The Contribution of 17beta-Hydroxysteroid Dehydrogenase Type 1 to the Estradiol-Estrone Ratio in Estrogen-Sensitive Breast Cancer Cells

Estrone and estradiol are both estrogens with estrone being the less potent form and estradiol being the most potent estrogen. The binding of the latter to cellular regulatory elements stimulates the proliferation of breast cancer cells. A high ratio of estradiol/estrone is related to increased cell proliferation, and is of great importance to understanding of breast cancer mechanisms. 17beta-hydroxysteroid dehydrogenase type 1 and type 2 play important roles in the activation of estrone and inactivation of estradiol. Breast cancer cells T47D, MCF-7, BT 20, and JEG 3 as control cells, were chosen to evaluate the contribution of these two enzymes to the ratio. Twenty four hours after addition of different concentrations of estrone and estradiol, the ratio stabilized to around 9/1 in breast cancer cell lines with high expression of type 1 (T47D, BT 20, and JEG 3), whereas it approached 1/5 in cells with low expression of type 1 (MCF-7). The estradiol/estrone concentration ratio was modified to 9/1 in MCF-7 and HEK-293 cells over-expressing type 1. In T47D and BT 20, this ratio was decreased from 9/1 to nearly 1/5 (19/81 and 17/83 respectively) after type 1 knockdown by specific siRNAs. Type 2 is mainly involved in the conversion of estradiol into estrone. This ratio was decreased from 9/1 to 7/3 after over-expression of type 2 in MCF-7 cells already over-expressing type 1. The ratio was further decreased by the addition of the oxidative cofactor, NAD, to the cell culture to facilitate the estradiol to estrone conversion catalyzed by type 2. These results demonstrate that the estradiol/estrone ratio is controlled by both type 1 and type 2 with an additional contribution by NAD, although type 1 is the first determining factor in the cellular environment compared with type 2 and cofactors. Moreover, kinetic studies were carried out in intact cells as a new approach, using HEK-293 cells over-expressing type 1 and T47D breast cancer cells

Mesoglycan connects Syndecan-4 and VEGFR2 through Annexin A1 and formyl peptide receptors to promote angiogenesis in vitro.

Mesoglycan is a mixture of glycosaminoglycans (GAG) with fibrinolytic effects and the potential to enhance skin wound repair. Here, we have used endothelial cells isolated from Wild Type (WT) and Syndecan-4 null (Sdc4-/-) C57BL/6 mice to demonstrate that mesoglycan promotes cell motility and in vitro angiogenesis acting on the co-receptor Syndecan-4 (SDC4). This latter is known to participate in the formation and release of extracellular vesicles (EVs). We characterized EVs released by HUVECs and assessed their effect on angiogenesis. Particularly, we focused on Annexin A1 (ANXA1) containing EVs, since they may contribute to tube formation via interactions with Formyl peptide receptors (FPRs). In our model, the bond ANXA1-FPRs stimulates the release of vascular endothelial growth factor (VEGF-A) that interacts with vascular endothelial receptor-2 (VEGFR2) and activates the pathway enhancing cell motility in an autocrine manner, as shown by Wound-Healing/invasion assays, and the induction of Endothelial to Mesenchymal Transition (EndMT). Thus, we have shown for the first time that mesoglycan exerts its pro-angiogenic effects in the healing process triggering the activation of the three interconnected molecular axis: mesoglycan-SDC4, EVs-ANXA1-FPRs and VEGF-A-VEGFR2

Severely Impaired Learning and Altered Neuronal Morphology in Mice Lacking NMDA Receptors in Medium Spiny Neurons

The striatum is composed predominantly of medium spiny neurons (MSNs) that integrate excitatory, glutamatergic inputs from the cortex and thalamus, and modulatory dopaminergic inputs from the ventral midbrain to influence behavior. Glutamatergic activation of AMPA, NMDA, and metabotropic receptors on MSNs is important for striatal development and function, but the roles of each of these receptor classes remain incompletely understood. Signaling through NMDA-type glutamate receptors (NMDARs) in the striatum has been implicated in various motor and appetitive learning paradigms. In addition, signaling through NMDARs influences neuronal morphology, which could underlie their role in mediating learned behaviors. To study the role of NMDARs on MSNs in learning and in morphological development, we generated mice lacking the essential NR1 subunit, encoded by the Grin1 gene, selectively in MSNs. Although these knockout mice appear normal and display normal 24-hour locomotion, they have severe deficits in motor learning, operant conditioning and active avoidance. In addition, the MSNs from these knockout mice have smaller cell bodies and decreased dendritic length compared to littermate controls. We conclude that NMDAR signaling in MSNs is critical for normal MSN morphology and many forms of learning

Downsizing a human inflammatory protein to a small molecule with equal potency and functionality

A significant challenge in chemistry is to rationally reproduce the functional potency of a protein in a small molecule, which is cheaper to manufacture, non-immunogenic, and also both stable and bioavailable. Synthetic peptides corresponding to small bioactive protein surfaces do not form stable structures in water and do not exhibit the functional potencies of proteins. Here we describe a novel approach to growing small molecules with protein-like potencies from a functionally important amino acid of a protein. A 77-residue human inflammatory protein (complement C3a) important in innate immunity is rationally transformed to equipotent small molecules, using peptide surrogates that incorporate a turn-inducing heterocycle with correctly positioned hydrogen-bond-accepting atoms. Small molecule agonists (molecular weigh

Accessible High-Throughput Virtual Screening Molecular Docking Software for Students and Educators

We survey low cost high-throughput virtual screening (HTVS) computer programs for instructors who wish to demonstrate molecular docking in their courses. Since HTVS programs are a useful adjunct to the time consuming and expensive wet bench experiments necessary to discover new drug therapies, the topic of molecular docking is core to the instruction of biochemistry and molecular biology. The availability of HTVS programs coupled with decreasing costs and advances in computer hardware have made computational approaches to drug discovery possible at institutional and non-profit budgets. This paper focuses on HTVS programs with graphical user interfaces (GUIs) that use either DOCK or AutoDock for the prediction of DockoMatic, PyRx, DockingServer, and MOLA since their utility has been proven by the research community, they are free or affordable, and the programs operate on a range of computer platforms

The Raf-1 inhibitor GW5074 and dexamethasone suppress sidestream smoke-induced airway hyperresponsiveness in mice

© 2008 Lei et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution Licens

The Raf-1 inhibitor GW5074 and dexamethasone suppress sidestream smoke-induced airway hyperresponsiveness in mice

<p>Abstract</p> <p>Background</p> <p>Sidestream smoke is closely associated with airway inflammation and hyperreactivity. The present study was designed to investigate if the Raf-1 inhibitor GW5074 and the anti-inflammatory drug dexamethasone suppress airway hyperreactivity in a mouse model of sidestream smoke exposure.</p> <p>Methods</p> <p>Mice were repeatedly exposed to smoke from four cigarettes each day for four weeks. After the first week of the smoke exposure, the mice received either dexamethasone intraperitoneally every other day or GW5074 intraperitoneally every day for three weeks. The tone of the tracheal ring segments was recorded with a myograph system and concentration-response curves were obtained by cumulative administration of agonists. Histopathology was examined by light microscopy.</p> <p>Results</p> <p>Four weeks of exposure to cigarette smoke significantly increased the mouse airway contractile response to carbachol, endothelin-1 and potassium. Intraperitoneal administration of GW5074 or dexamethasone significantly suppressed the enhanced airway contractile responses, while airway epithelium-dependent relaxation was not affected. In addition, the smoke-induced infiltration of inflammatory cells and mucous gland hypertrophy were attenuated by the administration of GW5074 or dexamethasone.</p> <p>Conclusion</p> <p>Sidestream smoke induces airway contractile hyperresponsiveness. Inhibition of Raf-1 activity and airway inflammation suppresses smoking-associated airway hyperresponsiveness.</p

Specific requirement of NMDA receptors for long-term memory consolidation in Drosophila ellipsoid body

In humans and many other animals, memory consolidation occurs through multiple temporal phases and usually involves more than one neuroanatomical brain system. Genetic dissection of Pavlovian olfactory learning in Drosophila melanogaster has revealed multiple memory phases, but the predominant view holds that all memory phases occur in mushroom body neurons. Here, we demonstrate an acute requirement for NMDA receptors (NMDARs) outside of the mushroom body during long-term memory (LTM) consolidation. Targeted dsRNA-mediated silencing of Nmdar1 and Nmdar2 (also known as dNR1 or dNR2, respectively) in cholinergic R4m-subtype large-field neurons of the ellipsoid body specifically disrupted LTM consolidation, but not retrieval. Similar silencing of functional NMDARs in the mushroom body disrupted an earlier memory phase, leaving LTM intact. Our results clearly establish an anatomical site outside of the mushroom body involved with LTM consolidation, thus revealing both a distributed brain system subserving olfactory memory formation and the existence of a system-level memory consolidation in Drosophila