WorldWideScience

Sample records for active erythropoietin receptor

  1. Identification of tyrosine residues within the intracellular domain of the erythropoietin receptor crucial for STAT5 activation.

    Gobert, S.; Chretien, S; Gouilleux, F; Muller, O.; Pallard, C; Dusanter-Fourt, I; Groner, B; Lacombe, C.; Gisselbrecht, S; Mayeux, P

    1996-01-01

    FDCP-1 cells are hematopoietic progenitor cells which require interleukin-3 for survival and proliferation. FDCP-1 cells stably transfected with the murine erythropoietin receptor cDNA survive and proliferate in the presence of erythropoietin. Erythropoietin induces the activation of the short forms (80 kDa) of STAT5 in the cells. Erythropoietin-induced activation of STAT5 was strongly reduced in cells expressing mutated variants of the erythropoietin receptors in which tyrosine residues in t...

  2. Soluble Erythropoietin Receptor Contributes to Erythropoietin Resistance in End-Stage Renal Disease

    Khankin, Eliyahu V.; Walter P Mutter; Hector Tamez; Hai-Tao Yuan; S Ananth Karumanchi; Ravi Thadhani

    2010-01-01

    BACKGROUND: Erythropoietin is a growth factor commonly used to manage anemia in patients with chronic kidney disease. A significant clinical challenge is relative resistance to erythropoietin, which leads to use of successively higher erythropoietin doses, failure to achieve target hemoglobin levels, and increased risk of adverse outcomes. Erythropoietin acts through the erythropoietin receptor (EpoR) present in erythroblasts. Alternative mRNA splicing produces a soluble form of EpoR (sEpoR) ...

  3. Biotinylated recombinant human erythropoietins: Bioactivity and utility as receptor ligand

    Wojchowski, D.M.; Caslake, L. (Pennsylvania State Univ., University Park (USA))

    1989-08-15

    Recombinant human erythropoietin labeled covalently with biotin at sialic acid moieties has been prepared, and has been shown to possess high biological activity plus utility as a receptor ligand. Initially, the effects on biological activity of covalently attaching biotin to erythropoietin alternatively at carboxylate, amino, or sialic acid groups were compared. Biotinylation of erythropoietin at carboxylate groups using biotin-amidocaproyl hydrazide plus 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide led to substantial biological inactivation, although biotinylated molecules retained detectable activity when prepared at low stoichiometries. Biotinylation at amino groups using sulfosuccinimidyl 6-(biotinamido) hexanoate resulted in a high level of biological inactivation with little, if any, retention of biological activity, regardless of labeling stoichiometries. Biotinylation at sialic acid moieties using periodate and biotinamidocaproyl hydrazide proceeded efficiently (greater than 95% and 80% labeling efficiencies for human urinary and recombinant erythropoietin, respectively) and yielded stably biotinylated erythropoietin molecules possessing comparably high biological activity (ie, 45% of the activity of unmodified hormone). Utility of recombinant biotin-(sialyl)-erythropoietin (in combination with 125I-streptavidin) in the assay of cell surface receptors was demonstrated using two distinct murine erythroleukemia cell lines, Friend 745 and Rauscher Red 1. The densities and affinities of specific hormone binding sites were 116 +/- 4 sites, 3.3 +/- 0.4 nmol/L kd and 164 +/- 5 sites, 2.7 +/- 0.4 nmol/L kd, respectively. It is predicted that the present development of biotin-(sialyl)-erythropoietin as a chemically and biologically stable, bioactive ligand will assist in advancing an understanding of the regulated expression and physicochemistry of the human and murine erythropoietin receptors.

  4. A dimeric peptide with erythropoiesis-stimulating activity uniquely affects erythropoietin receptor ligation and cell surface expression.

    Verma, Rakesh; Green, Jennifer M; Schatz, Peter J; Wojchowski, Don M

    2016-08-01

    Erythropoiesis-stimulating agents (ESAs) that exert long-acting antianemia effects have been developed recently, but their mechanisms are poorly understood. Analyses reveal unique erythropoietin receptor (EPOR)-binding properties for one such ESA, the synthetic EPOR agonist peginesatide. Compared with recombinant human EPO and darbepoietin, peginesatide exhibited a slow on rate, but sustained EPOR residency and resistant displacement. In EPO-dependent human erythroid progenitor UT7epo cells, culture in peginesatide unexpectedly upmodulated endogenous cell surface EPOR levels with parallel increases in full-length EPOR-68K levels. These unique properties are suggested to contribute to the durable activity of this (and perhaps additional) dimeric peptide hematopoietic growth factor receptor agonist. PMID:27174804

  5. Efficacy,safety and tolerance of continuous erythropoietin receptor activator intravenous administration on anemia correction in dialysis patients with chronic renal anemia

    钱家麒

    2013-01-01

    Objective To evaluate the efficacy,safety and toler-ance of continuous erythropoietin receptor activator(CE-RA) once every 2 weeks intravenous injection on anemia correction in dialysis patients compared to Epoetin-β(EPO-β) administration. Methods An open label,

  6. Once-Monthly Continuous Erythropoietin Receptor Activator (C.E.R.A.) in Patients with Hemodialysis-Dependent Chronic Kidney Disease: Pooled Data from Phase III Trials

    Locatelli, Francesco; Choukroun, Gabriel; Truman, Matt; Wiggenhauser, Alfons; Fliser, Danilo

    2016-01-01

    Introduction Erythropoiesis-stimulating agents and iron are commonly used in patients with chronic kidney disease with the aim of correcting anemia and maintaining stable hemoglobin levels. We analyzed pooled data from 13 studies with similar designs included in the Umbrella Continuous Erythropoietin Receptor Activator (C.E.R.A.) program to investigate the effects of continuous erythropoiesis receptor activator in clinically relevant subgroups of patients with chronic kidney disease and to de...

  7. Erythropoietin receptor signaling is membrane raft dependent.

    Kathy L McGraw

    Full Text Available Upon erythropoietin (Epo engagement, Epo-receptor (R homodimerizes to activate JAK2 and Lyn, which phosphorylate STAT5. Although recent investigations have identified key negative regulators of Epo-R signaling, little is known about the role of membrane localization in controlling receptor signal fidelity. Here we show a critical role for membrane raft (MR microdomains in creation of discrete signaling platforms essential for Epo-R signaling. Treatment of UT7 cells with Epo induced MR assembly and coalescence. Confocal microscopy showed that raft aggregates significantly increased after Epo stimulation (mean, 4.3±1.4(SE vs. 25.6±3.2 aggregates/cell; p≤0.001, accompanied by a >3-fold increase in cluster size (p≤0.001. Raft fraction immunoblotting showed Epo-R translocation to MR after Epo stimulation and was confirmed by fluorescence microscopy in Epo stimulated UT7 cells and primary erythroid bursts. Receptor recruitment into MR was accompanied by incorporation of JAK2, Lyn, and STAT5 and their activated forms. Raft disruption by cholesterol depletion extinguished Epo induced Jak2, STAT5, Akt and MAPK phosphorylation in UT7 cells and erythroid progenitors. Furthermore, inhibition of the Rho GTPases Rac1 or RhoA blocked receptor recruitment into raft fractions, indicating a role for these GTPases in receptor trafficking. These data establish a critical role for MR in recruitment and assembly of Epo-R and signal intermediates into discrete membrane signaling units.

  8. Activation of erythropoietin receptors by Friend viral gp55 and by erythropoietin and down-modulation by the murine Fv-2r resistance gene

    The leukemogenic membrane glycoprotein (gp55) encoded by Friend spleen focus-forming virus appears to bind to erythropoietin receptors (EpoR) to stimulate erythroblastosis. To directly compare the effects of gp55 with erythropoietin (Epo), the authors produced retrovirions that encode either gp55, Epo, or EpoR. After infection with EpoR virus, interleukin 3-dependent DA-3 cells bound 125I-labeled Epo and grew without interleukin 3 in the presence of Epo. These latter cells, but not parental DA-3 cells, became factor-independent after superinfection either with Epo virus or with Friend spleen focus-forming virus. In addition, Epo virus caused a disease in mice that mimicked Friend erythroleukemia. Although Fv-2r homozygotes are susceptible to all other retroviral diseases, they are resistant to both Epo viral and Friend viral erythroleukemia. These results indicate that both gp55 and Epo stimulate EpoR and that the Fv-2 gene encodes a protein that controls response to these ligands. However, the Fv-2 protein is not EpoR because the corresponding genes map to opposite ends of mouse chromosome 9. These results have important implications for understanding signal transduction by EpoR and the role of host genetic variation in controlling susceptibility to an oncogenic protein

  9. Dose equivalence between continuous erythropoietin receptor activator (CERA), Darbepoetin and Epoetin in patients with advanced chronic kidney disease

    Vega, A; Abad, S; Verdalles, U; Aragoncillo, I; Velazquez, K; Quiroga, B; Escudero, V; López-Gómez, JM

    2014-01-01

    Background: Anemia is a prevalent situation in patients with chronic kidney disease (CKD) and can be well managed with erythropoiesis-stimulating agents (ESAs). Continuous erythropoietin receptor activator (CERA) has a long half-life that allows to be administered once monthly. The lowest recommended dose for patients with non dialysis CKD is 120 μg per month. The objectives were to assess the efficacy of subcutaneous monthly dosing of CERA in CKD stages 4 and 5 not on dialysis, and to determine the equivalent dose to epoetin β and darbepoetin α. Methods: This is a cohort study. A 30-patient group that ESAs was changed to CERA (μg/month) was used as treatment group. We used the following clinically-based equivalent dosing: epoetin β (IU/week) and darbepoetin α (μg/week): 3000/15= 50; 4000/20=75; 6000/30=100; 8000/40=150. Another group of 30 patients with similar characteristics was used as control group and received the same epoetin β and darbepoetin α doses. Results: The mean CERA initial dose and at 6 months was 81.9 ± 35.2 and 82.0 ± 37.82 μg/month (p=0.37). The mean erythropoietin resistance index (ERI) and hemoglobin at baseline and at 6 months in the CERA group and in the control group were not statistically significant. Conclusion: Monthly dosing treatment with CERA is safe and effective. A dose of 75-100 μg/month is enough to maintain stable levels of hemoglobin. Hippokratia 2014; 18 (4): 315-318. PMID:26052197

  10. AMPK is Involved in Mediation of Erythropoietin Influence on Metabolic Activity and Reactive Oxygen Species Production in White Adipocytes

    Wang, Li; Di, Lijun; Noguchi, Constance Tom

    2014-01-01

    Erythropoietin, discovered for its indispensable role during erythropoiesis, has been used in the therapy for selected red blood cell disorders in erythropoietin-deficient patients. The biological activities of erythropoietin have been found to extend to non-erythroid tissues due to the expression of erythropoietin receptor. We previously demonstrated that erythropoietin promotes metabolic activity and white adipocytes browning to increase mitochondrial function and energy expenditure via per...

  11. Erythropoietin and erythropoietin receptor expression in the guinea pig inner ear

    Cayé-Thomasen, Per; Wagner, Niels; Lidegaard Frederiksen, Birgitte;

    2005-01-01

    The erythropoietin receptor (EPOR) is expressed in the brain and erythropoietin (EPO) has been shown to have neurotrophic and neuroprotective functions in the central nervous system and in the retina. These findings may be applied to the inner ear, pending EPO receptor presence. Accordingly, this...

  12. Expression of constitutively active erythropoietin receptor in pyramidal neurons of cortex and hippocampus boosts higher cognitive functions in mice

    Hassouna Imam

    2011-04-01

    Full Text Available Abstract Background Erythropoietin (EPO and its receptor (EPOR are expressed in the developing brain and their transcription is upregulated in adult neurons and glia upon injury or neurodegeneration. We have shown neuroprotective effects and improved cognition in patients with neuropsychiatric diseases treated with EPO. However, the critical EPO targets in brain are unknown, and separation of direct and indirect effects has remained difficult, given the role of EPO in hematopoiesis and brain oxygen supply. Results Here we demonstrate that mice with transgenic expression of a constitutively active EPOR isoform (cEPOR in pyramidal neurons of cortex and hippocampus exhibit enhancement of spatial learning, cognitive flexibility, social memory, and attentional capacities, accompanied by increased impulsivity. Superior cognitive performance is associated with augmented long-term potentiation of cEPOR expressing neurons in hippocampal slices. Conclusions Active EPOR stimulates neuronal plasticity independent of any hematopoietic effects and in addition to its neuroprotective actions. This property of EPOR signaling should be exploited for defining novel strategies to therapeutically enhance cognitive performance in disease conditions.

  13. Evaluation of functional erythropoietin receptor status in skeletal muscle in vivo

    Christensen, Britt; Lundby, Carsten; Jessen, Niels;

    2012-01-01

    myosin light chain 3 and one of desmin/actin were decreased, while three isoforms of creatine kinase and two of glyceraldehyd-3-phosphate dehydrogenase were increased. Conclusions/Significance: Acute exposure to recombinant human erythropoietin is not associated by detectable activation of the Epo-R or......Background: Erythropoietin receptors have been identified in human skeletal muscle tissue, but downstream signal transduction has not been investigated. We therefore studied in vivo effects of systemic erythropoietin exposure in human skeletal muscle. Methodology/Principal Findings: The protocols...... involved 1) acute effects of a single bolus injection of erythropoietin followed by consecutive muscle biopsies for 1-10 hours, and 2) a separate study with prolonged administration for 16 days with biopsies obtained before and after. The presence of erythropoietin receptors in muscle tissue as well as...

  14. Functional erythropoietin receptors on human tumor cells

    Erythropoietin (EPO) is the principal regulator of red blood cell survival, growth and maturation and has achieved great clinical utility for the correction of anemia associated with renal failure, cancer and chemotherapy, and stem cell transplantation. EPO increasingly is being recognized as a pleiotrophic growth factor, having actions on nonhematopoietic cells as well. Both EPO and erythropoietin receptor (EPO-R) expression have been associated with cells of the endothelium, retina, central nervous system, gastrointestinal tract and female reproductive system. The role of EPO in these nonhematopoietic sites is not thoroughly understood and in some instances may be site-specific. Promotion of angiogenesis and blood vessel integrity, increased cell proliferation, prevention of apoptosis, and protection against ischemic damage in the presence of hypoxia have all been described as possible functions of EPO in one or more of these cell types. On the other hand, EPO-R also have been identified on a variety of tumor cells (while in some cases not on the adjacent normal tissue), and several reports have suggested a role for EPO in the direct stimulation of cancer cell growth in vivo and in vitro. Among those tumor cells on which we and others have identified functional EPO-R are breast and ovarian cancer cells. Additionally, the work presented here describes the first evidence that transformed prostate epithelial cells, prostate cancer cell lines, and both normal and cancerous prostate tissue express EPO-R. All of the EPO-R bearing prostate cell lines tested underwent a significant dose-dependent proliferative response to EPO, and EPO triggered intracellular signaling in the cells as evidenced by protein phosphorylation. The results implicate EPO in the biology of both normal and malignant prostate cells and suggest the need for careful evaluation of the use of recombinant EPO as a therapeutic agent in prostate cancer

  15. Development of Radiolabeled Recombinant Erythropoietin for Receptor Studies

    The study of human erythropoietin receptor is of great importance in evolution, carcinogenesis as well as endocrine research. In this study the production and quality control of human recombinant erythropoietin (EPO) has been reported. EPO was successively labeled with [67Ga]-gallium chloride after conjugation with freshly prepared cyclic DTPA-dianhydride (ccDTPA). The best results of the conjugation were obtained by the addition of 0.5 ml of a EPO pharmaceutical solution (in phosphate buffer, pH=8) to a glass tube pre-coated with DTPA-dianhydride (0.01 mg) at 25degreeC with continuous mild stirring for 30 min followed by HPLC/RTLC control and biological biodistribution in normal rats. Under optimized conditions, radio-thin layer chromatography (RTLC), instant thin layer chromatography (ITLC) and high performance liquid chromatography (HPLC) showed overall radiochemical purity of higher than 96%(specific activity=300-500 MBq/mg, labeling efficiency 77%). Preliminary in vivo studies in normal rat specimen demonstrated a high liver, lung spleen, kidney as well as long bones uptake of the tracer; the fact which is consistent with the reported EPO receptor sites. This tracer can be used in nuclear medicine and biological studies for evaluation of EPO in vitro and/or in vivo.

  16. Phagocyte respiratory burst activates macrophage erythropoietin signalling to promote acute inflammation resolution.

    Luo, Bangwei; Wang, Jinsong; Liu, Zongwei; Shen, Zigang; Shi, Rongchen; Liu, Yu-Qi; Liu, Yu; Jiang, Man; Wu, Yuzhang; Zhang, Zhiren

    2016-01-01

    Inflammation resolution is an active process, the failure of which causes uncontrolled inflammation which underlies many chronic diseases. Therefore, endogenous pathways that regulate inflammation resolution are fundamental and of wide interest. Here, we demonstrate that phagocyte respiratory burst-induced hypoxia activates macrophage erythropoietin signalling to promote acute inflammation resolution. This signalling is activated following acute but not chronic inflammation. Pharmacological or genetical inhibition of the respiratory burst suppresses hypoxia and macrophage erythropoietin signalling. Macrophage-specific erythropoietin receptor-deficient mice and chronic granulomatous disease (CGD) mice, which lack the capacity for respiratory burst, display impaired inflammation resolution, and exogenous erythropoietin enhances this resolution in WT and CGD mice. Mechanistically, erythropoietin increases macrophage engulfment of apoptotic neutrophils via PPARγ, promotes macrophage removal of debris and enhances macrophage migration to draining lymph nodes. Together, our results provide evidences of an endogenous pathway that regulates inflammation resolution, with important implications for treating inflammatory conditions. PMID:27397585

  17. Identification of the receptor for erythropoietin on human and murine erythroleukemia cells and modulation by phorbol ester and dimethyl sulfoxide.

    Broudy, V C; Lin, N.; Egrie, J; de Haën, C; Weiss, T.; Papayannopoulou, T; Adamson, J W

    1988-01-01

    Erythropoietin, a glycoprotein that regulates erythropoiesis, initiates its biological effects by binding to a cell-surface receptor. Little is known about the structure of the erythropoietin receptor and the events that follow binding of erythropoietin to its receptor, in part because of the difficulty of obtaining sufficient quantities of cells that express the erythropoietin receptor. We used both iodinated and metabolically labeled erythropoietin to characterize the receptor on a variety ...

  18. The role of erythropoietin and erythropoietin receptor in malignant laryngeal tumors.

    Vukelic, Jelena; Dobrila-Dintinjana, Renata; Jonjic, Nives; Dekanic, Andrea; Ilijic, Vjekoslav

    2013-12-01

    Erythropoietin (Epo) is a glycoprotein hormone responsible for erythropoiesis. Its effect is realized by binding erythropoietin receptor (EpoR) expressed on erythroid progenitor cells. Hypoxia is the main stimulus for the secretion of erythropoietin. Anemia is an independent negative prognostic factor for survival in patients with malignant diseases. Synthetic forms of erythropoietin are used in clinical oncology practice to increase the level of hemoglobin. As well as endogenous they can bind to EpoR. Considering the fact that most effects of synthetic Epo are negative, the role of endogenous Epo/EpoR has become an extremely important issue. The authors do not agree on most items related to the effects of exogenous Epo and EpoR in patients with head and neck carcinomas. We are investigating the expression of Epo/EpoR in the tissue of malignant laryngeal carcinoma. Our hypothesis is that less differentiated laryngeal carcinomas will have a higher level of endogenous Epo/EpoR expression. Therefore, in patients with positive Epo/EpoR we expect shorter survival and poorer locoregional disease control. We anticipate that our hypothesis may help to provide the role of endogenous Epo/EpoR in patients with malignant tumors of the larynx. If the assumptions of this study are confirmed, the patients with laryngeal carcinomas whose tumor cells express Epo/EpoR should not be considered for the treatment of anemia with recombinant erythropoietin in any case. We also point out that our research will expand the knowledge of the biology of laryngeal tumor cells and that the results could be utilized as basic knowledge in development of future therapeutic strategies. PMID:24134826

  19. Erythropoietin.

    Bunn, H Franklin

    2013-03-01

    During the past century, few proteins have matched erythropoietin (Epo) in capturing the imagination of physiologists, molecular biologists, and, more recently, physicians and patients. Its appeal rests on its commanding role as the premier erythroid cytokine, the elegant mechanism underlying the regulation of its gene, and its remarkable impact as a therapeutic agent, arguably the most successful drug spawned by the revolution in recombinant DNA technology. This concise review will begin with a synopsis of the colorful history of this protein, culminating in its purification and molecular cloning. It then covers in more detail the contemporary understanding of Epo's physiology as well as its structure and interaction with its receptor. A major part of this article focuses on the regulation of the Epo gene and the discovery of HIF, a transcription factor that plays a cardinal role in molecular adaptation to hypoxia. In the concluding section, a synopsis of Epo's role in disorders of red blood cell production will be followed by an assessment of the remarkable impact of Epo therapy in the treatment of anemias, as well as concerns that provide a strong impetus for the development of even safer and more effective treatment. PMID:23457296

  20. Erythropoietin

    Miskowiak, Kamilla W; Vinberg, Maj; Harmer, Catherine J;

    2012-01-01

    Current pharmacological treatments for depression have a significant treatment-onset-response delay, an insufficient efficacy for many patients and fail to reverse cognitive dysfunction. Erythropoietin (EPO) has neuroprotective and neurotrophic actions and improves cognitive function in animal...

  1. Erythropoietin regulates Treg cells in asthma through TGFβ receptor signaling.

    Wan, Guoshi; Wei, Bing

    2015-01-01

    Asthma is a chronic inflammatory disorder of the airways, the development of which is suppressed by regulatory T cells (Treg). Erythropoietin (EPO) is originally defined as a hematopoietic growth factor. Recently, the anti-inflammatory effects of EPO in asthma have been acknowledged. However, the underlying mechanisms remain ill-defined. Here, we showed that EPO treatment significantly reduced the severity of an ovalbumin (OVA)-induced asthma in mice, seemingly through promoting Foxp3-mediated activation of Treg cells in OVA-treated mouse lung. The activation of Treg cells resulted from increases in transforming growth factor β1 (TGFβ1), which were mainly produced by M2 macrophages (M2M). In vitro, Co-culture with M2M increased Foxp3 levels in Treg cells and the Treg cell number, in a TGFβ receptor signaling dependent manner. Moreover, elimination of macrophages abolished the therapeutic effects of EPO in vivo. Together, our data suggest that EPO may increase M2M, which activate Treg cells through TGFβ receptor signaling to mitigate the severity of asthma. PMID:26807178

  2. Effects of erythropoietin and its receptor on nervous system

    Ping Wang; Wei Zhou

    2006-01-01

    OBJECTIVE: To investigate the effects of erythropoietin (EPO) and its receptor (EPOR) on nervous system, and its possible mechanism.DATA SOURCES: By inputting the key words "erythropoietin ,nervous system", we performed a search of Medline for English articles, which were published during September 1996 to August 2006, about EPO and EPOR in nervous system.STUDY SELECTION: The materials were selected firstly, literatures were chosen for treatment group and control group and those obviously non-randomized studies were excluded. The full texts of the left literatures were searched. Inclusive criteria: ① Randomized controlled study. ②Experimental or clinical studies (parallel control group included). ③Treatment group was recombinant human erythropoietin(rHuEPO)-treated group. Exclusive criteria: repetitive study.DATA EXTRACTION: A number of 380 randomized or non-randomized articles about the effect of EPO on nervous system were collected, and 49 experiments or clinical trials met the inclusive criteria. Among 331 exclusive articles, 237 were non-randomized or repetitive studies and 94 were review articles. DATA SYNTHESIS: Forty-nine experiments or clinical trials confirmed that EPO and EPOR were expressed in the central nervous system (CNS) and peripheral nervous system(PNS) of gnawer, primate and human being; rHuEPO had obvious neuroprotective effects on brain hypoxia, brain ischemia, experimental intracranial hemorrhage, brain trauma, experimental autoimmune encephalomyelitis, human immunodeficiency virus (HIV)-related sensory neuropathy, distal axonopathy, experimental diabetic neuropathy and acute spinal injury models. Its mechanism maybe involve anti-excitatory toxicity, preventing the production of nitric oxide (NO), lessening inflammatory reaction, resisting apoptosis, maintaining vascular integrity, promoting angiogenesis, promoting the proliferation and differentiation of neural stem cells and progenitor cells and so on. Exogenous EPO could be

  3. Erythropoietin.

    Jelkmann, Wolfgang

    2016-01-01

    Total hemoglobin (Hb) mass is an important determinant of aerobic power. The glycoprotein erythropoietin (Epo) promotes the production of red blood cells (RBCs). The present article reviews the regulation of erythropoiesis and ways of its manipulation. The various Epos, e.g. recombinant human (rh)Epo and (epoetin), and their long-acting analogues can be misused by cheating athletes, but the drugs are detectable by chemical tests, because their glycan isoform structures differ from those of endogenous Epo. Still, anti-doping control has become more difficult, since additional erythropoiesis-stimulating agents have become available (Epo mimetics, activin inhibitors, and small-molecule chemical drugs activating EPO expression). A major problem is created by hypoxia-inducible factor (HIF) stabilizers (e.g. α-ketoglutarate competitors and Co2+ salt) which activate HIFs and thus increase EPO expression. Direct EPO transfer is theoretically also possible but medically little advanced. To overcome weaknesses of direct testing of biological fluids, the World Anti-Doping Agency has implemented the Athlete Biological Passport for continuous monitoring of RBC parameters of athletes. Blood doping is assumed when distinct parameters (blood Hb concentration and reticulocytes) change in a nonphysiological way. PMID:27348128

  4. The Impact of Tumor Expression of Erythropoietin Receptors and Erythropoietin on Clinical Outcome of Esophageal Cancer Patients Treated With Chemoradiation

    Background: To investigate the impact of tumor erythropoietin receptors (Epo-R) and erythropoietin (Epo) expression in 64 patients with Stage III esophageal cancer receiving or not receiving erythropoietin during chemoradiation. Materials and Methods: The impact of tumor Epo-R expression, Epo expression, and 10 additional factors (age, Karnofsky-Performance-Score [KPS], tumor length, T and N stage, histology and grading, hemoglobin during radiotherapy, erythropoietin administration, surgery) on overall survival (OS) and locoregional control (LC) was evaluated. Results: Improved OS was associated with low (≤20%) Epo expression (p = 0.049), KPS >80 (p 0.008), T3 stage (p = 0.010), hemoglobin ≥12 g/dL (p < 0.001), and surgery (p = 0.010). Erythropoietin receptor expression showed a trend (p = 0.09). Locoregional control was associated with T stage (p = 0.005) and hemoglobin (p < 0.001), almost with erythropoietin administration (p = 0.06). On multivariate analyses, OS was associated with KPS (p = 0.045) and hemoglobin (p = 0.032), LC with hemoglobin (p < 0.001). Patients having low expression of both Epo-R and Epo had better OS (p = 0.003) and LC (p = 0.043) than others. Two-year OS was nonsignificantly better (p = 0.25) in patients with low Epo-R expression receiving erythropoietin (50%) than in those with higher Epo-R expression receiving erythropoietin (21%), low Epo-R expression/no erythropoietin administration (29%), or higher Epo-R expression/no erythropoietin administration (18%). Two-year LC rates were, respectively, 65%, 31%, 26%, and 29% (p = 0.20). Results for Epo expression were similar. Conclusions: Higher Epo-R expression or Epo expression seemed to be associated with poorer outcomes. Patients with low expression levels receiving erythropoietin seemed to do better than patients with higher expression levels or not receiving erythropoietin. The data need to be confirmed in a larger series of patients

  5. A new agonist of the erythropoietin receptor, Epobis, induces neurite outgrowth and promotes neuronal survival

    Pankratova, Stanislava; Gu, Bing; Kiryushko, Darya;

    2012-01-01

    Apart from its hematopoietic activity, erythropoietin (EPO) is also known as a tissue-protective cytokine. In the brain, EPO and its receptor are up-regulated in response to insult and exert pro-survival effects. EPO binds to its receptor (EPOR) via high- and low-affinity binding sites (Sites 1 and...... 2, respectively), inducing conformational changes in the receptor, followed by the activation of downstream signaling cascades. Based on the crystal structure of the EPO:EPOR(2) complex, we designed a peptide, termed Epobis, whose sequence encompassed amino acids from binding Site 1. The present...

  6. Functional significance of erythropoietin receptor on tumor cells

    Kodetthoor B Udupa

    2006-01-01

    Erythropoietin (Epo) is the regulator of red blood cell formation. Its receptor (EpoR) is now found in many cells and tissues of the body. EpoR is also shown to occur in tumor cells and Epo enhances the proliferation of these cells through cell signaling. EpoR antagonist can reduce the growth of the tumor in vivo. In view of our current knowledge of Epo, its recombinant forms and receptor,use of Epo in cancer patients to enhance the recovery of hematocrit after chemotherapy treatment has to be carefully evaluated.

  7. A dot assay for the erythropoietin receptor using human recombinant sup 125 I-erythropoietin

    Vannucchi, A.M.; Grossi, A.; Rafanelli, D.; Vannucchi, L.; Ferrini, P.R. (Univ. and USL 10/D, Florence (Italy))

    1989-10-01

    A dot assay was developed for the detection of membrane receptor(s) for erythropoietin (Ep). A relatively homogeneous population of cells bearing the receptor for Ep was generated in the spleen of mice made anemic with phenylhydrazine and crude membrane extracts were prepared from spleen cell suspensions. Aliquots of the membrane extracts were applied to microdishes of nitrocellulose in a volume of 4 microliters. After free reactive sites were blocked, the microdishes were incubated for 2 h at 37 degrees C with {sup 125}I-labeled human recombinant Ep ({sup 125}I-rEp), and nitrocellulose bound radioactivity was determined thereafter. Reproducible curves were obtained, and a significant correlation between bound radioactivity and the amount of membrane proteins applied to the nitrocellulose dishes was found. Specific binding was saturable, reaching a plateau at 2.5 nM. Binding parameters of nitrocellulose-immobilized receptor were not significantly different from the values calculated using intact cells. No appreciable binding of {sup 125}I-rEp to control membranes at low Ep-receptor content was observed. Among a panel of growth factors, only unlabeled rEp was able to compete for the binding of {sup 125}I-rEp to nitrocellulose-immobilized membrane proteins in a dose-dependent fashion. The technique described herein may be of use in the study of the Ep receptor and as an assay for its purification. Moreover, it may also be of general application in the study of receptor-ligand interactions.

  8. Physician Education: The Erythropoietin Receptor and Signal Transduction.

    Yoshimura; Arai

    1996-01-01

    ERYTHROPOIETIN (EPO): Erythropoietin (EPO) is a hormone that promotes the proliferation and differentiation of erythroid progenitor cells and regulates the number of erythrocytes in peripheral blood. EPO is produced mainly by the kidneys, and transcription of the EPO gene is promoted by a reduction in the oxygen concentration in the blood. The existence of EPO was suggested near the end of the 19th century by the discovery that hypoxia increases the production of red blood cells. EPO was identified as a serum factor in the 1950s, and in 1970 Miyake and coworkers succeeded in purifying it by using the urine of patients with aplastic anemia as a starting material. The human EPO gene was cloned in 1985 using a partial amino acid sequence from this purified EPO, and it is well known that recombinant EPO is currently used as a drug to treat anemia associated with chronic renal failure and other illnesses. ACTION OF EPO: When human bone marrow cells are cultured in a semisolid medium containing EPO, they form small erythroblast colonies in five to seven days, and by day 10 large erythroblast colonies appear that resemble fireworks ("burst" colonies). The original cells in the former colonies are called colony forming units-erythroid (CFU-E) or late-stage erythroblast progenitor cells and in the latter colonies they are called burst forming units-erythroid (BFU-E) or early-stage erythroblast progenitor cells. As shown in Figure 1, red blood cells are produced through differentiation from stem cells to BFU-E, CFU-E, and erythroblasts. Although EPO acts on both BFU-E and CFU-E cells, CFU-E cells show greater sensitivity to EPO, and other factors such as stem cell factor (SCF), interleukin (IL)-3, IL-4, and granulocyte macrophage colony-stimulating factor (GM-CSF) must be present together with EPO for BFU-E cell proliferation. In erythroblasts beyond the CFU-E stage, sensitivity to EPO decreases as the cells mature. THE EPO RECEPTOR AND THE CYTOKINE RECEPTOR FAMILY: The EPO

  9. An extra high dose of erythropoietin fails to support the proliferation of erythropoietin dependent cell lines

    ABE, Satoshi; Sasaki, Ryuzo; Masuda, Seiji

    2011-01-01

    Erythropoietin is responsible for the red blood cell formation by stimulating the proliferation and the differentiation of erythroid precursor cells. Erythropoietin triggers the conformational change in its receptor thereby induces the phosphorylation of JAK2. In this study, we show that an extra high dose of erythropoietin, however, fails to activate the erythropoietin receptor, to stimulate the phosphorylation of JAK2 and to support the cell proliferation of Ep-FDC-P2 cell. Moreover, high d...

  10. Human Erythropoietin Dimers with Markedly Enhanced in vivo Activity

    Sytkowski, Arthur J.; Dotimas Lunn, Elizabeth; Davis, Kerry Lynn; Feldman, Laurie; Siekman, Suvia

    1998-02-01

    Human erythropoietin, a widely used and important therapeutic glycoprotein, has a relatively short plasma half-life due to clearance by glomerular filtration as well as by other mechanisms. We hypothesized that an erythropoietin species with a larger molecular size would exhibit an increased plasma half-life and, potentially, an enhanced biological activity. We now report the production of biologically active erythropoietin dimers and trimers by chemical crosslinking of the conventional monomeric form. We imparted free sulfhydryl residues to a pool of erythropoietin monomer by chemical modification. A second pool was reacted with another modifying reagent to yield monomer with male-imido groups. Upon mixing these two pools, covalently linked dimers and trimers were formed that were biologically active in vitro. The plasma half-life of erythropoietin dimers in rabbits was >24 h compared with 4 h for the monomers. Importantly, erythropoietin dimers were biologically active in vivo as shown by their ability to increase the hematocrits of mice when injected subcutaneously. In addition, the dimers exhibited >26-fold higher activity in vivo than did the monomers and were very effective after only one dose. Dimeric and other oligomeric forms of Epo may have an important role in therapy.

  11. Erythropoietin receptor is expressed on human peripheral blood T and B lymphocytes and monocytes and is modulated by recombinant human erythropoietin treatment.

    Lisowska, Katarzyna A; Debska-Slizień, Alicja; Bryl, Ewa; Rutkowski, Bolesław; Witkowski, Jacek M

    2010-08-01

    Erythropoietin receptor (EPO-R) appears on the cell surface in the early stages of erythropoiesis. It has also been found on endothelial cells and polymorphonuclear leukocytes, suggesting erythropoietin (EPO) role beyond erythropoiesis itself. Earlier reports have shown that treatment with recombinant human erythropoietin (rhEPO) in chronic renal failure (CRF) patients improves interleukin-2 production and restores the T lymphocyte function. We decided to investigate possible expression of EPO-R on circulating peripheral blood lymphocytes and monocytes of CRF patients in order to assess the possibility of rhEPO direct action on these cells. Flow cytometry was used for detection and quantification of EPO-R, and reverse transcription polymerase chain reaction for detection of the EPO receptor mRNA. Our results show for the first time the existence of EPO-R on cell surface of human T and B lymphocytes and monocytes as well as at the transcriptional activity of the EPO-R gene in these cells, both in healthy and CRF individuals. We have also found significant differences between the numbers of EPO-R molecules on T and B lymphocytes of CRF patients not treated and treated with rhEPO and healthy control. Discovery of EPO-R expression on human lymphocytes suggests that EPO is probably able to directly modulate some signaling pathways important for these cells. PMID:20528849

  12. Redundant and selective roles for erythropoietin receptor tyrosines in erythropoiesis in vivo.

    Longmore, G D; You, Y; Molden, J; Liu, K D; Mikami, A; Lai, S Y; Pharr, P; Goldsmith, M A

    1998-02-01

    Cytokine receptors have been shown in cell culture systems to use phosphotyrosine residues as docking sites for certain signal transduction intermediates. Studies using various cellular backgrounds have yielded conflicting information about the importance of such residues. The present studies were undertaken to determine whether or not tyrosine residues within the erythropoietin receptor (EPOR) are essential for biologic activity during hematopoiesis in vivo. A variant of the EPOR was constructed that contains both a substitution (R129C) causing constitutive receptor activation as well as replacement of all eight cytoplasmic tyrosines by phenylalanines (cEPORYF). A comparison between animals exposed to recombinant retroviruses expressing cEPOR and cEPORYF showed that efficient red blood cell (RBC) development in vivo is dependent on the pressence of tyrosine residues in the cytoplasmic domain of the EPOR. In addition, an inefficient EPOR tyrosine independent pathway supporting RBC development was detected. Tyrosine add-back mutants showed that multiple individual tyrosines have the capacity to restore full erythropoietic potential to the EPOR as determined in whole animals. The analysis of primary erythroid progenitors transduced with the various cEPOR tyrosine mutants and tyrosine add-backs showed that only tyrosine 343 (Y1) and tyrosine 479 (Y8) were capable of supporting immature burst-forming unit-erythroid progenitor development. Thus, this receptor is characterized by striking functional redundancy of tyrosines in a biologically relevant context. However, selective tyrosine residues may be uniquely important for early signals supporting erythroid development. PMID:9446647

  13. Discovery and Characterization of Nonpeptidyl Agonists of the Tissue-Protective Erythropoietin Receptor.

    Miller, James L; Church, Timothy J; Leonoudakis, Dmitri; Lariosa-Willingham, Karen; Frigon, Normand L; Tettenborn, Connie S; Spencer, Jeffrey R; Punnonen, Juha

    2015-08-01

    Erythropoietin (EPO) and its receptor are expressed in a wide variety of tissues, including the central nervous system. Local expression of both EPO and its receptor is upregulated upon injury or stress and plays a role in tissue homeostasis and cytoprotection. High-dose systemic administration or local injection of recombinant human EPO has demonstrated encouraging results in several models of tissue protection and organ injury, while poor tissue availability of the protein limits its efficacy. Here, we describe the discovery and characterization of the nonpeptidyl compound STS-E412 (2-[2-(4-chlorophenoxy)ethoxy]-5,7-dimethyl-[1,2,4]triazolo[1,5-a]pyrimidine), which selectively activates the tissue-protective EPO receptor, comprising an EPO receptor subunit (EPOR) and the common β-chain (CD131). STS-E412 triggered EPO receptor phosphorylation in human neuronal cells. STS-E412 also increased phosphorylation of EPOR, CD131, and the EPO-associated signaling molecules JAK2 and AKT in HEK293 transfectants expressing EPOR and CD131. At low nanomolar concentrations, STS-E412 provided EPO-like cytoprotective effects in primary neuronal cells and renal proximal tubular epithelial cells. The receptor selectivity of STS-E412 was confirmed by a lack of phosphorylation of the EPOR/EPOR homodimer, lack of activity in off-target selectivity screening, and lack of functional effects in erythroleukemia cell line TF-1 and CD34(+) progenitor cells. Permeability through artificial membranes and Caco-2 cell monolayers in vitro and penetrance across the blood-brain barrier in vivo suggest potential for central nervous system availability of the compound. To our knowledge, STS-E412 is the first nonpeptidyl, selective activator of the tissue-protective EPOR/CD131 receptor. Further evaluation of the potential of STS-E412 in central nervous system diseases and organ protection is warranted. PMID:26018904

  14. Cytoprotective doses of erythropoietin or carbamylated erythropoietin have markedly different procoagulant and vasoactive activities

    Coleman, TR; Westenfelder, C; Togel, FE; Yang, Y; Hu, ZM; Swenson, L; Leuvenink, HGD; Ploeg, RJ; d'Uscio, LV; Katusic, ZS; Ghezzi, P; Zanetti, A; Kaushansky, K; Fox, NE; Cerami, A; Brines, M

    2006-01-01

    Recombinant human erythropoietin (rhEPO) is receiving increasing attention as a potential therapy for prevention of injury and restoration of function in nonhematopoietic tissues. However, the minimum effective dose required to mimic and augment these normal paracrine functions of erythropoietin (EP

  15. Action of erythropoietin in vitro on rabbit reticulocyte membrane Ca2+-ATPase activity.

    Lawrence, W D; Davis, P J; Blas, S D

    1987-01-01

    The mechanism of action of erythropoietin is thought to require specific interaction with the target cell surface and involve alteration of cellular calcium metabolism. Using the rabbit reticulocyte membrane as a model of the immature red cell membrane, we investigated the effects of human recombinant erythropoietin on membrane Ca2+-ATPase (calcium pump) activity in vitro. Erythropoietin in a concentration range of 0.025 to 3.0 U/ml progressively decreased membrane Ca2+-ATPase activity by up ...

  16. Packing Density of the Erythropoietin Receptor Transmembrane Domain Correlates with Amplification of Biological Responses

    Becker, Verena [German Cancer Research Center, Heidelberg; Sengupta, D [University of Heidelberg; Ketteler, Robin [German Cancer Research Center, Heidelberg; Ullmann, G. Matthias [University of Bayreuth; Smith, Jeremy C [ORNL; Klingmuller, Ursula [German Cancer Research Center, Heidelberg

    2008-10-01

    The formation of signal-promoting dimeric or oligomeric receptor complexes at the cell surface is modulated by self-interaction of their transmembrane (TM) domains. To address the importance of TM domain packing density for receptor functionality, we examined a set of asparagine mutants in the TM domain of the erythropoietin receptor (EpoR). We identified EpoR-T242N as a receptor variant that is present at the cell surface similar to wild-type EpoR but lacks visible localization in vesicle-like structures and is impaired in efficient activation of specific signaling cascades. Analysis by a molecular modeling approach indicated an increased interhelical distance for the EpoR-T242N TM dimer. By employing the model, we designed additional mutants with increased or decreased packing volume and confirmed a correlation between packing volume and biological responsiveness. These results propose that the packing density of the TM domain provides a novel layer for fine-tuned regulation of signal transduction and cellular decisions.

  17. Erythropoietin regulates Treg cells in asthma through TGFβ receptor signaling

    Wan, Guoshi; Wei, Bing

    2015-01-01

    Asthma is a chronic inflammatory disorder of the airways, the development of which is suppressed by regulatory T cells (Treg). Erythropoietin (EPO) is originally defined as a hematopoietic growth factor. Recently, the anti-inflammatory effects of EPO in asthma have been acknowledged. However, the underlying mechanisms remain ill-defined. Here, we showed that EPO treatment significantly reduced the severity of an ovalbumin (OVA)-induced asthma in mice, seemingly through promoting Foxp3-mediate...

  18. Erythropoietin, ferritin, haptoglobin, hemoglobin and transferrin receptor in metabolic syndrome: a case control study

    Hämäläinen Päivi

    2012-09-01

    Full Text Available Abstract Background Increased ferritin concentrations are associated with metabolic syndrome (MetS. The association between ferritin as well as hemoglobin level and individual MetS components is unclear. Erythropoietin levels in subjects with MetS have not been determined previously. The aim of this study was to compare serum erythropoietin, ferritin, haptoglobin, hemoglobin, and transferrin receptor (sTFR levels between subjects with and without MetS and subjects with individual MetS components. Methods A population based cross-sectional study of 766 Caucasian, middle-aged subjects (341 men and 425 women from five age groups born in Pieksämäki, Finland who were invited to a health check-up in 2004 with no exclusion criteria. Laboratory analyzes of blood samples collected in 2004 were done during year 2010. MetS was defined by National Cholesterol Education Program criteria. Results 159 (53% men and 170 (40% women of study population met MetS criteria. Hemoglobin and ferritin levels as well as erythropoietin and haptoglobin levels were higher in subjects with MetS (p  Conclusion Subjects with MetS have elevated hemoglobin, ferritin, erythropoietin and haptoglobin concentrations. Higher hemoglobin levels are related to all components of MetS. Higher ferritin levels associate with TG, abdominal obesity, elevated glucose or low high density cholesterol. Haptoglobin levels associate with blood pressure or elevated glucose. However, erythropoietin levels are related only with abdominal obesity. Higher serum erythropoietin concentrations may suggest underlying adipose tissue hypoxemia in MetS.

  19. Expression of platelet-derived growth factor BB, erythropoietin and erythropoietin receptor in canine and feline osteosarcoma

    Meyer, F.R.L.; Steinborn, R.; Grausgruber, H.; Wolfesberger, B.; Walter, I.

    2015-01-01

    The discovery of expression of the erythropoietin receptor (EPO-R) on neoplastic cells has led to concerns about the safety of treating anaemic cancer patients with EPO. In addition to its endocrine function, the receptor may play a role in tumour progression through an autocrine mechanism. In this study, the expression of EPO, EPO-R and platelet-derived growth factor BB (PDGF-BB) was analysed in five feline and 13 canine osteosarcomas using immunohistochemistry (IHC) and reverse transcription polymerase chain reaction (RT-PCR). EPO expression was positive in all tumours by IHC, but EPO mRNA was only detected in 38% of the canine and 40% of the feline samples. EPO-R was expressed in all samples by quantitative RT-PCR (RT-qPCR) and IHC. EPO-R mRNA was expressed at higher levels in all feline tumours, tumour cell lines, and kidney when compared to canine tissues. PDGF-BB expression was variable by IHC, but mRNA was detected in all samples. To assess the functionality of the EPO-R on tumour cells, the proliferation of canine and feline osteosarcoma cell lines was evaluated after EPO administration using an alamarBlue assay and Ki67 immunostaining. All primary cell lines responded to EPO treatment in at least one of the performed assays, but the effect on proliferation was very low indicating only a weak responsiveness of EPO-R. In conclusion, since EPO and its receptor are expressed by canine and feline osteosarcomas, an autocrine or paracrine tumour progression mechanism cannot be excluded, although in vitro data suggest a minimal role of EPO-R in osteosarcoma cell proliferation. PMID:26189892

  20. Systemic and cerebral vascular endothelial growth factor levels increase in murine cerebral malaria along with increased calpain and caspase activity and can be reduced by erythropoietin treatment

    Casper eHempel

    2014-06-01

    Full Text Available The pathogenesis of cerebral malaria includes compromised microvascular perfusion, increased inflammation, cytoadhesion and endothelial activation. These events cause blood-brain barrier disruption and neuropathology and can be associated with the vascular endothelial growth factor (VEGF signalling pathway. We studied this pathway in mice infected with Plasmodium berghei ANKA causing murine cerebral malaria with or without the use of erythropoietin as adjunct therapy. ELISA and western blotting was used for quantification of VEGF and relevant proteins in brain and plasma. Cerebral malaria increased levels of VEGF in brain and plasma and decreased plasma levels of soluble VEGF receptor 2. Erythropoietin treatment normalised VEGF receptor 2 levels and reduced brain VEGF levels. Hypoxia-inducible factor (HIF-1α was significantly upregulated whereas cerebral HIF-2α and erythropoietin levels remained unchanged. Furthermore, we noticed increased caspase-3 and calpain activity in terminally ill mice, as measured by protease-specific cleavage of α-spectrin and p35. In conclusion, we detected increased cerebral and systemic VEGF as well as HIF-1α, which in the brain were reduced to normal in erythropoietin-treated mice. Also caspase and calpain activity was reduced markedly in erythropoietin-treated mice.

  1. Expression and characterization of erythropoietin receptors on normal human bone marrow cells

    Hoshino, S.; Teramura, M.; Takahashi, M.; Motoji, T.; Oshimi, K.; Ueda, M.; Mizoguchi, H.

    1989-05-01

    We studied the specific binding of /sup 125/I-labeled bioactive recombinant human erythropoietin (Epo) to human bone marrow mononuclear cells (BMNC) obtained from normal subjects. The /sup 125/I-labeled Epo bound specifically to the BMNC. Scatchard analysis of the data showed two classes of binding sites; one high affinity (Kd 0.07 nM) and the other low affinity (Kd 0.38 nM). The number of Epo binding sites per BMNC was 46 +/- 16 high-affinity receptors and 91 +/- 51 low-affinity receptors. The specific binding was displaced by unlabeled Epo, but not by other growth factors. Receptor internalization was observed significantly at 37 degrees C, but was prevented by the presence of 0.2% sodium azide. These findings indicate that human BMNC possess two classes of specific Epo receptors with characteristics of a hormone-receptor association.

  2. Problem-Solving Test: Expression Cloning of the Erythropoietin Receptor

    Szeberenyi, Jozsef

    2008-01-01

    Terms to be familiar with before you start to solve the test: cytokines, cytokine receptors, cDNA library, cDNA synthesis, poly(A)[superscript +] RNA, primer, template, reverse transcriptase, restriction endonucleases, cohesive ends, expression vector, promoter, Shine-Dalgarno sequence, poly(A) signal, DNA helicase, DNA ligase, topoisomerases,…

  3. Comparison of real time RT-PCR and flow cytometry methods for evaluation of biological activity of recombinant human erythropoietin

    Sepehrizadeh Z; Tabatabaei Yazdi M; Zarrini GH; Hashemi Bozchlou S; Khoshakhlagh P

    2008-01-01

    Background: Evaluation of bioactivity of recombinant erythropoietin is essential for pharmaceutical industry, quality control authorities and researchers. The purpose of this study was to compare real time RT-PCR and flow cytometry for the assay of biological activity of recombinant erythropoietin. Methods: Three concentrations of recombinant erythropoietin BRP (80, 40 and 20 IU/ml) were injected subcutaneously to mice. After 4 days the blood was collected and used for reticulocyte counts by ...

  4. Simulated microgravity induce apoptosis and down-regulation of erythropoietin receptor of UT-7/EPO cells

    Zou, Li-xue; Cui, Shao-yan; Zhong, Jian; Yi, Zong-chun; Sun, Yan; Fan, Yu-bo; Zhuang, Feng-yuan

    2010-11-01

    Hematopoietic progenitor cell proliferation can be alternated on either spaceflight or under simulated microgravity experiments on the ground; however, the underlying mechanism remains largely unknown. In the present study, we have demonstrated that exposure of human erythropoietin (EPO)-dependent leukemia cell line UT-7/EPO cells to conditions of simulated microgravity with a rotary culture instrument significantly inhibited the cellular proliferation rate. Adding higher concentrations of EPO to the culture medium failed to improve the inhibitory status. Cell apoptosis was detected by fluorescence staining of cell nuclei and a flow cytometry assay using Annexin V/PI double staining. This microgravity-induced apoptosis in UT-7/EPO cells could be blocked by a pancaspase inhibitor Z-VAD-FMK. Immunoblotting demonstrated that rotary culture resulted in a reduction of the expression of Bcl-xL, an anti-apoptotic protein, and the cleavage of caspase-3. Furthermore, rotary culture reduced surface localization and protein content, as well as the mRNA expression of erythropoietin receptor (EPOR) of UT-7/EPO. Take together, the findings indicated that simulated microgravity may induce mitochondrial related apoptosis of UT-7/EPO cell through depressing the EPO-EPOR pathway.

  5. Upregulation of erythropoietin receptor in UT-7/EPO cells inhibits simulated microgravity-induced cell apoptosis

    Zou, Li-xue; Cui, Shao-yan; Zhong, Jian; Yi, Zong-chun; Sun, Yan; Fan, Yu-bo; Zhuang, Feng-yuan

    2011-07-01

    Hematopoietic progenitor cell proliferation can be altered in either spaceflight or under simulated microgravity experiments on the ground, however, the underlying mechanism remains unknown. Our previous study showed that exposure of the human erythropoietin (EPO)-dependent leukemia cell line UT-7/EPO to conditions of simulated microgravity significantly inhibited the cellular proliferation rate and induced cell apoptosis. We postulated that the downregulation of the erythropoietin receptor (EPOR) expression in UT-7/EPO cells under simulated microgravity may be a possible reason for microgravity triggered apoptosis. In this paper, a human EPOR gene was transferred into UT-7/EPO cells and the resulting expression of EPOR on the surface of UT-7/EPO cells increased approximately 61% ( p < 0.05) as selected by the antibiotic G418. It was also shown through cytometry assays and morphological observations that microgravity-induced apoptosis markedly decreased in these UT-7/EPO-EPOR cells. Thus, we concluded that upregulation of EPOR in UT-7/EPO cells could inhibit the simulated microgravity-induced cell apoptosis in this EPO dependent cell line.

  6. Erythropoietin signaling promotes transplanted progenitor cell survival

    Jia, Yi; Warin, Renaud; Yu, Xiaobing; Epstein, Reed; Noguchi, Constance Tom

    2009-01-01

    We examine the potential for erythropoietin signaling to promote donor cell survival in a model of myoblast transplantation. Expression of a truncated erythropoietin receptor in hematopoietic stem cells has been shown to promote selective engraftment in mice. We previously demonstrated expression of endogenous erythropoietin receptor on murine myoblasts, and erythropoietin treatment can stimulate myoblast proliferation and delay differentiation. Here, we report that enhanced erythropoietin re...

  7. Targeting the erythropoietin receptor on glioma cells reduces tumour growth

    Peres, Elodie A.; Valable, Samuel [CERVOxy team ' Hypoxia and cerebrovascular pathophysiology' , UMR 6232 CI-NAPS, Universite de Caen Basse-Normandie, Universite Paris-Descartes, CNRS, CEA. G.I.P. CYCERON, Caen (France); Guillamo, Jean-Sebastien [CERVOxy team ' Hypoxia and cerebrovascular pathophysiology' , UMR 6232 CI-NAPS, Universite de Caen Basse-Normandie, Universite Paris-Descartes, CNRS, CEA. G.I.P. CYCERON, Caen (France); Departement de Neurologie, CHU de Caen (France); Marteau, Lena [CERVOxy team ' Hypoxia and cerebrovascular pathophysiology' , UMR 6232 CI-NAPS, Universite de Caen Basse-Normandie, Universite Paris-Descartes, CNRS, CEA. G.I.P. CYCERON, Caen (France); Bernaudin, Jean-Francois [Service d' Histologie-Biologie Tumorale, ER2UPMC, Universite Paris 6, Hopital Tenon, Paris (France); Roussel, Simon [CERVOxy team ' Hypoxia and cerebrovascular pathophysiology' , UMR 6232 CI-NAPS, Universite de Caen Basse-Normandie, Universite Paris-Descartes, CNRS, CEA. G.I.P. CYCERON, Caen (France); Lechapt-Zalcman, Emmanuele [CERVOxy team ' Hypoxia and cerebrovascular pathophysiology' , UMR 6232 CI-NAPS, Universite de Caen Basse-Normandie, Universite Paris-Descartes, CNRS, CEA. G.I.P. CYCERON, Caen (France); Service d' Anatomie Pathologique, CHU de Caen (France); Bernaudin, Myriam [CERVOxy team ' Hypoxia and cerebrovascular pathophysiology' , UMR 6232 CI-NAPS, Universite de Caen Basse-Normandie, Universite Paris-Descartes, CNRS, CEA. G.I.P. CYCERON, Caen (France); Petit, Edwige, E-mail: epetit@cyceron.fr [CERVOxy team ' Hypoxia and cerebrovascular pathophysiology' , UMR 6232 CI-NAPS, Universite de Caen Basse-Normandie, Universite Paris-Descartes, CNRS, CEA. G.I.P. CYCERON, Caen (France)

    2011-10-01

    Hypoxia has been shown to be one of the major events involved in EPO expression. Accordingly, EPO might be expressed by cerebral neoplastic cells, especially in glioblastoma, known to be highly hypoxic tumours. The expression of EPOR has been described in glioma cells. However, data from the literature remain descriptive and controversial. On the basis of an endogenous source of EPO in the brain, we have focused on a potential role of EPOR in brain tumour growth. In the present study, with complementary approaches to target EPO/EPOR signalling, we demonstrate the presence of a functional EPO/EPOR system on glioma cells leading to the activation of the ERK pathway. This EPO/EPOR system is involved in glioma cell proliferation in vitro. In vivo, we show that the down-regulation of EPOR expression on glioma cells reduces tumour growth and enhances animal survival. Our results support the hypothesis that EPOR signalling in tumour cells is involved in the control of glioma growth.

  8. Targeting the erythropoietin receptor on glioma cells reduces tumour growth

    Hypoxia has been shown to be one of the major events involved in EPO expression. Accordingly, EPO might be expressed by cerebral neoplastic cells, especially in glioblastoma, known to be highly hypoxic tumours. The expression of EPOR has been described in glioma cells. However, data from the literature remain descriptive and controversial. On the basis of an endogenous source of EPO in the brain, we have focused on a potential role of EPOR in brain tumour growth. In the present study, with complementary approaches to target EPO/EPOR signalling, we demonstrate the presence of a functional EPO/EPOR system on glioma cells leading to the activation of the ERK pathway. This EPO/EPOR system is involved in glioma cell proliferation in vitro. In vivo, we show that the down-regulation of EPOR expression on glioma cells reduces tumour growth and enhances animal survival. Our results support the hypothesis that EPOR signalling in tumour cells is involved in the control of glioma growth.

  9. Photoaffinity labeling of the erythropoietin receptor and its identification in a ligand-free form

    Hosoi, Takayuki; Sawyer, S.T.; Krantz, S.B. (Vanderbilt Univ. School of Medicine, Nashville, TN (USA))

    1991-01-01

    Pure human recombinant erythropoietin (EP) was acylated through a primary amino residue with a cross-linking reagent, N-((3-((4-((p-azido-m-({sup 125}I)iodophenyl)azo)benzoyl)amino)propanoyl)oxy)-succinimide (Denny-Jaffe reagent), which is photoreactive and cleavable at the azo residue. The resulting conjugated hormone (DJ-EP) was purified from unmodified EP by reverse-phase high-pressure liquid chromatography and maintained its capacity to bind to receptors for EP on erythroid progenitor cells. The receptor for EP was previously identified as two related proteins of 100 and 85 kDa molecular mass by chemical cross-linking to {sup 125}I-EP. Recently, D'Andrea and co-workers cloned a cDNA that codes for a protein of 55-66 kDa, which is thought to be the EP receptor. In this report, cross-linking to the receptor through the monofunctional DJ-EP labeled the same 140- and 125-kDa molecular mass bands cross-linked with {sup 125}I-EP and disuccinimidyl suberate. Furthermore, cleavage of the azo bond of the DJ-EP receptor complex by sodium dithionite demonstrated that proteins of 105 and 90 kDa were labeled in ligand-free form by DJ-EP. This result demonstrates that artifactual cross-linking of multiple proteins or other artifacts of cross-linking do not explain the difference in molecular mass of the EP receptor identified by cross-linking and the receptor identified by expression cloning.

  10. Erythropoietin in cardiac disease : New features of an old drug

    Ruifrok, Willem-Peter T.; de Boer, Rudolf A.; Westenbrink, B. Daan; van Veldhuisen, Dirk J.; van Gilst, Wiek H.

    2008-01-01

    Erythropoietin is a haematopoietic hormone with extensive non-haematopoietic effects. The discovery of an erythropoietin receptor outside the haematopoietic system has fuelled the research into the beneficial effects of erythropoietin for various conditions, predominantly in cardiovascular disease.

  11. Expression of functionally active sialylated human erythropoietin in plants

    Jez, Jakub; Castilho, Alexandra; Grass, Josephine; Vorauer-Uhl, Karola; Sterovsky, Thomas; Altmann, Friedrich; Steinkellner, Herta

    2013-01-01

    Recombinant human erythropoietin (rhEPO), a glycohormone, is one of the leading biopharmaceutical products. The production of rhEPO is currently restricted to mammalian cell expression systems because of rhEPO's highly complex glycosylation pattern, which is a major determinant for drug-efficacy. Here we evaluate the ability of plants to produce different glycoforms of rhEPO. cDNA constructs were delivered to Nicotiana benthamiana (N. benthamiana) and transiently expressed by a viral based ex...

  12. Lenalidomide Stabilizes the Erythropoietin Receptor by Inhibiting the E3 Ubiquitin Ligase RNF41.

    Basiorka, Ashley A; McGraw, Kathy L; De Ceuninck, Leentje; Griner, Lori N; Zhang, Ling; Clark, Justine A; Caceres, Gisela; Sokol, Lubomir; Komrokji, Rami S; Reuther, Gary W; Wei, Sheng; Tavernier, Jan; List, Alan F

    2016-06-15

    In a subset of patients with non-del(5q) myelodysplastic syndrome (MDS), lenalidomide promotes erythroid lineage competence and effective erythropoiesis. To determine the mechanism by which lenalidomide promotes erythropoiesis, we investigated its action on erythropoietin receptor (EpoR) cellular dynamics. Lenalidomide upregulated expression and stability of JAK2-associated EpoR in UT7 erythroid cells and primary CD71+ erythroid progenitors. The effects of lenalidomide on receptor turnover were Type I cytokine receptor specific, as evidenced by coregulation of the IL3-Rα receptor but not c-Kit. To elucidate this mechanism, we investigated the effects of lenalidomide on the E3 ubiquitin ligase RNF41. Lenalidomide promoted EpoR/RNF41 association and inhibited RNF41 auto-ubiquitination, accompanied by a reduction in EpoR ubiquitination. To confirm that RNF41 is the principal target responsible for EpoR stabilization, HEK293T cells were transfected with EpoR and/or RNF41 gene expression vectors. Steady-state EpoR expression was reduced in EpoR/RNF41 cells, whereas EpoR upregulation by lenalidomide was abrogated, indicating that cellular RNF41 is a critical determinant of drug-induced receptor modulation. Notably, shRNA suppression of CRBN gene expression failed to alter EpoR upregulation, indicating that drug-induced receptor modulation is independent of cereblon. Immunohistochemical staining showed that RNF41 expression decreased in primary erythroid cells of lenalidomide-responding patients, suggesting that cellular RNF41 expression merits investigation as a biomarker for lenalidomide response. Our findings indicate that lenalidomide has E3 ubiquitin ligase inhibitory effects that extend to RNF41 and that inhibition of RNF41 auto-ubiquitination promotes membrane accumulation of signaling competent JAK2/EpoR complexes that augment Epo responsiveness. Cancer Res; 76(12); 3531-40. ©2016 AACR. PMID:27197154

  13. Erythropoietin receptor expression is a potential prognostic factor in human lung adenocarcinoma.

    Anita Rózsás

    Full Text Available Recombinant human erythropoietins (rHuEPOs are used to treat cancer-related anemia. Recent preclinical studies and clinical trials, however, have raised concerns about the potential tumor-promoting effects of these drugs. Because the clinical significance of erythropoietin receptor (EPOR signaling in human non-small cell lung cancer (NSCLC also remains controversial, our aim was to study whether EPO treatment modifies tumor growth and if EPOR expression has an impact on the clinical behavior of this malignancy. A total of 43 patients with stage III-IV adenocarcinoma (ADC and complete clinicopathological data were included. EPOR expression in human ADC samples and cell lines was measured by quantitative real-time polymerase chain reaction. Effects of exogenous rHuEPOα were studied on human lung ADC cell lines in vitro. In vivo growth of human ADC xenografts treated with rHuEPOα with or without chemotherapy was also assessed. In vivo tumor and endothelial cell (EC proliferation was determined by 5-bromo-2'-deoxy-uridine (BrdU incorporation and immunofluorescent labeling. Although EPOR mRNA was expressed in all of the three investigated ADC cell lines, rHuEPOα treatment (either alone or in combination with gemcitabine did not alter ADC cell proliferation in vitro. However, rHuEPOα significantly decreased tumor cell proliferation and growth of human H1975 lung ADC xenografts. At the same time, rHuEPOα treatment of H1975 tumors resulted in accelerated tumor endothelial cell proliferation. Moreover, in patients with advanced stage lung ADC, high intratumoral EPOR mRNA levels were associated with significantly increased overall survival. This study reveals high EPOR level as a potential novel positive prognostic marker in human lung ADC.

  14. Widespread Expression of Erythropoietin Receptor in Brain and Its Induction by Injury

    Ott, Christoph; Martens, Henrik; Hassouna, Imam; Oliveira, Bárbara; Erck, Christian; Zafeiriou, Maria-Patapia; Peteri, Ulla-Kaisa; Hesse, Dörte; Gerhart, Simone; Altas, Bekir; Kolbow, Tekla; Stadler, Herbert; Kawabe, Hiroshi; Zimmermann, Wolfram-Hubertus; Nave, Klaus-Armin; Schulz-Schaeffer, Walter; Jahn, Olaf; Ehrenreich, Hannelore

    2015-01-01

    Erythropoietin (EPO) exerts potent neuroprotective, neuroregenerative and procognitive functions. However, unequivocal demonstration of erythropoietin receptor (EPOR) expression in brain cells has remained difficult since previously available anti-EPOR antibodies (EPOR-AB) were unspecific. We report here a new, highly specific, polyclonal rabbit EPOR-AB directed against different epitopes in the cytoplasmic tail of human and murine EPOR and its characterization by mass spectrometric analysis of immuno-precipitated endogenous EPOR, Western blotting, immunostaining and flow cytometry. Among others, we applied genetic strategies including overexpression, Lentivirus-mediated conditional knockout of EpoR and tagged proteins, both on cultured cells and tissue sections, as well as intracortical implantation of EPOR-transduced cells to verify specificity. We show examples of EPOR expression in neurons, oligodendroglia, astrocytes and microglia. Employing this new EPOR-AB with double-labeling strategies, we demonstrate membrane expression of EPOR as well as its localization in intracellular compartments such as the Golgi apparatus. Moreover, we show injury-induced expression of EPOR. In mice, a stereotactically applied stab wound to the motor cortex leads to distinct EpoR expression by reactive GFAP-expressing cells in the lesion vicinity. In a patient suffering from epilepsy, neurons and oligodendrocytes of the hippocampus strongly express EPOR. To conclude, this new analytical tool will allow neuroscientists to pinpoint EPOR expression in cells of the nervous system and to better understand its role in healthy conditions, including brain development, as well as under pathological circumstances, such as upregulation upon distress and injury. PMID:26349059

  15. Macrophages as novel targets for erythropoietin

    Lifshitz, L; Tabak, G; Mittelman, M.; Gassmann, M.; Neumann, D.

    2010-01-01

    BackgroundOur original demonstration of immunomodulatory effects of erythropoietin in multiple myeloma, led us to the search of the cells in the immune system that are direct targets to erythropoietin. The finding that lymphocytes do not express erythropoietin receptors, has led to the hypothesis that other cells act as direct targets and thus mediate the erythropoietin effects. Having found erythropoietin effects on dendritic cells thus led to the question of whether macrophages act as targe...

  16. Agonists of the tissue-protective erythropoietin receptor in the treatment of Parkinson's disease.

    Punnonen, Juha; Miller, James L; Collier, Timothy J; Spencer, Jeffrey R

    2015-01-01

    Parkinson's disease (PD) is a neurodegenerative disease affecting more than a million people in the USA alone. While there are effective symptomatic treatments for PD, there is an urgent need for new therapies that slow or halt the progressive death of dopaminergic neurons. Significant progress has been made in understanding the pathophysiology of PD, which has substantially facilitated the discovery efforts to identify novel drugs. The tissue-protective erythropoietin (EPO) receptor, EPOR/CD131, has emerged as one promising target for disease-modifying therapies. Recombinant human EPO (rhEPO), several variants of EPO, EPO-mimetic peptides, cell-based therapies using cells incubated with or expressing EPO, gene therapy vectors encoding EPO, and small molecule EPO mimetic compounds all show potential as therapeutic candidates. Agonists of the EPOR/CD131 receptor demonstrate potent anti-apoptotic, antioxidant, and anti-inflammatory effects and protect neurons, including dopaminergic neurons, from diverse insults in vitro and in vivo. When delivered directly to the striatum, rhEPO protects dopaminergic neurons in animal models of PD. Early-stage clinical trials testing systemic rhEPO have provided encouraging results, while additional controlled studies are required to fully assess the potential of the treatment. Poor CNS availability of proteins and challenges related to invasive delivery limit delivery of EPO protein. Several variants of EPO and small molecule agonists of the EPO receptors are making progress in preclinical studies and may offer solutions to these challenges. While EPO was initially discovered as the primary modulator of erythropoiesis, the discovery and characterization of the tissue-protective EPOR/CD131 receptor offer an opportunity to selectively target the neuroprotective receptor as an approach to identify disease-modifying treatments for PD. PMID:25832721

  17. Study of the erythropoiesis activity of nano-encapsulated forms of erythropoietin

    Zhanagul Khasenbekova

    2014-01-01

    Full Text Available Introduction: The recombinant human erythropoietin (rhEPO is used in the treatment of anemia. In order to improve its pharmacokinetic properties, nanoparticles of biodegradable polymers of natural or synthetic origin were used. The aim of this study was to investigate the effect of new nano-encapsulated forms of recombinant human erythropoietin for oral use on the erythropoiesis in the cyclophosphamide immunosuppression model. Material and methods: The CHOpE immortalized cells culture (a primary producer of rhEPO "Vector" in Russia was used. The following biodegradable polymers were chosen: 0.05% and 0.005% carbopol, 0.05% and 0.005% kollidon, and 0.05% and 0.005% pectin. Immunosuppression was obtained by a single dose of i.p. injection of cyclophosphamide (250 mg/kg in white mice (18-20 g. During the next 5 days, the nano-encapsulated erythropoietin (100 ED/mouse was administered orally to each mouse. After 5 and 10 days, the cell count of the number of blood reticulocytes and the myelogram of bone marrow were performed. The control group of mice received injections of Eprex. Results: On the 5th day of the experiment, the highest level of reticulocyte was observed in the samples of erythropoietin with kollidon (0.05% and pectin (0.005% nanoparticles. On the 10th day, the highest activity was observed in the samples of erythropoietin substance with pectin at 0.05% and 0.005% concentrations. The levels of reticulocytes in these groups reached 13.53% and 14.55%, respectively. The results of the myelogram during immunosuppression showed some activity of erythropoietin in conjunction with both concentrations of pectin when a two-fold increase in the number of erythroblasts was observed on the 5th day. High degrees of erythrokaryocytes in the state of mitosis were observed in the 0.05% pectin samples. Similar results were observed in equivalent groups of control animals on the 10th day of the experiment, which is compatible with the data on Eprex

  18. Erythropoietin -induced proliferation of gastric mucosal cells

    Itoh, Kazuro; Sawasaki, Yoshio; Takeuchi, Kyoko; Kato, Shingo; Imai, Nobuhiro; Kato, Yoichiro; Shibata, Noriyuki; KOBAYASHI, MAKIO; Moriguchi, Yoshiyuki; Higuchi, Masato; Ishihata, Fumio; Sudoh, Yushi; Miura, Soichiro

    2006-01-01

    AIM: To analyze the localization of erythropoietin receptor on gastric specimens and characterize the effects of erythropoietin on the normal gastric epithelial proliferation using a porcine gastric epithelial cell culture model.

  19. Lenalidomide induces lipid raft assembly to enhance erythropoietin receptor signaling in myelodysplastic syndrome progenitors.

    Kathy L McGraw

    Full Text Available Anemia remains the principal management challenge for patients with lower risk Myelodysplastic Syndromes (MDS. Despite appropriate cytokine production and cellular receptor display, erythropoietin receptor (EpoR signaling is impaired. We reported that EpoR signaling is dependent upon receptor localization within lipid raft microdomains, and that disruption of raft integrity abolishes signaling capacity. Here, we show that MDS erythroid progenitors display markedly diminished raft assembly and smaller raft aggregates compared to normal controls (p = 0.005, raft number; p = 0.023, raft size. Because lenalidomide triggers raft coalescence in T-lymphocytes promoting immune synapse formation, we assessed effects of lenalidomide on raft assembly in MDS erythroid precursors and UT7 cells. Lenalidomide treatment rapidly induced lipid raft formation accompanied by EpoR recruitment into raft fractions together with STAT5, JAK2, and Lyn kinase. The JAK2 phosphatase, CD45, a key negative regulator of EpoR signaling, was displaced from raft fractions. Lenalidomide treatment prior to Epo stimulation enhanced both JAK2 and STAT5 phosphorylation in UT7 and primary MDS erythroid progenitors, accompanied by increased STAT5 DNA binding in UT7 cells, and increased erythroid colony forming capacity in both UT7 and primary cells. Raft induction was associated with F-actin polymerization, which was blocked by Rho kinase inhibition. These data indicate that deficient raft integrity impairs EpoR signaling, and provides a novel strategy to enhance EpoR signal fidelity in non-del(5q MDS.

  20. Erythropoietin in Brain Development and Beyond

    Mawadda Alnaeeli; Li Wang; Barbora Piknova; Heather Rogers; Xiaoxia Li; Constance Tom Noguchi

    2012-01-01

    Erythropoietin is known as the requisite cytokine for red blood cell production. Its receptor, expressed at a high level on erythroid progenitor/precursor cells, is also found on endothelial, neural, and other cell types. Erythropoietin and erythropoietin receptor expression in the developing and adult brain suggest their possible involvement in neurodevelopment and neuroprotection. During ischemic stress, erythropoietin, which is hypoxia inducible, can contribute to brain homeostasis by incr...

  1. In-vivo detection of the erythropoietin receptor in tumours using positron emission tomography

    Fuge, Felix; Doleschel, Dennis; Rix, Anne; Gremse, Felix; Lederle, Wiltrud; Kiessling, Fabian [RWTH Aachen University, Department for Experimental Molecular Imaging (ExMI), Medical Faculty, Aachen (Germany); Wessner, Axel [Roche Diagnostics GmbH, R and D RPD Protein Chemistry, Penzberg (Germany); Winz, Oliver; Mottaghy, Felix [University Clinic RWTH Aachen, Clinic for Nuclear Medicine, Aachen (Germany)

    2014-09-09

    Recombinant human erythropoietin (rhuEpo) is used clinically to treat anaemia. However, rhuEpo-treated cancer patients show decreased survival rates and erythropoietin receptor (EpoR) expression has been found in patient tumour tissue. Thus, rhuEpo application might promote EpoR{sup +} tumour progression. We therefore developed the positron emission tomography (PET)-probe {sup 68}Ga-DOTA-rhuEpo and evaluated its performance in EpoR{sup +} A549 non-small-cell lung cancer (NSCLC) xenografts. {sup 68}Ga-DOTA-rhuEpo was generated by coupling DOTA-hydrazide to carbohydrate side-chains of rhuEpo. Biodistribution was determined in tumour-bearing mice 0.5, 3, 6, and 9 h after probe injection. Competition experiments were performed by co-injecting {sup 68}Ga-DOTA-rhuEpo and rhuEpo in five-fold excess. Probe specificity was further evaluated histologically using Epo-Cy5.5 stainings. The blood half-life of {sup 68}Ga-DOTA-rhuEpo was 2.6 h and the unbound fraction was cleared by the liver and kidney. After 6 h, the highest tumour to muscle ratio was reached. The highest {sup 68}Ga-DOTA-rhuEpo accumulation was found in liver (10.06 ± 6.26%ID/ml), followed by bone marrow (1.87 ± 0.53%ID/ml), kidney (1.58 ± 0.39 %ID/ml), and tumour (0.99 ± 0.16%ID/ml). EpoR presence in these organs was histologically confirmed. Competition experiments showed significantly (p < 0.05) lower PET-signals in tumour and bone marrow at 3 and 6 h. {sup 68}Ga-DOTA-rhuEpo shows favourable pharmacokinetic properties and detects EpoR specifically. Therefore, it might become a valuable radiotracer to monitor EpoR status in tumours and support decision-making in anaemia therapy. (orig.)

  2. The effect of erythropoietin on normal and neoplastic cells

    Elliott S; Sinclair AM

    2012-01-01

    Steve Elliott, Angus M SinclairOncology Research, Amgen, Thousand Oaks, CA, USAAbstract: Erythropoietin (Epo) is an essential hormone that binds and activates the Epo receptor (EpoR) resident on the surface of erythroid progenitor cells, thereby promoting erythropoiesis. Recombinant human erythropoietin has been used successfully for over 20 years to treat anemia in millions of patients. In addition to erythropoiesis, Epo has also been reported to have other effects, such as tissue protection...

  3. Erythropoietin Pathway: A Potential Target for the Treatment of Depression.

    Ma, Chongyang; Cheng, Fafeng; Wang, Xueqian; Zhai, Changming; Yue, Wenchao; Lian, Yajun; Wang, Qingguo

    2016-01-01

    During the past decade, accumulating evidence from both clinical and experimental studies has indicated that erythropoietin may have antidepressant effects. In addition to the kidney and liver, many organs have been identified as secretory tissues for erythropoietin, including the brain. Its receptor is expressed in cerebral and spinal cord neurons, the hypothalamus, hippocampus, neocortex, dorsal root ganglia, nerve axons, and Schwann cells. These findings may highlight new functions for erythropoietin, which was originally considered to play a crucial role in the progress of erythroid differentiation. Erythropoietin and its receptor signaling through JAK2 activate multiple downstream signaling pathways including STAT5, PI3K/Akt, NF-κB, and MAPK. These factors may play an important role in inflammation and neuroprogression in the nervous system. This is particularly true for the hippocampus, which is possibly related to learning, memory, neurocognitive deficits and mood alterations. Thus, the influence of erythropoietin on the downstream pathways known to be involved in the treatment of depression makes the erythropoietin-related pathway an attractive target for the development of new therapeutic approaches. Focusing on erythropoietin may help us understand the pathogenic mechanisms of depression and the molecular basis of its treatment. PMID:27164096

  4. Pleiotrophic actions of erythropoietin

    Feldman, Laurie; Sytkowski, Arthur J.

    2003-01-01

    Erythropoietin is the prime regulator of red blood cell production. However, recent evidence suggests that the hormone has multiple effects outside the hematopoietic system. Functional receptors have been identified on a wide variety of normal and malignant cell types, and numerous biologic effects of the hormone on these cells have been observed both in vitro and in vivo. These findings are causing a reassessment of the understanding of erythropoietin physiology. Moreover, there are importan...

  5. Erythropoietin: Current Status

    Bunn, Howard Franklin

    1990-01-01

    Understanding the regulation of red blood cell production has been greatly enhanced by the cloning and expression of the gene for human erythropoietin (Epo) and its receptor. The availability of recombinant human erythropoietin (rhEpo) for administration to patients has ushered in a new era in molecular medicine. Intravenous or subcutaneous administration of rhEpo can reliably cure the anemia of chronic renal failure and may be effective in the treatment of anemias secondary to chronic inflam...

  6. Clinical significance of erythropoietin receptor expression in oral squamous cell carcinoma

    Hypoxic tumors are refractory to radiation and chemotherapy. High expression of biomarkers related to hypoxia in head and neck cancer is associated with a poorer prognosis. The present study aimed to evaluate the clinicopathological significance of erythropoietin receptor (EPOR) expression in oral squamous cell carcinoma (OSCC). The study included 256 patients who underwent primary surgical resection between October 1996 and August 2005 for treatment of OSCC without previous radiotherapy and/or chemotherapy. Clinicopathological information including gender, age, T classification, N classification, and TNM stage was obtained from clinical records and pathology reports. The mRNA and protein expression levels of EPOR in OSCC specimens were evaluated by Q-RT-PCR, Western blotting and immunohistochemistry assays. We found that EPOR were overexpressed in OSCC tissues. The study included 17 women and 239 men with an average age of 50.9 years (range, 26–87 years). The mean follow-up period was 67 months (range, 2–171 months). High EPOR expression was significantly correlated with advanced T classification (p < 0.001), advanced TNM stage (p < 0.001), and positive N classification (p = 0.001). Furthermore, the univariate analysis revealed that patients with high tumor EPOR expression had a lower 5-year overall survival rate (p = 0.0011) and 5-year disease-specific survival rate (p = 0.0017) than patients who had low tumor levels of EPOR. However, the multivariate analysis using Cox’s regression model revealed that only the T and N classifications were independent prognostic factors for the 5-year overall survival and 5-year disease-specific survival rates. High EPOR expression in OSCC is associated with an aggressive tumor behavior and poorer prognosis in the univariate analysis among patients with OSCC. Thus, EPOR expression may serve as a treatment target for OSCC in the future

  7. Chronic Treatment With an Erythropoietin Receptor Ligand Prevents Chronic Kidney Disease-Induced Enlargement of Myocardial Infarct Size.

    Nishizawa, Keitaro; Yano, Toshiyuki; Tanno, Masaya; Miki, Takayuki; Kuno, Atsushi; Tobisawa, Toshiyuki; Ogasawara, Makoto; Muratsubaki, Shingo; Ohno, Kouhei; Ishikawa, Satoko; Miura, Tetsuji

    2016-09-01

    Chronic kidney disease (CKD) is known to increase myocardial infarct size after ischemia/reperfusion. However, a strategy to prevent the CKD-induced myocardial susceptibility to ischemia/reperfusion injury has not been developed. Here, we examined whether epoetin β pegol, a continuous erythropoietin receptor activator (CERA), normalizes myocardial susceptibility to ischemia/reperfusion injury by its effects on protective signaling and metabolomes in CKD. CKD was induced by 5/6 nephrectomy in rats (subtotal nephrectomy, SNx), whereas sham-operated rats served controls (Sham). Infarct size as percentage of area at risk after 20-minutes coronary occlusion/2-hour reperfusion was larger in SNx than in Sham: 60.0±4.0% versus 43.9±2.2%. Administration of CERA (0.6 μg/kg SC every 7 days) for 4 weeks reduced infarct size in SNx (infarct size as percentage of area at risk=36.9±3.9%), although a protective effect was not detected for the acute injection of CERA. Immunoblot analyses revealed that myocardial phospho-Akt-Ser473 levels under baseline conditions and on reperfusion were lower in SNx than in Sham, and CERA restored the Akt phosphorylation on reperfusion. Metabolomic analyses showed that glucose 6-phosphate and glucose 1-phosphate were reduced and malate:aspartate ratio was 1.6-fold higher in SNx than in Sham, suggesting disturbed flux of malate-aspartate shuttle by CKD. The CERA improved the malate:aspartate ratio in SNx to the control level. In H9c2 cells, mitochondrial Akt phosphorylation by insulin-like growth factor-1 was attenuated by malate-aspartate shuttle inhibition. In conclusion, the results suggest that a CERA prevents CKD-induced susceptibility of the myocardium to ischemia/reperfusion injury by restoration of Akt-mediated signaling possibly via normalized malate-aspartate shuttle flux. PMID:27456523

  8. Identification of the receptor for erythropoietin by cross-linking to Friend virus-infected erythroid cells

    Erythropoietin (Epo) is a glycoprotein hormone that regulates erythroid development and interacts with surface receptors on developing erythroid cells. In this laboratory, a cell system with a relatively pure population of erythroid cells that respond to Epo has been developed. Immature erythroid cells are obtained from the spleens of mice infected with the anemia strain of Friend virus. The binding of 125I-labeled Epo (125-Epo) to plasma membranes from these cells was studied in this investigation. 125I-Epo binding reached equilibrium within 20 min at 370C. Twenty percent of the receptors bound 125I-Epo with a K/sub d/ of 0.08 x 10-9 M, while the remaining receptors bound the hormone with a k/sub d/ of 0.6 x 10-9 M. In this study, a receptor for Epo was identified by cross-linking 125I-Epo to the receptor in intact cells and plasma membrane preparations using disuccinimidyl suberate. Polyacrylamide gel electrophoresis revealed two labeled bands of 100 and 85 kDa. The 85-kDa band was more heavily labeled (65%) than the 100-kDa band. Both bands were equally decreased when increasing amounts of unlabeled Epo were included in the binding mixture, indicating a specific interaction of 175I-Epo with the receptor

  9. An amphipathic motif at the transmembrane-cytoplasmic junction prevents autonomous activation of the thrombopoietin receptor.

    Staerk, Judith; Lacout, Catherine; Sato, Takeshi; Smith, Steven O.; Vainchenker, William; Constantinescu, Stefan

    2006-01-01

    Ligand binding to the thrombopoietin receptor (TpoR) is thought to impose a dimeric receptor conformation(s) leading to hematopoietic stem cell renewal, megakaryocyte differentiation, and platelet formation. Unlike other cytokine receptors, such as the erythropoietin receptor, TpoR contains an amphipathic KWQFP motif at the junction between the transmembrane (TM) and cytoplasmic domains. We show here that a mutant TpoR (delta5TpoR), where this sequence was deleted, is constitutively active. I...

  10. Erythropoietin Receptor Positive Circulating Progenitor Cells and Endothelial Progenitor Cells in Patients with Different Stages of Diabetic Retinopathy

    Liu-mei Hu; Guo-xu Xu; Guo-tong XU; Wei-ye Li; Xia Lei; Bo Ma; Yu Zhang; Yan Yan; Ya-lan Wu; Ge-zhi Xu; Wen Ye; Ling Wang

    2011-01-01

    Objective To investigate the possible involvement of erythropoietin (EPO)/erythropoietin receptor(EPOR) system in neovascularization and vascular regeneration in diabetic retinopathy (DR).Methods EPOR positive circulating progenitor cells (CPCs: CD34+) and endothelial progenitor cells (EPCs: CD34+KDR+) were assessed by flow cytometry in type 2 diabetic patients with different stages of DR. The cohort consisted of age- and sex-matched control patients without diabetes (n=7), non-prolif-erative DR (NPDR, n=7), proliferative DR (PDR, n=8), and PDR complicated with diabetic nephropathy (PDR-DN, n=7). Results The numbers of EPOR+ CPCs and EPOR+ EPCs were reduced remarkably in NPDR compared with the control group (both P<0.01), whereas rebounded in PDR and PDR-DN groups in varying degrees. Similar changes were observed in respect of the proportion of EPOR+ CPCs in CPCs (NPDR vs.control, P< 0.01) and that of EPOR+ EPCs in EPCs (NPDR vs. control, P< 0.05). Conclusion Exogenous EPO, mediated via the EPO/EPOR system of EPCs, may alleviate the im-paired vascular regeneration in NPDR, whereas it might aggravate retinal neovascularization in PDR due to a rebound of EPOR+ EPCs associated with ischemia.

  11. Prognostic Impact of Erythropoietin Expression and Erythropoietin Receptor Expression on Locoregional Control and Survival of Patients Irradiated for Stage II/III Non-Small-Cell Lung Cancer

    Purpose: Prognostic factors can guide the physician in selecting the optimal treatment for an individual patient. This study investigates the prognostic value of erythropoietin (EPO) and EPO receptor (EPO-R) expression of tumor cells for locoregional control and survival in non-small-cell lung cancer (NSCLC) patients. Methods and Materials: Fourteen factors were investigated in 62 patients irradiated for stage II/III NSCLC, as follows: age, gender, Karnofsky performance score (KPS), histology, grading, TNM/American Joint Committee on Cancer (AJCC) stage, surgery, chemotherapy, pack years (average number of packages of cigarettes smoked per day multiplied by the number of years smoked), smoking during radiotherapy, hemoglobin levels during radiotherapy, EPO expression, and EPO-R expression. Additionally, patients with tumors expressing both EPO and EPO-R were compared to those expressing either EPO or EPO-R and to those expressing neither EPO nor EPO-R. Results: On univariate analysis, improved locoregional control was associated with AJCC stage II cancer (p 70 (p = 0.08), an N stage of 0 to 1 (p = 0.07), and no EPO-R expression (p = 0.10). On multivariate analysis, AJCC stage II and no EPO expression remained significant. No smoking during radiotherapy was almost significant. On univariate analysis, improved survival was associated with N stage 0 to 1 (p = 0.009), surgery (p = 0.039), hemoglobin levels of ≥12 g/d (p = 0.016), and no EPO expression (p = 0.001). On multivariate analysis, N stage 0 to 1 and no EPO expression maintained significance. Hemoglobin levels of ≥12 g/d were almost significant. On subgroup analyses, patients with tumors expressing both EPO and EPO-R had worse outcomes than those expressing either EPO or EPO-R and those expressing neither EPO nor RPO-R. Conclusions: EPO expression of tumor cells was an independent prognostic factor for locoregional control and survival in patients irradiated for NSCLC. EPO-R expression showed a trend

  12. Effects of Recombinant Erythropoietin on Breast Cancer-Initiating Cells

    Tiffany M. Phillips

    2007-12-01

    Full Text Available BACKGROUND: Cancer anemia causes fatigue and correlates with poor treatment outcome. Erythropoietin has been introduced in an attempt to correct these defects. However, five recent clinical trials reported a negative impact of erythropoietin on survival and/or tumor control, indicating that experimental evaluation of a possible direct effect of erythropoietin on cancer cells is required. Cancer recurrence is thought to rely on the proliferation of cancer initiating cells (CICs. In breast cancer, CICs can be identified by phenotypic markers and their fate is controlled by the Notch pathway. METHODS: In this study, we investigated the effect of erythropoietin on CICs in breast cancer cell lines. Levels of erythropoietin receptor (EpoR, CD24, CD44, Jagged-1 expression, activation of Notch-1 were assessed by flow cytometry. Self-renewing capacity of CICs was investigated in sphere formation assays. RESULTS: EpoR expression was found on the surface of CICs. Recombinant human Epo (rhEpo increased the numbers of CICs and self-renewing capacity in a Notch-dependent fashion by induction of Jagged-1. Inhibitors of the Notch pathway and P13-kinase blocked both effects. CONCLUSIONS: Erythropoietin functionally affects CICs directly. Our observation may explain the negative impact of recombinant Epo on local control and survival of cancer patients with EpoR-positive tumors.

  13. Effects of Erythropoietin on the Bone Microenvironment

    McGee, SJ; Havens, AM; Shiozawa, Y.; Jung, Y.; Taichman, RS

    2011-01-01

    It has well been established that blood and bone share a unique, regulatory relationship with one another, though the specifics of this relationship still remain unanswered. Erythropoietin (Epo) is known primarily for its role as a hematopoietic hormone. However, after the discovery of Epo receptor (Epo-R) outside the hematopoietic tissues, Epo has been avidly studied for its possible non-hematopoietic effects. It has been proposed that Epo interacts with bone both directly, by activating bon...

  14. Role of erythropoietin in the angiogenic activity of bone marrow endothelial cells of MGUS and multiple myeloma patients.

    Lamanuzzi, Aurelia; Saltarella, Ilaria; Ferrucci, Arianna; Ria, Roberto; Ruggieri, Simona; Racanelli, Vito; Rao, Luigia; Annese, Tiziana; Nico, Beatrice; Vacca, Angelo; Ribatti, Domenico

    2016-03-22

    Increasing evidences suggest several biological roles for erythropoietin and its receptor (Epo and EpoR), unrelated to erythropoiesis, including angiogenesis. Here, we detected the expression of EpoR in bone marrow-derived endothelial cells from monoclonal gammopathy of undetermined significance (MGUS) and multiple myeloma (MM) patients (MGECs and MMECs, respectively) and assessed whether Epo plays a role in MGECs- and MMECs-mediated angiogenesis. We show that EpoR is expressed by both MGECs and MMECs even though at a higher level in the first ones. Both EC types respond to rHuEpo in terms of cell proliferation, whereas other responses, including activation of JAK2/STAT5 and PI3K/Akt pathways, cell migration and capillarogenesis are enhanced by Epo in MGECs, but not in MMECs. In addition, the conditioned media of both Epo-treated cells induce a strong angiogenic response in vivo in the chorioallantoic membrane assay, comparable to that of vascular endothelial growth factor (VEGF). Overall, these data highlight the effect of Epo on MGECs- and MMECs-mediated angiogenesis: MGECs are more responsive to Epo treatment than MMECs, probably because over-angiogenic phenotype of MMECs is already activated by their autocrine/paracrine loops occurring in the "angiogenic switch" from MGUS. PMID:26919105

  15. Role of erythropoietin in the angiogenic activity of bone marrow endothelial cells of MGUS and multiple myeloma patients

    Ferrucci, Arianna; Ria, Roberto; Ruggieri, Simona; Racanelli, Vito; Rao, Luigia; Annese, Tiziana; Nico, Beatrice; Vacca, Angelo; Ribatti, Domenico

    2016-01-01

    Increasing evidences suggest several biological roles for erythropoietin and its receptor (Epo and EpoR), unrelated to erythropoiesis, including angiogenesis. Here, we detected the expression of EpoR in bone marrow-derived endothelial cells from monoclonal gammopathy of undetermined significance (MGUS) and multiple myeloma (MM) patients (MGECs and MMECs, respectively) and assessed whether Epo plays a role in MGECs- and MMECs-mediated angiogenesis. We show that EpoR is expressed by both MGECs and MMECs even though at a higher level in the first ones. Both EC types respond to rHuEpo in terms of cell proliferation, whereas other responses, including activation of JAK2/STAT5 and PI3K/Akt pathways, cell migration and capillarogenesis are enhanced by Epo in MGECs, but not in MMECs. In addition, the conditioned media of both Epo-treated cells induce a strong angiogenic response in vivo in the chorioallantoic membrane assay, comparable to that of vascular endothelial growth factor (VEGF). Overall, these data highlight the effect of Epo on MGECs- and MMECs-mediated angiogenesis: MGECs are more responsive to Epo treatment than MMECs, probably because over-angiogenic phenotype of MMECs is already activated by their autocrine/paracrine loops occurring in the “angiogenic switch” from MGUS. PMID:26919105

  16. Transferrin receptor number, synthesis, and endocytosis during erythropoietin-induced maturation of Friend virus-infected erythroid cells

    Erythropoietin (EP) responsive Friend virus-infected erythroid cells had 200,000 steady-state binding sites for transferrin at 370C when isolated from the spleens of Friend virus-infected mice. Upon culture of these cells with EP, the synthesis of transferrin receptors increased 4- to 7-fold and the number of transferrin-binding sites per cell doubled after 24 h. However, the rate of uptake of 59Fe from transferrin remained constant at approximately 35,000 atoms of 59Fe per minute per cell during this period in culture. The amount of 125I-transferrin internalized during the steady-state binding did not change during this culture period while the transferrin bound to the surface increased 3-fold. At all stages of erythroid maturation, the maximum rate of endocytosis was determined to be 18,000 molecules of transferrin per minute per cell, and the interval that 125I-transferrin remains in the interior of the cell was calculated to be 6.9 min. After 48 h of culture with EP, the number of steady-state transferrin-binding sites was reduced in part due to the sequestration of surface receptors within the cell. The uptake of iron from transferrin was limited by the level of endocytosis of transferrin during the initial phase of culture and the number of transferrin receptors at the cell surface during the latter stages of erythroid maturation of these cells

  17. Effects of erythropoietin on memory-relevant neurocircuitry activity and recall in mood disorders

    Miskowiak, K W; Macoveanu, J; Vinberg, M;

    2016-01-01

    OBJECTIVE: Erythropoietin (EPO) improves verbal memory and reverses subfield hippocampal volume loss across depression and bipolar disorder (BD). This study aimed to investigate with functional magnetic resonance imaging (fMRI) whether these effects were accompanied by functional changes in memory......MRI at 3T, mood ratings, and blood tests at baseline and week 14. During fMRI, participants performed a picture encoding task followed by postscan recall. RESULTS: Sixty-two patients had complete data (EPO: N = 32, saline: N = 30). EPO improved picture recall and increased encoding-related activity in...... cohort. The effects of EPO were not correlated with change in mood, red blood cells, blood pressure, or medication. CONCLUSION: The findings highlight enhanced encoding-related dlPFC and temporo-parietal activity as key neuronal underpinnings of EPO-associated memory improvement....

  18. Research Advances in Expression and Function of Erythropoietin and Erythropoietin Receptor in Tumors%促红细胞生成素及其受体在肿瘤中的表达及作用研究进展

    孔令英

    2011-01-01

    促红细胞生成素(EPO)最早被发现只在胎肝及成人肾脏中合成,可以促进红系集落形成单位的有丝分裂和增殖,并向形态可识别的前体细胞分化.研究发现,EPO及其受体(EPO-R)在多种不同非造血器官及组织中表达,并发挥组织保护、免疫调节、促进血管增生等作用.临床上已经成功地将重组的促红细胞生成素用于治疗肿瘤相关贫血等疾病.最新多项研究发现,在多种恶性肿瘤组织细胞中存在EPO及EPO-R的表达.%Erythropoietin was firstly found to be synthesized by fetal hepatocytes and human kidney, and it could stimulate the mitosis, proliferation, and differentiation into recognizable blood precursors of colonyforming unit-erythrocyte. Recent researches have shown EPO and its receptor( erythropoietin receptor, EPOR )express in many nonhematopoietic organs and multiple tissues, and exert tissue-protective,immunoregulatory and angiogenic effects in human body. Recombinant human erythropoietin( rh-EPO )has been successfully used in clinic to treat malignancy-associated anemia and other diseases. A series of studies have reported that EPO and EPO-R also express in various tumor cells.

  19. The gene for erythropoietin receptor is expressed in multipotential hematopoietic and embryonal stem cells: evidence for differentiation stage-specific regulation.

    Heberlein, C; Fischer, K D; Stoffel, M; Nowock, J; Ford, A.; Tessmer, U.; Stocking, C

    1992-01-01

    The principal regulator of erythropoiesis is the glycoprotein erythropoietin, which interacts with a specific cell surface receptor (EpoR). A study aimed at analyzing EpoR gene regulation has shown that both pluripotent embryonal stem cells and early multipotent hematopoietic cells express EpoR transcripts. Commitment to nonerythroid lineages (e.g., macrophage or lymphocytic) results in the shutdown of EpoR gene expression, whereas commitment to the erythroid lineage is concurrent with or fol...

  20. Functional regions of the mouse thrombopoietin receptor cytoplasmic domain: evidence for a critical region which is involved in differentiation and can be complemented by erythropoietin.

    Porteu, F; Rouyez, M C; Cocault, L; Bénit, L; Charon, M; Picard, F; Gisselbrecht, S; Souyri, M; Dusanter-Fourt, I

    1996-01-01

    Thrombopoietin (TPO) is the major regulator of growth and differentiation of megakaryocytes. To identify functionally important regions in the cytoplasmic domain of the TPO receptor, mpl, we introduced wild-type mpl and deletion mutants of murine mpl into the granulocyte-macrophage colony-stimulating factor (GM-CSF)- or erythropoietin (EPO)-dependent human cell line UT7. TPO induced differentiation of UT7-Wtmpl cells, not parental UT7 cells, along the megakaryocytic lineage, as evidenced by d...

  1. Biology of erythropoietin.

    Lacombe, C; Mayeux, P

    1998-08-01

    Erythropoietin (Epo) controls the proliferation, differentiation and survival of the erythroid progenitors. This cytokine was cloned in 1985 and rapidly became used for treatment of anemia of renal failure, opening the way to the first clinical trials of a hematopoietic growth factor. The clonage of one chain of the Epo receptor followed in 1989, thereby opening the research on intracellular signal transduction induced by Epo. Epo is synthesized mainly by the kidney and the liver and sequences required for tissue-specific expression have been localized in the Epo gene. A 3'enhancer is responsible for hypoxia-inducible Epo gene expression. HIF-1 alpha and beta proteins bind to this enhancer. Gene regulation by hypoxia is widespread in many cells and involves numerous genes in addition to the Epo gene. The Epo receptor belongs to the cytokine receptor family and includes a p66 chain which is dimerized upon Epo activation; two accessory proteins defined by cross-linking remain to be characterized. Epo binding induces the stimulation of Jak2 tyrosine kinase. Jak2 activation leads to the tyrosine phosphorylation of several proteins including the Epo receptor itself. As a result, different intracellular pathways are activated: Ras/MAP kinase, phosphatidylinositol 3-kinase and STAT transcription factors. However, the exact mechanisms by which the proliferation and/or the differentiation of erythroid cells are regulated after Epo stimulation are not known. Furthermore, target disruption of both Epo and Epo receptor showed that Epo was not involved in the commitment of the erythroid lineage and seemed to act mainly as a survival factor. PMID:9793257

  2. Recombinant human erythropoietin reduces plasminogen activator inhibitor and ameliorates pro-inflammatory responses following trauma

    M Mojtahedzadeh

    2011-05-01

    Full Text Available "n  "n Background and the purpose of the study: Besides its hematopoietic effects, erythropoietin (EPO by mobilization of iron and modulation of some inflammatory cytokines has antioxidant and anti-inflammatory properties. The purpose of this study was to evaluate these effects of erythropoietin and its impact on organ function in traumatized patients. "n Methods: Twenty-six ICU-admitted traumatized patients within 24 hrs after trauma were randomly assigned to the EPO (received EPO, 300 units/Kg/day and Control (not received EPO groups. The inflammatory biomarkers including Tumor Necrosis Factor alpha (TNF-α, Interleukin 1 (IL-1, Plasminogen Activator Inhibitor 1 (PAI-1 and Nitrotyrosine were recorded at the admission, 3, 6 and 9 days thereafter. Acute Physiology and Chronic Health Evaluation (APACHE II and Sequential Organ Failure Assessment (SOFA scores were also recorded. "n Results: Among 12 patients (EPO group TNF-α level at the day of 9 (P=0.046, and within EPO group at the days of 3 (P=0.026 ameliorate, 6 (P=0.016, and 9 (P=0.052 were significantly lowered. Level of IL-1 and PAI-1 decreased significantly at days of 3, 6 and 9 post intervention. Also there were significant differences between two groups in the SOFA score during three measured time intervals (the first, third and seventh days. "n Conclusion: From the results of this study it seems that injection of erythrocyte stimulating agent is well tolerated and inhibits the inflammatory response and oxidative stress following trauma.

  3. Characterization of a monoclonal antibody to human erythropoietin.

    Weiss, T L; Kavinsky, C J; Goldwasser, E

    1982-01-01

    Hybrid cells that synthesize a monospecific antibody directed toward erythropoietin have been produced by the fusion of mouse plasmacytoma cells with spleen cells from rats immunized against human erythropoietin. The antibody binds the alpha and beta forms and the asialo alpha form of erythropoietin to the same extent. It is an immunoglobulin of the IgG class and binds only erythropoietin in an impure preparation of the hormone. Biologically active unlabeled erythropoietin competes with biolo...

  4. Erythropoietin Activates Mitochondrial Biogenesis and Couples Red Cell Mass to Mitochondrial Mass in the Heart

    RATIONALE: Erythropoietin (EPO) is often administered to cardiac patients with anemia, particularly from chronic kidney disease, and stimulation of erythropoiesis may stabilize left ventricular and renal function by recruiting protective effects beyond the correction of anemia. O...

  5. Identification of Cell Type-Specific Differences in Erythropoietin Receptor Signaling in Primary Erythroid and Lung Cancer Cells

    Salopiata, Florian; Depner, Sofia; Wäsch, Marvin; Böhm, Martin E.; Mücke, Oliver; Plass, Christoph; Lehmann, Wolf D.; Kreutz, Clemens; Timmer, Jens; Klingmüller, Ursula

    2016-01-01

    Lung cancer, with its most prevalent form non-small-cell lung carcinoma (NSCLC), is one of the leading causes of cancer-related deaths worldwide, and is commonly treated with chemotherapeutic drugs such as cisplatin. Lung cancer patients frequently suffer from chemotherapy-induced anemia, which can be treated with erythropoietin (EPO). However, studies have indicated that EPO not only promotes erythropoiesis in hematopoietic cells, but may also enhance survival of NSCLC cells. Here, we verified that the NSCLC cell line H838 expresses functional erythropoietin receptors (EPOR) and that treatment with EPO reduces cisplatin-induced apoptosis. To pinpoint differences in EPO-induced survival signaling in erythroid progenitor cells (CFU-E, colony forming unit-erythroid) and H838 cells, we combined mathematical modeling with a method for feature selection, the L1 regularization. Utilizing an example model and simulated data, we demonstrated that this approach enables the accurate identification and quantification of cell type-specific parameters. We applied our strategy to quantitative time-resolved data of EPO-induced JAK/STAT signaling generated by quantitative immunoblotting, mass spectrometry and quantitative real-time PCR (qRT-PCR) in CFU-E and H838 cells as well as H838 cells overexpressing human EPOR (H838-HA-hEPOR). The established parsimonious mathematical model was able to simultaneously describe the data sets of CFU-E, H838 and H838-HA-hEPOR cells. Seven cell type-specific parameters were identified that included for example parameters for nuclear translocation of STAT5 and target gene induction. Cell type-specific differences in target gene induction were experimentally validated by qRT-PCR experiments. The systematic identification of pathway differences and sensitivities of EPOR signaling in CFU-E and H838 cells revealed potential targets for intervention to selectively inhibit EPO-induced signaling in the tumor cells but leave the responses in erythroid

  6. Identification of Cell Type-Specific Differences in Erythropoietin Receptor Signaling in Primary Erythroid and Lung Cancer Cells.

    Merkle, Ruth; Steiert, Bernhard; Salopiata, Florian; Depner, Sofia; Raue, Andreas; Iwamoto, Nao; Schelker, Max; Hass, Helge; Wäsch, Marvin; Böhm, Martin E; Mücke, Oliver; Lipka, Daniel B; Plass, Christoph; Lehmann, Wolf D; Kreutz, Clemens; Timmer, Jens; Schilling, Marcel; Klingmüller, Ursula

    2016-08-01

    Lung cancer, with its most prevalent form non-small-cell lung carcinoma (NSCLC), is one of the leading causes of cancer-related deaths worldwide, and is commonly treated with chemotherapeutic drugs such as cisplatin. Lung cancer patients frequently suffer from chemotherapy-induced anemia, which can be treated with erythropoietin (EPO). However, studies have indicated that EPO not only promotes erythropoiesis in hematopoietic cells, but may also enhance survival of NSCLC cells. Here, we verified that the NSCLC cell line H838 expresses functional erythropoietin receptors (EPOR) and that treatment with EPO reduces cisplatin-induced apoptosis. To pinpoint differences in EPO-induced survival signaling in erythroid progenitor cells (CFU-E, colony forming unit-erythroid) and H838 cells, we combined mathematical modeling with a method for feature selection, the L1 regularization. Utilizing an example model and simulated data, we demonstrated that this approach enables the accurate identification and quantification of cell type-specific parameters. We applied our strategy to quantitative time-resolved data of EPO-induced JAK/STAT signaling generated by quantitative immunoblotting, mass spectrometry and quantitative real-time PCR (qRT-PCR) in CFU-E and H838 cells as well as H838 cells overexpressing human EPOR (H838-HA-hEPOR). The established parsimonious mathematical model was able to simultaneously describe the data sets of CFU-E, H838 and H838-HA-hEPOR cells. Seven cell type-specific parameters were identified that included for example parameters for nuclear translocation of STAT5 and target gene induction. Cell type-specific differences in target gene induction were experimentally validated by qRT-PCR experiments. The systematic identification of pathway differences and sensitivities of EPOR signaling in CFU-E and H838 cells revealed potential targets for intervention to selectively inhibit EPO-induced signaling in the tumor cells but leave the responses in erythroid

  7. Comparison of real time RT-PCR and flow cytometry methods for evaluation of biological activity of recombinant human erythropoietin

    Sepehrizadeh Z

    2008-05-01

    Full Text Available Background: Evaluation of bioactivity of recombinant erythropoietin is essential for pharmaceutical industry, quality control authorities and researchers. The purpose of this study was to compare real time RT-PCR and flow cytometry for the assay of biological activity of recombinant erythropoietin. Methods: Three concentrations of recombinant erythropoietin BRP (80, 40 and 20 IU/ml were injected subcutaneously to mice. After 4 days the blood was collected and used for reticulocyte counts by flow cytometry and also for the RNA extraction. Real time RT-PCR amplification was carried out for β-globin. Results and conclusion: There was a significant correlation between the total RNA amounts (R2= 0.9995, relative quantity of β-globin mRNA (R2= 0.984 and reticulocyte counts (R2= 0.9742 with rhEpo concentrations. Total RNA and quantitative RT-PCR showed significant dose dependent results as well the reticulocyte counts by flow cytometry for the biological activity assay of rhEpo and so these methods could be considered as alternatives for flow cytometry.

  8. Nonclinical evaluation of the potential for mast cell activation by an erythropoietin analog.

    Weaver, James L; Boyne, Michael; Pang, Eric; Chimalakonda, Krishna; Howard, Kristina E

    2015-09-15

    The erythropoietin analog peginesatide was withdrawn from marketing due to unexpected severe anaphylactic reactions associated with administration of the multi-use formulation. The adverse events occurred rapidly following the first ever administration of the drug with most affected patients becoming symptomatic in less than 30min. This is most consistent with an anaphylactoid reaction due to direct activation of mast cells. Laboratory evaluation was undertaken using rat peritoneal mast cells as the model system. Initial studies showed that high concentrations of the formulated drug as well as formulated vehicle alone could cause mast cell degranulation as measured by histamine release. The purified active drug was not able to cause histamine release whereas the vehicle filtrate and lab created drug vehicle were equally potent at causing histamine release. Individual formulations of vehicle leaving one component out showed that histamine release was due to phenol. Dose response studies with phenol showed a very sharp dose response curve that was similar in three buffer systems. Cellular analysis by flow cytometry showed that the histamine release was not due to cell death, and that changes in light scatter parameters consistent with degranulation were rapidly observed. Limited testing with primary human mast cells showed a similar dose response of histamine release with exposure to phenol. To provide in vivo confirmation, rats were injected with vehicle formulated with various concentrations of phenol via a jugular vein cannula. Significant release of histamine was detected in blood samples taken 2min after dosing at the highest concentrations tested. PMID:26079829

  9. Erythropoietin Modulates Cerebral and Serum Degradation Products from Excess Calpain Activation following Prenatal Hypoxia-Ischemia.

    Jantzie, Lauren L; Winer, Jesse L; Corbett, Christopher J; Robinson, Shenandoah

    2016-01-01

    Preterm infants suffer central nervous system (CNS) injury from hypoxia-ischemia and inflammation - termed encephalopathy of prematurity. Mature CNS injury activates caspase and calpain proteases. Erythropoietin (EPO) limits apoptosis mediated by activated caspases, but its role in modulating calpain activation has not yet been investigated extensively following injury to the developing CNS. We hypothesized that excess calpain activation degrades developmentally regulated molecules essential for CNS circuit formation, myelination and axon integrity, including neuronal potassium-chloride co-transporter (KCC2), myelin basic protein (MBP) and phosphorylated neurofilament (pNF), respectively. Further, we predicted that post-injury EPO treatment could mitigate CNS calpain-mediated degradation. Using prenatal transient systemic hypoxia-ischemia (TSHI) in rats to mimic CNS injury from extreme preterm birth, and postnatal EPO treatment with a clinically relevant dosing regimen, we found sustained postnatal excess cortical calpain activation following prenatal TSHI, as shown by the cleavage of alpha II-spectrin (αII-spectrin) into 145-kDa αII-spectrin degradation products (αII-SDPs) and p35 into p25. Postnatal expression of the endogenous calpain inhibitor calpastatin was also reduced following prenatal TSHI. Calpain substrate expression following TSHI, including cortical KCC2, MBP and NF, was modulated by postnatal EPO treatment. Calpain activation was reflected in serum levels of αII-SDPs and KCC2 fragments, and notably, EPO treatment also modulated KCC2 fragment levels. Together, these data indicate that excess calpain activity contributes to the pathogenesis of encephalopathy of prematurity. Serum biomarkers of calpain activation may detect ongoing cerebral injury and responsiveness to EPO or similar neuroprotective strategies. PMID:26551007

  10. Erythropoietin blockade inhibits the induction of tumor angiogenesis and progression.

    Matthew E Hardee

    Full Text Available BACKGROUND: The induction of tumor angiogenesis, a pathologic process critical for tumor progression, is mediated by multiple regulatory factors released by tumor and host cells. We investigated the role of the hematopoietic cytokine erythropoietin as an angiogenic factor that modulates tumor progression. METHODOLOGY/PRINCIPAL FINDINGS: Fluorescently-labeled rodent mammary carcinoma cells were injected into dorsal skin-fold window chambers in mice, an angiogenesis model that allows direct, non-invasive, serial visualization and real-time assessment of tumor cells and neovascularization simultaneously using intravital microscopy and computerized image analysis during the initial stages of tumorigenesis. Erythropoietin or its antagonist proteins were co-injected with tumor cells into window chambers. In vivo growth of cells engineered to stably express a constitutively active erythropoietin receptor EPOR-R129C or the erythropoietin antagonist R103A-EPO were analyzed in window chambers and in the mammary fat pads of athymic nude mice. Co-injection of erythropoietin with tumor cells or expression of EPOR-R129C in tumor cells significantly stimulated tumor neovascularization and growth in window chambers. Co-injection of erythropoietin antagonist proteins (soluble EPOR or anti-EPO antibody with tumor cells or stable expression of antagonist R103A-EPO protein secreted from tumor cells inhibited angiogenesis and impaired tumor growth. In orthotopic tumor xenograft studies, EPOR-R129C expression significantly promoted tumor growth associated with increased expression of Ki67 proliferation antigen, enhanced microvessel density, decreased tumor hypoxia, and increased phosphorylation of extracellular-regulated kinases ERK1/2. R103A-EPO antagonist expression in mammary carcinoma cells was associated with near-complete disruption of primary tumor formation in the mammary fat pad. CONCLUSIONS/SIGNIFICANCE: These data indicate that erythropoietin is an

  11. Nonclinical evaluation of the potential for mast cell activation by an erythropoietin analog

    Weaver, James L., E-mail: James.Weaver@fda.hhs.gov [Division of Applied Regulatory Science, OCP/OTS/CDER/FDA, Silver Spring, MD (United States); Boyne, Michael, E-mail: mboyne@biotechlogic.com [Division of Pharmaceutical Analysis, OTR/OPQ/CDER/FDA, Silver Spring, MD (United States); Pang, Eric, E-mail: Eric.Pang@fda.hhs.gov [Division of Applied Regulatory Science, OCP/OTS/CDER/FDA, Silver Spring, MD (United States); Chimalakonda, Krishna, E-mail: Krishna.Chimalakonda@fda.hhs.gov [Division of Applied Regulatory Science, OCP/OTS/CDER/FDA, Silver Spring, MD (United States); Howard, Kristina E., E-mail: Kristina.Howard@fda.hhs.gov [Division of Applied Regulatory Science, OCP/OTS/CDER/FDA, Silver Spring, MD (United States)

    2015-09-15

    The erythropoietin analog peginesatide was withdrawn from marketing due to unexpected severe anaphylactic reactions associated with administration of the multi-use formulation. The adverse events occurred rapidly following the first ever administration of the drug with most affected patients becoming symptomatic in less than 30 min. This is most consistent with an anaphylactoid reaction due to direct activation of mast cells. Laboratory evaluation was undertaken using rat peritoneal mast cells as the model system. Initial studies showed that high concentrations of the formulated drug as well as formulated vehicle alone could cause mast cell degranulation as measured by histamine release. The purified active drug was not able to cause histamine release whereas the vehicle filtrate and lab created drug vehicle were equally potent at causing histamine release. Individual formulations of vehicle leaving one component out showed that histamine release was due to phenol. Dose response studies with phenol showed a very sharp dose response curve that was similar in three buffer systems. Cellular analysis by flow cytometry showed that the histamine release was not due to cell death, and that changes in light scatter parameters consistent with degranulation were rapidly observed. Limited testing with primary human mast cells showed a similar dose response of histamine release with exposure to phenol. To provide in vivo confirmation, rats were injected with vehicle formulated with various concentrations of phenol via a jugular vein cannula. Significant release of histamine was detected in blood samples taken 2 min after dosing at the highest concentrations tested. - Highlights: • Peginesatide caused severe anaphylactoid reactions in 0.2% of patients. • Both formulated drug and vehicle cause degranulation of rat mast cells. • Phenol was identified as the vehicle component causing degranulation. • Human mast cells show similar dose response to phenol as rat mast cells

  12. Human recombinant erythropoietin promotes differentiation of murine megakaryocytes in vitro.

    Ishibashi, T.; Koziol, J A; Burstein, S A

    1987-01-01

    To determine if erythropoietin affects megakaryocytopoiesis, we measured acetylcholinesterase (AchE) activity, a marker of the murine megakaryocytic lineage, after the addition of human recombinant erythropoietin to serumless murine bone marrow cultures. Erythropoietin increased AchE activity substantially. Moreover, when the hormone was added to serumless cultures of 426 isolated single megakaryocytes derived from megakaryocytic colonies, erythropoietin induced a significant increase in the ...

  13. The glucocorticoid receptor cooperates with the erythropoietin receptor and c-Kit to enhance and sustain proliferation of erythroid progenitors in vitro

    W. Zauner; G. Mellitzer; P. Steinlein (Peter); G. Fritsch; K. Huber; H. Beug (Hartmut); B. Löwenberg (Bob); M.M. von Lindern (Marieke)

    1999-01-01

    textabstractAlthough erythropoietin (Epo) is essential for the production of mature red blood cells, the cooperation with other factors is required for a proper balance between progenitor proliferation and differentiation. In avian erythroid progenitors, steroid hormone

  14. Enhanced erythropoietin and suppression of γ-glutamyl trans-peptidase (GGT) activity in murine lymphoma following administration of vanadium

    Administration of vanadium as ammonium mono-vanadate (0.005 μg/0.1 ml/mouse/day) was found to reduce the tumor cell proliferation in the host mice bearing Dalton's lymphoma. The high activity of γ-glutamyl trans-peptidase (CCT), a neoplastic marker, was seen in the host cells bearing lymphoma. Vanadium effectively prevented an increase in activity of γ-glutamyl trans-peptidase and maintained a sustained low activity of this enzyme. In addition, an improvement of the hematological aspects of the mice and almost fourfold elevation of erythropoietin (Epo) was obtained following vanadium treatment. This in Epo activity may play a vital role in regulating the growth of cellular neoplasia. The present study further confirms the anti-tumorigenic potential of vanadium in the control of tumor progression in lymphoma via modulating several factors involving erythropoiesis and may emerge as a new chemo-preventive agent for the future. (author)

  15. Erythropoietin receptor in human skeletal muscle and the effects of acute and long-term injections with recombinant human erythropoietin on the skeletal muscle

    Lundby, Carsten; Hellsten, Ylva; Jensen, Mie B. F.;

    2008-01-01

    potential effects of Epo in human skeletal muscle, two separate studies were conducted: one to study the acute effects of a single Epo injection on skeletal muscle gene expression and plasma hormones and another to study the effects of long-term (14 wk) Epo treatment on skeletal muscle structure. Subjects...... (n = 11) received a single Epo injection of 15,000 IU (double blinded, cross over, placebo). A single Epo injection reduced myoglobin and increased transferrin receptor and MRF-4 mRNA content within 10 h after injection. Plasma hormones remained unaltered. Capillarization and fiber hypertrophy was...

  16. Erythropoietin during hypoglycaemia in type 1 diabetes

    Kristensen, Peter Lommer; Høi-Hansen, Thomas; Olsen, Niels Vidiendal;

    2009-01-01

    AIMS: Preservation of cognitive function during hypoglycaemic episodes is crucial for patients with insulin-treated diabetes to avoid severe hypoglycaemic events. Erythropoietin has neuroprotective potential. However, the role of erythropoietin during hypoglycaemia is unclear. The aim of the study...... was to explore plasma erythropoietin response to hypoglycaemia and the relationship to basal renin-angiotensin system (RAS) activity and cognitive function. METHODS: We performed a single-blinded, controlled, cross-over study with induced hypoglycaemia or maintained glycaemic level. Nine patients with...... type 1 diabetes with high and nine with low activity in RAS were studied. Hypoglycaemia was induced using a standardized insulin-infusion. RESULTS: Overall, erythropoietin concentrations increased during hypoglycaemia. In the high RAS group erythropoietin rose 29% (p=0.032) whereas no significant...

  17. [Overview of erythropoietin].

    Lacombe, C; Mayeux, P; Casadevall, N

    1991-01-01

    Erythropoietin (Epo) is a glycoprotein that promotes the proliferation and differentiation of erythrocyte precursors. The major site of Epo production is the kidney and the liver is the main extra renal site of Epo production. Epo producing cells were identified by in situ hybridization, in the kidney, they are peritubular cells, most likely endothelial cells of the cortex and outer medulla; in the liver, they are mainly hepatocytes. The Epo secretion is stimulated by hypoxia, which is detected by an oxygen sensor. The Epo receptor is a multimeric protein, one chain which binds Epo has been cloned. However the structure of the Epo receptor is still puzzling, and one or more accessory chains remain to be identified. Since the clonage of the Epo gene, recombinant Epo has been available and allowed the treatment of patients with renal diseases with a constant efficacy. PMID:1662784

  18. Erythropoietin use and abuse.

    John, M Joseph; Jaison, Vineeth; Jain, Kunal; Kakkar, Naveen; Jacob, Jubbin J

    2012-03-01

    Recombinant human erythropoietin (rhEPO) is arguably the most successful therapeutic application of recombinant DNA technology till date. It was isolated in 1977 and the gene decoded in 1985. Since then, it has found varied applications, especially in stimulating erythropoiesis in anemia due to chronic conditions like renal failure, myelodysplasia, infections like HIV, in prematurity, and in reducing peri-operative blood transfusions. The discovery of erythropoietin receptor (EPO-R) and its presence in non-erythroid cells has led to several areas of research. Various types of rhEPO are commercially available today with different dosage schedules and modes of delivery. Their efficacy in stimulating erythropoiesis is dose dependent and differs according to the patient's disease and nutritional status. EPO should be used carefully according to guidelines as unsolicited use can result in serious adverse effects. Because of its capacity to improve oxygenation, it has been abused by athletes participating in endurance sports and detecting this has proved to be a challenge. PMID:22470858

  19. Erythropoietin use and abuse

    M Joseph John

    2012-01-01

    Full Text Available Recombinant human erythropoietin (rhEPO is arguably the most successful therapeutic application of recombinant DNA technology till date. It was isolated in 1977 and the gene decoded in 1985. Since then, it has found varied applications, especially in stimulating erythropoiesis in anemia due to chronic conditions like renal failure, myelodysplasia, infections like HIV, in prematurity, and in reducing peri-operative blood transfusions. The discovery of erythropoietin receptor (EPO-R and its presence in non-erythroid cells has led to several areas of research. Various types of rhEPO are commercially available today with different dosage schedules and modes of delivery. Their efficacy in stimulating erythropoiesis is dose dependent and differs according to the patient′s disease and nutritional status. EPO should be used carefully according to guidelines as unsolicited use can result in serious adverse effects. Because of its capacity to improve oxygenation, it has been abused by athletes participating in endurance sports and detecting this has proved to be a challenge.

  20. Erythropoietin and radiotherapy; Erythropoietine et radiotherapie

    Le Fur, E.; Albarghach, M.N.; Pradier, O. [CHU de Morvan, Dept. de radiotherapie, 29 - Brest (France)

    2010-01-15

    Erythropoietin (E.P.O.) is a glycoprotein hormone. This hormone is a growth factor for red blood cells precursors in the bone marrow. The decrease of oxygen partial pressure, a reduced number of erythrocytes caused by bleeding or excessive destruction, or increased tissues oxygen requirements lead to increased secretion of E.P.O.. Its action takes place on bone marrow erythroblastic cells through specific receptors. E.P.O. stimulates the proliferation of red cell precursors stem cells in the bone marrow, thus increasing their production in one to two weeks. The effectiveness of E.P.O. at increasing haemoglobin and improving patients quality of life has been demonstrated by several studies. However, its use in radiotherapy remains controversial. While tumour hypoxia caused by anaemia is a factor of radio resistance and thus a source of local failure, tumour expression of E.P.O. receptors presents a significant risk for tumour progression and neo-angiogenesis, which would be increased during the administration of E.P.O.. The purpose of this article is to answer the question: is there a place for E.P.O. in combination with radiotherapy in the management of cancer?

  1. Dimer formation of receptor activator of nuclear factor κB induces incomplete osteoclast formation

    Receptor activator of nuclear factor κB-ligand (RANKL) transduces a differentiation signal appropriate to osteoclasts likely through induction a receptor homotrimer; however, biological importance of RANK-trimerizarion is unknown. To address the signaling mechanism of the RANK receptor, we analyzed the effect of two different types of homodimer inducers RANK-TM-FKBP36v and hEpoR-RANK-TM on osteoclastogenesis. Dimerizing component FKBP36v or extracellular portion of human erythropoietin receptor (hEpoR) was fused to RANK lacking the extracellular domain, and the dimerization of this fusion protein was induced by addition of the chemical inducer of dimerization AP20187 or erythropoietin, respectively. Such treatment resulted in induction of TRAP-activity, a marker of osteoclast in a dose dependent manner, with an efficiency equivalent to that of induction by RANKL. However, dimerized-RANK-induced osteoclasts showed relatively low levels of multinucleation, pit forming activity, and expression of calcitonin receptor and cathepsin K, compared with osteoclasts which were induced in the presence of RANKL. As expression of nuclear factor of activated T cells 1 (NFATc1) was also reduced in dimerized-RANK-induced osteoclasts, RANK oligomerization by RANKL is a critical event to generate fully matured osteoclasts through upregulation of NFATc1

  2. Sexual dimorphism of erythropoietin-degrading activity in mouse submaxillary gland extracts

    Tam, R.C.; Bedwell, J.; Cotes, P.M.; Reed, P.J.

    1989-02-01

    In the course of investigation of submaxillary gland (SG) extracts from mice as a possible source of extra-renal erythropoietin (EPO) we have extended our previous studies of the degradation of EPO added to SG and kidney extracts. The discrepancy between estimates of EPO obtained with two radioimmunoassays (RIAs) differing only in time of incubation with /sup 125/I-labeled recombinant human EPO (r-HuEPO) (20 h and 72 h) has been used as an indicator of tracer degradation occurring during the RIA incubation. Degradation of /sup 125/I-labeled r-HuEPO by male mouse SG extracts was not prevented by addition of inhibitors of monodeiodinases or proteolytic enzymes. Degradation of added /sup 125/I-labeled r-HuEPO was monitored using gel filtration fast protein liquid chromatography. SG extracts from male and androgen-treated female mice both degraded tracer r-HuEPO to a greater extent than extracts from female mice. Tracer degradation increased with time and tissue concentration and could give rise to invalid estimates of EPO in SG extracts by RIA. In contrast, none of the kidney extracts degraded r-HuEPO. Recovery of mouse serum EPO added to and incubated with male mouse SG or kidney extracts was 13% and 93%, respectively, estimated by RIA under conditions that excluded degradation of the RIA tracer antigen.

  3. Coexpression of Kit and the receptors for erythropoietin, interleukin 6 and GM-CSF on hemopoietic cells

    M.O. de Jong (Marg); Y. Westerman (Yvonne); G. Wagemaker (Gerard); A.W. Wognum (Albert)

    1997-01-01

    textabstractThe detection of functional growth factor (GF) receptors on subpopulations of hemopoietic cells may provide a further dissection of immature cell subsets. Since little information is available about coexpression of different GF receptors at the level of sing

  4. Erythropoietin Protects Rat Brain Injury from Carbon Monoxide Poisoning by Inhibiting Toll-Like Receptor 4/NF-kappa B-Dependent Inflammatory Responses.

    Pang, Li; Zhang, Nan; Dong, Ning; Wang, Da-Wei; Xu, Da-Hai; Zhang, Ping; Meng, Xiang-Wei

    2016-04-01

    Inflammatory responses play critical roles in carbon monoxide (CO) poisoning-induced cerebral injury. The present study investigated whether erythropoietin (EPO) modulates the toll-like receptor 4 (TLR4) and nuclear factor-kappa B (NF-κB) inflammatory signaling pathways in brain injury after acute CO poisoning. EPO (2500 and 5000 U/kg) was injected subcutaneously twice a day after acute CO poisoning for 2 days. At 48 h after treatment, the expression levels of TLR4 and NF-κB as well as the levels of inflammatory cytokines in the hippocampal tissues were measured. Our results showed that CO poisoning induced a significant upregulation of TLR4, NF-κB, and inflammatory cytokines in the injured rat hippocampal tissues. Treatment with EPO remarkably suppressed the gene and protein expression levels of TLR4 and NF-κB, as well as the concentrations of TNF-α, IL-1β, and IL-6 in the hippocampal tissues. EPO treatment ameliorated CO poisoning-induced histological edema and neuronal necrosis. These results suggested that EPO protected against CO poisoning-induced brain damage by inhibiting the TLR4-NF-κB inflammatory signaling pathway. PMID:26521252

  5. Site-specific antibodies to human erythropoietin directed toward the NH2-terminal region.

    Sue, J M; Sytkowski, A. J.

    1983-01-01

    Site-specific antibodies to human erythropoietin have been raised in rabbits immunized with a synthetic polypeptide composed of the putative 26 NH2-terminal amino acids of the hormone. The immunogenic peptide was coupled to bovine serum albumin. Antibodies specific for peptide were detected by enzyme-linked immunosorbent assay. They immunoprecipitated both highly purified 125I-labeled erythropoietin and biologically active erythropoietin. The immunoprecipitation of 125I-labeled erythropoietin...

  6. Erythropoietin activates two distinct signaling pathways required for the initiation and the elongation of c-myc

    Chen, C.; Sytkowski, A. J.

    2001-01-01

    Erythropoietin (Epo) stimulation of erythroid cells results in the activation of several kinases and a rapid induction of c-myc expression. Protein kinase C is necessary for Epo up-regulation of c-myc by promoting elongation at the 3'-end of exon 1. PKCepsilon mediates this signal. We now show that Epo triggers two signaling pathways to c-myc. Epo rapidly up-regulated Myc protein in BaF3-EpoR cells. The phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 blocked Myc up-regulation in a concentration-dependent manner but had no effect on the Epo-induced phosphorylation of ERK1 and ERK2. LY294002 also had no effect on Epo up-regulation of c-fos. MEK1 inhibitor PD98059 blocked both the c-myc and the c-fos responses to Epo. PD98059 and the PKC inhibitor H7 also blocked the phosphorylation of ERK1 and ERK2. PD98059 but not LY294002 inhibited Epo induction of ERK1 and ERK2 phosphorylation in normal erythroid cells. LY294002 blocked transcription of c-myc at exon 1. PD98059 had no effect on transcription from exon 1 but, rather, blocked Epo-induced c-myc elongation at the 3'-end of exon 1. These results identify two Epo signaling pathways to c-myc, one of which is PI3K-dependent operating on transcriptional initiation, whereas the other is mitogen-activated protein kinase-dependent operating on elongation.

  7. Generation and phenotypic analysis of a transgenic line of rabbits secreting active recombinant human erythropoietin in the milk

    Mikuš, Tomáš; Poplštein, M.; Sedláková, J.; Landa, Vladimír; Jeníková, Gabriela; Trefil, P.; Lidický, J.; Malý, Petr

    2004-01-01

    Roč. 13, č. 5 (2004), s. 487-498. ISSN 0962-8819 R&D Projects: GA ČR GA304/03/0090 Institutional research plan: CEZ:AV0Z5052915 Keywords : erythropoietin, mammary gland, transgenic rabbit Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.107, year: 2004

  8. Erythropoietin receptor is not a surrogate marker for tumor hypoxia and does not correlate with survival in head and neck squamous cell carcinomas

    Background and purpose: To evaluate erythropoietin receptor (EPOR) expression in human head and neck squamous cell carcinomas and correlate this to the presence of tumor hypoxia and treatment outcome. Patients and methods: Eighty-five patients with locally advanced tumors of the head and neck were included. Of these, 34 were given the hypoxia marker pimonidazole i.v. 2 h prior to biopsy taking. Contiguous paraffin embedded biopsies were stained for EPOR expression and, if administered, for pimonidazole binding. Immunohistochemical staining for EPOR was interpreted semiquantitatively according to a composite scale, ranging from 0 to 200. Pimonidazole positivity was quantitatively analyzed in a semiautomatic way. Results: Diffuse weak-to-moderate cytoplasmic and membrane EPOR immunostaining was observed in 80 of 85 biopsies (94%) and staining scores ranged from 0 to 198 (median 100). No correlations were found between EPOR expression, and the primary tumor site, T-stage or N-stage. Also, There was no association between EPOR expression and treatment outcome. The degree of tumor hypoxia represented by the relative area of pimonidazole binding varied between 0 and 26% (median 7%). Contiguous biopsy sections showed a lack of colocalization between EPOR and pimonidazole binding. Conclusion: EPOR expression was demonstrated in the majority of the head and neck tumors. No colocalization was found between EPOR expression and pimonidazole binding indicating that the presence or absence of hypoxia did not necessarily indicate a distinct pattern of EPOR expression. The level of EPOR expression was not of prognostic significance in patients with head and neck cancer, although small effects of EPOR cannot be excluded because of the sample size of this study

  9. Research Advances in Expression and Functions of Erythropoietin and Erythropoietin Receptor in Cancers%促红细胞生成素及其受体在恶性肿瘤组织中的表达及功能的研究进展

    李梅; 吕跃; 陈晓勤

    2008-01-01

    促红细胞生成素(erythropoietin,EPO)最早被发现在红系细胞增殖、分化中发挥主要调节作用.研究发现在多种不同非造血器官及组织中有EPO及促红细胞生成素受体(erythropoietin receptor,EPO-R)的表达,并发挥促血管形成及组织保护效应.最近的多项研究发现,EPO及EPO-R广泛表达于多种恶性肿瘤细胞,EPO/EPO-R的自分泌/旁分泌通路与肿瘤微血管形成、刺激肿瘤细胞增殖、抑制凋亡及对放化疗的敏感性有关.确切机制需进一步研究.重组人促红细胞生成素(recombinant human erythropoietin,rh-EPO)在临床上已广泛用于治疗肿瘤相关贫血.研究证实其能增加血红蛋白水平,减少红细胞输注,同时改善患者生活质量.但亦有随机试验报道了rh-EPO治疗的患者相对安慰剂组患者无进展生存期下降.我们对EPO及其受体在非造血组织尤其是肿瘤组织中的表达、功能及相关机制的研究进展作简要概述.

  10. Effect of recombinant human erythropoietin administration on lipid peroxidation and antioxidant enzyme(s activities in preterm infants.

    Akisu M

    2001-12-01

    Full Text Available In the present investigation, we studied the effect of recombinant human erythropoietin (r-HuEPO on serum malondialdehyde (MDA as an index of lipid peroxidation, related to iron-catalyzed free radical reaction and erythrocyte superoxide dismutase (SOD, catalase (CAT, and glutathione peroxidase (GPX activities in very-low-birth weight (VLBW infants. Forty premature infants, at gestational ages were less than 33 weeks and birthweights were less than 1,500 g, were enrolled in the study. The study population was randomly divided into 2 groups. Twenty infants in Group 1 (treatment group were given r-HuEPO, and 20 infants in Group 2 served as the control. r-HuEPO treatment (750 U/kg a week was initiated on the 10th day of life and continued for 6 weeks. Preterm infants given erythrocyte transfusions during the study were excluded from the results. Serum ferritin and MDA levels, and erythrocyte superoxide dismutase (SOD, catalase (CAT, and glutathione peroxidase (GPX activities were analyzed at the end of the first week of life (at the beginning of the study. Subsequently, serum ferritin, and MDA levels were measured at the end of the 3rd and the 6th week. SOD, CAT, and GPX activities in the hemolysate were analyzed at the end of the 4th week. Six infants in the control group and 1 infant in the r-HuEPO group received transfusions through the end of the study, and these infants were excluded from the results. Significantly decreased serum ferritin concentrations were found in the r-HuEPO group compared to those in the control group both at the end of the 3rd and the 6th week (P < 0.05, and P < 0.01, respectively. In addition, serum MDA levels were also significantly reduced in Group 1 compared to control both at the end of the 3rd and the 6th week (P < 0.01 and P < 0.05, respectively. A good correlation was found between serum MDA and ferritin levels in Group 1. When the 2 groups were compared with respect to activities of SOD, CAT, and GPX at the end

  11. Erythropoietin Pathway: A Potential Target for the Treatment of Depression

    Chongyang Ma; Fafeng Cheng; Xueqian Wang; Changming Zhai; Wenchao Yue; Yajun Lian; Qingguo Wang

    2016-01-01

    During the past decade, accumulating evidence from both clinical and experimental studies has indicated that erythropoietin may have antidepressant effects. In addition to the kidney and liver, many organs have been identified as secretory tissues for erythropoietin, including the brain. Its receptor is expressed in cerebral and spinal cord neurons, the hypothalamus, hippocampus, neocortex, dorsal root ganglia, nerve axons, and Schwann cells. These findings may highlight new functions for ery...

  12. High glucose stimulates the expression of erythropoietin in rat glomerular epithelial cells

    Lim, Seul Ki; Park, Soo Hyun

    2011-01-01

    It has been reported that the levels of erythropoietin are associated with diabetes mellitus. Glomerular epithelial cells, located in the renal cortex, play an important role in the regulation of kidney function and hyperglycemia-induced cell loss of glomerular epithelial cells is implicated in the onset of diabetic nephropathy. This study investigated the effect of high glucose on erythropoietin and erythropoietin receptor expression in rat glomerular epithelial cells. We found that 25 mM D-...

  13. Erythropoietin is involved in hemoprotein syntheses in developing human decidua.

    Shiota, Mitsuru; Yasuda, Yoshiko; Shimaoka, Masao; Tsuritani, Mitsuhiro; Koike, Eiji; Oiki, Masaaki; Matsubara, Junko; Taketani, Shigeru; Murakami, Hitoshi; Yamasaki, Harufumi; Okumoto, Katsumi; Hoshiai, Hiroshi

    2013-03-01

    Before establishment of feto-placental circulation, decidua can synthesize hemoproteins to maintain oxygen homeostasis in situ. Using the human decidua of induced abortions ranging from 5 to 8 weeks of gestation, we determined the expression levels of erythropoietin, erythropoietin receptor, cytoglobin, myoglobin, embryonic-, fetal- and adult hemoglobin mRNA by quantitative RT-PCR analysis and identified their proteins by Western blot and immunohistochemical analyses. Erythropoietin signaling was demonstrated in phosphatidylinositol-3-kinase/protein kinase B pathway by Western blot, and the transcriptional factors for erythroid and non-erythroid heme synthesis were examined by RT-PCR analysis. In decidua, erythropoietin and its receptor mRNAs, erythropoietin receptor protein and phosphatidylinositol-3-kinase, were expressed with a peak at 6 weeks of gestation. Moreover, the decidua during 5 to 8 weeks of gestation expressed embryonic, fetal and adult hemoglobins additionally cytoglobin and myoglobin at transcriptional and protein levels. The heme portion of these hemoproteins is considered to be synthesized by non-erythroid δ-aminolevulinate synthase. These hemoproteins were discernible especially in decidual cells concomitant with cytotrophoblast cells and macrophage in these developing decidua. Considering the different capacity for oxygen binding and dissociation among hemoglobins with the oxygen storage capacity for cytoglobin and myoglobin, these hemoproteins appear to play a role in oxygen demand in decidua in situ before development of feto-placental circulation under the control of erythropoietin signaling. PMID:23480354

  14. Cell encoding recombinant human erythropoietin

    Beck, A.K.; Withy, R.M.; Zabrecky, J.R.; Masiello, N.C.

    1990-09-04

    This patent describes a C127 cell transformed with a recombinant DNA vector. It comprises: a DNA sequence encoding human erythropoietin, the transformed cell being capable of producing N-linked and O-linked glycosylated human erythropoietin.

  15. Erythropoietin has a mitogenic and positive chemotactic effect on endothelial cells

    Anagnostou, A.; Kessimian, N.; Steiner, M. (Memorial Hospital of Rhode Island, Pawtucket (USA) Brown Univ. Program in Medicine, Providence, RH (USA)); Lee, Eun Sun (Memorial Hospital of Rhode Island, Pawtucket (USA)); Levinson, R. (Brown Univ. Program in Medicine, Providence, RI (USA))

    1990-08-01

    Erythropoietin is known to be a hematopoietic growth factor with a singularly specific action on the proliferation and differentiation of erythroid progenitor cells. The authors have observed a dose-dependent proliferative action of human recombinant erythropoietin on human umbilical vein endothelial cells and bovine adrenal capillary endothelial cells. Binding studies with radioiodinated recombinant human erythropoietin revealed a large number ({approx}27,000) of an apparent single class of receptors with an affinity in the 10{sup {minus}9} M range. Linkage of the radiolabeled ligand to its receptor via a bifunctional crosslinking agent allowed them to identify an endothelial cell protein of 45 kDa as the principal receptor associated with this mitogenic effect of erythropoietin. Recombinant human erythropoietin also enhanced the migration of endothelial cells.

  16. Erythropoietin has a mitogenic and positive chemotactic effect on endothelial cells

    Erythropoietin is known to be a hematopoietic growth factor with a singularly specific action on the proliferation and differentiation of erythroid progenitor cells. The authors have observed a dose-dependent proliferative action of human recombinant erythropoietin on human umbilical vein endothelial cells and bovine adrenal capillary endothelial cells. Binding studies with radioiodinated recombinant human erythropoietin revealed a large number (∼27,000) of an apparent single class of receptors with an affinity in the 10-9 M range. Linkage of the radiolabeled ligand to its receptor via a bifunctional crosslinking agent allowed them to identify an endothelial cell protein of 45 kDa as the principal receptor associated with this mitogenic effect of erythropoietin. Recombinant human erythropoietin also enhanced the migration of endothelial cells

  17. Erythropoietin and diabetes mellitus

    Kenneth; Maiese

    2015-01-01

    Erythropoietin(EPO) is a 30.4 k Da growth factor and cytokine that governs cell proliferation, immune modulation, metabolic homeostasis, vascular function, and cytoprotection. EPO is under investigation for the treatment of variety of diseases, but appears especially suited for the treatment of disorders of metabolism that include diabetes mellitus(DM). DM and the com-plications of this disease impact a significant portion of the global population leading to disability and death with currently limited therapeutic options. In addition to its utility for the treatment of anemia, EPO can improve cardiac function, reduce fatigue, and improve cognition in patients with DM as well as regulate cellular energy metabolism, obesity, tissue repair and regeneration, apoptosis, and autophagy in experimental models of DM. Yet, EPO can have adverse effects that involve the vasculature system and unchecked cellular proliferation. Critical to the cytoprotective capacity and the potential for a positive clinical outcome with EPO are the control of signal transduction pathways that include protein kinase B, the mechanistic target of rapamycin, Wnt signaling, mammalian forkhead transcription factors of the O class, silent mating type information regulation 2 homolog 1(Saccharomyces cerevisiae), and AMP activated protein kinase. Therapeutic strategies that can specifically target and control EPO and its signaling pathways hold great promise for the development of new and effective clinical treatments for DM and the complications of this disorder.

  18. Correlation between erythropoietic activity and body growth rate in hypertransfused polycythemic growing rats as the result of an erythropoietin-dependent operating mechanism

    The established relationship between erythropoietic activity and body growth rate in the polycythemic growing rat could be the result of either an erythropoietin (EPO)-dependent or an EPO-independent operating mechanism. The present study was thus undertaken to elucidate the nature of the aforementioned mechanism by assessing the ratio between plasma immunoreactive EPO (iEPO) concentration and erythropoietic activity in young hypertransfused rats for different body growth rates. Red blood cell (RBC)-59Fe uptake was about 75% in 21-day-old rats; it rapidly decreased with time when the animals were placed on a protein-free diet, approaching a level of about 1% by the 10th day of protein starvation. Over the same period plasma iEPO decreased from 55 mU/ml to 7 mU/ml. Body growth rate was 0. Following this ''protein depletion period'' the rats received diets containing different amounts of casein (''protein repletion period'') added isocalorically to the protein-free diet to elicit a rise in body growth rate. Statistically significant relationships (p less than 0.001) were found between dietary casein concentration and body growth rate (r = 0.991), dietary casein concentration and RBC-59Fe uptake (r = 0.991), dietary casein concentration and plasma iEPO level (r = 0.992), body growth rate and RBC-59Fe (r = 0.986), and body growth rate and plasma iEPO level (r = 0.994) in hypertransfused polycythemic rats during the protein repletion period. These findings suggest that the correlation between erythropoietic activity and growth rate in the growing rat is the result of an erythropoietin-dependent operating mechanism, which appears to be independent of the ratio tissue oxygen supply/tissue oxygen demand

  19. Mechanism for the activation of glutamate receptors

    Scientists at the NIH have used a technique called cryo-electron microscopy to determine a molecular mechanism for the activation and desensitization of ionotropic glutamate receptors, a prominent class of neurotransmitter receptors in the brain and spina

  20. A Simple Three-Step Method for Design and Affinity Testing of New Antisense Peptides: An Example of Erythropoietin

    Nikola Štambuk

    2014-05-01

    Full Text Available Antisense peptide technology is a valuable tool for deriving new biologically active molecules and performing peptide–receptor modulation. It is based on the fact that peptides specified by the complementary (antisense nucleotide sequences often bind to each other with a higher specificity and efficacy. We tested the validity of this concept on the example of human erythropoietin, a well-characterized and pharmacologically relevant hematopoietic growth factor. The purpose of the work was to present and test simple and efficient three-step procedure for the design of an antisense peptide targeting receptor-binding site of human erythropoietin. Firstly, we selected the carboxyl-terminal receptor binding region of the molecule (epitope as a template for the antisense peptide modeling; Secondly, we designed an antisense peptide using mRNA transcription of the epitope sequence in the 3'→5' direction and computational screening of potential paratope structures with BLAST; Thirdly, we evaluated sense–antisense (epitope–paratope peptide binding and affinity by means of fluorescence spectroscopy and microscale thermophoresis. Both methods showed similar Kd values of 850 and 816 µM, respectively. The advantages of the methods were: fast screening with a small quantity of the sample needed, and measurements done within the range of physicochemical parameters resembling physiological conditions. Antisense peptides targeting specific erythropoietin region(s could be used for the development of new immunochemical methods. Selected antisense peptides with optimal affinity are potential lead compounds for the development of novel diagnostic substances, biopharmaceuticals and vaccines.

  1. Carbamylated Erythropoietin: A Prospective Drug Candidate for Neuroprotection

    Jianmin Chen; Zheng Yang; Xiao Zhang

    2016-01-01

    Carbamylated erythropoietin (cEpo), which is neuroprotective but lacks hematopoietic activity, has been attracting rising concerns. However, the cellular and molecular mechanisms involved in the process of neuroprotection of cEpo are not well known. Based on several recent reports, the neuroprotective effects of cEpo are illustrated, and signaling pathways involved in the different effects of erythropoietin and cEpo are discussed. These newly reported researches may shed new light on the deve...

  2. EPOR-Based Purification and Analysis of Erythropoietin Mimetic Peptides from Human Urine by Cys-Specific Cleavage and LC/MS/MS

    Vogel, Matthias; Thomas, Andreas; Schänzer, Wilhelm; Thevis, Mario

    2015-09-01

    The development of a new class of erythropoietin mimetic agents (EMA) for treating anemic conditions has been initiated with the discovery of oligopeptides capable of dimerizing the erythropoietin (EPO) receptor and thus stimulating erythropoiesis. The most promising amino acid sequences have been mounted on various different polymeric structures or carrier molecules to obtain highly active EPO-like drugs exhibiting beneficial and desirable pharmacokinetic profiles. Concomitant with creating new therapeutic options, erythropoietin mimetic peptide (EMP)-based drug candidates represent means to artificially enhance endurance performance and necessitate coverage by sports drug testing methods. Therefore, the aim of the present study was to develop a strategy for the comprehensive detection of EMPs in doping controls, which can be used complementary to existing protocols. Three model EMPs were used to provide proof-of-concept data. Following EPO receptor-facilitated purification of target analytes from human urine, the common presence of the cysteine-flanked core structure of EMPs was exploited to generate diagnostic peptides with the aid of a nonenzymatic cleavage procedure. Sensitive detection was accomplished by targeted-SIM/data-dependent MS2 analysis. Method characterization was conducted for the EMP-based drug peginesatide concerning specificity, linearity, precision, recovery, stability, ion suppression/enhancement, and limit of detection (LOD, 0.25 ng/mL). Additionally, first data for the identification of the erythropoietin mimetic peptides EMP1 and BB68 were generated, demonstrating the multi-analyte testing capability of the presented approach.

  3. Erythropoietin Action in Stress Response, Tissue Maintenance and Metabolism

    Yuanyuan Zhang

    2014-06-01

    Full Text Available Erythropoietin (EPO regulation of red blood cell production and its induction at reduced oxygen tension provides for the important erythropoietic response to ischemic stress. The cloning and production of recombinant human EPO has led to its clinical use in patients with anemia for two and half decades and has facilitated studies of EPO action. Reports of animal and cell models of ischemic stress in vitro and injury suggest potential EPO benefit beyond red blood cell production including vascular endothelial response to increase nitric oxide production, which facilitates oxygen delivery to brain, heart and other non-hematopoietic tissues. This review discusses these and other reports of EPO action beyond red blood cell production, including EPO response affecting metabolism and obesity in animal models. Observations of EPO activity in cell and animal model systems, including mice with tissue specific deletion of EPO receptor (EpoR, suggest the potential for EPO response in metabolism and disease.

  4. EPO-independent functional EPO receptor in breast cancer enhances estrogen receptor activity and promotes cell proliferation

    Highlights: • New anti-human EPOR antibody confirms full-length EPOR expression in breast cancer cells. • Proliferation of breast cancer cells is not affected by rhEPO treatment in vitro. • EPOR knockdown impairs proliferation of ERa positive breast cancer cells. • EPOR knockdown reduces AKT phosphorylation and ERa activity. - Abstract: The main function of Erythropoietin (EPO) and its receptor (EPOR) is the stimulation of erythropoiesis. Recombinant human EPO (rhEPO) is therefore used to treat anemia in cancer patients. However, clinical trials have indicated that rhEPO treatment might promote tumor progression and has a negative effect on patient survival. In addition, EPOR expression has been detected in several cancer forms. Using a newly produced anti-EPOR antibody that reliably detects the full-length isoform of the EPOR we show that breast cancer tissue and cells express the EPOR protein. rhEPO stimulation of cultured EPOR expressing breast cancer cells did not result in increased proliferation, overt activation of EPOR (receptor phosphorylation) or a consistent activation of canonical EPOR signaling pathway mediators such as JAK2, STAT3, STAT5, or AKT. However, EPOR knockdown experiments suggested functional EPO receptors in estrogen receptor positive (ERα+) breast cancer cells, as reduced EPOR expression resulted in decreased proliferation. This effect on proliferation was not seen in ERα negative cells. EPOR knockdown decreased ERα activity further supports a mechanism by which EPOR affects proliferation via ERα-mediated mechanisms. We show that EPOR protein is expressed in breast cancer cells, where it appears to promote proliferation by an EPO-independent mechanism in ERα expressing breast cancer cells

  5. EPO-independent functional EPO receptor in breast cancer enhances estrogen receptor activity and promotes cell proliferation

    Reinbothe, Susann; Larsson, Anna-Maria; Vaapil, Marica; Wigerup, Caroline [Department of Laboratory Medicine, Translational Cancer Research, Medicon Village, Lund University, SE-223 81 Lund (Sweden); CREATE Health, Sackler Faculty of Medicine, Tel-Aviv University, Tel-Aviv (Israel); Sun, Jianmin [Department of Laboratory Medicine, Translational Cancer Research, Medicon Village, Lund University, SE-223 81 Lund (Sweden); Jögi, Annika [Department of Laboratory Medicine, Translational Cancer Research, Medicon Village, Lund University, SE-223 81 Lund (Sweden); CREATE Health, Sackler Faculty of Medicine, Tel-Aviv University, Tel-Aviv (Israel); Neumann, Drorit [Department of Cell and Developmental Biology, Sackler Faculty of Medicine, Tel-Aviv University, Tel-Aviv (Israel); Rönnstrand, Lars [Department of Laboratory Medicine, Translational Cancer Research, Medicon Village, Lund University, SE-223 81 Lund (Sweden); Påhlman, Sven, E-mail: sven.pahlman@med.lu.se [Department of Laboratory Medicine, Translational Cancer Research, Medicon Village, Lund University, SE-223 81 Lund (Sweden); CREATE Health, Sackler Faculty of Medicine, Tel-Aviv University, Tel-Aviv (Israel)

    2014-02-28

    Highlights: • New anti-human EPOR antibody confirms full-length EPOR expression in breast cancer cells. • Proliferation of breast cancer cells is not affected by rhEPO treatment in vitro. • EPOR knockdown impairs proliferation of ERa positive breast cancer cells. • EPOR knockdown reduces AKT phosphorylation and ERa activity. - Abstract: The main function of Erythropoietin (EPO) and its receptor (EPOR) is the stimulation of erythropoiesis. Recombinant human EPO (rhEPO) is therefore used to treat anemia in cancer patients. However, clinical trials have indicated that rhEPO treatment might promote tumor progression and has a negative effect on patient survival. In addition, EPOR expression has been detected in several cancer forms. Using a newly produced anti-EPOR antibody that reliably detects the full-length isoform of the EPOR we show that breast cancer tissue and cells express the EPOR protein. rhEPO stimulation of cultured EPOR expressing breast cancer cells did not result in increased proliferation, overt activation of EPOR (receptor phosphorylation) or a consistent activation of canonical EPOR signaling pathway mediators such as JAK2, STAT3, STAT5, or AKT. However, EPOR knockdown experiments suggested functional EPO receptors in estrogen receptor positive (ERα{sup +}) breast cancer cells, as reduced EPOR expression resulted in decreased proliferation. This effect on proliferation was not seen in ERα negative cells. EPOR knockdown decreased ERα activity further supports a mechanism by which EPOR affects proliferation via ERα-mediated mechanisms. We show that EPOR protein is expressed in breast cancer cells, where it appears to promote proliferation by an EPO-independent mechanism in ERα expressing breast cancer cells.

  6. Endocrine effects of erythropoietin.

    Carlson, H E; Graber, M L; Gelato, M C; Hershman, J M

    1995-06-01

    Uremic men may manifest a variety of hormonal abnormalities, including decreased serum concentrations of testosterone and thyroid hormones and increased serum levels of growth hormone and prolactin. Some previous investigations have reported that erythropoietin therapy may reverse these hormonal changes. To investigate this possibility further, we measured serum prolactin, testosterone, LH, FSH, TSH, free thyroxine, triiodothyronine, growth hormone and IGF-I in 21 generally elderly male hemodialysis patients before and during erythropoietin therapy; many of the patients also received an anabolic steroid or metoclopramide treatment. Despite a significant erythropoietic response in a majority of the subjects, no significant changes were seen in any of the hormonal parameters other than a small decrease in serum growth hormone concentrations. Advanced age and chronic illness in our patients may have played a role in limiting the hormonal response reported by others. PMID:8593965

  7. Erythropoietin and diabetes mellitus

    Maiese, Kenneth

    2015-01-01

    Erythropoietin (EPO) is a 30.4 kDa growth factor and cytokine that governs cell proliferation, immune modulation, metabolic homeostasis, vascular function, and cytoprotection. EPO is under investigation for the treatment of variety of diseases, but appears especially suited for the treatment of disorders of metabolism that include diabetes mellitus (DM). DM and the complications of this disease impact a significant portion of the global population leading to disability and death with currentl...

  8. Erythropoietin binding protein from mammalian serum

    Clemons, G.K.

    1997-04-29

    Purified mammalian erythropoietin binding-protein is disclosed, and its isolation, identification, characterization, purification, and immunoassay are described. The erythropoietin binding protein can be used for regulation of erythropoiesis by regulating levels and half-life of erythropoietin. A diagnostic kit for determination of level of erythropoietin binding protein is also described. 11 figs.

  9. Erythropoietin binding protein from mammalian serum

    Clemons, Gisela K. (Berkeley, CA)

    1997-01-01

    Purified mammalian erythropoietin binding-protein is disclosed, and its isolation, identification, characterization, purification, and immunoassay are described. The erythropoietin binding protein can be used for regulation of erythropoiesis by regulating levels and half-life of erythropoietin. A diagnostic kit for determination of level of erythropoietin binding protein is also described.

  10. Erythropoietin receptor expression of rat brain at different developmental maturity and the effects of lipopolysaccharide on erythropoietin receptor expression%不同成熟度大鼠脑组织促红细胞生成素受体的表达及脂多糖对其表达的影响

    郭佳佳; 张彦华; 段佳佳; 徐发林

    2014-01-01

    目的探讨不同成熟度大鼠脑组织促红细胞生成素(EPO)受体(EPO-R)的表达及脂多糖(LPS)对不同成熟度大鼠脑组织EPO-R表达的影响。方法2日龄(Postnatal day 2,P2)新生SD大鼠,随机分为A、B两组:A为正常对照组,B为LPS组,分别腹腔注射等容积0.9%氯化钠注射液或0.6 mg/(kg·d)的 LPS,连续应用5天(P2~P6),并续养至12日龄(P12)。A组于P2、P7、P12及B组于P7、P12各时间点随机抽取8只新生大鼠取脑,以矢状缝为标志分为左右半脑,右侧脑用酶联免疫吸附试验(ELISA)检测脑组织EPO-R蛋白水平,左侧脑用反转录聚合酶链式反应(RT-PCR)检测EPO-R mRNA水平。结果(1)随大鼠成熟度的增加,正常对照组大鼠脑EPO-R蛋白(P2:13.73±2.04、P7:11.01±3.36、P12:10.52±2.35)及EPO-R mRNA(P2:0.44±0.05、P7:0.39±0.03、P12:0.38±0.04)表达下调,P2时表达水平与P7比较,差异有显著性(P0.05);(2)LPS组EPO-R蛋白与EPO-R mRNA表达较正常对照组上调,7日龄时正常对照组与LPS组比较,差异有显著性(P0.05)。结论随大鼠成熟度增加,脑组织EPO-R表达下调;LPS可上调大鼠脑组织EPO-R表达。%ObjectiveTo investigate the Erythropoietin Receptor(EPO-R) expression of rat brain at different developmental maturity and the effects of infection on EPO-R expression.Method Postnatal day 2 (P2) newborn SD rats were randomly divided into 2 groups:Control group(equal volume of saline) and Lipopolysaccharide (LPS) group[0.6 mg/(kg·d) LPS].The newborn rats in each group were administered corresponding drugs respectively at the corresponding time by intraperitoneal injection for 5 consecutive days,and continued to raise until P12. Every 8 newborn rats in A groups on P2,P7 and P12 and B group on P7 and P12 were selected randomly , the brains was divided into left and right hemispheres marked by sagittal suture.ELISA method were adopted

  11. NMDA Receptor Activation by Spontaneous Glutamatergic Neurotransmission

    Espinosa, Felipe; Kavalali, Ege T.

    2009-01-01

    Under physiological conditions N-methyl-d-aspartate (NMDA) receptor activation requires coincidence of presynaptic glutamate release and postsynaptic depolarization due to the voltage-dependent block of these receptors by extracellular Mg2+. Therefore spontaneous neurotransmission in the absence of action potential firing is not expected to lead to significant NMDA receptor activation. Here we tested this assumption in layer IV neurons in neocortex at their resting membrane potential (approxi...

  12. Hormone activation of baculovirus expressed progesterone receptors.

    Elliston, J F; Beekman, J M; Tsai, S Y; O'Malley, B W; Tsai, M J

    1992-03-15

    Human and chicken progesterone receptors (A form) were overproduced in a baculovirus expression system. These recombinant progesterone receptors were full-length bound progesterone specifically and were recognized by monoclonal antibodies, AB52 and PR22, specific for human and chicken progesterone receptor, respectively. In gel retardation studies, binding of recombinant human and chicken progesterone receptors to their progesterone response element (PRE) was specific and was enhanced in the presence of progesterone. Binding of human progesterone receptor to the PRE was also enhanced in the presence of the antiprogestin, RU486, but very little effect was observed in the presence of estradiol, dexamethasone, testosterone, and vitamin D. In our cell-free transcription system, human progesterone receptor induced transcription in a receptor-dependent and hormone-activable manner. Receptor-stimulated transcription required the presence of the PRE in the test template and could be specifically inhibited by excess PRE oligonucleotides. Furthermore, chicken progesterone receptor also induced in vitro transcription in a hormone-activable manner. These results demonstrate that steroid receptors overexpressed in a baculovirus expression system are functional and exhibit steroid-responsive binding and transcription. These observations support our present understanding of the mechanism of steroid receptor-regulated gene expression and provide a technological format for studies of the role of hormone and antihormone in altering gene expression. PMID:1544902

  13. NMDA receptor activation by spontaneous glutamatergic neurotransmission.

    Espinosa, Felipe; Kavalali, Ege T

    2009-05-01

    Under physiological conditions N-methyl-D-aspartate (NMDA) receptor activation requires coincidence of presynaptic glutamate release and postsynaptic depolarization due to the voltage-dependent block of these receptors by extracellular Mg(2+). Therefore spontaneous neurotransmission in the absence of action potential firing is not expected to lead to significant NMDA receptor activation. Here we tested this assumption in layer IV neurons in neocortex at their resting membrane potential (approximately -67 mV). In long-duration stable recordings, we averaged a large number of miniature excitatory postsynaptic currents (mEPSCs, >100) before or after application of dl-2 amino 5-phosphonovaleric acid, a specific blocker of NMDA receptors. The difference between the two mEPSC waveforms showed that the NMDA current component comprises approximately 20% of the charge transfer during an average mEPSC detected at rest. Importantly, the contribution of the NMDA component was markedly enhanced at membrane potentials expected for the depolarized up states (approximately -50 mV) that cortical neurons show during slow oscillations in vivo. In addition, partial block of the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor component of the mEPSCs did not cause a significant reduction in the NMDA component, indicating that potential AMPA receptor-driven local depolarizations did not drive NMDA receptor activity at rest. Collectively these results indicate that NMDA receptors significantly contribute to signaling at rest in the absence of dendritic depolarizations or concomitant AMPA receptor activity. PMID:19261712

  14. The discovery of erythropoietin.

    Erslev, A J

    1993-01-01

    A personal vignette of life as a resident and fellow at the Yale New Haven Hospital in the early 1950s follows. John Peters and his associates created a superb renal center at Yale New Haven, and they instilled in me a respect for quantitative measurements and a love for simple physiologic concepts. The environment was ideal for clinical and laboratory research, and it enabled me to show the existence of a regulatory erythropoietic hormone. I consider it a tribute to Dr. Peters that erythropoietin was later found to be produced by the kidneys and that it, as a recombinant drug, has helped ameliorate the anemia of uremia. PMID:8324267

  15. The effect of erythropoietin on platelet function and fibrinolysis in chronic renal failure

    Stenver, Doris Irene; Jeppesen, L; Nielsen, B;

    1994-01-01

    The influence of erythropoietin therapy on platelet function and fibrinolysis was evaluated in 12 anemic hemodialysis patients. Six months of therapy with human erythropoietin (50 to 80 IU/kg initially) raised the hemoglobin level to 10.8 g/dl but did not increase platelet activity in vivo as mea...

  16. Designing a small molecule erythropoietin mimetic.

    Guarnieri, Frank

    2015-01-01

    Erythropoietin (EPO) is a protein made by the kidneys in response to low red blood cell count that is secreted into the bloodstream and binds to a receptor on hematopoietic stem cells in the bone marrow inducing them to become new red blood cells. EPO made with recombinant DNA technology was brought to market in the 1980s to treat anemia caused by kidney disease and cancer chemotherapy. Because EPO infusion was able to replace blood transfusions in many cases, it rapidly became a multibillion dollar per year drug and as the first biologic created with recombinant technology it launched the biotech industry. For many years intense research was focused on creating a small molecule orally available EPO mimetic. The Robert Wood Johnson (RWJ) group seemed to definitively establish that only large peptides with a minimum of 60 residues could replace EPO, as anything less was not a full agonist. An intense study of the published work led me to hypothesize that the size of the mimetic is not the real issue, but the symmetry making and breaking of the EPO receptor induced by the ligand is the key to activating the stem cells. This analysis meant that residues in the binding site of the receptor deemed absolutely essential for ligand binding and activation from mutagenesis experiments, were probably not really that important. My fundamental hypotheses were: (a) the symmetric state of the homodimeric receptor is the most stable state and thus must be the off-state, (b) a highly localized binding site exists at a pivot point where the two halves of the receptor meet, (c) small molecules can be created that have high potency for this site that will be competitive with EPO and thus can displace the protein-protein interaction, (d) small symmetric molecules will stabilize the symmetric off-state of the receptor, and (e) a key asymmetry in the small molecule will stabilize a mirror image asymmetry in the receptor resulting in the stabilization of the on-state and proliferation of

  17. Erythropoietin Signaling Promotes Invasiveness of Human Head and Neck Squamous Cell Carcinoma

    Ahmed Mohyeldin

    2005-05-01

    Full Text Available Erythropoietin (Epo is used for managing anemia in cancer patients. However, recent studies have raised concerns for this practice. We investigated the expression and function of Epo and the erythropoietin receptor (EpoR in tumor biopsies and cell lines from human head and neck cancer. Epo responsiveness of the cell lines was assessed by Epoetin-α-induced tyrosine phosphorylation of the Janus kinase 2 (JAK2 protein kinase. Transmigration assays across Matrigel-coated filters were used to examine the effects of Epoetin-A on cell invasiveness. In 32 biopsies, we observed a significant association between disease progression and expression of Epo and its receptor, EpoR. Expression was highest in malignant cells, particularly within hypoxic and infiltrating tumor regions. Although both Epo and EpoR were expressed in human head and neck carcinoma cell lines, only EpoR was upregulated by hypoxia. Epoetin-α treatment induced prominent JAK2 phosphorylation and enhanced cell invasion. Inhibition of JAK2 phosphorylation reduced both basal and Epo-induced invasiveness. Our findings support a role for autocrine or paracrine Epo signaling in the malignant progression and local invasiveness of head and neck cancer. This mechanism may also be activated by recombinant Epo therapy and could potentially produce detrimental effects in rhEpo-treated cancer patients.

  18. Androgen insensitivity syndrome: gonadal androgen receptor activity

    To determine whether abnormalities of the androgen receptor previously observed in skin fibroblasts from patients with androgen insensitivity syndrome also occur in the gonads of affected individuals, androgen receptor activity in the gonads of a patient with testicular feminization syndrome was investigated. Using conditions for optimal recovery of androgen receptor from human testes established by previous studies, we detected the presence of a high-affinity (dissociation constant . 3.2 X 10(-10) mol/L), low-capacity (4.2 X 10(-12) mol/mg DNA), androgen-binding protein when tritium-labeled R1881 was incubated at 4 degrees C with nuclear extracts from the gonads of control patients or from a patient with testicular feminization syndrome but not when incubated at 37 degrees C. Thus this patient has an androgen receptor with a temperature lability similar to that of receptors from normal persons

  19. 21 CFR 864.7250 - Erythropoietin assay.

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Erythropoietin assay. 864.7250 Section 864.7250... assay. (a) Identification. A erythropoietin assay is a device that measures the concentration of erythropoietin (an enzyme that regulates the production of red blood cells) in serum or urine. This...

  20. Biased Signaling of Protease-activated Receptors

    PeishenZhao; NigelWilliamBunnett

    2014-01-01

    In addition to their role in protein degradation and digestion, proteases can also function as hormone-like signaling molecules that regulate vital patho-physiological processes, including inflammation, hemostasis, pain and repair mechanisms. Certain proteases can signal to cells by cleaving protease-activated receptors (PARs), a family of four G protein-coupled receptors. PARs are expressed by almost all cell types, control important physiological and disease-relevant processes, and are an e...

  1. [Erythropoietin and drug resistance in breast and ovarian cancers].

    Szenajch, Jolanta M; Synowiec, Agnieszka E

    2016-01-01

    Recombinant human erythropoietin (rhEPO) is used in breast and ovarian cancer patients to alleviate cancer- and chemotherapy-related anemia. Some clinical trials have reported that rhEPO may adversely impact survival and increase the risk of thrombovascular events in patients with breast cancer but not with ovarian cancer. The latter may potentially benefit the most from rhEPO treatment due to the nephrotoxic and myelosuppresive effects of standard platinum-based chemotherapy used in ovarian cancer disease. However, over the last decade the preclinical data have revealed that EPO is not only the principal growth factor and the hormone which regulates erythropoiesis, but also a cytokine with a pleiotropic activity which also can affect cancer cells. EPO can stimulate survival, ability to form metastases and drug resistance not only in continuous breast- and ovarian cancer cell lines but also in breast cancer stem-like cells. EPO receptor (EPOR) can also be constitutively active in both these cancers and, in breast cancer cells, may act in an interaction with estrogen receptor (ER) and epidermal growth factor receptor-2 (HER-2). EPOR, by an EPO-independent mechanism, promotes proliferation of breast cancer cells in cooperation with estrogen receptor, resulting in decreased effectiveness of tamoxifen treatment. In another interaction, as a result of the molecular antagonism between EPOR and HER2, rhEPO protects breast cancer cells against trastuzumab. Both clinical and preclinical evidence strongly suggest the urgent need to reevaluate the traditional use of rhEPO in the oncology setting. PMID:27321103

  2. Erythropoietin reduces storage lesions and decreases apoptosis indices in blood bank red blood cells

    Oscar Andrés Penuela

    2016-02-01

    Full Text Available ABSTRACT Background: Recent evidence shows a selective destruction of the youngest circulating red blood cells (neocytolysis trigged by a drop in erythropoietin levels. Objective: The aim of this study was to evaluate the effect of recombinant human erythropoietin beta on the red blood cell storage lesion and apoptosis indices under blood bank conditions. Methods: Each one of ten red blood cell units preserved in additive solution 5 was divided in two volumes of 100 mL and assigned to one of two groups: erythropoietin (addition of 665 IU of recombinant human erythropoietin and control (isotonic buffer solution was added. The pharmacokinetic parameters of erythropoietin were estimated and the following parameters were measured weekly, for six weeks: Immunoreactive erythropoietin, hemolysis, percentage of non-discocytes, adenosine triphosphate, glucose, lactate, lactate dehydrogenase, and annexin-V/esterase activity. The t-test or Wilcoxon's test was used for statistical analysis with significance being set for a p-value 6 weeks under blood bank conditions, with persistent supernatant concentrations of erythropoietin during the entire storage period. Adenosine triphosphate was higher in the Erythropoietin Group in Week 6 (4.19 ± 0.05 µmol/L vs. 3.53 ± 0.02 µmol/L; p-value = 0.009. The number of viable cells in the Erythropoietin Group was higher than in the Control Group (77% ± 3.8% vs. 71% ± 2.3%; p-value <0.05, while the number of apoptotic cells was lower (9.4% ± 0.3% vs. 22% ± 0.8%; p-value <0.05. Conclusions: Under standard blood bank conditions, an important proportion of red blood cells satisfy the criteria of apoptosis. Recombinant human erythropoietin beta seems to improve storage lesion parameters and mitigate apoptosis.

  3. ERK activation causes epilepsy by stimulating NMDA receptor activity

    Nateri, Abdolrahman S.; Raivich, Gennadij; Gebhardt, Christine; Da Costa, Clive; Naumann, Heike; Vreugdenhil, Martin; Makwana, Milan; Brandner, Sebastian; Adams, Ralf H.; Jefferys, John G. R.; Kann, Oliver; Behrens, Axel

    2007-01-01

    The ERK MAPK signalling pathway is a highly conserved kinase cascade linking transmembrane receptors to downstream effector mechanisms. To investigate the function of ERK in neurons, a constitutively active form of MEK1 (caMEK1) was conditionally expressed in the murine brain, which resulted in ERK activation and caused spontaneous epileptic seizures. ERK activation stimulated phosphorylation of eukaryotic translation initiation factor 4E (eIF4E) and augmented NMDA receptor 2B (NR2B) protein ...

  4. NMDA receptor activity in neuropsychiatric disorders

    ShaheenELakhan

    2013-06-01

    Full Text Available N-Methyl-D-aspartate (NMDA receptors play a variety of physiologic roles and their proper signaling is essential for cellular homeostasis. Any disruption in this pathway, leading to either enhanced or decreased activity, may result in the manifestation of neuropsychiatric pathologies such as schizophrenia, mood disorders, substance induced psychosis, Huntington's disease, Alzheimer's disease, and neuropsychiatric systemic lupus erythematosus. Here, we explore the notion that the overlap in activity of at least one biochemical pathway, the NMDA receptor pathway, may be the link to understanding the overlap in psychotic symptoms between diseases. This review intends to present a broad overview of those neuropsychiatric disorders for which alternations in NMDA receptor activity is prominent thus suggesting that continued direction of pharmaceutical intervention to this pathway may present a viable option for managing symptoms.

  5. Mechanism of FGF receptor dimerization and activation

    Sarabipour, Sarvenaz; Hristova, Kalina

    2016-01-01

    Fibroblast growth factors (fgfs) are widely believed to activate their receptors by mediating receptor dimerization. Here we show, however, that the FGF receptors form dimers in the absence of ligand, and that these unliganded dimers are phosphorylated. We further show that ligand binding triggers structural changes in the FGFR dimers, which increase FGFR phosphorylation. The observed effects due to the ligands fgf1 and fgf2 are very different. The fgf2-bound dimer structure ensures the smallest separation between the transmembrane (TM) domains and the highest possible phosphorylation, a conclusion that is supported by a strong correlation between TM helix separation in the dimer and kinase phosphorylation. The pathogenic A391E mutation in FGFR3 TM domain emulates the action of fgf2, trapping the FGFR3 dimer in its most active state. This study establishes the existence of multiple active ligand-bound states, and uncovers a novel molecular mechanism through which FGFR-linked pathologies can arise.

  6. No evidence for protective erythropoietin alpha signalling in rat hepatocytes

    Frede Stilla

    2009-04-01

    Full Text Available Abstract Background Recombinant human erythropoietin alpha (rHu-EPO has been reported to protect the liver of rats and mice from ischemia-reperfusion injury. However, direct protective effects of rHu-EPO on hepatocytes and the responsible signalling pathways have not yet been described. The aim of the present work was to study the protective effect of rHu-EPO on warm hypoxia-reoxygenation and cold-induced injury to hepatocytes and the rHu-EPO-dependent signalling involved. Methods Loss of viability of isolated rat hepatocytes subjected to hypoxia/reoxygenation or incubated at 4°C followed by rewarming was determined from released lactate dehydrogenase activity in the absence and presence of rHu-EPO (0.2–100 U/ml. Apoptotic nuclear morphology was assessed by fluorescence microscopy using the nuclear fluorophores H33342 and propidium iodide. Erythropoietin receptor (EPOR, EPO and Bcl-2 mRNAs were quantified by real time PCR. Activation of JAK-2, STAT-3 and STAT-5 in hepatocytes and rat livers perfused in situ was assessed by Western blotting. Results In contrast to previous in vivo studies on ischemia-reperfusion injury to the liver, rHu-EPO was without any protective effect on hypoxic injury, hypoxia-reoxygenation injury and cold-induced apoptosis to isolated cultured rat hepatocytes. EPOR mRNA was identified in these cells but specific detection of the EPO receptor protein was not possible due to the lack of antibody specificity. Both, in the cultured rat hepatocytes (10 U/ml for 15 minutes and in the rat liver perfused in situ with rHu-EPO (8.9 U/ml for 15 minutes no evidence for EPO-dependent signalling was found as indicated by missing effects of rHu-EPO on phosphorylation of JAK-2, STAT-3 and STAT-5 and on the induction of Bcl-2 mRNA. Conclusion Together, these results indicate the absence of any protective EPO signalling in rat hepatocytes. This implies that the protection provided by rHu-EPO in vivo against ischemia-reperfusion and

  7. Haemostatic aspects of recombinant human erythropoietin in colorectal surgery

    Poulsen, K A; Qvist, N; Winther, K;

    1998-01-01

    OBJECTIVE: To find out whether recombinant human erythropoietin (r-HuEPO) given perioperatively has any effect on haemostatic activity in patients undergoing elective colorectal resection. DESIGN: A placebo-controlled double-blind study. SETTING: Odense university hospital, Denmark. SUBJECTS: 24...

  8. Anxiolytic activity of adenosine receptor activation in mice.

    Jain, N; Kemp, N; Adeyemo, O; Buchanan, P; Stone, T W

    1995-10-01

    1. Purine analogues have been examined for anxiolytic- and anxiogenic-like activity in mice, by use of the elevated plus-maze. 2. The selective A1 receptor agonist, N6-cyclopentyladenosine (CPA) had marked anxiolytic-like activity at 10 and 50 microg kg(-1), with no effect on locomotor performance at these doses. 3. The A1 selective adenosine receptor antagonist, 1,3-dipropyl-8-cyclopentylxanthine (CPX) had no significant effect on anxiety-related measures or locomotor behaviour, but blocked the anxiolytic-like activity of CPA. The hydrophilic xanthine, 8-(p-sulphophenyl) theophylline did not prevent anxiolysis by CPA. 4. Caffeine had anxiogenic-like activity at 30 mg kg(-1) which was prevented by CPA at 50 micro kg(-1). 5. The A2 receptor agonist, N6-[2-(3,5-dimethoxyphenyl)-2(2-methylphenyl)-ethyl]adenosine (DPMA) had no effect on anxiety behaviour but depressed locomotor activity at the highest dose tested of 1 mg kg(-1). The A2 receptor antagonist, 1,3-dimethyl-l-propargylxanthine (DMPX) had no effect on anxiety-related measures or locomotion and did not modify the anxiolytic-like activity of CPA. 6. Administration of DPMA in combination with anxiolytic doses of CPA prevented the anxiolytic-like activity of the latter. 7. The results suggest that the selective activation of central A1 adenosine receptors induces anxiolytic-like behaviour, while the activation of A2 sites causes locomotor depression and reduces the effects of A1 receptor activation. The absence of any effect of CPX alone suggests that the receptors involved in modulating behaviour in the elevated plus-maze in mice are not activated tonically by endogenous adenosine. PMID:8640355

  9. Recombinant erythropoietin for the anaemia of patients with advanced Gastrointestinal Stromal Tumours (GIST receiving imatinib: an active agent only in non progressive patients

    Duffaud Florence

    2012-09-01

    Full Text Available Abstract Recombinant erythropoietin for the anaemia of patients with advanced Gastrointestinal Stromal Tumours (GIST receiving imatinib : an active agent only in non progressive patients. Background Imatinib is a standard treatment for advanced/metastatic GIST and in adjuvant setting. Anaemia is frequently observed in patients with advanced GIST, and is one of the most frequent side effects of imatinib with grade 3–4 anaemia in 10% of patients. Whether EPO treatment is useful in the management of GIST patients receiving imatinib treatment is unknown. Methods A retrospective study of EPO treatment in GIST patients receiving imatinib was undertaken in 4 centres. Thirty four patients received EPO treatment among the 319 GIST patients treated with imatinib in clinical trials or with compassionate use between 2001 and 2003. The efficacy of EPO on the anaemia of patients with GIST treated with imatinib was analyzed. Results There were 18 males and 16 females with a median age of 59 years. Median WHO-PS was 1. Primary tumour sites were mainly gastric (32% and small bowel (29%. Sites of metastases were mainly liver (82% and peritoneum (79%. The median delay between the initiation of imatinib treatment and EPO was 58 days (range 0–553. Median haemoglobin (Hb level prior to EPO was 9 g/dL (range 6,9-11,8 and 11,7 g/dL (range 6,8-14,4 after 2 months. An increase of more than 2 g/dL was observed in 18 (53% of patients. None of the 7 patients who progressed (PD under imatinib treatment (400 mg/day experienced HB response, as compared to 66% (18/27 of the remaining patients (PR + SD (p = 0,002. Primary tumour site, liver metastases, peritoneal metastases, age, gender did not correlate with HB response to EPO. Response to EPO was observed in 2/11 patients receiving high-dose imatinib (800 mg/day vs 16/23 of others. Using logistic regression, only PD before EPO treatment was retained as a predictive factor for EPO response. Conclusion EPO enables to

  10. CERAPP: Collaborative estrogen receptor activity prediction project

    Mansouri, Kamel; Abdelaziz, Ahmed; Rybacka, Aleksandra;

    2016-01-01

    Background: Humans are exposed to thousands of man-made chemicals in the environment. Some chemicals mimic natural endocrine hormones and, thus, have the potential to be endocrine disruptors. Most of these chemicals have never been tested for their ability to interact with the estrogen receptor (ER......). Risk assessors need tools to prioritize chemicals for evaluation in costly in vivo tests, for instance, within the U.S. EPA Endocrine Disruptor Screening Program. oBjectives: We describe a large-scale modeling project called CERAPP (Collaborative Estrogen Receptor Activity Prediction Project) and...

  11. Spongian diterpenoids inhibit androgen receptor activity

    Yang, Yu Chi; Labros G Meimetis; Tien, Amy H; Mawji, Nasrin R.; Carr, Gavin; Wang, Jun; Andersen, Raymond J.; Sadar, Marianne D.

    2013-01-01

    Androgen receptor (AR) is a ligand-activated transcription factor and a validated drug target for all stages of prostate cancer. Antiandrogens compete with physiological ligands for AR ligand-binding domain (LBD). High-throughput screening of a marine natural product library for small molecules that inhibit AR transcriptional activity yielded the furanoditerpenoid spongia-13(16),-14-dien-19-oic acid, designated terpene 1 (T1). Characterization of T1 and the structurally related semi-synthetic...

  12. Hepatic erythropoietin response in cirrhosis

    Risør, Louise M; Fenger, Mogens; Olsen, Niels V;

    2016-01-01

    Background Erythropoietin (EPO) is produced in the liver during fetal life, but after birth the production shifts to the kidneys. The liver maintains a production capacity of 10% of the total EPO-production, but can be up-regulated to 100%. Previous studies have demonstrated both elevated and...

  13. Fatty acids activate a chimera of the clofibric acid-activated receptor and the glucocorticoid receptor.

    Göttlicher, M; Widmark, E; Q. Li; Gustafsson, J.A.

    1992-01-01

    Peroxisome proliferators such as clofibric acid, nafenopin, and WY-14,643 have been shown to activate PPAR (peroxisome proliferator-activated receptor), a member of the steroid nuclear receptor superfamily. We have cloned the cDNA from the rat that is homologous to that from the mouse [Issemann, I. & Green, S. (1990) Nature (London) 347, 645-650], which encodes a 97% similar protein with a particularly well-conserved putative ligand-binding domain. To search for physiologically occurring acti...

  14. Erythropoietin ameliorates podocyte injury in advanced diabetic nephropathy in the db/db mouse.

    Loeffler, Ivonne; Rüster, Christiane; Franke, Sybille; Liebisch, Marita; Wolf, Gunter

    2013-09-15

    Podocyte damage and accumulation of advanced glycation end products (AGEs) are characteristics of diabetic nephropathy (DN). The pathophysiology of AGE-challenged podocytes, such as hypertrophy, apoptosis, and reduced cell migration, is closely related to the induction of the cell cycle inhibitor p27(Kip1) and to the inhibition of neuropilin 1 (NRP1). We have previously demonstrated that treatment with erythropoietin is associated with protective effects for podocytes in vitro. db/db mice with overt DN aged 15-16 wk were treated with either placebo, epoetin-β, or continuous erythropoietin receptor activator (CERA) for 2 wk. db/db mice compared with nondiabetic db/m control mice revealed the expected increases in body weight, blood glucose, albumin-to-creatinine ratio, and AGE accumulation. Whereas there were no differences in body weight, hyperglycemia and AGEs were observed among diabetic mice that received epoetin-β compared with CERA and placebo treatment, indicating that epoetin-β/CERA treatment does not interfere with the development of diabetes in this model. However, the albumin-to-creatinine ratio was significantly lower in db/db mice treated with epoetin-β or CERA. Furthermore, kidney weights in db/db mice were increased compared with db/m control mice, indicating renal hypertrophy, whereas the increase in renal weight in epoetin-β- or CERA-treated db/db mice was significantly lower than in placebo-treated control mice. Induction of p27(Kip1) and suppression of NRP1 were significantly reduced in the epoetin-β treatment group versus the CERA treatment group. Furthermore, erythropoietin treatment diminished the diabetes-induced podocyte loss. Together, independently from hematopoetic effects, epoetin-β or CERA treatment was associated with protective changes in DN, especially that NRP1 and p27(Kip1) expressions as well as numbers of podocytes returned to normal levels. Our data show, for the first time, that medication of overt DN with erythropoietin

  15. Human Receptor Activation by Aroclor 1260, a Polychlorinated Biphenyl Mixture

    Wahlang, Banrida; Falkner, K. Cameron; Clair, Heather B.; Al-Eryani, Laila; Prough, Russell A.; States, J. Christopher; Coslo, Denise M.; Omiecinski, Curtis J.; Cave, Matthew C.

    2014-01-01

    Polychlorinated biphenyls (PCBs) are persistent environmental toxicants, present in 100% of U.S. adults and dose-dependently associated with obesity and non-alcoholic fatty liver disease (NAFLD). PCBs are predicted to interact with receptors previously implicated in xenobiotic/energy metabolism and NAFLD. These receptors include the aryl hydrocarbon receptor (AhR), pregnane xenobiotic receptor (PXR), constitutive androstane receptor (CAR), peroxisome proliferator-activated receptors (PPARs), ...

  16. RELAXIN ACTIVATES PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR GAMMA

    Singh, Sudhir; Bennett, Robert G

    2009-01-01

    Relaxin is a polypeptide hormone that triggers multiple signaling pathways through its receptor RXFP1. Many of relaxin’s functions, including vascular and antifibrotic effects, are similar to those induced by activation of PPARγ. In this study, we tested the hypothesis that relaxin signaling through RXFP1 would activate PPARγ activity. In cells overexpressing RXFP1 (HEK-RXFP1), relaxin increased transcriptional activity through a PPAR response element (PPRE) in a concentration-dependent manne...

  17. Functional significance of erythropoietin in renal cell carcinoma

    One of the molecules regulated by the transcription factor, hypoxia inducible factor (HIF), is the hypoxia-responsive hematopoietic factor, erythropoietin (EPO). This may have relevance to the development of renal cell carcinoma (RCC), where mutations of the von Hippel-Lindau (VHL) gene are major risk factors for the development of familial and sporadic RCC. VHL mutations up-regulate and stabilize HIF, which in turn activates many downstream molecules, including EPO, that are known to promote angiogenesis, drug resistance, proliferation and progression of solid tumours. HIFs typically respond to hypoxic cellular environment. While the hypoxic microenvironment plays a critical role in the development and progression of tumours in general, it is of special significance in the case of RCC because of the link between VHL, HIF and EPO. EPO and its receptor, EPOR, are expressed in many cancers, including RCC. This limits the use of recombinant human EPO (rhEPO) to treat anaemia in cancer patients, because the rhEPO may be stimulatory to the cancer. EPO may also stimulate epithelial-mesenchymal transition (EMT) in RCC, and pathological EMT has a key role in cancer progression. In this mini review, we summarize the current knowledge of the role of EPO in RCC. The available data, either for or against the use of EPO in RCC patients, are equivocal and insufficient to draw a definitive conclusion

  18. Renal mesangial cell cultures as a model for study of erythropoietin production.

    Kurtz, Armin; Jelkmann, W; Sinowatz, F.; Bauer, Christian

    1983-01-01

    Mesangial cells derived from isolated glomeruli of rat kidney were grown as homogeneous cell lines in culture. They released, into the culture medium, erythropoietin that had free terminal galactosyl residues and was therefore not active in vivo. The production of erythropoietin by these cells was significantly enhanced by either lowering the PO2 in the incubation atmosphere or by adding cobalt chloride to the culture medium. Therefore, mesangial cells in culture may be considered as an in vi...

  19. Anxiolytic activity of adenosine receptor activation in mice.

    Jain, N; Kemp, N; Adeyemo, O; Buchanan, P.; Stone, T W

    1995-01-01

    1. Purine analogues have been examined for anxiolytic- and anxiogenic-like activity in mice, by use of the elevated plus-maze. 2. The selective A1 receptor agonist, N6-cyclopentyladenosine (CPA) had marked anxiolytic-like activity at 10 and 50 microg kg(-1), with no effect on locomotor performance at these doses. 3. The A1 selective adenosine receptor antagonist, 1,3-dipropyl-8-cyclopentylxanthine (CPX) had no significant effect on anxiety-related measures or locomotor behaviour, but blocked ...

  20. Synthesis and biological evaluation of 125I-erythropoietin as a potential radiopharmaceutical agent for tumours

    Erythropoietin (EPO) is a glycoprotein hormone responsible for regulating erythropoiesis. Expression of EPO and EPO receptors (EPOr) has recently been demonstrated in some neoplastic cell lines and tumours, suggesting a potential new target for therapy. In this work, EPO was labeled with iodine-125 using the lactoperoxidase method, known to prevent damage to protein during radioiodination, and labeling conditions were optimized. In vitro stability studies have shown that 125I-EPO is radiochemically stable for 20 days after radiolabeling. In vitro cell binding studies have demonstrated very low binding (125I-EPO. In mice with induced melanoma, only a residual fixation in the tumour was evident. Further studies are warranted on other tumoral cell lines to better understand the binding process and internalization into cells. Studies on EPO labeled with carbon-11 could be valuable, since there is a greater chance of preserving the biological activity of the protein using this method. (author)

  1. Clinical study on relationship between recombinant human erythropoietin in treating patients with cancer-related anemia and its relation to serum erythropoietin and transferrin receptor%重组人促红细胞生成素治疗肿瘤相关性贫血及其与血清促红细胞生成素、转铁蛋白受体关系的临床研究

    丁瑞敏

    2015-01-01

    目的:分析肿瘤相关性贫血经重组人促红细胞生成素治疗的效果及血清促红细胞生成素、转铁蛋白受体检测的指导价值。方法:选取2012年6月-2013年12月期间,本院收治的肿瘤相关性贫血患者为研究对象(72例),并选取同期收治的单纯性缺铁性贫血患者40例为常模组。经放射免疫分析法和ELISA抗体法分别测定血清促红细胞生成素和血清转铁蛋白受体水平。结果:玉组、域组患者治疗后的红细胞积压和血红蛋白水平均有改善,P0. 05. After 8 weeks of treat-ment targets difference compared to before treatment, P<0. 05. Conclusion:serum erythropoietin and Transferrin receptor levels is important for the determination of treatment on tumor-related anemia guide value.

  2. Relaxin Family Peptide Receptor 1 (RXFP1) Activation Stimulates the Peroxisome Proliferator-Activated Receptor Gamma

    Singh, Sudhir; Bennett, Robert G

    2009-01-01

    Relaxin (Rlx) has antifibrotic effects in a number of tissues. Many of these effects are similar to those induced by the activators of peroxisome proliferator-activated receptor γ (PPARγ), raising the possibility that a mechanism for Rlx’s antifibrotic effects may involve activation of the PPARγ pathway. This study investigates the effect of Rlx on PPARs and their mechanism of upregulation. It shows that Rlx stimulates ligand-independent PPAR activation in a dose-dependent manner. The combine...

  3. Recombinant erythropoietin in clinical practice

    Ng, T; Marx, G.; Littlewood, T; Macdougall, I

    2003-01-01

    The introduction of recombinant human erythropoietin (RHuEPO) has revolutionised the treatment of patients with anaemia of chronic renal disease. Clinical studies have demonstrated that RHuEPO is also useful in various non-uraemic conditions including haematological and oncological disorders, prematurity, HIV infection, and perioperative therapies. Besides highlighting both the historical and functional aspects of RHuEPO, this review discusses the applications of RHuEPO in clinical practice a...

  4. Preoperative erythropoietin in spine surgery

    Colomina, Maria J.; Bagó, Juan; Pellisé, Ferran; Godet, Carmen; Villanueva, Carlos

    2004-01-01

    Spine surgery may be associated with profuse intraoperative bleeding that often requires blood transfusions. In recent years several techniques have been developed to avoid allogenic transfusions and their potential complications to surgical patients. In this study we review and analyse the role of preoperative recombinant human erythropoietin (rHuEPO) administration in spine surgery as a blood conservation strategy. Between 1998 and 2002, a total of 250 patients scheduled for spine surgery w...

  5. Erythropoietin in the Neurology ICU

    Robertson, Claudia; Sadrameli, Saeed

    2013-01-01

    Erythropoietin (EPO) is an approved drug that is used in the treatment of chronic anemia associated with chronic renal failure. In the Neuro ICU, there are two potential uses for treatment with EPO. Anemia is common in patients with acute neurological disorders and may be a cause of secondary insults. Studies of EPO to treat anemia associated with critical illness have not conclusively shown a beneficial risk/benefit ratio. The relatively small reduction in transfusion requirement with EPO in...

  6. Monoclonal antibodies to the human insulin receptor that activate glucose transport but not insulin receptor kinase activity

    Forsayeth, J.R.; Caro, J.F.; Sinha, M.K.; Maddux, B.A.; Goldfine, I.D.

    1987-05-01

    Three mouse monoclonal antibodies were produced that reacted with the ..cap alpha.. subunit of the human insulin receptor. All three both immunoprecipitated /sup 125/I-labeled insulin receptors from IM-9 lymphocytes and competitively inhibited /sup 125/I-labeled insulin binding to its receptor. Unlike insulin, the antibodies failed to stimulate receptor autophosphorylation in both intact IM-9 lymphocytes and purified human placental insulin receptors. Moreover, unlike insulin, the antibodies failed to stimulate receptor-mediated phosphorylation of exogenous substrates. However, like insulin, two of the three antibodies stimulated glucose transport in isolated human adipocytes. One antibody, on a molar basis, was as potent as insulin. These studies indicate, therefore, that monoclonal antibodies to the insulin receptor can mimic a major function of insulin without activating receptor kinase activity. They also raise the possibility that certain actions of insulin such as stimulation of glucose transport may not require the activation of receptor kinase activity.

  7. Monoclonal antibodies to the human insulin receptor that activate glucose transport but not insulin receptor kinase activity

    Three mouse monoclonal antibodies were produced that reacted with the α subunit of the human insulin receptor. All three both immunoprecipitated 125I-labeled insulin receptors from IM-9 lymphocytes and competitively inhibited 125I-labeled insulin binding to its receptor. Unlike insulin, the antibodies failed to stimulate receptor autophosphorylation in both intact IM-9 lymphocytes and purified human placental insulin receptors. Moreover, unlike insulin, the antibodies failed to stimulate receptor-mediated phosphorylation of exogenous substrates. However, like insulin, two of the three antibodies stimulated glucose transport in isolated human adipocytes. One antibody, on a molar basis, was as potent as insulin. These studies indicate, therefore, that monoclonal antibodies to the insulin receptor can mimic a major function of insulin without activating receptor kinase activity. They also raise the possibility that certain actions of insulin such as stimulation of glucose transport may not require the activation of receptor kinase activity

  8. EPO's alter ego: erythropoietin has multiple actions.

    Lappin, Terence R; Maxwell, A Peter; Johnston, Patrick G

    2002-01-01

    Many cancer patients suffer from anemia, which has a major detrimental effect on their quality of life. Recombinant human erythropoietin (rHuEPO) is now widely used in cancer patients, as it improves hematocrit, lowers blood transfusion requirements, and improves quality of life. Recent research indicates that EPO has pleiotropic effects on the body well beyond the maintenance of red cell mass, but the mechanisms involved in relieving fatigue and improving quality of life in cancer patients are poorly understood. EPO receptors (EPO-Rs) have been detected in many different cells and tissues, providing evidence for autocrine, paracrine, and endocrine functions of EPO. Apart from its endocrine function, EPO may have a generalized role as an antiapoptotic agent that is associated with enhancement of muscle tone, mucosal status, and gonadal and cognitive function. The recent discovery of EPO-Rs in breast tumor vasculature, while raising important questions about the possible effects of pharmacological doses of rHuEPO on tumor cells, also suggests that the receptors could provide a useful target for drugs attached to EPO. PMID:12456956

  9. Protease activated receptors (PARS) mediation in gyroxin biological activity

    Gyroxin is a serine protease enzyme from the South American rattlesnake (Crotalus durissus terrificus) venom; it is only partially characterized and has multiple activities. Gyroxin induces blood coagulation, blood pressure decrease and a neurotoxic behavior named barrel rotation. The mechanisms involved in this neurotoxic activity are not known. Whereas gyroxin is a member of enzymes with high potential to become a new drug with clinical applications such as thrombin, batroxobin, ancrod, tripsyn and kalicrein, it is important to find out how gyroxin works. The analysis on agarose gel electrophoresis and circular dichroism confirmed the molecules' integrity and purity. The gyroxin intravenous administration in mice proved its neurotoxicity (barrel rotation). In vivo studies employing intravital microscopy proved that gyroxin induces vasodilation with the participation of protease activated receptors (PARs), nitric oxide and Na+K+ATPase. The leukocytes' adherence and rolling counting indicated that gyroxin has no pro inflammatory activity. Gyroxin induced platelet aggregation, which was blocked by inhibitors of PAR1 and PAR4 receptors (SCH 79797 and tcY-NH2, respectively). Finally, it was proved that the gyroxin temporarily alter the permeability of the blood brain barrier (BBB). Our study has shown that both the protease-activated receptors and nitric oxide are mediators involved in the biological activities of gyroxin. (author)

  10. Quantifying Agonist Activity at G Protein-coupled Receptors

    Ehlert, Frederick J.; Suga, Hinako; Griffin, Michael T.

    2011-01-01

    When an agonist activates a population of G protein-coupled receptors (GPCRs), it elicits a signaling pathway that culminates in the response of the cell or tissue. This process can be analyzed at the level of a single receptor, a population of receptors, or a downstream response. Here we describe how to analyze the downstream response to obtain an estimate of the agonist affinity constant for the active state of single receptors.

  11. Cannabinoids go nuclear: evidence for activation of peroxisome proliferator-activated receptors

    O'Sullivan, S E

    2007-01-01

    Cannabinoids act at two classical cannabinoid receptors (CB1 and CB2), a 7TM orphan receptor and the transmitter-gated channel transient receptor potential vanilloid type-1 receptor. Recent evidence also points to cannabinoids acting at members of the nuclear receptor family, peroxisome proliferator-activated receptors (PPARs, with three subtypes α, β (δ) and γ), which regulate cell differentiation and lipid metabolism. Much evidence now suggests that endocannabinoids are natural activators o...

  12. Model for growth hormone receptor activation based on subunit rotation within a receptor dimer

    Brown, Richard J.; Adams, Julian J.; Pelekanos, Rebecca A.; Wan, Yu; McKinstry, William J.; Palethorpe, Kathryn; Seeber, Ruth M.; Monks, Thea A.; Eidne, Karin A.; Parker, Michael W.; Waters, Michael J. (UWA); (St. Vincent); (Queensland)

    2010-07-13

    Growth hormone is believed to activate the growth hormone receptor (GHR) by dimerizing two identical receptor subunits, leading to activation of JAK2 kinase associated with the cytoplasmic domain. However, we have reported previously that dimerization alone is insufficient to activate full-length GHR. By comparing the crystal structure of the liganded and unliganded human GHR extracellular domain, we show here that there is no substantial change in its conformation on ligand binding. However, the receptor can be activated by rotation without ligand by inserting a defined number of alanine residues within the transmembrane domain. Fluorescence resonance energy transfer (FRET), bioluminescence resonance energy transfer (BRET) and coimmunoprecipitation studies suggest that receptor subunits undergo specific transmembrane interactions independent of hormone binding. We propose an activation mechanism involving a relative rotation of subunits within a dimeric receptor as a result of asymmetric placement of the receptor-binding sites on the ligand.

  13. DNA Damage Stress and Inhibition of Jak2-V617F Cause Its Degradation and Synergistically Induce Apoptosis through Activation of GSK3β

    Nagao, Toshikage; Oshikawa, Gaku; Wu, Nan; Kurosu, Tetsuya; Miura, Osamu

    2011-01-01

    The cytoplasmic tyrosine kinase Jak2 plays a crucial role in cytokine receptor signaling in hematopoietic cells. The activated Jak2-V617F mutant is present in most cases of BCR/ABL-negative myeloproliferative neoplasms and constitutively activates downstream signals from homodimeric cytokine receptors, such as the erythropoietin receptor (EpoR). Here we examine the effects of DNA damage stress on Jak2 or Jak2-V617F and on induction of apoptosis in hematopoietic cells. Etoposide or doxorubicin...

  14. Cytoprotective effect of recombinant human erythropoietin produced in transgenic tobacco plants.

    Farooqahmed S Kittur

    Full Text Available Asialo-erythropoietin, a desialylated form of human erythropoietin (EPO lacking hematopoietic activity, is receiving increased attention because of its broader protective effects in preclinical models of tissue injury. However, attempts to translate its protective effects into clinical practice is hampered by unavailability of suitable expression system and its costly and limit production from expensive mammalian cell-made EPO (rhuEPO(M by enzymatic desialylation. In the current study, we took advantage of a plant-based expression system lacking sialylating capacity but possessing an ability to synthesize complex N-glycans to produce cytoprotective recombinant human asialo-rhuEPO. Transgenic tobacco plants expressing asialo-rhuEPO were generated by stably co-expressing human EPO and β1,4-galactosyltransferase (GalT genes under the control of double CaMV 35S and glyceraldehyde-3-phosphate gene (GapC promoters, respectively. Plant-produced asialo-rhuEPO (asialo-rhuEPO(P was purified by immunoaffinity chromatography. Detailed N-glycan analysis using NSI-FTMS and MS/MS revealed that asialo-rhuEPO(P bears paucimannosidic, high mannose-type and complex N-glycans. In vitro cytoprotection assays showed that the asialo-rhuEPO(P (20 U/ml provides 2-fold better cytoprotection (44% to neuronal-like mouse neuroblastoma cells from staurosporine-induced cell death than rhuEPO(M (21%. The cytoprotective effect of the asialo-rhuEPO(P was found to be mediated by receptor-initiated phosphorylation of Janus kinase 2 (JAK2 and suppression of caspase 3 activation. Altogether, these findings demonstrate that plants are a suitable host for producing cytoprotective rhuEPO derivative. In addition, the general advantages of plant-based expression system can be exploited to address the cost and scalability issues related to its production.

  15. Thyroid hormone receptor β mutants: Dominant negative regulators of peroxisome proliferator-activated receptor γ action

    Araki, Osamu; Ying, Hao; Furuya, Fumihiko; Zhu, Xuguang; Cheng, Sheue-yann

    2005-01-01

    Thyroid hormone (T3) and peroxisome proliferators have overlapping metabolic effects in the maintenance of lipid homeostasis. Their actions are mediated by their respective receptors: thyroid hormone receptors (TR) and peroxisome proliferator-activated receptors (PPAR). We recently found that a dominantly negative TRβ mutant (PV) that causes a genetic disease, resistance to thyroid hormone, acts to repress the ligand (troglitazone)-mediated transcriptional activity of PPARγ in cultured thyroi...

  16. ARA 290, a Nonerythropoietic Peptide Engineered from Erythropoietin, Improves Metabolic Control and Neuropathic Symptoms in Patients with Type 2 Diabetes

    Brines, Michael; Dunne, Ann N; van Velzen, Monique; Proto, Paolo L; Ostenson, Claes-Goran; Kirk, Rita I; Petropoulos, Ioannis N; Javed, Saad; Malik, Rayaz A; Cerami, Anthony; Dahan, Albert

    2014-01-01

    Although erythropoietin ameliorates experimental type 2 diabetes with neuropathy, serious side effects limit its potential clinical use. ARA 290, a nonhematopoietic peptide designed from the structure of erythropoietin, interacts selectively with the innate repair receptor that mediates tissue protection. ARA 290 has shown efficacy in preclinical and clinical studies of metabolic control and neuropathy. To evaluate the potential activity of ARA 290 in type 2 diabetes and painful neuropathy, subjects were enrolled in this phase 2 study. ARA 290 (4 mg) or placebo were self-administered subcutaneously daily for 28 d and the subjects followed for an additional month without further treatment. No potential safety issues were identified. Subjects receiving ARA 290 exhibited an improvement in hemoglobin A1c (Hb A1c) and lipid profiles throughout the 56 d observation period. Neuropathic symptoms as assessed by the PainDetect questionnaire improved significantly in the ARA 290 group. Mean corneal nerve fiber density (CNFD) was reduced significantly compared with normal controls and subjects with a mean CNFD >1 standard deviation from normal showed a significant increase in CNFD compared with no change in the placebo group. These observations suggest that ARA 290 may benefit both metabolic control and neuropathy in subjects with type 2 diabetes and deserves continued clinical evaluation. PMID:25387363

  17. The insulin receptor C-terminus is involved in regulation of the receptor kinase activity.

    Kaliman, P; Baron, V; Alengrin, F; Takata, Y; Webster, N J; Olefsky, J M; Van Obberghen, E

    1993-09-21

    During the insulin receptor activation process, ligand binding and autophosphorylation induce two distinct conformational changes in the C-terminal domain of the receptor beta-subunit. To analyze the role of this domain and the involvement of the C-terminal autophosphorylation sites (Tyr1316 and Tyr1322) in receptor activation, we used (i) antipeptide antibodies against three different C-terminal sequences (1270-1281, 1294-1317, and 1309-1326) and (ii) an insulin receptor mutant (Y/F2) where Tyr1316 and Tyr1322 have been replaced by Phe. We show that the autophosphorylation-induced C-terminal conformational change is preserved in the Y/F2 receptor, indicating that this change is not induced by phosphorylation of the C-terminal sites but most likely by phosphorylation of the major sites in the kinase domain (Tyr1146, Tyr1150, and Tyr1151). Binding of antipeptide antibodies to the C-terminal domain modulated (activated or inhibited) both mutant and wild-type receptor-mediated phosphorylation of poly(Glu/Tyr). In contrast to the wild-type receptor, Y/F2 exhibited the same C-terminal configuration before and after insulin binding, evidencing that mutation of Tyr1316 and Tyr1322 introduced conformational changes in the C-terminus. Finally, the mutant receptor was 2-fold more active than the wild-type receptor for poly(Glu/Tyr) phosphorylation. In conclusion, the whole C-terminal region of the insulin receptor beta-subunit is likely to exert a regulatory influence on the receptor kinase activity. Perturbations of the C-terminal region, such as binding of antipeptides or mutation of Tyr1316 and Tyr1322, provoke alterations at the receptor kinase level, leading to activation or inhibition of the enzymic activity. PMID:7690586

  18. The WSXWS motif in cytokine receptors is a molecular switch involved in receptor activation

    Dagil, Robert; Knudsen, Maiken J.; Olsen, Johan Gotthardt;

    2012-01-01

    The prolactin receptor (PRLR) is activated by binding of prolactin in a 2:1 complex, but the activation mechanism is poorly understood. PRLR has a conserved WSXWS motif generic to cytokine class I receptors. We have determined the nuclear magnetic resonance solution structure of the membrane prox...

  19. In vitro neuroprotective action of recombinant rat erythropoietin produced by astrocyte cell lines and comparative studies with erythropoietin produced by Chinese hamster ovary cells

    Masuda, Seiji; Kada, Emi; Nagao, Masaya; Sasaki, Ryuzo

    1999-01-01

    In the central nervous system, astrocytes produce erythropoietin (Epo) and neurons express its receptor. To examine whether or not the brain Epo protects the in vitro cultured neurons from glutamate-induced cell death, we established rat astrocyte cell lines containing the plasmid for production of recombinant rat Epo. Epo partially purified from the culture medium showed a neuroprotective effect similar to that of rat Epo produced by Chinese hamster ovary (CHO) cells. Comparison was made in ...

  20. Recombinant human erythropoietin in sports: a review

    Rafael Maia de Almeida Bento

    2003-06-01

    Full Text Available Erythropoietin is an endogenous hormone of glicoproteic nature secreted by the kidneys and is the main regulator of the erythropoiesis. An alteration in its production generates a disturbance in the plasmatic concentration giving rise to several types of pathologies related to the hematopoietic system. The recombinant forms of erythropoietin have indiscriminately been used by athletes, mainly in endurance sports, by increasing the erythrocytes concentration, generating a better delivery of oxygen to the muscle tissue. The administration of recombinant erythropoietin was prohibited by the International Olympic Committee and its use considered as doping. This review has the intention to describe the physical, biological and pharmacokinetic properties of the endogenous erythropoietin, as well as its recombinant form, describing also its use in sports and the process of searching methodologies for its detection in doping control.

  1. Activation of glucocorticoid receptors increases 5-HT2A receptor levels

    Trajkovska, Viktorija; Kirkegaard, Lisbeth; Krey, Gesa; Marcussen, Anders Bue; Thomsen, Morten Skøtt; Chourbaji, Sabine; Brandwein, Christiane; Ridder, Stephanie; Halldin, Christer; Gass, Peter; Knudsen, Gitte M; Aznar, Susana

    2009-01-01

    Major depression is associated with both dysregulation of the hypothalamic pituitary adrenal axis and serotonergic deficiency, not the least of the 5-HT2A receptor. However, how these phenomena are linked to each other, and whether a low 5-HT2A receptor level is a state or a trait marker of...... depression is unknown. In mice with altered glucocorticoid receptor (GR) expression we investigated 5-HT2A receptor levels by Western blot and 3H-MDL100907 receptor binding. Serotonin fibre density was analyzed by stereological quantification of serotonin transporter immunopositive fibers. To establish an...... effect of GR activation on 5-HT2A levels, mature organotypic hippocampal cultures were exposed to corticosterone with or without GR antagonist mifepristone and mineralocorticoid receptor (MR) antagonist spironolactone. In GR under-expressing mice, hippocampal 5-HT2A receptor protein levels were decreased...

  2. Recent Trends in Erythropoietin-mediated Neuroprotection

    McPherson, Ronald J.; Juul, Sandra E.

    2007-01-01

    Fifteen years of evidence have established that the cytokine erythropoietin offers promise as a treatment for brain injury. In particular, neonatal brain injury may be reduced or prevented by early treatment with recombinant erythropoietin. Extreme prematurity and perinatal asphyxia are common conditions associated with poor neurodevelopmental outcomes including cerebral palsy, mental retardation, hearing or visual impairment, and attention deficit hyperactivity disorder. When high doses of e...

  3. Recombinant human erythropoietin in sports: a review

    Rafael Maia de Almeida Bento; Lúcia Menezes Pinto Damasceno; Francisco Radler Aquino Neto

    2003-01-01

    Erythropoietin is an endogenous hormone of glicoproteic nature secreted by the kidneys and is the main regulator of the erythropoiesis. An alteration in its production generates a disturbance in the plasmatic concentration giving rise to several types of pathologies related to the hematopoietic system. The recombinant forms of erythropoietin have indiscriminately been used by athletes, mainly in endurance sports, by increasing the erythrocytes concentration, generating a better delivery of ox...

  4. Cell death sensitization of leukemia cells by opioid receptor activation

    Friesen, Claudia; Roscher, Mareike; Hormann, Inis; Fichtner, Iduna; Alt, Andreas; Hilger, Ralf A.; Debatin, Klaus-Michael; Miltner, Erich

    2013-01-01

    Cyclic AMP (cAMP) regulates a number of cellular processes and modulates cell death induction. cAMP levels are altered upon stimulation of specific G-protein-coupled receptors inhibiting or activating adenylyl cyclases. Opioid receptor stimulation can activate inhibitory Gi-proteins which in turn block adenylyl cyclase activity reducing cAMP. Opioids such as D,L-methadone induce cell death in leukemia cells. However, the mechanism how opioids trigger apoptosis and activate caspases in leukemia cells is not understood. In this study, we demonstrate that downregulation of cAMP induced by opioid receptor activation using the opioid D,L-methadone kills and sensitizes leukemia cells for doxorubicin treatment. Enhancing cAMP levels by blocking opioid-receptor signaling strongly reduced D,L-methadone-induced apoptosis, caspase activation and doxorubicin-sensitivity. Induction of cell death in leukemia cells by activation of opioid receptors using the opioid D,L-methadone depends on critical levels of opioid receptor expression on the cell surface. Doxorubicin increased opioid receptor expression in leukemia cells. In addition, the opioid D,L-methadone increased doxorubicin uptake and decreased doxorubicin efflux in leukemia cells, suggesting that the opioid D,L-methadone as well as doxorubicin mutually increase their cytotoxic potential. Furthermore, we found that opioid receptor activation using D,L-methadone alone or in addition to doxorubicin inhibits tumor growth significantly in vivo. These results demonstrate that opioid receptor activation via triggering the downregulation of cAMP induces apoptosis, activates caspases and sensitizes leukemia cells for doxorubicin treatment. Hence, opioid receptor activation seems to be a promising strategy to improve anticancer therapies. PMID:23633472

  5. Recombinant Human Erythropoietin Protects Myocardial Cells from Apoptosis via the Janus-Activated Kinase 2/Signal Transducer and Activator of Transcription 5 Pathway in Rats with Epilepsy

    Bao-Xin Ma, MD

    2015-12-01

    Conclusions: These results indicate that myocardial cell apoptosis may contribute to myocardial injury in epilepsy. EPO protects myocardial cells from apoptosis via the JAK2/STAT5 pathway in rats with experimental epilepsy, whereas CEPO exerts antiapoptotic activity perhaps via a pathway independent of JAK2/STAT5 signaling.

  6. Comparison of the activation kinetics of the M3 acetylcholine receptor and a constitutively active mutant receptor in living cells.

    Hoffmann, Carsten; Nuber, Susanne; Zabel, Ulrike; Ziegler, Nicole; Winkler, Christiane; Hein, Peter; Berlot, Catherine H; Bünemann, Moritz; Lohse, Martin J

    2012-08-01

    Activation of G-protein-coupled receptors is the first step of the signaling cascade triggered by binding of an agonist. Here we compare the activation kinetics of the G(q)-coupled M(3) acetylcholine receptor (M(3)-AChR) with that of a constitutively active mutant receptor (M(3)-AChR-N514Y) using M(3)-AChR constructs that report receptor activation by changes in the fluorescence resonance energy transfer (FRET) signal. We observed a leftward shift in the concentration-dependent FRET response for acetylcholine and carbachol with M(3)-AChR-N514Y. Consistent with this result, at submaximal agonist concentrations, the activation kinetics of M(3)-AChR-N514Y were significantly faster, whereas at maximal agonist concentrations the kinetics of receptor activation were identical. Receptor deactivation was significantly faster with carbachol than with acetylcholine and was significantly delayed by the N514Y mutation. Receptor-G-protein interaction was measured by FRET between M(3)-AChR-yellow fluorescent protein (YFP) and cyan fluorescent protein (CFP)-Gγ(2). Agonist-induced receptor-G-protein coupling was of a time scale similar to that of receptor activation. As observed for receptor deactivation, receptor-G-protein dissociation was slower for acetylcholine than that for carbachol. Acetylcholine-stimulated increases in receptor-G-protein coupling of M(3)-AChR-N514Y reached only 12% of that of M(3)-AChR and thus cannot be kinetically analyzed. G-protein activation was measured using YFP-tagged Gα(q) and CFP-tagged Gγ(2). Activation of G(q) was significantly slower than receptor activation and indistinguishable for the two agonists. However, G(q) deactivation was significantly prolonged for acetylcholine compared with that for carbachol. Consistent with decreased agonist-stimulated coupling to G(q), agonist-stimulated G(q) activation by M(3)-AChR-N514Y was not detected. Taken together, these results indicate that the N514Y mutation produces constitutive activation of M(3

  7. The anemia of microgravity and recumbency. Role of sympathetic neural control of erythropoietin production

    Robertson, David; Krantz, Sanford B.; Biaggioni, Italo

    We hypothesize that reduced sympathetic stimulation of erythropoietin production may maintain the anemia which develops in virtually all space travellers. We tested this hypothesis in a human model of reduced sympathetic activity. Thirty-three patients with the Bradbury-Eggleston syndrome were divided into three groups according to their hemoglobin (Hgb) level. Patients with low Hgb had lower upright norepinephrine and lower upright renin. Patients with anemia also had inappropriately low plasma erythropoietin levels. We administered recombinant erythropoietin (Epogen) 25-50 units/kg s.c. 3 times per week and found that the anemia seen in autonomic failure could be reversed by this treatment. These results support the hypothesis that erythropoiesis is modulated by the sympathetic nervous system and that such mechanisms may also operate in the microgravity environment where sympathetic activity is reduced.

  8. The Orphan Nuclear Receptor TR4 Is a Vitamin A-activated Nuclear Receptor

    Zhou, X. Edward; Suino-Powell, Kelly M.; Xu, Yong; Chan, Cee-Wah; Tanabe, Osamu; Kruse, Schoen W.; Reynolds, Ross; Engel, James Douglas; Xu, H. Eric (Michigan-Med); (Van Andel)

    2015-11-30

    Testicular receptors 2 and 4 (TR2/4) constitute a subgroup of orphan nuclear receptors that play important roles in spermatogenesis, lipid and lipoprotein regulation, and the development of the central nervous system. Currently, little is known about the structural features and the ligand regulation of these receptors. Here we report the crystal structure of the ligand-free TR4 ligand binding domain, which reveals an autorepressed conformation. The ligand binding pocket of TR4 is filled by the C-terminal half of helix 10, and the cofactor binding site is occupied by the AF-2 helix, thus preventing ligand-independent activation of the receptor. However, TR4 exhibits constitutive transcriptional activity on multiple promoters, which can be further potentiated by nuclear receptor coactivators. Mutations designed to disrupt cofactor binding, dimerization, or ligand binding substantially reduce the transcriptional activity of this receptor. Importantly, both retinol and retinoic acid are able to promote TR4 to recruit coactivators and to activate a TR4-regulated reporter. These findings demonstrate that TR4 is a ligand-regulated nuclear receptor and suggest that retinoids might have a much wider regulatory role via activation of orphan receptors such as TR4.

  9. Incorporation of Ortho- and Meta-Tyrosine Into Cellular Proteins Leads to Erythropoietin-Resistance in an Erythroid Cell Line

    Esztella Mikolás

    2014-04-01

    Full Text Available Background/Aims: Erythropoietin-resistance is an unsolved concern in the treatment of renal anaemia. We aimed to investigate the possible role of ortho- and meta-tyrosine - the hydroxyl free radical products of L-phenylalanine - in the development of erythropoietin-resistance. Methods: TF-1 erythroblast cell line was used. Cell concentration was determined on day 1; 2 and 3 by two independent observers simultaneously in Bürker cell counting chambers. Protein concentration was determined with colorimetric method. Para-, ortho- and meta-tyrosine levels were measured using reverse phase-HPLC with fluorescence detection. Using Western blot method activating phosphorylation of STAT5 and ERK1/2 were investigated. Results: We found a time- and concentration-dependent decrease of erythropoietin-induced proliferative activity in case of ortho- and meta-tyrosine treated TF-1 erythroblasts, compared to the para-tyrosine cultured cells. Decreased erythropoietin-response could be regained with a competitive dose of para-tyrosine. Proteins of erythroblasts treated by ortho- or meta-tyrosine had lower para-tyrosine and higher ortho- or meta-tyrosine content. Activating phosphorylation of ERK and STAT5 due to erythropoietin was practically prevented by ortho- or meta-tyrosine treatment. Conclusion: According to this study elevated ortho- and meta-tyrosine content of erythroblasts may lead to the dysfunction of intracellular signaling, resulting in erythropoietin-hyporesponsiveness.

  10. Kit transduced signals counteract erythroid maturation by MAPK-dependent modulation of erythropoietin signaling and apoptosis induction in mouse fetal liver.

    Haas, N; Riedt, T; Labbaf, Z; Baßler, K; Gergis, D; Fröhlich, H; Gütgemann, I; Janzen, V; Schorle, H

    2015-05-01

    Signaling by the stem cell factor receptor Kit in hematopoietic stem and progenitor cells is functionally associated with the regulation of cellular proliferation, differentiation and survival. Expression of the receptor is downregulated upon terminal differentiation in most lineages, including red blood cell terminal maturation, suggesting that omission of Kit transduced signals is a prerequisite for the differentiation process to occur. However, the molecular mechanisms by which Kit signaling preserves the undifferentiated state of progenitor cells are not yet characterized in detail. In this study, we generated a mouse model for inducible expression of a Kit receptor carrying an activating mutation and studied its effects on fetal liver hematopoiesis. We found that sustained Kit signaling leads to expansion of erythroid precursors and interferes with terminal maturation beyond the erythroblast stage. Primary KIT(D816V) erythroblasts stimulated to differentiate fail to exit cell cycle and show elevated rates of apoptosis because of insufficient induction of survival factors. They further retain expression of progenitor cell associated factors c-Myc, c-Myb and GATA-2 and inefficiently upregulate erythroid transcription factors GATA-1, Klf1 and Tal1. In KIT(D816V) erythroblasts we found constitutive activation of the mitogen-activated protein kinase (MAPK) pathway, elevated expression of the src kinase family member Lyn and impaired Akt activation in response to erythropoietin. We demonstrate that the block in differentiation is partially rescued by MAPK inhibition, and completely rescued by the multikinase inhibitor Dasatinib. These results show that a crosstalk between Kit and erythropoietin receptor signaling cascades exists and that continuous Kit signaling, partly mediated by the MAPK pathway, interferes with this crosstalk. PMID:25323585

  11. Continuous production of erythropoietin by an established human renal carcinoma cell line: development of the cell line

    Establishment of a stable, transformed human renal carcinoma cell line that produces erythropoietin in vitro and has maintained this function continuously since 1981 and for > 150 passages in monolayer culture was accomplished by transplantation of human renal clear cell carcinoma tissue from a patient with erythrocytosis into an immunosuppressed athymic mouse. In addition to its immunocrossreactivity with native human urinary erythropoietin, the tumor erythropoietin demonstrates biological activity in the in vitro mouse erythroid colony-forming unit assay and in tumor-bearing nude mice. The cloned renal carcinoma cell line has an abnormal human karyotype and has ultrastructural features characteristic of human renal clear cell carcinoma. This cell line provides a reproducible model system for the production of an erythropoietin-like material and for the study of its synthesis and secretion

  12. The insulin receptor activation process involves localized conformational changes.

    Baron, V; Kaliman, P; Gautier, N; Van Obberghen, E

    1992-11-15

    The molecular process by which insulin binding to the receptor alpha-subunit induces activation of the receptor beta-subunit with ensuing substrate phosphorylation remains unclear. In this study, we aimed at approaching this molecular mechanism of signal transduction and at delineating the cytoplasmic domains implied in this process. To do this, we used antipeptide antibodies to the following sequences of the receptor beta-subunit: (i) positions 962-972 in the juxtamembrane domain, (ii) positions 1247-1261 at the end of the kinase domain, and (iii) positions 1294-1317 and (iv) positions 1309-1326, both in the receptor C terminus. We have previously shown that insulin binding to its receptor induces a conformational change in the beta-subunit C terminus. Here, we demonstrate that receptor autophosphorylation induces an additional conformational change. This process appears to be distinct from the one produced by ligand binding and can be detected in at least three different beta-subunit regions: the juxtamembrane domain, the kinase domain, and the C terminus. Hence, the cytoplasmic part of the receptor beta-subunit appears to undergo an extended conformational change upon autophosphorylation. By contrast, the insulin-induced change does not affect the juxtamembrane domain 962-972 nor the kinase domain 1247-1261 and may be limited to the receptor C terminus. Further, we show that the hormone-dependent conformational change is maintained in a kinase-deficient receptor due to a mutation at lysine 1018. Therefore, during receptor activation, the ligand-induced change could precede ATP binding and receptor autophosphorylation. We propose that insulin binding leads to a transient receptor form that may allow ATP binding and, subsequently, autophosphorylation. The second conformational change could unmask substrate-binding sites and stabilize the receptor in an active conformation. PMID:1331080

  13. Interaction of chemokines with their receptors--from initial chemokine binding to receptor activating steps

    Thiele, Stefanie; Rosenkilde, Mette Marie

    2014-01-01

    The human chemokine system comprises 19 seven-transmembrane helix (7TM) receptors and 45 endogenous chemokines that often interact with each other in a promiscuous manner. Due to the chemokine system's primary function in leukocyte migration, it has a central role in immune homeostasis and...... interactions possibly occur, resulting in a multi-step process, as recently proposed for other 7TM receptors. Overall, the N-terminus of chemokine receptors is pivotal for binding of all chemokines. During receptor activation, differences between the two major chemokine subgroups occur, as CC-chemokines mainly...

  14. Chronic hyperammonemia induces tonic activation of NMDA receptors in cerebellum.

    ElMlili, Nisrin; Boix, Jordi; Ahabrach, Hanan; Rodrigo, Regina; Errami, Mohammed; Felipo, Vicente

    2010-02-01

    Reduced function of the glutamate--nitric oxide (NO)--cGMP pathway is responsible for some cognitive alterations in rats with hyperammonemia and hepatic encephalopathy. Hyperammonemia impairs the pathway in cerebellum by increasing neuronal nitric oxide synthase (nNOS) phosphorylation in Ser847 by calcium-calmodulin-dependent protein kinase II (CaMKII), reducing nNOS activity, and by reducing nNOS amount in synaptic membranes, which reduces its activation following NMDA receptors activation. The reason for increased CaMKII activity in hyperammonemia remains unknown. We hypothesized that it would be as a result of increased tonic activation of NMDA receptors. The aims of this work were to assess: (i) whether tonic NMDA activation receptors is increased in cerebellum in chronic hyperammonemia in vivo; and (ii) whether this tonic activation is responsible for increased CaMKII activity and reduced activity of nNOS and of the glutamate--NO--cGMP pathway. Blocking NMDA receptors with MK-801 increases cGMP and NO metabolites in cerebellum in vivo and in slices from hyperammonemic rats. This is because of reduced phosphorylation and activity of CaMKII, leading to normalization of nNOS phosphorylation and activity. MK-801 also increases nNOS in synaptic membranes and reduces it in cytosol. This indicates that hyperammonemia increases tonic activation of NMDA receptors leading to reduced activity of nNOS and of the glutamate--NO--cGMP pathway. PMID:20002515

  15. Redefining the concept of protease-activated receptors: cathepsin S evokes itch via activation of Mrgprs

    Reddy, Vemuri B.; Sun, Shuohao; Azimi, Ehsan; Elmariah, Sarina B.; Dong, Xinzhong; Lerner, Ethan A.

    2015-01-01

    Sensory neurons expressing Mas-related G protein coupled receptors (Mrgprs) mediate histamine-independent itch. We show that the cysteine protease cathepsin S activates MrgprC11 and evokes receptor-dependent scratching in mice. In contrast to its activation of conventional protease-activated receptors, cathepsin S mediated activation of MrgprC11 did not involve the generation of a tethered ligand. We demonstrate further that different cysteine proteases selectively activate specific mouse and...

  16. Recombinant human erythropoietin (rHuEPO): more than just the correction of uremic anemia.

    Buemi, Michele; Aloisi, Carmela; Cavallaro, Emanuela; Corica, Francesco; Floccari, Fulvio; Grasso, Giovanni; Lasco, Antonino; Pettinato, Giuseppina; Ruello, Antonella; Sturiale, Alessio; Frisina, Nicola

    2002-01-01

    Hematopoiesis is controlled by numerous interdependent humoral and endocrine factors. Erythropoietin (EPO), a hydrophobic sialoglycoproteic hormone, plays a crucial role in the regulation of hematopoiesis, and induces proliferation, maturation and differentiation of the erythroid cell line precursors. Thanks to recombinant DNA techniques, different recombinant hormones can now be produced at low cost and in large amounts. This has led to greater understanding of the pathophysiological factors regulating hematopoiesis. This in turn, hasprompted the search for new therapeutic approaches. EPO might also be used to treat patients with different types of anemia: uremics, newborns, patients with anemia from cancer or myeloproliferative disease, thalassemia, bone marrow transplants, chronic infectious diseases. Besides erythroid cells, EPO affects other blood cell lines, such as myeloid cells, lymphocytes and megakaryocytes. It can also enhance polymorphonuclear cell phagocytosis and reduce macrophage activation, thus modulating the inflammatory process. Hematopoietic and endothelial cells probably have the same origin, and the discovery of eyrthropoietin receptors also on mesangial, myocardial and smooth muscle cells has prompted research into the non-erythropoietic function of the hormone. EPO has an important, direct, hemodynamic and vasoactive effect, which does not depend only on an increase in hematocrit and viscosity. Moreover, EPO and its receptors have been found in the brain, suggesting a role in preventing neuronal death. Finally, the recently discovered interaction between EPO and vascular endothelial growth factor (VEGF), and the ability of EPO to stimulate endothelial cell mitosis and motility may be of importance in neovascularization and wound healing. PMID:12018644

  17. Human receptor activation by aroclor 1260, a polychlorinated biphenyl mixture.

    Wahlang, Banrida; Falkner, K Cameron; Clair, Heather B; Al-Eryani, Laila; Prough, Russell A; States, J Christopher; Coslo, Denise M; Omiecinski, Curtis J; Cave, Matthew C

    2014-08-01

    Polychlorinated biphenyls (PCBs) are persistent environmental toxicants, present in 100% of U.S. adults and dose-dependently associated with obesity and non-alcoholic fatty liver disease (NAFLD). PCBs are predicted to interact with receptors previously implicated in xenobiotic/energy metabolism and NAFLD. These receptors include the aryl hydrocarbon receptor (AhR), pregnane xenobiotic receptor (PXR), constitutive androstane receptor (CAR), peroxisome proliferator-activated receptors (PPARs), liver-X-receptor (LXRα), and farnesoid-X-receptor (FXR). This study evaluates Aroclor 1260, a PCB mixture with congener composition mimicking that of human adipose tissue, and selected congeners, as potential ligands for these receptors utilizing human hepatoma-derived (HepG2) and primate-derived (COS-1) cell lines, and primary human hepatocytes. Aroclor 1260 (20 μg/ml) activated AhR, and PCB 126, a minor component, was a potent inducer. Aroclor 1260 activated PXR in a simple concentration-dependent manner at concentrations ≥10 μg/ml. Among the congeners tested, PCBs 138, 149, 151, 174, 183, 187, and 196 activated PXR. Aroclor 1260 activated CAR2 and CAR3 variants at lower concentrations and antagonize CAR2 activation by the CAR agonist, CITCO, at higher concentrations (≥20 μg/ml). Additionally, Aroclor 1260 induced CYP2B6 in primary hepatocytes. At subtoxic doses, Aroclor 1260 did not activate LXR or FXR and had no effect on LXR- or FXR-dependent induction by the agonists T0901317 or GW4064, respectively. Aroclor 1260 (20 μg/ml) suppressed PPARα activation by the agonist nafenopin, although none of the congeners tested demonstrated significant inhibition. The results suggest that Aroclor 1260 is a human AhR, PXR and CAR3 agonist, a mixed agonist/antagonist for CAR2, and an antagonist for human PPARα. PMID:24812009

  18. Tonic activation of presynaptic GABAB receptors on rat pallidosubthalamic terminals

    Lei CHEN; Wing-ho YUNG

    2005-01-01

    Aim: The subthalamic nucleus plays a critical role in the regulation of movement,and abnormal activity of its neurons is associated with some basal ganglia motor symptoms. We examined the presence of functional presynaptic GABAB receptors on pallidosubthalamic terminals and tested whether they were tonically active in the in vitro subthalamic slices. Methods: Whole-cell patch-clamp recordings were applied to acutely prepared rat subthalamic nucleus slices. The effects of specific GABAB agonist and antagonist on action potential-independent inhibitory postsynapfic currents (IPSCs), as well as holding current, were examined.Results: Superfusion of baclofen, a GABAB receptor agonist, significantly reduced the frequency of GABAA receptor-mediated miniature IPSCs (mIPSCs), in a Cd2+-sensitive manner, with no effect on the amplitude, indicating presynaptic inhibition on GABA release. In addition, baclofen induced a weak outward current only in a minority of subthalamic neurons. Both the pre- and post-synaptic effects of baclofen were prevented by the specific GABAB receptor antagonist,CGP55845. Furthermore, CGP55845 alone increased the frequency of mIPSCs,but had no effect on the holding current. Conclusion: These findings suggest the functional dominance of presynaptic GABAB receptors on the pallidosubthalamic terminals over the postsynaptic GABAB receptors on subthalamic neurons.Furthermore, the presynaptic, but not the postsynaptic, GABAB receptors are tonically active, suggesting that the presynaptic GABAB receptors in the subthalamic nucleus are potential therapeutic target for the treatment of Parkinson disease.

  19. Skeletal muscle alterations and exercise performance decrease in erythropoietin-deficient mice: a comparative study

    Mille-Hamard Laurence; Billat Veronique L; Henry Elodie; Bonnamy Blandine; Joly Florence; Benech Philippe; Barrey Eric

    2012-01-01

    Abstract Background Erythropoietin (EPO) is known to improve exercise performance by increasing oxygen blood transport and thus inducing a higher maximum oxygen uptake (VO2max). Furthermore, treatment with (or overexpression of) EPO induces protective effects in several tissues, including the myocardium. However, it is not known whether EPO exerts this protective effect when present at physiological levels. Given that EPO receptors have been identified in skeletal muscle, we hypothesized that...

  20. Erythropoietin binding sites in human foetal tissues

    Pekonen, F.; Rosenloef, K.; Rutanen, E.-M.

    1987-01-01

    Using /sup 125/I labelled recombinant DNA human erythropoietin (EP), we have explored the presence and properties of EP binding sites in foetal human tissues. The EP binding site is present in the foetal liver already during the first trimester of pregnancy. The binding site has a equilibrium association constant of 4.1-6.2 x 10/sup 9/l/mol and is specific for EP. The cross-reactivities of FSH, TSH, hCG, insulin and renin substrate were less than 0.01%. The EP binding capacity of foetal liver was 5.4-16 fmol/mg membrane protein. In foetal lung tissue, a slight EP binding activity was observed, whereas foetal spleen, muscle, brain, thyroid and placental tissues were virtually devoid of EP binding capacity. The same level of binding was reached at 37 deg. C in 1 h and at 4 deg. C in 24 h. The binding was pH-dependent with maximal specific binding at pH 7.7. SDS-PAGE gel electrophoresis analysis of covalently cross-linked /sup 125/I-EP to foetal liver membranes suggested that the EP binding site was composed of two subunits with an apparent mol wt of 41000 and 86000 dalton, respectively.

  1. Erythropoietin binding sites in human foetal tissues

    Using 125I labelled recombinant DNA human erythropoietin (EP), we have explored the presence and properties of EP binding sites in foetal human tissues. The EP binding site is present in the foetal liver already during the first trimester of pregnancy. The binding site has a equilibrium association constant of 4.1-6.2 x 109l/mol and is specific for EP. The cross-reactivities of FSH, TSH, hCG, insulin and renin substrate were less than 0.01%. The EP binding capacity of foetal liver was 5.4-16 fmol/mg membrane protein. In foetal lung tissue, a slight EP binding activity was observed, whereas foetal spleen, muscle, brain, thyroid and placental tissues were virtually devoid of EP binding capacity. The same level of binding was reached at 37 deg. C in 1 h and at 4 deg. C in 24 h. The binding was pH-dependent with maximal specific binding at pH 7.7. SDS-PAGE gel electrophoresis analysis of covalently cross-linked 125I-EP to foetal liver membranes suggested that the EP binding site was composed of two subunits with an apparent mol wt of 41000 and 86000 dalton, respectively. (author)

  2. Erythropoietin upregulation in pulmonary arterial hypertension.

    Karamanian, Vanesa A; Harhay, Michael; Grant, Gregory R; Palevsky, Harold I; Grizzle, William E; Zamanian, Roham T; Ihida-Stansbury, Kaori; Taichman, Darren B; Kawut, Steven M; Jones, Peter L

    2014-06-01

    The pathophysiologic alterations of patients with pulmonary arterial hypertension (PAH) are diverse. We aimed to determine novel pathogenic pathways from circulating proteins in patients with PAH. Multianalyte profiling (MAP) was used to measure 90 specifically selected antigens in the plasma of 113 PAH patients and 51 control patients. Erythropoietin (EPO) functional activity was assessed via in vitro pulmonary artery endothelial cell networking and smooth muscle cell proliferation assays. Fifty-eight patients had idiopathic PAH, whereas 55 had other forms of PAH; 5 had heritable PAH, 18 had connective tissue disease (15 with scleroderma and 3 with lupus erythematosis), 13 had portopulmonary hypertension, 6 had PAH associated with drugs or toxins, and 5 had congenital heart disease. The plasma-antigen profile of PAH revealed increased levels of several novel biomarkers, including EPO. Immune quantitative and histochemical studies revealed that EPO not only was significantly elevated in the plasma of PAH patients but also promoted pulmonary artery endothelial cell network formation and smooth muscle cell proliferation. MAP is a hypothesis-generating approach to identifying novel pathophysiologic pathways in PAH. EPO is upregulated in the circulation and lungs of patients with PAH and may affect endothelial and smooth muscle cell proliferation. PMID:25006446

  3. Hemopoietic cell precursor responses to erythropoietin in plasma clot cultures

    Kennedy, W.L.

    1979-01-01

    The time dependence of the response of mouse bone marrow cells to erythropoietin (Ep) in vitro was studied. Experiments include studies on the Ep response of marrow cells from normal, plethoric, or bled mice. Results with normal marrow reveal: (1) Not all erythroid precursors (CFU-E) are alike in their response to Ep. A significant number of the precursors develop to a mature erythroid colony after very short Ep exposures, but they account for only approx. 13% of the total colonies generated when Ep is active for 48 hrs. If Ep is active more than 6 hrs, a second population of erythroid colonies emerges at a nearly constant rate until the end of the culture. Full erythroid colony production requires prolonged exposure to erythropoietin. (2) The longer erythropoietin is actively present, the larger the number of erythroid colonies that reach 17 cells or more. Two distinct populations of immediate erythroid precursors are also present in marrow from plethoric mice. In these mice, total colony numbers are equal to or below those obtained from normal mice. However, the population of fast-responding CFU-E is consistently decreased to 10 to 20% of that found in normal marrow. The remaining colonies are formed from plethoric marrow at a rate equal to normal marrow. With increasing Ep exposures, the number of large colonies produced increases. From the marrow of bled mice, total erythroid colony production is equal to or above that of normal marrow. Two populations of colony-forming cells are again evident, with the fast-responding CFU-E being below normal levels. The lack of colonies from this group was compensated in bled mice by rapid colony production in the second population. A real increase in numbers of precursors present in this pool increased the rate of colony production in culture to twice that of normal marrow. The number of large colonies obtained from bled mice was again increased as the Ep exposure was lengthened. (ERB)

  4. Helix 11 Dynamics is Critical for Constitutive Androstane Receptor Activity

    Wright, Edward; Busby, Scott A.; Wisecarver, Sarah; Vincent, Jeremy; Griffin, Patrick R.; Fernandez, Elias J.

    2011-01-01

    The constitutive androstane receptor (CAR) transactivation can occur in the absence of exogenous ligand and this activity is enhanced by agonists TCPOBOP and meclizine. We use biophysical and cell-based assays to show that increased activity of CAR(TCPOBOP) relative to CAR(meclizine) corresponds to a higher affinity of CAR(TCPOBOP) for the steroid receptor coactivator-1. Additionally, steady-state fluorescence spectra suggest conformational differences between CAR(TCPOBOP):RXR and CAR(meclizi...

  5. Heterodimeric interaction between retinoid X receptor alpha and orphan nuclear receptor OR1 reveals dimerization-induced activation as a novel mechanism of nuclear receptor activation.

    Wiebel, F F; Gustafsson, J.A.

    1997-01-01

    OR1 is a member of the steroid/thyroid hormone nuclear receptor superfamily which has been described to mediate transcriptional responses to retinoids and oxysterols. On a DR4 response element, an OR1 heterodimer with the nuclear receptor retinoid X receptor alpha (RXR alpha) has been described to convey transcriptional activation in both the absence and presence of the RXR ligand 9-cis retinoic acid, the mechanisms of which have remained unclear. Here, we dissect the effects of RXR alpha and...

  6. Retinoic Acid-mediated Nuclear Receptor Activation and Hepatocyte Proliferation

    Bushue, Nathan; Wan, Yu-Jui Yvonne

    2016-01-01

    Due to their well-known differentiation and apoptosis-inducing abilities, retinoic acid (RA) and its analogs have strong anti-cancer efficacy in human cancers. However, in vivo RA is a liver mitogen. While speculation has persisted that RA-mediated signaling is likely involved in hepatocyte proliferation during liver regeneration, direct evidence is still required. Findings in support of this proposition include observations that a release of retinyl palmitate (the precursor of RA) occurs in liver stellate cells following liver injury. Nevertheless, the biological action of this released vitamin A is virtually unknown. More likely is that the released vitamin A is converted to RA, the biological form, and then bound to a specific receptor (retinoid x receptor; RXRα), which is most abundantly expressed in the liver. Considering the mitogenic effects of RA, the RA-activated RXRα would likely then influence hepatocyte proliferation and liver tissue repair. At present, the mechanism by which RA stimulates hepatocyte proliferation is largely unknown. This review summarizes the activation of nuclear receptors (peroxisome proliferator activated receptor-α, pregnane x receptor, constitutive androstane receptor, and farnesoid x receptor) in an RXRα dependent manner to induce hepatocyte proliferation, providing a link between RA and its proliferative role.

  7. Environmental polycyclic aromatic hydrocarbons affect androgen receptor activation in vitro

    Vinggaard, Anne Marie; Hnida, Christina; Larsen, John Christian

    2000-01-01

    of certain PAHs to activate the Ah receptor was assessed in H4IIE liver cancer cells, stably transfected with a luciferase reporter gene system. The positive control 2, 3,7, 8-tetrachlorodibenzodioxin (TCDD) caused a 13-14-fold induction of luciferase activity reaching maximum activity at 0.1 nM. DB...

  8. Environmental polycyclic aromatic hydrocarbons affect androgen receptor activation in vitro

    Vinggaard, Anne Marie; Hnida, Christina; Larsen, John Christian

    of certain PAHs to activate the Ah receptor was assessed in H4IIE liver cancer cells, stably transfected with a luciferase reporter gene system. The positive control 2, 3,7, 8-tetrachlorodibenzodioxin (TCDD) caused a 13-14-fold induction of luciferase activity reaching maximum activity at 0.1 nM. DB...

  9. Modulation of β-catenin signaling by glucagon receptor activation.

    Jiyuan Ke

    Full Text Available The glucagon receptor (GCGR is a member of the class B G protein-coupled receptor family. Activation of GCGR by glucagon leads to increased glucose production by the liver. Thus, glucagon is a key component of glucose homeostasis by counteracting the effect of insulin. In this report, we found that in addition to activation of the classic cAMP/protein kinase A (PKA pathway, activation of GCGR also induced β-catenin stabilization and activated β-catenin-mediated transcription. Activation of β-catenin signaling was PKA-dependent, consistent with previous reports on the parathyroid hormone receptor type 1 (PTH1R and glucagon-like peptide 1 (GLP-1R receptors. Since low-density-lipoprotein receptor-related protein 5 (Lrp5 is an essential co-receptor required for Wnt protein mediated β-catenin signaling, we examined the role of Lrp5 in glucagon-induced β-catenin signaling. Cotransfection with Lrp5 enhanced the glucagon-induced β-catenin stabilization and TCF promoter-mediated transcription. Inhibiting Lrp5/6 function using Dickkopf-1(DKK1 or by expression of the Lrp5 extracellular domain blocked glucagon-induced β-catenin signaling. Furthermore, we showed that Lrp5 physically interacted with GCGR by immunoprecipitation and bioluminescence resonance energy transfer assays. Together, these results reveal an unexpected crosstalk between glucagon and β-catenin signaling, and may help to explain the metabolic phenotypes of Lrp5/6 mutations.

  10. Cloning, constitutive activity and expression profiling of two receptors related to relaxin receptors in Drosophila melanogaster.

    Van Hiel, Matthias B; Vandersmissen, Hans Peter; Proost, Paul; Vanden Broeck, Jozef

    2015-06-01

    Leucine-rich repeat containing G protein-coupled receptors (LGRs) comprise a cluster of transmembrane proteins, characterized by the presence of a large N-terminal extracellular domain. This receptor group can be classified into three subtypes. Belonging to the subtype C LGRs are the mammalian relaxin receptors LGR7 (RXFP1) and LGR8 (RXFP2), which mediate important reproductive and other processes. We identified two related receptors in the genome of the fruit fly and cloned their open reading frames into an expression vector. Interestingly, dLGR3 demonstrated constitutive activity at very low doses of transfected plasmid, whereas dLGR4 did not show any basal activity. Both receptors exhibited a similar expression pattern during development, with relatively high transcript levels during the first larval stage. In addition, both receptors displayed higher expression in male adult flies as compared to female flies. Analysis of the tissue distribution of both receptor transcripts revealed a high expression of dLGR3 in the female fat body, while the expression of dLGR4 peaked in the midgut of both the wandering and adult stage. PMID:25064813

  11. DMPD: Receptor tyrosine kinases and the regulation of macrophage activation. [Dynamic Macrophage Pathway CSML Database

    Full Text Available 14726496 Receptor tyrosine kinases and the regulation of macrophage activation. Cor...(.csml) Show Receptor tyrosine kinases and the regulation of macrophage activation. PubmedID 14726496 Title Receptor tyrosine kinases

  12. Nuclear receptor corepressor-dependent repression of peroxisome-proliferator-activated receptor delta-mediated transactivation

    Krogsdam, Anne-M; Nielsen, Curt A F; Neve, Søren;

    2002-01-01

    delta-RXR alpha heterodimer bound to an acyl-CoA oxidase (ACO)-type peroxisome-proliferator response element recruited a glutathione S-transferase-NCoR fusion protein in a ligand-independent manner. Contrasting with most other nuclear receptors, PPAR delta was found to interact equally well......The nuclear receptor corepressor (NCoR) was isolated as a peroxisome-proliferator-activated receptor (PPAR) delta interacting protein using the yeast two-hybrid system. NCoR interacted strongly with the ligand-binding domain of PPAR delta, whereas interactions with the ligand-binding domains...

  13. Transcriptional activation of nuclear estrogen receptor and progesterone receptor and its regulation.

    Xin, Qi-Liang; Qiu, Jing-Tao; Cui, Sheng; Xia, Guo-Liang; Wang, Hai-Bin

    2016-08-25

    Estrogen receptor (ER) and progesterone receptor (PR) are two important members of steroid receptors family, an evolutionarily conserved family of transcription factors. Upon binding to their ligands, ER and PR enter cell nucleus to interact with specific DNA element in the context of chromatin to initiate the transcription of diverse target genes, which largely depends on the timely recruitment of a wide range of cofactors. Moreover, the interactions between steroid hormones and their respective receptors also trigger post-translational modifications on these receptors to fine-tune their transcriptional activities. Besides the well-known phosphorylation modifications on tyrosine and serine/threonine residues, recent studies have identified several other covalent modifications, such as ubiquitylation and sumoylation. These post-translational modifications of steroid receptors affect its stability, subcellular localization, and/or cofactor recruitment; eventually influence the duration and extent of transcriptional activation. This review is to focus on the recent research progress on the transcriptional activation of nuclear ER and PR as well as their physiological functions in early pregnancy, which may help us to better understand related female reproductive diseases. PMID:27546504

  14. Monitoring leptin activity using the chicken leptin receptor.

    Hen, Gideon; Yosefi, Sera; Ronin, Ana; Einat, Paz; Rosenblum, Charles I; Denver, Robert J; Friedman-Einat, Miriam

    2008-05-01

    We report on the construction of a leptin bioassay based on the activation of chicken leptin receptor in cultured cells. A human embryonic kidney (HEK)-293 cell line, stably transfected with the full-length cDNA of chicken leptin receptor together with a STAT3-responsive reporter gene specifically responded to recombinant human and Xenopus leptins. The observed higher sensitivity of chicken leptin receptor to the former is in agreement with the degree of sequence similarity among these species (about 60 and 38% identical amino acids between humans and chickens, and between humans and Xenopus respectively). The specific activation of signal transduction through the chicken leptin receptor, shown here for the first time, suggests that the transition of Gln269 (implicated in the Gln-to-Pro Zucker fatty mutation in rats) to Glu in chickens does not impair its activity. Analysis of leptin-like activity in human serum samples of obese and lean subjects coincided well with leptin levels determined by RIA. Serum samples of pre- and post partum cows showed a tight correlation with the degree of adiposity. However, specific activation of the chicken leptin receptor in this assay was not observed with serum samples from broiler or layer chickens (representing fat and lean phenotypes respectively) or with those from turkey. Similar leptin receptor activation profiles were observed with cells transfected with human leptin receptor. Further work is needed to determine whether the lack of leptin-like activity in the chicken serum samples is due to a lack of leptin in this species or simply to a serum level of leptin that is below the detection threshold. PMID:18434362

  15. Identification of COUP-TFII Orphan Nuclear Receptor as a Retinoic Acid-Activated Receptor

    Kruse, Schoen W; Suino-Powell, Kelly; Zhou, X Edward; Kretschman, Jennifer E; Reynolds, Ross; Vonrhein, Clemens; Xu, Yong; Wang, Liliang; Tsai, Sophia Y; Tsai, Ming-Jer; Xu, H Eric [Baylor; (Van Andel); (Globel Phasing); (Grand Valley)

    2010-01-12

    The chicken ovalbumin upstream promoter-transcription factors (COUP-TFI and II) make up the most conserved subfamily of nuclear receptors that play key roles in angiogenesis, neuronal development, organogenesis, cell fate determination, and metabolic homeostasis. Although the biological functions of COUP-TFs have been studied extensively, little is known of their structural features or aspects of ligand regulation. Here we report the ligand-free 1.48 {angstrom} crystal structure of the human COUP-TFII ligand-binding domain. The structure reveals an autorepressed conformation of the receptor, where helix {alpha}10 is bent into the ligand-binding pocket and the activation function-2 helix is folded into the cofactor binding site, thus preventing the recruitment of coactivators. In contrast, in multiple cell lines, COUP-TFII exhibits constitutive transcriptional activity, which can be further potentiated by nuclear receptor coactivators. Mutations designed to disrupt cofactor binding, dimerization, and ligand binding, substantially reduce the COUP-TFII transcriptional activity. Importantly, retinoid acids are able to promote COUP-TFII to recruit coactivators and activate a COUP-TF reporter construct. Although the concentration needed is higher than the physiological levels of retinoic acids, these findings demonstrate that COUP-TFII is a ligand-regulated nuclear receptor, in which ligands activate the receptor by releasing it from the autorepressed conformation.

  16. The pleiotropic effects of erythropoietin in the central nervous system.

    Buemi, M; Cavallaro, E; Floccari, F; Sturiale, A; Aloisi, C; Trimarchi, M; Corica, F; Frisina, N

    2003-03-01

    Erythropoietin (Epo) is a hydrophobic sialoglycoproteic hormone produced by the kidney and responsible for the proliferation, maturation, and differentiation of the precursors of the erythroid cell line. Human recombinant erythropoietin (rHuEpo) is used to treat different types of anemia, not only in uremic patients but also in newborns with anemia of prematurity, in patients with cancer-related anemia or myeloproliferative disease, thalassemias, bone marrow transplants, or those with chronic infectious diseases. The pleiotropic functions of Epo are well known. It has been shown that this hormone can modulate the inflammatory and immune response, has direct hemodynamic and vasoactive effects, could be considered a proangiogenic factor because of its interaction with vascular endothelial growth factor, and its ability to stimulate mitosis and motility of endothelial cells. The multifunctional role of Epo has further been confirmed by the discovery in the central nervous system of a specific Epo/Epo receptor (EpoR) system. Both Epo and EpoR are expressed by astrocytes and neurons and Epo is present in the cerebrospinal fluid (CSF). Therefore, novel functions of Epo, tissue-specific regulation, and the mechanisms of action have been investigated. In this review we have tried to summarize the current data on the role of Epo on brain function. We discuss the different sites of cerebral expression and mechanisms of regulation of Epo and its receptor and its role in the development and maturation of the brain. Second, we discuss the neurotrophic and neuroprotective function of Epo in different conditions of neuronal damage, such as hypoxia, cerebral ischemia, and subarachnoid hemorrhage, and the consequent possibility that rHuEpo therapy could soon be used in clinical practice to limit neuronal damage induced by these diseases. PMID:12638727

  17. Protective effects of erythropoietin pretreatment on myocardium with hypoxia/reoxygenation injury in rats

    QIN Chuan; XIAO Ying-bin; ZHONG Qan-jin; CHEN Lin; WANG Xue-feng

    2004-01-01

    To establish the rat model with myocardial hypoxia/reoxygenation (H/R) injury, and investigatethe protective effect of EPO pretreatment on the myocardium. Methods: Sixty male adult Wistar rats were randomly divided in-to 3 groups: control group, H/R group, and EPO group, 20 in each group. The rats in EPO group accepted injection of 5 000U/kg recombinant human erythropoietin (RHuEPO) through vein, and the other rats accepted the injection of the same volumeof saline. Twenty-four hours after the injection, rats in the EPO and H/R groups were put into the hypoxia environment for 12h and then returned to the normoxic environment for 2 h, and then the samples of blood and myocardium were collected. Serummyocardial enzyme activity, apoptosis, ultrastructure, myocardial MDA contents, EPO receptor (EPOR) expression in cardiacmyocytes and cardiac functions were tested. Results: EPOR expression was positive in cardiac myocytes of adult rat according to the result of immunohistochemitry assaying. Compared to those in H/R group, rats in EPO group presented lighter injury ofmyocardial ultrastructure, the reduction of serum myocardial enzyme activity, inhibition of apoptosis, the better recovery ofcardiac functions, and the Ness production of oxygen-derived free radicals. Conclusion: Adult rat cardiac myocytes could ex-press EPOR, and EPO pretreatment produced protective effects on myocardium with H/R injury.

  18. Flavonoids with M1 Muscarinic Acetylcholine Receptor Binding Activity

    Meyyammai Swaminathan

    2014-06-01

    Full Text Available Muscarinic acetylcholine receptor-active compounds have potential for the treatment of Alzheimer’s disease. In this study, a series of natural and synthetic flavones and flavonols was assayed in vitro for their ability to inhibit radioligand binding at human cloned M1 muscarinic receptors. Several compounds were found to possess competitive binding affinity (Ki = 40–110 µM, comparable to that of acetylcholine (Ki = 59 µM. Despite the fact that these compounds lack a positively-charged ammonium group under physiological conditions, molecular modelling studies suggested that they bind to the orthosteric site of the receptor, mainly through non-polar interactions.

  19. A mouse model of adult-onset anaemia due to erythropoietin deficiency.

    Yamazaki, Shun; Souma, Tomokazu; Hirano, Ikuo; Pan, Xiaoqing; Minegishi, Naoko; Suzuki, Norio; Yamamoto, Masayuki

    2013-01-01

    Erythropoietin regulates erythropoiesis in a hypoxia-inducible manner. Here we generate inherited super-anaemic mice (ISAM) as a mouse model of adult-onset anaemia caused by erythropoietin deficiency. ISAM express erythropoietin in the liver but lack erythropoietin production in the kidney. Around weaning age, when the major erythropoietin-producing organ switches from the liver to the kidney, ISAM develop anaemia due to erythropoietin deficiency, which is curable by administration of recombinant erythropoietin. In ISAM severe chronic anaemia enhances transgenic green fluorescent protein and Cre expression driven by the complete erythropoietin-gene regulatory regions, which facilitates efficient labelling of renal erythropoietin-producing cells. We show that the majority of cortical and outer medullary fibroblasts have the innate potential to produce erythropoietin, and also reveal a new set of erythropoietin target genes. ISAM are a useful tool for the evaluation of erythropoiesis-stimulating agents and to trace the dynamics of erythropoietin-producing cells. PMID:23727690

  20. Multiple autophosphorylation sites of the epidermal growth factor receptor are essential for receptor kinase activity and internalization. Contrasting significance of tyrosine 992 in the native and truncated receptors

    Sorkin, A; Helin, K; Waters, C M; Carpenter, G; Beguinot, L

    1992-01-01

    similar to a kinase-negative receptor. Mutation of tyrosine residue Y992 alone in the context of full length EGF receptor, however, did not affect receptor internalization or kinase activity toward phospholipase C-gamma 1. These data indicate that tyrosine 992 is critical for substrate phosphorylation and...... internalization only in the context of the truncated receptor, and that minor autophosphorylation sites, such as Y992, may act as compensatory regulatory sties in the absence of the major EGF receptor autophosphorylation sites....

  1. Peroxisome Proliferator-Activated Receptor and Vitamin D Receptor Signaling Pathways in Cancer Cells

    Peroxisome proliferator-activated receptors (PPARs) are members of the superfamily of nuclear hormone receptors, which respond to specific ligands such as polyunsaturated fatty acids by altering gene expression. Three subtypes of this receptor have been discovered, each evolving to achieve different biological functions. Like other nuclear receptors, the transcriptional activity of PPARs is affected not only by ligand-stimulation, but also by cross-talk with other molecules. For example, both PPARs and the RXRs are ligand-activated transcription factors that coordinately regulate gene expression. In addition, PPARs and vitamin D receptor (VDR) signaling pathways regulate a multitude of genes that are of importance for cellular functions including cell proliferation and cell differentiation. Interaction of the PPARs and VDR signaling pathways has been shown at the level of molecular cross-regulation of their transcription factor. A variety of ligands influencing the PPARs and VDR signaling pathways have been shown to reveal chemopreventive potential by mediating tumor suppressive activities in human cancers. Use of these compounds may represent a potential novel strategy to prevent cancers. This review summarizes the roles of the PPARs and the VDR in pathogenesis and progression of cancer

  2. Peroxisome Proliferator-Activated Receptor and Vitamin D Receptor Signaling Pathways in Cancer Cells

    Yasuko Kitagishi

    2013-10-01

    Full Text Available Peroxisome proliferator-activated receptors (PPARs are members of the superfamily of nuclear hormone receptors, which respond to specific ligands such as polyunsaturated fatty acids by altering gene expression. Three subtypes of this receptor have been discovered, each evolving to achieve different biological functions. Like other nuclear receptors, the transcriptional activity of PPARs is affected not only by ligand-stimulation, but also by cross-talk with other molecules. For example, both PPARs and the RXRs are ligand-activated transcription factors that coordinately regulate gene expression. In addition, PPARs and vitamin D receptor (VDR signaling pathways regulate a multitude of genes that are of importance for cellular functions including cell proliferation and cell differentiation. Interaction of the PPARs and VDR signaling pathways has been shown at the level of molecular cross-regulation of their transcription factor. A variety of ligands influencing the PPARs and VDR signaling pathways have been shown to reveal chemopreventive potential by mediating tumor suppressive activities in human cancers. Use of these compounds may represent a potential novel strategy to prevent cancers. This review summarizes the roles of the PPARs and the VDR in pathogenesis and progression of cancer.

  3. Peroxisome Proliferator-Activated Receptor and Vitamin D Receptor Signaling Pathways in Cancer Cells

    Matsuda, Satoru, E-mail: smatsuda@cc.nara-wu.ac.jp; Kitagishi, Yasuko [Department of Food Science and Nutrition, Nara Women’s University, Kita-Uoya Nishimachi, Nara 630-8506 (Japan)

    2013-10-21

    Peroxisome proliferator-activated receptors (PPARs) are members of the superfamily of nuclear hormone receptors, which respond to specific ligands such as polyunsaturated fatty acids by altering gene expression. Three subtypes of this receptor have been discovered, each evolving to achieve different biological functions. Like other nuclear receptors, the transcriptional activity of PPARs is affected not only by ligand-stimulation, but also by cross-talk with other molecules. For example, both PPARs and the RXRs are ligand-activated transcription factors that coordinately regulate gene expression. In addition, PPARs and vitamin D receptor (VDR) signaling pathways regulate a multitude of genes that are of importance for cellular functions including cell proliferation and cell differentiation. Interaction of the PPARs and VDR signaling pathways has been shown at the level of molecular cross-regulation of their transcription factor. A variety of ligands influencing the PPARs and VDR signaling pathways have been shown to reveal chemopreventive potential by mediating tumor suppressive activities in human cancers. Use of these compounds may represent a potential novel strategy to prevent cancers. This review summarizes the roles of the PPARs and the VDR in pathogenesis and progression of cancer.

  4. Enhanced brain release of erythropoietin, cytokines and NO during carotid clamping.

    Carelli, Stephana; Ghilardi, Giorgio; Bianciardi, Paola; Latorre, Elisa; Rubino, Federico; Bissi, Marina; Di Giulio, Anna Maria; Samaja, Michele; Gorio, Alfredo

    2016-02-01

    Although effective and safe, carotid endarterectomy (CEA) implies a reduced blood flow to the brain and likely an ischemia/reperfusion event. The high rate of uneventful outcomes associated with CEA suggests the activation of brain endogenous protection mechanisms aimed at limiting the possible ischemia/reperfusion damage. This study aims at assessing whether CEA triggers protective mechanisms such as brain release of erythropoietin and nitric oxide. CEA was performed in 12 patients; blood samples were withdrawn simultaneously from the surgically exposed ipsilateral jugular and leg veins before, during (2 and 40 min) and after clamp removal (2 min). Plasma antioxidant capacity, carbonylated proteins, erythropoietin, nitrates and nitrites (NOx) were determined. No changes in intraoperative EEG, peripheral and transcranial blood oxygen saturation were detectable, and no patients showed any neurologic sign after the intervention. Antioxidant capacity and protein carbonylation in plasma were unaffected. Differently, erythropoietin, VEGF, TNF-α and NOx increased during clamping in the jugular blood (2 and 40 min), while no changes were observed in the peripheral circulation. These results show that blood erythropoietin, VEGF, TNF-α, and NOx increased in the brain during uncomplicated CEA. This may represent an endogenous self-activated neuroprotective mechanism aimed at the prevention of ischemia/reperfusion damage. PMID:26494654

  5. Erythropoietin in heart failure : pathology and protection

    Westenbrink, Berend Daan

    2008-01-01

    Anemia is common in chronic heart failure (CHF) patients and related to impaired survival. The etiology of anemia in CHF-patients is often unknown. We hypothesized that dysregulation of erythropoietin (EPO) synthesis by the kidney or an altered sensitivity of the bone marrow to EPO might represent c

  6. High-dose erythropoietin for tissue protection

    Lund, Anton; Lundby, Carsten; Olsen, Niels Vidiendal

    2014-01-01

    BACKGROUND: The discovery of potential anti-apoptotic and cytoprotective effects of recombinant human erythropoietin (rHuEPO) has led to clinical trials investigating the use of high-dose, short-term rHuEPO therapy for tissue protection in conditions such as stroke and myocardial infarction...

  7. Erythropoietin: ready for prime-time cardioprotection.

    Riksen, N.P.; Hausenloy, D.J.; Yellon, D.M.

    2008-01-01

    To improve clinical outcomes in patients presenting with an acute myocardial infarction, new strategies to limit infarct size and postinfarct remodelling are warranted. Recent animal studies have revealed that erythropoietin has the potential to achieve both these goals. Even more intriguing is the

  8. Neurotrophin receptors expression and JNK pathway activation in human astrocytomas

    Neurotrophins are growth factors that regulate cell growth, differentiation and apoptosis in the nervous system. Their diverse actions are mediated through two different transmembrane – receptor signaling systems: Trk receptor tyrosine kinases (TrkA, TrkB, TrkC) and p75NTR neurotrophin receptor. Trk receptors promote cell survival and differentiation while p75NTR induces, in most cases, the activity of JNK-p53-Bax apoptosis pathway or suppresses intracellular survival signaling cascades. Robust Trk activation blocks p75NTR -induced apoptosis by suppressing the JNK-p53-Bax pathway. The aim of this exploratory study was to investigate the expression levels of neurotrophin receptors, Trks and p75NTR, and the activation of JNK pathway in human astrocytomas and in adjacent non-neoplastic brain tissue. Formalin-fixed paraffin-embedded serial sections from 33 supratentorial astrocytomas (5 diffuse fibrillary astrocytomas, WHO grade II; 6 anaplastic astrocytomas, WHO grade III; 22 glioblastomas multiforme, WHO grade IV) were immunostained following microwave pretreatment. Polyclonal antibodies against TrkA, TrkB, TrkC and monoclonal antibodies against p75NTR and phosphorylated forms of JNK (pJNK) and c-Jun (pc-Jun) were used. The labeling index (LI), defined as the percentage of positive (labeled) cells out of the total number of tumor cells counted, was determined. Moderate to strong, granular cytoplasmic immunoreactivity for TrkA, TrkB and TrkC receptors was detected in greater than or equal to 10% of tumor cells in the majority of tumors independently of grade; on the contrary, p75NTR receptor expression was found in a small percentage of tumor cells (~1%) in some tumors. The endothelium of tumor capillaries showed conspicuous immunoreactivity for TrkB receptor. Trk immunoreactivity seemed to be localized in some neurons and astrocytes in non-neoplastic tissue. Phosphorylated forms of JNK (pJNK) and c-Jun (pc-Jun) were significantly co-expressed in a tumor grade

  9. Development of a new radioimmunoassay for erythropoietin using recombinant erythropoietin

    Mason-Garcia, M.; Beckman, B.S.; Brookins, J.W.; Powell, J.S.; Lanham, W.; Blaisdell, S.; Keay, L.; Li, S.C.; Fisher, J.W. (Tulane Univ. School of Medicine, New Orleans, LA (USA))

    1990-11-01

    The development of a 24 hour radioimmunoassay for erythropoietin (EPO) using EPO derived from recombinant DNA as both immunogen and ligand is described in the present paper. Mixed breed rabbits immunized with 10 micrograms/kg of EPO derived from a stably transfected cell line (MD) produced antibodies to EPO with high titer (up to 1:896,000 final dilution in the tube), high affinity (8.4 x 10(11) liter/M), and good specificity. Purified EPO from the above source or from AmGen Biologicals (AG) were successfully radioiodinated with the chloramine-T method and used as ligand in the radioimmunoassay. Standard dose-response curves prepared with EPO from both commercial sources were not significantly different and showed a sensitivity of 0.75 to 0.96 mU/tube. The dose-response curves in both systems also showed parallelism with serially diluted serum from a patient with aplastic anemia. Within-assay and between-assay precision were determined by assaying multiple replicates of a serum pool. Recovery of exogenous EPO added to a serum pool averaged 97% for both systems. The range of normal human serum EPO was determined by assaying the sera of 153 hematologically-normal adult subjects and was found to be 1.1 to 27.3 mU/ml for MD EPO and 0.5 to 16.7 mU/ml for AG EPO. Sera from several patients with hematologic abnormalities were also assayed, including those of 36 patients with anemia of end-stage renal disease (mean +/- SEM, 29.5 +/- 4.0 mU/ml; P less than 0.01). In conclusion, this new, more rapid and sensitive radioimmunoassay system can be used to measure EPO levels in sera from normal human subjects and patients with several types of anemia, and should also be very useful in therapeutic drug monitoring of patients receiving EPO from various commercial sources.

  10. Structural mechanism of glutamate receptor activation and desensitization.

    Meyerson, Joel R; Kumar, Janesh; Chittori, Sagar; Rao, Prashant; Pierson, Jason; Bartesaghi, Alberto; Mayer, Mark L; Subramaniam, Sriram

    2014-10-16

    Ionotropic glutamate receptors are ligand-gated ion channels that mediate excitatory synaptic transmission in the vertebrate brain. To gain a better understanding of how structural changes gate ion flux across the membrane, we trapped rat AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) and kainate receptor subtypes in their major functional states and analysed the resulting structures using cryo-electron microscopy. We show that transition to the active state involves a 'corkscrew' motion of the receptor assembly, driven by closure of the ligand-binding domain. Desensitization is accompanied by disruption of the amino-terminal domain tetramer in AMPA, but not kainate, receptors with a two-fold to four-fold symmetry transition in the ligand-binding domains in both subtypes. The 7.6 Å structure of a desensitized kainate receptor shows how these changes accommodate channel closing. These findings integrate previous physiological, biochemical and structural analyses of glutamate receptors and provide a molecular explanation for key steps in receptor gating. PMID:25119039

  11. Comparison of in vivo biological activity of recombinant human erythropoietin in two species of mice%重组人促红素在2种品系小鼠体内的生物学活性的比较

    张素慧; 王巧旭; 徐超瑾; 程小芹; 唐黎明

    2013-01-01

    目的:对中国药典2010年版三部附录XB重组人促红素(rHuEPO)体内生物学活性测定方法中规定的动物品系进行探讨研究.方法:选用ICR小鼠、BALB/C小鼠作为实验动物,测定19批进口重组人促红素的体内生物学活性,从动物品系、性别的角度进行比较.结果:19批重组人促红素在雌性或雄性ICR小鼠、BALB/C小鼠体内生物学活性之间的差异无统计学意义;每组ICR小鼠的性别为雌雄各半时,体内生物学活性合标示量符合药典规定,但可信限率(FL)超过45%,结果不可靠.结论:ICR小鼠与BALB/C小鼠均可用于重组人促红素体内生物学活性测定,采用单一性别的动物进行试验结果更为可靠.%Objective:To explore animal species used in bioassay of recombinant human erythropoietin (rHuEPO) in Appendix XB of Chinese Pharmacopoeia Volume Ⅲ (2010 Edition).Methods:ICR mice and BALB/C mice were selected as the experiment animal,and in vivo biological activities of nineteen batches of rHuEPO were determined and compared in terms of animal species and sex.Results:The results indicated that in vivo biological activities of 19 batches of rHuEPO in BALB/C mice were similar to those in ICR mice of either sex.In groups of ICR mice of half males and half females,the in vivo biological activity was in accordance with the label claim standardized in the Chinese Pharmacopoeia ; however,the confidence limit rate was high (FL > 45 %),indicating that the result was unreliable.Conclusion:Both ICR mice and BALB/C mice can be applied for in vivo bioassay of rHuEPO.Results will be more reliable if data are from the group of single sex mice.

  12. Abelson tyrosine kinase links PDGFbeta receptor activation to cytoskeletal regulation of NMDA receptors in CA1 hippocampal neurons

    Beazely Michael A

    2008-12-01

    Full Text Available Abstract Background We have previously demonstrated that PDGF receptor activation indirectly inhibits N-methyl-D-aspartate (NMDA currents by modifying the cytoskeleton. PDGF receptor ligand is also neuroprotective in hippocampal slices and cultured neurons. PDGF receptors are tyrosine kinases that control a variety of signal transduction pathways including those mediated by PLCγ. In fibroblasts Src and another non-receptor tyrosine kinase, Abelson kinase (Abl, control PDGF receptor regulation of cytoskeletal dynamics. The mechanism whereby PDGF receptor regulates cytoskeletal dynamics in central neurons remains poorly understood. Results Intracellular applications of active Abl, but not heat-inactivated Abl, decreased NMDA-evoked currents in isolated hippocampal neurons. This mimics the effects of PDGF receptor activation in these neurons. The Abl kinase inhibitor, STI571, blocked the inhibition of NMDA currents by Abl. We demonstrate that PDGF receptors can activate Abl kinase in hippocampal neurons via mechanisms similar to those observed previously in fibroblasts. Furthermore, PDGFβ receptor activation alters the subcellular localization of Abl. Abl kinase is linked to actin cytoskeletal dynamics in many systems. We show that the inhibition of NMDA receptor currents by Abl kinase is blocked by the inclusion of the Rho kinase inhibitor, Y-27632, and that activation of Abl correlates with an increase in ROCK tyrosine phosphorylation. Conclusion This study demonstrates that PDGFβ receptors act via an interaction with Abl kinase and Rho kinase to regulated cytoskeletal regulation of NMDA receptor channels in CA1 pyramidal neurons.

  13. Epidermis-type lipoxygenase 3 regulates adipocyte differentiation and peroxisome proliferator-activated receptor gamma activity

    Hallenborg, Philip; Jørgensen, Claus; Petersen, Rasmus K;

    2010-01-01

    The nuclear receptor peroxisome proliferator-activated receptor gamma (PPAR gamma) is essential for adipogenesis. Although several fatty acids and their derivatives are known to bind and activate PPAR gamma, the nature of the endogenous ligand(s) promoting the early stages of adipocyte differenti...

  14. Binding of erythropoietin to CFU-E derived from fetal mouse liver cells

    Fukamachi, H.; Saito, T.; Tojo, A.; Kitamura, T.; Urabe, A.; Takaku, F.

    1987-09-01

    The binding of recombinant erythropoietin (EPO) to fetal mouse liver cells (FMLC) was investigated using a radioiodinated derivative which retained full biological activity. FMLC were fractionated using a preformed Percoll density gradient. Using the fractionated FMLC, the ability to form CFU-E colonies in a semisolid culture was examined, and the binding of (/sup 125/I)EPO was measured. The highest specific binding of (/sup 125/I)EPO was observed in a fraction with a density between 1.062 and 1.076 g/ml. The same fraction showed the highest ability to form CFU-E-derived colonies. After suspension culture of FMLC with EPO for 2 days, differentiated erythroid cells with higher density markedly increased. The specific binding of (/sup 125/I)EPO to these cells almost disappeared with differentiation. Scatchard analysis with cells of the CFU-E-enriched fraction showed a nonlinear curve, suggesting the existence of two classes of binding sites. One binding site was high-affinity (Kd1 = 0.41 nM), and the other low-affinity (Kd2 = 3.13 nM). These results suggest that the expression of EPO receptors on the erythroid cells is highest in CFU-E.

  15. A lentiviral gene therapy strategy for the in vitro production of feline erythropoietin.

    Natalia Vapniarsky

    Full Text Available Nonregenerative anemia due to chronic renal failure is a common problem in domestic cats. Unfortunately, administration of recombinant human erythropoietin often only improves anemia temporarily due to antibody development. In this in vitro study, feline erythropoietin cDNA was cloned from feline renal tissue and utilized in the construction of a replication-defective lentiviral vector. The native recombinant feline erythropoietin (rfEPO sequence was confirmed by sequencing. Upon viral vector infection of human 293H cells, Crandall Renal Feline Kidney cell line and primary feline peripheral blood mononuclear cells, bioactive rfEPO protein was produced. The presence of cellular rfEPO cDNA was confirmed by standard PCR, production of abundant rfEPO mRNA was confirmed by real-time PCR, and secretion of rfEPO protein was demonstrated by Western blot analyses, while rfEPO protein bioactivity was confirmed via an MTT proliferation bioassay. This in vitro study demonstrates the feasibility of a replication-defective lentiviral vector delivery system for the in vitro production of biologically active feline erythropoietin. Anemic cats with chronic renal failure represent a potential in vivo application of a lentiviral gene therapy system.

  16. Tyrosines 868, 966, and 972 in the Kinase Domain of JAK2 Are Autophosphorylated and Required for Maximal JAK2 Kinase Activity

    Argetsinger, Lawrence S.; Stuckey, Jeanne A.; Robertson, Scott A.; Koleva, Rositsa I.; Cline, Joel M.; Marto, Jarrod A.; Myers, Martin G.; Carter-Su, Christin

    2010-01-01

    Janus kinase 2 (JAK2) is activated by a majority of cytokine family receptors including receptors for GH, leptin, and erythropoietin. To identify novel JAK2-regulatory and/or -binding sites, we set out to identify autophosphorylation sites in the kinase domain of JAK2. Two-dimensional phosphopeptide mapping of in vitro autophosphorylated JAK2 identified tyrosines 868, 966, and 972 as sites of autophosphorylation. Phosphorylated tyrosines 868 and 972 were also identified by mass spectrometry a...

  17. Polycythemia in transgenic mice expressing the human erythropoietin gene

    Erythropoietin is a glycoprotein hormone that regulates mammalian erythropoiesis. To study the expression of the human erythropoietin gene, EPO, 4 kilobases of DNA encompassing the gene with 0.4 kilobase of 5' flanking sequence and 0.7 kilobase of 3' flanking sequence was microinjected into fertilized mouse eggs. Transgenic mice were generated that are polycythemic, with increased erythrocytic indices in peripheral blood, increased numbers of erythroid precursors in hematopoietic tissue, and increased serum erythropoietin levels. Transgenic homozygotes show a greater degree of polycythemia than do heterozygotes as well as striking extramedullary erythropoiesis. Human erythropoietin RNA was found not only in fetal liver, adult liver, and kidney but also in all other transgenic tissues analyzed. Anemia induced increased human erythropoietin RNA levels in liver but not kidney. These transgenic mice represent a unique model of polycythemia due to increased erythropoietin levels

  18. New GABA amides activating GABA A-receptors

    Peter Raster; Andreas Späth; Svetlana Bultakova; Pau Gorostiza; Burkhard König; Piotr Bregestovski

    2013-01-01

    We have prepared a series of new and some literature-reported GABA-amides and determined their effect on the activation of GABAA-receptors expressed in CHO cells. Special attention was paid to the purification of the target compounds to remove even traces of GABA contaminations, which may arise from deprotection steps in the synthesis. GABA-amides were previously reported to be partial, full or superagonists. In our hands these compounds were not able to activate GABAA-receptor channels in wh...

  19. Beyond anaemia management: evolving role of erythropoietin therapy in neurological disorders, multiple myeloma and tumour hypoxia models.

    Boogaerts, Marc; Mittelman, Moshe; Vaupel, Peter

    2005-01-01

    Recombinant human erythropoietin (epoetin) has become the standard of care in the treatment of anaemia resulting from cancer and its treatment, and chronic kidney disease. The discovery that erythropoietin and its receptor are located in regions outside the erythropoietic system has led to interest in the potential role of epoetin in other tissues, such as the central nervous system. Animal studies have shown that systemically applied epoetin can cross the blood-brain barrier, where it reduces tissue injury associated with stroke, blunt trauma and experimental autoimmune encephalomyelitis. Pilot studies in humans have shown that epoetin treatment given within 8 h of stroke reduces infarct size and results in a significantly better outcome when compared with placebo treatment. Studies also suggest that epoetin has the potential to improve cognitive impairment associated with adjuvant chemotherapy in patients with cancer. Anaemia is a major factor causing tumour hypoxia, a condition that can promote changes within neoplastic cells that further tumour survival and malignant progression and also reduces the effectiveness of several anticancer therapies including radiotherapy and oxygen-dependent cytotoxic agents. Use of epoetin to prevent or correct anaemia has the potential to reduce tumour hypoxia and improve treatment outcome. Several therapeutic studies in anaemic animals with experimental tumours have shown a beneficial effect of epoetin on delaying tumour growth. Furthermore, clinical observations in patients with multiple myeloma and animal studies have suggested that epoetin has an antimyeloma effect, mediated via the immune system through activation of CD8+ T cells. Therefore, the role of epoetin may go well beyond that of increasing haemoglobin levels in anaemic patients, although additional studies are required to confirm these promising results. PMID:16244507

  20. Mechanisms of Activation of Receptor Tyrosine Kinases: Monomers or Dimers

    Ichiro N. Maruyama

    2014-04-01

    Full Text Available Receptor tyrosine kinases (RTKs play essential roles in cellular processes, including metabolism, cell-cycle control, survival, proliferation, motility and differentiation. RTKs are all synthesized as single-pass transmembrane proteins and bind polypeptide ligands, mainly growth factors. It has long been thought that all RTKs, except for the insulin receptor (IR family, are activated by ligand-induced dimerization of the receptors. An increasing number of diverse studies, however, indicate that RTKs, previously thought to exist as monomers, are present as pre-formed, yet inactive, dimers prior to ligand binding. The non-covalently associated dimeric structures are reminiscent of those of the IR family, which has a disulfide-linked dimeric structure. Furthermore, recent progress in structural studies has provided insight into the underpinnings of conformational changes during the activation of RTKs. In this review, I discuss two mutually exclusive models for the mechanisms of activation of the epidermal growth factor receptor, the neurotrophin receptor and IR families, based on these new insights.

  1. Calcium-sensing receptor activation depresses synaptic transmission

    Phillips, Cecilia G.; Harnett, Mark T.; Chen, Wenyan; Smith, Stephen M.

    2008-01-01

    At excitatory synapses, decreases in cleft [Ca] arising from activity-dependent transmembrane Ca flux reduce the probability of subsequent transmitter release. Intense neural activity, induced by physiological and pathological stimuli, disturb the external microenvironment reducing extracellular [Ca] ([Ca]o) and thus may impair neurotransmission. Increases in [Ca]o activate the extracellular calcium sensing receptor (CaSR) which in turn inhibits non-selective cation channels (NSCC) at the maj...

  2. Kallikrein activates bradykinin B2 receptors in absence of kininogen.

    Biyashev, Dauren; Tan, Fulong; Chen, Zhenlong; Zhang, Kai; Deddish, Peter A; Erdös, Ervin G; Hecquet, Claudie

    2006-03-01

    Kallikreins cleave plasma kininogens to release the bioactive peptides bradykinin (BK) or kallidin (Lys-BK). These peptides then activate widely disseminated B2 receptors with consequences that may be either noxious or beneficial. We used cultured cells to show that kallikrein can bypass kinin release to activate BK B2 receptors directly. To exclude intermediate kinin release or kininogen uptake from the cultured medium, we cultured and maintained cells in medium entirely free of animal proteins. We compared the responses of stably transfected Chinese hamster ovary (CHO) cells that express human B2 receptors (CHO B2) and cells that coexpress angiotensin I-converting enzyme (ACE) as well (CHO AB). We found that BK (1 nM or more) and tissue kallikrein (1-10 nM) both significantly increased release of arachidonic acid beyond unstimulated baseline level. An enzyme-linked immunoassay for kinin established that kallikrein did not release a kinin from CHO cells. We confirmed the absence of kininogen mRNA with RT-PCR to rule out kininogen synthesis by CHO cells. We next tested an ACE inhibitor for enhanced BK receptor activation in the absence of kinin release and synthesized an ACE-resistant BK analog as a control for these experiments. Enalaprilat (1 microM) potentiated kallikrein (100 nM) in CHO AB cells but was ineffective in CHO B2 cells that do not bear ACE. We concluded that kallikrein activated B2 receptors without releasing a kinin. Furthermore, inhibition of ACE enhanced the receptor activation by kallikrein, an action that may contribute to the manifold therapeutic effects of ACE inhibitors. PMID:16272198

  3. Allosteric activation mechanism of the cys-loop receptors

    Yong-chang CHANG; Wen WU; Jian-liang ZHANG; Yao HUANG

    2009-01-01

    Binding of a neurotransmitter to its ionotropic receptor opens a distantly located ion channel, a process termed allosteric activation. Here we review recent advances in the molecular mechanism by which the cys-loop receptors are activated with emphasis on the best studied nicotinic acetylcholine receptors (nAChRs). With a combination of affinity labeling, mutagenesis, electrophysiology, kinetic modeling, electron microscopy (EM), and crystal structure analysis, the allosteric activation mechanism is emerging. Specifically, the binding domain and gating domain are interconnected by an allosteric activation network. Agonist binding induces conformational changes, resulting in the rotation of a β sheet of amino-terminal domain and outward movement of loop 2, loop F, and cys-loop, which are coupled to the M2-M3 linker to pull the channel to open. However, there are still some controversies about the movement of the channel-lining domain M2. Nine angstrom resolution EM structure of a nAChR imaged in the open state suggests that channel opening is the result of rotation of the M2 domain. In contrast, recent crystal structures of bacterial homologues of the cys-loop receptor family in apparently open state have implied an M2 tilting model with pore dilation and quaternary twist of the whole pentameric receptor. An elegant study of the nAChR using protonation scanning of M2 domain supports a similar pore dilation activation mechanism with minimal rotation of M2. This remains to be validated with other approaches including high resolution structure determination of the mammalian cys-loop receptors in the open state.

  4. Antierythropoietin Antibodies in Hemodialysis Patients Treated with Recombinant Erythropoietin

    Savaş ÖZTÜRK; Alper GÜMÜŞ; Vecihi MEMİLİ; Muhammet Emin DÜZ; Egemen CEBECİ; Macit KOLDAŞ; Rümeyza KAZANCIOĞLU

    2014-01-01

    OBJECTIVE: Erythropoietin resistance is a serious problem in patients treated with recombinant erythropoietin. Antierythropoietin antibodies are considered to be one of the causes of this resistance. MATERIAL and ME THODS: We investigated antierythropoietin antibodies in chronic hemodialysis patients and compared the results with healthy controls by means of establishing an ELISA method. A total of 121 chronic hemodialysis patients receiving recombinant erythropoietin were included in the ...

  5. Effects of Recombinant Erythropoietin on Breast Cancer-Initiating Cells

    Tiffany M. Phillips; Kwanghee Kim; Erina Vlashi; McBride, William H.; Frank Pajonk

    2007-01-01

    BACKGROUND: Cancer anemia causes fatigue and correlates with poor treatment outcome. Erythropoietin has been introduced in an attempt to correct these defects. However, five recent clinical trials reported a negative impact of erythropoietin on survival and/or tumor control, indicating that experimental evaluation of a possible direct effect of erythropoietin on cancer cells is required. Cancer recurrence is thought to rely on the proliferation of cancer initiating cells (CICs). In breast can...

  6. Antierythropoietin Antibodies in Hemodialysis Patients Treated with Recombinant Erythropoietin

    Savaş ÖZTÜRK

    2014-05-01

    Full Text Available OBJECTIVE: Erythropoietin resistance is a serious problem in patients treated with recombinant erythropoietin. Antierythropoietin antibodies are considered to be one of the causes of this resistance. MATERIAL and ME THODS: We investigated antierythropoietin antibodies in chronic hemodialysis patients and compared the results with healthy controls by means of establishing an ELISA method. A total of 121 chronic hemodialysis patients receiving recombinant erythropoietin were included in the study. The patients were subdivided according to the type of recombinant erythropoietin (erythropoietin-α or erythropoietin-β they had been treated with in the last six months. RESULTS: The absorbance values of patients were compared with the absorbance values of the control group by a specific and reproducible method. LOD (limit of detection and LOQ (limit of quantitation values were also calculated. The difference in the absorbance values between the therapy and control groups was statistically significant. CONCLUSION: Both erythropoietin-α and erythropoietin-β induce production of antibodies against erythropoietin. Anti rh-EPO antibodies may play a role in EPO resistance.

  7. Interaction of the C-terminal acidic domain of the insulin receptor with histone modulates the receptor kinase activity.

    Baron, V; Kaliman, P; Alengrin, F; Van Obberghen, E

    1995-04-01

    In this study, we investigated the role of the insulin receptor domain 1270-1280, an acid-rich sequence located in the receptor C-terminus. Antipeptide IgG raised against this sequence were obtained and used to analyze their effect on receptor function. Antipeptide IgG inhibited receptor autophosphorylation at Tyr1146, Tyr1150 and Tyr1151. These sites are known to be key modulators of the receptor activity. Autophosphorylation at other sites may also have been inhibited. The antipeptide antibody decreased the receptor kinase activity measured with poly(Glu80Tyr20) and a synthetic peptide corresponding to the proreceptor sequence 1142-1158. We provide evidence that the effect of the antibody on substrate phosphorylation may result from the control of the phosphorylation level of the receptor. Concerning the action of the antipeptide IgG on the receptor kinase activity, histone did not behave similarly to poly(Glu80Tyr20). The antibody recognizing sequence 1270-1280 competed with histone for an overlapping binding site. Histone also modulated insulin receptor autophosphorylation, supporting the idea that interference with domain 1270-1280 alters the receptor kinase. Our data suggest that the acidic region including residues 1270-1280 of the insulin receptor C-terminus is involved in the following events: (a) receptor binding with histone, an exogenous substrate of the receptor kinase, and (b) the regulation of receptor autophosphorylation and kinase activity. Based on these observations, we would like to propose that this insulin receptor domain could interact with cellular proteins modulating the receptor kinase. PMID:7744039

  8. Systemic and cerebral vascular endothelial growth factor levels increase in murine cerebral malaria along with increased Calpain and caspase activity and can be reduced by erythropoietin treatment

    Hempel, Casper; Hoyer, Nils; Kildemoes, Anna;

    2014-01-01

    . Furthermore, we noticed increased caspase-3 and calpain activity in terminally ill mice, as measured by protease-specific cleavage of α-spectrin and p35. In conclusion, we detected increased cerebral and systemic VEGF as well as HIF-1α, which in the brain were reduced to normal in EPO-treated mice. Also...

  9. Sphingosine-1-phosphate receptor 1 reporter mice reveal receptor activation sites in vivo

    Kono, Mari; Tucker, Ana E.; Tran, Jennifer; Bergner, Jennifer B.; Turner, Ewa M; Proia, Richard L.

    2014-01-01

    Activation of the GPCR sphingosine-1-phosphate receptor 1 (S1P1) by sphingosine-1-phosphate (S1P) regulates key physiological processes. S1P1 activation also has been implicated in pathologic processes, including autoimmunity and inflammation; however, the in vivo sites of S1P1 activation under normal and disease conditions are unclear. Here, we describe the development of a mouse model that allows in vivo evaluation of S1P1 activation. These mice, known as S1P1 GFP signaling mice, produce a ...

  10. The aryl hydrocarbon receptor and glucocorticoid receptor interact to activate human metallothionein 2A

    Although the aryl hydrocarbon receptor (AHR) and glucocorticoid receptor (GR) play essential roles in mammalian development, stress responses, and other physiological events, crosstalk between these receptors has been the subject of much debate. Metallothioneins are classic glucocorticoid-inducible genes that were reported to increase upon treatment with AHR agonists in rodent tissues and cultured human cells. In this study, the mechanism of human metallothionein 2A (MT2A) gene transcription activation by AHR was investigated. Cotreatment with 3-methylcholanthrene and dexamethasone, agonists of AHR and GR respectively, synergistically increased MT2A mRNA levels in HepG2 cells. MT2A induction was suppressed by RNA interference against AHR or GR. Coimmunoprecipitation experiments revealed a physical interaction between AHR and GR proteins. Moreover, chromatin immunoprecipitation assays indicated that AHR was recruited to the glucocorticoid response element in the MT2A promoter. Thus, we provide a novel mechanism whereby AHR modulates expression of human MT2A via the glucocorticoid response element and protein–protein interactions with GR. - Highlights: • Aryl hydrocarbon receptor forms a complex with glucocorticoid receptor in cells. • Human metallothionein gene is regulated by the AHR and GR interaction. • AHR–GR complex binds to glucocorticoid response element in metallothionein gene. • We demonstrated a novel transcriptional mechanism via AHR and GR interaction

  11. The aryl hydrocarbon receptor and glucocorticoid receptor interact to activate human metallothionein 2A

    Sato, Shoko, E-mail: satosho@rs.tus.ac.jp [Laboratory of Nutrition, Graduate School of Agricultural Science, Tohoku University, Sendai 981-8555 (Japan); Shirakawa, Hitoshi, E-mail: shirakah@m.tohoku.ac.jp [Laboratory of Nutrition, Graduate School of Agricultural Science, Tohoku University, Sendai 981-8555 (Japan); Tomita, Shuhei, E-mail: tomita@med.tottori-u.ac.jp [Division of Molecular Pharmacology, Department of Pathophysiological and Therapeutic Science, Yonago 683-8503 (Japan); Tohkin, Masahiro, E-mail: tohkin@phar.nagoya-cu.ac.jp [Department of Medical Safety Science, Graduate School of Pharmaceutical Science, Nagoya City University, Nagoya 267-8603 (Japan); Gonzalez, Frank J., E-mail: gonzalef@mail.nih.gov [Laboratory of Metabolism, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892 (United States); Komai, Michio, E-mail: mkomai@m.tohoku.ac.jp [Laboratory of Nutrition, Graduate School of Agricultural Science, Tohoku University, Sendai 981-8555 (Japan)

    2013-11-15

    Although the aryl hydrocarbon receptor (AHR) and glucocorticoid receptor (GR) play essential roles in mammalian development, stress responses, and other physiological events, crosstalk between these receptors has been the subject of much debate. Metallothioneins are classic glucocorticoid-inducible genes that were reported to increase upon treatment with AHR agonists in rodent tissues and cultured human cells. In this study, the mechanism of human metallothionein 2A (MT2A) gene transcription activation by AHR was investigated. Cotreatment with 3-methylcholanthrene and dexamethasone, agonists of AHR and GR respectively, synergistically increased MT2A mRNA levels in HepG2 cells. MT2A induction was suppressed by RNA interference against AHR or GR. Coimmunoprecipitation experiments revealed a physical interaction between AHR and GR proteins. Moreover, chromatin immunoprecipitation assays indicated that AHR was recruited to the glucocorticoid response element in the MT2A promoter. Thus, we provide a novel mechanism whereby AHR modulates expression of human MT2A via the glucocorticoid response element and protein–protein interactions with GR. - Highlights: • Aryl hydrocarbon receptor forms a complex with glucocorticoid receptor in cells. • Human metallothionein gene is regulated by the AHR and GR interaction. • AHR–GR complex binds to glucocorticoid response element in metallothionein gene. • We demonstrated a novel transcriptional mechanism via AHR and GR interaction.

  12. Can erythropoietin improve tumor oxygenation?

    Kelleher, D.K.; Thews, O.; Vaupel, P. [Mainz Univ. (Germany). Inst. of Physiology and Pathophysiology

    1998-12-01

    Tumor growth, oxygenation and radiosensitivity were investigated in a series of studies in which anemia was induced in rats either by the development of a hemorrhagic ascites or by a single dose of carboplatin, which resulted in reductions in the hemoglobin concentration of 30%. The development of both the tumor- and chemotherapy-induced forms of anemia could be prevented by the s.c. administration of recombinant human erythropoietin (rhEPO; 1000 IU/kg, 3 times per week over 14 days). Seven days before pO{sub 2} measurements, DS-sarcomas were implanted s.c. on the hind foot dorsum. With both anemia models, tumor growth did not differ between anemic animals and animals treated with rhEPO. Tumor oxygenation was measured polarographically using O{sub 2}-sensitive needle electrodes and pO{sub 2} histography. In anemic animals, tumor oxygenation was poorer compared to untreated controls. The reduction could be partially reversed by rhEPO treatment, but not fully compensated. These findings suggested that rhEPO treatment can improve tumor oxygenation by increasing the O{sub 2} availability to tumor tissue. Further experiments therefore assessed the possibility of enhancing the efficacy of a single radiation dose (10 Gy) by rhEPO treatment of anemic animals. While anemic animals showed decreased radiosensitivity, prevention of anemia by rhEPO treatment resulted in a significant increase in tumor radiosensitivity, although again a full recovery to radiosensitivity levels found in non-anemic animals was not achieved. (orig.) [Deutsch] Um den Einfluss einer Anaemie auf das Wachstum, die Gewebeoxygenierung und die Strahlensensibilitaet von Tumoren der Ratte zu untersuchen, wurde entweder durch Erzeugung eines haemorrhagischen Tumoraszites oder durch einmalige Gabe von Carboplatin (45 mg/kg i.v.) die Haemoglobinkonzentration des Blutes um 30% erniedrigt (normochrome normozytaere Anaemie). In einer zweiten Versuchsgruppe konnte sowohl beim Vorliegen eines haemorrhagischen

  13. Resistance of dialyzed patients to erythropoietin

    Michelle Teodoro Alves

    2015-06-01

    Full Text Available Resistance to recombinant human erythropoietin is a common condition in dialyzed patients with chronic kidney disease and is associated with more hospitalizations, increased mortality and frequent blood transfusions. The main cause of hyporesponsiveness to recombinant human erythropoietin in these patients is iron deficiency. However, a high proportion of patients does not respond to treatment, even to the use of intravenous iron, which indicates the presence of other important causes of resistance. In addition to the iron deficiency, the most common causes of resistance include inflammation, infection, malnutrition, inadequate dialysis, and hyperparathyroidism, although other factors may be associated. In the presence of adequate iron stores, other causes should be investigated and treated appropriately.

  14. Influence of phasic and tonic dopamine release on receptor activation

    Dreyer, Jakob Kristoffer Kisbye; Herrik, Kjartan F; Berg, Rune W; Hounsgaard, Jørn D

    2010-01-01

    Tonic and phasic dopamine release is implicated in learning, motivation, and motor functions. However, the relationship between spike patterns in dopaminergic neurons, the extracellular concentration of dopamine, and activation of dopamine receptors remains unresolved. In the present study, we...... develop a computational model of dopamine signaling that give insight into the relationship between the dynamics of release and occupancy of D(1) and D(2) receptors. The model is derived from first principles using experimental data. It has no free parameters and offers unbiased estimation of the...

  15. The angiotensin II type 1 receptor antagonist Losartan binds and activates bradykinin B2 receptor signaling

    Bonde, Marie Mi; Olsen, Kristine Boisen; Erikstrup, Niels;

    2011-01-01

    The angiotensin II type 1 receptor (AT1R) blocker (ARB) Losartan has cardioprotective effects during ischemia-reperfusion injury and inhibits reperfusion arrhythmias -effects that go beyond the benefits of lowering blood pressure. The renin-angiotensin and kallikrein-kinin systems are intricately...... connected and some of the cardioprotective effects of Losartan are abolished by blocking the bradykinin B2 receptor (B2R) signaling. In this study, we investigated the ability of six clinically available ARBs to specifically bind and activate the B2R. First, we investigated their ability to activate...... phosphoinositide (PI) hydrolysis in COS-7 cells transiently expressing the B2R. We found that only Losartan activated the B2R, working as a partial agonist compared to the endogenous ligand bradykinin. This effect was blocked by the B2R antagonist HOE 140. A competitive binding analysis revealed that Losartan does...

  16. Peroxisome proliferator-activated receptors, metabolic syndrome and cardiovascular disease

    Azhar, Salman

    2010-01-01

    Metabolic syndrome (MetS) is a constellation of risk factors including insulin resistance, central obesity, dyslipidemia and hypertension that markedly increase the risk of Type 2 diabetes (T2DM) and cardiovascular disease (CVD). The peroxisome proliferators-activated receptor (PPAR) isotypes, PPARα, PPARδ/β and PPARγ are ligand-activated nuclear transcription factors, which modulate the expression of an array of genes that play a central role in regulating glucose, lipid and cholesterol meta...

  17. Steroid signaling activation and intracellular localization of sex steroid receptors

    Giraldi, Tiziana; Giovannelli, Pia; Di Donato, Marzia; Castoria, Gabriella; Migliaccio, Antimo; Auricchio, Ferdinando

    2010-01-01

    In addition to stimulating gene transcription, sex steroids trigger rapid, non-genomic responses in the extra-nuclear compartment of target cells. These events take place within seconds or minutes after hormone administration and do not require transcriptional activity of sex steroid receptors. Depending on cell systems, activation of extra-nuclear signaling pathways by sex steroids fosters cell cycle progression, prevents apoptosis, leads to epigenetic modifications and increases cell migrat...

  18. Erythropoietin Does Not Enhance Skeletal Muscle Protein Synthesis Following Exercise in Young and Older Adults

    Lamon, Séverine; Zacharewicz, Evelyn; Arentson-Lantz, Emily; Gatta, Paul A. Della; Ghobrial, Lobna; Gerlinger-Romero, Frederico; Garnham, Andrew; Paddon-Jones, Douglas; Russell, Aaron P.

    2016-01-01

    Purpose: Erythropoietin (EPO) is a renal cytokine that is primarily involved in hematopoiesis while also playing a role in non-hematopoietic tissues expressing the EPO-receptor (EPOR). The EPOR is present in human skeletal muscle. In mouse skeletal muscle, EPO stimulation can activate the AKT serine/threonine kinase 1 (AKT) signaling pathway, the main positive regulator of muscle protein synthesis. We hypothesized that a single intravenous EPO injection combined with acute resistance exercise would have a synergistic effect on skeletal muscle protein synthesis via activation of the AKT pathway. Methods: Ten young (24.2 ± 0.9 years) and 10 older (66.6 ± 1.1 years) healthy subjects received a primed, constant infusion of [ring-13C6] L-phenylalanine and a single injection of 10,000 IU epoetin-beta or placebo in a double-blind randomized, cross-over design. 2 h after the injection, the subjects completed an acute bout of leg extension resistance exercise to stimulate skeletal muscle protein synthesis. Results: Significant interaction effects in the phosphorylation levels of the members of the AKT signaling pathway indicated a differential activation of protein synthesis signaling in older subjects when compared to young subjects. However, EPO offered no synergistic effect on vastus lateralis mixed muscle protein synthesis rate in young or older subjects. Conclusions: Despite its ability to activate the AKT pathway in skeletal muscle, an acute EPO injection had no additive or synergistic effect on the exercise-induced activation of muscle protein synthesis or muscle protein synthesis signaling pathways. PMID:27458387

  19. Activation of serotonin receptors promotes microglial injury-induced motility but attenuates phagocytic activity

    Krabbe, Grietje; Matyash, Vitali; Pannasch, Ulrike; Mamer, Lauren; Boddeke, Hendrikus W. G. M.; Kettenmann, Helmut

    2012-01-01

    Microglia, the brain immune cell, express several neurotransmitter receptors which modulate microglial functions. In this project we studied the impact of serotonin receptor activation on distinct microglial properties as serotonin deficiency not only has been linked to a number of psychiatric disea

  20. An improved ivermectin-activated chloride channel receptor for inhibiting electrical activity in defined neuronal populations

    Lynagh, Timothy Peter; Lynch, Joseph W

    2010-01-01

    for surgically implanted stimulus delivery methods and their use of nonhuman receptors. A third silencing method, an invertebrate glutamate-gated chloride channel receptor (GluClR) activated by ivermectin, solves the stimulus delivery problem as ivermectin is a safe, well tolerated drug that reaches...

  1. Change in Erythropoietin Pharmacokinetics Following Hematopoietic Transplantation

    Widness, JA; Schmidt, RL; Hohl, RJ; Goldman, FD; Al-Huniti, NH; Freise, KJ; Veng-Pedersen, P.

    2007-01-01

    Pre-clinical studies have demonstrated that bone marrow ablation has a profound effect in decreasing erythropoietin (EPO) elimination. The study’s objective was to determine in humans if EPO pharmacokinetics (PKs) are perturbed following bone marrow ablation. EPO PK studies were performed in eight subjects, aged 4 to 61 years, undergoing fully myeloablative hematopoietic stem cell transplantation. Serial PK studies using intravenous injection of recombinant human EPO (92±2.0 U/kg) (mean±SEM) ...

  2. Erythropoietin, uncertainty principle and cancer related anaemia

    Otavio CLARK; Adams, Jared R; Bennett, Charles L.; Djulbegovic, Benjamin

    2002-01-01

    Background This study was designed to evaluate if erythropoietin (EPO) is effective in the treatment of cancer related anemia, and if its effect remains unchanged when data are analyzed according to various clinical and methodological characteristics of the studies. We also wanted to demonstrate that cumulative meta-analysis (CMA) can be used to resolve uncertainty regarding clinical questions. Methods Systematic Review (SR) of the published literature on the role of EPO in cancer-related ane...

  3. Screening of selected pesticides for oestrogen receptor activation in vitro

    Vinggaard, Anne; Breinholt, Vibeke; Larsen, John Christian

    1999-01-01

    Twenty pesticides were tested for their ability to activate the oestrogen receptor in vitro using an,MCF7 cell proliferation assay and a Yeast Oestrogen Screen. The fungicides fenarimol, triadimefon, and triadimenol were identified as weak oestrogen receptor agonists, which at 10 mu M induces a 2.......0, 2.4, and 1.9-fold increase in proliferation of human MCF7 breast cancer cells (E3 clone). The relative proliferation efficiency (RPE) was 43-69%, indicating partial agonism at the oestrogen receptor. Several pesticides did not have any effect oil the proliferation response after 6 days of exposure......, including. chlorpyrifos, diuron, iprodion, linuron, pentachlorphenol, prochloraz, propioconazol, propyzamine, quintozen, tetrachorvinphos and tetradifon. Some pesticides resulted in a negligible proliferation response, which was nor statistically significant under the present experimental conditions. These...

  4. Screening of selected pesticides for oestrogen receptor activation in vitro

    Vinggaard, Anne; Breinholt, Vibeke; Larsen, John Christian

    1999-01-01

    Twenty pesticides were tested for their ability to activate the oestrogen receptor in vitro using an,MCF7 cell proliferation assay and a Yeast Oestrogen Screen. The fungicides fenarimol, triadimefon, and triadimenol were identified as weak oestrogen receptor agonists, which at 10 mu M induces a 2.......0, 2.4, and 1.9-fold increase in proliferation of human MCF7 breast cancer cells (E3 clone). The relative proliferation efficiency (RPE) was 43-69%, indicating partial agonism at the oestrogen receptor. Several pesticides did not have any effect oil the proliferation response after 6 days of exposure......, including. chlorpyrifos, diuron, iprodion, linuron, pentachlorphenol, prochloraz, propioconazol, propyzamine, quintozen, tetrachorvinphos and tetradifon. Some pesticides resulted in a negligible proliferation response, which was nor statistically significant under the present experimental conditions...

  5. Erythropoietin and the use of a transgenic model of erythropoietin-deficient mice

    Pichon A

    2016-04-01

    Full Text Available Aurélien Pichon,1–3 Florine Jeton,1,2 Raja El Hasnaoui-Saadani,4 Luciana Hagström,5 Thierry Launay,6 Michèle Beaudry,1 Dominique Marchant,1 Patricia Quidu,1 Jose-Luis Macarlupu,7 Fabrice Favret,8 Jean-Paul Richalet,1,2 Nicolas Voituron1,2 1Laboratory “Hypoxia and Lung” EA 2363, University Paris 13, Sorbonne Paris Cité, Bobigny Cedex, 2Laboratory of Excellence GR-Ex, Paris, 3Laboratory MOVE EA 6314, FSS, Poitiers University, Poitiers, France; 4Research Unit, College of Medicine, Princess Noura University, Riyadh, Saudi Arabia; 5Laboratório Interdisciplinar de Biociências, Universidade de Brasília, Brasília, Brazil; 6Unité de Biologie Intégrative des Adaptations à l'Exercice, University Paris Saclay and Genopole®, University Sorbonne-Paris-Cité, Paris, France; 7High Altitude Unit, Laboratories for Research and Development, Universidad Peruana Cayetano Heredia, Lima, Peru; 8Laboratory “Mitochondrie, Stress Oxydant et Protection Musculaire” EA 3072, University of Strasbourg, Strasbourg, France Abstract: Despite its well-known role in red blood cell production, it is now accepted that erythropoietin (Epo has other physiological functions. Epo and its receptors are expressed in many tissues, such as the brain and heart. The presence of Epo/Epo receptors in these organs suggests other roles than those usually assigned to this protein. Thus, the aim of this review is to describe the effects of Epo deficiency on adaptation to normoxic and hypoxic environments and to suggest a key role of Epo on main physiological adaptive functions. Our original model of Epo-deficient (Epo-TAgh mice allowed us to improve our knowledge of the possible role of Epo in O2 homeostasis. The use of anemic transgenic mice revealed Epo as a crucial component of adaptation to hypoxia. Epo-TAgh mice survive well in hypoxic conditions despite low hematocrit. Furthermore, Epo plays a key role in neural control of ventilatory acclimatization and response to

  6. Glycine Receptor α2 Subunit Activation Promotes Cortical Interneuron Migration

    Ariel Avila

    2013-08-01

    Full Text Available Glycine receptors (GlyRs are detected in the developing CNS before synaptogenesis, but their function remains elusive. This study demonstrates that functional GlyRs are expressed by embryonic cortical interneurons in vivo. Furthermore, genetic disruption of these receptors leads to interneuron migration defects. We discovered that extrasynaptic activation of GlyRs containing the α2 subunit in cortical interneurons by endogenous glycine activates voltage-gated calcium channels and promotes calcium influx, which further modulates actomyosin contractility to fine-tune nuclear translocation during migration. Taken together, our data highlight the molecular events triggered by GlyR α2 activation that control cortical tangential migration during embryogenesis.

  7. Conserved phosphorylation sites in the activation loop of the Arabidopsis phytosulfokine receptor PSKR1 differentially affect kinase and receptor activity

    Hartmann, Jens; Linke, Dennis; Bönniger, Christine; Tholey, Andreas; Sauter, Margret

    2015-01-01

    PSK (phytosulfokine) is a plant peptide hormone perceived by a leucine-rich repeat receptor kinase. Phosphosite mapping of epitope-tagged PSKR1 (phytosulfokine receptor 1) from Arabidopsis thaliana plants identified Ser696 and Ser698 in the JM (juxtamembrane) region and probably Ser886 and/or Ser893 in the AL (activation loop) as in planta phosphorylation sites. In vitro-expressed kinase was autophosphorylated at Ser717 in the JM, and at Ser733, Thr752, Ser783, Ser864, Ser911, Ser958 and Thr9...

  8. Paraneoplastic Erythrocytosis of Colon Cancer, with Serum Erythropoietin within the Normal Reference Range

    Kitayama, Hiromitsu; Kondo, Tomohiro; SUGIYAMA, Junko; Hirayama, Michiaki; Oyamada, Yumiko; Tsuji, Yasushi

    2016-01-01

    Patient: Female, 75 Final Diagnosis: Erythropoietin-secreting colon cancer Symptoms: None Medication: — Clinical Procedure: Immunohistochemistry Specialty: Hematology Objective: Rare disease Background: Paraneoplastic erythrocytosis can be brought on by ectopic erythropoietin production usually in kidney, brain, and liver tumor with increase of serum erythropoietin level. We report here a paraneoplastic erythrocytosis of colon cancer with serum erythropoietin within the normal reference, whic...

  9. Conserved phosphorylation sites in the activation loop of the Arabidopsis phytosulfokine receptor PSKR1 differentially affect kinase and receptor activity.

    Hartmann, Jens; Linke, Dennis; Bönniger, Christine; Tholey, Andreas; Sauter, Margret

    2015-12-15

    PSK (phytosulfokine) is a plant peptide hormone perceived by a leucine-rich repeat receptor kinase. Phosphosite mapping of epitope-tagged PSKR1 (phytosulfokine receptor 1) from Arabidopsis thaliana plants identified Ser(696) and Ser(698) in the JM (juxtamembrane) region and probably Ser(886) and/or Ser(893) in the AL (activation loop) as in planta phosphorylation sites. In vitro-expressed kinase was autophosphorylated at Ser(717) in the JM, and at Ser(733), Thr(752), Ser(783), Ser(864), Ser(911), Ser(958) and Thr(998) in the kinase domain. The LC-ESI-MS/MS spectra provided support that up to three sites (Thr(890), Ser(893) and Thr(894)) in the AL were likely to be phosphorylated in vitro. These sites are evolutionarily highly conserved in PSK receptors, indicative of a conserved function. Site-directed mutagenesis of the four conserved residues in the activation segment, Thr(890), Ser(893), Thr(894) and Thr(899), differentially altered kinase activity in vitro and growth-promoting activity in planta. The T899A and the quadruple-mutated TSTT-A (T890A/S893A/T894A/T899A) mutants were both kinase-inactive, but PSKR1(T899A) retained growth-promoting activity. The T890A and S893A/T894A substitutions diminished kinase activity and growth promotion. We hypothesize that phosphorylation within the AL activates kinase activity and receptor function in a gradual and distinctive manner that may be a means to modulate the PSK response. PMID:26472115

  10. Erythropoietin and the brain: from neurodevelopment to neuroprotection.

    Buemi, M; Cavallaro, E; Floccari, F; Sturiale, A; Aloisi, C; Trimarchi, M; Grasso, G; Corica, F; Frisina, N

    2002-09-01

    It is now widely known that erythropoietin (Epo) does not only affect the haematopoietic system, but it can be considered a multifunctional trophic factor with an effect on the general homoeostasis of the entire organism. The recent discovery of a specific Epo/Epo-receptor system in the central nervous system (CNS) and cerebrospinal fluid, independently of the haematopoietic system, has further paved the way for new studies aimed at investigating the different sites of cerebral expression of Epo and its receptor, the regulation of their expression and, finally, the effects that this hormone has on the development and maturation of the brain. A further aim has been to investigate how it influences CNS homoeostasis and neurotransmission in adult brain. Attention has also been focused on the neurotrophic and neuroprotective function of Epo in different conditions of neuronal damage, such as hypoxia, cerebral ischaemia and subarachnoid haemorrhage, and therefore on the possibility that human recombinant Epo therapy could soon be used in clinical practice, also to limit neuronal damage induced by these diseases. PMID:12193153

  11. Environmental phthalate monoesters activate pregnane X receptor-mediated transcription

    Phthalate esters, widely used as plasticizers in the manufacture of products made of polyvinyl chloride, induce reproductive and developmental toxicities in rodents. The mechanism that underlies these effects of phthalate exposure, including the potential role of members of the nuclear receptor superfamily, is not known. The present study investigates the effects of phthalates on the pregnane X receptor (PXR), which mediates the induction of enzymes involved in steroid metabolism and xenobiotic detoxification. The ability of phthalate monoesters to activate PXR-mediated transcription was assayed in a HepG2 cell reporter assay following transfection with mouse PXR (mPXR), human PXR (hPXR), or the hPXR allelic variants V140M, D163G, and A370T. Mono-2-ethylhexyl phthalate (MEHP) increased the transcriptional activity of both mPXR and hPXR (5- and 15-fold, respectively) with EC50 values of 7-8 μM. mPXR and hPXR were also activated by monobenzyl phthalate (MBzP, up to 5- to 6-fold) but were unresponsive to monomethyl phthalate and mono-n-butyl phthalate (M(n)BP) at the highest concentrations tested (300 μM). hPXR-V140M and hPXR-A370T exhibited patterns of phthalate responses similar to the wild-type receptor. By contrast, hPXR-D163G was unresponsive to all phthalate monoesters tested. Further studies revealed that hPXR-D163G did respond to rifampicin, but required approximately 40-fold higher concentrations than wild-type receptor, suggesting that the ligand-binding domain D163G variant has impaired ligand-binding activity. The responsiveness of PXR to activation by phthalate monoesters demonstrated here suggests that these ubiquitous environmental chemicals may, in part, exhibit their endocrine disruptor activities by altering PXR-regulated steroid hormone metabolism with potential adverse health effects in exposed individuals

  12. Hyaluronic acid induces activation of the κ-opioid receptor.

    Barbara Zavan

    Full Text Available INTRODUCTION: Nociceptive pain is one of the most common types of pain that originates from an injury involving nociceptors. Approximately 60% of the knee joint innervations are classified as nociceptive. The specific biological mechanism underlying the regulation of nociceptors is relevant for the treatment of symptoms affecting the knee joint. Intra-articular administration of exogenous hyaluronic acid (HA in patients with osteoarthritis (OA appears to be particularly effective in reducing pain and improving patient function. METHODS: We performed an in vitro study conducted in CHO cells that expressed a panel of opioid receptors and in primary rat dorsal root ganglion (DRG neurons to determine if HA induces the activation of opioid peptide receptors (OPr using both aequorin and the fluorescent dye Fura-2/AM. RESULTS: Selective agonists and antagonists for each OPr expressed on CHO cells were used to test the efficacy of our in vitro model followed by stimulation with HA. The results showed that HA induces stimulatory effects on the κ receptor (KOP. These effects of HA were also confirmed in rat DRG neurons, which express endogenously the OPr. CONCLUSIONS: HA activates the KOP receptor in a concentration dependent manner, with a pEC(50 value of 7.57.

  13. Allosteric receptor activation by the plant peptide hormone phytosulfokine.

    Wang, Jizong; Li, Hongju; Han, Zhifu; Zhang, Heqiao; Wang, Tong; Lin, Guangzhong; Chang, Junbiao; Yang, Weicai; Chai, Jijie

    2015-09-10

    Phytosulfokine (PSK) is a disulfated pentapeptide that has a ubiquitous role in plant growth and development. PSK is perceived by its receptor PSKR, a leucine-rich repeat receptor kinase (LRR-RK). The mechanisms underlying the recognition of PSK, the activation of PSKR and the identity of the components downstream of the initial binding remain elusive. Here we report the crystal structures of the extracellular LRR domain of PSKR in free, PSK- and co-receptor-bound forms. The structures reveal that PSK interacts mainly with a β-strand from the island domain of PSKR, forming an anti-β-sheet. The two sulfate moieties of PSK interact directly with PSKR, sensitizing PSKR recognition of PSK. Supported by biochemical, structural and genetic evidence, PSK binding enhances PSKR heterodimerization with the somatic embryogenesis receptor-like kinases (SERKs). However, PSK is not directly involved in PSKR-SERK interaction but stabilizes PSKR island domain for recruitment of a SERK. Our data reveal the structural basis for PSKR recognition of PSK and allosteric activation of PSKR by PSK, opening up new avenues for the design of PSKR-specific small molecules. PMID:26308901

  14. Central nervous system frontiers for the use of erythropoietin

    Olsen, Niels Vidiendal

    2003-01-01

    Recombinant human erythropoietin (r-HuEPO; epoetin alfa) is well established as safe and effective for the treatment of anemia. In addition to the erythropoietic effects of endogenous erythropoietin (EPO), recent evidence suggests that it may elicit a neuroprotective effect in the central nervous...

  15. The unfulfilled promise of erythropoietin in patients with MI

    van der Meer, Peter; van Veldhuisen, Dirk J.

    2011-01-01

    Over the past 5-10 years, the positive effects of erythropoietin on myocardial infarction have been reported in several experimental, animal models. However, the results of the REVEAL study call into question the beneficial effects of erythropoietin in humans presenting with myocardial infarction.

  16. Neurohumoral activation in heart failure: the role of adrenergic receptors

    Patricia C. Brum

    2006-09-01

    Full Text Available Heart failure (HF is a common endpoint for many forms of cardiovascular disease and a significant cause of morbidity and mortality. The development of end-stage HF often involves an initial insult to the myocardium that reduces cardiac output and leads to a compensatory increase in sympathetic nervous system activity. Acutely, the sympathetic hyperactivity through the activation of beta-adrenergic receptors increases heart rate and cardiac contractility, which compensate for decreased cardiac output. However, chronic exposure of the heart to elevated levels of catecholamines released from sympathetic nerve terminals and the adrenal gland may lead to further pathologic changes in the heart, resulting in continued elevation of sympathetic tone and a progressive deterioration in cardiac function. On a molecular level, altered beta-adrenergic receptor signaling plays a pivotal role in the genesis and progression of HF. beta-adrenergic receptor number and function are decreased, and downstream mechanisms are altered. In this review we will present an overview of the normal beta-adrenergic receptor pathway in the heart and the consequences of sustained adrenergic activation in HF. The myopathic potential of individual components of the adrenergic signaling will be discussed through the results of research performed in genetic modified animals. Finally, we will discuss the potential clinical impact of beta-adrenergic receptor gene polymorphisms for better understanding the progression of HF.A insuficiência cardíaca (IC é a via final comum da maioria das doenças cardiovasculares e uma das maiores causas de morbi-mortalidade. O desenvolvimento do estágio final da IC freqüentemente envolve um insulto inicial do miocárdio, reduzindo o débito cardíaco e levando ao aumento compensatório da atividade do sistema nervoso simpático (SNS. Existem evidências de que apesar da exposição aguda ser benéfica, exposições crônicas a elevadas concentra

  17. Implications of compound heterozygous insulin receptor mutations in congenital muscle fibre type disproportion myopathy for the receptor kinase activation

    Klein, H H; Müller, R; Vestergaard, H;

    1999-01-01

    We studied insulin receptor kinase activation in two brothers with congenital muscle fibre type disproportion myopathy and compound heterozygous mutations of the insulin receptor gene, their parents, and their unaffected brother. In the father who has a heterozygote Arg1174-->Gln mutation, in situ...... receptors to become insulin-dependently activated. The mother carries a point mutation at the last base pair in exon 17 which, due to abnormal alternative splicing, could lead to normally transcribed receptor or truncated receptor lacking the kinase region. Kinase activation was normal in the mother...... receptors in the mother's skeletal muscle are transcribed almost exclusively from the non-mutated allele. The mutation in exon 17 could lead to reduced transcription or rapid degradation of a predominantly transcribed truncated gene product or both....

  18. Umbilical Cord Serum Erythropoietin Levels and Maternal Smoking in Pregnancy

    Soner Sazak

    2012-01-01

    Full Text Available Objective. To evaluate the effect of maternal smoking during pregnancy on levels of umbilical cord erythropoietin. Methods. Erythropoietin levels were measured in umbilical cord sera of 60 newborns who were delivered vaginally at term. There were 20 (33% smoking and 40 (67% nonsmoking mothers. Results. Mean cord serum erythropoietin levels were significantly lower in the nonsmokers (nonsmokers, 24 ± 9 IU/L; smokers, 61 ± 46 IU/L; P<.001. There was a significant positive correlation between the number of cigarettes smoked per day and cord serum erythropoietin levels (r, 0.58; P≤.05. Conclusions. Smoking during pregnancy is associated with increased levels of umbilical cord erythropoietin at birth. This may indicate a risk of fetal hypoxia and growth restriction. Education and encouragement of cessation of smoking during pregnancy are important to avoid associated fetal and maternal morbidity and mortality.

  19. Activation of Penile Proadipogenic Peroxisome Proliferator-Activated Receptor with an Estrogen: Interaction with Estrogen Receptor Alpha during Postnatal Development

    Mahmoud M. Mansour

    2008-01-01

    Full Text Available Exposure to the estrogen receptor alpha (ER ligand diethylstilbesterol (DES between neonatal days 2 to 12 induces penile adipogenesis and adult infertility in rats. The objective of this study was to investigate the in vivo interaction between DES-activated ER and the proadipogenic transcription factor peroxisome proliferator-activated receptor gamma (PPAR. Transcripts for PPARs , , and and 1a splice variant were detected in Sprague-Dawley normal rat penis with PPAR predominating. In addition, PPAR1b and PPAR2 were newly induced by DES. The PPAR transcripts were significantly upregulated with DES and reduced by antiestrogen ICI 182, 780. At the cellular level, PPAR protein was detected in urethral transitional epithelium and stromal, endothelial, neuronal, and smooth muscular cells. Treatment with DES activated ER and induced adipocyte differentiation in corpus cavernosum penis. Those adipocytes exhibited strong nuclear PPAR expression. These results suggest a biological overlap between PPAR and ER and highlight a mechanism for endocrine disruption.

  20. Mechanism of A2 adenosine receptor activation. I. Blockade of A2 adenosine receptors by photoaffinity labeling

    Lohse, M.J.; Klotz, K.N.; Schwabe, U.

    1991-04-01

    It has previously been shown that covalent incorporation of the photoreactive adenosine derivative (R)-2-azido-N6-p-hydroxy-phenylisopropyladenosine ((R)-AHPIA) into the A1 adenosine receptor of intact fat cells leads to a persistent activation of this receptor, resulting in a reduction of cellular cAMP levels. In contrast, covalent incorporation of (R)-AHPIA into human platelet membranes, which contain only stimulatory A2 adenosine receptors, reduces adenylate cyclase stimulation via these receptors. This effect of (R)-AHPIA is specific for the A2 receptor and can be prevented by the adenosine receptor antagonist theophylline. Binding studies indicate that up to 90% of A2 receptors can be blocked by photoincorporation of (R)-AHPIA. However, the remaining 10-20% of A2 receptors are sufficient to mediate an adenylate cyclase stimulation of up to 50% of the control value. Similarly, the activation via these 10-20% of receptors occurs with a half-life that is only 2 times longer than that in control membranes. This indicates the presence of a receptor reserve, with respect to both the extent and the rate of adenylate cyclase stimulation. These observations require a modification of the models of receptor-adenylate cyclase coupling.

  1. Erythropoietin and cerebral vascular protection: role of nitric oxide

    Anantha Vijay R SANTHANAM; Zvonimir S KATUSIC

    2006-01-01

    Cerebral vasospasm after subarachnoid hemorrhage (SAH) is a major clinical problem causing cerebral ischemia and infarction.The pathogenesis of vasospasm is related to a number of pathological processes including endothelial damage and alterations in vasomotor function leading to narrowing of artefial diameter and a subsequent decrease in cerebral blood flow.Discovery of the tissue protective effects of erythropoietin (EPO) stimulated the search for therapeutic application of EPO for the prevention and treatment of cerebrovascular disease.Recent studies have identified the role of EP0 in vascular protection mediated by the preservation of endothelial cell integrity and stimulation of angiogenesis.In this review, we discuss the EPO-induced activation of endothelial nitric oxide (NO) synthase and its contribution to the prevention of cerebral vasospasm.

  2. Allosterism and Structure in Thermally Activated Transient Receptor Potential Channels.

    Diaz-Franulic, Ignacio; Poblete, Horacio; Miño-Galaz, Germán; González, Carlos; Latorre, Ramón

    2016-07-01

    The molecular sensors that mediate temperature changes in living organisms are a large family of proteins known as thermosensitive transient receptor potential (TRP) ion channels. These membrane proteins are polymodal receptors that can be activated by cold or hot temperatures, depending on the channel subtype, voltage, and ligands. The stimuli sensors are allosterically coupled to a pore domain, increasing the probability of finding the channel in its ion conductive conformation. In this review we first discuss the allosteric coupling between the temperature and voltage sensor modules and the pore domain, and then discuss the thermodynamic foundations of thermo-TRP channel activation. We provide a structural overview of the molecular determinants of temperature sensing. We also posit an anisotropic thermal diffusion model that may explain the large temperature sensitivity of TRP channels. Additionally, we examine the effect of several ligands on TRP channel function and the evidence regarding their mechanisms of action. PMID:27297398

  3. Monocyte Signal Transduction Receptors in Active and Latent Tuberculosis

    Magdalena Druszczynska

    2013-01-01

    Full Text Available The mechanisms that promote either resistance or susceptibility to TB disease remain insufficiently understood. Our aim was to compare the expression of cell signaling transduction receptors, CD14, TLR2, CD206, and β2 integrin LFA-1 on monocytes from patients with active TB or nonmycobacterial lung disease and healthy individuals with M.tb latency and uninfected controls to explain the background of the differences between clinical and subclinical forms of M.tb infection. A simultaneous increase in the expression of the membrane bound mCD14 receptor and LFA-1 integrin in patients with active TB may be considered a prodrome of breaking immune control by M.tb bacilli in subjects with the latent TB and absence of clinical symptoms.

  4. Activation of the chicken gonadotropin-inhibitory hormone receptor reduces gonadotropin releasing hormone receptor signaling.

    Shimizu, Mamiko; Bédécarrats, Grégoy Y

    2010-06-01

    Gonadotropin-inhibitory hormone (GnIH) is a hypothalamic peptide from the RFamide peptide family that has been identified in multiple avian species. Although GnIH has clearly been shown to reduce LH release from the anterior pituitary gland, its mechanism of action remains to be determined. The overall objectives of this study were (1) to characterize the GnIH receptor (GnIH-R) signaling pathway, (2) to evaluate potential interactions with gonadotropin releasing hormone type III receptor (GnRH-R-III) signaling, and (3) to determine the molecular mechanisms by which GnIH and GnRH regulate pituitary gonadotrope function during a reproductive cycle in the chicken. Using real-time PCR, we showed that in the chicken pituitary gland, GnIH-R mRNA levels fluctuate in an opposite manner to GnRH-R-III, with higher and lower levels observed during inactive and active reproductive stages, respectively. We demonstrated that the chicken GnIH-R signals by inhibiting adenylyl cyclase cAMP production, most likely by coupling to G(alphai). We also showed that this inhibition is sufficient to significantly reduce GnRH-induced cAMP responsive element (CRE) activation in a dose-dependent manner, and that the ratio of GnRH/GnIH receptors is a significant factor. We propose that in avian species, sexual maturation is characterized by a change in GnIH/GnRH receptor ratio, resulting in a switch in pituitary sensitivity from inhibitory (involving GnIH) to stimulatory (involving GnRH). In turn, decreasing GnIH-R signaling, combined with increasing GnRH-R-III signaling, results in significant increases in CRE activation, possibly initiating gonadotropin synthesis. PMID:20350548

  5. Serum Erythropoietin Levels in Pediatric Hematologic Disorders and Impact of Recombinant Human Erythropoietin Use

    Mualla Çetin

    2009-06-01

    Full Text Available Objective: In anemic patients, the correlation between serum erythropoietin (sEpo level and the severity of anemia has been reported previously. However, in different anemia groups, different sEpo levels are measured in patients with similar hemoglobin levels and the etiology of this situation could not be explained. Methods: We evaluated hemoglobin and sEpo levels in 31 iron deficiency anemia, 26 Fanconi anemia (FA, 21 thallasemia intermedia (TI, 15 acute lymphoblastic leukemia (ALL patients at presentation and 12 healthy controls. Results: In all disease groups, an inverse linear correlation was shown between hemoglobin and logarhytmic sEpo level. The covariance analyses according to corrected hemoglobin levels exhibited the highest sEpo level in FA, followed by ALL, TI and iron deficiency anemia, sequentialy. Conclusion: There was no statisticaly significant difference of sEpo levels in FA patients in terms of androgen treatment and this finding supports that androgen affects erythropoisis directly, and has no effect on erythropoietin. The results indicate that there is no erythropoietin deficiency in the anemia of these patients and the admnistration of exogenous erythropoietin offers no clinical benefit.

  6. Thyrotropin Receptor Activation Increases Hyaluronan Production in Preadipocyte Fibroblasts

    Zhang, Lei; Bowen, Timothy; Grennan-Jones, Fiona; Paddon, Carol; Giles, Peter; Webber, Jason; Steadman, Robert; Ludgate, Marian

    2009-01-01

    The thyrotropin receptor (TSHR) is expressed during lineage-specific differentiation (e.g. adipogenesis) and is activated by TSH, thyroid-stimulating antibodies, and gain-of-function mutations (TSHR*). Comparison of gene expression profiles of nonmodified human preadipocytes (n = 4) with the parallel TSHR* population revealed significant up-regulation of 27 genes including hyaluronan (HA) synthases (HAS) 1 and 2. The array data were confirmed by quantitative PCR of HAS1 and HAS2 and enzyme-li...

  7. Insect Repellents: Modulators of Mosquito Odorant Receptor Activity

    Bohbot, Jonathan D.; Joseph C. Dickens

    2010-01-01

    Background DEET, 2-undecanone (2-U), IR3535 and Picaridin are widely used as insect repellents to prevent interactions between humans and many arthropods including mosquitoes. Their molecular action has only recently been studied, yielding seemingly contradictory theories including odorant-dependent inhibitory and odorant-independent excitatory activities on insect olfactory sensory neurons (OSNs) and odorant receptor proteins (ORs). Methodology/Principal Findings Here we characterize the act...

  8. A novel hydroxyfuroic acid compound as an insulin receptor activator – structure and activity relationship of a prenylindole moiety to insulin receptor activation

    Tsai Henry J

    2009-07-01

    Full Text Available Abstract Background Diabetes Mellitus is a chronic disease and many patients of which require frequent subcutaneous insulin injection to maintain proper blood glucose levels. Due to the inconvenience of insulin administration, an orally active insulin replacement has long been a prime target for many pharmaceutical companies. Demethylasterriquinone (DMAQ B1, extracted from tropical fungus, Pseudomassaria sp., has been reported to be an orally effective agent at lowering circulating glucose levels in diabetic (db/db mice; however, the cytotoxicity associated with the quinone moiety has not been addressed thus far. Methods A series of hydroxyfuroic acid compounds were synthesized and tested for their efficacies at activating human insulin receptor. Cytotoxicity to Chinese hamster ovary cells, selectivities over insulin-like growth factor-1 (IGF-1, epidermal growth factor (EGF, and fibroblast growth factor (FGF receptors were examined in this study. Result and Conclusion This study reports a new non-quinone DMAQ B1 derivative, a hydroxyfuroic acid compound (D-410639, which is 128 fold less cytotoxic as DMAQ B1 and as potent as compound 2, a DMAQ B1 synthetic derivative from Merck, at activating human insulin receptor. D-410639 has little activation potential on IGF-1 receptor but is a moderate inhibitor to EGF receptor. Structure and activity relationship of the prenylindole moiety to insulin receptor activation is discussed.

  9. Interplay between Endothelin and Erythropoietin in Astroglia: The Role in Protection against Hypoxia

    Richard Schäfer; Lars Mueller; Reinhild Buecheler; Barbara Proksch; Matthias Schwab; Christoph H. Gleiter; Lusine Danielyan

    2014-01-01

    We show that, under in vitro conditions, the vulnerability of astroglia to hypoxia is reflected by alterations in endothelin (ET)-1 release and capacity of erythropoietin (EPO) to regulate ET-1 levels. Exposure of cells to 24 h hypoxia did not induce changes in ET-1 release, while 48–72 h hypoxia resulted in increase of ET-1 release from astrocytes that could be abolished by EPO. The endothelin receptor type A (ETA) antagonist BQ123 increased extracellular levels of ET-1 in human fetal astrog...

  10. Ultrastructural and biochemical analysis of fibrinogen receptors on activated thrombocytes

    The present studies have been concerned with the role of fibrinogen and its receptor, GP IIb/IIIa, during the activation and early aggregation of pigeon thrombocytes. Thrombocytes were surface labeled with 125I then separated on SDS-PAGE. Analysis by gel autoradiography revealed major bands at MW 145 kd and 98 kd, which corresponded to human GPIIb and GPIIIa. Immunologic similarity of the pigeon and human receptor components was established by dot blot analysis using polyclonal antibodies directed against human GPIIb and GPIIIa. Pigeon fibrinogen, isolated by plasma precipitation with PEG-1000 and purified over Sepharose 4B, was used to study receptor-ligand interaction. Separation of pigeon fibrinogen on SDS-PAGE resulted in three peptides having apparent MW of 62kd, 55kd, and 47kd which are comparable to human fibrinogen. Further similarity of human and pigeon fibrinogen was verified by immonodiffusion against an antibody specific for the human protein. The role of fibrinogen and its receptor in thrombocyte function was established by turbidimetric aggregation using thrombin as an agonist under conditions requiring Ca++ and fibrinogen

  11. Neutron activation analysis in receptor modeling in environmental chemistry

    The release of air pollutants in high enough concentrations can have adverse health effects on the general public, particularly in urban areas. To develop strategies to manage the quality of air pollution, it is necessary to understand the flow of materials through the system, the sources and sinks, transformation, and transport pathways of the species of interest. An approach that is extensively being used in our laboratories, as well as in many others, is the use of receptor modeling. These models are based on the concepts of conservation of mass and a mass balance analysis, which in turn is based on the characterization of airborne particles into their constituents. From these methods, sources contributing to air quality are identified from the perspective of the receptor or ambient sampler. Separation of the ambient particles containing trace elements into distinct size classes allows the study of various sources to be identified. Another reason for the rapid development of receptor models has been the concurrent development of a variety of analytical techniques that permit greatly improved analyses to provide the needed input data. Two of the most powerful methods are instrumental neutron activation analysis (INAA) and X-ray fluorescence (XRF), including particle-induced X-ray emission. By using these methods along with sophisticated receptor modeling and size fractionation, the authors have been able to identify specific sources of local and regional air pollution

  12. Response of anaemia in rheumatoid arthritis to treatment with subcutaneous recombinant human erythropoietin.

    Gudbjörnsson, B; Hällgren, R; Wide, L; Birgegård, G

    1992-01-01

    Eleven patients with chronic inflammatory arthritides and haemoglobin concentrations less than 105 g/l with symptoms from their anaemia were treated with a dose of 250 IU/kg/week of recombinant human erythropoietin for six weeks. The treatment was given as subcutaneous injections five days a week. All patients had active inflammatory disease. Nine patients responded to treatment with an increase in haemoglobin of more than 15 g/l. The mean (SD) haemoglobin concentration increased from 93.0 (8...

  13. Increased Levels of Erythropoietin in Nipple Aspirate Fluid and in Ductal Cells from Breast Cancer Patients

    Mannello, Ferdinando; Fabbri, Laura; Ciandrini, Eleonora; Tonti, Gaetana A.

    2008-01-01

    Background: Erythropoietin (Epo) is an important regulator of erythropoiesis, and controls proliferation and differentiation of both erythroid and non-erythroid tissues. Epo is actively synthesized by breast cells during lactation, and also plays a role in breast tissues promoting hypoxia-induced cancer initiation. Our aims are to perform an exploratory investigation on the Epo accumulation in breast secretions from healthy and cancer patients and its localization in breast cancer cells. Meth...

  14. Neural correlates of improved executive function following erythropoietin treatment in mood disorders

    Miskowiak, K W; Vinberg, M; Glerup, L;

    2016-01-01

    magnetic resonance imaging (fMRI) study investigated the effects of EPO on neural circuitry activity during working memory (WM) performance. METHOD: Patients with treatment-resistant major depression, who were moderately depressed, or with BD in partial remission, were randomized to eight weekly infusions......BACKGROUND: Cognitive dysfunction in depression and bipolar disorder (BD) is insufficiently targeted by available treatments. Erythropoietin (EPO) increases neuroplasticity and may improve cognition in mood disorders, but the neuronal mechanisms of these effects are unknown. This functional...

  15. Dynamic correlation networks in human peroxisome proliferator-activated receptor-γ nuclear receptor protein.

    Fidelak, Jeremy; Ferrer, Silvia; Oberlin, Michael; Moras, Dino; Dejaegere, Annick; Stote, Roland H

    2010-10-01

    Peroxisome proliferator-activated receptor-γ nuclear receptor (PPAR-γ) belongs to the superfamily of nuclear receptor proteins that function as ligand-dependent transcription factors and plays a specific physiological role as a regulator of lipid metabolism. A number of experimental studies have suggested that allostery plays an important role in the functioning of PPAR-γ. Here we use normal-mode analysis of PPAR-γ to characterize a network of dynamically coupled amino acids that link physiologically relevant binding surfaces such as the ligand-dependent activation domain AF-2 with the ligand binding site and the heterodimer interface. Multiple calculations were done in both the presence and absence of the agonist rosiglitazone, and the differences in dynamics were characterized. The global dynamics of the ligand binding domain were affected by the ligand, and in particular, changes to the network of dynamically correlated amino acids were observed with only small changes in conformation. These results suggest that changes in dynamic couplings can be functionally significant with respect to the transmission of allosteric signals. PMID:20496064

  16. Determinants of Red Cell Distribution Width (RDW) in Cardiorenal Patients : RDW is Not Related to Erythropoietin Resistance

    Emans, Mireille E.; van der Putten, Karien; van Rooijen, Karlijn L.; Kraaijenhagen, Rob J.; Swinkels, Dorine; van Solinge, Wouter W.; Cramer, Maarten J.; Doevendans, Pieter A. F. M.; Braam, Branko; Gaillard, Carlo A. J. M.

    2011-01-01

    Background: Studies have shown that red cell distribution width (RDW) is related to outcome in chronic heart failure (CHF). The pathophysiological process is unknown. We studied the relationship between RDW and erythropoietin (EPO) resistance, and related factors such as erythropoietic activity, fun

  17. Detecting constitutive activity and protean agonism at cannabinoid-2 receptor.

    Beltramo, Massimiliano; Brusa, Rossella; Mancini, Isabella; Scandroglio, Paola

    2010-01-01

    Since the cannabinoid system is involved in regulating several physiological functions such as locomotor activity, cognition, nociception, food intake, and inflammatory reaction, it has been the subject of intense study. Research on the pharmacology of this system has enormously progressed in the last 20years. One intriguing aspect that emerged from this research is that cannabinoid receptors (CBs) express a high level of constitutive activity. Investigation on this particular aspect of receptor pharmacology has largely focused on CB1, the CB subtype highly expressed in several brain regions. More recently, research on constitutive activity on the other CB subtype, CB2, was stimulated by the increasing interest on its potential as target for the treatment of various pathologies (e.g., pain and inflammation). There are several possible implications of constitutive activity on the therapeutic action of both agonists and antagonists, and consequently, it is important to have valuable methods to study this aspect of CB2 pharmacology. In the present chapter, we describe three methods to study constitutive activity at CB2: two classical methods relying on the detection of changes in cAMP level and GTPγS binding and a new one based on cell impedance measurement. In addition, we also included a section on detection of protean agonism, which is an interesting pharmacological phenomenon strictly linked to constitutive activity. PMID:21036225

  18. Bioluminescence imaging of estrogen receptor activity during breast cancer progression.

    Vantaggiato, Cristina; Dell'Omo, Giulia; Ramachandran, Balaji; Manni, Isabella; Radaelli, Enrico; Scanziani, Eugenio; Piaggio, Giulia; Maggi, Adriana; Ciana, Paolo

    2016-01-01

    Estrogen receptors (ER) are known to play an important regulatory role in mammary gland development as well as in its neoplastic transformation. Although several studies highlighted the contribution of ER signaling in the breast transformation, little is known about the dynamics of ER state of activity during carcinogenesis due to the lack of appropriate models for measuring the extent of receptor signaling in time, in the same animal. To this aim, we have developed a reporter mouse model for the non-invasive in vivo imaging of ER activity: the ERE-Luc reporter mouse. ERE-Luc is a transgenic mouse generated with a firefly luciferase (Luc) reporter gene driven by a minimal promoter containing an estrogen responsive element (ERE). This model allows to measure receptor signaling in longitudinal studies by bioluminescence imaging (BLI). Here, we have induced sporadic mammary cancers by treating systemically ERE-Luc reporter mice with DMBA (9,10-dimethyl 1,2-benzanthracene) and measured receptor signaling by in vivo imaging in individual animals from early stage until a clinically palpable tumor appeared in the mouse breast. We showed that DMBA administration induces an increase of bioluminescence in the whole abdominal area 6 h after treatment, the signal rapidly disappears. Several weeks later, strong bioluminescence is observed in the area corresponding to the mammary glands. In vivo and ex vivo imaging analysis demonstrated that this bioluminescent signal is localized in the breast area undergoing neoplastic transformation. We conclude that this non-invasive assay is a novel relevant tool to identify the activation of the ER signaling prior the morphological detection of the neoplastic transformation. PMID:27069764

  19. Activation of glycine receptors modulates spontaneous epileptiform activity in the immature rat hippocampus

    Chen, Rongqing; Okabe, Akihito; Sun, Haiyan; Sharopov, Salim; Hanganu-Opatz, Ileana L; Kolbaev, Sergei N; Fukuda, Atsuo; Luhmann, Heiko J; Kilb, Werner

    2014-01-01

    While the expression of glycine receptors in the immature hippocampus has been shown, no information about the role of glycine receptors in controlling the excitability in the immature CNS is available. Therefore, we examined the effect of glycinergic agonists and antagonists in the CA3 region of an intact corticohippocampal preparation of the immature (postnatal days 4–7) rat using field potential recordings. Bath application of 100 μm taurine or 10 μm glycine enhanced the occurrence of recurrent epileptiform activity induced by 20 μm 4-aminopyridine in low Mg2+ solution. This proconvulsive effect was prevented by 3 μm strychnine or after incubation with the loop diuretic bumetanide (10 μm), suggesting that it required glycine receptors and an active NKCC1-dependent Cl− accumulation. Application of higher doses of taurine (≥1 mm) or glycine (100 μm) attenuated recurrent epileptiform discharges. The anticonvulsive effect of taurine was also observed in the presence of the GABAA receptor antagonist gabazine and was attenuated by strychnine, suggesting that it was partially mediated by glycine receptors. Bath application of the glycinergic antagonist strychnine (0.3 μm) induced epileptiform discharges. We conclude from these results that in the immature hippocampus, activation of glycine receptors can mediate both pro- and anticonvulsive effects, but that a persistent activation of glycine receptors is required to suppress epileptiform activity. In summary, our study elucidated the important role of glycine receptors in the control of neuronal excitability in the immature hippocampus. PMID:24665103

  20. Peroxisome Proliferator–Activated Receptors and The Metabolic Syndrome

    Anna Meiliana

    2009-04-01

    Full Text Available BACKGROUND: Obesity is a growing threat to global health by virtue of its association with insulin resistance, inflammation, hypertension, and dyslipidemia, collectively known as the metabolic syndrome (MetS. The nuclear receptors PPARα and PPARγ are therapeutic targets for hypertriglyceridemia and insulin resistance, respectively, and drugs that modulate these receptors are currently in clinical use. More recent work on the PPARδ has uncovered a dual benefit for both hypertriglyceridemia and insulin resistance, highlighting the broad potential of PPARs in the treatment of metabolic disease. CONTENT: We have learned much about PPARs, the metabolic fat sensors, and the molecular pathways they regulate. Through their distinct tissue distribution and specific target gene activation, the three PPARs together control diverse aspects of fatty acid metabolism, energy balance, insulin sensitivity glucose homeostasis, inflammation, hypertension and atherosclerosis. These studies have advanced our understanding of the etiology for the MetS. Mechanisms revealed by these studies highlight the importance of emerging concepts, such as the endocrine function of adipose tissue, tissue-tissue cross-talk and lipotoxicity, in the pathogenesis of type 2 diabetes mellitus and CVD. SUMMARY: The elucidation of key regulators of energy balance and insulin signaling have revolutionized our understanding of fat and sugar metabolism and their intimate link. The three ‘lipidsensing’ (PPARα, PPARγ and PPARδ exemplify this connection, regulating diverse aspects of lipid and glucose homeostasis, and serving as bonafide therapeutic targets. KEYWORDS: peroxisome proliferator, activated receptor, metabolic syndrome.

  1. Anemia of renal failure. Use of erythropoietin.

    Humphries, J E

    1992-05-01

    Chronic renal failure is almost invariably accompanied by symptomatic anemia. It has been demonstrated that the primary cause of this anemia is inadequate production of erythropoietin by the diseased kidneys. The isolation of erythropoietin, followed by the cloning and expression of the human erythropoietin gene, made possible clinical trials of rHuEPO in uremic patients. rHuEPO produced dramatic increases in the hematocrit in almost all patients treated and also ameliorated many symptoms, such as lethargy, dizziness, and poor appetite, that had long been attributed to the effect of uremic toxins. Adverse effects of treatment with rHuEPO noted in the early clinical trials included hypertension, seizures, arteriovenous fistula or shunt thrombosis, and hyperkalemia. Further study of rHuEPO has shown that many of these side effects may be no more frequent in patients receiving rHuEPO than in other uremic patients not receiving rHuEPO. Reduction of the rHuEPO dosage and subcutaneous administration produce less rapid increases in the hematocrit and may lessen the incidence and severity of these side effects. rHuEPO therapy places great demands on both the body's iron stores and the capacity to rapidly transfer iron from storage sites to the erythroid progenitor cells. Thus, almost all patients treated with rHuEPO become iron deficient and require oral or parenteral iron replacement. Response to rHuEPO in uremic patients is diminished if the anemia is complicated by iron deficiency, inflammatory disorders, aluminum overload, or deficiency of folate or vitamin B12. rHuEPO therapy is safe and effective in the treatment of the anemia of chronic renal failure. The use of rHuEPO leads to enhanced quality of life and eliminates the need for red cell transfusions. In addition to hemodialysis patients, predialysis patients and those on CAPD benefit from and are candidates for rHuEPO therapy. PMID:1578966

  2. Identification of Modulators of the Nuclear Receptor Peroxisome Proliferator-Activated Receptor α (PPARα) in a Mouse Liver Gene Expression Compendium

    The nuclear receptor family member peroxisome proliferator-activated receptor α (PPARα) is activated by therapeutic hypolipidemic drugs and environmentally-relevant chemicals to regulate genes involved in lipid transport and catabolism. Chronic activation of PPARα in rodents inc...

  3. Bronchoconstrictor effect of thrombin and thrombin receptor activating peptide in guinea-pigs in vivo

    Cicala, Carla; Bucci, Mariarosaria; De Dominicis, Gianfranco; Harriot, Pat; Sorrentino, Ludovico; Cirino, Giuseppe

    1999-01-01

    Several thrombin cellular effects are dependent upon stimulation of proteinase activated receptor-1 (PAR-1) localized over the cellular surface. Following activation by thrombin, a new N-terminus peptide is unmasked on PAR-1 receptor, which functions as a tethered ligand for the receptor itself. Synthetic peptides called thrombin receptor activating peptides (TRAPs), corresponding to the N-terminus residue unmasked, reproduce several thrombin cellular effects, but are devoid of catalytic acti...

  4. Mechanistic insights into activation and SOCS3-mediated inhibition of myeloproliferative neoplasm-associated JAK2 mutants from biochemical and structural analyses

    Varghese, Leila N; Ungureanu, Daniela; Liau, Nicholas P.D.; Young, Samuel N.; Laktyushin, Artem; Hammaren, Henrik; Lucet, Isabelle S; Nicola, Nicos A.; Silvennoinen, Olli; Babon, Jeffrey J.; Murphy, James M.

    2014-01-01

    JAK2 (Janus kinase 2) initiates the intracellular signalling cascade downstream of cell surface receptor activation by cognate haematopoietic cytokines, including erythropoietin and thrombopoietin. The pseudokinase (JH2) domain of JAK2 negatively regulates the catalytic activity of the adjacent tyrosine kinase (JH1) domain and mutations within the pseudokinase domain underlie human myeloproliferative neoplasms, including polycythaemia vera and essential thrombocytosis. To date, the mechanism ...

  5. D1 dopamine receptor activity of anti-parkinsonian drugs.

    Fici, G J; Wu, H; VonVoigtlander, P F; Sethy, V H

    1997-01-01

    Clinical and preclinical investigations suggest that stimulation of D1 dopamine receptors may be responsible for dyskinesias induced by dopamine agonist treatment of Parkinson's Disease (PD), and that these dyskinesias may be decreased by treatment with a D1 antagonist (clozapine). Therefore, the effects of dopamine agonists and antagonists have been investigated in a primary cerebellar granule cell model of cAMP formation that seems to be highly responsive to the D1 receptors. SKF 38393, lisuride, apomorphine, pergolide, dopamine, bromocriptine and 7-OH-DPAT showed concentration-dependent increases in cAMP formation, with EC50s (in microM) of 0.013, 0.053, 0.25, 1.04, 2.18, 50.9 and 54.4, respectively. SKF 38393, apomorphine, dopamine and pergolide had similar intrinsic activity (100%), while the intrinsic activities of 7-OH-DPAT, bromocriptine and lisuride were 28.0%, 20.7% and 17.2%, respectively. SCH 23390, a selective D1 dopamine receptor antagonist, blocked an increase in cAMP formation produced by EC50 concentrations of all of the dopamine agonists investigated in this study. Clozapine concentration-dependently blocked pergolide-induced increases in cAMP and was approximately 1700-fold less potent than SCH 23390 (IC50: 0.97 microM and 0.56 nM, respectively). U-95666A (1-1000 microM), selective for the D2 receptors, showed no significant effect on cAMP, while pramipexole (0.1-100 microM), a D3 preferring agonist, did not elevate cAMP. These data suggest that primary cerebellar granule cell cultures are an excellent model for measuring D1 dopamine receptor-mediated changes in cellular cAMP. The results are discussed with reference to the relationship between the D1 receptor-stimulated increase in cAMP formation and the induction of dyskinesia in humans by these anti-parkinsonian drugs. PMID:9126882

  6. In vivo metabolism of recombinant human erythropoietin in the rat

    Spivak, J.L.; Hogans, B.B.

    1989-01-01

    We compared the in vivo plasma clearance and organ accumulation in anesthetized rats of 125I-labeled, recombinant human erythropoietin and 125I-labeled, desialylated recombinant erythropoietin. The immediate volume of distribution of 125I-labeled, recombinant erythropoietin approximated that of the plasma volume. Its plasma clearance was multiexponential, with an initial rapid distribution phase (t1/2 = 53 minutes) and a slower elimination phase (t1/2 = 180 minutes). Organ accumulation of labeled recombinant erythropoietin, as compared with 125I-labeled human albumin, was negligible until 30 minutes after injection when small amounts appeared in the kidneys and bone marrow. Only 24% of the 125I-labeled, desialylated recombinant erythropoietin was recovered immediately after injection, and 96% of the hormone was cleared from the plasma with a t1/2 of 2.0 minutes. The bulk of the desialylated hormone accumulated in the liver where it was rapidly catabolized and its breakdown products released back into the plasma. Significantly, in contrast to unmodified erythropoietin, there was also early accumulation of desialylated hormone in the kidneys, marrow, and spleen. Desialylated orosomucoid but not orosomucoid, yeast mannan, or dextran sulfate 500 inhibited the rapid plasma clearance and hepatic accumulation of desialylated erythropoietin. Oxidation of the desialylated hormone restored its plasma recovery and clearance to normal but rendered it biologically inactive, and accumulation in organs other than the kidney was negligible.

  7. Methamphetamine Increases Locomotion and Dopamine Transporter Activity in Dopamine D5 Receptor-Deficient Mice

    Hayashizaki, Seiji; Hirai, Shinobu; Ito, Yumi; Honda, Yoshiko; Arime, Yosefu; Sora, Ichiro; Okado, Haruo; Kodama, Tohru; Takada, Masahiko

    2013-01-01

    Dopamine regulates the psychomotor stimulant activities of amphetamine-like substances in the brain. The effects of dopamine are mediated through five known dopamine receptor subtypes in mammals. The functional relevance of D5 dopamine receptors in the central nervous system is not well understood. To determine the functional relevance of D5 dopamine receptors, we created D5 dopamine receptor-deficient mice and then used these mice to assess the roles of D5 dopamine receptors in the behaviora...

  8. "Effects of recombinant human erythropoietin high mimicking abuse doses on oxidative stress processes in rats".

    Bianchi, Sara; Fusi, Jonathan; Franzoni, Ferdinando; Giovannini, Luca; Galetta, Fabio; Mannari, Claudio; Guidotti, Emanuele; Tocchini, Leonardo; Santoro, Gino

    2016-08-01

    Although many studies highlight how long-term moderate dose of Recombinant Human Erythropoietin (rHuEPO) treatments result in beneficial and antioxidants effects, few studies take into account the effects that short-term high doses of rHuEPO (mimicking abuse conditions) might have on the oxidative stress processes. Thus, the aim of this study was to investigate the in vivo antioxidant activity of rHuEPO, administered for a short time and at high doses to mimic its sports abuse as doping. Male Wistar healthy rats (n=36) were recruited for the study and were treated with three different concentrations of rHuEPO: 7.5, 15, 30μg/kg. Plasma concentrations of erythropoietin, 8-epi Prostaglandin F2α, plasma and urinary concentrations of NOx were evaluated with specific assay kit, while hematocrit levels were analyzed with an automated cell counter. Antioxidant activity of rHuEPO was assessed analyzing the possible variation of the plasma scavenger capacity against hydroxylic and peroxylic radicals by TOSC (Total Oxyradical Scavenging Capacity) assay. Statistical analyses showed higher hematocrit values, confirmed by a statistically significant increase of plasmatic EPO concentration. An increase in plasma scavenging capacity against peroxyl and hydroxyl radicals, in 8-isoprostane plasmatic concentrations and in plasmatic and urinary levels of NOX were also found in all the treated animals, though not always statistically significant. Our results confirm the literature data regarding the antioxidant action of erythropoietin administered at low doses and for short times, whereas they showed an opposite incremental oxidative stress action when erythropoietin is administered at high doses. PMID:27470373

  9. Stimulation of cannabinoid receptor 2 (CB2 suppresses microglial activation

    Fernandez Francisco

    2005-12-01

    Full Text Available Abstract Background Activated microglial cells have been implicated in a number of neurodegenerative disorders, including Alzheimer's disease (AD, multiple sclerosis (MS, and HIV dementia. It is well known that inflammatory mediators such as nitric oxide (NO, cytokines, and chemokines play an important role in microglial cell-associated neuron cell damage. Our previous studies have shown that CD40 signaling is involved in pathological activation of microglial cells. Many data reveal that cannabinoids mediate suppression of inflammation in vitro and in vivo through stimulation of cannabinoid receptor 2 (CB2. Methods In this study, we investigated the effects of a cannabinoid agonist on CD40 expression and function by cultured microglial cells activated by IFN-γ using RT-PCR, Western immunoblotting, flow cytometry, and anti-CB2 small interfering RNA (siRNA analyses. Furthermore, we examined if the stimulation of CB2 could modulate the capacity of microglial cells to phagocytise Aβ1–42 peptide using a phagocytosis assay. Results We found that the selective stimulation of cannabinoid receptor CB2 by JWH-015 suppressed IFN-γ-induced CD40 expression. In addition, this CB2 agonist markedly inhibited IFN-γ-induced phosphorylation of JAK/STAT1. Further, this stimulation was also able to suppress microglial TNF-α and nitric oxide production induced either by IFN-γ or Aβ peptide challenge in the presence of CD40 ligation. Finally, we showed that CB2 activation by JWH-015 markedly attenuated CD40-mediated inhibition of microglial phagocytosis of Aβ1–42 peptide. Taken together, these results provide mechanistic insight into beneficial effects provided by cannabinoid receptor CB2 modulation in neurodegenerative diseases, particularly AD.

  10. Behavioral meaningful opioidergic stimulation activates kappa receptor gene expression

    E. Teodorov

    2012-10-01

    Full Text Available The periaqueductal gray (PAG has been reported to be a location for opioid regulation of pain and a potential site for behavioral selection in females. Opioid-mediated behavioral and physiological responses differ according to the activity of opioid receptor subtypes. The present study investigated the effects of the peripheral injection of the kappa-opioid receptor agonist U69593 into the dorsal subcutaneous region of animals on maternal behavior and on Oprk1 gene activity in the PAG of female rats. Female Wistar rats weighing 200-250 g at the beginning of the study were randomly divided into 2 groups for maternal behavior and gene expression experiments. On day 5, pups were removed at 7:00 am and placed in another home cage that was distant from their mother. Thirty minutes after removing the pups, the dams were treated with U69593 (0.15 mg/kg, sc or 0.9% saline (up to 1 mL/kg and after 30 min were evaluated in the maternal behavior test. Latencies in seconds for pup retrieval, grouping, crouching, and full maternal behavior were scored. The results showed that U69593 administration inhibited maternal behavior (P < 0.05 because a lower percentage of kappa group dams showed retrieval of first pup, retrieving all pups, grouping, crouching and displaying full maternal behavior compared to the saline group. Opioid gene expression was evaluated using real-time reverse-transcription polymerase chain reaction (RT-PCR. A single injection of U69593 increased Oprk1 PAG expression in both virgin (P < 0.05 and lactating female rats (P < 0.01, with no significant effect on Oprm1 or Oprd1 gene activity. Thus, the expression of kappa-opioid receptors in the PAG may be modulated by single opioid receptor stimulation and behavioral meaningful opioidergic transmission in the adult female might occur simultaneously to specific changes in gene expression of kappa-opioid receptor subtype. This is yet another alert for the complex role of the opioid system in

  11. Behavioral meaningful opioidergic stimulation activates kappa receptor gene expression

    The periaqueductal gray (PAG) has been reported to be a location for opioid regulation of pain and a potential site for behavioral selection in females. Opioid-mediated behavioral and physiological responses differ according to the activity of opioid receptor subtypes. The present study investigated the effects of the peripheral injection of the kappa-opioid receptor agonist U69593 into the dorsal subcutaneous region of animals on maternal behavior and on Oprk1 gene activity in the PAG of female rats. Female Wistar rats weighing 200-250 g at the beginning of the study were randomly divided into 2 groups for maternal behavior and gene expression experiments. On day 5, pups were removed at 7:00 am and placed in another home cage that was distant from their mother. Thirty minutes after removing the pups, the dams were treated with U69593 (0.15 mg/kg, sc) or 0.9% saline (up to 1 mL/kg) and after 30 min were evaluated in the maternal behavior test. Latencies in seconds for pup retrieval, grouping, crouching, and full maternal behavior were scored. The results showed that U69593 administration inhibited maternal behavior (P < 0.05) because a lower percentage of U69593 group dams showed retrieval of first pup, retrieving all pups, grouping, crouching and displaying full maternal behavior compared to the saline group. Opioid gene expression was evaluated using real-time reverse-transcription polymerase chain reaction (RT-PCR). A single injection of U69593 increased Oprk1 PAG expression in both virgin (P < 0.05) and lactating female rats (P < 0.01), with no significant effect on Oprm1 or Oprd1 gene activity. Thus, the expression of kappa-opioid receptors in the PAG may be modulated by single opioid receptor stimulation and behavioral meaningful opioidergic transmission in the adult female might occur simultaneously to specific changes in gene expression of kappa-opioid receptor subtype. This is yet another alert for the complex role of the opioid system in female

  12. Behavioral meaningful opioidergic stimulation activates kappa receptor gene expression

    Teodorov, E. [Centro de Matemática, Computação e Cognição, Universidade Federal do ABC, São Paulo, SP (Brazil); Ferrari, M.F.R. [Departamento de Genética e Biologia Evolutiva, Instituto de Biociências, Universidade de São Paulo, São Paulo, SP (Brazil); Fior-Chadi, D.R. [Departamento de Fisiologia, Instituto de Biociências, Universidade de São Paulo, São Paulo, SP (Brazil); Camarini, R. [Departamento de Farmacologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo, SP (Brazil); Felício, L.F. [Departamento de Patologia, Faculdade de Medicina Veterinária e Zootecnia, Universidade de São Paulo, São Paulo, SP (Brazil)

    2012-06-01

    The periaqueductal gray (PAG) has been reported to be a location for opioid regulation of pain and a potential site for behavioral selection in females. Opioid-mediated behavioral and physiological responses differ according to the activity of opioid receptor subtypes. The present study investigated the effects of the peripheral injection of the kappa-opioid receptor agonist U69593 into the dorsal subcutaneous region of animals on maternal behavior and on Oprk1 gene activity in the PAG of female rats. Female Wistar rats weighing 200-250 g at the beginning of the study were randomly divided into 2 groups for maternal behavior and gene expression experiments. On day 5, pups were removed at 7:00 am and placed in another home cage that was distant from their mother. Thirty minutes after removing the pups, the dams were treated with U69593 (0.15 mg/kg, sc) or 0.9% saline (up to 1 mL/kg) and after 30 min were evaluated in the maternal behavior test. Latencies in seconds for pup retrieval, grouping, crouching, and full maternal behavior were scored. The results showed that U69593 administration inhibited maternal behavior (P < 0.05) because a lower percentage of U69593 group dams showed retrieval of first pup, retrieving all pups, grouping, crouching and displaying full maternal behavior compared to the saline group. Opioid gene expression was evaluated using real-time reverse-transcription polymerase chain reaction (RT-PCR). A single injection of U69593 increased Oprk1 PAG expression in both virgin (P < 0.05) and lactating female rats (P < 0.01), with no significant effect on Oprm1 or Oprd1 gene activity. Thus, the expression of kappa-opioid receptors in the PAG may be modulated by single opioid receptor stimulation and behavioral meaningful opioidergic transmission in the adult female might occur simultaneously to specific changes in gene expression of kappa-opioid receptor subtype. This is yet another alert for the complex role of the opioid system in female

  13. Epoetin beta pegol, but not recombinant erythropoietin, retains its hematopoietic effect in vivo in the presence of the sialic acid-metabolizing enzyme sialidase.

    Aizawa, Ken; Kawasaki, Ryohei; Tashiro, Yoshihito; Hirata, Michinori; Endo, Koichi; Shimonaka, Yasushi

    2016-08-01

    Erythropoiesis-stimulating agents (ESAs) are widely used for treating chronic kidney disease (CKD)-associated anemia. The biological activity of ESAs is mainly regulated by the number of sialic acid-containing carbohydrates on the erythropoietin (EPO) peptide. Sialidase, a sialic acid-metabolizing enzyme that accumulates in CKD patients, is suspected of contributing to shortening the circulation half-life of ESAs. Epoetin beta pegol (continuous erythropoietin receptor activator; C.E.R.A.), is an EPO integrated with methoxypolyethylene glycol (PEG). It has been suggested that C.E.R.A. may exert a favorable therapeutic effect, even under conditions of elevated sialidase; however, no detailed investigation of the pharmacological profile of C.E.R.A. in the presence of sialidase has been reported. In the present study, we injected C.E.R.A. or EPO pre-incubated with sialidase into rats, and assessed the hematopoietic effect by reticulocyte count. The hematopoietic effect of C.E.R.A., but not EPO, was preserved after sialidase treatment, despite the removal of sialic acid. Proliferation of EPO-dependent leukemia cells (AS-E2) was significantly increased by desialylated C.E.R.A. and EPO compared to non-treated C.E.R.A. or EPO. In conclusion, we show that C.E.R.A. exerts a favorable hematopoietic effect even under conditions of elevated sialidase. Our findings may contribute to a better understanding of CKD and more effective therapeutic approaches based on a patient's profile of anemia. PMID:27084258

  14. Erythropoietin in cancer: the new face of an old friend.

    Megalakaki, C

    2008-01-01

    During the last decade, anemia, a very common situation in patients with malignant diseases, either associated with chemotherapy or not, is being treated with recombinant erythropoietin (rEPO). Recent experimental findings have elucidated the role of EPO as a strongly anti-apoptotic agent in multiple non-erythroid and neoplastic tissues. The discovery of probably functional EPO receptors (EPOR) on malignant cells, hinting that EPO may act as a tumor growth factor, raised embarrassing thoughts regarding the routine administration of erythropoiesis-stimulating agents (ESAs). In addition, the results of a few clinical trials showing a negative impact on overall survival of rEPO-treated cancer patients, although strongly criticized for several methodological pitfalls, led the FDA to force a "black label" warning concerning the use of rEPO and to recommend that physicians should use the lowest possible dose of ESAs in chemotherapy-treated cancer patients. This recommendation comes in accord with the recent guidelines of European Organisation for Research and Treatment of Cancer (EORTC) which are reviewed in this paper, along with the structure of EPO and EPOR, the role of EPO on normal and malignant cells and the clinical applications of EPO. PMID:18404780

  15. The non-haematopoietic biological effects of erythropoietin.

    Arcasoy, Murat O

    2008-04-01

    In the haematopoietic system, the principal function of erythropoietin (Epo) is the regulation of red blood cell production, mediated by its specific cell surface receptor (EpoR). Following the cloning of the Epo gene (EPO) and characterization of the selective haematopoietic action of Epo in erythroid lineage cells, recombinant Epo forms (epoetin-alfa, epoetin-beta and the long-acting analogue darbepoetin-alfa) have been widely used for treatment of anaemia in chronic kidney disease and chemotherapy-induced anaemia in cancer patients. Ubiquitous EpoR expression in non-erythroid cells has been associated with the discovery of diverse biological functions for Epo in non-haematopoietic tissues. During development, Epo-EpoR signalling is required not only for fetal liver erythropoiesis, but also for embryonic angiogenesis and brain development. A series of recent studies suggest that endogenous Epo-EpoR signalling contributes to wound healing responses, physiological and pathological angiogenesis, and the body's innate response to injury in the brain and heart. Epo and its novel derivatives have emerged as major tissue-protective cytokines that are being investigated in the first human studies involving neurological and cardiovascular diseases. This review focuses on the scientific evidence documenting the biological effects of Epo in non-haematopoietic tissues and discusses potential future applications of Epo and its derivatives in the clinic. PMID:18324962

  16. High doses of recombinant erythropoietin stimulate platelet production in mice

    McDonald, T.P.; Cottrell, M.B.; Clift, R.E.; Cullen, W.C.; Lin, F.K.

    1987-07-01

    Previously, recombinant erythropoietin (rEpo) was shown to increase the number and size of megakaryocytic colonies in vitro, and in vivo it elevates the number of megakaryocytes in mouse spleens. To test the hypothesis that rEpo would stimulate platelet production in mice, both normal mice and mice in rebound-thrombocytosis were injected with rEpo and the %35S incorporation into platelets was measured. A thrombocytopoiesis-stimulating factor (TSF or thrombopoietin) was used as a positive control. rEpo increased isotopic incorporation into platelets of both normal mice and mice in rebound-thrombocytosis, as did TSF, but required large doses (15 U rEpo/mouse). In other mice, hematocrits, platelet counts, platelet sizes, and 24-hr %35S incorporation into platelets were measured 2 days after injection of two equally divided doses of either rEpo or TSF. Significant increases in both platelet sizes and %35S incorporation into platelets were found after injections of 15 U rEpo/mouse or 2.3 U TSF/mouse. These data indicate that rEpo, at high doses, will stimulate platelet production in mice, and may suggest molecular similarities between rEpo and TSF and their ability to compete for common receptor sites on megakaryocytes and their progenitor cells.

  17. Characterization of human endothelial cell urokinase-type plasminogen activator receptor protein and messenger RNA

    Barnathan, E S; Kuo, A; Karikó, K;

    1990-01-01

    Human umbilical vein endothelial cells in culture (HUVEC) express receptors for urokinase-type plasminogen activators (u-PA). The immunochemical nature of this receptor and its relationship to u-PA receptors expressed by other cell types is unknown. Cross-linking active site-blocked u-PA to HUVEC...

  18. Dopamine receptor activation increases HIV entry into primary human macrophages.

    Peter J Gaskill

    Full Text Available Macrophages are the primary cell type infected with HIV in the central nervous system, and infection of these cells is a major component in the development of neuropathogenesis and HIV-associated neurocognitive disorders. Within the brains of drug abusers, macrophages are exposed to increased levels of dopamine, a neurotransmitter that mediates the addictive and reinforcing effects of drugs of abuse such as cocaine and methamphetamine. In this study we examined the effects of dopamine on HIV entry into primary human macrophages. Exposure to dopamine during infection increased the entry of R5 tropic HIV into macrophages, irrespective of the concentration of the viral inoculum. The entry pathway affected was CCR5 dependent, as antagonizing CCR5 with the small molecule inhibitor TAK779 completely blocked entry. The effect was dose-dependent and had a steep threshold, only occurring above 108 M dopamine. The dopamine-mediated increase in entry required dopamine receptor activation, as it was abrogated by the pan-dopamine receptor antagonist flupenthixol, and could be mediated through both subtypes of dopamine receptors. These findings indicate that the effects of dopamine on macrophages may have a significant impact on HIV pathogenesis. They also suggest that drug-induced increases in CNS dopamine may be a common mechanism by which drugs of abuse with distinct modes of action exacerbate neuroinflammation and contribute to HIV-associated neurocognitive disorders in infected drug abusers.

  19. NMDA receptor subunit expression and PAR2 receptor activation in colospinal afferent neurons (CANs during inflammation induced visceral hypersensitivity

    Caudle Robert M

    2009-09-01

    Full Text Available Abstract Background Visceral hypersensitivity is a clinical observation made when diagnosing patients with functional bowel disorders. The cause of visceral hypersensitivity is unknown but is thought to be attributed to inflammation. Previously we demonstrated that a unique set of enteric neurons, colospinal afferent neurons (CANs, co-localize with the NR1 and NR2D subunits of the NMDA receptor as well as with the PAR2 receptor. The aim of this study was to determine if NMDA and PAR2 receptors expressed on CANs contribute to visceral hypersensitivity following inflammation. Recently, work has suggested that dorsal root ganglion (DRG neurons expressing the transient receptor potential vanilloid-1 (TRPV1 receptor mediate inflammation induced visceral hypersensitivity. Therefore, in order to study CAN involvement in visceral hypersensitivity, DRG neurons expressing the TRPV1 receptor were lesioned with resiniferatoxin (RTX prior to inflammation and behavioural testing. Results CANs do not express the TRPV1 receptor; therefore, they survive following RTX injection. RTX treatment resulted in a significant decrease in TRPV1 expressing neurons in the colon and immunohistochemical analysis revealed no change in peptide or receptor expression in CANs following RTX lesioning as compared to control data. Behavioral studies determined that both inflamed non-RTX and RTX animals showed a decrease in balloon pressure threshold as compared to controls. Immunohistochemical analysis demonstrated that the NR1 cassettes, N1 and C1, of the NMDA receptor on CANs were up-regulated following inflammation. Furthermore, inflammation resulted in the activation of the PAR2 receptors expressed on CANs. Conclusion Our data show that inflammation causes an up-regulation of the NMDA receptor and the activation of the PAR2 receptor expressed on CANs. These changes are associated with a decrease in balloon pressure in response to colorectal distension in non-RTX and RTX lesioned

  20. Evidence that adiponectin receptor 1 activation exacerbates ischemic neuronal death

    Thundyil John

    2010-08-01

    Full Text Available Abstract Background- Adiponectin is a hormone produced in and released from adipose cells, which has been shown to have anti-diabetic and anti-inflammatory actions in peripheral cells. Two cell surface adiponectin receptors (ADRs mediate the majority of the known biological actions of adiponectin. Thus far, ADR expression in the brain has been demonstrated in the arcuate and the paraventricular nucleus of hypothalamus, where its activation affects food intake. Recent findings suggest that levels of circulating adiponectin increase after an ischemic stroke, but the role of adiponectin receptor activation in stroke pathogenesis and its functional outcome is unclear. Methods- Ischemic stroke was induced in C57BL/6 mice by middle cerebral artery occlusion (MCAO for 1 h, followed by reperfusion. Primary cortical neuronal cultures were established from individual embryonic neocortex. For glucose deprivation (GD, cultured neurons were incubated in glucose-free Locke's medium for 6, 12 or 24 h. For combined oxygen and glucose deprivation (OGD, neurons were incubated in glucose-free Locke's medium in an oxygen-free chamber with 95% N2/5% CO2 atmosphere for either 3, 6, 9, 12 or 24 h. Primary neurons and brain tissues were analysed for Adiponectin and ADRs using reverse transcriptase polymerase chain reaction (RT-PCR, immunoblot and immunochemistry methods. Results- Cortical neurons express ADR1 and ADR2, and that the levels of ADR1 are increased in neurons in response to in vitro or in vivo ischemic conditions. Neurons treated with either globular or trimeric adiponectin exhibited increased vulnerability to oxygen and glucose deprivation which was associated with increased activation of a pro-apoptotic signaling cascade involving p38 mitogen-activated protein kinase (p38MAPK and AMP-activated protein kinase (AMPK. Conclusions- This study reveals a novel pathogenic role for adiponectin and adiponectin receptor activation in ischemic stroke. We show that

  1. Protein kinase C phosphorylation sensitizes but does not activate the capsaicin receptor transient receptor potential vanilloid 1 (TRPV1)

    Bhave, Gautam; HU, HUI-JUAN; Glauner, Kathi S.; Zhu, Weiguo; Wang, Haibin; Brasier, D. J.; Oxford, Gerry S; Gereau, Robert W.

    2003-01-01

    Protein kinase C (PKC) modulates the function of the capsaicin receptor transient receptor potential vanilloid 1 (TRPV1). This modulation manifests as increased current when the channel is activated by capsaicin. In addition, studies have suggested that phosphorylation by PKC might directly gate the channel, because PKC-activating phorbol esters induce TRPV1 currents in the absence of applied ligands. To test whether PKC both modulates and gates the TRPV1 function by direct phosphorylation, w...

  2. Influence of metabotropic glutamate receptor agonists on the inhibitory effects of adenosine A1 receptor activation in the rat hippocampus

    de Mendonça, Alexandre; Ribeiro, J. A.

    1997-01-01

    Glutamate and other amino acids are the main excitatory neurotransmitters in many brain regions, including the hippocampus, by activating ion channel-coupled glutamate receptors, as well as metabotropic receptors linked to G proteins and second messenger systems. Several conditions which promote the release of glutamate, like frequency stimulation and hypoxia, also lead to an increase in the extracellular levels of the important neuromodulator, adenosine. We studied whether the activation of ...

  3. The glucagon-like peptide 1 receptor agonist enhances intrinsic peroxisome proliferator-activated receptor γ activity in endothelial cells

    Onuma, Hirohisa; Inukai, Kouichi, E-mail: kinukai@ks.kyorin-u.ac.jp; Kitahara, Atsuko; Moriya, Rie; Nishida, Susumu; Tanaka, Toshiaki; Katsuta, Hidenori; Takahashi, Kazuto; Sumitani, Yoshikazu; Hosaka, Toshio; Ishida, Hitoshi

    2014-08-22

    Highlights: • PPARγ activation was involved in the GLP-1-mediated anti-inflammatory action. • Exendin-4 enhanced endogenous PPARγ transcriptional activity in HUVECs. • H89, a PKA inhibitor, abolished GLP-1-induced PPARγ enhancement. • The anti-inflammatory effects of GLP-1 may be explained by PPARγ activation. - Abstract: Recent studies have suggested glucagon-like peptide-1 (GLP-1) signaling to exert anti-inflammatory effects on endothelial cells, although the precise underlying mechanism remains to be elucidated. In the present study, we investigated whether PPARγ activation is involved in the GLP-1-mediated anti-inflammatory action on endothelial cells. When we treated HUVEC cells with 0.2 ng/ml exendin-4, a GLP-1 receptor agonist, endogenous PPARγ transcriptional activity was significantly elevated, by approximately 20%, as compared with control cells. The maximum PPARγ activity enhancing effect of exendin-4 was observed 12 h after the initiation of incubation with exendin-4. As H89, a PKA inhibitor, abolished GLP-1-induced PPARγ enhancement, the signaling downstream from GLP-1 cross-talk must have been involved in PPARγ activation. In conclusion, our results suggest that GLP-1 has the potential to induce PPARγ activity, partially explaining the anti-inflammatory effects of GLP-1 on endothelial cells. Cross-talk between GLP-1 signaling and PPARγ activation would have major impacts on treatments for patients at high risk for cardiovascular disease.

  4. Erythropoietin treatment enhances muscle mitochondrial capacity in humans

    Plenge, Ulla; Belhage, Bo; Guadalupe-Grau, Amelia;

    2012-01-01

    Erythropoietin (Epo) treatment has been shown to induce mitochondrial biogenesis in cardiac muscle along with enhanced mitochondrial capacity in mice. We hypothesized that recombinant human Epo (rhEpo) treatment enhances skeletal muscle mitochondrial oxidative phosphorylation (OXPHOS) capacity in...

  5. Depressed serum erythropoietin in pregnant women with elevated blood lead.

    Graziano, J H; Slavkovic, V; Factor-Litvak, P; Popovac, D; Ahmedi, X; Mehmeti, A

    1991-01-01

    During the course of a prospective study of lead exposure and pregnancy outcome in 1,502 women, we tested the hypothesis that environmental lead exposure is associated with depressed serum erythropoietin concentration. At mid-pregnancy and at delivery, blood samples were stratified by hemoglobin concentration; within each hemoglobin stratum, sera of women with the lowest and highest whole blood lead concentrations were selected for serum erythropoietin analysis. Analysis of variance revealed that women with higher blood lead levels had inappropriately low serum erythropoietin at both mid-pregnancy and at delivery. Thus, depressed serum erythropoietin appears to indicate lead nephrotoxicity, and it may also be responsible for the anemia associated with lead poisoning. PMID:1772259

  6. Effect of Erythropoietin in Infants with the Anemia of Prematurity

    A. Sh. Farhat; A. Mohammadzadeh; F. Naseri

    2004-01-01

    Recombinant human erythropoietin (Epo) is known to accelerate erythropoesis in preterm infants. This study was designed to assess the effect of Epo in treatment of anemia of prematurity .Preterm infants with Hct

  7. Synthesis and biological evaluation of {sup 125}I-erythropoietin as a potential radiopharmaceutical agent for tumours

    Clemente, Goncalo dos Santos, E-mail: goncaloclemente@uc.pt [University of Coimbra (Portugal). Inst. of Nuclear Sciences Applied to Health; Duarte, Vera Lucia Serra [Polytechnical Institute of Lisbon (Portugal). Higher School of Health Technology

    2011-01-15

    Erythropoietin (EPO) is a glycoprotein hormone responsible for regulating erythropoiesis. Expression of EPO and EPO receptors (EPOr) has recently been demonstrated in some neoplastic cell lines and tumours, suggesting a potential new target for therapy. In this work, EPO was labeled with iodine-125 using the lactoperoxidase method, known to prevent damage to protein during radioiodination, and labeling conditions were optimized. In vitro stability studies have shown that {sup 125}I-EPO is radiochemically stable for 20 days after radiolabeling. In vitro cell binding studies have demonstrated very low binding (<2%) of EPO to normal and neoplastic cell lines tested. As expected, the biodistribution in healthy mice exhibited comparatively high rates of fixation in the organs of the excretory system. Thyroid also proved to be a critical organ which may indicate in vivo dissociation of {sup 125}I-EPO. In mice with induced melanoma, only a residual fixation in the tumour was evident. Further studies are warranted on other tumoral cell lines to better understand the binding process and internalization into cells. Studies on EPO labeled with carbon-11 could be valuable, since there is a greater chance of preserving the biological activity of the protein using this method. (author)

  8. Muscarinic receptor activation elicits sustained, recurring depolarizations in reticulospinal neurons.

    Smetana, R W; Alford, S; Dubuc, R

    2007-05-01

    In lampreys, brain stem reticulospinal (RS) neurons constitute the main descending input to the spinal cord and activate the spinal locomotor central pattern generators. Cholinergic nicotinic inputs activate RS neurons, and consequently, induce locomotion. Cholinergic muscarinic agonists also induce locomotion when applied to the brain stem of birds. This study examined whether bath applications of muscarinic agonists could activate RS neurons and initiate motor output in lampreys. Bath applications of 25 microM muscarine elicited sustained, recurring depolarizations (mean duration of 5.0 +/- 0.5 s recurring with a mean period of 55.5 +/- 10.3 s) in intracellularly recorded rhombencephalic RS neurons. Calcium imaging experiments revealed that muscarine induced oscillations in calcium levels that occurred synchronously within the RS neuron population. Bath application of TTX abolished the muscarine effect, suggesting the sustained depolarizations in RS neurons are driven by other neurons. A series of lesion experiments suggested the caudal half of the rhombencephalon was necessary. Microinjections of muscarine (75 microM) or the muscarinic receptor (mAchR) antagonist atropine (1 mM) lateral to the rostral pole of the posterior rhombencephalic reticular nucleus induced or prevented, respectively, the muscarinic RS neuron response. Cells immunoreactive for muscarinic receptors were found in this region and could mediate this response. Bath application of glutamatergic antagonists (6-cyano-7-nitroquinoxaline-2,3-dione/D-2-amino-5-phosphonovaleric acid) abolished the muscarine effect, suggesting that glutamatergic transmission is needed for the effect. Ventral root recordings showed spinal motor output coincides with RS neuron sustained depolarizations. We propose that unilateral mAchR activation on specific cells in the caudal rhombencephalon activates a circuit that generates synchronous sustained, recurring depolarizations in bilateral populations of RS neurons. PMID

  9. Involvement of Activating NK Cell Receptors and Their Modulation in Pathogen Immunity

    Francesco Marras

    2011-01-01

    Full Text Available Natural Killer (NK cells are endowed with cell-structure-sensing receptors providing inhibitory protection from self-destruction (inhibitory NK receptors, iNKRs, including killer inhibitory receptors and other molecules and rapid triggering potential leading to functional cell activation by Toll-like receptors (TLRs, cytokine receptors, and activating NK cell receptors including natural cytotoxicity receptors (NCRs, i.e., NKp46, NKp46, and NKp44. NCR and NKG2D recognize ligands on infected cells which may be endogenous or may directly bind to some structures derived from invading pathogens. In this paper, we address the known direct or indirect interactions between activating receptors and pathogens and their expression during chronic HIV and HCV infections.

  10. Cannabinoid activation of peroxisome proliferator-activated receptors: potential for modulation of inflammatory disease.

    O'Sullivan, S E; Kendall, D A

    2010-08-01

    Cannabinoids act via cell surface G protein-coupled receptors (CB(1) and CB(2)) and the ion channel receptor TRPV1. Evidence has now emerged suggesting that an additional target is the peroxisome proliferator-activated receptor (PPAR) family of nuclear receptors. There are three PPAR subtypes alpha, delta (also known as beta) and gamma, which regulate cell differentiation, metabolism and immune function. The major endocannabinoids, anandamide and 2-arachidonoylglycerol, and ajulemic acid, a structural analogue of the phytocannabinoid Delta(9)-tetrahydrocannabinol (THC), have anti-inflammatory properties mediated by PPARgamma. Other cannabinoids which activate PPARgamma include N-arachidonoyl-dopamine, THC, cannabidiol, HU210, WIN55212-2 and CP55940. The endogenous acylethanolamines, oleoylethanolamide and palmitoylethanolamide regulate feeding and body weight, stimulate fat utilization and have neuroprotective effects mediated through PPARalpha. Other endocannabinoids that activate PPARalpha include anandamide, virodhamine and noladin ether. There is, as yet, little direct evidence for interactions of cannabinoids with PPARdelta. There is a convergence of effects of cannabinoids, acting via cell surface and nuclear receptors, on immune cell function which provides promise for the targeted therapy of a variety of immune, particularly neuroinflammatory, diseases. PMID:19833407

  11. In vitro neuronal network activity in NMDA receptor encephalitis

    Jantzen Sabine U

    2013-02-01

    Full Text Available Abstract Background Anti-NMDA-encephalitis is caused by antibodies against the N-methyl-D-aspartate receptor (NMDAR and characterized by a severe encephalopathy with psychosis, epileptic seizures and autonomic disturbances. It predominantly occurs in young women and is associated in 59% with an ovarian teratoma. Results We describe effects of cerebrospinal fluid (CSF from an anti-N-methyl-D-aspartate receptor (NMDAR encephalitis patient on in vitro neuronal network activity (ivNNA. In vitro NNA of dissociated primary rat cortical populations was recorded by the microelectrode array (MEA system. The 23-year old patient was severely affected but showed an excellent recovery following multimodal immunomodulatory therapy and removal of an ovarian teratoma. Patient CSF (pCSF taken during the initial weeks after disease onset suppressed global spike- and burst rates of ivNNA in contrast to pCSF sampled after clinical recovery and decrease of NMDAR antibody titers. The synchrony of pCSF-affected ivNNA remained unaltered during the course of the disease. Conclusion Patient CSF directly suppresses global activity of neuronal networks recorded by the MEA system. In contrast, pCSF did not regulate the synchrony of ivNNA suggesting that NMDAR antibodies selectively regulate distinct parameters of ivNNA while sparing their functional connectivity. Thus, assessing ivNNA could represent a new technique to evaluate functional consequences of autoimmune encephalitis-related CSF changes.

  12. Erythropoietin in Myocardial Infarction: Experimental Evidence and Clinical Studies

    Andreas Stein; Ilka Ott

    2011-01-01

    Erythropoietin produced mainly in the kidney is the main regulator of erythropoiesis. Experimental studies identified additional, non-haematopoietic, protective effects during myocardial ischemia and reperfusion due to inhibition of apoptosis, stimulation of vasculogenesis and progenitor cell mobilisation. Based on these findings, five prospective, randomised, clinical trials have been performed. A short term regimen of erythropoietin was applied during PCI in patients with STEMI up to a cumu...

  13. Erythropoietin and hypothermia for hypoxic-ischemic encephalopathy

    Rogers, EE; Bonifacio, SL; Glass, HC; Juul, SE; Chang, T.; Mayock, DE; Durand, DJ; Song, D.; Barkovich, AJ; Ballard, RA; Wu, YW

    2014-01-01

    © 2014 Elsevier Inc. All rights reserved. Background Erythropoietin is neuroprotective in animal models of neonatal hypoxic-ischemic encephalopathy. We previously reported a phase I safety and pharmacokinetic study of erythropoietin in neonates. This article presents the neurodevelopmental follow-up of infants who were enrolled in the phase I clinical trial. Methods We enrolled 24 newborns with hypoxic-ischemic encephalopathy in a dose-escalation study. Patients received up to six doses of er...

  14. Regulation of platelet activating factor receptor coupled phosphoinositide-specific phospholipase C activity

    Morrison, W.J.

    1988-01-01

    The major objectives of this study were two-fold. The first was to establish whether binding of platelet activating factor (PAF) to its receptor was integral to the stimulation of polyphosphoinositide-specific phospholipase C (PLC) in rabbit platelets. The second was to determine regulatory features of this receptor-coupled mechanism. ({sup 3}H)PAF binding demonstrated two binding sites, a high affinity site with a inhibitory constant (Ki) of 2.65 nM and a low affinity site with a Ki of 0.80 {mu}M. PAF receptor coupled activation of phosphoinositide-specific PLC was studied in platelets which were made refractory, by short term pretreatments, to either PAF or thrombin. Saponin-permeabilized rabbit platelets continue to regulate the mechanism(s) coupling PAF receptors to PLC stimulation. However, TRP{gamma}S and GDP{beta}S, which affect guanine nucleotide regulatory protein functions, were unable to modulate the PLC activity to any appreciable extent as compared to PAF. The possible involvement of protein kinase C (PKC) activation in regulating PAF-stimulated PLC activity was studied in rabbit platelets pretreated with staurosporine followed by pretreatments with PAF or phorbol 12-myristate 13-acetate (PMA).

  15. Plasminogen activation by receptor-bound urokinase. A kinetic study with both cell-associated and isolated receptor

    Ellis, V; Behrendt, N; Danø, K

    1991-01-01

    The specific cellular receptor for urokinase-type plasminogen activator (uPA) is found on a variety of cell types and has been postulated to play a central role in the mediation of pericellular proteolytic activity. We have studied the kinetics of plasminogen (Plg) activation catalyzed by uPA spe...

  16. Recombinant human erythropoietin for repair of white matter damage

    Wei Zhou; Xiao Rong; Li Tao; Weineng Lu

    2011-01-01

    Erythropoietin has been shown to exhibit neuroprotective effects in animal models. A neonatal rat model of hypoxic-ischemic white matter damage was established via bilateral carotid artery ligation in 4-day-old Sprague-Dawley rats. The rats were subsequently treated with recombinant human erythropoietin to observe pathological changes in the brain and long-term neurobehavioral functions before and after intervention. Results showed that the number of myelin basic protein-positive cells, which reflected myelin/oligodendrocyte damage, significantly increased, although the number of amyloid precursor protein-positive cells, which reflected axonal injury, significantly decreased in periventricular white matter at 72 hours and 7 days following erythropoietin intervention. The number of glial fibrillary acidic protein-positive cells, indicating astrocytic damage, significantly decreased in periventricular white matter of erythropoietin-treated rats at 48 hours, 72 hours, 7 days, and 26 days. Following erythropoietin intervention in the 30-day-old rats, head-turning time in the slope test was shortened and open-field test scores increased. These results suggested that erythropoietin promoted repair of white matter damage, as well as improved neurobehavioral functions in a rat model of hypoxic-ischemic injury.

  17. Identification of key residues involved in the activation and signaling properties of dopamine D3 receptor.

    Kota, Kokila; Kuzhikandathil, Eldo V; Afrasiabi, Milad; Lacy, Brett; Kontoyianni, Maria; Crider, A Michael; Song, Daniel

    2015-09-01

    The dopamine D3 receptor exhibits agonist-dependent tolerance and slow response termination (SRT) signaling properties that distinguish it from the closely-related D2 receptors. While amino acid residues important for D3 receptor ligand binding have been identified, the residues involved in activation of D3 receptor signaling and induction of signaling properties have not been determined. In this paper, we used cis and trans isomers of a novel D3 receptor agonist, 8-OH-PBZI, and site-directed mutagenesis to identify key residues involved in D3 receptor signaling function. Our results show that trans-8-OH-PBZI, but not cis-8-OH-PBZI, elicit the D3 receptor tolerance and SRT properties. We show that while both agonists require a subset of residues in the orthosteric binding site of D3 receptors for activation of the receptor, the ability of the two isomers to differentially induce tolerance and SRT is mediated by interactions with specific residues in the sixth transmembrane helix and third extracellular loop of the D3 receptor. We also show that unlike cis-8-OH-PBZI, which is a partial agonist at the dopamine D2S receptor and full agonist at dopamine D2L receptor, trans-8-OH-PBZI is a full agonist at both D2S and D2L receptors. The different effect of the two isomers on D3 receptor signaling properties and D2S receptor activation correlated with differential effects of the isomers on agonist-induced mouse locomotor activity. The two isomers of 8-OH-PBZI represent novel pharmacological tools for in silico D3 and D2 receptor homology modeling and for determining the role of D3 receptor tolerance and SRT properties in signaling and behavior. PMID:26116441

  18. Basic conditions for radioimmunoassay of erythropoietin, and plasma levels of erythropoietin in normal subjects and anemic patients

    Mizoguchi, Hideaki; Ohta, Kazuo; Suzuki, Toshiaki; Murakami, Akihiko; Ueda, Masatsugu; Sasaki, Ryuzou; Chiba, Hideo

    1987-02-01

    We have developed a specific and sensitive radioimmunoassay for erythropoietin. The sensitivity of our assay is 0.5 mU or 5 mU/ml and is sufficient to detect normal plasma erythropoietin levels. The mean plasma erythropoietin titer of normal Japanese with our radioimmunoassay was found to be 21.9 +- 12.0 mU/ml (n = 199). The validity of the method was further confirmed by the observations that the plasma erythropoietin titers were inversely related to hemoglobin levels in patients with nonuremic anemias, lower in uremic patients than in patients with nonuremic anemias with similar hemoglobin levels, markedly elevated in patients with aplastic anemia and pure red cell aplasia, and in a low normal range in patients with polycythemia vera.

  19. Convergence on a Distinctive Assembly Mechanism by Unrelated Families of Activating Immune Receptors

    Feng, Jianwen; Garrity, David; Call, Matthew E.; Moffett, Howell; Wucherpfennig, Kai W.

    2005-01-01

    Activating receptors in cells of hematopoetic origin include members of two unrelated protein families, the immunoglobulin (Ig) and C type lectins, which differ even in the orientation of the transmembrane (TM) domains. We examined assembly of four receptors with diverse function: the NK receptors KIR2DS and NKG2C/CD94, the Fc receptor for IgA, and the GPVI collagen receptor. For each of the four different receptors studied here, assembly results in the formation of a three-helix interface in...

  20. Pharmacological Effects of Erythropoietin and its Derivative Carbamyl erythropoietin in Cerebral White Matter Injury

    Liu, Wei

    Periventricular leukomalacia (PVL) is the predominant form of brain injury in the premature infant and the most common cause of cerebral palsy, yet no therapy currently exists for this serious human disorder. As PVL often occurs in preterm infants suffering from cerebral hypoxia/ischemia with or without prior exposure to maternal-fetal infection/inflammation, we used hypoxia/ischemia with or without lipopolysaccharide (LPS) injection, to produce clinically relevant PVL-like lesions in the white matter in postnatal day six (P6) mice. We studied the white matter pathology under different conditions, such as different durations of hypoxia and different doses of LPS, to evaluate the effects of those etiological factors on neonatal white matter injury. Distinct related pathological events were investigated at different time points during the progression of PVL. We used immunohistochemistry, histological analysis, and electron microscopy (EM) to study demylination that occurs in the white matter area, which is consistent with the pathology of human PVL. Previous studies have shown that erythropoietin (EPO) and its derivative carbamylated EPO (CEPO) are neuroprotective in various experimental models of brain injury. However, none of these studies investigated their efficacy against white matter injury using appropriate animal models of PVL. We produced unilateral or bilateral white matter injury in P6 mice using unilateral carotid ligation (UCL) followed by hypoxia (6% oxygen, 35 min) or by UCL/hypoxia plus LPS injection, respectively. We administered a single intraperitoneal (i.p.) dose of EPO or CEPO (5000 IU/kg) immediately after the insult, and found both drugs to provide significant protection against white matter injury in PVL mice compared to vehicle-treated groups. In addition, EPO and CEPO treatments attenuated neurobehavioral dysfunctions in an acute manner after PVL injury. EPO and CEPO have relatively few adverse effects, and thus may be a therapeutic agent

  1. Monoclonal antibodies to the human insulin receptor that activate glucose transport but not insulin receptor kinase activity.

    Forsayeth, J R; Caro, J F; Sinha, M K; Maddux, B A; Goldfine, I D

    1987-01-01

    Three mouse monoclonal antibodies were produced that reacted with the alpha subunit of the human insulin receptor. All three both immunoprecipitated 125I-labeled insulin receptors from IM-9 lymphocytes and competitively inhibited 125I-labeled insulin binding to its receptor. Unlike insulin, the antibodies failed to stimulate receptor autophosphorylation in both intact IM-9 lymphocytes and purified human placental insulin receptors. Moreover, unlike insulin, the antibodies failed to stimulate ...

  2. Association between recombinant human erythropoietin and quality of life and exercise capacity of patients receiving haemodialysis. Canadian Erythropoietin Study Group.

    1990-01-01

    OBJECTIVE--To determine whether recombinant human erythropoietin improves the quality of life and exercise capacity of anaemic patients receiving haemodialysis. DESIGN--A double blind, randomised, placebo controlled study. SETTING--Eight Canadian university haemodialysis centres. PATIENTS--118 Patients receiving haemodialysis aged 18-75 with haemoglobin concentrations less than 90 g/l, no causes of anaemia other than erythropoietin deficiency, and no other serious diseases. INTERVENTIONS--Pat...

  3. Topical Rosiglitazone Treatment Improves Ulcerative Colitis by Restoring Peroxisome Proliferator-Activated Receptor-gamma Activity

    Pedersen, G.; Brynskov, Jørn

    2010-01-01

    OBJECTIVES: Impaired epithelial expression of peroxisome proliferator-activated receptor-gamma (PPAR gamma) has been described in animal colitis models and briefly in patients with ulcerative colitis, but the functional significance in humans is not well defined. We examined PPAR gamma expression...

  4. Diabetes or peroxisome proliferator-activated receptor alpha agonist increases mitochondrial thioesterase I activity in heart

    Peroxisome proliferator-activated receptor alpha (PPAR alpha) is a transcriptional regulator of the expression of mitochondrial thioesterase I (MTE-I) and uncoupling protein 3 (UCP3), which are induced in the heart at the mRNA level in response to diabetes. Little is known about the regulation of pr...

  5. Activation of Protease-Activated Receptor 2 Induces VEGF Independently of HIF-1

    Rasmussen, Jeppe Grøndahl; Riis, Simone Elkjær; Frøbert, Ole; Yang, Sufang; Kastrup, Jens; Zachar, Vladimir; Simonsen, Ulf; Fink, Trine

    2012-01-01

    Background Human adipose stem cells (hASCs) can promote angiogenesis through secretion of proangiogenic factors such as vascular endothelial growth factor (VEGF). In other cell types, it has been shown that induction of VEGF is mediated by both protease activated receptor 2 (PAR2) and hypoxia inducible factor 1(HIF-1). The present study hypothesized that PAR2 stimulation through activation of kinase signaling cascades lead to induction of HIF-1 and secretion of VEGF. Methodology/Principal Findings Immunohistochemistry revealed the expression of PAR2 receptors on the surface of hASCs. Blocking the PAR2 receptors with a specific antibody prior to trypsin treatment showed these receptors are involved in trypsin-evoked increase in VEGF secretion from hASCs. Blocking with specific kinase inhibitors suggested that that activation of MEK/ERK and PI3-kinase/Akt pathways are involved in trypsin-eveoked induction of VEGF. The effect of the trypsin treatment on the transcription of VEGF peaked at 6 hours after the treatment and was comparable to the activation observed after keeping hASCs for 24 hours at 1% oxygen. In contrast to hypoxia, trypsin alone failed to induce HIF-1 measured with ELISA, while the combination of trypsin and hypoxia had an additive effect on both VEGF transcription and secretion, results which were confirmed by Western blot. Conclusion In hASCs trypsin and hypoxia induce VEGF expression through separate pathways. PMID:23049945

  6. New Target Genes for the Peroxisome Proliferator-Activated Receptor-γ (PPARγ Antitumour Activity: Perspectives from the Insulin Receptor

    Daniela P. Foti

    2009-01-01

    Full Text Available The insulin receptor (IR plays a crucial role in mediating the metabolic and proliferative functions triggered by the peptide hormone insulin. There is considerable evidence that abnormalities in both IR expression and function may account for malignant transformation and tumour progression in some human neoplasias, including breast cancer. PPARγ is a ligand-activated, nuclear hormone receptor implicated in many pleiotropic biological functions related to cell survival and proliferation. In the last decade, PPARγ agonists—besides their known action and clinical use as insulin sensitizers—have proved to display a wide range of antineoplastic effects in cells and tissues expressing PPARγ, leading to intensive preclinical research in oncology. PPARγ and activators affect tumours by different mechanisms, involving cell proliferation and differentiation, apoptosis, antiinflammatory, and antiangiogenic effects. We recently provided evidence that PPARγ and agonists inhibit IR by non canonical, DNA-independent mechanisms affecting IR gene transcription. We conclude that IR may be considered a new PPARγ “target” gene, supporting a potential use of PPARγ agonists as antiproliferative agents in selected neoplastic tissues that overexpress the IR.

  7. Erythropoietin-Like Effects of Dihydroartemisinin in Wistar Albino Rats

    Utoh-Nedosa U. Anastasia

    2011-01-01

    Full Text Available Artemisinin drugs were active during the intra-erythrocytic stage of malaria parasite infection. The activity of artemisinin and synthetic endoperoxides was related to their interaction with heme. The electrophillic intermediate formed from artemisinin in the presence of heme alkylates the protein portion of hemoglobin preferentially to the heme portion. Problem statement: Since there might be an interaction between artemisinin and the heme of the blood, we studied the effects of 5-day and 7-day oral Dihydroartemisinin (DHA treatments with 5 dosage regimens of dihydroartemisinin on the blood and six vital organs of Wistar albino rats. Approach: The dosages of DHA tested on 5 test adult Wistar albino rats (weight = 106-140 grams were 1, 2, 60 or 80 mg Kg-1 rat weight of DHA by oral intubation for 5 or 7 days. Four rats of similar weight which served as controls in each experiment were given distilled water equivalents of the administered doses of DHA. Another group of 5 test rats and four control rats (weight 75-90 gms were given 1 mg kg-1 rat weight of DHA or distilled water for 5 or 7 days and were allowed to rest for one week after which the treatment was repeated. Results: The findings of the study showed that Dihydroartemisinin (DHA had erythropoietin-like properties. In the study DHA produced dose, repetition and time dependent statistically significant increases in the Packed Cell Volume (PCV (PConclusion: This result suggested that the administered DHA inhibited its own stimulated statistically significant increases in the PCV and the WBC of the treated rats through an inhibitory (negative feed-back effect. The structure and composition of the blood cell types like the presence of large numbers of reticlocytes and left-shifted neutrophils in the blood samples of 5-day DHA -treated rats but not in those of 7-day DHA treated rats indicated that new haemopoiesis was actively going on in the first 5 days of DHA treatment but had slowed down

  8. Regeneration in the nervous system with erythropoietin

    Maiese, Kenneth

    2015-01-01

    Globally, greater than 30 million individuals are afflicted with disorders of the nervous system accompanied by tens of thousands of new cases annually with limited, if any, treatment options. Erythropoietin (EPO) offers an exciting and novel therapeutic strategy to address both acute and chronic neurodegenerative disorders. EPO governs a number of critical protective and regenerative mechanisms that can impact apoptotic and autophagic programmed cell death pathways through protein kinase B (Akt), sirtuins, mammalian forkhead transcription factors, and wingless signaling. Translation of the cytoprotective pathways of EPO into clinically effective treatments for some neurodegenerative disorders has been promising, but additional work is necessary. In particular, development of new treatments with erythropoiesis-stimulating agents such as EPO brings several important challenges that involve detrimental vascular outcomes and tumorigenesis. Future work that can effectively and safely harness the complexity of the signaling pathways of EPO will be vital for the fruitful treatment of disorders of the nervous system. PMID:26549969

  9. Infantile pyknocytosis: effectiveness of erythropoietin treatment

    Eleonora Buzzi

    2013-04-01

    Full Text Available Infantile pyknocytosis is a rare form of neonatal haemolytic anaemia with unusual red cell morphology. Anaemia is mostly severe and red blood cells transfusion is often needed. In this report, we have described a male child aged 10 days, born at 37 weeks + 3 days, who presented neonatal jaundice and severe anaemia. After a careful peripheral blood smear examination, infantile pyknocytosis was diagnosed. A treatment with recombinant subcutaneous erythropoietin (1,000 UI/prokg/week in conjunction with iron supplementation (6 mg/kg/day was started. The therapy was reduced 6 weeks after the beginning and discontinued 4 weeks after the reaching of a steady state of the haemoglobin values. After 12 months of follow up, the patient showed no anaemia and pyknocytosis.

  10. In vivo stimulatory effect of erythropoietin on endothelial nitric oxide synthase in cerebral arteries.

    Santhanam, Anantha Vijay R; Smith, Leslie A; Nath, Karl A; Katusic, Zvonimir S

    2006-08-01

    The discovery of tissue protective effects of erythropoietin has stimulated significant interest in erythropoietin (Epo) as a novel therapeutic approach to vascular protection. The present study was designed to determine the cerebral vascular effects of recombinant Epo in vivo. Recombinant adenoviral vectors (10(9) plaque-forming units/animal) encoding genes for human erythropoietin (AdEpo) and beta-galactosidase (AdLacZ) were injected into the cisterna magna of rabbits. After 48 h, basilar arteries were harvested for analysis of vasomotor function, Western blotting, and measurement of cGMP levels. Gene transfer of AdEpo increased the expressions of recombinant Epo and its receptor in the basilar arteries. Arteries exposed to recombinant Epo demonstrated attenuation of contractile responses to histamine (10(-9) to 10(-5) mol/l) (P < 0.05, n = 5). Endothelium-dependent relaxations to acetylcholine (10(-9) to 10(-5) mol/l) were significantly augmented (P < 0.05, n = 5), whereas endothelium-independent relaxations to a nitric oxide (NO) donor 2-(N,N-diethylamino)diazenolate-2-oxide sodium salt remained unchanged in AdEpo-transduced basilar arteries. Transduction with AdEpo increased the protein expression of endothelial NO synthase (eNOS) and phosphorylated the S1177 form of the enzyme. Basal levels of cGMP were significantly elevated in arteries transduced with AdEpo consistent with increased NO production. Our studies suggest that in cerebral circulation, Epo enhances endothelium-dependent vasodilatation mediated by NO. This effect could play an important role in the vascular protective effect of Epo. PMID:16565320

  11. Management of anemia in patients undergoing curative radiotherapy. Erythropoietin, transfusions, or better nothing?

    Dunst, J. [Dept. of Radiotherapy, Martin Luther Univ. Halle-Wittenberg, Halle (Germany)

    2004-11-01

    Background and results: anemia is a well-known risk factor for decreased local control and survival in patients undergoing curative radiotherapy. There is clear evidence from recent clinical investigations that anemia is an independent risk factor and hemoglobin (Hb) levels during radiotherapy are important (and not pretreatment Hb levels). The most likely explanation for the prognostic impact is the association with tumor hypoxia. An ''optimal'' Hb range with regard to tumor oxygenation seems to exist, and Hb levels < 11 g/dl and > {proportional_to}15 g/dl impair tumor oxygenation but have (over a broader range) no significant impact on normal tissue oxygenation. There is some evidence from retrospective and prospective studies that the response to radiotherapy and the prognosis, especially in cervical cancers, might be improved if the Hb levels during radiotherapy can be maintained in the optimal range, either by transfusions or by erythropoietin. The effect of any antianemic therapy should be analyzed according to whether or not treatment was successful with regard to achieving optimal Hb levels during irradiation. Erythropoietin is probably more effective in steadily increasing and stabilizing Hb levels, but bears the risk of overcorrection of Hb levels. The clinical relevance of erythropoietin receptors on tumor cells remains questionable. Conclusions: treatment of anemia with the objective of improving local control and survival in radiotherapy patients is probably more difficult and sophisticated than coping with symptoms of anemia or improving quality of life. Nevertheless, the potential of antianemic treatment is high on the basis of experimental and clinical data, and further clinical trials are warranted. (orig.)

  12. Peroxisome Proliferator-Activated Receptor-γ in Thyroid Autoimmunity

    Silvia Martina Ferrari

    2015-01-01

    Full Text Available Peroxisome proliferator-activated receptor- (PPAR- γ expression has been shown in thyroid tissue from patients with thyroiditis or Graves’ disease and furthermore in the orbital tissue of patients with Graves’ ophthalmopathy (GO, such as in extraocular muscle cells. An increasing body of evidence shows the importance of the (C-X-C motif receptor 3 (CXCR3 and cognate chemokines (C-X-C motif ligand (CXCL9, CXCL10, and CXCL11, in the T helper 1 immune response and in inflammatory diseases such as thyroid autoimmune disorders. PPAR-γ agonists show a strong inhibitory effect on the expression and release of CXCR3 chemokines, in vitro, in various kinds of cells, such as thyrocytes, and in orbital fibroblasts, preadipocytes, and myoblasts from patients with GO. Recently, it has been demonstrated that rosiglitazone is involved in a higher risk of heart failure, stroke, and all-cause mortality in old patients. On the contrary, pioglitazone has not shown these effects until now; this favors pioglitazone for a possible use in patients with thyroid autoimmunity. However, further studies are ongoing to explore the use of new PPAR-γ agonists in the treatment of thyroid autoimmune disorders.

  13. Erythropoietin enhances hippocampal long-term potentiation and memory

    El-Kordi Ahmed

    2008-09-01

    Full Text Available Abstract Background Erythropoietin (EPO improves cognition of human subjects in the clinical setting by as yet unknown mechanisms. We developed a mouse model of robust cognitive improvement by EPO to obtain the first clues of how EPO influences cognition, and how it may act on hippocampal neurons to modulate plasticity. Results We show here that a 3-week treatment of young mice with EPO enhances long-term potentiation (LTP, a cellular correlate of learning processes in the CA1 region of the hippocampus. This treatment concomitantly alters short-term synaptic plasticity and synaptic transmission, shifting the balance of excitatory and inhibitory activity. These effects are accompanied by an improvement of hippocampus dependent memory, persisting for 3 weeks after termination of EPO injections, and are independent of changes in hematocrit. Networks of EPO-treated primary hippocampal neurons develop lower overall spiking activity but enhanced bursting in discrete neuronal assemblies. At the level of developing single neurons, EPO treatment reduces the typical increase in excitatory synaptic transmission without changing the number of synaptic boutons, consistent with prolonged functional silencing of synapses. Conclusion We conclude that EPO improves hippocampus dependent memory by modulating plasticity, synaptic connectivity and activity of memory-related neuronal networks. These mechanisms of action of EPO have to be further exploited for treating neuropsychiatric diseases.

  14. Full and partial agonists of thromboxane prostanoid receptor unveil fine tuning of receptor superactive conformation and G protein activation.

    Valérie Capra

    Full Text Available The intrahelical salt bridge between E/D(3.49 and R(3.50 within the E/DRY motif on helix 3 (H3 and the interhelical hydrogen bonding between the E/DRY and residues on H6 are thought to be critical in stabilizing the class A G protein-coupled receptors in their inactive state. Removal of these interactions is expected to generate constitutively active receptors. This study examines how neutralization of E(3.49/6.30 in the thromboxane prostanoid (TP receptor alters ligand binding, basal, and agonist-induced activity and investigates the molecular mechanisms of G protein activation. We demonstrate here that a panel of full and partial agonists showed an increase in affinity and potency for E129V and E240V mutants. Yet, even augmenting the sensitivity to detect constitutive activity (CA with overexpression of the receptor or the G protein revealed resistance to an increase in basal activity, while retaining fully the ability to cause agonist-induced signaling. However, direct G protein activation measured through bioluminescence resonance energy transfer (BRET indicates that these mutants more efficiently communicate and/or activate their cognate G proteins. These results suggest the existence of additional constrains governing the shift of TP receptor to its active state, together with an increase propensity of these mutants to agonist-induced signaling, corroborating their definition as superactive mutants. The particular nature of the TP receptor as somehow "resistant" to CA should be examined in the context of its pathophysiological role in the cardiovascular system. Evolutionary forces may have favored regulation mechanisms leading to low basal activity and selected against more highly active phenotypes.

  15. The Structure of the GM-CSF Receptor Complex Reveals a Distinct Mode of Cytokine Receptor Activation

    Hansen, Guido; Hercus, Timothy R.; McClure, Barbara J.; Stomski, Frank C.; Dottore, Mara; Powell, Jason; Ramshaw, Hayley; Woodcock, Joanna M.; Xu, Yibin; Guthridge, Mark; McKinstry, William J.; Lopez, Angel F.; Parker, Michael W. (SVIMR-A); (Hanson)

    2008-08-11

    Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a pleiotropic cytokine that controls the production and function of blood cells, is deregulated in clinical conditions such as rheumatoid arthritis and leukemia, yet offers therapeutic value for other diseases. Its receptors are heterodimers consisting of a ligand-specific {alpha} subunit and a {beta}c subunit that is shared with the interleukin (IL)-3 and IL-5 receptors. How signaling is initiated remains an enigma. We report here the crystal structure of the human GM-CSF/GM-CSF receptor ternary complex and its assembly into an unexpected dodecamer or higher-order complex. Importantly, mutagenesis of the GM-CSF receptor at the dodecamer interface and functional studies reveal that dodecamer formation is required for receptor activation and signaling. This unusual form of receptor assembly likely applies also to IL-3 and IL-5 receptors, providing a structural basis for understanding their mechanism of activation and for the development of therapeutics.

  16. Complement Component C3 Binds to Activated Normal Platelets without Preceding Proteolytic Activation and Promotes Binding to Complement Receptor 1

    Osama A Hamad; Nilsson, Per H.; Wouters, Diana; Lambris, John D.; Ekdahl, Kristina N.; Nilsson, Bo

    2010-01-01

    It has been reported that complement is activated on the surface of activated platelets, despite the presence of multiple regulators of complement activation. To reinvestigate the mechanisms by which activated platelets bind to complement components, the presence of complement proteins on the surfaces of nonactivated and thrombin receptor-activating peptide-activated platelets was analyzed by flow cytometry and Western blot analyses. C1q, C4, C3, and C9 were found to bind to thrombin receptor...

  17. TAM receptors affect adult brain neurogenesis by negative regulation of microglial cell activation1

    Ji, Rui; Tian, Shifu; Lu, Helen J.; LU, QINGJUN; Yan ZHENG; Wang, Xiaomin; Ding, Jixiang; Li, Qiutang; Lu, Qingxian

    2013-01-01

    TAM tyrosine kinases play multiple functional roles including regulation of the target genes important in homeostatic regulation of cytokine receptors or Toll-like receptor-mediated signal transduction pathways. Here, we show that TAM receptors affect adult hippocampal neurogenesis and loss of TAM receptors impair hippocampal neurogenesis, largely attributed to exaggerated inflammatory responses by microglia characterized by increased MAP kinase and NF-κB activation and elevated production of...

  18. Structural motifs of importance for the constitutive activity of the orphan 7TM receptor EBI2: analysis of receptor activation in the absence of an agonist

    Benned-Jensen, Tau; Rosenkilde, Mette M

    2008-01-01

    The Epstein-Barr induced receptor 2 (EBI2) is a lymphocyte-expressed orphan seven transmembrane-spanning (7TM) receptor that signals constitutively through Galphai, as shown, for instance by guanosine 5'-O-(3-thio)triphosphate incorporation. Two regions of importance for the constitutive activity...

  19. Signaling Mechanism of Cannabinoid Receptor-2 Activation-Induced β-Endorphin Release.

    Gao, Fang; Zhang, Ling-Hong; Su, Tang-Feng; Li, Lin; Zhou, Rui; Peng, Miao; Wu, Cai-Hua; Yuan, Xiao-Cui; Sun, Ning; Meng, Xian-Fang; Tian, Bo; Shi, Jing; Pan, Hui-Lin; Li, Man

    2016-08-01

    Activation of cannabinoid receptor-2 (CB2) results in β-endorphin release from keratinocytes, which then acts on primary afferent neurons to inhibit nociception. However, the underlying mechanism is still unknown. The CB2 receptor is generally thought to couple to Gi/o to inhibit cAMP production, which cannot explain the peripheral stimulatory effects of CB2 receptor activation. In this study, we found that in a keratinocyte cell line, the Gβγ subunits from Gi/o, but not Gαs, were involved in CB2 receptor activation-induced β-endorphin release. Inhibition of MAPK kinase, but not PLC, abolished CB2 receptor activation-induced β-endorphin release. Also, CB2 receptor activation significantly increased intracellular Ca(2+). Treatment with BAPTA-AM or thapsigargin blocked CB2 receptor activation-induced β-endorphin release. Using a rat model of inflammatory pain, we showed that the MAPK kinase inhibitor PD98059 abolished the peripheral effect of the CB2 receptor agonist on nociception. We thus present a novel mechanism of CB2 receptor activation-induced β-endorphin release through Gi/o-Gβγ-MAPK-Ca(2+) signaling pathway. Our data also suggest that stimulation of MAPK contributes to the peripheral analgesic effect of CB2 receptor agonists. PMID:26108183

  20. Human bradykinin B(2) receptor is activated by kallikrein and other serine proteases.

    Hecquet, C; Tan, F; Marcic, B M; Erdös, E G

    2000-10-01

    Bradykinin (BK) and kallidin (Lys-BK), liberated from kininogens by kallikreins, are ligands of the BK B(2) receptor. We investigated whether kallikreins, besides releasing peptide agonist, could also activate the receptor directly. We studied the effect of porcine and human recombinant tissue kallikrein and plasma kallikrein on [Ca(2+)](i) mobilization and [(3)H]arachidonic acid release from cultured cells stably transfected to express human BK B(2) receptor (CHO/B(2), MDCK/B(2), HEK/B(2)), and endothelial cells were used as control cells. As with BK, the actions of kallikrein were blocked by the B(2) antagonist, HOE 140. Kallikrein was inactive on cells lacking B(2) receptor. Kallikrein and BK desensitized the receptor homologously but there was no cross-desensitization. Furthermore, 50 nM human cathepsin G and 50 nM trypsin also activated the receptor; this also was blocked by HOE 140. Experiments excluded a putative kinin release by proteases. [(3)H]AA release by BK was reduced by 40% by added kininase I (carboxypeptidase M); however, receptor activation by tissue kallikrein, trypsin, or cathepsin G was not affected. Prokallikrein and inhibited kallikrein were inactive, suggesting cleavage of a peptide bond in the receptor. Kallikreins were active on mutated B(2) receptor missing the 19 N-terminal amino acids, suggesting a type of activation different from that of thrombin receptor. Paradoxically, tissue kallikreins decreased the [(3)H]BK binding to the receptor with a low K(D) (3 nM) and inhibited it 78%. Thus, kallikreins and some other proteases activate human BK B(2) receptor directly, independent of BK release. The BK B(2) receptor may belong to a new group of serine protease-activated receptors. PMID:10999954

  1. Peroxisome Proliferator-Activated Receptors in Female Reproduction and Fertility

    Carta, Gaspare; Artini, Paolo Giovanni

    2016-01-01

    Reproductive functions may be altered by the exposure to a multitude of endogenous and exogenous agents, drug or environmental pollutants, which are known to affect gene transcription through the peroxisome proliferator-activated receptors (PPARs) activation. PPARs act as ligand activated transcription factors and regulate metabolic processes such as lipid and glucose metabolism, energy homeostasis, inflammation, and cell proliferation and differentiation. All PPARs isotypes are expressed along the hypothalamic-pituitary-gonadal axis and are strictly involved in reproductive functions. Since female fertility and energy metabolism are tightly interconnected, the research on female infertility points towards the exploration of potential PPARs activating/antagonizing compounds, mainly belonging to the class of thiazolidinediones (TZDs) and fibrates, as useful agents for the maintenance of metabolic homeostasis in women with ovarian dysfunctions. In the present review, we discuss the recent evidence about PPARs expression in the hypothalamic-pituitary-gonadal axis and their involvement in female reproduction. Finally, the therapeutic potential of their manipulation through several drugs is also discussed.

  2. Family C 7TM receptor dimerization and activation

    Bonde, Marie Mi; Sheikh, Søren P; Hansen, Jakob Lerche

    2006-01-01

    The family C seven transmembrane (7TM) receptors constitutes a small and especially well characterized subfamily of the large 7TM receptor superfamily. Approximately 50% of current prescription drugs target 7TM receptors, this biologically important family represents the largest class of drug-tar...

  3. Exercise reduces adipose tissue via cannabinoid receptor type 1 which is regulated by peroxisome proliferator-activated receptor-delta

    Yan, Zhen Cheng; Liu, Dao Yan; Zhang, Li Li;

    2007-01-01

    Obesity is one major cardiovascular risk factor. We tested effects of endurance exercise on cannabinoid receptor type 1 (CB1) and peroxisome proliferator-activated receptor-delta (PPAR-delta)-dependent pathways in adipose tissue. Male Wistar rats were randomly assigned to standard laboratory chow...... or a high-fat diet without and with regular endurance exercise. Exercise in rats on high-fat diet significantly reduced visceral fat mass, blood pressure, and adipocyte size (each p...

  4. Distinct RNA motifs are important for coactivation of steroid hormone receptors by steroid receptor RNA activator (SRA)

    Lanz, Rainer B.; Razani, Bahram; Goldberg, Aaron D.; O'Malley, Bert W.

    2002-01-01

    Steroid receptor RNA activator (SRA) is an RNA transcript that functions as a eukaryotic transcriptional coactivator for steroid hormone receptors. We report here the isolation and functional characterization of distinct RNA substructures within the SRA molecule that constitute its coactivation function. We used comparative sequence analysis and free energy calculations to systematically study SRA RNA subdomains for identification of structured regions and base pairings, and we used site-dire...

  5. Calcium is the switch in the moonlighting dual function of the ligand-activated receptor kinase phytosulfokine receptor 1

    Muleya, Victor

    2014-09-23

    Background: A number of receptor kinases contain guanylate cyclase (GC) catalytic centres encapsulated in the cytosolic kinase domain. A prototypical example is the phytosulfokine receptor 1 (PSKR1) that is involved in regulating growth responses in plants. PSKR1 contains both kinase and GC activities however the underlying mechanisms regulating the dual functions have remained elusive. Findings: Here, we confirm the dual activity of the cytoplasmic domain of the PSKR1 receptor. We show that mutations within the guanylate cyclase centre modulate the GC activity while not affecting the kinase catalytic activity. Using physiologically relevant Ca2+ levels, we demonstrate that its GC activity is enhanced over two-fold by Ca2+ in a concentration-dependent manner. Conversely, increasing Ca2+ levels inhibits kinase activity up to 500-fold at 100 nM Ca2+. Conclusions: Changes in calcium at physiological levels can regulate the kinase and GC activities of PSKR1. We therefore propose a functional model of how calcium acts as a bimodal switch between kinase and GC activity in PSKR1 that could be relevant to other members of this novel class of ligand-activated receptor kinases.

  6. Report on isoelectric focusing trial of erythropoietin profiling in two cancerpatients during chemotherapy and darbepoetin treatment:

    Matos, Erika; Debeljak, Nataša; Komel, Radovan; Možina, Barbara; Borštnar, Simona; Španinger, Klemen; Sytkowski, Arthur J.

    2011-01-01

    The level of erythropoietin, main regulator of erythropoiesis, is affected by hypoxia, anaemia, application of recombinant erythropoietin, chemotherapy and others. Isoelectric focusing (IEF) combined with double immunobloting is a method that enables distinct analysis of endogenous and recombinant erythropoietin isoforms. Aim of our study was to set up analysis of treatment effects on the pattern of endogenous erythropoietin in anaemic breast cancer patient. Urine and blood samples were colle...

  7. Anemia induces accumulation of erythropoietin mRNA in the kidney and liver.

    Bondurant, M. C.; Koury, M J

    1986-01-01

    Regulation of the production of erythropoietin occurs in the kidney and liver largely through control of accumulation of erythropoietin mRNA. Erythropoietin mRNA was first detected in kidneys at 1.5 h postanemia and reached a plateau value at least 200-fold above the control value by 4 to 8 h. A 20-base sequence immediately upstream from the reported erythropoietin mRNA initiation site is complementary to a hypervariable sequence in 18S rRNA.

  8. Der Einfluss hypoxisch induzierter zerebraler Faktoren auf die renale Synthese von Erythropoietin

    Klaus, Janina

    2010-01-01

    The influence of hypoxic-induced cerebral factors on renal synthesis of erythropoietin. A study in rats Starting point. While the structure and function of erythropoietin are well documented, the mechanisms of the regulation of the renal synthesis of erythropoietin are poorly understood. Especially, the description of the localization and function of the O2-sensitive sensor regulating in its turn the renal synthesis of erythropoietin is insufficient. A body of evidence suggests that extrar...

  9. Receptor activity modifying proteins (RAMPs) interact with the VPAC1 receptor: evidence for differential RAMP modulation of multiple signalling pathways

    Full text: Receptor activity modifying proteins (RAMP) constitute a family of three accessory proteins that affect the expression and/or phenotype of the calcitonin receptor (CTR) or CTR-like receptor (CRLR). In this study we screened a range of class II G protein-coupled receptors (PTH1, PTH2, GHRH, VPAC1, VPAC2 receptors) for possible RAMP interactions by measurement of receptor-induced translocation of c-myc tagged RAMP1 or HA tagged RAMP3. Of these, only the VPAC1 receptor caused significant translocation of c-myc-RAMP1 or HA-RAMP3 to the cell surface. Co-transfection of VPAC1 and RAMPs did not alter 125I-VIP binding and specificity. VPAC1 receptor function was subsequently analyzed through parallel determinations of cAMP accumulation and phosphoinositide (PI) hydrolysis in the presence and absence of each of the three RAMPs. In contrast to CTR-RAMP interaction, where there was an increase in cAMP Pharmacologisand a decrease in PI hydrolysis, VPAC1-RAMP interaction was characterized by a specific increase in agonist-mediated PI hydrolysis when co-transfected with RAMP2. This change was due to an enhancement of Emax with no change in EC50 value for VIP. No significant change in cAMP accumulation was observed. This is the first demonstration of an interaction of RAMPs with a G protein-coupled receptor outside the CTR family and may suggest a more generalized role for RAMPs in modulating G protein-coupled receptor signaling. Copyright (2001) Australasian Society of Clinical and Experimental Pharmacologists and Toxicologists

  10. Significance of AT1 receptor independent activation of mineralocorticoid receptor in murine diabetic cardiomyopathy.

    Yuji Nagatomo

    Full Text Available BACKGROUND: Diabetes mellitus (DM has deleterious influence on cardiac performance independent of coronary artery disease and hypertension. The objective of the present study was to investigate the role of the renin-angiotensin-aldosterone system, especially angiotensin II type 1a receptor (AT1aR and mineralocorticoid receptor (MR signaling, in left ventricular (LV dysfunction induced by diabetes mellitus (DM. METHODS AND RESULTS: DM was induced by intraperitoneal injection of streptozotocin (200 mg/kg BW in wild-type (WT or AT1aR knockout (KO male mice, and they were bred during 6 or 12 weeks. Some KO mice were administered the MR antagonist eplerenone (100 mg/kg body weight. At 6 weeks, LV diastolic function was impaired in WT-DM, but preserved in KO-DM. At that time point MR mRNA expression was upregulated, NADPH oxidase subunit (p47phox and glutathione peroxidase (GPx1 mRNA expression were upregulated, the staining intensities of LV tissue for 4-hydroxy-2-nonenal was stronger in immunohistochemistry, the number of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL positive cells was increased, Bcl-2 protein expression was significantly downregulated, and the expression of SERCA2a and phosphorylated phospholamban was depressed in WT-DM, while these changes were not seen in KO-DM. At 12 weeks, however, these changes were also noted in KO-DM. Eplerenone arrested those changes. The plasma aldosterone concentration was elevated in WT-DM but not in KO-DM at 6 weeks. It showed 3.7-fold elevation at 12 weeks even in KO-DM, which suggests "aldosterone breakthrough" phenomenon. However, the aldosterone content in LV tissue was unchanged in KO-DM. CONCLUSIONS: DM induced diastolic dysfunction was observed even in KO at 12 weeks, which was ameliorated by minelarocorticoid receptor antagonist, eplerenone. AT1-independent MR activation in the LV might be responsible for the pathogenesis of diabetic cardiomyopathy.

  11. Repressive effects of resveratrol on androgen receptor transcriptional activity.

    Wen-feng Shi

    Full Text Available BACKGROUND: The chemopreventive effects of resveratrol (RSV on prostate cancer have been well established; the androgen receptor (AR plays pivotal roles in prostatic tumorigenesis. However, the exact underlying molecular mechanisms about the effects of RSV on AR have not been fully elucidated. A model system is needed to determine whether and how RSV represses AR transcriptional activity. METHODOLOGY: The AR cDNA was first cloned into the retroviral vector pOZ-N and then integrated into the genome of AR-negative HeLa cells to generate the AR(+ cells. The constitutively expressed AR was characterized by monitoring hormone-stimulated nuclear translocation, DNA binding, and transcriptional activation, with the AR(- cells serving as controls. AR(+ cells were treated with RSV, and both AR protein levels and AR transcriptional activity were measured simultaneously. Chromatin immunoprecipitation (ChIP assays were used to detect the effects of RSV on the recruitment of AR to its cognate element (ARE. RESULTS: AR in the AR (+ stable cell line functions in a manner similar to that of endogenously expressed AR. Using this model system we clearly demonstrated that RSV represses AR transcriptional activity independently of any effects on AR protein levels. However, neither the hormone-mediated nucleus translocation nor the AR/ARE interaction was affected by RSV treatment. CONCLUSION: We demonstrated unambiguously that RSV regulates AR target gene expression, at least in part, by repressing AR transcriptional activity. Repressive effects of RSV on AR activity result from mechanisms other than the affects of AR nuclear translocation or DNA binding.

  12. Effects of Sulfonylureas on Peroxisome Proliferator-Activated Receptor γ Activity and on Glucose Uptake by Thiazolidinediones

    Kyeong Won Lee; Yun Hyi Ku; Min Kim; Byung Yong Ahn; Sung Soo Chung; Kyong Soo Park

    2011-01-01

    Background Sulfonylurea primarily stimulates insulin secretion by binding to its receptor on the pancreatic β-cells. Recent studies have suggested that sulfonylureas induce insulin sensitivity through peroxisome proliferator-activated receptor γ (PPARγ), one of the nuclear receptors. In this study, we investigated the effects of sulfonylurea on PPARγ transcriptional activity and on the glucose uptake via PPARγ. Methods Transcription reporter assays using Cos7 cells were performed to determine...

  13. Interplay between Endothelin and Erythropoietin in Astroglia: The Role in Protection against Hypoxia

    Richard Schäfer

    2014-02-01

    Full Text Available We show that, under in vitro conditions, the vulnerability of astroglia to hypoxia is reflected by alterations in endothelin (ET-1 release and capacity of erythropoietin (EPO to regulate ET-1 levels. Exposure of cells to 24 h hypoxia did not induce changes in ET-1 release, while 48–72 h hypoxia resulted in increase of ET-1 release from astrocytes that could be abolished by EPO. The endothelin receptor type A (ETA antagonist BQ123 increased extracellular levels of ET-1 in human fetal astroglial cell line (SV-FHAS. The survival and proliferation of rat primary astrocytes, neural precursors, and neurons upon hypoxic conditions were increased upon administration of BQ123. Hypoxic injury and aging affected the interaction between the EPO and ET systems. Under hypoxia EPO decreased ET-1 release from astrocytes, while ETA receptor blockade enhanced the expression of EPO mRNA and EPO receptor in culture-aged rat astroglia. The blockade of ETA receptor can increase the availability of ET-1 to the ETB receptor and can potentiate the neuroprotective effects of EPO. Thus, the new therapeutic use of combined administration of EPO and ETA receptor antagonists during hypoxia-associated neurodegenerative disorders of the central nervous system (CNS can be suggested.

  14. Trans-activation by thyroid hormone receptors: functional parallels with steroid hormone receptors.

    Thompson, C C; Evans, R M

    1989-01-01

    The effects of thyroid hormones are mediated through nuclear receptor proteins that modulate the transcription of specific genes in target cells. We previously isolated cDNAs encoding two different mammalian thyroid hormone receptors, one from human placenta (hTR beta) and the other from rat brain (rTR alpha), and showed that their in vitro translation products bind thyroid hormones with the characteritistic affinities of the native thyroid hormone receptor. We now demonstrate that both of th...

  15. Modulation of Opioid Receptor Ligand Affinity and Efficacy Using Active and Inactive State Receptor Models

    Anand, Jessica P.; Purington, Lauren C.; Pogozheva, Irina D.; Traynor, John R.; Mosberg, Henry I.

    2012-01-01

    Mu opioid receptor (MOR) agonists are widely used for the treatment of pain; however chronic use results in the development of tolerance and dependence. It has been demonstrated that co-administration of a MOR agonist with a delta opioid receptor (DOR) antagonist maintains the analgesia associated with MOR agonists, but with reduced negative side effects. Using our newly refined opioid receptor models for structure-based ligand design, we have synthesized several pentapeptides with tailored a...

  16. Can Specific Protein-Lipid Interactions Stabilize an Active State of the Beta 2 Adrenergic Receptor?

    Neale, Chris; Herce, Henry D; Pomès, Régis; García, Angel E

    2015-10-20

    G-protein-coupled receptors are eukaryotic membrane proteins with broad biological and pharmacological relevance. Like all membrane-embedded proteins, their location and orientation are influenced by lipids, which can also impact protein function via specific interactions. Extensive simulations totaling 0.25 ms reveal a process in which phospholipids from the membrane's cytosolic leaflet enter the empty G-protein binding site of an activated β2 adrenergic receptor and form salt-bridge interactions that inhibit ionic lock formation and prolong active-state residency. Simulations of the receptor embedded in an anionic membrane show increased lipid binding, providing a molecular mechanism for the experimental observation that anionic lipids can enhance receptor activity. Conservation of the arginine component of the ionic lock among Rhodopsin-like G-protein-coupled receptors suggests that intracellular lipid ingression between receptor helices H6 and H7 may be a general mechanism for active-state stabilization. PMID:26488656

  17. Activation of 5-hyrdoxytryptamine 7 receptors within the rat nucleus tractus solitarii modulates synaptic properties.

    Matott, Michael P; Kline, David D

    2016-03-15

    Serotonin (5-HT) is a potent neuromodulator with multiple receptor types within the cardiorespiratory system, including the nucleus tractus solitarii (nTS) - the central termination site of visceral afferent fibers. The 5-HT7 receptor facilitates cardiorespiratory reflexes through its action in the brainstem and likely in the nTS. However, the mechanism and site of action for these effects is not clear. In this study, we examined the expression and function of 5-HT7 receptors in the nTS of Sprague-Dawley rats. 5-HT7 receptor mRNA and protein were identified across the rostrocaudal extent of the nTS. To determine 5-HT7 receptor function, we examined nTS synaptic properties following 5-HT7 receptor activation in monosynaptic nTS neurons in the in vitro brainstem slice preparation. Application of 5-HT7 receptor agonists altered tractus solitarii evoked and spontaneous excitatory postsynaptic currents which were attenuated with a selective 5-HT7 receptor antagonist. 5-HT7 receptor-mediated changes in excitatory postsynaptic currents were also altered by block of 5-HT1A and GABAA receptors. Interestingly, 5-HT7 receptor activation also reduced the amplitude but not frequency of GABAA-mediated inhibitory currents. Together these results indicate a complex role for 5-HT7 receptors in the nTS that mediate its diverse effects on cardiorespiratory parameters. PMID:26779891

  18. Triclocarban mediates induction of xenobiotic metabolism through activation of the constitutive androstane receptor and the estrogen receptor alpha.

    Mei-Fei Yueh

    Full Text Available Triclocarban (3,4,4'-trichlorocarbanilide, TCC is used as a broad-based antimicrobial agent that is commonly added to personal hygiene products. Because of its extensive use in the health care industry and resistance to degradation in sewage treatment processes, TCC has become a significant waste product that is found in numerous environmental compartments where humans and wildlife can be exposed. While TCC has been linked to a range of health and environmental effects, few studies have been conducted linking exposure to TCC and induction of xenobiotic metabolism through regulation by environmental sensors such as the nuclear xenobiotic receptors (XenoRs. To identify the ability of TCC to activate xenobiotic sensors, we monitored XenoR activities in response to TCC treatment using luciferase-based reporter assays. Among the XenoRs in the reporter screening assay, TCC promotes both constitutive androstane receptor (CAR and estrogen receptor alpha (ERα activities. TCC treatment to hUGT1 mice resulted in induction of the UGT1A genes in liver. This induction was dependent upon the constitutive active/androstane receptor (CAR because no induction occurred in hUGT1Car(-/- mice. Induction of the UGT1A genes by TCC corresponded with induction of Cyp2b10, another CAR target gene. TCC was demonstrated to be a phenobarbital-like activator of CAR in receptor-based assays. While it has been suggested that TCC be classified as an endocrine disruptor, it activates ERα leading to induction of Cyp1b1 in female ovaries as well as in promoter activity. Activation of ERα by TCC in receptor-based assays also promotes induction of human CYP2B6. These observations demonstrate that TCC activates nuclear xenobiotic receptors CAR and ERα both in vivo and in vitro and might have the potential to alter normal physiological homeostasis. Activation of these xenobiotic-sensing receptors amplifies gene expression profiles that might represent a mechanistic base for

  19. Sulpiride and the role of dopaminergic receptor blockade in the antipsychotic activity of neuroleptics

    It is now generally recognized that dopamine receptors excist in the CNS as different subtypes: D1 receptors, associated with adenylyl cyclase activity, and D2 receptor, uncoupled to a cyclic APM generating system. In order to understand the role of D1 and D2 receptors in the antipsychotic action of neuroleptics, we have performed subchronic treatment with haloperidol, a drug which acts on D1 receptors, and sulpiride, a selective antagonist to D2 receptors. Long-term treatment with haloperidol does not induce significant supersensitivity of the D2 receptors. In fact under these conditions 3H-(-)-sulpiride binding, which is a marker of D2 receptor function, does not increase in rat striatum, while the long-term administration of sulpiride, itself produces supersensitivity of D2 receptors. Moreover, sulpiride does not induce supersensitivity of the D1 receptors, characterized by 3H-spiroperidol binding. These data suggest that both types of dopamine receptors may be involved in the clinical antipsychotic effects of neuroleptics. Unilateral leison of the nigrostriatal dopaminergic pathway produces an increase of striatal dopaminergic receptors, measured either by 3H-spiroperidol and 3H-(-)-sulpiride binding. These findings suggest that D1 and D2 receptors are present in postsynaptic membranes while it is still not known whether they exist in the same cellular elements. (author)

  20. The role of ECL2 in CGRP receptor activation: a combined modelling and experimental approach

    Woolley, Michael. J.; Watkins, Harriet A.; Taddese, Bruck; Karakullukcu, Z. Gamze; Barwell, James; Smith, Kevin J.; Hay, Debbie L.; Poyner, David R.; Reynolds, Christopher A.; Conner, Alex C.

    2013-01-01

    The calcitonin gene-related peptide (CGRP) receptor is a complex of a calcitonin receptor-like receptor (CLR), which is a family B G-protein-coupled receptor (GPCR) and receptor activity modifying protein 1. The role of the second extracellular loop (ECL2) of CLR in binding CGRP and coupling to Gs was investigated using a combination of mutagenesis and modelling. An alanine scan of residues 271–294 of CLR showed that the ability of CGRP to produce cAMP was impaired by point mutations at 13 residues; most of these also impaired the response to adrenomedullin (AM). These data were used to select probable ECL2-modelled conformations that are involved in agonist binding, allowing the identification of the likely contacts between the peptide and receptor. The implications of the most likely structures for receptor activation are discussed. PMID:24047872

  1. Investigating real-time activation of adenosine receptors by bioluminescence resonance energy transfer technique

    Huang, Yimei; Yang, Hongqin; Zheng, Liqin; Chen, Jiangxu; Wang, Yuhua; Li, Hui; Xie, Shusen

    2013-02-01

    Adenosine receptors play important roles in many physiological and pathological processes, for example regulating myocardial oxygen consumption and the release of neurotransmitters. The activations of adenosine receptors have been studied by some kinds of techniques, such as western blot, immunohistochemistry, etc. However, these techniques cannot reveal the dynamical response of adenosine receptors under stimulation. In this paper, bioluminescence resonance energy transfer technique was introduced to study the real-time activation of adenosine receptors by monitoring the dynamics of cyclic adenosine monophosphate (cAMP) level. The results showed that there were significant differences between adenosine receptors on real-time responses under stimulation. Moreover, the dynamics of cAMP level demonstrated that competition between adenosine receptors existed. Taken together, our study indicates that monitoring the dynamics of cAMP level using bioluminescence resonance energy transfer technique could be one potential approach to investigate the mechanism of competitions between adenosine receptors.

  2. Role of receptors in Bacillus thuringiensis crystal toxin activity.

    Pigott, Craig R; Ellar, David J

    2007-06-01

    Bacillus thuringiensis produces crystalline protein inclusions with insecticidal or nematocidal properties. These crystal (Cry) proteins determine a particular strain's toxicity profile. Transgenic crops expressing one or more recombinant Cry toxins have become agriculturally important. Individual Cry toxins are usually toxic to only a few species within an order, and receptors on midgut epithelial cells have been shown to be critical determinants of Cry specificity. The best characterized of these receptors have been identified for lepidopterans, and two major receptor classes have emerged: the aminopeptidase N (APN) receptors and the cadherin-like receptors. Currently, 38 different APNs have been reported for 12 different lepidopterans. Each APN belongs to one of five groups that have unique structural features and Cry-binding properties. While 17 different APNs have been reported to bind to Cry toxins, only 2 have been shown to mediate toxin susceptibly in vivo. In contrast, several cadherin-like proteins bind to Cry toxins and confer toxin susceptibility in vitro, and disruption of the cadherin gene has been associated with toxin resistance. Nonetheless, only a small subset of the lepidopteran-specific Cry toxins has been shown to interact with cadherin-like proteins. This review analyzes the interactions between Cry toxins and their receptors, focusing on the identification and validation of receptors, the molecular basis for receptor recognition, the role of the receptor in resistant insects, and proposed models to explain the sequence of events at the cell surface by which receptor binding leads to cell death. PMID:17554045

  3. Mechanisms Governing the Activation and Trafficking of Yeast G Protein-coupled Receptors

    Stefan, Christopher J.; Overton, Mark C.; Blumer, Kendall J.

    1998-01-01

    We have addressed the mechanisms governing the activation and trafficking of G protein-coupled receptors (GPCRs) by analyzing constitutively active mating pheromone receptors (Ste2p and Ste3p) of the yeast Saccharomyces cerevisiae. Substitution of the highly conserved proline residue in transmembrane segment VI of these receptors causes constitutive signaling. This proline residue may facilitate folding of GPCRs into native, inactive conformations, and/or mediate a...

  4. Endocytosis of activated receptors and clathrin-coated pit formation: deciphering the chicken or egg relationship

    1996-01-01

    The fundamental mechanisms by which receptors once targeted for endocytosis are found in coated pits is an important yet unresolved question. Specifically, are activated receptors simply trapped on encountering preexisting coated pits, subsequently being rapidly internalized? Or do the receptors themselves, by active recruitment, gather soluble coat and cytosolic components and initiate the rapid assembly of new coated pits that then mediate their internalization? To explore this question, we...

  5. Nuclear factor RIP140 modulates transcriptional activation by the estrogen receptor.

    Cavaillès, V; Dauvois, S; L'Horset, F; Lopez, G; Hoare, S.; Kushner, P J; Parker, M G

    1995-01-01

    A conserved region in the hormone-dependent activation domain AF2 of nuclear receptors plays an important role in transcriptional activation. We have characterized a novel nuclear protein, RIP140, that specifically interacts in vitro with this domain of the estrogen receptor. This interaction was increased by estrogen, but not by anti-estrogens and the in vitro binding capacity of mutant receptors correlates with their ability to stimulate transcription. RIP140 also interacts with estrogen re...

  6. Tryptophan at the transmembrane–cytosolic junction modulates thrombopoietin receptor dimerization and activation

    Defour, Jean-Philippe; Itaya, Miki; Gryshkova, Vitalina; Brett, Ian C.; Pecquet, Christian; Sato, Takeshi; Smith, Steven O.; Stefan N. Constantinescu

    2013-01-01

    Dimerization of single-pass membrane receptors is essential for activation. In the human thrombopoietin receptor (TpoR), a unique amphipathic RWQFP motif separates the transmembrane (TM) and intracellular domains. Using a combination of mutagenesis, spectroscopy, and biochemical assays, we show that W515 of this motif impairs dimerization of the upstream TpoR TM helix. TpoR is unusual in that a specific residue is required for this inhibitory function, which prevents receptor self-activation....

  7. Adipocyte insulin receptor activity maintains adipose tissue mass and lifespan.

    Friesen, Max; Hudak, Carolyn S; Warren, Curtis R; Xia, Fang; Cowan, Chad A

    2016-08-01

    Type 2 diabetes follows a well-defined progressive pathogenesis, beginning with insulin resistance in metabolic tissues such as the adipose. Intracellular signaling downstream of insulin receptor activation regulates critical metabolic functions of adipose tissue, including glucose uptake, lipogenesis, lipolysis and adipokine secretion. Previous studies have used the aP2 promoter to drive Cre recombinase expression in adipose tissue. Insulin receptor (IR) knockout mice created using this aP2-Cre strategy (FIRKO mice) were protected from obesity and glucose intolerance. Later studies demonstrated the promiscuity of the aP2 promoter, casting doubts upon the tissue specificity of aP2-Cre models. It is our goal to use the increased precision of the Adipoq promoter to investigate adipocyte-specific IR function. Towards this end we generated an adipocyte-specific IR knockout (AIRKO) mouse using an Adipoq-driven Cre recombinase. Here we report AIRKO mice are less insulin sensitive throughout life, and less glucose tolerant than wild-type (WT) littermates at the age of 16 weeks. In contrast to WT littermates, the insulin sensitivity of AIRKO mice is unaffected by age or dietary regimen. At any age, AIRKO mice are comparably insulin resistant to old or obese WT mice and have a significantly reduced lifespan. Similar results were obtained when these phenotypes were re-examined in FIRKO mice. We also found that the AIRKO mouse is protected from high-fat diet-induced weight gain, corresponding with a 90% reduction in tissue weight of major adipose depots compared to WT littermates. Adipose tissue mass reduction is accompanied by hepatomegaly and increased hepatic steatosis. These data indicate that adipocyte IR function is crucial to systemic energy metabolism and has profound effects on adiposity, hepatic homeostasis and lifespan. PMID:27246738

  8. Kallikrein Promotes Inflammation in Human Dental Pulp Cells Via Protease-Activated Receptor-1.

    Hayama, Tomomi; Kamio, Naoto; Okabe, Tatsu; Muromachi, Koichiro; Matsushima, Kiyoshi

    2016-07-01

    Plasma kallikrein (KLKB1), a serine protease, cleaves high-molecular weight kininogen to produce bradykinin, a potent vasodilator and pro-inflammatory peptide. In addition, KLKB1 activates plasminogen and other leukocyte and blood coagulation factors and processes pro-enkephalin, prorenin, and C3. KLKB1 has also been shown to cleave protease-activated receptors in vascular smooth muscle cells to regulate the expression of epidermal growth factor receptor. In this study, we investigated KLKB1-dependent inflammation and activation of protease-activated receptor-1 in human dental pulp cells. These cells responded to KLKB1 stimulation by increasing intracellular Ca(2+) , upregulating cyclooxygenase-2, and secreting prostaglandin E2 . Remarkably, SCH79797, an antagonist of protease-activated receptor-1, blocked these effects. Thus, these data indicate that KLKB1 induces inflammatory reactions in human dental tissues via protease-activated receptor 1. J. Cell. Biochem. 117: 1522-1528, 2016. © 2015 Wiley Periodicals, Inc. PMID:26566265

  9. Endothelial Cells Promote Pigmentation through Endothelin Receptor B Activation.

    Regazzetti, Claire; De Donatis, Gian Marco; Ghorbel, Houda Hammami; Cardot-Leccia, Nathalie; Ambrosetti, Damien; Bahadoran, Philippe; Chignon-Sicard, Bérengère; Lacour, Jean-Philippe; Ballotti, Robert; Mahns, Andre; Passeron, Thierry

    2015-12-01

    Findings of increased vascularization in melasma lesions and hyperpigmentation in acquired bilateral telangiectatic macules suggested a link between pigmentation and vascularization. Using high-magnification digital epiluminescence dermatoscopy, laser confocal microscopy, and histological examination, we showed that benign vascular lesions of the skin have restricted but significant hyperpigmentation compared with the surrounding skin. We then studied the role of microvascular endothelial cells in regulating skin pigmentation using an in vitro co-culture model using endothelial cells and melanocytes. These experiments showed that endothelin 1 released by microvascular endothelial cells induces increased melanogenesis signaling, characterized by microphthalmia-associated transcription factor phosphorylation, and increased tyrosinase and dopachrome tautomerase levels. Immunostaining for endothelin 1 in vascular lesions confirmed the increased expression on the basal layer of the epidermis above small vessels compared with perilesional skin. Endothelin acts through the activation of endothelin receptor B and the mitogen-activated protein kinase, extracellular signal-regulated kinase (ERK)1/2, and p38, to induce melanogenesis. Finally, culturing of reconstructed skin with microvascular endothelial cells led to increased skin pigmentation that could be prevented by inhibiting EDNRB. Taken together these results demonstrated the role of underlying microvascularization in skin pigmentation, a finding that could open new fields of research for regulating physiological pigmentation and for treating pigmentation disorders such as melasma. PMID:26308584

  10. Tyrosine kinase JAK1 is associated with the granulocyte-colony-stimulating factor receptor and both become tyrosine-phosphorylated after receptor activation.

    Nicholson, S. E.; Oates, A. C.; Harpur, A G; Ziemiecki, A; Wilks, A F; Layton, J E

    1994-01-01

    Granulocyte-colony-stimulating factor (G-CSF) stimulates the proliferation and differentiation of cells of the neutrophil lineage by interaction with a specific receptor. Early signal transduction events following G-CSF receptor activation were studied. We detected tyrosine phosphorylation of both the G-CSF receptor and the protein tyrosine kinase JAK1 following G-CSF binding to the human G-CSF receptor. In vitro, the kinase activity of JAK1 was increased by G-CSF stimulation. Coimmunoprecipi...

  11. Selenoprotein W controls epidermal growth factor receptor surface expression, activation and degradation via receptor ubiquitination

    Epidermal growth factor (EGF) receptor (EGFR) is the founding member of the ErbB family of growth factor receptors that modulate a complex network of intracellular signaling pathways controlling growth, proliferation and differentiation. Selenoprotein W (SEPW1) is a diet-regulated, highly conserved...

  12. Plasma soluble urokinase plasminogen activator receptor in children with urinary tract infection

    Wittenhagen, Per; Andersen, Jesper Brandt; Hansen, Anita; Lindholm, Lone; Rønne, Frederik Malmborg; Theil, Jørn; Tvede, Michael; Eugen-Olsen, Jesper

    2011-01-01

    In this prospective study we investigated the role of plasma levels of soluble urokinase plasminogen activator receptor (suPAR) in children with urinary tract infection.......In this prospective study we investigated the role of plasma levels of soluble urokinase plasminogen activator receptor (suPAR) in children with urinary tract infection....

  13. The adipogenic acetyltransferase Tip60 targets activation function 1 of peroxisome proliferator-activated receptor gamma

    van Beekum, Olivier; Brenkman, Arjan B; Grøntved, Lars;

    2008-01-01

    proteins with which this nuclear receptor interacts under specific conditions. Here we identify the HIV-1 Tat-interacting protein 60 (Tip60) as a novel positive regulator of PPARgamma transcriptional activity. Using tandem mass spectrometry, we found that PPARgamma and the acetyltransferase Tip60 interact......-mediated reduction of Tip60 protein impairs differentiation of 3T3-L1 preadipocytes. Taken together, these findings qualify the acetyltransferase Tip60 as a novel adipogenic factor....

  14. The neuroprotective role of erythropoietin in the management of acute ischaemic stroke: from bench to bedside.

    Ntaios, G; Savopoulos, C; Chatzinikolaou, A; Hatzitolios, A I

    2008-12-01

    Recombinant human erythropoietin was produced soon after the discovery of the erythropoietin gene in 1985 and since then, it is used in various clinical conditions such as chronic renal failure. Moreover, experimental studies have shown that erythropoietin exerts neuroprotective action as well. Recently, a clinical trial yielded promising results concerning the use of erythropoietin in stroke management. In this review, we summarize the main data which suggest that recombinant human erythropoietin and its analogues may indeed have a role in stroke treatment. PMID:18513348

  15. Modulation of receptors and adenylate cyclase activity during sucrose feeding, food deprivation, and cold exposure

    Thermogenesis in brown adipose tissue (BAT) serves as a regulator of body temperature and weight maintenance. Thermogenesis can be stimulated by catecholamine activation of adenylate cyclase through the β-adrenergic receptor. To investigate the effects of sucrose feeding, food deprivation, and cold exposure on the β-adrenergic pathway, adenylate cyclase activity and β-adrenergic receptors were assessed in rat BAT after 2 wk of sucrose feeding, 2 days of food deprivation, or 2 days of cold exposure. β-Adrenergic receptors were identified in BAT using [125I]iodocyanopindolol. Binding sites had the characteristics of mixed β1- and β2-type adrenergic receptors at a ratio of 60/40. After sucrose feeding or cold exposure, there was the expected increase in BAT mitochondrial mass as measured by total cytochrome-c oxidase activity but a decrease in β-adrenergic receptor density due to a loss of the β1-adrenergic subtype. This BAT β-adrenergic receptor downregulation was tissue specific, since myocardial β-adrenergic receptors were unchanged with either sucrose feeding or cold exposure. Forskolin-stimulated adenylate cyclase activity increased in BAT after sucrose feeding or cold exposure but not after food deprivation. These data suggest that in BAT, sucrose feeding or cold exposure result in downregulation of β-adrenergic receptors and that isoproterenol-stimulated adenylate cyclase activity was limited by receptor availability

  16. Modulation of receptors and adenylate cyclase activity during sucrose feeding, food deprivation, and cold exposure

    Scarpace, P.J.; Baresi, L.A.; Morley, J.E. (Veterans Administration Medical Center, Los Angeles, CA (USA) Univ. of California, Los Angeles (USA))

    1987-12-01

    Thermogenesis in brown adipose tissue (BAT) serves as a regulator of body temperature and weight maintenance. Thermogenesis can be stimulated by catecholamine activation of adenylate cyclase through the {beta}-adrenergic receptor. To investigate the effects of sucrose feeding, food deprivation, and cold exposure on the {beta}-adrenergic pathway, adenylate cyclase activity and {beta}-adrenergic receptors were assessed in rat BAT after 2 wk of sucrose feeding, 2 days of food deprivation, or 2 days of cold exposure. {beta}-Adrenergic receptors were identified in BAT using ({sup 125}I)iodocyanopindolol. Binding sites had the characteristics of mixed {beta}{sub 1}- and {beta}{sub 2}-type adrenergic receptors at a ratio of 60/40. After sucrose feeding or cold exposure, there was the expected increase in BAT mitochondrial mass as measured by total cytochrome-c oxidase activity but a decrease in {beta}-adrenergic receptor density due to a loss of the {beta}{sub 1}-adrenergic subtype. This BAT {beta}-adrenergic receptor downregulation was tissue specific, since myocardial {beta}-adrenergic receptors were unchanged with either sucrose feeding or cold exposure. Forskolin-stimulated adenylate cyclase activity increased in BAT after sucrose feeding or cold exposure but not after food deprivation. These data suggest that in BAT, sucrose feeding or cold exposure result in downregulation of {beta}-adrenergic receptors and that isoproterenol-stimulated adenylate cyclase activity was limited by receptor availability.

  17. Antileishmanial activity of the estrogen receptor modulator raloxifene.

    Juliana Q Reimão

    2014-05-01

    Full Text Available BACKGROUND: The treatment of leishmaniasis relies mostly on parenteral drugs with potentially serious adverse effects. Additionally, parasite resistance in the treatment of leishmaniasis has been demonstrated for the majority of drugs available, making the search for more effective and less toxic drugs and treatment regimens a priority for the control of leishmaniasis. The aims of this study were to evaluate the antileishmanial activity of raloxifene in vitro and in vivo and to investigate its mechanism of action against Leishmania amazonensis. METHODOLOGY/PRINCIPAL FINDINGS: Raloxifene was shown to possess antileishmanial activity in vitro against several species with EC50 values ranging from 30.2 to 38.0 µM against promastigotes and from 8.8 to 16.2 µM against intracellular amastigotes. Raloxifene's mechanism of action was investigated through transmission electron microscopy and labeling with propidium iodide, DiSBAC2(3, rhodamine 123 and monodansylcadaverine. Microscopic examinations showed that raloxifene treated parasites displayed autophagosomes and mitochondrial damage while the plasma membrane remained continuous. Nonetheless, plasma membrane potential was rapidly altered upon raloxifene treatment with initial hyperpolarization followed by depolarization. Loss of mitochondrial membrane potential was also verified. Treatment of L. amazonensis-infected BALB/c mice with raloxifene led to significant decrease in lesion size and parasite burden. CONCLUSIONS/SIGNIFICANCE: The results of this work extend the investigation of selective estrogen receptor modulators as potential candidates for leishmaniasis treatment. The antileishmanial activity of raloxifene was demonstrated in vitro and in vivo. Raloxifene produces functional disorder on the plasma membrane of L. amazonensis promastigotes and leads to functional and morphological disruption of mitochondria, which culminate in cell death.

  18. Sex-dependent regulation of hypoxic ventilation in mice and humans is mediated by erythropoietin

    Soliz, Jorge; Thomsen, Jonas Juhl; Soulage, Christophe;

    2009-01-01

    Acclimatization to hypoxic exposure relies on an elevated ventilation and erythropoietic activity. We recently proposed that erythropoietin (Epo) links both responses: apart from red blood cell production, cerebral and plasma Epo interact with the central and peripheral respiratory centers. Knowing...... that women cope better than men with reduced oxygen supply (as observed at high altitude), we analyzed the hypoxic ventilatory response in Epo-overexpressing transgenic male and female mice with high Epo levels in brain and plasma (Tg6) or in wild-type animals injected with recombinant human Epo (rh...

  19. A bacterial tyrosine phosphatase inhibits plant pattern recognition receptor activation

    Perception of pathogen-associated molecular patterns (PAMPs) by surface-localised pattern-recognition receptors (PRRs) is a key component of plant innate immunity. Most known plant PRRs are receptor kinases and initiation of PAMP-triggered immunity (PTI) signalling requires phosphorylation of the PR...

  20. Clinical Trial of Erythropoietin in Young Children With Cerebral Palsy.

    Cho, Kye Hee; Min, Kyunghoon; Lee, Seung Hoon; Lee, SunHee; An, SeongSoo A; Kim, MinYoung

    2016-09-01

    This study was conducted to assess the safety and efficacy of recombinant human erythropoietin in young children with cerebral palsy aged between 6 months and 3 years. All participants received subcutaneous recombinant human erythropoietin and 8 weeks of rehabilitation therapy. Adverse events, changes of vital signs, and hematologic tests were monitored up to 8 weeks postinjection. Functional measures of development at 4 and 8 weeks postinjection were compared with baseline values, and improvements were compared with those of an age-matched historical control group. Nine participants completed the trial from June 2012 to February 2015. No adverse events were related to recombinant human erythropoietin. Erythropoiesis was noted, although within normal range. Functional improvements were observed in all participants (P cerebral palsy. PMID:27233796

  1. Estrogen Receptor β Activation Rapidly Modulates Male Sexual Motivation through the Transactivation of Metabotropic Glutamate Receptor 1a.

    Seredynski, Aurore L; Balthazart, Jacques; Ball, Gregory F; Cornil, Charlotte A

    2015-09-23

    In addition to the transcriptional activity of their liganded nuclear receptors, estrogens, such as estradiol (E2), modulate cell functions, and consequently physiology and behavior, within minutes through membrane-initiated events. The membrane-associated receptors (mERs) underlying the acute effects of estrogens on behavior have mostly been documented in females where active estrogens are thought to be of ovarian origin. We determined here, by acute intracerebroventricular injections of specific agonists and antagonists, the type(s) of mERs that modulate rapid effects of brain-derived estrogens on sexual motivation in male Japanese quail. Brain aromatase blockade acutely inhibited sexual motivation. Diarylpropionitrile (DPN), an estrogen receptor β (ERβ)-specific agonist, and to a lesser extent 17α-estradiol, possibly acting through ER-X, prevented this effect. In contrast, drugs targeting ERα (PPT and MPP), GPR30 (G1 and G15), and the Gq-mER (STX) did not affect sexual motivation. The mGluR1a antagonist LY367385 significantly inhibited sexual motivation but mGluR2/3 and mGluR5 antagonists were ineffective. LY367385 also blocked the behavioral restoration induced by E2 or DPN, providing functional evidence that ERβ interacts with metabotropic glutamate receptor 1a (mGluR1a) signaling to acutely regulate male sexual motivation. Together these results show that ERβ plays a key role in sexual behavior regulation and the recently uncovered cooperation between mERs and mGluRs is functional in males where it mediates the acute effects of estrogens produced centrally in response to social stimuli. The presence of an ER-mGluR interaction in birds suggests that this mechanism emerged relatively early in vertebrate history and is well conserved. Significance statement: The membrane-associated receptors underlying the acute effects of estrogens on behavior have mostly been documented in females, where active estrogens are thought to be of ovarian origin. Using acute

  2. Characterization of human endothelial cell urokinase-type plasminogen activator receptor protein and messenger RNA

    Barnathan, E S; Kuo, A; Karikó, K; Rosenfeld, L; Murray, S C; Behrendt, N; Rønne, E; Weiner, D; Henkin, J; Cines, D B

    1990-01-01

    Human umbilical vein endothelial cells in culture (HUVEC) express receptors for urokinase-type plasminogen activators (u-PA). The immunochemical nature of this receptor and its relationship to u-PA receptors expressed by other cell types is unknown. Cross-linking active site-blocked u-PA to HUVEC...... endothelial cell cDNA library using the polymerase chain reaction (PCR) and oligonucleotide primers corresponding to the DNA sequence of the receptor cloned from transformed human fibroblasts (Roldan et al, EMBO J 9:467, 1990). The size of the cDNA (approximately 1,054 base pairs, bp) and the presence of a...

  3. The G Protein-Coupled Estrogen Receptor Agonist G-1 Inhibits Nuclear Estrogen Receptor Activity and Stimulates Novel Phosphoproteomic Signatures.

    Smith, L Cody; Ralston-Hooper, Kimberly J; Ferguson, P Lee; Sabo-Attwood, Tara

    2016-06-01

    Estrogen exerts cellular effects through both nuclear (ESR1 and ESR2) and membrane-bound estrogen receptors (G-protein coupled estrogen receptor, GPER); however, it is unclear if they act independently or engage in crosstalk to influence hormonal responses. To investigate each receptor's role in proliferation, transcriptional activation, and protein phosphorylation in breast cancer cells (MCF-7), we employed selective agonists for ESR1 propyl-pyrazole-triol (PPT), ESR2 diarylpropionitrile (DPN), and GPER (G-1) and also determined the impact of xenoestrogens bisphenol-A (BPA) and genistein on these effects. As anticipated, 17β-estradiol (E2), PPT, DPN, BPA, and genistein each enhanced proliferation and activation of an ERE-driven reporter gene whereas G-1 had no significant impact. However, G-1 significantly reduced E2-, PPT-, DPN-, BPA-, and genistein-induced proliferation and ERE activation at doses greater than 500 nM indicating that G-1 mediated inhibition is not ESR isotype specific. As membrane receptors initiate cascades of phosphorylation events, we performed a global phosphoproteomic analysis on cells exposed to E2 or G-1 to identify potential targets of receptor crosstalk via downstream protein phosphorylation targets. Of the 211 phosphorylated proteins identified, 40 and 13 phosphoproteins were specifically modified by E2 and G-1, respectively. Subnetwork enrichment analysis revealed several processes related to cell cycle were specifically enriched by G-1 compared with E2. Further there existed a number of newly identified proteins that were specifically phosphorylated by G-1. These phosphorylation networks highlight specific proteins that may modulate the inhibitory effects of G-1 and suggest a novel role for interference with nuclear receptor activity driven by E2 and xenoestrogens. PMID:27026707

  4. Group III metabotropic glutamate receptors and D1-like and D2-like dopamine receptors interact in the rat nucleus accumbens to influence locomotor activity.

    David, Hélène N; Abraini, Jacques H

    2002-03-01

    Evidence for functional interactions between metabotropic glutamate (mGlu) receptors and dopamine (DA) neurotransmission is now clearly established. In the present study, we investigated interactions between group III mGlu receptors and D1- and D2-like receptors in the nucleus accumbens (NAcc). Administration, into the NAcc, of the selective group III mGlu receptor agonist, AP4, resulted in an increase in locomotor activity, which was blocked by pretreatment with the group III mGlu receptor antagonist, MPPG. In addition, pretreatment with AP4 further blocked the increase in motor activity induced by the D1-like receptor agonist, SKF 38393, but potentiated the locomotor responses induced by either the D2-like receptor agonist, quinpirole, or coinfusion of SKF 38393 and quinpirole. MPPG reversed the effects of AP4 on the motor responses induced by D1-like and/or D2-like receptor activation. These results confirm that glutamate transmission may control DA-dependent locomotor function through mGlu receptors and further indicate that group III mGlu receptors oppose the behavioural response produced by D1-like receptor activation and favour those produced by D2-like receptor activation. PMID:11906529

  5. Oligomerization of opioid receptors with β2-adrenergic receptors: A role in trafficking and mitogen-activated protein kinase activation

    Jordan, B. A.; Trapaidze, N.; Gomes, I; Nivarthi, R.; Devi, L.A.

    2000-01-01

    G-protein-coupled receptors (GPCRs) have recently joined the list of cell surface receptors that dimerize. Dimerization has been shown to alter the ligand-binding, signaling, and trafficking properties of these receptors. Recent studies have shown that GPCRs heterodimerize with closely related members, resulting in the modulation of their function. In this study, we have attempted to determine whether members of GPCR superfamilies that couple to different families of G...

  6. Regulatory network of inflammation downstream of proteinase-activated receptors

    Hurst Robert E

    2007-03-01

    Full Text Available Abstract Background Protease-activated receptors (PAR are present in the urinary bladder, and their expression is altered in response to inflammation. PARs are a unique class of G protein-coupled that carry their own ligands, which remain cryptic until unmasked by proteolytic cleavage. Although the canonical signal transduction pathway downstream of PAR activation and coupling with various G proteins is known and leads to the rapid transcription of genes involved in inflammation, the effect of PAR activation on the downstream transcriptome is unknown. We have shown that intravesical administration of PAR-activating peptides leads to an inflammatory reaction characterized by edema and granulocyte infiltration. Moreover, the inflammatory response to intravesical instillation of known pro-inflammatory stimuli such as E. coli lipopolysaccharide (LPS, substance P (SP, and antigen was strongly attenuated by PAR1- and to a lesser extent by PAR2-deficiency. Results Here, cDNA array experiments determined inflammatory genes whose expression is dependent on PAR1 activation. For this purpose, we compared the alteration in gene expression in wild type and PAR1-/- mice induced by classical pro-inflammatory stimuli (LPS, SP, and antigen. 75 transcripts were considered to be dependent on PAR-1 activation and further annotated in silico by Ingenuity Pathways Analysis (IPA and gene ontology (GO. Selected transcripts were target validated by quantitative PCR (Q-PCR. Among PAR1-dependent transcripts, the following have been implicated in the inflammatory process: b2m, ccl7, cd200, cd63, cdbpd, cfl1, dusp1, fkbp1a, fth1, hspb1, marcksl1, mmp2, myo5a, nfkbia, pax1, plaur, ppia, ptpn1, ptprcap, s100a10, sim2, and tnfaip2. However, a balanced response to signals of injury requires a transient cellular activation of a panel of genes together with inhibitory systems that temper the overwhelming inflammation. In this context, the activation of genes such as dusp1 and

  7. Serum erythropoietin levels by radioimmunoassay in polycythaemia

    Birgegaard, G.; Miller, O.; Caro, J.; Erslev, A. (Cardeza Foundation for Hematological Research, Jefferson University, Philadelphia, Pa.)

    1982-01-01

    A radioimmunoassay (RIA) method for erythropoietin (Epo) was developed and validated against the polycythaemic mouse assay. The correlation was good, with a r=0.94. Several other criteria of specificity were also filled by the RIA, which had a lower detection limit of 5 mU/ml. The mean serum-Epo level in 6 patients with secondary polycythaemia, 50.2 +- 26.2 mU/ml, was significantly higher than in a group of 11 normal subjects, 28.7 +- 7.2 mU/ml (P<0.0002). However, the Epo level in 31 polycythaemia vera (PV) patients, M = 21.9 +- 6.6 mU/ml, was not significantly different from normal (P = 0.006). Since previous studies with bioassay of heat-treated and concentrated plasma samples have shown a decreased serum-Epo level in PV, Epo levels were measured before and after heat treatment and concentration of samples from normals and polycythaemics. It was found that the levels of immunoreactive material increased after heat treatment and 40 times concentration in samples from normals and patients with secondary polycythaemias, but decreased in PV. We conclude that the Epo levels in serum in the low range measured by our and previous RIA:s probably are not true Epo levels but are partly due to an unspecific serum effect, that was removed by heat treatment.

  8. Inappropriate erythropoietin secretion in polycythemia vera

    Chikkappa, G. (Brookhaven National Lab., Upton, NY); Burlington, H.; Chanana, A.D.; Chandra, P.; Cronkite, E.P.; Reincke, U.; Zanjani, E.

    1977-01-01

    A patient with classical polycythemia vera (PV) was found to have an inappropriately elevated serum erythropoietin (Ep) level. Investigations did not reveal any lesion or blood abnormality known to be associated with excessive Ep production and erythrocytosis. Sudden withdrawal of blood to reduce the Hb and Hct from 18.5 gm% and 56% to 13.6 gm% and 41.5%, respectively, resulted in an increment of serum Ep to abnormal level. With iron treatment there was a brisk return of Hb and Hct to prebleeding levels which was associated with reduction in the serum Ep. The inverse relationship between the EP and Hb or Hct is inconsistent with the presence of excessive Ep-producing lesion. These results suggested that the threshold for Ep secretion from normal Ep-secreting tissue to Hb and Hct levels is set at an abnormal level. This patient's marrow cells when cultured in vitro in the absence of Ep, unlike other PV patients' (except one) marrow cells, did not grow erythroid colonies. In the presence of Ep, however, the colonies comparable to those formed from normal marrow cultures were obtained. These results suggested that his marrow erythropoietic cells were neither Ep independent nor Ep-hyperresponsive, as has been suggested by some investigators for erythropoiesis in PV. This patient presents phenomena that hitherto have not been reported.

  9. Increased Synthesis of Liver Erythropoietin with CKD.

    de Seigneux, Sophie; Lundby, Anne-Kristine Meinild; Berchtold, Lena; Berg, Anders H; Saudan, Patrick; Lundby, Carsten

    2016-08-01

    Anemia of CKD seems to be related to impaired production of renal erythropoietin (Epo). The glycosylation pattern of Epo depends on the synthesizing cell and thus, can indicate its origin. We hypothesized that synthesis of Epo from nonkidney cells increases to compensate for insufficient renal Epo production during CKD. We determined plasma Epo levels and Epo glycosylation patterns in 33 patients with CKD before undergoing dialysis and nine patients with CKD undergoing dialysis. We compared these values with values obtained in healthy volunteers and other controls. Although patients with CKD before undergoing dialysis had median (interquartile range) Epo levels higher than those of healthy controls (13.8 IU/L; interquartile range, 10.0-20.7 IU/L versus 8.4 IU/L; interquartile range, 7.6-9.0 IU/L; PPMI), Epo glycosylation in patients with CKD before undergoing dialysis (PMI=36.1±11.7%) differed from that in healthy controls (PMI=9.2±3.8%; PPMI=53.9±10.6%; P>0.05), which contains mainly liver-derived Epo. Furthermore, glycosylation modification correlated with eGFR loss. These results suggest that patients with CKD maintain persistent Epo synthesis despite declining renal function, and this maintenance may result in part from increased liver Epo synthesis. PMID:26757994

  10. Erythropoietin, uncertainty principle and cancer related anaemia

    Bennett Charles L

    2002-09-01

    Full Text Available Abstract Background This study was designed to evaluate if erythropoietin (EPO is effective in the treatment of cancer related anemia, and if its effect remains unchanged when data are analyzed according to various clinical and methodological characteristics of the studies. We also wanted to demonstrate that cumulative meta-analysis (CMA can be used to resolve uncertainty regarding clinical questions. Methods Systematic Review (SR of the published literature on the role of EPO in cancer-related anemia. A cumulative meta-analysis (CMA using a conservative approach was performed to determine the point in time when uncertainty about the effect of EPO on transfusion-related outcomes could be considered resolved. Participants: Patients included in randomized studies that compared EPO versus no therapy or placebo. Main outcome measures: Number of patients requiring transfusions. Results Nineteen trials were included. The pooled results indicated a significant effect of EPO in reducing the number of patients requiring transfusions [odds ratio (OR = 0.41; 95%CI: 0.33 to 0.5; p 11.5 g/dl. We identified 1995 as the point in time when a statistically significant effect of EPO was demonstrated and after which we considered that uncertainty about EPO efficacy was resolved. Conclusion EPO is effective in the treatment of anemia in cancer patients. This could have already been known in 1995 if a CMA had been performed at that time.

  11. Erythropoietin, uncertainty principle and cancer related anaemia

    Clark, Otavio; Adams, Jared R; Bennett, Charles L; Djulbegovic, Benjamin

    2002-01-01

    Background This study was designed to evaluate if erythropoietin (EPO) is effective in the treatment of cancer related anemia, and if its effect remains unchanged when data are analyzed according to various clinical and methodological characteristics of the studies. We also wanted to demonstrate that cumulative meta-analysis (CMA) can be used to resolve uncertainty regarding clinical questions. Methods Systematic Review (SR) of the published literature on the role of EPO in cancer-related anemia. A cumulative meta-analysis (CMA) using a conservative approach was performed to determine the point in time when uncertainty about the effect of EPO on transfusion-related outcomes could be considered resolved. Participants: Patients included in randomized studies that compared EPO versus no therapy or placebo. Main outcome measures: Number of patients requiring transfusions. Results Nineteen trials were included. The pooled results indicated a significant effect of EPO in reducing the number of patients requiring transfusions [odds ratio (OR) = 0.41; 95%CI: 0.33 to 0.5; p 11.5 g/dl. We identified 1995 as the point in time when a statistically significant effect of EPO was demonstrated and after which we considered that uncertainty about EPO efficacy was resolved. Conclusion EPO is effective in the treatment of anemia in cancer patients. This could have already been known in 1995 if a CMA had been performed at that time. PMID:12270068

  12. Activation of transforming potential of the human insulin receptor gene

    A retrovirus containing part of the human insulin receptor (hIR) gene was constructed by replacing ros sequences in the avian sarcoma virus UR2 with hIR cDNA sequences coding for 46 amino acids of the extracellular domain and the entire transmembrane and cytoplasmic domains of the β subunit of hIR. The resulting virus, named UIR, contains the hIR sequence fused to the 5' portion of the UR2 gag gene coding for p19. UIR is capable of transforming chicken embryo fibroblasts and promoting formation of colonies in soft agar; however, it does not form tumors in vivo. A variant that arose from the parental UIR is capable of efficiently inducing sarcomas in vivo. UIR-transformed cells exhibit higher rates of glucose uptake and growth than normal cells. The 4-kilobase UIR genome codes for a membrane-associated, glycosylated gag-hIR fusion protein of 75 kDa designated P75/sup gag-hir/. P75/sup gag-hir/ contains a protein tyrosine kinase activity that is capable of undergoing autophosphorylation and of phosphorylating foreign substrates in vitro; it is phosphorylated at both serine and tyrosine residues in vivo

  13. Activation of transforming potential of the human insulin receptor gene

    Wang, L.H.; Lin, B.; Jong, S.M.J.; Dixon, D.; Ellis, L.; Roth, R.A.; Rutter, W.J.

    1987-08-01

    A retrovirus containing part of the human insulin receptor (hIR) gene was constructed by replacing ros sequences in the avian sarcoma virus UR2 with hIR cDNA sequences coding for 46 amino acids of the extracellular domain and the entire transmembrane and cytoplasmic domains of the ..beta.. subunit of hIR. The resulting virus, named UIR, contains the hIR sequence fused to the 5' portion of the UR2 gag gene coding for p19. UIR is capable of transforming chicken embryo fibroblasts and promoting formation of colonies in soft agar; however, it does not form tumors in vivo. A variant that arose from the parental UIR is capable of efficiently inducing sarcomas in vivo. UIR-transformed cells exhibit higher rates of glucose uptake and growth than normal cells. The 4-kilobase UIR genome codes for a membrane-associated, glycosylated gag-hIR fusion protein of 75 kDa designated P75/sup gag-hir/. P75/sup gag-hir/ contains a protein tyrosine kinase activity that is capable of undergoing autophosphorylation and of phosphorylating foreign substrates in vitro; it is phosphorylated at both serine and tyrosine residues in vivo

  14. DCC constrains tumour progression via its dependence receptor activity.

    Castets, Marie; Broutier, Laura; Molin, Yann; Brevet, Marie; Chazot, Guillaume; Gadot, Nicolas; Paquet, Armelle; Mazelin, Laetitia; Jarrosson-Wuilleme, Loraine; Scoazec, Jean-Yves; Bernet, Agnès; Mehlen, Patrick

    2012-02-23

    The role of deleted in colorectal carcinoma (DCC) as a tumour suppressor has been a matter of debate for the past 15 years. DCC gene expression is lost or markedly reduced in the majority of advanced colorectal cancers and, by functioning as a dependence receptor, DCC has been shown to induce apoptosis unless engaged by its ligand, netrin-1 (ref. 2). However, so far no animal model has supported the view that the DCC loss-of-function is causally implicated as predisposing to aggressive cancer development. To investigate the role of DCC-induced apoptosis in the control of tumour progression, here we created a mouse model in which the pro-apoptotic activity of DCC is genetically silenced. Although the loss of DCC-induced apoptosis in this mouse model is not associated with a major disorganization of the intestines, it leads to spontaneous intestinal neoplasia at a relatively low frequency. Loss of DCC-induced apoptosis is also associated with an increase in the number and aggressiveness of intestinal tumours in a predisposing APC mutant context, resulting in the development of highly invasive adenocarcinomas. These results demonstrate that DCC functions as a tumour suppressor via its ability to trigger tumour cell apoptosis. PMID:22158121

  15. Antipeptide antibody that specifically inhibits insulin receptor autophosphorylation and protein kinase activity

    Two site-specific antibodies that immunoprecipitate the human insulin receptor have been prepared by immunizing rabbits with chemically synthesized peptides derived from the cDNA-predicted amino acid sequence of the β subunit of the proreceptor. Antibodies to the carboxyl terminus (AbP5) and to a domain around tyrosine-960 (AbP4) specifically recognize the β subunit of the receptor on immunoblots. Both antibodies immunoprecipitated 125I-labeled insulin-receptor complexes and the autophosphorylated receptor. Although neither antibody inhibited insulin binding to the receptor, both insulin-dependent autophosphorylation and exogenous substrate phosphorylation were inhibited by AbP4. Inhibition by AbP4 was dependent upon the phosphorylation state of the receptor; it was not detected when the receptor was autophosphorylated prior to addition of AbP4. AbP4 did not inhibit activity of the related epidermal growth factor (EGF)-receptor tyrosine protein kinase nor did it inhibit the activity of cAMP-dependent kinase or protein kinase C. The observation that an antibody directed to residues 952-967 of the proreceptor neutralizes the protein kinase activity of the β subunit suggest that this region may play a critical role in the function of the hormone-dependent, protein tyrosine-specific kinase activity of the insulin receptor

  16. A Molecular Mechanism for Sequential Activation of a G Protein-Coupled Receptor

    Grundmann, Manuel; Tikhonova, Irina G; Hudson, Brian D; Smith, Nicola J; Mohr, Klaus; Ulven, Trond; Milligan, Graeme; Kenakin, Terry; Kostenis, Evi

    2016-01-01

    Ligands targeting G protein-coupled receptors (GPCRs) are currently classified as either orthosteric, allosteric, or dualsteric/bitopic. Here, we introduce a new pharmacological concept for GPCR functional modulation: sequential receptor activation. A hallmark feature of this is a stepwise ligand...... binding mode with transient activation of a first receptor site followed by sustained activation of a second topographically distinct site. We identify 4-CMTB (2-(4-chlorophenyl)-3-methyl-N-(thiazol-2-yl)butanamide), previously classified as a pure allosteric agonist of the free fatty acid receptor 2, as...... mutational and pharmacological perturbations along with computational methods, and propose a kinetic model applicable to the analysis of sequential receptor activation. We envision this form of dynamic agonism as a common principle of nature to spatiotemporally encode cellular information....

  17. Different response to human recombinant erythropoietin in patients undergoing hemodialysis treatment

    Petković Nenad

    2013-01-01

    Full Text Available Introduction. Treatment with recombinant human erythropoietin enabled the correction of anemia in the patients on regular hemodialysis but large individual differences in the dose required to achieve the target hemoglobin level were observed. In this study the erythropoietin resistance index was calculated in patients on hemodialysis in order to examine variations in the response to erythropoietin and factors that influence it. Material and Methods. The study included 48 patients (25 males of mean age 67.5 years, who had been on regular hemodialysis in Šamac for 43.9 months on average. All were treated with erythropoietin from the beginning of hemodialysis treatment. Their response to erythropoietin therapy was estimated by the erythropoietin resistance index. Results. The use of erythropoietin enabled the correction of anemia but different doses were needed to achieve and maintain the target hemoglobin level. The individual weekly dose of erythropoietin ranged from 15 U/kg/week to 244 U/kg/week and the erythropoietin resistance index ranged from 0.13 U/kg/week/g/l to 2.46 U/kg/week/g/l. A satisfactory erythropoietin response with erythropoietin resistance index below 0.5 U/kg/week/g/l was found in 14 (30% patients, while 19 (40% patients had this index above 0.7 U/kg/week/g/l and 10 (21% above 0.9 U/kg/week/g/l. Multivariate linear regression analysis detected C-reactive protein as a significant predictor of erythropoietin resistance index. Conclusion. Target hemoglobin levels were achieved and maintained by different doses of erythropoietin in individual patients, which resulted in great individual differences in response as estimated by the erythropoietin resistance index. Multivariate analysis indicated C-reactive protein as a variable significantly associated with this index.

  18. Quantitative structure-activity relationships of selective antagonists of glucagon receptor using QuaSAR descriptors.

    Manoj Kumar, Palanivelu; Karthikeyan, Chandrabose; Hari Narayana Moorthy, Narayana Subbiah; Trivedi, Piyush

    2006-11-01

    In the present paper, quantitative structure activity relationship (QSAR) approach was applied to understand the affinity and selectivity of a novel series of triaryl imidazole derivatives towards glucagon receptor. Statistically significant and highly predictive QSARs were derived for glucagon receptor inhibition by triaryl imidazoles using QuaSAR descriptors of molecular operating environment (MOE) employing computer-assisted multiple regression procedure. The generated QSAR models revealed that factors related to hydrophobicity, molecular shape and geometry predominantly influences glucagon receptor binding affinity of the triaryl imidazoles indicating the relevance of shape specific steric interactions between the molecule and the receptor. Further, QSAR models formulated for selective inhibition of glucagon receptor over p38 mitogen activated protein (MAP) kinase of the compounds in the series highlights that the same structural features, which influence the glucagon receptor affinity, also contribute to their selective inhibition. PMID:17077558

  19. Function of the cytoplasmic tail of human calcitonin receptor-like receptor in complex with receptor activity-modifying protein 2

    Receptor activity-modifying protein 2 (RAMP2) enables calcitonin receptor-like receptor (CRLR) to form an adrenomedullin (AM)-specific receptor. Here we investigated the function of the cytoplasmic C-terminal tail (C-tail) of human (h)CRLR by co-transfecting its C-terminal mutants into HEK-293 cells stably expressing hRAMP2. Deleting the C-tail from CRLR disrupted AM-evoked cAMP production or receptor internalization, but did not affect [125I]AM binding. We found that CRLR residues 428-439 are required for AM-evoked cAMP production, though deleting this region had little effect on receptor internalization. Moreover, pretreatment with pertussis toxin (100 ng/mL) led to significant increases in AM-induced cAMP production via wild-type CRLR/RAMP2 complexes. This effect was canceled by deleting CRLR residues 454-457, suggesting Gi couples to this region. Flow cytometric analysis revealed that CRLR truncation mutants lacking residues in the Ser/Thr-rich region extending from Ser449 to Ser467 were unable to undergo AM-induced receptor internalization and, in contrast to the effect on wild-type CRLR, overexpression of GPCR kinases-2, -3 and -4 failed to promote internalization of CRLR mutants lacking residues 449-467. Thus, the hCRLR C-tail is crucial for AM-evoked cAMP production and internalization of the CRLR/RAMP2, while the receptor internalization is dependent on the aforementioned GPCR kinases, but not Gs coupling.

  20. The effects of erythropoietin signaling on telomerase regulation in non-erythroid malignant and non-malignant cells

    Highlights: • We assumed that some of erythropoietin adverse effects may be mediated by telomerase activity. • EPO administration increased telomerase activity, cells proliferation and migration. • The inhibition of telomerase modestly repressed the proliferative effect of erythropoietin. • Telomere shortening caused by long term inhibition of the enzyme totally abolished that effect. • This effect was mediated via the Lyn–AKT axis and not by the canonical JAK2–STAT pathway. - Abstract: Treatment with erythropoietin (EPO) in several cancers is associated with decreased survival due to cancer progression. Due to the major importance of telomerase in cancer biology we hypothesized that some of these effects may be mediated through EPO effect on telomerase. For this aim we explored the possible effects of EPO on telomerase regulation, cell migration and chemosensitivity in non-erythroid malignant and non-malignant cells. Cell proliferation, telomerase activity (TA) and cell migration increased in response to EPO. EPO had no effect on cancer cells sensitivity to cisplatinum and on the cell cycle status. The inhibition of telomerase modestly repressed the proliferative effect of EPO. Telomere shortening caused by long term inhibition of the enzyme abolished the effect of EPO, suggesting that EPO effects on cancer cells are related to telomere dynamics. TA was correlated with the levels of Epo-R. The increase in TA was mediated post-translationally through the Lyn-Src and not the canonical JAK2 pathway

  1. The effects of erythropoietin signaling on telomerase regulation in non-erythroid malignant and non-malignant cells

    Uziel, Orit, E-mail: Oritu@clalit.org.il [Felsenstein Medical Research Center, Sackler School of Medicine, Tel-Aviv University, Ramat-Aviv (Israel); Kanfer, Gil [Felsenstein Medical Research Center, Sackler School of Medicine, Tel-Aviv University, Ramat-Aviv (Israel); Dep. of Human Molecular Genetics and Biochemistry, Sackler School of Medicine, Tel-Aviv University, Ramat-Aviv (Israel); Beery, Einat [Felsenstein Medical Research Center, Sackler School of Medicine, Tel-Aviv University, Ramat-Aviv (Israel); Yelin, Dana; Shepshelovich, Daniel [Medicine A, Sackler School of Medicine, Tel-Aviv University, Ramat-Aviv (Israel); Bakhanashvili, Mary [Unit of Infectious Diseases, Sheba Medical Center, Tel-Hashomer (Israel); Nordenberg, Jardena [Felsenstein Medical Research Center, Sackler School of Medicine, Tel-Aviv University, Ramat-Aviv (Israel); Dep. of Human Molecular Genetics and Biochemistry, Sackler School of Medicine, Tel-Aviv University, Ramat-Aviv (Israel); Endocrinology Laboratory, Beilinson Medical Center, Petah-Tikva (Israel); Lahav, Meir [Felsenstein Medical Research Center, Sackler School of Medicine, Tel-Aviv University, Ramat-Aviv (Israel); Medicine A, Sackler School of Medicine, Tel-Aviv University, Ramat-Aviv (Israel)

    2014-07-18

    Highlights: • We assumed that some of erythropoietin adverse effects may be mediated by telomerase activity. • EPO administration increased telomerase activity, cells proliferation and migration. • The inhibition of telomerase modestly repressed the proliferative effect of erythropoietin. • Telomere shortening caused by long term inhibition of the enzyme totally abolished that effect. • This effect was mediated via the Lyn–AKT axis and not by the canonical JAK2–STAT pathway. - Abstract: Treatment with erythropoietin (EPO) in several cancers is associated with decreased survival due to cancer progression. Due to the major importance of telomerase in cancer biology we hypothesized that some of these effects may be mediated through EPO effect on telomerase. For this aim we explored the possible effects of EPO on telomerase regulation, cell migration and chemosensitivity in non-erythroid malignant and non-malignant cells. Cell proliferation, telomerase activity (TA) and cell migration increased in response to EPO. EPO had no effect on cancer cells sensitivity to cisplatinum and on the cell cycle status. The inhibition of telomerase modestly repressed the proliferative effect of EPO. Telomere shortening caused by long term inhibition of the enzyme abolished the effect of EPO, suggesting that EPO effects on cancer cells are related to telomere dynamics. TA was correlated with the levels of Epo-R. The increase in TA was mediated post-translationally through the Lyn-Src and not the canonical JAK2 pathway.

  2. Dopamine receptor activation modulates GABA neuron migration from the basal forebrain to the cerebral cortex.

    Crandall, James E; McCarthy, Deirdre M; Araki, Kiyomi Y; Sims, John R; Ren, Jia-Qian; Bhide, Pradeep G

    2007-04-01

    GABA neurons of the cerebral cortex and other telencephalic structures are produced in the basal forebrain and migrate to their final destinations during the embryonic period. The embryonic basal forebrain is enriched in dopamine and its receptors, creating a favorable environment for dopamine to influence GABA neuron migration. However, whether dopamine receptor activation can influence GABA neuron migration is not known. We show that dopamine D1 receptor activation promotes and D2 receptor activation decreases GABA neuron migration from the medial and caudal ganglionic eminences to the cerebral cortex in slice preparations of embryonic mouse forebrain. Slice preparations from D1 or D2 receptor knock-out mouse embryos confirm the findings. In addition, D1 receptor electroporation into cells of the basal forebrain and pharmacological activation of the receptor promote migration of the electroporated cells to the cerebral cortex. Analysis of GABA neuron numbers in the cerebral wall of the dopamine receptor knock-out mouse embryos further confirmed the effects of dopamine receptor activation on GABA neuron migration. Finally, dopamine receptor activation mobilizes striatal neuronal cytoskeleton in a manner consistent with the effects on neuronal migration. These data show that impairing the physiological balance between D1 and D2 receptors can alter GABA neuron migration from the basal forebrain to the cerebral cortex. The intimate relationship between dopamine and GABA neuron development revealed here may offer novel insights into developmental disorders such as schizophrenia, attention deficit or autism, and fetal cocaine exposure, all of which are associated with dopamine and GABA imbalance. PMID:17409246

  3. The orphan nuclear receptor Rev-Erbalpha is a peroxisome proliferator-activated receptor (PPAR) gamma target gene and promotes PPARgamma-induced adipocyte differentiation

    Fontaine, Coralie; Dubois, Guillaume; Duguay, Yannick;

    2003-01-01

    Rev-Erbalpha (NR1D1) is an orphan nuclear receptor encoded on the opposite strand of the thyroid receptor alpha gene. Rev-Erbalpha mRNA is induced during adipocyte differentiation of 3T3-L1 cells, and its expression is abundant in rat adipose tissue. Peroxisome proliferator-activated receptor gamma...

  4. Gemfibrozil, a lipid lowering drug, inhibits the activation of primary human microglia via peroxisome proliferator-activated receptor β

    Jana, Malabendu; Pahan, Kalipada

    2012-01-01

    Microglial activation participates in the pathogenesis of various neuroinflammatory and neurodegenerative diseases. However, mechanisms by which microglial activation could be controlled are poorly understood. Peroxisome proliferator-activated receptors (PPAR) are transcription factors belonging to the nuclear receptor super family with diverse effect. This study underlines the importance of PPARβ/δ in mediating the anti-inflammatory effect of gemfibrozil, an FDA-approved lipid-lowering drug,...

  5. Activation of the A2A adenosine G-protein-coupled receptor by conformational selection.

    Ye, Libin; Van Eps, Ned; Zimmer, Marco; Ernst, Oliver P; Prosser, R Scott

    2016-05-12

    Conformational selection and induced fit are two prevailing mechanisms to explain the molecular basis for ligand-based activation of receptors. G-protein-coupled receptors are the largest class of cell surface receptors and are important drug targets. A molecular understanding of their activation mechanism is critical for drug discovery and design. However, direct evidence that addresses how agonist binding leads to the formation of an active receptor state is scarce. Here we use (19)F nuclear magnetic resonance to quantify the conformational landscape occupied by the adenosine A2A receptor (A2AR), a prototypical class A G-protein-coupled receptor. We find an ensemble of four states in equilibrium: (1) two inactive states in millisecond exchange, consistent with a formed (state S1) and a broken (state S2) salt bridge (known as 'ionic lock') between transmembrane helices 3 and 6; and (2) two active states, S3 and S3', as identified by binding of a G-protein-derived peptide. In contrast to a recent study of the β2-adrenergic receptor, the present approach allowed identification of a second active state for A2AR. Addition of inverse agonist (ZM241385) increases the population of the inactive states, while full agonists (UK432097 or NECA) stabilize the active state, S3', in a manner consistent with conformational selection. In contrast, partial agonist (LUF5834) and an allosteric modulator (HMA) exclusively increase the population of the S3 state. Thus, partial agonism is achieved here by conformational selection of a distinct active state which we predict will have compromised coupling to the G protein. Direct observation of the conformational equilibria of ligand-dependent G-protein-coupled receptor and deduction of the underlying mechanisms of receptor activation will have wide-reaching implications for our understanding of the function of G-protein-coupled receptor in health and disease. PMID:27144352

  6. Oral administration of protease inhibits enterotoxigenic Escherichia coli receptor activity in piglet small intestine.

    Mynott, T L; Luke, R K; Chandler, D S

    1996-01-01

    The virulence of enterotoxigenic Escherichia coli (ETEC) is attributed to their ability to adhere via fimbrial adhesins to specific receptors located on the intestinal mucosa. A novel approach to preventing ETEC induced diarrhoea would be to prevent attachment of ETEC to intestine by proteolytically modifying the receptor attachment sites. This study aimed to examine the effect of bromelain, a proteolytic extract obtained from pineapple stems, on ETEC receptor activity in porcine small intest...

  7. Identification of Essential Cannabinoid-binding Domains: STRUCTURAL INSIGHTS INTO EARLY DYNAMIC EVENTS IN RECEPTOR ACTIVATION*

    Shim, Joong-Youn; Bertalovitz, Alexander C.; Kendall, Debra A.

    2011-01-01

    The classical cannabinoid agonist HU210, a structural analog of (−)-Δ9-tetrahydrocannabinol, binds to brain cannabinoid (CB1) receptors and activates signal transduction pathways. To date, an exact molecular description of the CB1 receptor is not yet available. Utilizing the minor binding pocket of the CB1 receptor as the primary ligand interaction site, we explored HU210 binding using lipid bilayer molecular dynamics (MD) simulations. Among the potential ligand contact residues, we identifie...

  8. Functional Characterization of Nuclear Receptor and Co-activator Binding Loci in the Human Genome

    Maguire, Kim, (Thesis)

    2010-01-01

    Steroid hormones, such as oestrogen, mediate their effects via activation of oestrogen regulated genes using nuclear receptors. Selective oestrogen receptor modulators (SERMs), such as Tamoxifen, are used to treat oestrogen responsive breast cancers, hctioning to act as oestrogen antagonists, preventing the oestrogen receptor biding DNA and blocking gene expression. However, Tamoxifen has been identified as an oestrogen agonist in other tissues which can often lead to secondary tumors in the ...

  9. Pharmacology and toxicology of fibrates as hypolipidemic drugs mediated by nuclear receptor peroxisome proliferator—activated receptor

    SugaT

    2002-01-01

    PPAR(peroxisome proliferator-activated receptor) is a family of nuclear receptor.In recent years,it has been focused for the discovery and development of new drugs which are mediated by PPARs.Fibrate hypolipidemic drugs are the specific and potent ligands to PPAR alpha and have been widely used for the treatment of hyperlipidemia.But these drugs induce hepatocarcinogenesis in rodent animals after the long-term administration.However,there are species differences on these phenomena which are not seen in mammals ioncluding human.To clarify the mechanism of carcinogenesis by these drugs in important for the evaluation of safety of these drugs in human.

  10. Prenatal Allospecific NK Cell Tolerance Hinges on Instructive Allorecognition through the Activating Receptor during Development.

    Alhajjat, Amir M; Strong, Beverly S; Lee, Amanda E; Turner, Lucas E; Wadhwani, Ram K; Ortaldo, John R; Heusel, Jonathan W; Shaaban, Aimen F

    2015-08-15

    Little is known about how the prenatal interaction between NK cells and alloantigens shapes the developing NK cell repertoire toward tolerance or immunity. Specifically, the effect on NK cell education arising from developmental corecognition of alloantigens by activating and inhibitory receptors with shared specificity is uncharacterized. Using a murine prenatal transplantation model, we examined the manner in which this seemingly conflicting input affects NK cell licensing and repertoire formation in mixed hematopoietic chimeras. We found that prenatal NK cell tolerance arose from the elimination of phenotypically hostile NK cells that express an allospecific activating receptor without coexpressing any allospecific inhibitory receptors. Importantly, the checkpoint for the system appeared to occur centrally within the bone marrow during the final stage of NK cell maturation and hinged on the instructive recognition of allogeneic ligand by the activating receptor rather than through the inhibitory receptor as classically proposed. Residual nondeleted hostile NK cells expressing only the activating receptor exhibited an immature, anergic phenotype, but retained the capacity to upregulate inhibitory receptor expression in peripheral sites. However, the potential for this adaptive change to occur was lost in developmentally mature chimeras. Collectively, these findings illuminate the intrinsic process in which developmental allorecognition through the activating receptor regulates the emergence of durable NK cell tolerance and establishes a new paradigm to fundamentally guide future investigations of prenatal NK cell-allospecific education. PMID:26136432

  11. Activity of recombinant trypsin isoforms on human proteinase-activated receptors (PAR): mesotrypsin cannot activate epithelial PAR-1, -2, but weakly activates brain PAR-1

    Grishina, Zoryana; Ostrowska, Ewa; Halangk, Walter; Sahin-Tóth, Miklós; Reiser, Georg

    2005-01-01

    Trypsin-like serine proteinases trigger signal transduction pathways through proteolytic cleavage of proteinase-activated receptors (PARs) in many tissues. Three members, PAR-1, PAR-2 and PAR-4, are trypsin substrates, as trypsinolytic cleavage of the extracellular N terminus produces receptor activation. Here, the ability of the three human pancreatic trypsin isoforms (cationic trypsin, anionic trypsin and mesotrypsin (trypsin IV)) as recombinant proteins was tested on PARs.Using fura 2 [Ca2...

  12. UVB increases urokinase-type plasminogen activator receptor (uPAR) expression.

    Marschall, C; Lengyel, E; Nobutoh, T; Braungart, E; Douwes, K; Simon, A; Magdolen, V; Reuning, U; Degitz, K

    1999-07-01

    Keratinocytes synthesize and secrete urokinase-type plasminogen activator, which binds to its specific receptor on keratinocytes. When bound to urokinase-type plasminogen activator receptor, urokinase-type plasminogen activator proteolytically converts surface bound plasminogen to plasmin, which in turn cleaves many extracellular components leading to pericellular proteolysis. The activation of the urokinase system has been observed during re-epithelialization of skin wounds and in lesions of the autoimmune blistering skin disease pemphigus. As pemphigus is photoinducible, we investigated the effect of ultraviolet B on urokinase-type plasminogen activator and urokinase-type plasminogen activator receptor expression in the epidermal keratinocyte cell line A431. Ultraviolet B increased cellular and secreted urokinase-type plasminogen activator protein (enzyme-linked immunosorbent assay) and urokinase-type plasminogen activator receptor cell surface expression (flow cytometry) 24 h postirradiation. Northern blot analysis indicated that ultraviolet B increased urokinase-type plasminogen activator receptor mRNA. Compared with a more rapid mRNA induction by epidermal growth factor (maximal after 4 h) the ultraviolet B response was maximal after 24 h and prolonged up to 36 h. The mRNA induction was not dependent on protein synthesis as judged by cycloheximide incubation. Ultraviolet B did not influence urokinase-type plasminogen activator receptor mRNA stability (actinomycin D incubation). A transiently transfected chloramphenicol acetyltransferase-reporter construct containing a -398/+51 urokinase-type plasminogen activator receptor promoter fragment was activated when cells were exposed to ultraviolet B. This induction was almost completely abolished by mutating a -182/-176 AP-1 binding sequence. Ultraviolet B increased the binding capacity at this AP-1 motif in electrophoretic mobility shift assays. These data identify a distinct transcriptional mechanism by which

  13. Identification of essential cannabinoid-binding domains: structural insights into early dynamic events in receptor activation.

    Shim, Joong-Youn; Bertalovitz, Alexander C; Kendall, Debra A

    2011-09-23

    The classical cannabinoid agonist HU210, a structural analog of (-)-Δ(9)-tetrahydrocannabinol, binds to brain cannabinoid (CB1) receptors and activates signal transduction pathways. To date, an exact molecular description of the CB1 receptor is not yet available. Utilizing the minor binding pocket of the CB1 receptor as the primary ligand interaction site, we explored HU210 binding using lipid bilayer molecular dynamics (MD) simulations. Among the potential ligand contact residues, we identified residues Phe-174(2.61), Phe-177(2.64), Leu-193(3.29), and Met-363(6.55) as being critical for HU210 binding by mutational analysis. Using these residues to guide the simulations, we determined essential cannabinoid-binding domains in the CB1 receptor, including the highly sought after hydrophobic pocket important for the binding of the C3 alkyl chain of classical and nonclassical cannabinoids. Analyzing the simulations of the HU210-CB1 receptor complex, the CP55940-CB1 receptor complex, and the (-)-Δ(9)-tetrahydrocannabinol-CB1 receptor complex, we found that the positioning of the C3 alkyl chain and the aromatic stacking between Trp-356(6.48) and Trp-279(5.43) is crucial for the Trp-356(6.48) rotamer change toward receptor activation through the rigid-body movement of H6. The functional data for the mutant receptors demonstrated reductions in potency for G protein activation similar to the reductions seen in ligand binding affinity for HU210. PMID:21795705

  14. Oxidative stress during erythropoietin hyporesponsiveness anemia at end stage renal disease: Molecular and biochemical studies.

    Khalil, Samar K M; Amer, H A; El Behairy, Adel M; Warda, Mohamad

    2016-05-01

    Inflammation and oxidative stress are two faces of one coin in end stage renal disease patients (ESRD) on maintenance hemodialysis. Their interconnection induces anemia complicated with erythropoietin hyporesponsiveness. The biochemical bases behind the resistance to erythropoietin therapy with frequent hemoglobinemia, oxidative stress and iron status have not been fully understood. Here two equal groups (40 patients each) of responders and non-responders to recombinant human erythropoietin therapy (higher than 300 IU/kg/wk of epoetin) were investigated. Hematological and biochemical analyses of collected blood and serum samples were performed along with serum electrophoretic protein footprinting. The leukocytic DNA fragmentation was used to evaluate the degree of oxidative insult. The good responders showed lower erythrocyte malondialdehyde (E-MDA) level and less DNA fragmentation of circulating leukocytes than poor responders with elevated hemoglobin, albumin, A/G ratio, total iron, and ferritin levels. Contrariwise, lower erythrocyte superoxide dismutase (E-SOD) and catalase activities in EPO poor responder group were noticed. Neither other serum constituents nor electrophoretic protein pattern showed any difference between the two groups. There were higher levels of inflammatory markers, interleukin-6 (IL6) and C-reactive protein (CRP) in EPO poor responder than good responder. The negative correlations between Hb and both IL6 and CRP levels in the present data remotely indicate a positive correlation between inflammatory markers and severity of anemia. A direct correlation between Hb and antioxidant enzymes (E-SOD and catalase) was noticed, while inverse correlation with E-MDA was recorded. The study proved that oral supplementation of vitamin C to ESRD patients might mitigate the previously elevated serum MDA level in these patients. PMID:27222740

  15. Oxidative stress during erythropoietin hyporesponsiveness anemia at end stage renal disease: Molecular and biochemical studies

    Khalil, Samar K.M.; Amer, H.A.; El Behairy, Adel M.; Warda, Mohamad

    2016-01-01

    Inflammation and oxidative stress are two faces of one coin in end stage renal disease patients (ESRD) on maintenance hemodialysis. Their interconnection induces anemia complicated with erythropoietin hyporesponsiveness. The biochemical bases behind the resistance to erythropoietin therapy with frequent hemoglobinemia, oxidative stress and iron status have not been fully understood. Here two equal groups (40 patients each) of responders and non-responders to recombinant human erythropoietin therapy (higher than 300 IU/kg/wk of epoetin) were investigated. Hematological and biochemical analyses of collected blood and serum samples were performed along with serum electrophoretic protein footprinting. The leukocytic DNA fragmentation was used to evaluate the degree of oxidative insult. The good responders showed lower erythrocyte malondialdehyde (E-MDA) level and less DNA fragmentation of circulating leukocytes than poor responders with elevated hemoglobin, albumin, A/G ratio, total iron, and ferritin levels. Contrariwise, lower erythrocyte superoxide dismutase (E-SOD) and catalase activities in EPO poor responder group were noticed. Neither other serum constituents nor electrophoretic protein pattern showed any difference between the two groups. There were higher levels of inflammatory markers, interleukin-6 (IL6) and C-reactive protein (CRP) in EPO poor responder than good responder. The negative correlations between Hb and both IL6 and CRP levels in the present data remotely indicate a positive correlation between inflammatory markers and severity of anemia. A direct correlation between Hb and antioxidant enzymes (E-SOD and catalase) was noticed, while inverse correlation with E-MDA was recorded. The study proved that oral supplementation of vitamin C to ESRD patients might mitigate the previously elevated serum MDA level in these patients.

  16. Receptor interconversion model of hormone action. 3. Estrogen receptor mediated repression of reporter gene activity in A431 cells.

    Nag, A; Park, I; Krust, A; Smith, R G

    1990-03-20

    The chicken estrogen receptor exists in three interconvertible forms, two of which bind estradiol with high affinity and one which lacks the capacity to bind estradiol. Interconversion is regulated by reactions involving ATP/Mg2+. By cotransfecting into A431 cells estrogen receptor cDNA in an expression vector together with the pA2 (-821/-87) tk-CAT vitellogenin construct, we demonstrate that constitutive expression of chloramphenicol acetyltransferase (CAT) activity can be regulated either by selection of ligand or by modifying phosphorylation reactions in the recipient cells. In the presence of estrogen receptors, constitutive expression of CAT activity is inhibited in three situations: (i) in the absence of an estrogenic ligand; (ii) in the presence of an anti-estrogen; and (iii) in the presence of an estrogenic ligand together with 12-O-tetradecanoylphorbol 13-acetate (TPA). Estrogen receptor mediated repression of constitutive CAT activity is not observed with the pA2 (-331/-87) tk-CAT construct, indicating that DNA sequences required for repression are located between -821 and -331 base pairs upstream of the transcription initiation site. PMID:2346742

  17. Functional residues essential for the activation of the CB1 cannabinoid receptor.

    Shim, Joong-Youn; Padgett, Lea

    2013-01-01

    Recently developed X-ray crystal structures of active state G-protein-coupled receptors have opened the way for detailed examination of the movement of the transmembrane (TM) helices and the specific residues involved in the receptor activation upon ligand binding. Previous modeling studies have indicated that the brain cannabinoid (CB1) receptor binds with a ligand at least in part through a hydrophobic tail on the ligand. This interaction is believed to be similar to the rotameric toggle switch proposed for the β2 adrenergic receptor (β2AR). In the present study, an active state model for the CB1 receptor, guided by the X-ray structure of the active state for β2AR, was constructed with HU210 bound as a ligand. Molecular dynamics (MD) simulations were employed to provide a smooth progression between inactive and active states of the receptor. This model was compared with our previously published CB1 receptor model to identify the functional residues that play key roles in triggering receptor conformational changes upon agonist binding. Movements in TM helices and functional residues are discussed. W279(5.43), contributing to an inward movement of the fifth TM helix (TM5) to the helical core, could serve as another rotameric switch for receptor activation. V282(5.46), interacting with the ligand's hydrophobic C3 alkyl chain, appears to play a key role in TM5 inward movement centered at L286(5.50) and subsequent coupling to V204(3.40). V204(3.40), closely interacting with the TM5 and TM6 hydrophobic patch residues in the middle of the receptor, particularly I290(5.54) and L352(6.44), appears to facilitate helical rearrangements, leading to the breakage of the ionic lock and the rotameric change of Y397(7.53), which are key features of the active state. PMID:23332708

  18. β-Arrestin biosensors reveal a rapid, receptor-dependent activation/deactivation cycle.

    Nuber, Susanne; Zabel, Ulrike; Lorenz, Kristina; Nuber, Andreas; Milligan, Graeme; Tobin, Andrew B; Lohse, Martin J; Hoffmann, Carsten

    2016-03-31

    (β-)Arrestins are important regulators of G-protein-coupled receptors (GPCRs). They bind to active, phosphorylated GPCRs and thereby shut off 'classical' signalling to G proteins, trigger internalization of GPCRs via interaction with the clathrin machinery and mediate signalling via 'non-classical' pathways. In addition to two visual arrestins that bind to rod and cone photoreceptors (termed arrestin1 and arrestin4), there are only two (non-visual) β-arrestin proteins (β-arrestin1 and β-arrestin2, also termed arrestin2 and arrestin3), which regulate hundreds of different (non-visual) GPCRs. Binding of these proteins to GPCRs usually requires the active form of the receptors plus their phosphorylation by G-protein-coupled receptor kinases (GRKs). The binding of receptors or their carboxy terminus as well as certain truncations induce active conformations of (β-)arrestins that have recently been solved by X-ray crystallography. Here we investigate both the interaction of β-arrestin with GPCRs, and the β-arrestin conformational changes in real time and in living human cells, using a series of fluorescence resonance energy transfer (FRET)-based β-arrestin2 biosensors. We observe receptor-specific patterns of conformational changes in β-arrestin2 that occur rapidly after the receptor-β-arrestin2 interaction. After agonist removal, these changes persist for longer than the direct receptor interaction. Our data indicate a rapid, receptor-type-specific, two-step binding and activation process between GPCRs and β-arrestins. They further indicate that β-arrestins remain active after dissociation from receptors, allowing them to remain at the cell surface and presumably signal independently. Thus, GPCRs trigger a rapid, receptor-specific activation/deactivation cycle of β-arrestins, which permits their active signalling. PMID:27007855

  19. GABA-A receptor-mediated signaling alters the structure of spontaneous activity in the developing retina

    Wang, Chih-Tien; Blankenship, Aaron G.; Anishchenko, Anastasia; Elstrott, Justin; Fikhman, Michael; Nakanishi, Shigetada; Feller, Marla B

    2007-01-01

    Ambient GABA modulates firing patterns in adult neural circuits by tonically activating extrasynaptic GABA-A receptors. Here, we demonstrate that during a developmental period when activation of GABA-A receptors causes membrane depolarization, tonic activation of GABA-A receptors blocks all spontaneous activity recorded in retinal ganglion cells (RGCs) and starburst amacrine cells (SACs). Bath application of the GABA-A receptor agonist muscimol blocked spontaneous correlated increases in intr...

  20. Direct activation of Transient Receptor Potential Vanilloid 1(TRPV1) by Diacylglycerol (DAG)

    Oh Seog; Kim Yong; Park Chul-Kyu; Zhu Mei; Jung Sung; Woo Dong; Lee C Justin

    2008-01-01

    Abstract The capsaicin receptor, known as transient receptor potential channel vanilloid subtype 1 (TRPV1), is activated by a wide range of noxious stimulants and putative ligands such as capsaicin, heat, pH, anandamide, and phosphorylation by protein kinase C (PKC). However, the identity of endogenous activators for TRPV1 under physiological condition is still debated. Here, we report that diacylglycerol (DAG) directly activates TRPV1 channel in a membrane-delimited manner in rat dorsal root...

  1. Modulation of visceral pain and inflammation by protease-activated receptors

    Vergnolle, Nathalie

    2004-01-01

    The gastrointestinal (GI) tract is exposed to a large array of proteases, under both physiological and pathophysiological conditions. The discovery of G protein-coupled receptors activated by proteases, the protease-activated receptors (PARs), has highlighted new signaling functions for proteases in the GI tract, particularly in the domains of inflammation and pain mechanisms. Activation of PARs by selective peptidic agonists in the intestine or the pancreas leads to inflammatory events and c...

  2. Interactions between Human Liver Fatty Acid Binding Protein and Peroxisome Proliferator Activated Receptor Selective Drugs

    Tony Velkov

    2013-01-01

    Fatty acid binding proteins (FABPs) act as intracellular shuttles for fatty acids as well as lipophilic xenobiotics to the nucleus, where these ligands are released to a group of nuclear receptors called the peroxisome proliferator activated receptors (PPARs). PPAR mediated gene activation is ultimately involved in maintenance of cellular homeostasis through the transcriptional regulation of metabolic enzymes and transporters that target the activating ligand. Here we show that liver- (L-) FA...

  3. Quercetin Directly Interacts with Vitamin D Receptor (VDR): Structural Implication of VDR Activation by Quercetin

    Lee, Ki-Young; Choi, Hye-Seung; Choi, Ho-Sung; Chung, Ka Young; Lee, Bong-Jin; Maeng, Han-Joo; Seo, Min-Duk

    2016-01-01

    The vitamin D receptor (VDR) is a member of the nuclear receptor (NR) superfamily. The VDR binds to active vitamin D3 metabolites, which stimulates downstream transduction signaling involved in various physiological activities such as calcium homeostasis, bone mineralization, and cell differentiation. Quercetin is a widely distributed flavonoid in nature that is known to enhance transactivation of VDR target genes. However, the detailed molecular mechanism underlying VDR activation by quercet...

  4. Enzymatic Regulation of Steroidogenesis and Nuclear Receptor Activation : Special Focus on Vitamin D and Sex Hormones

    LUNDQVIST, JOHAN

    2011-01-01

    Enzyme-catalyzed reactions are important to regulate steroidogenesis and nuclear receptor activation. The present investigation examines the role of steroid metabolism catalyzed by CYP7B1 for regulation of hormone receptor activation and the effects of vitamin D on enzymatic regulation of steroidogenesis. The study reports data indicating that CYP7B1 can regulate estrogenic signaling by converting estrogens into inactive or less active metabolites. Similar results were obtained for CYP7B1-med...

  5. A2A adenosine receptor antagonism enhances synaptic and motor effects of cocaine via CB1 cannabinoid receptor activation.

    Alessandro Tozzi

    Full Text Available BACKGROUND: Cocaine increases the level of endogenous dopamine (DA in the striatum by blocking the DA transporter. Endogenous DA modulates glutamatergic inputs to striatal neurons and this modulation influences motor activity. Since D2 DA and A2A-adenosine receptors (A2A-Rs have antagonistic effects on striatal neurons, drugs targeting adenosine receptors such as caffeine-like compounds, could enhance psychomotor stimulant effects of cocaine. In this study, we analyzed the electrophysiological effects of cocaine and A2A-Rs antagonists in striatal slices and the motor effects produced by this pharmacological modulation in rodents. PRINCIPAL FINDINGS: Concomitant administration of cocaine and A2A-Rs antagonists reduced glutamatergic synaptic transmission in striatal spiny neurons while these drugs failed to produce this effect when given in isolation. This inhibitory effect was dependent on the activation of D2-like receptors and the release of endocannabinoids since it was prevented by L-sulpiride and reduced by a CB1 receptor antagonist. Combined application of cocaine and A2A-R antagonists also reduced the firing frequency of striatal cholinergic interneurons suggesting that changes in cholinergic tone might contribute to this synaptic modulation. Finally, A2A-Rs antagonists, in the presence of a sub-threshold dose of cocaine, enhanced locomotion and, in line with the electrophysiological experiments, this enhanced activity required activation of D2-like and CB1 receptors. CONCLUSIONS: The present study provides a possible synaptic mechanism explaining how caffeine-like compounds could enhance psychomotor stimulant effects of cocaine.

  6. Glucose-Sensing Receptor T1R3: A New Signaling Receptor Activated by Glucose in Pancreatic β-Cells.

    Kojima, Itaru; Nakagawa, Yuko; Hamano, Kunihisa; Medina, Johan; Li, Longfei; Nagasawa, Masahiro

    2015-01-01

    Subunits of the sweet taste receptors T1R2 and T1R3 are expressed in pancreatic β-cells. Compared with T1R3, mRNA expression of T1R2 is considerably lower. At the protein level, expression of T1R2 is undetectable in β-cells. Accordingly, a major component of the sweet taste-sensing receptor in β-cells may be a homodimer of T1R3 rather than a heterodimer of T1R2/T1R3. Inhibition of this receptor by gurmarin or deletion of the T1R3 gene attenuates glucose-induced insulin secretion from β-cells. Hence the T1R3 homodimer functions as a glucose-sensing receptor (GSR) in pancreatic β-cells. When GSR is activated by the T1R3 agonist sucralose, elevation of intracellular ATP concentration ([ATP]i) is observed. Sucralose increases [ATP]i even in the absence of ambient glucose, indicating that sucralose increases [ATP]i not simply by activating glucokinase, a rate-limiting enzyme in the glycolytic pathway. In addition, sucralose augments elevation of [ATP]i induced by methylsuccinate, suggesting that sucralose activates mitochondrial metabolism. Nonmetabolizable 3-O-methylglucose also increases [ATP]i and knockdown of T1R3 attenuates elevation of [ATP]i induced by high concentration of glucose. Collectively, these results indicate that the T1R3 homodimer functions as a GSR; this receptor is involved in glucose-induced insulin secretion by activating glucose metabolism probably in mitochondria. PMID:25947913

  7. Expression of protease-activated receptor 1 and 2 and anti-tubulogenic activity of protease-activated receptor 1 in human endothelial colony-forming cells.

    Tiago M Fortunato

    Full Text Available Endothelial colony-forming cells (ECFCs are obtained from the culture of human peripheral blood mononuclear cell (hPBMNC fractions and are characterised by high proliferative and pro-vasculogenic potential, which makes them of great interest for cell therapy. Here, we describe the detection of protease-activated receptor (PAR 1 and 2 amongst the surface proteins expressed in ECFCs. Both receptors are functionally coupled to extracellular signal-regulated kinase (ERK 1 and 2, which become activated and phosphorylated in response to selective PAR1- or PAR2-activating peptides. Specific stimulation of PAR1, but not PAR2, significantly inhibits capillary-like tube formation by ECFCs in vitro, suggesting that tubulogenesis is negatively regulated by proteases able to stimulate PAR1 (e.g. thrombin. The activation of ERKs is not involved in the regulation of tubulogenesis in vitro, as suggested by use of the MEK inhibitor PD98059 and by the fact that PAR2 stimulation activates ERKs without affecting capillary tube formation. Both qPCR and immunoblotting showed a significant downregulation of vascular endothelial growth factor 2 (VEGFR2 in response to PAR1 stimulation. Moreover, the addition of VEGF (50-100 ng/ml but not basic Fibroblast Growth Factor (FGF (25-100 ng/ml rescued tube formation by ECFCs treated with PAR1-activating peptide. Therefore, we propose that reduction of VEGF responsiveness resulting from down-regulation of VEGFR2 is underlying the anti-tubulogenic effect of PAR1 activation. Although the role of PAR2 remains elusive, this study sheds new light on the regulation of the vasculogenic activity of ECFCs and suggests a potential link between adult vasculogenesis and the coagulation cascade.

  8. Stress reticulocytes lose transferrin receptors by an extrinsic process involving spleen and macrophages.

    Rhodes, Melissa M; Koury, Stephen T; Kopsombut, Prapaporn; Alford, Catherine E; Price, James O; Koury, Mark J

    2016-09-01

    As they mature into erythrocytes during normal erythropoiesis, reticulocytes lose surface transferrin receptors before or concurrently with reticulin. Exosome release accounts for most of the loss of transferrin receptors from reticulocytes. During erythropoietic stress, reticulocytes are released early from hematopoietic tissues and have increased reticulin staining and transferrin receptors. Flow cytometry of dually stained erythrocytes of mice recovering from phlebotomy demonstrated delayed loss of reticulin and transferrin receptors during in vitro maturation compared to in vivo maturation, indicating that an in vivo process extrinsic to the reticulocytes facilitates their maturation. Splenectomy or macrophage depletion by liposomal clodronate inhibited in vivo maturation of reticulocytes and increased the numbers of reticulin-negative, transferrin receptor-positive cells during and after recovery from phlebotomy. This reticulin-negative, transferrin receptor-positive population was rarely found in normal mice. Transmission electron microscopy demonstrated that the reticulin-negative, transferrin receptor-positive cells were elongated and discoid erythrocytes, but they had intracellular and surface structures that appeared to be partially degraded organelles. The results indicate that maturation of circulating stress reticulocytes is enhanced by an extrinsic process that occurs in the spleen and involves macrophage activity. Complete loss of reticulin with incomplete loss of surface transferrin receptors in this process produces a reticulin-negative, transferrin receptor-positive erythrocyte population that has potential utility for detecting prior erythropoietic stresses including bleeding, hemolysis and erythropoietin administration, even after recovery has been completed. Am. J. Hematol. 91:875-882, 2016. © 2016 Wiley Periodicals, Inc. PMID:27194638

  9. The role of transmembrane segment II in 7TM receptor activation

    Benned-Jensen, Tau; Rosenkilde, M M

    2009-01-01

    , accumulating evidence emphasize that this is not the case. In this review, we focus on TM-II with an emphasis on position II:20/2.60, and present data from structure-activity studies on a range of Family A 7TM receptors including chemokine, ghrelin and melanocortin receptors in addition to the orphan EBI2...

  10. Tumor-Suppressive Activity of Lunatic Fringe in Prostate through Differential Modulation of Notch Receptor Activation

    Shubing Zhang

    2014-02-01

    Full Text Available Elevated Notch ligand and receptor expression has been associated with aggressive forms of prostate cancer, suggesting a role for Notch signaling in regulation of prostate tumor initiation and progression. Here, we report a critical role for Lunatic Fringe (Lfng, which encodes an O-fucosylpeptide 3-ß-N-acetylglucosaminyltransferase known to modify epidermal growth factor repeats of Notch receptor proteins, in regulation of prostate epithelial differentiation and proliferation, as well as in prostate tumor suppression. Deletion of Lfng in mice caused altered Notch activation in the prostate, associated with elevated accumulation of Notch1, Notch2, and Notch4 intracellular domains, decreased levels of the putative Notch3 intracellular fragment, as well as increased expression of Hes1, Hes5, and Hey2. Loss of Lfng resulted in expansion of the basal layer, increased proliferation of both luminal and basal cells, and ultimately, prostatic intraepithelial neoplasia. The Lfng-null prostate showed down-regulation of prostatic tumor suppressor gene NKX3.1 and increased androgen receptor expression. Interestingly, expression of LFNG and NKX3.1 were positively correlated in publically available human prostate cancer data sets. Knockdown of LFNG in DU-145 prostate cancer cells led to expansion of CD44+CD24− and CD49f+CD24− stem/progenitor-like cell population associated with enhanced prostatosphere-forming capacity. Taken together, these data revealed a tumor-suppressive role for Lfng in the prostate through differential regulation of Notch signaling.

  11. Erythropoietin production by PDGFR-β(+) cells.

    Gerl, Katharina; Nolan, Karen A; Karger, Christian; Fuchs, Michaela; Wenger, Roland H; Stolt, Claus C; Willam, Carsten; Kurtz, Armin; Kurt, Birgül

    2016-08-01

    PDGFR-β-expressing cells of the kidneys are considered as a relevant site of erythropoietin (EPO) production. The origin of these cells, their contribution to renal EPO production, and if PDGFR-β-positive cells in other organs are also capable to express EPO are less clear. We addressed these questions in mice, in which hypoxia-inducible transcription factors were stabilized in PDGFR-β(+) cells by inducible deletion of the von Hippel-Lindau (Vhl) protein. Vhl deletion led to a 600-fold increase of plasma EPO concentration, 170-fold increase of renal EPO messenger RNA (mRNA) levels, and an increase of hematocrit values up to 70 %. Intrarenal localization of EPO-expressing cells coincided with the zonal heterogeneity and distribution of cells expressing PDGFR-β. Amongst a variety of extrarenal organs only adrenal glands showed significant EPO mRNA expression after Vhl deletion in PDGFR-β(+) cells. EPO mRNA, plasma EPO, and hematocrit fell to subnormal values if HIF-2α, but not HIF-1α, was deleted either alone or in combination with Vhl in PDGFR-β(+) cells. Treatment of mice with a prolyl-hydroxylase inhibitor caused an increase of EPO mRNA abundance and plasma EPO concentrations in wild-type mice and in mice lacking HIF-1α in PDGFR-β(+) cells but exerted no effect in mice lacking HIF-2α in PDGFR-β(+) cells. These findings suggest that PDGFR-β(+) cells are the only relevant site of EPO expression in the kidney and that HIF-2 is the essential transcription factor triggering EPO expression therein. Moreover, our findings suggest that PDGFR-β(+) cells elaborating EPO might arise from the metanephric mesenchyme, rather than from the neural crest. PMID:27220347

  12. DMPD: Proximal effects of Toll-like receptor activation in dendritic cells. [Dynamic Macrophage Pathway CSML Database

    Full Text Available 17142025 Proximal effects of Toll-like receptor activation in dendritic cells. Watt...) (.svg) (.html) (.csml) Show Proximal effects of Toll-like receptor activation in dendritic cells. PubmedID... 17142025 Title Proximal effects of Toll-like receptor activation in dendritic ce

  13. P2Y2 receptor activation decreases blood pressure and increases renal Na+ excretion

    Rieg, Timo; Gerasimova, Maria; Boyer, José L.; Insel, Paul A.; Vallon, Volker

    2011-01-01

    ATP and UTP are endogenous agonists of P2Y2/4 receptors. To define the in vivo effects of P2Y2 receptor activation on blood pressure and urinary excretion, we compared the response to INS45973, a P2Y2/4 receptor agonist and UTP analog, in wild-type (WT) and P2Y2 receptor knockout (P2Y2−/−) mice. INS45973 was administered intravenously as a bolus injection or continuous infusion to determine effects on blood pressure and renal function, respectively. Within seconds, bolus application of INS459...

  14. PROKR2 missense mutations associated with Kallmann syndrome impair receptor signalling activity.

    Monnier, Carine; Dodé, Catherine; Fabre, Ludovic; Teixeira, Luis; Labesse, Gilles; Pin, Jean-Philippe; Hardelin, Jean-Pierre; Rondard, Philippe

    2009-01-01

    Kallmann syndrome (KS) combines hypogonadism due to gonadotropin-releasing hormone deficiency, and anosmia or hyposmia, related to defective olfactory bulb morphogenesis. In a large series of KS patients, ten different missense mutations (p.R85C, p.R85H, p.R164Q, p.L173R, p.W178S, p.Q210R, p.R268C, p.P290S, p.M323I, p.V331M) have been identified in the gene encoding the G protein-coupled receptor prokineticin receptor-2 (PROKR2), most often in the heterozygous state. Many of these mutations were, however, also found in clinically unaffected individuals, thus raising the question of their actual implication in the KS phenotype. We reproduced each of the ten mutations in a recombinant murine Prokr2, and tested their effects on the signalling activity in transfected HEK-293 cells, by measuring intracellular calcium release upon ligand-activation of the receptor. We found that all mutated receptors except one (M323I) had decreased signalling activities. These could be explained by different defective mechanisms. Three mutations (L173R, W178S, P290S) impaired cell surface-targeting of the receptor. One mutation (Q210R) abolished ligand-binding. Finally, five mutations (R85C, R85H, R164Q, R268C, V331M) presumably impaired G protein-coupling of the receptor. In addition, when wild-type and mutant receptors were coexpressed in HEK-293 cells, none of the mutant receptors that were retained within the cells did affect cell surface-targeting of the wild-type receptor, and none of the mutant receptors properly addressed at the plasma membrane did affect wild-type receptor signalling activity. This argues against a dominant negative effect of the mutations in vivo. PMID:18826963

  15. Activation of peroxisome proliferator-activated receptor-α enhances fatty acid oxidation in human adipocytes

    Highlights: → PPARα activation increased mRNA expression levels of adipocyte differentiation marker genes and GPDH activity in human adipocytes. → PPARα activation also increased insulin-dependent glucose uptake in human adipocytes. → PPARα activation did not affect lipid accumulation in human adipocytes. → PPARα activation increased fatty acid oxidation through induction of fatty acid oxidation-related genes in human adipocytes. -- Abstract: Peroxisome proliferator-activated receptor-α (PPARα) is a key regulator for maintaining whole-body energy balance. However, the physiological functions of PPARα in adipocytes have been unclarified. We examined the functions of PPARα using human multipotent adipose tissue-derived stem cells as a human adipocyte model. Activation of PPARα by GW7647, a potent PPARα agonist, increased the mRNA expression levels of adipocyte differentiation marker genes such as PPARγ, adipocyte-specific fatty acid-binding protein, and lipoprotein lipase and increased both GPDH activity and insulin-dependent glucose uptake level. The findings indicate that PPARα activation stimulates adipocyte differentiation. However, lipid accumulation was not changed, which is usually observed when PPARγ is activated. On the other hand, PPARα activation by GW7647 treatment induced the mRNA expression of fatty acid oxidation-related genes such as CPT-1B and AOX in a PPARα-dependent manner. Moreover, PPARα activation increased the production of CO2 and acid soluble metabolites, which are products of fatty acid oxidation, and increased oxygen consumption rate in human adipocytes. The data indicate that activation of PPARα stimulates both adipocyte differentiation and fatty acid oxidation in human adipocytes, suggesting that PPARα agonists could improve insulin resistance without lipid accumulation in adipocytes. The expected effects of PPARα activation are very valuable for managing diabetic conditions accompanied by obesity, because PPAR

  16. Treatment of anemia of nephrotic syndrome with recombinant erythropoietin

    Gansevoort, RT; Vaziri, ND; deJong, PE

    1996-01-01

    Nephrotic syndrome has been recently shown to cause erythropoietin (EPO) deficiency in humans and experimental models. However, efficacy and safety of recombinant EPO (rEPO) in the treatment of the associated anemia has not been previously investigated. We report a patient with nephrotic syndrome an

  17. Timing of erythropoietin treatment for cardioprotection in ischemia/reperfusion

    Lipsic, E; van der Meer, P; Henning, RH; Suurmeijer, AJH; Boddeus, KM; van Veldhuisen, DJ; van Gilst, WH; Schoemaker, RG

    2004-01-01

    Erythropoietin (EPO) is a hormone known to stimulate hematopoiesis. However, recent research suggests additional properties of EPO, such as protection against ischemia/reperfusion (I/R) injury in various tissues. We studied the effect of timing of EPO administration on cardioprotection during I/R in

  18. RECOMBINANT-HUMAN-ERYTHROPOIETIN IN PATIENTS WITH MYELODYSPLASTIC SYNDROMES

    SCHOUTEN, HC; VELLENGA, E; VANRHENEN, DJ; DEWOLF, JTM; COPPENS, PJW; BLIJHAM, GH

    1991-01-01

    As anemia is frequently the main problem in myelodysplastic syndromes (MDS), we studied the efficacy of human erythropoietin (rhEpo) in stimulating the erythroid lineage in 14 patients, starting with 40 U/kg three times a week and doubling the dose every 6 weeks until a response was observed. The hi

  19. Production and analysis of a biosimilar erythropoietin in Egypt

    Ebied WM

    2014-05-01

    Full Text Available Wael M Ebied,1 Hytham M Ahmed,2 Fawzy A Elbarbry31SEDICO Pharmaceuticals, Merck & Co External Partner, 6th of October City, Cairo, 2Pharmaceutical Analysis Department, Faculty of Pharmacy, Damanhour University, Damanhour, Egypt; 3Pharmaceutical Sciences, School of Pharmacy, Pacific University Oregon, Hillsboro, OR, USAAbstract: Although management of chronic diseases has been a major challenge for health care systems in developed and developing countries, biopharmaceuticals have been successful in treating many life-threatening conditions. However, the high cost of these agents restricts their availability to countries where patients and/or health care systems are able to afford them. Licensing these biopharmaceuticals as biosimilars after expiration of their patents might increase access to such medicines at an affordable price in developing countries. South Egypt Drug Industries Company (SEDICO is an Egyptian pharmaceutical company that has had the opportunity to manufacture some of these drugs. SEDICO biotechnology products, such as insulin, erythropoietin, streptokinase, angiokinase, follicle-stimulating hormone, aprotinin, filgrastim, and somatropin, have been available on the Egyptian market for more than 6 years. For this paper, erythropoietin, which has been investigated over a number of years, was chosen as a representative example of SEDICO biotechnology products. Our findings confirm that SEDICO erythropoietin can compete with the originator epoetins on the Egyptian market with high quality and at a lower cost.Keywords: biosimilars, developing countries, insulin, human growth hormone, erythropoietin, epoetin, Egypt

  20. Activating Effect of Benzbromarone, a Uricosuric Drug, on Peroxisome Proliferator-Activated Receptors

    Chiyoko Kunishima

    2007-01-01

    Full Text Available Benzbromarone, a uricosuric drug, reportedly causes hepatic hypertrophy accompanied by proliferation of peroxisomes in rats. To elucidate the mechanisms underlying induction of peroxisome proliferation by benzbromarone, we examined binding affinity for peroxisome proliferator-activated receptor α (PPARα and γ (PPARγ, and effects on the binding activity of PPARs with peroxisome proliferation-responsive element (PPRE and expression of the PPARs target protein. Binding affinity of benzbromarone for PPARα and PPARγ was examined by reporter gene assay. Binding activity of PPARs with PPRE was determined by electric mobility shift assay, and expression of lipoprotein lipase (LPL and acyl-CoA synthetase (ACS by Western blot method. Benzbromarone displayed affinity for PPARα and PPARγ, and promoted binding of PPARs to PPRE. Furthermore, cultured cells with benzbromarone added showed upregulated expression of LPL and ACS. These results suggest that benzbromarone induces peroxisome proliferation in hepatocytes by binding to PPARs, and controls expression of proteins related to lipid metabolism.

  1. Activating effect of benzbromarone, a uricosuric drug, on peroxisome proliferator-activated receptors.

    Kunishima, Chiyoko; Inoue, Ikuo; Oikawa, Toshihiro; Nakajima, Hiromu; Komoda, Tsugikazu; Katayama, Shigehiro

    2007-01-01

    Benzbromarone, a uricosuric drug, reportedly causes hepatic hypertrophy accompanied by proliferation of peroxisomes in rats. To elucidate the mechanisms underlying induction of peroxisome proliferation by benzbromarone, we examined binding affinity for peroxisome proliferator-activated receptor alpha (PPARalpha) and gamma (PPARgamma), and effects on the binding activity of PPARs with peroxisome proliferation-responsive element (PPRE) and expression of the PPARs target protein. Binding affinity of benzbromarone for PPARalpha and PPARgamma was examined by reporter gene assay. Binding activity of PPARs with PPRE was determined by electric mobility shift assay, and expression of lipoprotein lipase (LPL) and acyl-CoA synthetase (ACS) by Western blot method. Benzbromarone displayed affinity for PPARalpha and PPARgamma, and promoted binding of PPARs to PPRE. Furthermore, cultured cells with benzbromarone added showed upregulated expression of LPL and ACS. These results suggest that benzbromarone induces peroxisome proliferation in hepatocytes by binding to PPARs, and controls expression of proteins related to lipid metabolism. PMID:18274627

  2. The low density lipoprotein receptor-related protein/alpha2-macroglobulin receptor regulates cell surface plasminogen activator activity on human trophoblast cells.

    Zhang, J C; Sakthivel, R; Kniss, D; Graham, C H; Strickland, D K; McCrae, K R

    1998-11-27

    The low density lipoprotein receptor-related protein/alpha2-macroglobulin receptor (LRP/alpha2MR) mediates the internalization of numerous ligands, including prourokinase (pro-UK) and complexes between two-chain urokinase (tc-u-PA) and plasminogen activator inhibitor type-1 (PAI-1). It has been suggested that through its ability to internalize these ligands, LRP/alpha2MR may regulate the expression of plasminogen activator activity on cell surfaces; this hypothesis, however, has not been experimentally confirmed. To address this issue, we assessed the ability of LRP/alpha2MR to regulate plasminogen activator activity on human trophoblast cells, which express both LRP/alpha2MR and the urokinase receptor (uPAR). Trophoblasts internalized and degraded exogenous 125I-pro-UK (primarily following its conversion to tc-u-PA and incorporation into tc-u-PA.PAI complexes) in an LRP/alpha2MR-dependent manner, which was inhibited by the LRP/alpha2MR receptor-associated protein. Receptor-associated protein also caused a approximately 50% reduction in cell surface plasminogen activator activity and delayed the regeneration of unoccupied uPAR by cells on which uPAR were initially saturated with pro-UK. Identical effects were caused by anti-LRP/alpha2MR antibodies. These results demonstrate that LRP/alpha2MR promotes the expression of cell surface plasminogen activator activity on trophoblasts by facilitating the clearance of tc-u-PA.PAI complexes and regeneration of unoccupied cell surface uPAR. PMID:9822706

  3. GABA-B receptor activation and conflict behavior

    Ketelaars, C.E.J.; Bollen, E.L.; Rigter, H.; Bruinvels, J.

    1988-01-01

    Baclofen and oxazepam enhance extinction of conflict behavior in the Geller-Seifter test while baclofen and diazepam release punished behavior in Vogel's conflict test. In order to investigate the possibility that the effect of the selective GABA-B receptor agonist baclofen is mediated indirectly via the GABA-A/benzodiazepine receptor complex, the effect of pretreatment of rats with baclofen on (/sup 3/H)-diazepam binding to washed and unwashed cortical and cerebellar membranes of rats has been studied. Baclofen pretreatment increase Bmax in washed cerebellar membranes when bicuculline was present in the incubation mixture. No effect was seen in cortical membranes. The present results render it unlikely that the effect of baclofen on extinction of conflict behavior and punished drinking is mediated via the GABA-A/benzodiazepine receptor complex. 50 references, 1 figure, 4 tables.

  4. GABA-B receptor activation and conflict behavior

    Baclofen and oxazepam enhance extinction of conflict behavior in the Geller-Seifter test while baclofen and diazepam release punished behavior in Vogel's conflict test. In order to investigate the possibility that the effect of the selective GABA-B receptor agonist baclofen is mediated indirectly via the GABA-A/benzodiazepine receptor complex, the effect of pretreatment of rats with baclofen on [3H]-diazepam binding to washed and unwashed cortical and cerebellar membranes of rats has been studied. Baclofen pretreatment increase Bmax in washed cerebellar membranes when bicuculline was present in the incubation mixture. No effect was seen in cortical membranes. The present results render it unlikely that the effect of baclofen on extinction of conflict behavior and punished drinking is mediated via the GABA-A/benzodiazepine receptor complex. 50 references, 1 figure, 4 tables

  5. Structural basis for AMPA receptor activation and ligand selectivity

    Hogner, A; Kastrup, Jette Sandholm Jensen; Jin, R;

    2002-01-01

    structures reveal that AMPA agonists with an isoxazole moiety adopt different binding modes in the receptor, dependent on the substituents of the isoxazole. Br-HIBO displays selectivity among different AMPA receptor subunits, and the design and structure determination of the S1S2J-Y702F mutant in complex...... with Br-HIBO and ACPA have allowed us to explain the molecular mechanism behind this selectivity and to identify key residues for ligand recognition. The agonists induce the same degree of domain closure as AMPA, except for Br-HIBO, which shows a slightly lower degree of domain closure. An excellent...... the functional studies on the full-length receptor, form a powerful platform for the design of new selective agonists....

  6. P2X7 receptor antagonist activity of the anti-allergic agent oxatomide.

    Yoshida, Kazuki; Ito, Masaaki; Matsuoka, Isao

    2015-11-15

    Activation of the P2X7 receptor by extracellular ATP is associated with various immune responses including allergic inflammation. Anti-allergic agents, such as H1-antihistamines, are known to inhibit the effects of different chemical mediators such as acetylcholine and platelet-activating factor. Therefore, we hypothesized that some anti-allergic agents might affect P2X7 receptor function. Using N18TG2 and J774 cells, which express functional P2X7 receptors, the effects of several anti-allergic agents on P2X7 receptor function were investigated by monitoring the ATP-induced increase in intracellular Ca(2+) concentrations ([Ca(2+)]i). Among the various agents tested, oxatomide significantly inhibited P2X7 receptor-mediated [Ca(2+)]i elevation in a concentration-dependent manner without affecting the P2Y2 receptor-mediated response in both N18TG2 and J774 cells. Consistently, oxatomide inhibited P2X7 receptor-mediated membrane current and downstream responses such as mitogen-activated protein kinase activation, inflammation-related gene induction, and cell death. In addition, oxatomide inhibited P2X7 receptor-mediated degranulation in mouse bone marrow-derived mast cells. Whole cell patch clamp analyses in HEK293 cells expressing human, mouse, and rat P2X7 receptors revealed that the inhibitory effect of oxatomide on ATP-induced current was most prominent for the human P2X7 receptor and almost non-existent for the rat P2X7 receptor. The potent inhibitory effects of oxatomide on human P2X7 receptor-mediated function were confirmed in RPMI8226 human B cell-like myeloma cells, which endogenously express the P2X7 receptor. Our results demonstrated that the antihistamine oxatomide also acts as a P2X7 receptor antagonist. Future studies should thus evaluate whether P2X7 receptor antagonism contributes to the anti-allergic effects of oxatomide. PMID:26463039

  7. Peroxisome proliferator-activated receptoractivation and excess energy burning in hepatocarcinogenesis.

    Misra, Parimal; Reddy, Janardan K

    2014-03-01

    Peroxisome proliferator-activated receptor-α (PPARα) modulates the activities of all three interlinked hepatic fatty acid oxidation systems, namely mitochondrial and peroxisomal β-oxidation and microsomal ω-oxidation pathways. Hyperactivation of PPARα, by both exogenous and endogenous activators up-regulates hepatic fatty acid oxidation resulting in excess energy burning in liver contributing to the development of liver cancer in rodents. Sustained PPARα signaling disproportionately increases H2O2-generating fatty acid metabolizing enzymes as compared to H2O2-degrading enzymes in liver leading to enhanced generation of DNA damaging reactive oxygen species, progressive endoplasmic reticulum stress and inflammation. These alterations also contribute to increased liver cell proliferation with changes in apoptosis. Thus, reactive oxygen species, oxidative stress and hepatocellular proliferation are likely the main contributing factors in the pathogenesis of hepatocarcinogenesis, mediated by sustained PPARα activation-related energy burning in liver. Furthermore, the transcriptional co-activator Med1, a key subunit of the Mediator complex, is essential for PPARα signaling in that both PPARα-null and Med1-null hepatocytes are unresponsive to PPARα activators and fail to give rise to liver tumors when chronically exposed to PPARα activators. PMID:24291192

  8. Utilizing the folate receptor for active targeting of cancer nanotherapeutics

    Grant L. Zwicke

    2012-12-01

    Full Text Available The development of specialized nanoparticles for use in the detection and treatment of cancer is increasing. Methods are being proposed and tested that could target treatments more directly to cancer cells, which could lead to higher efficacy and reduced toxicity, possibly even eliminating the adverse effects of damage to the immune system and the loss of quick replicating cells. In this mini-review we focus on recent studies that employ folate nanoconjugates to target the folate receptor. Folate receptors are highly overexpressed on the surface of many tumor types. This expression can be exploited to target imaging molecules and therapeutic compounds directly to cancerous tissues.

  9. Erythropoietin overrides the triggering effect of DNA platination products in a mouse model of Cisplatin-induced neuropathy

    Egensperger Rupert

    2009-07-01

    Full Text Available Abstract Background Cisplatin mediates its antineoplastic activity by formation of distinct DNA intrastrand cross links. The clinical efficacy and desirable dose escalations of cisplatin are restricted by the accumulation of DNA lesions in dorsal root ganglion (DRG cells leading to sensory polyneuropathy (PNP. We investigated in a mouse model by which mechanism recombinant erythropoietin (rhEPO protects the peripheral nervous system from structural and functional damage caused by cisplatin treatment with special emphasis on DNA damage burden. Results A cumulative dose of 16 mg cisplatin/kg resulted in clear electrophysiological signs of neuropathy, which were significantly attenuated by concomitant erythropoietin (cisplatin 32,48 m/s ± 1,68 m/s; cisplatin + rhEPO 49,66 m/s ± 1,26 m/s; control 55,01 m/s ± 1,88 m/s; p Conclusion The protective effect of recombinant erythropoietin is not mediated by reducing the burden of DNA platination in the target cells, but it is likely to be due to a higher resistance of the target cells to the adverse effect of DNA damage. The increased frequency of intact mitochondria might also contribute to this protective role.

  10. Noradrenergic modulation of basolateral amygdala neuronal activity: opposing influences of alpha-2 and beta receptor activation.

    Buffalari, Deanne M; Grace, Anthony A

    2007-11-01

    Substantial data exists demonstrating the importance of the amygdala and the locus ceruleus (LC) in responding to stress, aversive memory formation, and the development of stress-related disorders; however, little is known about the effects of norepinephrine (NE) on amygdala neuronal activity in vivo. The basolateral nucleus of the amygdala (BLA) receives dense NE projections from the LC, NE increases in the BLA in response to stress, and the BLA can also modulate the LC via reciprocal projections. These experiments examined the effects of noradrenergic agents on spontaneous and evoked responses of BLA neurons. NE iontophoresis inhibited spontaneous firing and decreased the responsiveness of BLA neurons to electrical stimulation of entorhinal cortex and sensory association cortex (Te3). Confirmed BLA projection neurons exhibited exclusively inhibitory responses to NE. Systemic administration of propranolol, a beta-receptor antagonist, decreased the spontaneous firing rate and potentiated the NE-evoked inhibition of BLA neurons. In addition, iontophoresis of the alpha-2 agonist clonidine, footshock administration, and LC stimulation mimicked the effects of NE iontophoresis on spontaneous activity. Furthermore, the effects of LC stimulation were partially blocked by systemic administration of alpha 2 and beta receptor antagonists. This is the first study to demonstrate the actions of directly applied and stimulus-evoked NE in the BLA in vivo, and provides a mechanism by which beta receptors can mediate the important behavioral consequences of NE within the BLA. The interaction between these two structures is particularly relevant with regard to their known involvement in stress responses and stress-related disorders. PMID:17989300

  11. The Hinge Region of Human Thyroid-Stimulating Hormone (TSH) Receptor Operates as a Tunable Switch between Hormone Binding and Receptor Activation

    Majumdar, Ritankar; Dighe, Rajan R.

    2012-01-01

    The mechanism by which the hinge regions of glycoprotein hormone receptors couple hormone binding to activation of downstream effecters is not clearly understood. In the present study, agonistic (311.62) and antagonistic (311.87) monoclonal antibodies (MAbs) directed against the TSH receptor extracellular domain were used to elucidate role of the hinge region in receptor activation. MAb 311.62 which identifies the LRR/Cb-2 junction (aa 265-275), increased the affinity of TSHR for the hormone ...

  12. Roles of activin receptor-like kinase 7 signaling and its target, peroxisome proliferator-activated receptor γ, in lean and obese adipocytes

    Yogosawa, Satomi; Izumi, Tetsuro

    2013-01-01

    We recently discovered a novel signaling pathway involving activin receptor-like kinase 7 (ALK7), one of the type I transforming growth factor-β receptors. ALK7 and activated Smads 2, 3, and 4 inhibit the master regulators of adipogenesis, CCAAT/enhancer-binding protein α (C/EBPα) and peroxisome proliferator-activated receptor γ (PPARγ) specifically in differentiated adipocytes, but surprisingly increase both the adipocyte size and lipid content by suppressing lipolysis. Here, we show that, a...

  13. Chronic regulation of colonic epithelial secretory function by activation of G protein-coupled receptors.

    Toumi, F

    2011-02-01

    Enteric neurotransmitters that act at G protein-coupled receptors (GPCRs) are well known to acutely promote epithelial Cl(-) and fluid secretion. Here we examined if acute GPCR activation might have more long-term consequences for epithelial secretory function.

  14. Direct activation of GABAA receptors by substances in the organic acid fraction of Japanese sake.

    Izu, Hanae; Shigemori, Kensuke; Eguchi, Masaya; Kawane, Shuhei; Fujii, Shouko; Kitamura, Yuji; Aoshima, Hitoshi; Yamada, Yasue

    2017-01-01

    We investigated the effect of substances present in Japanese sake on the response of ionotropic γ-aminobutyric acid (GABA)A receptors expressed in Xenopus oocytes. Sake was fractionated by ion-exchange chromatography. The fraction containing organic acids (OA fraction) showed agonist activities on the GABAA receptor. OA fractions from sake were analyzed by capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS). Of the 64 compounds identified, 13 compounds showed GABAA receptor agonist activities. Especially, l-lactic acid showed high agonist activity and its EC50 value was 37μM. Intraperitoneal injections of l-lactic acid, gluconic acid, and pyruvic acid (10, 10, and 5mg/kg BW, respectively), which showed agonistic activity on the GABAA receptor, led to significant anxiolytic effects during an elevated plus-maze test in mice. PMID:27507485

  15. Effects related to gene-gene interactions of peroxisome proliferator-activated receptor on essential hypertension

    俞浩

    2013-01-01

    Objective To explore the impact of the gene-gene interaction among the single nucleotide polymorphisms(SNPs) of peroxisome proliferator-activated receptorα/δ/γ on essential hypertension(EH).Methods

  16. Systemic administration of erythropoietin inhibits retinopathy in RCS rats.

    Weiyong Shen

    Full Text Available OBJECTIVE: Royal College of Surgeons (RCS rats develop vasculopathy as photoreceptors degenerate. The aim of this study was to examine the effect of erythropoietin (EPO on retinopathy in RCS rats. METHODS: Fluorescein angiography was used to monitor retinal vascular changes over time. Changes in retinal glia and vasculature were studied by immunostaining. To study the effects of EPO on retinal pathology, EPO (5000 IU/kg was injected intraperitoneally in 14 week old normal and RCS rats twice a week for 4 weeks. Changes in the retinal vasculature, glia and microglia, photoreceptor apoptosis, differential expression of p75 neurotrophin receptor (p75NTR, pro-neurotrophin 3 (pro-NT3, tumour necrosis factor-α (TNFα, pigment epithelium derived factor (PEDF and vascular endothelial growth factor-A (VEGF-A, the production of CD34(+ cells and mobilization of CD34(+/VEGF-R2(+ cells as well as recruitment of CD34(+ cells into the retina were examined after EPO treatment. RESULTS: RCS rats developed progressive capillary dropout and subretinal neovascularization which were accompanied by retinal gliosis. Systemic administration of EPO stabilized the retinal vasculature and inhibited the development of focal vascular lesions. Further studies showed that EPO modulated retinal gliosis, attenuated photoreceptor apoptosis and p75NTR and pro-NT3 upregulation, promoted the infiltration of ramified microglia and stimulated VEGF-A expression but had little effect on TNFα and PEDF expression. EPO stimulated the production of red and white blood cells and CD34(+ cells along with effective mobilization of CD34(+/VEGF-R2(+ cells. Immunofluorescence study demonstrated that EPO enhanced the recruitment of CD34+ cells into the retina. CONCLUSIONS: Our results suggest that EPO has therapeutic potentials in treatment of neuronal and vascular pathology in retinal disease. The protective effects of EPO on photoreceptors and the retinal vasculature may involve multiple

  17. Differential Requirement of the Extracellular Domain in Activation of Class B G Protein-coupled Receptors.

    Zhao, Li-Hua; Yin, Yanting; Yang, Dehua; Liu, Bo; Hou, Li; Wang, Xiaoxi; Pal, Kuntal; Jiang, Yi; Feng, Yang; Cai, Xiaoqing; Dai, Antao; Liu, Mingyao; Wang, Ming-Wei; Melcher, Karsten; Xu, H Eric

    2016-07-15

    G protein-coupled receptors (GPCRs) from the secretin-like (class B) family are key players in hormonal homeostasis and are important drug targets for the treatment of metabolic disorders and neuronal diseases. They consist of a large N-terminal extracellular domain (ECD) and a transmembrane domain (TMD) with the GPCR signature of seven transmembrane helices. Class B GPCRs are activated by peptide hormones with their C termini bound to the receptor ECD and their N termini bound to the TMD. It is thought that the ECD functions as an affinity trap to bind and localize the hormone to the receptor. This in turn would allow the hormone N terminus to insert into the TMD and induce conformational changes of the TMD to activate downstream signaling. In contrast to this prevailing model, we demonstrate that human class B GPCRs vary widely in their requirement of the ECD for activation. In one group, represented by corticotrophin-releasing factor receptor 1 (CRF1R), parathyroid hormone receptor (PTH1R), and pituitary adenylate cyclase activating polypeptide type 1 receptor (PAC1R), the ECD requirement for high affinity hormone binding can be bypassed by induced proximity and mass action effects, whereas in the other group, represented by glucagon receptor (GCGR) and glucagon-like peptide-1 receptor (GLP-1R), the ECD is required for signaling even when the hormone is covalently linked to the TMD. Furthermore, the activation of GLP-1R by small molecules that interact with the intracellular side of the receptor is dependent on the presence of its ECD, suggesting a direct role of the ECD in GLP-1R activation. PMID:27226600

  18. Artificial sweeteners and salts producing a metallic taste sensation activate TRPV1 receptors.

    Riera, Céline E; Vogel, Horst; Simon, Sidney A; le Coutre, Johannes

    2007-08-01

    Throughout the world many people use artificial sweeteners (AS) for the purpose of reducing caloric intake. The most prominently used of these molecules include saccharin, aspartame (Nutrasweet), acesulfame-K, and cyclamate. Despite the caloric advantage they provide, one key concern in their use is their aversive aftertaste that has been characterized on a sensory level as bitter and/or metallic. Recently, it has been shown that the activation of particular T2R bitter taste receptors is partially involved with the bitter aftertaste sensation of saccharin and acesulfame-K. To more fully understand the biology behind these phenomena we have addressed the question of whether AS could stimulate transient receptor potential vanilloid-1 (TRPV1) receptors, as these receptors are activated by a large range of structurally different chemicals. Moreover, TRPV1 receptors and/or their variants are found in taste receptor cells and in nerve terminals throughout the oral cavity. Hence, TRPV1 activation could be involved in the AS aftertaste or even contribute to the poorly understood metallic taste sensation. Using Ca(2+) imaging on TRPV1 receptors heterologously expressed in the human embryonic kidney (HEK) 293 cells and on dissociated primary sensory neurons, we find that in both systems, AS activate TRPV1 receptors, and, moreover, they sensitize these channels to acid and heat. We also found that TRPV1 receptors are activated by CuSO(4), ZnSO(4), and FeSO(4), three salts known to produce a metallic taste sensation. In summary, our results identify a novel group of compounds that activate TRPV1 and, consequently, provide a molecular mechanism that may account for off tastes of sweeteners and metallic tasting salts. PMID:17567713

  19. Erythropoietin amplifies stroke-induced oligodendrogenesis in the rat.

    Li Zhang

    Full Text Available BACKGROUND: Erythropoietin (EPO, a hematopoietic cytokine, enhances neurogenesis and angiogenesis during stroke recovery. In the present study, we examined the effect of EPO on oligodendrogenesis in a rat model of embolic focal cerebral ischemia. METHODOLOGY AND PRINCIPAL FINDINGS: Recombinant human EPO (rhEPO at a dose of 5,000 U/kg (n = 18 or saline (n = 18 was intraperitoneally administered daily for 7 days starting 24 h after stroke onset. Treatment with rhEPO augmented actively proliferating oligodendrocyte progenitor cells (OPCs measured by NG2 immunoreactive cells within the peri-infarct white matter and the subventricular zone (SVZ, but did not protect against loss of myelinating oligodendrocytes measured by cyclic nucleotide phosphodiesterase (CNPase positive cells 7 days after stroke. However, 28 and 42 days after stroke, treatment with rhEPO significantly increased myelinating oligodendrocytes and myelinated axons within the peri-infarct white matter. Using lentivirus to label subventricular zone (SVZ neural progenitor cells, we found that in addition to the OPCs generated in the peri-infarct white matter, SVZ neural progenitor cells contributed to rhEPO-increased OPCs in the peri-infarct area. Using bromodeoxyuridine (BrdU for birth-dating cells, we demonstrated that myelinating oligodendrocytes observed 28 days after stroke were derived from OPCs. Furthermore, rhEPO significantly improved neurological outcome 6 weeks after stroke. In vitro, rhEPO increased differentiation of adult SVZ neural progenitor cells into oligodendrocytes and enhanced immature oligodendrocyte cell proliferation. CONCLUSIONS: Our in vivo and in vitro data indicate that EPO amplifies stroke-induced oligodendrogenesis that could facilitate axonal re-myelination and lead to functional recovery after stroke.

  20. Activation of mitogen activated protein kinases via complement receptor type 2

    LUO Min-hua 罗敏华; CHEN Ming-liang 陈明亮; Heribert Stoiber; Manfred P Dierich

    2004-01-01

    Background Complement receptor type 2 (CR2) is the receptor for C3d and C3dg and for Epstein-Barr virus. The aim of our study was to explore whether CR2 can independently mediate the activation of mitogen-activated protein kinases (MAPKs, including ERK, JNK, and p38MAPK), and to highlight the molecular mechanism of CD4+ cell deletion in AIDS.Results FACS results showed that the positive rates of HOS-CR2 and HOS-CD4CR2 cells were greater than 96%, and Western blot showed that the CR2 expression levels on HOS-CR2 and HOS-CD4CR2 cells were high. Activation and blocking tests of MAPKs (ERK, JNK, and p38MAPK) were carried out in HOS-CR2, HOS-CD4, and HOS-CD4CR2 cells. The activation of MAPKs in HOS-CR2 cells stimulated with PMA (100 ng/ml) and NHS (10%) was identical. The activation of MAPKs increased at 5 minutes, reached a peak at 10 minutes, and decreased to baseline within 30 minutes, all in a time-dependent manner; the activation of MAPKs was blocked by anti-CR2 McAb, PD98059 (inhibitor of ERK), and Wortmanin (inhibitor of PI-3K), respectively. In HOS-CD4 cells, MAPKs were activated by HIV-gp160. In HOS-CD4CR2 cells, MAPK activation was induced by HIV-gp160, 10% NHS, and HIV-gp160+10%NHS; phosphorylation of p38MAPK was dramatically induced by HIV-gp160+NHS, and lasted for 1 hour. The cell proliferation results showed that HIV-gp160 inhibited the proliferation of HOS-CD4 and HOS-CD4CR2 cells (P<0.01) and that NHS enhanced the effect of HIV-gp160 (P<0.01).Conclusions The activation of MAPKs is independently mediated by CR2 and that anti-CR2 McAb, PD98059, and Wortmanin block the activation of MAPKs, respectively. The results of the signal transduction and cell proliferation assays of HOS-CD4CR2 cells show that CR2 plays a role in the pathogenesis of HIV infection, especially in the inhibition of CD4+ cell proliferation.

  1. Thrombin functions as an inflammatory mediator through activation of its receptor

    1996-01-01

    A rat model of inflammation was used to investigate the biological effects of thrombin. The thrombin-specific inhibitor Hirulog markedly attentuated the carrageenin-induced edema of the paw of the rat. Injection of thrombin into the paw also produced edema. The effect of thrombin was due to activation of its receptor; a thrombin receptor activating peptide (TRAP) reproduced the effects of thrombin in causing edema. TRAP also increased vascular permeability as demonstrated by extravasation of ...

  2. Therapy for acute pancreatitis with platelet-activating factor receptor antagonists

    Chen, Chong; Xia, Shi-hai; Chen, Hong; Li, Xiao-Hong

    2008-01-01

    Acute pancreatitis (AP) causes release of platelet-activating factor (PAF), which induces systemic effects that contribute to circulatory disturbances and multiple organ failure. PAF is a cell surface secretion of bioactive lipid, which could produce physiological and pathological effects by binding to its cell surface receptor called platelet-activating factor receptor (PAF-R). Studies showed that PAF participates in the occurrence and development of AP and administration of platelet-activat...

  3. Receptor activator of nuclear factor kappa B ligand and osteoprotegerin levels in gingival crevicular fluid

    Fatemeh Sarlati; Mandana Sattari; Shilan Razzaghi; Malihe Nasiri

    2012-01-01

    Background: Osteoclastogenesis is coordinated by the interaction of three members of the tumor necrosis factor (TNF) superfamily: Osteoprotegerin (OPG)/receptor activator of nuclear factor kappa B ligand (RANKL)/receptor activator of nuclear factor kappa B (RANK). The aim of this study was to investigate RANKL and OPG levels, and their relative ratio in gingival crevicular fluid (GCF) of patients with chronic and aggressive periodontitis, as well as healthy controls. Materials and Methods:...

  4. Farnesoid X Receptor Inhibits the Transcriptional Activity of Carbohydrate Response Element Binding Protein in Human Hepatocytes

    Caron, Sandrine; Huaman Samanez, Carolina; Dehondt, Hélène; Ploton, Maheul; Briand, Olivier; Lien, Fleur; Dorchies, Emilie; Dumont, Julie; Postic, Catherine; Cariou, Bertrand; Lefebvre, Philippe; Staels, Bart

    2013-01-01

    The glucose-activated transcription factor carbohydrate response element binding protein (ChREBP) induces the expression of hepatic glycolytic and lipogenic genes. The farnesoid X receptor (FXR) is a nuclear bile acid receptor controlling bile acid, lipid, and glucose homeostasis. FXR negatively regulates hepatic glycolysis and lipogenesis in mouse liver. The aim of this study was to determine whether FXR regulates the transcriptional activity of ChREBP in human hepatocytes and to unravel the...

  5. Evaluation on Potential Contributions of Protease Activated Receptors Related Mediators in Allergic Inflammation

    Huiyun Zhang; Xiaoning Zeng; Shaoheng He

    2014-01-01

    Protease activated receptors (PARs) have been recognized as a distinctive four-member family of seven transmembrane G protein-coupled receptors (GPCRs) that can be cleaved by certain serine proteases. In recent years, there has been considerable interest in the role of PARs in allergic inflammation, the fundamental pathologic changes of allergy, but the potential roles of PARs in allergy remain obscure. Since many of these proteases are produced and actively involved in the pathologic process...

  6. Kallikrein activates bradykinin B2 receptors in the absence of kininogen

    Biyashev, Dauren; Tan, Fulong; Chen, Zhenlong; Zhang, Kai; Deddish, Peter A.; Erdös, Ervin G.; Hecquet, Claudie

    2005-01-01

    Kallikreins cleave plasma kininogens to release the bioactive peptides bradykinin (BK) or kallidin (Lys-BK). These peptides then activate widely disseminated B2 receptors with consequences that may be either noxious or beneficial. We used cultured cells to show that kallikrein can bypass kinin release to activate BK B2 receptors directly. To exclude intermediate kinin release or kininogen uptake from the culture medium, we cultured and maintained cells in medium entirely free of animal protei...

  7. Constitutive Activity among Orphan Class-A G Protein Coupled Receptors

    Martin, Adam L.; Michael A Steurer; Aronstam, Robert S.

    2015-01-01

    The purpose of this study was to evaluate the extent of constitutive activity among orphan class-A G protein coupled receptors within the cAMP signaling pathway. Constitutive signaling was revealed by changes in gene expression under control of the cAMP response element. Gene expression was measured in Chinese hamster ovary cells transiently co-transfected with plasmids containing a luciferase reporter and orphan receptor. Criteria adopted for defining constitutive activation were: 1) 200% el...

  8. Identification of Functionally Relevant Lysine Residues That Modulate Human Farnesoid X Receptor Activation

    Sun, An-Qiang; Luo, Yuhuan; Backos, Donald S.; Xu, Shuhua; Balasubramaniyan, Natarajan; Reigan, Philip; Suchy, Frederick J.

    2013-01-01

    Base amino acid lysine residues play an important role in regulation of nuclear receptors [e.g., farnesyl X receptor (FXR)], leading to enhanced or suppressed biologic activity. To understand the molecular mechanisms and the subsequent effects in modulating FXR functions in diverse biologic processes, we individually replaced eight highly conserved lysine residues of human FXR (hFXR) with arginine. The effects of each mutated FXR on target gene activation, subcellular localization, protein-pr...

  9. Defective lysosomal targeting of activated fibroblast growth factor receptor 3 in achondroplasia

    Cho, Jay Y.; Guo, Changsheng; Torello, Monica; Lunstrum, Gregory P.; Iwata, Tomoko; Deng, Chuxia; Horton, William A.

    2003-01-01

    Mutations of fibroblast growth factor receptor 3 (FGFR3) are responsible for achondroplasia (ACH) and related dwarfing conditions in humans. The pathogenesis involves constitutive activation of FGFR3, which inhibits proliferation and differentiation of growth plate chondrocytes. Here we report that activating mutations in FGFR3 increase the stability of the receptor. Our results suggest that the mutations disrupt c-Cbl-mediated ubiquitination that serves as a targeting signal for lysosomal de...

  10. Steroid receptor RNA activator (SRA1): unusual bifaceted gene products with suspected relevance to breast cancer

    Leygue, Etienne

    2007-01-01

    The steroid receptor RNA activator (SRA) is a unique modulator of steroid receptor transcriptional activity, as it is able to mediate its coregulatory effects as a RNA molecule. Recent findings, however, have painted a more complex picture of the SRA gene (SRA1) products. Indeed, even though SRA was initially thought to be noncoding, several RNA isoforms have now been found to encode an endogenous protein (SRAP), which is well conserved among Chordata. Although the function of SRAP remains la...

  11. The role of κ-opioid receptor activation in mediating antinociception and addiction

    Wang, Yu-hua; Sun, Jian-feng; Tao, Yi-min; Chi, Zhi-Qiang; Liu, Jing-gen

    2010-01-01

    The κ-opioid receptor (KOR), a member of the opioid receptor family, is widely expressed in the central nervous system and peripheral tissues. Substantial evidence has shown that activation of KOR by agonists and endogenous opioid peptides in vivo may produce a strong analgesic effect that is free from the abuse potential and the adverse side effects of μ-opioid receptor (MOR) agonists, such as morphine. In addition, activation of the KOR has also been shown to exert an inverse effect on morp...

  12. Multiple activities of insect repellents on odorant receptors in mosquitoes

    Several lines of evidence suggest that insect repellent molecules reduce mosquito-host contacts by interacting with odorants and odorant receptors (ORs) ultimately affecting olfactory-driven behaviors. We describe the molecular effects of ten insect repellents and a pyrethroid insecticide with known...

  13. Activation of the Retinoid X Receptor Modulates Angiotensin II-Induced Smooth Muscle Gene Expression and Inflammation in Vascular Smooth Muscle Cells

    Lehman, Allison M.B.; Montford, John R.; Horita, Henrick; Ostriker, Allison C.; Weiser-Evans, Mary C. M.; Nemenoff, Raphael A.; Furgeson, Seth B.

    2014-01-01

    The retinoid X receptor (RXR) partners with numerous nuclear receptors, such as the peroxisome proliferator activated receptor (PPAR) family, liver X receptors (LXRs), and farnesoid X receptor (FXR). Although each heterodimer can be activated by specific ligands, a subset of these receptors, defined as permissive nuclear receptors, can also be activated by RXR agonists known as rexinoids. Many individual RXR heterodimers have beneficial effects in vascular smooth muscle cells (SMCs). Because ...

  14. Activation of Nuclear Receptors RAR, RXR, and LXR Does Not Reduce Cuprizone-Induced Demyelination in Mice

    Davina Kruczek; Tim Clarner; Cordian Beyer; Markus Kipp; Jörg Mey

    2015-01-01

    Experiments with animal models of multiple sclerosis have shown that the expression of retinoid X receptors (RXR) increases during demyelination and that RXR is involved in the regulation of remyelination. After ligand binding RXRs form heterodimeric transcription factors with other nuclear receptor (NR) families including the retinoic acid receptors (RAR) and liver X receptors (LXR). We tested whether activation of these nuclear receptor complexes reduces pathological demyelination using the...

  15. Increased amphetamine-induced locomotor activity, sensitization and accumbal dopamine release in M5 muscarinic receptor knockout mice

    Schmidt, Lene S.; Miller, Anthony D.; Lester, Deranda B.; Bay-Richter, Cecilie; Schülein, Christina; Schmidt, Henriette F.; Wess, Jürgen; Blaha, Charles D.; Woldbye, David P.D.; Fink-Jensen, Anders; Wortwein, Gitta

    2009-01-01

    Muscarinic M5 receptors are the only muscarinic receptor subtype expressed by dopamine-containing neurons of the ventral tegmental area. These cells play an important role for the reinforcing properties of psychostimulants and M5 receptors modulate their activity. Previous studies showed that M5 receptor knockout (M5−/−) mice are less sensitive to the reinforcing properties of addictive drugs. Here we investigate the role of M5 receptors in the effects of amphetamine and cocaine on locomotor ...

  16. The DEAD-Box Protein DP103 (Ddx20 or Gemin-3) Represses Orphan Nuclear Receptor Activity via SUMO Modification

    Lee, Martin B.; Lebedeva, Lioudmila A.; Suzawa, Miyuki; Wadekar, Subhagya A.; Desclozeaux, Marion; Ingraham, Holly A.

    2005-01-01

    Structural analysis of nuclear receptor subfamily V orphan nuclear receptors suggests that ligand-independent mechanisms must regulate this subclass of receptors. Here, we report that steroidogenic factor 1 (SF-1) and liver receptor homolog 1 are repressed via posttranslational SUMO modification at conserved lysines within the hinge domain. Indeed, mutating these lysines or adding the SUMO isopeptidase SENP1 dramatically increased both native and Gal4-chimera receptor activities. The mechanis...

  17. Structure-activity relationships for the antifungal activity of selective estrogen receptor antagonists related to tamoxifen.

    Arielle Butts

    Full Text Available Cryptococcosis is one of the most important invasive fungal infections and is a significant contributor to the mortality associated with HIV/AIDS. As part of our program to repurpose molecules related to the selective estrogen receptor modulator (SERM tamoxifen as anti-cryptococcal agents, we have explored the structure-activity relationships of a set of structurally diverse SERMs and tamoxifen derivatives. Our data provide the first insights into the structural requirements for the antifungal activity of this scaffold. Three key molecular characteristics affecting anti-cryptococcal activity emerged from our studies: 1 the presence of an alkylamino group tethered to one of the aromatic rings of the triphenylethylene core; 2 an appropriately sized aliphatic substituent at the 2 position of the ethylene moiety; and 3 electronegative substituents on the aromatic rings modestly improved activity. Using a cell-based assay of calmodulin antagonism, we found that the anti-cryptococcal activity of the scaffold correlates with calmodulin inhibition. Finally, we developed a homology model of C. neoformans calmodulin and used it to rationalize the structural basis for the activity of these molecules. Taken together, these data and models provide a basis for the further optimization of this promising anti-cryptococcal scaffold.

  18. Activated HER-receptors in predicting outcome of ER-positive breast cancer patients treated with adjuvant endocrine therapy

    Larsen, Mathilde Skaarup; Bjerre, Karsten; Lykkesfeldt, Anne Elisabeth; Giobbie-Hurder, Anita; Lænkholm, Anne Vibeke; Henriksen, Katrine L; Ejlertsen, Bent Laursen; Rasmussen, Birgitte

    2012-01-01

    The four human epidermal growth factor receptors (HER1-4) are involved in growth stimulation and may play a role in endocrine resistance. The receptors form dimers, leading to activation by mutual phosphorylation. Our purpose was to explore the role of the activated receptors (pHER1, pHER2, pHER3...

  19. Expression and analysis of the glycosylation properties of recombinant human erythropoietin expressed in Pichia pastoris

    Ser Huy Teh; Mun Yik Fong; Zulqarnain Mohamed

    2011-01-01

    The Pichia pastoris expression system was used to produce recombinant human erythropoietin, a protein synthesized by the adult kidney and responsible for the regulation of red blood cell production. The entire recombinant human erythropoietin (rhEPO) gene was constructed using the Splicing by Overlap Extension by PCR (SOE-PCR) technique, cloned and expressed through the secretory pathway of the Pichia expression system. Recombinant erythropoietin was successfully expressed in P. pastoris. The...

  20. Anti-Erythropoietin Antibody Associated Pure Red Cell Aplasia Resolved after Liver Transplantation

    Hung, Annie K.; Jennifer Guy; Behler, Caroline M.; Lee, Eugene E.

    2015-01-01

    Patients undergoing antiviral therapy for chronic hepatitis C often develop anemia secondary to ribavirin and interferon. Recombinant erythropoietin has been used to improve anemia associated with antiviral therapy and to minimize dose reductions, which are associated with decreased rates of sustained virologic response. A rare potential side effect of recombinant erythropoietin is anti-erythropoietin antibody associated pure red cell aplasia. In chronic kidney disease patients with this enti...