Paradoxical interactions among estrogen receptors, estrogens and SERMS: mutual annihilation and synergy

The phenomenon of mutual annihilation of action between 17β estradiol (E₂) and a selective estrogen receptor modulator (SERM), previously described in prepubertal rat diaphysis, epiphysis and uterus, has been investigated in ROS 17/2.8 rat osteoblastic cells and in transiently co-transfected cells in culture. In ROS 17/2.8 cells, the estrogen-induced marker enzyme creatine kinase B (CKB) was stimulated by raloxifene, tamoxifen and tamoxifen methiodide to a specific activity equal to or greater than that induced by 10 nM E₂. However, when a fully inhibitory dose of any of these SERMS was given simultaneously with E₂, no stimulation of CK activity resulted. Therefore, SERMS can be full agonists when acting alone, but complete antagonists to a super-physiological dose of estrogen. It is expected that excess tamoxifen would prevent the action of a SERM, but that the agonist activity of a SERM is abolished by 1000-fold less estrogen is a phenomenon without obvious explanation by classical pharmacology of competitive inhibition. To probe the mechanism of this interaction further, a ckb-CAT reporter plasmid, plus the human receptor expression plasmid, HEO, was transfected transiently into several cell types. In MCF-7 cells, a 1:10 ratio of E₂ to tamoxifen produced mutual annihilation, but the same ratio in ROS 17/2.8 or HeLa cells led to synergistic stimulation. In HeLa cells, co-transfected with the more efficient wild-type estrogen receptor plasmid, HEGO, synergy was demonstrated only at sub-saturation levels of HEGO. We speculate that, in the presence of estradiol and a SERM, not only active homodimers would be formed, but also hetero-dimers of estrogen-liganded and tamoxifen-liganded receptor monomers, depending on the molar ratio of their ligands and their relative affinities. The resulting hetero-dimer conformation would change the specific receptor surface for interactions with the growing number of co-activators and co-repressors, structural changes which could help to explain the mutual annihilation and synergy phenomena and their cell selectivity.     

Cholesterol, linoleic acid or/and tyrosine yield different spectra of products when oxidized alone or in a mixture: studies in various oxidative systems

Identification of reliable biomarkers for oxidative stress for the prediction of the early development of pathological conditions is essential. The detection of biomarkers for oxidative stress such as degradation products of polyunsaturated fatty acid (PUFA), oxysterols, and oxidized proteins, as indicators of oxidative stress are in use, but suffers from insufficient specificity, accuracy and reliability. The overall aim of the present study was to develop new markers which will not only provide information about the presence and level of oxidative stress in biological systems but also on the type of reactive oxygen species (ROS) involved and their metabolic consequences. In the first stage of the study, we compared the level and type of oxidized products formed when different ROS were applied onto three major biomolecules, i.e. cholesterol, linoleic acid (LH) and tyrosine, representing sterols, PUFA and protein, when each compounds was exposed alone or in a mixture to the ROS [copper ions, 2,2-azobis(2-amidinopropane) dihydrochloride (AAPH) and hypochlorous acid (HOCl)]. It was found that different types of oxidants resulted in the formation of different types of oxidation products. Furthermore, oxidation pattern differs when the substrates (cholesterol, PUFA or amino acid) were present alone or in a mixture. As biological systems such as lipoproteins and cell membranes are composed of the above studied molecules, the need for simultaneous detection of the major oxidized products is requires for better characterization of the oxidative stress outcome.     

Downstream of kinase, p62(dok), is a mediator of Fc gamma IIB inhibition of Fc epsilon RI signaling

The low-affinity receptor for IgG, Fc gamma RIIB, is expressed widely in the immune system and functions to attenuate Ag-induced immune responses. In mast cells, coaggregation of Fc gamma RIIB with the high-affinity IgE receptor, Fc epsilon RI, leads to inhibition of Ag-induced degranulation and cytokine production. Fc gamma RIIB inhibitory activity requires a conserved motif within the Fc gamma RIIB cytoplasmic domain termed the immunoreceptor tyrosine-based inhibition motif. When coaggregated with an activating receptor (e.g., Fc epsilon RI, B cell Ag receptor), Fc gamma RIIB is rapidly phosphorylated on tyrosine and recruits the SH2 domain-containing inositol 5-phosphatase (SHIP). However, the mechanisms by which SHIP mediates Fc gamma RIIB inhibitory function in mast cells remain poorly defined. In this report we demonstrate that Fc gamma RIIB coaggregation with Fc epsilon RI stimulates enhanced SHIP tyrosine phosphorylation and association with Shc and p62(dok). Concurrently, enhanced p62(dok) tyrosine phosphorylation and association with RasGAP are observed, suggesting that SHIP may mediate Fc gamma RIIB inhibitory function in mast cells via recruitment of p62(dok) and RasGAP. Supporting this hypothesis, recruitment of p62(dok) to Fc epsilon RI is sufficient to inhibit Fc epsilon RI-induced calcium mobilization and extracellular signal-regulated kinase 1/2 activation. Interestingly, both the amino-terminal pleckstrin homology and phosphotyrosine binding domains and the carboxyl-terminal proline/tyrosine-rich region of p62(dok) can mediate inhibition, suggesting activation of parallel downstream signaling pathways that converge at extracellular signal-regulated kinase 1/2 activation. Finally, studies using gene-ablated mice indicate that p62(dok) is dispensable for Fc gamma RIIB inhibitory signaling in mast cells. Taken together, these data suggest a role for p62(dok) as a mediator of Fc gamma RIIB inhibition of Fc epsilon RI signal transduction in mast cells.     

Cultured mouse marrow cell lines: Interactions between fibroblastoid cells and monocytes

Continuous cell lines were derived from primary cultures of adherent bone marrow cells from SJL/J, BALB/c, C3H/eb, RF, and nude-ICR mice. All these lines readily assumed a pure fibroblastoid appearance with the exception of the BALB/c line (MBA-14), which retained both fibroblastoid and monocytoid cells. This particular line could promote the proliferation of myeloid progenitors (CFU-C) in short-term bone-marrow cultures. The two cell types that composed the MBA-14 cell line were successfully isolated and grown separately; the monocytes as the 14M and 14M1 cell lines and the fibroblastoid cells as the 14F clones. The latter were found to be preadipocytes and accumulated fat in the absence of added hydrocortisone, in medium supplemented with fetal calf serum. Growth of the monocyte lines (14M and 14M1) was dependent upon the mononuclear phagocyte stimulator CSF-1. In the parent MBA-14 cell line the growth of monocytes seemed to depend upon stimulating factor(s) produced by the fibroblastoid cells. The 14M1 monocytes were able to process and degrade antigen as efficiently as primary macrophages. Furthermore, processed antigen produced by 14M1 cells evoked proliferative response by antigen-primed lymph-node cells. In addition to these immunological functions the 14M1 cells were capable of modulating the colony-stimulating activity and degree of adipogenesis exhibited by the fibroblastoid cells. These interactions between monocytes and fibroblastoid cells may constitute part of the mechanism controlling the activity of the hematopoietic microenvironment.     

Haplotypes and linkage disequilibrium at the phenylalanine hydroxylase locus, PAH, in a global representation of populations

Because defects in the phenylalanine hydroxylase gene (PAH) cause phenylketonuria (PKU), PAH was studied for normal polymorphisms and linkage disequilibrium soon after the gene was cloned. Studies in the 1980s concentrated on European populations in which PKU was common and showed that haplotype-frequency variation exists between some regions of the world. In European populations, linkage disequilibrium generally was found not to exist between RFLPs at opposite ends of the gene but was found to exist among the RFLPs clustered at each end. We have now undertaken the first global survey of normal variation and disequilibrium across the PAH gene. Four well-mapped single-nucleotide polymorphisms (SNPs) spanning approximately 75 kb, two near each end of the gene, were selected to allow linkage disequilibrium across most of the gene to be examined. These SNPs were studied as PCR-RFLP markers in samples of, on average, 50 individuals for each of 29 populations, including, for the first time, multiple populations from Africa and from the Americas. All four sites are polymorphic in all 29 populations. Although all but 5 of the 16 possible haplotypes reach frequencies >5% somewhere in the world, no haplotype was seen in all populations. Overall linkage disequilibrium is highly significant in all populations, but disequilibrium between the opposite ends is significant only in Native American populations and in one African population. This study demonstrates that the physical extent of linkage disequilibrium can differ substantially among populations from different regions of the world, because of both ancient genetic drift in the ancestor common to a large regional group of modern populations and recent genetic drift affecting individual populations.     

Improving 3D Genome Reconstructions Using Orthologous and Functional Constraints

The study of the 3D architecture of chromosomes has been advancing rapidly in recent years. While a number of methods for 3D reconstruction of genomic models based on Hi-C data were proposed, most of the analyses in the field have been performed on different 3D representation forms (such as graphs). Here, we reproduce most of the previous results on the 3D genomic organization of the eukaryote Saccharomyces cerevisiae using analysis of 3D reconstructions. We show that many of these results can be reproduced in sparse reconstructions, generated from a small fraction of the experimental data (5% of the data), and study the properties of such models. Finally, we propose for the first time a novel approach for improving the accuracy of 3D reconstructions by introducing additional predicted physical interactions to the model, based on orthologous interactions in an evolutionary-related organism and based on predicted functional interactions between genes. We demonstrate that this approach indeed leads to the reconstruction of improved models.     

Inferring phylogenetic networks by the maximum parsimony criterion: a case study

Horizontal gene transfer (HGT) may result in genes whose evolutionary histories disagree with each other, as well as with the species tree. In this case, reconciling the species and gene trees results in a network of relationships, known as the "phylogenetic network" of the set of species. A phylogenetic network that incorporates HGT consists of an underlying species tree that captures vertical inheritance and a set of edges which model the "horizontal" transfer of genetic material. In a series of papers, Nakhleh and colleagues have recently formulated a maximum parsimony (MP) criterion for phylogenetic networks, provided an array of computationally efficient algorithms and heuristics for computing it, and demonstrated its plausibility on simulated data. In this article, we study the performance and robustness of this criterion on biological data. Our findings indicate that MP is very promising when its application is extended to the domain of phylogenetic network reconstruction and HGT detection. In all cases we investigated, the MP criterion detected the correct number of HGT events required to map the evolutionary history of a gene data set onto the species phylogeny. Furthermore, our results indicate that the criterion is robust with respect to both incomplete taxon sampling and the use of different site substitution matrices. Finally, our results show that the MP criterion is very promising in detecting HGT in chimeric genes, whose evolutionary histories are a mix of vertical and horizontal evolution. Besides the performance analysis of MP, our findings offer new insights into the evolution of 4 biological data sets and new possible explanations of HGT scenarios in their evolutionary history.     

Maximum likelihood of phylogenetic networks

Bioinformatics (2006) 22(21), 2604–2611 The authors would like to apologize for errors of graph misplacementin Figures 4–6, and an     

Feeding live prey to zoo animals: response of zoo visitors in Switzerland

In summer 2007, with the help of a written questionnaire, the attitudes of more than 400 visitors to the zoological garden of Zurich, Switzerland, toward the idea of feeding live insects to lizards, live fish to otters, and live rabbits to tigers were investigated. The majority of Swiss zoo visitors agreed with the idea of feeding live prey (invertebrates and vertebrates) to zoo animals, both off‐ and on‐exhibit, except in the case of feeding live rabbits to tigers on‐exhibit. Women and frequent visitors of the zoo disagreed more often with the on‐exhibit feeding of live rabbits to tigers. Study participants with a higher level of education were more likely to agree with the idea of feeding live invertebrates and vertebrates to zoo animals off‐exhibit. In comparison to an earlier study undertaken in Scotland, zoo visitors in Switzerland were more often in favor of the live feeding of vertebrates. Feeding live prey can counter the loss of hunting skills of carnivores and improve the animals' well‐being. However, feeding enrichments have to strike a balance between optimal living conditions of animals and the quality of visitor experience. Our results show that such a balance can be found, especially when live feeding of mammals is carried out off‐exhibit. A good interpretation of food enrichment might help zoos to win more support for the issue, and for re‐introduction programs and conservation. Zoo Biol 29:344–350, 2010. © 2009 Wiley‐Liss, Inc.