Dopaminergic Activation of Estrogen Receptors Induces Fos Expression within Restricted Regions of the Neonatal Female Rat Brain

Steroid receptor activation in the developing brain influences a variety of cellular processes that endure into adulthood, altering both behavior and physiology. Recent data suggests that dopamine can regulate expression of progestin receptors within restricted regions of the developing rat brain by activating estrogen receptors in a ligand-independent manner. It is unclear whether changes in neuronal activity induced by dopaminergic activation of estrogen receptors are also region specific. To investigate this question, we examined where the dopamine D1-like receptor agonist, SKF 38393, altered Fos expression via estrogen receptor activation. We report that dopamine D1-like receptor agonist treatment increased Fos protein expression within many regions of the developing female rat brain. More importantly, prior treatment with an estrogen receptor antagonist partially reduced D1-like receptor agonist-induced Fos expression only within the bed nucleus of the stria terminalis and the central amygdala. These data suggest that dopaminergic activation of estrogen receptors alters neuronal activity within restricted regions of the developing rat brain. This implies that ligand-independent activation of estrogen receptors by dopamine might organize a unique set of behaviors during brain development in contrast to the more wide spread ligand activation of estrogen receptors by estrogen

Apoptosis of human melanoma cells induced by inhibition of B-RAFV600E involves preferential splicing of bimS

Bim is known to be critical in killing of melanoma cells by inhibition of the RAF/MEK/ERK pathway. However, the potential role of the most potent apoptosis-inducing isoform of Bim, BimS, remains largely unappreciated. Here, we show that inhibition of the mutant B-RAFV600E triggers preferential splicing to produce BimS, which is particularly important in induction of apoptosis in B-RAFV600E melanoma cells. Although the specific B-RAFV600E inhibitor PLX4720 upregulates all three major isoforms of Bim, BimEL, BimL, and BimS, at the protein and mRNA levels in B-RAFV600E melanoma cells, the increase in the ratios of BimS mRNA to BimEL and BimL mRNA indicates that it favours BimS splicing. Consistently, enforced expression of B-RAFV600E in wild-type B-RAF melanoma cells and melanocytes inhibits BimS expression. The splicing factor SRp55 appears necessary for the increase in BimS splicing, as SRp55 is upregulated, and its inhibition by small interfering RNA blocks induction of BimS and apoptosis induced by PLX4720. The PLX4720-induced, SRp55-mediated increase in BimS splicing is also mirrored in freshly isolated B-RAFV600E melanoma cells. These results identify a key mechanism for induction of apoptosis by PLX4720, and are instructive for sensitizing melanoma cells to B-RAFV600E inhibitors

Using a human cardiovascular-respiratory model to characterize cardiac tamponade and pulsus paradoxus

<p>Abstract</p> <p>Background</p> <p>Cardiac tamponade is a condition whereby fluid accumulation in the pericardial sac surrounding the heart causes elevation and equilibration of pericardial and cardiac chamber pressures, reduced cardiac output, changes in hemodynamics, partial chamber collapse, pulsus paradoxus, and arterio-venous acid-base disparity. Our large-scale model of the human cardiovascular-respiratory system (H-CRS) is employed to study mechanisms underlying cardiac tamponade and pulsus paradoxus. The model integrates hemodynamics, whole-body gas exchange, and autonomic nervous system control to simulate pressure, volume, and blood flow.</p> <p>Methods</p> <p>We integrate a new pericardial model into our previously developed H-CRS model based on a fit to patient pressure data. Virtual experiments are designed to simulate pericardial effusion and study mechanisms of pulsus paradoxus, focusing particularly on the role of the interventricular septum. Model differential equations programmed in C are solved using a 5^th-order Runge-Kutta numerical integration scheme. MATLAB is employed for waveform analysis.</p> <p>Results</p> <p>The H-CRS model simulates hemodynamic and respiratory changes associated with tamponade clinically. Our model predicts effects of effusion-generated pericardial constraint on chamber and septal mechanics, such as altered right atrial filling, delayed leftward septal motion, and prolonged left ventricular pre-ejection period, causing atrioventricular interaction and ventricular desynchronization. We demonstrate pericardial constraint to markedly accentuate normal ventricular interactions associated with respiratory effort, which we show to be the distinct mechanisms of pulsus paradoxus, namely, series and parallel ventricular interaction. Series ventricular interaction represents respiratory variation in right ventricular stroke volume carried over to the left ventricle via the pulmonary vasculature, whereas parallel interaction (via the septum and pericardium) is a result of competition for fixed filling space. We find that simulating active septal contraction is important in modeling ventricular interaction. The model predicts increased arterio-venous CO₂due to hypoperfusion, and we explore implications of respiratory pattern in tamponade.</p> <p>Conclusion</p> <p>Our modeling study of cardiac tamponade dissects the roles played by septal motion, atrioventricular and right-left ventricular interactions, pulmonary blood pooling, and the depth of respiration. The study fully describes the physiological basis of pulsus paradoxus. Our detailed analysis provides biophysically-based insights helpful for future experimental and clinical study of cardiac tamponade and related pericardial diseases.</p

Estimates of DNA damage by the comet assay in the direct-developing frog Eleutherodactylus johnstonei (Anura, Eleutherodactylidae)

The aim of this study was to use the Comet assay to assess genetic damage in the direct-developing frog Eleutherodactylus johnstonei. A DNA diffusion assay was used to evaluate the effectiveness of alkaline, enzymatic and alkaline/enzymatic treatments for lysing E. johnstonei blood cells and to determine the amount of DNA strand breakage associated with apoptosis and necrosis. Cell sensitivity to the mutagens bleomycin (BLM) and 4-nitro-quinoline-1-oxide (4NQO) was also assessed using the Comet assay, as was the assay reproducibility. Alkaline treatment did not lyse the cytoplasmic and nuclear membranes of E. johnstonei blood cells, whereas enzymatic digestion with proteinase K (40 μg/mL) yielded naked nuclei. The contribution of apoptosis and necrosis (assessed by the DNA diffusion assay) to DNA damage was estimated to range from 0% to 8%. BLM and 4NQO induced DNA damage in E. johnstonei blood cells at different concentrations and exposure times. Dose-effect curves with both mutagens were highly reproducible and showed consistently low coefficients of variation (CV ≤ 10%). The results are discussed with regard to the potential use of the modified Comet assay for assessing the exposure of E. johnstonei to herbicides in ecotoxicological studies

Interplay between estrogen receptor and AKT in estradiol-induced alternative splicing.

BACKGROUND: Alternative splicing is critical for generating complex proteomes in response to extracellular signals. Nuclear receptors including estrogen receptor alpha (ERα) and their ligands promote alternative splicing. The endogenous targets of ERα:estradiol (E2)-mediated alternative splicing and the influence of extracellular kinases that phosphorylate ERα on E2-induced splicing are unknown. METHODS: MCF-7 and its anti-estrogen derivatives were used for the majority of the assays. CD44 mini gene was used to measure the effect of E2 and AKT on alternative splicing. ExonHit array analysis was performed to identify E2 and AKT-regulated endogenous alternatively spliced apoptosis-related genes. Quantitative reverse transcription polymerase chain reaction was performed to verify alternative splicing. ERα binding to alternatively spliced genes was verified by chromatin immunoprecipitation assay. Bromodeoxyuridine incorporation-ELISA and Annexin V labeling assays were done to measure cell proliferation and apoptosis, respectively. RESULTS: We identified the targets of E2-induced alternative splicing and deconstructed some of the mechanisms surrounding E2-induced splicing by combining splice array with ERα cistrome and gene expression array. E2-induced alternatively spliced genes fall into at least two subgroups: coupled to E2-regulated transcription and ERα binding to the gene without an effect on rate of transcription. Further, AKT, which phosphorylates both ERα and splicing factors, influenced ERα:E2 dependent splicing in a gene-specific manner. Genes that are alternatively spliced include FAS/CD95, FGFR2, and AXIN-1. E2 increased the expression of FGFR2 C1 isoform but reduced C3 isoform at mRNA level. E2-induced alternative splicing of FAS and FGFR2 in MCF-7 cells correlated with resistance to FAS activation-induced apoptosis and response to keratinocyte growth factor (KGF), respectively. Resistance of MCF-7 breast cancer cells to the anti-estrogen tamoxifen was associated with ERα-dependent overexpression of FGFR2, whereas resistance to fulvestrant was associated with ERα-dependent isoform switching, which correlated with altered response to KGF. CONCLUSION: E2 may partly alter cellular proteome through alternative splicing uncoupled to its effects on transcription initiation and aberration in E2-induced alternative splicing events may influence response to anti-estrogens.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are

The Na(+)/Ca(2+) exchanger NCKX4 governs termination and adaptation of the mammalian olfactory response

Sensory perception requires accurate encoding of stimulus information by sensory receptor cells. We identified NCKX4, a potassium-dependent Na(+)/Ca(2+) exchanger, as being necessary for rapid response termination and proper adaptation of vertebrate olfactory sensory neurons (OSNs). Nckx4(-/-) (also known as Slc24a4) mouse OSNs displayed substantially prolonged responses and stronger adaptation. Single-cell electrophysiological analyses revealed that the majority of Na(+)-dependent Ca(2+) exchange in OSNs relevant to sensory transduction is a result of NCKX4 and that Nckx4(-/-) mouse OSNs are deficient in encoding action potentials on repeated stimulation. Olfactory-specific Nckx4(-/-) mice had lower body weights and a reduced ability to locate an odorous source. These results establish the role of NCKX4 in shaping olfactory responses and suggest that rapid response termination and proper adaptation of peripheral sensory receptor cells tune the sensory system for optimal perception

Diabetes Alters Intracellular Calcium Transients in Cardiac Endothelial Cells

Diabetic cardiomyopathy (DCM) is a diabetic complication, which results in myocardial dysfunction independent of other etiological factors. Abnormal intracellular calcium ([Ca2+]i) homeostasis has been implicated in DCM and may precede clinical manifestation. Studies in cardiomyocytes have shown that diabetes results in impaired [Ca2+]i homeostasis due to altered sarcoplasmic reticulum Ca2+ ATPase (SERCA) and sodium-calcium exchanger (NCX) activity. Importantly, altered calcium homeostasis may also be involved in diabetes-associated endothelial dysfunction, including impaired endothelium-dependent relaxation and a diminished capacity to generate nitric oxide (NO), elevated cell adhesion molecules, and decreased angiogenic growth factors. However, the effect of diabetes on Ca2+ regulatory mechanisms in cardiac endothelial cells (CECs) remains unknown. The objective of this study was to determine the effect of diabetes on [Ca2+]i homeostasis in CECs in the rat model (streptozotocin-induced) of DCM. DCM-associated cardiac fibrosis was confirmed using picrosirius red staining of the myocardium. CECs isolated from the myocardium of diabetic and wild-type rats were loaded with Fura-2, and UTP-evoked [Ca2+]i transients were compared under various combinations of SERCA, sarcoplasmic reticulum Ca2+ ATPase (PMCA) and NCX inhibitors. Diabetes resulted in significant alterations in SERCA and NCX activities in CECs during [Ca2+]i sequestration and efflux, respectively, while no difference in PMCA activity between diabetic and wild-type cells was observed. These results improve our understanding of how diabetes affects calcium regulation in CECs, and may contribute to the development of new therapies for DCM treatment

Examining mindfulness and its relation to self-differentiation and alexithymia

Published online first in 10 July 2013Research supports the association between mindfulness, emotion regulation, stress reduction, and interpersonal/relational wellness. The present study evaluated the potential effect of mindfulness on some indicators of psychological imbalance such as low self-differentiation and alexithymia. In this cross-sectional study, a sample of 168 undergraduates (72 % women) completed measures of perceived mindfulness (CAMS-R and PHLMS), self-differentiation (SIPI), and alexithymia (TAS-20). Results revealed positive correlations between the different dimensions of mindfulness and negative correlations between those dimensions, selfdifferentiation, and alexithymia. The dimensions of quality of mindfulness and acceptance were mediators in the relationship between self-differentiation and alexithymia. A nonsignificant interaction between gender and alexithymia was found. All mindfulness dimensions, but self-differentiation, contributed to explain the allocation of the non-alexithymic group. These results indicate that mindfulness seems to be a construct with great therapeutic and research potential at different levels, suggesting that some aspects of mindfulness seem to promote a better self-differentiation and prevent alexithymia