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Sample records for idoxifene antagonises e2-dependent

  1. Selected reaction monitoring LC-MS determination of idoxifene and its pyrrolidinone metabolite in human plasma using robotic high-throughput, sequential sample injection.

    PubMed

    Onorato, J M; Henion, J D; Lefebvre, P M; Kiplinger, J P

    2001-01-01

    The generation of large numbers of samples during early drug discovery has increased the demand for rapid and selective methods of analysis. Liquid chromatography-tandem mass spectrometry (LC-MS-MS), because of its sensitivity, selectivity, and robustness, has emerged as a powerful tool in the pharmaceutical industry for many analytical needs. This work presents a high-throughput selected reaction monitoring LC-MS bioanalytical method for the determination of idoxifene, a selective estrogen receptor modulator, and its pyrrolidinone metabolite in clinical human plasma samples. The described method uses short, small-bore columns, high flow rates, and elevated HPLC column temperatures to perform LC separations of idoxifene and its metabolite within 10 s/sample. Sequential injections were accomplished with a 215/889 multiple probe liquid handler (Gilson, Inc.), which aspirates eight samples simultaneously and performs its rinse cycle parallel to sample injection, resulting in minimum lag time between injections. This high-throughput method was applied to the determination of idoxifene and its metabolite in clinical human plasma samples. Sample preparation employed liquid/liquid extraction in the 96-well format. Method validation included determination of intra- and interassay accuracy and precision values, recovery studies, autosampler stability, and freeze-thaw stability. The LOQ obtained was 10 ng/mL for idoxifene and 30 ng/mL for the metabolite. Using idoxifene-d5 as an internal standard, idoxifene showed acceptable accuracy and precision values at QC level 1 (QC1, 15 ng/mL), level 2 (QC2, 100 ng/mL), and level 3 (QC3, 180 ng/mL) (85.0% accuracy +/- 12.0% precision, 95.1 +/- 4.9%, and 90.3 +/- 4.7%, respectively). The pyrrolidinone metabolite also showed acceptable accuracy and precision values (using no internal standard for quantitation) at QC1 (60 ng/mL), QC2 (100 ng/mL), and QC3 (180 ng/mL) (104.9 +/- 14.4%, 91.1 +/- 13.0%, and 90.8 +/- 12.2%, respectively). The

  2. Naloxone Antagonises Soman-induced Central Respiratory Depression in Rats.

    PubMed

    Škrbić, Ranko; Stojiljković, Miloš P; Ćetković, Slavko S; Dobrić, Silva; Jeremić, Dejan; Vulović, Maja

    2016-12-19

    The influence of naloxone on respiration impaired by the highly toxic organophosphate nerve agent soman in anaesthetized rats was investigated. Soman, administered in a dose that was ineffective in blocking the electrically induced contractions of the phrenic nerve-diaphragm preparation in situ, induced a complete block of the spontaneous respiratory movements of the diaphragm, indicating the domination of central over the peripheral effects. Naloxone dose-dependently antagonised the soman-induced respiratory blockade. Atropine, at a dose that was per se ineffective in counteracting soman-induced respiratory depression, potentiated the protective effects of naloxone and completely restored respiration. Naloxone remained completely ineffective in antagonising respiratory depression induced by the muscarinic receptor agonist the oxotremorine. It is assumed that naloxone antagonises soman-induced respiratory inhibition by blocking endogenous opioidergic respiratory control pathways that are independent of the stimulation of muscarinic receptors. This article is protected by copyright. All rights reserved.

  3. Comparison between liquid chromatography-time of-flight mass spectrometry and selected reaction monitoring liquid chromatography-mass spectrometry for quantitative determination of idoxifene in human plasma.

    PubMed

    Zhang, H; Henion, J

    2001-06-05

    This study compares HPLC electrospray time-of-flight mass spectrometry and selected reaction monitoring (SRM) LC-MS for high throughput quantitative determination of a small molecule drug in biological samples. A high throughput LC-MS method was developed for quantitatative determination of idoxifene in human plasma and the evaluation was accomplished with the cross-validation of the developed LC-MS method between the time-of-flight mass spectrometer, and a triple quadrupole mass spectrometer operated in the SRM mode. A simple one-step semi-automated 96-well liquid-liquid extraction procedure was used to prepare 96 samples in approximately 30 min and a rapid gradient was used to shorten the LC run time. Time-of-flight mass spectrometry provides acquisition of full-scan mass spectra and extracted ion current chromatograms, which may be extracted from the total ion current chromatogram for peak area determination. The limit of quantitation for idoxifene in human plasma obtained with the time-of-flight mass spectrometer was 5 ng/ml based on 100-microl aliquots of human plasma, and the linear dynamic range was from 5 ng/ml to 2000 ng/ml. The quantitative LC-MS results from the time-of-flight mass spectrometer demonstrated that precision did not exceed 7.1% and accuracy did not exceed 1.7% with reference to quality control samples at three concentration levels in replicates of six. In contrast, the limit of quantitation for idoxifene in human plasma using a tandem triple quadrupole mass spectrometer was 0.5 ng/ml with a linear dynamic range to 1000 ng/ml. The results from the triple quadrupole instrument show that the precision did not exceed 2.2% and accuracy did not exceed 2.9%. The overall results suggest time-of-flight mass spectrometry may be a viable technique for high throughput bioanalytical work for the quantitative determination of a representative small molecule drug in the low ng/ml range in human plasma.

  4. Atypical antipsychotics, clozapine and sulpiride do not antagonise amphetamine-induced stereotyped locomotion.

    PubMed

    Moore, S; Kenyon, P

    1994-02-01

    An automated tracking system which converted an animal's path between quadrants of a circular open field into a series of trips was used to analyse stereotyped locomotion in amphetamine treated rats. Amphetamine (3.5 mg/kg) increased the horizontal distance moved and the number and proportion of thigmotaxic trips around the perimeter of the apparatus (length 4 trips). To investigate the hypothesis that classic antipsychotics, but not atypical antipsychotics, would antagonise the repetitive boundary patrolling associated with amphetamine-induced hyperactivity, animals were pretreated with haloperidol (0.01, 0.025, 0.05, 0.075 mg/kg), clozapine (5, 10, 20 mg/kg) or (+/-)sulpiride (10, 20, 50 mg/kg) 30 min before 3.5 mg/kg amphetamine. The results showed that the classic antipsychotic haloperidol antagonised both hyperactivity and the increased proportion of length 4 trips. In marked contrast, the atypical antipsychotics clozapine and sulpiride antagonised hyperactivity but did not reduce the proportion of length 4 trips. The inability of atypical antipsychotics to reduce the repetitive boundary patrolling associated with amphetamine-induced hyperactivity is consistent with the action of these drugs on other forms of amphetamine-induced stereotyped behaviour, and indicates that locomotor routes under amphetamine are stereotyped. The measurement of trip lengths provides a sensitive tool for examining drug action on the spatial distribution of open field locomotion.

  5. Standing Up Slowly Antagonises Initial Blood Pressure Decrease in Older Adults with Orthostatic Hypotension.

    PubMed

    de Bruïne, Eline S; Reijnierse, Esmee M; Trappenburg, Marijke C; Pasma, Jantsje H; de Vries, Oscar J; Meskers, Carel G M; Maier, Andrea B

    2017-01-01

    Orthostatic hypotension (OH) is common in older adults and associated with increased morbidity and mortality, loss of independence and high health-care costs. Standing up slowly is a recommended non-pharmacological intervention. However, the effectiveness of this advice has not been well studied. The aim of this study was to investigate whether standing up slowly antagonises posture-related blood pressure (BP) decrease in a clinically relevant population of geriatric outpatients. In this cross-sectional study, 24 community-dwelling older adults referred to a geriatric outpatient clinic and diagnosed with OH were included. BP was measured continuously during 3 consecutive transitions from supine to standing position during normal, slow and fast transition. The relative BP decrease at 0-15 s after slow transition was significantly lower than after normal transition (p = 0.003 for both systolic BP and diastolic BP) and fast transition (p = 0.045 for systolic BP; diastolic BP: non-significant). The relative diastolic BP decrease at 60-180 s after normal transition was significantly lower than after fast transition (p = 0.029). Standing up slowly antagonises BP decrease predominantly during the first 15 s of standing up in a clinically relevant population of geriatric outpatients diagnosed with OH. Results support the non-pharmacological intervention in clinical practice to counteract OH. © 2016 S. Karger AG, Basel.

  6. Baclofen antagonises intravenous self-administration of gamma-hydroxybutyric acid in mice.

    PubMed

    Fattore, L; Cossu, G; Martellotta, M C; Deiana, S; Fratta, W

    2001-07-20

    gamma-Hydroxybutyric acid (GHB) is a widely used recreational drug known to exert positive reinforcing effects in animals and humans. The GABA(B) receptor agonist baclofen has been proved to possess antimotivational effect and to inhibit alcohol, cocaine, heroin and nicotine intake. In the present study we evaluated the effect of baclofen on i.v. self-administration of GHB in drug-naive mice under a fixed-ratio (FR-1) schedule of reinforcement and nose-poking-like response as operandum. Results show that baclofen was able to completely prevent GHB seeking behaviour, decreasing the rate of responding to basal values, without showing any reinforcing properties when made contingent on nose-poking response. Our findings demonstrate that baclofen antagonises GHB i.v. self-administration, supporting an important role for the GABA(B) receptor in reward-related mechanisms underlying addictive behaviour.

  7. Piracetam and TRH analogues antagonise inhibition by barbiturates, diazepam, melatonin and galanin of human erythrocyte D-glucose transport.

    PubMed

    Naftalin, Richard J; Cunningham, Philip; Afzal-Ahmed, Iram

    2004-06-01

    1 Nootropic drugs increase glucose uptake into anaesthetised brain and into Alzheimer's diseased brain. Thyrotropin-releasing hormone, TRH, which has a chemical structure similar to nootropics increases cerebellar uptake of glucose in murine rolling ataxia. This paper shows that nootropic drugs like piracetam (2-oxo 1 pyrrolidine acetamide) and levetiracetam and neuropeptides like TRH antagonise the inhibition of glucose transport by barbiturates, diazepam, melatonin and endogenous neuropeptide galanin in human erythrocytes in vitro. 2 The potencies of nootropic drugs in opposing scopolamine-induced memory loss correlate with their potencies in antagonising pentobarbital inhibition of erythrocyte glucose transport in vitro (P<0.01). Less potent nootropics, D-levetiracetam and D-pyroglutamate, have higher antagonist Ki's against pentobarbital inhibition of glucose transport than more potent L-stereoisomers (P<0.001). 3 Piracetam and TRH have no direct effects on net glucose transport, but competitively antagonise hypnotic drug inhibition of glucose transport. Other nootropics, like aniracetam and levetiracetam, while antagonising pentobarbital action, also inhibit glucose transport. Analeptics like bemigride and methamphetamine are more potent inhibitors of glucose transport than antagonists of hypnotic action on glucose transport. 4 There are similarities between amino-acid sequences in human glucose transport protein isoform 1 (GLUT1) and the benzodiazepine-binding domains of GABAA (gamma amino butyric acid) receptor subunits. Mapped on a 3D template of GLUT1, these homologies suggest that the site of diazepam and piracetam interaction is a pocket outside the central hydrophilic pore region. 5 Nootropic pyrrolidone antagonism of hypnotic drug inhibition of glucose transport in vitro may be an analogue of TRH antagonism of galanin-induced narcosis.

  8. Piracetam and TRH analogues antagonise inhibition by barbiturates, diazepam, melatonin and galanin of human erythrocyte D-glucose transport

    PubMed Central

    Naftalin, Richard J; Cunningham, Philip; Afzal-Ahmed, Iram

    2004-01-01

    Nootropic drugs increase glucose uptake into anaesthetised brain and into Alzheimer's diseased brain. Thyrotropin-releasing hormone, TRH, which has a chemical structure similar to nootropics increases cerebellar uptake of glucose in murine rolling ataxia. This paper shows that nootropic drugs like piracetam (2-oxo 1 pyrrolidine acetamide) and levetiracetam and neuropeptides like TRH antagonise the inhibition of glucose transport by barbiturates, diazepam, melatonin and endogenous neuropeptide galanin in human erythrocytes in vitro. The potencies of nootropic drugs in opposing scopolamine-induced memory loss correlate with their potencies in antagonising pentobarbital inhibition of erythrocyte glucose transport in vitro (P<0.01). Less potent nootropics, D-levetiracetam and D-pyroglutamate, have higher antagonist Ki's against pentobarbital inhibition of glucose transport than more potent L-stereoisomers (P<0.001). Piracetam and TRH have no direct effects on net glucose transport, but competitively antagonise hypnotic drug inhibition of glucose transport. Other nootropics, like aniracetam and levetiracetam, while antagonising pentobarbital action, also inhibit glucose transport. Analeptics like bemigride and methamphetamine are more potent inhibitors of glucose transport than antagonists of hypnotic action on glucose transport. There are similarities between amino-acid sequences in human glucose transport protein isoform 1 (GLUT1) and the benzodiazepine-binding domains of GABAA (gamma amino butyric acid) receptor subunits. Mapped on a 3D template of GLUT1, these homologies suggest that the site of diazepam and piracetam interaction is a pocket outside the central hydrophilic pore region. Nootropic pyrrolidone antagonism of hypnotic drug inhibition of glucose transport in vitro may be an analogue of TRH antagonism of galanin-induced narcosis. PMID:15148255

  9. The vav oncogene antagonises EGFR signalling and regulates adherens junction dynamics during Drosophila eye development.

    PubMed

    Martín-Bermudo, Maria-Dolores; Bardet, Pierre-Luc; Bellaïche, Yohanns; Malartre, Marianne

    2015-04-15

    Organ shaping and patterning depends on the coordinated regulation of multiple processes. The Drosophila compound eye provides an excellent model to study the coordination of cell fate and cell positioning during morphogenesis. Here, we find that loss of vav oncogene function during eye development is associated with a disorganised retina characterised by the presence of additional cells of all types. We demonstrate that these defects result from two distinct roles of Vav. First, and in contrast to its well-established role as a positive effector of the EGF receptor (EGFR), we show that readouts of the EGFR pathway are upregulated in vav mutant larval eye disc and pupal retina, indicating that Vav antagonises EGFR signalling during eye development. Accordingly, decreasing EGFR signalling in vav mutant eyes restores retinal organisation and rescues most vav mutant phenotypes. Second, using live imaging in the pupal retina, we observe that vav mutant cells do not form stable adherens junctions, causing various defects, such as recruitment of extra primary pigment cells. In agreement with this role in junction dynamics, we observe that these phenotypes can be exacerbated by lowering DE-Cadherin or Cindr levels. Taken together, our findings establish that Vav acts at multiple times during eye development to prevent excessive cell recruitment by limiting EGFR signalling and by regulating junction dynamics to ensure the correct patterning and morphogenesis of the Drosophila eye. © 2015. Published by The Company of Biologists Ltd.

  10. Tobramycin and bicarbonate synergise to kill planktonic Pseudomonas aeruginosa, but antagonise to promote biofilm survival.

    PubMed

    Kaushik, Karishma S; Stolhandske, Jake; Shindell, Orrin; Smyth, Hugh D; Gordon, Vernita D

    2016-01-01

    Increasing antibiotic resistance and the declining rate at which new antibiotics come into use create a need to increase the efficacy of existing antibiotics. The aminoglycoside tobramycin is standard-of-care for many types of Pseudomonas aeruginosa infections, including those in the lungs of cystic fibrosis (CF) patients. P. aeruginosa is a nosocomial and opportunistic pathogen that, in planktonic form, causes acute infections and, in biofilm form, causes chronic infections. Inhaled bicarbonate has recently been proposed as a therapy to improve antimicrobial properties of the CF airway surface liquid and viscosity of CF mucus. Here we measure the effect of combining tobramycin and bicarbonate against P. aeruginosa, both lab strains and CF clinical isolates. Bicarbonate synergises with tobramycin to enhance killing of planktonic bacteria. In contrast, bicarbonate antagonises with tobramycin to promote better biofilm growth. This suggests caution when evaluating bicarbonate as a therapy for CF lungs infected with P. aeruginosa biofilms. We analyse tobramycin and bicarbonate interactions using an interpolated surface methodology to measure the dose-response function. These surfaces allow more accurate estimation of combinations yielding synergy and antagonism than do standard isobolograms. By incorporating predictions based on Loewe additivity theory, we can consolidate information on a wide range of combinations that produce a complex dose-response surface, into a single number that measures the net effect. This tool thus allows rapid initial estimation of the potential benefit or harm of a therapeutic combination. Software code is freely made available as a resource for the community.

  11. A type III effector antagonises death receptor signalling during bacterial gut infection

    PubMed Central

    Pearson, Jaclyn S; Giogha, Cristina; Ong, Sze Ying; Kennedy, Catherine L; Kelly, Michelle; Robinson, Keith S; Wong, Tania; Mansell, Ashley; Riedmaier, Patrice; Oates, Clare VL; Zaid, Ali; Mühlen, Sabrina; Crepin, Valerie F; Marches, Olivier; Ang, Ching-Seng; Williamson, Nicholas A; O’Reilly, Lorraine A; Bankovacki, Aleksandra; Nachbur, Ueli; Infusini, Giuseppe; Webb, Andrew I; Silke, John; Strasser, Andreas; Frankel, Gad; Hartland, Elizabeth L

    2013-01-01

    Successful infection by enteric bacterial pathogens depends on the ability of the bacteria to colonise the gut, replicate in host tissues and disseminate to other hosts. Pathogens such as Salmonella, Shigella and enteropathogenic and enterohaemorrhagic E. coli (EPEC and EHEC), utilise a type III secretion system (T3SS) to deliver virulence effector proteins into host cells during infection that promote colonisation and interfere with antimicrobial host responses 1-3. Here we report that the T3SS effector NleB1 from EPEC binds to host cell death domain containing proteins and thereby inhibits death receptor signalling. Protein interaction studies identified FADD, TRADD and RIPK1 as binding partners of NleB1. NleB1 expressed ectopically or injected by the bacterial T3SS prevented Fas ligand or TNF-induced formation of the canonical death inducing signalling complex (DISC) and proteolytic activation of caspase-8, an essential step in death receptor induced apoptosis. This inhibition depended on the N-GlcNAc transferase activity of NleB1, which specifically modified Arg117 in the death domain of FADD. The importance of the death receptor apoptotic pathway to host defence was demonstrated using mice deficient in the FAS signalling pathway, which showed delayed clearance of the EPEC-like mouse pathogen Citrobacter rodentium and reversion to virulence of an nleB mutant. The activity of NleB suggests that EPEC and other attaching and effacing (A/E) pathogens antagonise death receptor induced apoptosis of infected cells, thereby blocking a major antimicrobial host response. PMID:24025841

  12. c-Myc Antagonises the Transcriptional Activity of the Androgen Receptor in Prostate Cancer Affecting Key Gene Networks.

    PubMed

    Barfeld, Stefan J; Urbanucci, Alfonso; Itkonen, Harri M; Fazli, Ladan; Hicks, Jessica L; Thiede, Bernd; Rennie, Paul S; Yegnasubramanian, Srinivasan; DeMarzo, Angelo M; Mills, Ian G

    2017-04-01

    Prostate cancer (PCa) is the most common non-cutaneous cancer in men. The androgen receptor (AR), a ligand-activated transcription factor, constitutes the main drug target for advanced cases of the disease. However, a variety of other transcription factors and signaling networks have been shown to be altered in patients and to influence AR activity. Amongst these, the oncogenic transcription factor c-Myc has been studied extensively in multiple malignancies and elevated protein levels of c-Myc are commonly observed in PCa. Its impact on AR activity, however, remains elusive. In this study, we assessed the impact of c-Myc overexpression on AR activity and transcriptional output in a PCa cell line model and validated the antagonistic effect of c-MYC on AR-targets in patient samples. We found that c-Myc overexpression partially reprogrammed AR chromatin occupancy and was associated with altered histone marks distribution, most notably H3K4me1 and H3K27me3. We found c-Myc and the AR co-occupy a substantial number of binding sites and these exhibited enhancer-like characteristics. Interestingly, c-Myc overexpression antagonised clinically relevant AR target genes. Therefore, as an example, we validated the antagonistic relationship between c-Myc and two AR target genes, KLK3 (alias PSA, prostate specific antigen), and Glycine N-Methyltransferase (GNMT), in patient samples. Our findings provide unbiased evidence that MYC overexpression deregulates the AR transcriptional program, which is thought to be a driving force in PCa. Crown Copyright © 2017. Published by Elsevier B.V. All rights reserved.

  13. TRAIL-activated EGFR by Cbl-b-regulated EGFR redistribution in lipid rafts antagonises TRAIL-induced apoptosis in gastric cancer cells.

    PubMed

    Xu, Ling; Zhang, Ye; Liu, Jing; Qu, Jinglei; Hu, Xuejun; Zhang, Fan; Zheng, Huachuan; Qu, Xiujuan; Liu, Yunpeng

    2012-11-01

    Most gastric cancer cells are resistant to tumour necrosis factor-related apoptosis-inducing ligand (TRAIL). Since TRAIL resistance is associated with lipid rafts, in which both death receptors and epidermal growth factor receptors (EGFR) are enriched, our aim is to identify how lipid raft-regulated receptor redistribution influences the sensitivity of TRAIL in gastric cancer cells. In TRAIL-resistant gastric cancer cells, TRAIL did not induce effective death-inducing signalling complex (DISC) formation in lipid rafts, accompanied with EGFR translocation into lipid rafts, and activation of EGFR pathway. Knockdown of casitas B-lineage lymphoma-b (Cbl-b) enhanced TRAIL-induced apoptosis by promoting DISC formation in lipid rafts. However, knockdown of Cbl-b also enhanced EGFR translocation into lipid rafts and EGFR pathway activation induced by TRAIL. Either using inhibitors of EGFR or depletion of EGFR with small interfering RNA (siRNA) prevented EGFR pathway activation, and thus increased TRAIL-induced apoptosis, especially in Cbl-b knockdown clones. Taken together, TRAIL-induced EGFR activation through Cbl-b-regulated EGFR redistribution in lipid rafts antagonised TRAIL-induced apoptosis. The contribution of DISC formation and the inhibition of EGFR signal triggered in lipid rafts are both essential for increasing the sensitivity of gastric cancer cells to TRAIL.

  14. [Case control trial on putative factors antagonising the successful project course of MD thesis projects].

    PubMed

    Scharfenberg, J; Schaper, K; Krummenauer, F

    2015-05-01

    Award of the degree MD has special relevance in Germany since the underlying research project can be started during the qualification for admission to doctoral training. This leads to a large number of thesis projects with a not always sufficiently pronounced enthusiasm and thus poor chances of success. Accordingly a case control study was undertaken in the Department of Human Medicine, Witten/Herdecke University to investigate reported drop-outs of thesis projects. In autumn 2012 all students in the clinical phases of human medicine education were surveyed using a self-conceived questionnaire on previously initiated or terminated thesis projects, "terminated" is defined as the unsuccessful ending of a project after working for at least 3 months. Individually reported thesis terminations were evaluated using defined items in a 4-stage Likert scale regarding thesis plan and project, subsequently, graduate students who successfully completed a project received the same questionnaire. The items possibly corresponding to process determinants were averaged to a total of 7 dimensions prior to the analysis; the resulting scores were normalised in value ranges 0.0 to 1.0 (1.0 = optimal project situation) whereby individual items could be included in several scores. By means of 5 items a primary endpoint from the faculty's perspective on "compliance with formal procedures" was aggregated; by means of a two-sided Wilcoxon test at the 5 % level students with unsuccessful and successful courses were compared along the corresponding scores. 181 of 276 students from 7 study semesters participated in the screening; details of 17 terminations and 23 currently successful courses could be evaluated in the case control study. For significant differences (p < 0.001) between unsuccessful and successful courses in the primary endpoint, median scores of 0.17 (0.07-0.50) versus 0.73 (0.53-0.83) were estimated. There were differences between unsuccessful and (as yet) successful courses, especially with regard to the aspects "compliance with formal procedures". Thus a recommendation can be derived in the sense of a stricter and, if necessary, sanctioning demand for formal procedures such as early reporting of thesis projects to the responsible committees. A weakness is the low number of evaluable self-reported drop-outs as well as the overall moderate response rate. Georg Thieme Verlag KG Stuttgart · New York.

  15. Vitamin D Antagonises the Suppressive Effect of Inflammatory Cytokines on CTLA-4 Expression and Regulatory Function

    PubMed Central

    Jeffery, Louisa E.; Qureshi, Omar S.; Gardner, David; Hou, Tie Z.; Briggs, Zoe; Soskic, Blagoje; Baker, Jennifer; Raza, Karim; Sansom, David M.

    2015-01-01

    The immune suppressive protein CTLA-4 is constitutively expressed by Tregs and induced in effector T cells upon activation. Its crucial role in adaptive immunity is apparent from the fatal autoimmune pathology seen in CTLA-4 knockout mice. However, little is known regarding factors that regulate CTLA-4 expression and their effect upon its function to remove CD80 and CD86 from antigen presenting cells by transendocytosis. Th17 cells are emerging as significant players in autoimmunity as well as other diseases. Therefore, in this study we have examined the effects of Th17 polarising conditions on CTLA-4 expression and function in human T cells and show that Th17 conditions can suppress the expression of CTLA-4 and its transendocytic function. In contrast to Th17 cells, vitamin D is inversely associated with autoimmune disease. We have previously shown a striking ability of 1,25 dihydroxyvitamin D3 (1,25(OH)2D3) to enhance CTLA-4, however, its effects upon B7 transendocytosis and its activity in the context of inflammation remained unknown. Here we show that induction of CTLA-4 by 1,25(OH)2D3 can actually be enhanced in the presence of Th17 polarising cytokines. Furthermore, its transendocytic function was maintained such that T cells generated in the presence of Th17 conditions and 1,25(OH)2D3 were highly effective at capturing CTLA-4 ligands from antigen presenting cells and suppressing T cell division. Taken together, these data reveal an inhibitory effect of Th17 polarising conditions upon CTLA-4-mediated regulation and show that 1,25(OH)2D3 counteracts this effect. Given the importance of CTLA-4-mediated suppression in the control of autoimmune diseases, our novel data highlight the importance of vitamin D in inflammatory settings. PMID:26134669

  16. WNT16 antagonises excessive canonical WNT activation and protects cartilage in osteoarthritis.

    PubMed

    Nalesso, Giovanna; Thomas, Bethan Lynne; Sherwood, Joanna Claire; Yu, Jing; Addimanda, Olga; Eldridge, Suzanne Elizabeth; Thorup, Anne-Sophie; Dale, Leslie; Schett, Georg; Zwerina, Jochen; Eltawil, Noha; Pitzalis, Costantino; Dell'Accio, Francesco

    2017-01-01

    Both excessive and insufficient activation of WNT signalling results in cartilage breakdown and osteoarthritis. WNT16 is upregulated in the articular cartilage following injury and in osteoarthritis. Here, we investigate the function of WNT16 in osteoarthritis and the downstream molecular mechanisms. Osteoarthritis was induced by destabilisation of the medial meniscus in wild-type and WNT16-deficient mice. Molecular mechanisms and downstream effects were studied in vitro and in vivo in primary cartilage progenitor cells and primary chondrocytes. The pathway downstream of WNT16 was studied in primary chondrocytes and using the axis duplication assay in Xenopus. WNT16-deficient mice developed more severe osteoarthritis with reduced expression of lubricin and increased chondrocyte apoptosis. WNT16 supported the phenotype of cartilage superficial-zone progenitor cells and lubricin expression. Increased osteoarthritis in WNT16-deficient mice was associated with excessive activation of canonical WNT signalling. In vitro, high doses of WNT16 weakly activated canonical WNT signalling, but, in co-stimulation experiments, WNT16 reduced the capacity of WNT3a to activate the canonical WNT pathway. In vivo, WNT16 rescued the WNT8-induced primary axis duplication in Xenopus embryos. In osteoarthritis, WNT16 maintains a balanced canonical WNT signalling and prevents detrimental excessive activation, thereby supporting the homeostasis of progenitor cells. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.

  17. Slmb antagonises the aPKC/Par-6 complex to control oocyte and epithelial polarity.

    PubMed

    Morais-de-Sá, Eurico; Mukherjee, Avik; Lowe, Nick; St Johnston, Daniel

    2014-08-01

    The Drosophila anterior-posterior axis is specified when the posterior follicle cells signal to polarise the oocyte, leading to the anterior/lateral localisation of the Par-6/aPKC complex and the posterior recruitment of Par-1, which induces a microtubule reorganisation that localises bicoid and oskar mRNAs. Here we show that oocyte polarity requires Slmb, the substrate specificity subunit of the SCF E3 ubiquitin ligase that targets proteins for degradation. The Par-6/aPKC complex is ectopically localised to the posterior of slmb mutant oocytes, and Par-1 and oskar mRNA are mislocalised. Slmb appears to play a related role in epithelial follicle cells, as large slmb mutant clones disrupt epithelial organisation, whereas small clones show an expansion of the apical domain, with increased accumulation of apical polarity factors at the apical cortex. The levels of aPKC and Par-6 are significantly increased in slmb mutants, whereas Baz is slightly reduced. Thus, Slmb may induce the polarisation of the anterior-posterior axis of the oocyte by targeting the Par-6/aPKC complex for degradation at the oocyte posterior. Consistent with this, overexpression of the aPKC antagonist Lgl strongly rescues the polarity defects of slmb mutant germline clones. The role of Slmb in oocyte polarity raises an intriguing parallel with C. elegans axis formation, in which PAR-2 excludes the anterior PAR complex from the posterior cortex to induce polarity, but its function can be substituted by overexpressing Lgl.

  18. Van-Gogh-like 2 antagonises the canonical WNT pathway and is methylated in colorectal cancers.

    PubMed

    Piazzi, G; Selgrad, M; Garcia, M; Ceccarelli, C; Fini, L; Bianchi, P; Laghi, L; D'Angelo, L; Paterini, P; Malfertheiner, P; Chieco, P; Boland, C R; Bazzoli, F; Ricciardiello, L

    2013-04-30

    Aberrant activation of the canonical WNT signaling is a feature of colorectal cancer (CRC). Van-Gogh-like 2 (VANGL2) belongs to the non-canonical WNT pathway whose activation inhibits canonical WNT signaling. In this study, we investigated the role of VANGL2 and its epigenetic regulation in CRC. Van-Gogh-like 2 expression and promoter methylation after 5-aza-2'-deoxycytidine (5-aza) treatment were evaluated in CRC cells. DNA samples from 418 sporadic CRCs were tested for VANGL2 promoter methylation and microsatellite instability (MSI). Proliferation, colony formation and activation of the WNT pathway were tested in cells after VANGL2 overexpression. Van-Gogh-like 2 mRNA was significantly higher in 5-aza-treated RKO, LOVO and SW48, whereas no differences were found in SW480. Van-Gogh-like 2 was fully methylated in RKO, SW48, HCT116, DLD1 and Caco2; partially methylated in LOVO, LS174T and SW837; and unmethylated in SW480, SW620 and HT29. Higher expression of VANGL2 mRNA was found in the unmethylated cell lines. In CRC specimens (8.93% MSI), methylated VANGL2 was associated with MSI, higher grade, proximal colon location and BRAF mutation. Van-Gogh-like 2 overexpression in SW480 significantly decreased proliferation, colony formation and β-catenin levels. Van-Gogh-like 2 is frequently methylated in MSI-CRCs with BRAF mutation and may act as a tumour suppressor gene, counteracting WNT/β-catenin signaling.

  19. Van-Gogh-like 2 antagonises the canonical WNT pathway and is methylated in colorectal cancers

    PubMed Central

    Piazzi, G; Selgrad, M; Garcia, M; Ceccarelli, C; Fini, L; Bianchi, P; Laghi, L; D'Angelo, L; Paterini, P; Malfertheiner, P; Chieco, P; Boland, C R; Bazzoli, F; Ricciardiello, L

    2013-01-01

    Background: Aberrant activation of the canonical WNT signaling is a feature of colorectal cancer (CRC). Van-Gogh-like 2 (VANGL2) belongs to the non-canonical WNT pathway whose activation inhibits canonical WNT signaling. In this study, we investigated the role of VANGL2 and its epigenetic regulation in CRC. Methods: Van-Gogh-like 2 expression and promoter methylation after 5-aza-2′-deoxycytidine (5-aza) treatment were evaluated in CRC cells. DNA samples from 418 sporadic CRCs were tested for VANGL2 promoter methylation and microsatellite instability (MSI). Proliferation, colony formation and activation of the WNT pathway were tested in cells after VANGL2 overexpression. Results: Van-Gogh-like 2 mRNA was significantly higher in 5-aza-treated RKO, LOVO and SW48, whereas no differences were found in SW480. Van-Gogh-like 2 was fully methylated in RKO, SW48, HCT116, DLD1 and Caco2; partially methylated in LOVO, LS174T and SW837; and unmethylated in SW480, SW620 and HT29. Higher expression of VANGL2 mRNA was found in the unmethylated cell lines. In CRC specimens (8.93% MSI), methylated VANGL2 was associated with MSI, higher grade, proximal colon location and BRAF mutation. Van-Gogh-like 2 overexpression in SW480 significantly decreased proliferation, colony formation and β-catenin levels. Conclusion: Van-Gogh-like 2 is frequently methylated in MSI-CRCs with BRAF mutation and may act as a tumour suppressor gene, counteracting WNT/β-catenin signaling. PMID:23579212

  20. Small Molecules Antagonise the MIA-Fibronectin Interaction in Malignant Melanoma

    PubMed Central

    Yip, King Tuo; Zhong, Xue Yin; Seibel, Nadia; Pütz, Stefanie; Autzen, Jasmin; Gasper, Raphael; Hofmann, Eckhard; Scherkenbeck, Jürgen; Stoll, Raphael

    2016-01-01

    Melanoma inhibitory activity (MIA), an extracellular protein highly expressed by malignant melanoma cells, plays an important functional role in melanoma development, progression, and metastasis. After its secretion, MIA directly interacts with extracellular matrix proteins, such as fibronectin (FN). By this mechanism, MIA actively facilitates focal cell detachment from surrounding structures and strongly promotes tumour cell invasion and migration. Hence, the molecular understanding of MIA’s function provides a promising target for the development of new strategies in malignant melanoma therapy. Here, we describe for the first time the discovery of small molecules that are able to disrupt the MIA-FN complex by selectively binding to a new druggable pocket, which we could identify on MIA by structural analysis and fragment-based screening. Our findings may inspire novel drug discovery efforts aiming at a therapeutically effective treatment of melanoma by targeting MIA. PMID:27151361

  1. Co-culture with potentially probiotic microorganisms antagonises virulence factors of Clostridium difficile in vitro.

    PubMed

    Trejo, Fernando M; Pérez, Pablo F; De Antoni, Graciela L

    2010-06-01

    Toxigenic strains of Clostridium difficile were co-cultured with different strains of bifidobacteria and lactobacilli. Spent culture supernatants were tested for biological activity on cultured Vero cells. Co-culture of C. difficile with some potentially probiotic strains lead to a reduction of the biological activity of spent culture supernatants. The observed effects cannot be ascribed either to secreted factors from the probiotic strains or to toxin adsorption by bacterial cells. Immunological assays showed that there was significant diminution of both clostridial toxins (TcdA and TcdB) in spent culture supernatants of co-cultures as compared with pure clostridial cultures. Even though co-cultured clostridial cells showed a slight increase of intracellular toxins, this increase did not completely explains the reduction of toxin concentration in culture supernatants. The evidence suggests that the antagonism could be due to the diminution of the synthesis and/or secretion of both clostridial toxins. Our findings provide new insights into the possible mechanisms involved in the protective effect of probiotics in the context of C. difficile infection.

  2. Alpha fetoprotein antagonises benzyl isothiocyanate inhibition of the malignant behaviors of hepatocellular carcinoma cells

    PubMed Central

    Zhu, Mingyue; Li, Wei; Guo, Junli; Lu, Yan; Dong, Xu; Lin, Bo; Chen, Yi; Zhang, Xueer; Li, Mengsen

    2016-01-01

    Benzyl isothiocyanate (BITC) is a dietary isothiocyanate derived from cruciferous vegetables. Recent studies showed that BITC inhibited the growth of many cancer cells, including hepatocellular carcinoma (HCC) cells. Alpha-fetoprotein (AFP) is a important molecule for promoting progression of HCC, in the present investigation, we explore the influence of AFP on the role of BITC in the malignant behaviours of HCC cells, and the potential underlying mechanisms. We found thatBITC inhibited viability, migration, invasion and induced apoptosis of human liver cancer cell lines, Bel 7402(AFP producer) and HLE(non-AFP producer) cells in vitro. The role of BITC involve in promoting actived-caspase-3 and PARP-1 expression, and enhancing caspase-3 activity but decreasing MMP-2/9, survivin and CXCR4 expression. AFP antagonized the effect of BITC. This study suggests that BITC induced significant reductions in the viability of HCC cell lines. BITC may activate caspase-3 signal and inhibit the expression of growth- and metastasis-related proteins; AFP is an pivotal molecule for the HCC chemo-resistance of BITC. PMID:27716619

  3. Tumor cyclooxygenase-2/prostaglandin E2-dependent promotion of FOXP3 expression and CD4+ CD25+ T regulatory cell activities in lung cancer.

    PubMed

    Sharma, Sherven; Yang, Seok-Chul; Zhu, Li; Reckamp, Karen; Gardner, Brian; Baratelli, Felicita; Huang, Min; Batra, Raj K; Dubinett, Steven M

    2005-06-15

    Cyclooxygenase (COX)-2 and its product prostaglandin (PG) E2 underlie an immunosuppressive network that is important in the pathogenesis of non-small cell lung cancer. CD4+ CD25+ T regulatory (Treg) cells play an important role in maintenance of immunologic self-tolerance. CD4+ CD25+ Treg cell activities increase in lung cancer and appear to play a role in suppressing antitumor immune responses. Definition of the pathways controlling Treg cell activities will enhance our understanding of limitation of the host antitumor immune responses. Tumor-derived COX-2/PGE2 induced expression of the Treg cell-specific transcription factor, Foxp3, and increased Treg cell activity. Assessment of E-prostanoid (EP) receptor requirements revealed that PGE2-mediated induction of Treg cell Foxp3 gene expression was significantly reduced in the absence of the EP4 receptor and ablated in the absence of the EP2 receptor expression. In vivo, COX-2 inhibition reduced Treg cell frequency and activity, attenuated Foxp3 expression in tumor-infiltrating lymphocytes, and decreased tumor burden. Transfer of Treg cells or administration of PGE2 to mice receiving COX-2 inhibitors reversed these effects. We conclude that inhibition of COX-2/PGE2 suppresses Treg cell activity and enhances antitumor responses.

  4. Women with high BMI: should they be managed differently due to antagonising action of leptin in labour?

    PubMed

    Wuntakal, Rekha; Kaler, Mandeep; Hollingworth, Tony

    2013-06-01

    Leptin - a protein hormone is synthesised in the adipose tissue in humans. Its level therefore should be directly proportional to the amount of adipose tissue in the body. There is evidence that leptin may be responsible for various complications in obese and morbidly obese women in labour by its effect on the myometrium causing uterine smooth muscle relaxation (causes less Ca(2+) flux in myometrium). By doing this, we believe it opposes oxytocin effect on the myometrium which in fact promotes uterine smooth muscle contractions (causes more Ca(2+) flux in myometrium). The opposing action of these two hormone may contribute to the dysfunctional labour process, prolonged first stage of labour, increase in operative vaginal delivery in second stage of labour and increase in caesarean section rate both in first and second stage of labour in obese women. Also, there is increased incidence of postdated pregnancy, induction of labour and atonic postpartum haemorrhage in obese and morbidly obese women. Does this mean labour should be managed differently in women with high BMI? Copyright © 2013 Elsevier Ltd. All rights reserved.

  5. Extensive meiotic asynapsis in mice antagonises meiotic silencing of unsynapsed chromatin and consequently disrupts meiotic sex chromosome inactivation.

    PubMed

    Mahadevaiah, Shantha K; Bourc'his, Déborah; de Rooij, Dirk G; Bestor, Timothy H; Turner, James M A; Burgoyne, Paul S

    2008-07-28

    Chromosome synapsis during zygotene is a prerequisite for the timely homologous recombinational repair of meiotic DNA double-strand breaks (DSBs). Unrepaired DSBs are thought to trigger apoptosis during midpachytene of male meiosis if synapsis fails. An early pachytene response to asynapsis is meiotic silencing of unsynapsed chromatin (MSUC), which, in normal males, silences the X and Y chromosomes (meiotic sex chromosome inactivation [MSCI]). In this study, we show that MSUC occurs in Spo11-null mouse spermatocytes with extensive asynapsis but lacking meiotic DSBs. In contrast, three mutants (Dnmt3l, Msh5, and Dmc1) with high levels of asynapsis and numerous persistent unrepaired DSBs have a severely impaired MSUC response. We suggest that MSUC-related proteins, including the MSUC initiator BRCA1, are sequestered at unrepaired DSBs. All four mutants fail to silence the X and Y chromosomes (MSCI failure), which is sufficient to explain the midpachytene apoptosis. Apoptosis does not occur in mice with a single additional asynapsed chromosome with unrepaired meiotic DSBs and no disturbance of MSCI.

  6. Treatment with para-chlorophenylalanine antagonises the emetic response and the serotonin-releasing actions of cisplatin in cancer patients.

    PubMed Central

    Alfieri, A. B.; Cubeddu, L. X.

    1995-01-01

    To test the role of serotonin in chemotherapy-induced nausea and emesis, ten cancer patients were pretreated with the serotonin synthesis inhibitor para-chlorophenylalanine (PCPA). PCPA (2 g 8 hourly for 2 or 3 days prior to cisplatin) reduced the spontaneous urinary excretion of 5-hydroxyindoleacetic acid (5-HIAA), inhibited the increase in urinary 5-HIAA induced by cisplatin and markedly attenuated the acute period of nausea and vomiting associated with the cytotoxic drug. These results indicate that gastrointestinal serotonin mediates cisplatin-induced emesis and that the amount of serotonin released by cisplatin is a major factor in determining the severity of the acute period of emesis experienced by the patient. PMID:7533519

  7. Kicking against the PRCs – A Domesticated Transposase Antagonises Silencing Mediated by Polycomb Group Proteins and Is an Accessory Component of Polycomb Repressive Complex 2

    PubMed Central

    Perera, Pumi; Mora-García, Santiago; de Leau, Erica; Thornton, Harry; de Alves, Flavia Lima; Rapsilber, Juri; Yang, Suxin; James, Geo Velikkakam; Schneeberger, Korbinian; Finnegan, E. Jean; Turck, Franziska; Goodrich, Justin

    2015-01-01

    The Polycomb group (PcG) and trithorax group (trxG) genes play crucial roles in development by regulating expression of homeotic and other genes controlling cell fate. Both groups catalyse modifications of chromatin, particularly histone methylation, leading to epigenetic changes that affect gene activity. The trxG antagonizes the function of PcG genes by activating PcG target genes, and consequently trxG mutants suppress PcG mutant phenotypes. We previously identified the ANTAGONIST OF LIKE HETEROCHROMATIN PROTEIN1 (ALP1) gene as a genetic suppressor of mutants in the Arabidopsis PcG gene LIKE HETEROCHROMATIN PROTEIN1 (LHP1). Here, we show that ALP1 interacts genetically with several other PcG and trxG components and that it antagonizes PcG silencing. Transcriptional profiling reveals that when PcG activity is compromised numerous target genes are hyper-activated in seedlings and that in most cases this requires ALP1. Furthermore, when PcG activity is present ALP1 is needed for full activation of several floral homeotic genes that are repressed by the PcG. Strikingly, ALP1 does not encode a known chromatin protein but rather a protein related to PIF/Harbinger class transposases. Phylogenetic analysis indicates that ALP1 is broadly conserved in land plants and likely lost transposase activity and acquired a novel function during angiosperm evolution. Consistent with this, immunoprecipitation and mass spectrometry (IP-MS) show that ALP1 associates, in vivo, with core components of POLYCOMB REPRESSIVE COMPLEX 2 (PRC2), a widely conserved PcG protein complex which functions as a H3K27me3 histone methyltransferase. Furthermore, in reciprocal pulldowns using the histone methyltransferase CURLY LEAF (CLF), we identify not only ALP1 and the core PRC2 components but also plant-specific accessory components including EMBRYONIC FLOWER 1 (EMF1), a transcriptional repressor previously associated with PRC1-like complexes. Taken together our data suggest that ALP1 inhibits PcG silencing by blocking the interaction of the core PRC2 with accessory components that promote its HMTase activity or its role in inhibiting transcription. ALP1 is the first example of a domesticated transposase acquiring a novel function as a PcG component. The antagonistic interaction of a modified transposase with the PcG machinery is novel and may have arisen as a means for the cognate transposon to evade host surveillance or for the host to exploit features of the transposition machinery beneficial for epigenetic regulation of gene activity. PMID:26642436

  8. Kicking against the PRCs - A Domesticated Transposase Antagonises Silencing Mediated by Polycomb Group Proteins and Is an Accessory Component of Polycomb Repressive Complex 2.

    PubMed

    Liang, Shih Chieh; Hartwig, Ben; Perera, Pumi; Mora-García, Santiago; de Leau, Erica; Thornton, Harry; de Lima Alves, Flavia; de Alves, Flavia Lima; Rappsilber, Juri; Rapsilber, Juri; Yang, Suxin; James, Geo Velikkakam; Schneeberger, Korbinian; Finnegan, E Jean; Turck, Franziska; Goodrich, Justin

    2015-12-01

    The Polycomb group (PcG) and trithorax group (trxG) genes play crucial roles in development by regulating expression of homeotic and other genes controlling cell fate. Both groups catalyse modifications of chromatin, particularly histone methylation, leading to epigenetic changes that affect gene activity. The trxG antagonizes the function of PcG genes by activating PcG target genes, and consequently trxG mutants suppress PcG mutant phenotypes. We previously identified the ANTAGONIST OF LIKE HETEROCHROMATIN PROTEIN1 (ALP1) gene as a genetic suppressor of mutants in the Arabidopsis PcG gene LIKE HETEROCHROMATIN PROTEIN1 (LHP1). Here, we show that ALP1 interacts genetically with several other PcG and trxG components and that it antagonizes PcG silencing. Transcriptional profiling reveals that when PcG activity is compromised numerous target genes are hyper-activated in seedlings and that in most cases this requires ALP1. Furthermore, when PcG activity is present ALP1 is needed for full activation of several floral homeotic genes that are repressed by the PcG. Strikingly, ALP1 does not encode a known chromatin protein but rather a protein related to PIF/Harbinger class transposases. Phylogenetic analysis indicates that ALP1 is broadly conserved in land plants and likely lost transposase activity and acquired a novel function during angiosperm evolution. Consistent with this, immunoprecipitation and mass spectrometry (IP-MS) show that ALP1 associates, in vivo, with core components of POLYCOMB REPRESSIVE COMPLEX 2 (PRC2), a widely conserved PcG protein complex which functions as a H3K27me3 histone methyltransferase. Furthermore, in reciprocal pulldowns using the histone methyltransferase CURLY LEAF (CLF), we identify not only ALP1 and the core PRC2 components but also plant-specific accessory components including EMBRYONIC FLOWER 1 (EMF1), a transcriptional repressor previously associated with PRC1-like complexes. Taken together our data suggest that ALP1 inhibits PcG silencing by blocking the interaction of the core PRC2 with accessory components that promote its HMTase activity or its role in inhibiting transcription. ALP1 is the first example of a domesticated transposase acquiring a novel function as a PcG component. The antagonistic interaction of a modified transposase with the PcG machinery is novel and may have arisen as a means for the cognate transposon to evade host surveillance or for the host to exploit features of the transposition machinery beneficial for epigenetic regulation of gene activity.

  9. A tarantula-venom peptide that antagonises the TRPA1 nociceptor ion channel by binding to the S1-S4 gating domain

    PubMed Central

    Gui, Junhong; Liu, Boyi; Cao, Guan; Lipchik, Andrew M.; Perez, Minervo; Dekan, Zoltan; Mobli, Mehdi; Daly, Norelle L.; Alewood, Paul F.; Parker, Laurie L.; King, Glenn F.; Zhou, Yufeng; Jordt, Sven-Eric; Nitabach, Michael N.

    2014-01-01

    Background The venoms of predators such as spiders, scorpions, cone snails, sea anemones, and snakes, have been an excellent source of pharmacological diversity for drug discovery and as pharmacological tools for elucidating the structure, function, and physiological properties of ion channels. Here we describe the first known peptide antagonist of the nociceptor ion channel transient receptor potential ankyrin 1 (TRPA1). Results We constructed a recombinant cDNA library encoding ∼100 diverse GPI-anchored peptide toxins (t-toxins) derived from spider venoms and screened this library by co-expression in Xenopus oocytes with TRPA1. This screen resulted in identification of protoxin-I (ProTx-I), a 35-residue peptide from the venom of the Peruvian green-velvet tarantula, Thrixopelma pruriens, as the first known high-affinity peptide TRPA1 antagonist. Interestingly, ProTx-I was previously identified as an antagonist of voltage-gated sodium (NaV) channels. To identify the surfaces of ProTx-I by which it binds to these distinct ion channel types, we constructed a t-toxin library of ProTx-I alanine-scanning mutants and screened this library against NaV1.2 and TRPA1. This revealed distinct partially overlapping surfaces of ProTx-I by which it binds to these two ion channels, and whose specific chemical features explain its higher affinity for NaV1.2 than for TRPA1. Importantly, this mutagenesis yielded two novel ProTx-I variants that are only active against either TRPA1or NaV1.2, but not both. By testing its activity against chimeric channels, we identified the extracellular loops of the TRPA1 S1-S4 gating domain as the ProTx-I binding site. Conclusions These studies establish screening of t-toxin libraries of native and mutated toxins, which we term “toxineering”, as a generally applicable method for isolation of novel ion channel modifiers and for design of ion channel modifiers with altered target selectivity. They also suggest that ProTx-I will be a valuable pharmacological reagent for addressing the biophysical mechanisms of TRPA1 gating, the physiology and pathophysiology of TRPA1 function in nociceptors, and for potential clinical application in the context of pain and inflammation. PMID:24530065

  10. The N-methyl-D-aspartate receptor channel blockers memantine, MRZ 2/579 and other amino-alkyl-cyclohexanes antagonise 5-HT(3) receptor currents in cultured HEK-293 and N1E-115 cell systems in a non-competitive manner.

    PubMed

    Rammes, G; Rupprecht, R; Ferrari, U; Zieglgänsberger, W; Parsons, C G

    2001-06-22

    The type 3 serotonin (5-HT(3)) receptor is a ligand-gated ion channel. In concentration-clamp experiments, we investigated the effects of the uncompetitive N-methyl-D-aspartate (NMDA) receptor antagonists memantine, amantadine and MRZ 2/579 on 5-HT receptors stabley expressed in HEK-293 cells and on native 5-HT(3) receptors in the N1E-115 cell line. All agents antagonized serotonin (10 microM)-induced inward currents with similar potency to that reported for NMDA receptors. This effect was characterized by inducing a pronounced receptor desensitization, and was probably non-competitive and voltage-independent. In contrast, (S)-ketamine was much weaker as an antagonist of 5-HT(3) receptors than NMDA receptors. Similar effects on 5-HT(3) receptors have been reported previously for a variety of anti-depressants and it is possible that the clinical anti-depressant effects reported for both memantine and amantadine are mediated, at least in part, by antagonistic effects at 5-HT(3) receptors.

  11. In vivo application of 3D-line skeleton graph analysis (LSGA) technique with high-resolution magnetic resonance imaging of trabecular bone structure.

    PubMed

    Pothuaud, Laurent; Newitt, David C; Lu, Ying; MacDonald, Brian; Majumdar, Sharmila

    2004-05-01

    Over the last several years magnetic resonance (MR) imaging has emerged as a means of measuring in vivo 3D trabecular bone structure. In particular, MR based diagnosis could be used to complement standard bone mineral density (BMD) methods for assessing osteoporosis and evaluating longitudinal changes. The aim of this study was to demonstrate the feasibility of using the 3D-LSGA technique for the evaluation of trabecular bone structure of high-resolution MR images, particularly for assessing longitudinal changes, in vivo. First, the reproducibility of topological 3D-LSGA based measurements was evaluated in a set of seven volunteers, and coefficients of variations ranged from 3.5% to 6%. Second, high-resolution MR images of the radius in 30 postmenopausal women from a placebo controlled drug study (Idoxifene), divided into placebo ( n=9) and treated ( n=21) groups, were obtained at baseline (BL) and after 1 year of treatment (follow-up, FU). In addition, dual X-ray absorptiometry (DXA) measures of BMD were obtained in the distal radius. Standard morphological measurements based on the mean intercept length (MIL) technique as well as 3D-LSGA based measurements were applied to the 3D MR images. Significant changes from BL to FU were detected, in the treated group, using the topological 3D-LSGA based measurements, morphological measures of volume of connected trabeculae and App Tb.N from MIL analysis. The duration of the study was short, and the number of patients remaining in the study was small, hence these results cannot be interpreted with regard to a true therapeutic response. Furthermore, the site (wrist) and the drug (idoxifene) are not optimal for follow-up study. However, this paper demonstrated the feasibility of using 3D-LSGA based evaluation coupled with in vivo high-resolution MR imaging as a complementary approach for the monitoring of trabecular bone changes in individual subjects.

  12. Growth control: re-examining Zyxin's role in the hippo pathway.

    PubMed

    Harvey, Kieran F

    2015-03-16

    The Hippo pathway is a conserved regulator of organ growth that computes information from the cellular microenvironment. A new study examines the role of the Hippo pathway protein Zyxin and finds that it antagonises Expanded to modulate F-actin and organ size. Copyright © 2015 Elsevier Ltd. All rights reserved.

  13. Sub-Saharan Africa Report

    DTIC Science & Technology

    1987-01-08

    English 29 Nov 86 p 3 KENYA [Article by Dough Charaggu] : The Finance Minister,; [ Text ] Professor. George Saitoti, yesterday signed an aid...the plan had been effected, sour relations between the two bodies and farmers would have antagonised ’farmers and the Government. Mr Mlchoma said

  14. Antagonism by exifone, a new cognitive enhancing agent, of the amnesias induced by four benzodiazepines in mice.

    PubMed

    Porsolt, R D; Lenègre, A; Avril, I; Doumont, G

    1988-01-01

    Exifone is a novel compound proposed for treating cognitive decline associated with age and shows corrective effects in animal models of memory dysfunction. The present experiments examined the antagonism by exifone of the amnesias induced in mice in a passive avoidance test by four benzodiazepines: bromazepam, diazepam, lorazepam and triazolam. Subsequent experiments investigated the specificity of exifone's antagonism of benzodiazepine-induced amnesia by examining its interaction with the effects of the benzodiazepines in the staircase test (anxiolytic/sedative activity) and the electroshock test (anticonvulsant activity). The results indicated that exifone clearly antagonised the amnesias induced by the four benzodiazepines, but was without intrinsic effects in the staircase or electroshock test and did not antagonise the effects of the benzodiazepines in these two tests. These results suggest that exifone might be useful for decreasing the amnesias induced by commonly used benzodiazepines without affecting their anxiolytic or anticonvulsant activity.

  15. The action of a dopamine (DA1) receptor agonist, fenoldopam in human vasculature in vivo and in vitro.

    PubMed Central

    Hughes, A; Thom, S; Martin, G; Redman, D; Hasan, S; Sever, P

    1986-01-01

    This study was designed to investigate dopaminergic mechanisms in human vasculature using the selective vascular dopamine receptor agonist fenoldopam in vivo and in vitro. In vivo, forearm blood flow was measured plethysmographically and in vitro isolated rings of human blood vessels from a variety of sites were used for tissue bath studies. Intra-arterial fenoldopam markedly increased forearm blood flow, this effect was antagonised by (R) sulpiride, a vascular dopamine (DA1) antagonist, but not by metoclopramide, a neuronal (DA2) antagonist, or by guanethidine, an adrenergic neurone blocking agent. In vitro, fenoldopam relaxed preconstricted human renal, mesenteric and lumbar arteries, but not saphenous vein in a concentration dependent manner. (RS) sulpiride and SCH 23390 competitively antagonised this effect. These studies demonstrate the presence of a vasodilatory vascular dopamine receptor in man both in vivo and in vitro. PMID:2878679

  16. Effect of metoclopramide in guinea-pig ileum longitudinal muscle: evidence against dopamine-mediation.

    PubMed Central

    Zar, M A; Ebong, O; Bateman, D N

    1982-01-01

    The investigation examines the hypothesis that metoclopramide-induced potentiation of gastrointestinal motility is mediated through dopamine receptors. In vitro studies on the longitudinal muscle of the guinea-pig ileum were performed. Metoclopramide, in concentrations comparable with those seen in plasma after therapeutic doses in man, selectively potentiated the cholinergic response Dopamine (1-100 microM) inhibited cholinergic transmission by inhibiting neuronal acetylcholine release. The inhibitory action of dopamine was antagonised by phentolamine, and alpha-adrenoceptor antagonist, but not by the dopamine receptor antagonists metoclopramide or primozide. Bromocriptine inhibited cholinergic responses by a postsynaptic mechanism which was not antagonised by metoclopramide, primozide, or phentolamine. The results are consistent with the view that metoclopramide-induced potentiation of gastrointestinal motility does not involve local dopamine receptors. PMID:7056499

  17. Antagonism of bromocriptine-induced cage climbing behaviour in mice by the selective D-2 dopamine receptor antagonists, metoclopramide and molindone.

    PubMed

    Balsara, J J; Nandal, N V; Gada, V P; Bapat, T R; Chandorkar, A G

    1986-01-01

    Bromocriptine (5-30 mg/kg, ip), 2 hr after administration, induced cage climbing behaviour in mice. Pretreatment with haloperidol, an antagonist of both D-1 and D-2 dopamine receptors, metoclopramide and molindone, the selective D-2 dopamine receptor antagonists, effectively antagonised bromocriptine-induced climbing behaviour. The results indicate that bromocriptine most probably induces climbing behaviour in mice by stimulating the postsynaptic striatal D-2 dopamine receptors.

  18. Effects in cats of atipamezole, flumazenil and 4-aminopyridine on stress-related neurohormonal and metabolic responses induced by medetomidine, midazolam and ketamine.

    PubMed

    Ueoka, Naotami; Hikasa, Yoshiaki

    2015-08-01

    This study aimed to investigate the antagonistic effects of a fixed dose of atipamezole (ATI), flumazenil (FLU) and 4-aminopyridine (4AP), both alone and in various combinations, on key stress-related neurohormonal and metabolic changes induced by medetomidine (MED), midazolam (MID) and ketamine (KET) in healthy cats. Seven cats were used consistently in eight investigation groups. Cats were administered a mixture of 0.05 mg/kg MED and 0.5 mg/kg MID followed 10 mins later by 10 mg/kg KET intramuscularly. Twenty minutes after KET injection, the cats were intravenously injected with either a physiological saline solution at 0.1 ml/kg (control) or one of the seven variations of experimental drugs, alone or in combination: ATI, FLU, 4AP, ATI + FLU, FLU + 4AP, ATI + 4AP and ATI + FLU + 4AP. Blood samples were collected 10 times during the 24 h test period. Plasma glucose, insulin, cortisol, epinephrine, norepinephrine and non-esterified fatty acid levels were measured. The administration of MED + MID + KET resulted in hyperglycaemia and decreases in epinephrine, norepinephrine, cortisol and non-esterified fatty acid levels. FLU or 4AP alone or FLU + 4AP did not effectively antagonise the effects induced by MED + MID + KET but enhanced the hyperglycaemia. ATI alone was effective in antagonising these effects. Compared with non-ATI regimens, combinations with ATI were more effective in antagonising the effects induced by MED + MID + KET; however, ATI + FLU + 4AP caused large increases in cortisol, epinephrine and norepinephrine concentrations. ATI, both alone and in combination, is effective in antagonising the neurohormonal and metabolic effects of MED + MID + KET in cats. However, ATI + FLU + 4AP is not suitable because of large stress-related hormonal responses.

  19. Studies on the interactions of copper and cannabis.

    PubMed

    Singh, P P; Das, P K

    1978-04-11

    The action of copper (CuSO4, 5 mg/kg, oral) on selected neuropharmacological actions of cannabis resin (CI, oral) was studied on albino rats and mice. Copper potentiated the barbiturate hypnosis-potentiating activity of CI in albino rats and mice and had no effect on hypothermic activity in albino rats. Single doses of copper partially inhibited tolerance to barbiturate hypnosis-potentiation activity and markedly delayed the development of tolerance to hypothermic activity of CI. Oral as well as i.c.v. copper (CuSO4, 0.1 microgram) in single dose antagonised the tolerance to hypothermic activity of cannabis or THC for one to two weeks. Copper-CI interaction could be antagonised by penicillamine. Zinc (ZnSO4, 5 mg/kg, oral) had an action similar to that of copper in antagonising the development of tolerance to the hypothermic activity of CI, but magnesium (MgSO4, 5 mg/kg, i.p.) was devoid of any such action. Studies indicate that, although copper has no significant neuropharmacological action, it interacts with CI activity, especially in tolerant rats, in effects on hypothermia. The site of action of copper is possibly the hypothalamus, where it inhibits the processes of tolerance development to CI on the noradrenergic neurone.

  20. 2-(4-Amino-3-methylphenyl)-5-fluorobenzothiazole is a ligand and shows species-specific partial agonism of the aryl hydrocarbon receptor

    SciTech Connect

    Bazzi, Rana; Bradshaw, Tracey D.; Rowlands, J. Craig; Stevens, Malcolm F.G.; Bell, David R.

    2009-05-15

    2-(4-Amino-3-methylphenyl)-5-fluorobenzothiazole (5F 203) and related compounds are a series of anti-cancer candidate pharmaceuticals, that have been shown to activate the AhR. We show that these compounds are high-affinity ligands for the rat AhR, but a quantitative assay for their ability to induce CYP1A1 RNA in H4IIEC3 cells, a measure of activation of the AhR, showed a poor relationship between affinity for the AhR and ability to induce CYP1A1 RNA. 5F 203, an agonist with low potency, was able to antagonise the induction of CYP1A1 RNA by TCDD, while IH 445, a potent agonist, did not antagonise the induction of CYP1A1 RNA by TCDD, and Schild analysis confirmed 5F 203 to be a potent antagonist of the induction of CYP1A1 RNA by TCDD in H4IIEC3 cells. In contrast, several benzothiazoles show potent induction of CYP1A1 RNA in human MCF-7 cells, and 5F 203 is unable to detectably antagonise the induction of CYP1A1 RNA in MCF-7 cells, showing a species difference in antagonism. Evaluation of the anti-proliferative activity of benzothiazoles showed that the ability to agonise the AhR correlated with growth inhibition both in H4IIEC3 cells for a variety of benzothiazoles, and between H4IIEC3 and MCF-7 cells for 5F 203, suggesting an important role of agonism of the AhR in the anti-proliferative activity of benzothiazoles.

  1. Functional role of alpha2-adrenoceptor subtypes in the cooling-enhanced vasoconstriction of isolated cutaneous digital veins of the horse.

    PubMed

    Zerpa, Hector; Berhane, Yoel; Elliott, Jonathan; Bailey, Simon R

    2010-02-10

    Cooling-enhanced contractile responses in cutaneous arteries have been reported to involve the activation of alpha(2C)-adrenoceptors, but their role in cutaneous veins is not clearly understood. The aim was to pharmacologically characterize the subtype of postsynaptic alpha(2)-adrenoceptors in the equine digital vein mediating contraction at two temperatures. The increase in isometric tension of endothelium-denuded equine digital vein in response to UK-14304 was studied in the absence and presence of relatively selective alpha(2)-adrenoceptor antagonists at temperatures of either 30 degrees C (the peripheral digit temperature of horses maintained in a thermoneutral environment) or 22 degrees C. The response to UK-14304 was enhanced by cooling and antagonised by RX-821002 (alpha(2)-adrenoceptor non-selective; 30 degrees C: apparent pK(b)=8.5; 22 degrees C: pK(b)=8.2) and yohimbine (alpha(2)-adrenoceptor non-selective; 30 degrees C: apparent pK(b)=7.2; 22 degrees C: apparent pK(b)=7.4). The response at 30 degrees C was non-surmountably antagonised by BRL-44408 (alpha(2A)-adrenoceptor-selective; apparent pK(b)=8.9) and MK-912 (alpha(2C)-adrenoceptor-selective: apparent pK(b)=9.9). JP-1302 (alpha(2C)-adrenoceptor-selective) surmountably antagonised the response with a low potency (apparent pK(b)=5.6) at 30 degrees C. The response at 22 degrees C was surmountably antagonised by: BRL-44408 (alpha(2A)-adrenoceptor-selective; apparent pK(b)=6.5); MK-912 (alpha(2C)-adrenoceptor-selective; pK(b)=9.7) and JP-1302 (alpha(2C)-adrenoceptor-selective; apparent pK(b)=7.5). ARC-239 (alpha(2B)-adrenoceptor-selective) did not affect the response at either temperature. The apparent potency of the different antagonists and their non-surmountable effect, suggests that the UK-14304 response at 30 degrees C could be mediated by alpha(2A)-adrenoceptors and alpha(2C)-adrenoceptors. By contrast, the alpha(2C)-adrenoceptor appears to be the main alpha(2)-adrenoceptor mediating the augmented

  2. Co-transfection of decorin and interleukin-10 modulates pro-fibrotic extracellular matrix gene expression in human tenocyte culture

    NASA Astrophysics Data System (ADS)

    Abbah, Sunny A.; Thomas, Dilip; Browne, Shane; O'Brien, Timothy; Pandit, Abhay; Zeugolis, Dimitrios I.

    2016-02-01

    Extracellular matrix synthesis and remodelling are driven by increased activity of transforming growth factor beta 1 (TGF-β1). In tendon tissue repair, increased activity of TGF-β1 leads to progressive fibrosis. Decorin (DCN) and interleukin 10 (IL-10) antagonise pathological collagen synthesis by exerting a neutralising effect via downregulation of TGF-β1. Herein, we report that the delivery of DCN and IL-10 transgenes from a collagen hydrogel system supresses the constitutive expression of TGF-β1 and a range of pro-fibrotic extracellular matrix genes.

  3. [Drug or plant substances which antagonize venoms or potentiate antivenins].

    PubMed

    Chippaux, J P; Rakotonirina, V S; Rakotonirina, A; Dzikouk, G

    1997-01-01

    Dendroaspis jamesoni (Elapidae) and Echis oceliatus (Viperidae) are responsible for most of severe evenomation in Cameroon. Toxicity of venoms of these two species has been measured using mice according to the method of Spearman & Kàrber. The effect on experimental envenomation of various drugs (atropine, promethazine, neostigmine, hydrocortisone, pentosane sulfuric polyester, heparin, tranexamic acid and aminocaproic acid) and plant extracts (Schumanniophyton magnificum, Bidens pilosa, Securidaca longepedunculata and Garcinia lucida) has been observed associated or not with the antivenom lpser Afrique (SAV). The venom of D. jamesoni contains neurotoxins agonizing and antagonising acetylcholine. The toxicity of the venom did not depend on the route of injection. Atropine, promethazine, neostigmine and hydrocortisone protected animals against a venom dose up to 2 LD50. Moreover, atropine and promethazine potentiated the SAV. Similar results have been obtained with extracts from S. magnificum and B. pilosa. The venom of E. ocellatus induces haemorrhage and necrosis. The toxicity increased by 3-fold when the venom was injected through intravenous or intraperitoneal route, compared to intramuscular route. Pentosane sulfuric polyester and tranexamic acid protected mice against doses up to 3 LD50. Pentosane sulfuric polyester, hydrocortisone, heparin and aminocaproic acid increased the SAV protective titre by 50%. However, tried plant extracts weakly antagonised the venom and did not potentiate the SAV.

  4. The role of mouse Akt2 in insulin-dependent suppression of adipocyte lipolysis in vivo

    PubMed Central

    Koren, Shlomit; DiPilato, Lisa M.; Emmett, Matthew J.; Shearin, Abigail L.; Chu, Qingwei; Monks, Bob; Birnbaum, Morris J.

    2015-01-01

    Aim/hypothesis The release of fatty acids from adipocytes, i.e. lipolysis, is maintained under tight control, primarily by the opposing actions of catecholamines and insulin. A widely accepted model is that insulin antagonises catecholamine-dependent lipolysis through phosphorylation and activation of cAMP phosphodiesterase 3B (PDE3B) by the serine-threonine protein kinase Akt (protein kinase B). Recently, this hypothesis has been challenged, as in cultured adipocytes insulin appears, under some conditions, to suppress lipolysis independently of Akt. Methods To address the requirement for Akt2, the predominant isoform expressed in classic insulin target tissues, in the suppression of fatty acid release in vivo, we assessed lipolysis in mice lacking Akt2. Results In the fed state and following an oral glucose challenge, Akt2 null mice were glucose intolerant and hyperinsulinaemic, but nonetheless exhibited normal serum NEFA and glycerol levels, suggestive of normal suppression of lipolysis. Furthermore, insulin partially inhibited lipolysis in Akt2 null mice during an insulin tolerance test (ITT) and hyperinsulinaemic–euglycaemic clamp, respectively. In support of these in vivo observations, insulin antagonised catecholamine-induced lipolysis in primary brown fat adipocytes from Akt2-deficient nice. Conclusion These data suggest that suppression of lipolysis by insulin in hyperinsulinaemic states can take place in the absence of Akt2. PMID:25740694

  5. The role of mouse Akt2 in insulin-dependent suppression of adipocyte lipolysis in vivo.

    PubMed

    Koren, Shlomit; DiPilato, Lisa M; Emmett, Matthew J; Shearin, Abigail L; Chu, Qingwei; Monks, Bob; Birnbaum, Morris J

    2015-05-01

    The release of fatty acids from adipocytes, i.e. lipolysis, is maintained under tight control, primarily by the opposing actions of catecholamines and insulin. A widely accepted model is that insulin antagonises catecholamine-dependent lipolysis through phosphorylation and activation of cAMP phosphodiesterase 3B (PDE3B) by the serine-threonine protein kinase Akt (protein kinase B). Recently, this hypothesis has been challenged, as in cultured adipocytes insulin appears, under some conditions, to suppress lipolysis independently of Akt. To address the requirement for Akt2, the predominant isoform expressed in classic insulin target tissues, in the suppression of fatty acid release in vivo, we assessed lipolysis in mice lacking Akt2. In the fed state and following an oral glucose challenge, Akt2 null mice were glucose intolerant and hyperinsulinaemic, but nonetheless exhibited normal serum NEFA and glycerol levels, suggestive of normal suppression of lipolysis. Furthermore, insulin partially inhibited lipolysis in Akt2 null mice during an insulin tolerance test (ITT) and hyperinsulinaemic-euglycaemic clamp, respectively. In support of these in vivo observations, insulin antagonised catecholamine-induced lipolysis in primary brown fat adipocytes from Akt2-deficient mice. These data suggest that suppression of lipolysis by insulin in hyperinsulinaemic states can take place in the absence of Akt2.

  6. Nicotine-induced conditioned taste aversion in the rat: effects of ethanol.

    PubMed

    Korkosz, Agnieszka; Scinska, Anna; Taracha, Ewa; Plaznik, Adam; Kukwa, Andrzej; Kostowski, Wojciech; Bienkowski, Przemyslaw

    2006-05-10

    It has been shown that small doses of ethanol antagonise the discriminative stimulus properties of nicotine in the rat. The aim of the present study was to evaluate whether ethanol could antagonise the aversive stimulus effects of nicotine. Wistar rats were trained to associate nicotine injections with a novel tasting fluid (0.1% saccharin) in the conditioned taste aversion procedure. Nicotine (0.3 mg/kg, s.c.) was injected 5 min after the end of a 20-min exposure to the saccharin solution. Ethanol (0.25-0.5 g/kg, i.p.) was administered 5 or 50 min before nicotine. In general, ethanol did not inhibit nicotine-induced conditioned taste aversion. Contrary to the findings in drug discrimination studies, a slight but significant enhancement of nicotine-induced taste aversion conditioning was observed after ethanol pre-treatment. Blood ethanol levels were measured in a separate group of rats. Maximal blood ethanol levels after i.p. administration of 0.25 or 0.5 g/kg ethanol exceeded 20 and 80 mg%, respectively. Concluding, the present results may indicate that ethanol does not attenuate nicotine-induced conditioned taste aversion in the rat.

  7. Uncoupling of the dynamics of host-pathogen interaction uncovers new mechanisms of viral interferon antagonism at the single-cell level.

    PubMed

    Rand, Ulfert; Hillebrand, Upneet; Sievers, Stephanie; Willenberg, Steffi; Köster, Mario; Hauser, Hansjörg; Wirth, Dagmar

    2014-07-01

    Antiviral defence in mammals is mediated through type-I interferons (IFNs). Viruses antagonise this process through expression of IFN antagonist proteins (IAPs). Understanding and modelling of viral escape mechanisms and the dynamics of IAP action has the potential to facilitate the development of specific and safe drugs. Here, we describe the dynamics of interference by selected viral IAPs, NS1 from Influenza A virus and NS3/4A from Hepatitis C virus. We used Tet-inducible IAP gene expression to uncouple this process from virus-driven dynamics. Stochastic activation of the IFN-β gene required the use of single-cell live imaging to define the efficacy of the inhibitors during the virus-induced signalling processes. We found significant correlation between the onset of IAP expression and halted IFN-β expression in cells where IFN-β induction had already occurred. These data indicate that IAPs not only prevent antiviral signalling prior to IFN-β induction, but can also stop the antiviral response even after it has been activated. We found reduced NF-κB activation to be the underlying mechanism by which activated IFN expression can be blocked. This work demonstrates a new mechanism by which viruses can antagonise the IFN response.

  8. [Clinical evaluation of three medetomidine--midazolam--ketamine combinations for neutering of ferrets (Mustela putorius furo)].

    PubMed

    Schernthaner, Anita; Lendl, Christine; Busch, Raymonde; Henke, Julia

    2008-01-01

    33 ferrets (Mustela putorius furo, 11 females, 22 males, ASA I-II) were neutered in a combination anaesthesia with medetomidine, midazolam and ketamine. The animals were randomized into 3 groups. All animals received 20 microg/kg BW medetomidine and 0.5 mg/kg BW midazolam. The three groups differed regarding dosis and way of application of ketamine (IM10 = 10 mg/kg BW intramuscularly; IM07 = 7 mg/kg BW intramuscularly; SC10 = 10 mg/kg BW subcutaneously). After 30 minutes anaesthesia was partially antagonised with 100 microg/kg BW atipamezole i.m.. Sedation, muscle relaxation, analgesia, and overall anaesthetic impression were compared by a scoring protocol. Reactions to painful stimuli of clamping the spermatic cord or the ovarial ligament including the A. ovarica were judged, too. All animals lost their righting reflex and could be placed in dorsal recumbency. Induction and recovery time were significantly the shortest in study group IM10 with 1.73 +/- 0.3 and 9.73 +/- 4.6 min respectively. Recovery was significantly prolonged in group SC10 with 30.27 +/- 15.6 min. The MMK-anaesthesia with 10 mg/kg ketamine i.m. is very useful for neutering ferrets. Respiratory depression and bradycardia typically for medetomidine were seen in all three combinations, but quickly reversed after partial antagonisation. Induction and intubation, followed by inhalation anaesthesia, were possible with all three regimes.

  9. Nicotine increases the resistance of lung cancer cells to cisplatin through enhancing Bcl-2 stability

    PubMed Central

    Nishioka, T; Luo, L-Y; Shen, L; He, H; Mariyannis, A; Dai, W; Chen, C

    2014-01-01

    Background: Nicotine is able to activate mitogenic signalling pathways, which promote cell growth or survival as well as increase chemoresistance of cancer cells. However, the underlying mechanisms are not fully understood. Methods: In this study, we used immunoblotting and immunoprecipitation methods to test the ubiquitination and degradation of Bcl-2 affected by nicotine in lung cancer cells. Apoptotic assay was also used to measure the antagonising effect of nicotine on cisplatin-mediated cytotoxicity. Results: We demonstrated that the addition of nicotine greatly attenuated Bcl-2 ubiquitination and degradation, which further desensitised lung cancer cells to cisplatin-induced cytotoxicity. In this process, Bcl-2 was persistently phosphorylated in the cells cotreated with nicotine and cisplatin. Furthermore, Akt was proven to be responsible for sustained activation of Bcl-2 by nicotine, which further antagonised cisplatin-mediated apoptotic signalling. Conclusions: Our study suggested that nicotine activates its downstream signalling to interfere with the ubiquitination process and prevent Bcl-2 from being degraded in lung cancer cells, resulting in the increase of chemoresistance. PMID:24548862

  10. Interaction of desipramine and amitriptyline with adrenergic mechanisms in the human iris in vivo.

    PubMed

    Szabadi, E; Gaszner, P; Bradshaw, C M

    1981-01-01

    Mydriatic responses of the pupil were evoked by locally instilled noradrenaline and methoxamine in eight healthy volunteers. The effects of three single oral doses (25 mg, 50 mg and 100 mg) of amitriptyline and desipramine were compared on the mydriatic responses. Both antidepressants potentiated the mydriasis evoked by noradrenaline; desipramine appeared to be approximately four times more potent than amitriptyline. Both antidepressants antagonised the mydriasis evoked by noradrenaline; desipramine appeared to be approximately four times more potent than amitriptyline. Both antidepressants antagonised the mydriasis evoked by methoxamine, amitriptyline being approximately twice as potent as desipramine. It is suggested that the potentiation of the response to noradrenaline may reflect the blockade of the uptake of noradrenaline into adrenergic nerve terminals, whereas the antagonism of the response to methoxamine may reflect the blockade of postsynaptic alpha-adrenoceptors by the antidepressants. It is argued that the interaction of the antidepressants with adrenergic mechanisms could explain why amitriptyline, a potent anticholinergic agent, causes no significant change in resting pupil diameter, while desipramine, a relatively weaker anticholinergic agent, produces a significant mydriasis.

  11. Inhibition of Interferon Induction and Action by the Nairovirus Nairobi Sheep Disease Virus/Ganjam Virus

    PubMed Central

    Holzer, Barbara; Bakshi, Siddharth; Bridgen, Anne; Baron, Michael D.

    2011-01-01

    The Nairoviruses are an important group of tick-borne viruses that includes pathogens of man (Crimean Congo hemorrhagic fever virus) and livestock animals (Dugbe virus, Nairobi sheep disease virus (NSDV)). NSDV is found in large parts of East Africa and the Indian subcontinent (where it is known as Ganjam virus). We have investigated the ability of NSDV to antagonise the induction and actions of interferon. Both pathogenic and apathogenic isolates could actively inhibit the induction of type 1 interferon, and also blocked the signalling pathways of both type 1 and type 2 interferons. Using transient expression of viral proteins or sections of viral proteins, these activities all mapped to the ovarian tumour-like protease domain (OTU) found in the viral RNA polymerase. Virus infection, or expression of this OTU domain in transfected cells, led to a great reduction in the incorporation of ubiquitin or ISG15 protein into host cell proteins. Point mutations in the OTU that inhibited the protease activity also prevented it from antagonising interferon induction and action. Interestingly, a mutation at a peripheral site, which had little apparent effect on the ability of the OTU to inhibit ubiquitination and ISG15ylation, removed the ability of the OTU to block the induction of type 1 and the action of type 2 interferons, but had a lesser effect on the ability to block type 1 interferon action, suggesting that targets other than ubiquitin and ISG15 may be involved in the actions of the viral OTU. PMID:22163042

  12. Regulation of tyrosine phosphatases in the adventitia during vascular remodelling

    SciTech Connect

    Micke, Patrick; Hackbusch, Daniel; Mercan, Sibel; Stawowy, Philipp; Ostman, Arne; Kappert, Kai

    2009-05-15

    Protein tyrosine phosphatases (PTPs) are regulators of growth factor signalling in vascular remodelling. The aim of this study was to evaluate PTP expression in the context of PDGF-signalling in the adventitia after angioplasty. Utilising a rat carotid artery model, the adventitial layers of injured and non-injured vessels were laser microdissected. The mRNA expression of the PDGF {beta}-receptor, the ligands PDGF-A/B/C/D and the receptor-antagonising PTPs (DEP-1, TC-PTP, SHP-2, PTP1B) were determined and correlated to vascular morphometrics, proliferation markers and PDGF {beta}-receptor phosphorylation. The levels of the PDGF {beta}-receptor, PDGF-C and PDGF-D were upregulated concurrently with the antagonising PTPs DEP-1 and TC-PTP at day 8, and normalised at day 14 after vessel injury. Although the proliferation parameters were time-dependently altered in the adventitial layer, the phosphorylation of the PDGF {beta}-receptor remained unchanged. The expression dynamics of specific PTPs indicate a regulatory role of PDGF-signalling also in the adventitia during vascular remodelling.

  13. Reversal of amphetamine-induced behaviours by MDL 100,907, a selective 5-HT2A antagonist.

    PubMed

    Moser, P C; Moran, P M; Frank, R A; Kehne, J H

    1996-01-01

    MDL 100,907 is a potent and selective antagonist of the 5-HT2A receptor which, unlike other antagonists at this receptor, has little affinity for the 5-HT2C receptor. We have investigated the antipsychotic potential of MDL 100,907 by examining its ability to antagonise different behavioural effects of amphetamine in rats. MDL 100,907 reversed the locomotor stimulant effects of amphetamine in rats without itself having any effect on locomotor activity. It also antagonised the disruptive effects of amphetamine on the development of latent inhibition. In contrast, MDL 100,907 had no effect on the discriminative stimulus properties of amphetamine, nor did it affect the ability of amphetamine to reduce the threshold required to sustain rewarding brain stimulation in the ventral tegmental area. This profile is different from that of typical and atypical neuroleptics, and also from other 5-HT2 receptor antagonists, which lack the selectivity of MDL 100,907. These results suggest that MDL 100,907 may have a unique interaction with dopaminergic systems and support the further development of selective 5-HT2 receptor antagonists as a novel therapeutic strategy for schizophrenia.

  14. GPR30 Signaling and Regulation in Breast Cancer

    DTIC Science & Technology

    2011-04-01

    GPCR ), GPR30, was shown to elicit E2-dependent signaling in SKBr3 cells, which lack both classical E2 receptors [4]. In 2005, our group and...we have determined by immunofluorescent antibody staining that they are GPR30 positive. We also used a PyMT cell line derived from the tumor of a

  15. Actions of thienyl analogs of baclofen in the guinea-pig isolated ileum.

    PubMed

    Ong, J; Kerr, D I; Berthelot, P; Vaccher, C; Flouquet, N; Debaert, M

    1992-08-06

    In guinea-pig isolated ileal preparations, the 5-methylthien-2-yl (5d), 5-bromothien-2-yl (5f) and 5-chlorothien-2-yl (5h) analogs of baclofen depressed twitch responses to field stimulation in a dose-dependent manner. These actions were reversibly and competitively antagonised by 2-hydroxysaclofen but not by naloxone, phentolamine, propranolol or theophylline. The relative potencies (EC50 values) were baclofen (10 microM) greater than 5h (40 microM) greater than 5d (80 microM) greater than 5f (120 microM). These analogs represent a novel class of specific GABAB receptor agonists which, like baclofen, should readily enter the brain.

  16. Targeting cytokines as a treatment for patients with sepsis: A lost cause or a strategy still worthy of pursuit?

    PubMed

    Brown, K Alun; Brown, Geraint A; Lewis, Sion M; Beale, Richard; Treacher, David F

    2016-07-01

    Despite often knowing the aetiology of sepsis and its clinical course there has not been the anticipated advances in treatment strategies. Cytokines are influential mediators of immune/inflammatory reactions and in patients with sepsis high circulating levels are implicated in the onset and perpetuation of organ failure. Antagonising the activities of pro-inflammatory cytokines enhances survival in animal models of sepsis but, so far, such a therapeutic strategy has not improved patient outcome. This article addresses the questions of why encouraging laboratory findings have failed to be translated into successful treatments of critically ill patients and whether modifying cytokine activity still remains a promising avenue for therapeutic advance in severe sepsis. In pursuing this task we have selected reports that we believe provide an incisive, critical and balanced view of the topic. Copyright © 2016 Elsevier B.V. All rights reserved.

  17. Ban the sunset? Nonpropositional content and regulation of pharmaceutical advertising.

    PubMed

    Biegler, Paul; Vargas, Patrick

    2013-01-01

    The risk that direct-to-consumer advertising of prescription pharmaceuticals (DTCA) may increase inappropriate medicine use is well recognized. The U.S. Food and Drug Administration addresses this concern by subjecting DTCA content to strict scrutiny. Its strictures are, however, heavily focused on the explicit claims made in commercials, what we term their "propositional content." Yet research in social psychology suggests advertising employs techniques to influence viewers via nonpropositional content, for example, images and music. We argue that one such technique, evaluative conditioning, is operative in DTCA. We further argue that evaluative conditioning fosters unjustified beliefs about drug safety and efficacy, antagonising the autonomy of viewers' choices about advertised medicines. We conclude that current guidelines are deficient in failing to account for evaluative conditioning, and that more research and debate are needed to determine the permissibility of this and other forms of nonpropositional persuasion.

  18. NMDA receptor antagonism with novel indolyl, 2-(1,1-Dimethyl-1,3-dihydro-benzo[e]indol-2-ylidene)-malonaldehyde, reduces seizures duration in a rat model of epilepsy

    PubMed Central

    Rothan, Hussin A.; Amini, Elham; Faraj, Fadihl L.; Golpich, Mojtaba; Teoh, Teow Chong; Gholami, Khadijeh; Yusof, Rohana

    2017-01-01

    N-methyl-D-aspartate receptors (NMDAR) play a central role in epileptogensis and NMDAR antagonists have been shown to have antiepileptic effects in animals and humans. Despite significant progress in the development of antiepileptic therapies over the previous 3 decades, a need still exists for novel therapies. We screened an in-house library of small molecules targeting the NMDA receptor. A novel indolyl compound, 2-(1,1-Dimethyl-1,3-dihydro-benzo[e]indol-2-ylidene)-malonaldehyde, (DDBM) showed the best binding with the NMDA receptor and computational docking data showed that DDBM antagonised the binding sites of the NMDA receptor at lower docking energies compared to other molecules. Using a rat electroconvulsive shock (ECS) model of epilepsy we showed that DDBM decreased seizure duration and improved the histological outcomes. Our data show for the first time that indolyls like DDBM have robust anticonvulsive activity and have the potential to be developed as novel anticonvulsants. PMID:28358047

  19. Noggin4 is a long-range inhibitor of Wnt8 signalling that regulates head development in Xenopus laevis.

    PubMed

    Eroshkin, Fedor M; Nesterenko, Alexey M; Borodulin, Alexander V; Martynova, Natalia Yu; Ermakova, Galina V; Gyoeva, Fatima K; Orlov, Eugeny E; Belogurov, Alexey A; Lukyanov, Konstantin A; Bayramov, Andrey V; Zaraisky, Andrey G

    2016-03-14

    Noggin4 is a Noggin family secreted protein whose molecular and physiological functions remain unknown. In this study, we demonstrate that in contrast to other Noggins, Xenopus laevis Noggin4 cannot antagonise BMP signalling; instead, it specifically binds to Wnt8 and inhibits the Wnt/β -catenin pathway. Live imaging demonstrated that Noggin4 diffusivity in embryonic tissues significantly exceeded that of other Noggins. Using the Fluorescence Recovery After Photobleaching (FRAP) assay and mathematical modelling, we directly estimated the affinity of Noggin4 for Wnt8 in living embryos and determined that Noggin4 fine-tune the Wnt8 posterior-to-anterior gradient. Our results suggest a role for Noggin4 as a unique, freely diffusing, long-range inhibitor of canonical Wnt signalling, thus explaining its ability to promote head development.

  20. Insulin-like growth factor I reverses interleukin-1beta inhibition of insulin secretion, induction of nitric oxide synthase and cytokine-mediated apoptosis in rat islets of Langerhans.

    PubMed

    Mabley, J G; Belin, V; John, N; Green, I C

    1997-11-10

    We have previously observed that treatment of rat islets of Langerhans with interleukin-1beta for 12 h results in nitric oxide-dependent inhibition of insulin secretion, while 48 h treatment increased rates of islet cell death by apoptosis. Here, we demonstrate that interleukin-1beta-mediated nitric oxide formation and inhibition of insulin secretion are significantly reduced by 24 h pretreatment of rat islets of Langerhans with insulin-like growth factor I (IGF-I). IGF-I decreased cytokine induction of nitric oxide synthase in islets. Use of an arginine analogue in culture or IGF-I pretreatment of islets were also effective in protecting islets against cytokine-mediated apoptotic cell death. We conclude that IGF-I antagonises inhibitory and cytotoxic effects of cytokines in rat islets.

  1. Radiotherapy and wound healing.

    PubMed

    Devalia, Haresh L; Mansfield, Lucy

    2008-03-01

    This review article discusses basic radiation physics and effects of radiation on wounds. It examines various postulated hypothesis on the role of circulatory decrease and radiation-induced direct cellular damage. The new concept related to the radiation pathogenesis proposes that there is a cascade of cytokines initiated immediately after the radiation. Sustained activation of myofibroblasts in the wound accounts for its chronicity. Recent advances highlight that transforming growth factor beta1 is the master switch in pathogenesis of radiation fibrosis. This articles overviews its role and summarises the available evidences related to radiation damage. The goal of this article was to provide its modern understanding, as future research will concentrate on antagonising the effects of cytokines to promote wound healing.

  2. Do GABA uptake inhibitors herald a new generation of GABA-ergic drugs for trigeminal neuralgia?

    PubMed

    Hon, Ng Chee

    2004-10-01

    Anticonvulsant drugs are the initial treatment of choice in trigeminal neuralgia. Historically, carbamazepine is and remains the gold standard. Many similarities between the pathophysiologic phenomena observed in epilepsy models and neuropathic pain models justify the rationale for the use of anticonvulsants in the management of neuropathic pain disorders. We have evidence that there is a high intensity of Gamma-aminobutyric acid transporters in the trigeminal sensory nuclei, and that Gamma-aminobutyric transporters expression was increased in the spinal trigeminal nucleus of rats after facial carrageenan injections. These are correlated with increased Gamma-aminobutyric acid uptake in the synaptosomal preparation from the spinal trigeminal nucleus of rats that received the injections and can be antagonised with glial uptake inhibitors. We conclude that Gamma-aminobutyric acid uptake inhibitors may prove to be a novel generation of GABAergic drugs for trigeminal neuralgia.

  3. Neuropharmacological actions of panchagavya formulation containing Emblica officinalis Gaerth and Glycyrrhiza glabra Linn in mice.

    PubMed

    Achliya, Girish S; Wadodkar, Sudhir G; Avinash, K Dorle

    2004-05-01

    A panchagavya Ayurvedic formulation containing E. officinalis, G. glabra, and cow's ghee was evaluated for its effect on pentobarbital-induced sleeping time, pentylenetetrazol-induced seizures, maximal electroshock-induced seizures, spontaneous motor activity, rota-rod performance (motor coordination) and antagonism to amphetamine in mice. The formulation (300, 500 mg/kg, po) produced a significant prolongation of pentobarbital-induced sleeping time and reduced spontaneous locomotor activity. The formulation also significantly antagonised the amphetamine induced hyper-locomotor activity (500, 750 mg/kg, po) and protected mice against tonic convulsions induced by maximal electroshock (500, 750 mg/kg, po). The formulation slightly prolonged the phases of seizure activity but did not protect mice against lethality induced by pentylenetetrazole. The formulation did not show neurotoxicity. The results suggest that the panchagavya formulation is sedative in nature.

  4. Mu-receptor binding in physiological media: comparison with isolated tissue data.

    PubMed

    Carroll, J A; Miller, L; Shaw, J S; Downes, C P

    1984-12-01

    Mu-receptor affinities have been determined for a number of opioid drugs using a combination of isolated tissue and receptor binding techniques. The affinities of antagonists and partial agonists were determined by their ability to antagonise responses to [D-Ala2,MePhe4,Glyol5]enkephalin (GLYOL) on the rat vas deferens preparation. There was little correlation between these results and affinities measured by displacement of [3H]-GLYOL from guinea-pig brain membranes incubated in 50mM Tris buffer. By contrast, affinities measured by displacement of [3H]-naloxone from rat brain membranes incubated in a Krebs/HEPES buffer containing a non-hydrolysable analogue of GTP, agreed very closely with the isolated tissue data.

  5. [Presynaptic histamine H3-receptors exist on cardiac sympathetic terminals of guinea pig].

    PubMed

    Luo, X X

    1995-07-01

    This is the first time to report the existence of new presynaptic inhibitory autoreceptors--histamine H3-receptors in guinea pig myocardium. We found that (R)-alpha-methylhistamine (alpha-MeHA), a selective histamine H3-receptor agonist, attenuates the sympathetic inotropic response of isolated guinea pig atria elicited by electrical field stimulation. This inhibition was associated with a marked reduction in endogenous norepinephrine release. The above phenomenon was antagonised by selective histamine H3-receptor antagonists, and inhibited by pretreatment with N ethylmeleimide. The cardiac sympathetic response could be attenuated or facilitated by increase or decrease of endogenous histamine. Our findings indicate that the endogenous histamine might be involved in the modulation of cardiac sympathetic neurotransmission by interacting with histamine H3-receptors and the receptors are probably coupled to a G(o)/Gi protein.

  6. The unrecognised difficult extubation: a call for vigilance.

    PubMed

    Antoine, J; Hussain, Z; El-Sayed, I; Apfel, C C

    2010-09-01

    Tracheal extubation remains a critical and often overlooked period of difficult airway management. A 66-year-old man, scheduled for C5-C7 anterior fusion, with an easy view of the vocal cords, presented with a sublaryngeal obstruction that required a reduced tracheal tube size. Despite correct tube placement, intra-operative ventilation remained difficult. At the end of surgery a pulsatile tracheal compression was fibreopticially observed above the carina. After discussion with the attending otolaryngologist, neuromuscular blockade was antagonised and the patient was able to maintain normal minute volumes while spontaneously ventilating. With the otolaryngologist present, and with the patient conscious, the trachea was successfully extubated over an airway exchange catheter. A subsequent CT scan revealed an impingement of the trachea by the innominate artery and a mildly ectatic ascending and descending aorta that, in conjunction with tracheomalacia and neuromuscular blockade, could explain the observed signs and symptoms.

  7. Chlorambucil-sensitive and -resistant lymphoid cells display different responses to the histone deacetylase inhibitor, sodium butyrate.

    PubMed

    Kwa, Faith A A; Cole-Sinclair, Merrole; Kapuscinski, Miroslav

    2010-12-17

    Clinical chemoresistance is a frequent complication of alkylating agent treatment of malignant tumours. Chromatin remodelling using histone deacetylase inhibitors (e.g., sodium butyrate, NaBu) may increase target cell chemosensitivity. Apoptotic responses and expression of chromatin modifying enzymes in lymphoid cell lines, LP-1 and NCI-H929, to chlorambucil (CLB) and/or NaBu were examined in this study. NaBu augmented the apoptotic response in CLB-resistant LP-1 cells but antagonised it in CLB-sensitive NCI-H929 cells. CLB increased expression of methyltransferase I and histone acetyltransferase I in both cell lines while NaBu had only small effect. CLB-induced increased gene expression was attenuated by NaBu in CLB-sensitive NCI-H929 cells but not in resistant LP-1 cells. These results suggest that chromatin modifying agents may have differential effects on cells depending on their chemosensitivity.

  8. Influence of the Gut Microflora and of Biliary Constituents on Morphological Changes in the Small Intestine in Obstructive Jaundice

    PubMed Central

    Quraishy, M. Saeed; Chescoe, Dawn; Mullervy, Jenny; Coates, Marie; Hinton, Richard H.

    1996-01-01

    Increased amounts of intestinal endotoxin are absorbed in obstructive jaundice. The precise mechanism is not known but the increased absorption may arise from alterations in the luminal contents, in the intestinal flora, in the gut wall or in interactions between all three. To examine the effects of the intestinal flora we have compared the morphological changes in the small intestine in obstructive jaundice in germ free and conventional rats while the effects of bile constituents have been examined by addition of bile constituents to the diet of bile duct ligated rats. Changes in the intestine were examined, histologically, by enzyme histochemistry, and by transmission and scanning electron microscopy. The results showed no differences in response between germ free and conventional rats. Feeding of diets containing bile salts exacerbated the lesion. Feeding of diets containing cholesterol, however, reduced the degree of intestinal changes produced by cholestasis and completely antagonised the increase in damage caused by feeding of bile salts. PMID:9187547

  9. Noggin4 is a long-range inhibitor of Wnt8 signalling that regulates head development in Xenopus laevis

    PubMed Central

    Eroshkin, Fedor M.; Nesterenko, Alexey M.; Borodulin, Alexander V.; Martynova, Natalia Yu.; Ermakova, Galina V.; Gyoeva, Fatima K.; Orlov, Eugeny E.; Belogurov, Alexey A.; Lukyanov, Konstantin A.; Bayramov, Andrey V.; Zaraisky, Andrey G.

    2016-01-01

    Noggin4 is a Noggin family secreted protein whose molecular and physiological functions remain unknown. In this study, we demonstrate that in contrast to other Noggins, Xenopus laevis Noggin4 cannot antagonise BMP signalling; instead, it specifically binds to Wnt8 and inhibits the Wnt/β -catenin pathway. Live imaging demonstrated that Noggin4 diffusivity in embryonic tissues significantly exceeded that of other Noggins. Using the Fluorescence Recovery After Photobleaching (FRAP) assay and mathematical modelling, we directly estimated the affinity of Noggin4 for Wnt8 in living embryos and determined that Noggin4 fine-tune the Wnt8 posterior-to-anterior gradient. Our results suggest a role for Noggin4 as a unique, freely diffusing, long-range inhibitor of canonical Wnt signalling, thus explaining its ability to promote head development. PMID:26973133

  10. Neuropharmacological study of hetero[2,1]benzothiazepine derivatives analogues of tianeptine.

    PubMed

    Sánchez-Mateo, Candelaria C; Darias, Victoriano; Expósito-Orta, M Auxiliadora; Albertos, Luz M

    2003-01-01

    Neuropharmacological studies were conducted in mice with a number of hetero[2,1]benzothiazepine derivatives, analogues of tianeptine. Seven of the 12 compounds under study potentiated the actions of 5-hydroxytryptophan (5-HTP, 50 mg/kg i.p.) and/or antagonised the hypothermia induced by high doses of apomorphine. Moreover, some of them inhibited the head twitches induced by 5-HTP (250 mg/kg i.p.) and the stereotyped behaviour and/or climbing behaviour of low doses of apomorphine. These compounds also produced a slight inhibition of exploratory behaviour in the holeboard test. On the other hand, no significant muscle relaxant, anticonvulsant and anxiolytic activities were observed at any dose employed. Together, these data suggest that some of the compounds under study exert antidepressant and neuroleptic effects in mice with no muscle relaxant, anxiolytic and anticonvulsant activities.

  11. Buprenorphine in combination with naloxone at a ratio of 15:1 does not enhance antinociception from buprenorphine in healthy cats.

    PubMed

    Slingsby, L S; Murrell, J C; Taylor, P M

    2012-06-01

    Naloxone can enhance the antinociceptive/analgesic effects of buprenorphine in humans and rats. The antinociceptive effects of a patented 15:1 buprenorphine:naloxone combination was investigated in cats using a thermal and mechanical nociceptive model. Twelve cats received buprenorphine 10 μg/kg, naloxone 0.67 μg/kg or a buprenorphine-naloxone combination intramuscularly in a randomised cross over study. Using thermal and mechanical analgesiometry validated in the cat, pre-treatment baselines were measured. Following test drug administration, thresholds were studied for the next 24h. Naloxone did not enhance the thermal antinociceptive effect of buprenorphine. The results from this study are in agreement with previously published work showing that naloxone antagonises the effects of clinically analgesic doses of buprenorphine. Mechanical nociceptive thresholds were not affected by buprenorphine. Copyright © 2011 Elsevier Ltd. All rights reserved.

  12. Alarm pheromone induces stress analgesia via an opioid system in the honeybee.

    PubMed

    Núñez, J; Almeida, L; Balderrama, N; Giurfa, M

    1997-12-31

    Changes of the stinging response threshold of Apis mellifera scutellata were measured on foragers fixed on a holder and stimulated with an electric shock as a noxious stimulus. The threshold of responsiveness to the noxious stimulus increased when bees were previously stimulated with isopentyl acetate, which is a main component of the alarm pheromone of the sting chamber. This effect is antagonised by previous injection of naloxone-hydrochloride (Endo Laboratories Inc.). Results suggest that in the honeybee an endogenous opioid system activated by isopentyl acetate is responsible for modulation of perception for nociceptive stimuli. The resulting stress-induced analgesia in the defender bee would reduce its probability of withdrawal thus increasing its efficiency against enemies.

  13. Alkylation of Staurosporine to Derive a Kinase Probe for Fluorescence Applications

    PubMed Central

    Disney, Alexander J. M.; Kellam, Barrie

    2016-01-01

    Abstract The natural product staurosporine is a high‐affinity inhibitor of nearly all mammalian protein kinases. The labelling of staurosporine has proven effective as a means of generating protein kinase research tools. Most tools have been generated by acylation of the 4′‐methylamine of the sugar moiety of staurosporine. Herein we describe the alkylation of this group as a first step to generate a fluorescently labelled staurosporine. Following alkylation, a polyethylene glycol linker was installed, allowing subsequent attachment of fluorescein. We report that this fluorescein–staurosporine conjugate binds to cAMP‐dependent protein kinase in the nanomolar range. Furthermore, its binding can be antagonised with unmodified staurosporine as well as ATP, indicating it targets the ATP binding site in a similar fashion to native staurosporine. This reagent has potential application as a screening tool for protein kinases of interest. PMID:27008372

  14. Alkylation of Staurosporine to Derive a Kinase Probe for Fluorescence Applications.

    PubMed

    Disney, Alexander J M; Kellam, Barrie; Dekker, Lodewijk V

    2016-05-06

    The natural product staurosporine is a high-affinity inhibitor of nearly all mammalian protein kinases. The labelling of staurosporine has proven effective as a means of generating protein kinase research tools. Most tools have been generated by acylation of the 4'-methylamine of the sugar moiety of staurosporine. Herein we describe the alkylation of this group as a first step to generate a fluorescently labelled staurosporine. Following alkylation, a polyethylene glycol linker was installed, allowing subsequent attachment of fluorescein. We report that this fluorescein-staurosporine conjugate binds to cAMP-dependent protein kinase in the nanomolar range. Furthermore, its binding can be antagonised with unmodified staurosporine as well as ATP, indicating it targets the ATP binding site in a similar fashion to native staurosporine. This reagent has potential application as a screening tool for protein kinases of interest.

  15. Bimodal antagonism of PKA signalling by ARHGAP36

    PubMed Central

    Eccles, Rebecca L.; Czajkowski, Maciej T.; Barth, Carolin; Müller, Paul Markus; McShane, Erik; Grunwald, Stephan; Beaudette, Patrick; Mecklenburg, Nora; Volkmer, Rudolf; Zühlke, Kerstin; Dittmar, Gunnar; Selbach, Matthias; Hammes, Annette; Daumke, Oliver; Klussmann, Enno; Urbé, Sylvie; Rocks, Oliver

    2016-01-01

    Protein kinase A is a key mediator of cAMP signalling downstream of G-protein-coupled receptors, a signalling pathway conserved in all eukaryotes. cAMP binding to the regulatory subunits (PKAR) relieves their inhibition of the catalytic subunits (PKAC). Here we report that ARHGAP36 combines two distinct inhibitory mechanisms to antagonise PKA signalling. First, it blocks PKAC activity via a pseudosubstrate motif, akin to the mechanism employed by the protein kinase inhibitor proteins. Second, it targets PKAC for rapid ubiquitin-mediated lysosomal degradation, a pathway usually reserved for transmembrane receptors. ARHGAP36 thus dampens the sensitivity of cells to cAMP. We show that PKA inhibition by ARHGAP36 promotes derepression of the Hedgehog signalling pathway, thereby providing a simple rationale for the upregulation of ARHGAP36 in medulloblastoma. Our work reveals a new layer of PKA regulation that may play an important role in development and disease. PMID:27713425

  16. Bimodal antagonism of PKA signalling by ARHGAP36.

    PubMed

    Eccles, Rebecca L; Czajkowski, Maciej T; Barth, Carolin; Müller, Paul Markus; McShane, Erik; Grunwald, Stephan; Beaudette, Patrick; Mecklenburg, Nora; Volkmer, Rudolf; Zühlke, Kerstin; Dittmar, Gunnar; Selbach, Matthias; Hammes, Annette; Daumke, Oliver; Klussmann, Enno; Urbé, Sylvie; Rocks, Oliver

    2016-10-07

    Protein kinase A is a key mediator of cAMP signalling downstream of G-protein-coupled receptors, a signalling pathway conserved in all eukaryotes. cAMP binding to the regulatory subunits (PKAR) relieves their inhibition of the catalytic subunits (PKAC). Here we report that ARHGAP36 combines two distinct inhibitory mechanisms to antagonise PKA signalling. First, it blocks PKAC activity via a pseudosubstrate motif, akin to the mechanism employed by the protein kinase inhibitor proteins. Second, it targets PKAC for rapid ubiquitin-mediated lysosomal degradation, a pathway usually reserved for transmembrane receptors. ARHGAP36 thus dampens the sensitivity of cells to cAMP. We show that PKA inhibition by ARHGAP36 promotes derepression of the Hedgehog signalling pathway, thereby providing a simple rationale for the upregulation of ARHGAP36 in medulloblastoma. Our work reveals a new layer of PKA regulation that may play an important role in development and disease.

  17. Separate and combined effects of the social drugs on psychomotor performance.

    PubMed

    Kerr, J S; Sherwood, N; Hindmarch, I

    1991-01-01

    Ten female subjects (five smokers and five non-smokers) performed a choice reaction time task (CRT), a compensatory tracking task (CTT), a short-term memory task (STM) and were tested for their critical flicker fusion threshold (CFF) at set points over 4 h after the administration of each possible combination of nicotine (2 mg gum or placebo), caffeine (250 mg capsule or placebo) and alcohol (30 g or placebo). Memory and motor function were shown to be facilitated by nicotine or caffeine, and the debilitating effects of alcohol were frequently antagonised by either drug. In spite of the differences in their neuropharmacological actions, combinations of nicotine, caffeine and alcohol may be compared through their effects on common information processing mechanisms involved in psychomotor performance.

  18. Effects of intra-ventrolateral periaqueductal grey palmitoylethanolamide on thermoceptive threshold and rostral ventromedial medulla cell activity.

    PubMed

    de Novellis, Vito; Luongo, Livio; Guida, Francesca; Cristino, Luigia; Palazzo, Enza; Russo, Roberto; Marabese, Ida; D'Agostino, Giuseppe; Calignano, Antonio; Rossi, Francesca; Di Marzo, Vincenzo; Maione, Sabatino

    2012-02-15

    Palmitoylethanolamide (PEA), a peroxisome proliferator-activated receptor-α (PPAR-α) ligand, exerts antinociceptive and anti-inflammatory effects. PEA (3 and 6 nmol) was microinjected in the ventrolateral periaqueductal grey (VL PAG) of male rats and effects on nociceptive responses and ongoing and tail flick-related activities of rostral ventromedial medulla (RVM) ON and OFF cells were recorded. Intra-PAG microinjection of PEA reduced the ongoing activity of ON and OFF cells and produced an increase in the latency of the nociceptive reaction. These effects were prevented by a selective PPAR-α antagonist, GW6471 and by a large-conductance Ca(2+)-activated K(+) channel inhibitor, charybdotoxin. Cannabinoid 1 (CB(1)) receptor blockade by AM251 increased the PEA-induced effect both on the ongoing activity of the ON cell and on the latency to tail flick without affecting the effect of PEA on the OFF cell. Conversely, a transient receptor potential vanilloid type 1 (TRPV(1)) blocker, I-RTX, had no effect on the ON cell activity and tail flick latency, whereas it blocked the PEA-induced decrease in ongoing activity of the OFF cell. PEA decreased the burst and increased the latency of tail flick-evoked onset of ON cell activity in a manner antagonised by GW6471 and charybdotoxin. AM251 and I-RTX, instead, enhanced these latter effects. In conclusion, intra-VL PAG PEA induces antinociceptive effects associated with a decrease in RVM ON and OFF cell activities. PPAR-α receptors mediate, and CB(1) and TRPV(1) receptors antagonise, PEA-induced effects within the PAG-RVM circuitry.

  19. desHis¹Glu⁹-glucagon-[mPEG] and desHis¹Glu⁹(Lys³⁰PAL)-glucagon: long-acting peptide-based PEGylated and acylated glucagon receptor antagonists with potential antidiabetic activity.

    PubMed

    Irwin, Nigel; Franklin, Zara J; O'Harte, Finbarr P M

    2013-06-05

    Glucagon is hormone secreted from the pancreatic alpha-cells that is involved in blood glucose regulation. As such, antagonism of glucagon receptor signalling represents an exciting approach for treating diabetes. To harness these beneficial metabolic effects, two novel glucagon analogues, desHis¹Glu⁹-glucagon-[mPEG] and desHis¹Glu⁹(Lys³⁰PAL)-glucagon, has been evaluated for potential glucagon receptor antagonistic properties. Both novel peptides were completely resistant to enzymatic breakdown and significantly (P<0.05 to P<0.001) inhibited glucagon-mediated elevations of cAMP production in glucagon receptor transfected cells. Similarly, desHis¹Glu⁹-glucagon-[mPEG] and desHis¹Glu⁹(Lys³⁰PAL)-glucagon effectively antagonised glucagon-induced increases of insulin secretion from BRIN BD11 cells. When administered acutely to normal, high fat fed or ob/ob mice, both analogues had no significant effects on overall blood glucose or plasma insulin levels when compared to saline treated controls. However, desHis¹Glu⁹-glucagon-[mPEG] significantly (P<0.05) annulled glucagon-induced increases in blood glucose and plasma insulin levels in normal mice and had similar non-significant tendencies in high fat and ob/ob mice. In addition, desHis¹Glu⁹(Lys³⁰PAL)-glucagon effectively (P<0.05 to P<0.001) antagonised glucagon-mediated elevations of blood glucose levels in high fat fed and ob/ob mice, but was less efficacious in normal mice. Further studies confirmed the significant persistent glucagon receptor antagonistic properties of both novel enzyme-resistant analogues 4h post administration in normal mice. These studies emphasise the potential of longer-acting peptide-based glucagon receptor antagonists, and particularly acylated versions, for the treatment of diabetes. Copyright © 2013 Elsevier B.V. All rights reserved.

  20. Beta blockers in heart failure haemodynamics, clinical effects and modes of action

    PubMed Central

    de Milliano, P.A.R.; Tijssen, J.G.P.; van Zwieten, P.A.; Lie, K.I.

    2001-01-01

    Treatment for heart failure may be directed at relieving symptoms and/or improving prognosis. One of the primary aims of research in heart failure is to alter the progressive decline in pump function and thereby improve prognosis. For many years, diuretics have been known as therapeutics in heart failure and they are very effective in symptom relief. Vasodilators and inotropes also have beneficial effects on symptom relief especially in the acute phase through changes in cardiac output, filling pressures and renal perfusion. However, although these treatments produce short-term relief, none have been shown to influence the disease process and thereby improve mortality. Indeed, many of these drugs may even lead to untoward long-term clinical outcomes as has been shown for example for milrinone and ibopamine. There is overwhelming evidence that drugs interfering with the neurohormonal activation in heart failure not only produce symptomatic relief but are also capable of attenuating disease progression with concomitant reductions in both morbidity and mortality. About a decade ago, convincing and large-scale evidence showed that ACE inhibitors produced favourable effects by antagonising the activated renin-angiotensin system. More recently, β-blockers, which antagonise the activated sympathetic system, were shown to be beneficial in the long term in moderate severe heart failure in terms of significant improvements in both morbidity and mortality. The RALES study further amplified the concept that drugs that interact in the neurohormonal system have beneficial effects. In this study, spironolactone, a weak, potassium-sparing diuretic counteracting aldosterone showed a reduction in mortality in more severe forms of heart failure. PMID:25696756

  1. Olvanil: a non-pungent TRPV1 activator has anti-emetic properties in the ferret.

    PubMed

    Chu, Kit-Man; Ngan, Man-Piu; Wai, Man-Keung; Yeung, Chi-Kong; Andrews, Paul L R; Percie du Sert, Nathalie; Rudd, John A

    2010-02-01

    Anti-emetic drugs such as the tachykinin NK(1) receptor antagonists are useful to control emesis induced by diverse challenges. Evidence suggests pungent capsaicin-like TRPV1 activators also have broad inhibitory anti-emetic activity. However, pungent compounds are associated with undesirable effects including adverse actions on the cardiovascular system and on temperature homeostasis. In the present investigations using the ferret, we examine if the non-pungent vanilloid, olvanil, has useful anti-emetic properties without adversely affecting behaviour, blood pressure or temperature control. Olvanil (0.05-5 mg/kg, s.c.) was compared to the pungent vanilloid, resiniferatoxin (RTX; 0.1 mg/kg, s.c.), and to the anandamide reuptake inhibitor, AM404 (10 mg/kg, s.c.), for a potential to inhibit emesis induced by apomorphine (0.25 mg/kg, s.c.), copper sulphate (50 mg/kg, intragastric), and cisplatin (10 mg/kg, i.p.). Changes in blood pressure and temperature were also recorded using radiotelemetry implants. In peripheral administration studies, RTX caused transient hypertension, hypothermia and reduced food and water intake, but also significantly inhibited emesis induced by apomorphine, copper sulphate, or cisplatin. Olvanil did not have a similar adverse profile, and antagonised apomorphine- and cisplatin-induced emesis but not that induced by copper sulphate. AM404 reduced only emesis induced by cisplatin without affecting other parameters measured. Following intracerebral administration only olvanil antagonised cisplatin-induced emesis, but this was associated with transient hypothermia. In conclusion, olvanil demonstrated clear anti-emetic activity in the absence of overt cardiovascular, homeostatic, or behavioural effects associated with the pungent vanilloid, RTX. Our studies indicate that non-pungent vanilloids may have a useful spectrum of anti-emetic properties via central and/or peripheral mechanisms after peripheral administration.

  2. Anti-ischemic properties of a new spiro-cyclic benzopyran activator of the cardiac mito-KATP channel.

    PubMed

    Calderone, Vincenzo; Testai, Lara; Martelli, Alma; Rapposelli, Simona; Digiacomo, Maria; Balsamo, Aldo; Breschi, Maria C

    2010-01-01

    Many activators of K(ATP) channels exhibit cardioprotective effects, mainly mediated by channels expressed on mitochondria (mito-K(ATP)). Previous results showed anti-ischemic effects of the spiro-cyclic derivative A, on isolated rat hearts. In this work this molecule was more extensively studied and diazoxide was used as reference mito-K(ATP) opener. The studies were performed on an in vivo rat model of myocardial infarct and on heart-derived H9c2 cells exposed to an anoxic environment. The mechanism of action was further investigated on isolated rat heart mitochondria. In the model of myocardial infarct compound A and diazoxide produced significant cardioprotective effects, antagonised by the selective mito-K(ATP) blocker 5-hydroxydecanoic acid (5-HD). Compound A, like diazoxide, produced modest and non-significant hypotensive responses, while the hyperglycaemic effects of diazoxide were not observed for the new compound. Protective effects of compound A and diazoxide were also recorded in H9c2 cells and again were inhibited by 5-HD. Compound A and diazoxide caused swelling of cardiac mitochondria, in agreement with the profile of mito-K(ATP) openers. Both compounds evoked concentration-dependent Ca2+-release from Ca2+-preloaded mitochondria, prevented mitochondrial Ca2+-uptake and caused mitochondrial membrane depolarisation. These effects were antagonised by ATP, the endogenous K(ATP) inhibitor. In conclusion, compound A exhibits a promising profile of an anti-ischemic agent, with a mechanism likely to be linked to the activation of mito-K(ATP) channels, and, because of its chemical characteristics such as structural rigidity and chirality due to the spiro-cyclic moiety, represents an interesting template for development of analogues further improved in activity and selectivity.

  3. Antipsychotic-induced hyperprolactinaemia, hypogonadism and osteoporosis in the treatment of schizophrenia.

    PubMed

    O'Keane, Veronica

    2008-03-01

    Treatment of schizophrenic illness usually involves the long-term administration of antipsychotic drugs. Most antipsychotic agents antagonise the actions of endogenous dopamine (DA) at DA-2 receptors in the brain. The relative affinity for, and binding time to, DA-2 receptors was considered to be one of the key determinants of the antipsychotic potency of classical antipsychotic drugs. Some newer atypical antipsychotics, of which clozapine is the prototype, have a relatively poor affinity for DA-2 receptors; whereas other atypical antipsychotics are potent DA-2 antagonists. The propensity of antipsychotic agents to cause hyperprolactinaemia is related to their potency in antagonising DA-2 receptors on the anterior pituitary. In our studies, bone loss was consistently related to DA-2 antagonist potency of antipsychotic drugs, rather than their classification using conventional 'typical' versus 'atypical' systems. It is established that hyperprolactinaemia causes suppression of the reproductive endocrine axis and consequent bone mineral density (BMD) loss. Results from our group and others have demonstrated that a similar pathophysiological process is occurring in individuals with antipsychotic-induced hyperprolactinaemia. We found high rates of osteoporosis and osteopenia in those taking long-term antipsychotic drugs, and this was related to the dose and duration of treatment. Bone loss was associated with hypogonadism in male and female groups. Young Caucasian women appear to be particularly vulnerable to developing hyperprolactinaemia and the associated hypogonadism and bone loss. The occurrence of menstrual dysfunction should alert clinical suspicions of hyperprolactinaemia and bone de-mineralisation. Lastly, there are no published trials examining the effects of hormone replacement on BMD in those taking long-term antipsychotic drugs, but preliminary findings from our studies suggest that active management of bone loss in those with antipsychotic-associated bone

  4. Amelioration of glycerol-induced acute renal failure in the rat with 8-cyclopentyl-1,3-dipropylxanthine.

    PubMed Central

    Kellett, R.; Bowmer, C. J.; Collis, M. G.; Yates, M. S.

    1989-01-01

    1. Previous studies have shown that 8-phenyltheophylline (8-PT), a non-selective antagonist at adenosine A1- and A2-receptors, can ameliorate the severity of glycerol-induced acute renal failure (ARF) in the rat. In the present study we have examined the effects of an antagonist with selectivity for adenosine A1-receptors (8-cyclopentyl-1,3-dipropylxanthine, CPX) on the development of ARF. 2. In the anaesthetised rat 8-PT (4 mg kg-1, i.v.) and CPX (0.1 mg kg-1, i.v.) antagonised adenosine-evoked responses which are thought to be mediated via A1-receptors (bradycardia and decrease in renal blood flow). The agonist dose-ratio produced by CPX was equal to or greater than that found with 8-PT (heart rate and renal blood flow respectively). The hypotensive response to adenosine which is predominantly due to A2-receptor activation was also antagonised by 8-PT, whereas CPX was a much less effective antagonist of this response. 3. Administration of CPX (0.1 mg kg-1, i.v.; twice daily for two days) significantly attenuated the increase in plasma levels of urea and creatinine, the increased kidney weight and the renal tubule damage observed in rats 2 days following induction of ARF with intramuscular glycerol injection. In addition treatment with CPX significantly enhanced the clearances of inulin and p-aminohippurate. 4. After glycerol injection, the mortality rate over 7 days in untreated and vehicle-treated rats was 43% and 21% respectively. In contrast, all animals treated with CPX survived over the 7 day observation period. 5. These results support the suggestion that adenosine is an important factor in the development of ARF and indicate that this effect of the purine is likely to be mediated via an adenosine A1-receptor. PMID:2590769

  5. Muscarinic acetylcholine receptor-mediated effects in slices from human epileptogenic cortex.

    PubMed

    Gigout, S; Wierschke, S; Lehmann, T-N; Horn, P; Dehnicke, C; Deisz, R A

    2012-10-25

    Acetylcholine has been implicated in higher cortical functions such as learning, memory and cognition, yet the cellular effects of muscarinic acetylcholine receptor (mAChR) activation are poorly understood in the human cortex. Here we investigated the effect of the mAChR agonist carbachol (CCh) and various mAChR antagonists in human cortical slices (from tissue removed during neurosurgical treatment of epilepsy) by intracellular and extracellular recordings. CCh increased neuronal firing, which was antagonised by atropine (non-selective mAChR antagonist) and pirenzepine (M(1)/M(4) mAChRs antagonist) when applied before or after CCh application. AF-DX 116 (M(2)/M(4) mAChRs antagonist) had no effect on CCh-induced increase of firing. CCh also reduced evoked excitatory postsynaptic potentials (EPSP), and the CCh-induced depression of EPSP was fully reversed by atropine. Pirenzepine reversed the depression of CCh on EPSP, but failed to prevent the depression when applied before CCh. AF-DX 116 prevented the CCh-induced depression of evoked EPSP when applied before CCh. CCh also depressed GABAergic transmission and this effect was antagonised by AF-DX 116. Xanomeline (M(1)/M(4) mAChR agonist) increased neuronal firing and decreased EPSP, but had no effect on GABAergic transmission. Reduction (with linopirdine) and enhancement (with retigabine) of the M-current (mediated by K(V)7 channels), increased and decreased neuronal firing, respectively, but had marginal effects on the evoked EPSP. Our results indicate that three pharmacologically distinct mAChRs modulate neuronal firing, glutamatergic and GABAergic transmissions in the human epileptogenic neocortex. The data are discussed towards possible implications of altered mAChR signalling in hyperexcitability and cognitive functions in the human neocortex. Copyright © 2012 IBRO. Published by Elsevier Ltd. All rights reserved.

  6. Effects of depressant amino acids and antagonists on an in vitro spinal cord preparation from the adult rat.

    PubMed

    Long, S K; Evans, R H; Krijzer, F

    1989-07-01

    A mature sacrococcygeal in vitro spinal preparation from the rat has been used to demonstrate effects of neutral amino acids and their antagonists. gamma-Aminobutanoate (GABA), glycine and taurine (0.5-5 mM) produced dose-dependent depression of spontaneous paroxysmal activity generated in Mg2+ -free medium. The depressant effect of GABA was antagonised selectively by picrotoxin (25-50 microM) and the depressant effects of glycine and taurine were antagonised selectively by strychnine (0.2 microM). Glycine (0.5-5 mM) had a dose-dependent depolarizing action which was present at the central ends of isolated ventral roots. gamma-Aminobutanoate and taurine, had only weak depolarizing actions on ventral root fibres. Depolarizing responses to glycine showed a marked fading. Reduction in the fading appeared to be responsible for a paradoxical potentiation of glycine-induced depolarizations, which occurred in the presence of strychnine (0.2-2 microM). Strychnine (2-10 microM), picrotoxin (10-50 microM) or bicuculline (10 microM) had little or no effect on the amplitude, duration or latency of the monosynaptic component of ventral root reflexes evoked by supramaximal stimulation of dorsal roots (DR-VRP). However all three antagonists introduced slow, NMDA receptor mediated, components to these ventral root potentials. Picrotoxin and bicuculline, but not strychnine, reversibly depressed the dorsal root potential evoked from an adjacent dorsal root (DR-DRP). The depressant actions of 2-amino-5-phosphonopentanoate (AP5), kynurenate and 3-((+/-)-2-carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP) revealed both NMDA and non-NMDA receptor mediated components in the dorsal root potential.

  7. Involvement of α1B-adrenoceptors in the anti-immobility effect of imipramine in the tail suspension test.

    PubMed

    Ribeiro, Carlos Alberto S; Pupo, André S

    2015-03-05

    Imipramine is a tricyclic antidepressant inhibitor of norepinephrine and serotonin neuronal reuptake. The roles of specific α1-adrenoceptor subtypes that might be targeted by the increased synaptic levels of noradrenaline induced by imipramine are not well understood. This study investigates the α1-adrenoceptor subtypes involved in the anti-immobility effect of imipramine in the mouse tail suspension test. The anti-immobility effect of imipramine (32mg/kg, i.p.) was significantly antagonised by the non-subtype-selective α1-adrenoceptor antagonist prazosin (0.5 and 1.0mg/kg, i.p.). Neither the selective α1A-adrenoceptor antagonist 5-methyl-3-[3-[3-[4-[2-(2,2,2,-trifluroethoxy)phenyl]-1-piperazinyl]propyl]-2,4-(1H,3H)-pyrimidinedione (RS-100329, 0.5 and 1.0mg/kg) nor the selective α1D-adrenoceptor antagonist 8-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-8-azaspiro[4.5]decane-7,9-dione dihydrochloride, (BMY-7378, up to 1.0mg/kg, i.p.) affected the anti-immobility effect of imipramine. However, the anti-immobility effect of imipramine was significantly antagonised by the selective α1B-adrenoceptor antagonist (2S)-4-(4-amino-6,7-dimethoxy-2-quinazolinyl)-2-[[(1,1-dimethylethyl)amino]carbonyl]-1-piperazinecarboxylate (L-765,314). In addition, mice treated only with RS-100329 or BMY-7378, but not with L-765,314, showed reduced immobility times in comparison to mice treated with vehicle. These results indicate that the selective antagonism of α1A- and α1D-adrenoceptors results in antidepressant-like effects and that the α1B-subtype is the main target for the increased levels of noradrenaline caused by imipramine.

  8. Actions of 5-hydroxytryptophan to inhibit and disinhibit mouse behaviour in the light/dark test.

    PubMed

    Cheng, C H; Costall, B; Kelly, M E; Naylor, R J

    1994-04-01

    The involvement of 5-HT receptors in behavioural responding to an aversive situation was investigated in the mouse light/dark test. The administration of 5-hydroxytryptophan (5-HTP) (12.5-50 mg/kg i.p.) increased brain 5-HT turnover and inhibited mouse behaviour in the light/dark test box. The 5-HT2C/5-HT2A receptor antagonists methysergide (1.0 and 5.0 mg/kg i.p.) and ritanserin (0.1-1.0 mg/kg i.p.) antagonised (methysergide) or reversed (ritanserin) the effects of 5-HTP to an increased exploration of the light compartment; a low dose of the 5-HT3 receptor antagonist ondansetron (0.01 mg/kg i.p.) had a similar effect. The disinhibitory effect of the 5-HTP/ritanserin interaction was antagonised by the 5-HT3/5-HT4 receptor antagonists SDZ205-557 (0.001-0.1 mg/kg) and a high dose of tropisetron (1.0 mg/kg i.p.) but not by ondansetron (1.0 mg/kg i.p.). At these doses tropisetron and ondansetron had no effect in their own right. Thus the dominant effect of 5-HTP in the mouse is to inhibit behaviour, a response mediated via 5-HT2C/5-HT2A and 5-HT3 receptors. A 5-HT4 receptor may effect an opposing disinhibitory potential as revealed by ritanserin.

  9. HOXC6 Is transcriptionally regulated via coordination of MLL histone methylase and estrogen receptor in an estrogen environment.

    PubMed

    Ansari, Khairul I; Hussain, Imran; Shrestha, Bishakha; Kasiri, Sahba; Mandal, Subhrangsu S

    2011-08-12

    Homeobox (HOX)-containing gene HOXC6 is a critical player in mammary gland development and milk production, and is overexpressed in breast and prostate cancers. We demonstrated that HOXC6 is transcriptionally regulated by estrogen (E2). HOXC6 promoter contains two putative estrogen response elements (EREs), termed as ERE1(1/2) and ERE2(1/2). Promoter analysis using luciferase-based reporter assay demonstrated that both EREs are responsive to E2, with ERE1(1/2) being more responsive than ERE2(1/2). Estrogen receptors (ERs) ERα and ERβ bind to these EREs in an E2-dependent manner, and antisense-mediated knockdown of ERs suppressed the E2-dependent activation of HOXC6 expression. Similarly, knockdown of histone methylases MLL2 and MLL3 decreased the E2-mediated activation of HOXC6. However, depletion of MLL1 or MLL4 showed no significant effect. MLL2 and MLL3 were bound to the HOXC6 EREs in an E2-dependent manner. In contrast, MLL1 and MLL4 that were bound to the HOXC6 promoter in the absence of E2 decreased upon exposure to E2. MLL2 and MLL3 play key roles in histone H3 lysine-4 trimethylation and in the recruitment of general transcription factors and RNA polymerase II in the HOXC6 promoter during E2-dependent transactivation. Nuclear receptor corepressors N-CoR and SAFB1 were bound in the HOXC6 promoter in the absence of E2, and that binding was decreased upon E2 treatment, indicating their critical roles in suppressing HOXC6 gene expression under nonactivated conditions. Knockdown of either ERα or ERβ abolished E2-dependent recruitment of MLL2 and MLL3 into the HOXC6 promoter, demonstrating key roles of ERs in the recruitment of these mixed lineage leukemias into the HOXC6 promoter. Overall, our studies demonstrated that HOXC6 is an E2-responsive gene, and that histone methylases MLL2 and MLL3, in coordination with ERα and ERβ, transcriptionally regulate HOXC6 in an E2-dependent manner.

  10. HOXC6 is transcriptionally regulated via coordination of MLL histone methylase and estrogen receptor under estrogen environment

    PubMed Central

    Ansari, Khairul I.; Hussain, Imran; Shrestha, Bishakha; Kasiri, Sahba; Mandal, Subhrangsu S.

    2011-01-01

    Homeobox containing gene HOXC6 is a critical player in mammary gland development, milk production and is overexpressed in breast and prostate cancer. We demonstrated that HOXC6 is transcriptionally regulated by estrogen (E2). HOXC6 promoter contains two putative estrogen-response elements (EREs), termed as ERE11/2 and ERE21/2. Promoter analysis using luciferase based reporter assay demonstrated that both EREs are responsive to E2, ERE11/2 being more responsive than ERE21/2. Estrogen receptors, ERα and ERβ, bind to these EREs in an E2-dependent manner and antisense-mediated knockdown of ERs suppressed the E2-dependent activation of HOXC6 expression. Similarly, knockdown of histone methylases, MLL2 and MLL3, decreased E2-mediated activation of HOXC6. However, depletion of MLL1 or MLL4 showed no significant effect. MLL2 and MLL3 were bound to the HOXC6 EREs in an E2-dependent manner. In contrast, MLL1 and MLL4 that were bound to the HOXC6 promoter in the absence of E2, decreased upon exposure to E2. MLL2 and MLL3 play key roles in histone H3K4-trimethylation and recruitment of general transcription factors and RNAP II in the HOXC6 promoter during E2-dependent transactivation. Nuclear receptor corepressors N-CoR and SAFB1 were bound in the HOXC6 promoter in absence of E2 and that binding were decreased upon E2-treatment indicating their critical roles in suppressing HOXC6 gene expression under non-activated condition. Knockdown of either ERα or ERβ abolished E2-dependent recruitment of MLL2 and MLL3 into the HOXC6 promoter demonstrating key roles of ERs in recruitment of these MLLs into HOXC6 promoter. Overall, our studies demonstrated that HOXC6 is an estrogen-responsive gene and histone methylases MLL2 and MLL3, in coordination with ERα and ERβ, transcriptionally regulate HOXC6 in an E2-dependent manner. PMID:21683083

  11. Can Diabetes Change the Intrinsic Subtype Specificity of Breast Cancer?

    DTIC Science & Technology

    2008-09-01

    Carroll JS, Liu XS, Brodsky AS, et al. Chromosome-wide mapping of estrogen receptor binding reveals long-range regulation requiring the forkhead...type A associated with better prognosis. A transcription factor network comprising GATA-3, FOXA1 and ERα dictates estrogen (E2) dependence of...CLASSIFICATION OF: 17. LIMITATION OF ABSTRACT 18 . NUMBER OF PAGES 19a. NAME OF RESPONSIBLE PERSON Harikrishna Nakshatri a. REPORT U b. ABSTRACT U

  12. BIG3 Inhibits the Estrogen-Dependent Nuclear Translocation of PHB2 via Multiple Karyopherin-Alpha Proteins in Breast Cancer Cells

    PubMed Central

    Kim, Nam-Hee; Yoshimaru, Tetsuro; Chen, Yi-An; Matsuo, Taisuke; Komatsu, Masato; Miyoshi, Yasuo; Tanaka, Eiji; Sasa, Mitsunori; Mizuguchi, Kenji; Katagiri, Toyomasa

    2015-01-01

    We recently reported that brefeldin A-inhibited guanine nucleotide-exchange protein 3 (BIG3) binds Prohibitin 2 (PHB2) in cytoplasm, thereby causing a loss of function of the PHB2 tumor suppressor in the nuclei of breast cancer cells. However, little is known regarding the mechanism by which BIG3 inhibits the nuclear translocation of PHB2 into breast cancer cells. Here, we report that BIG3 blocks the estrogen (E2)-dependent nuclear import of PHB2 via the karyopherin alpha (KPNA) family in breast cancer cells. We found that overexpressed PHB2 interacted with KPNA1, KPNA5, and KPNA6, thereby leading to the E2-dependent translocation of PHB2 into the nuclei of breast cancer cells. More importantly, knockdown of each endogenous KPNA by siRNA caused a significant inhibition of E2-dependent translocation of PHB2 in BIG3-depleted breast cancer cells, thereby enhancing activation of estrogen receptor alpha (ERα). These data indicated that BIG3 may block the KPNAs (KPNA1, KPNA5, and KPNA6) binding region(s) of PHB2, thereby leading to inhibition of KPNAs-mediated PHB2 nuclear translocation in the presence of E2 in breast cancer cells. Understanding this regulation of PHB2 nuclear import may provide therapeutic strategies for controlling E2/ERα signals in breast cancer cells. PMID:26052702

  13. Clozapine enhances disruption of prepulse inhibition after sub-chronic dizocilpine- or phencyclidine-treatment in Wistar rats.

    PubMed

    Schwabe, Kerstin; Brosda, Jan; Wegener, Nico; Koch, Michael

    2005-02-01

    Sensitisation (i.e. progressive enhancement) of behavioural abnormalities induced by repeated treatment with non-competitive NMDA receptor antagonists in animals is considered an animal model for schizophrenia. Here, male Wistar rats were treated for 11 days with either dizocilpine (0.1 mg/kg), phencyclidine (PCP, 2 mg/kg), or saline and tested for prepulse inhibition (PPI) of the acoustic startle response (ASR). The aims of this study were twofold: First, we tested whether sensitisation of PPI deficits previously found in Sprague-Dawley rats were also found in Wistar rats, and, second, whether these effects can be ameliorated by the atypical antipsychotic clozapine. PPI is a paradigm for the assessment of sensorimotor gating (and its deficits) and is impaired in schizophrenic patients. After the sub-chronic treatment the rats were tested drug-free (day 12), and on the following days after drug challenge by PCP (2 mg/kg), combinations of PCP (2 mg/kg) and clozapine (5 and 10 mg/kg), or clozapine (5 mg/kg) alone. PPI was significantly reduced by both NMDA receptor antagonists. This effect was not further enhanced by the daily treatment. Startle magnitude was increased after eight days of dizocilpine-treatment only, indicating sensitisation of startle-potentiation by this drug. Testing the rats drug-free on day 12 revealed enhanced PPI and reduced startle (compared to the matching test on day 0) irrespective of previous treatment. Drug challenge with PCP (2 mg/kg) again reduced PPI in all groups. Clozapine (5 and 10 mg/kg) failed to antagonise the PPI-disruptive effects of PCP and even enhanced the PCP-induced PPI-deficits in rats pretreated with PCP or dizocilpine. These findings suggest: (1) that PPI and startle are influenced differently by non-competitive NMDA receptor antagonists, (2) that PCP and dizocilpine reduce PPI in Wistar rats, but do not lead to a sensitisation of this effect; and (3) that under the present schedule of treatments, the antipsychotic

  14. Immunosuppressants in advanced clinical development for organ transplantation and selected autoimmune diseases.

    PubMed

    Kovarik, John M; Burtin, Pascale

    2003-05-01

    Immunosuppressants dampen the immune response or restore balance among immune system components. They are primarily used to prevent allograft rejection after organ transplantation and to prevent or treat disease flares in autoimmune diseases. Immunosuppressants available at present include the calcineurin inhibitors (cyclosporin, tacrolimus), antimetabolites (azathioprine, leflunomide, methotrexate, mycophenolate mofetil), antiproliferatives (sirolimus), monoclonal antibodies to T lymphocyte (basiliximab, daclizumab, muromonab-CD3) and anticytokines (anakinra, etanercept, infliximab). The immunosuppressive market grows at a rate of > 10% yearly, with total sales in 2001 of US$2.7 billion. Immunotherapy in transplantation and autoimmune diseases is tending towards the use of multi-drug regimens tailored for the individual patient. At least 23 new immunosuppressants are currently in advanced clinical testing or preregistration, and can be divided into three groups. First, emerging drugs targeting intracellular ligands in immune cells are primarily analogues of currently-marketed agents, which attempt to provide improved pharmaceutical or safety profiles compared with the prototype compound. They are largely being developed in organ transplantation. Second, emerging drugs targeting cell surface ligands on immune cells attempt to antagonise novel molecular sites to interfere with immune cell activation via costimulatory signals, immune cell adhesion to tissues or the vasculature and immune cell trafficking. These agents are being primarily developed in rheumatoid arthritis, psoriasis and/or multiple sclerosis. Finally, emerging drugs acting as anticytokines, which largely follow on from the success of those on the market, by antagonising the function of tumour necrosis factor or a narrow selection of interleukins. All are being assessed in rheumatoid arthritis. Drug development of immunosuppressants is increasingly attempting to intervene in disease progression over

  15. Mechanisms of trophoblast migration, endometrial angiogenesis in preeclampsia: The role of decorin

    PubMed Central

    Lala, Peeyush K.; Nandi, Pinki

    2016-01-01

    abstract The objective of the present review is to synthesize the information on the cellular and molecular players responsible for maintaining a homeostatic balance between a naturally invasive human placenta and the maternal uterus in pregnancy; to review the roles of decorin (DCN) as a molecular player in this homeostasis; to list the common maladies associated with a break-down in this homeostasis, resulting from a hypo-invasive or hyper-invasive placenta, and their underlying mechanisms. We show that both the fetal components of the placenta, represented primarily by the extravillous trophoblast, and the maternal component represented primarily by the decidual tissue and the endometrial arterioles, participate actively in this balance. We discuss the process of uterine angiogenesis in the context of uterine arterial changes during normal pregnancy and preeclampsia. We compare and contrast trophoblast growth and invasion with the processes involved in tumorigenesis with special emphasis on the roles of DCN and raise important questions that remain to be addressed. Decorin (DCN) is a small leucine-rich proteoglycan produced by stromal cells, including dermal fibroblasts, chondrocytes, chorionic villus mesenchymal cells and decidual cells of the pregnant endometrium. It contains a 40 kDa protein core having 10 leucine-rich repeats covalently linked with a glycosaminoglycan chain. Biological functions of DCN include: collagen assembly, myogenesis, tissue repair and regulation of cell adhesion and migration by binding to ECM molecules or antagonising multiple tyrosine kinase receptors (TKR) including EGFR, IGF-IR, HGFR and VEGFR-2. DCN restrains angiogenesis by binding to thrombospondin-1, TGFβ, VEGFR-2 and possibly IGF-IR. DCN can halt tumor growth by antagonising oncogenic TKRs and restraining angiogenesis. DCN actions at the fetal-maternal interface include restraint of trophoblast migration, invasion and uterine angiogenesis. We demonstrate that DCN

  16. Mechanisms of trophoblast migration, endometrial angiogenesis in preeclampsia: The role of decorin.

    PubMed

    Lala, Peeyush K; Nandi, Pinki

    2016-03-03

    The objective of the present review is to synthesize the information on the cellular and molecular players responsible for maintaining a homeostatic balance between a naturally invasive human placenta and the maternal uterus in pregnancy; to review the roles of decorin (DCN) as a molecular player in this homeostasis; to list the common maladies associated with a break-down in this homeostasis, resulting from a hypo-invasive or hyper-invasive placenta, and their underlying mechanisms. We show that both the fetal components of the placenta, represented primarily by the extravillous trophoblast, and the maternal component represented primarily by the decidual tissue and the endometrial arterioles, participate actively in this balance. We discuss the process of uterine angiogenesis in the context of uterine arterial changes during normal pregnancy and preeclampsia. We compare and contrast trophoblast growth and invasion with the processes involved in tumorigenesis with special emphasis on the roles of DCN and raise important questions that remain to be addressed. Decorin (DCN) is a small leucine-rich proteoglycan produced by stromal cells, including dermal fibroblasts, chondrocytes, chorionic villus mesenchymal cells and decidual cells of the pregnant endometrium. It contains a 40 kDa protein core having 10 leucine-rich repeats covalently linked with a glycosaminoglycan chain. Biological functions of DCN include: collagen assembly, myogenesis, tissue repair and regulation of cell adhesion and migration by binding to ECM molecules or antagonising multiple tyrosine kinase receptors (TKR) including EGFR, IGF-IR, HGFR and VEGFR-2. DCN restrains angiogenesis by binding to thrombospondin-1, TGFβ, VEGFR-2 and possibly IGF-IR. DCN can halt tumor growth by antagonising oncogenic TKRs and restraining angiogenesis. DCN actions at the fetal-maternal interface include restraint of trophoblast migration, invasion and uterine angiogenesis. We demonstrate that DCN overexpression in

  17. Estradiol-17β increases 12- and 15-lipoxygenase (type2) expression and activity and reactive oxygen species in human umbilical vascular smooth muscle cells.

    PubMed

    Somjen, Dalia; Kohen, Fortune; Limor, Rona; Sharon, Orli; Knoll, Esther; Many, Ariel; Stern, Naftali

    2016-10-01

    The net vascular effect of estrogens on the vasculature is still under debate. Here we tested the effects of estradiol- 17β (E2) as well as estrogen-receptor subtype specific and non-specific agonists and antagonists on the expression and eicosanoid production of lipoxygenase (LO) enzymes expressed in culture human umbilical vascular smooth muscle cells (VSMC), the platelet type 12LO and 15LO type 2. E2 increased 12 and 15LO mRNA expression by 2-3 folds and elicited an acute 50% increase 12 and 15 hydroxyeicosatetraenoic acid (HETE) production. Neither estrogen receptor ERα nor ERβ-specific agonists were able to reproduce the induction of LO expression, but E2-induced expression was effectively blocked by ER non-specific and receptor subtype specific antagonists. Because 12 and 15HETE can increase reactive oxygen species in other cell types, we tested the possibility that E2 could raise ROS through LO. Indeed, E2 as well as the LO products 12 and 15HETE increased reactive oxygen species (ROS) in VSMC. E2-dependent and HETE-induced ROS could be blocked by NAD (P) H-oxidase inhibitors and by the ER general antagonist ICI. E2-induced ROS was partially (∼50%) blocked by the LO inhibitor baicalein, but the LO blocker had no effect on 12 or 15HETE- induced ROS formation, thus suggesting that part of E2-dependent ROS generation resulted from E2-induced 12 and 15HETE. Collectively these findings unveil an unrecognized effect of E2 in human VSMC, to induce 12 and 15LO type 2 expression and activity and suggest that E2-dependent ROS formation in VSMC may be partially mediated by the induction of 12 and 15HETE. Copyright © 2016 Elsevier Ltd. All rights reserved.

  18. Lysosomal Function Is Involved in 17β-Estradiol-Induced Estrogen Receptor α Degradation and Cell Proliferation

    PubMed Central

    Marino, Maria; Acconcia, Filippo

    2014-01-01

    The homeostatic control of the cellular proteome steady-state is dependent either on the 26S proteasome activity or on the lysosome function. The sex hormone 17β-estradiol (E2) controls a plethora of biological functions by binding to the estrogen receptor α (ERα), which is both a nuclear ligand-activated transcription factor and also an extrinsic plasma membrane receptor. Regulation of E2-induced physiological functions (e.g., cell proliferation) requires the synergistic activation of both transcription of estrogen responsive element (ERE)-containing genes and rapid extra-nuclear phosphorylation of many different signalling kinases (e.g., ERK/MAPK; PI3K/AKT). Although E2 controls ERα intracellular content and activity via the 26S proteasome-mediated degradation, biochemical and microscopy-based evidence suggests a possible cross-talk among lysosomes and ERα activities. Here, we studied the putative localization of endogenous ERα to lysosomes and the role played by lysosomal function in ERα signalling. By using confocal microscopy and biochemical assays, we report that ERα localizes to lysosomes and to endosomes in an E2-dependent manner. Moreover, the inhibition of lysosomal function obtained by chloroquine demonstrates that, in addition to 26S proteasome-mediated receptor elimination, lysosome-based degradation also contributes to the E2-dependent ERα breakdown. Remarkably, the lysosome function is further involved in those ERα activities required for E2-dependent cell proliferation while it is dispensable for ERα-mediated ERE-containing gene transcription. Our discoveries reveal a novel lysosome-dependent degradation pathway for ERα and show a novel biological mechanism by which E2 regulates ERα cellular content and, as a consequence, cellular functions. PMID:24736371

  19. The yin and yang of cannabis-induced psychosis: the actions of Δ(9)-tetrahydrocannabinol and cannabidiol in rodent models of schizophrenia.

    PubMed

    Arnold, J C; Boucher, A A; Karl, T

    2012-01-01

    The link between cannabis and psychosis has often been debated with polarized views on the topic. There is substantial epidemiological evidence showing that cannabis increases the risk of psychosis, whereas other research suggests that schizophrenia patients self-medicate with the substance. These conflicting accounts may at least be partially explained by the two phytocannabinoids cannabidiol (CBD) and Δ(9)-tetrahydrocannabinol (THC) and their opposing actions on schizophrenia-related symptoms. In the present review we will first focus on how traditional rodent models of schizophrenia have been used to improve our understanding of the propsychotic actions of THC and the antipsychotic actions of CBD. We will also review novel rodent models used to address genetic vulnerability to cannabis-induced schizophrenia and show that specific genes are being uncovered that modulate cannabinoid action (e.g. the schizophrenia susceptibility gene neuregulin 1). We will also review rodent studies that have addressed interactions between THC and CBD. These animal studies underscore great complexity with some studies showing that CBD antagonises the neurobehavioural effects of THC, while others show the opposite, that CBD potentiates the actions of THC. Various mechanisms are put forth to explain these divergent effects such as CBD antagonism at central CB1 receptors or that CBD inhibits proteins that regulate THC disposition and metabolism (e.g. the ABC transporter, P-glycoprotein).

  20. A tale of two cannabinoids: the therapeutic rationale for combining tetrahydrocannabinol and cannabidiol.

    PubMed

    Russo, Ethan; Guy, Geoffrey W

    2006-01-01

    This study examines the current knowledge of physiological and clinical effects of tetrahydrocannabinol (THC) and cannabidiol (CBD) and presents a rationale for their combination in pharmaceutical preparations. Cannabinoid and vanilloid receptor effects as well as non-receptor mechanisms are explored, such as the capability of THC and CBD to act as anti-inflammatory substances independent of cyclo-oxygenase (COX) inhibition. CBD is demonstrated to antagonise some undesirable effects of THC including intoxication, sedation and tachycardia, while contributing analgesic, anti-emetic, and anti-carcinogenic properties in its own right. In modern clinical trials, this has permitted the administration of higher doses of THC, providing evidence for clinical efficacy and safety for cannabis based extracts in treatment of spasticity, central pain and lower urinary tract symptoms in multiple sclerosis, as well as sleep disturbances, peripheral neuropathic pain, brachial plexus avulsion symptoms, rheumatoid arthritis and intractable cancer pain. Prospects for future application of whole cannabis extracts in neuroprotection, drug dependency, and neoplastic disorders are further examined. The hypothesis that the combination of THC and CBD increases clinical efficacy while reducing adverse events is supported.

  1. Antagonistic activities of Klp10A and Orbit regulate spindle length, bipolarity and function in vivo.

    PubMed

    Laycock, Joseph E; Savoian, Matthew S; Glover, David M

    2006-06-01

    The metaphase-spindle steady-state length occurs as spindle microtubules ;flux', incorporating new subunits at their plus ends, while simultaneously losing subunits from their minus ends. Orbit/Mast/CLASP is required for tubulin subunit addition at kinetochores, and several kinesins regulate spindle morphology and/or flux by serving as microtubule depolymerases. Here, we use RNA interference in S2 cells to examine the relationship between Orbit and the four predicted kinesin-type depolymerases encoded by the Drosophila genome (Klp10A, Klp59C, Klp59D and Klp67A). Single depletion of Orbit results in monopolar spindles, mitotic arrest and a subsequent increase in apoptotic cells. These phenotypes are rescued by co-depleting Klp10A but none of the other three depolymerases. Spindle bipolarity is restored by preventing the spindle collapse seen in cells that lack Orbit, leading to functional spindles that are similar to controls in shape and length. We conclude that Klp10A exclusively antagonises Orbit in the regulation of bipolar spindle formation and maintenance.

  2. Identification of small molecule inhibitors of the Lin28-mediated blockage of pre-let-7g processing.

    PubMed

    Lightfoot, Helen L; Miska, Eric A; Balasubramanian, Shankar

    2016-11-02

    The protein Lin28 and microRNA let-7 play critical roles in mammalian development and human disease. Lin28 inhibits let-7 biogenesis through direct interaction with let-7 precursors (pre-let-7). Accumulating evidence in vitro and in vivo suggests this interaction plays a dominant role in embryonic stem cell self-renewal and tumorigenesis. Thus the Lin28-let-7 interaction might be an attractive drug target, if not for the well-known difficulties in targeting protein-RNA interactions with drugs. The identification and development of suitable probe molecules to further elucidate therapeutic potential, as well as mechanistic details of this pathway will be valuable. We report the development and application of a biophysical high-throughput screening assay for the identification of small molecule inhibitors of the Lin28-pre-let-7 interaction. A library of pharmacologically active small molecules was screened and several small molecule inhibitors were identified and biochemically validated. Of these four validated inhibitors, two compounds successfully restored processing of pre-let-7g in the presence of Lin28, validating the concept. Thus, we have identified examples of small molecule inhibitors of the interaction between Lin28 and pre-let-7. This study provides a proof of concept for small molecule inhibitors that antagonise the effects of Lin28 and enhance processing of let-7 miRNA.

  3. Functional assessment of neuronal cannabinoid receptors in the muscular layers of human ileum and colon.

    PubMed

    Manara, L; Croci, T; Guagnini, F; Rinaldi-Carmona, M; Maffrand, J P; Le Fur, G; Mukenge, S; Ferla, G

    2002-04-01

    The notion that specific receptors account for the ability of natural and synthetic cannabinoids to alter physiological functions, prompted this study aimed at assessing their functional presence in the human gut. The effects have been studied of cannabinoids and selective antagonists of their receptors on chemically or electrically evoked contractions in preparations of human intestinal smooth muscle in vitro. Atropine prevented the contractions of longitudinal and circular muscle strips of ileum and colon induced by carbachol or electrical field stimulation; tetrodotoxin abolished only the latter which suggests they do involve activation of cholinergic neurons. The synthetic cannabinoid (+)WIN 55,212-2 had no effect on carbachol contractions, but in a concentration-dependent fashion prevented those elicited by electrical field stimulation - which were insensitive to the putative endogenous cannabinoid anandamide - more potently in longitudinal than in circular strips. The selective CB1 receptor antagonist SR141716, which had no effect in the absence of (+)WIN 55,212-2, competitively antagonised its inhibition of electrical field stimulation contractions, unlike the selective CB2 antagonist SR144528. Cannabinoid CB1 receptors are functionally present in the human ileum and colon; their pharmacological activation apparently results in inhibition of excitatory cholinergic pathways subserving smooth muscle contraction.

  4. Novel interactions between the HTLV antisense proteins HBZ and APH-2 and the NFAR protein family: Implications for the HTLV lifecycles

    PubMed Central

    Murphy, Jane; Hall, William W.; Ratner, Lee; Sheehy, Noreen

    2016-01-01

    The human T-cell leukaemia virus type 1 and type 2 (HTLV-1/HTLV-2) antisense proteins HBZ and APH-2 play key roles in the HTLV lifecycles and persistence in the host. Nuclear Factors Associated with double-stranded RNA (NFAR) proteins NF90/110 function in the lifecycles of several viruses and participate in host innate immunity against infection and oncogenesis. Using GST pulldown and co-immunoprecipitation assays we demonstrate specific novel interactions between HBZ/APH-2 and NF90/110 and characterised the protein domains involved. Moreover we show that NF90/110 significantly enhance Tax mediated LTR activation, an effect that was abolished by HBZ but enhanced by APH-2. Additionally we found that HBZ and APH-2 modulate the promoter activity of survivin and are capable of antagonising NF110-mediated survivin activation. Thus interactions between HTLV antisense proteins and the NFAR protein family have an overall positive impact on HTLV infection. Hence NFARs may represent potential therapeutic targets in HTLV infected cells. PMID:27110706

  5. Novel interactions between the HTLV antisense proteins HBZ and APH-2 and the NFAR protein family: Implications for the HTLV lifecycles.

    PubMed

    Murphy, Jane; Hall, William W; Ratner, Lee; Sheehy, Noreen

    2016-07-01

    The human T-cell leukaemia virus type 1 and type 2 (HTLV-1/HTLV-2) antisense proteins HBZ and APH-2 play key roles in the HTLV lifecycles and persistence in the host. Nuclear Factors Associated with double-stranded RNA (NFAR) proteins NF90/110 function in the lifecycles of several viruses and participate in host innate immunity against infection and oncogenesis. Using GST pulldown and co-immunoprecipitation assays we demonstrate specific novel interactions between HBZ/APH-2 and NF90/110 and characterised the protein domains involved. Moreover we show that NF90/110 significantly enhance Tax mediated LTR activation, an effect that was abolished by HBZ but enhanced by APH-2. Additionally we found that HBZ and APH-2 modulate the promoter activity of survivin and are capable of antagonising NF110-mediated survivin activation. Thus interactions between HTLV antisense proteins and the NFAR protein family have an overall positive impact on HTLV infection. Hence NFARs may represent potential therapeutic targets in HTLV infected cells. Copyright © 2016 Elsevier Inc. All rights reserved.

  6. Suppression of TAK1 pathway by shear stress counteracts the inflammatory endothelial cell phenotype induced by oxidative stress and TGF-β1

    PubMed Central

    Lee, Ee Soo; Boldo, Llorenç Solé; Fernandez, Bernadette O.; Feelisch, Martin; Harmsen, Martin C.

    2017-01-01

    Endothelial dysfunction is characterised by aberrant redox signalling and an inflammatory phenotype. Shear stress antagonises endothelial dysfunction by increasing nitric oxide formation, activating anti-inflammatory pathways and suppressing inflammatory pathways. The TAK1 (MAP3K7) is a key mediator of inflammation and non-canonical TGF-β signalling. While the individual roles of TAK1, ERK5 (MAPK7) and TGF-β pathways in endothelial cell regulation are well characterised, an integrative understanding of the orchestration of these pathways and their crosstalk with the redox system under shear stress is lacking. We hypothesised that shear stress counteracts the inflammatory effects of oxidative stress and TGF-β1 on endothelial cells by restoring redox balance and repressing the TAK1 pathway. Using human umbilical vein endothelial cells, we here show that TGF-β1 aggravates oxidative stress-mediated inflammatory activation and that shear stress activates ERK5 signalling while attenuating TGF-β signalling. ERK5 activation restores redox balance, but fails to repress the inflammatory effect of TGF-β1 which is suppressed upon TAK1 inhibition. In conclusion, shear stress counteracts endothelial dysfunction by suppressing the pro-inflammatory non-canonical TGF-β pathway and by activating the ERK5 pathway which restores redox signalling. We propose that a pharmacological compound that abates TGF-β signalling and enhances ERK5 signalling may be useful to counteract endothelial dysfunction. PMID:28209993

  7. The effect of interleukin 17 and Toll-like receptor 2 on CD11b expression and apoptosis of neutrophils in zymosaninduced arthritis and paw oedema

    PubMed Central

    Milanova, Viktoriya; Ivanovska, Nina

    2014-01-01

    We investigated the effect of interleukin (IL)-17 on Ly6G+ cell apoptosis in zymosan-induced arthritis (ZIA) and oedema (ZIO). Zymosan injection at the ankle joint caused swelling and coincided with histological joint alterations and IL-17A expression in areas with cell infiltrates. Flow cytometry of blood demonstrated increased frequencies of Ly6G+CD11b+ cells and their decreased apoptosis in ZIA. Annexin V+ neutrophils had lower CD11b expression, unlike Annexin V− cells. Cell survival for 12 hours was affected neither by IL-17 nor by zymosan alone, while both stimuli diminished Annexin V+ cell frequencies and up-regulated CD11b on Annexin V− cells. Interleukin 17 antagonised to the effects of zymosan in 24-hour cultures. The administration of IL-17 in ZIO increased paw thickness, enlarged the blood Ly6G+ pool, elevated CD11b expression and decreased apoptosis. We suggest that altered neutrophil apoptosis in arthritis can be overcome by anti-IL-17 therapy combined with an inhibition of Toll-like receptor (TLR) 2 and CD11b signalling. PMID:26155114

  8. Transient receptor potential vanilloid-1-mediated calcium responses are inhibited by the alkylamine antihistamines dexbrompheniramine and chlorpheniramine.

    PubMed

    Sadofsky, Laura R; Campi, Barbara; Trevisani, Marcello; Compton, Steven J; Morice, Alyn H

    2008-12-01

    American guidelines, unlike European guidelines, support the use of antihistamines as a first line of treatment for some causes of chronic cough. Transient receptor potential vanilloid-1 (TRPV1) is an ion channel activated by the tussive agents capsaicin, resiniferatoxin, and protons. It is predominantly expressed by C-fiber and some Adelta -fiber sensory neurons and is thought to be a cough receptor. By measuring increases in intracellular calcium as an indicator of TRPV1 activation, the authors sought to determine whether antihistamines could antagonise TRPV1 permanently expressed in HEK and Pro5 cells and TRPV1 endogenously expressed in rat dorsal root ganglia neurons. In human TRPV1-expressing HEK cells (hTRPV1-HEK), diphenhydramine and fexofenadine failed to inhibit capsaicin-triggered calcium responses. However, both dexbrompheniramine and chlorpheniramine significantly inhibited capsaicin-evoked responses in hTRPV1-HEK. Dexbrompheniramine also inhibited activation of rat TRPV1 expressed in HEK and Pro5 cells, without interfering with TRPA1 and proteinase-activated receptor-2 (PAR(2)) activation. Finally, in rat dorsal root ganglia neuron preparations, dexbrompheniramine dose-dependently inhibited capsaicin-evoked calcium responses. Thus, the inhibition of TRPV1 activation by dexbrompheniramine may provide one potential mechanism whereby this antihistamine exerts its therapeutic effect in chronic cough.

  9. Identification of the Beer Component Hordenine as Food-Derived Dopamine D2 Receptor Agonist by Virtual Screening a 3D Compound Database

    NASA Astrophysics Data System (ADS)

    Sommer, Thomas; Hübner, Harald; El Kerdawy, Ahmed; Gmeiner, Peter; Pischetsrieder, Monika; Clark, Timothy

    2017-03-01

    The dopamine D2 receptor (D2R) is involved in food reward and compulsive food intake. The present study developed a virtual screening (VS) method to identify food components, which may modulate D2R signalling. In contrast to their common applications in drug discovery, VS methods are rarely applied for the discovery of bioactive food compounds. Here, databases were created that exclusively contain substances occurring in food and natural sources (about 13,000 different compounds in total) as the basis for combined pharmacophore searching, hit-list clustering and molecular docking into D2R homology models. From 17 compounds finally tested in radioligand assays to determine their binding affinities, seven were classified as hits (hit rate = 41%). Functional properties of the five most active compounds were further examined in β-arrestin recruitment and cAMP inhibition experiments. D2R-promoted G-protein activation was observed for hordenine, a constituent of barley and beer, with approximately identical ligand efficacy as dopamine (76%) and a Ki value of 13 μM. Moreover, hordenine antagonised D2-mediated β-arrestin recruitment indicating functional selectivity. Application of our databases provides new perspectives for the discovery of bioactive food constituents using VS methods. Based on its presence in beer, we suggest that hordenine significantly contributes to mood-elevating effects of beer.

  10. Target Acquired: Progress and Promise of Targeted Therapeutics in the Treatment of Prostate Cancer.

    PubMed

    Stuchbery, Ryan; Kurganovs, Natalie J; McCoy, Patrick J; Nelson, Colleen C; Hayes, Vanessa M; Corcoran, Niall M; Hovens, Christopher M

    2015-01-01

    Cancer is fundamentally a genomic disease caused by mutations or rearrangements in the DNA or epigenetic machinery of a patient. An emerging field in cancer treatment targets key aberrations arising from the mutational landscape of an individual patient's disease rather than employing a cancer-wide cytotoxic therapy approach. In prostate cancer in particular, where there is an observed variation in response to standard treatments between patients with disease of a similar pathological stage and grade, mutationdirected treatment may grow to be a viable tool for clinicians to tailor more effective treatments. This review will describe a number of mutations across multiple forms of cancer that have been successfully antagonised by targeted therapeutics including their identification, the development of targeted compounds to combat them and the development of resistance to these therapies. This review will continue to examine these same mutations in the treatment and management of prostate cancer; the prevalence of targetable mutations in prostate cancer, recent clinical trials of targeted-agents and the potential or limitations for their use.

  11. Selected Lactobacillus strains isolated from sugary and milk kefir reduce Salmonella infection of epithelial cells in vitro.

    PubMed

    Zavala, L; Golowczyc, M A; van Hoorde, K; Medrano, M; Huys, G; Vandamme, P; Abraham, A G

    2016-09-01

    The isolation of potentially probiotic strains and the subsequent study of their properties are very important steps to gain insight in the health benefits ascribed to sugary and milk kefir. The aim of the present study was to characterise fifteen Lactobacillus strains isolated from these beverages by determining some surface properties and their ability to antagonise enterocyte cell damage after Salmonella infection in vitro. Lactobacillus surface properties were determined by hydrophobicity, autoaggregation, and coaggregation assays with Salmonella. In addition, lactobacilli adhesion to Caco-2/TC-7 cells and the effect on Salmonella invasion were evaluated. Finally, the disassembly of F-actin cytoskeleton on intestinal epithelial cells was assayed in vitro when Salmonella infection was performed in the presence of selected Lactobacillus strains. Ten out of the 15 strains showed a high adhesion capacity to Caco-2/TC-7 cells. Most of the strains were hydrophilic and non-autoaggregating. Strains isolated from sugary kefir were non-coaggregating with Salmonella, while strains Lactobacillus paracasei CIDCA 83120, 83121, 83123, 83124, 8339, 83102 isolated from milk kefir were able to coaggregate after 1 h. L. paracasei CIDCA 8339 and Lactobacillus kefiri CIDCA 83102 were able to diminish Salmonella invasion to the enterocytes. An antagonistic effect on cytoskeleton disruption elicited by the pathogen was also demonstrated. Our results suggest that both strains isolated from milk kefir could be considered as appropriate probiotic candidates.

  12. First isolation and antinociceptive activity of a lipid transfer protein from noni (Morinda citrifolia) seeds.

    PubMed

    Campos, Dyély C O; Costa, Andrea S; Lima, Amanda D R; Silva, Fredy D A; Lobo, Marina D P; Monteiro-Moreira, Ana Cristina O; Moreira, Renato A; Leal, Luzia K A M; Miron, Diogo; Vasconcelos, Ilka M; Oliveira, Hermógenes D

    2016-05-01

    In this study a novel heat-stable lipid transfer protein, designated McLTP1, was purified from noni (Morinda citrifolia L.) seeds, using four purification steps which resulted in a high-purified protein yield (72 mg McLTP1 from 100g of noni seeds). McLTP1 exhibited molecular masses of 9.450 and 9.466 kDa, determined by electrospray ionisation mass spectrometry. The N-terminal sequence of McLTP1 (AVPCGQVSSALSPCMSYLTGGGDDPEARCCAGV), as analysed by NCBI-BLAST database, revealed a high degree of identity with other reported plant lipid transfer proteins. In addition, this protein proved to be resistant to pepsin, trypsin and chymotrypsin digestion. McLTP1 given intraperitoneally (1, 2, 4 and 8 mg/kg) and orally (8 mg/kg) caused an inhibition of the writhing response induced by acetic acid in mice. This protein displayed thermostability, retaining 100% of its antinociceptive activity after 30 min incubation at 80 °C. Pretreatment of mice with McLTP1 (8 mg/kg, i.p. and p.o.) also decreased neurogenic and inflammatory phases of nociception in the formalin test. Naloxone (2 mg/kg, i.p.) antagonised the antinociceptive effect of McLTP1 suggesting that the opioid mechanisms mediate the analgesic properties of this protein.

  13. Arginine methylation-dependent reader-writer interplay governs growth control by E2F-1

    PubMed Central

    Zheng, Shunsheng; Moehlenbrink, Jutta; Lu, Yi-Chien; Zalmas, Lykourgos-Panagiotis; Sagum, Cari A.; Carr, Simon; McGouran, Joanna F.; Alexander, Leila; Fedorov, Oleg; Munro, Shonagh; Kessler, Benedikt; Bedford, Mark T.; Yu, Qiang; La Thangue, Nicholas B.

    2014-01-01

    Summary The mechanisms that underlie and dictate the different biological outcomes of E2F-1 activity have yet to be elucidated. We describe the residue-specific methylation of E2F-1 by the asymmetric dimethylating protein arginine methyltransferase (PRMT) 1 and symmetric dimethylating PRMT5, and relate the marks to different functional consequences of E2F-1 activity. Methylation by PRMT1 hinders methylation by PRMT5, which augments E2F-1-dependent apoptosis, whereas PRMT5-dependent methylation favours proliferation by antagonising methylation by PRMT1. The ability of E2F-1 to prompt apoptosis in DNA damaged cells coincides with enhanced PRMT1 methylation. In contrast, cyclin A binding to E2F-1 impedes PRMT1 methylation and augments PRMT5 methylation, thus ensuring that E2F-1 is locked into its cell cycle progression mode. The Tudor domain protein p100-TSN reads the symmetric methylation mark, and binding of p100-TSN down-regulates E2F-1 apoptotic activity. Our results define an exquisite level of precision in the reader-writer interplay that governs the biological outcome of E2F-1 activity. PMID:24076217

  14. Mechanism of multi-site phosphorylation from a ROCK-I:RhoE complex structure

    PubMed Central

    Komander, David; Garg, Ritu; Wan, Paul T C; Ridley, Anne J; Barford, David

    2008-01-01

    The ROCK-I serine/threonine protein kinase mediates the effects of RhoA to promote the formation of actin stress fibres and integrin-based focal adhesions. ROCK-I phosphorylates the unconventional G-protein RhoE on multiple N- and C-terminal sites. These phosphorylation events stabilise RhoE, which functions to antagonise RhoA-induced stress fibre assembly. Here, we provide a molecular explanation for multi-site phosphorylation of RhoE from the crystal structure of RhoE in complex with the ROCK-I kinase domain. RhoE interacts with the C-lobe αG helix of ROCK-I by means of a novel binding site remote from its effector region, positioning its N and C termini proximal to the ROCK-I catalytic site. Disruption of the ROCK-I:RhoE interface abolishes RhoE phosphorylation, but has no effect on the ability of RhoE to disassemble stress fibres. In contrast, mutation of the RhoE effector region attenuates RhoE-mediated disruption of the actin cytoskeleton, indicating that RhoE exerts its inhibitory effects on ROCK-I through protein(s) binding to its effector region. We propose that ROCK-I phosphorylation of RhoE forms part of a feedback loop to regulate RhoA signalling. PMID:18946488

  15. A proximal E-box modulates NGF effects on rat PPT-A promoter activity in cultured dorsal root ganglia neurones.

    PubMed

    Gerrard, Lesley; Howard, Mark; Paterson, Trevor; Thippeswamy, Thimmasettappa; Quinn, John P; Haddley, Kate

    2005-10-01

    The rat preprotachykinin A (rtPPTA) promoter fragment spanning -865+92, relative to the major transcriptional start, has previously been demonstrated to be nerve growth factor (NGF) responsive in primary cultures of rat dorsal root ganglion (DRG) neurones [Harrison, P.T., Dalziel, R.G., Ditchfield, N.A., Quinn, J.P., 1999. Neuronal-specific and nerve growth factor-inducible expression directed by the preprotachykinin-A promoter delivered by an adeno-associated virus vector. Neuroscience 94, 997-1003]. In this communication, we demonstrate that an E box element at -60, in part, regulates the activity of this rtPPT-A promoter fragment in DRG neurones in response to NGF. Differential regulation of the promoter is observed in the presence or absence of NGF when the E Box site is present. Under basal conditions binding of proteins to this -60 element may antagonise promoter activity. Hence, in the absence of NGF, mutation of the -60 E box increased reporter gene expression. Further, comparison of levels of reporter gene expression supported by both WT and mutated promoter indicate that in the presence of NGF the -60 E box element also plays a role as an activator domain. This represents a novel mechanism for NGF regulation of rtPPT-A. Similarly, an important role for this signalling pathway was observed in neonate rat DRG neuronal cultures, which require NGF for their survival, namely mutation of the -60 element resulted in higher levels of reporter gene expression.

  16. Time for tea: mood, blood pressure and cognitive performance effects of caffeine and theanine administered alone and together.

    PubMed

    Rogers, Peter J; Smith, Jessica E; Heatherley, Susan V; Pleydell-Pearce, C W

    2008-01-01

    Although both contain behaviourally significant concentrations of caffeine, tea is commonly perceived to be a less stimulating drink than coffee. At least part of the explanation for this may be that theanine, which is present in tea but not coffee, has relaxing effects. There is also some evidence that theanine affects cognitive performance, and it has been found to reduce blood pressure in hypertensive rats. To study the subjective, behavioural and blood pressure effects of theanine and caffeine administered alone and together, in doses relevant to the daily tea consumption of regular tea drinkers. In a randomised, double-blind, placebo-controlled study, healthy adult participants (n = 48) received either 250-mg caffeine, 200-mg theanine, both or neither of these. They completed ratings of mood, including anxiety, and alertness, and had their blood pressure measured before and starting 40 min after drug administration. Anxiety was also assessed using a visual probe task. Caffeine increased self-rated alertness and jitteriness and blood pressure. Theanine antagonised the effect of caffeine on blood pressure but did not significantly affect jitteriness, alertness or other aspects of mood. Theanine also slowed overall reaction time on the visual probe task. Theanine is a physiologically and behaviourally active compound and, while it is unclear how its effects might explain perceived differences between tea and coffee, evidence suggests that it may be useful for reducing raised blood pressure.

  17. The efficacy of novel B cell biologics as the future of SLE treatment: a review.

    PubMed

    Kamal, Ameer; Khamashta, Munther

    2014-11-01

    Systemic lupus erythematosus (SLE) is a chronic autoimmune inflammatory disease with wide ranging multi-systemic effects. Current understanding centralises B cells in SLE pathogenesis with clinical features resulting from autoantibody formation, immune complex deposition, antigen presentation and cytokine activation. Existing standard of care therapies generates adverse side effects; secondary to corticosteroid use and untargeted immunosuppression. The inability to uphold remission and abolish the disease process, in addition to the increasing numbers of patients seen with refractory disease with these therapies, has provoked the development of novel B cell biologics targeting specific pathogenic pathways fundamental to the SLE disease process. Current evidence highlighting the efficacy of Rituximab, Ocrelizumab and Epratuzumab in inducing B cell depletion and achieving disease amelioration through specific B cell surface receptor antagonism is discussed. We review the efficacy of Atacicept, Briobacept and Belimumab in antagonising B lymphocyte stimulator (BLyS) and A proliferation inducing ligand (APRIL), two stimulatory cytokines crucial to B cell survival, growth and function. Two large multicentre randomised controlled trials, BLISS-52 and BLISS-76, have led to FDA approval of Belimumab. Following this breakthrough, other anti-BLyS therapies, Blisibimod and Tabalumab, are currently under Phase III evaluation. Similarly, murine models and Phase I/II trials have demonstrated significant efficacy of Rituximab, Epratuzumab, Briobacept and Atacicept as potential future therapies and we now eagerly await results from Phase III trials. Future research must compare the efficacy of different biologics amongst different patient subpopulations and SLE manifestations, in order to develop clinically and cost effective therapies.

  18. Reversal by phenytoin of carbamazepine-induced water intoxication: a pharmacokinetic interaction.

    PubMed Central

    Perucca, E; Richens, A

    1980-01-01

    The hypothesis that phenytoin may antagonise the antidiuretic effect of carbamazepine has been examined by comparing the free water clearance response to a standard water load in 36 patients stabilised on different drug regimes. The diuretic response to the water load was significantly greater in patients receiving chronic treatment with carbamazepine and phenytoin in combination than in matched control subjects receiving carbamazepine as a single drug. Acute administration of phenytoin (1,100 mg), however, had no significant influence on carbamazepine-induced antidiuresis. Evidence is presented that reversal of the antidiuretic effect of carbamazepine by chronic phenytoin administration is secondary to a marked reduction of the serum carbamazepine concentration during combined therapy. These results suggest that the risk of developing water intoxication is greater in patients receiving carbamazepine alone than in those receiving phenytoin in combination. Since the antidiuretic effect is correlated with the serum carbamazepine concentration rather than with the prescribed daily dose, monitoring the serum level of the drug is likely to provide the best rational approach to the prevention of excessive water retention. PMID:7205298

  19. Actin dynamics tune the integrated stress response by regulating eukaryotic initiation factor 2α dephosphorylation

    PubMed Central

    Chambers, Joseph E; Dalton, Lucy E; Clarke, Hanna J; Malzer, Elke; Dominicus, Caia S; Patel, Vruti; Moorhead, Greg; Ron, David; Marciniak, Stefan J

    2015-01-01

    Four stress-sensing kinases phosphorylate the alpha subunit of eukaryotic translation initiation factor 2 (eIF2α) to activate the integrated stress response (ISR). In animals, the ISR is antagonised by selective eIF2α phosphatases comprising a catalytic protein phosphatase 1 (PP1) subunit in complex with a PPP1R15-type regulatory subunit. An unbiased search for additional conserved components of the PPP1R15-PP1 phosphatase identified monomeric G-actin. Like PP1, G-actin associated with the functional core of PPP1R15 family members and G-actin depletion, by the marine toxin jasplakinolide, destabilised the endogenous PPP1R15A-PP1 complex. The abundance of the ternary PPP1R15-PP1-G-actin complex was responsive to global changes in the polymeric status of actin, as was its eIF2α-directed phosphatase activity, while localised G-actin depletion at sites enriched for PPP1R15 enhanced eIF2α phosphorylation and the downstream ISR. G-actin's role as a stabilizer of the PPP1R15-containing holophosphatase provides a mechanism for integrating signals regulating actin dynamics with stresses that trigger the ISR. DOI: http://dx.doi.org/10.7554/eLife.04872.001 PMID:25774599

  20. Modulatory effect of intestinal polyamines and trace amines on the spontaneous phasic contractions of the isolated ileum and colon rings of mice

    PubMed Central

    Sánchez, Manuel; Suárez, Lorena; Andrés, María Teresa; Flórez, Blanca Henar; Bordallo, Javier; Riestra, Sabino; Cantabrana, Begoña

    2017-01-01

    ABSTRACT Background: Gastrointestinal motility modulatory factors include substances of the intestinal content, such as polyamines and trace amines (TAs), the focus of this study. Methods: The amines of food, intestinal content and from faecal bacteria of Swiss mice were determined by HPLC and functionally characterised in isolated distal ileum and medial colon rings. Results: Mouse food and intestinal content contain polyamines (spermidine>putrescine>spermine) and TAs (isoamylamine>cadaverine). Intestinal bacteria mainly produce putrescine and cadaverine. The amines inhibited the spontaneous motility of the ileum (0.1-3 mM) and colon rings (0.01-3 mM, with lower IC50), with: spermine~isoamylamine~spermidine. Spermine inhibition was tetrodotoxin (TTX)-insensitive, while isoamylamine was TTX-sensitive, suggesting neural control. Mainly in the ileum, isoamylamine (3 mM) elicited acute effects modified by TTX, atropine and propranolol, and suppressed by spermine (3 mM), not being localized at the smooth muscle level. The amines assayed (3 mM), except putrescine and cadaverine in the ileum and isoamylamine in the colon, antagonised acetylcholine (ACh, 0.1 mM)-elicited phasic contractions. Isoamylamine and spermine in colon relaxed KCl (100 mM)-elicited tonic contractions, suggesting an effect on smooth muscle, but did not justify the suppression of motility caused by spermine and isoamylamine. Conclusions: Polyamines and TAs of the intestinal content might act on chemosensors and modulate intestinal peristalsis. PMID:28659731

  1. Neuropharmacology of 5-hydroxytryptamine

    PubMed Central

    Richard Green, A

    2006-01-01

    This review outlines the history of our knowledge of the neuropharmacology of 5-hydroxytryptamine (5-HT; serotonin), focusing primarily on the work of U.K. scientists. The existence of a vasoconstrictive substance in the blood has been known for over 135 years. The substance was named serotonin and finally identified as 5-HT in 1949. The presence of 5-HT in the brain was reported by Gaddum in 1954 and it was Gaddum who also demonstrated that the action of 5-HT (in the gut) was antagonised by the potent hallucinogen lysergic acid diethylamide. This provoked the notion that 5-HT played a pivotal role in the control of mood and subsequent investigations have generally confirmed this hypothesis. Over the last 50 years a good understanding has been gained of the mechanisms involved in control of the storage, synthesis and degradation of 5-HT in the brain. Knowledge has also been gained on control of the functional activity of this monoamine, often by the use of behavioural models. A considerable literature also now exists on the mechanisms by which many of the drugs used to treat psychiatric illness alter the functional activity of 5-HT, particularly the drugs used to treat depression. Over the last 20 years the number of identified 5-HT receptor subtypes has increased from 2 to 14, or possibly more. A major challenge now is to utilise this knowledge to develop receptor-specific drugs and use the information gained to better treat central nervous system disorders. PMID:16402098

  2. Mitochondrial mechanisms of endothelial dysfunction.

    PubMed

    Szewczyk, Adam; Jarmuszkiewicz, Wieslawa; Koziel, Agnieszka; Sobieraj, Izabela; Nobik, Wioletta; Lukasiak, Agnieszka; Skup, Agata; Bednarczyk, Piotr; Drabarek, Beata; Dymkowska, Dorota; Wrzosek, Antoni; Zablocki, Krzysztof

    2015-08-01

    Endothelial cells play an important physiological role in vascular homeostasis. They are also the first barrier that separates blood from deeper layers of blood vessels and extravascular tissues. Thus, they are exposed to various physiological blood components as well as challenged by pathological stimuli, which may exert harmful effects on the vascular system by stimulation of excessive generation of reactive oxygen species (ROS). The major sources of ROS are NADPH oxidase and mitochondrial respiratory chain complexes. Modulation of mitochondrial energy metabolism in endothelial cells is thought to be a promising target for therapy in various cardiovascular diseases. Uncoupling protein 2 (UCP2) is a regulator of mitochondrial ROS generation and can antagonise oxidative stress-induced endothelial dysfunction. Several studies have revealed the important role of UCP2 in hyperglycaemia-induced modifications of mitochondrial function in endothelial cells. Additionally, potassium fluxes through the inner mitochondrial membrane, which are involved in ROS synthesis, affect the mitochondrial volume and change both the mitochondrial membrane potential and the transport of calcium into the mitochondria. In this review, we concentrate on the mitochondrial role in the cytoprotection phenomena of endothelial cells. Copyright © 2015 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.

  3. Inhibition of NR2B-Containing N-methyl-D-Aspartate Receptors (NMDARs) in Experimental Autoimmune Encephalomyelitis, a Model of Multiple Sclerosis

    PubMed Central

    Farjam, Mojtaba; Beigi Zarandi, Faegheh Baha'addini; Farjadian, Shirin; Geramizadeh, Bita; Nikseresht, Ali Reza; Panjehshahin, Mohammad Reza

    2014-01-01

    Neurodegeneration is the pathophysiological basis for permanent neurological disabilities in multiple sclerosis (MS); thus neuroprotection is emerging as a therapeutic approach in MS research. Modulation of excitotoxicity by inhibition of NMDARs has been suggested for neuroprotection, but selective antagonisation of the NR2B subtype of these receptors, a subtype believed to play a more pivotal role in neurodegeneration, has not been tested in MS. In this study inhibition of NR2B-containing NMDAR was evaluated on the animal model of MS, experimental autoimmune encephalomyelitis (EAE). EAE induction was done using MOG in C57BL/6 mice. Therapeutic administration of different doses of highly selective NR2B-containing NMDAR inhibitor (RO25-6981) was compared with memantine (non-selective NMDAR antagonist) and vehicle. Neurological deficits in EAE animals were more efficiently decreased by selective inhibition of NR2B-containing NMDARs. Histological studies of the spinal cords also showed decreased inflammation, myelin degradation and neuro-axonal degeneration when RO25-6981was administered with higher doses. The effects were dose dependent. Regarding the role of NR2B-containing NMDARs in excitotoxicity, selective inhibition of these receptor subtypes seems to modulate the neurological disabilities and pathological changes in EAE. Further elucidation of the exact mechanism of action as well as more experimental studies can suggest NR2B-containing NMDAR inhibition as a potentially effective treatment strategy for slowing down the clinical deterioration of disability in MS. PMID:25237366

  4. Inhibition of NR2B-Containing N-methyl-D-Aspartate Receptors (NMDARs) in Experimental Autoimmune Encephalomyelitis, a Model of Multiple Sclerosis.

    PubMed

    Farjam, Mojtaba; Beigi Zarandi, Faegheh Baha'addini; Farjadian, Shirin; Geramizadeh, Bita; Nikseresht, Ali Reza; Panjehshahin, Mohammad Reza

    2014-01-01

    Neurodegeneration is the pathophysiological basis for permanent neurological disabilities in multiple sclerosis (MS); thus neuroprotection is emerging as a therapeutic approach in MS research. Modulation of excitotoxicity by inhibition of NMDARs has been suggested for neuroprotection, but selective antagonisation of the NR2B subtype of these receptors, a subtype believed to play a more pivotal role in neurodegeneration, has not been tested in MS. In this study inhibition of NR2B-containing NMDAR was evaluated on the animal model of MS, experimental autoimmune encephalomyelitis (EAE). EAE induction was done using MOG in C57BL/6 mice. Therapeutic administration of different doses of highly selective NR2B-containing NMDAR inhibitor (RO25-6981) was compared with memantine (non-selective NMDAR antagonist) and vehicle. Neurological deficits in EAE animals were more efficiently decreased by selective inhibition of NR2B-containing NMDARs. Histological studies of the spinal cords also showed decreased inflammation, myelin degradation and neuro-axonal degeneration when RO25-6981was administered with higher doses. The effects were dose dependent. Regarding the role of NR2B-containing NMDARs in excitotoxicity, selective inhibition of these receptor subtypes seems to modulate the neurological disabilities and pathological changes in EAE. Further elucidation of the exact mechanism of action as well as more experimental studies can suggest NR2B-containing NMDAR inhibition as a potentially effective treatment strategy for slowing down the clinical deterioration of disability in MS.

  5. Analgesia after feline ovariohysterectomy under midazolam-medetomidine-ketamine anaesthesia with buprenorphine or butorphanol, and carprofen or meloxicam: a prospective, randomised clinical trial.

    PubMed

    Polson, Sally; Taylor, Polly M; Yates, David

    2012-08-01

    One hundred female cats undergoing routine ovariohysterectomy under midazolam-medetomidine-ketamine anaesthesia were included in a blinded, randomised, prospective clinical study to compare postoperative analgesia produced by four analgesic drug combinations given preoperatively (n = 25 per group). A secondary aim was to assess the effects in kittens and pregnant animals. Buprenorphine 180 µg/m(2) or butorphanol 6 mg/m(2) were given with either carprofen 4 mg/kg (groups BUPC and BUTC, respectively) or meloxicam 0.3 mg/kg (groups BUPM or BUTM, respectively). Medetomidine was not antagonised. A simple, descriptive scale (SDS; 0-4), a dynamic and interactive visual analogue scale (DIVAS; 0-100 mm) and mechanical nociceptive thresholds (MT; 2.5-mm diameter probe) were used to evaluate postoperative pain. All pain scores were low (DIVAS <10 mm, SDS <2 and MT >10 N) and there were no significant differences between the groups. It was concluded that all protocols provided adequate analgesia and when used with midazolam-medetomidine-ketamine are effective for routine feline ovariohysterectomy.

  6. Contributions of peripheral and central opioid receptors to antinociception in rat muscle pain models.

    PubMed

    Sánchez, Eva Ma; Bagües, Ana; Martín, Ma Isabel

    2010-10-01

    Administration of hypertonic saline (HS) is an accepted model to study muscular pain. HS-induced nociceptive responses were tested in masseter, already described, and in two new pain models of spinally innervated muscles (gastrocnemius and triceps) developed in rats at our laboratory. HS administration in the masseter induced vigorous hindpaw shaking and in the gastrocnemius or triceps, paw withdrawal or flexing. Participation of the central and peripheral opioid receptors in HS-induced pain is compared in these muscles: masseter, innervated by trigeminal nerve, and gastrocnemius and triceps by spinal nerves. Morphine and loperamide were used to reveal peripheral and central components of opioid analgesia. Both agonists reduced HS-induced nociceptive behaviours in the masseter and were antagonised by the opioid antagonist naloxone and by naloxone methiodide, an opioid receptor antagonist that poorly penetrates the blood-brain barrier. Unexpectedly, in the gastrocnemius and triceps, morphine, but not loperamide, decreased the nociceptive behaviour and this effect was only reversed by naloxone. So, peripheral opioid receptors seem to participate in HS-induced masseter pain, whereas only central opioid receptors reduced the nociception in gastrocnemius and triceps. Our results suggest that the use of peripheral opioids can be more advantageous than central opioids for treatment of orofacial muscular pain.

  7. How do general anaesthetics work?

    NASA Astrophysics Data System (ADS)

    Antkowiak, Bernd

    2001-05-01

    Almost a century ago, Meyer and Overton discovered a linear relationship between the potency of anaesthetic agents to induce general anaesthesia and their ability to accumulate in olive oil. Similar correlations between anaesthetic potency and lipid solubility were later reported from investigations on various experimental model systems. However, exceptions to the Meyer-Overton correlation exist in all these systems, indicating that lipid solubility is an important, but not the sole determinant of anaesthetic action. In the mammalian central nervous system, most general anaesthetics act at multiple molecular sites. It seems likely that not all of these effects are involved in anaesthesia. GABAA- and NMDA-receptor/ion channels have already been identified as relevant targets. However, further mechanisms, such as a blockade of Na+ channels and an activation of K+ channels, also come into play. A comparison of different anaesthetics seems to show that each compound has its own spectrum of molecular actions and thus shows specific, fingerprint-like effects on different levels of neuronal activity. This may explain why there is no known compound that specifically antagonises general anaesthesia. General anaesthesia is a multidimensional phenomenon. Unconsciousness, amnesia, analgesia, loss of sensory processing and the depression of spinal motor reflexes are important components. It was not realised until very recently that different molecular mechanisms might underlie these different components. These findings challenge traditional views, such as the assumption that one anaesthetic can be freely replaced by another.

  8. Quantitative electrocortical changes in the rat induced by phencyclidine and other stimulants.

    PubMed

    Neal, H; Bond, A

    1985-04-01

    An automated technique for the continuous analysis of different frequency bands of the electrocorticogram (ECoG) of the rat has been used to quantify the actions of phencyclidine (PCP) and various other stimulant drugs. It has been demonstrated that phencyclidine, etoxadrol and LY154045 produced similar changes in the individual frequency bands whereas amphetamine and apomorphine had different profiles of activity. The phencyclidine-like compounds exhibited extremely strong stimulation of the ECoG with very large increases recorded in high frequency (15-50 Hz) activity and reductions in all other frequency bands. Various compounds have been used in an attempt to antagonise the changes in the ECoG. Chlorpromazine caused a slight shift in the dose-response curves as did chlordiazepoxide when used with phencyclidine. The GABA agonists, THIP and muscimol, had no effect on the stimulation of the ECoG. In contrast another presumed GABA agonist, baclofen, proved to be the most effective agent for blocking the stimulation induced by phencyclidine. The role of the GABAB receptor in the action of phencyclidine is discussed.

  9. Sex-change chemicals and their influence on the brain.

    PubMed

    Harris, C A

    2001-11-16

    The potential for man-made chemicals to mimic or antagonise natural hormones is a controversial issue, but one for which increasing amounts of evidence are being gathered worldwide. The controversy surrounds not so much the matter of whether these chemicals can mimic hormones in vitro--this phenomenon has been widely accepted in the scientific world - but more whether, as a result, they can disrupt reproduction in a wildlife situation. It has, nevertheless, been acknowledged that many wildlife populations are exhibiting reproductive and/or developmental abnormalities such as intersex gonads in wild roach populations in the U.K. and various reproductive disorders in alligators in Lake Apopka, Florida. However, the causative agents for many of these effects are difficult to specify, due to the extensive mixtures of chemicals--each of which may act via different pathways--to which wild populations are exposed, together with the wide variability observed even in natural (uncontaminated) habitats. As a result, any information detailing fundamental mechanism of action of the so-called endocrine disrupting chemicals (EDCs) is of use in determining whether or not these chemicals, as they are present in the environment, may in fact be capable of causing some of the effects observed in wildlife over recent years.

  10. Oestrogen receptors β1 and βcx have divergent roles in breast cancer survival and lymph node metastasis

    PubMed Central

    Rosin, G; de Boniface, J; Karthik, G M; Frisell, J; Bergh, J; Hartman, J

    2014-01-01

    Background: The expression of oestrogen receptor (ER) α characterises a subset of breast cancers associated with good response to endocrine therapy. However, the clinical significance of the second ER, ERβ1, and its splice variant ERβcx is still unclear. Methods: We here report an assessment of ERα, ERβ1 and ERβcx by immunohistochemistry using quantitative digital image analysis of 340 primary tumours and corresponding sentinel lymph nodes. Results: No differences were seen in ER levels in primary tumours vs lymph node metastases. ERβ1 and ERβcx were equally distributed among age groups and tumour histological grades. Loss of ERβ1 in the primary tumour was strongly associated with poor survival. Its prognostic impact was particularly evident in young patients and in high-grade tumours. The worst outcome was seen in the tumours lacking both ERα and ERβ1. ERβcx expression in the primary tumour correlated with a higher risk of lymph node metastasis, and with poor survival when expressed in sentinel node lymphocytes. Conclusions: Our study reveals highly significant although antagonising roles of ERβ1 and ERβcx in breast cancer. Consequently, we suggest that the histopathological assessment of ERβ1 is of value as a prognostic and potentially predictive biomarker. PMID:25025959

  11. Being, belonging and bestowing: differing degrees of community involvement amongst rural elders in England and Wales.

    PubMed

    Curry, Nigel; Fisher, Rhiannon

    2013-12-01

    Government exhortations for full community involvement in their locales in England and Wales have particular significance for rural elders as the literature suggests that they are amongst the most active in this sphere. But different people have different propensities to 'join in'. A taxonomy of different degrees of involvement in community is derived: antagonising, absenting, being, belonging, bestowing and communing. Being, belonging and bestowing are explored empirically, because they are the elements of principal concern to policy, making use of surveys of rural elders in six locations in England and Wales. A minority of respondents undertook bestowing activity (actively assisting others or taking part in voluntary activity) but most felt there were benefits from being (just living in a place) or belonging (having an identity with place) to a community. Of those who did not join in, most claimed that this was because they did not want to. Education and qualifications had the strongest association with 'joining in', although income and wealth per se had little impact. Two lessons for government community policy are important. First, many older people do not wish to 'join in', and second, the variables that tended to be associated with participation may not be that easy to influence through community policy.

  12. Shedding (far-red) light on phytochrome mechanisms and responses in land plants.

    PubMed

    Possart, Anja; Fleck, Christian; Hiltbrunner, Andreas

    2014-03-01

    In order to monitor ambient light conditions, plants rely on functionally diversified photoreceptors. Among these, phytochromes perceive red (R) and far-red (FR) light. FR light does not constitute a photosynthetic energy source; it however influences adaptive and developmental processes. In seed plants, phytochrome A (phyA) acts as FR receptor and mediates FR high irradiance responses (FR-HIRs). It exerts a dual role by promoting e.g. germination and seedling de-etiolation in canopy shade and by antagonising shade avoidance growth. Even though cryptogam plants such as mosses and ferns do not have phyA, they show FR-induced responses. In the present review we discuss the mechanistic basis of phyA-dependent FR-HIRs as well as their dual role in seed plants. We compare FR responses in seed plants and cryptogam plants and conclude on different potential concepts for the detection of canopy shade. Scenarios for the evolution of FR perception and responses are discussed.

  13. Valerian extract Ze 911 inhibits postsynaptic potentials by activation of adenosine A1 receptors in rat cortical neurons.

    PubMed

    Vissiennon, Z; Sichardt, K; Koetter, U; Brattström, A; Nieber, K

    2006-06-01

    In this study we evaluated the adenosine A1 receptor-mediated effect of valerian extract (Ze 911) on postsynaptic potentials (PSPs) in pyramidal cells of the rat cingulate cortex in a slice preparation. We first observed that N6-cyclopentyladenosine (CPA, 0.01 - 10 microM), an adenosine A1 receptor agonist, inhibited PSPs in a concentration-dependent manner. The CPA (10 microM)-induced inhibition was antagonized by 1,3-dipropyl-8-cyclopentylxanthine (DPCPX, 0.1 microM), an adenosine A1 receptor antagonist. Ze 911 concentration dependently (0.1 - 15 mg/mL) inhibited PSPs in the presence of the adenosine A2A receptor antagonist 1,3,7-trimethyl-8-(3-chlorostyryl)xanthine (CSC, 0.2 microM) and adenosine deaminase (1 U/mL). The maximal inhibition induced by 10 mg/mL was completely antagonised by DPCPX (0.1 microM), an A1 receptor blocker. The data suggest that activation of adenosine A1 receptors is involved in the pharmacological effects of the valerian extract Ze 911.

  14. Glucocorticoid pretreatment increases toxicity due to peroxides in alveolar epithelial-like cell lines.

    PubMed

    Walther, Udo I; Stets, Regine

    2009-02-04

    In previous experiments an increase in zinc-mediated toxicity was found after pretreatment of alveolar epithelial type II-like cells with glucocorticoids. In this work toxicity of two peroxides (tertiary butyl hydroperoxide [tBHP], hydrogene peroxide [HP]) was assessed in L2 and A549 cells compared to dexamethasone (DEX) pretreated cells. Pretreatment of cells with 7.5micromol/l DEX for 72h decreased cellular glutathione content in both cell lines. Furthermore compared to not pretreated cells toxicity of both peroxides was increased in A549 cells, while in L2 cells only toxicity of tBHP was significantly increased by the glucocorticoid pretreatment. HP toxicity only showed a tendency to be increased in L2 cells after DEX pretreatment. The results point to a glucocorticoid-dependent increased oxidative stress of alveolar epithelial type II cells as antagonised by antioxidative enzymes such as catalase and/or preferentially by the glutathione system. This furthermore should be considered for all glucocorticoid applications in vivo as well.

  15. Interaction between μ-opioid and 5-HT1A receptors in the regulation of panic-related defensive responses in the rat dorsal periaqueductal grey.

    PubMed

    Rangel, Marcel P; Zangrossi, Hélio; Roncon, Camila M; Graeff, Frederico G; Audi, Elisabeth A

    2014-12-01

    A wealth of evidence indicates that the activation of 5-HT1A and 5-HT2A receptors in the dorsal periaqueductal grey matter (dPAG) inhibits escape, a panic-related defensive behaviour. Results that were previously obtained with the elevated T-maze test of anxiety/panic suggest that 5-HT1A and μ-opioid receptors in this midbrain area work together to regulate this response. To investigate the generality of this finding, we assessed whether the same cooperative mechanism is engaged when escape is evoked by a different aversive stimulus electrical stimulation of the dPAG. Administration of the μ-receptor blocker CTOP into the dPAG did not change the escape threshold, but microinjection of the μ-receptor agonist DAMGO (0.3 and 0.5 nmol) or the 5-HT1A receptor agonist 8-OHDPAT (1.6 nmol) increased this index, indicating a panicolytic-like effect. Pretreatment with CTOP antagonised the anti-escape effect of 8-OHDPAT. Additionally, combined administration of subeffective doses of DAMGO and 8-OHDPAT increased the escape threshold, indicating drug synergism. Therefore, regardless of the aversive nature of the stimulus, μ-opioid and 5-HT1A receptors cooperatively act to regulate escape behaviour. A better comprehension of this mechanism might allow for new therapeutic strategies for panic disorder. © The Author(s) 2014.

  16. Structure of a stapled peptide antagonist bound to nutlin-resistant Mdm2.

    PubMed

    Chee, Sharon Min Qi; Wongsantichon, Jantana; Soo Tng, Quah; Robinson, Robert; Joseph, Thomas L; Verma, Chandra; Lane, David P; Brown, Christopher J; Ghadessy, Farid J

    2014-01-01

    As key negative regulator of the p53 tumour suppressor, Mdm2 is an attractive therapeutic target. Small molecules such as Nutlin have been developed to antagonise Mdm2, resulting in p53-dependent death of tumour cells. We have recently described a mutation in Mdm2 (M62A), which precludes binding of Nutlin, but not p53. This Nutlin-resistant variant is not, however, refractory to binding and inhibition by stapled peptide antagonists targeting the same region of Mdm2. A detailed understanding of how stapled peptides are recalcitrant to Mdm2 mutations conferring Nutlin-resistance will aid in the further development of potent Mdm2 antagonists. Here, we report the 2.00 Å crystal structure of a stapled peptide antagonist bound to Nutlin resistant Mdm2. The stapled peptide relies on an extended network of interactions along the hydrophobic binding cleft of Mdm2 for high affinity binding. Additionally, as seen in other stapled peptide structures, the hydrocarbon staple itself contributes to binding through favourable interactions with Mdm2. The structure highlights the intrinsic plasticity present in both Mdm2 and the hydrocarbon staple moiety, and can be used to guide future iterations of both small molecules and stapled peptides for improved antagonists of Mdm2.

  17. Effect of thalidomide on the skeletal muscle in experimental heart failure.

    PubMed

    Vescovo, Giorgio; Ravara, Barbara; Angelini, Annalisa; Sandri, Marco; Carraro, Ugo; Ceconi, Claudio; Dalla Libera, Luciano

    2002-08-01

    Tumour Necrosis Factor alpha (TNFalpha) has been shown to contribute to heart failure (CHF) progression. We have tried to antagonise the detrimental effects of TNFalpha on skeletal muscle apoptosis, by using thalidomide, a drug that inhibits its biosynthesis. CHF was induced in 20 rats by injecting monocrotaline, which determines right ventricle (RV) failure. After 2 weeks, when CHF developed, 12 rats were treated with thalidomide 3.5.mg/kg per day for 2 weeks. Eight had saline and served as CHF controls. Thalidomide failed to decrease TNFalpha and its second messenger sphingosine (SPH), but was able to prevent the shift toward the fast myosin heavy chains. In the Tibialis Anterior muscle of the thalidomide group, the degree of atrophy, the number of apoptotic nuclei and the levels of caspases, were similar to those of the CHF controls. Thalidomide, at the doses used in this study, which are the same employed for the treatment of tubercolosis, leprosy, AIDS and cancer in humans, did not lower either TNFalpha or SPH and only marginally influenced the apoptosis-induced muscle atrophy. Since other TNFalpha blockers are under investigation for improving the clinical status of patients with CHF, the present data could be relevant in the design of randomised clinical trials in humans.

  18. Identification of the Beer Component Hordenine as Food-Derived Dopamine D2 Receptor Agonist by Virtual Screening a 3D Compound Database

    PubMed Central

    Sommer, Thomas; Hübner, Harald; El Kerdawy, Ahmed; Gmeiner, Peter; Pischetsrieder, Monika; Clark, Timothy

    2017-01-01

    The dopamine D2 receptor (D2R) is involved in food reward and compulsive food intake. The present study developed a virtual screening (VS) method to identify food components, which may modulate D2R signalling. In contrast to their common applications in drug discovery, VS methods are rarely applied for the discovery of bioactive food compounds. Here, databases were created that exclusively contain substances occurring in food and natural sources (about 13,000 different compounds in total) as the basis for combined pharmacophore searching, hit-list clustering and molecular docking into D2R homology models. From 17 compounds finally tested in radioligand assays to determine their binding affinities, seven were classified as hits (hit rate = 41%). Functional properties of the five most active compounds were further examined in β-arrestin recruitment and cAMP inhibition experiments. D2R-promoted G-protein activation was observed for hordenine, a constituent of barley and beer, with approximately identical ligand efficacy as dopamine (76%) and a Ki value of 13 μM. Moreover, hordenine antagonised D2-mediated β-arrestin recruitment indicating functional selectivity. Application of our databases provides new perspectives for the discovery of bioactive food constituents using VS methods. Based on its presence in beer, we suggest that hordenine significantly contributes to mood-elevating effects of beer. PMID:28281694

  19. Role of activin-A in cigarette smoke-induced inflammation and COPD.

    PubMed

    Verhamme, Fien M; Bracke, Ken R; Amatngalim, Gimano D; Verleden, Geert M; Van Pottelberge, Geert R; Hiemstra, Pieter S; Joos, Guy F; Brusselle, Guy G

    2014-04-01

    Activin-A is a pleiotropic cytokine belonging to the transforming growth factor-β superfamily and has been implicated in asthma and pulmonary fibrosis. However, the role of activin-A and its endogenous inhibitor, follistatin, in the pathogenesis of chronic obstructive pulmonary disease (COPD) is unknown. We first quantified activin-A and follistatin in the lungs of air- or cigarette smoke-exposed mice and in the lungs of patients with COPD by immunohistochemistry, ELISA and quantitative real-time PCR. We subsequently studied the effect of cigarette smoke on primary human bronchial epithelial cells in vitro. Next, activin-A signalling was antagonised in vivo by administration of follistatin in mice exposed to air or cigarette smoke for 4 weeks. Protein levels of activin-A were increased in the airway epithelium of patients with COPD compared with never-smokers and smokers. Cigarette smoke-exposed human bronchial epithelial cells expressed higher levels of activin-A and lower levels of follistatin. Both mRNA and protein levels of activin-A were increased in the lungs of cigarette smoke-exposed mice, whereas follistatin levels were reduced upon cigarette smoke exposure. Importantly, administration of follistatin attenuated the cigarette smoke-induced increase of inflammatory cells and mediators in the bronchoalveolar lavage fluid in mice. These results suggest that an imbalance between activin-A and follistatin contributes to the pathogenesis of cigarette smoke-induced inflammation and COPD.

  20. Selective inhibition of miR-21 by phage display screened peptide

    PubMed Central

    Bose, Debojit; Nahar, Smita; Rai, Manish Kumar; Ray, Arjun; Chakraborty, Kausik; Maiti, Souvik

    2015-01-01

    miRNAs are nodal regulators of gene expression and deregulation of miRNAs is causally associated with different diseases, including cancer. Modulation of miRNA expression is thus of therapeutic importance. Small molecules are currently being explored for their potential to downregulate miRNAs. Peptides have shown to have better potency and selectivity toward their targets but their potential in targeting and modulating miRNAs remain unexplored. Herein, using phage display we found a very selective peptide against pre-miR-21. Interestingly, the peptide has the potential to downregulate miR-21, by binding to pre-miR-21 and hindering Dicer processing. It is selective towards miR-21 inside the cell. By antagonising miR-21 function, the peptide is able to increase the expression of its target proteins and thereby increase apoptosis and suppress cell proliferation, invasion and migration. This peptide can further be explored for its anti-cancer activity in vivo and may be even extended to clinical studies. PMID:25824952

  1. Control of mammalian germ cell entry into meiosis.

    PubMed

    Feng, Chun-Wei; Bowles, Josephine; Koopman, Peter

    2014-01-25

    Germ cells are unique in undergoing meiosis to generate oocytes and sperm. In mammals, meiosis onset is before birth in females, or at puberty in males, and recent studies have uncovered several regulatory steps involved in initiating meiosis in each sex. Evidence suggests that retinoic acid (RA) induces expression of the critical pre-meiosis gene Stra8 in germ cells of the fetal ovary, pubertal testis and adult testis. In the fetal testis, CYP26B1 degrades RA, while FGF9 further antagonises RA signalling to suppress meiosis. Failsafe mechanisms involving Nanos2 may further suppress meiosis in the fetal testis. Here, we draw together the growing knowledge relating to these meiotic control mechanisms, and present evidence that they are co-ordinately regulated and that additional factors remain to be identified. Understanding this regulatory network will illuminate not only how the foundations of mammalian reproduction are laid, but also how mis-regulation of these steps can result in infertility or germline tumours.

  2. Physiological impact of abnormal lipoxin A₄ production on cystic fibrosis airway epithelium and therapeutic potential.

    PubMed

    Higgins, Gerard; Ringholz, Fiona; Buchanan, Paul; McNally, Paul; Urbach, Valérie

    2015-01-01

    Lipoxin A4 has been described as a major signal for the resolution of inflammation and is abnormally produced in the lungs of patients with cystic fibrosis (CF). In CF, the loss of chloride transport caused by the mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel gene results in dehydration, mucus plugging, and reduction of the airway surface liquid layer (ASL) height which favour chronic lung infection and neutrophil based inflammation leading to progressive lung destruction and early death of people with CF. This review highlights the unique ability of LXA4 to restore airway surface hydration, to stimulate airway epithelial repair, and to antagonise the proinflammatory program of the CF airway, circumventing some of the most difficult aspects of CF pathophysiology. The report points out novel aspects of the cellular mechanism involved in the physiological response to LXA4, including release of ATP from airway epithelial cell via pannexin channel and subsequent activation of and P2Y11 purinoreceptor. Therefore, inadequate endogenous LXA4 biosynthesis reported in CF exacerbates the ion transport abnormality and defective mucociliary clearance, in addition to impairing the resolution of inflammation, thus amplifying the vicious circle of airway dehydration, chronic infection, and inflammation.

  3. MIT(1), a black mamba toxin with a new and highly potent activity on intestinal contraction.

    PubMed

    Schweitz, H; Pacaud, P; Diochot, S; Moinier, D; Lazdunski, M

    1999-11-19

    Mamba intestinal toxin (MIT(1)) isolated from Dendroaspis polylepis venom is a 81 amino acid polypeptide cross-linked by five disulphide bridges. MIT(1) has a very potent action on guinea-pig intestinal contractility. MIT(1) (1 nM) potently contracts longitudinal ileal muscle and distal colon, and this contraction is equivalent to that of 40 mM K(+). Conversely MIT(1) relaxes proximal colon again as potently as 40 mM K(+). The MIT(1)-induced effects are antagonised by tetrodotoxin (1 microM) in proximal and distal colon but not in longitudinal ileum. The MIT(1)-induced relaxation of the proximal colon is reversibly inhibited by the NO synthase inhibitor L-NAME (200 microM). (125)I-labelled MIT(1) binds with a very high affinity to both ileum and brain membranes (K(d)=1.3 pM and 0.9 pM, and B(max)=30 fmol/mg and 26 fmol/mg, respectively). MIT(1) is a very highly selective toxin for a receptor present both in the CNS and in the smooth muscle and which might be an as yet unidentified K(+) channel.

  4. Baclofen prevents drug-induced reinstatement of extinguished nicotine-seeking behaviour and nicotine place preference in rodents.

    PubMed

    Fattore, Liana; Spano, Maria Sabrina; Cossu, Gregorio; Scherma, Maria; Fratta, Walter; Fadda, Paola

    2009-07-01

    The gamma-aminobutyric acid(GABA)-B receptor agonist baclofen is known to reduce drug intake in both animals and humans and to prevent reinstatement of cocaine-, opioid-, and alcohol-seeking in rats after a period of extinction, but its effect on nicotine reinstatement is unknown. This study investigated the effect of baclofen on nicotine-seeking reinstatement both using the extinction/reinstatement model of nicotine self-administration and conditioned place preference (CPP). Results showed that in rats previously trained to intravenously self-administer nicotine (30 microg/kg/inf) under a FR-1 schedule of reinforcement, acute nicotine (0.15 mg/kg) priming effectively reinstates nicotine-seeking behaviour following extinction. At doses used in this study (up to 2.5 mg/kg) baclofen alone did not affect locomotor activity and did not reinstate responding. However, baclofen dose-dependently attenuated drug-induced reinstatement of nicotine-seeking in rats. Moreover, baclofen (1.25 mg/kg) completely blocked nicotine-induced reinstatement of extinguished nicotine (0.3 mg/kg) CPP in mice. Altogether, our results showed that baclofen is able to antagonise reinstatement of nicotine-seeking and CPP triggered by nicotine primings, suggesting its potential clinical utility as an anti-relapse agent.

  5. Interactions between GABA and 5-hydroxytryptamine in the guinea-pig ileum.

    PubMed

    Ong, J; Kerr, D I

    1983-10-28

    In isolated segments of the guinea-pig ileum, there was: (a) an early, short-lived (less than 20 s) depression by gamma-aminobutyric acid (GABA) of contractile responses to 5-hydroxytryptamine (5-HT), acetylcholine(ACh), or nicotine, also seen with 3-amino-1-propanesulphonic acid (3APS) or muscimol in place of GABA, and sensitive to bicuculline, picrotoxinin or piretanide, and (b) a delayed, longer-lasting (30 s-1 min) depression of responses to 5-HT and nicotine, but not exogenously applied ACh, also seen with baclofen and only antagonised by delta-aminovaleric acid (DAVA). At 25 degrees C, all these effects were still observed but slowed, whilst at 37 degrees C after cold storage (6 degrees C) overnight, the early, short-lived depression was reduced or eliminated, yet the delayed depression was enhanced. It is concluded that the early, short-lived depression is mediated through GABAA-receptor sites, and the delayed, longer-lasting depression through GABAB-receptor sites on neurones of the myenteric plexus; effects consistent with GABA being a neurotransmitter in the enteric nervous system.

  6. BH3 profiling and a toolkit of BH3-mimetic drugs predict anti-apoptotic dependence of cancer cells

    PubMed Central

    Butterworth, Michael; Pettitt, Andrew; Varadarajan, Shankar; Cohen, Gerald M

    2016-01-01

    Background: Anti-apoptotic BCL-2 family members antagonise apoptosis by sequestering their pro-apoptotic counterparts. The balance between the different BCL-2 family members forms the basis of BH3 profiling, a peptide-based technique used to predict chemosensitivity of cancer cells. Recent identification of cell-permeable, selective inhibitors of BCL-2, BCL-XL and MCL-1, further facilitates the determination of the BCL-2 family dependency of cancer cells. Methods: We use BH3 profiling in combination with cell death analyses using a chemical inhibitor toolkit to assess chemosensitivity of cancer cells. Results: Both BH3 profiling and the inhibitor toolkit effectively predict chemosensitivity of cells addicted to a single anti-apoptotic protein but a combination of both techniques is more instructive when cell survival depends on more than one anti-apoptotic protein. Conclusions: The inhibitor toolkit provides a rapid, inexpensive and simple means to assess the chemosensitivity of tumour cells and in conjunction with BH3 profiling offers much potential in personalising cancer therapy. PMID:26954718

  7. SPAR1/RTEL1 maintains genomic stability by suppressing homologous recombination

    PubMed Central

    Barber, Louise J.; Youds, Jillian L.; Ward, Jordan D.; McIlwraith, Michael J.; O’Neil, Nigel J.; Petalcorin, Mark I.R.; Martin, Julie S.; Collis, Spencer J.; Cantor, Sharon B.; Auclair, Melissa; Tissenbaum, Heidi; West, Stephen C.; Rose, Ann M.; Boulton, Simon J.

    2013-01-01

    SUMMARY Inappropriate homologous recombination (HR) can cause gross chromosomal rearrangements that in mammalian cells may lead to tumorigenesis. In yeast, the Srs2 protein is an anti-recombinase that eliminates inappropriate recombination events, but the functional equivalent of Srs2 in higher eukaryotes has proven to be elusive. In this work, we identify C. elegans SPAR-1 as a functional analogue of Srs2 and describe its vertebrate counterpart, SPAR1/RTEL1, which is required for genome stability and tumour avoidance. We find that spar-1 mutant worms and SPAR1 knockdown human cells share characteristic phenotypes with yeast srs2 mutants, including inviability upon deletion of the sgs1/BLM homologue, hyper-recombination, and DNA damage sensitivity. In vitro, purified human SPAR1 antagonises HR by promoting the disassembly of D loop recombination intermediates in a reaction dependent upon ATP hydrolysis. We propose that loss of HR control following deregulation of SPAR1/RTEL1 may be a critical event that drives genome instability and cancer. PMID:18957201

  8. Gastric pentadecapeptide BPC 157 counteracts morphine-induced analgesia in mice.

    PubMed

    Boban Blagaic, A; Turcic, P; Blagaic, V; Dubovecak, M; Jelovac, N; Zemba, M; Radic, B; Becejac, T; Stancic Rokotov, D; Sikiric, P

    2009-12-01

    Previously, the gastric pentadecapeptide BPC 157, (PL 14736, Pliva) has been shown to have several beneficial effects, it exert gastroprotective, anti-inflammatory actions, stimulates would healing and has therapeutic value in inflammatory bowel disease. The present study aimed to study the effect of naloxone and BPC 157 on morphine-induced antinociceptive action in hot plate test in the mouse. It was found that naloxone and BPC 157 counteracted the morphine (16 mg/kg s.c.) - analgesia. Naloxone (10 mg/kg s.c.) immediately antagonised the analgesic action and the reaction time returned to the basic values, the development of BPC 157-induced action (10 pg/kg, 10 ng/kg, 10 microg/kg i.p.) required 30 minutes. When haloperidol, a central dopamine-antagonist (1 mg/kg i.p.), enhanced morphine-analgesia, BPC 157 counteracted this enhancement and naloxone reestablished the basic values of pain reaction. BPC 157, naloxone, and haloperidol per se failed to exert analgesic action. In summary, interaction between dopamine-opioid systems was demonstrated in analgesia, BPC 157 counteracted the haloperidol-induced enhancement of the antinociceptive action of morphine, indicating that BPC acts mainly through the central dopaminergic system.

  9. Behavioral interactions between ethanol and imidazodiazepines with high affinities for benzodiazepine receptors

    SciTech Connect

    Lister, R.G.

    1988-01-01

    The intrinsic effect of two imidazodiazepines RO 15-3505 and RO 17-1812 on the behavior of mice in a holeboard test were investigated. The interactions of these two drugs with ethanol were also studied. RO 15-3505 failed to significantly alter either exploratory head-dipping or locomotor activity when administered alone but doses of 0.75 and 1.5 mg/kg reversed the reduction in the number of head-dips caused by ethanol and partially reversed ethanol's locomotor stimulant action. In contrast, RO 17-1812 increased locomotor activity when administered alone, and enhanced the reduction in exploration caused by ethanol. Neither RO 15-3505 nor RO 17-1812 altered blood alcohol concentrations suggesting a pharmacodynamic basis for these interactions. The results suggest that in the holeboard test the interactions of imidazodiazepines with ethanol are related to the nature of their interaction with benzodiazepine receptors, inverse agonists antagonising and agonists enhancing ethanol's effects on exploration.

  10. Epigenetics and Peripheral Artery Disease.

    PubMed

    Golledge, Jonathan; Biros, Erik; Bingley, John; Iyer, Vikram; Krishna, Smriti M

    2016-04-01

    The term epigenetics is usually used to describe inheritable changes in gene function which do not involve changes in the DNA sequence. These typically include non-coding RNAs, DNA methylation and histone modifications. Smoking and older age are recognised risk factors for peripheral artery diseases, such as occlusive lower limb artery disease and abdominal aortic aneurysm, and have been implicated in promoting epigenetic changes. This brief review describes studies that have associated epigenetic factors with peripheral artery diseases and investigations which have examined the effect of epigenetic modifications on the outcome of peripheral artery diseases in mouse models. Investigations have largely focused on microRNAs and have identified a number of circulating microRNAs associated with human peripheral artery diseases. Upregulating or antagonising a number of microRNAs has also been reported to limit aortic aneurysm development and hind limb ischemia in mouse models. The importance of DNA methylation and histone modifications in peripheral artery disease has been relatively little studied. Whether circulating microRNAs can be used to assist identification of patients with peripheral artery diseases and be modified in order to improve the outcome of peripheral artery disease will require further investigation.

  11. Arpin downregulation in breast cancer is associated with poor prognosis

    PubMed Central

    Lomakina, Maria E; Lallemand, François; Vacher, Sophie; Molinie, Nicolas; Dang, Irene; Cacheux, Wulfran; Chipysheva, Tamara A; Ermilova, Valeria D; de Koning, Leanne; Dubois, Thierry; Bièche, Ivan; Alexandrova, Antonina Y; Gautreau, Alexis

    2016-01-01

    Background: The Arp2/3 complex is required for cell migration and invasion. The Arp2/3 complex and its activators, such as the WAVE complex, are deregulated in diverse cancers. Here we investigate the expression of Arpin, the Arp2/3 inhibitory protein that antagonises the WAVE complex. Methods: We used qRT–PCR and reverse phase protein arrays in a patient cohort with known clinical parameters and outcome, immunofluorescence in breast biopsy cryosections and breast cancer cell lines. Results: Arpin was downregulated at the mRNA and protein levels in mammary carcinoma cells. Arpin mRNA downregulation was associated with poor metastasis-free survival (MFS) on univariate analysis (P=0.022). High expression of the NCKAP1 gene that encodes a WAVE complex subunit was also associated with poor MFS on univariate analysis (P=0.0037) and was mutually exclusive with Arpin low. Arpin low or NCKAP1 high was an independent prognosis factor on multivariate analysis (P=0.0012) and was strongly associated with poor MFS (P=0.000064). Conclusions: Loss of the Arp2/3 inhibitory protein Arpin produces a similar poor outcome in breast cancer as high expression of the NCKAP1 subunit of the Arp2/3 activatory WAVE complex. PMID:26867158

  12. Physiological and molecular characterization of compost bacteria antagonistic to soilborne plant pathogens.

    PubMed

    Mohamed, Rowida; Groulx, Emma; Defilippi, Stefanie; Erak, Tamara; Tambong, James T; Tweddell, Russell J; Tsopmo, Apollinaire; Avis, Tyler J

    2017-02-08

    Disease suppressive composts have the potential to mitigate the risks associated with chemical pesticides. One of the main characteristics responsible for the suppressive nature of composts is their microbiological populations. In order to gain insight into the determinants responsible for their suppressive effects, composts were assayed (i) to isolate and identify beneficial antagonistic bacteria, (ii) to quantify their antifungal and anti-oomycetal activities, (iii) to extract inhibitory compounds produced by the bacteria, and (iv) to identify antimicrobial lipopeptides produced by these bacteria. The antagonistic bacteria belonged to the Arthrobacter, Pseudomonas, Bacillus, Brevibacillus, Paenibacillus, and Rummeliibacillus genera and had the ability to antagonise the growth of Fusarium sambucinum, Verticillium dahliae, and/or Pythium sulcatum. These bacteria produced antimicrobial compounds that affected the mycelial growth and/or conidial germination of the pathogens. Mass spectrometry analyses showed the presence of various antimicrobial lipopeptides in Bacillus and Bacillus-related spp. extracts, demonstrating that they are, at least in part, responsible for the antagonistic activity of the bacteria. Results from this work provide greater insight into some of the biological, biochemical, and physiological determinants of suppressiveness in composts involved in the control of plant pathogens.

  13. The secretagogue effect of the poison from Centruroides limpidus limpidus on the pancreas of mice and the antagonistic action of the Bouvardia ternifolia extract.

    PubMed

    Jiménez-Ferrer, E; Reynosa-Zapata, I; Pérez-Torres, Y; Tortoriello, J

    2005-01-01

    In Mexican traditional medicine the plant species Bouvardia ternifolia is used as remedy to treat patients who have been stung by scorpions. In the preceding study, the methanol extract from the roots of this plant was capable of reducing the poisonous effect of Centruroides limpidus limpidus on mice. The poisoning from scorpion C. limpidus limpidus includes manifestations associated with the pancreatitis. This study evaluated the effect produced by the hexane and methanol extract from the root of B. ternifolia upon the acutely inflamed pancreas induced by the venom of C. limpidus limpidus on rats, and the release of amylase in the isolated pancreas of mice. The intravenous administration of venom induced the extravasation of labelled albumin, in a dose dependant manner. The pre-administration of both extracts of Bouvardia ternifolia reduced significantly (p < 0.05) the extravasation by 60%. Upon measuring the secretagogue effect of the venom in the isolated pancreas of mice, the EC50 of the venom was 3.76 x 10(-3) mg ml(-1), whilst in the presence of the methanol and hexane extracts, this EC50 was 9.13 x 10(-3) mg ml(-1) and 0.01629 mg ml(-1). In conclusion, the C. limpidus limpidus venom possesses a secretagogue effect of amylase on the pancreas of mice and produces an inflamed pancreas which is effectively antagonised by the hexane and methanol extracts from the roots of B. ternifolia.

  14. An in vitro investigation of Lactococcus lactis antagonizing cariogenic bacterium Streptococcus mutans.

    PubMed

    Tong, Zhongchun; Zhou, Lin; Li, Jie; Kuang, Rong; Lin, Yuan; Ni, Longxing

    2012-04-01

    The present study tested the antagonism between Lactococcus lactis and Streptococcus mutans and evaluated the feasibility of the application of L. lactis for the inhibition of S. mutans in the oral cavity. Competition assays on plates were employed to determine whether L. lactis antagonises S. mutans under different nutritional conditions, and real-time reverse-transcriptase PCR was used to evaluate the effects of metabolites of S. mutans on the bacteriocin nisin genes in L. lactis. Furthermore, the colonisation and effects of L. lactis on the surface of a tooth were examined by scanning electron microscopy. L. lactis competitively inhibited S. mutans growth under nutritional deficiency, and the metabolites of S. mutans, including several exogenous molecular signals, enhanced the expression of genes related to nisin synthesis, nisA, nisB and nisI. Additionally, L. lactis effectively colonised the surface of tooth enamel, which showed substantially less decay with L. lactis adhesion compared to S. mutans adhesion. These findings suggest avenues of research into a new strategy to reduce major cariogenic S. mutans adhesion on the surfaces of teeth. Copyright © 2011 Elsevier Ltd. All rights reserved.

  15. Induction of metamorphosis from the larval to the polyp stage is similar in Hydrozoa and a subgroup of Scyphozoa (Cnidaria, Semaeostomeae)

    NASA Astrophysics Data System (ADS)

    Siefker, Barbara; Kroiher, Michael; Berking, Stefan

    2000-12-01

    Larvae of cnidarians need an external cue for metamorphosis to start. The larvae of various hydrozoa, in particular of Hydractinia echinata, respond to Cs+, Li+, NH4 + and seawater in which the concentration of Mg2+ ions is reduced. They further respond to the phorbolester, tetradecanoyl-phorbol-13-acetate (TPA) and the diacylglycerol (DAG) diC8, which both are argued to stimulate a protein kinase C. The only well-studied scyphozoa, Cassiopea spp., respond differently, i.e. to TPA and diC8 only. We found that larvae of the scyphozoa Aurelia aurita, Chrysaora hysoscella and Cyanea lamarckii respond to all the compounds mentioned. Trigonelline ( N-methylnicotinic acid), a metamorphosis inhibitor found in Hydractinia larvae, is assumed to act by delivering a methyl group for transmethylation processes antagonising metamorphosis induction in Chrysaora hysoscella and Cyanea lamarckii. The three species tested are scyphozoa belonging to the subgroup of semaeostomeae, while Cassiopea spp. belong to the rhizostomeae. The results obtained may contribute to the discussion concerning the evolution of cnidarians and may help to clarify whether the way metamorphosis can be induced in rhizostomeae as a whole is different from that in hydrozoa and those scyphozoa belonging to the subgroup semaeostomeae.

  16. Modulatory effect of intestinal polyamines and trace amines on the spontaneous phasic contractions of the isolated ileum and colon rings of mice.

    PubMed

    Sánchez, Manuel; Suárez, Lorena; Andrés, María Teresa; Flórez, Blanca Henar; Bordallo, Javier; Riestra, Sabino; Cantabrana, Begoña

    2017-01-01

    Background: Gastrointestinal motility modulatory factors include substances of the intestinal content, such as polyamines and trace amines (TAs), the focus of this study. Methods: The amines of food, intestinal content and from faecal bacteria of Swiss mice were determined by HPLC and functionally characterised in isolated distal ileum and medial colon rings. Results: Mouse food and intestinal content contain polyamines (spermidine>putrescine>spermine) and TAs (isoamylamine>cadaverine). Intestinal bacteria mainly produce putrescine and cadaverine. The amines inhibited the spontaneous motility of the ileum (0.1-3 mM) and colon rings (0.01-3 mM, with lower IC50), with: spermine~isoamylamine~spermidine. Spermine inhibition was tetrodotoxin (TTX)-insensitive, while isoamylamine was TTX-sensitive, suggesting neural control. Mainly in the ileum, isoamylamine (3 mM) elicited acute effects modified by TTX, atropine and propranolol, and suppressed by spermine (3 mM), not being localized at the smooth muscle level. The amines assayed (3 mM), except putrescine and cadaverine in the ileum and isoamylamine in the colon, antagonised acetylcholine (ACh, 0.1 mM)-elicited phasic contractions. Isoamylamine and spermine in colon relaxed KCl (100 mM)-elicited tonic contractions, suggesting an effect on smooth muscle, but did not justify the suppression of motility caused by spermine and isoamylamine. Conclusions: Polyamines and TAs of the intestinal content might act on chemosensors and modulate intestinal peristalsis.

  17. Role of the Drosophila Non-Visual ß-Arrestin Kurtz in Hedgehog Signalling

    PubMed Central

    Molnar, Cristina; Ruiz-Gómez, Ana; Martín, Mercedes; Rojo-Berciano, Susana; Mayor, Federico; de Celis, Jose F.

    2011-01-01

    The non-visual ß-arrestins are cytosolic proteins highly conserved across species that participate in a variety of signalling events, including plasma membrane receptor degradation, recycling, and signalling, and that can also act as scaffolding for kinases such as MAPK and Akt/PI3K. In Drosophila melanogaster, there is only a single non-visual ß-arrestin, encoded by kurtz, whose function is essential for neuronal activity. We have addressed the participation of Kurtz in signalling during the development of the imaginal discs, epithelial tissues requiring the activity of the Hedgehog, Wingless, EGFR, Notch, Insulin, and TGFβ pathways. Surprisingly, we found that the complete elimination of kurtz by genetic techniques has no major consequences in imaginal cells. In contrast, the over-expression of Kurtz in the wing disc causes a phenotype identical to the loss of Hedgehog signalling and prevents the expression of Hedgehog targets in the corresponding wing discs. The mechanism by which Kurtz antagonises Hedgehog signalling is to promote Smoothened internalization and degradation in a clathrin- and proteosomal-dependent manner. Intriguingly, the effects of Kurtz on Smoothened are independent of Gprk2 activity and of the activation state of the receptor. Our results suggest fundamental differences in the molecular mechanisms regulating receptor turnover and signalling in vertebrates and invertebrates, and they could provide important insights into divergent evolution of Hedgehog signalling in these organisms. PMID:21437272

  18. Total Spinal Block after Thoracic Paravertebral Block

    PubMed Central

    Beyaz, Serbülent Gökhan; Özocak, Hande; Ergönenç, Tolga; Erdem, Ali Fuat; Palabıyık, Onur

    2014-01-01

    Thoracic paravertebral block (TPVB) can be performed with or without general anaesthesia for various surgical procedures. TPVB is a popular anaesthetic technique due to its low side effect profile and high analgesic potency. We used 20 mL of 0.5% levobupivacaine for a single injection of unilateral TPVB at the T7 level with neurostimulator in a 63 year old patient with co-morbid disease who underwent cholecystectomy. Following the application patient lost consciousness, and was intubated. Haemodynamic instability was normalised with rapid volume replacement and vasopressors. Anaesthetic drugs were stopped at the end of the surgery and muscle relaxant was antagonised. Return of mucle strenght was shown with neuromuscular block monitoring. Approximately three hours after TPVB, spontaneous breathing started and consciousness returned. A total spinal block is a rare and life-threatening complication. A total spinal block is a complication of spinal anaesthesia, and it can also occur after peripheral blocks. Clinical presentation is characterised by hypotension, bradicardia, apnea, and cardiac arrest. An early diagnosis and appropriate treatment is life saving. In this case report, we want to present total spinal block after TPVB. PMID:27366387

  19. An investigation of the effect of the prostaglandin EP2 receptor agonist, butaprost, on the human isolated myometrium from pregnant and non-pregnant women.

    PubMed

    Duckworth, N; Marshall, K; Clayton, J K

    2002-02-01

    The aim of this study was to compare the effect of two known spasmogens, oxytocin and the stable thromboxane receptor mimetic, U46619, on human myometrium treated with the prostaglandin E receptor (EP2) agonist, butaprost (selective for the EP2 receptor). Strips of myometrium from pregnant and non-pregnant donors were set up in a superfusion apparatus. Butaprost was administered as a bolus dose and via infusion. During the infusion of 10(-6) M butaprost, spasmogens were administered as bolus doses. Butaprost caused dose-related inhibition of myometrial activity when administered as a bolus dose (3-100 nmol) and concentration-dependent inhibition during infusion studies (10(-8)-10(-5 )M). Butaprost (10(-6 )M) attenuated the response to U46619 (0.l-10 nmol) in pregnant myometrium, but this difference was not statistically significant. Responses of pregnant myometrium to oxytocin (0.01-0.1 nmol) were significantly attenuated (P<0.05) in the presence of butaprost (10(-6)M). Butaprost physiologically antagonised the oxytocin response, possibly by increasing intracellular cAMP levels. This antagonism was much more marked than that seen with butaprost and U46619. It is unclear why these two types of antagonism differ and this effect is currently being investigated further using other prostanoid and non-prostanoid agents.

  20. Dual targeting of Angiopoetin-2 and VEGF potentiates effective vascular normalisation without inducing empty basement membrane sleeves in xenograft tumours

    PubMed Central

    Coutelle, O; Schiffmann, L M; Liwschitz, M; Brunold, M; Goede, V; Hallek, M; Kashkar, H; Hacker, U T

    2015-01-01

    Background: Effective vascular normalisation following vascular endothelial growth factor (VEGF) inhibition is associated with endothelial cell regression leaving empty basement membrane sleeves (BMS). These long-lived BMS permit the rapid regrowth of tumour vasculature upon treatment cessation and promote resistance to VEGF-targeting drugs. Previous attempts at removing BMS have failed. Angiopoietin-2 (Ang2) is a vascular destabilizing factor that antagonises normalisation. We hypothesised that Ang2 inhibition could permit vascular normalisation at significantly reduced doses of VEGF inhibition, avoiding excessive vessel regression and the formation of empty BMS. Methods: Mice xenografted with human colorectal cancer cells (LS174T) were treated with low (0.5 mg kg−1) or high (5 mg kg−1) doses of the VEGF-targeting antibody bevacizumab with or without an Ang2 blocking peptibody L1-10. Tumour growth, BMS formation and normalisation parameters were examined including vessel density, pericyte coverage, adherence junctions, leakiness, perfusion, hypoxia and proliferation. Results: Dual targeting of VEGF and Ang2 achieved effective normalisation at only one-tenth of the dose required with bevacizumab alone. Pericyte coverage, vascular integrity, adherence junctions and perfusion as prerequisites for improved access of chemotherapy were improved without inducing empty BMS that facilitate rapid vascular regrowth. Conclusions: Dual targeting of VEGF and Ang2 can potentiate the effectiveness of VEGF inhibitors and avoid the formation of empty BMS. PMID:25562438

  1. Sesamin ameliorates doxorubicin-induced cardiotoxicity: involvement of Sirt1 and Mn-SOD pathway.

    PubMed

    Su, Suwen; Li, Qian; Liu, Yi; Xiong, Chen; Li, Junxia; Zhang, Rong; Niu, Yujie; Zhao, Lijuan; Wang, Yongli; Guo, Huicai

    2014-01-13

    Oxidative stress caused by doxorubicin (DOX) is believed to be a major underlying molecular mechanism of DOX-induced cardiotoxicity. Sesamin (Ses), an active component extracted from sesame seeds, exhibits antioxidative and anti-inflammatory effects. In the present study, possible protective mechanisms of Ses on DOX-induced cardiotoxicity were investigated in rats and cultured H9C2 cells. We demonstrated that Ses exhibits a significant protective effect on cardiac tissue in animal and cell models of DOX-induced cardiac injury. Moreover, Ses can ameliorate DOX-induced oxidative stress and mitochondrial damage. Further studies suggested that Ses is able to up-regulate the protein expression of Mn-SOD in normal rats and to restore the decreased expression of Mn-SOD in DOX-induced cardiac injury rats. Exposure to Ses or DOX alone slightly increased the protein expression of Sirt1; however, a more remarkable increase in Sirt1 protein level was detected in the Ses+DOX group. Treatment with a pan-sirtuin inhibitor (nicotinamide) or a Sirt1-specific inhibitor (EX-527) partially antagonised the effect of Ses on DOX-induced mitochondrial damage and completely abolished the effect of Ses on Mn-SOD expression. These findings indicate that the protective mechanisms of Ses on DOX-induced cardiotoxicity are involved in the alleviation of oxidative stress injury and Mn-SOD dysfunction, partially via the activation of Sirt1.

  2. Phaeobacter inhibens as biocontrol agent against Vibrio vulnificus in oyster models.

    PubMed

    Porsby, Cisse Hedegaard; Gram, Lone

    2016-08-01

    Molluscan shellfish can cause food borne diseases and here we investigated if addition of Vibrio-antagonising bacteria could reduce Vibrio vulnificus in model oyster systems and prevent its establishment in live animals. Phaeobacter inhibens, which produces an antibacterial compound, tropodithietic acid (TDA), inhibited V. vulnificus as did pure TDA (MIC of 1-3.9 μM). P. inhibens DSM 17395 (at 10(6) cfu/ml) eradicated 10(5) cfu/ml V. vulnificus CMCP6 (a rifampicin resistant variant) from a co-culture oyster model system (oyster juice) whereas the pathogen grew to 10(7) cfu/ml when co-cultured with a TDA negative Phaeobacter mutant. P. inhibens grew well in oyster juice to 10(8) CFU/ml and sterile filtered samples from these cultures were inhibitory to Vibrio spp. P. inhibens established itself in live European flat oysters (Ostrea edulis) and remained at 10(5) cfu/g for five days. However, the presence of P. inhibens could not prevent subsequently added V. vulnificus from entering the live animals, likely because of too low levels of the biocontrol strain. Whilst the oyster model studies provided indication that P. inhibens DSM 17395 could be a good candidate as biocontrol agent against V. vulnificus further optimization is need in the actual animal rearing situation.

  3. Nicholas Culpeper (1616-1654): London's first general practitioner?

    PubMed

    Farthing, Michael J G

    2015-08-01

    Nicholas Culpeper is often regarded as an ill-disciplined, maverick, mid-17th century herbalist and the father of contemporary alternative medicine. There are elements of this statement that have some truth but to dismiss his contribution to the development of health provision in London at the time would be a great injustice. Culpeper did not complete his apprenticeship as an apothecary and was not a formally trained physician, but he developed a clinical practice for the poor of London, indistinguishable from the role of the present day general practitioner. Observers at the time recognised his concern and compassion and his commitment to treat the whole patient and not just the disease. His enduring contribution was his translation from Latin of the physicians' Pharmacopoeia Londinensis which could be regarded as the first major step towards the demystification of medicine. Culpeper's London Dispensatory and the many other medical treatises that followed were affordable and widely available to the common man. Culpeper antagonised both apothecaries and physicians because he breached the regulations of the day by accepting patients directly. So perhaps Culpeper was, de facto, London's first general practitioner, at least 150 years before the role was formally recognised in the Apothecaries Act 1815.

  4. An antiapoptotic Bcl-2 family protein index predicts the response of leukaemic cells to the pan-Bcl-2 inhibitor S1

    PubMed Central

    Zhang, Z; Liu, Y; Song, T; Xue, Z; Shen, X; Liang, F; Zhao, Y; Li, Z; Sheng, H

    2013-01-01

    Background: Bcl-2-like members have been found to be inherently overexpressed in many types of haematologic malignancies. The small-molecule S1 is a BH3 mimetic and a triple inhibitor of Bcl-2, Mcl-1 and Bcl-XL. Methods: The lethal dose 50 (LD50) values of S1 in five leukaemic cell lines and 41 newly diagnosed leukaemia samples were tested. The levels of Bcl-2 family members and phosphorylated Bcl-2 were semiquantitatively measured by western blotting. The interactions between Bcl-2 family members were tested by co-immunoprecipitation. The correlation between the LD50 and expression levels of Bcl-2 family members, alone or in combination, was analysed. Results: S1 exhibited variable sensitivity with LD50 values ranging >2 logs in both established and primary leukaemic cells. The ratio of pBcl-2/(Bcl-2+Mcl-1) could predict the S1 response. Furthermore, we demonstrated that pBcl-2 antagonised S1 by sequestering the Bak and Bim proteins that were released from Mcl-1, andpBcl-2/Bak, pBcl-2/Bax and pBcl-2/Bim complexes cannot be disrupted by S1. Conclusion: A predictive index was obtained for the novel BH3 mimetic S1. The shift of proapoptotic proteins from being complexed with Mcl-1 to being complexed with pBcl-2 was revealed for the first time, which is the mechanism underlying the index value described herein. PMID:23558901

  5. The physiological and pharmacological role of presynaptic alpha- and beta-adrenoceptors in man.

    PubMed Central

    Brown, M J; Struthers, A D; Burrin, J M; Di Silvio, L; Brown, D C

    1985-01-01

    Two studies were performed each in six normal volunteers in order to find evidence of either a physiological or pharmacological role of presynaptic alpha- and presynaptic beta-adrenoceptors in man. In Study 1 subjects received a 60 min infusion of guanfacine 3 mg (alpha 2-adrenoceptor agonist) preceded by either idazoxan (alpha 2-adrenoceptor antagonist) or vehicle. Guanfacine reduced plasma noradrenaline concentration by approximately 30% and this fall was not antagonised by the alpha 2-receptor antagonist. The 30-fold increase in plasma growth hormone, measured as a marker of the central action of guanfacine, was almost completely blocked by idazoxan. A comparison of the drug concentrations of idazoxan and guanfacine, together with their relative affinities for alpha 2-adrenoceptors, suggested that the idazoxan could not block the peripheral actions of guanfacine and that these were responsible for the fall in plasma noradrenaline concentration. In Study 2 adrenaline 0.05 micrograms kg-1 min-1 was infused for 80 min preceded by either idazoxan or vehicle. After vehicle, adrenaline caused no change in plasma noradrenaline concentration whereas it rose approximately 25% after administration of idazoxan. This was probably due to unmasking of presynaptic beta-adrenoceptor stimulation by adrenaline when the opposing inhibitory autoreceptor was blocked. PMID:2868743

  6. The influence of microglia activation on the efficacy of amitriptyline, doxepin, milnacipran, venlafaxine and fluoxetine in a rat model of neuropathic pain.

    PubMed

    Zychowska, Magdalena; Rojewska, Ewelina; Makuch, Wioletta; Przewlocka, Barbara; Mika, Joanna

    2015-02-15

    The analgesic properties of antidepressants are often used in the treatment of neuropathy; however their influence on glial cells in maintaining neuropathic pain is unknown. Our studies examined the neuropathic pain-relieving properties after intraperitoneal injection of amitriptyline, doxepin, milnacipran, venlafaxine and fluoxetine 7 days after sciatic nerve injury (CCI) in rats and its influence on microglia/macrophages (IBA-1) and astroglia (GFAP) activation in the spinal cord and dorsal root ganglia (DRG) using Western blot. All tested antidepressants significantly reduced CCI-induced allodynia but hyperalgesia was only antagonised by fluoxetine, doxepine and venlafaxine. The strongest analgesia was observed after fluoxetine administration. Western blot analysis showed the upregulation of the IBA-1 in the lumbar spinal cord and DRG after amitriptyline or milnacipran administration in CCI-exposed rats, whereas after fluoxetine the downregulation was observed. The administration of doxepin did not change the IBA-1 protein level in both studied structures; however venlafaxine decreased the IBA-1 only in the DRG. No changes in the GFAP level in both structures were observed after any of listed above antidepressants administration. Chronic minocycline treatment enhanced amitriptyline and milnacipran, but did not fluoxetine analgesia under neuropathic pain in rats. Our results suggest that nerve injury-induced pain is related with the activation of microglia, which is diminished by fluoxetine treatment in the neuropathic pain model.

  7. Asthma.

    PubMed Central

    Calverley, P. M.

    1996-01-01

    Bronchial asthma is now recognised to be a major cause of morbidity and even mortality in people of all ages. Two important ideas have changed our approach to asthma management. The first is understanding that asthma is a chronic inflammatory disorder which needs regular treatment with anti-inflammatory drugs such as inhaled corticosteroids to prevent further attacks. The second development is the availability of prescribable peak flow meters, which allows both confident diagnosis and early prediction of relapse. Asthma management guidelines provide a logical treatment framework for most patients, but a few difficult cases still consume large amounts of medical time. The commonest problem is one of compliance with treatment which may respond to patient education, although this is not universally so. Other problems include misdiagnosis, acid reflux and, rarely, true corticosteroid-resistant asthma. Several potentially important new treatments have been developed. These include longer acting anticholinergic drugs, drugs with bronchodilator and some anti-inflammatory properties which antagonise or inhibit the production of leukotrienes, sub-types of phosphodiesterase inhibitor with anti-inflammatory properties and immunosuppressive drugs such as cyclosporin. Ultimately these new treatments must be rigorously tested and integrated into a care plan that remains centred on patient education. PMID:8746278

  8. Inter-population variability in the reproductive morphology of the shore crab (Carcinus maenas): evidence of endocrine disruption in a marine crustacean?

    PubMed

    Brian, Jayne V

    2005-04-01

    Environmental contaminants that are capable of causing endocrine disrupting effects are currently a major cause for concern. These chemicals are known to influence the reproductive development of vertebrates by mimicking or antagonising the actions of endogenous hormones. However, little is known regarding their potential effects on invertebrates. Here we examine variations in the reproductive morphology of the shore crab (Carcinus maenas) for evidence of endocrine disruption. Crabs were collected from a number of sites comprising a putative gradient of exposure to endocrine disrupting chemicals. Patterns of inter-population variability in the expression of sexually dimorphic traits were then examined for evidence of hormone disruption. Extensive variability was detected and patterns of chelal morphology were consistent with the gradient of endocrine disruption. However, overall, the patterns of morphological variability were not consistent with hormonally-mediated effects. This suggests that shore crabs are not susceptible to the same type of endocrine disrupting effects that have been detected in vertebrates, which are most commonly mediated via the oestrogen receptor. However, the potential for androgenic effects on crustacean morphology are discussed.

  9. Physiological Impact of Abnormal Lipoxin A4 Production on Cystic Fibrosis Airway Epithelium and Therapeutic Potential

    PubMed Central

    Higgins, Gerard; McNally, Paul; Urbach, Valérie

    2015-01-01

    Lipoxin A4 has been described as a major signal for the resolution of inflammation and is abnormally produced in the lungs of patients with cystic fibrosis (CF). In CF, the loss of chloride transport caused by the mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channel gene results in dehydration, mucus plugging, and reduction of the airway surface liquid layer (ASL) height which favour chronic lung infection and neutrophil based inflammation leading to progressive lung destruction and early death of people with CF. This review highlights the unique ability of LXA4 to restore airway surface hydration, to stimulate airway epithelial repair, and to antagonise the proinflammatory program of the CF airway, circumventing some of the most difficult aspects of CF pathophysiology. The report points out novel aspects of the cellular mechanism involved in the physiological response to LXA4, including release of ATP from airway epithelial cell via pannexin channel and subsequent activation of and P2Y11 purinoreceptor. Therefore, inadequate endogenous LXA4 biosynthesis reported in CF exacerbates the ion transport abnormality and defective mucociliary clearance, in addition to impairing the resolution of inflammation, thus amplifying the vicious circle of airway dehydration, chronic infection, and inflammation. PMID:25866809

  10. Clozapine: dopamine D1 receptor agonism in the prefrontal cortex as the code to decipher a Rosetta stone of antipsychotic drugs.

    PubMed

    Ahlenius, S

    1999-05-01

    A large number of ligand binding studies have shown that clozapine has a number of receptor affinities, including those of the dopamine (DA) D1 and D2 receptor families. The study of intrinsic efficacy at these receptors is less straight-forward. In the experiments summarised here, evidence is presented that clozapine behaves as an agonist at DA D1 receptors. Thus, the hypothermia produced by clozapine (2.5 mg kg(-1)) in the rat is fully antagonised by either of the selective DA D1 receptor antagonists SCH-23390 (0.1 mg kg(-1)) or NNC-687 (4 mg kg(-1)). These results provide an intriguing explanation for the clinical profile of clozapine as an atypical antipsychotic drug. Thus, there are supporting clinical and laboratory observations implicating DA D1 receptors in the prefrontal cortex in cognitive functions. Finally, clozapine displays features with regard to extrapyramidal motor mechanisms, and seizure thresholds, that could be explained by its properties as a DA D1 receptor agonist.

  11. Betulin binds to gamma-aminobutyric acid receptors and exerts anticonvulsant action in mice.

    PubMed

    Muceniece, Ruta; Saleniece, Kristine; Rumaks, Juris; Krigere, Liga; Dzirkale, Zane; Mezhapuke, Rudolfs; Zharkova, Olga; Klusa, Vija

    2008-10-01

    The lupane type pentacyclic triterpenes: lupeol, betulin, and betulinic acid are widely distributed natural compounds. Recently, pharmaceutical compositions from plant extracts (family Marcgraviaceae) containing betulinic acid, have been patented as anxiolytic remedies. To extend our knowledge of the CNS effects of the triterpenes, we suggest here that the chemically related lupeol, betulin and betulinic acid may interact with the brain neurotransmitter gamma-aminobutyric acid (GABA) receptors in vitro and in vivo. Using radioligand receptor-binding assay, we showed that only betulin bound to the GABA(A)-receptor sites in mice brain in vitro and antagonised the GABA(A)-receptor antagonist bicuculline-induced seizures in mice after intracisternal and intraperitoneal administration. Neither betulinic acid nor lupeol bound to GABA(A) receptor nor did they inhibit bicuculline-induced seizures in vivo. These findings demonstrate for the first time the CNS effects of betulin in vivo, and they also show distinct GABA(A)-receptor-related properties of lupane type triterpenes. These findings may open new avenues in understanding the central effects of betulin, and they also indicate possibilities for novel drug design on the basis of betulin structure.

  12. Cloning and pharmacological characterisation of the guinea pig 5-ht5A receptor.

    PubMed

    Thomas, David R; Larminie, Christopher G; Lyons, Helen R; Fosberry, Andrew; Hill, Matthew J; Hayes, Philip D

    2004-06-28

    The guinea pig 5-hydroxytryptamine(5A) (gp5-ht(5A)) receptor was cloned from guinea pig brain using degenerate polymerase chain reaction (PCR) and shows 88%, 85% and 84% amino acid sequence identity versus the human, rat and mouse 5-ht(5A) receptors, respectively. The receptor was transiently expressed in human embryonic kidney (HEK) 293 cells. [(3)H]-Lysergic acid diethylamide (LSD) bound saturably to gp5-ht(5A)/HEK293 membranes with a K(d) of 2.3+/-0.1 nM and B(max) of 15.7+/-3.4 pmol/mg protein. The receptor binding profile, determined by competition with [(3)H]LSD, correlated well with that for the human 5-ht(5A) receptor. 5-HT stimulated [(35)S]GTPgammaS binding to gp5-ht(5A)/HEK293 membranes (pEC(50) 8.1+/-0.2), and the response was surmountably antagonised by methiothepin and ritanserin, giving apparent pK(B) values of 8.0 and 7.2, respectively. The 5-HT response was absent using membranes prepared from gp5-ht(5A)/HEK293 cells pretreated with pertussis toxin (PTX). These data suggest that the gp5-ht(5A) receptor couples to G(i)-proteins in this expression system and shows a similar pharmacological profile to that for the human 5-ht(5A) receptor.

  13. Characterization of a new synthetic isoflavonoid with inverse agonist activity at the central benzodiazepine receptor.

    PubMed

    Lopes, Daniele V S; Caruso, Rodrigo R B; Castro, Newton G; Costa, Paulo R R; da Silva, Alcides J M; Noël, François

    2004-07-14

    Research aimed at developing selective drugs acting on gamma-aminobutyric acid (GABA)A receptors introduced compounds from diverse chemical classes unrelated to the 1,4-benzodiazepines, including flavonoids. These studies also revealed the potential use of inverse agonists as cognition-enhancing agents. Here we report pharmacological properties of the novel synthetic isoflavonoid 2-methoxy-3,8,9-trihydroxy coumestan (PCALC36). PCALC36 displaced [3H]flunitrazepam binding to rat brain synaptosomes with an IC50 of 13.8 microM. Scatchard analysis of the effect of PCALC36 showed a concentration-dependent reduction of the Bmax of [3H]flunitrazepam, without a marked change in Kd. This effect could be reversed by diluting and washing the preparation. Addition of 20-microM GABA shifted to the right the inhibition curve of PCALC36 on [3H]flunitrazepam binding (IC50 ratio of 0.68), which is characteristic for inverse agonists. PCALC36 produced little change in the GABAergic tonic currents recorded by whole-cell patch clamp in cultured rat hippocampal neurones, but it caused a 20% reduction in miniature inhibitory postsynaptic current amplitude and completely antagonised the full (direct) agonist midazolam in a quickly reversible manner. The data suggest that the coumestan backbone can be useful for developing novel ligands at the GABAA receptor.

  14. 'Smashed by the National Health'? A Closer Look at the Demise of the Pioneer Health Centre, Peckham.

    PubMed

    Conford, Philip

    2016-04-01

    The Pioneer Health Centre, based in South London before and after the Second World War, remains a source of interest for advocates of a positive approach to health promotion in contrast with the treatment of those already ill. Its closure in 1950 for lack of funds has been blamed on the then recently established National Health Service, but this article argues that such an explanation is over-simplified and ignores a number of other factors. The Centre had struggled financially during the 1930s and tried to gain support from the Medical Research Council. The Council appeared interested in the Centre before the war, but was less sympathetic in the 1940s. Around the time of its closure and afterwards, the Centre was also involved in negotiations with London County Council; these failed because the Centre's directors would not accept the changes which the Council would have needed to make. Unpublished documents reveal that the Centre's directors were uncompromising and that their approach to the situation antagonised their colleagues. Changes in medical science also worked against the Centre. The success of sulphonamide drugs appeared to render preventive medicine less significant, while the development of statistical techniques cast doubt on the Centre's experimental methods. The Centre was at the heart of the nascent organic farming movement, which opposed the rapid growth of chemical cultivation. But what might be termed 'chemical triumphalism' was on the march in both medicine and agriculture, and the Centre was out of tune with the mood of the times.

  15. The Role of Dopamine and Glutamate Modulation in Huntington Disease

    PubMed Central

    Mittal, Sumeer K.; Eddy, Clare

    2013-01-01

    Background: Huntington disease (HD) is an inherited neuropsychiatric condition with progressive neurodegenerative changes, mainly affecting the striatum. Pathological processes within the striatum are likely to lead to alterations in dopamine and glutamate activity in frontostriatal circuitry, resulting in characteristic motor, behavioural and cognitive symptoms. Methods: We conducted a systematic literature search in order to identify and review randomised, double-blinded, placebo-controlled trials of anti-dopaminergic and anti-glutamatergic therapy in HD. Results: Ten studies satisfied our selection criteria. These studies investigated a range of agents which act to antagonise dopamine (tetrabenazine, typical and atypical antipsychotics) or glutamate (amantadine, riluzole) transmission. Discussion: Although most agents showed efficacy in terms of amelioration of chorea, the available evidence did not allow us to identify a universally effective treatment. One difficulty associated with analysing the available evidence was a high prevalence of side effects, which prevented the full therapeutic potential of the medications from being adequately investigated. A further limitation is that many studies evaluated treatment effectiveness only in relation to patients' motor symptoms, even though behavioural and cognitive changes may negatively impact patients' quality of life. There is a clear need for further higher-level evidence addressing the effects of dopaminergic and glutamatergic agents on global functioning in HD. PMID:22713410

  16. The chick embryo as a model for the effects of prenatal exposure to alcohol on craniofacial development.

    PubMed

    Kiecker, Clemens

    2016-07-15

    Prenatal exposure to ethanol results in fetal alcohol spectrum disorder (FASD), a syndrome characterised by a broad range of clinical manifestations including craniofacial dysmorphologies and neurological defects. The characterisation of the mechanisms by which ethanol exerts its teratogenic effects is difficult due to the pleiotropic nature of its actions. Different experimental model systems have been employed to investigate the aetiology of FASD. Here, I will review studies using these different model organisms that have helped to elucidate how ethanol causes the craniofacial abnormalities characteristic of FASD. In these studies, ethanol was found to impair the prechordal plate-an important embryonic signalling centre-during gastrulation and to negatively affect the induction, migration and survival of the neural crest, a cell population that generates the cartilage and most of the bones of the skull. At the cellular level, ethanol appears to inhibit Sonic hedgehog signalling, alter levels of retionoic acid activity, trigger a Ca(2+)-CamKII-dependent pathway that antagonises WNT signalling, affect cytoskeletal dynamics and increase oxidative stress. Embryos of the domestic chick Gallus gallus domesticus have played a central role in developing a working model for the effects of ethanol on craniofacial development because they are easily accessible and because key steps in craniofacial development are particularly well established in the avian embryo. I will finish this review by highlighting some potential future avenues of fetal alcohol research. Copyright © 2016 Elsevier Inc. All rights reserved.

  17. Amantadin e tremor, a 5-hydroxytryptamine-mediated response?

    PubMed

    Cox, B; Tha, S J

    1975-02-01

    Amantadine-induced tremor has been investigated using mice. Experiments with, mebanazine, reserpine, diethyldithiocarbamate, and p-chlorophenylalanine suggest that the tremorgenic action of amantadine is influenced by a balance between three putative central nervous system (CNS) transmitters: noradrenaline, dopamine and 5-hydroxytryptamine (5-HT). Drugs which reduce the concentration of the catecholamines in brain increase amantadine induced tremor. p-Chlorophenylalanine, which specifically depletes brain 5-HT, antagonises amantadine-induced tremor. An ED50 (tremor) dose of amantadine decreases the concentration of 5-hydroxy-indoleacetic acid (5-HIAA) in rat brain, particularly when this elevated due to pretreatment with 5-hydroxytryptophan. Neither inhibition of monoamine oxidase nor reduction of 5-HT-reuptake appear to be responsible for this decrease. Experiments on rat fundus suggest that amantadine increased the sensitivity of receptors to 5-HT. A similar mechanism of action in the CNS could explain both the tremor and the decrease in brain 5-HIAA. The possible relevance of these findings is discussed with respect to the known anti-Parkinson action of amantadine.

  18. Neurotoxicity of chlorpromazine and modulation by amantadine as a function of mouse strain.

    PubMed

    Messiha, F S

    1991-01-01

    The separate and combined effects of successive administration of amantadine, 100 mg/kg, i.p., and chlorpromazine, 0.2 mg/kg, i.p., on motor activity and whole brain levels of certain biogenic amines and major metabolites were studied in four strains of mice. These were the albino ICR, the inbred BALB/C, C57BL/6 and the hybrid CDF-I mice. Amantadine produced a strain-dependent behavioral stimulation subsequent the fourth dose. This was apparent in ICR and C57BL/6 mouse strains and was followed by a behavioral depression phase occurring during the night in C57BL/6 mice which was antagonized by chlorpromazine. Administration of chlorpromazine alone affected only CDF-1 mouse mobility. Chlorpromazine reduced only ICR mouse brain dopamine without concomitant changes in major acid metabolites. Repeated administration of amantadine prior to chlorpromazine negated this effect. Chlorpromazine enhancement of BALB/C brain serotonin and 5-hydroxyindoleacetic acid was antagonised by pretreatment with amantadine. This antagonism was also evident in BALB/C mouse brain dihydroxyphenylacetic acid. The results suggest genotypic-dependent behavioral and cerebral effects by the drugs studied. The antagonism between amantadine and chlorpromazine on brain amines may explain the therapeutic efficacy of amantadine in modulating chlorpromazine-induced extrapyramidal disorders.

  19. The Spectrin cytoskeleton regulates the Hippo signalling pathway

    PubMed Central

    Fletcher, Georgina C; Elbediwy, Ahmed; Khanal, Ichha; Ribeiro, Paulo S; Tapon, Nic; Thompson, Barry J

    2015-01-01

    The Spectrin cytoskeleton is known to be polarised in epithelial cells, yet its role remains poorly understood. Here, we show that the Spectrin cytoskeleton controls Hippo signalling. In the developing Drosophila wing and eye, loss of apical Spectrins (alpha/beta-heavy dimers) produces tissue overgrowth and mis-regulation of Hippo target genes, similar to loss of Crumbs (Crb) or the FERM-domain protein Expanded (Ex). Apical beta-heavy Spectrin binds to Ex and co-localises with it at the apical membrane to antagonise Yki activity. Interestingly, in both the ovarian follicular epithelium and intestinal epithelium of Drosophila, apical Spectrins and Crb are dispensable for repression of Yki, while basolateral Spectrins (alpha/beta dimers) are essential. Finally, the Spectrin cytoskeleton is required to regulate the localisation of the Hippo pathway effector YAP in response to cell density human epithelial cells. Our findings identify both apical and basolateral Spectrins as regulators of Hippo signalling and suggest Spectrins as potential mechanosensors. PMID:25712476

  20. Presynaptic actions of 4-aminopyridine and gamma-aminobutyric acid on rat sympathetic ganglia in vitro.

    PubMed

    Galvan, M; Grafe, P; ten Bruggencate, G

    1980-11-01

    Responses to bath-applications of 4-aminopyridine(4-AP) and gamma-aminobutyric acid (GABA) were recorded intracellularly from neurones in the rat isolated superior cervical ganglion. 4-aminopyridine (0.1-1.0 mmol/l) usually induced spontaneous action potentials and excitatory postsynaptic potentials (EPSPs), which were blocked by hexamethonium. Membrane potential was unchanged; spike duration was slightly increased. Vagus nerve B- and C-fibre potentials were prolonged. In 4-AP solution (0.2-0.3 mmol/l), GABA (0.1 mmol/l), 3-aminopropanesulphonic acid or muscimol evoked "bursts" of spikes and EPSPs in addition to a neuronal depolarization. These "bursts", which were not elicited by glycine, glutamate, taurine or (+/-)-baclofen, were completely antagonised by hexamethonium, tetrodotoxin or bicuculline methochloride. It is concluded that: (a) 4-AP has a potent presynaptic action on sympathetic ganglia; (b) presynaptic actions of GABA can be recorded postsynaptically in the presence of 4-AP; and (c) the presynaptic GABA-receptors revealed in this condition are similar to those on the postsynaptic membrane.

  1. Naturally-Occurring Glucosinolates, Glucoraphanin and Glucoerucin, are Antagonists to Aryl Hydrocarbon Receptor as Their Chemopreventive Potency.

    PubMed

    Abdull Razis, Ahmad Faizal; Noor, Noramaliza Mohd

    2015-01-01

    As a cytosolic transcription factor, the aryl hydrocarbon (Ah) receptor is involved in several patho- physiological events leading to immunosuppression and cancer; hence antagonists of the Ah receptor may possess chemoprevention properties. It is known to modulate carcinogen-metabolising enzymes, for instance the CYP1 family of cytochromes P450 and quinone reductase, both important in the biotransformation of many chemical carcinogens via regulating phase I and phase II enzyme systems. Utilising chemically-activated luciferase expression (CALUX) assay it was revealed that intact glucosinolates, glucoraphanin and glucoerucin, isolated from Brassica oleracea L. var. acephala sabellica and Eruca sativa ripe seeds, respectively, are such antagonists. Both glucosinolates were poor ligands for the Ah receptor; however, they effectively antagonised activation of the receptor by the avid ligand benzo[a]pyrene. Indeed, intact glucosinolate glucoraphanin was a more potent antagonist to the receptor than glucoerucin. It can be concluded that both glucosinolates effectively act as antagonists for the Ah receptor, and this may contribute to their established chemoprevention potency.

  2. Novel, potent and selective inhibitors of protein kinase C show oral anti-inflammatory activity.

    PubMed

    Nixon, J S; Bishop, J; Bradshaw, D; Davis, P D; Hill, C H; Elliott, L H; Kumar, H; Lawton, G; Lewis, E J; Mulqueen, M

    1991-01-01

    Clarification of the precise role of protein kinase C (PKC) in cellular functional responses has been hampered by a lack of potent, selective inhibitors. The structural lead provided by staurosporine, a potent but non-selective protein kinase (PK) inhibitor, was used to derive a series of bis(indolyl)maleimides of which the most potent, Ro 31-8425 (I50: PKC = 8 nM) showed 350-fold selectivity for PKC over cAMP-dependent protein kinase. Ro 31-8425 antagonised cellular processes triggered by phorbol esters (potent, specific PKC activators) and inhibited the allogeneic mixed lymphocyte reaction, suggesting a role for PKC in T-cell activation. Methylation of the primary amine in Ro 31-8425 produced an analogue. Ro 31-8830 which, when administered orally, produced a dose-dependent inhibition of a phorbol ester-induced paw oedema in mice (minimum effective dose = 15 mg/kg). Ro 31-8830 also selectively inhibited the secondary inflammation in a developing adjuvant arthritis model in the rat. The results presented here suggest that these selective inhibitors of PKC may have therapeutic value in the treatment of T-cell-mediated autoimmune diseases.

  3. A Cell Cycle Role for the Epigenetic Factor CTCF-L/BORIS

    PubMed Central

    Alonso-Lecue, Pilar; Abraira, Cristina; Delgado, M. Dolores; Gandarillas, Alberto

    2012-01-01

    CTCF is a ubiquitous epigenetic regulator that has been proposed as a master keeper of chromatin organisation. CTCF-like, or BORIS, is thought to antagonise CTCF and has been found in normal testis, ovary and a large variety of tumour cells. The cellular function of BORIS remains intriguing although it might be involved in developmental reprogramming of gene expression patterns. We here unravel the expression of CTCF and BORIS proteins throughout human epidermis. While CTCF is widely distributed within the nucleus, BORIS is confined to the nucleolus and other euchromatin domains. Nascent RNA experiments in primary keratinocytes revealed that endogenous BORIS is present in active transcription sites. Interestingly, BORIS also localises to interphase centrosomes suggesting a role in the cell cycle. Blocking the cell cycle at S phase or mitosis, or causing DNA damage, produced a striking accumulation of BORIS. Consistently, ectopic expression of wild type or GFP- BORIS provoked a higher rate of S phase cells as well as genomic instability by mitosis failure. Furthermore, down-regulation of endogenous BORIS by specific shRNAs inhibited both RNA transcription and cell cycle progression. The results altogether suggest a role for BORIS in coordinating S phase events with mitosis. PMID:22724006

  4. Review article: the potential mechanisms of action of rifaximin in the management of inflammatory bowel diseases.

    PubMed

    Sartor, R B

    2016-01-01

    Gut microbiota dysbiosis contributes to the pathogenesis of inflammatory bowel diseases (IBD). Although the microbiota's role in IBD pathogenesis, specifically Crohn's disease (CD), provides a rationale for antibiotic treatment, antibiotic use in CD remains controversial. Rifaximin, traditionally identified as a nonsystemic bactericidal antibiotic, may be therapeutically beneficial for inducing CD remission. To examine the role of rifaximin in the management of IBD and its potential mechanisms of action. A literature search using the following strategy: ('inflammatory bowel disease' OR 'Crohn's' OR 'ulcerative'), 'rifaximin' AND ('barrier' OR 'translocation' OR 'adhesion' OR 'internalization' OR 'pregnane X'), AND 'pregnane X' AND ('Crohn's' OR 'ulcerative colitis' OR 'inflammatory bowel disease'). In vitro data suggest rifaximin mediates changes in epithelial cell physiology and reduces bacterial attachment and internalisation. In experimental colitis models, rifaximin antagonised the effects of tumour necrosis factor-α on intestinal epithelial cells by activating pregnane X receptor, which inhibits nuclear factor-κB-mediated proinflammatory mediators and induces detoxification genes (e.g. multidrug resistance 1 and cytochrome P450 3A4). Rifaximin also inhibits bacterial translocation into the mesenteric lymph nodes. Accumulating evidence suggests that mechanisms of action of rifaximin in IBD may not be limited to direct bactericidal activity; therefore, rifaximin could potentially be redefined as a gut environment modulator. © 2015 John Wiley & Sons Ltd.

  5. Identification of the Beer Component Hordenine as Food-Derived Dopamine D2 Receptor Agonist by Virtual Screening a 3D Compound Database.

    PubMed

    Sommer, Thomas; Hübner, Harald; El Kerdawy, Ahmed; Gmeiner, Peter; Pischetsrieder, Monika; Clark, Timothy

    2017-03-10

    The dopamine D2 receptor (D2R) is involved in food reward and compulsive food intake. The present study developed a virtual screening (VS) method to identify food components, which may modulate D2R signalling. In contrast to their common applications in drug discovery, VS methods are rarely applied for the discovery of bioactive food compounds. Here, databases were created that exclusively contain substances occurring in food and natural sources (about 13,000 different compounds in total) as the basis for combined pharmacophore searching, hit-list clustering and molecular docking into D2R homology models. From 17 compounds finally tested in radioligand assays to determine their binding affinities, seven were classified as hits (hit rate = 41%). Functional properties of the five most active compounds were further examined in β-arrestin recruitment and cAMP inhibition experiments. D2R-promoted G-protein activation was observed for hordenine, a constituent of barley and beer, with approximately identical ligand efficacy as dopamine (76%) and a Ki value of 13 μM. Moreover, hordenine antagonised D2-mediated β-arrestin recruitment indicating functional selectivity. Application of our databases provides new perspectives for the discovery of bioactive food constituents using VS methods. Based on its presence in beer, we suggest that hordenine significantly contributes to mood-elevating effects of beer.

  6. Safety profile of anakinra in the management of rheumatologic, metabolic and autoinflammatory disorders.

    PubMed

    Lopalco, Giuseppe; Rigante, Donato; Giannini, Margherita; Galeazzi, Mauro; Lapadula, Giovanni; Iannone, Florenzo; Cantarini, Luca

    2016-01-01

    Anakinra is a biologic response modifier that competitively antagonises the biologic effects of interleukin-1, the ancestor pleiotropic proinflammatory cytokine produced by numerous cell types, found in excess in the serum, synovial fluid and any involved tissues of patients with many inflammatory diseases. The magnitude of the risk of different infections, including Mycobacterium tuberculosis (Mtb) infection, associated with the large use of anakinra in many rheumatologic, metabolic or autoinflammatory disorders is still unknown. In addition, it is unclear whether this effect is modified by the concomitant use of antirheumatic drugs and corticosteroids. The rates of development of Mtb disease in patients treated with anakinra due to rheumatoid arthritis, systemic autoinflammatory diseases, Schnitzler's syndrome, Behçet's disease, adult-onset Still disease, systemic juvenile idiopathic arthritis, gout and diabetes mellitus have been usually very low. However, clinicians must carefully weigh the benefits of biological drugs against their risks, particularly in patients prone to infections. Additional data are needed to understand whether this risk of Mtb infection and reactivation are representative of a class effect related to biologics or whether anakinra bears specifically an intrinsic lower risk in comparison with other biologic drugs.

  7. Evaluation of two sedation protocols for use before diagnostic intra-articular anaesthesia in lame dogs.

    PubMed

    Van Vynckt, D; Samoy, Y; Polis, I; Bosmans, T; Verschooten, F; Van Ryssen, B

    2011-12-01

    To assess the influence of two sedation protocols on the degree of lameness in dogs. Fifty lame dogs were allocated to one of two sedation protocols. Group ACPM (acepromazine + methadone; n=25) was sedated with acepromazine and methadone. Group MED (medetomidine antagonised with atipamezole; n=25) was sedated with medetomidine and reversed with atipamezole. Each dog was evaluated for lameness before and after sedation using videotapes. Four experienced clinicians allocated global lameness scores before and after sedation to each dog using a numerical rating scale. In 80% of the dogs in group ACPM and in 72% in group MED lameness was not affected by the sedation. In 12% of the dogs in group ACPM and 20% of the dogs in group MED the observers noticed an increase of lameness of 1 or 2 degrees on a scale of 0 to 10. In 8% of the dogs in both groups lameness decreased with 1 degree. A possible diagnostic test for investigation of obscure lameness is intra-articular anaesthesia. Sedation is necessary to allow intra-articular injection. This study provided evidence that the effect of sedation with the proposed protocols on the degree of lameness is negligible. © 2011 British Small Animal Veterinary Association.

  8. Decrease in sensitisation rate and intestinal anaphylactic response after nitric oxide synthase inhibition in a food hypersensitivity model.

    PubMed Central

    Fargeas, M J; Theodorou, V; Weirich, B; Fioramonti, J; Buéno, L

    1996-01-01

    BACKGROUND--Although nitric oxide (NO) has been found to have a role in gut inflammation and to modulate immunoglobulin production, little is known about its part in food hypersensitivities. AIM--This study aimed to evaluate the role of NO through the inhibition of constitutive and inducible NO synthase (cNOS and iNOS respectively) on the sensitisation process (antibody titres) and on intestinal anaphylactic responses (colonic hypersecretion upon antigen challenge). ANIMALS AND METHODS--Guinea pigs sensitised to cow's milk proteins were treated either during the sensitisation period or before antigen challenge by N-nitro-L-arginine methyl ester (L-NAME) (inhibiting both cNOS and iNOS) or amino-guanidine (selective iNOS inhibitor). RESULTS--Chronic treatment by L-NAME or aminoguanidine reduced antibody titres and the secretory response to antigen challenge. In contrast, only L-NAME administered before challenge was able to antagonise the hypersecretion induced by the challenge. CONCLUSIONS--NO generated by iNOS has a role in the sensitisation process: iNOS inhibition results in lower rates of antibodies leading to a reduced secretory response upon challenge. In contrast, blockade of colonic hypersecretion by L-NAME but not by aminoguanidine suggests that NO via cNOS is a key mediator in intestinal anaphylactic reactions. PMID:8707095

  9. [Is primary, secondary or tertiary prevention of coronary heart disease possible by means of oestrogens or gestagens?].

    PubMed

    Husmann, F

    2002-04-01

    Clinical trials in primary prevention of coronary atherosclerosis have demonstrated that estrogen application in postmenopausal women may reduce the relative risk by approximately 50 % when initiated at the time of decreasing endogenous estradiol production. Multifactorial estrogen actions (i. e. vasodilatation, inhibition of vascular smooth muscle cell proliferation) could explain these observations. Based on some investigations in secondary and tertiary prevention of coronary atherosclerosis in postmenopausal women it may be concluded that oestrogens alone or in combination with certain progestins may have a protective effect in reducing the rate of reinfarctions. From an epidemiological point of view it was emphasized that these studies were not randomised and therefore not suitable for drawing conclusions generally valid in all postmenopausal women. The HERS-study was initiated in order to get reliable informations. However, the results of this study are not comparable with any other investigation as in the HERS-study for the first time a continuous combined oestrogen/progestin application was chosen. The authors pointed out that several positive oestrogen actions may be antagonised by progestins as the oestrogen receptor density could be down regulated by progestins. The HERS study shows indeed no increase in reinfarctions and a decrease in total mortality after long term treatment. Consequently, the study shows that there is a demand to strongly emphasise the significance of primary prevention.

  10. CNQX and DNQX block non-NMDA synaptic transmission but not NMDA-evoked locomotion in lamprey spinal cord.

    PubMed

    Alford, S; Grillner, S

    1990-01-08

    The motor pattern underlying locomotion in the lamprey is activated and maintained by excitatory amino acid neurotransmission. The quinoxalinediones 6,7-dinitroquinoxaline-2,3-dione (DNQX) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) are potent and selective antagonists of non-N-methyl-D-aspartate (NMDA) receptors in the mammalian central nervous system. In the lamprey, these compounds are now shown to block fast excitatory synaptic potentials elicited in neurones of the spinal ventral horn. They selectively antagonise responses to the application of selective kainate and quisqualate receptor agonists (kainate and alpha-amino-3-hydroxy-5-methyl-4-isoxalone (AMPA)) but do not influence NMDA receptor-mediated responses. Additionally, it is shown that the activation of NMDA receptors is sufficient to elicit and maintain fictive locomotion after blockade of non-NMDA receptors with either DNQX or CNQX. Conversely, activation of quisqualate receptors with AMPA, but not quisqualate leads to fictive locomotion with properties much like that activated by kainate.

  11. A novel autoregulatory loop between the Gcn2-Atf4 pathway and L-Proline metabolism controls stem cell identity

    PubMed Central

    D'Aniello, C; Fico, A; Casalino, L; Guardiola, O; Di Napoli, G; Cermola, F; De Cesare, D; Tatè, R; Cobellis, G; Patriarca, E J; Minchiotti, G

    2015-01-01

    Increasing evidence indicates that metabolism is implicated in the control of stem cell identity. Here, we demonstrate that embryonic stem cell (ESC) behaviour relies on a feedback loop that involves the non-essential amino acid L-Proline (L-Pro) in the modulation of the Gcn2-Eif2α-Atf4 amino acid starvation response (AAR) pathway that in turn regulates L-Pro biosynthesis. This regulatory loop generates a highly specific intrinsic shortage of L-Pro that restricts proliferation of tightly packed domed-like ESC colonies and safeguards ESC identity. Indeed, alleviation of this nutrient stress condition by exogenously provided L-Pro induces proliferation and modifies the ESC phenotypic and molecular identity towards that of mesenchymal-like, invasive pluripotent stem cells. Either pharmacological inhibition of the prolyl-tRNA synthetase by halofuginone or forced expression of Atf4 antagonises the effects of exogenous L-Pro. Our data provide unprecedented evidence that L-Pro metabolism and the nutrient stress response are functionally integrated to maintain ESC identity. PMID:25857264

  12. Neuroprotection by Exendin-4 Is GLP-1 Receptor Specific but DA D3 Receptor Dependent, Causing Altered BrdU Incorporation in Subventricular Zone and Substantia Nigra

    PubMed Central

    Harkavyi, A.; Rampersaud, N.; Whitton, P. S.

    2013-01-01

    Glucagon-like peptide-1 receptor (GLP-1R) activation by exendin-4 (EX-4) is effective in preclinical models of Parkinson's disease (PD) and appears to promote neurogenesis even in severely lesioned rats. In the present study, we determined the effects of EX-4 on cellular BrdU incorporation in the rat subventricular zone (SVZ) and substantia nigra (SN). We also determined the specificity of this effect with the GLP-1R antagonist EX-(9-39) as well as the potential role of dopamine (DA) D3 receptors. Rats were administered 6-OHDA and 1 week later given EX-4 alone, with EX-(9-39) or nafadotride (D3 antagonist) and BrdU. Seven days later, rats were challenged with apomorphine to evaluate circling. Extracellular DA was measured using striatal microdialysis and subsequently tissue DA measured. Tyrosine hydroxylase and BrdU were verified using immunohistochemistry. Apomorphine circling was reversed by EX-4 in lesioned rats, an effect reduced by EX-4, while both EX-(9-39) and NAF attenuated this. 6-OHDA decreased extracellular and tissue DA, both reversed by EX-4 but again attenuated by EX-(9-39) or NAF. Analysis of BrdU+ cells in the SVZ revealed increases in 6-OHDA-treated rats which were reversed by EX-4 and antagonised by either EX-(9-39) or NAF, while in the SN the opposite profile was seen. PMID:26316987

  13. Role of capsaicin sensitive nerves in epidermal growth factor effects on gastric mucosal injury and blood flow

    PubMed Central

    Kang, J; Teng, C; Chen, F; Wee, A

    1998-01-01

    Background—Epidermal growth factor (EGF) and capsaicin protect against experimental gastric mucosal injury. Capsaicin exerts its gastroprotective effect by stimulating afferent neurones leading to release of calcitonin gene related peptide (CGRP) which causes gastric hyperaemia. EGF also causes gastric hyperaemia but whether it acts via capsaicin sensitive neurones is unknown. 
Aims—To assess the influence of: (1) capsaicin desensitisation on EGF effects on gastric mucosal injury and gastric mucosal blood flow; and (2) close arterial infusion of hCGRP8-37, a CGRP antagonist, on EGF effects on gastric mucosal blood flow. 
Methods—The absolute ethanol induced gastric mucosal injury model in the rat was used. Gastric mucosal damage was assessed by planimetry and light microscopy. Gastric mucosal blood flow was measured by laser Doppler flowmetry in a gastric chamber preparation. 
Results—Capsaicin desensitisation abolished the gastroprotective and gastric hyperaemic effects of EGF. Close arterial infusion of hCGRP8-37 antagonised the hyperaemic effect of both capsaicin and EGF. 
Conclusion—Results show that EGF may exert its gastroprotective and gastric hyperaemic effects via capsaicin sensitive afferent neurones. 

 Keywords: capsaicin; epidermal growth factor; gastric mucosal injury; gastric mucosal blood flow; calcitonin gene related peptide antagonist; rat PMID:9577339

  14. Histamine and betahistine in the treatment of vertigo: elucidation of mechanisms of action.

    PubMed

    Lacour, M; Sterkers, O

    2001-01-01

    The aim of this review is to provide clinicians with a picture of the mechanisms by which: histamine and histaminergic agonists act on the vestibular system both peripherally and centrally; and histaminergic agonists and antagonists interfere with the recovery process after peripheral vestibular lesion. We have focused on betahistine, a structural analogue of histamine with weak histamine H(1) receptor agonist and more potent H(3) receptor antagonist properties, to review the currently available data on the role of the histaminergic system in the recovery process after peripheral vestibular deficits and the effects of histamine analogues in the clinical treatment of vertigo. This review provides new insights into the basic mechanisms by which betahistine improves vestibular compensation in animal models of unilateral vestibular dysfunction, and elucidates particularly the mechanisms of action of this substance at the level of the CNS. It is proposed that betahistine may reduce peripherally the asymmetric functioning of the sensory vestibular organs in addition to increasing vestibulocochlear blood flow by antagonising local H(3) heteroreceptors. Betahistine acts centrally by enhancing histamine synthesis within tuberomammillary nuclei of the posterior hypothalamus and histamine release within vestibular nuclei through antagonism of H(3) autoreceptors. This mechanism, together with less specific effects of betahistine on alertness regulation through cerebral H(1) receptors, should promote and facilitate central vestibular compensation. Elucidation of the mechanisms of action of betahistine is of particular interest for the treatment of vestibular and cochlear disorders and vertigo.

  15. Anti-inflammatory, immunomodulatory and antinociceptive activity of Terminalia arjuna Roxb bark powder in mice and rats.

    PubMed

    Halder, Sumita; Bharal, Nidhi; Mediratta, Pramod K; Kaur, Inderjeet; Sharma, Krishna K

    2009-07-01

    Terminalia arjuna back powder (400 mg/kg, po) significantly reduced formalin-indued paw oedema at 24 h but not carrageenan-induced paw oedema. It significantly increased the anti-SRBC antibody titre in the secondary phase of immune response. The same dose significantly reduced the duration of licks and bites in both phases of formalin-induced pain response and showed significant increase in tail flick latency at higher dose (800 mg/kg, po). These effects of T. arjuna were antagonised by pretreatment with naloxone (1 mg/kg, ip). In another series of experiments, mice pretreated with morphine for three days in increasing doses (10, 15, 20 mg/kg, ip; twice daily) showed a decreased response in antinociceptive activity of morphine (5 mg/kg, ip). Further, cross tolerance was observed with T. arjuna (800 mg/kg, po) in morphine tolerant animals. These findings support the hypothesis that T. arjuna has anti-inflammatory potential against some phlogistic agents along with some immunomodulatory activity and also has antinococeptive action probably mediated via central opioid receptors.

  16. Integrated β-catenin, BMP, PTEN, and Notch signalling patterns the nephron

    PubMed Central

    Lindström, Nils O; Lawrence, Melanie L; Burn, Sally F; Johansson, Jeanette A; Bakker, Elvira RM; Ridgway, Rachel A; Chang, C-Hong; Karolak, Michele J; Oxburgh, Leif; Headon, Denis J; Sansom, Owen J; Smits, Ron; Davies, Jamie A; Hohenstein, Peter

    2015-01-01

    The different segments of the nephron and glomerulus in the kidney balance the processes of water homeostasis, solute recovery, blood filtration, and metabolite excretion. When segment function is disrupted, a range of pathological features are presented. Little is known about nephron patterning during embryogenesis. In this study, we demonstrate that the early nephron is patterned by a gradient in β-catenin activity along the axis of the nephron tubule. By modifying β-catenin activity, we force cells within nephrons to differentiate according to the imposed β-catenin activity level, thereby causing spatial shifts in nephron segments. The β-catenin signalling gradient interacts with the BMP pathway which, through PTEN/PI3K/AKT signalling, antagonises β-catenin activity and promotes segment identities associated with low β-catenin activity. β-catenin activity and PI3K signalling also integrate with Notch signalling to control segmentation: modulating β-catenin activity or PI3K rescues segment identities normally lost by inhibition of Notch. Our data therefore identifies a molecular network for nephron patterning. DOI: http://dx.doi.org/10.7554/eLife.04000.001 PMID:25647637

  17. Integrated β-catenin, BMP, PTEN, and Notch signalling patterns the nephron.

    PubMed

    Lindström, Nils O; Lawrence, Melanie L; Burn, Sally F; Johansson, Jeanette A; Bakker, Elvira R M; Ridgway, Rachel A; Chang, C-Hong; Karolak, Michele J; Oxburgh, Leif; Headon, Denis J; Sansom, Owen J; Smits, Ron; Davies, Jamie A; Hohenstein, Peter

    2015-02-03

    The different segments of the nephron and glomerulus in the kidney balance the processes of water homeostasis, solute recovery, blood filtration, and metabolite excretion. When segment function is disrupted, a range of pathological features are presented. Little is known about nephron patterning during embryogenesis. In this study, we demonstrate that the early nephron is patterned by a gradient in β-catenin activity along the axis of the nephron tubule. By modifying β-catenin activity, we force cells within nephrons to differentiate according to the imposed β-catenin activity level, thereby causing spatial shifts in nephron segments. The β-catenin signalling gradient interacts with the BMP pathway which, through PTEN/PI3K/AKT signalling, antagonises β-catenin activity and promotes segment identities associated with low β-catenin activity. β-catenin activity and PI3K signalling also integrate with Notch signalling to control segmentation: modulating β-catenin activity or PI3K rescues segment identities normally lost by inhibition of Notch. Our data therefore identifies a molecular network for nephron patterning.

  18. [Pharmacogenetics, pharmacogenomics and individualised antiepileptic therapy].

    PubMed

    Herranz, J L

    2006-10-10

    Despite the large number of antiepileptic drugs (AED) currently on the market, in 20-30% of patients it is still not possible to achieve a total control of the seizures or to prevent the appearance of idiosyncratic side effects. In this study the pharmacokinetic and pharmacodynamic factors involved in pharmacoresistance in epilepsies are analysed, with special attention given to the polymorphisms of metabolising enzymes or their inducers, particularly the hepatic cytochrome CYP, the over-expression of membrane-transporting proteins -P-glycoprotein (PGP), MRP- and polymorphisms in the ion channels. Idiosyncratic effects are produced due to an imbalance between the production of toxic metabolites and the individual capacity to detoxify them. Pharmacogenetics and pharmacogenomics can identify certain personal, biochemical, enzymatic and genetic characteristics that are of use in selecting the most effective AED with the lowest risk of idiosyncratic effects for each person. Although new AED that can help reduce the number of medication resistant patients must be introduced, at the same other therapeutic strategies have to be developed with the aid of pharmacogenomics; more specifically there is a need for AED that are not transported by PGP o MRP, or substances that antagonise those membrane transporters and make it possible for the AED to reach the site where they are to act. Furthermore, the detoxification capacity of each individual must be known in order to be able to minimise the risk of idiosyncratic side effects.

  19. Development of Novel Activin-Targeted Therapeutics

    PubMed Central

    Chen, Justin L; Walton, Kelly L; Al-Musawi, Sara L; Kelly, Emily K; Qian, Hongwei; La, Mylinh; Lu, Louis; Lovrecz, George; Ziemann, Mark; Lazarus, Ross; El-Osta, Assam; Gregorevic, Paul; Harrison, Craig A

    2015-01-01

    Soluble activin type II receptors (ActRIIA/ActRIIB), via binding to diverse TGF-β proteins, can increase muscle and bone mass, correct anemia or protect against diet-induced obesity. While exciting, these multiple actions of soluble ActRIIA/IIB limit their therapeutic potential and highlight the need for new reagents that target specific ActRIIA/IIB ligands. Here, we modified the activin A and activin B prodomains, regions required for mature growth factor synthesis, to generate specific activin antagonists. Initially, the prodomains were fused to the Fc region of mouse IgG2A antibody and, subsequently, “fastener” residues (Lys45, Tyr96, His97, and Ala98; activin A numbering) that confer latency to other TGF-β proteins were incorporated. For the activin A prodomain, these modifications generated a reagent that potently (IC50 5 nmol/l) and specifically inhibited activin A signaling in vitro, and activin A-induced muscle wasting in vivo. Interestingly, the modified activin B prodomain inhibited both activin A and B signaling in vitro (IC50 ~2 nmol/l) and in vivo, suggesting it could serve as a general activin antagonist. Importantly, unlike soluble ActRIIA/IIB, the modified prodomains did not inhibit myostatin or GDF-11 activity. To underscore the therapeutic utility of specifically antagonising activin signaling, we demonstrate that the modified activin prodomains promote significant increases in muscle mass. PMID:25399825

  20. PP2ACdc55’s role in reductional chromosome segregation during achiasmate meiosis in budding yeast is independent of its FEAR function

    PubMed Central

    Kerr, Gary W.; Wong, Jin Huei; Arumugam, Prakash

    2016-01-01

    PP2ACdc55 is a highly conserved serine-threonine protein phosphatase that is involved in diverse cellular processes. In budding yeast, meiotic cells lacking PP2ACdc55 activity undergo a premature exit from meiosis I which results in a failure to form bipolar spindles and divide nuclei. This defect is largely due to its role in negatively regulating the Cdc Fourteen Early Anaphase Release (FEAR) pathway. PP2ACdc55 prevents nucleolar release of the Cdk (Cyclin-dependent kinase)-antagonising phosphatase Cdc14 by counteracting phosphorylation of the nucleolar protein Net1 by Cdk. CDC55 was identified in a genetic screen for monopolins performed by isolating suppressors of spo11Δ spo12Δ lethality suggesting that Cdc55 might have a role in meiotic chromosome segregation. We investigated this possibility by isolating cdc55 alleles that suppress spo11Δ spo12Δ lethality and show that this suppression is independent of PP2ACdc55’s FEAR function. Although the suppressor mutations in cdc55 affect reductional chromosome segregation in the absence of recombination, they have no effect on chromosome segregation during wild type meiosis. We suggest that Cdc55 is required for reductional chromosome segregation during achiasmate meiosis and this is independent of its FEAR function. PMID:27455870

  1. Immunomodulation with dendritic cells and donor lymphocyte infusion converge to induce graft vs neuroblastoma reactions without GVHD after allogeneic bone marrow transplantation

    PubMed Central

    Ash, S; Stein, J; Askenasy, N; Yaniv, I

    2010-01-01

    Background: Mounting evidence points to the efficacy of donor lymphocyte infusion (DLI) and immunisation with tumour-pulsed dendritic cells (DC) in generating graft vs leukaemia reactions after allogeneic bone marrow transplantation (BMT). We assessed the efficacy of DLI and DC in generating potent graft vs neuroblastoma tumour (GVT) reactions following allogeneic BMT. Methods: Mice bearing congenic (H2Ka) Neuro-2a tumours were grafted with allogeneic (H2Kb) T-cell-depleted bone marrow cells. Tumour-pulsed donor DC (DCNeuro2a) were inoculated (on day +7) in conjunction with donor (H2Kb) and haploidentical (H2Ka/b) lymphocytes. Results: Murine Neuro-2a cells elicit immune reactions as efficient as B lymphoma in major histocompatibility complex antigen-disparate mice. Lymphopenia induced by conditioning facilitates GVT, and transition to adaptive immunity is enhanced by simultaneous infusion of and DCNeuro2a and lymphocytes devoid of graft vs host (GVH) activity (H2Ka/b). In variance, the efficacy of DC-mediated immunomodulation was diminished by severe graft vs host disease (GVHD), showing mechanistic dissociation and antagonising potential to GVT. Conclsions: The GVHD is not a prerequisite to induce GVT reactivity after allogeneic BMT, but is rather detrimental to induction of anti-tumour immunity by DC-mediated immunomodulation. Simultaneous inoculation of tumour-pulsed donor DC and DLI synergise in stimulation of potent GVT reactions to the extent of eradication of established NB tumours. PMID:20978501

  2. Phorate can reverse P450 metabolism-based herbicide resistance in Lolium rigidum.

    PubMed

    Busi, Roberto; Gaines, Todd Adam; Powles, Stephen

    2017-02-01

    Organophosphate insecticides can inhibit specific cytochrome P450 enzymes involved in metabolic herbicide resistance mechanisms, leading to synergistic interactions between the insecticide and the herbicide. In this study we report synergistic versus antagonistic interactions between the organophosphate insecticide phorate and five different herbicides observed in a population of multiple herbicide-resistant Lolium rigidum. Phorate synergised with three different herbicide modes of action, enhancing the activity of the ALS inhibitor chlorsulfuron (60% LD50 reduction), the VLCFAE inhibitor pyroxasulfone (45% LD50 reduction) and the mitosis inhibitor trifluralin (70% LD50 reduction). Conversely, phorate antagonised the two thiocarbamate herbicides prosulfocarb and triallate with a 12-fold LD50 increase. We report the selective reversal of P450-mediated metabolic multiple resistance to chlorsulfuron and trifluralin in the grass weed L. rigidum by synergistic interaction with the insecticide phorate, and discuss the putative mechanistic basis. This research should encourage diversity in herbicide use patterns for weed control as part of a long-term integrated management effort to reduce the risk of selection of metabolism-based multiple herbicide resistance in L. rigidum. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

  3. Coumestrol induces senescence through protein kinase CKII inhibition-mediated reactive oxygen species production in human breast cancer and colon cancer cells.

    PubMed

    Lee, Young-Hoon; Yuk, Heung Joo; Park, Ki-Hun; Bae, Young-Seuk

    2013-11-01

    An inhibitor of the protein kinase CKII (CKII) was purified from leaves of Glycine max (L.) Merrill and was identified as coumestrol by structural analysis. Coumestrol inhibited the phosphotransferase activity of CKII toward β-casein, with an IC50 of about 5 μM. It acted as a competitive inhibitor with respect to ATP as a substrate, with an apparent Ki value of 7.67 μM. Coumestrol at 50μM resulted in 50% and 30% growth inhibition of human breast cancer MCF-7 and colorectal cancer HCT116 cells, respectively. Coumestrol promoted senescence through the p53-p21(Cip1/WAF1) pathway by inducing reactive oxygen species (ROS) production in MCF-7 and HCT116 cells. The ROS scavenger N-acetyl-l-cysteine (NAC), NADPH oxidase inhibitor apocynin and p22(phox) siRNA almost completely abolished this event. Overexpression of CKIIα antagonised cellular senescence mediated by coumestrol, indicating that this compound induced senescence via a CKII-dependent pathway. Since senescence is an important tumour suppression process in vivo, these results suggest that coumestrol can function by inhibiting oncogenic disease, at least in part, through CKII inhibition-mediated cellular senescence. Copyright © 2013 Elsevier Ltd. All rights reserved.

  4. The antimicrobial effect of colistin methanesulfonate on Mycobacterium tuberculosis in vitro.

    PubMed

    van Breda, Shane Vontelin; Buys, Antoinette; Apostolides, Zeno; Nardell, Edward Anthony; Stoltz, Anton Carel

    2015-07-01

    Polymyxins have previously been described to have activity against Mycobacterium tuberculosis (MTB), but further research was abandoned due to systemic toxicity concerns to achieve the required MIC. Colistin methanesulfonate (CMS), a polymyxin, is well tolerated when inhaled directly into the lungs, resulting in high local concentrations. We report here for the first time, MIC and MBC data for CMS determined by the microtiter Alamar Blue assay (MABA). We also determined how the MIC would be affected by the presence of pulmonary surfactant (PS) and if any synergy with isoniazid (INH) and rifampicin (RIF) exists. The effect of CMS on the ultrastructure of MTB was also determined. The MIC for CMS was 16 mg/L, while the MBC was 256 mg/L. MIC for CMS in PS was antagonised by eight fold. For synergy, indifference was determined while time-kill assays revealed a greater killing effect when CMS was used together with INH. Ultrastructure analysis suggests that the disruption of the outer polysaccharide layer of MTB by CMS may lead to enhanced uptake of INH. Our findings may provide insight for further investigations of CMS against MTB.

  5. Potential Role of Caffeine in the Treatment of Parkinson’s Disease

    PubMed Central

    Roshan, Mohsin H.K.; Tambo, Amos; Pace, Nikolai P.

    2016-01-01

    Parkinson’s disease [PD] is the second most common neurodegenerative disorder after Alzheimer’s disease, affecting 1% of the population over the age of 55. The underlying neuropathology seen in PD is characterised by progressive loss of dopaminergic neurons in the substantia nigra pars compacta with the presence of Lewy bodies. The Lewy bodies are composed of aggregates of α-synuclein. The motor manifestations of PD include a resting tremor, bradykinesia, and muscle rigidity. Currently there is no cure for PD and motor symptoms are treated with a number of drugs including levodopa [L-dopa]. These drugs do not delay progression of the disease and often provide only temporary relief. Their use is often accompanied by severe adverse effects. Emerging evidence from both in vivo and in vitro studies suggests that caffeine may reduce parkinsonian motor symptoms by antagonising the adenosine A2A receptor, which is predominately expressed in the basal ganglia. It is hypothesised that caffeine may increase the excitatory activity in local areas by inhibiting the astrocytic inflammatory processes but evidence remains inconclusive. In addition, the co-administration of caffeine with currently available PD drugs helps to reduce drug tolerance, suggesting that caffeine may be used as an adjuvant in treating PD. In conclusion, caffeine may have a wide range of therapeutic effects which are yet to be explored, and therefore warrants further investigation in randomized clinical trials. PMID:27563362

  6. Estrogen receptor ERα plays a major role in ethanol-evoked myocardial oxidative stress and dysfunction in conscious female rats.

    PubMed

    Yao, Fanrong; Abdel-Rahman, Abdel A

    2016-02-01

    Our previous studies showed that ethanol elicited estrogen (E2)-dependent myocardial oxidative stress and dysfunction. In the present study we tested the hypothesis that E2 signaling via the estrogen receptor (ER), ERα, mediates this myocardial detrimental effect of alcohol. To achieve this goal, conscious female rats in proestrus phase (highest endogenous E2 level) received a selective ER antagonist (200 μg/kg; intra-venous [i.v.]) for ERα (MPP), ERβ (PHTPP) or GPER (G15) or saline 30 min before ethanol (1 g/kg; i.v.) or saline infusion. ERα blockade virtually abrogated ethanol-evoked myocardial dysfunction and hypotension, while ERβ blockade had little effect on the hypotensive response, but caused delayed attenuation of the ethanol-evoked reductions in left ventricular developed pressure and the rate of left ventricle pressure rise. GPER blockade caused delayed attenuation of all cardiovascular effects of ethanol. All three antagonists attenuated the ethanol-evoked increases in myocardial catalase and ALDH2 activities, Akt, ERK1/2, p38, eNOS, and nNOS phosphorylation, except for a lack of effect of PHTPP on p38. Finally, all three ER antagonists attenuated ethanol-evoked elevation in myocardial ROS, but this effect was most notable with ERα blockade. In conclusion, ERα plays a greater role in, and might serve as a molecular target for ameliorating, the E2-dependent myocardial oxidative stress and dysfunction caused by ethanol.

  7. MLL Histone Methylases Regulate Expression of HDLR-SR-B1 in Presence of Estrogen and Control Plasma Cholesterol in Vivo

    PubMed Central

    Ansari, Khairul I.; Kasiri, Sahba; Hussain, Imran; Bobzean, Samara A. Morris; Perrotti, Linda I.

    2013-01-01

    High-density lipoprotein receptors scavenger receptor class B type I [HDLR-SR-B1 (SR-B1)] is a key player in reverse cholesterol transport and maintaining blood cholesterol. We demonstrated that human SR-B1 is transcriptionally activated by 17β-estradiol (E2) in HEPG2 and JAR cells. SR-B1 promoter contains multiple estrogen response elements (ERE half-sites) along with some Sp1 binding sites. Knockdown of estrogen receptor (ER)α and ERβ down-regulated E2-induced SR-B1 expression. ERs were bound to SR-B1 promoter EREs in an E2-dependent manner. Along with ERs, mixed-lineage leukemia (MLL) histone methylases, especially MLL1 and MLL2, play key roles in E2-mediated SR-B1 activation. MLL1 and MLL2 bind to SR-B1 promoter in an E2-dependent manner and control the assembly of transcription pre-initiation complex and RNA polymerase II (RNAPII) recruitment. ERs and MLLs play critical roles in determining the cholesterol uptake by steroidogenic tissues/cells, and their knockdown suppressed the E2-induced cholesterol uptake efficiencies of the cells. Intriguingly, MLL2 knockdown in mice resulted in a 33% increase in plasma cholesterol level and also reduced SR-B1 expression in mice liver, demonstrating its crucial functions in controlling plasma cholesterol in vivo. PMID:23192982

  8. Novel interactions between the HTLV antisense proteins HBZ and APH-2 and the NFAR protein family: Implications for the HTLV lifecycles

    SciTech Connect

    Murphy, Jane; Hall, William W.; Ratner, Lee; Sheehy, Noreen

    2016-07-15

    The human T-cell leukaemia virus type 1 and type 2 (HTLV-1/HTLV-2) antisense proteins HBZ and APH-2 play key roles in the HTLV lifecycles and persistence in the host. Nuclear Factors Associated with double-stranded RNA (NFAR) proteins NF90/110 function in the lifecycles of several viruses and participate in host innate immunity against infection and oncogenesis. Using GST pulldown and co-immunoprecipitation assays we demonstrate specific novel interactions between HBZ/APH-2 and NF90/110 and characterised the protein domains involved. Moreover we show that NF90/110 significantly enhance Tax mediated LTR activation, an effect that was abolished by HBZ but enhanced by APH-2. Additionally we found that HBZ and APH-2 modulate the promoter activity of survivin and are capable of antagonising NF110-mediated survivin activation. Thus interactions between HTLV antisense proteins and the NFAR protein family have an overall positive impact on HTLV infection. Hence NFARs may represent potential therapeutic targets in HTLV infected cells. - Highlights: • This study demonstrates for the first time interactions between NF90/110 and the HTLV antisense proteins HBZ and APH-2. • We show that NF90/110 significantly enhance LTR activation by the HTLV Tax protein, an effect that is abolished by HBZ but enhanced by APH-2. • The study shows that even though the HTLV antisense proteins activate survivin expression they antagonize the ability of NF90/110 to do so. • Overall we found that NF90/110 positively regulate HTLV infection and as such might represent a therapeutic target in infected cells.

  9. The role of TRPM8 in the Guinea-pig bladder-cooling reflex investigated using a novel TRPM8 antagonist.

    PubMed

    Gardiner, Jennifer C; Kirkup, Anthony J; Curry, John; Humphreys, Sian; O'Regan, Paul; Postlethwaite, Michael; Young, Kimberley C; Kitching, Linda; Ethell, Brian T; Winpenny, David; McMurray, Gordon

    2014-10-05

    Patients with overactive bladder often exhibit abnormal bladder contractions in response to intravesical cold saline (positive ice-water test). The molecular entity involved in cold sensation within the urinary bladder is unknown, but a potential candidate is the ion channel, transient receptor potential (melastatin)-8 (TRPM8). The objective of the present study was to investigate the role of TRPM8 in a bladder-cooling reflex evoked in anaesthetised guinea-pigs that is comparable to the positive ice-water test seen in patients. Guinea-pig TRPM8 was cloned from L6 dorsal root ganglia (DRG) and expressed in HEK293 cells. Functional agonist- and cold-induced Ca2+ influx and electrophysiology assays were performed in these cells, and for comparison in HEK293 cells expressing human TRPM8, using a novel TRPM8 antagonist, the S-enantiomer of 1-phenylethyl 4-(benzyloxy)-3-methoxybenzyl (2-aminoethyl) carbamate hydrochloride (PBMC). Potency data from these assays was used to calculate intravenous infusion protocols for targeted plasma concentrations of PBMC in studies on micturition reflexes evoked by intravesical infusion of menthol or cold saline in anaesthetised guinea-pigs. Tissue expression of TRPM8 in guinea-pig bladder, urethra and in dorsal root ganglia neurones traced from the bladder was also investigated. TRPM8 mRNA and protein were detected in L6 dorsal root ganglia, bladder urothelium and smooth muscle. PBMC antagonised in vitro activation of human and guinea-pig TRPM8 and reversed menthol and cold-induced facilitation of the micturition reflex at plasma concentrations consistent with in vitro potencies. The present data suggest that the bladder-cooling reflex in the guinea-pig involves TRPM8. The potential significance of TRPM8 in bladder disease states deserves future investigation. Copyright © 2014 Elsevier B.V. All rights reserved.

  10. SSR504734 enhances basal expression of prepulse inhibition but exacerbates the disruption of prepulse inhibition by apomorphine

    PubMed Central

    Singer, Philipp; Zhang, Weining; Yee, Benjamin K.

    2013-01-01

    Rationale Inhibition of glycine transporter 1 (GlyT1) elevates extracellular glycine and can thus increase N-methyl-D-aspartate receptor (NMDAR) excitability in the brain. The potent GlyT1 inhibitor, SSR504734, has also been shown to potentiate the behavioural effects of direct and indirect dopamine agonists. Thus, an acute systemic dose of SSR504734 was sufficient to exacerbate the motor-stimulant effect of the dopamine releaser amphetamine in C57BL/6 mice, even though SSR504734 alone exerted no significant effect on motor activity. Objectives Here, we explore if SSR504734 might modulate dopamine-dependent sensory gating in the paradigm of prepulse inhibition (PPI) of the acoustic startle reflex. Methods Experiment 1 characterized the effect of SSR504734 (10 and 30 mg/kg i.p.) on PPI expression when administered alone. Experiments 2 and 3 investigated the impact of SSR504734 when administered in conjunction with the dopamine receptor agonist, apomorphine (1 and 2 mg/kg s.c.), which is known to reliably disrupt PPI. Results When administered alone, acute SSR504734 enhanced PPI only at 30 mg/kg – a dose that has been shown to improve cognitive functions including working memory, which has been linked to enhanced NMDAR function resulting from the elevation of extracellular glycine. However, this effect did not allow SSR504734 to antagonise the PPI-disruptive effect of apomorphine. At the lower dose of 10 mg/kg – that was insufficient to enhance PPI when administered alone - SSR504734 even exacerbated the deleterious effect of apomorphine on PPI. Conclusions The therapeutic potential of GlyT1 inhibition against distinct behavioural/cognitive deficiency might require different magnitudes of GlyT1 inhibition. [246 words] PMID:23736281

  11. Silicon central pattern generators for cardiac diseases

    PubMed Central

    Nogaret, Alain; O'Callaghan, Erin L; Lataro, Renata M; Salgado, Helio C; Meliza, C Daniel; Duncan, Edward; Abarbanel, Henry D I; Paton, Julian F R

    2015-01-01

    Cardiac rhythm management devices provide therapies for both arrhythmias and resynchronisation but not heart failure, which affects millions of patients worldwide. This paper reviews recent advances in biophysics and mathematical engineering that provide a novel technological platform for addressing heart disease and enabling beat-to-beat adaptation of cardiac pacing in response to physiological feedback. The technology consists of silicon hardware central pattern generators (hCPGs) that may be trained to emulate accurately the dynamical response of biological central pattern generators (bCPGs). We discuss the limitations of present CPGs and appraise the advantages of analog over digital circuits for application in bioelectronic medicine. To test the system, we have focused on the cardio-respiratory oscillators in the medulla oblongata that modulate heart rate in phase with respiration to induce respiratory sinus arrhythmia (RSA). We describe here a novel, scalable hCPG comprising physiologically realistic (Hodgkin–Huxley type) neurones and synapses. Our hCPG comprises two neurones that antagonise each other to provide rhythmic motor drive to the vagus nerve to slow the heart. We show how recent advances in modelling allow the motor output to adapt to physiological feedback such as respiration. In rats, we report on the restoration of RSA using an hCPG that receives diaphragmatic electromyography input and use it to stimulate the vagus nerve at specific time points of the respiratory cycle to slow the heart rate. We have validated the adaptation of stimulation to alterations in respiratory rate. We demonstrate that the hCPG is tuneable in terms of the depth and timing of the RSA relative to respiratory phase. These pioneering studies will now permit an analysis of the physiological role of RSA as well as its any potential therapeutic use in cardiac disease. PMID:25433077

  12. Silicon central pattern generators for cardiac diseases.

    PubMed

    Nogaret, Alain; O'Callaghan, Erin L; Lataro, Renata M; Salgado, Helio C; Meliza, C Daniel; Duncan, Edward; Abarbanel, Henry D I; Paton, Julian F R

    2015-02-15

    Cardiac rhythm management devices provide therapies for both arrhythmias and resynchronisation but not heart failure, which affects millions of patients worldwide. This paper reviews recent advances in biophysics and mathematical engineering that provide a novel technological platform for addressing heart disease and enabling beat-to-beat adaptation of cardiac pacing in response to physiological feedback. The technology consists of silicon hardware central pattern generators (hCPGs) that may be trained to emulate accurately the dynamical response of biological central pattern generators (bCPGs). We discuss the limitations of present CPGs and appraise the advantages of analog over digital circuits for application in bioelectronic medicine. To test the system, we have focused on the cardio-respiratory oscillators in the medulla oblongata that modulate heart rate in phase with respiration to induce respiratory sinus arrhythmia (RSA). We describe here a novel, scalable hCPG comprising physiologically realistic (Hodgkin-Huxley type) neurones and synapses. Our hCPG comprises two neurones that antagonise each other to provide rhythmic motor drive to the vagus nerve to slow the heart. We show how recent advances in modelling allow the motor output to adapt to physiological feedback such as respiration. In rats, we report on the restoration of RSA using an hCPG that receives diaphragmatic electromyography input and use it to stimulate the vagus nerve at specific time points of the respiratory cycle to slow the heart rate. We have validated the adaptation of stimulation to alterations in respiratory rate. We demonstrate that the hCPG is tuneable in terms of the depth and timing of the RSA relative to respiratory phase. These pioneering studies will now permit an analysis of the physiological role of RSA as well as its any potential therapeutic use in cardiac disease. © 2014 The Authors. The Journal of Physiology © 2014 The Physiological Society.

  13. The neuroprotective action of the mood stabilizing drugs lithium chloride and sodium valproate is mediated through the up-regulation of the homeodomain protein Six1

    SciTech Connect

    Plant, Kathryn E.; Anderson, Elizabeth; Simecek, Nicole; Brown, Richard; Forster, Sam; Spinks, Jenny; Toms, Nick; Gibson, G. Gordon; Lyon, Jon; Plant, Nick

    2009-02-15

    The mood stabilizing agents lithium chloride (LiCl) and sodium valproate (VPA) have recently gained interest as potential neuroprotective therapeutics. However, exploitation of these therapeutic applications is hindered by both a lack of molecular understanding of the mode of action, and a number of sub-optimal properties, including a relatively small therapeutic window and variable patient response. Human neuroblastoma cells (SH-SY5Y) were exposed to 1 mM lithium chloride or 1 mM sodium valproate for 6 h or 72 h, and transcriptomes measured by Affymetrix U133A/B microarray. Statistically significant gene expression changes were identified using SAM software, with selected changes confirmed at transcript (TaqMan) and protein (Western blotting) levels. Finally, anti-apoptotic action was measured by an in vitro fluorescent assay. Exposure of SH-SY5Y cells to therapeutically relevant concentrations of either lithium chloride or sodium valproate elicited 936 statistically significant changes in gene expression. Amongst these changes we observed a large (maximal 31.3-fold) increase in the expression of the homeodomain protein Six1, and have characterized the time- and dose-dependent up-regulation of this gene in response to both drugs. In addition, we demonstrate that, like LiCl or VPA treatment, Six1 over-expression protects SH-SY5Y cells from staurosporine-induced apoptosis via the blockade of caspsase-3 activation, whereas removal of Six1 protein via siRNA antagonises the ability of LiCl and VPA to protect SH-SY5Y cells from STS-induced apoptosis. These results provide a novel mechanistic rationale underlying the neuroprotective mechanism of LiCl and VPA, suggesting exciting possibilities for the development of novel therapeutic agents against neurodegenerative diseases such as Alzheimer's or Parkinsonism.

  14. Structural insights into the interaction between the Cripto CFC domain and the ALK4 receptor.

    PubMed

    Calvanese, Luisa; Saporito, Angela; Oliva, Romina; D' Auria, Gabriella; Pedone, Carlo; Paolillo, Livio; Ruvo, Menotti; Marasco, Daniela; Falcigno, Lucia

    2009-03-01

    The protein Cripto is the founding member of the extra-cellular EGF-CFC growth factors, which are composed of two adjacent cysteine-rich domains: the EGF-like and the CFC. Members of the EGF-CFC family play key roles in embryonic development and are also implicated in tumourigenesis. Cripto is highly over-expressed in many tumours, while it is poorly detectable in normal tissues. Although both Cripto domains are involved in its tumourigenic activity, the CFC domain appears to play a crucial role. Indeed, through this domain, Cripto interferes with the onco-suppressive activity of Activins, either by blocking the Activin receptor ALK4 or by antagonising proteins of the TGF-beta family. We have undertaken the chemical synthesis and the structural characterisation of human CFC Cripto domain. Using a combined NMR and computational approach, supported by binding studies by SPR, we have investigated the molecular basis of the interaction between h-CFC and ALK4. Binding studies indicate that the synthetic h-CFC interacts with the ALK4 receptor with a K(D) in micro M range, whereas it does not recognise the ActRIIB receptor. The NMR study shows that the h-CFC overall topology is determined by the presence of three disulfide bridges and that residues H120 and W124 are located between the first strand and the first loop with the side chains externally exposed. A model of the CFC-ALK4 complex has also been obtained by molecular docking and shows that all residues indicated by prior mutagenesis studies can contribute to the ALK4-CFC interaction at the protein-protein interface.

  15. Novel interactions of the mineralocorticoid receptor.

    PubMed

    Fuller, Peter J

    2015-06-15

    The mineralocorticoid receptor (MR) differs from the other steroid receptors in that it responds to two physiological ligands, aldosterone and cortisol. In epithelial tissues, aldosterone selectivity is determined by 11β-hydroxysteroid dehydrogenase type II. In other tissues cortisol is the primary ligand; in some tissues cortisol may act as an antagonist. To better target MR, an understanding of the structural determinants of tissue and ligand-specific MR activation is required. Our focus is on interactions of the ligand-binding domain (LBD) with ligand, the N-terminal domain and putative co-regulatory molecules. Molecular modelling has identified a region in the LBD of the MR and indeed other steroid receptors that critically defines ligand-specificity for aldosterone and cortisol, yet is not part of the ligand-binding pocket. An interaction between the N-terminus and LBD observed in the MR is aldosterone-dependent but is unexpectedly antagonised by cortisol. The structural basis of this interaction has been defined. We have identified proteins which interact in the presence of either aldosterone or cortisol but not both. These have been confirmed as coactivators of the full-length hMR. The structural basis of this interaction has been determined for tesmin, a ligand-discriminant coactivator of the MR. The successful identification of the structural basis of antagonism and of ligand-specific interactions of the MR may provide the basis for the development of novel MR ligands with tissue specificity. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  16. miR-193a-3p interaction with HMGB1 downregulates human endothelial cell proliferation and migration

    PubMed Central

    Khoo, Cheen P.; Roubelakis, Maria G.; Schrader, Jack B.; Tsaknakis, Grigorios; Konietzny, Rebecca; Kessler, Benedikt; Harris, Adrian L.; Watt, Suzanne M.

    2017-01-01

    Circulating endothelial colony forming cells (ECFCs) contribute to vascular repair where they are a target for therapy. Since ECFC proliferative potential is increased in cord versus peripheral blood and to define regulatory factors controlling this proliferation, we compared the miRNA profiles of cord blood and peripheral blood ECFC-derived cells. Of the top 25 differentially regulated miRNAs selected, 22 were more highly expressed in peripheral blood ECFC-derived cells. After validating candidate miRNAs by q-RT-PCR, we selected miR-193a-3p for further investigation. The miR-193a-3p mimic reduced cord blood ECFC-derived cell proliferation, migration and vascular tubule formation, while the miR-193a-3p inhibitor significantly enhanced these parameters in peripheral blood ECFC-derived cells. Using in silico miRNA target database analyses combined with proteome arrays and luciferase reporter assays of miR-193a-3p mimic treated cord blood ECFC-derived cells, we identified 2 novel miR-193a-3p targets, the high mobility group box-1 (HMGB1) and the hypoxia upregulated-1 (HYOU1) gene products. HMGB1 silencing in cord blood ECFC-derived cells confirmed its role in regulating vascular function. Thus, we show, for the first time, that miR-193a-3p negatively regulates human ECFC vasculo/angiogenesis and propose that antagonising miR-193a-3p in less proliferative and less angiogenic ECFC-derived cells will enhance their vasculo/angiogenic function. PMID:28276476

  17. Dicumarol, an inhibitor of ADP-ribosylation of CtBP3/BARS, fragments golgi non-compact tubular zones and inhibits intra-golgi transport.

    PubMed

    Mironov, Alexander A; Colanzi, Antonino; Polishchuk, Roman S; Beznoussenko, Galina V; Mironov, Alexander A; Fusella, Aurora; Di Tullio, Giuseppe; Silletta, Maria Giuseppina; Corda, Daniela; De Matteis, Maria Antonietta; Luini, Alberto

    2004-07-01

    Dicumarol (3,3'-methylenebis[4-hydroxycoumarin]) is an inhibitor of brefeldin-A-dependent ADP-ribosylation that antagonises brefeldin-A-dependent Golgi tubulation and redistribution to the endoplasmic reticulum. We have investigated whether dicumarol can directly affect the morphology of the Golgi apparatus. Here we show that dicumarol induces the breakdown of the tubular reticular networks that interconnect adjacent Golgi stacks and that contain either soluble or membrane-associated cargo proteins. This results in the formation of 65-120-nm vesicles that are sometimes invaginated. In contrast, smaller vesicles (45-65 nm in diameter, a size consistent with that of coat-protein-I-dependent vesicles) that excluded cargo proteins from their lumen are not affected by dicumarol. All other endomembranes are largely unaffected by dicumarol, including Golgi stacks, the ER, multivesicular bodies and the trans-Golgi network. In permeabilized cells, dicumarol activity depends on the function of CtBP3/BARS protein and pre-ADP-ribosylation of cytosol inhibits the breakdown of Golgi tubules by dicumarol. In functional experiments, dicumarol markedly slows down intra-Golgi traffic of VSV-G transport from the endoplasmic reticulum to the medial Golgi, and inhibits the diffusional mobility of both galactosyl transferase and VSV-G tagged with green fluorescent protein. However, it does not affect: transport from the trans-Golgi network to the cell surface; Golgi-to-endoplasmic reticulum traffic of ERGIC58; coat-protein-I-dependent Golgi vesiculation by AlF4 or ADP-ribosylation factor; or ADP-ribosylation factor and beta-coat protein binding to Golgi membranes. Thus the ADP-ribosylation inhibitor dicumarol induces the selective breakdown of the tubular components of the Golgi complex and inhibition of intra-Golgi transport. This suggests that lateral diffusion between adjacent stacks has a role in protein transport through the Golgi complex.

  18. Corticosterone and dopamine D2/D3 receptors mediate the motivation for voluntary wheel running in C57BL/6J mice.

    PubMed

    Ebada, Mohamed Elsaed; Kendall, David A; Pardon, Marie-Christine

    2016-09-15

    Physical exercise can improve cognition but whether this is related to motivation levels is unknown. Voluntary wheel running is a rewarding activity proposed as a model of motivation to exercise. To question the potential effects of exercise motivation on subsequent behaviour, we used a pharmacological approach targeting some reward mechanisms. The stress hormone corticosterone has rewarding effects mediated by activation of low affinity glucocorticoid receptors (GR). To investigate whether corticosterone synthesis motivates exercise via activation of GRs and subsequently, impacts on behaviour, we treated C57BL/6J mice acutely with the inhibitor of corticosterone synthesis metyrapone (35mg/kg) or repeatedly with the GR antagonist mifepristone (30mg/kg) prior to 1-h running wheel sessions. To investigate whether reducing motivation to exercise impacts on behaviour, we antagonised running-induced dopamine D2/D3 receptors activation with sulpiride (25 or 50mg/kg) and assessed locomotor, anxiety-related and memory performance after 20 running sessions over 4 weeks. We found that corticosterone synthesis contributes to running levels, but the maintenance of running behaviour was not mediated by activation of GRs. Intermittent exercise was not associated with changes in behavioural or cognitive performance. The persistent reduction in exercise levels triggered by sulpiride also had limited impact on behavioural performance, although the level of performance for some behaviours was related to the level of exercise. Altogether, these findings indicate that corticosterone and dopamine D2/D3 receptor activation contribute to the motivation for wheel running, but suggest that motivation for exercise is not a sufficient factor to alter behaviour in healthy mice.

  19. miR-193a-3p interaction with HMGB1 downregulates human endothelial cell proliferation and migration.

    PubMed

    Khoo, Cheen P; Roubelakis, Maria G; Schrader, Jack B; Tsaknakis, Grigorios; Konietzny, Rebecca; Kessler, Benedikt; Harris, Adrian L; Watt, Suzanne M

    2017-03-09

    Circulating endothelial colony forming cells (ECFCs) contribute to vascular repair where they are a target for therapy. Since ECFC proliferative potential is increased in cord versus peripheral blood and to define regulatory factors controlling this proliferation, we compared the miRNA profiles of cord blood and peripheral blood ECFC-derived cells. Of the top 25 differentially regulated miRNAs selected, 22 were more highly expressed in peripheral blood ECFC-derived cells. After validating candidate miRNAs by q-RT-PCR, we selected miR-193a-3p for further investigation. The miR-193a-3p mimic reduced cord blood ECFC-derived cell proliferation, migration and vascular tubule formation, while the miR-193a-3p inhibitor significantly enhanced these parameters in peripheral blood ECFC-derived cells. Using in silico miRNA target database analyses combined with proteome arrays and luciferase reporter assays of miR-193a-3p mimic treated cord blood ECFC-derived cells, we identified 2 novel miR-193a-3p targets, the high mobility group box-1 (HMGB1) and the hypoxia upregulated-1 (HYOU1) gene products. HMGB1 silencing in cord blood ECFC-derived cells confirmed its role in regulating vascular function. Thus, we show, for the first time, that miR-193a-3p negatively regulates human ECFC vasculo/angiogenesis and propose that antagonising miR-193a-3p in less proliferative and less angiogenic ECFC-derived cells will enhance their vasculo/angiogenic function.

  20. Resveratrol protects against doxorubicin-induced cardiotoxicity in aged hearts through the SIRT1-USP7 axis

    PubMed Central

    Sin, Thomas K; Tam, Bjorn T; Yung, Benjamin Y; Yip, Shea P; Chan, Lawrence W; Wong, Cesar S; Ying, Michael; Rudd, John A; Siu, Parco M

    2015-01-01

    A compromised cardiac function is often seen in elderly cancer patients receiving doxorubicin therapy. The present study tested the hypothesis that acute intervention with resveratrol, a natural anti-oxidant found in grapes and red wine, reduces the cardiotoxicity of doxorubicin through restoration of sirtuin 1 (SIRT1) deacetylase activity, and attenuation of the catabolic/apoptotic pathways orchestrated by USP7, a p53 deubiquitinating protein, using young (aged 2 months) and old (aged 10 months) senescence-accelerated mice prone 8 (SAMP8). Animals were randomised to receive saline, doxorubicin, and doxorubicin in combination with resveratrol, in the presence or absence of SIRT1 inhibitors, sirtinol or EX527. Resveratrol alone, but not in combination with either of the SIRT1 inhibitors, suppressed the doxorubicin-induced impairment of cardiac systolic function in aged animals. Doxorubicin reduced SIRT1 deacetylase activity, and elevated proteasomal activity and USP7; it also increased the protein level of p300 and ubiquitinated proteins in hearts from aged SAMP8. These doxorubicin-induced alterations were prevented by resveratrol, whereas the protective action of resveratrol was antagonised by sirtinol and EX527. In young SAMP8 hearts, resveratrol attenuated the doxorubicin-induced increases in acetylation of Foxo1 and transactivation of MuRF-1, whereas these mitigations were not found after treatment with SIRT1 inhibitors. However, the protein contents of acetylated Foxo1 and MuRF-1 were not affected by any of the drugs studied in aged SAMP8 hearts. Resveratrol also ameliorated the augmentation of pro-apoptotic markers including p53, Bax, caspase 3 activity and apoptotic DNA fragmentation induced by doxorubicin in hearts from aged animals, whereas these reductions were diminished by combined treatment with SIRT1 inhibitors. These data demonstrate that resveratrol ameliorates doxorubicin-induced cardiotoxicity in aged hearts through the restoration of SIRT1

  1. Evaluation of trypanocidal activity of combinations of anti-sleeping sickness drugs with cysteine protease inhibitors.

    PubMed

    Steverding, Dietmar

    2015-01-01

    Chemotherapy of human African trypanosomiasis (HAT) is unsatisfactory because only a few drugs, with serious side effects and poor efficacy, are available. As drug combination regimes often achieve greater therapeutic efficacy than monotherapies, here the trypanocidal activity of the cysteine protease inhibitor K11777 in combination with current anti-HAT drugs using bloodstream forms of Trypanosoma brucei was investigated. Isobolographic analysis was used to determine the interaction between cysteine protease inhibitors (K11777, CA-074Me and CAA0225) and anti-HAT drugs (suramin, pentamidine, melarsoprol and eflornithine). Bloodstream forms of T. brucei were incubated in culture medium containing cysteine protease inhibitors or anti-HAT drugs alone or in combination at a 1:1 fixed-dose ratio. After 48 h incubation, live cells were counted, the 50% growth inhibition values determined and combination indices calculated. The general cytotoxicity of drug combinations was evaluated with human leukaemia HL-60 cells. Combinations of K11777 with suramin, pentamidine and melarsoprol showed antagonistic effects while with eflornithine a synergistic effect was observed. Whereas eflornithine antagonises with CA-074Me, an inhibitor inactivating the targeted TbCATL only under reducing conditions, it synergises with CAA0255, an inhibitor structurally related to CA-074Me which inactivates TbCATL independently of thiols. These findings indicate an essential role of thiols for the synergistic interaction between K11777 and eflornithine. Encouragingly, the K11777/eflornithine combination displayed higher trypanocidal than cytotoxic activity. The results of this study suggest that the combination of the cysteine protease inhibitor K11777 and eflornithine display promising synergistic trypanocidal activity that warrants further investigation of the drug combination as possible alternative treatment of HAT.

  2. Anxiolytic activity evaluation of four medicinal plants from Cameroon.

    PubMed

    Bum, E Ngo; Soudi, S; Ayissi, E R; Dong, C; Lakoulo, N H; Maidawa, F; Seke, P F E; Nanga, L D; Taiwe, G S; Dimo, T; Njikam, Njifutie; Rakotonirina, A; Rakotonirina, S V; Kamanyi, A

    2011-01-01

    Afrormosia laxiflora (A. laxiflora), Chenopodium ambrosioides (C. ambrosioides), Microglossa pyrifolia (M. pyrifolia) and Mimosa pudica (M. pudica) are plants used in traditional medicine in Cameroon to treat insomnia, epilepsy, anxiety, and agitation. They were evaluated for their anxiolytic like activity in mice. Animal models (elevated plus maze and stress-induced hyperthermia tests) were used. The four plants showed anxiolytic activity. In stress-induced hyperthermia test, A. laxiflora, C. ambrosioides, M. pyrifolia and M. pudica significantly antagonised the increase of temperature. ΔT° decreased from 0.75°C in the control group to 0.36°C at the dose of 110 mg/kg for A. laxiflora; from 1°C in the control group to -1.1°C at the dose of 120 mg/kg for C. ambrosioides; from 1.7°C in the control group to 0.2°C at the dose of 128 mg/kg for M. pyrifolia and from 1.3°C in the control group to 0.5°C at the dose of 180 mg/kg for M. pudica. In the elevated plus maze test, the four plants increased the number of entries into, percentage of entries into, and percentage of time in open arms. A. laxiflora, C. ambrosioides and M. pudica also reduced the percentage of entries and time in closed arms. In addition, C. ambrosioides, M. pyrifolia and M. pudica showed antipyretic activity by reducing the body temperature. The results suggested that C. ambrosioides, M. pyrifolia and M. pudica posses anxiolytic-like and antipyretic activities while A. laxiflora possesses only anxiolytic-like properties. These plants could be helpful in the treatment of anxiety and fever in traditional medicine in Cameroon.

  3. Identification and Functional Analysis of a Novel Tryptophyllin Peptide from the Skin of the Red-eye Leaf Frog, Agalychnis callidryas

    PubMed Central

    Wang, Ran; Zhou, Yu; Chen, Tianbao; Zhou, Mei; Wang, Lei; Shaw, Chris

    2015-01-01

    Amphibian skin has proved repeatedly to be a largely untapped source of bioactive peptides and this is especially true of members of the Phyllomedusinae subfamily of frogs native to South and Central America. Tryptophyllins are a group of peptides mainly found in the skin of members of this genus. In this study, a novel tryptophyllin (TPH) type 3 peptide, named AcT-3, has been isolated and structurally-characterised from the skin secretion and lyophilised skin extract of the red-eye leaf frog, Agalychnis callidryas. The peptide was identified in and purified from the skin secretion by reverse-phase HPLC. MALDI-TOF mass spectrometry and MS/MS fragmentation sequencing established its primary structure as: pGlu-Gly-Lys-Pro-Tyr-Trp-Pro-Pro-Pro-Phe-Leu-Pro-Glu, with a non-protonated molecular mass of 1538.19Da. The mature peptide possessed the canonical N-terminal pGlu residue that arises from post-translational modification of a Gln residue. The deduced open-reading frame consisted of 63 amino acid residues encoding a highly-conserved signal peptide of approximately 22 amino acid residues, an intervening acidic spacer peptide domain, a single AcT-3 encoding domain and a C terminal processing site. A synthetic replicate of AcT-3 was found to antagonise the effect of BK on rat tail artery smooth muscle and to contract the intestinal smooth muscle preparations. It was also found that AcT-3 could dose-dependently inhibit the proliferation of human prostate cancer cell lines after 72h incubation. PMID:25561903

  4. Uridine Triphosphate Thio Analogues Inhibit Platelet P2Y12 Receptor and Aggregation

    PubMed Central

    Gündüz, Dursun; Tanislav, Christian; Sedding, Daniel; Parahuleva, Mariana; Santoso, Sentot; Troidl, Christian; Hamm, Christian W.; Aslam, Muhammad

    2017-01-01

    Platelet P2Y12 is an important adenosine diphosphate (ADP) receptor that is involved in agonist-induced platelet aggregation and is a valuable target for the development of anti-platelet drugs. Here we characterise the effects of thio analogues of uridine triphosphate (UTP) on ADP-induced platelet aggregation. Using human platelet-rich plasma, we demonstrate that UTP inhibits P2Y12 but not P2Y1 receptors and antagonises 10 µM ADP-induced platelet aggregation in a concentration-dependent manner with an IC50 value of ~250 µM. An eight-fold higher platelet inhibitory activity was observed with a 2-thio analogue of UTP (2S-UTP), with an IC50 of 30 µM. The 4-thio analogue (4S-UTP) with an IC50 of 7.5 µM was 33-fold more effective. A three-fold decrease in inhibitory activity, however, was observed by introducing an isobutyl group at the 4S- position. A complete loss of inhibition was observed with thio-modification of the γ phosphate of the sugar moiety, which yields an enzymatically stable analogue. The interaction of UTP analogues with P2Y12 receptor was verified by P2Y12 receptor binding and cyclic AMP (cAMP) assays. These novel data demonstrate for the first time that 2- and 4-thio analogues of UTP are potent P2Y12 receptor antagonists that may be useful for therapeutic intervention. PMID:28146050

  5. The effect of obestatin on anxiety-like behaviour in mice.

    PubMed

    Szakács, Júlia; Csabafi, Krisztina; Lipták, Nándor; Szabó, Gyula

    2015-10-15

    Obestatin is a 23 amino acid-peptide, derived from the same preproghrelin-gene as ghrelin. Obestatin was originally reported as a ghrelin antagonist with anorexigenic activity, but later it was proven to be involved in multiple processes including sleep, memory retention, anxiety, morphine-induced analgesia and withdrawal. In the present study, in male CFLP mice, by using computerised open field (OF) and elevated plus maze (EPM) tests we have investigated the behavioural effects of the acute intracerebroventricular (icv) administration of obestatin alone, and following ghrelin receptor blockage with [d-Lys3]-Growth Hormone Releasing Peptide-6 ([d-Lys3]- GHRP6) or corticotropin-releasing hormone (CRH) receptor 1 antagonism with antalarmin. Plasma corticosterone levels were measured for each treatment group by using chemofluorescent assay. Our results in the EPM test showed that obestatin reduced the percent of time spent in the open arms. The basal locomotor activity (ambulation distance and time, rearing and jumping) was not influenced significantly neither in the obestatin-treated groups, nor in those receiving pre-treatment with antalarmin or [d-Lys3]-GHRP6. The percentage of central ambulation distance however was decreased by obestatin, while the percentage of time spent in the central zone showed a decreasing tendency. The administration of antalarmin or [d-Lys3]-GHRP6 have both reversed the effect of obestatin on central ambulation. Plasma corticosterone levels were elevated by obestatin, which effect was antagonised by the injection of antalarmin. These are the first results to indicate that obestatin exerts anxiogenic-like effect in mice, which might be mediated through ghrelin receptor and CRH activation.

  6. The ghrelin/obestatin balance in the physiological and pathological control of growth hormone secretion, body composition and food intake.

    PubMed

    Hassouna, R; Zizzari, P; Tolle, V

    2010-07-01

    Ghrelin and obestatin are two gastrointestinal peptides obtained by post-translational processing of a common precursor, preproghrelin. Ghrelin is an orexigenic and adipogenic peptide and a potent growth hormone secretagogue (GHS) modified by the enzyme ghrelin-O-acyl-transferase to bind and activate its receptor, the GHS-R. The ghrelin/GHS-R pathway is complex and the effects of ghrelin on GH secretion, adiposity and food intake appear to be relayed by distinct mechanisms involving different transduction signals and constitutive activity for the GH-R, different cofactors as modulators of endogenous ghrelin signalling and/or alternative ghrelin receptors. The discovery of obestatin in 2005 brought an additional level of complexity to this fascinating system. Obestatin was initially identified as an anorexigenic peptide and as the cognate ligand for GPR39, but its effect on food intake and its ability to activate GPR39 are still controversial. Although several teams failed to reproduce the anorexigenic actions of obestatin, this peptide has been shown to antagonise GH secretion and food intake induced by ghrelin and could be an interesting pharmacological tool to counteract the actions of ghrelin. Ghrelin and obestatin immunoreactivities are recovered in the blood with an ultradian pulsatility and their concentrations in plasma vary with the nutritional status of the body. It is still a matter of debate whether both hormones are regulated by independent mechanisms and whether obestatin is a physiologically relevant peptide. Nevertheless, a significant number of studies show that the ghrelin/obestatin ratio is modified in anorexia nervosa and obesity. This suggests that the ghrelin/obestatin balance could be essential to adapt the body's response to nutritional challenges. Although measuring ghrelin and obestatin in plasma is challenging because many forms of the peptides circulate, more sensitive and selective assays to detect the different preproghrelin

  7. PGE2 receptor EP3 inhibits water reabsorption and contributes to polyuria and kidney injury in a streptozotocin-induced mouse model of diabetes.

    PubMed

    Hassouneh, Ramzi; Nasrallah, Rania; Zimpelmann, Joe; Gutsol, Alex; Eckert, David; Ghossein, Jamie; Burns, Kevin D; Hébert, Richard L

    2016-06-01

    The first clinical manifestation of diabetes is polyuria. The prostaglandin E2 (PGE2) receptor EP3 antagonises arginine vasopressin (AVP)-mediated water reabsorption and its expression is increased in the diabetic kidney. The purpose of this work was to study the contribution of EP3 to diabetic polyuria and renal injury. Male Ep 3 (-/-) (also known as Ptger3 (-/-)) mice were treated with streptozotocin (STZ) to generate a mouse model of diabetes and renal function was evaluated after 12 weeks. Isolated collecting ducts (CDs) were microperfused to study the contribution of EP3 to AVP-mediated fluid reabsorption. Ep 3 (-/-)-STZ mice exhibited attenuated polyuria and increased urine osmolality compared with wild-type STZ (WT-STZ) mice, suggesting enhanced water reabsorption. Compared with WT-STZ mice, Ep 3 (-/-)-STZ mice also had increased protein expression of aquaporin-1, aquaporin-2, and urea transporter A1, and reduced urinary AVP excretion, but increased medullary V2 receptors. In vitro microperfusion studies indicated that Ep 3 (-/-) and WT-STZ CDs responded to AVP stimulation similarly to those of wild-type mice, with a 60% increase in fluid reabsorption. In WT non-injected and WT-STZ mice, EP3 activation with sulprostone (PGE2 analogue) abrogated AVP-mediated water reabsorption; this effect was absent in mice lacking EP3. A major finding of this work is that Ep 3 (-/-)-STZ mice showed blunted renal cyclooxygenase-2 protein expression, reduced renal hypertrophy, reduced hyperfiltration and reduced albuminuria, as well as diminished tubular dilation and nuclear cysts. Taken together, the data suggest that EP3 contributes to diabetic polyuria by inhibiting expression of aquaporins and that it promotes renal injury during diabetes. EP3 may prove to be a promising target for more selective management of diabetic kidney disease.

  8. PPAR-α and -γ but not -δ agonists inhibit airway inflammation in a murine model of asthma: in vitro evidence for an NF-κB-independent effect

    PubMed Central

    Trifilieff, Alexandre; Bench, Anne; Hanley, Marcus; Bayley, Debbie; Campbell, Emma; Whittaker, Paul

    2003-01-01

    Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors that have been proposed to regulate inflammation by antagonising the nuclear factor-κB (NF-κB) signalling pathway. We investigated the role of PPARs using synthetic agonists in murine models of airway inflammation, and addressed the possible effect on NF-κB signalling in vitro using a human epithelial cell line, A549. Sensitised BALB/c mice exposed to an aerosol solution of ovalbumin had an increased number of airway eosinophils, neutrophils and lymphocytes. When given intranasally an hour before the aerosol challenge, a PPAR-α (GW 9578) and PPAR-γ (GI 262570) selective agonist as well as a dual PPAR-α/γ (GW 2331) agonist selectively inhibited allergen-induced bronchoalveolar lavage eosinophil and lymphocyte but not neutrophil influx. In contrast, a PPAR-δ agonist (GW 501516) was inactive. When given intranasally an hour before challenge, PPAR-α and PPAR-γ selective agonists as well as a dual PPAR-α/γ agonist did not inhibit lipopolysaccharide-induced bronchoalveolar lavage neutrophil influx or tumour necrosis factor-α (TNF-α) and KC production. In A549 cells, selective agonists for PPAR-α, -γ and -δ did not inhibit intracellular adhesion molecule-1 expression following stimulation with proinflammatory cytokines. In addition, IL-8 release and the activation of an NF-κB-responsive reporter gene construct were inhibited only at micromolar concentrations, suggesting that these effects were not PPAR-mediated. Our in vivo data show that agonists of PPAR-α and -γ, but not -δ, inhibit allergen-induced bronchoalveolar lavage eosinophil and lymphocyte influx. In vitro data suggest that this effect might not be mediated by antagonism of the NF-κB pathway. PMID:12746235

  9. Identification and functional analysis of a novel tryptophyllin peptide from the skin of the red-eye leaf frog, Agalychnis callidryas.

    PubMed

    Wang, Ran; Zhou, Yu; Chen, Tianbao; Zhou, Mei; Wang, Lei; Shaw, Chris

    2015-01-01

    Amphibian skin has proved repeatedly to be a largely untapped source of bioactive peptides and this is especially true of members of the Phyllomedusinae subfamily of frogs native to South and Central America. Tryptophyllins are a group of peptides mainly found in the skin of members of this genus. In this study, a novel tryptophyllin (TPH) type 3 peptide, named AcT-3, has been isolated and structurally-characterised from the skin secretion and lyophilised skin extract of the red-eye leaf frog, Agalychnis callidryas. The peptide was identified in and purified from the skin secretion by reverse-phase HPLC. MALDI-TOF mass spectrometry and MS/MS fragmentation sequencing established its primary structure as: pGlu-Gly-Lys-Pro-Tyr-Trp-Pro-Pro-Pro-Phe-Leu-Pro-Glu, with a non-protonated molecular mass of 1538.19Da. The mature peptide possessed the canonical N-terminal pGlu residue that arises from post-translational modification of a Gln residue. The deduced open-reading frame consisted of 63 amino acid residues encoding a highly-conserved signal peptide of approximately 22 amino acid residues, an intervening acidic spacer peptide domain, a single AcT-3 encoding domain and a C terminal processing site. A synthetic replicate of AcT-3 was found to antagonise the effect of BK on rat tail artery smooth muscle and to contract the intestinal smooth muscle preparations. It was also found that AcT-3 could dose-dependently inhibit the proliferation of human prostate cancer cell lines after 72h incubation.

  10. Molecular characterisation of Porcine rubulavirus (PorPV) isolates from different outbreaks in Mexico.

    PubMed

    Cuevas-Romero, S; Rivera-Benítez, J F; Blomström, A-L; Ramliden, M; Hernández-Baumgarten, E; Hernández-Jáuregui, P; Ramírez-Mendoza, H; Berg, M

    2016-02-01

    Since the report of the initial outbreak of Porcine rubulavirus (PorPV) infection in pigs, only one full-length genome from 1984 (PorPV-LPMV/1984) has been characterised. To investigate the overall genetic variation, full-length gene nucleotide sequences of current PorPV isolates were obtained from different clinical cases of infected swine. Genome organisation and sequence analysis of the encoded proteins (NP, P, F, M, HN and L) revealed high sequence conservation of the NP protein and the expression of the P and V proteins in all PorPV isolates. The V protein of one isolate displayed a mutation that has been implicated to antagonise the antiviral immune responses of the host. The M protein indicated a variation in a short region that could affect the electrostatic charge and the interaction with the membrane. One PorPV isolate recovered from the lungs showed a mutation at the cleavage site (HRKKR) of the F protein that could represent an important factor to determine the tissue tropism and pathogenicity of this virus. The HN protein showed high sequence identity through the years (up to 2013). Additionally, a number of sequence motifs of very high amino acid conservation among the PorPV isolates important for polymerase activity of the L protein have been identified. In summary, genetic comparisons and phylogenetic analyses indicated that three different genetic variants of PorPV are currently spreading within the swine population, and a new generation of circulating virus with different characteristics has begun to emerge.

  11. FGF23 activates injury-primed renal fibroblasts via FGFR4-dependent signalling and enhancement of TGF-β autoinduction.

    PubMed

    Smith, Edward R; Holt, Stephen G; Hewitson, Tim D

    2017-09-15

    Bone-derived fibroblast growth factor 23 (FGF23) is an important endocrine regulator of mineral homeostasis with effects transduced by cognate FGF receptor (FGFR)1-α-Klotho complexes. Circulating FGF23 levels rise precipitously in patients with kidney disease and portend worse renal and cardiovascular outcomes. De novo expression of FGF23 has been found in the heart and kidney following injury but its significance remains unclear. Studies showing that exposure to chronically high FGF23 concentrations activates hypertrophic gene programs in the cardiomyocyte has spawned intense interest in other pathological off-target effects of FGF23 excess. In the kidney, observational evidence points to a concordance of ectopic renal FGF23 expression and the activation of local transforming growth factor (TGF)-β signalling. Although we have previously shown that FGF23 activates injury-primed renal fibroblasts in vitro, our understanding of the mechanism underpinning these effects was incomplete. Here we show that in the absence of α-Klotho, FGF23 augments pro-fibrotic signalling cascades in injury-primed renal fibroblasts via activation of FGFR4 and upregulation of the calcium transporter, transient receptor potential cation channel 6. The resultant rise in intracellular calcium and production of mitochondrial reactive oxygen species induced expression of NFAT responsive-genes and enhanced TGF-β1 autoinduction through non-canonical JNK-dependent pathways. Reconstitution with transmembrane α-Klotho, or its soluble ectodomain, restored classical Egr signalling and antagonised FGF23-driven myofibroblast differentiation. Thus, renal FGF23 may amplify local myofibroblast activation in injury and perpetuate pro-fibrotic signalling. These findings strengthen the rationale for exploring therapeutic inhibition of FGFR4 or restoration of α-Klotho as upstream regulators of off-target FGF23 effects. Copyright © 2017 The Author(s). Published by Elsevier Ltd.. All rights reserved.

  12. The endocrine disrupting potential of monosodium glutamate (MSG) on secretion of the glucagon-like peptide-1 (GLP-1) gut hormone and GLP-1 receptor interaction.

    PubMed

    Shannon, Maeve; Green, Brian; Willars, Gary; Wilson, Jodie; Matthews, Natalie; Lamb, Joanna; Gillespie, Anna; Connolly, Lisa

    2017-01-04

    Monosodium glutamate (MSG) is a suspected obesogen with epidemiological evidence positively correlating consumption to increased body mass index and higher prevalence of metabolic syndrome. ELISA and high content analysis (HCA) were employed to examine the disruptive effects of MSG on the secretion of enteroendocrine hormone glucagon-like peptide-1 (GLP-1) and GLP-1 receptor (GLP-1R), respectively. Following 3h MSG exposure of the enteroendocrine pGIP/neo: STC-1 cell line model (500μg/ml) significantly increased GLP-1 secretion (1.8 fold; P≤0.001), however, 72h exposure (500μg/ml) caused a 1.8 fold decline (P≤0.05). Also, 3h MSG exposure (0.5-500μg/ml) did not induce any cytotoxicity (including multiple pre-lethal markers) but 72h exposure at 250-500μg/ml, decreased cell number (11.8-26.7%; P≤0.05), increased nuclear area (23.9-29.8%; P≤0.001) and decreased mitochondrial membrane potential (13-21.6%; P≤0.05). At 500μg/ml, MSG increased mitochondrial mass by 16.3% (P≤0.01). MSG did not agonise or antagonise internalisation of the GLP-1R expressed recombinantly in U2OS cells, following GLP-1 stimulation. In conclusion, 72h exposure of an enteroendocrine cell line at dietary levels of MSG, results in pre-lethal cytotoxicity and decline in GLP-1 secretion. These adverse events may play a role in the pathogenesis of obesity as outlined in the obesogen hypothesis by impairing GLP-1 secretion, related satiety responses and glucose-stimulated insulin release.

  13. FGF stimulation of the Erk1/2 signalling cascade triggers transition of pluripotent embryonic stem cells from self-renewal to lineage commitment.

    PubMed

    Kunath, Tilo; Saba-El-Leil, Marc K; Almousailleakh, Marwa; Wray, Jason; Meloche, Sylvain; Smith, Austin

    2007-08-01

    Pluripotent embryonic stem (ES) cells must select between alternative fates of self-replication and lineage commitment during continuous proliferation. Here, we delineate the role of autocrine production of fibroblast growth factor 4 (Fgf4) and associated activation of the Erk1/2 (Mapk3/1) signalling cascade. Fgf4 is the major stimulus activating Erk in mouse ES cells. Interference with FGF or Erk activity using chemical inhibitors or genetic ablations does not impede propagation of undifferentiated ES cells. Instead, such manipulations restrict the ability of ES cells to commit to differentiation. ES cells lacking Fgf4 or treated with FGF receptor inhibitors resist neural and mesodermal induction, and are refractory to BMP-induced non-neural differentiation. Lineage commitment potential of Fgf4-null cells is restored by provision of FGF protein. Thus, FGF enables rather than antagonises the differentiation activity of BMP. The key downstream role of Erk signalling is revealed by examination of Erk2-null ES cells, which fail to undergo either neural or mesodermal differentiation in adherent culture, and retain expression of pluripotency markers Oct4, Nanog and Rex1. These findings establish that Fgf4 stimulation of Erk1/2 is an autoinductive stimulus for naïve ES cells to exit the self-renewal programme. We propose that the Erk cascade directs transition to a state that is responsive to inductive cues for germ layer segregation. Consideration of Erk signalling as a primary trigger that potentiates lineage commitment provides a context for reconciling disparate views on the contribution of FGF and BMP pathways during germ layer specification in vertebrate embryos.

  14. Iron depletion suppresses mTORC1-directed signalling in intestinal Caco-2 cells via induction of REDD1.

    PubMed

    Watson, Ailsa; Lipina, Christopher; McArdle, Harry J; Taylor, Peter M; Hundal, Harinder S

    2016-05-01

    Iron is an indispensable micronutrient that regulates many aspects of cell function, including growth and proliferation. These processes are critically dependent upon signalling via the mammalian or mechanistic target of rapamycin complex 1 (mTORC1). Herein, we test whether iron depletion induced by cell incubation with the iron chelator, deferoxamine (DFO), mediates its effects on cell growth through mTORC1-directed signalling and protein synthesis. We have used Caco-2 cells, a well-established in vitro model of human intestinal epithelia. Iron depletion increased expression of iron-regulated proteins (TfR, transferrin receptor and DMT1, divalent metal transporter, as predicted, but it also promoted a marked reduction in growth and proliferation of Caco-2 cells. This was strongly associated with suppressed mTORC1 signalling, as judged by reduced phosphorylation of mTOR substrates, S6K1 and 4E-BP1, and diminished protein synthesis. The reduction in mTORC1 signalling was tightly coupled with increased expression and accumulation of REDD1 (regulated in DNA damage and development 1) and reduced phosphorylation of Akt and TSC2. The increase in REDD1 abundance was rapidly reversed upon iron repletion of cells but was also attenuated by inhibitors of gene transcription, protein phosphatase 2A (PP2A) and by REDD1 siRNA--strategies that also antagonised the loss in mTORC1 signalling associated with iron depletion. Our findings implicate REDD1 and PP2A as crucial regulators of mTORC1 activity in iron-depleted cells and indicate that their modulation may help mitigate atrophy of the intestinal mucosa that may occur in response to iron deficiency. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  15. Preclinical pharmacology of bilastine, a new selective histamine H1 receptor antagonist: receptor selectivity and in vitro antihistaminic activity.

    PubMed

    Corcóstegui, Reyes; Labeaga, Luis; Innerárity, Ana; Berisa, Agustin; Orjales, Aurelio

    2005-01-01

    This study aimed to establish the receptor selectivity and antihistaminic activity of bilastine, a new selective antihistamine receptor antagonist. In vitro experiments were conducted using a receptor binding screening panel and guinea-pig and rat tissues. Antihistaminic activity was determined using H1 receptor binding studies and in vitro H1 antagonism studies conducted in guinea-pig tissues and human cell lines. Receptor selectivity was established using a receptor binding screening panel and a receptor antagonism screening conducted in guinea-pig, rat and rabbit tissues. Inhibition of inflammatory mediators was determined through the Schultz-Dale reaction in sensitised guinea-pig ileum. Bilastine binds to histamine H1-receptors as indicated by its displacement of [3H]-pyrilamine from H1-receptors expressed in guinea-pig cerebellum and human embryonic kidney (HEK) cell lines. The studies conducted on guinea-pig smooth muscle demonstrated the capability of bilastine to antagonise H1-receptors. Bilastine is selective for histamine H1-receptors as shown in receptor-binding screening conducted to determine the binding capacity of bilastine to 30 different receptors. The specificity of its H1-receptor antagonistic activity was also demonstrated in a series of in vitro experiments conducted on guinea-pig and rat tissues. The results of these studies confirmed the lack of significant antagonism against serotonin, bradykinin, leukotriene D4, calcium, muscarinic M3-receptors, alpha1-adrenoceptors, beta2-adrenoceptors, and H2- and H3-receptors. The results of the in vitro Schultz-Dale reaction demonstrated that bilastine also has anti-inflammatory activity. These preclinical studies provide evidence that bilastine has H1- antihistamine activity, with high specificity for H1-receptors, and poor or no affinity for other receptors. Bilastine has also been shown to have anti-inflammatory properties.

  16. A review of therapies for diabetic macular oedema and rationale for combination therapy

    PubMed Central

    Amoaku, W M K; Saker, S; Stewart, E A

    2015-01-01

    Diabetic macular oedema (DMO) is responsible for significant visual impairment in diabetic patients. The primary cause of DMO is fluid leakage resulting from increased vascular permeability through contributory anatomical and biochemical changes. These include endothelial cell (EC) death or dysfunction, pericyte loss or dysfunction, thickened basement membrane, loss or dysfunction of glial cells, and loss/change of EC Glycocalyx. The molecular changes include increased reactive oxygen species, pro-inflammatory changes: advanced glycation end products, intracellular adhesion molecule-1, Complement 5–9 deposition and cytokines, which result in increased paracellular permeability, tight junction disruption, and increased transcellular permeability. Laser photocoagulation has been the mainstay of treatment until recently when pharmacological treatments were introduced. The current treatments for DMO target reducing vascular leak in the macula once it has occurred, they do not attempt to treat the underlying pathology. These pharmacological treatments are aimed at antagonising vascular endothelial growth factor (VEGF) or non-VEGF inflammatory pathways, and include intravitreal injections of anti-VEGFs (ranibizumab, aflibercept or bevacizumab) or steroids (fluocinolone, dexamethasone or triamcinolone) as single therapies. The available evidence suggests that each individual treatment modality in DMO does not result in a completely dry macula in most cases. The ideal treatment for DMO should improve vision and improve morphological changes in the macular (eg, reduce macular oedema) for a significant duration, reduced adverse events, reduced treatment burden and costs, and be well tolerated by patients. This review evaluates the individual treatments available as monotherapies, and discusses the rationale and potential for combination therapy in DMO. A comprehensive review of clinical trials related to DMO and their outcomes was completed. Where phase III randomised

  17. The antipsychotics clozapine and olanzapine increase plasma glucose and corticosterone levels in rats: comparison with aripiprazole, ziprasidone, bifeprunox and F15063.

    PubMed

    Assié, Marie-Bernadette; Carilla-Durand, Elisabeth; Bardin, Laurent; Maraval, Mireille; Aliaga, Monique; Malfètes, Nathalie; Barbara, Michèle; Newman-Tancredi, Adrian

    2008-09-11

    Several novel antipsychotics activate serotonin 5-HT1A receptors as well as antagonising dopamine D2/3 receptors. Such a pharmacological profile is associated with a lowered liability to produce extrapyramidal side effects and enhanced efficacy in treating negative and cognitive symptoms of schizophrenia. However, 5-HT1A receptor agonists increase plasma corticosterone and many antipsychotics disturb the regulation of glucose. Here, we compared the influence on plasma glucose and corticosterone of acute treatments with 'new generation' antipsychotics which target dopamine D2/3 receptors and 5-HT1A receptors, with that of atypical antipsychotics, and with haloperidol. Olanzapine and clozapine, antipsychotics that are known to produce weight gain and diabetes in humans, both at 10 mg/kg p.o., substantially increased plasma glucose (from 0.8 to 1.7 g/l) at 1 h after administration, an effect that returned to control levels after 4 h. In comparison, F15063 (40 mg/kg p.o.) was without effect at any time point. Olanzapine and clozapine dose-dependently increased plasma glucose concentrations as did SLV313 and SSR181507. Haloperidol and risperidone had modest effects whereas aripiprazole, ziprasidone and bifeprunox, antipsychotics that are not associated with metabolic dysfunction in humans, and F15063 had little or no influence on plasma glucose. The same general pattern of response was found for plasma corticosterone levels. The present data provide the first comparative study of conventional, atypical and 'new generation' antipsychotics on glucose and corticosterone levels in rats. A variety of mechanisms likely underlie the hyperglycemia and corticosterone release observed with clozapine and olanzapine, whilst the balance of dopamine D2/3/5-HT1A interaction may contribute to the less favourable impact of SLV313 and SSR181507 compared with that of bifeprunox and F15063.

  18. Milk-derived bioactive peptides inhibit human endothelial-monocyte interactions via PPAR-γ dependent regulation of NF-κB.

    PubMed

    Marcone, Simone; Haughton, Karen; Simpson, Paul J; Belton, Orina; Fitzgerald, Desmond J

    2015-01-01

    Milk-derived bioactive peptides retain many biological properties and have therapeutic effects in cardiovascular disorders such as atherosclerosis. Under inflammatory conditions the expression of endothelial cells adhesion molecules is induced, increasing monocyte adhesion to human vessel wall, a critical step in the pathogenesis of atherosclerosis. In the present work we explored the effects of milk-derived bioactive peptides on the expression of the inflammatory phenotype of human endothelial cells and their effects on monocyte adherence to endothelial cells. Treatment of endothelial cells with milk-derived hydrolysate inhibited their production of inflammatory proteins MCP-1 and IL-8 and expression of VCAM-1, ICAM-1 and E-selectin. Milk derived hydrolysate also attenuated the adhesion of human monocytes to activated endothelial cells. The effect was similar to that obtained in endothelial cells treated with troglitazone, a ligand of peroxisome proliferators-activator receptor-gamma (PPAR-γ). PPAR-γ is a transcription factor which when activated antagonises the pro-inflammatory capability of nuclear factor κB (NF-κB). We further examined whether the effects of milk-derived hydrolysates on endothelial cells may be mediated through NF-κB activation via a PPAR-γ dependent mechanism. The specific PPAR-γ inhibitor, GW9662 blocked the effects of the hydrolysate on the NF-κB-mediated chemokines and adhesion molecules expression in endothelial cells. These results suggest that milk-derived bioactive peptides work as anti-atherogenic agents through the inhibition of endothelial-dependent adhesive interactions with monocytes by inhibiting the NF-κB pathway through a PPAR-γ dependent mechanism.

  19. Nutrition health issues in self-reported postpartum depression

    PubMed Central

    Mortimore, Denise; Snow, Sarah

    2011-01-01

    Aim In this retrospective survey women with and without self-reported postpartum depression (PPD) were compared in regards to consumption-frequency of foods and supplements rich in nutrients beneficial to nervous system (NS) health, in regards to consumption-frequency of compounds which may counteract the effect of the above and in regards to nutritional support provided to them during a pregnancy between 2003 and 2008. Background Postpartum depression (PPD) is defined as a major depressive episode that begins within 1 month of delivery and is experienced by roughly 13% of mothers. Patients and methods Four Hundred participants were recruited through the internet. Data gathered via multiple choice questionnaires was statistically analyzed using SPSS and Statistical software; statistical procedures included discriminant analysis, Pearson's product moment correlation, independent t-test and cross-tabulations. Results Out of 400 participants 83 (20.8%) were affected by self-reported depression after a pregnancy between 2003 and 2008. Depressed subjects consumed oily fish and offal significantly more often than non depressed subjects. Depression was more prevalent among women with vegetarian diets. No significant difference concerning food group intake or the ratios between foods rich in nutrients beneficial to NS health and foods rich in compounds antagonising their effect were found between depressed and non depressed subjects. Iron supplementation correlated positively with zinc supplementation in both groups. Roughly 70% of women reported to have received no information about n-3 fatty acid fish oils during pregnancy; informed subjects consumed fish oils more often. The majority of subjects with self-reported depression described nutritional support during pregnancy as inadequate. Conclusion Within this Austrian sample, the prevalence rate of postpartum depression was high; while the consumption of oily fish and vegetarian diets negatively correlated with

  20. General pharmacological properties of a new non-opiate antitussive: zipeprol (3024 CERM). I. Action on respiratory function and acute toxicity.

    PubMed

    Rispat, G; Burgi, H; Cosnier, D; Duchêne-Marullaz, P; Streichenberger, G

    1976-04-01

    1-(2-Methoxy-2-phenyl)-ethyl-4-(2-hydroxy-3-methoxy-3-phenyl)-propyl-iperazine-dihydrochloride (zipeprol, Respilene) is a substance of non-phenanthrenic chemical structure. In the cat, it antagonised cough induced by stimulation of the superior laryngeal nerve or by direct mechanical excitation of the sensitive tracheo-bronchial receptors. The efficacy of zipeprol after enteral administration made it possible both to establish good intestinal absorption and to rank it favourably in relation to several major antitussive reference products; codeine, codethyline, dextromethorphan, diphenhydramine and pentoxyverine. The activity of zipeprol was superior or equal to that of all these substances, excdept codeine. The antitussive properties appeared to be due to a central action. Other properties have been demonstrated which suggest at least a supplementary mechanism in the inhibition of cough, in addition to the central action. These consisted of slight antihistamine and anticholinergic properties, marked local-anesthetic potency and bronchospasmolytic activity. This latter property was demonstrated by the inhibition of histamine and serotonin induced bronchospasm in the guinea-pig. In vitro, using human sputum, zipeprol had a mucolytic action, shown by a decrease in sputum vis viscosity and lysis of DNA and AMPS fibrils. In the dog, at high doses, zipeprol unlike codeine, did not inhibit central stimulation of respiration by hypercapnia, in addition no modification of ventilatory dynamics or blood gases was seen. On the basis of these results, zipeprol can be considered as possessing no respiratory depressant effect even in the upper ranges of its antitussive doses.

  1. Occurrence of a unique protein toxin from the Indian King Cobra (Ophiophagus hannah) venom.

    PubMed

    Gomes, A; De, P; Dasgupta, S C

    2001-01-01

    A unique (lethal-cardiotoxic-hemorrhagic) protein toxin (Toxin CM55) was isolated and purified from Indian King Cobra (Ophiophagus hannah) venom by CM-sephadex ion exchange chromatography and reverse phase HPLC. The purified toxin had an SDS-molecular weight of 22 +/- 0.5 kD. UV absorption spectra of Toxin CM55 showed a peak at 280 nm, whereas when excited at 280 nm fluorescence, Toxin CM55 showed an E(max) at 333.4 nm. Toxin CM55 had an LD(50) of 28.28 microg/20 g (i. v.) in albino mice. The cardiotoxic action of the toxin was established on isolated guinea pig/rabbit heart and guinea pig auricle. In rats, Toxin CM55 caused ECG abnormalities including widened QRS complex and monomorphic ventricular tachycardia suggesting that the possible site of action of Toxin CM55 was the ventricle. Toxin CM55 produced significant vasoconstriction on peripheral blood vessels. It produced significant contraction of isolated guinea pig ileum, rat fundus and rat uterus, which was completely antagonised by methysergide. The toxin was found to release a significant amount of serotonin from rabbit platelets. Toxin CM55 produced cutaneous hemorrhage in albino mice, which was also produced in reserpine and p-chloro phenylalanine pretreated animals. Rabbit antiserum was raised against Toxin CM55, which gave prominent bands in immunogel diffusion and immunoelectrophoresis. The antiserum provided 2 LD(50) protection against Toxin CM55-induced lethality in mice and also neutralised 3 MHD hemorrhagic dose of the toxin.

  2. Bolus injection of human UII in conscious rats evokes a biphasic haemodynamic response

    PubMed Central

    Gardiner, Sheila M; March, Julie E; Kemp, Philip A; Bennett, Terence

    2004-01-01

    A biphasic cardiovascular response to bolus i.v. injection of human urotensin II (hUII, 3 nmol kg−1) in conscious, male, Sprague–Dawley (SD) rats was identified and underlying mechanisms were explored. Initially (0–5 min) there was tachycardia, hypotension and mesenteric and hindquarters vasodilatation; later (30–120 min), tachycardia, hindquarters vasodilatation and a modest rise in blood pressure occurred. Pretreatment with indomethacin or NG nitro-L-arginine methylester (L-NAME) reduced the mesenteric vasodilator response to hUII, and abolished the late tachycardia and hindquarters vasodilatation. Indomethacin also abolished the hypotension and early hindquarters vasodilatation, and substantially reduced the initial tachycardia. Indomethacin and L-NAME together prevented all haemodynamic responses to hUII. Inhibition of inducible NOS had no effect on responses to hUII, whereas inhibition of neuronal NOS reduced the delayed tachycardic response to hUII but did not significantly affect the vasodilatation. Only the initial tachycardic response to hUII was antagonised by propranolol. In spontaneously hypertensive rats (SHR), the initial haemodynamic responses to hUII were qualitatively similar to those in SD rats, although there was also a modest renal vasodilatation. The secondary response comprised a smaller tachycardia and a small rise in blood pressure, with no significant hindquarters vasodilatation. Haemodynamic responses to hUII were not enhanced by endothelin and angiotensin receptor antagonism in either SD rats or in SHRs. One interpretation of these results is that the primary response to bolus injection of hUII is prostanoid- or prostanoid- and NO-mediated (mesenteric vasodilatation) and that this triggers secondary events, which are dependent on eNOS (hindquarters vasodilatation) and neuronal NOS (tachycardia). PMID:15339862

  3. Wfs1-deficient mice display impaired behavioural adaptation in stressful environment.

    PubMed

    Luuk, Hendrik; Plaas, Mario; Raud, Sirli; Innos, Jürgen; Sütt, Silva; Lasner, Helena; Abramov, Urho; Kurrikoff, Kaido; Kõks, Sulev; Vasar, Eero

    2009-03-17

    Wfs1-deficient mice were generated by disrupting the 8th exon of Wfs1 gene. Reproduction rates of homozygous Wfs1-deficient mice were slightly below the expected values, they displayed intolerance to glucose and overall lower body weight. The present behavioural study was performed in female Wfs1-deficient mice due to their milder metabolic disturbances. Non-fasting blood glucose levels did not differ between homozygous Wfs1-deficient mice and wild-type littermates. While there was no difference in baseline plasma corticosterone, exposure to stress induced a nearly three-fold elevation of corticosterone in Wfs1-deficient mice in relation to wild-type littermates. Wfs1-deficient mice did not display obvious shortcomings in sensory and motor functioning as exemplified by intact responses in conditioned learning paradigms and rota-rod test. Locomotor activity of Wfs1-deficient mice was significantly lower only in brightly lit environment. Short-term isolation had a significant anxiogenic-like effect on the behaviour of Wfs1-deficient mice in dark/light exploration test. Lower exploratory activity of Wfs1-deficient mice in the plus-maze was antagonised by pre-treatment with diazepam (1 mg/kg), a GABA(A) receptor agonist. Wfs1-deficient mice displayed increased anxiety-like behaviour in hyponeophagia test. The locomotor stimulatory effects of amphetamine (2.5-7.5 mg/kg) and apomorphine (3 mg/kg) were significantly attenuated and facilitated, respectively, in Wfs1-deficient mice. There were no differences between Wfs1-deficient mice and wild-types in forced swimming behaviour and conditioned fear responses. Subtle impairments in reversal learning were apparent in Wfs1-deficient mice in the Morris water maze. Altogether, the present study demonstrates impaired behavioural adaptation of Wfs1-deficient mice in stress-inducing situations. It is likely that Wfs1 protein plays a major role in the behavioural adaptation mechanisms to novel and stressful environments.

  4. Betamethasone, progesterone and RU-486 (mifepristone) exert similar effects on connexin expression in trophoblast-derived HTR-8/SVneo cells.

    PubMed

    Cervellati, F; Pavan, B; Lunghi, L; Manni, E; Fabbri, E; Mascoli, C; Biondi, C; Patella, A; Vesce, F

    2011-01-01

    Connexins (Cx) are membrane proteins able to influence cell trophoblast responses, such as proliferation, differentiation, migration and invasiveness. Likewise, glucocorticoids are also known to modulate many factors involved in implantation, including trophoblast gap-junction intercellular communication, although their influence on pregnancy is controversial. In order to investigate the effects of betamethasone, a synthetic glucocorticoid, on Cx and glucocorticoid receptor (GR) expression and localisation, as well as on cell proliferation, the extravillous trophoblast-derived HTR-8/SVneo cell line was used as a model. The results, confirmed by means of immunofluorescence, demonstrate that betamethasone selectively modifies GR and Cx expression, enhancing the GRα isoform without affecting GRβ, and inhibiting Cx40 expression whilst increasing that of Cx43 and Cx45. Furthermore, betamethasone was shown to exert an inhibitory action on cell proliferation. In this model the abortion drug RU-486 (mifepristone), reported to be a GR antagonist, did not counteract this effect of betamethasone. On the contrary, it induced responses similar to those of the hormone. Knowing that RU-486 is also a potent progesterone-receptor antagonist, the effect of progesterone alone and in combination with the drug on Cx expression and cell proliferation was then tested. Progesterone showed the same effect as betamethasone on Cx expression, but it did not affect proliferation. Based on these results, neither the abortion effects of RU-486 nor the protective action of betamethasone and progesterone are exerted by modulation of Cx. RU-486 did not antagonise the progesterone effect, suggesting that its abortive action does not involve alteration of trophoblast Cx expression.

  5. ProNodal acts via FGFR3 to govern duration of Shh expression in the prechordal mesoderm

    PubMed Central

    Ellis, Pamela S.; Burbridge, Sarah; Soubes, Sandrine; Ohyama, Kyoji; Ben-Haim, Nadav; Chen, Canhe; Dale, Kim; Shen, Michael M.; Constam, Daniel; Placzek, Marysia

    2015-01-01

    The secreted glycoprotein sonic hedgehog (Shh) is expressed in the prechordal mesoderm, where it plays a crucial role in induction and patterning of the ventral forebrain. Currently little is known about how Shh is regulated in prechordal tissue. Here we show that in the embryonic chick, Shh is expressed transiently in prechordal mesoderm, and is governed by unprocessed Nodal. Exposure of prechordal mesoderm microcultures to Nodal-conditioned medium, the Nodal inhibitor CerS, or to an ALK4/5/7 inhibitor reveals that Nodal is required to maintain both Shh and Gsc expression, but whereas Gsc is largely maintained through canonical signalling, Nodal signals through a non-canonical route to maintain Shh. Further, Shh expression can be maintained by a recombinant Nodal cleavage mutant, proNodal, but not by purified mature Nodal. A number of lines of evidence suggest that proNodal acts via FGFR3. ProNodal and FGFR3 co-immunoprecipitate and proNodal increases FGFR3 tyrosine phosphorylation. In microcultures, soluble FGFR3 abolishes Shh without affecting Gsc expression. Further, prechordal mesoderm cells in which Fgfr3 expression is reduced by Fgfr3 siRNA fail to bind to proNodal. Finally, targeted electroporation of Fgfr3 siRNA to prechordal mesoderm in vivo results in premature Shh downregulation without affecting Gsc. We report an inverse correlation between proNodal-FGFR3 signalling and pSmad1/5/8, and show that proNodal-FGFR3 signalling antagonises BMP-mediated pSmad1/5/8 signalling, which is poised to downregulate Shh. Our studies suggest that proNodal/FGFR3 signalling governs Shh duration by repressing canonical BMP signalling, and that local BMPs rapidly silence Shh once endogenous Nodal-FGFR3 signalling is downregulated. PMID:26417042

  6. Effects of opioid agonist and antagonist in dams exposed to morphine during the perinatal period.

    PubMed

    Sobor, Melinda; Timár, Julia; Riba, Pál; Friedmann, Tamás; Király, Kornél P; Gyarmati, Susanna; Al-Khrasani, Mahmoud; Fürst, Susanna

    2011-01-15

    The aim of the present work was to further analyse the features of opioid dependence following chronic morphine treatment during pregnancy and lactation. Dams from the day of mating were treated either with saline or with morphine (10mg/kg) subcutaneously once daily. Physical and behavioural signs of morphine withdrawal were investigated both in the early postpartum period (maternal behaviour) and after weaning (physical signals, locomotion, anxiety-like behaviour). Maternal behaviour was evaluated after acute challenge with naloxone (3 mg/kgs.c.) or morphine (10 mg/kgs.c.) and morphine plus naloxone (10 mg/kgs.c. and 3 mg/kgs.c., respectively). After weaning sensitivity to the rewarding effect of morphine was measured by conditioned place preference and to the aversive effect of naloxone by conditioned place aversion tests. The intensity of physical and behavioural indices of dependence was also investigated by precipitation of withdrawal with naloxone (10 mg/kgs.c) after weaning. Naloxone impaired the maternal behaviour in morphine-treated dams but not in saline-ones. Acute challenge with morphine impaired maternal responsiveness both in saline and in morphine-treated dams, this effect of morphine, however could be completely antagonised by naloxone only in the saline-treated but not in the morphine-treated ones. Significantly increased sensitivity to the rewarding stimulus of morphine and more pronounced aversion to naloxone were observed in morphine-treated dams. Naloxone precipitated only moderate physical withdrawal signals in morphine-treated dams, while anxiety and locomotor activity after administration of naloxone (behavioural withdrawal) were not changed in them. In summary chronic, moderate dose morphine treatment during pregnancy and lactation resulted in only mild dependence, but it affected opioid-receptor sensitivity and presumably disrupted the functioning of endogenous opioid system. Copyright © 2010 Elsevier Inc. All rights reserved.

  7. BCR and chemokine responses upon anti-IgM and anti-IgD stimulation in chronic lymphocytic leukaemia.

    PubMed

    Haerzschel, Andrea; Catusse, Julie; Hutterer, Evelyn; Paunovic, Manuela; Zirlik, Katja; Eibel, Hermann; Krenn, Peter W; Hartmann, Tanja N; Burger, Meike

    2016-12-01

    Dysregulation of B cell receptor (BCR) signalling is a hallmark of chronic lymphocytic leukaemia (CLL) pathology, and targeting BCR pathway kinases has brought great therapeutic advances. Activation of the BCR in lymphoid organs has been associated with CLL cell proliferation and survival, leading to progressive disease. While these responses are mediated predominantly by IgM, the role of IgD is less clear. Seeking to uncover downstream consequences of individual and combined stimulation of the two BCR isotypes, we found an amplification of IgD expression and IgD-mediated calcium signalling by previous stimulation of IgM in CLL. Furthermore, no heterologous downmodulation of the isotypes, as observed in healthy donors, was present. Only marginal downregulation of the expression of various chemokine receptors by α-IgM and α-IgD stimulation was found as compared to normal B cells. Consistently, calcium responses of CLL cells to different chemokines were only weakly affected by preceding BCR activation. In contrast, migration towards the two homeostatic chemokines CXCL12 and CCL21 was differentially regulated by IgM and IgD. While IgM activation reduced migration of CLL cells towards CXCL12, but not CCL21, IgD activation predominantly impacted on CCL21 but not CXCL12-mediated chemotaxis. This indicates that the preference for one chemokine over the other may depend on the functional presence of the two isotypes in CLL. Inhibitors against the kinases Syk, Lyn, and Btk antagonised both BCR- and chemokine-induced calcium signals.

  8. Fatty acids and lymphocyte functions.

    PubMed

    Calder, P C; Yaqoob, P; Thies, F; Wallace, F A; Miles, E A

    2002-01-01

    The immune system acts to protect the host against pathogenic invaders. However, components of the immune system can become dysregulated such that their activities are directed against host tissues, so causing damage. Lymphocytes are involved in both the beneficial and detrimental effects of the immune system. Both the level of fat and the types of fatty acid present in the diet can affect lymphocyte functions. The fatty acid composition of lymphocytes, and other immune cells, is altered according to the fatty acid composition of the diet and this alters the capacity of those cells to produce eicosanoids, such as prostaglandin E2, which are involved in immunoregulation. A high fat diet can impair lymphocyte function. Cell culture and animal feeding studies indicate that oleic, linoleic, conjugated linoleic, gamma-linolenic, dihomo-gamma-linolenic, arachidonic, alpha-linolenic, eicosapentaenoic and docosahexaenoic acids can all influence lymphocyte proliferation, the production of cytokines by lymphocytes, and natural killer cell activity. High intakes of some of these fatty acids are necessary to induce these effects. Among these fatty acids the long chain n-3 fatty acids, especially eicosapentaenoic acid, appear to be the most potent when included in the human diet. Although not all studies agree, it appears that fish oil, which contains eicosapentaenoic acid, down regulates the T-helper 1-type response which is associated with chronic inflammatory disease. There is evidence for beneficial effects of fish oil in such diseases; this evidence is strongest for rheumatoid arthritis. Since n-3 fatty acids also antagonise the production of inflammatory eicosanoid mediators from arachidonic acid, there is potential for benefit in asthma and related diseases. Recent evidence indicates that fish oil may be of benefit in some asthmatics but not others.

  9. Single cycle structure-based humanization of an anti-nerve growth factor therapeutic antibody.

    PubMed

    Covaceuszach, Sonia; Marinelli, Sara; Krastanova, Ivet; Ugolini, Gabriele; Pavone, Flaminia; Lamba, Doriano; Cattaneo, Antonino

    2012-01-01

    Most forms of chronic pain are inadequately treated by present therapeutic options. Compelling evidence has accumulated, demonstrating that Nerve Growth Factor (NGF) is a key modulator of inflammatory and nociceptive responses, and is a promising target for the treatment of human pathologies linked to chronic and inflammatory pain. There is therefore a growing interest in the development of therapeutic molecules antagonising the NGF pathway and its nociceptor sensitization actions, among which function-blocking anti-NGF antibodies are particularly relevant candidates.In this respect, the rat anti-NGF αD11 monoclonal antibody (mAb) is a potent antagonist, able to effectively antagonize rodent and human NGF in a variety of in vitro and in vivo systems. Here we show that mAb αD11 displays a significant analgesic effect in two different models of persistent pain in mice, with a remarkable long-lasting activity. In order to advance αD11 mAb towards its clinical application in man, anti-NGF αD11 mAb was humanized by applying a novel single cycle strategy based on the a priori experimental determination of the crystal and molecular structure of the parental Fragment antigen-binding (Fab). The humanized antibody (hum-αD11) was tested in vitro and in vivo, showing that the binding mode and the NGF neutralizing biological activities of the parental antibody are fully preserved, with even a significant affinity improvement. The results firmly establish hum-αD11 as a lead candidate for clinical applications in a therapeutic area with a severe unmet medical need. More generally, the single-cycle structure-based humanization method represents a considerable improvement over the standard humanization methods, which are intrinsically empirical and require several refinement cycles.

  10. EV-077 in vitro inhibits platelet aggregation in type-2 diabetics on aspirin.

    PubMed

    Sakariassen, Kjell S; Femia, Eti A; Daray, Federico M; Podda, Gian M; Razzari, Cristina; Pugliano, Mariateresa; Errasti, Andrea E; Armesto, Arnaldo R; Nowak, Wanda; Alberts, Pēteris; Meyer, Jean-Philippe; Sorensen, Alexandra S; Cattaneo, Marco; Rothlin, Rodolfo P

    2012-11-01

    This study aimed to characterize the in vitro effect of EV-077, a compound that antagonises the binding of prostanoids and isoprostanes to the thromboxane receptor (TP) and inhibits the thromboxane synthase (TS), on platelet aggregation of patients with type-2 diabetes and coronary artery disease (CAD) on chronic aspirin treatment. The effect of EV-077 on 8-iso-PGE(2)-mediated TP receptor contraction of human arteries was also investigated. Fifty-two type-2 diabetics with CAD on chronic aspirin (100 mg) treatment were studied. Arachidonic acid-induced platelet aggregation was measured by impedance aggregometry in platelet-rich plasma (PRP) and whole blood anticoagulated with hirudin, and by light transmission aggregometry in citrate-anticoagulated PRP following 10-min in vitro exposure to EV-077 (100 nmol/l) or control. The effect of EV-077 was measured on isometric contraction of 24 human umbilical arteries induced by isoprostane 8-iso-PGE(2). Arachidonic acid (1 mmol/l) induced substantial aggregation in hirudin-anticoagulated whole blood (63 ± 4 AU), which was significantly reduced by in vitro exposure to EV-077 (38 ± 3 AU, P<0.001). Virtually no arachidonic acid-induced aggregation in citrate-anticoagulated or hirudin-anticoagulated PRP was observed. EV-077 potently, competitively and reversibly inhibited TP mediated contraction of umbilical arteries by 8-iso-PGE(2) (P<0.01). Aspirin did not completely inhibit arachidonic acid-induced platelet aggregation in whole blood from type-2 diabetics with CAD. This aggregation is likely induced by prostanoids and/or isoprostanes produced by leukocytes, because it was significantly reduced by EV-077. The TP receptor-mediated contraction of human arteries induced by isoprostane 8-iso-PGE(2) was effectively inhibited by EV-077. Copyright © 2012 Elsevier Ltd. All rights reserved.

  11. A saturated N-acylethanolamine other than N-palmitoyl ethanolamine with anti-inflammatory properties: a neglected story...

    PubMed

    Dalle Carbonare, M; Del Giudice, E; Stecca, A; Colavito, D; Fabris, M; D'Arrigo, A; Bernardini, D; Dam, M; Leon, A

    2008-05-01

    N-acylethanolamines, which include the endocannabinoid anandamide and the cannabinoid receptor-inactive saturated compounds N-palmitoyl ethanolamine and N-stearoyl ethanolamine, are ethanolamines of long-chain fatty acids degraded by fatty acid amide hydrolase (FAAH) known to accumulate in degenerating tissues and cells. Whilst much evidence supports a protective anti-inflammatory role of both anandamide and N-palmitoyl ethanolamine, very little information is available with regard to the bioactivity of N-stearoyl ethanolamine. Employing a murine model of passive IgE-induced cutaneous anaphylaxis, we have found that N-stearoyl ethanolamine is endowed with marked anti-inflammatory properties in vivo, supporting the hypothesis that endogenous N-stearoyl ethanolamine is, in analogy to N-palmitoyl ethanolamine, a bioactive signalling lipid capable of downregulating allergic inflammation in the skin. This effect, although mimicked by synthetic, non-selective, CB(1)/CB(2) receptor agonists, such as WIN55, 212-2, was not sensitive to CB(1) or CB(2) receptor antagonists, but rather was fully reversed by capsazepine, a competitive antagonist of the TRPV1 receptor. Moreover, CB(1) receptor antagonists, although effective in antagonising the WIN55,212-2-induced hypothermia, did not reduce the anti-inflammatory effect of WIN55,212-2, whilst CB(2) receptor antagonists, per se inactive, potentiated the WIN55,212-2 effect, suggesting an involvement of non-CB(1)/CB(2) receptors in the anti-inflammatory action of WIN55,212-2. All this, together with demonstration of FAAH as a major regulator of the in vivo concentrations of saturated N-stearoyl ethanolamine, in addition to N-palmitoyl ethanolamine, raise the speculation that pharmacological treatments with saturated N-acylethanolamines such as N-stearoyl ethanolamine, or alternatively FAAH inhibitors able to increase their local concentration, rather than selective CB receptor agonists, might be of promising therapeutic benefit in

  12. Do thyroid hormones function in insects?

    PubMed

    Davey, K G

    2000-01-01

    Earlier work demonstrated that phenoxy-phenyl compounds such as fenoxycarb and thyroxine mimicked the effects of JH III in causing a reduction in volume of the follicle cells of Locusta migratoria. While these compounds were only moderately effective, a derivative of thyroxine, 3,3',5-triiodothyronine (T3) was as effective as JH III, and T3 has been shown to bind to the same membrane receptor and activate the same pathway as JH III. The current paper shows that other thyroxine derivatives vary in activity. 3,3', 5'-Triiodothyronine (reverse T3) is inactive. 3,5-Diiodothyronine (T2) is more active than JH III, while its relatives (iodines at 3', 5' or at 3,3') are inactive. When follicles are exposed in vitro to rhodamine conjugated T3, the fluorescent compound can be seen to enter the cells and accumulate there: this process is inhibited by cycloheximide or by a temperature of 0 degrees C. The accumulation is antagonised by JH III but not JH I (which does not bind to the JH III membrane receptor) and by an antiserum raised against the putative membrane receptor protein. The action of T3, but not T2, is inhibited by 6-n-propyl-2-thiouracil or by aurothioglucose, both known to inhibit deiodinases. The activity of T3, but not of T2, increases with time of exposure to the follicle cells. These facts suggest that T3 enters the cells by receptor mediated endocytosis and is converted to a more active compound. Immunoreactivity to T3, but not thyroxine, can be detected in the haemolymph of locusts, and the titre varies slightly with the gonotrophic cycle. The food shows immunoreactivity for both thyroxine and T3. These findings suggest that thyroid hormones are ingested by locusts and have the potential to be used as hormonal signals in the control of egg production.

  13. Nutrition health issues in self-reported postpartum depression.

    PubMed

    Hogg-Kollars, Sabine; Mortimore, Denise; Snow, Sarah

    2011-01-01

    In this retrospective survey women with and without self-reported postpartum depression (PPD) were compared in regards to consumption-frequency of foods and supplements rich in nutrients beneficial to nervous system (NS) health, in regards to consumption-frequency of compounds which may counteract the effect of the above and in regards to nutritional support provided to them during a pregnancy between 2003 and 2008. Postpartum depression (PPD) is defined as a major depressive episode that begins within 1 month of delivery and is experienced by roughly 13% of mothers. Four Hundred participants were recruited through the internet. Data gathered via multiple choice questionnaires was statistically analyzed using SPSS and Statistical software; statistical procedures included discriminant analysis, Pearson's product moment correlation, independent t-test and cross-tabulations. Out of 400 participants 83 (20.8%) were affected by self-reported depression after a pregnancy between 2003 and 2008. Depressed subjects consumed oily fish and offal significantly more often than non depressed subjects. Depression was more prevalent among women with vegetarian diets. No significant difference concerning food group intake or the ratios between foods rich in nutrients beneficial to NS health and foods rich in compounds antagonising their effect were found between depressed and non depressed subjects. Iron supplementation correlated positively with zinc supplementation in both groups. Roughly 70% of women reported to have received no information about n-3 fatty acid fish oils during pregnancy; informed subjects consumed fish oils more often. The majority of subjects with self-reported depression described nutritional support during pregnancy as inadequate. Within this Austrian sample, the prevalence rate of postpartum depression was high; while the consumption of oily fish and vegetarian diets negatively correlated with depression, Patient information positively correlated with the

  14. Sulfasalazine reduces bile acid induced apoptosis in human hepatoma cells and perfused rat livers

    PubMed Central

    Rust, C; Bauchmuller, K; Bernt, C; Vennegeerts, T; Fickert, P; Fuchsbichler, A; Beuers, U

    2006-01-01

    Background Bile acid induced apoptosis in hepatocytes can be antagonised by nuclear factor κB (NFκB) dependent survival pathways. Sulfasalazine modulates NFκB in different cell types. We aimed to determine the effects of sulfasalazine and its metabolites sulfapyridine and 5‐aminosalicylic acid (5‐ASA) on bile acid induced apoptosis in hepatocytes. Methods Apoptosis was determined by caspase assays and immunoblotting, NFκB activation by electrophoretic mobility shift assay and reporter gene assays, generation of reactive oxygen species (ROS) fluorometrically, bile secretion gravimetrically, and bile acid uptake radiochemically and by gas chromatography in HepG2‐Ntcp cells and isolated perfused rat livers. Results Glycochenodeoxycholic acid (GCDCA 75 µmol/l) induced apoptosis was reduced by sulfasalazine dose dependently (1–1000 µmol/l) in HepG2‐Ntcp cells whereas its metabolites 5‐ASA and sulfapyridine had no effect. Sulfasalazine significantly reduced GCDCA induced activation of caspases 9 and 3. In addition, sulfasalazine activated NFκB and decreased GCDCA induced generation of ROS. Bile acid uptake was competitively inhibited by sulfasalazine. In perfused rat livers, GCDCA (25 µmol/l) induced liver injury and extensive hepatocyte apoptosis were significantly reduced by simultaneous administration of 100 µmol/l sulfasalazine: lactate dehydrogenase and glutamate‐pyruvate transaminase activities were reduced by 82% and 87%, respectively, and apoptotic hepatocytes were observed only occasionally. GCDCA uptake was reduced by 45 (5)% when sulfasalazine was coadministered. However, when 50% of GCDCA (12.5 µmol/l) was administered alone, marked hepatocyte apoptosis and liver injury were again observed, questioning the impact of reduced GCDCA uptake for the antiapoptotic effect of sulfasalazine. Conclusion Sulfasalazine is a potent inhibitor of GCDCA induced hepatocyte apoptosis in vitro and in the intact liver. PMID:16322111

  15. T-Cell Protein Tyrosine Phosphatase Attenuates STAT3 and Insulin Signaling in the Liver to Regulate Gluconeogenesis

    PubMed Central

    Fukushima, Atsushi; Loh, Kim; Galic, Sandra; Fam, Barbara; Shields, Ben; Wiede, Florian; Tremblay, Michel L.; Watt, Matthew J.; Andrikopoulos, Sofianos; Tiganis, Tony

    2010-01-01

    OBJECTIVE Insulin-induced phosphatidylinositol 3-kinase (PI3K)/Akt signaling and interleukin-6 (IL-6)-instigated JAK/STAT3-signaling pathways in the liver inhibit the expression of gluconeogenic genes to decrease hepatic glucose output. The insulin receptor (IR) and JAK1 tyrosine kinases and STAT3 can serve as direct substrates for the T-cell protein tyrosine phosphatase (TCPTP). Homozygous TCPTP-deficiency results in perinatal lethality prohibiting any informative assessment of TCPTP's role in glucose homeostasis. Here we have used Ptpn2+/− mice to investigate TCPTP's function in glucose homeostasis. RESEARCH DESIGN AND METHODS We analyzed insulin sensitivity and gluconeogenesis in chow versus high-fat–fed (HFF) Ptpn2+/− and Ptpn2+/+ mice and insulin and IL-6 signaling and gluconeogenic gene expression in Ptpn2+/− and Ptpn2+/+ hepatocytes. RESULTS HFF Ptpn2+/− mice exhibited lower fasted blood glucose and decreased hepatic glucose output as determined in hyperinsulinemic euglycemic clamps and by the decreased blood glucose levels in pyruvate tolerance tests. The reduced hepatic glucose output coincided with decreased expression of the gluconeogenic genes G6pc and Pck1 and enhanced hepatic STAT3 phosphorylation and PI3K/Akt signaling in the fasted state. Insulin-induced IR-β–subunit Y1162/Y1163 phosphorylation and PI3K/Akt signaling and IL-6–induced STAT3 phosphorylation were also enhanced in isolated Ptpn2+/− hepatocytes. The increased insulin and IL-6 signaling resulted in enhanced suppression of G6pc and Pck1 mRNA. CONCLUSIONS Liver TCPTP antagonises both insulin and STAT3 signaling pathways to regulate gluconeogenic gene expression and hepatic glucose output. PMID:20484139

  16. Frequency specific activity in subthalamic nucleus correlates with hand bradykinesia in Parkinson's disease

    PubMed Central

    Tan, Huiling; Pogosyan, Alek; Anzak, Anam; Foltynie, Thomas; Limousin, Patricia; Zrinzo, Ludvic; Ashkan, Keyoumars; Bogdanovic, Marko; Green, Alexander L.; Aziz, Tipu; Brown, Peter

    2013-01-01

    Local field potential recordings made from the basal ganglia of patients undergoing deep brain stimulation have suggested that frequency specific activity is involved in determining the rate of force development and the peak force at the outset of a movement. However, the extent to which the basal ganglia might be involved in motor performance later on in a sustained contraction is less clear. We therefore recorded from the subthalamic nucleus region (STNr) in patients with Parkinson's disease (PD) as they made maximal voluntary grips. Relative to age-matched controls they had more rapid force decrement when contraction was meant to be sustained and prolonged release reaction time and slower rate of force offset when they were supposed to release the grip. These impairments were independent from medication status. Increased STNr power over 5–12 Hz (in the theta/alpha band) independently predicted better performance—reduced force decrement, shortened release reaction time and faster rate of force offset. In contrast, lower mean levels and progressive reduction of STNr power over 55–375 Hz (high gamma/high frequency) over the period when contraction was meant to be sustained were both strongly associated with greater force decrement over time. Higher power over 13–23 Hz (low beta) was associated with more rapid force decrement during the period when grip should have been sustained, and with a paradoxical shortening of the release reaction time. These observations suggest that STNr activities at 5–12 Hz and 55–375 Hz are necessary for optimal grip performance and that deficiencies of such activities lead to motor impairments. In contrast, increased levels of 13–25 Hz activity both promote force decrement and shorten the release reaction time, consistent with a role in antagonising (and terminating) voluntary movement. Frequency specific oscillatory activities in the STNr impact on motor performance from the beginning to the end of a voluntary grip

  17. ‘Smashed by the National Health’? A Closer Look at the Demise of the Pioneer Health Centre, Peckham

    PubMed Central

    Conford, Philip

    2016-01-01

    The Pioneer Health Centre, based in South London before and after the Second World War, remains a source of interest for advocates of a positive approach to health promotion in contrast with the treatment of those already ill. Its closure in 1950 for lack of funds has been blamed on the then recently established National Health Service, but this article argues that such an explanation is over-simplified and ignores a number of other factors. The Centre had struggled financially during the 1930s and tried to gain support from the Medical Research Council. The Council appeared interested in the Centre before the war, but was less sympathetic in the 1940s. Around the time of its closure and afterwards, the Centre was also involved in negotiations with London County Council; these failed because the Centre’s directors would not accept the changes which the Council would have needed to make. Unpublished documents reveal that the Centre’s directors were uncompromising and that their approach to the situation antagonised their colleagues. Changes in medical science also worked against the Centre. The success of sulphonamide drugs appeared to render preventive medicine less significant, while the development of statistical techniques cast doubt on the Centre’s experimental methods. The Centre was at the heart of the nascent organic farming movement, which opposed the rapid growth of chemical cultivation. But what might be termed ‘chemical triumphalism’ was on the march in both medicine and agriculture, and the Centre was out of tune with the mood of the times. PMID:26971599

  18. Hippocampal long-term potentiation that is elicited by perforant path stimulation or that occurs in conjunction with spatial learning is tightly controlled by beta-adrenoreceptors and the locus coeruleus.

    PubMed

    Hansen, Niels; Manahan-Vaughan, Denise

    2015-11-01

    The noradrenergic system, driven by locus coeruleus (LC) activation, plays a key role in the regulating and directing of changes in hippocampal synaptic efficacy. The LC releases noradrenaline in response to novel experience and LC activation leads to an enhancement of hippocampus-based learning, and facilitates synaptic plasticity in the form of long-term depression (LTD) and long-term potentiation (LTP) that occur in association with spatial learning. The predominant receptor for mediating these effects is the β-adrenoreceptor. Interestingly, the dependency of synaptic plasticity on this receptor is different in the hippocampal subfields whereby in the CA1 in vivo, LTP, but not LTD requires β-adrenoreceptor activation, whereas in the mossy fiber synapse LTP and LTD do not depend on this receptor. By contrast, synaptic plasticity that is facilitated by spatial learning is highly dependent on β-adrenoreceptor activation in both hippocampal subfields. Here, we explored whether LTP induced by perforant-path (pp) stimulation in vivo or that is facilitated by spatial learning depends on β-adrenoreceptors. We found that under both LTP conditions, antagonising the receptors disabled the persistence of LTP. β-adrenoreceptor-antagonism also prevented spatial learning. Strikingly, activation of the LC before high-frequency stimulation (HFS) of the pp prevented short-term potentiation but not LTP, and LC stimulation after pp-HFS-induced depotentiation of LTP. This depotentiation was prevented by β-adrenoreceptor-antagonism. These data suggest that β-adrenoreceptor-activation, resulting from noradrenaline release from the LC during enhanced arousal and learning, comprises a mechanism whereby the duration and degree of LTP is regulated and fine tuned. This may serve to optimize the creation of a spatial memory engram by means of LTP and LTD. This process can be expected to support the special role of the dentate gyrus as a crucial subregional locus for detecting and

  19. Interleukin-27 exhibited anti-inflammatory activity during Plasmodium berghei infection in mice.

    PubMed

    Fazalul Rahiman, S S; Basir, R; Talib, H; Tie, T H; Chuah, Y K; Jabbarzare, M; Chong, W C; Mohd Yusoff, M A; Nordin, N; Yam, M F; Abdullah, W O; Abdul Majid, R

    2013-12-01

    Interleukin-27 (IL-27) has a pleiotropic role either as a pro-inflammatory or anti-inflammatory cytokine in inflammatory related diseases. The role and involvement of IL-27 during malaria was investigated and the effects of modulating its release on the production of major inflammatory cytokines and the histopathological consequences in major affected organs during the infection were evaluated. Results showed that IL-27 concentration was significantly elevated throughout the infection but no positive correlation with the parasitaemia development observed. Augmentation of IL-27 significantly elevated the release of anti-inflammatory cytokine, IL-10 whereas antagonising and neutralising IL-27 produced the opposite. A significant elevation of pro-inflammatory cytokines (IFN-γ and IL-6) was also observed, both during augmentation and inhibition of IL-27. Thus, it is suggested that IL-27 exerts an anti-inflammatory activity in the Th1 type response by signalling the production of IL-10 during malaria. Histopathological examination showed sequestration of PRBC in the microvasculature of major organs in malarial mice. Other significant histopathological changes include hyperplasia and hypertrophy of the Kupffer cells in the liver, hyaline membrane formation in lung tissue, enlargement of the white and red pulp followed by the disappearance of germinal centre of the spleen, and tubular vacuolation of the kidney tissues. In conclusion, it is suggested that IL-27 may possibly acts as an anti-inflammatory cytokine during the infection. Modulation of its release produced a positive impact on inflammatory cytokine production during the infection, suggesting its potential in malaria immunotherapy, in which the host may benefit from its inhibition.

  20. Transcription and activity of 5-fluorouracil converting enzymes in fluoropyrimidine resistance in colon cancer in vitro.

    PubMed

    Mader, R M; Sieder, A E; Braun, J; Rizovski, B; Kalipciyan, M; Mueller, M W; Jakesz, R; Rainer, H; Steger, G G

    1997-12-01

    Cellular resistance to 5-fluorouracil (5-FU) is not completely understood. Since 5-FU shares the pyrimidine pathway with the physiological pyrimidines, we investigated the relationship between fluoropyrimidine metabolism, nucleic acid uptake and cytotoxicity of 5-FU in eight colon tumour cell lines including 5-FU-resistant subclones. The cytotoxicity of 5-FU was increased up to 423-fold when the anabolites 5-fluorouridine (FUrd), 5-fluorodeoxyuridine (FdUrd), and 5-fluorodeoxyuridine monophosphate (FdUMP) were compared with the parent drug in vitro. The enzymes uridine phosphorylase and thymidine phosphorylase were predictive for the cytotoxicity of 5-FU in 5/7 cell lines. Inhibition of uridine phosphorylase and thymidine phosphorylase by antisense strategies effectively antagonised 5-FU, abolishing 84% and 79% of its toxicity. The importance of thymidine phosphorylase was supported by a highly restricted enzyme activity in 5-FU-resistant cells. In 5-FU naive cells, a stimulating effect of 5-FU on thymidylate synthase mRNA and ribonucleotide reductase mRNA expression was observed. In these cells, antisense oligonucleotides to ribonucleotide reductase significantly reduced cell growth. Downregulation of ribonucleotide reductase mRNA in 5-FU-resistant subclones suggests different mechanisms in primary and secondary resistance to 5-FU. Most of the intracellular 5-FU was selectively incorporated into RNA (range: 45-91%) and generally spared DNA (range: 0.2-11%). In synthesising our data, we conclude that drug resistance could be overwhelmed through bypassing limiting steps in the activation of 5-FU. In the majority of colonic tumours, the activity of uridine phosphorylase and thymidine phosphorylase may have prognostic relevance for the cytotoxicity of 5-FU in vitro.

  1. Presynaptic histamine H2 receptors modulate the sympathetic nerve transmission in the isolated rat vas deferens; no role for H3-receptors.

    PubMed

    Poli, E; Todorov, S; Pozzoli, C; Bertaccini, G

    1994-10-01

    The modulatory activity mediated by histamine receptors on the sympathetic nerve transmission was investigated in the rat vas deferens. Agonists and antagonists acting at the different histamine receptor subtypes (H1, H2 and H3) were tested on electrically-driven preparations in vitro. Low-frequency stimulation (0.1 Hz) evoked muscle contractions almost completely-sustained by ATP release, while at high-frequency stimulation (5-10 Hz) norepinephrine was mainly involved. The H1 receptor agonists, pyridilethylamine and 2-(2 aminoethyl)thiazole, enhanced the electrically evoked twitch responses, but not contractions induced by exogenously-applied norepinephrine and ATP. These effects were prevented by the H1-blocking drugs, mepyramine and phenyramine, but only at high concentrations (10 mumol/l). All these H1-antagonists strongly enhanced muscle response to electrical stimulation. The H2 receptor agonists, dimaprit, amthamine and impromidine, reduced the contractions evoked by field stimulation, but not by exogenously applied norepinephrine and ATP, the effect being antagonised by H2-blocking drugs, ranitidine and famotidine. The H3 receptor agonist, R(alpha)-methylhistamine, reduced the electrically evoked muscle contractions, the effect being not modified by the selective H3-blocking drug, thioperamide, but prevented by famotidine. These data suggest that rat vas deferens contains presynaptic histamine H2 receptors, able to mediate inhibitory effects on the sympathetic transmission, while histamine H3 receptors are apparently not involved. On the contrary, the role of H1 is still unclear, since both agonists and antagonists may have the same effects.

  2. Interplay of Ca(2+) and Mg (2+) in sodium-calcium exchanger and in other Ca(2+)-binding proteins: magnesium, watchdog that blocks each turn if able.

    PubMed

    Levitsky, Dmitri O; Takahashi, Masayuki

    2013-01-01

    Sodium-calcium exchange across plasma membrane is regulated by intracellular calcium ions. The sodium-calcium exchanger (NCX1) is activated by successive saturation of numerous Ca(2+)-binding sites located in the intracellular loop of the protein. The progressive saturation of the binding domain CBD12 by Ca(2+) results in a series of conformational changes of CBD12 as well as of entire NCX1 molecule. Like other soluble and membrane Ca(2+)-binding proteins, NCX1 can also be regulated by Mg(2+) that antagonises Ca(2+) at the level of divalent cation-binding sites. This chapter summarises data on Mg(2+) impacts in the cells. Regulatory action of Mg(2+) on intracellular Ca(2+)-dependent processes can be achieved due to changes of its cytoplasmic level, which take place in the range of [Mg(2+)](i) from 0.5 to 3 mM. Under normal conditions, these changes are ensured by activation of plasmalemmal Mg(2+) transport systems and by variations in ATP level in cytoplasm. In heart and in brain, some pathological conditions, such as hypoxia, ischemia and ischemia followed by reperfusion, are associated with an important increase in intracellular Ca(2+). The tissue damage due to Ca(2+) overload may be prevented by Mg(2+). The protective actions of Mg(2+) can be achieved due to its ability to compete with Ca(2+) for the binding sites in a number of proteins responsible for the rise in intracellular free Ca(2+), including NCX1, in case when the reverse mode of Na(+)/Ca(2+) exchange becomes predominant. Saturation of CBD12 by Mg(2+) results in important changes of NCX1 conformation. Modulating actions of Mg(2+) on the conformation of NCX1 were detected at a narrow range of Mg(2+) concentration, from 0.5 to 1 mM. These data support an idea that variations of intracellular Mg(2+) could modify transmembrane Ca(2+) movements ensured by NCX1.

  3. Inhibitory Effect of Memantine on Streptozotocin-Induced Insulin Receptor Dysfunction, Neuroinflammation, Amyloidogenesis, and Neurotrophic Factor Decline in Astrocytes.

    PubMed

    Rajasekar, N; Nath, Chandishwar; Hanif, Kashif; Shukla, Rakesh

    2016-12-01

    Our earlier studies showed that insulin receptor (IR) dysfunction along with neuroinflammation and amyloidogenesis played a major role in streptozotocin (STZ)-induced toxicity in astrocytes. N-methyl-D-aspartate (NMDA) receptor antagonist-memantine shows beneficial effects in Alzheimer's disease (AD) pathology. However, the protective molecular and cellular mechanism of memantine in astrocytes is not properly understood. Therefore, the present study was undertaken to investigate the effect of memantine on insulin receptors, neurotrophic factors, neuroinflammation, and amyloidogenesis in STZ-treated astrocytes. STZ (100 μM) treatment for 24 h in astrocytes resulted significant decrease in brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), and insulin-degrading enzyme (IDE) expression in astrocytes. Treatment with memantine (1-10 μM) improved STZ-induced neurotrophic factor decline (BDNF, GDNF) along with IR dysfunction as evidenced by a significant increase in IR protein expression, phosphorylation of IRS-1, Akt, and GSK-3 α/β in astrocytes. Further, memantine attenuated STZ-induced amyloid precursor protein (APP), β-site APP-cleaving enzyme-1 and amyloid-β1-42 expression and restored IDE expression in astrocytes. In addition, memantine also displays protective effects against STZ-induced astrocyte activation showed by reduction of inflammatory markers, nuclear factor kappa-B translocation, glial fibrillary acidic protein, cyclooxygenase-2, tumor necrosis factor-α level, and oxidative-nitrostative stress. The results suggest that besides the NMDA receptor antagonisic activity, effect on astroglial IR and neurotrophic factor may also be an important factor in the beneficial effect of memantine in AD pathology. Graphical Abstract Novel neuroprotective mechanisms of memenatine in streptozotocin-induced toxicity in astrocytes.

  4. SB-699551-A (3-cyclopentyl-N-[2-(dimethylamino)ethyl]-N-[(4'-{[(2-phenylethyl)amino]methyl}-4-biphenylyl)methyl]propanamide dihydrochloride), a novel 5-ht5A receptor-selective antagonist, enhances 5-HT neuronal function: Evidence for an autoreceptor role for the 5-ht5A receptor in guinea pig brain.

    PubMed

    Thomas, David R; Soffin, Ellen M; Roberts, Claire; Kew, James N C; de la Flor, Raul M; Dawson, Lee A; Fry, Victoria A; Coggon, Sara A; Faedo, Stefania; Hayes, Philip D; Corbett, David F; Davies, Ceri H; Hagan, Jim J

    2006-09-01

    This study utilised the selective 5-ht(5A) receptor antagonist, SB-699551-A (3-cyclopentyl-N-[2-(dimethylamino)ethyl]-N-[(4'-{[(2-phenylethyl)amino]methyl}-4-biphenylyl)methyl]propanamide dihydrochloride), to investigate 5-ht5A receptor function in guinea pig brain. SB-699551-A competitively antagonised 5-HT-stimulated [35S]GTPgammaS binding to membranes from human embryonic kidney (HEK293) cells transiently expressing the guinea pig 5-ht5A receptor (pA2 8.1+/-0.1) and displayed 100-fold selectivity versus the serotonin transporter and those 5-HT receptor subtypes (5-HT(1A/B/D), 5-HT2A/C and 5-HT7) reported to modulate central 5-HT neurotransmission in the guinea pig. In guinea pig dorsal raphe slices, SB-699551-A (1 microM) did not alter neuronal firing per se but attenuated the 5-CT-induced depression in serotonergic neuronal firing in a subpopulation of cells insensitive to the 5-HT1A receptor-selective antagonist WAY-100635 (100 nM). In contrast, SB-699551-A (100 or 300 nM) failed to affect both electrically-evoked 5-HT release and 5-CT-induced inhibition of evoked release measured using fast cyclic voltammetry in vitro. SB-699551-A (0.3, 1 and 3 mg/kg s.c.) did not modulate extracellular levels of 5-HT in the guinea pig frontal cortex in vivo. However, when administered in combination with WAY-100635 (0.3 mg/kg s.c.), SB-699551-A (0.3, 1 or 3 mg/kg s.c.) produced a significant increase in extracellular 5-HT levels. These studies provide evidence for an autoreceptor role for the 5-ht5A receptor in guinea pig brain.

  5. Prostanoid receptors mediating contraction in rat, macaque and human bladder smooth muscle in vitro.

    PubMed

    Root, James A; Davey, Dorren A; Af Forselles, Kerry J

    2015-12-15

    Selective prostaglandin EP1 antagonists have been suggested for the treatment of bladder dysfunction. This study assessed the contractile prostanoid receptor subtypes in human and non-human bladder in vitro. Classical tissue bath studies were conducted using bladder strips exposed to prostanoid agonists and antagonists. Prostaglandin E2 (PGE2) contracted rat, macaque and human bladder smooth muscle strips (pEC50 7.91±0.06 (n=7), 6.40±0.13 (n=7), and 6.07±0.11 (n=5), respectively). The EP1 receptor antagonist, PF2907617 (300nM), caused a rightward shift of the PGE2 concentration-response curve in the rat bladder only (pKB 8.40±0.15, n=3). PGE2 responses in rat and macaque bladders, but not human, were antagonised by the EP3 antagonist CJ24979 (1µM). Sulprostone, a mixed EP1/EP3/FP receptor agonist, induced potent contractions of rat bladder muscle (pEC50 7.94±0.31, n=6). The FP receptor agonist, prostaglandin F2α (PGF2α), induced bladder contraction in all species tested, but with a lower potency in rat. The selective FP receptor agonist latanoprost caused potent contractions of macaque and human bladder strips only. SQ29548, a selective TP antagonist, and GW848687X, a mixed EP1/TP antagonist caused rightward shifts of the concentration-response curves to the selective TP agonist, U46619 (pKB estimates 8.53±0.07 and 7.56±0.06, n=3, respectively). Responses to U46619 were absent in rat preparations. These data suggest significant species differences exist in bladder contractile prostanoid receptor subtypes. We conclude that the EP1 subtype does not represent the best approach to the clinical treatment of bladder disorders targeting inhibition of smooth muscle contraction.

  6. Activation of muscarinic receptors increases the activity of the granule neurones of the rat dorsal cochlear nucleus--a calcium imaging study.

    PubMed

    Kőszeghy, Áron; Vincze, János; Rusznák, Zoltán; Fu, Yuhong; Paxinos, George; Csernoch, László; Szücs, Géza

    2012-06-01

    Acetylcholine modulates the function of the cochlear nucleus via several pathways. In this study, the effects of cholinergic stimulation were studied on the cytoplasmic Ca(2+) concentration of granule neurones of the rat dorsal cochlear nucleus (DCN). Ca(2+) transients were recorded in Oregon-Green-BAPTA 1-loaded brain slices using a calcium imaging technique. For the detection, identification and characterisation of the Ca(2+) transients, a wavelet analysis-based method was developed. Granule cells were identified on the basis of their size and localisation. The action potential-coupled character of the Ca(2+) transients of the granule cells was established by recording fluorescence changes and electrical activity simultaneously. Application of the cholinergic agonist carbamyl-choline (CCh) significantly increased the frequency of the Ca(2+) transients (from 0.37 to 6.31 min(-1), corresponding to a 17.1-fold increase; n = 89). This effect was antagonised by atropine, whereas CCh could still evoke an 8.3-fold increase of the frequency of the Ca(2+) transients when hexamethonium was present. Using immunolabelling, the expression of both type 1 and type 3 muscarinic receptors (M1 and M3 receptors, respectively) was demonstrated in the granule cells. Application of 1,1-dimethyl-4-diphenylacetoxypiperidinium iodide (an M3-specific antagonist) prevented the onset of the CCh effect, whereas an M1-specific antagonist (pirenzepine) was less effective. We conclude that cholinergic stimulation increases the activity of granule cells, mainly by acting on their M3 receptors. The modulation of the firing activity of the granule cells, in turn, may modify the firing of projection neurones and may adjust signal processing in the entire DCN.

  7. Health care delivery model in epilepsy to reduce treatment gap: World Health Organization study from a rural tribal population of India.

    PubMed

    Nizamie, S Haque; Akthar, Sayeed; Banerjee, Indrajeet; Goyal, Nishant

    2009-04-01

    To design and develop an effective health care delivery model in epilepsy to reduce the treatment gap in a rural tribal community in India. This study was conducted in tribal dominated Namkum Block (114,068 population) of Ranchi, Ranchi District, Jharkhand state, India and carried out as four-staged program-first stage consisted of separate training programs (to 6 volunteer health workers, traditional practitioners of community including 267 faith healers and qualified practitioners), second stage consisted of awareness campaign programs, third stage consisted of diagnosis, treatment delivery and follow-up in once a month camps with free medication and final stage consisted of continued follow-up after the end of study by local practitioners. Health volunteers identified 787 probable cases in the community, 453 attended the camps, and 318 were diagnosed and treated for epilepsy in the camp. Treatment gap was 95% on the initial assessment. 213 epileptic patients enrolled in the study completed 12 months treatment and more than 75% were seizure free at the end of the study. Eighty percent of patients' care-givers and their family members were satisfied with the care provided. At the end of study, local medical practitioners continued to do the follow-up of study participants to ensure continuity of care although results of further follow-up are not included in the present study. A four-staged program in epilepsy treatment delivery model was successful. Voluntary health workers from the community can be effectively trained to identify cases and persuade them to seek treatment. The delivery model should include intensive health awareness campaign, training of doctors and other health care providers, free supply of AEDs (Antiepileptic drugs), continuous follow-up for compliance and side-effects of the drug and tactful dealing with indigenous practitioners and faith healers without antagonising them.

  8. Responses to noradrenaline in human subcutaneous resistance arteries are mediated by both alpha 1- and alpha 2-adrenoceptors.

    PubMed Central

    Nielsen, H.; Mortensen, F. V.; Mulvany, M. J.

    1990-01-01

    1. In vitro experiments in a microvascular myograph were designed to characterize postjunctional alpha-adrenoceptors of human subcutaneous resistance arteries (normalised internal diameter 143-313 microns). 2. Both the alpha 1-selective agonist phenylephrine in the presence of 0.3 microM yohimbine and the alpha 2-selective agonist B-HT 933 in the presence of 0.3 microM prazosin elicited prominent and concentration-dependent contractions. The maximum response to phenylephrine and B-HT 933 was not different from the response to high K physiological salt solution (125 mM K+), and the pD2 values (-log EC50) were 5.90 and 6.11, respectively. 3. In the presence of the alpha 2-selective antagonist yohimbine (0.3 microM), the alpha 1-selective antagonist prazosin competitively antagonised the responses to phenylephrine; the pA2 of prazosin for the receptor which mediated the response to phenylephrine was 8.41. 4. Blockade of either alpha 2-adrenoceptors with 0.1 microM yohimbine or alpha 1-adrenoceptors with 0.1 microM prazosin caused shifts to the right of the noradrenaline concentration-response curves and the shifts in pD2 were 0.69 and 0.61, respectively. The combination of the two antagonists at the above-mentioned concentrations caused a marked, parallel shift to the right of the noradrenaline concentration-response curve, the shift of the pD2 was 2.68. 5. These results suggest that activation of both alpha 1- and alpha 2-adrenoceptors produces contractions in human subcutaneous resistance arteries, and that responses to noradrenaline in these vessels are mediated by both alpha-adrenoceptor subtypes. PMID:1970494

  9. Pharmacology and clinical efficacy of desloratadine as an anti-allergic and anti-inflammatory drug.

    PubMed

    Agrawal, D K

    2001-03-01

    Desloratadine is a biologically active metabolite of the second-generation antihistamine loratadine. Desloratadine is a highly selective peripheral H1 receptor antagonist that is significantly more potent than loratadine. Results of in vitro and in vivo studies have suggested that desloratadine has anti-allergic effects that are unrelated to its ability to antagonise the effects of histamine. Desloratadine inhibits the expression of cell adhesion molecules, inhibits the generation and release of inflammatory mediators and cytokines, attenuates eosinophil chemotaxis, adhesion and superoxide generation. Studies in animals indicate that desloratadine does not cross the blood-brain barrier and therefore does not cause sedation and does not impair cognition or psychomotor performance. Desloratadine has an excellent overall safety profile. It has no effect on QRS and QTc intervals and does not cause arrhythmias. Desloratadine is not associated with any significant changes in gastrointestinal function. In clinical studies, oral desloratadine is rapidly absorbed and bioavailability is not affected by ingestion with food or grapefruit juice. The half-life of desloratadine in humans is 27 h; the linear kinetic profile is unaltered by race or gender. Desloratadine is not a substrate for P-glycoprotein or organic anion transport polypeptide and the drug does not appear to be metabolised to a significant extent by the cytochrome P450 CYP3A4 pathway. It therefore may be safely administered with ketoconazole, erythromycin, fluoxetine, or azithromycin. Clinically, desloratadine effectively controls both nasal and non-nasal symptoms of seasonal allergic rhinitis (SAR), including nasal congestion. Desloratadine also provides significant relief of SAR symptoms in patients with co-existing asthma and is effective in the treatment of chronic idiopathic urticaria. Desloratadine improves quality of life and is well-tolerated.

  10. In vitro interaction of some drug combinations to inhibit rapidly growing mycobacteria isolates from cats and dogs and these isolates' susceptibility to cefovecin and clofazimine.

    PubMed

    Bennie, C J M; To, J L K; Martin, P A; Govendir, M

    2015-01-01

    To investigate whether selected drug combinations used to treat rapidly growing mycobacteria (RGM) have drug-drug interactions that affect efficacy and to investigate each isolate's susceptibility to cefovecin and clofazimine, individually. In vitro susceptibility testing of bacterial isolates. Initially, five feline isolates and one canine isolate from both Mycobacterium fortuitum and M. smegmatis clusters (n = 12) underwent microbroth susceptibility testing to individual drugs to establish minimum inhibitory concentrations (MIC) of cefovecin, ciprofloxacin, clarithromycin, clofazimine, doxycycline, enrofloxacin, trimethoprim and sulfanilamide (the latter two as a combination). Checkerboard assays were then performed for susceptible M. smegmatis isolates for the following combinations: clarithromycin (one isolate only) versus enrofloxacin, clarithromycin vs doxycycline, clarithromycin vs trimethoprim/sulfanilamide; enrofloxacin vs doxycycline (six isolates); enrofloxacin vs trimethoprim/sulfanilamide (six isolates). Susceptible M. fortuitum isolates were tested against enrofloxacin versus doxycycline (four isolates only). All six M. fortuitum isolates were susceptible to enrofloxacin, but only four of six were susceptible to doxycycline. All six M. smegmatis isolates were susceptible to doxycycline, enrofloxacin and trimethoprim/sulfanilamide. A single isolate from the 12, a M. smegmatis isolate, was susceptible to clarithromycin. The fractional inhibitory concentration of each drug ranged from 0.64 to 1.84, indicating that neither synergism nor antagonism was evident. All 12 isolates were resistant to cefovecin. The clofazimine MIC50 to inhibit isolate growth was approximately 3.3 μg/mL for both strains. Drugs commonly used for treatment of RGM, when tested as combinations, do not appear to antagonise one another in vitro. Cefovecin is not efficacious for treatment of RGM infections. © 2015 Australian Veterinary Association.

  11. Trypanosoma brucei DHFR-TS Revisited: Characterisation of a Bifunctional and Highly Unstable Recombinant Dihydrofolate Reductase-Thymidylate Synthase

    PubMed Central

    Gibson, Marc W.; Dewar, Simon; Ong, Han B.; Sienkiewicz, Natasha

    2016-01-01

    Bifunctional dihydrofolate reductase–thymidylate synthase (DHFR-TS) is a chemically and genetically validated target in African trypanosomes, causative agents of sleeping sickness in humans and nagana in cattle. Here we report the kinetic properties and sensitivity of recombinant enzyme to a range of lipophilic and classical antifolate drugs. The purified recombinant enzyme, expressed as a fusion protein with elongation factor Ts (Tsf) in ThyA- Escherichia coli, retains DHFR activity, but lacks any TS activity. TS activity was found to be extremely unstable (half-life of 28 s) following desalting of clarified bacterial lysates to remove small molecules. Stability could be improved 700-fold by inclusion of dUMP, but not by other pyrimidine or purine (deoxy)-nucleosides or nucleotides. Inclusion of dUMP during purification proved insufficient to prevent inactivation during the purification procedure. Methotrexate and trimetrexate were the most potent inhibitors of DHFR (Ki 0.1 and 0.6 nM, respectively) and FdUMP and nolatrexed of TS (Ki 14 and 39 nM, respectively). All inhibitors showed a marked drop-off in potency of 100- to 1,000-fold against trypanosomes grown in low folate medium lacking thymidine. The most potent inhibitors possessed a terminal glutamate moiety suggesting that transport or subsequent retention by polyglutamylation was important for biological activity. Supplementation of culture medium with folate markedly antagonised the potency of these folate-like inhibitors, as did thymidine in the case of the TS inhibitors raltitrexed and pemetrexed. PMID:27175479

  12. Modulation of membrane currents and mechanical activity by niflumic acid in rat vascular smooth muscle.

    PubMed

    Kirkup, A J; Edwards, G; Green, M E; Miller, M; Walker, S D; Weston, A H

    1996-12-12

    The effects of niflumic acid on whole-cell membrane currents and mechanical activity were examined in the rat portal vein. In freshly dispersed portal vein cells clamped at -60 mV in caesium (Cs+)-containing solutions, niflumic acid (1-100 microM) inhibited calcium (Ca2+)-activated chloride currents (IC1(Ca)) induced by caffeine (10 mM) and by noradrenaline (10 microM). In a potassium (K+)-containing solution and at a holding potential of - 10 mV, niflumic acid (10-100 microM) induced an outward K+ current (IK(ATP)) which was sensitive to glibenclamide (10-30 microM). At concentrations < 30 microM and at a holding potential of -2 mV, niflumic acid had no effect on the magnitude of the caffeine- or noradrenaline-stimulated current (IBK(Ca)) carried by the large conductance, Ca(2+)-sensitive K+ channel (BKCa). However, at a concentration of 100 microM, niflumic acid significantly inhibited IBK(Ca)) evoked by caffeine (10 mM) but not by NS1619 (1-(2'-hydroxy-5'-trifluoromethylphenyl)-5-trifluoromethyl-2(3 H) benzimidazolone; 20 microM). In Cs(+)-containing solutions, niflumic acid (10-100 microM) did not inhibit voltage-sensitive Ca2+ currents. In intact portal veins, niflumic acid (1-300 microM) inhibited spontaneous mechanical activity, an action which was partially antagonised by glibenclamide (1-10 microM), and contractions produced by noradrenaline (10 microM), an effect which was glibenclamide-insensitive. It is concluded that inhibition of ICl(Ca) and stimulation of IK(ATP) both contribute to the mechano-inhibitory actions of niflumic acid in the rat portal vein.

  13. Differential expression of angiotensin II type 1 and type 2 receptors at the maternal-fetal interface: potential roles in early placental development.

    PubMed

    Tower, C L; Lui, S; Charlesworth, N R; Smith, S D; Aplin, J D; Jones, R L

    2010-12-01

    Angiotensin II (Ang II) is locally generated in the placenta and regulates syncytial transport, vascular contractility and trophoblast invasion. It acts through two receptor subtypes, AGTR1 and AGTR2 (AT1 and AT2), which typically mediate antagonising actions. The objectives of this study are to characterise the cellular distribution of AGTR1 and AGTR2 at the maternal-fetal interface and explore the effects on cytotrophoblast turnover. Low levels of AGTR2 mRNA were detected in first trimester placental homogenates using real-time PCR. Immunohistochemistry using polyclonal antibodies against AGTR1 and AGTR2 detected the receptors in first trimester placenta, decidua basalis and villous tip outgrowths in culture. Serial staining with cytokeratin-7 was used to identify extravillous trophoblasts (EVTs). AGTR1 was found in the syncytiotrophoblast microvillous membrane, in a subpopulation of villous cytotrophoblasts, and in Hofbauer cells. AGTR1 was strongly upregulated in cytotrophoblasts in cell columns and villous tip outgrowths, but was absent in interstitial and endovascular EVTs within the decidua. AGTR2 immunostaining was present in Hofbauer cells and villous cytotrophoblasts, but was absent from syncytiotrophoblast. Faint staining was detected in cell column cytotrophoblasts and villous outgrowths, but not in EVTs within the decidua. Both receptors were detected in placental homogenates by western blotting. Ang II significantly increased proliferation of cytotrophoblasts in both villous explants and villous tip outgrowths, but did not affect apoptosis. Blockade of AGTR1 and AGTR2 together abrogated this effect. This study shows specific expression patterns for AGTR1 and AGTR2 in distinct trophoblast populations at the maternal-fetal interface and suggests that Ang II plays a role in placental development and generation of EVTs.

  14. Taurine-like GABA aminotransferase inhibitors prevent rabbit brain slices against oxygen-glucose deprivation-induced damage.

    PubMed

    Ricci, Lorenzo; Valoti, Massimo; Sgaragli, Giampietro; Frosini, Maria

    2012-06-01

    The activation of the GABAergic system has been shown to protect brain tissues against the damage that occurs after cerebral ischaemia. On the other hand, the taurine analogues (±)Piperidine-3-sulphonic- (PSA), 2-aminoethane phosphonic- (AEP), 2-(N-acetylamino) cyclohexane sulfonic-acids (ATAHS) and 2-aminobenzene sulfonate-acids (ANSA) have been reported to block GABA metabolism by inhibiting rabbit brain GABA aminotransferase and to increase GABA content in rabbit brain slices. The present investigation explored the neuroprotection provided by GABA, Vigabatrin (VIGA) and taurine analogues in the course of oxygen-glucose deprivation and reperfusion induced damage of rabbit brain slices. Tissue damage was assessed by measuring the release of glutamate and lactate dehydrogenase (LDH) during reperfusion and by determining final tissue water gain, measured as the index of cell swelling. GABA (30-300 μM) and VIGA (30-300 μM) significantly antagonised LDH and glutamate release, as well as tissue water gain caused by oxygen-glucose deprivation and reperfusion. Lower (1-10 μM) or higher concentrations (up to 3,000 μM) were ineffective. ANSA, PSA and ATAHS significantly reduced glutamate and LDH release and tissue water gain in a range of concentrations between 30 and 300 μM. Lower (0-10 μM) or higher (up to 3,000 μM) concentrations were ineffective. Both mechanisms suggest hormetic ("U-shaped") effects. These results indicate that the GABAergic system activation performed directly by GABA or indirectly through GABA aminotransferase inhibition is a promising approach for protecting the brain against ischemia and reperfusion-induced damage.

  15. A Novel Nuclear Trafficking Module Regulates the Nucleocytoplasmic Localization of the Rabies Virus Interferon Antagonist, P Protein*

    PubMed Central

    Oksayan, Sibil; Wiltzer, Linda; Rowe, Caitlin L.; Blondel, Danielle; Jans, David A.; Moseley, Gregory W.

    2012-01-01

    Regulated nucleocytoplasmic transport of proteins is central to cellular function and dysfunction during processes such as viral infection. Active protein trafficking into and out of the nucleus is dependent on the presence within cargo proteins of intrinsic specific modular signals for nuclear import (nuclear localization signals, NLSs) and export (nuclear export signals, NESs). Rabies virus (RabV) phospho (P) protein, which is largely responsible for antagonising the host anti-viral response, is expressed as five isoforms (P1–P5). The subcellular trafficking of these isoforms is thought to depend on a balance between the activities of a dominant N-terminal NES (N-NES) and a distinct C-terminal NLS (C-NLS). Specifically, the N-NES-containing isoforms P1 and P2 are cytoplasmic, whereas the shorter P3–P5 isoforms, which lack the N-NES, are believed to be nuclear through the activity of the C-NLS. Here, we show for the first time that RabV P contains an additional strong NLS in the N-terminal region (N-NLS), which, intriguingly, overlaps with the N-NES. This arrangement represents a novel nuclear trafficking module where the N-NLS is inactive in P1 but becomes activated in P3, concomitant with truncation of the N-NES, to become the principal targeting signal conferring nuclear accumulation. Understanding this unique switch arrangement of overlapping, co-regulated NES/NLS sequences is vital to delineating the critical role of RabV P protein in viral infection. PMID:22700958

  16. Single Cycle Structure-Based Humanization of an Anti-Nerve Growth Factor Therapeutic Antibody

    PubMed Central

    Covaceuszach, Sonia; Marinelli, Sara; Krastanova, Ivet; Ugolini, Gabriele; Pavone, Flaminia; Lamba, Doriano; Cattaneo, Antonino

    2012-01-01

    Most forms of chronic pain are inadequately treated by present therapeutic options. Compelling evidence has accumulated, demonstrating that Nerve Growth Factor (NGF) is a key modulator of inflammatory and nociceptive responses, and is a promising target for the treatment of human pathologies linked to chronic and inflammatory pain. There is therefore a growing interest in the development of therapeutic molecules antagonising the NGF pathway and its nociceptor sensitization actions, among which function-blocking anti-NGF antibodies are particularly relevant candidates. In this respect, the rat anti-NGF αD11 monoclonal antibody (mAb) is a potent antagonist, able to effectively antagonize rodent and human NGF in a variety of in vitro and in vivo systems. Here we show that mAb αD11 displays a significant analgesic effect in two different models of persistent pain in mice, with a remarkable long-lasting activity. In order to advance αD11 mAb towards its clinical application in man, anti-NGF αD11 mAb was humanized by applying a novel single cycle strategy based on the a priori experimental determination of the crystal and molecular structure of the parental Fragment antigen-binding (Fab). The humanized antibody (hum-αD11) was tested in vitro and in vivo, showing that the binding mode and the NGF neutralizing biological activities of the parental antibody are fully preserved, with even a significant affinity improvement. The results firmly establish hum-αD11 as a lead candidate for clinical applications in a therapeutic area with a severe unmet medical need. More generally, the single-cycle structure-based humanization method represents a considerable improvement over the standard humanization methods, which are intrinsically empirical and require several refinement cycles. PMID:22403636

  17. The effect of ethanol on spermatogenesis and fertility in male Sprague-Dawley rats pretreated with acetylsalicylic acid.

    PubMed

    Dare, W N; Noronha, C C; Kusemiju, O T; Okanlawon, O A

    2002-12-01

    Prenatal alcohol is associated with a variety of developmental abnormalies, including neuroanatomical, physical and behavioural features. This study was designed to determine the effects of administration of alcohol, exemplified mostly by ethanol (15 ml/kg, 25%, v/v) and acetylsalicylic acid (ASA, 15 mg/kg) as a representative of nonsteroidal anti-inflammatory drugs, individually and their combination (ethanol 15 ml/kg, 25%, v/v ASA 15 mg/kg) on semen quality and fertility after paternal intraperitoneal administration in Sprague-Dawley rats. In the combination study, the rats received ASA about 1 hour before ethanol administration. The combination experiments were conducted to determine if the effects of ethanol can be prevented by pre-treatment with acetyl salicylic acid (ASA) which has been reported to antagonise the rate-depressant effects of ethanol. All animals received this treatment for ten weeks. Semen parameters were determined and compared with controls. The result showed that when given alone, ethanol significantly reduced the sperm density and percentage of motile spermatozoa relative to controls. Pre-treatment with ASA failed to stop the decrease in sperm density and percentage motility caused by ethanol. Moreover none of the experimental male rats was able to fertilize the females exposed to them despite successful mating demonstrated by the presence of sperm plug. The present study demonstrates that chronic consumption of ethanol or ingestion of ASA has toxic effect on spermatozoa and impairs fertility in male Sprague-Dawley rats. Moreover, pre-treatment with ASA has no effect on the deleterious effects caused by ethanol.

  18. Sigma 1 receptor activation regulates brain-derived neurotrophic factor through NR2A-CaMKIV-TORC1 pathway to rescue the impairment of learning and memory induced by brain ischaemia/reperfusion.

    PubMed

    Xu, Qian; Ji, Xue-Fei; Chi, Tian-Yan; Liu, Peng; Jin, Ge; Gu, Shao-Li; Zou, Li-Bo

    2015-05-01

    Sigma-1 receptor (Sig-1R) agonists showed anti-amnesic properties in Alzheimer's disease models and anti-inflammatory properties in cerebrum ischaemia models. The agonist of Sig-1R was reported to up-regulate brain-derived neurotrophic factor (BDNF) levels in the hippocampus of mice. Here, we investigate whether the activation of Sig-1R attenuates the learning and memory impairment induced by ischaemia/reperfusion and how it affects the expression of BDNF. Bilateral common carotid artery occlusion (BCCAO) was induced for 20 min in C57BL/6 mice. Sig-1R agonist, PRE084, sigma 1/2 non-selective agonist, DTG, Sig-1R antagonist and BD1047 were injected once daily throughout the experiment. Behavioural tests were performed from day 8. On day 22 after BCCAO, mice were sacrificed for biochemical analysis. PRE084 and DTG ameliorated learning and memory impairments in the Y maze, novel object recognition, and water maze tasks and prevented the decline of synaptic proteins and BDNF expression in the hippocampus of BCCAO mice. Furthermore, PRE084 and DTG up-regulated the level of NMDA receptor 2A (NR2A), calcium/calmodulin-dependent protein kinase type IV (CaMKIV) and CREB-specific co-activator transducer of regulated CREB activity 1 (TORC1). Additionally, the effects of PRE084 and DTG were antagonised by the co-administration of BD1047. Sig-1R activation showed an attenuation in the ischaemia/reperfusion model and the activation of Sig-1R increased the expression of BDNF, possibly through the NR2A-CaMKIV-TORC1 pathway, and Sig-1R agonists might function as neuroprotectant agents in vascular dementia.

  19. Adult-onset deletion of Pten increases islet mass and beta cell proliferation in mice

    PubMed Central

    Yang, Kai-Ting; Bayan, Jennifer-Ann; Zeng, Ni; Aggarwal, Richa; He, Lina; Peng, Zhechu; Kassa, Anketse; Kim, Melissa; Luo, Zhiou; Shi, Zhenrong; Medina, Vivian; Boddupally, Keerthi; Stiles, Bangyan L.

    2013-01-01

    Aims/hypothesis Adult beta cells have a diminished ability to proliferate. Phosphatase and tensin homologue (PTEN) is a lipid phosphatase that antagonises the function of the mitogenic phosphatidylinositol 3-kinase (PI3K) pathway. The objective of this study was to understand the role of PTEN and PI3K signalling in the maintenance of beta cells postnatally. Methods We developed a Ptenlox/lox; Rosa26lacZ; RIP-CreER+ model that permitted us to induce Pten deletion by treatment with tamoxifen in mature animals. We evaluated islet mass and function as well as beta cell proliferation in 3- and 12-month-old mice treated with tamoxifen (Pten deleted) vs mice treated with vehicle (Pten control). Results Deletion of Pten in juvenile (3-month-old) beta cells significantly induced their proliferation and increased islet mass. The expansion of islet mass occurred concomitantly with the enhanced ability of the Pten-deleted mice to maintain euglycaemia in response to streptozotocin treatment. In older mice (>12 months of age), deletion of Pten similarly increased islet mass and beta cell proliferation. This novel finding suggests that PTEN-regulated mechanisms may override the age-onset diminished ability of beta cells to respond to mitogenic stimulation. We also found that proteins regulating G1/S cell-cycle transition, such as cyclin D1, cyclin D2, p27 and p16, were altered when PTEN was lost, suggesting that they may play a role in PTEN/PI3K-regulated beta cell proliferation in adult tissue. Conclusions/interpretation The signals regulated by the PTEN/PI3K pathway are important for postnatal maintenance of beta cells and regulation of their proliferation in adult tissues. PMID:24162585

  20. The actions of some cannabinoid receptor ligands in the rat isolated mesenteric artery

    PubMed Central

    White, Richard; Robin Hiley, C

    1998-01-01

    The actions of a number of cannabinoid receptor ligands were investigated using the myograph-mounted rat isolated mesenteric artery. Anandamide, CP 55,940, HU-210, palmitoylethanolamide and WIN 55,212-2 all caused concentration-dependent relaxations of methoxamine-precontracted vessels which were not affected by removal of the endothelium.Precontracting vessels with 60 mM KCl instead of methoxamine greatly reduced the vasorelaxant effects of anandamide and palmitoylethanolamide. High K+ solution caused a modest decrease in the relaxant potency of CP 55,940 and HU-210, and had no effect on relaxations induced by WIN 55,212-2.Relaxations of methoxamine-induced tone by anandamide, CP 55,940 and HU-210, but not palmitoylethanolamide and WIN 55,212-2, were attenuated by the cannabinoid receptor antagonist, SR 141716A. Relaxation of vessels contracted with 60 mM KCl by CP 55,940 was also sensitive to SR 141716A.Anandamide and CP 55,940 caused small but concentration-dependent contractions in resting vessels in the absence of extracellular calcium. These were not sensitive to SR 141716A. Palmitoylethanolamide and WIN 55,212-2 produced smaller contractions only at higher concentrations.Anandamide and CP 55,940, but not palmitoylethanolamide and WIN 55,212-2, caused concentration-dependent inhibition of the phasic contractions induced by methoxamine in calcium-free conditions, but only anandamide caused inhibition of contractions to caffeine under such conditions. These inhibitory effects were not antagonised by SR 141716A.The present study provides the first detailed investigation of the actions of cannabinoid agonists on vascular smooth muscle. Our results show that these compounds exert both receptor-dependent and -independent effects on agonist-induced calcium mobilization in the rat isolated mesenteric artery. PMID:9806337

  1. Long-term surgical anaesthesia with isoflurane in human habituated Nile Crocodiles.

    PubMed

    Stegmann, George F; Williams, Catherine J A; Franklin, Craig; Wang, Tobias; Axelsson, Michael

    2017-02-24

    A suitable long-term anaesthetic technique was required for implantation of physiological sensors and telemetric devices in sub-adult Nile crocodiles (Crocodylus niloticus) to allow the collection of physiological data. Five Nile crocodiles with a median body mass of 24 kg were used. After manual capture, they were blindfolded and 0.2 mL (1 mg/mL) medetomidine was administered intramuscularly in four of the animals which had an estimated body mass between 20 kg and 30 kg. One crocodile with an estimated body mass of 50 kg received 0.5 mL. For induction, 5 mL propofol (10 mg/mL) was injected intravenously into the occipital sinus. Additional doses were given when required to ensure adequate anaesthesia. Anaesthesia was maintained with 1.5% isoflurane. Ventilation was controlled. Local anaesthesia was administered for surgical incision and external placement of the radio transmitter. Medetomidine was antagonised with atipamezole at the end of surgery. Median heart rate during surgery was 22 beats/min, at extubation 32 beats per min and 30 beats per min the following day at the same body temperature as under anaesthesia. Median body temperature of the animals increased from 27.3 °C to 27.9 °C during anaesthesia, as room temperature increased from 24.5 °C to 29.0 °C during surgery. Anaesthesia was successfully induced with intramuscular medetomidine and intravenous propofol and was maintained with isoflurane for the placement of telemetric implants. Intraoperative analgesia was supplemented with lidocaine infiltration. Perioperative physiological parameters remained stable and within acceptable clinical limits. Multiple factors appear to influence these variables during the recovery period, including residual anaesthetic effects, environmental temperature and physical activity.

  2. Genistein-induced fluid accumulation in ovariectomised rats' uteri is associated with increased cystic fibrosis transmembrane regulator expression

    PubMed Central

    Chinigarzadeh, Asma; Kassim, Normadiah M.; Muniandy, Sekaran; Salleh, Naguib

    2014-01-01

    OBJECTIVE: High genistein doses have been reported to induce fluid accumulation in the uteri of ovariectomised rats, although the mechanism underlying this effect remains unknown. Because genistein binds to the oestrogen receptor and the cystic fibrosis transmembrane regulator mediates uterine fluid secretion, we hypothesised that this genistein effect involves both the oestrogen receptor and cystic fibrosis transmembrane regulator. METHODS: Ovariectomised adult female Sprague-Dawley rats were treated with 25, 50, or 100 mg/kg/day genistein for three consecutive days with and without the ER antagonist ICI 182780. One day after the final drug injection, the animals were humanely sacrificed, and the uteri were removed for histology and cystic fibrosis transmembrane regulator mRNA and protein expression analysis using real-time polymerase chain reaction and Western blotting, respectively. The cystic fibrosis transmembrane regulator protein distribution was analysed visually by immunohistochemistry. RESULTS: The histological analysis revealed an increase in the circumference of the uterine lumen with increasing doses of genistein, which was suggestive of fluid accumulation. Moreover, genistein stimulated a dose-dependent increase in the expression of cystic fibrosis transmembrane regulator protein and mRNA, and high-intensity cystic fibrosis transmembrane regulator immunostaining was observed at the apical membrane of the luminal epithelium following 50 and 100 mg/kg/day genistein treatment. The genistein-induced increase in uterine luminal circumference and cystic fibrosis transmembrane regulator expression was antagonised by treatment with ICI 182780. CONCLUSION: Genistein-induced luminal fluid accumulation in ovariectomised rats' uteri involves the oestrogen receptor and up-regulation of cystic fibrosis transmembrane regulator expression, and these findings reveal the mechanism underlying the effect of this compound on changes in fluid volume in the uterus after

  3. Genistein-induced fluid accumulation in ovariectomised rats' uteri is associated with increased cystic fibrosis transmembrane regulator expression.

    PubMed

    Chinigarzadeh, Asma; Kassim, Normadiah M; Muniandy, Sekaran; Salleh, Naguib

    2014-02-01

    High genistein doses have been reported to induce fluid accumulation in the uteri of ovariectomised rats, although the mechanism underlying this effect remains unknown. Because genistein binds to the oestrogen receptor and the cystic fibrosis transmembrane regulator mediates uterine fluid secretion, we hypothesised that this genistein effect involves both the oestrogen receptor and cystic fibrosis transmembrane regulator. Ovariectomised adult female Sprague-Dawley rats were treated with 25, 50, or 100 mg/kg/day genistein for three consecutive days with and without the ER antagonist ICI 182780. One day after the final drug injection, the animals were humanely sacrificed, and the uteri were removed for histology and cystic fibrosis transmembrane regulator mRNA and protein expression analysis using real-time polymerase chain reaction and Western blotting, respectively. The cystic fibrosis transmembrane regulator protein distribution was analysed visually by immunohistochemistry. The histological analysis revealed an increase in the circumference of the uterine lumen with increasing doses of genistein, which was suggestive of fluid accumulation. Moreover, genistein stimulated a dose-dependent increase in the expression of cystic fibrosis transmembrane regulator protein and mRNA, and high-intensity cystic fibrosis transmembrane regulator immunostaining was observed at the apical membrane of the luminal epithelium following 50 and 100 mg/kg/day genistein treatment. The genistein-induced increase in uterine luminal circumference and cystic fibrosis transmembrane regulator expression was antagonised by treatment with ICI 182780. Genistein-induced luminal fluid accumulation in ovariectomised rats' uteri involves the oestrogen receptor and up-regulation of cystic fibrosis transmembrane regulator expression, and these findings reveal the mechanism underlying the effect of this compound on changes in fluid volume in the uterus after menopause.

  4. Functional Dissection of Caenorhabditis elegans CLK-2/TEL2 Cell Cycle Defects during Embryogenesis and Germline Development

    PubMed Central

    Moser, Sandra C.; von Elsner, Sophie; Büssing, Ingo; Alpi, Arno; Schnabel, Ralf; Gartner, Anton

    2009-01-01

    CLK-2/TEL2 is essential for viability from yeasts to vertebrates, but its essential functions remain ill defined. CLK-2/TEL2 was initially implicated in telomere length regulation in budding yeast, but work in Caenorhabditis elegans has uncovered a function in DNA damage response signalling. Subsequently, DNA damage signalling defects associated with CLK-2/TEL2 have been confirmed in yeast and human cells. The CLK-2/TEL2 interaction with the ATM and ATR DNA damage sensor kinases and its requirement for their stability led to the proposal that CLK-2/TEL2 mutants might phenocopy ATM and/or ATR depletion. We use C. elegans to dissect developmental and cell cycle related roles of CLK-2. Temperature sensitive (ts) clk-2 mutants accumulate genomic instability and show a delay of embryonic cell cycle timing. This delay partially depends on the worm p53 homolog CEP-1 and is rescued by co-depletion of the DNA replication checkpoint proteins ATL-1 (C. elegans ATR) and CHK-1. In addition, clk-2 ts mutants show a spindle orientation defect in the eight cell stages that lead to major cell fate transitions. clk-2 deletion worms progress through embryogenesis and larval development by maternal rescue but become sterile and halt germ cell cycle progression. Unlike ATL-1 depleted germ cells, clk-2–null germ cells do not accumulate DNA double-strand breaks. Rather, clk-2 mutant germ cells arrest with duplicated centrosomes but without mitotic spindles in an early prophase like stage. This germ cell cycle arrest does not depend on cep-1, the DNA replication, or the spindle checkpoint. Our analysis shows that CLK-2 depletion does not phenocopy PIKK kinase depletion. Rather, we implicate CLK-2 in multiple developmental and cell cycle related processes and show that CLK-2 and ATR have antagonising functions during early C. elegans embryonic development. PMID:19360121

  5. The receptor for advanced glycation end products RAGE is involved in corneal healing.

    PubMed

    Nass, Norbert; Trau, Stefanie; Paulsen, Friedrich; Kaiser, Delia; Kalinski, Thomas; Sel, Saadettin

    2017-05-01

    Impaired corneal healing is still a major cause of blindness. As RAGE (receptor for advanced glycation endproducts) is involved in inflammation and wound healing in other tissues, we here investigated its relevance for corneal wound healing. Corneal re-epithelialization after alkaline injury was analysed in an ex-vivo approach with cultured, enucleated eyes from mice either of the C57Bl/6 NChR genotype (RAGE+/+) and mice of the same strain lacking the RAGE gene (RAGE-/-). The wound area was determined time dependently by fluorescence imaging using fluorescein staining. The eyes of RAGE-/- mice showed a significantly slower re-epithelialization than eyes of the RAGE+/- and the RAGE+/+ genotype. In immunohistochemistry, RAGE expression was increased in wounded corneas whereas the abundance of the RAGE ligand HMGB1 was unaffected, but an increase in S100b-like proteins was revealed upon injury. However, neither the addition of the RAGE agonist HMGB1 or an HMGB1 antagonising antibody nor bovine S100b protein to the culture medium of the wounded eyes had an effect on corneal wound closure in ex-vivo. Further gene expression analysis by RT-PCR demonstrated an increase in RAGE expression on the mRNA level, no significant regulation of HMGB1 and a differential regulation of the S100 gene family after alkaline burn of the cornea. In conclusion, RAGE is clearly involved in corneal re-epithelialization most probably mediated by signalling via S100 proteins. Copyright © 2017 Elsevier GmbH. All rights reserved.

  6. The paratransgenic potential of Lactobacillus kunkeei in the honey bee Apis mellifera.

    PubMed

    Rangberg, A; Mathiesen, G; Amdam, G V; Diep, D B

    2015-01-01

    The honey bee (Apis mellifera) is a domestic insect of high value to human societies, as a crop pollinator in agriculture and a model animal in scientific research. The honey bee, however, has experienced massive mortality worldwide due to the phenomenon Colony Collapse Disorder (CCD), resulting in alarming prospects for crop failure in Europe and the USA. The reasons for CCD are complex and much debated, but several honey bee pathogens are believed to be involved. Paratransgenesis is a Trojan horse strategy, where endogenous microorganisms are used to express effector molecules that antagonise pathogen development. For use in honey bees, paratransgenesis must rely on a set of criteria that the candidate paratransgenic microorganism must fulfil in order to obtain a successful outcome: (1) the candidate must be genetically modifiable to express effector molecules; (2) the modified organism should have no adverse effects on honey bee health upon reintroduction; and (3) it must survive together with other non-pathogenic bee-associated microorganisms. Lactic acid bacteria (LAB) are common gut bacteria in vertebrates and invertebrates, and some have naturally beneficial properties in their host. In the present work we aimed to find a potential paratransgenic candidate within this bacterial group for use in honey bees. Among isolated LAB associated with bee gut microbiota, we found the fructophilic Lactobacillus kunkeei to be the most predominant species during foraging seasons. Four genetically different strains of L. kunkeei were selected for further assessment. We demonstrated (1) that L. kunkeei is transformable; (2) that the transformed cells had no obvious adverse effect on honey bee survival; and (3) that transformed cells survived well in the gut environment of bees upon reintroduction. Our study demonstrates that L. kunkeei fulfils the three criteria for paratransgenesis and can be a suitable candidate for further research on this strategy in honey bees.

  7. Inhibitory effects of imidazoline receptor ligands on basal and kainic acid-induced neurotoxic signalling in mice.

    PubMed

    Keller, Benjamin; García-Sevilla, Jesús A

    2016-09-01

    This in vivo study assessed the potential of the imidazoline receptor (IR) ligands moxonidine (selective I1-IR), BU224 (selective I2-IR) and LSL61122 (mixed I1/I2-IR) to dampen excitotoxic signalling induced by kainic acid (KA; 45 mg/kg) in the mouse brain (hippocampus and cerebral cortex). KA triggered a strong behavioural syndrome (seizures; maximal at 60-90 minutes) and sustained stimulation (at 72 hours with otherwise normal mouse behaviour) of pro-apoptotic c-Jun-N-terminal kinases (JNK) and calpain with increased cleavage of p35 into neurotoxic p25 (cyclin-dependent kinase 5 [Cdk5] activators) in mouse hippocampus. Pretreatment (five days) with LSL61122 (10 mg/kg), but not moxonidine (1 mg/kg) or BU224 (20 mg/kg), attenuated the KA-induced behavioural syndrome, and all three IR ligands inhibited JNK and calpain activation, as well as p35/p25 cleavage after KA in the hippocampus (effects also observed after acute IR drug treatments). Efaroxan (I1-IR, 10 mg/kg) and idazoxan (I2-IR, 10 mg/kg), postulated IR antagonists, did not antagonise the effects of moxonidine and LSL61122 on KA targets (these IR ligands showed agonistic properties inhibiting pro-apoptotic JNK). Brain subcellular preparations revealed reduced synaptosomal postsynaptic density-95 protein contents (a mediator of JNK activation) and indicated increased p35/Cdk5 complexes (with pro-survival functions) after treatment with moxonidine, BU224 and LSL61122. These results showed that I1- and I2-IR ligands (moxonidine and BU224), and especially the mixed I1/I2-IR ligand LSL61122, are partly neuroprotective against KA-induced excitotoxic signalling. These findings suggest a therapeutic potential of IR drugs in disorders associated with glutamate-mediated neurodegeneration. © The Author(s) 2016.

  8. BRCA1-associated exclusion of 53BP1 from DNA damage sites underlies temporal control of DNA repair

    PubMed Central

    Chapman, J. Ross; Sossick, Alex J.; Boulton, Simon J.; Jackson, Stephen P.

    2012-01-01

    Summary Following irradiation, numerous DNA-damage-responsive proteins rapidly redistribute into microscopically visible subnuclear aggregates, termed ionising-radiation-induced foci (IRIF). How the enrichment of proteins on damaged chromatin actually relates to DNA repair remains unclear. Here, we use super-resolution microscopy to examine the spatial distribution of BRCA1 and 53BP1 proteins within single IRIF at subdiffraction-limit resolution, yielding an unprecedented increase in detail that was not previously apparent by conventional microscopy. Consistent with a role for 53BP1 in promoting DNA double-strand break repair by non-homologous end joining, 53BP1 enrichment in IRIF is most prominent in the G0/G1 cell cycle phases, where it is enriched in dense globular structures. By contrast, as cells transition through S phase, the recruitment of BRCA1 into the core of IRIF is associated with an exclusion of 53BP1 to the focal periphery, leading to an overall reduction of 53BP1 occupancy at DNA damage sites. Our data suggest that the BRCA1-associated IRIF core corresponds to chromatin regions associated with repair by homologous recombination, and the enrichment of BRCA1 in IRIF represents a temporal switch in the DNA repair program. We propose that BRCA1 antagonises 53BP1-dependent DNA repair in S phase by inhibiting its interaction with chromatin proximal to damage sites. Furthermore, the genomic instability exhibited by BRCA1-deficient cells might result from a failure to efficiently exclude 53BP1 from such regions during S phase. PMID:22553214

  9. Functional evaluation of the receptors mediating vasoconstriction of rat aorta by trace amines and amphetamines.

    PubMed

    Broadley, Kenneth J; Fehler, Martina; Ford, William R; Kidd, Emma J

    2013-09-05

    Trace amines including β-phenylethylamine (β-PEA) and amphetamines classically exert pharmacological actions via indirect sympathomimetic mechanisms. However, there is evidence for other mechanisms and this study explores the receptors mediating vasoconstriction in rat aorta. β-PEA, d-amphetamine, MDMA, cathinone and methylphenidate caused concentration-dependent contractions of rat isolated aortic rings which were unaffected by prazosin (1 μM), ICI-118,551 (1 μM), cocaine (10 μM) and pargyline (10 μM), to inhibit α1- and β2-adrenoceptors, neuronal transport and monoamine oxidase (MAO), respectively. Octopamine concentration-response curves, however, were shifted to the right. In the presence of the inhibitors, the rate of onset of octopamine contractions was slowed. Lineweaver-Burk analysis of the kinetics of the response generated different KM values for octopamine in the absence (2.35 × 10(-6)M) and presence (6.09 × 10(-5)M) of inhibitors, indicating mediation by different receptors. Tryptamine-induced vasoconstriction also resisted blockade by adrenergic inhibitors and the 5-HT1A, 1B, 1D and 5-HT2A receptor antagonists, methiothepin (50 nM) and ketanserin (30 nM), respectively. Trace amines and amphetamines therefore exert vasoconstriction independently of adrenoceptors, neuronal transport and 5-HT receptor activation. There was no evidence of tachyphylaxis or cross-tachyphylaxis of the vasoconstriction to these amines. Tyramine was a partial agonist and in its presence, β-PEA, d-amphetamine and octopamine were antagonised indicating that they all act through a common receptor for which tyramine serves as an antagonist. We conclude that the vasoconstriction is via TAAR-1, because of structural similarities between amines, ability to stimulate recombinant trace amine-associated receptor 1 (TAAR-1) and the presence of TAAR-1 in rat aorta. © 2013 Elsevier B.V. All rights reserved.

  10. Anti-Plasmodial Polyvalent Interactions in Artemisia annua L. Aqueous Extract – Possible Synergistic and Resistance Mechanisms

    PubMed Central

    Suberu, John O.; Gorka, Alexander P.; Jacobs, Lauren; Roepe, Paul D.; Sullivan, Neil

    2013-01-01

    Artemisia annua hot water infusion (tea) has been used in in vitro experiments against P. falciparum malaria parasites to test potency relative to equivalent pure artemisinin. High performance liquid chromatography (HPLC) and mass spectrometric analyses were employed to determine the metabolite profile of tea including the concentrations of artemisinin (47.5±0.8 mg L-1), dihydroartemisinic acid (70.0±0.3 mg L-1), arteannuin B (1.3±0.0 mg L-1), isovitexin (105.0±7.2 mg L-1) and a range of polyphenolic acids. The tea extract, purified compounds from the extract, and the combination of artemisinin with the purified compounds were tested against chloroquine sensitive and chloroquine resistant strains of P. falciparum using the DNA-intercalative SYBR Green I assay. The results of these in vitro tests and of isobologram analyses of combination effects showed mild to strong antagonistic interactions between artemisinin and the compounds (9-epi-artemisinin and artemisitene) extracted from A. annua with significant (IC50 <1 μM) anti-plasmodial activities for the combination range evaluated. Mono-caffeoylquinic acids, tri-caffeoylquinic acid, artemisinic acid and arteannuin B showed additive interaction while rosmarinic acid showed synergistic interaction with artemisinin in the chloroquine sensitive strain at a combination ratio of 1:3 (artemisinin to purified compound). In the chloroquine resistant parasite, using the same ratio, these compounds strongly antagonised artemisinin anti-plasmodial activity with the exception of arteannuin B, which was synergistic. This result would suggest a mechanism targeting parasite resistance defenses for arteannuin B’s potentiation of artemisinin. PMID:24244716

  11. E2/Estrogen Receptor/Sjogren Syndrome-Associated Autoantigen Relieves Coactivator Activator-Induced G1/S Arrest To Promote Breast Tumorigenicity

    PubMed Central

    Kang, Yun Kyoung; Jung, Sung Yun; Qin, Jun; Li, Chao; Tsai, Sophia Y.; Tsai, Ming-Jer

    2014-01-01

    Coactivator activator (CoAA) is a dual-functional coregulator that regulates steroid receptor-mediated transcription and alternative splicing. Previously, we have shown that CoAA has tumor-suppressive potential in tumorigenic human kidney cells. Here, we uncover a molecular mechanism by which Sjogren syndrome-associated autoantigen (SSA), an estrogen receptor (ER) coactivator, induces MYC oncogene by removing repressive CoAA through E2-dependent degradation of CoAA and promotes G1/S transition of the cell cycle as well as anchorage-independent growth capability of breast cancer cells. We also show that E2 and ER enhance the E3 ligase activity of SSA to modulate CoAA through splicing isoform-selective ubiquitylation. We propose this as one potential molecular basis for the reduced tumor incidence in autoimmune disease patients and suggest SSA as a potential therapeutic target to treat breast cancer. PMID:24567374

  12. Induction of promyelocytic leukemia (PML) oncogenic domains (PODs) by papillomavirus

    SciTech Connect

    Nakahara, Tomomi; Lambert, Paul F.

    2007-09-30

    Promyelocytic leukemia oncogenic domains (PODs), also called nuclear domain 10 (ND10), are subnuclear structures that have been implicated in a variety of cellular processes as well as the life cycle of DNA viruses including papillomaviruses. In order to investigate the interplay between papillomaviruses and PODs, we analyzed the status of PODs in organotypic raft cultures of human keratinocytes harboring HPV genome that support the differentiation-dependent HPV life cycle. The number of PODs per nucleus was increased in the presence of HPV genomes selectively within the poorly differentiated layers but was absent in the terminally differentiated layers of the stratified epithelium. This increase in PODs was correlated with an increase in abundance of post-translationally modified PML protein. Neither the E2-dependent transcription nor viral DNA replication was reliant upon the presence of PML. Implications of these findings in terms of HPV's interaction with its host are discussed.

  13. Involvement of Brd4 in different steps of the papillomavirus life cycle

    PubMed Central

    Iftner, Thomas; Haedicke-Jarboui, Juliane; Wu, Shwu-Yuan; Chiang, Cheng-Ming

    2017-01-01

    Bromodomain-containing protein 4 (Brd4) is a cellular chromatin-binding factor and transcriptional regulator that recruits sequence-specific transcription factors and chromatin modulators to control target gene transcription. Papillomaviruses (PVs) have evolved to hijack Brd4’s activity in order to create a facilitating environment for the viral life cycle. Brd4, in association with the major viral regulatory protein E2, is involved in multiple steps of the PV life cycle including replication initiation, viral gene transcription, and viral genome segregation and maintenance. Phosphorylation of Brd4, regulated by casein kinase II (CK2) and protein phosphatase 2A (PP2A), is critical for viral gene transcription as well as E1- and E2-dependent origin replication. Thus, pharmacological agents regulating Brd4 phosphorylation and inhibitors blocking phospho-Brd4 functions are promising candidates for therapeutic intervention in treating human papillomavirus (HPV) infections as well as associated disease. PMID:27965149

  14. A transcriptional repressive role for epithelial-specific ETS factor ELF3 on oestrogen receptor alpha in breast cancer cells.

    PubMed

    Gajulapalli, Vijaya Narasihma Reddy; Samanthapudi, Venkata Subramanyam Kumar; Pulaganti, Madhusudana; Khumukcham, Saratchandra Singh; Malisetty, Vijaya Lakhsmi; Guruprasad, Lalitha; Chitta, Suresh Kumar; Manavathi, Bramanandam

    2016-04-15

    Oestrogen receptor-α (ERα) is a ligand-dependent transcription factor that primarily mediates oestrogen (E2)-dependent gene transcription required for mammary gland development. Coregulators critically regulate ERα transcription functions by directly interacting with it. In the present study, we report that ELF3, an epithelial-specific ETS transcription factor, acts as a transcriptional repressor of ERα. Co-immunoprecipitation (Co-IP) analysis demonstrated that ELF3 strongly binds to ERα in the absence of E2, but ELF3 dissociation occurs upon E2 treatment in a dose- and time-dependent manner suggesting that E2 negatively influences such interaction. Domain mapping studies further revealed that the ETS (E-twenty six) domain of ELF3 interacts with the DNA binding domain of ERα. Accordingly, ELF3 inhibited ERα's DNA binding activity by preventing receptor dimerization, partly explaining the mechanism by which ELF3 represses ERα transcriptional activity. Ectopic expression of ELF3 decreases ERα transcriptional activity as demonstrated by oestrogen response elements (ERE)-luciferase reporter assay or by endogenous ERα target genes. Conversely ELF3 knockdown increases ERα transcriptional activity. Consistent with these results, ELF3 ectopic expression decreases E2-dependent MCF7 cell proliferation whereas ELF3 knockdown increases it. We also found that E2 induces ELF3 expression in MCF7 cells suggesting a negative feedback regulation of ERα signalling in breast cancer cells. A small peptide sequence of ELF3 derived through functional interaction between ERα and ELF3 could inhibit DNA binding activity of ERα and breast cancer cell growth. These findings demonstrate that ELF3 is a novel transcriptional repressor of ERα in breast cancer cells. Peptide interaction studies further represent a novel therapeutic option in breast cancer therapy. © 2016 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

  15. Kisspeptin regulates tuberoinfundibular dopaminergic neurones and prolactin secretion in an oestradiol-dependent manner in male and female rats.

    PubMed

    Ribeiro, A B; Leite, C M; Kalil, B; Franci, C R; Anselmo-Franci, J A; Szawka, R E

    2015-02-01

    Prolactin (PRL) secretion is inhibited by hypothalamic dopamine. Kisspeptin controls luteinising hormone (LH) secretion and is also involved in PRL regulation. We further investigated the effect of kisspeptin-10 (Kp-10) on the activity of tuberoinfundibular dopaminergic (TIDA) neurones and the role of oestradiol (E2 ) in this mechanism. Female and male rats were injected with i.c.v. Kp-10 and evaluated for PRL release and the activity of dopamine terminals in the median eminence (ME) and neurointermediate lobe of the pituitary (NIL). Kp-10 at the doses of 0.6 and 3 nmol increased plasma PRL and decreased 4-dihydroxyphenylacetic acid (DOPAC) levels in the ME and NIL of ovariectomised (OVX), E2 -treated rats but had no effect in OVX. In gonad-intact males, 3 nmol Kp-10 increased PRL secretion and decreased DOPAC levels in the ME but not in the NIL. Castrated males treated with either testosterone or E2 also displayed increased PRL secretion and reduced ME DOPAC in response to Kp-10, whereas castrated rats receiving oil or dihydrotestosterone were unresponsive. By contrast, the LH response to Kp-10 was not E2 -dependent in either females or males. Additionally, immunohistochemical double-labelling demonstrated that TIDA neurones of male rats contain oestrogen receptor (ER)-α, with a higher proportion of neurones expressing ERα than in dioestrous females. The dopaminergic neurones of periventricular hypothalamic nucleus displayed much lower ERα expression. Thus, TIDA neurones express ERα in male and female rats, and kisspeptin increases PRL secretion through inhibition of TIDA neurones in an E2 -dependent manner in both sexes. These findings provide new evidence about the role of kisspeptin in the regulation of dopamine and PRL. © 2014 British Society for Neuroendocrinology.

  16. Mixed lineage leukaemia histone methylases 1 collaborate with ERα to regulate HOXA10 expression in AML

    PubMed Central

    Yao, Jie; Fang, Li-Chao; Yang, Zai-Lin; Huang, Hui; Li, Yan; Deng, Jun; Zheng, Junsong

    2014-01-01

    HOXA10, a homeobox-containing gene involved in definitive haematopoiesis, which implicated in the pathogenesis of AML (acute myeloid leukaemia), has been studied extensively. But the regulatory mechanism that drives HOXA10 expression is still unclear. In the present paper, HOXA10 regulated by MLL1 (mixed lineage leukaemia histone methylase 1) with an epigenetic way has been demonstrated. The HOXA10 promoter contains several EREs (oestrogen response elements), including ERE1 and ERE2, which are close to the transcription start site, and are associated with E2-mediated activation of HOXA10. It has been shown that knockdown of the ERα (oestrogen receptor α) suppresses E2-mediated activation of HOXA10. Similarly, knockdown of MLL1 suppresses activation of HOXA10 and is bound to the ERE of HOXA10 promoter in an E2-dependent manner by forming complex with ERα. Knockdown of ERα affects the E2-dependent binding of MLL1 into HOXA10 EREs, suggesting critical roles of ERα in recruiting MLL on the HOXA10 promoter. More interestingly, the methylation status of histone protein H3K4 (H3 at lysine 4) with E2 is much higher than without E2 treatment in leukaemia cell. On the contrary, the methylation status of HOXA10 promoter with E2 treatment is much lower, which elevate the HOXA10 expression. Moreover, with ERα knockdown, the H3K4 methylation level is also decrease in myeloid cell. Overall, it has been clearly demonstrated that HOXA10 is transcriptionally regulated by MLL1, which, in coordination with ERα, plays a critical role in this process with epigenetic way and suggests a potential anti-E2 treatment of AML. PMID:25307539

  17. Estrogen receptor α L429 and A430 regulate 17β-estradiol-induced cell proliferation via CREB1.

    PubMed

    Pesiri, Valeria; Totta, Pierangela; Segatto, Marco; Bianchi, Fabrizio; Pallottini, Valentina; Marino, Maria; Acconcia, Filippo

    2015-12-01

    17β-Estradiol (E2)-dependent cell proliferation requires both estrogen receptor α (ERα)-based integrated control of gene transcription and kinase pathways activation. Such coordination of intracellular E2:ERα-dependent signaling mechanisms is finely tuned by receptor association with specific partner proteins. Recently, we identified the leucine (L) 429 and alanine (A) 430 within the ERα ligand binding domain as important residues for receptor non-covalent interaction to ubiquitinated species [i.e., ERα ubiquitin-binding surface (ERα UBS)] and for E2-induced ERα activation. To date, if these two ERα amino acids are involved in the control of E2-dependent pathways required for cell proliferation is unknown. Here, by using stably expressing ERα mutated in L429 and A430 (i.e., L429A,A430G-LAAG) cell lines, we show that L429 and A430 are critical for E2-induced cell proliferation, PI3K/AKT pathway activation, and ERα-mediated transcriptional changes. Moreover, we demonstrate that these two receptor structural determinants direct the E2-induced PI3K/AKT/CREB1 pathway activation and CREB1-mediated transcriptional activity that in turn control the hormone-induced cell proliferation. As a whole, our data demonstrate for the first time that the ERα UBS contributes to the modulation of E2-induced ERα-mediated cell proliferation and provide a novel connection between the receptor structure and the functional molecular mechanisms by which E2:ERα complex can regulate cell processes.

  18. Estrogen mediated epithelial proliferation in the uterus is directed by stromal Fgf10 and Bmp8a.

    PubMed

    Chung, Daesuk; Gao, Fei; Jegga, Anil G; Das, Sanjoy K

    2015-01-15

    To define endometrial stromal-derived paracrine mediators that participate in estradiol-17β (E2)-induced epithelial proliferation, microarray analysis of gene expression was carried out in mouse uterine epithelial-stromal co-culture systems under the condition of E2 or vehicle (control). Our results demonstrated gene alteration by E2: in epithelial cells, we found up-regulation of 119 genes and down-regulation of 28 genes, while in stroma cells we found up-regulation of 144 genes and down-regulation of 184 genes. A functional enrichment analysis of the upregulated epithelial genes implicated them for proliferation, while upregulated stromal genes were associated with extracellular functions. Quantitative RT-PCR and in situ hybridization results confirmed differential gene expression in both cell cultures and ovariectomized uteri after the above treatments. Based on our identification of stromal secretory factors, we found evidence that suppression by siRNA specifically for Bmp8a and/or Fgf10 in the stromal layer caused significant inhibition of proliferation by E2 in the co-culture system, suggesting Bmp8a and Fgf10 act as paracrine mediators during E2-dependent control of uterine proliferation. The localization of receptors and receptor activation signaling in epithelial cells in both the co-culture system and uteri was consistent with their involvement in ligand-receptor signaling. Interestingly, loss of Bmp8a or Fgf10 also caused abrogation of E2-regulated epithelial receptor signaling in co-culture systems, suggesting that stroma-derived Fgf10 and Bmp8a are responsible for epithelial communication. Overall, stromal Fgf10 and Bmp8a serve as potential paracrine factors for E2-dependent regulation of epithelial proliferation in the uterus.

  19. Aromatase Inhibitor-Associated Bone Fractures: A Case-Cohort GWAS and Functional Genomics

    PubMed Central

    Liu, Mohan; Goss, Paul E.; Ingle, James N.; Kubo, Michiaki; Furukawa, Yoichi; Batzler, Anthony; Jenkins, Gregory D.; Carlson, Erin E.; Nakamura, Yusuke; Schaid, Daniel J.; Chapman, Judy-Anne W.; Shepherd, Lois E.; Ellis, Matthew J.; Khosla, Sundeep; Wang, Liewei

    2014-01-01

    Bone fractures are a major consequence of osteoporosis. There is a direct relationship between serum estrogen concentrations and osteoporosis risk. Aromatase inhibitors (AIs) greatly decrease serum estrogen levels in postmenopausal women, and increased incidence of fractures is a side effect of AI therapy. We performed a discovery case-cohort genome-wide association study (GWAS) using samples from 1071 patients, 231 cases and 840 controls, enrolled in the MA.27 breast cancer AI trial to identify genetic factors involved in AI-related fractures, followed by functional genomic validation. Association analyses identified 20 GWAS single nucleotide polymorphism (SNP) signals with P < 5E-06. After removal of signals in gene deserts and those composed entirely of imputed SNPs, we applied a functional validation “decision cascade” that resulted in validation of the CTSZ-SLMO2-ATP5E, TRAM2-TMEM14A, and MAP4K4 genes. These genes all displayed estradiol (E2)-dependent induction in human fetal osteoblasts transfected with estrogen receptor-α, and their knockdown altered the expression of known osteoporosis-related genes. These same genes also displayed SNP-dependent variation in E2 induction that paralleled the SNP-dependent induction of known osteoporosis genes, such as osteoprotegerin. In summary, our case-cohort GWAS identified SNPs in or near CTSZ-SLMO2-ATP5E, TRAM2-TMEM14A, and MAP4K4 that were associated with risk for bone fracture in estrogen receptor-positive breast cancer patients treated with AIs. These genes displayed E2-dependent induction, their knockdown altered the expression of genes related to osteoporosis, and they displayed SNP genotype-dependent variation in E2 induction. These observations may lead to the identification of novel mechanisms associated with fracture risk in postmenopausal women treated with AIs. PMID:25148458

  20. Effects of the aryl hydrocarbon receptor agonist 3-methylcholanthrene on the 17β-estradiol regulated mRNA transcriptome of the rat uterus.

    PubMed

    Helle, Janina; Keiler, Annekathrin M; Zierau, Oliver; Dörfelt, Peggy; Vollmer, Günter; Lehmann, Leane; Chittur, Sridar V; Tenniswood, Martin; Welsh, JoEllen; Kretzschmar, Georg

    2017-07-01

    Polycyclic aromatic hydrocarbons (PAHs) are products of incomplete combustion of organic compounds, abundant in exhaust fumes and cigarette smoke. They act by binding to the aryl hydrocarbon receptor (AHR) which induces expression of phase 1 and phase 2 enzymes in the liver. PAH induced AHR activation may also lead to adverse effects by modulating other pathways, for example estrogen receptor (ER) signaling in the female reproductive tract. We have investigated the effects of the PAH 3-methylcholanthrene (3-MC) on 17β-estradiol (E2) dependent signaling in the uterus of ovariectomized rats to characterize the cross talk between AHR and ER on an mRNA transcriptome wide scale. A standard three day uterotrophic assay was performed in young adult Lewis rats. Treatment induced effects were analyzed using histology, immunohistochemistry and gene expression analysis by microarray and qPCR. 3-MC shows broad E2 antagonistic effects on uterine mRNA transcription of the vast majority of E2 regulated genes, significantly altering prostaglandin biosynthesis, complement activation, coagulation pathways and other inflammatory response pathways. The regulation of ER expression in the uterus, but not the regulation of E2 metabolism in the liver, was identified as a potentially important factor in mediating this general antiestrogenic effect. The regulation of prostaglandin biosynthesis by E2 is important for inflammation-like events during pregnancy including the initiation of birth. Our results suggest that adverse effects of PAHs on prostaglandin related pathways are likely caused by the interference with E2 signaling, specifically by inhibiting the E2 mediated downregulation of PGF2α. Characterization of the generalized antagonistic effect of 3-MC on E2 dependent signaling in the rat uterus thus contributes to a better understanding of molecular mechanisms of the toxicity of PAHs in female reproductive organs. Copyright © 2017 Elsevier Ltd. All rights reserved.

  1. Modulation of non-adrenergic, non-cholinergic neural bronchoconstriction in guinea-pig airways via GABAB-receptors.

    PubMed

    Belvisi, M G; Ichinose, M; Barnes, P J

    1989-08-01

    1. Evidence suggests that gamma-aminobutyric acid (GABA) and its receptors are present in the peripheral nervous system. We have now investigated the effect of GABA and related substances on non-adrenergic, non-cholinergic (NANC) neurally-evoked bronchoconstriction in the anaesthetised guinea-pig. 2. Bilateral vagal stimulation (5 V, 5 ms, 3 or 5 Hz) for 30 s, after propranolol (1 mg kg-1 i.v.) and atropine (1 mg kg-1 i.v.) evoked a NANC bronchoconstrictor response manifest as a mean tracheal pressure rise of 21.9 +/- 1.04 cmH2O (n = 70). The bronchoconstrictor response was reproducible for any given animal. 3. GABA (10 micrograms-10 mg kg-1 i.v.) did not alter basal tracheal pressure but reduced the NANC bronchoconstrictor response to vagal stimulation in a dose-dependent manner (ED50 = 186 micrograms kg-1 with a maximal inhibition of 74 +/- 3.4% at 10 mg kg-1). Neither the opioid antagonist naloxone (1 mg kg-1 i.v.) nor the alpha-adrenoceptor antagonist phentolamine (2.5 mg kg-1 i.v.) had any significant effect on the inhibitory response produced by GABA (500 micrograms kg-1). 4. GABA-induced inhibition was not antagonised by the GABAA-antagonist bicuculline (2 mg kg-1 i.v.). 5. The GABAB-agonist baclofen (10 micrograms-3 mg kg-1 i.v.) caused a dose-dependent inhibition of the NANC response (ED50 = 100 micrograms kg-1 with a maximal inhibition of 35.5 +/- 2.8% at 3 mg kg-1). The GABAA-agonist, 4,5,6,7-tetrahydroisoxazolo[5,4-C] pyridin-3-ol (THIP), also inhibited the NANC bronchoconstrictor response. However, the dose of THIP required for this effect was high (3 mg kg- ') and the effect ( <10% inhibition) was small. 6. Substance P (SP; 5upgkg-1 or 25pgkg-1), produced a bronchoconstrictor response equivalent to that produced by NANC vagal stimulation. This response was significantly increased by injection of GABA. Baclofen had no significant effect on responses evoked by exogenous SP. 7. We conclude that GABA inhibits the release of transmitter from NANC nerves

  2. Activity of adenosine diphosphates and triphosphates on a P2YT-type receptor in brain capillary endothelial cells

    PubMed Central

    Simon, J; Vigne, P; Eklund, K M; Michel, A D; Carruthers, A M; Humphrey, P P A; Frelin, C; Barnard, E A

    2001-01-01

    A P2Y (nucleotide) receptor activity in a clonal population (B10) of rat brain capillary endothelial cells is coupled to inhibition of adenylyl cyclase and has functional similarities to the P2YT (previously designated ‘P2T') receptor for ADP of blood platelets. However, the only P2Y receptor which was detectable in a previous study of B10 cells by mRNA analysis was the P2Y1 receptor, which elsewhere shows no transduction via cyclic nucleotides. We have sought here to clarify these issues. The inhibition of forskolin-stimulated adenylyl cyclase induced by purified nucleotides was measured on B10 cells. The EC50 value for 2-methylthioADP (2-MeSADP) was 2.2 nM and, surprisingly, 2-MeSATP was an almost equally strong agonist (EC50=3.5 nM). ATP and 2-ClATP were weak partial agonists (EC50=26 μM and 10 μM respectively) and under appropriate conditions could antagonise the activity on 2-MeSADP. A known selective antagonist of the platelet P2YT receptor, 2-propylthioadenosine-5′-(β,γ)-difluoromethylene) triphosphonate (AR-C 66096), was a competitive antagonist of this B10 cell receptor, with pKB=7.6. That ligand is inactive at the P2Y1 receptor in the same cells. Conversely, the competitive P2Y1 receptor antagonists, the 3′, 5′- and 2′, 5′-adenosine bis-monophosphates, are, instead, weak agonists at the adenylyl cyclase-inhibitory receptor. The inhibition of adenylyl cyclase by 2-MeSADP was completely abolished by pertussis toxin. In summary, these brain endothelial cells possess a P2YT-type receptor in addition to the P2Y1 receptor. The two have similarities in agonist profiles but are clearly distinguishable by antagonists and by their second messenger activations. The possible relationships between the B10 and platelet P2YT receptors are discussed. PMID:11156575

  3. The suitability of concentration addition for predicting the effects of multi-component mixtures of up to 17 anti-androgens with varied structural features in an in vitro AR antagonist assay

    SciTech Connect

    Ermler, Sibylle; Scholze, Martin; Kortenkamp, Andreas

    2011-12-15

    The risks associated with human exposures to chemicals capable of antagonising the effects of endogenous androgens have attracted considerable recent interest. Exposure is typically to large numbers of chemicals with androgen receptor (AR) antagonist activity, yet there is limited evidence of the combined effects of multi-component mixtures of these chemicals. A few in vitro studies with mixtures of up to six AR antagonists suggest that the concept of concentration addition (CA) provides good approximations of experimentally observed mixture effects, but studies with larger numbers of anti-androgens, and with more varied structural features, are missing. Here we show that the mixture effects of up to 17 AR antagonists, comprising compounds as diverse as UV-filter substances, parabens, perfluorinated compounds, bisphenol-A, benzo({alpha})pyrene, synthetic musks, antioxidants and polybrominated biphenyls, can be predicted well on the basis of the anti-androgenicity of the single components using the concept of CA. We tested these mixtures in an in vitro AR-dependent luciferase reporter gene assay, based on MDA-kb2 cells. The effects of further mixtures, composed of four and six anti-androgens, could be predicted accurately by CA. However, there was a shortfall from expected additivity with a ten-component mixture at two different mixture ratios, but attempts to attribute these deviations to differential expression of hormone-metabolising CYP isoforms did not produce conclusive results. CA provides good approximations of in vitro mixture effects of anti-androgens with varying structural features. -- Highlights: Black-Right-Pointing-Pointer Humans are exposed to a large number of androgen receptor antagonists. Black-Right-Pointing-Pointer There is limited evidence of the combined effects of anti-androgenic chemicals. Black-Right-Pointing-Pointer We modelled the predictability of combined effects of up to 17 anti-androgens. Black-Right-Pointing-Pointer We tested the

  4. Resveratrol protects against doxorubicin-induced cardiotoxicity in aged hearts through the SIRT1-USP7 axis.

    PubMed

    Sin, Thomas K; Tam, Bjorn T; Yung, Benjamin Y; Yip, Shea P; Chan, Lawrence W; Wong, Cesar S; Ying, Michael; Rudd, John A; Siu, Parco M

    2015-04-15

    Doxorubicin induced functional deteriorations and elevations of USP7-related apoptotic/catabolic signalling in the senescent heart Resveratrol protects against doxorubicin-induced alterations through the restoration of SIRT1 deacetylase activity A compromised cardiac function is often seen in elderly cancer patients receiving doxorubicin therapy. The present study tested the hypothesis that acute intervention with resveratrol, a natural anti-oxidant found in grapes and red wine, reduces the cardiotoxicity of doxorubicin through restoration of sirtuin 1 (SIRT1) deacetylase activity, and attenuation of the catabolic/apoptotic pathways orchestrated by USP7, a p53 deubiquitinating protein, using young (aged 2 months) and old (aged 10 months) senescence-accelerated mice prone 8 (SAMP8). Animals were randomised to receive saline, doxorubicin, and doxorubicin in combination with resveratrol, in the presence or absence of SIRT1 inhibitors, sirtinol or EX527. Resveratrol alone, but not in combination with either of the SIRT1 inhibitors, suppressed the doxorubicin-induced impairment of cardiac systolic function in aged animals. Doxorubicin reduced SIRT1 deacetylase activity, and elevated proteasomal activity and USP7; it also increased the protein level of p300 and ubiquitinated proteins in hearts from aged SAMP8. These doxorubicin-induced alterations were prevented by resveratrol, whereas the protective action of resveratrol was antagonised by sirtinol and EX527. In young SAMP8 hearts, resveratrol attenuated the doxorubicin-induced increases in acetylation of Foxo1 and transactivation of MuRF-1, whereas these mitigations were not found after treatment with SIRT1 inhibitors. However, the protein contents of acetylated Foxo1 and MuRF-1 were not affected by any of the drugs studied in aged SAMP8 hearts. Resveratrol also ameliorated the augmentation of pro-apoptotic markers including p53, Bax, caspase 3 activity and apoptotic DNA fragmentation induced by doxorubicin in

  5. Resveratrol protects against doxorubicin-induced cardiotoxicity in aged hearts through the SIRT1-USP7 axis.

    PubMed

    Sin, Thomas K; Tam, Bjorn T; Yung, Benjamin Y; Yip, Shea P; Chan, Lawrence W; Wong, Cesar S; Ying, Michael; Rudd, John A; Siu, Parco M

    2015-01-13

    A compromised cardiac function is often seen in elderly cancer patients receiving doxorubicin therapy. The present study tested the hypothesis that acute intervention with resveratrol, a natural antioxidant found in grapes and red wine, reduces the cardiotoxicity of doxorubicin through restoration of SIRT1 deacetylase activity, and attenuation of catabolic/apoptotic pathways orchestrated by USP7, a p53 deubiquitinating protein, using young (2 month-old) and old (10 month-old) senescence-accelerated mice (prone 8; SAMP8). Animals were randomised to receive saline, doxorubicin, and doxorubicin in combination with resveratrol, in the presence or absence of SIRT1 inhibitors, sirtinol or EX527. Resveratrol alone, but not in combination with either SIRT1 inhibitors, suppressed the doxorubicin-induced impairment of cardiac systolic function in aged animals. Doxorubicin reduced SIRT1 deacetylase activity, and elevated proteasomal activity and USP7; it also increased the protein level of p300 and ubiquitinated proteins in hearts from aged SAMP8 mice. These doxorubicin-induced alterations were prevented by resveratrol, but the protective action of resveratrol was antagonised by sirtinol and EX527. In young SAMP8 hearts, resveratrol attenuated the doxorubicin-induced increase in acetylation of Foxo1 and transactivation of MuRF-1 whereas these mitigations were not found with the treatment of SIRT1 inhibitors. However, the protein content of acetylated Foxo1 and MuRF-1 were not affected by any of the drugs studied in aged SAMP8 hearts. Resveratrol also ameliorated the augmentations of pro-apoptotic markers including p53, Bax, caspase 3 activity and apoptotic DNA fragmentation induced by doxorubicin in hearts from aged animals, whereas these reductions were diminished by combined treatment with SIRT1 inhibitors. These data demonstrate that resveratrol ameliorates doxorubicin-induced cardiotoxicity in the aged hearts through the restoration of SIRT1 activity to attenuate USP7

  6. Kinetics of pro-inflammatory cytokines, interleukin-10, and virus neutralising antibodies during acute ephemeral fever virus infections in Brahman cattle.

    PubMed

    Barigye, R; Melville, L F; Davis, S; Walsh, S; Hunt, N; Hunt, R; Elliot, N

    2015-12-15

    While fever and inflammation are hallmark features of bovine ephemeral fever (BEF), the cytokine networks that underlie the acute phase of the disease have not been empirically defined in cattle. This study characterised the plasma kinetics of proinflammatory cytokines (IL-1β, IL-6, TNF-α) and IL-10 during acute BEF and elucidated on the relationship between the onset of the virus neutralizing antibody response and resolution of viraemia in natural BEF virus (BEFV) infections in cattle. Plasma from three BEFV-infected and three uninfected cattle was tested for the study cytokines by a cELISA, viraemia monitored by qRT-PCR, and virus neutralizing antibody titres determined using a standard protocol. Unlike the negative controls, plasma concentrations of IL-1β, TNF-α, IL-6, and IL-10 were consistently increased in the three virus-infected animals. Two of the infected heifers were recumbent and pyrexic on the first day of monitoring and increased cytokine production was already in progress by the time viraemia was detected in all the three infected animals. In all the virus-infected heifers, IL-1β was the most strongly expressed cytokine, IL-6 and IL-10 manifested intermediate plasma concentrations while TNF-α was the least expressed and demonstrated bi-phasic peaks three and five days after the onset of pyrexia. In two of the BEFV-infected heifers, viraemia resolved on the day of seroconversion while in the other infected animal, viral RNA was detectable up to three days after seroconversion. The present data document variable increase in plasma IL-1β, IL-6, TNF-α, and IL-10 during natural BEFV infections and the fact that upregulation of all but TNF-α precedes seroconversion. In addition to virus neutralising antibodies, it is likely that cytokine-mediated cellular mechanisms may be required for resolution of viraemia in BEF. Considering the anti-inflammatory properties of IL-10, its upregulation may potentially antagonise the fever response in BEFV

  7. Academisation of nursing: An ethnography of social transformations in Chile.

    PubMed

    Ayala, Ricardo A; Fealy, Gerard M; Vanderstraeten, Raf; Bracke, Piet

    2014-04-01

    Nursing in Chile is considered to be the leading example of professional development in Latin America - nurses must undertake five years of university education on a full-time programme. Academisation of nursing education is a key aspect in the evolution into professional status. The consequences of education, however, are commonly related to the replication of social institutions and structures that perpetuate social inequalities. The study's aim was to comprehend the consequences of nursing academisation and its relationships with the social transformations which that country has witnessed. We draw upon ethnographic data, gathered between 2010 and 2011 in a 500-bed, high-quality university hospital in Chile. Participants were nurses ranging from beginners to experienced professionals and recruited from wards representing technically expert nursing and caring-oriented nursing. The data were organised to allow the development of concepts and patterns, using the Grounded Theory approach. Despite the fact that Chilean nursing originated from the educated elite class, today's nurses share a middle-class consciousness, and a sense of class distinction is encouraged throughout academic training - the 'eliteness' of professional groups. This discourse antagonises middle-class people who 'should' adopt a professional-class identity. A tension among nurses surfaced, based on a competition for a scarce resource: social mobility. Furthermore, an antagonist stratification between university-trained nurses and auxiliary nurses has developed, and in the process the title 'nurse' and the practice of 'nursing' have been monopolised by university-trained nurses, resulting in a relationship of domination-subordination. The academisation process followed by Chilean nursing is rooted in the social-class transformations of that country. Such process has been ineffective in preventing social inequalities, resulting in the reproduction of earlier historical class differences in nursing

  8. Neuroprotective action of raloxifene against hypoxia-induced damage in mouse hippocampal cells depends on ERα but not ERβ or GPR30 signalling.

    PubMed

    Rzemieniec, J; Litwa, E; Wnuk, A; Lason, W; Gołas, A; Krzeptowski, W; Kajta, M

    2015-02-01

    Raloxifene is the selective estrogen receptor modulator (SERM) currently used in clinical practice to activate estrogen receptors (ERs) in bone tissue and to antagonise ERs in breast and uterine cancers. Little is known, however, about mechanisms of action of raloxifene on hypoxia-induced neuronal cell damage. The aim of the present study was to investigate the neuroprotective potential of raloxifene against hypoxia-induced damage of mouse hippocampal cells in primary cultures, with a particular focus on raloxifene interactions with the classical nuclear ERs (ERα, ERβ) and the recently identified membrane ER G-protein-coupled receptor 30 (GPR30). In this study, 18 h of hypoxia increased hypoxia inducible factor 1 alpha (Hif1α) mRNA expression and induced apoptotic processes, such as loss of the mitochondrial membrane potential, activation of caspase-3 and fragmentation of cell nuclei based on Hoechst 33342 staining. These effects were accompanied by reduced ATPase and intracellular esterase activities as well as substantial lactate dehydrogenase (LDH) release from cells exposed to hypoxia. Our study demonstrated strong neuroprotective and anti-apoptotic caspase-3-independent actions of raloxifene in hippocampal cells exposed to hypoxia. Raloxifene also inhibited the hypoxia-induced decrease in Erα mRNA expression and attenuated the hypoxia-induced rise in Erβ and Gpr30 mRNA expression levels. Impact of raloxifene on hypoxia-affected Erα mRNA was mirrored by fluctuations in the protein level of the receptor as demonstrated by Western blot and immunofluorescent labelling. Raloxifene-induced changes in Erβ mRNA expression level were in parallel with ERβ immunofluorescent labeling. However, changes in Gpr30 mRNA level were not reflected by changes in the protein levels measured either by ELISA, Western blot or immunofluorescent staining at 24h post-treatment. Using specific siRNAs, we provided evidence for a key involvement of ERα, but not ERβ or GPR30 in

  9. A P2X receptor from the tardigrade species Hypsibius dujardini with fast kinetics and sensitivity to zinc and copper.

    PubMed

    Bavan, Selvan; Straub, Volko A; Blaxter, Mark L; Ennion, Steven J

    2009-01-20

    Orthologs of the vertebrate ATP gated P2X channels have been identified in Dictyostelium and green algae, demonstrating that the emergence of ionotropic purinergic signalling was an early event in eukaryotic evolution. However, the genomes of a number of animals including Drosophila melanogaster and Caenorhabditis elegans, both members of the Ecdysozoa superphylum, lack P2X-like proteins, whilst other species such as the flatworm Schistosoma mansoni have P2X proteins making it unclear as to what stages in evolution P2X receptors were lost. Here we describe the functional characterisation of a P2X receptor (HdP2X) from the tardigrade Hypsibius dujardini demonstrating that purinergic signalling is preserved in some ecdysozoa. ATP (EC50 approximately 44.5 microM) evoked transient inward currents in HdP2X with millisecond rates of activation and desensitisation. HdP2X is antagonised by pyridoxal-phosphate-6-azophenyl-2',4' disulfonic acid (IC50 15.0 microM) and suramin (IC50 22.6 microM) and zinc and copper inhibit ATP-evoked currents with IC50 values of 62.8 microM and 19.9 microM respectively. Site-directed mutagenesis showed that unlike vertebrate P2X receptors, extracellular histidines do not play a major role in coordinating metal binding in HdP2X. However, H306 was identified as playing a minor role in the actions of copper but not zinc. Ivermectin potentiated responses to ATP with no effect on the rates of current activation or decay. The presence of a P2X receptor in a tardigrade species suggests that both nematodes and arthropods lost their P2X genes independently, as both traditional and molecular phylogenies place the divergence between Nematoda and Arthropoda before their divergence from Tardigrada. The phylogenetic analysis performed in our study also clearly demonstrates that the emergence of the family of seven P2X channels in human and other mammalian species was a relatively recent evolutionary event that occurred subsequent to the split between

  10. A P2X receptor from the tardigrade species Hypsibius dujardini with fast kinetics and sensitivity to zinc and copper

    PubMed Central

    Bavan, Selvan; Straub, Volko A; Blaxter, Mark L; Ennion, Steven J

    2009-01-01

    Background Orthologs of the vertebrate ATP gated P2X channels have been identified in Dictyostelium and green algae, demonstrating that the emergence of ionotropic purinergic signalling was an early event in eukaryotic evolution. However, the genomes of a number of animals including Drosophila melanogaster and Caenorhabditis elegans, both members of the Ecdysozoa superphylum, lack P2X-like proteins, whilst other species such as the flatworm Schistosoma mansoni have P2X proteins making it unclear as to what stages in evolution P2X receptors were lost. Here we describe the functional characterisation of a P2X receptor (HdP2X) from the tardigrade Hypsibius dujardini demonstrating that purinergic signalling is preserved in some ecdysozoa. Results ATP (EC50 ~44.5 μM) evoked transient inward currents in HdP2X with millisecond rates of activation and desensitisation. HdP2X is antagonised by pyridoxal-phosphate-6-azophenyl-2',4' disulfonic acid (IC50 15.0 μM) and suramin (IC50 22.6 μM) and zinc and copper inhibit ATP-evoked currents with IC50 values of 62.8 μM and 19.9 μM respectively. Site-directed mutagenesis showed that unlike vertebrate P2X receptors, extracellular histidines do not play a major role in coordinating metal binding in HdP2X. However, H306 was identified as playing a minor role in the actions of copper but not zinc. Ivermectin potentiated responses to ATP with no effect on the rates of current activation or decay. Conclusion The presence of a P2X receptor in a tardigrade species suggests that both nematodes and arthropods lost their P2X genes independently, as both traditional and molecular phylogenies place the divergence between Nematoda and Arthropoda before their divergence from Tardigrada. The phylogenetic analysis performed in our study also clearly demonstrates that the emergence of the family of seven P2X channels in human and other mammalian species was a relatively recent evolutionary event that occurred subsequent to the split between

  11. Signalling pathways activated by 5-HT(1B)/5-HT(1D) receptors in native smooth muscle and primary cultures of rabbit renal artery smooth muscle cells.

    PubMed

    Hinton, J M; Hill, P; Jeremy, J; Garland, C

    2000-01-01

    The potential of primary cultures of rabbit renal artery vascular smooth muscle cells (VSMCs) was assessed as a means to investigate the signalling pathways linked to 5-hydroxytryptamine (5-HT) 5-HT(1B)/5-HT(1D) receptors in native arteries. In renal artery segments denuded of endothelium, incubated with ketanserin and prazosin (each 1 microM), and prestimulated with 20 mM K(+) Krebs buffer, 5-HT and CP 93,129, a 5-HT(1B) receptor agonist, evoked concentration-dependent contractions. GR 127935, a 5-HT(1B)/5-HT(1D) receptor antagonist, significantly antagonised 5-HT-evoked contractions at nanomolar concentrations. Reverse transcription polymerase chain reaction (RT-PCR) of mRNA from smooth muscle cells from the isolated renal artery and from primary cultures of VSMCs from the same artery expressed mRNA transcripts for the 5-HT(1B) receptor and the 5-HT(1D) receptor in both preparations. The sequence of the PCR fragments corresponded to the known sequence for these receptors. Application of 5-HT evoked a concentration-dependent, pertussis toxin (PTx)-sensitive reduction in cyclic AMP in both cultured cells and intact artery (cyclic AMP concentration reduced by 65.53 +/- 3.33 and 52.65 +/- 5.34% from basal with 10 microM 5-HT, respectively). The effect of 10 microM 5-HT on cAMP was increased in the presence of 20 mM K(+) (reduced by 82.50 +/- 2.50 and 87.54 +/- 3.97%, respectively). In intact arteries, contraction through 5-HT(1B)/5-HT(1D) receptors was significantly attenuated by inhibitors of phosphatidylinositol 3-kinase (wortmannin) and activated mitogen-activated protein kinase (MAPK), MEK (U0126). In the cultured VSMCs, activated MAPK was identified by immunocytochemistry and immunoblotting after stimulation with 5-HT, but only if 20 mM K(+) was present at the onset of stimulation. These data provide the first direct evidence that 5-HT(1B)/5-HT(1B) receptors are linked to the activation of MAPK and indicate that primary cultures of renal VSMCs could provide a

  12. Effects of ceruletide and haloperidol on the hypothalamo-pituitary beta-endorphin system and brain beta-endorphin contents in the rat: with special reference to effects of ceruletide in chronically haloperidol-treated rats.

    PubMed

    Hagino, Y; Okuwa, M; Moroji, T

    1991-01-01

    Subcutaneous (sc) administration of 200 micrograms/kg ceruletide (CER), a decapeptide chemically related CCK-8, and 5 mg/kg haloperidol (HLP) to rats increased the plasma immunoreactive beta-endorphin (ir-beta-END) level. The combined injection of CER and haloperidol caused higher plasma ir-beta-END levels than either drug alone. High plasma ir-beta-END levels returned to control levels on the 2nd day. Prior intraperitoneal (ip) administration of a CCK receptor antagonist, L-364,718 (3 mg/kg), but not proglumide (400 mg/kg, ip), inhibited CER-induced, but not HLP-induced, elevation in plasma ir-beta-END levels. The dopamine agonist, bromocriptine (1 mg/kg, ip) decreased plasma ir-beta-END levels, but had not effect on CER-induced elevation in plasma ir-beta-END levels, whereas bromocriptine-induced reduction in plasma ir-beta-END levels was antagonised by HLP. CER injection to chronically HLP-treated rats caused a greater elevation of plasma ir-beta-END levels compared to saline-injected rats. In contrast to the acute experiment, plasma ir-beta-END levels remained elevated over a period of 24 h. In the acute experiment, CER, HLP or the combined treatment with these two drugs had no effect on ir-beta-END contents in the pituitary gland and brain. In the chronic experiment, HLP increased the adenohypophyseal and septal ir-beta-END contents and decreased the hippocampal ir-beta-END contents 24 h after the final HLP injection. CER caused a small reduction only in the hippocampal ir-beta-END contents of CER-injected rats 15 min after injection. When determined on the 2nd day, however, the increases in the adenohypophyseal and septal ir-beta-END contents and the decrease in the hippocampal ir-beta-END contents observed in CER-injected rats were of the same magnitude as those of rats not given the CER injection. These findings indicate that CER stimulates the release of ir-beta-END from the adenohypophysis through CCK-A receptors and that elevated plasma ir

  13. Vorapaxar for secondary prevention of thrombotic events for patients with previous myocardial infarction: a prespecified subgroup analysis of the TRA 2°P-TIMI 50 trial.

    PubMed

    Scirica, Benjamin M; Bonaca, Marc P; Braunwald, Eugene; De Ferrari, Gaetano M; Isaza, Daniel; Lewis, Basil S; Mehrhof, Felix; Merlini, Piera A; Murphy, Sabina A; Sabatine, Marc S; Tendera, Michal; Van de Werf, Frans; Wilcox, Robert; Morrow, David A

    2012-10-13

    Vorapaxar inhibits platelet activation by antagonising thrombin-mediated activation of the protease-activated receptor 1 on human platelets. The effect of adding other antiplatelet drugs to aspirin for long-term secondary prevention of thrombotic events in stable patients with previous myocardial infarction is uncertain. We tested this effect in a subgroup of patients from the Thrombin Receptor Antagonist in Secondary Prevention of Atherothrombotic Ischemic Events (TRA 2°P)-TIMI 50 trial. In TRA 2°P-TIMI 50--a randomised, placebo-controlled, parallel trial--we randomly assigned patients with a history of atherothrombosis to receive vorapaxar (2·5 mg daily) or matching placebo in a 1:1 ratio. Patients, and those giving treatment, assessing outcomes, and analysing results were masked to treatment allocation. Patients with a qualifying myocardial infarction within the previous 2 weeks to 12 months were analysed as a pre-defined subgroup. The primary efficacy endpoint was cardiovascular death, myocardial infarction, or stroke, analysed by intention to treat. We analysed events by Kaplan-Meier analysis and compared groups with a Cox proportional hazard model. TRA 2°P-TIMI 50 is registered at ClinicalTrials.gov (NCT00526474). 17,779 of 26,449 patients had a qualifying myocardial infarction and were assigned treatment (8898 to vorapaxar and 8881 to placebo). Median follow-up was 2·5 years (IQR 2·0-2·9). Cardiovascular death, myocardial infarction, or stroke occurred in 610 of 8898 patients in the vorapaxar group and 750 of 8881 in the placebo group (3-year Kaplan-Meier estimates 8·1%vs 9·7%, HR 0·80, 95% CI 0·72-0·89; p<0·0001). Moderate or severe bleeding was more common in the vorapaxar group versus the placebo group (241/8880 [3·4%, 3-year Kaplan-Meier estimate] vs 151/8849 [2·1%, 3-year Kaplan-Meier estimate], HR 1·61, 95% CI 1·31-1·97; p<0·0001). Intracranial haemorrhage occurred in 43 of 8880 patients (0·6%, 3-year Kaplan-Meier estimate) with

  14. Identification in human airways smooth muscle cells of the prostanoid receptor and signalling pathway through which PGE2 inhibits the release of GM-CSF.

    PubMed

    Clarke, Deborah L; Belvisi, Maria G; Catley, Matthew C; Yacoub, Magdi H; Newton, Robert; Giembycz, Mark A

    2004-04-01

    1. The prostanoid receptor(s) on human airways smooth muscle (HASM) cells that mediates the inhibitory effect of PGE(2) on interleukin (IL)-1 beta-induced granulocyte/macrophage colony-stimulating factor (GM-CSF) release has been classified. 2. IL-1 beta evoked the release of GM-CSF from HASM cells, which was suppressed by PGE(2), 16,16-dimethyl PGE(2) (nonselective), misoprostol (EP(2)/EP(3)-selective), ONO-AE1-259 and butaprost (both EP(2)-selective) with pIC(50) values of 8.61, 7.13, 5.64, 8.79 and 5.43, respectively. EP-receptor agonists that have selectivity for the EP(1)-(17-phenyl-omega-trinor PGE(2)) and EP(3)-receptor (sulprostone) subtypes as well as cicaprost (IP-selective), PGD(2), PGF(2 alpha) and U-46619 (TP-selective) were poorly active or inactive at concentrations up to 10 microM. 3. AH 6809, a drug that can be used to selectively block EP(2)-receptors in HASM cells, antagonised the inhibitory effect of PGE(2), 16,16-dimethyl PGE(2) and ONO-AE1-259 with apparent pA(2) values of 5.85, 6.09 and 6.1 respectively. In contrast, the EP(4)-receptor antagonists, AH 23848B and L-161,982, failed to displace to the right the concentration-response curves that described the inhibition of GM-CSF release evoked by PGE(2) and ONO-AE1-259. 4. Inhibition of GM-CSF release by PGE(2) and 8-Br-cAMP was abolished in cells infected with an adenovirus vector encoding an inhibitor protein of cAMP-dependent protein kinase (PKA) but not by H-89, a purported small molecule inhibitor of PKA. 5. We conclude that prostanoid receptors of the EP(2)-subtype mediate the inhibitory effect of PGE(2) on GM-CSF release from HASM cells by recruiting a PKA-dependent pathway. In addition, the data illustrate that caution should be exercised when using H-89 in studies designed to assess the role of PKA in biological processes.

  15. The effects of LPS on adhesion and migration of human dental pulp stem cells in vitro.

    PubMed

    Li, Dongmei; Fu, Lei; Zhang, Yaqing; Yu, Qing; Ma, Fengle; Wang, Zhihua; Luo, Zhirong; Zhou, Zeyuan; Cooper, Paul R; He, Wenxi

    2014-10-01

    The aim of the present study was to investigate the effects of lipopolysaccharide (LPS) on the migration and adhesion of human dental pulp stem cells (hDPSCs) and the associated intracellular signalling pathways. hDPSCs obtained from impacted third molars were exposed to LPS and in vitro cell adhesion and migration were evaluated. The effects of LPS on gene expression of adhesion molecules and chemotactic factors were investigated using quantitative real-time reverse-transcriptase polymerase chain (qRT-PCR). The potential involvement of nuclear factor NF-kappa-B (NF-κB) or mitogen-activated protein kinase (MAPK) signalling pathways in the migration and adhesion of hDPSCs induced by LPS was assessed using a transwell cell migration assay and qRT-PCR. LPS promoted the adhesion of hDPSCs at 1μg/mL and 10μg/mL concentrations, 1μg/mL LPS showing the greater effect. Transwell cell migration assay demonstrated that LPS increased migration of hDPSCs at 1μg/mL concentration while decreasing it significantly at 10μg/mL. The mRNA expressions of adhesion molecules and chemotactic factors were enhanced significantly after stimulation with 1μg/mL LPS. Specific inhibitors for NF-κB and extracellular signal regulated kinases (ERK), c-Jun N-terminal kinase (JNK), and P38, markedly antagonised LPS-induced adhesion and migration of hDPSCs and also significantly abrogated LPS-induced up-regulation of adhesion molecules and chemotactic factors. In addition, specific inhibitors of SDF-1/CXCR4, AMD3100 significantly diminished LPS-induced migration of hDPSCs. LPS at specific concentrations can promote cell adhesion and migration in hDPSCs via the NF-κB and MAPK pathways by up-regulating the expression of adhesion molecules and chemotactic factors. LPS may influence pulp healing through enhancing the adhesion and migration of human dental pulp stem cells when it enters into pulp during pulp exposure or deep caries. Copyright © 2014 Elsevier Ltd. All rights reserved.

  16. The effects of bupropion, a new antidepressant drug, and diazepam, and their interaction in man

    PubMed Central

    Hamilton, M. J.; Bush, M.; Smith, P.; Peck, A. W.

    1982-01-01

    approximating to the four clinical frequencies revealed no differences between active drug(s) and lactose at 2 h 45 min after treatment. A possible increase in fast activity occurred after bupropion 6 h 15 min post-treatment. 7 It was concluded that bupropion 100 mg administered as a single dose, while not producing significant changes in the variables measured, when co-administered with single 5 mg doses of diazepam antagonised the functional impairment and drowsiness which follow that drug. PMID:6817770

  17. Expression of P. falciparum var Genes Involves Exchange of the Histone Variant H2A.Z at the Promoter

    PubMed Central

    Petter, Michaela; Lee, Chin Chin; Byrne, Timothy J.; Boysen, Katja E.; Volz, Jennifer; Ralph, Stuart A.; Cowman, Alan F.; Brown, Graham V.; Duffy, Michael F.

    2011-01-01

    Plasmodium falciparum employs antigenic variation to evade the human immune response by switching the expression of different variant surface antigens encoded by the var gene family. Epigenetic mechanisms including histone modifications and sub-nuclear compartmentalization contribute to transcriptional regulation in the malaria parasite, in particular to control antigenic variation. Another mechanism of epigenetic control is the exchange of canonical histones with alternative variants to generate functionally specialized chromatin domains. Here we demonstrate that the alternative histone PfH2A.Z is associated with the epigenetic regulation of var genes. In many eukaryotic organisms the histone variant H2A.Z mediates an open chromatin structure at promoters and facilitates diverse levels of regulation, including transcriptional activation. Throughout the asexual, intraerythrocytic lifecycle of P. falciparum we found that the P. falciparum ortholog of H2A.Z (PfH2A.Z) colocalizes with histone modifications that are characteristic of transcriptionally-permissive euchromatin, but not with markers of heterochromatin. Consistent with this finding, antibodies to PfH2A.Z co-precipitate the permissive modification H3K4me3. By chromatin-immunoprecipitation we show that PfH2A.Z is enriched in nucleosomes around the transcription start site (TSS) in both transcriptionally active and silent stage-specific genes. In var genes, however, PfH2A.Z is enriched at the TSS only during active transcription in ring stage parasites. Thus, in contrast to other genes, temporal var gene regulation involves histone variant exchange at promoter nucleosomes. Sir2 histone deacetylases are important for var gene silencing and their yeast ortholog antagonises H2A.Z function in subtelomeric yeast genes. In immature P. falciparum parasites lacking Sir2A or Sir2B high var transcription levels correlate with enrichment of PfH2A.Z at the TSS. As Sir2A knock out parasites mature the var genes are

  18. Tianeptine. A review of its pharmacodynamic and pharmacokinetic properties, and therapeutic efficacy in depression and coexisting anxiety and depression.

    PubMed

    Wilde, M I; Benfield, P

    1995-03-01

    Tianeptine is a novel antidepressant agent, both structurally (modified tricyclic) and in terms of its pharmacodynamic profile. Unlike other antidepressant agents, tianeptine stimulates the uptake of serotonin (5-hydroxytryptamine; 5-HT) in rat brain synaptosomes and rat and human platelets, increases 5-hydroxyindoleacetic acid (5-HIAA) levels in cerebral tissue and plasma, and reduces serotonergic-induced behaviour. Tianeptine reduces the hypothalamic-pituitary-adrenal response to stress, antagonises stress-induced behavioural deficits and prevents changes in cerebral morphology. The antidepressant efficacy of tianeptine, as shown in 2 trials of patients with major depression or depressed bipolar disorder with or without melancholia, is greater than that of placebo. In patients with major depression without melancholia or psychotic features, depressed bipolar disorder or dysthymic disorder, the antidepressant efficacy of short term (4 weeks to 3 months) tianeptine therapy appears to be similar to that of amitriptyline, imipramine and fluoxetine and may be superior to that of maprotiline in patients with coexisting depression and anxiety. However, submaximal dosages of amitriptyline and maprotiline were used in these studies. Preliminary evidence suggests that tianeptine may also be effective in patients with endogenous depression. Progressive therapeutic improvements have been observed with up to 1 year of tianeptine treatment, and long term therapy may reduce the rate of relapse or recurrence. Tianeptine is effective in the treatment of depression in elderly and post-alcohol-withdrawal patient subgroups. Tianeptine was more effective in reducing psychic anxiety than placebo in patients with major depression or depressed bipolar disorder with or without melancholia. The overall anxiolytic properties of tianeptine in patients with coexisting depression and anxiety appear to be similar to those of amitriptyline, imipramine and fluoxetine and may be superior to those

  19. SB-656104-A, a novel selective 5-HT7 receptor antagonist, modulates REM sleep in rats.

    PubMed

    Thomas, David R; Melotto, Sergio; Massagrande, Mario; Gribble, Andrew D; Jeffrey, Phillip; Stevens, Alexander J; Deeks, Nigel J; Eddershaw, Peter J; Fenwick, Susan H; Riley, Graham; Stean, Tania; Scott, Claire M; Hill, Matthew J; Middlemiss, Derek N; Hagan, Jim J; Price, Gary W; Forbes, Ian T

    2003-06-01

    1 (6-((R)-2-[2-[4-(4-Chloro-phenoxy)-piperidin-1-yl]-ethyl]-pyrrolidine-1-sulphonyl)-1H-indole hydrochloride) (SB-656104-A), a novel 5-hydroxytryptamine (5-HT(7)) receptor antagonist, potently inhibited [(3)H]-SB-269970 binding to the human cloned 5-HT(7(a)) (pK(i) 8.7+/-0.1) and 5-HT(7(b)) (pK(i) 8.5+/-0.2) receptor variants and the rat native receptor (pK(i) 8.8+/-0.2). The compound displayed at least 30-fold selectivity for the human 5-HT(7(a)) receptor versus other human cloned 5-HT receptors apart from the 5-HT(1D) receptor ( approximately 10-fold selective). 2 SB-656104-A antagonised competitively the 5-carboxamidotryptamine (5-CT)-induced accumulation of cyclic AMP in h5-HT(7(a))/HEK293 cells with a pA(2) of 8.5. 3 Following a constant rate iv infusion to steady state in rats, SB-656104 had a blood clearance (CL(b)) of 58+/-6 ml min(-1) kg(-1) and was CNS penetrant with a steady-state brain : blood ratio of 0.9 : 1. Following i.p. administration to rats (10 mg kg(-1)), the compound displayed a t(1/2) of 1.4 h with mean brain and blood concentrations (at 1 h after dosing) of 0.80 and 1.0 micro M, respectively. 4 SB-656104-A produced a significant reversal of the 5-CT-induced hypothermic effect in guinea pigs, a pharmacodynamic model of 5-HT(7) receptor interaction in vivo (ED(50) 2 mg kg(-1)). 5 SB-656104-A, administered to rats at the beginning of the sleep period (CT 0), significantly increased the latency to onset of rapid eye movement (REM) sleep at 30 mg kg(-1) i.p. (+93%) and reduced the total amount of REM sleep at 10 and 30 mg kg(-1) i.p. with no significant effect on the latency to, or amount of, non-REM sleep. SB-269970-A produced qualitatively similar effects in the same study. 6 In summary, SB-656104-A is a novel 5-HT(7) receptor antagonist which has been utilised in the present study to provide further evidence for a role for 5-HT(7) receptors in the modulation of REM sleep.

  20. [Behavioural studies during the gestational-lactation period in morphine treated rats].

    PubMed

    Sobor, Melinda; Timár, Júlia; Riba, Pál; Király, Kornél P; Al-Khrasani, Mahmoud; Gyarmati, Zsuzsanna; Fürst, Zsuzsanna

    2013-12-01

    Opioids impair the maternal behaviour of experimental animals. The effect of morphine on maternal behaviour in rat dams treated chronically with morphine during the whole pregnancy and lactation has not been yet analysed systematically. The aim of our work was to investigate the behavioural effects of moderate dose morphine administered constantly in the whole perinatal period in rats. Nulliparous female rats were treated with 10 mg/kg morphine s.c. once daily, from the day of mating. Maternal behaviour was observed, the effects of acute morphine treatment on the maternal behaviour and whether this effect could be antagonised by naloxone were also investigated. Physical and other behavioural (anxiety-like signals in elevated plus maze, changes in locomotor activity) withdrawal signs precipitated by naloxone were registered. After weaning sensitivity to the rewarding effect of morphine was measured by conditioned place preference and to the aversive effect of naloxone by conditioned place aversion tests. Antinociceptive test on tail-flick apparatus was performed to investigate the changes in morphine antinociceptive effects due to chronic morphine treatment. Maternal behaviour was significantly impaired in morphine-treated dams. This effect of morphine lasted c.a. 2-3 hours a day, it showed dose-dependency and was enhanced in MO-treated group (sensitisation). Only weak physical and no other behavioural (anxiety-like behaviour or hypolocomotion) withdrawal signs were precipitated by naloxone. The positive reinforcing effect of morphine and aversive effect of naloxone were markedly increased on conditioned place paradigm. Significant antinociceptive tolerance was not seen. Although human drug abuse can be hardly modelling under experimental circumstances, our constant, relatively moderate dose morphine treatment administered once daily during the whole pregnancy and lactation resulted in several subtle behavioural changes in dams. In perinatally opioid

  1. Pharmacological characterisation of the first non-peptide bradykinin B2 receptor agonist FR 190997: an in vitro study on human, rabbit and pig vascular B2 receptors.

    PubMed

    Rizzi, A; Rizzi, C; Amadesi, S; Calo', G; Varani, K; Inamura, N; Regoli, D

    1999-10-01

    FR 190997, a new kinin B2 receptor agonist of non-peptide nature, has been studied in three isolated vessels: the human umbilical vein (hUV), the rabbit jugular vein (rbJV), and the pig coronary artery (pCA). Bradykinin (BK) contracts the hUV and rbJV through smooth muscle B2 receptors, while it relaxes the pCA through endothelial receptors of the B2 type. Contractions of the hUV and rbJV in response to FR 190997 show slow onset and are not reproducible compared to the rapid and reproducible effect of BK. They reach only 70% and 30% of the BK-induced maximal contractions in the hUV and rbJV, respectively. The effects of FR 190997 are antagonised by HOE 140 and this antagonist shows similar pK(B) values against BK and FR 190997, indicating that the non-peptide agent interacts with the kinin B2 receptor. FR 190997 is inactive as relaxant of the pCA; in this tissue, it acts as a pure and competitive antagonist, with a pK(B) value of 7.6, while HOE 140 acts as an insurmountable antagonist (pK(B) 9.3). When tested as an antagonist, FR 190997 inhibits also the contractile effects of BK in the hUV (pK(B) 7.8) and in the rbJV (pK(B) 7.6). FR 190997 is selective for the B2 receptor since it does not interact with the B1, and is specific since it does not affect the contraction evoked by 5-hydroxytryptamine, endothelin-1, and noradrenaline in the hUV, or the relaxation induced by substance P in the pCA. FR 190997 shows therefore different pharmacological profiles in various preparations, acting as a partial agonist in the hUV and especially in the rbJV and as a pure antagonist in the pCA. This new compound could be of interest in understanding how non-peptide agonists may activate receptors for peptides.

  2. Cav1.2 channel current block by the PKA inhibitor H-89 in rat tail artery myocytes via a PKA-independent mechanism: Electrophysiological, functional, and molecular docking studies.

    PubMed

    Fusi, Fabio; Trezza, Alfonso; Spiga, Ottavia; Sgaragli, Giampietro; Bova, Sergio

    2017-09-15

    To characterize the role of cAMP-dependent protein kinase (PKA) in regulating vascular Ca(2+) current through Cav1.2 channels [ICa1.2], we have documented a marked capacity of the isoquinoline H-89, widely used as a PKA inhibitor, to reduce current amplitude. We hypothesized that the ICa1.2 inhibitory activity of H-89 was mediated by mechanisms unrelated to PKA inhibition. To support this, an in-depth analysis of H-89 vascular effects on both ICa1.2 and contractility was undertaken by performing whole-cell patch-clamp recordings and functional experiments in rat tail main artery single myocytes and rings, respectively. H-89 inhibited ICa1.2 with a pIC50 (M) value of about 5.5, even under conditions where PKA activity was either abolished by both the PKA antagonists KT5720 and protein kinase inhibitor fragment 6-22 amide or enhanced by the PKA stimulators 6-Bnz-cAMP and 8-Br-cAMP. Inhibition of ICa1.2 by H-89 appeared almost irreversible upon washout, was charge carrier- and voltage-dependent, and antagonised by the Cav1.2 channel agonist (S)-(-)-Bay K 8644. H-89 did not alter both potency and efficacy of verapamil, did not affect current kinetics or voltage-dependent activation, while shifting to the left the 50% voltage of inactivation in a concentration-dependent manner. H-89 docked at the α1C subunit in a pocket region close to that of (S)-(-)-Bay K 8644 docking, forming a hydrogen bond with the same, key amino acid residue Tyr-1489. Finally, both high K(+)- and (S)-(-)-Bay K 8644-induced contractions of rings were fully reverted by H-89. In conclusion, these results indicate that H-89 inhibited vascular ICa1.2 and, consequently, the contractile function through a PKA-independent mechanism. Therefore, caution is recommended when interpreting experiments where H-89 is used to inhibit vascular smooth muscle PKA. Copyright © 2017 Elsevier Inc. All rights reserved.

  3. Efficacy and safety of sugammadex in the reversal of deep neuromuscular blockade induced by rocuronium in patients with end-stage renal disease: A comparative prospective clinical trial.

    PubMed

    de Souza, Camila M; Tardelli, Maria A; Tedesco, Helio; Garcia, Natalia N; Caparros, Mario P; Alvarez-Gomez, Jose A; de Oliveira Junior, Itamar S

    2015-10-01

    Renal failure affects the pharmacology of nondepolarizing neuromuscular blockers making recovery of neuromuscular function unpredictable. Sugammadex antagonises rocuronium-induced neuromuscular blockade by encapsulating rocuronium, creating a stable complex molecule that is mainly excreted by the kidneys. Previous studies suggest that sugammadex is effective in reversing moderate neuromuscular block in the presence of renal failure, but no data are available regarding reversal of profound neuromuscular block in patients with renal failure. The objective of this study is to compare the efficacy and safety of sugammadex in reversing profound neuromuscular block induced by rocuronium in patients with end-stage renal disease and those with normal renal function. A prospective clinical trial. Two university hospitals, from 1 October 2011 to 31 January 2012. Forty patients undergoing kidney transplant: 20 with renal failure [creatinine clearance (ClCr) <30 ml min] and 20 control patients (ClCr >90 ml min). Neuromuscular monitoring was performed by acceleromyography and train-of-four (TOF) stimulation. Profound neuromuscular block (posttetanic count, one to three responses) was maintained during surgery. Sugammadex 4 mg kg was administered on completion of skin closure. Recovery of the TOF ratio to 0.9 was recorded. Monitoring of neuromuscular function continued in the postanesthesia care unit for a further 2 h. The efficacy of sugammadex was evaluated by the time taken for the TOF ratio to recover to 0.9. The safety of sugammadex was assessed by monitoring for recurrence of neuromuscular block every 15 min for 2 h. Secondary variables were time to recovery of TOF ratio to 0.7 and 0.8. After sugammadex administration, the mean time for recovery of the TOF ratio to 0.9 was prolonged in the renal failure group (5.6 ± 3.6 min) compared with the control group (2.7 ± 1.3 min, P = 0.003). No adverse events or evidence of recurrence of

  4. Comparative responses of molluscs and fish to environmental estrogens and an estrogenic effluent.

    PubMed

    Jobling, S; Casey, D; Rodgers-Gray, T; Oehlmann, J; Schulte-Oehlmann, U; Pawlowski, S; Baunbeck, T; Turner, A P; Tyler, C R

    2003-10-29

    It is now well established that there is a diverse array of chemicals discharged into the environment that can mimic or antagonise the action of hormones. These endocrine-disrupting chemicals (EDCs) can thus interact with physiological systems and cause alterations in development, growth and reproduction in wildlife that are exposed to them. As yet, however, there is little information on the relative sensitivities of different wildlife groups to these chemicals and/or mixtures of them (e.g. estrogenic effluents) and hence, there are fundamental shortfalls in our knowledge of the ecological importance of endocrine disruption in wildlife. In this study, the effects of exposure to individual estrogenic chemicals (17alpha-ethinylestradiol; EE2, bisphenol-A, and 4-tert octylphenol) and a mixture containing these chemicals (treated sewage effluent) on embryo production in the prosobranch mollusc, Potamopyrgus antipodarum, were studied and compared with the effects of EE2 and the same estrogenic effluent on vitellogenin induction and/or egg production in various species of freshwater fish (fathead minnow; Pimaphales promelas, rainbow trout (Oncorhynchus mykiss); Cyprinus carpio, carp; Cyprinus carpio). The lab-based studies demonstrated that all of the tested chemicals (known to be estrogenic and to cause reproductive effects in fish) also affected embryo production in P. antipodarum. Furthermore, exposure to EE2 induced similar reproductive responses in the snails as in the fathead minnow (Pimephales promelas), stimulating egg/embryo production at low doses (up to 1 ng/l in the minnow and 25 ng/l in the snail) and causing inhibitory effects at higher doses. A similar pattern of embryo production occurred in P. antipodarum when it was exposed to a graded concentration of treated sewage effluent containing mixtures of estrogenic EDCs and hence, the total number of new embryos produced by the snails increased steadily over the 9 weeks exposure period in treated snails

  5. The efficacy and adverse effects of dicobalt edetate in cyanide poisoning.

    PubMed

    Marrs, Timothy Clive; Thompson, John Paul

    2016-09-01

    Dicobalt edetate is one of a number of cobalt compounds that have been studied in the treatment of cyanide poisoning, their efficacy being based upon the fact that cyanide combines with cobalt to form relatively non-toxic complexes. Inorganic cobalt salts are quite toxic (cyanide and cobalt antagonise one another's toxicity) and complexes such as dicobalt edetate were studied with the aim of identifying compounds that were less acutely toxic, but which retained the antidotal properties of cobalt salts. The proprietary preparation, Kelocyanor™, contains free cobalt and glucose as well as dicobalt edetate. The aim of this study was to evaluate the published evidence for the efficacy and adverse effects of dicobalt edetate. A Pubmed search was undertaken for the period 1961-September 2015. The search terms were "dicobalt edetate", "cobalt edetate" and "Kelocyanor", which produced 24 relevant citations. A review of the references in four relevant books (L'intoxication cyanhydrique et son traitement, Clinical and Experimental Toxicology of Cyanides, Antidotes for Poisoning by Cyanide and Antidotes) produced three further relevant papers, making a total of 27 papers. Efficacy of dicobalt edetate: There is evidence from animal pharmacodynamic studies that dicobalt edetate is an effective cyanide antidote in experimental animals. Some 39 cases of human poisoning treated with dicobalt edetate have been reported, but in only nine cases were blood cyanide concentrations measured, although administration of dicobalt edetate procured survival in four of the seven patients with concentrations in the lethal range (>3.0 mg/L). It is unlikely that death in any of the adequately documented fatal cases was attributable to treatment failure with dicobalt edetate, as it is probable that they all had suffered anoxic brain damage before treatment could be initiated. Furthermore, in one case, acute gold toxicity contributed substantially to death. Adverse effects of dicobalt edetate

  6. SLO-1-Channels of Parasitic Nematodes Reconstitute Locomotor Behaviour and Emodepside Sensitivity in Caenorhabditis elegans slo-1 Loss of Function Mutants

    PubMed Central

    Schniederjans, Monika; Miltsch, Sandra M.; Krücken, Jürgen; Guest, Marcus; Holden-Dye, Lindy; Harder, Achim; von Samson-Himmelstjerna, Georg

    2011-01-01

    The calcium-gated potassium channel SLO-1 in Caenorhabditis elegans was recently identified as key component for action of emodepside, a new anthelmintic drug with broad spectrum activity. In this study we identified orthologues of slo-1 in Ancylostoma caninum, Cooperia oncophora, and Haemonchus contortus, all important parasitic nematodes in veterinary medicine. Furthermore, functional analyses of these slo-1 orthologues were performed using heterologous expression in C. elegans. We expressed A. caninum and C. oncophora slo-1 in the emodepside-resistant genetic background of the slo-1 loss-of-function mutant NM1968 slo-1(js379). Transformants expressing A. caninum slo-1 from C. elegans slo-1 promoter were highly susceptible (compared to the fully emodepside-resistant slo-1(js379)) and showed no significant difference in their emodepside susceptibility compared to wild-type C. elegans (p = 0.831). Therefore, the SLO-1 channels of A. caninum and C. elegans appear to be completely functionally interchangeable in terms of emodepside sensitivity. Furthermore, we tested the ability of the 5′ flanking regions of A. caninum and C. oncophora slo-1 to drive expression of SLO-1 in C. elegans and confirmed functionality of the putative promoters in this heterologous system. For all transgenic lines tested, expression of either native C. elegans slo-1 or the parasite-derived orthologue rescued emodepside sensitivity in slo-1(js379) and the locomotor phenotype of increased reversal frequency confirming the reconstitution of SLO-1 function in the locomotor circuits. A potent mammalian SLO-1 channel inhibitor, penitrem A, showed emodepside antagonising effects in A. caninum and C. elegans. The study combined the investigation of new anthelmintic targets from parasitic nematodes and experimental use of the respective target genes in C. elegans, therefore closing the gap between research approaches using model nematodes and those using target organisms. Considering the still

  7. Effects of prior treatment with salmeterol and formoterol on airway and systemic beta 2 responses to fenoterol.

    PubMed Central

    Grove, A; Lipworth, B J

    1996-01-01

    BACKGROUND: Previous studies have shown that both salmeterol and formoterol act as partial beta 2 receptor agonists in terms of antagonising the extrapulmonary responses to fenoterol in normal subjects. The aim of the present study was to extend previous observations in evaluating the effect of prior treatment with salmeterol and formoterol on bronchodilator responses to fenoterol, a full beta 2 receptor agonist, in patients with asthma. METHODS: Ten stable asthmatic patients of mean (SE) age 37 (3.7) years and forced expiratory volume in one second (FEV1) 59.5 (4.1)% of predicted completed the study. One hour after inhaling single doses of placebo, salmeterol 25 micrograms, or formoterol 12 micrograms, dose-response curves to repeated doses of inhaled fenoterol were constructed (cumulative doses of 100-3200 micrograms). Measurements of airway and systemic beta 2 receptor mediated responses were made at baseline, after inhalation of placebo, salmeterol, or formoterol, and after each dose of fenoterol. RESULTS: Salmeterol and formoterol produced significant bronchodilation compared with placebo (mean difference and 95% CI compared with placebo): FEV1, salmeterol 0.41 (95% CI 0.13 to 0.69) 1, formoterol 0.47 (95% CI 0.19 to 0.75) 1. Salmeterol and formoterol had no significant effect on systemic responses compared with placebo. There were no significant differences in peak airway responses to fenoterol after treatment with salmeterol or formoterol compared with placebo (mean (pooled SE)): FEV1, placebo 2.84 (0.03) 1, salmeterol 2.87 (0.03) 1, and formoterol 2.88 (0.03) 1. There were no significant differences in the area under the dose-response curve for any of the parameters during the dose-response curve following treatment with salmeterol or formoterol compared with placebo. There was no difference in the slope of the dose-response curves to fenoterol for FEV1 or forced expiratory flow (FEF25-75) after treatment with salmeterol or formoterol compared with placebo

  8. The mesolimbic nucleus accumbens is critically involved with the mediation of the motor inhibitory and facilitatory effects of dopamine agonists on mouse spontaneous climbing behaviour.

    PubMed

    Costall, B; Eniojukan, J F; Naylor, R J

    1983-12-23

    Mouse spontaneous climbing behaviour was dose-dependently inhibited by putative dopamine agonists administered subcutaneously (s.c.) or directly into the mesolimbic nucleus accumbens. ED50 values for s.c. administrations of apomorphine, bromocriptine, DPI, 3-PPP and DK118 (5-hydroxy-6-methyl-2-di-n-propylaminotetralin) were 0.01, 0.26, 0.17, 0.24 and 0.0004 mg/kg respectively, and for intra-accumbens apomorphine, DPI, bromocriptine, DK118 and 2-di-n-propylamino-5,6-dihydroxytetralin were 0.21, 0.22, 7.5, 0.00034 and 0.000034 micrograms respectively. Dose-dependent reduction in motor inhibitory potential/motor facilitation was also recorded for higher doses of apomorphine given s.c., or for apomorphine, DK118 and 2-di-n-propylamino-5,6-dihydroxytetralin given intra-accumbens (ED50 values to restore spontaneous climbing to control values were 4.1, 4.2 and 0.8 micrograms respectively). The motor inhibitory actions of apomorphine, 3-PPP, DK118 and 2-di-n-propylamino-5,6-dihydroxytetralin were antagonised by (-)-sulpiride, but not by yohimbine or prazosin. The actions of DPI were yohimbine-sensitive. Whilst the motor inhibition caused by s.c. bromocriptine was neuroleptic-sensitive, that observed on intra-accumbens injection was resistant to all antagonists. The intra-accumbens effectiveness of the dopamine agonists could not be mimicked by injections above the nucleus accumbens (into the head of the caudate-putamen complex) or below the nucleus accumbens (into the tuberculum olfactorium) (with the exception of the effectiveness of bromocriptine administered into the tuberculum olfactorium). It is suggested that the actions of 'dopamine agonist' to both inhibit and facilitate mouse spontaneous climbing behaviour involves an action in the mesolimbic nucleus accumbens. Whilst the mechanisms involved are generally neuroleptic-sensitive, alpha 2-adrenoceptor (for DPI) and other unidentified mechanisms (for bromocriptine) may also be important.

  9. A Short Receptor Downregulates JAK/STAT Signalling to Control the Drosophila Cellular Immune Response

    PubMed Central

    Pennetier, Delphine; Ubeda, Jean-Michel; Braun, Anne; Daburon, Virginie; Krzemień, Joanna; Bourbon, Henri-Marc; Zhou, Rui; Vincent, Alain; Crozatier, Michèle

    2010-01-01

    The posterior signalling centre (PSC), a small group of specialised cells, controls hemocyte (blood cell) homeostasis in the Drosophila larval hematopoietic organ, the lymph gland. This role of the PSC is very reminiscent of the “niche,” the micro-environment of hematopoietic stem cells in vertebrates. We have recently shown that the PSC acts in a non–cell-autonomous manner to maintain janus tyrosine kinase/signal transducers and activators of transcription (JAK/STAT) signalling in hematopoietic progenitors (prohemocytes), thereby preserving the multipotent character necessary for their differentiation into lamellocytes, a cryptic and dedicated immune cell type required to fight specific immune threats such as wasp parasitism. In this report, on the basis of a knock out generated by homologous recombination, we show that a short type I cytokine-related receptor CG14225/Latran is required for switching off JAK/STAT signalling in prohemocytes. This is a prerequisite to massive differentiation of lamellocytes upon wasp parasitisation. In vivo and cell culture assays indicate that Latran forms heteromers with Domeless, the Drosophila type I cytokine signalling receptor related to mammalian GP130, and antagonises Domeless activity in a dose-dependent manner. Our analysis further shows that a primary immune response to wasp parasitism is a strong decrease in cytokine mRNA levels in the lymph gland, followed by an increase in the latran/domeless ratio. We propose that this sequence of events culminates in the complete inhibition of residual JAK/STAT signalling by Latran. JAK/STAT activity has been associated with several human diseases including leukaemia while knock-out studies in mice point to a central role of this pathway in hematopoiesis and regulation of immune functions. The specific function of Drosophila Latran is, to our knowledge, the first in vivo example of a role for a nonsignalling receptor in controlling a dedicated immune response, and thus raises

  10. HMG-CoA reductase regulates CCL17-induced colon cancer cell migration via geranylgeranylation and RhoA activation

    SciTech Connect

    Al-Haidari, Amr A.; Syk, Ingvar; Thorlacius, Henrik

    2014-03-28

    Highlights: • Simvastatin blocked CCL17-induced and CCR4-dependent RhoA activation in HT29 cells. • CCL17/CCR4-mediated migration of colon cancer cells was antagonised by simvastatin. • Cell migration recovered by adding Mevalonate and geranylgeranyl pyrophosphate. • Targeting HMG-CoA reductase might be useful to inhibit colon cancer metastasis. - Abstract: Background: Simvastatin is widely used to lower cholesterol levels in patients with cardiovascular diseases, although accumulating evidence suggests that statins, such as simvastatin, also exert numerous anti-tumoral effects. Aim: The aim of this study was to examine the effect of simvastatin on colon cancer cell migration. Methods: Migration assays were performed to evaluate CCL17-induced colon cancer cell (HT-29) chemotaxis. In vitro tumor growth and apoptosis were assessed using a proliferation assay and annexin V assay, respectively. Active RhoA protein levels in CCL17-stimulated colon cancer cells were quantified using a G-LISA assay. Results: We found that simvastatin dose-dependently decreased CCL17-induced colon cancer cell migration. Simvastatin had no effect on colon cancer cell proliferation or apoptosis. Inhibition of beta chemokine receptor 4, CCR4, reduced CCL17-evoked activation of RhoA in colon cancer cells. Moreover, administration of mevalonate reversed the inhibitory effect of simvastatin on CCL17-induced colon cancer cell migration. Interestingly, co-incubation with geranylgeranyl pyrophosphate (GGPP) antagonized the inhibitory impact of simvastatin on colon cancer cell migration triggered by CCL17. Moreover, we observed that simvastatin decreased CCL17-induced activation of RhoA in colon cancer cells. Administration of mevalonate and GGPP reversed the inhibitory effect of simvastatin on CCL17-provoked RhoA activation in colon cancer cells. Conclusions: Taken together, our findings show for the first time that HMG-CoA reductase regulates CCL17-induced colon cancer cell migration via

  11. Inhibition of bradykinin-evoked trigeminal nerve stimulation by the non-peptide bradykinin B2 receptor antagonist WIN 64338 in vivo and in vitro.

    PubMed Central

    Hall, J. M.; Figini, M.; Butt, S. K.; Geppetti, P.

    1995-01-01

    1. This study investigated the effect of the recently described non-peptide bradykinin B2 receptor antagonist, WIN 64338 ([[4-[[2- [[bis(cyclohexylamino)methylene]amino]-3-(2-naphthalenyl)-1-oxopropyl] amino]phenyl]methyl]tributylphosphoniumchloride monohydrochloride), in experimental models of bradykinin-evoked sensory nerve stimulation. 2. In the rabbit isolated iris sphincter in vitro, bradykinin-evoked contractile responses are mediated via tachykinins released from peripheral endings of the trigeminal sensory nerve. WIN 64338 (1-10 microM) competitively antagonised contractile responses to bradykinin with a pKB estimate of 6.6 +/- 0.1 (n = 11). The antagonism was selective since WIN 64338 (10 microM) did not significantly inhibit submaximal contractile responses to the direct-acting spasmogens substance P (10 nM), neurokinin A (3 nM), substance P methyl ester (10 nM) or senktide (100 nM); nor by sensory non-adrenergic non-cholinergic nerve stimulation evoked by capsaicin (10 microM), or electrical field-stimulation (3, 10, 30 Hz) (P > 0.05; n = 3-11). 3. Topical application of bradykinin to the conjunctiva and to the nasal mucosa of the guinea-pig in vivo causes plasma extravasation predominantly via the release of tachykinins from peripheral endings of the trigeminal nerve. The increases in plasma extravasation (measured by extravasation of Evans blue dye) induced by bradykinin in the guinea-pig conjunctiva (20 nmol) and nasal mucosa (50 nmol) were markedly reduced (by 81 +/- 3% and 69 +/- 5%, respectively) following pretreatment with WIN 64338 (30 nmol kg-1, i.v.) (n = 5-6; P < 0.05), with almost complete inhibition at a higher dose of WIN 64338 (300 nmol kg-1, i.v.; n = 5-6). This inhibition was selective since at 300 nmol kg-1, WIN 64338 did not inhibit plasma extravasation evoked by substance P in the conjunctiva (5 nmol; P > 0.05; n = 6) or in the nasal mucosa (50 nmol; P > 0.05; n = 5). 4. This study demonstrates that WIN 64338 is a selective and

  12. Effects of piperine, the pungent component of black pepper, at the human vanilloid receptor (TRPV1)

    PubMed Central

    McNamara, Fergal N; Randall, Andrew; Gunthorpe, Martin J

    2005-01-01

    We have characterised the effects of piperine, a pungent alkaloid found in black pepper, on the human vanilloid receptor TRPV1 using whole-cell patch-clamp electrophysiology. Piperine produced a clear agonist activity at the human TRPV1 receptor yielding rapidly activating whole-cell currents that were antagonised by the competitive TRPV1 antagonist capsazepine and the non-competitive TRPV1 blocker ruthenium red. The current–voltage relationship of piperine-activated currents showed pronounced outward rectification (25±4-fold between −70 and +70 mV) and a reversal potential of 0.0±0.4 mV, which was indistinguishable from that of the prototypical TRPV1 agonist capsaicin. Although piperine was a less potent agonist (EC50=37.9±1.9 μM) than capsaicin (EC50=0.29±0.05 μM), it demonstrated a much greater efficacy (approximately two-fold) at TRPV1. This difference in efficacy did not appear to be related to the proton-mediated regulation of the receptor since a similar degree of potentiation was observed for responses evoked by piperine (230±20%, n=11) or capsaicin (284±32%, n=8) upon acidification to pH 6.5. The effects of piperine upon receptor desensitisation were also unable to explain this effect since piperine resulted in more pronounced macroscopic desensitisation (t1/2=9.9±0.7 s) than capsaicin (t1/2>20 s) and also caused greater tachyphylaxis in response to repetitive agonist applications. Overall, our data suggest that the effects of piperine at human TRPV1 are similar to those of capsaicin except for its propensity to induce greater receptor desensitisation and, rather remarkably, exhibit a greater efficacy than capsaicin itself. These results may provide insight into the TRPV1-mediated effects of piperine on gastrointestinal function. PMID:15685214

  13. Characterization of the postjunctional α2C-adrenoceptor mediating vasoconstriction to UK14304 in porcine pulmonary veins

    PubMed Central

    Görnemann, T; von Wenckstern, H; Kleuser, B; Villalón, C M; Centurión, D; Jähnichen, S; Pertz, H H

    2007-01-01

    Background and purpose: In terms of postjunctional α2-adrenoceptors in the pulmonary circulation, no evidence is available with regard to the receptor subtypes mediating vasoconstriction. Therefore, we characterized the α2-adrenoceptor subtypes mediating contraction in isolated porcine pulmonary veins. Experimental approach: α-adrenoceptor-mediated vasoconstriction was studied using a tissue bath protocol. mRNA profile and relative quantification of α2-adrenoceptor subtypes were determined in porcine pulmonary veins using reverse-transcriptase polymerase chain reaction (RT-PCR) and real-time PCR. Key results: In porcine pulmonary veins, noradrenaline, phenylephrine (α1-adrenoceptor agonist), UK14304 and clonidine (α2-adrenoceptor agonists) caused concentration-dependent contractions. The rank order of agonist potency was: NA≈UK14304≈clonidine > phenylephrine. UK14304 responses were antagonised by MK912 (noncompetitive antagonist parameter pD'2: 10.1), rauwolscine (pKB: 9.5), yohimbine (pKB: 9.1), WB4101 (pKB: 8.7), ARC239 (pKB: 7.5), prazosin (pKB: 7.1) and BRL44408 (pKB: 7.0). Antagonist potencies fitted best with radioligand binding data (pKi) at the human recombinant α2C-adrenoceptor (r2 = 0.96, P = 0.0001). Correlation with α2B-adrenoceptors was lower (r2 = 0.74, P > 0.01) and no correlation was obtained with α2A-adrenoceptors. Moreover, RT-PCR studies in porcine pulmonary veins showed mRNA signals for α2A- and α2C-adrenoceptors, but not for α2B-adrenoceptors, whilst real-time PCR studies indicated a prominent expression of α2C-adrenoceptor mRNA. Conclusions and Implications: Postjunctional α2C-adrenoceptors mediated contraction in porcine pulmonary veins. α1-Adrenoceptors also seem to be present in this tissue. Since α2-adrenoceptor responsiveness is increased when pulmonary vascular tone is elevated, α2C-adrenoceptor antagonists may be beneficial in diseases such as pulmonary hypertension or congestive heart failure. PMID:17375080

  14. Modifying effect of calcium/magnesium intake ratio and mortality: a population-based cohort study

    PubMed Central

    Dai, Qi; Shu, Xiao-Ou; Deng, Xinqing; Xiang, Yong-Bing; Li, Honglan; Yang, Gong; Shrubsole, Martha J; Ji, Butian; Cai, Hui; Chow, Wong-Ho; Gao, Yu-Tang; Zheng, Wei

    2013-01-01

    Objectives Magnesium (Mg) and calcium (Ca) antagonise each other in (re)absorption, inflammation and many other physiological activities. Based on mathematical estimation, the absorbed number of Ca or Mg depends on the dietary ratio of Ca to Mg intake. We hypothesise that the dietary Ca/Mg ratio modifies the effects of Ca and Mg on mortality due to gastrointestinal tract cancer and, perhaps, mortality due to diseases occurring in other organs or systems. Design Prospective studies. Setting Population-based cohort studies (The Shanghai Women's Health Study and the Shanghai Men's Health Study) conducted in Shanghai, China. Participants 74 942 Chinese women aged 40–70 years and 61 500 Chinese men aged 40–74 years participated in the study. Primary outcome measures All-cause mortality and disease-specific mortality. Results In this Chinese population with a low Ca/Mg intake ratio (a median of 1.7 vs around 3.0 in US populations), intakes of Mg greater than US Recommended Daily Allowance (RDA) levels (320 mg/day among women and 420 mg/day among men) were related to increased risks of total mortality for both women and men. Consistent with our hypothesis, the Ca/Mg intake ratio significantly modified the associations of intakes of Ca and Mg with mortality risk, whereas no significant interactions between Ca and Mg in relation to outcome were found. The associations differed by gender. Among men with a Ca/Mg ratio >1.7, increased intakes of Ca and Mg were associated with reduced risks of total mortality, and mortality due to coronary heart diseases. In the same group, intake of Ca was associated with a reduced risk of mortality due to cancer. Among women with a Ca/Mg ratio ≤1.7, intake of Mg was associated with increased risks of total mortality, and mortality due to cardiovascular diseases and colorectal cancer. Conclusions These results, if confirmed, may help to understand the optimal balance between Ca and Mg in the aetiology and prevention of these

  15. Actions of novel agonists, antagonists and antipsychotic agents at recombinant rat 5-HT6 receptors: a comparative study of coupling to G alpha s.

    PubMed

    Dupuis, Delphine S; Mannoury la Cour, Clotilde; Chaput, Christine; Verrièle, Laurence; Lavielle, Gilbert; Millan, Mark J

    2008-07-07

    Though 5-HT6 receptors are targets for the treatment of schizophrenia and other psychiatric disorders, the influence of drugs upon signal transduction has not been extensively characterized. Herein, we employed a Scintillation Proximity Assay (SPA)/antibody-immunocapture procedure of coupling to G alpha s to evaluate the interaction of a broad range of novel agonists, antagonists and antipsychotics at rat 5-HT(6) receptors stably expressed in HEK293 cells. Serotonin (pEC(50), 7.7) increased [35S]GTP gamma S binding to G alpha s by ca 2-fold without affecting binding to Gi/o or Gq. LSD (9.2), 5-MeODMT (7.9), 5-CT (7.0) and tryptamine (6.1) were likewise full agonists. In contrast, the novel sulfonyl derivatives, WAY181,187 (9.1) and WAY208,466 (7.8), behaved as partial agonists and attenuated the actions of 5-HT. SB271,046 and SB258,585 abolished activation of G alpha s by 5-HT with pKb values of 10.2 and 9.9, respectively, actions mimicked by the novel antagonist, SB399,885 (10.9). SB271,046 likewise blocked partial agonist properties of WAY181,187 and WAY208,466 with pKb values of 9.8 and 9.0, respectively. 5-HT-stimulated [35S]GTP gamma S binding to G alpha s was antagonised by various antipsychotics including olanzapine (7.8), asenapine (9.1) and SB737,050 (7.8), whereas aripiprazole and bifeprunox were inactive. Further, antagonist properties of clozapine (8.0) were mimicked by its major metabolite, N-desmethylclozapine (7.9). In conclusion, the novel ligands, WAY208,466 and WAY181,187, behaved as partial agonists at 5-HT6 receptors coupled to G alpha s, while SB399,885 was a potent antagonist. Though 5-HT6 receptor blockade is not indispensable for therapeutic efficacy, it may well play a role in the functional actions of certain antipsychotic agents.

  16. Potential Link between the Sphingosine-1-Phosphate (S1P) System and Defective Alveolar Macrophage Phagocytic Function in Chronic Obstructive Pulmonary Disease (COPD)

    PubMed Central

    Barnawi, Jameel; Tran, Hai; Jersmann, Hubertus; Pitson, Stuart; Roscioli, Eugene; Hodge, Greg; Meech, Robyn; Haberberger, Rainer; Hodge, Sandra

    2015-01-01

    patient-derived macrophages. Antagonising SIPR5 significantly improved phagocytosis. Conclusion Our results suggest a potential link between the S1P signalling system and defective macrophage phagocytic function in COPD and advise therapeutic targets. PMID:26485657

  17. Distinguishing subgroups among μ-opioid receptor agonists using Na(+),K(+)-ATPase as an effector mechanism.

    PubMed

    Masocha, Willias; González, Luis G; Agil, Ahmad

    2016-03-05

    We evaluated the effects of intracerebroventricular administration of ouabain on the antinociception induced by five μ-opioid receptor agonists in a tail flick test on female CD-1 mice and the effects of these μ-opioid receptor agonists on mice forebrain synaptosomal ouabain-sensitive Na(+),K(+)-ATPase activity. The subcutaneous administration of the μ-opioid receptor agonists tested produced a dose-dependent antinociceptive effect. The antinociception induced by morphine (1-32 mg/kg), levorphanol (0.4-6.4 mg/kg), and buprenorphine (0.02-0.64 mg/kg) was antagonised in a dose-dependent manner by ouabain (0.001-10 ng, i.c.v.), whilst the antinociception produced by fentanyl (0.02-0.16 mg/kg) and methadone (2-10 mg/kg) was not influenced significantly by ouabain (1-100 ng, i.c.v.). Incubation in vitro of forebrain synaptosomes with morphine (10(-9)-10(-4) M), levorphanol (10(-10)-10(-4) M), buprenorphine (10(-10)-10(-5) M), or fentanyl (10(-10)-10(-5) M) stimulated significantly ouabain-sensitive Na(+),K(+)-ATPase activity in a concentration-dependent way. The order of efficacy (using the Emax as a measure of intrinsic efficacy) was: morphine (29.83±0.56%)>levorphanol (18.61±1.26%)>buprenorphine (14.91±0.74%)>fentanyl (10.10±1.73%). On the other hand, methadone (10(-10)-10(-5) M) did not significantly modify the ouabain-sensitive Na(+),K(+)-ATPase activity (Emax=5.11±0.92%). These results suggest that Na(+),K(+)-ATPase activity is involved in the antinociceptive effects of morphine, levorphanol and buprenorphine, but not in that produced by fentanyl and methadone. Thus, we can conclude that at least two subgroups can be distinguished among the μ-opioid receptor agonists taking into consideration the role of Na(+),K(+)-ATPase in their antinociceptive effects.

  18. Identification of key residues that confer Rhodobacter sphaeroides LPS activity at horse TLR4/MD-2.

    PubMed

    Irvine, Katherine L; Gangloff, Monique; Walsh, Catherine M; Spring, David R; Gay, Nicholas J; Bryant, Clare E

    2014-01-01

    The molecular determinants underpinning how hexaacylated lipid A and tetraacylated precursor lipid IVa activate Toll-like receptor 4 (TLR4) are well understood, but how activation is induced by other lipid A species is less clear. Species specificity studies have clarified how TLR4/MD-2 recognises different lipid A structures, for example tetraacylated lipid IVa requires direct electrostatic interactions for agonism. In this study, we examine how pentaacylated lipopolysaccharide from Rhodobacter sphaeroides (RSLPS) antagonises human TLR4/MD-2 and activates the horse receptor complex using a computational approach and cross-species mutagenesis. At a functional level, we show that RSLPS is a partial agonist at horse TLR4/MD-2 with greater efficacy than lipid IVa. These data suggest the importance of the additional acyl chain in RSLPS signalling. Based on docking analysis, we propose a model for positioning of the RSLPS lipid A moiety (RSLA) within the MD-2 cavity at the TLR4 dimer interface, which allows activity at the horse receptor complex. As for lipid IVa, RSLPS agonism requires species-specific contacts with MD-2 and TLR4, but the R2 chain of RSLA protrudes from the MD-2 pocket to contact the TLR4 dimer in the vicinity of proline 442. Our model explains why RSLPS is only partially dependent on horse TLR4 residue R385, unlike lipid IVa. Mutagenesis of proline 442 into a serine residue, as found in human TLR4, uncovers the importance of this site in RSLPS signalling; horse TLR4 R385G/P442S double mutation completely abolishes RSLPS activity without its counterpart, human TLR4 G384R/S441P, being able to restore it. Our data highlight the importance of subtle changes in ligand positioning, and suggest that TLR4 and MD-2 residues that may not participate directly in ligand binding can determine the signalling outcome of a given ligand. This indicates a cooperative binding mechanism within the receptor complex, which is becoming increasingly important in TLR

  19. Identification of Key Residues That Confer Rhodobacter sphaeroides LPS Activity at Horse TLR4/MD-2

    PubMed Central

    Walsh, Catherine M.; Spring, David R.; Gay, Nicholas J.; Bryant, Clare E.

    2014-01-01

    The molecular determinants underpinning how hexaacylated lipid A and tetraacylated precursor lipid IVa activate Toll-like receptor 4 (TLR4) are well understood, but how activation is induced by other lipid A species is less clear. Species specificity studies have clarified how TLR4/MD-2 recognises different lipid A structures, for example tetraacylated lipid IVa requires direct electrostatic interactions for agonism. In this study, we examine how pentaacylated lipopolysaccharide from Rhodobacter sphaeroides (RSLPS) antagonises human TLR4/MD-2 and activates the horse receptor complex using a computational approach and cross-species mutagenesis. At a functional level, we show that RSLPS is a partial agonist at horse TLR4/MD-2 with greater efficacy than lipid IVa. These data suggest the importance of the additional acyl chain in RSLPS signalling. Based on docking analysis, we propose a model for positioning of the RSLPS lipid A moiety (RSLA) within the MD-2 cavity at the TLR4 dimer interface, which allows activity at the horse receptor complex. As for lipid IVa, RSLPS agonism requires species-specific contacts with MD-2 and TLR4, but the R2 chain of RSLA protrudes from the MD-2 pocket to contact the TLR4 dimer in the vicinity of proline 442. Our model explains why RSLPS is only partially dependent on horse TLR4 residue R385, unlike lipid IVa. Mutagenesis of proline 442 into a serine residue, as found in human TLR4, uncovers the importance of this site in RSLPS signalling; horse TLR4 R385G/P442S double mutation completely abolishes RSLPS activity without its counterpart, human TLR4 G384R/S441P, being able to restore it. Our data highlight the importance of subtle changes in ligand positioning, and suggest that TLR4 and MD-2 residues that may not participate directly in ligand binding can determine the signalling outcome of a given ligand. This indicates a cooperative binding mechanism within the receptor complex, which is becoming increasingly important in TLR

  20. Evaluation of the antidiarrhoeal activity of Byrsocarpus coccineus.

    PubMed

    Akindele, A J; Adeyemi, O O

    2006-11-03

    Based on its use in traditional African medicine, the antidiarrhoeal activity of the aqueous leaf extract of Byrsocarpus coccineus, Connaraceae, was evaluated on normal and castor oil-induced intestinal transit, castor oil-induced diarrhoea, enteropooling and gastric emptying. The extract (50, 100, 200 and 400 mg/kg, p.o.) produced a significant (P<0.05) dose dependent decrease in propulsion in the castor oil-induced intestinal transit in mice. The mean peristaltic index (%) for these doses of extract, control (distilled water; 10 ml/kg, p.o.) and morphine (10 mg/kg, s.c.) were 55.27+/-1.86, 53.12+/-3.73, 38.60+/-3.79, 30.25+/-1.27, 89.33+/-5.62 and 20.29+/-3.38, respectively. The effect of the extract at the highest dose was significantly (P<0.05) lower than that of the standard drug. This effect was antagonised by yohimbine (1 mg/kg, s.c.) but not by isosorbide dinitrate (IDN, 150 mg/kg, p.o.). At 200 mg/kg, the extract produced a significant decrease in propulsion in normal intestinal transit. In a dose dependent manner, it delayed the onset of diarrhoea, produced a significant decrease in the frequency of defaecation, severity of diarrhoea and protected the mice treated with castor oil. Mean diarrhoea scores were 30.83+/-1.72, 22.40+/-1.71, 21.43+/-1.32, 13.80+/-0.33, 18.00+/-3.94 and 7.67+/-2.41 for control, extract (50, 100, 200 and 400 mg/kg) and morphine, respectively. This effect was not antagonized by IDN. The extract (400 mg/kg) significantly decreased the volume (ml) of intestinal fluid secretion induced by castor oil (0.60+/-0.23) compared with 1.27+/-0.12 for control. However, there was no significant effect on gastric emptying. The results obtained suggest that Byrsocarpus coccineus possesses antidiarrhoeal activity due to its inhibitory effect on gastrointestinal propulsion, mediated through alpha(2) adrenoceptors, and also inhibition of fluid secretion. Preliminary phytochemical analysis revealed the presence of alkaloids, tannins, saponins

  1. Differential hypoglycaemic, anorectic, autonomic and emetic effects of the glucagon-like peptide receptor agonist, exendin-4, in the conscious telemetered ferret.

    PubMed

    Lu, Zengbing; Percie Du Sert, Nathalie; Chan, Sze Wa; Yeung, Chi-Kong; Lin, Ge; Yew, David T W; Andrews, Paul L R; Rudd, John A

    2014-12-10

    Rodents are incapable of emesis and consequently the emetic potential of glucagon-like peptide-1 receptor (GLP-1R) agonists in studies designed to assess a potential blood glucose lowering action of the compound was missed. Therefore, we investigated if the ferret, a carnivore with demonstrated translation capability in emesis research, would identify the emetic potential of the GLP-1R agonist, exendin-4, and any associated effects on gastric motor function, appetite and cardiovascular homeostasis. The biological activity of the GLP-1R ligands was investigated in vivo using a glucose tolerance test in pentobarbitone-anesthetised ferrets and in vitro using organ bath studies. Radiotelemetry was used to investigate the effect of exendin-4 on gastric myoelectric activity (GMA) and cardiovascular function in conscious ferrets; behaviour was also simultaneously assessed. Western blot was used to characterize GLP-1R distribution in the gastrointestinal and brain tissues. In anesthetised ferrets, exendin-4 (30 nmol/kg, s.c.) reduced experimentally elevated blood glucose levels by 36.3%, whereas the GLP-1R antagonist, exendin (9-39) (300 nmol/kg, s.c.) antagonised the effect and increased AUC0-120 by 31.0% when injected alone (P < 0.05). In animals with radiotelemetry devices, exendin-4 (100 nmol/kg, s.c.) induced emesis in 1/9 ferrets, but inhibited food intake and decreased heart rate variability (HRV) in all animals (P < 0.05). In the animals not exhibiting emesis, there was no effect on GMA, mean arterial blood pressure, heart rate, or core body temperature. In the ferret exhibiting emesis, there was a shift in the GMA towards bradygastria with a decrease in power, and a concomitant decrease in HRV. Western blot revealed GLP-1R throughout the gastrointestinal tract but exendin-4 (up to 300 nM) and exendin (9-39), failed to contract or relax isolated ferret gut tissues. GLP-1R were found in all major brain regions and the levels were comparable those in the vagus nerve

  2. α-Galactosylceramide suppresses murine eosinophil production through interferon-γ-dependent induction of NO synthase and CD95

    PubMed Central

    Gaspar-Elsas, Maria Ignez; Queto, Túlio; Masid-de-Brito, Daniela; Vieira, Bruno Marques; de Luca, Bianca; Cunha, Fernando Queiroz; Xavier-Elsas, Pedro

    2015-01-01

    , antagonised by LTD4, NSAIDs and aminoguanidine, and modified by dexamethasone. PMID:25752588

  3. Alterations in estrogen signalling pathways upon acquisition of anthracycline resistance in breast tumor cells

    PubMed Central

    Chewchuk, Simon; Guo, Baoqing; Parissenti, Amadeo Mark

    2017-01-01

    Intrinsic or acquired drug resistance is a major impediment to the successful treatment of women with breast cancer using chemotherapy. We have observed that MCF-7 breast tumor cells selected for resistance to doxorubicin or epirubicin (MCF-7DOX2 and MCF-7EPI cells, respectively) exhibited increased expression of several members of the aldo-keto reductase (AKR) gene family (in particular AKR1C3 and AKR1B10) relative to control MCF-7CC cells selected by propagation in the absence of drug. Normal cellular roles for the AKRs include the promotion of estrogen (E2) synthesis from estrone (E1) and the hydroxylation and detoxification of exogenous xenobiotics such as anthracycline chemotherapy drugs. While hydroxylation of anthracyclines strongly attenuates their cytotoxicity, it is unclear whether the enhanced AKR expression in the above anthracycline-resistant cells promotes E2 synthesis and/or alterations in E2 signalling pathways and whether such changes contribute to enhanced survival and anthracycline resistance. To determine the role of AKRs and E2 pathways in doxorubicin resistance, we examined changes in the expression of E2-related genes and proteins upon acquisition of doxorubicin resistance. We also assessed the effects of AKR overexpression or downregulation or the effects of activators or inhibitors of E2-dependent pathways on previously acquired resistance to doxorubicin. In this study we observed that the enhanced AKR expression upon acquisition of anthracycline resistance was, in fact, associated with enhanced E2 production. However, the expression of estrogen receptor α (ERα) was reduced by 2- to 5-fold at the gene transcript level and 2- to 20-fold at the protein level upon acquisition of anthracycline resistance. This was accompanied by an even stronger reduction in ERα phosphorylation and activity, including highly suppressed expression of two proteins under E2-dependent control (Bcl-2 and cyclin D1). The diminished Bcl-2 and cyclin D1 expression

  4. Association of Human Papillomavirus 16 E2 with Rad50-Interacting Protein 1 Enhances Viral DNA Replication

    PubMed Central

    Campos-León, Karen; Wijendra, Kalpanee; Siddiqa, Abida; Pentland, Ieisha; Feeney, Katherine M.; Knapman, Alison; Davies, Rachel

    2016-01-01

    ABSTRACT Rad50-interacting protein 1 (Rint1) associates with the DNA damage response protein Rad50 during the transition from the S phase to the G2/M phase and functions in radiation-induced G2 checkpoint control. It has also been demonstrated that Rint1 is essential in vesicle trafficking from the Golgi apparatus to the endoplasmic reticulum (ER) through an interaction with Zeste-White 10 (ZW10). We have isolated a novel interaction between Rint1 and the human papillomavirus 16 (HPV16) transcription and replication factor E2. E2 binds to Rint1 within its ZW10 interaction domain, and we show that in the absence of E2, Rint1 is localized to the ER and associates with ZW10. E2 expression results in a disruption of the Rint1-ZW10 interaction and an accumulation of nuclear Rint1, coincident with a significant reduction in vesicle movement from the ER to the Golgi apparatus. Interestingly, nuclear Rint1 and members of the Mre11/Rad50/Nbs1 (MRN) complex were found in distinct E2 nuclear foci, which peaked during mid-S phase, indicating that the recruitment of Rint1 to E2 foci within the nucleus may also result in the recruitment of this DNA damage-sensing protein complex. We show that exogenous Rint1 expression enhances E2-dependent virus replication. Conversely, the overexpression of a truncated Rint1 protein that retains the E2 binding domain but not the Rad50 binding domain acts as a dominant negative inhibitor of E2-dependent HPV replication. Put together, these experiments demonstrate that the interaction between Rint1 and E2 has an important function in HPV replication. IMPORTANCE HPV infections are an important driver of many epithelial cancers, including those within the anogenital and oropharyngeal tracts. The HPV life cycle is tightly regulated and intimately linked to the differentiation of the epithelial cells that it infects. HPV replication factories formed in the nucleus are locations where viral DNA is copied to support virus persistence and amplification

  5. An extracytoplasmic function sigma factor-dependent periplasmic glutathione peroxidase is involved in oxidative stress response of Shewanella oneidensis

    DOE PAGES

    Dai, Jingcheng; Wei, Hehong; Tian, Chunyuan; ...

    2015-01-01

    Background: Bacteria use alternative sigma factors (σs) to regulate condition-specific gene expression for survival and Shewanella harbors multiple ECF (extracytoplasmic function) σ genes and cognate anti-sigma factor genes. Here we comparatively analyzed two of the rpoE-like operons in the strain MR-1: rpoE-rseA-rseB-rseC and rpoE2-chrR. Results: RpoE was important for bacterial growth at low and high temperatures, in the minimal medium, and high salinity. The degP/htrA orthologue, required for growth of Escherichia coli and Pseudomonas aeruginosa at high temperature, is absent in Shewanella, while the degQ gene is RpoE-regulated and is required for bacterial growth at high temperature. RpoE2 was essentialmore » for the optimal growth in oxidative stress conditions because the rpoE2 mutant was sensitive to hydrogen peroxide and paraquat. The operon encoding a ferrochelatase paralogue (HemH2) and a periplasmic glutathione peroxidase (PgpD) was identified as RpoE2-dependent. PgpD exhibited higher activities and played a more important role in the oxidative stress responses than the cytoplasmic glutathione peroxidase CgpD under tested conditions. The rpoE2-chrR operon and the identified regulon genes, including pgpD and hemH2, are coincidently absent in several psychrophilic and/or deep-sea Shewanella strains. Conclusion: In S. oneidensis MR-1, the RpoE-dependent degQ gene is required for optimal growth under high temperature. The rpoE2 and RpoE2-dependent pgpD gene encoding a periplasmic glutathione peroxidase are involved in oxidative stress responses. But rpoE2 is not required for bacterial growth at low temperature and it even affected bacterial growth under salt stress, indicating that there is a tradeoff between the salt resistance and RpoE2-mediated oxidative stress responses.« less

  6. An extracytoplasmic function sigma factor-dependent periplasmic glutathione peroxidase is involved in oxidative stress response of Shewanella oneidensis

    SciTech Connect

    Dai, Jingcheng; Wei, Hehong; Tian, Chunyuan; Damron, Fredrick; Zhou, Jizhong; Qiu, Dongru

    2015-01-01

    Background: Bacteria use alternative sigma factors (σs) to regulate condition-specific gene expression for survival and Shewanella harbors multiple ECF (extracytoplasmic function) σ genes and cognate anti-sigma factor genes. Here we comparatively analyzed two of the rpoE-like operons in the strain MR-1: rpoE-rseA-rseB-rseC and rpoE2-chrR. Results: RpoE was important for bacterial growth at low and high temperatures, in the minimal medium, and high salinity. The degP/htrA orthologue, required for growth of Escherichia coli and Pseudomonas aeruginosa at high temperature, is absent in Shewanella, while the degQ gene is RpoE-regulated and is required for bacterial growth at high temperature. RpoE2 was essential for the optimal growth in oxidative stress conditions because the rpoE2 mutant was sensitive to hydrogen peroxide and paraquat. The operon encoding a ferrochelatase paralogue (HemH2) and a periplasmic glutathione peroxidase (PgpD) was identified as RpoE2-dependent. PgpD exhibited higher activities and played a more important role in the oxidative stress responses than the cytoplasmic glutathione peroxidase CgpD under tested conditions. The rpoE2-chrR operon and the identified regulon genes, including pgpD and hemH2, are coincidently absent in several psychrophilic and/or deep-sea Shewanella strains. Conclusion: In S. oneidensis MR-1, the RpoE-dependent degQ gene is required for optimal growth under high temperature. The rpoE2 and RpoE2-dependent pgpD gene encoding a periplasmic glutathione peroxidase are involved in oxidative stress responses. But rpoE2 is not required for bacterial growth at low temperature and it even affected bacterial growth under salt stress, indicating that there is a tradeoff between the salt resistance and RpoE2-mediated oxidative stress responses.

  7. Association of Human Papillomavirus 16 E2 with Rad50-Interacting Protein 1 Enhances Viral DNA Replication.

    PubMed

    Campos-León, Karen; Wijendra, Kalpanee; Siddiqa, Abida; Pentland, Ieisha; Feeney, Katherine M; Knapman, Alison; Davies, Rachel; Androphy, Elliot J; Parish, Joanna L

    2017-03-01

    Rad50-interacting protein 1 (Rint1) associates with the DNA damage response protein Rad50 during the transition from the S phase to the G2/M phase and functions in radiation-induced G2 checkpoint control. It has also been demonstrated that Rint1 is essential in vesicle trafficking from the Golgi apparatus to the endoplasmic reticulum (ER) through an interaction with Zeste-White 10 (ZW10). We have isolated a novel interaction between Rint1 and the human papillomavirus 16 (HPV16) transcription and replication factor E2. E2 binds to Rint1 within its ZW10 interaction domain, and we show that in the absence of E2, Rint1 is localized to the ER and associates with ZW10. E2 expression results in a disruption of the Rint1-ZW10 interaction and an accumulation of nuclear Rint1, coincident with a significant reduction in vesicle movement from the ER to the Golgi apparatus. Interestingly, nuclear Rint1 and members of the Mre11/Rad50/Nbs1 (MRN) complex were found in distinct E2 nuclear foci, which peaked during mid-S phase, indicating that the recruitment of Rint1 to E2 foci within the nucleus may also result in the recruitment of this DNA damage-sensing protein complex. We show that exogenous Rint1 expression enhances E2-dependent virus replication. Conversely, the overexpression of a truncated Rint1 protein that retains the E2 binding domain but not the Rad50 binding domain acts as a dominant negative inhibitor of E2-dependent HPV replication. Put together, these experiments demonstrate that the interaction between Rint1 and E2 has an important function in HPV replication.IMPORTANCE HPV infections are an important driver of many epithelial cancers, including those within the anogenital and oropharyngeal tracts. The HPV life cycle is tightly regulated and intimately linked to the differentiation of the epithelial cells that it infects. HPV replication factories formed in the nucleus are locations where viral DNA is copied to support virus persistence and amplification of

  8. Time course of the estradiol-dependent induction of oxytocin receptor binding in the ventromedial hypothalamic nucleus of the rat

    SciTech Connect

    Johnson, A.E.; Ball, G.F.; Coirini, H.; Harbaugh, C.R.; McEwen, B.S.; Insel, T.R. )

    1989-09-01

    Oxytocin (OT) transmission is involved in the steroid-dependent display of sexual receptivity in rats. One of the biochemical processes stimulated by the ovarian steroid 17 beta-estradiol (E2) that is relevant to reproduction is the induction of OT receptor binding in the ventromedial hypothalamic nucleus (VMN). The purpose of these experiments was to determine if E2-induced changes in OT receptor binding in the VMN occur within a time frame relevant to cyclic changes in ovarian steroid secretion. OT receptor binding was measured in the VMN of ovariectomized rats implanted for 0-96 h with E2-containing Silastic capsules. The rate of decay of OT receptor binding was measured in another group of animals 6-48 h after capsule removal. Receptors were labeled with the specific OT receptor antagonist ({sup 125}I)d(CH2)5(Tyr(Me)2,Thr4,Tyr-NH2(9))OVT, and binding was measured with quantitative autoradiographic methods. In addition, plasma E2 levels and uterine weights were assessed in animals from each treatment condition. Significant increases in E2-dependent OT receptor binding and uterine weight occurred within 24 h of steroid treatment. After E2 withdrawal, OT receptor binding and uterine weight decreased significantly within 24 h. These results are consistent with the hypothesis that steroid modulation of OT receptor binding is necessary for the induction of sexual receptivity.

  9. The Immunophilin-Like Protein XAP2 Is a Negative Regulator of Estrogen Signaling through Interaction with Estrogen Receptor α

    PubMed Central

    Berg, Petra; Korbonits, Marta; Pongratz, Ingemar

    2011-01-01

    XAP2 (also known as aryl hydrocarbon receptor interacting protein, AIP) is originally identified as a negative regulator of the hepatitis B virus X-associated protein. Recent studies have expanded the range of XAP2 client proteins to include the nuclear receptor family of transcription factors. In this study, we show that XAP2 is recruited to the promoter of ERα regulated genes like the breast cancer marker gene pS2 or GREB1 and negatively regulate the expression of these genes in MCF-7 cells. Interestingly, we show that XAP2 downregulates the E2-dependent transcriptional activation in an estrogen receptor (ER) isoform-specific manner: XAP2 inhibits ERα but not ERβ-mediated transcription. Thus, knockdown of intracellular XAP2 levels leads to increased ERα activity. XAP2 proteins, carrying mutations in their primary structures, loose the ability of interacting with ERα and can no longer regulate ER target gene transcription. Taken together, this study shows that XAP2 exerts a negative effect on ERα transcriptional activity and may thus prevent ERα-dependent events. PMID:21984905

  10. Involvement of Brd4 in different steps of the papillomavirus life cycle.

    PubMed

    Iftner, Thomas; Haedicke-Jarboui, Juliane; Wu, Shwu-Yuan; Chiang, Cheng-Ming

    2017-03-02

    Bromodomain-containing protein 4 (Brd4) is a cellular chromatin-binding factor and transcriptional regulator that recruits sequence-specific transcription factors and chromatin modulators to control target gene transcription. Papillomaviruses (PVs) have evolved to hijack Brd4's activity in order to create a facilitating environment for the viral life cycle. Brd4, in association with the major viral regulatory protein E2, is involved in multiple steps of the PV life cycle including replication initiation, viral gene transcription, and viral genome segregation and maintenance. Phosphorylation of Brd4, regulated by casein kinase II (CK2) and protein phosphatase 2A (PP2A), is critical for viral gene transcription as well as E1- and E2-dependent origin replication. Thus, pharmacological agents regulating Brd4 phosphorylation and inhibitors blocking phospho-Brd4 functions are promising candidates for therapeutic intervention in treating human papillomavirus (HPV) infections as well as associated disease. Copyright © 2016 Elsevier B.V. All rights reserved.

  11. Antisense transcript long noncoding RNA (lncRNA) HOTAIR is transcriptionally induced by estradiol.

    PubMed

    Bhan, Arunoday; Hussain, Imran; Ansari, Khairul I; Kasiri, Sahba; Bashyal, Aarti; Mandal, Subhrangsu S

    2013-10-09

    HOTAIR (HOX antisense intergenic RNA) is a long noncoding RNA (lncRNA) that is transcribed from the antisense strand of homeobox C gene locus in chromosome 12. HOTAIR coordinates with chromatin-modifying enzymes and regulates gene silencing. It is overexpressed in various carcinomas including breast cancer. Herein, we demonstrated that HOTAIR is crucial for cell growth and viability and its knockdown induced apoptosis in breast cancer cells. We also demonstrated that HOTAIR is transcriptionally induced by estradiol (E2). Its promoter contains multiple functional estrogen response elements (EREs). Estrogen receptors (ERs) along with various ER coregulators such as histone methylases MLL1 (mixed lineage leukemia 1) and MLL3 and CREB-binding protein/p300 bind to the promoter of HOTAIR in an E2-dependent manner. Level of histone H3 lysine-4 trimethylation, histone acetylation, and RNA polymerase II recruitment is enriched at the HOTAIR promoter in the presence of E2. Knockdown of ERs and MLLs downregulated the E2-induced HOTAIR expression. Thus, similar to protein-coding gene transcription, E2-induced transcription of antisense transcript HOTAIR is coordinated via ERs and ER coregulators, and this mechanism of HOTAIR overexpression potentially contributes towards breast cancer progression. Published by Elsevier Ltd.

  12. Activation of the MKL1/actin signaling pathway induces hormonal escape in estrogen-responsive breast cancer cell lines.

    PubMed

    Kerdivel, Gwenneg; Boudot, Antoine; Habauzit, Denis; Percevault, Frederic; Demay, Florence; Pakdel, Farzad; Flouriot, Gilles

    2014-06-05

    Estrogen receptor alpha (ERα) is generally considered to be a good prognostic marker because almost 70% of ERα-positive tumors respond to anti-hormone therapies. Unfortunately, during cancer progression, mammary tumors can escape from estrogen control, resulting in resistance to treatment. In this study, we demonstrate that activation of the actin/megakaryoblastic leukemia 1 (MKL1) signaling pathway promotes the hormonal escape of estrogen-sensitive breast cancer cell lines. The actin/MKL1 signaling pathway is silenced in differentiated ERα-positive breast cancer MCF-7 and T47D cell lines and active in ERα-negative HMT-3522 T4-2 and MDA-MB-231 breast cancer cells, which have undergone epithelial-mesenchymal transition. We showed that MKL1 activation in MCF-7 cells, either by modulating actin dynamics or using MKL1 mutants, down-regulates ERα expression and abolishes E2-dependent cell growth. Interestingly, the constitutively active form of MKL1 represses PR and HER2 expression in these cells and increases the expression of HB-EGF, TGFβ, and amphiregulin growth factors in an E2-independent manner. The resulting expression profile (ER-, PR-, HER2-) typically corresponds to the triple-negative breast cancer expression profile.

  13. Changes in hypothalamic monoamine oxidase activity in relation to 17 beta-estradiol and clomiphene citrate treatments in the teleost Channa punctatus during three seasons.

    PubMed

    Khan, I A; Joy, K P

    1991-03-01

    Hypothalamic monoamine oxidase (MAO) activity displayed biphasic responses to administration of estradiol-17 beta (E2) depending on season. Lower doses of 0.05 and 0.10 microgram/g BW of E2 had enhanced enzyme activity and higher doses (0.5 and 1.0 microgram/g BW) depressed it significantly in preparatory season. In prespawning phase, the lowest dose of 0.05 microgram/g BW of E2 was stimulatory and the higher doses were inhibitory to MAO activity. In postspawning season, lower doses (0.05 and 0.10 microgram/g BW) of E2 failed to elicit any significant change in enzyme activity whereas the higher doses (0.5 and 1.0 microgram/g BW) elevated it significantly. Administration of clomid (5, 10, and 20 micrograms/g BW) inhibited MAO activity in a dose-dependent manner during all the three seasons. The results show that E2 may participate in feedback control of gonadotropin secretion by modifying monoaminergic activity at the level of oxidative deamination by MAO.

  14. Tristetraprolin Represses Estrogen Receptor α Transactivation in Breast Cancer Cells*

    PubMed Central

    Barrios-García, Tonatiuh; Tecalco-Cruz, Angeles; Gómez-Romero, Vania; Reyes-Carmona, Sandra; Meneses-Morales, Iván; León-Del-Río, Alfonso

    2014-01-01

    Estrogen receptor α (ERα) mediates the effects of 17β-estradiol (E2) in normal mammary gland, and it is a key participant in breast cancer tumor development. ERα transactivation activity is mediated by the synergistic interaction of two domains designated AF1 and AF2. The function of AF2 is to recruit coactivator and corepressor proteins that allow ERα to oscillate between the roles of transcriptional activator and repressor. In contrast, the mechanism responsible for AF-1 transcriptional activity is not completely understood. In this study, we identified tristetraproline (TTP) as a novel ERα-associated protein. TTP expression in MCF7 cells repressed ERα transactivation and reduced MCF7 cell proliferation and the ability of the cells to form tumors in a mouse model. We show that TTP transcriptional activity is mediated through its recruitment to the promoter region of ERα target genes and its interaction with histone deacetylases, in particular with HDAC1. TTP expression attenuates the coactivating activity of SRC-1, suggesting that exchange between TTP and other coactivators may play an important role in fine-tuning ERα transactivation. These results indicate that TTP acts as a bona fide ERα corepressor and suggest that this protein may be a contributing factor in the development of E2-dependent tumors in breast cancer. PMID:24737323

  15. Analysis of The Cancer Genome Atlas sequencing data reveals novel properties of the human papillomavirus 16 genome in head and neck squamous cell carcinoma.

    PubMed

    Nulton, Tara J; Olex, Amy L; Dozmorov, Mikhail; Morgan, Iain M; Windle, Brad

    2017-03-14

    Human papillomavirus (HPV) DNA is detected in up to 80% of oropharyngeal carcinomas (OPC) and this HPV positive disease has reached epidemic proportions. To increase our understanding of the disease, we investigated the status of the HPV16 genome in HPV-positive head and neck cancers (HNC). Raw RNA-Seq and Whole Genome Sequence data from The Cancer Genome Atlas HNC samples were analyzed to gain a full understanding of the HPV genome status for these tumors. Several remarkable and novel observations were made following this analysis. Firstly, there are three main HPV genome states in these tumors that are split relatively evenly: An episomal only state, an integrated state, and a state in which the viral genome exists as a hybrid episome with human DNA. Secondly, none of the tumors expressed high levels of E6; E6*I is the dominant variant expressed in all tumors. The most striking conclusion from this study is that around three quarters of HPV16 positive HNC contain episomal versions of the viral genome that are likely replicating in an E1-E2 dependent manner. The clinical and therapeutic implications of these observations are discussed.

  16. Quantitative Adverse Outcome Pathways and Their ...

    EPA Pesticide Factsheets

    A quantitative adverse outcome pathway (qAOP) consists of one or more biologically based, computational models describing key event relationships linking a molecular initiating event (MIE) to an adverse outcome. A qAOP provides quantitative, dose–response, and time-course predictions that can support regulatory decision-making. Herein we describe several facets of qAOPs, including (a) motivation for development, (b) technical considerations, (c) evaluation of confidence, and (d) potential applications. The qAOP used as an illustrative example for these points describes the linkage between inhibition of cytochrome P450 19A aromatase (the MIE) and population-level decreases in the fathead minnow (FHM; Pimephales promelas). The qAOP consists of three linked computational models for the following: (a) the hypothalamic-pitutitary-gonadal axis in female FHMs, where aromatase inhibition decreases the conversion of testosterone to 17β-estradiol (E2), thereby reducing E2-dependent vitellogenin (VTG; egg yolk protein precursor) synthesis, (b) VTG-dependent egg development and spawning (fecundity), and (c) fecundity-dependent population trajectory. While development of the example qAOP was based on experiments with FHMs exposed to the aromatase inhibitor fadrozole, we also show how a toxic equivalence (TEQ) calculation allows use of the qAOP to predict effects of another, untested aromatase inhibitor, iprodione. While qAOP development can be resource-intensive, the quan

  17. Mig-6 Mouse Model of Endometrial Cancer.

    PubMed

    Kim, Tae Hoon; Yoo, Jung-Yoon; Jeong, Jae-Wook

    2017-01-01

    Endometrial cancer is a frequently occurring gynecological disorder. Estrogen-dependent endometrioid carcinoma is the most common type of gynecological cancer. One of the major pathologic phenomena of endometrial cancer is the loss of estrogen (E2) and progesterone (P4) control over uterine epithelial cell proliferation. P4 antagonizes the growth-promoting properties of E2 in the uterus. P4 prevents the development of endometrial cancer associated with unopposed E2 by blocking E2 actions. Mitogen inducible gene 6 (Mig-6, Errfi1, RALT, or gene 33) is an immediate early response gene that can be induced by various mitogens and common chronic stress stimuli. Mig-6 has been identified as an important component of P4-mediated inhibition of E2 signaling in the uterus. Decreased expression of MIG-6 is observed in human endometrial carcinomas. Transgenic mice with Mig-6 ablation in the uterus develop endometrial hyperplasia and E2-dependent endometrial cancer. Thus, MIG-6 has a tumor suppressor function in endometrial tumorigenesis. The following discussion summarizes our current knowledge of Mig-6 mouse models and their role in understanding the molecular mechanisms of endometrial tumorigenesis and in the development of therapeutic approaches for endometrial cancer.

  18. MicroRNA-34a Suppresses Cell Proliferation by Targeting LMTK3 in Human Breast Cancer MCF-7 Cell Line

    PubMed Central

    Zhao, Guoqing; Guo, Jun; Li, Dong; Jia, Chengyou; Yin, Wanzhong; Sun, Ran; Lv, Zhongwei

    2013-01-01

    Breast cancer remains the leading cause of cancer mortality in females, and about 70% of the primary breast cancer patients are diagnosed ERα-positive, which is the most common type of breast cancer. MicroRNA-34a (miR-34a) has been shown to be a master regulator of tumor suppression in many types of cancers including breast cancer. However, the role of miR-34a in ERα-positive breast cancer has not been elucidated. Here, we find that in MCF-7, which is an ERα-positive breast cancer cell line, miR-34a is remarkably downregulated after E2 treatment. Overexpression of miR-34a by lentivirus suppresses cell proliferation, S phase ratio, and tumor formation in an E2-dependent manner in vitro. According to the mRNA sequence, lemur tyrosine kinase 3 (LMTK3), which is an important regulator of estrogen receptor alpha (ERα), is a predicted target of miR-34a. This is confirmed by dual luciferase reporter assay and the decrease of LMTK3 mRNA and protein levels after overexpression of miR-34a. Moreover, miR-34a overexpression decreases AKT signaling pathway and increases ERα phosphorylation status. Taken together, these results suggest that miR-34a inhibits breast cancer proliferation by targeting LMTK3 and might be used as an anti-ERα agent in breast cancer therapy. PMID:24050776

  19. Intrauterine exposure to 17β-oestradiol (E2) impairs postnatal development in both female and male prostate in gerbil.

    PubMed

    Sanches, Bruno D A; Santos, Juliana M; Zani, Bruno C; Biancardi, Manoel F; Santos, Fernanda C A; Góes, Rejane M; Vilamaior, Patricia S L; Taboga, Sebastião R

    2017-07-30

    We employed histological techniques to assess the effects of intrauterine exposure to different dosages of E2 on male and female Mongolian gerbils on the postnatal development of the prostate. E2 promotes alterations this gland branches in the female, but not in males, even at low dosage, at higher dosages, acini of altered aspect are verified in the male and female prostate, as well as a decrease in branching number, reduced cell proliferation and staining for FGF10, simultaneously to the increased labelling for TGFβ1, which may account for alterations on branching of the prostate. The sensitivity of the female prostate to intrauterine exposure to E2, which can reflect the E2 dependence of female prostate development. This becomes alarming in view of the occurrence of prostate in female of several mammals and including women, and the possibility that low E2 dosage exposures considered safe to males provoke developmental alterations in female prostate. Copyright © 2017. Published by Elsevier Inc.

  20. Xanthohumol suppresses oestrogen-signalling in breast cancer through the inhibition of BIG3-PHB2 interactions

    PubMed Central

    Yoshimaru, Tetsuro; Komatsu, Masato; Tashiro, Etsu; Imoto, Masaya; Osada, Hiroyuki; Miyoshi, Yasuo; Honda, Junko; Sasa, Mitsunori; Katagiri, Toyomasa

    2014-01-01

    Xanthohumol (XN) is a natural anticancer compound that inhibits the proliferation of oestrogen receptor-α (ERα)-positive breast cancer cells. However, the precise mechanism of the antitumour effects of XN on oestrogen (E2)-dependent cell growth, and especially its direct target molecule(s), remain(s) largely unknown. Here, we focus on whether XN directly binds to the tumour suppressor protein prohibitin 2 (PHB2), forming a novel natural antitumour compound targeting the BIG3-PHB2 complex and acting as a pivotal modulator of E2/ERα signalling in breast cancer cells. XN treatment effectively prevented the BIG3-PHB2 interaction, thereby releasing PHB2 to directly bind to both nuclear- and cytoplasmic ERα. This event led to the complete suppression of the E2-signalling pathways and ERα-positive breast cancer cell growth both in vitro and in vivo, but did not suppress the growth of normal mammary epithelial cells. Our findings suggest that XN may be a promising natural compound to suppress the growth of luminal-type breast cancer. PMID:25483453

  1. Four closely-related RING-type E3 ligases, APD1-4, are involved in pollen mitosis II regulation in Arabidopsis.

    PubMed

    Luo, Guo; Gu, Hongya; Liu, Jingjing; Qu, Li-Jia

    2012-10-01

    Ubiquitination of proteins is one of the critical regulatory mechanisms in eukaryotes. In higher plants, protein ubiquitination plays an essential role in many biological processes, including hormone signaling, photomorphogenesis, and pathogen defense. However, the roles of protein ubiquitination in the reproductive process are not clear. In this study, we identified four plant-specific RING-finger genes designated Aberrant Pollen Development 1 (APD1) to APD4, as regulators of pollen mitosis II (PMII) in Arabidopsis thaliana (L.). The apd1 apd2 double mutant showed a significantly increased percentage of bicellular-like pollen at the mature pollen stage. Further downregulation of the APD3 and APD4 transcripts in apd1 apd2 by RNA interference (RNAi) resulted in more severe abnormal bicellular-like pollen phenotypes than in apd1 apd2, suggesting that cell division was defective in male gametogenesis. All of the four genes were expressed in multiple stages at different levels during male gametophyte development. Confocal analysis using green florescence fusion proteins (GFP) GFP-APD1 and GFP-APD2 showed that APDs are associated with intracellular membranes. Furthermore, APD2 had E2-dependent E3 ligase activity in vitro, and five APD2-interacting proteins were identified. Our results suggest that these four genes may be involved, redundantly, in regulating the PMII process during male gametogenesis. © 2012 Institute of Botany, Chinese Academy of Sciences.

  2. Chronic leptin treatment sensitizes MCF-7 breast cancer cells to estrogen.

    PubMed

    Valle, Adamo; Sastre-Serra, Jorge; Oliver, Jordi; Roca, Pilar

    2011-01-01

    Obesity is associated with an increased risk of estrogen-dependent breast cancer. The adipokine leptin, whose levels are chronically increased in obese people, has been shown to stimulate ER positive cancer cell growth. Considering previous evidence of a crosstalk between leptin and estrogen signaling, the objective of this study was to establish the influence of chronic leptin treatment on estrogen-dependent cell growth. To this aim, we use the estrogen receptor (ER) positive MCF-7 breast cancer cell line treated chronically with leptin and analyzed estrogen-dependent cell growth, ERs (ERα and ERβ) expression, ER-dependent transcriptional activity as well as cell survival to the antiestrogenic agents tamoxifen and ICI 182,780. Leptin signaling pathway kept activated after chronic stimulation (7 days) with leptin showing significant phosphorylation of JAK2 and STAT3 and higher cell proliferation rate. Chronic leptin at 100 ng/mL dose increased ERα to ERβ ratio and consistently enhanced estrogen-dependent transcriptional activity, increasing E2-dependent cell growth and resistance to antiestrogen agents. This study supports the existence of a crosstalk between leptin and estrogen, in which leptin might play an important role potentiating the mitogenic action of estrogen, probably by alteration of ERα to ERβ ratio. Copyright © 2011 S. Karger AG, Basel.

  3. NF-E2, FLI1 and RUNX1 collaborate at areas of dynamic chromatin to activate transcription in mature mouse megakaryocytes

    PubMed Central

    Zang, Chongzhi; Luyten, Annouck; Chen, Justina; Liu, X. Shirley; Shivdasani, Ramesh A.

    2016-01-01

    Mutations in mouse and human Nfe2, Fli1 and Runx1 cause thrombocytopenia. We applied genome-wide chromatin dynamics and ChIP-seq to determine these transcription factors’ (TFs) activities in terminal megakaryocyte (MK) maturation. Enhancers with H3K4me2-marked nucleosome pairs were most enriched for NF-E2, FLI and RUNX sequence motifs, suggesting that this TF triad controls much of the late MK program. ChIP-seq revealed NF-E2 occupancy near previously implicated target genes, whose expression is compromised in Nfe2-null cells, and many other genes that become active late in MK differentiation. FLI and RUNX were also the motifs most enriched near NF-E2 binding sites and ChIP-seq implicated FLI1 and RUNX1 in activation of late MK, including NF-E2-dependent, genes. Histones showed limited activation in regions of single TF binding, while enhancers that bind NF-E2 and either RUNX1, FLI1 or both TFs gave the highest signals for TF occupancy and H3K4me2; these enhancers associated best with genes activated late in MK maturation. Thus, three essential TFs co-occupy late-acting cis-elements and show evidence for additive activity at genes responsible for platelet assembly and release. These findings provide a rich dataset of TF and chromatin dynamics in primary MK and explain why individual TF losses cause thrombopocytopenia. PMID:27457419

  4. Co-transcriptional R-loops are the main cause of estrogen-induced DNA damage.

    PubMed

    Stork, Caroline Townsend; Bocek, Michael; Crossley, Madzia P; Sollier, Julie; Sanz, Lionel A; Chédin, Frédéric; Swigut, Tomek; Cimprich, Karlene A

    2016-08-23

    The hormone estrogen (E2) binds the estrogen receptor to promote transcription of E2-responsive genes in the breast and other tissues. E2 also has links to genomic instability, and elevated E2 levels are tied to breast cancer. Here, we show that E2 stimulation causes a rapid, global increase in the formation of R-loops, co-transcriptional RNA-DNA products, which in some instances have been linked to DNA damage. We show that E2-dependent R-loop formation and breast cancer rearrangements are highly enriched at E2-responsive genomic loci and that E2 induces DNA replication-dependent double-strand breaks (DSBs). Strikingly, many DSBs that accumulate in response to E2 are R-loop dependent. Thus, R-loops resulting from the E2 transcriptional response are a significant source of DNA damage. This work reveals a novel mechanism by which E2 stimulation leads to genomic instability and highlights how transcriptional programs play an important role in shaping the genomic landscape of DNA damage susceptibility.

  5. Co-transcriptional R-loops are the main cause of estrogen-induced DNA damage

    PubMed Central

    Stork, Caroline Townsend; Bocek, Michael; Crossley, Madzia P; Sollier, Julie; Sanz, Lionel A; Chédin, Frédéric; Swigut, Tomek; Cimprich, Karlene A

    2016-01-01

    The hormone estrogen (E2) binds the estrogen receptor to promote transcription of E2-responsive genes in the breast and other tissues. E2 also has links to genomic instability, and elevated E2 levels are tied to breast cancer. Here, we show that E2 stimulation causes a rapid, global increase in the formation of R-loops, co-transcriptional RNA-DNA products, which in some instances have been linked to DNA damage. We show that E2-dependent R-loop formation and breast cancer rearrangements are highly enriched at E2-responsive genomic loci and that E2 induces DNA replication-dependent double-strand breaks (DSBs). Strikingly, many DSBs that accumulate in response to E2 are R-loop dependent. Thus, R-loops resulting from the E2 transcriptional response are a significant source of DNA damage. This work reveals a novel mechanism by which E2 stimulation leads to genomic instability and highlights how transcriptional programs play an important role in shaping the genomic landscape of DNA damage susceptibility. DOI: http://dx.doi.org/10.7554/eLife.17548.001 PMID:27552054

  6. Ubiquitin-Activated Interaction Traps (UBAITs) identify E3 ligase binding partners.

    PubMed

    O'Connor, Hazel F; Lyon, Nancy; Leung, Justin W; Agarwal, Poonam; Swaim, Caleb D; Miller, Kyle M; Huibregtse, Jon M

    2015-12-01

    We describe a new class of reagents for identifying substrates, adaptors, and regulators of HECT and RING E3s. UBAITs (Ubiquitin-Activated Interaction Traps) are E3-ubiquitin fusion proteins and, in an E1- and E2-dependent manner, the C-terminal ubiquitin moiety forms an amide linkage to proteins that interact with the E3, enabling covalent co-purification of the E3 with partner proteins. We designed UBAITs for both HECT (Rsp5, Itch) and RING (Psh1, RNF126, RNF168) E3s. For HECT E3s, trapping of interacting proteins occurred in vitro either through an E3 thioester-linked lariat intermediate or through an E2 thioester intermediate, and both WT and active-site mutant UBAITs trapped known interacting proteins in yeast and human cells. Yeast Psh1 and human RNF126 and RNF168 UBAITs also trapped known interacting proteins when expressed in cells. Human RNF168 is a key mediator of ubiquitin signaling that promotes DNA double-strand break repair. Using the RNF168 UBAIT, we identify H2AZ--a histone protein involved in DNA repair--as a new target of this E3 ligase. These results demonstrate that UBAITs represent powerful tools for profiling a wide range of ubiquitin ligases.

  7. Neuroglobin upregulation induced by 17β-estradiol sequesters cytocrome c in the mitochondria preventing H2O2-induced apoptosis of neuroblastoma cells

    PubMed Central

    De Marinis, E; Fiocchetti, M; Acconcia, F; Ascenzi, P; Marino, M

    2013-01-01

    The sex steroid hormone 17β-estradiol (E2) upregulates the levels of neuroglobin (NGB), a new neuroprotectant globin, to elicit its neuroprotective effect against H2O2-induced apoptosis. Several mechanisms could be proposed to justify the NGB involvement in E2 prevention of stress-induced apoptotic cell death. Here, we evaluate the ability of E2 to modulate the intracellular NGB localization and the NGB interaction with mitochondrial cytochrome c following the H2O2-induced toxicity. Present results demonstrate that NGB is expressed in the nuclei, mitochondria, and cytosol of human neuroblastoma SK-N-BE cells. E2, but not H2O2 treatment of SK-N-BE cells, reallocates NGB mainly at the mitochondria and contemporarily reduces the number of apoptotic nuclei and the levels of cleaved caspase-3. Remarkably, the E2 treatment strongly increases NGB–cytochrome c association into mitochondria and reduces the levels of cytochrome c into the cytosol of SK-N-BE cells. Although both estrogen receptors (ERα and ERβ) are expressed in the nucleus, mitochondria, and cytosol of SK-N-BE cells, this E2 effect specifically requires the mitochondrial ERβ activity. As a whole, these data demonstrate that the interception of the intrinsic apoptotic pathway into mitochondria (i.e., the prevention of cytochrome c release) is one of the pivotal mechanisms underlying E2-dependent NGB neuroprotection against H2O2 toxicity. PMID:23429294

  8. Dynamin II is required for 17β-estradiol signaling and autophagy-based ERα degradation

    PubMed Central

    Totta, Pierangela; Busonero, Claudia; Leone, Stefano; Marino, Maria; Acconcia, Filippo

    2016-01-01

    17β-estradiol (E2) regulates diverse physiological effects, including cell proliferation, by binding to estrogen receptor α (ERα). ERα is both a transcription factor that drives E2-sensitive gene expression and an extra-nuclear localized receptor that triggers the activation of diverse kinase cascades. While E2 triggers cell proliferation, it also induces ERα degradation in a typical hormone-dependent feedback loop. Although ERα breakdown proceeds through the 26S proteasome, a role for lysosomes and for some endocytic proteins in controlling ERα degradation has been reported. Here, we studied the role of the endocytic protein dynamin II in E2-dependent ERα signaling and degradation. The results indicate that dynamin II siRNA-mediated knock-down partially prevents E2-induced ERα degradation through the inhibition of an autophagy-based pathway and impairs E2-induced cell proliferation signaling. Altogether, these data demonstrate that dynamin II is required for the E2:ERα signaling of physiological functions and uncovers a role for autophagy in the control of ERα turnover. PMID:27009360

  9. Xanthohumol suppresses oestrogen-signalling in breast cancer through the inhibition of BIG3-PHB2 interactions.

    PubMed

    Yoshimaru, Tetsuro; Komatsu, Masato; Tashiro, Etsu; Imoto, Masaya; Osada, Hiroyuki; Miyoshi, Yasuo; Honda, Junko; Sasa, Mitsunori; Katagiri, Toyomasa

    2014-12-08

    Xanthohumol (XN) is a natural anticancer compound that inhibits the proliferation of oestrogen receptor-α (ERα)-positive breast cancer cells. However, the precise mechanism of the antitumour effects of XN on oestrogen (E2)-dependent cell growth, and especially its direct target molecule(s), remain(s) largely unknown. Here, we focus on whether XN directly binds to the tumour suppressor protein prohibitin 2 (PHB2), forming a novel natural antitumour compound targeting the BIG3-PHB2 complex and acting as a pivotal modulator of E2/ERα signalling in breast cancer cells. XN treatment effectively prevented the BIG3-PHB2 interaction, thereby releasing PHB2 to directly bind to both nuclear- and cytoplasmic ERα. This event led to the complete suppression of the E2-signalling pathways and ERα-positive breast cancer cell growth both in vitro and in vivo, but did not suppress the growth of normal mammary epithelial cells. Our findings suggest that XN may be a promising natural compound to suppress the growth of luminal-type breast cancer.

  10. Clathrin Heavy Chain Interacts With Estrogen Receptor α and Modulates 17β-Estradiol Signaling

    PubMed Central

    Totta, Pierangela; Pesiri, Valeria; Enari, Masato; Marino, Maria

    2015-01-01

    17β-estradiol (E2)-induced signaling and control of estrogen receptor (ER)α degradation both play a major role in breast cancer cell proliferation. We recently reported the involvement of lysosomal function in both E2-dependent ERα breakdown and E2-induced cell proliferation and thus hypothesized a role for endocytic proteins in ERα signaling. An small interfering RNA screen identified proteins that regulate intracellular endocytic traffic and whose silencing alters E2-induced ERα degradation. One such protein was the clathrin heavy chain (CHC), whose role in E2:ERα signaling to cell proliferation is unknown. Here, we show that CHC physically interacts with ERα in the cytoplasm of breast cancer cells and regulates E2-induced cell proliferation. Surprisingly, the CHC:ERα interaction is required to sustain E2 signaling but is dispensable for ERα degradation. Our data also demonstrate that many membrane trafficking proteins contribute to the regulation of ERα degradation, thus unraveling the contribution of endocytic proteins in E2:ERα signaling. PMID:25860340

  11. Dietary clofibrate stimulates the formation and size of estradiol-induced breast tumors in female August-Copenhagen Irish (ACI) rats.

    PubMed

    Mesia-Vela, Sonia; Sanchez, Rosa I; Roberts, Kathleen G; Reuhl, Kenneth R; Conney, Allan H; Kauffman, Frederick C

    2008-04-03

    Administration of 0.4% clofibrate in the diet stimulated estradiol (E(2))-induced mammary carcinogenesis in the August-Copenhagen Irish (ACI) rat without having an effect on serum levels of E(2). This treatment stimulated by several-fold the NAD(P)H-dependent oxidative metabolism of E(2) and oleyl-CoA-dependent esterification of E(2) to 17beta-oleyl-estradiol by liver microsomes. Glucuronidation of E(2) by microsomal glucuronosyltransferase was increased moderately. In contrast, the activity of NAD(P)H quinone reductase 1 (NQO1), a representative monofunctional phase 2 enzyme, was significantly decreased in liver cytosol of rats fed clofibrate. Decreases in hepatic NQO1 in livers of animals fed clofibrate were noted before the appearance of mammary tumors. E(2) was delivered in cholesterol pellets implanted in 7-8-week-old female ACI rats. The animals received AIN-76A diet containing 0.4% clofibrate for 6, 12 or 28 weeks. Control animals received AIN-76A diet. Dietary clofibrate increased the number and size of palpable mammary tumors but did not alter the histopathology of the E(2)-induced mammary adenocarcinomas. Collectively, these results suggest that the stimulatory effect of clofibrate on hepatic esterification of E(2) with fatty acids coupled with the inhibition of protective phase 2 enzymes, may in part, enhance E(2)-dependent mammary carcinogenesis in the ACI rat model.

  12. Role of endogenous interleukin-1 receptor antagonist in regulating fever induced by localised inflammation in the rat

    PubMed Central

    Cartmell, T; Luheshi, G N; Hopkins, S J; Rothwell, N J; Poole, S

    2001-01-01

    Interleukin (IL)-1 is a mediator of host defence responses to inflammation and injury, including fever, but its sites of synthesis and action have not been fully elucidated. The actions of IL-1 are antagonised by IL-1 receptor antagonist (IL-1ra). The present study tested the hypothesis that IL-1 and IL-1ra are produced locally at sites of peripheral inflammation in rats, and that endogenous IL-1ra acts to limit the fever resulting from the inflammation. Injection of lipopolysaccharide (LPS; 100 μg kg−1) into a subcutaneous air pouch (i.po.) of rats induced a significant increase in body temperature. Virtually all (∼85 %) of the injected LPS was recovered from the pouch between 1 and 8 h (when the experiment was terminated) after injection of LPS, but LPS was undetectable (< 50 pg ml−1) in plasma at any time. Concentrations of immunoreactive IL-1α and IL-1β were increased significantly in the pouch at 1, 2, 3, 5 and 8 h after injection of LPS, corresponding with the rise in body temperature and the fever peak. The appearance of IL-1ra was delayed until 2 h. Thereafter, the concentrations of IL-1β and IL-1ra increased in parallel with the development of fever, while the concentrations of IL-1α remained constant. IL-1ra, but not IL-1α or IL-1β, was detected in significant quantities in the plasma of LPS-injected animals. Treatment of rats with an anti-IL-1ra serum (2 ml, i.po.) at the time of injection of LPS (10 or 100 μg kg−1, i.po.) abolished the appearance of IL-1ra in the circulation. Although neutralisation of endogenous IL-1ra did not affect the maximum body temperature reached after injection of submaximum (10 μg kg−1, i.po.) or maximum (100 μg kg−1, i.po.) doses of LPS, the duration of the fever was significantly prolonged, and was associated with a 3- to 4-fold increase in immunoreactive IL-1β concentrations in the pouch fluid, but not in the plasma, at the 8 h time point. These data show that effects of local (i.po.) injection of LPS

  13. Pharmacological classification of α1-adrenoceptors mediating contractions of rabbit isolated ear artery: comparison with rat isolated thoracic aorta

    PubMed Central

    Fagura, M S; Lydford, S J; Dougall, I G

    1997-01-01

    The present study attempted to classify pharmacologically the α1-adrenoceptor subtype(s) present in two isolated, vascular ring preparations, the rabbit ear artery and rat thoracic aorta. In the ear artery, the agonist effects of phenylephrine were antagonized by 5-methyl urapidil (pA2=7.90; Schild slope=0.85) and BMY 7378 (pA2=6.11; Schild slope=0.80) but not in a simple competitive manner. The shallow Schild slopes are consistent with the activation of a heterogeneous receptor population. Indeed the 5-methyl urapidil data set could be fitted to a two-receptor model yielding a high antagonist affinity (pKBH) estimate of 7.85 and a low affinity (pKBL) estimate of 6.03. The effects of clonidine in the ear artery were competitively antagonised by 5-methyl urapidil (pKB=7.91) and BMY 7378 (pKB=5.53). These data are consistent with contractions to clonidine being mediated by a single receptor subtype. In the aorta, the effects of phenylephrine were antagonized by 5-methyl urapidil (pA2=7.95; Schild slope=1.11) and BMY 7378 (pA2=9.08; Schild slope=0.73). Neither data set was consistent with a simple competitive interaction. The BMY 7378 data suggested again, that phenylephrine was acting at a heterogeneous receptor population. Subsequent analysis by the two-receptor model yielded a high affinity (pKBH) estimate of 8.95 and a low affinity (pKBL) estimate of 7.00. The alkylating agent, chloroethylclonidine (CEC) elicited concentration-dependent contractions in the ear artery with a potency (p[A]50) of 5.57. Pretreatment of this tissue with CEC (5 μM, 30 min incubation) had no effect on subsequent responses to phenylephrine. In contrast, in the aorta, CEC demonstrated no agonism but pretreatment with this agent (5 μM, 15 min incubation) caused a rightward shift and depression of subsequent phenylephrine concentration-effect curves. The affinity of clonidine in the rabbit ear artery (pKA=6.17) was found to be significantly different to its affinity in the

  14. Rosiglitazone and 15-deoxy-Δ12,14-prostaglandin J2, ligands of the peroxisome proliferator-activated receptor-γ (PPAR-γ), reduce ischaemia/reperfusion injury of the gut

    PubMed Central

    Cuzzocrea, Salvatore; Pisano, Barbara; Dugo, Laura; Ianaro, Angela; Patel, Nimesh S A; Paola, Rosanna Di; Genovese, Tiziana; Chatterjee, Prabal K; Rosa, Massimo Di; Caputi, Achille P; Thiemermann, Christoph

    2003-01-01

    proinflammatory cytokines (TNF-α and IL-1β) and (vi) histological evidence of gut injury. Administration of rosiglitazone and 15d-PGJ2 also markedly reduced the nitrotyrosine formation and the upregulation of ICAM-1 during reperfusion. In order to elucidate whether the protective effects of rosiglitazone and 15d-PGJ2 are related to the activation of the PPAR-γ receptor, we also investigated the effect of a PPAR-γ antagonist, bisphenol A diglycidyl ether (BADGE), on the protective effects of rosiglitazone and 15d-PGJ2. BADGE (1 mg kg−1 administered i.v. 30 min prior to the treatment of rosiglitazone or 15d-PGJ2) significantly antagonised the effect of the two PPAR-γ agonists and thus abolished the protective effect against gut I/R. These results demonstrate that the two PPAR-γ agonists, rosiglitazone and 15d-PGJ2, significantly reduce I/R injury of the intestine. PMID:12970094

  15. Mechanism of ginsenoside Rg1 renal protection in a mouse model of d-galactose-induced subacute damage.

    PubMed

    Fan, Yanling; Xia, Jieyu; Jia, Daoyong; Zhang, Mengsi; Zhang, Yanyan; Huang, Guoning; Wang, Yaping

    2016-09-01

    Context Ginseng is a widely used herbal medicine in China but its mechanism of action remains unclear. Objective The objectives of this work were to study the protective effect of ginsenoside Rg1 on subacute murine renal damage induced by d-galactose and its mechanism. Materials and methods C57BL/6J mice were injected with 120 mg/kg/d (sc) d-galactose for 1 week, followed by a combined treatment of Rg1 20 mg/kg/d (ip) and 120 mg/kg/d d-galactose (sc) for 5 weeks. Mice were injected with the 0.9% saline 0.2 mL/d (sc) and 120 mg/kg/d d-galactose (sc) for 6 weeks in the control group and the d-galactose group, respectively. After 6 weeks, urea, creatinine, uric acid, cystatin (Cys-C), senescence-associated β-galactosidase (SA-β-gal) staining positive kidney cells, superoxide dismutase (SOD), glutathione peroxidase (GSH-PX), malondialdehyde (MDA), glycation end products (AGEs) and 8-hydroxy-2 deoxyguanosine (8-OH-dG) were measured. Results Treatment with Rg1 ameliorated kidney function and aging state (urea from 17.19 ± 1.09 to 15.77 ± 1.22 mmol·L (-) (1), creatinine from 29.40 ± 5.72 to 22.60 ± 3.97 μmol·L (-) (1), uric acid from 86.80 ± 5.97 to 72.80 ± 10.61 μmol·L (-) (1), Cys-C from 0.23 ± 0.03 to 0.18 ± 0.05 mg·L (-) (1), ROD of SA-β-gal from 56.32 ± 10.48 to 26.78 ± 7.34, SOD from 150.22 ± 19.07 to 190.56 ± 15.83 U·(mg·prot) (-1), MDA from 9.28 ± 1.59 to 3.17 ± 0.82 nmol·(mg·prot) (-1), GSH-PX from 15.68 ± 2.11 to 20.32 ± 2.96 U·(mg·prot) (-1) as well as regulated glomerulus morphology (glomerulus diameter from 775.77 ± 18.41 to 695.04 ± 14.61 μm, renal capsule width from 39.56 ± 3.51 to 31.42 ± 2.70 μm, glomerulus basement membrane from 206.03 ± 16.22 to 157.27 ± 15.70 nm, podocyte slit from 55.21 ± 8.55 to 37.63 ± 6.65 nm). Conclusions Ginsenoside Rg1 can antagonise d

  16. Protective effect of Salvia miltiorrhiza and Carthamus tinctorius extract against lipopolysaccharide-induced liver injury

    PubMed Central

    Gao, Li-Na; Yan, Kuo; Cui, Yuan-Lu; Fan, Guan-Wei; Wang, Yue-Fei

    2015-01-01

    and caspase-3 was also decreased by DHI. Western blot analysis revealed that DHI antagonised LPS-stimulated decrease of Bcl-2 and increase of Bax protein expression. Furthermore, DHI inhibited LPS-induced IκBα and NF-κB p65 phosphorylation. CONCLUSION: DHI may be a multi-function protectant against acute hepatic injury in mice through its anti-inflammatory, anti-oxidative and anti-apoptotic activities. PMID:26290634

  17. Protective effect of Salvia miltiorrhiza and Carthamus tinctorius extract against lipopolysaccharide-induced liver injury.

    PubMed

    Gao, Li-Na; Yan, Kuo; Cui, Yuan-Lu; Fan, Guan-Wei; Wang, Yue-Fei

    2015-08-14

    decreased by DHI. Western blot analysis revealed that DHI antagonised LPS-stimulated decrease of Bcl-2 and increase of Bax protein expression. Furthermore, DHI inhibited LPS-induced IκBα and NF-κB p65 phosphorylation. DHI may be a multi-function protectant against acute hepatic injury in mice through its anti-inflammatory, anti-oxidative and anti-apoptotic activities.

  18. 3,4-Diaminopyridine masks the inhibition of noradrenaline release from chick sympathetic neurons via presynaptic alpha 2-adrenoceptors: insights into the role of N- and L-type calcium channels.

    PubMed

    Dolezal, V; Huang, H Y; Schobert, A; Hertting, G

    1996-05-20

    antagonised this effect. UK 14304 did not measurably inhibit the stimulation-evoked rise of intraterminal [Ca2+]i in the presence of 3,4-diaminopyridine but it produced an inhibition by 26% if nifedipine had been applied together with 3,4-diaminopyridine. Our observations show that, under control conditions, the stimulated release of [3H]noradrenaline is mainly associated with the opening of N-type channels, while in the presence of 3,4-diaminopyridine the contribution of L-type channels becomes more important. The alpha 2-adrenoceptor stimulation by UK 14304 inhibits the release of [3H]noradrenaline but, in the presence of 3,4-diaminopyridine, the inhibition of release can only be observed if the massive influx through L-type calcium channels is prevented. These data suggest that presynaptic alpha 2-adrenoceptors of chick sympathetic neurons preferentially influence the N-type calcium channels.

  19. 5-Hydroxytryptamine-inhibiting property of Feverfew: role of parthenolide content.

    PubMed

    Mittra, S; Datta, A; Singh, S K; Singh, A

    2000-12-01

    To study the mechanism of antimigraine activity of Tanacetum parthenium (Feverfew), its extracts and parthenolide, a component of Feverfew, by observing their effect on 5-HT storage and release, and stimulation of 5-HT2B and 5-HT2A receptors. Also to standardize a dosage form of Feverfew with respect to its parthenolide content. Isometric responses to 5-HT and an indirect acting serotonergic, d-fenfluramine, were obtained on rat fundus and ileum. In one set of experiments the effect of dichloromethane extract of Feverfew and parthenolide was observed on the above. The extract was then thermally degraded upto 10%, 23%, and 33% with respect to its parthenolide content by keeping at 60 degrees C and 75% relative humidity and the experiments were repeated. In another set of experiments rats were fed with 20 mg/kg Feverfew powder (equivalent to a human dose of 500 micrograms parthenolide per day) for 30 d or were i.p. injected with parthenolide (23.4 micrograms/day) for 7 d. In the same set of experiments one group of rats were fed with 15% and 77% degraded Feverfew powder in the same dose as mentioned above. After 30 days the effects of the above were observed on 5-HT and d-fenfluramine. Feverfew was specially formulated and tested for stability under accelerated conditions. Parthenolide (1 x 10(-5) mol/L) non-competitively antagonised the effects of d-fenfluramine but had no significant effect on 5-HT2B and 5-HT2A receptors in rat fundus and ileum at 30 min which turned significant on increasing the incubation time to 1.5 h, in rat fundus. Parthenolide (5 x 10(-5) mol/L) followed the same trend. However, Feverfew extract (1 x 10(-5) mol/L) potently and directly blocked 5-HT2B and 5-HT2A receptors and neuronally released 5-HT. At 5 x 10(-5) mol/L the extract potently and irreversibly blocked the above. Both parthenolide and Feverfew extract showed a time-dependency in their action. The extract when degraded thermally upto 10% could significantly block the 5-HT

  20. Activation of κ opioid receptors increases intrinsic excitability of dentate gyrus granule cells

    PubMed Central

    McDermott, Carmel M; Schrader, Laura A

    2011-01-01

    . Preincubation of the slice with the selective KOR antagonist, nor-binalthorphimine (BNI, 1 μm) inhibited the effect of U6 on the latency to first spike and spike half-width suggesting that these effects are mediated through KORs. The inclusion of GDP-βS (1 mm) in the recording pipette prevented all of the U6 effects, suggesting that all effects are mediated via a G-protein-dependent mechanism. Inclusion of the A-type K+ current blocker, 4-aminopyridine (4-AP, 5 mm) in the pipette also antagonised the effects of U6. Kv4.2 is one of the channel α subunits thought to be responsible for carrying the A-type K+ current. Incubation of hippocampus slices with U6 resulted in a decrease in the Kv4.2 subunit protein at the cell surface. These results are consistent with an increase in cell excitability in response to KOR activation and may reflect new possibilities for additional opioid functions. PMID:21606111

  1. Mutation of the palmitoylation site of estrogen receptor α in vivo reveals tissue-specific roles for membrane versus nuclear actions

    PubMed Central

    Adlanmerini, Marine; Solinhac, Romain; Abot, Anne; Fabre, Aurélie; Raymond-Letron, Isabelle; Guihot, Anne-Laure; Boudou, Frédéric; Sautier, Lucile; Vessières, Emilie; Kim, Sung Hoon; Lière, Philippe; Fontaine, Coralie; Krust, Andrée; Chambon, Pierre; Katzenellenbogen, John A.; Gourdy, Pierre; Shaul, Philip W.; Henrion, Daniel; Arnal, Jean-François; Lenfant, Françoise

    2014-01-01

    Estrogen receptor alpha (ERα) activation functions AF-1 and AF-2 classically mediate gene transcription in response to estradiol (E2). A fraction of ERα is targeted to plasma membrane and elicits membrane-initiated steroid signaling (MISS), but the physiological roles of MISS in vivo are poorly understood. We therefore generated a mouse with a point mutation of the palmitoylation site of ERα (C451A-ERα) to obtain membrane-specific loss of function of ERα. The abrogation of membrane localization of ERα in vivo was confirmed in primary hepatocytes, and it resulted in female infertility with abnormal ovaries lacking corpora lutea and increase in luteinizing hormone levels. In contrast, E2 action in the uterus was preserved in C451A-ERα mice and endometrial epithelial proliferation was similar to wild type. However, E2 vascular actions such as rapid dilatation, acceleration of endothelial repair, and endothelial NO synthase phosphorylation were abrogated in C451A-ERα mice. A complementary mutant mouse lacking the transactivation function AF-2 of ERα (ERα-AF20) provided selective loss of function of nuclear ERα actions. In ERα-AF20, the acceleration of endothelial repair in response to estrogen–dendrimer conjugate, which is a membrane-selective ER ligand, was unaltered, demonstrating integrity of MISS actions. In genome-wide analysis of uterine gene expression, the vast majority of E2-dependent gene regulation was abrogated in ERα-AF20, whereas in C451A-ERα it was nearly fully preserved, indicating that membrane-to-nuclear receptor cross-talk in vivo is modest in the uterus. Thus, this work genetically segregated membrane versus nuclear actions of a steroid hormone receptor and demonstrated their in vivo tissue-specific roles. PMID:24371309

  2. A Distinct Mechanism for Coactivator versus Corepressor Function by Histone Methyltransferase G9a in Transcriptional Regulation*

    PubMed Central

    Purcell, Daniel J.; Jeong, Kwang Won; Bittencourt, Danielle; Gerke, Daniel S.; Stallcup, Michael R.

    2011-01-01

    Histone methyltransferase G9a has been understood primarily as a corepressor of gene expression, but we showed previously that G9a positively regulates nuclear receptor-mediated transcription in reporter gene assays. Here, we show that endogenous G9a contributes to the estradiol (E2)-dependent induction of some endogenous target genes of estrogen receptor (ER)α in MCF-7 breast cancer cells while simultaneously limiting the E2-induced expression of other ERα target genes. Thus, G9a has a dual and selective role as a coregulator for ERα target genes. The ERα binding regions associated with the pS2 gene, which requires G9a for E2-induced expression, are transiently occupied by G9a at 15 min after beginning E2 treatment, suggesting that G9a coactivator function is by direct interaction with ERα target genes. Transient reporter gene assays with deletion mutants of G9a demonstrated that domains previously associated with the corepressor functions of G9a (C-terminal methyltransferase domain, ankyrin repeat domain, and cysteine-rich domain) were unnecessary for G9a coactivator function in ERα-mediated transcription. In contrast, the N-terminal domain of G9a was necessary and sufficient for enhancement of ERα-mediated transcription and for E2-induced occupancy of G9a on ERα binding sites associated with endogenous target genes of ERα. In addition to a previously identified activation domain, this region contains a previously uncharacterized ligand-dependent ERα binding function, indicating how G9a is recruited to the target genes. Therefore, the coactivator and corepressor functions of G9a involve different G9a domains and different molecular mechanisms. PMID:21984853

  3. Cyr61 promotes breast tumorigenesis and cancer progression

    SciTech Connect

    Tsai, Miaw-Sheue; Bogart, Daphne F.; Castaneda, Jessica M.; Li, Patricia; Lupu, Ruth

    2002-01-16

    Cyr61, a member of the CCN family of genes, is an angiogenic factor. We have shown that it is overexpressed in invasive and metastatic human breast cancer cells and tissues. Here, we investigated whether Cyr61 is necessary and/or sufficient to bypass the ''normal'' estrogen (E2) requirements for breast cancer cell growth. Our results demonstrate that under E2-depleted condition, Cyr61 is sufficient to induce MCF-7 cells grow in the absence of E2. MCF-7 cells transfected with Cyr61 (MCF-7/Cyr61) became E2-independent but still E2-responsive. On the other hand, MCF-7/vector cells remain E2-dependent. MCF-7/Cyr61 cells acquire an antiestrogen-resistant phenotype, one of the most common clinical occurrences during breast cancer progression. MCF-7/Cyr61 cells are anchorage-independent and capable of forming Matrigel outgrowth patterns in the absence of E2. ERa expression in MCF-7/Cyr61 cells is decreased although still functional. Additionally, MCF-7/Cyr61 cells are tumorigenic in ovariectomized athymic nude mice. The tumors resemble human invasive carcinomas with increased vascularization and overexpression of vascular endothelial growth factor (VEGF). Our results demonstrate that Cyr61 is a tumor-promoting factor and a key regulator of breast cancer progression. This study provides evidence that Cyr61 is sufficient to induce E2-independence and anti-E2 resistance, and to promote invasiveness in vitro, and to induce tumorigenesis in vivo, all of which are characteristics of an aggressive breast cancer phenotype.

  4. Non-invasive administration of 17β-estradiol rapidly increases aggressive behavior in non-breeding, but not breeding, male song sparrows.

    PubMed

    Heimovics, Sarah A; Ferris, Jennifer K; Soma, Kiran K

    2015-03-01

    17β-Estradiol (E2) acts in the brain via genomic and non-genomic mechanisms to influence physiology and behavior. There is seasonal plasticity in the mechanisms by which E2 activates aggression, and non-genomic mechanisms appear to predominate during the non-breeding season. Male song sparrows (Melospiza melodia) display E2-dependent territorial aggression throughout the year. Field studies show that song sparrow aggression during a territorial intrusion is similar in the non-breeding and breeding seasons, but aggression after an intrusion ends differs seasonally. Non-breeding males stop behaving aggressively within minutes whereas breeding males remain aggressive for hours. We hypothesize that this seasonal plasticity in the persistence of aggression relates to seasonal plasticity in E2 signaling. We used a non-invasive route of E2 administration to compare the non-genomic (within 20min) effects of E2 on aggressive behavior in captive non-breeding and breeding season males. E2 rapidly increased barrier contacts (attacks) during an intrusion by 173% in non-breeding season males only. Given that these effects were observed within 20min of E2 administration, they likely occurred via a non-genomic mechanism of action. The present data, taken together with past work, suggest that environmental cues associated with the non-breeding season influence the molecular mechanisms through which E2 influences behavior. In song sparrows, transient expression of aggressive behavior during the non-breeding season is highly adaptive: it minimizes energy expenditure and maximizes the amount of time available for foraging. In all, these data suggest the intriguing possibility that aggression in the non-breeding season may be activated by a non-genomic E2 mechanism due to the fitness benefits associated with rapid and transient expression of aggression.

  5. Evaluating the effect of stellar multiplicity on the point spread function of space-based weak lensing surveys

    NASA Astrophysics Data System (ADS)

    Kuntzer, T.; Courbin, F.; Meylan, G.

    2016-02-01

    The next generation of space-based telescopes used for weak lensing surveys will require exquisite point spread function (PSF) determination. Previously negligible effects may become important in the reconstruction of the PSF, in part because of the improved spatial resolution. In this paper, we show that unresolved multiple star systems can affect the ellipticity and size of the PSF and that this effect is not cancelled even when using many stars in the reconstruction process. We estimate the error in the reconstruction of the PSF due to the binaries in the star sample both analytically and with image simulations for different PSFs and stellar populations. The simulations support our analytical finding that the error on the size of the PSF is a function of the multiple stars distribution and of the intrinsic value of the size of the PSF, i.e. if all stars were single. Similarly, the modification of each of the complex ellipticity components (e1,e2) depends on the distribution of multiple stars and on the intrinsic complex ellipticity. Using image simulations, we also show that the predicted error in the PSF shape is a theoretical limit that can be reached only if large number of stars (up to thousands) are used together to build the PSF at any desired spatial position. For a lower number of stars, the PSF reconstruction is worse. Finally, we compute the effect of binarity for different stellar magnitudes and show that bright stars alter the PSF size and ellipticity more than faint stars. This may affect the design of PSF calibration strategies and the choice of the related calibration fields.

  6. Activation of protein synthesis in mouse uterine epithelial cells by estradiol-17β is mediated by a PKC-ERK1/2-mTOR signaling pathway.

    PubMed

    Wang, Yuxiang; Zhu, Liyin; Kuokkanen, Satu; Pollard, Jeffrey W

    2015-03-17

    The uterine epithelium of mice and humans undergoes cyclical waves of cell proliferation and differentiation under the regulation of estradiol-17β (E2) and progesterone (P4). These epithelial cells respond to E2 with increased protein and DNA synthesis, whereas P4 inhibits only the E2-induced DNA synthetic response. Here we show that E2 regulates protein synthesis in these epithelial cells through activating PKC that in turn stimulates ERK1/2 to phosphorylate and thereby activate the central regulator of protein synthesis mechanistic target of rapamycin (mTOR). This mTOR pathway is not inhibited by P4. Inhibitor studies with an estrogen receptor (ESR1) antagonist showed the dependence of this mTOR pathway on ESR1 but that once activated, a phosphorylation cascade independent of ESR1 propagates the pathway. E2 also stimulates an IGF1 receptor (IGF1R) to PI3 kinase to AKT to GSK-3β pathway required for activation of the canonical cell cycle machinery that is inhibited by P4. PKC activation did not stimulate this pathway nor does inhibition of PKC or ERK1/2 affect it. These studies therefore indicate a mechanism whereby DNA and protein synthesis are regulated by two ESR1-activated pathways that run in parallel with only the one responsible for the initiation of DNA synthesis blocked by P4. Inhibition of mTOR by rapamycin in vivo resulted in inhibition of E2-induced protein and DNA synthesis. Proliferative diseases of the endometrium such as endometriosis and cancer are common and E2 dependent. Thus, defining this mTOR pathway suggests that local (intrauterine or peritoneal) rapamycin administration might be a therapeutic option for these diseases.

  7. Genome-wide profiling of Hfq-binding RNAs uncovers extensive post-transcriptional rewiring of major stress response and symbiotic regulons in Sinorhizobium meliloti.

    PubMed

    Torres-Quesada, Omar; Reinkensmeier, Jan; Schlüter, Jan-Philip; Robledo, Marta; Peregrina, Alexandra; Giegerich, Robert; Toro, Nicolás; Becker, Anke; Jiménez-Zurdo, Jose I

    2014-01-01

    The RNA chaperone Hfq is a global post-transcriptional regulator in bacteria. Here, we used RNAseq to analyze RNA populations from the legume symbiont Sinorhizobium meliloti that were co-immunoprecipitated (CoIP-RNA) with a FLAG-tagged Hfq in five growth/stress conditions. Hfq-bound transcripts (1315) were largely identified in stressed bacteria and derived from small RNAs (sRNAs), both trans-encoded (6.4%) and antisense (asRNAs; 6.3%), and mRNAs (86%). Pull-down with Hfq recovered a small proportion of annotated S. meliloti sRNAs (14% of trans-sRNAs and 2% of asRNAs) suggesting a discrete impact of this protein in sRNA pathways. Nonetheless, Hfq selectively stabilized CoIP-enriched sRNAs, anticipating that these interactions are functionally significant. Transcription of 26 Hfq-bound sRNAs was predicted to occur from promoters recognized by the major stress σ factors σ(E2) or σ(H1/2). Recovery rates of sRNAs in each of the CoIP-RNA libraries suggest a large impact of Hfq-assisted riboregulation in S. meliloti osmoadaptation. Hfq directly targeted 18% of the predicted S. meliloti mRNAs, which encode functionally diverse proteins involved in transport and metabolism, σ(E2)-dependent stress responses, quorum sensing, flagella biosynthesis, ribosome, and membrane assembly or symbiotic nitrogen fixation. Canonical targeting of the 5' regions of two of the ABC transporter mRNAs by the homologous Hfq-binding AbcR1 and AbcR2 sRNAs leading to inhibition of protein synthesis was confirmed in vivo. We therefore provide a comprehensive resource for the systems-level deciphering of hitherto unexplored S. meliloti stress and symbiotic post-transcriptional regulons and the identification of Hfq-dependent sRNA-mRNA regulatory pairs.

  8. Novel Selective Estrogen Mimics for the Treatment of Tamoxifen-Resistant Breast Cancer

    PubMed Central

    Molloy, Mary Ellen; Perez White, Bethany E.; Gherezghiher, Teshome; Michalsen, Bradley T.; Xiong, Rui; Patel, Hitisha; Zhao, Huiping; Maximov, Philipp Y.; Jordan, V. Craig; Thatcher, Gregory R. J.; Tonetti, Debra A.

    2014-01-01

    Endocrine-resistant breast cancer is a major clinical obstacle. The use of 17β-estradiol (E2) has re-emerged as a potential treatment option following exhaustive use of tamoxifen (TAM) or aromatase inhibitors although side effects have hindered its clinical usage. Protein kinase C alpha (PKCα) expression was shown to be a predictor of disease outcome for patients receiving endocrine therapy and may predict a positive response to an estrogenic treatment. Here, we have investigated the use of novel benzothiophene selective estrogen mimics (SEMs) as an alternative to E2 for the treatment of TAM-resistant breast cancer. Following in vitro characterization of SEMs, a panel of clinically relevant PKCα-expressing, TAM-resistant models were used to investigate the antitumor effects of these compounds. SEM treatment resulted in growth inhibition and apoptosis of TAM-resistant cell lines in vitro. In vivo SEM treatment induced tumor regression of TAM-resistant T47D:A18/PKCα and T47D:A18-TAM1 tumor models. T47D:A18/PKCα tumor regression was accompanied by translocation of ERα to extranuclear sites, possibly defining a mechanism through which these SEMs initiate tumor regression. SEM treatment did not stimulate growth of E2-dependent T47D:A18/neo tumors. Additionally, unlike E2 or TAM, treatment with SEMs did not stimulate uterine weight gain. These findings suggest the further development of SEMs as a feasible therapeutic strategy for the treatment of endocrine-resistant breast cancer without the side effects associated with E2. PMID:25205655

  9. The E8∧E2 Gene Product of Human Papillomavirus Type 16 Represses Early Transcription and Replication but Is Dispensable for Viral Plasmid Persistence in Keratinocytes▿

    PubMed Central

    Lace, Michael J.; Anson, James R.; Thomas, Gregory S.; Turek, Lubomir P.; Haugen, Thomas H.

    2008-01-01

    A conserved E8∧E2 spliced mRNA is detected in keratinocytes transfected with human papillomavirus type 16 (HPV-16) plasmid DNA. Expression of HPV-16 E8∧E2 (16-E8∧E2) is independent of the major early promoter, P97, and is modulated by both specific splicing events and conserved cis elements in the upstream regulatory region in a manner that differs from transcriptional regulation of other early viral genes. Mutations that disrupt the predicted 16-E8∧E2 message also increase initial HPV-16 plasmid amplification 8- to 15-fold and major early gene (P97) transcription 4- to 5-fold over those of the wild type (wt). Expressing the 16-E8∧E2 gene product from the cytomegalovirus (CMV) promoter represses HPV-16 early gene transcription from P97 in a dose-dependent manner, as detected by RNase protection assays. When expressed from the CMV promoter, 16-E8∧E2 also inhibits the amplification of an HPV-16 plasmid and a heterologous simian virus 40 (SV40) ori plasmid that contains E2 binding sites in cis. In contrast, cotransfections with HPV-16 wt genomes that express physiologic levels of 16-E8∧E2 are sufficient to repress HPV-16 plasmid amplification but are limiting and insufficient for the repression of SV40 amplification. 16-E8∧E2-dependent repression of HPV-16 E1 expression is sufficient to account for this observed inhibition of initial HPV-16 plasmid amplification. Unlike with other papillomaviruses, primary human keratinocytes immortalized by the HPV-16 E8 mutant genome contain more than eightfold-higher levels of unintegrated plasmid than the wt, demonstrating that 16-E8∧E2 limits the viral copy number but is not required for plasmid persistence and maintenance. PMID:18753207

  10. The effect of sex hormones on peroxisome proliferator-activated receptor gamma expression and activity in mature adipocytes.

    PubMed

    Sato, Hiromi; Sugai, Hana; Kurosaki, Hiroshi; Ishikawa, Momoko; Funaki, Asami; Kimura, Yuki; Ueno, Koichi

    2013-01-01

    Peroxisome proliferator-activated receptor (PPAR) γ plays a major role in the regulation of lipid and carbohydrate metabolism. Pioglitazone is a PPARγ agonist that is widely used for the treatment of type 2 diabetes mellitus. However, female patients have been reported to experience stronger efficacy and adverse effects than male patients. This study evaluated the effects of sex hormones on PPARγ expression and activity in adipocytes. Mouse 3T3-L1 preadipocytes were used after being grown into matured adipocytes. The sex hormones 17β-estradiol (E2), testosterone (T), or 5α-androstan-17β-ol-3-one (dihydrotestosterone; DHT) were added to the matured adipocytes and the cells were then maintained for short (24-72 h) or long (1- or 2-weeks) periods. E2 significantly upregulated PPARγ protein expression in a concentration-dependent manner after extended exposure, whereas T and DHT did not have such an effect. When cells were co-treated with pioglitazone and E2, PPARγ protein expression significantly increased in an E2-dependent manner, whereas this expression seemed to be reduced by pioglitazone mono-treatment and co-treatment with DHT at higher concentrations. The secretion levels of adiponectin protein, a major indicator of PPARγ activity, were significantly decreased by DHT, but were not affected by E2. Finally a luciferase assay was performed using a PPAR response element-Luk reporter gene. Transcriptional activity was not changed by any of single sex hormone treatment, but was significantly downregulated by co-treatment with pioglitazone and DHT. Taken together, our results suggest that sex hormones may influence PPARγ expression and function, which may explain the observed sex-specific different effect of pioglitazone.

  11. Sex-specific response of rat costochondral cartilage growth plate chondrocytes to 17β-estradiol involves differential regulation of plasma membrane associated estrogen receptors.

    PubMed

    Elbaradie, Khairat B Y; Wang, Yun; Boyan, Barbara D; Schwartz, Zvi

    2013-05-01

    Both male and female rat growth plate chondrocytes express estrogen receptors (ERs); however 17β-estradiol (E2) induces membrane responses leading to activation of phospholipase A2 (PLA2), phospholipase C (PLC), prostaglandin E2 (PGE2) production, protein kinase C (PKC), and ultimately mitogen protein kinase (MAPK) only in female cells. This study investigated if these sex-specific responses are due to differences in the actual ERs or in downstream signaling. Western blots and flow cytometry of costochondral cartilage resting zone chondrocytes (RCs) showed 2-3 times more ERα in plasma membranes (PMs) from female cells than male cells. Tunicamycin blocked E2-dependent ER-translocation to the PM, indicating palmitoylation was required. Co-immunoprecipitation showed E2 induced complex formation between ER isoforms only in female RCs. To examine if the lack of response in PKC and PGE2 in males is due to differences in signaling, we examined involvement of ERs and the role of PLC and PLA2. Selective ERα (propylpyrazole triol, PPT) and ERβ (diarylproprionitrile, DPN) agonists activated PKC in female RCs only. The PLC inhibitor, U73122 blocked E2's effect on PKC and the cytosolic PLA2 inhibitor, AACOCF3 inhibited the effect on PGE2 in female RCs, confirming involvement of PLC and PLA2 in the mechanism. The PLC activator, m-3M3FβS activated PKC and PLAA peptide increased PGE2 levels in male and female RCs, showing that the signaling pathways are present. These data indicate that differences in membrane ER amount, localization, translocation and interaction are responsible for the sexual dimorphic response to E2.

  12. TSPYL5 SNPs: Association with Plasma Estradiol Concentrations and Aromatase Expression

    PubMed Central

    Liu, Mohan; Ingle, James N.; Fridley, Brooke L.; Buzdar, Aman U.; Robson, Mark E.; Kubo, Michiaki; Wang, Liewei; Batzler, Anthony; Jenkins, Gregory D.; Pietrzak, Tracy L.; Carlson, Erin E.; Goetz, Matthew P.; Northfelt, Donald W.; Perez, Edith A.; Williard, Clark V.; Schaid, Daniel J.; Nakamura, Yusuke

    2013-01-01

    We performed a discovery genome-wide association study to identify genetic factors associated with variation in plasma estradiol (E2) concentrations using DNA from 772 postmenopausal women with estrogen receptor (ER)-positive breast cancer prior to the initiation of aromatase inhibitor therapy. Association analyses showed that the single nucleotide polymorphisms (SNP) (rs1864729) with the lowest P value (P = 3.49E-08), mapped to chromosome 8 near TSPYL5. We also identified 17 imputed SNPs in or near TSPYL5 with P values < 5E-08, one of which, rs2583506, created a functional estrogen response element. We then used a panel of lymphoblastoid cell lines (LCLs) stably transfected with ERα with known genome-wide SNP genotypes to demonstrate that TSPYL5 expression increased after E2 exposure of cells heterozygous for variant TSPYL5 SNP genotypes, but not in those homozygous for wild-type alleles. TSPYL5 knockdown decreased, and overexpression increased aromatase (CYP19A1) expression in MCF-7 cells, LCLs, and adipocytes through the skin/adipose (I.4) promoter. Chromatin immunoprecipitation assay showed that TSPYL5 bound to the CYP19A1 I.4 promoter. A putative TSPYL5 binding motif was identified in 43 genes, and TSPYL5 appeared to function as a transcription factor for most of those genes. In summary, genome-wide significant SNPs in TSPYL5 were associated with elevated plasma E2 in postmenopausal breast cancer patients. SNP rs2583506 created a functional estrogen response element, and LCLs with variant SNP genotypes displayed increased E2-dependent TSPYL5 expression. TSPYL5 induced CYP19A1 expression and that of many other genes. These studies have revealed a novel mechanism for regulating aromatase expression and plasma E2 concentrations in postmenopausal women with ER(+) breast cancer. PMID:23518928

  13. Activation of protein synthesis in mouse uterine epithelial cells by estradiol-17β is mediated by a PKC–ERK1/2–mTOR signaling pathway

    PubMed Central

    Wang, Yuxiang; Zhu, Liyin; Kuokkanen, Satu; Pollard, Jeffrey W.

    2015-01-01

    The uterine epithelium of mice and humans undergoes cyclical waves of cell proliferation and differentiation under the regulation of estradiol-17β (E2) and progesterone (P4). These epithelial cells respond to E2 with increased protein and DNA synthesis, whereas P4 inhibits only the E2-induced DNA synthetic response. Here we show that E2 regulates protein synthesis in these epithelial cells through activating PKC that in turn stimulates ERK1/2 to phosphorylate and thereby activate the central regulator of protein synthesis mechanistic target of rapamycin (mTOR). This mTOR pathway is not inhibited by P4. Inhibitor studies with an estrogen receptor (ESR1) antagonist showed the dependence of this mTOR pathway on ESR1 but that once activated, a phosphorylation cascade independent of ESR1 propagates the pathway. E2 also stimulates an IGF1 receptor (IGF1R) to PI3 kinase to AKT to GSK-3β pathway required for activation of the canonical cell cycle machinery that is inhibited by P4. PKC activation did not stimulate this pathway nor does inhibition of PKC or ERK1/2 affect it. These studies therefore indicate a mechanism whereby DNA and protein synthesis are regulated by two ESR1-activated pathways that run in parallel with only the one responsible for the initiation of DNA synthesis blocked by P4. Inhibition of mTOR by rapamycin in vivo resulted in inhibition of E2-induced protein and DNA synthesis. Proliferative diseases of the endometrium such as endometriosis and cancer are common and E2 dependent. Thus, defining this mTOR pathway suggests that local (intrauterine or peritoneal) rapamycin administration might be a therapeutic option for these diseases. PMID:25733860

  14. Estradiol-dependent modulation of serotonergic markers in auditory areas of a seasonally breeding songbird.

    PubMed

    Matragrano, Lisa L; Sanford, Sara E; Salvante, Katrina G; Beaulieu, Michaël; Sockman, Keith W; Maney, Donna L

    2012-02-01

    Because no organism lives in an unchanging environment, sensory processes must remain plastic so that in any context, they emphasize the most relevant signals. As the behavioral relevance of sociosexual signals changes along with reproductive state, the perception of those signals is altered by reproductive hormones such as estradiol (E2). We showed previously that in white-throated sparrows, immediate early gene responses in the auditory pathway of females are selective for conspecific male song only when plasma E2 is elevated to breeding-typical levels. In this study, we looked for evidence that E2-dependent modulation of auditory responses is mediated by serotonergic systems. In female nonbreeding white-throated sparrows treated with E2, the density of fibers immunoreactive for serotonin transporter innervating the auditory midbrain and rostral auditory forebrain increased compared with controls. E2 treatment also increased the concentration of the serotonin metabolite 5-HIAA in the caudomedial mesopallium of the auditory forebrain. In a second experiment, females exposed to 30 min of conspecific male song had higher levels of 5-HIAA in the caudomedial nidopallium of the auditory forebrain than birds not exposed to song. Overall, we show that in this seasonal breeder, (a) serotonergic fibers innervate auditory areas; (b) the density of those fibers is higher in females with breeding-typical levels of E2 than in nonbreeding, untreated females; and (c) serotonin is released in the auditory forebrain within minutes in response to conspecific vocalizations. Our results are consistent with the hypothesis that E2 acts via serotonin systems to alter auditory processing.

  15. Mesenchymal Stromal Cells Induce Peculiar Alternatively Activated Macrophages Capable of Dampening Both Innate and Adaptive Immune Responses.

    PubMed

    Chiossone, Laura; Conte, Romana; Spaggiari, Grazia Maria; Serra, Martina; Romei, Cristina; Bellora, Francesca; Becchetti, Flavio; Andaloro, Antonio; Moretta, Lorenzo; Bottino, Cristina

    2016-07-01

    Mesenchymal stromal cells (MSCs) support hematopoiesis and exert immunoregulatory activities. Here, we analyzed the functional outcome of the interactions between MSCs and monocytes/macrophages. We showed that MSCs supported the survival of monocytes that underwent differentiation into macrophages, in the presence of macrophage colony-stimulating factor. However, MSCs skewed their polarization toward a peculiar M2-like functional phenotype (M(MSC) ), through a prostaglandin E2-dependent mechanism. M(MSC) were characterized by high expression of scavenger receptors, increased phagocytic capacity, and high production of interleukin (IL)-10 and transforming growth factor-β. These cytokines contributed to the immunoregulatory properties of M(MSC) , which differed from those of typical IL-4-induced macrophages (M2). In particular, interacting with activated natural killer (NK) cells, M(MSC) inhibited both the expression of activating molecules such as NKp44, CD69, and CD25 and the production of IFNγ, while M2 affected only IFNγ production. Moreover, M(MSC) inhibited the proliferation of CD8(+) T cells in response to allogeneic stimuli and induced the expansion of regulatory T cells (Tregs). Toll-like receptor engagement reverted the phenotypic and functional features of M(MSC) to those of M1 immunostimulatory/proinflammatory macrophages. Overall our data show that MSCs induce the generation of a novel type of alternatively activated macrophages capable of suppressing both innate and adaptive immune responses. These findings may help to better understand the role of MSCs in healthy tissues and inflammatory diseases including cancer, and provide clues for novel therapeutic approaches. Stem Cells 2016;34:1909-1921.

  16. Estradiol-dependent Catecholaminergic Innervation of Auditory Areas in a Seasonally Breeding Songbird

    PubMed Central

    Matragrano, Lisa L.; Sanford, Sara E.; Salvante, Katrina G.; Sockman, Keith W.; Maney, Donna L.

    2011-01-01

    A growing body of evidence suggests that gonadal steroids such as estradiol (E2) alter neural responses not only in brain regions associated with reproductive behavior, but also in sensory areas. Because catecholamine systems are involved in sensory processing and selective attention, and because they are sensitive to E2 in many species, they may mediate the neural effects of E2 in sensory areas. Here, we tested the effects of E2 on catecholaminergic innervation, synthesis, and activity in the auditory system of white-throated sparrows, a seasonally breeding songbird in which E2 promotes selective auditory responses to song. Non-breeding females with regressed ovaries were held on a winter-like photoperiod and implanted with silastic capsules containing either no hormone or E2. In one hemisphere of the brain, we used immunohistochemistry to quantify fibers immunoreactive for tyrosine hydroxylase or dopamine beta-hydroxylase in the auditory forebrain, thalamus, and midbrain. E2 treatment increased catecholaminergic innervation in the same areas of the auditory system in which E2 promotes selectivity for song. In the contralateral hemisphere, we quantified dopamine, norepinephrine and their metabolites in tissue punches using HPLC. Norepinephrine increased in the auditory forebrain, but not the midbrain, after E2 treatment. We found evidence of interhemispheric differences, both in immunoreactivity and catecholamine content did not depend on E2 treatment. Overall, our results show that increases in plasma E2 typical of the breeding season enhance catecholaminergic innervation and synthesis in some parts of the auditory system, raising the possibility that catecholamines play a role in E2-dependent auditory plasticity in songbirds. PMID:21714815

  17. Expression of alkyl hydroperoxide reductase is regulated negatively by OxyR1 and positively by RpoE2 sigma factor in Azospirillum brasilense Sp7.

    PubMed

    Singh, Sudhir; Dwivedi, Susheel Kumar; Singh, Vijay Shankar; Tripathi, Anil Kumar

    2016-10-01

    OxyR proteins are LysR-type transcriptional regulators, which play an important role in responding to oxidative stress in bacteria. Azospirillum brasilense Sp7 harbours two copies of OxyR. The inactivation of the oxyR1, the gene organized divergently to ahpC in A. brasilense Sp7, led to an increased tolerance to alkyl hydroperoxides, which was corroborated by an increase in alkyl hydroperoxide reductase (AhpC) activity, enhanced expression of ahpC :lacZ fusion and increased synthesis of AhpC protein in the oxyR1::km mutant. The upstream region of ahpC promoter harboured a putative OxyR binding site, T-N11-A. Mutation of T, A or both in the T-N11-Amotif caused derepression of ahpC in A. brasilense suggesting that T-N11-A might be the binding site for a negative regulator. Retardation of the electrophoretic mobility of the T-N11-A motif harbouring oxyR1-ahpC intergenic DNA by recombinant OxyR1, under reducing as well as oxidizing conditions, indicated that OxyR1 acts as a negative regulator of ahpC in A. brasilense. Sequence of the promoter of ahpC, predicted on the basis of transcriptional start site, and an enhanced expression of ahpC:lacZ fusion in chrR2::km mutant background suggested that ahpC promoter was RpoE2 dependent. Thus, this study shows that in A. brasilense Sp7, ahpC expression is regulated negatively by OxyR1 but is regulated positively by RpoE2, an oxidative-stress-responsive sigma factor. It also shows that OxyR1 regulates the expression RpoE1, which is known to play an important role during photooxidative stress in A. brasilense.

  18. In Vitro Biologic Activities of the Antimicrobials Triclocarban, Its Analogs, and Triclosan in Bioassay Screens: Receptor-Based Bioassay Screens

    PubMed Central

    Ahn, Ki Chang; Zhao, Bin; Chen, Jiangang; Cherednichenko, Gennady; Sanmarti, Enio; Denison, Michael S.; Lasley, Bill; Pessah, Isaac N.; Kültz, Dietmar; Chang, Daniel P.Y.; Gee, Shirley J.; Hammock, Bruce D.

    2008-01-01

    Background Concerns have been raised about the biological and toxicologic effects of the antimicrobials triclocarban (TCC) and triclosan (TCS) in personal care products. Few studies have evaluated their biological activities in mammalian cells to assess their potential for adverse effects. Objectives In this study, we assessed the activity of TCC, its analogs, and TCS in in vitro nuclear-receptor–responsive and calcium signaling bioassays. Materials and methods We determined the biological activities of the compounds in in vitro, cell-based, and nuclear-receptor–responsive bioassays for receptors for aryl hydrocarbon (AhR), estrogen (ER), androgen (AR), and ryanodine (RyR1). Results Some carbanilide compounds, including TCC (1–10 μM), enhanced estradiol (E2)-dependent or testosterone-dependent activation of ER- and AR-responsive gene expression up to 2.5-fold but exhibited little or no agonistic activity alone. Some carbanilides and TCS exhibited weak agonistic and/or antagonistic activity in the AhR-responsive bioassay. TCS exhibited antagonistic activity in both ER- and AR-responsive bioassays. TCS (0.1–10 μM) significantly enhanced the binding of [3H]ryanodine to RyR1 and caused elevation of resting cytosolic [Ca2+] in primary skeletal myotubes, but carbanilides had no effect. Conclusions Carbanilides, including TCC, enhanced hormone-dependent induction of ER- and AR-dependent gene expression but had little agonist activity, suggesting a new mechanism of action of endocrine-disrupting compounds. TCS, structurally similar to noncoplanar ortho-substituted poly-chlorinated biphenyls, exhibited weak AhR activity but interacted with RyR1 and stimulated Ca2+ mobilization. These observations have potential implications for human and animal health. Further investigations are needed into the biological and toxicologic effects of TCC, its analogs, and TCS. PMID:18795164

  19. Structural explanation of the rheology of a colloidal suspension under high dc electric fields

    NASA Astrophysics Data System (ADS)

    Espín, Manuel J.; Delgado, Ángel V.; González-Caballero, Fernando

    2006-04-01

    In this work we describe the electrorheology of suspensions consisting of hematite (α-Fe2O3) particles dispersed in silicone oil in the presence of large dc electric fields. If an electric field pulse is applied to the systems, it is possible to estimate the time that the electrorheological (ER) fluid takes to reach its final microstructure in the presence of the field. Our results indicate that response times of several seconds are typical, and that this time decreases with the field strength. Conventional shear-rate sweeps indicate the existence of a well-defined dynamic yield stress and a shear-thinning behavior. Interestingly, both the yield stress and the shear-thinning slope a [relating the viscosity, η , and the shear rate, γ˙ , as η=aγ˙-b+η(∞) ] show a linear dependence on the field strength, E , in disagreement with the E2 dependence often reported. This deviation is associated with changes in the conductivity of the dispersion medium with the field strength. A simple calculation of the interactions present in our ER fluid demonstrates that the ER behavior is entirely controlled by hydrodynamic (∝γ˙) and electrical forces (∝E) . This is confirmed by the collapse of all experimental results in a single master curve when the relative viscosity is plotted against the ratio γ˙/E . Careful attention has been paid in this work to the microstructure of the suspensions in the presence of both shear and electric fields simultaneously: the particles gather themselves on the walls of the electrorheological measurement cell, forming aggregates with cylindrical symmetry, shaped as rings or lamellas of solids. The electric field induced increase in viscosity is the consequence of the balance between two actions: that of the electric field, tending to keep particles together, and that of the shear field, forcing the flow of the liquid phase in the regions between rings or between rings and walls.

  20. Structural explanation of the rheology of a colloidal suspension under high dc electric fields.

    PubMed

    Espín, Manuel J; Delgado, Angel V; González-Caballero, Fernando

    2006-04-01

    In this work we describe the electrorheology of suspensions consisting of hematite (alpha-Fe2O3) particles dispersed in silicone oil in the presence of large dc electric fields. If an electric field pulse is applied to the systems, it is possible to estimate the time that the electrorheological (ER) fluid takes to reach its final microstructure in the presence of the field. Our results indicate that response times of several seconds are typical, and that this time decreases with the field strength. Conventional shear-rate sweeps indicate the existence of a well-defined dynamic yield stress and a shear-thinning behavior. Interestingly, both the yield stress and the shear-thinning slope alpha [relating the viscosity, eta, and the shear rate, .gamma, as eta=alphagamma(.-b) + eta (infinity)] show a linear dependence on the field strength, E, in disagreement with the E2 dependence often reported. This deviation is associated with changes in the conductivity of the dispersion medium with the field strength. A simple calculation of the interactions present in our ER fluid demonstrates that the ER behavior is entirely controlled by hydrodynamic (proportional to .gamma) and electrical forces (proportional to E). This is confirmed by the collapse of all experimental results in a single master curve when the relative viscosity is plotted against the ratio .gamma/E. Careful attention has been paid in this work to the microstructure of the suspensions in the presence of both shear and electric fields simultaneously: the particles gather themselves on the walls of the electrorheological measurement cell, forming aggregates with cylindrical symmetry, shaped as rings or lamellas of solids. The electric field induced increase in viscosity is the consequence of the balance between two actions: that of the electric field, tending to keep particles together, and that of the shear field, forcing the flow of the liquid phase in the regions between rings or between rings and walls.

  1. Mice Lacking the kf-1 Gene Exhibit Increased Anxiety- but not Despair-Like Behavior

    PubMed Central

    Tsujimura, Atsushi; Matsuki, Masato; Takao, Keizo; Yamanishi, Kiyofumi; Miyakawa, Tsuyoshi; Hashimoto-Gotoh, Tamotsu

    2008-01-01

    KF-1 was originally identified as a protein encoded by human gene with increased expression in the cerebral cortex of a patient with Alzheimer's disease. In mouse brain, kf-1 mRNA is detected predominantly in the hippocampus and cerebellum, and kf-1 gene expression is elevated also in the frontal cortex of rats after chronic antidepressant treatments. KF-1 mediates E2-dependent ubiquitination and may modulate cellular protein levels as an E3 ubiquitin ligase, though its target proteins are not yet identified. To elucidate the role of kf-1 in the central nervous system, we generated kf-1 knockout mice by gene targeting, using Cre-lox recombination. The resulting kf-1−/− mice were normal and healthy in appearance. Behavioral analyses revealed that kf-1−/− mice showed significantly increased anxiety-like behavior compared with kf-1+/+ littermates in the light/dark transition and elevated plus maze tests; however, no significant differences were observed in exploratory locomotion using the open field test or in behavioral despair using the forced swim and tail suspension tests. These observations suggest that KF-1 suppresses selectively anxiety under physiological conditions probably through modulating protein levels of its unknown target(s). Interestingly, kf-1−/− mice exhibited significantly increased prepulse inhibition, which is usually reduced in human schizophrenic patients. Thus, the kf-1−/− mice provide a novel animal model for elucidating molecular mechanisms of psychiatric diseases such as anxiety/depression, and may be useful for screening novel anxiolytic/antidepressant compounds. PMID:18958194

  2. Mycobacterium tuberculosis, but not vaccine BCG, specifically upregulates matrix metalloproteinase-1.

    PubMed

    Elkington, Paul T G; Nuttall, Robert K; Boyle, Joseph J; O'Kane, Cecilia M; Horncastle, Donna E; Edwards, Dylan R; Friedland, Jon S

    2005-12-15

    Pulmonary cavitation is fundamental to the global success of Mycobacterium tuberculosis. However, the mechanisms of this lung destruction are poorly understood. The biochemistry of lung matrix predicts matrix metalloproteinase (MMP) involvement in immunopathology. We investigated gene expression of all MMPs, proteins with a disintegrin and metalloproteinase domain, and tissue inhibitors of metalloproteinases in M. tuberculosis-infected human macrophages by real-time polymerase chain reaction. MMP secretion was measured by zymography and Western analysis, and expression in patients with pulmonary tuberculosis was localized by immunohistochemistry. MMP-1 and MMP-7 gene expression and secretion are potently upregulated by M. tuberculosis, and no increase in tissue inhibitor of metalloproteinase expression occurs to oppose their activity. Dexamethasone completely suppresses MMP-1 but not MMP-7 gene expression and secretion. In patients with active tuberculosis, macrophages express MMP-1 and MMP-7 adjacent to areas of tissue destruction. MMP-1 but not MMP-7 expression and secretion are relatively M. tuberculosis specific, are not upregulated by tuberculosis-associated cytokines, and are prostaglandin dependent. In contrast, the vaccine M. bovis bacillus Calmette-Guérin (BCG) does not stimulate MMP-1 secretion from human macrophages, although M. tuberculosis and BCG do upregulate MMP-7 equally. BCG-infected macrophages secrete reduced prostaglandin E2 concentrations compared with M. tuberculosis-infected macrophages, and prostaglandin pathway supplementation augments MMP-1 secretion from BCG-infected cells. M. tuberculosis specifically upregulates MMP-1 in a cellular model of human infection and in patients with tuberculosis. In contrast, vaccine BCG, which does not cause lung cavitation, does not upregulate prostaglandin E2-dependent MMP-1 secretion.

  3. Neuroglobin, a pro-survival player in estrogen receptor α-positive cancer cells

    PubMed Central

    Fiocchetti, M; Nuzzo, M T; Totta, P; Acconcia, F; Ascenzi, P; Marino, M

    2014-01-01

    Recently, we reported that human neuroglobin (NGB) is a new player in the signal transduction pathways that lead to 17β-estradiol (E2)-induced neuron survival. Indeed, E2 induces in neuron mitochondria the enhancement of NGB level, which in turn impairs the activation of a pro-apoptotic cascade. Nowadays, the existence of a similar pathway activated by E2 in non-neuronal cells is completely unknown. Here, the role of E2-induced NGB upregulation in tumor cells is reported. E2 induced the upregulation of NGB in a dose- and time-dependent manner in MCF-7, HepG2, SK-N-BE, and HeLa cells transfected with estrogen receptor α (ERα), whereas E2 was unable to modulate the NGB expression in the ERα-devoid HeLa cells. Both transcriptional and extranuclear ERα signals were required for the E2-dependent upregulation of NGB in MCF-7 and HepG2 cell lines. E2 stimulation modified NGB intracellular localization, inducing a significant reduction of NGB in the nucleus with a parallel increase of NGB in the mitochondria in both HepG2 and MCF-7 cells. Remarkably, E2 pretreatment did not counteract the H2O2-induced caspase-3 and poly (ADP-ribose) polymerase 1 (PARP-1) cleavage, as well as Bcl-2 overexpression in MCF-7 and HepG2 cells in which NGB was stably silenced by using shRNA lentiviral particles, highlighting the pivotal role of NGB in E2-induced antiapoptotic pathways in cancer cells. Present results indicate that the E2-induced NGB upregulation in cancer cells could represent a defense mechanism of E2-related cancers rendering them insensitive to oxidative stress. As a whole, these data open new avenues to develop therapeutic strategies against E2-related cancers. PMID:25299774

  4. The cooked meat derived genotoxic carcinogen 2-amino-3-methylimidazo[4,5-b]pyridine has potent hormone-like activity: mechanistic support for a role in breast cancer.

    PubMed

    Lauber, Sandra N; Gooderham, Nigel J

    2007-10-01

    The cooked meat-derived heterocyclic amine 2-amino-3-methylimidazo[4,5-b]pyridine (PhIP) is activated by CYP1A2 to the N-hydroxy metabolite, then esterified by acetyl transferase and sulfur transferase into unstable DNA-reactive products that can lead to mutation. The genotoxicity of PhIP has been implicated in its carcinogenicity. Yet, CYP1A2-null mice are still prone to PhIP-mediated cancer, inferring that alternative mechanisms must be operative in tumor induction. PhIP induces tumors of the breast, prostate, and colon in rats and lymphoma in mice. This profile of carcinogenicity is indicative of hormonal involvement. We recently reported that PhIP has potent estrogenic activity inducing transcription of estrogen (E2)-regulated genes, proliferation of E(2)-dependent cells, up-regulation of progesterone receptor, and stimulation of mitogen-activated protein kinase signaling. In this report, we show for the first time that PhIP at doses as low as of 10(-11) mol/L has direct effects on a rat pituitary lactotroph model (GH3 cells) and is able to induce cell proliferation and the synthesis and secretion of prolactin. This PhIP-induced pituitary cell proliferation and synthesis and secretion of prolactin can be attenuated by an estrogen receptor (ER) inhibitor, implying that PhIP effects on lactotroph responses are ERalpha mediated. In view of the strong association between estrogen, progesterone, prolactin, and breast cancer, the PhIP repertoire of hormone-like activities provides further mechanistic support for the tissue-specific carcinogenicity of the chemical. Furthermore, the recent epidemiology studies that report an association between consumption of cooked red meat and premenopausal and postmenopausal human breast cancer are consonant with these observations.

  5. Selective disruption of ER{alpha} DNA-binding activity alters uterine responsiveness to estradiol.

    PubMed

    Hewitt, Sylvia C; O'Brien, Jeanne E; Jameson, J Larry; Kissling, Grace E; Korach, Kenneth S

    2009-12-01

    In vitro models have been used to demonstrate that estrogen receptors (ERs) can regulate estrogen-responsive genes either by directly interacting with estrogen-responsive element (ERE) DNA motifs or by interacting with other transcription factors such as AP1. In this study, we evaluated estrogen (E(2))-dependent uterine gene profiles by microarray in the KIKO mouse, an in vivo knock-in mouse model that lacks the DNA-binding function of ERalpha and is consequently restricted to non-ERE-mediated responses. The 2- or 24-h E(2)-mediated uterine gene responses were distinct in wild-type (WT), KIKO, and alphaERKO genotypes, indicating that unique sets of genes are regulated by ERE and non-ERE pathways. After 2 h E(2) treatment, 38% of the WT transcripts were also regulated in the KIKO, demonstrating that the tethered mechanism does operate in this in vivo model. Surprisingly, 1438 E(2)-regulated transcripts were unique in the KIKO mouse and were not seen in either WT or alphaERKO. Pathway analyses revealed that some canonical pathways, such as the Jak/Stat pathway, were affected in a similar manner by E(2) in WT and KIKO. In other cases, however, the WT and KIKO differed. One example is the Wnt/beta-catenin pathway; this pathway was impacted, but different members of the pathway were regulated by E(2) or were regulated in a different manner, consistent with differences in biological responses. In summary, this study provides a comprehensive analysis of uterine genes regulated by E(2) via ERE and non-ERE pathways.

  6. Human Papillomavirus Type 18 cis-Elements Crucial for Segregation and Latency.

    PubMed

    Ustav, Mart; Castaneda, Fernando Rodriguez; Reinson, Tormi; Männik, Andres; Ustav, Mart

    2015-01-01

    Stable maintenance replication is characteristic of the latency phase of HPV infection, during which the viral genomes are actively maintained as extrachromosomal genetic elements in infected proliferating basal keratinocytes. Active replication in the S-phase and segregation of the genome into daughter cells in mitosis are required for stable maintenance replication. Most of our knowledge about papillomavirus genome segregation has come from studies of bovine papillomavirus type 1 (BPV-1), which have demonstrated that the E2 protein cooperates with cellular trans-factors and that E2 binding sites act as cis-regulatory elements in the viral genome that are essential for the segregation process. However, the genomic organization of the regulatory region in HPVs, and the properties of the viral proteins are different from those of their BPV-1 counterparts. We have designed a segregation assay for HPV-18 and used it to demonstrate that the E2 protein performs segregation in combination with at least two E2 binding sites. The cooperative binding of the E2 protein to two E2 binding sites is a major determinant of HPV-18 genome segregation, as demonstrated by the change in spacing between adjacent binding sites #1 and #2 in the HPV-18 Upstream Regulatory Region (URR). Duplication or triplication of the natural 4 bp 5'-CGGG-3' spacer between the E2 binding sites increased the cooperative binding of the E2 molecules as well as E2-dependent segregation. Removal of any spacing between these sites eliminated cooperative binding of the E2 protein and disabled segregation of the URR and HPV-18 genome. Transfer of these configurations of the E2 binding sites into viral genomes confirmed the role of the E2 protein and binding sites #1 and #2 in the segregation process. Additional analysis demonstrated that these sites also play an important role in the transcriptional regulation of viral gene expression from different HPV-18 promoters.

  7. Meclofenamate elicits a nephropreventing effect in a rat model of ischemic acute kidney injury by suppressing indoxyl sulfate production and restoring renal organic anion transporters

    PubMed Central

    Saigo, Chika; Nomura, Yui; Yamamoto, Yuko; Sagata, Masataka; Matsunaga, Rika; Jono, Hirofumi; Nishi, Kazuhiko; Saito, Hideyuki

    2014-01-01

    Indoxyl sulfate (IS), a putative low-molecular weight uremic toxin, is excreted in the urine under normal kidney function, but is retained in the circulation and tissues during renal dysfunction in acute kidney injury and chronic kidney disease. IS, which is one of the most potent inducers of oxidative stress in the kidney and cardiovascular system, is enzymatically produced in the liver from indole by cytochrome P450-mediated hydroxylation to indoxyl, followed by sulfotransferase-mediated sulfate conjugation. We used rat liver S9 fraction to identify inhibitors of IS production. After testing several compounds, including phytochemical polyphenols, we identified meclofenamate as a potent inhibitor of IS production with an apparent IC50 value of 1.34 μM. Ischemia/reperfusion (I/R) of rat kidney caused a marked elevation in the serum IS concentration 48 hours after surgery. However, intravenous administration of meclofenamate (10 mg/kg) significantly suppressed this increase in the serum level of IS. Moreover, IS concentrations in both kidney and liver were dramatically elevated by renal I/R treatment, but this increase was blocked by meclofenamate. Serum creatinine and blood urea nitrogen were markedly elevated in rats after renal I/R treatment, but these increases were significantly restored by administration of meclofenamate. Renal expression of both basolateral membrane-localized organic anion transporters rOAT1 and rOAT3 was downregulated by I/R treatment. However, expression of rOAT1 and rOAT3 recovered after administration of meclofenamate, which is associated with the inhibition of I/R-evoked elevation of prostaglandin E2. Our results suggest that meclofenamate inhibits hepatic sulfotransferase-mediated production of IS, thereby suppressing serum and renal accumulation of IS. Meclofenamate also prevents the prostaglandin E2-dependent downregulation of rOAT1 and rOAT3 expression. In conclusion, meclofenamate was found to elicit a nephropreventive effect in

  8. Antioxidant butylated hydroxyanisole inhibits estrogen-induced breast carcinogenesis in female ACI rats.

    PubMed

    Singh, Bhupendra; Mense, Sarah M; Remotti, Fabrizio; Liu, Xinhua; Bhat, Hari K

    2009-01-01

    Exposure to estrogens is suggested to be a risk factor in human breast cancer development. The mechanisms underlying estrogen-induced cancer have not been fully elucidated. Both estrogen receptor (ER)-mediated proliferative processes and ER-independent generation of oxidative stress are suggested to play important roles in estrogen-induced breast carcinogenesis. In the current study, we investigated the role of oxidative stress in breast carcinogenesis using the ACI rat model of mammary tumorigenesis. Female ACI rats were treated with 17beta-estradiol (E(2)), butylated hydroxyanisole (BHA), or a combination of E(2) + BHA for up to 240 days. Cotreatment of rats with E(2) + BHA reduced estrogen-induced breast tumor development with tumor incidence of 24%, a significant decrease relative to E(2) where tumor incidence was 82%. Proliferative changes in the breast tissue of E(2) + BHA-treated animals were similar to those observed in E(2)-treated animals. Tissue levels of 8-isoprostane, a marker of oxidant stress, as well as the activities of antioxidant enzymes including glutathione peroxidase, superoxide dismutase, and catalase were quantified in the breast tissues of rats treated with E(2) + BHA and compared to activity levels found in E(2)-treated animals and respective age-matched controls. Cotreatment with BHA inhibited E(2)-mediated increases in 8-isoprostane levels as well as activities of antioxidant enzymes. In summary, these data suggest that estrogen-mediated oxidant stress plays a critical role in the development of estrogen-dependent breast cancers and BHA inhibits E(2)-dependent breast carcinogenesis by decreasing oxidant stress.

  9. Modulation of 17β-Estradiol Signaling on Cellular Proliferation by Caveolin-2.

    PubMed

    Totta, Pierangela; Gionfra, Fabio; Busonero, Claudia; Acconcia, Filippo

    2016-06-01

    The sex hormone 17β-estradiol (E2) exerts pleiotropic effects by binding to the ligand-activated transcription factor estrogen receptor α (ERα). The E2:ERα complex regulates several physiological processes, including cell survival and proliferation, through transcriptional effects (i.e., estrogen responsive element [ERE]-based gene transcription) and non-transcriptional membrane-initiated effects (i.e., the activation of extra-nuclear signaling cascades), which derive from the activation of the pool of ERα that is localized to plasma membrane caveolae. Caveolae are ω-shaped membrane sub-domains that are composed of scaffold proteins named caveolins (i.e., caveolin-1, caveolin-2, and caveolin-3). Although caveolin-3 is exclusively expressed in muscles, caveolin-1 and caveolin-2 are co-expressed in all human tissues. From a functional point of view, caveolin-2 can operate both dependently on and independently of caveolin-1, which is the main coat component of caveolae. Interestingly, while a functional interplay between caveolin-1 and ERα has been reported in the control of E2-induced physiological effects, the role of caveolin-2 in E2:ERα signaling within the cell remains poorly understood. This study shows that siRNA-mediated caveolin-2 depletion in breast ductal carcinoma cells (MCF-7) reduces E2-induced ERα phosphorylation at serine residue 118 (S118), controls intracellular receptor levels, precludes ERα-mediated extra-nuclear activation of signaling pathways, reduces ERα transcriptional activity, and prevents cellular proliferation. Meanwhile, the impact of caveolin-1 depletion on ERα signaling in MCF-7 cells is shown to be similar to that elicited by siRNA-mediated caveolin-2 depletion. Altogether, these data demonstrate that caveolin-2 expression is necessary for the control of E2-dependent cellular proliferation.

  10. Naringenin and 17beta-estradiol coadministration prevents hormone-induced human cancer cell growth.

    PubMed

    Bulzomi, Pamela; Bolli, Alessandro; Galluzzo, Paola; Leone, Stefano; Acconcia, Filippo; Marino, Maria

    2010-01-01

    Flavonoids have been described as health-promoting, disease-preventing dietary components. In vivo and in vitro experiments also support a protective effect of flavonoids to reduce the incidence of certain hormone-responsive cancers. In particular, our previous results indicate that the flavanone naringenin (Nar), decoupling estrogen receptor alpha (ERalpha) action mechanisms, drives cancer cells to apoptosis. Because these studies were conducted in the absence of the endogenous hormone 17beta-estradiol (E2), the physiological relevance of these findings is not clear. We investigate whether the antiproliferative Nar effect persists in the presence of physiological E2 concentration (i.e. 10 nM), using both ERalpha-transfected (HeLa cells) and ERalpha-containing (HepG2 cells) cancer cell lines. Ligand saturation experiments indicate that Nar decreases the binding of E2 to ERalpha without impairing the estrogen response element (ERE)-driven reporter plasmid activity. In contrast, Nar stimulation prevents E2-induced extracellular regulated kinases (ERK1/2) and AKT activation and still induces the activation of p38, the proapoptotic member of mitogen-activating protein kinase (MAPK) family. As a consequence, Nar stimulation impedes the E2-induced transcription of cyclin D1 promoter and reverts the E2-induced cell proliferation, driving cancer cell to apoptosis. Thus, these results suggest that coexposure to this low-affinity, low-potency ligand for ERalpha specifically antagonizes the E2-induced ERalpha-dependent rapid signals by reducing the effect of the endogenous hormone in promoting cellular proliferation. As a whole, these data indicate that Nar is an excellent candidate as a chemopreventive agent in E2-dependent cancers.

  11. NMDA receptor blockade reduces TMJ-evoked activity of trigeminal subnucleus caudalis neurons in an estrogen-dependent manner

    PubMed Central

    Tashiro, A.; Okamoto, K.; Bereiter, D.A.

    2009-01-01

    Estrogen status is a risk factor in painful temporomandibular disorders (TMJD). Previously we reported that estradiol (E2) enhanced nociceptive processing of TMJ input by neurons in superficial laminae at the spinomedullary (Vc/C1-2) region; however, the mechanisms for this enhancement are not known. The present study determined if ionotropic glutamate receptors contribute to TMJ nociceptive processing in an E2-dependent manner. Ovariectomized (OvX) female rats were treated with high E2 (HE2) or low dose E2 (LE2) for 2 days and neural activity was recorded in laminae I-II at the Vc/C1-2 region. TMJ-responsive units were activated by adenosine triphosphate (ATP) injections into the joint space. ATP-evoked unit responses in HE2 rats were reduced significantly by topical application of the N-methyl-D-aspartate receptor antagonist, D(−) -2-amino-5-phosphonopentanoic acid (AP5) in a dose-related manner, while units from LE2 were not affected. Application of the non-NMDA receptor antagonist, 6,7-dinitroquinoxaline-2,3-dione (DNQX), inhibited the ATP-evoked responses in both groups. Spontaneous activity of TMJ units was not influenced by AP5, whereas it was reduced by DNQX similarly in both groups. The high threshold convergent cutaneous receptive field area of TMJ units was not changed by AP5, whereas DNQX caused a significant reduction in both groups. These results suggest that NMDA-dependent mechanisms contribute to the enhanced ATP-evoked responses of TMJ units in superficial laminae at the Vc/C1-2 region under high E2 conditions, while non-NMDA-dependent mechanisms modify the encoding properties of TMJ units independent of E2 status. PMID:19799971

  12. NMDA receptor blockade reduces temporomandibular joint-evoked activity of trigeminal subnucleus caudalis neurons in an estrogen-dependent manner.

    PubMed

    Tashiro, A; Okamoto, K; Bereiter, D A

    2009-12-29

    Estrogen status is a risk factor in painful temporomandibular disorders (TMJD). Previously we reported that estradiol (E2) enhanced nociceptive processing of TMJ input by neurons in superficial laminae at the spinomedullary (Vc/C(1-2)) region; however, the mechanisms for this enhancement are not known. The present study determined if ionotropic glutamate receptors contribute to TMJ nociceptive processing in an E2-dependent manner. Ovariectomized (OvX) female rats were treated with high E2 (HE2) or low dose E2 (LE2) for 2 days and neural activity was recorded in laminae I-II at the Vc/C(1-2) region. TMJ-responsive units were activated by ATP injections into the joint space. ATP-evoked unit responses in HE2 rats were reduced significantly by topical application of the N-methyl-D-aspartate receptor antagonist, D(-)-2-amino-5-phosphonopentanoic acid (AP5) in a dose-related manner, while units from LE2 were not affected. Application of the non-NMDA receptor antagonist, 6,7-dinitroquinoxaline-2,3-dione (DNQX), inhibited the ATP-evoked responses in both groups. Spontaneous activity of TMJ units was not influenced by AP5, whereas it was reduced by DNQX similarly in both groups. The high threshold convergent cutaneous receptive field area of TMJ units was not changed by AP5, whereas DNQX caused a significant reduction in both groups. These results suggest that NMDA-dependent mechanisms contribute to the enhanced ATP-evoked responses of TMJ units in superficial laminae at the Vc/C(1-2) region under high E2 conditions, while non-NMDA-dependent mechanisms modify the encoding properties of TMJ units independent of E2 status.

  13. Progesterone Antagonism of Neurite Outgrowth Depends on Microglial Activation via Pgrmc1/S2R

    PubMed Central

    Bali, N; Arimoto, J. M.; Morgan, T. E.

    2013-01-01

    Neuronal plasticity is regulated by the ovarian steroids estradiol (E2) and progesterone (P4) in many normal brain functions, as well as in acute response to injury and chronic neurodegenerative disease. In a female rat model of axotomy, the E2-dependent compensatory neuronal sprouting is antagonized by P4. To resolve complex glial-neuronal cell interactions, we used the “wounding-in-a-dish” model of neurons cocultured with astrocytes or mixed glia (microglia to astrocytes, 1:3). Although both astrocytes and mixed glia supported E2-enhanced neurite outgrowth, P4 antagonized E2-induced neurite outgrowth only with mixed glia, but not astrocytes alone. We now show that P4-E2 antagonism of neurite outgrowth is mediated by microglial expression of progesterone receptor (Pgr) membrane component 1 (Pgrmc1)/S2R, a putative nonclassical Pgr mediator with multiple functions. The P4-E2 antagonism of neurite outgrowth was restored by add-back of microglia to astrocyte-neuron cocultures. Because microglia do not express the classical Pgr, we examined the role of Pgrmc1, which is expressed in microglia in vitro and in vivo. Knockdown by siRNA-Pgrmc1 in microglia before add-back to astrocyte-neuron cocultures suppressed the P4-E2 antagonism of neurite outgrowth. Conditioned media from microglia restored the P4-E2 activity, but only if microglia were activated by lipopolysaccharide or by wounding. Moreover, the microglial activation was blocked by Pgmrc1-siRNA knockdown. These findings explain why nonwounded cultures without microglial activation lack P4 antagonism of E2-induced neurite outgrowth. We suggest that microglial activation may influence brain responses to exogenous P4, which is a prospective therapy in traumatic brain injury. PMID:23653459

  14. 17β-Estradiol suppresses visceral adipogenesis and activates brown adipose tissue-specific gene expression.

    PubMed

    Al-Qahtani, Saad Misfer; Bryzgalova, Galyna; Valladolid-Acebes, Ismael; Korach-André, Marion; Dahlman-Wright, Karin; Efendić, Suad; Berggren, Per-Olof; Portwood, Neil

    2017-01-01

    Both functional ovaries and estrogen replacement therapy (ERT) reduce the risk of type 2 diabetes (T2D). Understanding the mechanisms underlying the antidiabetic effects of 17β-estradiol (E2) may permit the development of a molecular targeting strategy for the treatment of metabolic disease. This study examines how the promotion of insulin sensitivity and weight loss by E2 treatment in high-fat-diet (HFD)-fed mice involve several anti-adipogenic processes in the visceral adipose tissue. Magnetic resonance imaging (MRI) revealed specific reductions in visceral adipose tissue volume in HFD+E2 mice, compared with HFD mice. This loss of adiposity was associated with diminished visceral adipocyte size and reductions in expression of lipogenic genes, adipokines and of the nuclear receptor nr2c2/tr4. Meanwhile, expression levels of adipose triglyceride lipase/pnpla2 and leptin receptor were increased. As mRNA levels of stat3, a transcription factor involved in brown adipose tissue differentiation, were also increased in visceral adipose, the expression of other brown adipose-specific markers was assessed. Both expression and immunohistochemical staining of ucp-1 were increased, and mRNA levels of dio-2, and of adrβ3, a regulator of ucp-1 expression during the thermogenic response, were increased. Furthermore, expression of cpt-1b, a brown adipose-specific gene involved in fatty acid utilization, was also increased. Methylation studies demonstrated that the methylation status of both dio-2 and adrβ3 was significantly reduced. These results show that improved glycemic control and weight loss due to E2 involve anti-adipogenic mechanisms which include suppressed lipogenesis and augmented fatty acid utilization, and in addition, the activation of brown adipose tissue-specific gene expression in association with E2-dependent epigenetic modifications in these genes.

  15. Long-term estrogen exposure promotes carcinogen bioactivation, induces persistent changes in gene expression, and enhances the tumorigenicity of MCF-7 human breast cancer cells

    PubMed Central

    Spink, Barbara C.; Bennett, James A.; Pentecost, Brian T.; Lostritto, Nicole; Englert, Neal A.; Benn, Geoffrey K.; Goodenough, Angela K.; Turesky, Robert J.; Spink, David C.

    2009-01-01

    The cumulative exposure to estrogens is an important determinant in the risk of breast cancer, yet the full range of mechanisms involving estrogens in the genesis and progression of breast cancer remains a subject of debate. Interactions of estrogens and environmental toxicants have received attention as putative factors contributing to carcinogenesis. Mechanistic studies have demonstrated interactions between estrogen receptor α (ERα) and the aryl hydrocarbon receptor (AhR), with consequences on the genes that they regulate. Many studies of ERα and AhR-mediated effects and crosstalk between them have focused on the initial molecular events. In this study, we investigated ERα- and AhR-mediated effects in long-term estrogen exposed (LTEE) MCF-7 human breast cancer cells, which were obtained by continuous culturing for at least 12 weeks in medium supplemented with 1 nM of 17β-estradiol (E2). With these LTEE cells and with parallel control cells cultured without E2 supplementation, we performed an extensive study of cytochrome P450 (CYP) induction, carcinogen bioactivation, global gene expression, and tumorigenicity in immunocompromised mice. We found that LTEE cells, in comparison with control cells, had higher levels of AhR mRNA and protein, greater responsiveness for AhR-regulated CYP1A1 and CYP1B1 induction, a 6-fold higher initial level of benzo(a)pyrene-DNA adducts as determined by liquid chromatography tandem mass spectrometry, marked differences in the expression of numerous genes, and a higher rate of E2-dependent tumor growth as xenografts. These studies indicate that LTEE causes adaptive responses in MCF-7 cells, which may reflect processes that contribute to the overall carcinogenic effect of E2. PMID:19619570

  16. Genome-wide profiling of Hfq-binding RNAs uncovers extensive post-transcriptional rewiring of major stress response and symbiotic regulons in Sinorhizobium meliloti

    PubMed Central

    Torres-Quesada, Omar; Reinkensmeier, Jan; Schlüter, Jan-Philip; Robledo, Marta; Peregrina, Alexandra; Giegerich, Robert; Toro, Nicolás; Becker, Anke; Jiménez-Zurdo, Jose I

    2014-01-01

    The RNA chaperone Hfq is a global post-transcriptional regulator in bacteria. Here, we used RNAseq to analyze RNA populations from the legume symbiont Sinorhizobium meliloti that were co-immunoprecipitated (CoIP-RNA) with a FLAG-tagged Hfq in five growth/stress conditions. Hfq-bound transcripts (1315) were largely identified in stressed bacteria and derived from small RNAs (sRNAs), both trans-encoded (6.4%) and antisense (asRNAs; 6.3%), and mRNAs (86%). Pull-down with Hfq recovered a small proportion of annotated S. meliloti sRNAs (14% of trans-sRNAs and 2% of asRNAs) suggesting a discrete impact of this protein in sRNA pathways. Nonetheless, Hfq selectively stabilized CoIP-enriched sRNAs, anticipating that these interactions are functionally significant. Transcription of 26 Hfq-bound sRNAs was predicted to occur from promoters recognized by the major stress σ factors σE2 or σH1/2. Recovery rates of sRNAs in each of the CoIP–RNA libraries suggest a large impact of Hfq-assisted riboregulation in S. meliloti osmoadaptation. Hfq directly targeted 18% of the predicted S. meliloti mRNAs, which encode functionally diverse proteins involved in transport and metabolism, σE2-dependent stress responses, quorum sensing, flagella biosynthesis, ribosome, and membrane assembly or symbiotic nitrogen fixation. Canonical targeting of the 5′ regions of two of the ABC transporter mRNAs by the homologous Hfq-binding AbcR1 and AbcR2 sRNAs leading to inhibition of protein synthesis was confirmed in vivo. We therefore provide a comprehensive resource for the systems-level deciphering of hitherto unexplored S. meliloti stress and symbiotic post-transcriptional regulons and the identification of Hfq-dependent sRNA–mRNA regulatory pairs. PMID:24786641

  17. IGF-I/EGF and E2 signaling crosstalk through IGF-IR conduit point affects breast cancer cell adhesion.

    PubMed

    Voudouri, Kallirroi; Nikitovic, Dragana; Berdiaki, Aikaterini; Kletsas, Dimitris; Karamanos, Nikos K; Tzanakakis, George N

    2016-12-01

    Epidermal growth factor (EGF)/insulin like growth factor-I (IGF-I) and Estradiol (E2) can regulate biological functions of hormone-dependent tumor cells. Fibronectin (FN) is a large glycoprotein abundantly expressed in breast cancer extracellular matrices (ECMs) postulated to be a marker of aggressiveness during cancer pathogenesis. In this study we demonstrate that IGF-I/EGF as well E2 strongly increase the adhesion of the MCF-7 breast cancer cells onto FN. Moreover, IGF-IR is necessary for the IGF-I-/EGF- and E2-induced cell adhesion. Erk1/2 inhibition abolished the IGF-I-/EGF-/E2-induced MCF-7 cell adhesion, suggesting that this regulation of cell adhesion is perpetrated through Erk1/2 downstream signaling. Erk1/2 signaling was shown to modulate IGF-IR status as its' inhibition attenuates both IGF-IR expression and activation. Notably, EGF and E2 enhanced the mRNA as well as protein expression of IGF-IR in MCF-7 cells. Confocal microscopy demonstrated that treatment of MCF-7 cells with IGF-I or EGF induced actin reorganization, which was attenuated with Erk1/2 inhibition. Interestingly, IGF-I treatment induced a co-localization of IGF-IR and FAK, which was evident mostly at the cell membranes of MCF-7 cells. In summary, IGF-IR was shown to be a convergence point for the IGF-/EGF- and E2-dependent MCF-7 cell adhesion onto FN.

  18. The Deacetylase SIRT1 Regulates the Replication Properties of Human Papillomavirus 16 E1 and E2.

    PubMed

    Das, Dipon; Smith, Nathan; Wang, Xu; Morgan, Iain M

    2017-05-15

    Human papillomaviruses (HPV) replicate their genomes in differentiating epithelium using the viral proteins E1 and E2 in association with host proteins. While the roles of E1 and E2 in this process are understood, the host factors involved and how they interact with and regulate E1-E2 are not. Our previous work identified the host replication and repair factor TopBP1 as an E2 partner protein essential for optimal E1-E2 replication and for the viral life cycle. The role of TopBP1 in host DNA replication is regulated by the class III deacetylase SIRT1; activation of the DNA damage response prevents SIRT1 deacetylation of TopBP1, resulting in a switch from DNA replication to repair functions for this protein and cell cycle arrest. Others have demonstrated an essential role for SIRT1 in regulation of the HPV31 life cycle; here, we report that SIRT1 can directly regulate HPV16 E1-E2-mediated DNA replication. SIRT1 is part of the E1-E2 DNA replication complex and is recruited to the viral origin of replication in an E1-E2-dependent manner. CRISPR/Cas9 was used to generate C33a clones with undetectable SIRT1 expression and lack of SIRT1 elevated E1-E2 DNA replication, in part due to increased acetylation and stabilization of the E2 protein in the absence of SIRT1. The results demonstrate that SIRT1 is a member of, and can regulate, the HPV16 replication complex. We discuss the potential role of this protein in the viral life cycle.IMPORTANCE HPV are causative agents in a number of human diseases, and currently only the symptoms of these diseases are treated. To identify novel therapeutic approaches for combating these diseases, the viral life cycle must be understood in more detail. This report demonstrates that a cellular enzyme, SIRT1, is part of the HPV16 DNA replication complex and is brought to the viral genome by the viral proteins E1 and E2. Using gene editing technology (CRISPR/Cas9), the SIRT1 gene was removed from cervical cancer cells. The consequence of this

  19. Human Cells Cultured under Physiological Oxygen Utilize Two Cap-binding Proteins to recruit Distinct mRNAs for Translation*

    PubMed Central

    Timpano, Sara; Uniacke, James

    2016-01-01

    Translation initiation is a focal point of translational control and requires the binding of eIF4E to the 5′ cap of mRNA. Under conditions of extreme oxygen depletion (hypoxia), human cells repress eIF4E and switch to an alternative cap-dependent translation mediated by a homolog of eIF4E, eIF4E2. This homolog forms a complex with the oxygen-regulated hypoxia-inducible factor 2α and can escape translation repression. This complex mediates cap-dependent translation under cell culture conditions of 1% oxygen (to mimic tumor microenvironments), whereas eIF4E mediates cap-dependent translation at 21% oxygen (ambient air). However, emerging evidence suggests that culturing cells in ambient air, or “normoxia,” is far from physiological or “normal.” In fact, oxygen in human tissues ranges from 1–11% or “physioxia.” Here we show that two distinct modes of cap-dependent translation initiation are active during physioxia and act on separate pools of mRNAs. The oxygen-dependent activities of eIF4E and eIF4E2 are elucidated by observing their polysome association and the status of mammalian target of rapamycin complex 1 (eIF4E-dependent) or hypoxia-inducible factor 2α expression (eIF4E2-dependent). We have identified oxygen conditions where eIF4E is the dominant cap-binding protein (21% normoxia or standard cell culture conditions), where eIF4E2 is the dominant cap-binding protein (1% hypoxia or ischemic diseases and cancerous tumors), and where both cap-binding proteins act simultaneously to initiate the translation of distinct mRNAs (1–11% physioxia or during development and stem cell differentiation). These data suggest that the physioxic proteome is generated by initiating translation of mRNAs via two distinct but complementary cap-binding proteins. PMID:27002144

  20. Molecular mechanisms that drive estradiol-dependent burst firing of Kiss1 neurons in the rostral periventricular preoptic area

    PubMed Central

    Zhang, Chunguang; Tonsfeldt, Karen J.; Qiu, Jian; Bosch, Martha A.; Kobayashi, Kazuto; Steiner, Robert A.; Kelly, Martin J.

    2013-01-01

    Kisspeptin (Kiss1) neurons in the rostral periventricular area of the third ventricle (RP3V) provide excitatory drive to gonadotropin-releasing hormone (GnRH) neurons to control fertility. Using whole cell patch clamp recording and single-cell (sc)RT-PCR techniques targeting Kiss1-CreGFP or tyrosine hydroxylase (TH)-EGFP neurons, we characterized the biophysical properties of these neurons and identified the critical intrinsic properties required for burst firing in 17β-estradiol (E2)-treated, ovariectomized female mice. One-fourth of the RP3V Kiss1 neurons exhibited spontaneous burst firing. RP3V Kiss1 neurons expressed a hyperpolarization-activated h-current (Ih) and a T-type calcium current (IT), which supported hyperpolarization-induced rebound burst firing. Under voltage clamp conditions, all Kiss1 neurons expressed a kinetically fast Ih that was augmented 3.4-fold by high (LH surge-producing)-E2 treatment. scPCR analysis of Kiss1 neurons revealed abundant expression of the HCN1 channel transcripts. Kiss1 neurons also expressed a Ni2+- and TTA-P2-sensitive IT that was augmented sixfold with high-E2 treatment. CaV3.1 mRNA was also highly expressed in these cells. Current clamp analysis revealed that rebound burst firing was induced in RP3V Kiss1 neurons in high-E2-treated animals, and the majority of Kiss1 neurons had a hyperpolarization threshold of −84.7 mV, which corresponded to the V½ for IT de-inactivation. Finally, Kiss1 neurons in the RP3V were hyperpolarized by μ- and κ-opioid and GABAB receptor agonists, suggesting that these pathways also contribute to rebound burst firing. Therefore, Kiss1 neurons in the RP3V express the critical channels and receptors that permit E2-dependent rebound burst firing and provide the biophysical substrate that drives the preovulatory surge of GnRH. PMID:24105416

  1. Human Cells Cultured under Physiological Oxygen Utilize Two Cap-binding Proteins to recruit Distinct mRNAs for Translation.

    PubMed

    Timpano, Sara; Uniacke, James

    2016-05-13

    Translation initiation is a focal point of translational control and requires the binding of eIF4E to the 5' cap of mRNA. Under conditions of extreme oxygen depletion (hypoxia), human cells repress eIF4E and switch to an alternative cap-dependent translation mediated by a homolog of eIF4E, eIF4E2. This homolog forms a complex with the oxygen-regulated hypoxia-inducible factor 2α and can escape translation repression. This complex mediates cap-dependent translation under cell culture conditions of 1% oxygen (to mimic tumor microenvironments), whereas eIF4E mediates cap-dependent translation at 21% oxygen (ambient air). However, emerging evidence suggests that culturing cells in ambient air, or "normoxia," is far from physiological or "normal." In fact, oxygen in human tissues ranges from 1-11% or "physioxia." Here we show that two distinct modes of cap-dependent translation initiation are active during physioxia and act on separate pools of mRNAs. The oxygen-dependent activities of eIF4E and eIF4E2 are elucidated by observing their polysome association and the status of mammalian target of rapamycin complex 1 (eIF4E-dependent) or hypoxia-inducible factor 2α expression (eIF4E2-dependent). We have identified oxygen conditions where eIF4E is the dominant cap-binding protein (21% normoxia or standard cell culture conditions), where eIF4E2 is the dominant cap-binding protein (1% hypoxia or ischemic diseases and cancerous tumors), and where both cap-binding proteins act simultaneously to initiate the translation of distinct mRNAs (1-11% physioxia or during development and stem cell differentiation). These data suggest that the physioxic proteome is generated by initiating translation of mRNAs via two distinct but complementary cap-binding proteins. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  2. Vitamin D and estrogen synergy in Vdr-expressing CD4(+) T cells is essential to induce Helios(+)FoxP3(+) T cells and prevent autoimmune demyelinating disease.

    PubMed

    Spanier, Justin A; Nashold, Faye E; Mayne, Christopher G; Nelson, Corwin D; Hayes, Colleen E

    2015-09-15

    Multiple sclerosis (MS) is a neurodegenerative disease resulting from an autoimmune attack on the axon-myelin unit. A female MS bias becomes evident after puberty and female incidence has tripled in the last half-century, implicating a female sex hormone interacting with a modifiable environmental factor. These aspects of MS suggest that many female MS cases may be preventable. Mechanistic knowledge of this hormone-environment interaction is needed to devise strategies to reduce female MS risk. We previously demonstrated that vitamin D3 (D3) deficiency increases and D3 supplementation decreases experimental autoimmune encephalomyelitis (EAE) risk in a female-biased manner. We also showed that D3 acts in an estrogen (E2)-dependent manner, since ovariectomy eliminated and E2 restored D3-mediated EAE protection. Here we probed the hypothesis that E2 and D3 interact synergistically within CD4(+) T cells to control T cell fate and prevent demyelinating disease. The E2 increased EAE resistance in wild-type (WT) but not T-Vdr(0) mice lacking Vdr gene function in CD4(+) T cells, so E2 action depended entirely on Vdr(+)CD4(+) T cells. The E2 levels were higher in WT than T-Vdr(0) mice, suggesting the Vdr(+)CD4(+) T cells produced E2 or stimulated its production. The E2 decreased Cyp24a1 and increased Vdr transcripts in T cells, prolonging the calcitriol half-life and increasing calcitriol responsiveness. The E2 also increased CD4(+)Helios(+)FoxP3(+) T regulatory (Treg) cells in a Vdr-dependent manner. Thus, CD4(+) T cells have a cooperative amplification loop involving E2 and calcitriol that promotes CD4(+)Helios(+)FoxP3(+) Treg cell development and is disrupted when the D3 pathway is impaired. The global decline in population D3 status may be undermining a similar cooperative E2-D3 interaction controlling Treg cell differentiation in women, causing a breakdown in T cell self tolerance and a rise in MS incidence.

  3. The aryl hydrocarbon receptor and estrogen receptor alpha differentially modulate nuclear factor erythroid-2-related factor 2 transactivation in MCF-7 breast cancer cells

    SciTech Connect

    Lo, Raymond; Matthews, Jason

    2013-07-15

    Nuclear factor erythroid-2-related factor 2 (NRF2; NFE2L2) plays an important role in mediating cellular protection against reactive oxygen species. NRF2 signaling is positively modulated by the aryl hydrocarbon receptor (AHR) but inhibited by estrogen receptor alpha (ERα). In this study we investigated the crosstalk among NRF2, AHR and ERα in MCF-7 breast cancer cells treated with the NRF2 activator sulforaphane (SFN), a dual AHR and ERα activator, 3,3′-diindolylmethane (DIM), 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or 17β-estradiol (E2). SFN-dependent increases in NADPH-dependent oxidoreductase 1 (NQO1) and heme oxygenase I (HMOX1) mRNA levels were significantly reduced after co-treatment with E2. E2-dependent repression of NQO1 and HMOX1 was associated with increased ERα but reduced p300 recruitment and reduced histone H3 acetylation at both genes. In contrast, DIM + SFN or TCDD + SFN induced NQO1 and HMOX1 mRNA expression to levels higher than SFN alone, which was prevented by RNAi-mediated knockdown of AHR. DIM + SFN but not TCDD + SFN also induced recruitment of ERα to NQO1 and HMOX1. However, the presence of AHR at NQO1 and HMOX1 restored p300 recruitment and histone H3 acetylation, thereby reversing the ERα-dependent repression of NRF2. Taken together, our study provides further evidence of functional interplay among NRF2, AHR and ERα signaling pathways through altered p300 recruitment to NRF2-regulated target genes. - Highlights: • We examined crosstalk among ERα, AHR, and NRF2 in MCF-7 breast cancer cells. • AHR enhanced the mRNA expression levels of two NRF2 target genes – HMOX1 and NQO1. • ERα repressed HMOX1 and NQO1 expression via decreased histone acetylation. • AHR prevented ERα-dependent repression of HMOX1 and NQO1.

  4. Mirtazapine : A Review of its Pharmacology and Therapeutic Potential in the Management of Major Depression.

    PubMed

    Davis, R; Wilde, M I

    1996-05-01

    and neurophysiological experiments as well as behavioural studies performed in rats support the α2-adrenoceptor antagonist properties of mirtazapine. Receptor binding studies have shown that mirtazapine has a high affinity for serotonin 5-HT2 and 5-HT3 receptors, central and peripheral histamine H1 receptors and a low affinity for 5-HT1, dopaminergic and muscarinic cholinergic receptors. Its activity at serotonin receptor subtypes has been confirmed in animal behaviour models. Mirtazapine activates 5-HT1 receptor-mediated serotonergic neurotransmission by enhancing the stimulatory effect of the noradrenergic system on serotonergic cell firing (an α1-adrenoceptor-mediated effect) as well as antagonising the inhibitory effect of the noradrenergic system on serotonin release (an α2-adrenoceptor-mediated effect). Electrophysiological experiments have demonstrated that mirtazapine enhances serotonergic transmission through blockade of presynaptic α2-adrenoceptors. The drug does not inhibit serotonin reuptake. The bioavailability of mirtazapine is approximately 50%. Peak plasma concentrations are reached within 2.2 to 3.1 hours after single oral doses of 15 to 75mg and are dose-dependent. Mirtazapine is extensively metabolised in the liver; up to 85% of the drug is eliminated in the urine (up to 4% as unchanged drug) and the remaining 15% is eliminated in the faeces. The mean elimination half-life of mirtazapine is approximately 22 hours, making it suitable for once-daily administration. In randomised double-blind comparative trials including patients with major depression, short term (5 to 6 weeks) therapy with mirtazapine was significantly more effective than placebo, as effective as amitriptyline, clomipramine and doxepin, and at least as effective as trazodone. Results from a meta-analysis of 5 comparative trials in which 60% of patients were hospitalised with severe depression [mean baseline 17-item Hamilton Depression Rating Scale (HAMD) score ≥25] revealed no

  5. [Role of α7 nicotinic acetylcholine receptor in attenuation of endotoxin induced delirium with dexmedetomidine in mice].

    PubMed

    Zhang, Xueyan; Li, Zhifeng; Sun, Xiaochen; Jin, Feng; Liu, Junting; Li, Jianguo

    2016-02-01

    , when compared with NS group, the mice in LPS group showed that they had the ability of cognition of new environment, but learning and memory abilities were lowered, and tension was increased. DEX could significantly attenuate the degree of delirium, however, the protection of DEX from the delirious syndrome was antagonized partly by α-BGT. (2) The new object recognition test results showed that compared with NS group, the ability of exploring new object was decreased in LPS group. DEX could significantly improve the exploration ability. However, DEX failed to control the delirious syndrome in α-BGT+DEX+LPS group. (3) The results of ELISA showed that the levels of TNF-α and NSE in serum were significantly increased in LPS groups as compared with that in NS group, and the levels of TNF-α and NSE were significantly decreased in DEX+LPS group. However, α-BGT could antagonise the protective effect of DEX [TNF-α (ng/L) in NS, LPS, DEX+LPS and α-BGT+DEX+LPS groups was 23.72±3.13, 808.78±87.86, 192.96±31.47, 829.99±80.98, respectively, and NSE (μg/L) was 8.70±0.74, 25.90±3.03, 18.10±2.14, and 23.12±2.21, respectively, all P < 0.01]. (4) The results of Western Blot showed that compared with NS group, the protein expression of ChAT in LPS group was significantly declined, and the protein expression of AChE was significantly increased. DEX could reverse the expressions of ChAT and AChT, however, α-BGT could reverse the protective effect of DEX [ChAT (gray value) in NS, LPS, DEX+LPS and α-BGT+DEX+LPS groups was 1.536±0.150, 0.381±0.138, 0.914±0.173, 0.628±0.088, respectively, and AChE (gray value) was 0.382±0.201, 1.843±0.325, 0.898±0.155, and 1.470±0.220, respectively, P < 0.05 or P<0.01]. Delirium syndrome may occur in mice with endotoxemia. DEX could attenuate endotoxemia-associated delirium syndrome through transforming central neurotransmitter, and its mechanism maybe related with α7nAChR.

  6. Topical antihistamines and mast cell stabilisers for treating seasonal and perennial allergic conjunctivitis.

    PubMed

    Castillo, Mayret; Scott, Neil W; Mustafa, Mohammad Z; Mustafa, Mohammed S; Azuara-Blanco, Augusto

    2015-06-01

    Seasonal/perennial allergic conjunctivitis is the most common allergic conjunctivitis, usually with acute manifestations when a person is exposed to allergens and with typical signs and symptoms including itching, redness, and tearing. The clinical signs and symptoms of allergic conjunctivitis are mediated by the release of histamine by mast cells. Histamine antagonists (also called antihistamines) inhibit the action of histamine by blocking histamine H1 receptors, antagonising the vasoconstrictor, and to a lesser extent, the vasodilator effects of histamine. Mast cell stabilisers inhibit degranulation and consequently the release of histamine by interrupting the normal chain of intracellular signals. Topical treatments include eye drops with antihistamines, mast cell stabilisers, non-steroidal anti-inflammatory drugs, combinations of the previous treatments, and corticosteroids. Standard treatment is based on topical antihistamines alone or topical mast cell stabilisers alone or a combination of treatments. There is clinical uncertainty about the relative efficacy and safety of topical treatment. The objective of this review was to assess the effects of topical antihistamines and mast cell stabilisers, alone or in combination, for use in treating seasonal and perennial allergic conjunctivitis. We searched CENTRAL (which contains the Cochrane Eyes and Vision Trials Register) (2014, Issue 7), Ovid MEDLINE, Ovid MEDLINE In-Process and Other Non-Indexed Citations, Ovid MEDLINE Daily, Ovid OLDMEDLINE (January 1946 to July 2014), EMBASE (January 1980 to July 2014), the metaRegister of Controlled Trials (mRCT) (www.controlled-trials.com), ClinicalTrials.gov (www.clinicaltrials.gov) and the World Health Organization (WHO) International Clinical Trials Registry Platform (ICTRP) (www.who.int/ictrp/search/en). We did not use any date or language restrictions in the electronic searches for trials. We last searched the electronic databases on 17 July 2014. We also searched the

  7. Prostaglandin production at the onset of ovine parturition is regulated by both estrogen-independent and estrogen-dependent pathways.

    PubMed

    Whittle, W L; Holloway, A C; Lye, S J; Gibb, W; Challis, J R

    2000-10-01

    4-OHA infusion. Placental trophoblast PGHS-II mRNA and protein expression were increased significantly after both cortisol and cortisol plus 4-OHA infusion. Endometrial PGHS-II mRNA and protein expression increased after cortisol infusion, but not after cortisol plus 4-OHA infusion. Plasma steroid and PG concentrations, uterine activity pattern, and intrauterine PGHS-II expression were not altered in either control group. We conclude that these data suggest distinct pathways of intrauterine PG synthesis: a cortisol-dependent/E2-independent mechanism within trophoblast tissue leading to elevations in fetal plasma PGE2, and an E2-dependent mechanism within maternal endometrium that leads to increased maternal plasma PGF2alpha and appears necessary for uterine activity and parturition.