogen receptor-alpha-dependent mechanism

Estrogen induces proliferation of uterine epithelium through a paracrine action of estrogen receptor (ERalpha) in the underlying stroma. In ovariectomized mice primed with progesterone, estrogen stimulates proliferation in both the epithelium and the stroma. We set out to test whether a paracrine mode of action is involved in estrogen-induced proliferation of the uterine stroma. Epithelial and mesenchymal tissues derived from uteri of neonatal ERalpha null mice (ERalphaKO) or wild-type mice were separated and recombined in all four possible configurations (ERalpha+ or ERalpha- epithelium with ERalpha+ or ERalpha- mesenchyme) and grafted into female athymic mice. After 5 wk, hosts were ovariectomized and challenged with hormone treatment, and cellular proliferation was monitored by thymidine autoradiography. Results showed that, although the full response of the epithelium was dependent on an ERalpha-positive mesenchyme, stromal cell proliferation was independent of tissue ERalpha. This latter observation suggests that the response of the stroma was due to a systemic factor induced in the ERalpha-positive hosts. To test this possibility, pieces of whole uterus from neonatal wild-type or ERalphaKO mice were grafted into syngeneic wild-type or ERalphaKO hosts. In these whole-uterus grafts, estradiol stimulated ERalphaKO uterine stroma when they were grown in wild-type hosts but not when grown in ERalphaKO hosts. The epithelium of whole-uterus ERalphaKO grafts did not respond to estrogen, regardless of the host phenotype. These observations suggest that treatment of progesterone-primed mice with estradiol stimulates production of a systemic factor that is capable of inducing uterine stromal cell proliferation and that this systemic factor is produced by an ERalpha-dependent mechanism.     

Orientation of the g-tensor axes of the Rieske subunit in the cytochrome bc1 complex

The orientation of the g-tensors of the Rieske iron-sulfur protein subunit was determined in a single crystal of the bovine mitochondrial cytochrome bc1 complex with stigmatellin in the Qo quinol binding site. The g-tensor principal axes are skewed with respect to the Fe-Fe and S-S atom direction in the 2Fe2S cluster, which is allowed by the lack of rigorous symmetry of the cluster. The asymmetric unit in the crystal is the active dimer, and the g-tensor axes have slightly different orientations relative to the iron-sulfur cluster in the two halves of the dimer. The g approximately 1.79 axis makes an average angle of 30 degrees with respect to the Fe-Fe direction and the g approximately 2.024 axis an average angle of 26 degrees with respect to the S-S direction. This assignment of the g-tensor axis directions indicates that conformations of the Rieske protein are likely the same in the cytochrome bc1 and b6f complexes and that the extent of motion of the Rieske head domain during the catalytic cycle has been highly conserved during evolution of these distantly related complexes.     

Amino Acid Patterns Within Short Consensus Repeats Define Conserved Duplicons Shared by Genes of the RCA Complex

Complement control proteins (CCPs) contain repeated protein domains, short consensus repeats (SCRs), which must be relevant to diverse functions such as complement activation, coagulation, viral binding, fetal implantation, and self-nonself recognition. Although SCRs share some discontinuous and imperfect motifs, there are many variable positions and indels making classification in subfamilies extremely difficult. Using domain-by-domain phylogenetic analysis, we have found that most domains can be classified into only 11 subfamilies, designated a, b, c, d, e, f, g, h, i, j, or k and identified by critical residues. Each particular CCP is characterized by the order of representatives of the subfamilies. Human complement receptor 1 (CR1) has ajefbkd repeated four times and followed by ch. The classification crosses CCPs and indicates that a particular CCP is a function of the mix of SCRs. The aje set is a feature of several CCPs including human CR1 and DAF and murine Crry and appears to be associated with the success or failure of implantation inter alia. This approach facilitates genomic analysis of available sequences and suggests a framework for the evolution of CCPs. Units of duplication range from single SCRs, to septamers such as efbkdaj, to extensive segments such as MCP-CR1L. Imperfections of duplication with subsequent deletion have contributed to diversification.     

Immunization with modified vaccinia virus Ankara-based recombinant vaccine against severe acute respiratory syndrome is associated with enhanced hepatitis in ferrets

Severe acute respiratory syndrome (SARS) caused by a newly identified coronavirus (SARS-CoV) is a serious emerging human infectious disease. In this report, we immunized ferrets (Mustela putorius furo) with recombinant modified vaccinia virus Ankara (rMVA) expressing the SARS-CoV spike (S) protein. Immunized ferrets developed a more rapid and vigorous neutralizing antibody response than control animals after challenge with SARS-CoV; however, they also exhibited strong inflammatory responses in liver tissue. Inflammation in control animals exposed to SARS-CoV was relatively mild. Thus, our data suggest that vaccination with rMVA expressing SARS-CoV S protein is associated with enhanced hepatitis.     

Glucocorticoid ligands specify different interactions with NF-kappaB by allosteric effects on the glucocorticoid receptor DNA binding domain

Glucocorticoids inhibit inflammation by acting through the glucocorticoid receptor (GR) and powerfully repressing NF-kappaB function. Ligand binding to the C-terminal of GR promotes the nuclear translocation of the receptor and binding to NF-kappaB through the GR DNA binding domain. We sought how ligand recognition influences the interaction between NF-kappaB and GR. Both dexamethasone (agonist) and RU486 (antagonist) promote efficient nuclear translocation, and we show occupancy of the same intranuclear compartment as NF-kappaB with both ligands. However, unlike dexamethasone, RU486 had negligible activity to inhibit NF-kappaB transactivation. This failure may stem from altered co-factor recruitment or altered interaction with NF-kappaB. Using both glutathione S-transferase pull-down and bioluminescence resonance energy transfer approaches, we identified a major glucocorticoid ligand effect on interaction between the GR and the p65 component of NF-kappaB, with RU486 inhibiting recruitment compared with dexamethasone. Using the bioluminescence resonance energy transfer assay, we found that RU486 efficiently recruited NCoR to the GR, unlike dexamethasone, which recruited SRC1. Therefore, RU486 promotes differential protein recruitment to both the C-terminal and DNA binding domain of the receptor. Importantly, using chromatin immunoprecipitation, we show that impaired interaction between GR and p65 with RU486 leads to reduced recruitment of the GR to the NF-kappaB-responsive region of the interleukin-8 promoter, again in contrast to dexamethasone that significantly increased GR binding. We demonstrate that ligand-induced conformation of the GR C-terminal has profound effects on the functional surface generated by the DNA binding domain of the GR. This has implications for understanding ligand-dependent interdomain communication.     

Estimating the true prevalence of Mycobacterium bovis in hunter-harvested white-tailed deer in Michigan

Apparent prevalence, although useful as a consistent index, may underestimate the true prevalence of disease. In Michigan, the ability to estimate the true prevalence of bovine tuberculosis (TB; caused by Mycobacterium bovis) in free-ranging white-tailed deer (Odocoileus virginianus) will become increasingly important to accurately assess progress towards eradication. Our objectives were threefold: to estimate the true prevalence of M. bovis in free-ranging deer in Michigan, to evaluate the effectiveness of existing TB surveillance methods, and to indirectly assess whether TB epidemiologic data from captive cervid herds can be meaningfully extrapolated to free-ranging populations. The study population consisted of all free-ranging deer submitted for TB testing in 2001 from six townships in northeastern Lower Michigan. Tissue samples of tonsil and cranial lymph nodes were collected bilaterally from all deer eligible for the study that did not have gross lesions suggestive of TB (n = 701). Samples were subjected to histopathologic, acid-fast (AF) staining, mycobacterial culture, and polymerase chain reaction (PCR) testing. Seven deer cultured positive for M. bovis that would not have been detected by current surveillance, yielding apparent and true prevalence estimates (95% confidence limits) of 2.7% (1.6, 3.8) and 3.6% (2.3, 4.9), respectively. The sensitivity, specificity, and positive and negative predictive values of the current surveillance protocol were 75, 100, 100, and 99%, respectively. Histologic lesions were present only in tonsils, and ranged from simple necrosis to caseation, suppuration, and granuloma formation. Acid-fast staining and PCR detected M. bovis in only one of the seven culture-positive deer. Our study provides the first estimate of the true prevalence of M. bovis in Michigan's free-ranging deer population and suggests modest underestimation of that prevalence by current surveillance. This study also suggests that caution is warranted when extrapolating epidemiologic data on TB in captive cervids to free-ranging populations and confirms the pivotal role of the tonsil in early infections.     

X-Ray Structure of Rhodobacter Capsulatus Cytochrome bc 1: Comparison with its Mitochondrial and Chloroplast Counterparts

Ubihydroquinone: cytochrome (cyt)c oxidoreductase, or cyt bc (1), is a widespread, membrane integral enzyme that plays a crucial role during photosynthesis and respiration. It is one of the major contributors of the electrochemical proton gradient, which is subsequently used for ATP synthesis. The simplest form of the cyt bc (1) is found in bacteria, and it contains only the three ubiquitously conserved catalytic subunits: the Fe-S protein, cyt b and cyt c (1). Here we present a preliminary X-ray structure of Rhodobacter capsulatus cyt bc (1) at 3.8 A and compare it to the available structures of its homologues from mitochondria and chloroplast. Using the bacterial enzyme structure, we highlight the structural similarities and differences that are found among the three catalytic subunits between the members of this family of enzymes. In addition, we discuss the locations of currently known critical mutations, and their implications in terms of the cyt bc (1) catalysis.     

Elimination of background in film-based applications

Erase-It Background Eliminator is a solution used directly on processed film to remove background or improve data resolution. Traditional methods, such as optimization of the scientific protocol or better estimation of exposure time, are tedious and uncertain. Nevertheless, autoradiography continues to be a simple, effective method to visualize data. Therefore, we evaluated the ability of Erase-It Working Solution to help solve background and resolution issues. To demonstrate the efficiency of the Background Eliminator, we analyzed the product's ability to remove signal evenly, performance on several brands of film, and usefulness with various detection methods. Even reduction of signal was demonstrated by performing densitometric analysis on film generated from a dot blot with serial dilutions of analyte. In addition, overexposed films from various suppliers were effectively treated to remove background and visualize data. Autoradiographs, generated with 32P-labeled probes, and chemiluminescent substrate were also processed resulting in clearer images. Our results demonstrate that film data can be treated quickly and conveniently without fear of artificial enhancement. We show the Background Eliminator to be a universal and timesaving tool to visualize results that otherwise may be difficult to interpret.