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Sample records for apelin receptor knockout

  1. Apelin, the novel endogenous ligand of the orphan receptor APJ, regulates cardiac contractility.

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    Szokodi, István; Tavi, Pasi; Földes, Gábor; Voutilainen-Myllylä, Sari; Ilves, Mika; Tokola, Heikki; Pikkarainen, Sampsa; Piuhola, Jarkko; Rysä, Jaana; Tóth, Miklós; Ruskoaho, Heikki

    2002-09-06

    The orphan receptor APJ and its recently identified endogenous ligand, apelin, exhibit high levels of mRNA expression in the heart. However, the functional importance of apelin in the cardiovascular system is not known. In isolated perfused rat hearts, infusion of apelin (0.01 to 10 nmol/L) induced a dose-dependent positive inotropic effect (EC50: 33.1+/-1.5 pmol/L). Moreover, preload-induced increase in dP/dt(max) was significantly augmented (Pzoniporide, a selective inhibitor of Na+-H+ exchange (NHE) isoform-1, and KB-R7943, a potent inhibitor of the reverse mode Na+-Ca2+ exchange (NCX), significantly suppressed the response to apelin (P<0.001). Perforated patch-clamp recordings showed that apelin did not modulate L-type Ca2+ current or voltage-activated K+ currents in isolated adult rat ventricular myocytes. Apelin mRNA was markedly downregulated in cultured neonatal rat ventricular myocytes subjected to mechanical stretch and in vivo in two models of chronic ventricular pressure overload. The present study provides the first evidence for the physiological significance of apelin in the heart. Our results show that apelin is one of the most potent endogenous positive inotropic substances yet identified and that the inotropic response to apelin may involve activation of PLC, PKC, and sarcolemmal NHE and NCX.

  2. Apelin ameliorates TNF-α-induced reduction of glycogen synthesis in the hepatocytes through G protein-coupled receptor APJ.

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    Jiaojiao Chu

    Full Text Available Apelin, a novel adipokine, is the specific endogenous ligand of G protein-coupled receptor APJ. Consistent with its putative role as an adipokine, apelin has been linked to states of insulin resistance. However, the function of apelin in hepatic insulin resistance, a vital part of insulin resistance, and its underlying mechanisms still remains unclear. Here we define the impacts of apelin on TNF-α-induced reduction of glycogen synthesis in the hepatocytes. Our studies indicate that apelin reversed TNF-α-induced reduction of glycogen synthesis in HepG2 cells, mouse primary hepatocytes and liver tissues of C57BL/6J mice by improving JNK-IRS1-AKT-GSK pathway. Moreover, Western blot revealed that APJ, but not apelin, expressed in the hepatocytes and liver tissues of mice. We found that F13A, a competitive antagonist for G protein-coupled receptor APJ, suppressed the effects of apelin on TNF-α-induced reduction of glycogen synthesis in the hepatocytes, suggesting APJ is involved in the function of apelin. In conclusion, we show novel evidence suggesting that apelin ameliorates TNF-α-induced reduction of glycogen synthesis in the hepatocytes through G protein-coupled receptor APJ. Apelin appears as a beneficial adipokine with anti-insulin resistance properties, and thus as a promising therapeutic target in metabolic disorders.

  3. The Role of Apelin on the Alleviative Effect of Angiotensin Receptor Blocker in Unilateral Ureteral Obstruction-Induced Renal Fibrosis

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    Masashi Nishida

    2012-03-01

    Full Text Available Background: Apelin is a selective endogenous ligand of the APJ receptor, which genetically has closest identity to the angiotensin II type 1 receptor (AT-1. The effects of the apelin/APJ system on renal fibrosis still remain unclear. Methods: We examined the effects of the apelin/APJ system on renal fibrosis during AT-1 blockade in a mouse unilateral ureteral obstruction (UUO model. Results: We obtained the following results: (1 At UUO day 7, mRNA expressions of apelin/APJ and phosphorylations of Akt/endothelial nitric oxide synthase (eNOS in the UUO kidney were increased compared to those in the nonobstructed kidney. (2 AT-1 blockade by the treatment with losartan resulted in a further increase of apelin mRNA as well as phosphorylations of Akt/eNOS proteins, and this was accompanied by alleviated renal interstitial fibrosis, decreased myofibroblast accumulation, and a decreased number of interstitial macrophages. (3 Blockade of the APJ receptor by the treatment with F13A during losartan administration completely abrogated the effects of losartan in the activation of the Akt/eNOS pathway and the amelioration of renal fibrosis. (4 Inhibition of NOS by the treatment with L-NAME also resulted in a further increase in renal fibrosis compared to the control group. Conclusion: These results suggest that increased nitric oxide production through the apelin/APJ/Akt/eNOS pathway may, at least in part, contribute to the alleviative effect of losartan in UUO-induced renal fibrosis.

  4. [Changes of apelin and its receptor in lung tissue of rats with pulmonary hypertension induced by monocrotaline].

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    Wang, Qing; Wang, Gui-Qin; Pang, Ling-Xia; Xue, Feng; Chen, Xing-Yan; Chen, Ran; Kong, Xiao-Xia; Gong, Yong-Sheng; Fan, Xiao-Fang

    2013-03-01

    To observe the change of apelin and its receptor (APJ) in the lung tissue of rats with pulmonary hypertension induced by monocrotaline and to explore its significance. Twenty-five male SD rats were randomly divided into control group (n = 10) and monocrotaline group (n = 15). On the twenty-first day after the rats were intraperitoneally injected 60 mg/kg monocrotaline for monocrotaline group or equal volume vehicle for control group, the mean pulmonary artery pressure was measured by right heart catheterization. Histopathological study of lung tissue was done with hematoxylin-eosin (HE) and Masson's trichrome staining. The concentration of apelin in the plasma was measured by radioimmunoassay. The expressions of apelin/APJ proteins and genes in lung tissue were measured respectively by Western blot and reverse transcription polymerase chain reaction (RT-PCR). The mean pulmonary arterial pressure, right ventricular hypertrophy, pulmonary vascular remodeling index, content of apelin protein in lung tissue of monocrotaline group were higher than those in control group. APJ protein and gene expression in monocrotaline group were significantly lower than those in control group (P pulmonary hypertension induced by monocrotaline.

  5. Angiotensin-Converting Enzyme 2 Metabolizes and Partially Inactivates Pyr-Apelin-13 and Apelin-17: Physiological Effects in the Cardiovascular System.

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    Wang, Wang; McKinnie, Shaun M K; Farhan, Maikel; Paul, Manish; McDonald, Tyler; McLean, Brent; Llorens-Cortes, Catherine; Hazra, Saugata; Murray, Allan G; Vederas, John C; Oudit, Gavin Y

    2016-08-01

    Apelin peptides mediate beneficial effects on the cardiovascular system and are being targeted as potential new drugs. However, apelin peptides have extremely short biological half-lives, and improved understanding of apelin peptide metabolism may lead to the discovery of biologically stable analogues with therapeutic potential. We examined the ability of angiotensin-converting enzyme 2 (ACE2) to cleave and inactivate pyr-apelin 13 and apelin 17, the dominant apelin peptides. Computer-assisted modeling shows a conserved binding of pyr-apelin 13 and apelin 17 to the ACE2 catalytic site. In ACE2 knockout mice, hypotensive action of pyr-apelin 13 and apelin 17 was potentiated, with a corresponding greater elevation in plasma apelin levels. Similarly, pharmacological inhibition of ACE2 potentiated the vasodepressor action of apelin peptides. Biochemical analysis confirmed that recombinant human ACE2 can cleave pyr-apelin 13 and apelin 17 efficiently, and apelin peptides are degraded slower in ACE2-deficient plasma. The biological relevance of ACE2-mediated proteolytic processing of apelin peptides was further supported by the reduced potency of pyr-apelin 12 and apelin 16 on the activation of signaling pathways and nitric oxide production from endothelial cells. Importantly, although pyr-apelin 13 and apelin 17 rescued contractile function in a myocardial ischemia-reperfusion model, ACE2 cleavage products, pyr-apelin 12 and 16, were devoid of these cardioprotective effects. We designed and synthesized active apelin analogues that were resistant to ACE2-mediated degradation, thereby confirming that stable apelin analogues can be designed as potential drugs. We conclude that ACE2 represents a major negative regulator of apelin action in the vasculature and heart. © 2016 American Heart Association, Inc.

  6. Apelin/APJ system: A bifunctional target for cardiac hypertrophy.

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    Lu, Liqun; Wu, Di; Li, Lanfang; Chen, Linxi

    2017-03-01

    Apelin acts as the endogenous ligand of G protein coupled receptors APJ. The apelin/APJ system is responsible for the occurrence and development of cardiovascular diseases. In recent years, apelin/APJ has been considered to play an important role in cardiac hypertrophy, but whether that role is beneficial or aggravating remains controversial. Apelin/APJ alleviates cardiac hypertrophy which is triggered by angiotensin II, oxidative stress and exercise. However, central administration of apelin induces cardiac hypertrophy. Peripheral administration of apelin also promotes the development of cardiac hypertrophy under non-pathological conditions. Furthermore, our laboratory discovers that apelin/APJ is able to induce hypertrophy of cardiomyocytes in vitro. The exact mechanism of apelin/APJ's dual effects in cardiac hypertrophy requires further study. In this paper, we review the controversies associated with apelin/APJ in cardiac hypertrophy and we elaborate the role of apelin/APJ in cardiac hypertrophy related-diseases including obesity, diabetes, hypertension, myocarditis and myocardial infarction. We conclude that further studies should emphasize more about the relationship between apelin/APJ and pathological hypertrophy especially in clinical patients. Moreover, apelin/APJ can be a promising therapeutic target for cardiac hypertrophy. Copyright © 2016. Published by Elsevier B.V.

  7. Effects of Apelin on Cardiovascular Aging.

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    Zhou, Ying; Wang, Yong; Qiao, Shubin; Yin, Liang

    2017-01-01

    Apelin is the endogenous ligand of APJ, the orphan G protein-coupled receptor. The apelin-APJ signal transduction pathway is widely expressed in the cardiovascular system and is an important factor in cardiovascular homeostasis. This signal transduction pathway has long been related to diseases with high morbidity in the elderly, such as atherosclerosis, coronary atherosclerotic heart disease, hypertension, calcific aortic valve disease, heart failure and atrial fibrillation. In this review, we discuss the apelin-APJ signal transduction pathway related to age-associated cardiovascular diseases.

  8. Promoting effects of the adipokine, apelin, on diabetic nephropathy.

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    Bao-hai Zhang

    Full Text Available Angiogenesis, increased glomerular permeability, and albuminuria are thought to contribute to the progression of diabetic nephropathy (DN. Apelin receptor (APLNR and the endogenous ligand of APLNR, apelin, induce the sprouting of endothelial cells in an autocrine or paracrine manner, which may be one of the mechanisms of DN. The aim of this study was to investigate the role of apelin in the pathogenesis of DN. Therefore, we observed apelin/APLNR expression in kidneys from patients with type 2 diabetes as well as the correlation between albuminuria and serum apelin in patients with type 2 diabetes. We also measured the proliferating, migrating, and chemotactic effects of apelin on glomerular endothelial cells. To measure the permeability of apelin in glomerular endothelial cells, we used transwells to detect FITC-BSA penetration through monolayered glomerular endothelial cells. The results showed that serum apelin was significantly higher in the patients with type 2 diabetes compared to healthy people (p<0.05, Fig. 1B and that urinary albumin was positively correlated with serum apelin (R = 0.78, p<0.05. Apelin enhanced the migration, proliferation, and chemotaxis of glomerular endothelial cells in a dose-dependent manner (p<0.05. Apelin also promoted the permeability of glomerular endothelial cells (p<0.05 and upregulated the expression of VEGFR2 and Tie2 in glomerular endothelial cells (p<0.05. These results indicated that upregulated apelin in type 2 diabetes, which may be attributed to increased fat mass, promotes angiogenesis in glomeruli to form abnormal vessels and that enhanced apelin increases permeability via upregulating the expression of VEGFR2 and Tie2 in glomerular endothelial cells.

  9. Apelin/APJ system: A novel potential therapy target for kidney disease.

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    Huang, Zhen; Wu, Lele; Chen, Linxi

    2018-05-01

    Apelin is an endogenous ligand of seven-transmembrane G protein-coupled receptor APJ. Apelin and APJ are distributed in various tissues, including the heart, lung, kidney, and even in tumor tissues. Studies show that apelin mRNA is highly expressed in the inner stripe of kidney outer medulla, which plays an important role in process of water and sodium balance. Additionally, more studies also indicate that apelin/APJ system exerts a broad range of activities in kidney. Therefore, we review the role of apelin/APJ system in kidney diseases such as renal fibrosis, renal ischemia/reperfusion injury, diabetic nephropathy, polycystic kidney disease, and hemodialysis (HD). Apelin/APJ system can improve renal interstitial fibrosis by reducing the deposition of extracellular matrix. Apelin/APJ system significantly reduces renal ischemia/reperfusion injury by inhibiting renal cell death. Apelin/APJ system involves the progression of diabetic nephropathy (DN). Apelin/APJ system also predicts the process of polycystic kidney disease. Besides, apelin/APJ system prevents some dialysis complications in HD patients. And apelin/APJ system alleviates chronic kidney disease (CKD) by inhibiting vascular calcification (VC). Overall, apelin/APJ system plays diversified roles in kidney disease and may be a potential target for the treatment of kidney disease. © 2017 Wiley Periodicals, Inc.

  10. Hepatic changes in metabolic gene expression in old ghrelin and ghrelin receptor knockout mice

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    Ghrelin knockout (GKO) and ghrelin receptor (growth hormone secretagogue receptor) knockout (GHSRKO) mice exhibit enhanced insulin sensitivity, but the mechanism is unclear. Insulin sensitivity declines with age and is inversely associated with accumulation of lipid in liver, a key glucoregulatory ...

  11. Central apelin-13 administration modulates hypothalamic control of feeding.

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    Ferrante, C; Orlando, G; Recinella, L; Leone, S; Chiavaroli, A; Di Nisio, C; Shohreh, R; Manippa, F; Ricciuti, A; Vacca, M; Brunetti, L

    2016-01-01

    The 77 amino prepropeptide apelin has been isolated from bovine stomach tissue and several smaller fragments, including apelin-13, showed high affinity for the orphan APJ receptor. The distribution of apelinergic fibers and receptors in the hypothalamus may suggest a role of apelin-13 on energy balance regulation, albeit the studies reporting the acute effects of apelin on feeding control are inconsistent. Considering the possible involvement of apelinergic system on hypothalamic appetite controlling network, in the present study we evaluated in the rat the effects of intrahypothalamic apelin-13 injection on food intake and the involvement of orexigenic and anorexigenic hypothalamic peptides and neurotransmitters. Eighteen rats (6 for each group of treatment) were injected into the ARC with either vehicle or apelin-13 (1-2 μg/rat). Food intake and hypothalamic peptide and neurotransmitter levels were evaluated 2 and 24 h after injection. Compared to vehicle, apelin-13 administration increased food intake both 2 and 24 h following treatment. This effect could be related to inhibited cocaine- and amphetamine-regulated transcript (CART) gene expression and serotonin (5-hydroxytryptamine, 5-HT) synthesis and release, and increased orexin A gene expression in the hypothalamus.

  12. Physiological roles of CNS muscarinic receptors gained from knockout mice

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    Thomsen, Morgane; Sørensen, Gunnar; Dencker, Ditte

    2017-01-01

    , knockout mice are likely to continue to provide valuable insights into brain physiology and pathophysiology, and advance the development of new medications for a range of conditions such as Alzheimer's disease, Parkinson's disease, schizophrenia, and addictions, as well as non-opioid analgesics...... receptors modulating neuronal activity and neurotransmitter release in many brain regions, shaping neuronal plasticity, and affecting functions ranging from motor and sensory function to cognitive processes. As gene targeting technology evolves including the use of conditional, cell type specific strains...

  13. Apelin Role in the Development of Glucose Metabolism Disorders (Review of the Literature and Our Own Researches

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    G.V. Demidenko

    2014-05-01

    Full Text Available The physiological and pathogenetic role of adipokine apelin, endogenous ligand of apelin (APJ or APLNR receptors in the development of glucose metabolism disorders has been analyzed. Established correlations of apelin with components of carbohydrate metabolism confirm the effect on glucose metabolism manifestations. Ambiguous data about apelin level at insulin resistance syndrome, prediabetes, diabetes mellitus type 2, hypertension require further detailed study. The close association of apelin with development of diabetes mellitus type 2 and prediction of cardiovascular events in patients with metabolic syndrome has been found.

  14. Bone phenotypes of P2 receptor knockout mice

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    Orriss, Isabel; Syberg, Susanne; Wang, Ning

    2011-01-01

    The action of extracellular nucleotides is mediated by ionotropic P2X receptors and G-protein coupled P2Y receptors. The human genome contains 7 P2X and 8 P2Y receptor genes. Knockout mice strains are available for most of them. As their phenotypic analysis is progressing, bone abnormalities have...... been observed in an impressive number of these mice: distinct abnormalities in P2X7-/- mice, depending on the gene targeting construct and the genetic background, decreased bone mass in P2Y1-/- mice, increased bone mass in P2Y2-/- mice, decreased bone resorption in P2Y6-/- mice, decreased bone...... formation and bone resorption in P2Y13-/- mice. These findings demonstrate the unexpected importance of extracellular nucleotide signalling in the regulation of bone metabolism via multiple P2 receptors and distinct mechanisms involving both osteoblasts and osteoclasts....

  15. Neuroprotective effect of the endogenous neural peptide apelin in cultured mouse cortical neurons

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    Zeng, Xiang Jun; Yu, Shan Ping; Zhang, Like; Wei, Ling

    2010-01-01

    The adipocytokine apelin and its G protein-coupled APJ receptor were initially isolated from a bovine stomach and have been detected in the brain and cardiovascular system. Recent studies suggest that apelin can protect cardiomyocytes from ischemic injury. Here, we investigated the effect of apelin on apoptosis in mouse primary cultures of cortical neurons. Exposure of the cortical cultures to a serum-free medium for 24 h induced nuclear fragmentation and apoptotic death; apelin-13 (1.0-5.0 nM) markedly prevented the neuronal apoptosis. Apelin neuroprotective effects were mediated by multiple mechanisms. Apelin-13 reduced serum deprivation (SD)-induced ROS generation, mitochondria depolarization, cytochrome c release and activation of caspase-3. Apelin-13 prevented SD-induced changes in phosphorylation status of Akt and ERK1/2. In addition, apelin-13 attenuated NMDA-induced intracellular Ca 2+ accumulation. These results indicate that apelin is an endogenous neuroprotective adipocytokine that may block apoptosis and excitotoxic death via cellular and molecular mechanisms. It is suggested that apelins may be further explored as a potential neuroprotective reagent for ischemia-induced brain damage.

  16. Apelin Protects Primary Rat Retinal Pericytes from Chemical Hypoxia-Induced Apoptosis

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    Li Chen

    2015-01-01

    Full Text Available Pericytes are a population of cells that participate in normal vessel architecture and regulate permeability. Apelin, as the endogenous ligand of G protein-coupled receptor APJ, participates in a number of physiological and pathological processes. To date, the effect of apelin on pericyte is not clear. Our study aimed to investigate the potential protection mechanisms of apelin, with regard to primary rat retinal pericytes under hypoxia. Immunofluorescence staining revealed that pericytes colocalized with APJ in the fibrovascular membranes dissected from proliferative diabetic retinopathy patients. In the in vitro studies, we first demonstrated that the expression of apelin/APJ was upregulated in pericytes under hypoxia, and apelin increased pericytes proliferation and migration. Moreover, knockdown of apelin in pericyte was achieved via lentivirus-mediated RNA interference. After the inhibition of apelin, pericytes proliferation was inhibited significantly in hypoxia culture condition. Furthermore, exogenous recombinant apelin effectively prevented hypoxia-induced apoptosis through downregulating active-caspase 3 expression and increasing the ratio of B cell lymphoma-2 (Bcl-2/Bcl-2 associated X protein (Bax in pericytes. These results suggest that apelin suppressed hypoxia-induced pericytes injury, which indicated that apelin could be a potential therapeutic target for retinal angiogenic diseases.

  17. Neuroprotective effect of the endogenous neural peptide apelin in cultured mouse cortical neurons

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    Zeng, Xiang Jun [Department of Pathophysiology, Capital Medical University, Beijing 100069 (China); Department of Anesthesiology, 101 Woodruff Circle, Suite 617, Emory University School of Medicine, Atlanta, GA 30322 (United States); Yu, Shan Ping [Department of Anesthesiology, 101 Woodruff Circle, Suite 617, Emory University School of Medicine, Atlanta, GA 30322 (United States); Zhang, Like [Department of Pathophysiology, Capital Medical University, Beijing 100069 (China); Wei, Ling, E-mail: lwei7@emory.edu [Department of Anesthesiology, 101 Woodruff Circle, Suite 617, Emory University School of Medicine, Atlanta, GA 30322 (United States)

    2010-07-01

    The adipocytokine apelin and its G protein-coupled APJ receptor were initially isolated from a bovine stomach and have been detected in the brain and cardiovascular system. Recent studies suggest that apelin can protect cardiomyocytes from ischemic injury. Here, we investigated the effect of apelin on apoptosis in mouse primary cultures of cortical neurons. Exposure of the cortical cultures to a serum-free medium for 24 h induced nuclear fragmentation and apoptotic death; apelin-13 (1.0-5.0 nM) markedly prevented the neuronal apoptosis. Apelin neuroprotective effects were mediated by multiple mechanisms. Apelin-13 reduced serum deprivation (SD)-induced ROS generation, mitochondria depolarization, cytochrome c release and activation of caspase-3. Apelin-13 prevented SD-induced changes in phosphorylation status of Akt and ERK1/2. In addition, apelin-13 attenuated NMDA-induced intracellular Ca{sup 2+} accumulation. These results indicate that apelin is an endogenous neuroprotective adipocytokine that may block apoptosis and excitotoxic death via cellular and molecular mechanisms. It is suggested that apelins may be further explored as a potential neuroprotective reagent for ischemia-induced brain damage.

  18. Effects of Weight Loss and Exercise on Apelin Serum Concentrations and Adipose Tissue Expression in Human Obesity

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    Joanna Krist

    2013-02-01

    Full Text Available Objective: Apelin is an adipokine which plays a role in the regulation of glucose homeostasis and may contribute to the link between increased adipose tissue mass and obesity related metabolic diseases. Here we investigate the role of omental and subcutaneous (SC adipose tissue apelin and its receptor APJ mRNA expression in human obesity and test the hypothesis that changes in circulating apelin are associated with reduced fat mass in three weight loss intervention studies. Methods: Apelin serum concentration was measured in 740 individuals in a cross-sectional (n = 629 study including a subgroup (n = 161 for which omental and SC apelin mRNA expression has been analyzed and in three interventions: 12 weeks exercise (n = 60, 6 months calorie-restricted diet (n = 19, 12 months after bariatric surgery (n = 32. Results: Apelin mRNA is significantly higher expressed in adipose tissue of patients with type 2 diabetes and correlates with circulating apelin, BMI, body fat, C-reactive protein, and insulin sensitivity. Obesity surgery-induced weight loss causes a significant reduction in omental and SC apelin expression. All interventions led to significantly reduced apelin serum concentrations which significantly correlate with improved insulin sensitivity, independently of changes in BMI. Conclusions: Reduced apelin expression and serum concentration may contribute to improved insulin sensitivity beyond significant weight loss.

  19. Effects of weight loss and exercise on apelin serum concentrations and adipose tissue expression in human obesity.

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    Krist, Joanna; Wieder, Katharina; Klöting, Nora; Oberbach, Andreas; Kralisch, Susan; Wiesner, Tobias; Schön, Michael R; Gärtner, Daniel; Dietrich, Arne; Shang, Edward; Lohmann, Tobias; Dreßler, Miriam; Fasshauer, Mathias; Stumvoll, Michael; Blüher, Matthias

    2013-01-01

    Apelin is an adipokine which plays a role in the regulation of glucose homeostasis and may contribute to the link between increased adipose tissue mass and obesity related metabolic diseases. Here we investigate the role of omental and subcutaneous (SC) adipose tissue apelin and its receptor APJ mRNA expression in human obesity and test the hypothesis that changes in circulating apelin are associated with reduced fat mass in three weight loss intervention studies. Apelin serum concentration was measured in 740 individuals in a cross-sectional (n = 629) study including a subgroup (n = 161) for which omental and SC apelin mRNA expression has been analyzed and in three interventions: 12 weeks exercise (n = 60), 6 months calorie-restricted diet (n = 19), 12 months after bariatric surgery (n = 32). Apelin mRNA is significantly higher expressed in adipose tissue of patients with type 2 diabetes and correlates with circulating apelin, BMI, body fat, C-reactive protein, and insulin sensitivity. Obesity surgery-induced weight loss causes a significant reduction in omental and SC apelin expression. All interventions led to significantly reduced apelin serum concentrations which significantly correlate with improved insulin sensitivity, independently of changes in BMI. Reduced apelin expression and serum concentration may contribute to improved insulin sensitivity beyond significant weight loss.

  20. Apelin-13 ameliorates cognitive impairments in 6-hydroxydopamine-induced substantia nigra lesion in rats.

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    Haghparast, Elham; Esmaeili-Mahani, Saeed; Abbasnejad, Mehdi; Sheibani, Vahid

    2018-04-01

    Although Parkinson's disease (PD) is well known with its motor deficits, the patients often suffer from cognitive dysfunction. Apelin, as the endogenous ligand of the APJ receptor, is found in several brain regions such as substantia nigra and mesolimbic pathway. However, the role of apelin in cognition and cognitive disorders has not been fully clarified. In this study the effects of apelin-13 were investigated on cognitive disorders in rat Parkinsonism experimental model. 6-hydroxydopamine (6-OHDA) was administrated into the substantia nigra. Apelin-13 (1, 2 and 3μg/rat) was administered into the substantia nigra one week after the 6-OHDA injection. Morris water maze (MWM), object location and novel object recognition tests were performed one month after the apelin injection. 6-OHDA-treated animals showed a significant impairment in cognitive functions which was revealed by the increased in the escape latency and traveled distance in MWM test and decreased in the exploration index in novel object recognition and object location tasks. Apelin-13 (3μg/rat) significantly attenuates the mentioned cognitive impairments in 6-OHDA-treated animals. In conclusion, the data support the pro-cognitive property of apelin-13 in 6-OHDA-induced cognitive deficit and provided a new pharmacological aspect of the neuropeptide apelin. Copyright © 2018 Elsevier Ltd. All rights reserved.

  1. Bone growth and turnover in progesterone receptor knockout mice.

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    Rickard, David J.; Iwaniec, Urszula T.; Evans, Glenda; Hefferan, Theresa E.; Hunter, Jaime C.; Waters, Katrina M.; Lydon, John P.; O' Malley, Bert W.; Khosla, Sundeep; Spelsberg, Thomas C.; Turner, Russell T.

    2008-05-01

    The role of progesterone receptor (PR) signaling in skeletal metabolism is controversial. To address whether signaling through the PR is necessary for normal bone growth and turnover, we performed histomorphometric and mCT analyses of bone from homozygous female PR knockout (PRKO) mice at 6, 12, and 26 weeks of age. These mice possess a null mutation of the PR locus, which blocks the gene expression of A and B isoforms of PR. Body weight gain, uterine weight gain and tibia longitudinal bone growth was normal in PRKO mice. In contrast, total and cortical bone mass were increased in long bones of post-pubertal (12 and 26-week-old) PRKO mice, whereas cancellous bone mass was normal in the tibia but increased in the humerus. The striking 57% decrease in cancellous bone from the proximal tibia metaphysis which occurred between 6 and 26 weeks in WT mice was abolished in PRKO mice. The improved bone balance in aging PRKO mice was associated with elevated bone formation and a tendency toward reduced osteoclast perimeter. Taken together, these findings suggest that PR signaling in mice attenuates the accumulation of cortical bone mass during adolescence and is required for early age-related loss of cancellous bone.

  2. Impaired water maze learning performance in mu-opioid receptor knockout mice.

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    Jang, Choon-Gon; Lee, Seok-Yong; Yoo, Ji-Hoon; Yan, Ji-Jing; Song, Dong-Keun; Loh, Horace H; Ho, Ing K

    2003-09-10

    Previous study has demonstrated that the lack of mu-opioid receptor decreased LTP in the dentate gyrus of the hippocampus, suggesting the possibility that the lack of mu-opioid receptor may accompany a change in learning and memory. However, no behavioral study has been undertaken to correlate LTP deficits with spatial memory impairment in mu-opioid receptor knockout mice. Therefore, the present study investigated the hypothesis that mu-opioid receptors contribute to learning and memory by using the Morris water maze, and comparing responses in wild type and mu-opioid receptor gene knockout mice. Our results indicated that mu-opioid receptor knockout mice showed a significant spatial memory impairment compared to wild type in the Morris water maze. This result suggests that the expression of mu-opioid receptor plays an important role in spatial learning and memory examined by Morris water maze.

  3. Apelin elevates blood pressure in ICR mice with L-NAME-induced endothelial dysfunction

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    NAGANO, KATSUMASA; ISHIDA, JUNJI; UNNO, MADOKA; MATSUKURA, TANOMU; FUKAMIZU, AKIYOSHI

    2013-01-01

    Apelin is the endogenous ligand of APJ, which belongs to the family of G protein-coupled receptors. Apelin and APJ are highly expressed in various cardiovascular tissues, including the heart, kidney and vascular endothelial and smooth muscle cells. Although apelin exerts hypotensive effects via activation of endothelial nitric oxide synthase (eNOS), the ability of apelin to regulate blood pressure under pathological conditions is poorly understood. In the current study, NG-nitro-L-arginine methyl ester (L-NAME), a potent NOS inhibitor, was administered chronically, to induce peripheral vascular damage in mice. L-NAME-treated mice exhibited hypertension, increased vascular cell adhesion molecule-1 and plasminogen activator inhibitor-1 mRNA levels in the aorta and impaired vasodilatation associated with decreased aortic eNOS expression, consistent with endothelial damage. Three days following withdrawal of L-NAME treatment, the blood pressure response to apelin stimulation was assessed. Although apelin reduced blood pressure in non-treated mice, it was found to transiently elevate blood pressure in L-NAME-treated mice. These results indicate that apelin functions as a vasopressor peptide under pathological conditions, including vascular endothelial dysfunction in mice. PMID:23525196

  4. Mu-opioid receptor knockout mice show diminished food-anticipatory activity

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    Kas, Martien J H; van den Bos, Ruud; Baars, Annemarie M; Lubbers, Marianne; Lesscher, Heidi M B; Hillebrand, Jacquelien J G; Schuller, Alwin G; Pintar, John E; Spruijt, Berry M

    We have previously suggested that during or prior to activation of anticipatory behaviour to a coming reward, mu-opioid receptors are activated. To test this hypothesis schedule induced food-anticipatory activity in mu-opioid receptor knockout mice was measured using running wheels. We hypothesized

  5. Impaired social behavior in 5-HT3A receptor knockout mice

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    Laura A Smit-Rigter

    2010-11-01

    Full Text Available The 5-HT3 receptor is a ligand-gated ion channel expressed on interneurons throughout the brain. So far, analysis of the 5-HT3A knockout mouse revealed changes in nociceptive processing and a reduction in anxiety related behavior. Recently, it was shown that the 5-HT3 receptor is also expressed on Cajal-Retzius cells which play a key role in cortical development and that knockout mice lacking this receptor showed aberrant growth of the dendritic tree of cortical layer II/III pyramidal neurons. Other mouse models in which serotonergic signaling was disrupted during development showed similar morphological changes in the cortex, and in addition, also deficits in social behavior. Here, we subjected male and female 5-HT3A knockout mice and their non-transgenic littermates to several tests of social behavior. We found that 5-HT3A knockout mice display impaired social communication in the social transmission of food preference task. Interestingly, we showed that in the social interaction test only female 5-HT3A knockout mice spent less time in reciprocal social interaction starting after 5 minutes of testing. Moreover, we observed differences in preference for social novelty for male and female 5-HT3A knockout mice during the social approach test. However, no changes in olfaction, exploratory activity and anxiety were detected. These results indicate that the 5-HT3A knockout mouse displays impaired social behavior with specific changes in males and females, reminiscent to other mouse models in which serotonergic signaling is disturbed in the developing brain.

  6. TNF receptor 1, IL-1 receptor, and iNOS genetic knockout mice are not protected from anthrax infection.

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    Kalns, John; Scruggs, Julie; Millenbaugh, Nancy; Vivekananda, Jeeva; Shealy, David; Eggers, Jeffrey; Kiel, Johnathan

    2002-03-22

    Anthrax produces at least two toxins that cause an intense systemic inflammatory response, edema, shock, and eventually death. The relative contributions of various elements of the immune response to mortality and course of disease progression are poorly understood. We hypothesized that knockout mice missing components of the immune system will have an altered response to infection. Parent strain mice and knockouts were challenged with LD95 of anthrax spores (5 x 10(6)) administered subcutaneously. Our results show that all genetic knockouts succumbed to anthrax infection at the same frequency as the parent. TNF antibody delayed death but TNF receptor 1 knockout had no effect. IL-1 receptor or iNOS knockouts died sooner. Anthrax was more abundant in the injection site of TNF-alpha and iNOS knockouts compared to parent suggesting that attenuated cellular response increases rate of disease progression. With the exception of edema and necrosis at the injection site pathological changes in internal organs were not observed. (C)2002 Elsevier Science (USA).

  7. Effects of dietary eicosapentaenoic acid (EPA supplementation in high-fat fed mice on lipid metabolism and apelin/APJ system in skeletal muscle.

    Directory of Open Access Journals (Sweden)

    Chantal Bertrand

    Full Text Available Various studies have shown that eicosapentaenoic acid (EPA has beneficial effects on obesity and associated disorders. Apelin, the ligand of APJ receptor also exerts insulin-sensitizing effects especially by improving muscle metabolism. EPA has been shown to increase apelin production in adipose tissue but its effects in muscle have not been addressed. Thus, the effects of EPA supplementation (36 g/kg EPA in high-fat diet (HFD (45% fat, 20% protein, 35% carbohydrate were studied in mice with focus on muscle lipid metabolism and apelin/APJ expression. Compared with HFD mice, HFD+EPA mice had significantly less weight gain, fat mass, lower blood glucose, insulinemia and hepatic steatosis after 10 weeks of diet. In addition, EPA prevented muscle metabolism alterations since intramuscular triglycerides were decreased and β-oxidation increased. In soleus muscles of HFD+EPA mice, apelin and APJ expression were significantly increased compared to HFD mice. However, plasma apelin concentrations in HFD and HFD+EPA mice were similar. EPA-induced apelin expression was confirmed in differentiated C2C12 myocytes but in this model, apelin secretion was also increased in response to EPA treatment. In conclusion, EPA supplementation in HFD prevents obesity and metabolic alterations in mice, especially in skeletal muscle. Since EPA increases apelin/APJ expression in muscle, apelin may act in a paracrine/autocrine manner to contribute to these benefical effects.

  8. Interactive effects of apelin, renin-angiotensin system and nitric oxide in treatment of obesity-induced type 2 diabetes mellitus in male albino rats.

    Science.gov (United States)

    Sabry, Maha Mohamed; Mahmoud, Manal Moustafa; Shoukry, Heba Samy; Rashed, Laila; Kamar, Samaa Samir; Ahmed, Mona Mohamed

    2018-03-22

    Apelin and its receptor (APJ) are involved in the regulation of a variety of pathophysiological processes. We studied the effect of apelin treatment on obesity-induced type 2 diabetes mellitus (T2DM) and possible interaction between apelin/APJ system and renin-angiotensin system (RAS). Forty eight male albino rats were divided into two groups: control group and diabetic group. Diabetic group was subdivided into: control diabetic, apelin-treated, apelin + losartan-treated, apelin + l-NAME-treated and losartan-treated diabetic subgroup. Administration of apelin-13 yielded an improvement of IR, dyslipidaemia, inflammation, oxidative stress with significant decrease in AT1R gene expression and significant increase in ACE2 gene expression in adipose tissues. Losartan + apelin yielded a further significant decrease in ATR1 gene expression, glycaemic indices, serum TGs and TPA versus Apelin only. Adding l-NAME in subgroup (2D) reversed the effect of apelin. We suggested that the beneficial effect of Apelin is mainly mediated by NO-activated pathway and/or ACE2/Ang (1-7) dependent pathway.

  9. Hematopoiesis in 5-Fluorouracil-Treated Adenosine A(3) Receptor Knock-Out Mice

    Czech Academy of Sciences Publication Activity Database

    Hofer, Michal; Pospíšil, Milan; Dušek, L.; Hoferová, Zuzana; Komůrková, Denisa

    2015-01-01

    Roč. 64, č. 2 (2015), s. 255-262 ISSN 0862-8408 Institutional support: RVO:68081707 Keywords : Adenosine A(3) receptor knock-out mice * Hematopoiesis * 5-fluorouracil-induced hematotoxicity Subject RIV: BO - Biophysics Impact factor: 1.643, year: 2015

  10. Beneficial Effects of Apelin on Vascular Function in Patients With Central Obesity.

    Science.gov (United States)

    Schinzari, Francesca; Veneziani, Augusto; Mores, Nadia; Barini, Angela; Di Daniele, Nicola; Cardillo, Carmine; Tesauro, Manfredi

    2017-05-01

    Patients with central obesity have impaired insulin-stimulated vasodilation and increased ET-1 (endothelin 1) vasoconstriction, which may contribute to insulin resistance and vascular damage. Apelin enhances insulin sensitivity and glucose disposal but also acts as a nitric oxide (NO)-dependent vasodilator and a counter-regulator of AT 1 (angiotensin [Ang] II type 1) receptor-induced vasoconstriction. We, therefore, examined the effects of exogenous (Pyr 1 )apelin on NO-mediated vasodilation and Ang II- or ET-1-dependent vasoconstrictor tone in obese patients. In the absence of hyperinsulinemia, forearm blood flow responses to graded doses of acetylcholine and sodium nitroprusside were not different during saline or apelin administration (both P >0.05). During intra-arterial infusion of regular insulin, however, apelin enhanced the vasodilation induced by both acetylcholine and nitroprusside (both P 0.05). In conclusion, in patients with central obesity, apelin has favorable effects not only to improve insulin-stimulated endothelium-dependent and endothelium-independent vasodilator responses but also to blunt Ang II- and ET-1-dependent vasoconstriction by a mechanism not involving NO. Taken together, our results suggest that targeting the apelin system might favorably impact some hemodynamic abnormalities of insulin-resistant states like obesity. © 2017 American Heart Association, Inc.

  11. Altered metabolism in the melatonin-related receptor (GPR50) knockout mouse.

    Science.gov (United States)

    Ivanova, Elena A; Bechtold, David A; Dupré, Sandrine M; Brennand, John; Barrett, Perry; Luckman, Simon M; Loudon, Andrew S I

    2008-01-01

    The X-linked orphan receptor GPR50 shares 45% homology with the melatonin receptors, yet its ligand and physiological function remain unknown. Here we report that mice lacking functional GPR50 through insertion of a lacZ gene into the coding sequence of GPR50 exhibit an altered metabolic phenotype. GPR50 knockout mice maintained on normal chow exhibit lower body weight than age-matched wild-type littermates by 10 wk of age. Furthermore, knockout mice were partially resistant to diet-induced obesity. When placed on a high-energy diet (HED) for 5 wk, knockout mice consumed significantly more food per unit body weight yet exhibited an attenuated weight gain and reduced body fat content compared with wild-type mice. Wheel-running activity records revealed that, although GPR50 knockout mice showed no alteration of circadian period, the overall levels of activity were significantly increased over wild types in both nocturnal and diurnal phases. In line with this, basal metabolic rate (O2 consumption, CO2 production, and respiratory quotient) was found to be elevated in knockout mice. Using in situ hybridization (wild-type mice) and beta-galactosidase activity (from LacZ insertion element in knockout mice), brain expression of GPR50 was found to be restricted to the ependymal layer of the third ventricle and dorsomedial nucleus of the hypothalamus. GPR50 expression was highly responsive to energy status, showing a significantly reduced expression following both fasting and 5 wk of HED. These data implicate GPR50 as an important regulator of energy metabolism.

  12. Memory Impairment in Estrogen Receptor ? Knockout Mice Through Accumulation of Amyloid-? Peptides

    OpenAIRE

    Hwang, Chul Ju; Yun, Hyung-Mun; Park, Kyung-Ran; Song, Ju Kyung; Seo, Hyun Ok; Hyun, Byung Kook; Choi, Dong Young; Yoo, Hwan-Soo; Oh, Ki-Wan; Hwang, Dae Yeun; Han, Sang-Bae; Hong, Jin Tae

    2014-01-01

    Estrogen has been known to reduce the development of Alzheimer?s disease (AD). However, exact mechanisms are not clear. We investigated whether estrogen can increase amyloid-beta (A?) degradation and affects A?-induced memory impairment in an estrogen deficiency model. Estrogen receptor alpha (ER?) knockout mice and wild-type mice were intracerebroventricular (ICV) infused with A? (300?pmol) for 2?weeks. Cognitive function was then assessed by the Morris water maze test and passive avoidance ...

  13. Altered sleep and affect in the neurotensin receptor 1 knockout mouse.

    Science.gov (United States)

    Fitzpatrick, Karrie; Winrow, Christopher J; Gotter, Anthony L; Millstein, Joshua; Arbuzova, Janna; Brunner, Joseph; Kasarskis, Andrew; Vitaterna, Martha H; Renger, John J; Turek, Fred W

    2012-07-01

    Sleep and mood disorders have long been understood to have strong genetic components, and there is considerable comorbidity of sleep abnormalities and mood disorders, suggesting the involvement of common genetic pathways. Here, we examine a candidate gene implicated in the regulation of both sleep and affective behavior using a knockout mouse model. Previously, we identified a quantitative trait locus (QTL) for REM sleep amount, REM sleep bout number, and wake amount in a genetically segregating population of mice. Here, we show that traits mapping to this QTL correlated with an expression QTL for neurotensin receptor 1 (Ntsr1), a receptor for neurotensin, a ligand known to be involved in several psychiatric disorders. We examined sleep as well as behaviors indicative of anxiety and depression in the NTSR1 knockout mouse. NTSR1 knockouts had a lower percentage of sleep time spent in REM sleep in the dark phase and a larger diurnal variation in REM sleep duration than wild types under baseline conditions. Following sleep deprivation, NTSR1 knockouts exhibited more wake and less NREM rebound sleep. NTSR1 knockouts also showed increased anxious and despair behaviors. Here we illustrate a link between expression of the Ntsr1 gene and sleep traits previously associated with a particular QTL. We also demonstrate a relationship between Ntsr1 and anxiety and despair behaviors. Given the considerable evidence that anxiety and depression are closely linked with abnormalities in sleep, the data presented here provide further evidence that neurotensin and Ntsr1 may be a component of a pathway involved in both sleep and mood disorders.

  14. Diacylglycerol lipase a knockout mice demonstrate metabolic and behavioral phenotypes similar to those of cannabinoid receptor 1 knockout mice

    Directory of Open Access Journals (Sweden)

    David R Powell

    2015-06-01

    Full Text Available After creating >4650 knockouts (KOs of independent mouse genes, we screened them by high-throughput phenotyping and found that cannabinoid receptor 1 (Cnr1 KO mice had the same lean phenotype published by others. We asked if our KOs of DAG lipase a or b (Dagla or Daglb, which catalyze biosynthesis of the endocannabinoid (EC 2-Arachidonoylglycerol (2-AG, or Napepld, which catalyzes biosynthesis of the EC anandamide, shared the lean phenotype of Cnr1 KO mice. We found that Dagla KO mice, but not Daglb or Napepld KO mice, were among the leanest of 3651 chow-fed KO lines screened. In confirmatory studies, chow- or high fat diet-fed Dagla and Cnr1 KO mice were leaner than wild type (WT littermates; when data from multiple cohorts of adult mice were combined, body fat was 47% and 45% lower in Dagla and Cnr1 KO mice, respectively, relative to WT values. In contrast, neither Daglb nor Napepld KO mice were lean. Weanling Dagla KO mice ate less than WT mice and had body weight similar to pair-fed WT mice, and adult Dagla KO mice had normal activity and VO2 levels, similar to Cnr1 KO mice. Our Dagla and Cnr1 KO mice also had low fasting insulin, triglyceride and total cholesterol levels, and after a glucose challenge had normal glucose but very low insulin levels. Dagla and Cnr1 KO mice also showed similar responses to a battery of behavioral tests. These data suggest: 1 the lean phenotype of young Dagla and Cnr1 KO mice is mainly due to hypophagia; 2 in pathways where ECs signal through Cnr1 to regulate food intake and other metabolic and behavioral phenotypes observed in Cnr1 KO mice, Dagla alone provides the 2-AG that serves as the EC signal; and 3 small molecule Dagla inhibitors with a pharmacokinetic profile similar to that of Cnr1 inverse agonists are likely to mirror the ability of these Cnr1 inverse agonists to lower body weight and improve glycemic control in obese patients with type 2 diabetes, but may also induce undesirable neuropsychiatric

  15. Increased cocaine self-administration in M4 muscarinic acetylcholine receptor knockout mice

    DEFF Research Database (Denmark)

    Schmidt, Lene Sørensen; Thomsen, Morgane; Weikop, Pia

    2011-01-01

    Rationale The reinforcing effects of cocaine are mediated by the mesolimbic dopamine system. Behavioral and neurochemical studies have shown that the cholinergic muscarinic M4 receptor subtype plays an important role in regulation of dopaminergic neurotransmission. Objectives Here we investigated...... for the first time the involvement of M4 receptors in the reinforcing effects of cocaine using chronic intravenous cocaine self-administration in extensively backcrossed M4 receptor knockout (M4 -/-) mice. Methods We evaluated acquisition of cocaine self-administration in experimentally naïve mice. Both cocaine...... self-administration and food-maintained operant behavior were evaluated under fixed ratio 1 (FR 1) and progressive ratio (PR) schedules of reinforcement. In addition, cocaine-induced dopamine release and cocaine-induced hyperactivity were evaluated. Results M4 -/- mice earned significantly more cocaine...

  16. Proximal gut mucosal epithelial homeostasis in aged IL-1 type I receptor knockout mice after starvation.

    Science.gov (United States)

    Song, Juquan; Wolf, Steven E; Wu, Xiao-Wu; Finnerty, Celeste C; Herndon, David N; Jeschke, Marc G

    2011-08-01

    Previous studies have shown that starvation induces small bowel atrophy, and that atrophy diminishes with aging. In this experiment, we assessed whether starvation-induced atrophy of proximal gut mucosa is associated with the Interleukin-1 receptor (IL-1R) signaling pathway in aged mice. Thirty 26-month-old IL-1R knockout mice and age-matched wild-type C57BL/6 mice were randomly divided into two groups: ad libitum fed and fasted. Mice were euthanized 12 or 48 hours after starvation. The proximal small bowel was harvested for morphologic analysis. Gut epithelial cell proliferation was detected using immunohistochemical staining for proliferating cell nuclear antigen (PCNA), and apoptosis was identified using terminal deoxyuridine nick-end labeling (TUNEL) staining. Aged IL-1R knockout mice were larger than aged-matched wild-type mice (P starvation (P starvation (P Starvation decreased cell proliferation in IL-1R knockout mice (P starvation increases atrophy and is associated with decreased cell proliferation rather than increased apoptosis. Copyright © 2011 Elsevier Inc. All rights reserved.

  17. Attenuated vasodilator effectiveness of protease-activated receptor 2 agonist in heterozygous par2 knockout mice.

    Directory of Open Access Journals (Sweden)

    John C Hennessey

    Full Text Available Studies of homozygous PAR2 gene knockout mice have described a mix of phenotypic effects in vitro and in vivo. However, there have been few studies of PAR2 heterozygous (wild-type/knockout; PAR2-HET mice. The phenotypes of many hemi and heterozygous transgenic mice have been described as intermediates between those of wild-type and knockout animals. In our study we aimed to determine the effects of intermediary par2 gene zygosity on vascular tissue responses to PAR2 activation. Specifically, we compared the vasodilator effectiveness of the PAR2 activating peptide 2-furoyl-LIGRLO-amide in aortas of wild-type PAR2 homozygous (PAR2-WT and PAR2-HET mice. In myographs under isometric tension conditions, isolated aortic rings were contracted by alpha 1-adrenoeceptor agonist (phenylephrine, and thromboxane receptor agonist (U46619 and then relaxation responses by the additions of 2-furoyl-LIGRLO-amide, acetylcholine, and nitroprusside were recorded. A Schild regression analysis of the inhibition by a PAR2 antagonist (GB-83 of PAR2 agonist-induced aortic ring relaxations was used to compare receptor expression in PAR2-WT to PAR2-HET. PAR2 mRNA in aortas was measured by quantitative real-time PCR. In aortas contracted by either phenylephrine or U46619, the maximum relaxations induced by 2-furoyl-LIGRLO-amide were less in PAR2-HET than in the gender-matched PAR2-WT. GB-83 was 3- to 4-fold more potent for inhibition of 2fly in PAR2-HET than in PAR2-WT. PAR2 mRNA content of aortas from PAR2-HET was not significantly different than in PAR2-WT. Acetylcholine- and nitroprusside-induced relaxations of aortas from PAR2-HET were not significantly different than in PAR2-WT and PAR2 knockout. An interesting secondary finding was that relaxations induced by agonists of PAR2 and muscarinic receptors were larger in females than in males. We conclude that the lower PAR2-mediated responses in PAR2-HET aortas are consistent with evidence of a lower quantity of functional

  18. Impairments in the initiation of maternal behavior in oxytocin receptor knockout mice.

    Directory of Open Access Journals (Sweden)

    Megan E Rich

    Full Text Available Oxytocin (Oxt acting through its single receptor subtype, the Oxtr, is important for the coordination of physiology and behavior associated with parturition and maternal care. Knockout mouse models have been helpful in exploring the contributions of Oxt to maternal behavior, including total body Oxt knockout (Oxt -/- mice, forebrain conditional Oxtr knockout (Oxtr FB/FB mice, and total body Oxtr knockout (Oxtr -/- mice. Since Oxtr -/- mice are unable to lactate, maternal behavior has only been examined in virgin females, or in dams within a few hours of parturition, and there have been no studies that have examined their anxiety-like and depression-like behavior following parturition. To improve our understanding of how the absence of Oxt signaling affects maternal behavior, mood and anxiety, we designed a study using Oxtr -/- mice that separated nursing behavior from other aspects of maternal care, such as licking and grooming by thelectomizing (i.e. removing the nipples of Oxtr +/+ mice and sham-thelectomizing Oxtr -/- mice, and pairing both genotypes with a wet nurse. We then measured pup abandonment, maternal behavior, and postpartum anxiety-like and depression-like behaviors. We hypothesized that genetic disruption of the Oxtr would impact maternal care, mood and anxiety. Specifically, we predicted that Oxtr -/- dams would have impaired maternal care and increased anxiety-like and depression-like behaviors in the postpartum period. We found that Oxtr -/- dams had significantly higher levels of pup abandonment compared to controls, which is consistent with previous work in Oxtr FB/FB mice. Interestingly, Oxtr -/- dams that initiated maternal care did not differ from wildtype controls in measures of maternal behavior. We also did not find any evidence of altered anxiety-like or depressive-like behavior in the postpartum period of Oxtr -/- dams. Thus, our data suggest that Oxt lowers the threshold for the initiation of maternal behavior.

  19. Impaired functional organization in the visual cortex of muscarinic receptor knock-out mice.

    Science.gov (United States)

    Groleau, Marianne; Nguyen, Hoang Nam; Vanni, Matthieu P; Huppé-Gourgues, Frédéric; Casanova, Christian; Vaucher, Elvire

    2014-09-01

    Acetylcholine modulates maturation and neuronal activity through muscarinic and nicotinic receptors in the primary visual cortex. However, the specific contribution of different muscarinic receptor subtypes in these neuromodulatory mechanisms is not fully understood. The present study evaluates in vivo the functional organization and the properties of the visual cortex of different groups of muscarinic receptor knock-out (KO) mice. Optical imaging of intrinsic signals coupled to continuous and episodic visual stimulation paradigms was used. Retinotopic maps along elevation and azimuth were preserved among the different groups of mice. However, compared to their wild-type counterparts, the apparent visual field along elevation was larger in M2/M4-KO mice but smaller in M1-KO. There was a reduction in the estimated relative receptive field size of V1 neurons in M1/M3-KO and M1-KO mice. Spatial frequency and contrast selectivity of V1 neuronal populations were affected only in M1/M3-KO and M1-KO mice. Finally, the neuronal connectivity was altered by the absence of M2/M4 muscarinic receptors. All these effects suggest the distinct roles of different subtypes of muscarinic receptors in the intrinsic organization of V1 and a strong involvement of the muscarinic transmission in the detectability of visual stimuli. Copyright © 2014 Elsevier Inc. All rights reserved.

  20. Apelin rs2235306 polymorphism is not related to metabolic ...

    African Journals Online (AJOL)

    Eman T. Mehanna

    2014-11-27

    Nov 27, 2014 ... Abstract Background: Apelin is an adipokine that was identified to play a role in the control of glucose homeostasis. Apelin rs2235306 gene polymorphism was linked to insulin resistance and poor glycemic control. Aim of the study: To assess the relation of apelin rs2235306 polymorphism with metabolic ...

  1. Promoting effect of adipocytokine, apelin, on hepatic injury in ...

    African Journals Online (AJOL)

    Marwa N. Emam

    2015-12-14

    Dec 14, 2015 ... olytic enzyme secretion that causes pancreatic acinar autolysis with progressive interstitial edema just one hour after injec- tion. It is used to induce experimental AP models in rats and mice.9. 2.4. Apelin-13 treated acute pancreatitis group. Apelin-13 (Apelin®, Phoenix Pharmaceutical, Belmont, CA,. USA) ...

  2. Apelin rs2235306 polymorphism is not related to metabolic ...

    African Journals Online (AJOL)

    Background: Apelin is an adipokine that was identified to play a role in the control of glucose homeostasis. Apelin rs2235306 gene polymorphism was linked to insulin resistance and poor glycemic control. Aim of the study: To assess the relation of apelin rs2235306 polymorphism with metabolic syndrome and its ...

  3. Raphe serotonin neuron-specific oxytocin receptor knockout reduces aggression without affecting anxiety-like behavior in male mice only

    Science.gov (United States)

    Pagani, Jerome H.; Williams Avram, Sarah K.; Cui, Zhenzhong; Song, June; Mezey, Éva; Senerth, Julia M.; Baumann, Michael H.; Young, W. Scott

    2015-01-01

    Serotonin and oxytocin influence aggressive and anxiety-like behaviors, though it is unclear how the two may interact. That the oxytocin receptor is expressed in the serotonergic raphe nuclei suggests a mechanism by which the two neurotransmitters may cooperatively influence behavior. We hypothesized that oxytocin acts on raphe neurons to influence serotonergically-mediated anxiety-like, aggressive and parental care behaviors. We eliminated expression of the oxytocin receptor in raphe neurons by crossing mice expressing Cre recombinase under control of the serotonin transporter promoter (Slc6a4) with our conditional oxytocin receptor knockout line. The knockout mice generated by this cross are normal across a range of behavioral measures: there are no effects for either sex on locomotion in an open-field, olfactory habituation/dishabituation or, surprisingly, anxiety-like behaviors in the elevated O and plus mazes. There was a profound deficit in male aggression: only one of 11 raphe oxytocin receptor knockouts showed any aggressive behavior, compared to eight of 11 wildtypes. In contrast, female knockouts displayed no deficits in maternal behavior or aggression. Our results show that oxytocin, via its effects on raphe neurons, is a key regulator of resident-intruder aggression in males but not maternal aggression. Furthermore, this reduction in male aggression is quite different from the effects reported previously after forebrain or total elimination of oxytocin receptors. Finally, we conclude that when constitutively eliminated, oxytocin receptors expressed by serotonin cells do not contribute to baseline anxiety-like behaviors or maternal care. PMID:25677455

  4. Isolation and characterization of coronary endothelial and smooth muscle cells from A1 adenosine receptor-knockout mice

    OpenAIRE

    Teng, Bunyen; Ansari, Habib R.; Oldenburg, Peter J.; Schnermann, J.; Mustafa, S. Jamal

    2005-01-01

    Mice have been used widely in in vivo and in vitro cardiovascular research. The availability of knockout mice provides further clues to the physiological significance of specific receptor subtypes. Adenosine A1 receptor (A1AR)-knockout (A1KO) mice and their wild-type (A1WT) controls were employed in this investigation. The heart and aortic arch were carefully removed and retroinfused with enzyme solution (1 mg/ml collagenase type I, 0.5 mg/ml soybean trypsin inhibitor, 3% BSA, and 2% antibiot...

  5. Comparative effects of chlorpyrifos in wild type and cannabinoid Cb1 receptor knockout mice

    Energy Technology Data Exchange (ETDEWEB)

    Baireddy, Praveena; Liu, Jing; Hinsdale, Myron; Pope, Carey, E-mail: carey.pope@okstate.edu

    2011-11-15

    Endocannabinoids (eCBs) modulate neurotransmission by inhibiting the release of a variety of neurotransmitters. The cannabinoid receptor agonist WIN 55.212-2 (WIN) can modulate organophosphorus (OP) anticholinesterase toxicity in rats, presumably by inhibiting acetylcholine (ACh) release. Some OP anticholinesterases also inhibit eCB-degrading enzymes. We studied the effects of the OP insecticide chlorpyrifos (CPF) on cholinergic signs of toxicity, cholinesterase activity and ACh release in tissues from wild type (+/+) and cannabinoid CB1 receptor knockout (-/-) mice. Mice of both genotypes (n = 5-6/treatment group) were challenged with CPF (300 mg/kg, 2 ml/kg in peanut oil, sc) and evaluated for functional and neurochemical changes. Both genotypes exhibited similar cholinergic signs and cholinesterase inhibition (82-95% at 48 h after dosing) in cortex, cerebellum and heart. WIN reduced depolarization-induced ACh release in vitro in hippocampal slices from wild type mice, but had no effect in hippocampal slices from knockouts or in striatal slices from either genotype. Chlorpyrifos oxon (CPO, 100 {mu}M) reduced release in hippocampal slices from both genotypes in vitro, but with a greater reduction in tissues from wild types (21% vs 12%). CPO had no significant in vitro effect on ACh release in striatum. CPF reduced ACh release in hippocampus from both genotypes ex vivo, but reduction was again significantly greater in tissues from wild types (52% vs 36%). In striatum, CPF led to a similar reduction (20-23%) in tissues from both genotypes. Thus, while CB1 deletion in mice had little influence on the expression of acute toxicity following CPF, CPF- or CPO-induced changes in ACh release appeared sensitive to modulation by CB1-mediated eCB signaling in a brain-regional manner. -- Highlights: Black-Right-Pointing-Pointer C57Bl/6 mice showed dose-related cholinergic toxicity following subcutaneous chlorpyrifos exposure. Black-Right-Pointing-Pointer Wild type and

  6. Receptor for advanced glycation end products (RAGE) knockout reduces fetal dysmorphogenesis in murine diabetic pregnancy.

    Science.gov (United States)

    Ejdesjö, Andreas; Brings, Sebastian; Fleming, Thomas; Fred, Rikard G; Nawroth, Peter P; Eriksson, Ulf J

    2016-07-01

    The receptor for Advanced Glycation End products (RAGE) is implicated in the pathogenesis of diabetic complications, but its importance in diabetic embryopathy is unclear. We therefore investigated the role of RAGE in diabetic embryopathy using streptozotocin induced diabetes in female wild type (WT) C57Bl/6N and RAGE knockout C57Bl/6N (RAGE(-/-)) mice, mated with control males of the same genotype. Maternal diabetes induced more fetal resorption and malformation (facial skeleton, neural tube) in the WT than in the RAGE(-/-) fetuses. Maternal plasma glucose and methylgyoxal concentrations, as well as embryonic N(ε)-carboxymethyl-lysine (CML) levels were increased to the same extent in diabetic WT and RAGE(-/-) pregnancy. However, maternal diabetes induced increased fetal hepatic isoprostane 8-iso-PGF2α levels (oxidative stress marker) and embryonic activation of NFκB in WT only (not in RAGE(-/-) embryos). The association between RAGE knockout and diminished embryonic dysmorphogenesis in diabetic pregnancy suggests that embryonic RAGE activation is involved in diabetic embryopathy. Copyright © 2016 Elsevier Inc. All rights reserved.

  7. Estrogens and Spermiogenesis: New Insights from Type 1 Cannabinoid Receptor Knockout Mice

    Directory of Open Access Journals (Sweden)

    Giovanna Cacciola

    2013-01-01

    Full Text Available Spermatogenesis is a complex mechanism which allows the production of male gametes; it consists of mitotic, meiotic, and differentiation phases. Spermiogenesis is the terminal differentiation process during which haploid round spermatids undergo several biochemical and morphological changes, including extensive remodelling of chromatin and nuclear shape. Spermiogenesis is under control of endocrine, paracrine, and autocrine factors, like gonadotropins and testosterone. More recently, emerging pieces of evidence are suggesting that, among these factors, estrogens may have a role. To date, this is a matter of debate and concern because of the agonistic and antagonistic estrogenic effects that environmental chemicals may have on animal and human with damaging outcome on fertility. In this review, we summarize data which fuel this debate, with a particular attention to our recent results, obtained using type 1 cannabinoid receptor knockout male mice as animal model.

  8. AVE 0991-angiotensin-(1-7) receptor agonist, inhibits atherogenesis in apoE-knockout mice.

    Science.gov (United States)

    Toton-Zuranska, J; Gajda, M; Pyka-Fosciak, G; Kus, K; Pawlowska, M; Niepsuj, A; Wolkow, P; Olszanecki, R; Jawien, J; Korbut, R

    2010-04-01

    Recent evidence shows that the renin-angiotensin system is a crucial player in atherosclerotic processes. It was also proved that Ang II promotes atherogenesis. Angiotensin-(1-7) [Ang-(1-7)] opposites Ang II action. Therefore, we would like to find out whether Ang-(1-7) receptor agonist: AVE 0991, could ameliorate atherosclerosis progression in an experimental model of atherosclerosis: apolipoprotein E (apoE) - knockout mice. AVE 0991 inhibited atherogenesis, measured both by "en face" method (7.63+/-1.6% vs. 14.6+/-2.1%) and "cross-section" method (47 235+/-7 546 microm(2) vs. 91 416+/-8 357 microm(2)). This is the first report showing the effect of AVE 0991 on atherogenesis in gene-targeted mice.

  9. Apelin-13 inhibits large-conductance Ca2+-activated K+ channels in cerebral artery smooth muscle cells via a PI3-kinase dependent mechanism.

    Directory of Open Access Journals (Sweden)

    Amit Modgil

    Full Text Available Apelin-13 causes vasoconstriction by acting directly on APJ receptors in vascular smooth muscle (VSM cells; however, the ionic mechanisms underlying this action at the cellular level remain unclear. Large-conductance Ca(2+-activated K(+ (BKCa channels in VSM cells are critical regulators of membrane potential and vascular tone. In the present study, we examined the effect of apelin-13 on BK(Ca channel activity in VSM cells, freshly isolated from rat middle cerebral arteries. In whole-cell patch clamp mode, apelin-13 (0.001-1 μM caused concentration-dependent inhibition of BK(Ca in VSM cells. Apelin-13 (0.1 µM significantly decreased BK(Ca current density from 71.25 ± 8.14 pA/pF to 44.52 ± 7.10 pA/pF (n=14 cells, P<0.05. This inhibitory effect of apelin-13 was confirmed by single channel recording in cell-attached patches, in which extracellular application of apelin-13 (0.1 µM decreased the open-state probability (NPo of BK(Ca channels in freshly isolated VSM cells. However, in inside-out patches, extracellular application of apelin-13 (0.1 µM did not alter the NPo of BK(Ca channels, suggesting that the inhibitory effect of apelin-13 on BKCa is not mediated by a direct action on BK(Ca. In whole cell patches, pretreatment of VSM cells with LY-294002, a PI3-kinase inhibitor, markedly attenuated the apelin-13-induced decrease in BK(Ca current density. In addition, treatment of arteries with apelin-13 (0.1 µM significantly increased the ratio of phosphorylated-Akt/total Akt, indicating that apelin-13 significantly increases PI3-kinase activity. Taken together, the data suggest that apelin-13 inhibits BK(Ca channel via a PI3-kinase-dependent signaling pathway in cerebral artery VSM cells, which may contribute to its regulatory action in the control of vascular tone.

  10. Hyperfunction of muscarinic receptor maintains long-term memory in 5-HT4 receptor knock-out mice.

    Directory of Open Access Journals (Sweden)

    Luis Segu

    Full Text Available Patients suffering from dementia of Alzheimer's type express less serotonin 4 receptors (5-HTR(4, but whether an absence of these receptors modifies learning and memory is unexplored. In the spatial version of the Morris water maze, we show that 5-HTR(4 knock-out (KO and wild-type (WT mice performed similarly for spatial learning, short- and long-term retention. Since 5-HTR(4 control mnesic abilities, we tested whether cholinergic system had circumvented the absence of 5-HTR(4. Inactivating muscarinic receptor with scopolamine, at an ineffective dose (0.8 mg/kg to alter memory in WT mice, decreased long-term but not short-term memory of 5-HTR(4 KO mice. Other changes included decreases in the activity of choline acetyltransferase (ChAT, the required enzyme for acetylcholine synthesis, in the septum and the dorsal hippocampus in 5-HTR(4 KO under baseline conditions. Training- and scopolamine-induced increase and decrease, respectively in ChAT activity in the septum in WT mice were not detected in the 5-HTR(4 KO animals. Findings suggest that adaptive changes in cholinergic systems may circumvent the absence of 5-HTR(4 to maintain long-term memory under baseline conditions. In contrast, despite adaptive mechanisms, the absence of 5-HTR(4 aggravates scopolamine-induced memory impairments. The mechanisms whereby 5-HTR(4 mediate a tonic influence on ChAT activity and muscarinic receptors remain to be determined.

  11. Alterations in apical dendrite bundling in the somatosensory cortex of 5-HT3A receptor knockout mice.

    NARCIS (Netherlands)

    Smit-Rigter, L.A.; Wadman, W.J.; van Hooft, J.A.

    2011-01-01

    In various species and areas of the cerebral cortex, apical dendrites of pyramidal neurons form clusters which extend through several layers of the cortex also known as dendritic bundles. Previously, it has been shown that 5-HT3A receptor knockout mice show hypercomplex apical dendrites of cortical

  12. Enhanced proliferation of astrocytes from beta(2)-adrenergic receptor knockout mice is influenced by the IGF system

    NARCIS (Netherlands)

    Chesik, Daniel; Glazenburg, Lisa; De Keyser, Jacques; Wilczak, Nadine

    In the present study, we investigated the IGF system in neonatal astrocytes derived from mice with a targeted disruption of the beta-2 adrenergic receptor (beta(2)AR). beta(2)AR knockout astrocytes demonstrated higher proliferation rates and increased expression of the astrogliotic marker GFAP, as

  13. Brain Region-Specific Effects of cGMP-Dependent Kinase II Knockout on AMPA Receptor Trafficking and Animal Behavior

    Science.gov (United States)

    Kim, Seonil; Pick, Joseph E.; Abera, Sinedu; Khatri, Latika; Ferreira, Danielle D. P.; Sathler, Matheus F.; Morison, Sage L.; Hofmann, Franz; Ziff, Edward B.

    2016-01-01

    Phosphorylation of GluA1, a subunit of AMPA receptors (AMPARs), is critical for AMPAR synaptic trafficking and control of synaptic transmission. cGMP-dependent protein kinase II (cGKII) mediates this phosphorylation, and cGKII knockout (KO) affects GluA1 phosphorylation and alters animal behavior. Notably, GluA1 phosphorylation in the KO…

  14. Regulation of dopamine presynaptic markers and receptors in the striatum of DJ-1 and Pink1 knockout rats

    Science.gov (United States)

    Sun, Jianjun; Kouranova, Evguenia; Cui, Xiaoxia; Mach, Robert H.; Xu, Jinbin

    2014-01-01

    Pathogenic autosomal recessive mutations in the DJ-1 (Park7) or the PTEN-induced putative kinase 1 (Pink1 or PARK6) genes are associated with familial Parkinson’s disease (PD). It is not well known regarding the pathological mechanisms involving the DJ-1 and Pink1 mutations. Here we characterized DJ-1 and Pink1 knockout rats both through expression profiling and using quantitative autoradiography to measure the densities of the dopamine D1, D2, D3 receptors, vesicular monoamine transporter type-2 (VMAT2) and dopamine transporter (DAT) in the striatum of transgenic rats and wild type controls. Expression profiling with a commercially available array of 84 genes known to be involved in PD indicated that only the target gene was significantly downregulated in each transgenic rat model. D1 receptor, VMAT2, and DAT were measured using [3H]SCH23390, [3H]dihydrotetrabenazine, and [3H]WIN35428, respectively. No significant changes were observed in the density of DAT in either model. Although the densities of VMAT2 and D1 receptor were unchanged in Pink1 knockout, but both were increased in DJ-1 knockout rats. The densities of D2 and D3 receptors, determined by mathematical analysis of binding of radioligands [3H]WC-10 and [3H]raclopride, were significantly increased in both knockout models. These distinctive changes in the expression of dopamine presynaptic markers and receptors in the striatum may reflect different compensatory regulation of dopamine system in DJ-1 versus Pink1 knockout rat models of familial PD. PMID:24157858

  15. Hypoxic Exercise Training Promotes apelin/APJ Expression in Skeletal Muscles of High Fat Diet-Induced Obese Mice.

    Science.gov (United States)

    Ji, Weixiu; Gong, Lijing; Wang, Jianxiong; He, Hui; Zhang, Ying

    2017-01-01

    Apelin, an endogenous ligand of the G-protein-coupled receptor APJ, is a novel myokine and may play a key role in regulating energy metabolism. The purpose of the present study was to investigate the effects of hypoxic exposure, exercise, and hypoxic exercise training on the expression of apelin and APJ in skeletal muscle of obese mice. Sixty two-months old C57BL/6J mice were randomly divided into two groups: Ten in normal diet group (N) and 50 in the high fat diet (HFD) groups. After two months of feeding, the HFD mice, whose body weight was 20% higher than the average weight of the N group, were selected as obese mice and further allocated into four groups: Control (C), Exercise (E), Hypoxia (H), and Exercise plus Hypoxia (E+H), at 8-9 mice/group. Besides body weight, measured variables in skeletal muscle were protein/mRNA levels of apelin/APJ, AMPKα-Thr172 phosphorylation, hypoxia inducible factor-1α (HIF-1α), mRNA levels of peroxisome proliferator-activated receptor α (PPARα), estrogen-related receptor (ERRα), and nuclear respiratory factor 1 (NRF1). Obese mice had significantly lower mRNA and protein expressions of apelin/ APJ in skeletal muscles than the normal body weight mice. After four weeks of interventions, hypoxic exercise training decreased body weight and increased mRNA and protein expressions of apelin and APJ, mRNA expression of ERRα, and protein expression of HIF-1α. These results indicate that changes of body weight may be associated with the levels of apelin/APJ expressions in skeletal muscle. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  16. Somatostatin receptor 1 and 5 double knockout mice mimic neurochemical changes of Huntington's disease transgenic mice.

    Directory of Open Access Journals (Sweden)

    Padmesh S Rajput

    Full Text Available Selective degeneration of medium spiny neurons and preservation of medium sized aspiny interneurons in striatum has been implicated in excitotoxicity and pathophysiology of Huntington's disease (HD. However, the molecular mechanism for the selective sparing of medium sized aspiny neurons and vulnerability of projection neurons is still elusive. The pathological characteristic of HD is an extensive reduction of the striatal mass, affecting caudate putamen. Somatostatin (SST positive neurons are selectively spared in HD and Quinolinic acid/N-methyl-D-aspartic acid induced excitotoxicity, mimic the model of HD. SST plays neuroprotective role in excitotoxicity and the biological effects of SST are mediated by five somatostatin receptor subtypes (SSTR1-5.To delineate subtype selective biological responses we have here investigated changes in SSTR1 and 5 double knockout mice brain and compared with HD transgenic mouse model (R6/2. Our study revealed significant loss of dopamine and cAMP regulated phosphoprotein of 32 kDa (DARPP-32 and comparable changes in SST, N-methyl-D-aspartic acid receptors subtypes, calbindin and brain nitric oxide synthase expression as well as in key signaling proteins including calpain, phospho-extracellular-signal-regulated kinases1/2, synapsin-IIa, protein kinase C-α and calcineurin in SSTR1/5(-/- and R6/2 mice. Conversely, the expression of somatostatin receptor subtypes, enkephalin and phosphatidylinositol 3-kinases were strain specific. SSTR1/5 appears to be important in regulating NMDARs, DARPP-32 and signaling molecules in similar fashion as seen in HD transgenic mice.This is the first comprehensive description of disease related changes upon ablation of G- protein coupled receptor gene. Our results indicate that SST and SSTRs might play an important role in regulation of neurodegeneration and targeting this pathway can provide a novel insight in understanding the pathophysiology of Huntington's disease.

  17. alpha7 Nicotinic acetylcholine receptor knockout selectively enhances ethanol-, but not beta-amyloid-induced neurotoxicity.

    Science.gov (United States)

    de Fiebre, Nancyellen C; de Fiebre, Christopher M

    2005-01-03

    The alpha7 subtype of nicotinic acetylcholine receptor (nAChR) has been implicated as a potential site of action for two neurotoxins, ethanol and the Alzheimer's disease related peptide, beta-amyloid. Here, we utilized primary neuronal cultures of cerebral cortex from alpha7 nAChR null mutant mice to examine the role of this receptor in modulating the neurotoxic properties of subchronic, "binge" ethanol and beta-amyloid. Knockout of the alpha7 nAChR gene selectively enhanced ethanol-induced neurotoxicity in a gene dosage-related fashion. Susceptibility of cultures to beta-amyloid induced toxicity, however, was unaffected by alpha7 nAChR gene null mutation. Further, beta-amyloid did not inhibit the binding of the highly alpha7-selective radioligand, [(125)I]alpha-bungarotoxin. On the other hand, in studies in Xenopus oocytes ethanol efficaciously inhibited alpha7 nAChR function. These data suggest that alpha7 nAChRs modulate the neurotoxic effects of binge ethanol, but not the neurotoxicity produced by beta-amyloid. It is hypothesized that inhibition of alpha7 nAChRs by ethanol provides partial protection against the neurotoxic properties of subchronic ethanol.

  18. Raphe serotonin neuron-specific oxytocin receptor knockout reduces aggression without affecting anxiety-like behavior in male mice only.

    Science.gov (United States)

    Pagani, J H; Williams Avram, S K; Cui, Z; Song, J; Mezey, É; Senerth, J M; Baumann, M H; Young, W S

    2015-02-01

    Serotonin and oxytocin influence aggressive and anxiety-like behaviors, though it is unclear how the two may interact. That the oxytocin receptor is expressed in the serotonergic raphe nuclei suggests a mechanism by which the two neurotransmitters may cooperatively influence behavior. We hypothesized that oxytocin acts on raphe neurons to influence serotonergically mediated anxiety-like, aggressive and parental care behaviors. We eliminated expression of the oxytocin receptor in raphe neurons by crossing mice expressing Cre recombinase under control of the serotonin transporter promoter (Slc6a4) with our conditional oxytocin receptor knockout line. The knockout mice generated by this cross are normal across a range of behavioral measures: there are no effects for either sex on locomotion in an open-field, olfactory habituation/dishabituation or, surprisingly, anxiety-like behaviors in the elevated O and plus mazes. There was a profound deficit in male aggression: only one of 11 raphe oxytocin receptor knockouts showed any aggressive behavior, compared to 8 of 11 wildtypes. In contrast, female knockouts displayed no deficits in maternal behavior or aggression. Our results show that oxytocin, via its effects on raphe neurons, is a key regulator of resident-intruder aggression in males but not maternal aggression. Furthermore, this reduction in male aggression is quite different from the effects reported previously after forebrain or total elimination of oxytocin receptors. Finally, we conclude that when constitutively eliminated, oxytocin receptors expressed by serotonin cells do not contribute to baseline anxiety-like behaviors or maternal care. © 2015 John Wiley & Sons Ltd and International Behavioural and Neural Genetics Society.

  19. Soy milk versus simvastatin for preventing atherosclerosis and left ventricle remodeling in LDL receptor knockout mice.

    Science.gov (United States)

    Santos, L; Davel, A P; Almeida, T I R; Almeida, M R; Soares, E A; Fernandes, G J M; Magalhães, S F; Barauna, V G; Garcia, J A D

    2017-02-20

    Functional food intake has been highlighted as a strategy for the prevention of cardiovascular diseases by reducing risk factors. In this study, we compared the effects of oral treatment with soy milk and simvastatin on dyslipidemia, left ventricle remodeling and atherosclerotic lesion of LDL receptor knockout mice (LDLr-/-) fed a hyperlipidic diet. Forty 3-month old male LDLr-/- mice were distributed into four groups: control group (C), in which animals received standard diet; HL group, in which animals were fed a hyperlipidic diet; HL+SM or HL+S groups, in which animals were submitted to a hyperlipidic diet plus soy milk or simvastatin, respectively. After 60 days, both soy milk and simvastatin treatment prevented dyslipidemia, atherosclerotic lesion progression and left ventricle hypertrophy in LDLr-/- mice. These beneficial effects of soy milk and simvastatin were associated with reduced oxidative stress and inflammatory state in the heart and aorta caused by the hyperlipidic diet. Treatment with soy milk was more effective in preventing HDLc reduction and triacylglycerol and VLDLc increase. On the other hand, simvastatin was more effective in preventing an increase in total cholesterol, LDLc and superoxide production in aorta, as well as CD40L both in aorta and left ventricle of LDLr-/-. In conclusion, our results suggest a cardioprotective effect of soy milk in LDLr-/- mice comparable to the well-known effects of simvastatin.

  20. Muscarinic acetylcholine receptor knockout mice show distinct synaptic plasticity impairments in the visual cortex

    Science.gov (United States)

    Origlia, Nicola; Kuczewski, Nicola; Aztiria, Eugenio; Gautam, Dinesh; Wess, Jürgen; Domenici, Luciano

    2006-01-01

    In the present report, we focused our attention on the role played by the muscarinic acetylcholine receptors (mAChRs) in different forms of long-term synaptic plasticity. Specifically, we investigated long-term potentiation (LTP) and long-term depression (LTD) expression elicited by theta-burst stimulation (TBS) and low-frequency stimulation (LFS), respectively, in visual cortical slices obtained from different mAChR knockout (KO) mice. A normal LTP was evoked in M1/M3 double KO mice, while LTP was impaired in the M2/M4 double KO animals. On the other hand, LFS induced LTD in M2/M4 double KO mice, but failed to do so in M1/M3 KO mice. Interestingly, LFS produced LTP instead of LTD in M1/M3 KO mice. Analysis of mAChR single KO mice revealed that LTP was affected only by the simultaneous absence of both M2 and M4 receptors. A LFS-dependent shift from LTD to LTP was also observed in slices from M1 KO mice, while LTD was simply abolished in slices from M3 KO mice. Using pharmacological tools, we showed that LTP in control mice was blocked by pertussis toxin, an inhibitor of Gi/o proteins, but not by raising intracellular cAMP levels. In addition, the inhibition of phospholipase C by U73122 induced the same shift from LTD to LTP after LFS observed in M1 single KO and M1/M3 double KO mice. Our results indicate that different mAChR subtypes regulate different forms of long-term synaptic plasticity in the mouse visual cortex, activating specific G proteins and downstream intracellular mechanisms. PMID:17023506

  1. Moderate Continuous Aerobic Exercise Training Improves Cardiomyocyte Contractility in Β1 Adrenergic Receptor Knockout Mice

    Directory of Open Access Journals (Sweden)

    Aurora Corrêa Rodrigues

    2018-02-01

    Full Text Available Abstract Background: The lack of cardiac β1-adrenergic receptors (β1-AR negatively affects the regulation of both cardiac inotropy and lusitropy, leading, in the long term, to heart failure (HF. Moderate-intensity aerobic exercise (MCAE is recommended as an adjunctive therapy for patients with HF. Objective: We tested the effects of MCAE on the contractile properties of left ventricular (LV myocytes from β1 adrenergic receptor knockout (β1ARKO mice. Methods: Four- to five-month-old male wild type (WT and β1ARKO mice were divided into groups: WT control (WTc and trained (WTt; and β1ARKO control (β1ARKOc and trained (β1ARKOt. Animals from trained groups were submitted to a MCAE regimen (60 min/day; 60% of maximal speed, 5 days/week on a treadmill, for 8 weeks. P ≤ 0.05 was considered significant in all comparisons. Results: The β1ARKO and exercised mice exhibited a higher (p < 0.05 running capacity than WT and sedentary ones, respectively. The β1ARKO mice showed higher body (BW, heart (HW and left ventricle (LVW weights, as well as the HW/BW and LVW/BW than WT mice. However, the MCAE did not affect these parameters. Left ventricular myocytes from β1ARKO mice showed increased (p < 0.05 amplitude and velocities of contraction and relaxation than those from WT. In addition, MCAE increased (p < 0.05 amplitude and velocities of contraction and relaxation in β1ARKO mice. Conclusion: MCAE improves myocyte contractility in the left ventricle of β1ARKO mice. This is evidence to support the therapeutic value of this type of exercise training in the treatment of heart diseases involving β1-AR desensitization or reduction.

  2. Multiple sweet receptors and transduction pathways revealed in knockout mice by temperature dependence and gurmarin sensitivity.

    Science.gov (United States)

    Ohkuri, Tadahiro; Yasumatsu, Keiko; Horio, Nao; Jyotaki, Masafumi; Margolskee, Robert F; Ninomiya, Yuzo

    2009-04-01

    Sweet taste transduction involves taste receptor type 1, member 2 (T1R2), taste receptor type 1, member 3 (T1R3), gustducin, and TRPM5. Because knockout (KO) mice lacking T1R3, gustducin's Galpha subunit (Galphagust), or TRPM5 exhibited greatly reduced, but not abolished responses of the chorda tympani (CT) nerve to sweet compounds, it is likely that multiple sweet transduction pathways exist. That gurmarin (Gur), a sweet taste inhibitor, inhibits some but not all mouse CT responses to sweet compounds supports the existence of multiple sweet pathways. Here, we investigated Gur inhibition of CT responses to sweet compounds as a function of temperature in KO mice lacking T1R3, Galphagust, or TRPM5. In T1R3-KO mice, responses to sucrose and glucose were Gur sensitive (GS) and displayed a temperature-dependent increase (TDI). In Galphagust-KO mice, responses to sucrose and glucose were Gur-insensitive (GI) and showed a TDI. In TRPM5-KO mice, responses to glucose were GS and showed a TDI. All three KO mice exhibited no detectable responses to SC45647, and their responses to saccharin displayed neither GS nor a TDI. For all three KO mice, the lingual application of pronase, another sweet response inhibitor, almost fully abolished responses to sucrose and glucose but did not affect responses to saccharin. These results provide evidence for 1) the existence of multiple transduction pathways underlying responses to sugars: a T1R3-independent GS pathway for sucrose and glucose, and a TRPM5-independent temperature sensitive GS pathway for glucose; 2) the requirement for Galphagust in GS sweet taste responses; and 3) the existence of a sweet independent pathway for saccharin, in mouse taste cells on the anterior tongue.

  3. Lectin-like oxidized low-density lipoprotein receptor-1 promotes endothelial dysfunction in LDL receptor knockout background.

    Science.gov (United States)

    Hofmann, Anja; Brunssen, Coy; Poitz, David M; Langbein, Heike; Strasser, Ruth H; Henle, Thomas; Ravens, Ursula; Morawietz, Henning

    2017-11-01

    Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is the major receptor for oxidized LDL in endothelial cells. LOX-1 is highly expressed in atherosclerotic plaques. The impact of LOX-1 on development of endothelial dysfunction in large vessels in absence or presence of atherosclerosis-prone conditions has not been studied to date. Mice with endothelial cell-specific LOX-1 overexpression (bLOX-1tg) were analyzed. Wild-type (WT) mice served as controls. In addition, bLOX-1tg mice were crossed with LDL receptor knockout (Ldlr -/- ) mice. All mice were fed a western-type diet (WD) or control diet (CD) for 20 weeks. Afterwards, endothelial function was analyzed ex vivo in thoracic aortas using a Mulvany myograph. WD induced hypertriglyceridemia (bLOX-1tg: 1.6-fold; WT: 1.4-fold) and hypercholesterolemia (P LDL-cholesterol (∼9-fold) compared to WT and bLOX-1tg mice on WD. Endothelial function in response to WD was impaired in bLOX-1tg/Ldlr -/- mice (Eff max : 56.7 ± 23.0%) compared to WT (Eff max : 88.2 ± 15.8%, P < 0.001), bLOX-1tg (Eff max : 76.7 ± 12.9%, P < 0.05) and Ldlr -/- mice (Eff max : 70.1 ± 13.1%, P < 0.05). No differences between WT, bLOX-1tg and Ldlr -/- mice were detectable when comparing all genotypes. Endothelial LOX-1 overexpression in an atherosclerosis-prone background impairs endothelial function, proving its importance in the development of atherosclerosis. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. Involvement of multiple taste receptors in umami taste: analysis of gustatory nerve responses in metabotropic glutamate receptor 4 knockout mice.

    Science.gov (United States)

    Yasumatsu, Keiko; Manabe, Tomohiro; Yoshida, Ryusuke; Iwatsuki, Ken; Uneyama, Hisayuki; Takahashi, Ichiro; Ninomiya, Yuzo

    2015-02-15

    The taste receptor T1R1 + T1R3 heterodimer and metabotropic glutamate receptors (mGluR) may function as umami taste receptors. Here, we used mGluR4 knockout (mGluR4-KO) mice and examined the function of mGluR4 in peripheral taste responses of mice. The mGluR4-KO mice showed reduced responses to glutamate and L-AP4 (mGluR4 agonist) in the chorda tympani and glossopharyngeal nerves without affecting responses to other taste stimuli. Residual glutamate responses in mGluR4-KO mice were suppressed by gurmarin (T1R3 blocker) and AIDA (group I mGluR antagonist). The present study not only provided functional evidence for the involvement of mGluR4 in umami taste responses, but also suggested contributions of T1R1 + T1R3 and mGluR1 receptors in glutamate responses. Umami taste is elicited by L-glutamate and some other amino acids and is thought to be initiated by G-protein-coupled receptors. Proposed umami receptors include heterodimers of taste receptor type 1, members 1 and 3 (T1R1 + T1R3), and metabotropic glutamate receptors 1 and 4 (mGluR1 and mGluR4). Accumulated evidences support the involvement of T1R1 + T1R3 in umami responses in mice. However, little is known about the in vivo function of mGluR in umami taste. Here, we examined taste responses of the chorda tympani (CT) and the glossopharyngeal (GL) nerves in wild-type mice and mice genetically lacking mGluR4 (mGluR4-KO). Our results indicated that compared to wild-type mice, mGluR4-KO mice showed significantly smaller gustatory nerve responses to glutamate and L-(+)-2-amino-4-phosphonobutyrate (an agonist for group III mGluR) in both the CT and GL nerves without affecting responses to other taste stimuli. Residual glutamate responses in mGluR4-KO mice were not affected by (RS)-alpha-cyclopropyl-4-phosphonophenylglycine (an antagonist for group III mGluR), but were suppressed by gurmarin (a T1R3 blocker) in the CT and (RS)-1-aminoindan-1,5-dicarboxylic acid (an antagonist for group I mGluR) in the CT and GL nerve

  5. Effects of muscarinic receptor antagonists on cocaine discrimination in wild-type mice and in muscarinic receptor M1, M2, and M4 receptor knockout mice.

    Science.gov (United States)

    Joseph, Lauren; Thomsen, Morgane

    2017-06-30

    Muscarinic M 1 /M 4 receptor stimulation can reduce abuse-related effects of cocaine and may represent avenues for treating cocaine addiction. Muscarinic antagonists can mimic and enhance effects of cocaine, including discriminative stimulus (S D ) effects, but the receptor subtypes mediating those effects are not known. A better understanding of the complex cocaine/muscarinic interactions is needed to evaluate and develop potential muscarinic-based medications. Here, knockout mice lacking M 1 , M 2 , or M 4 receptors (M 1 -/- , M 2 -/- , M 4 -/- ), as well as control wild-type mice and outbred Swiss-Webster mice, were trained to discriminate 10mg/kg cocaine from saline. Muscarinic receptor antagonists with no subtype selectivity (scopolamine), or preferential affinity at the M 1 , M 2 , or M 4 subtype (telenzepine, trihexyphenidyl; methoctramine, AQ-RA 741; tropicamide) were tested alone and in combination with cocaine. In intact animals, antagonists with high affinity at M 1 /M 4 receptors partially substituted for cocaine and increased the S D effect of cocaine, while M 2 -preferring antagonists did not substitute, and reduced the S D effect of cocaine. The cocaine-like effects of scopolamine were absent in M 1 -/- mice. The cocaine S D attenuating effects of methoctramine were absent in M 2 -/- mice and almost absent in M 1 -/- mice. The findings indicate that the cocaine-like S D effects of muscarinic antagonists are primarily mediated through M 1 receptors, with a minor contribution of M 4 receptors. The data also support our previous findings that stimulation of M 1 receptors and M 4 receptors can each attenuate the S D effect of cocaine, and show that this can also be achieved by blocking M 2 autoreceptors, likely via increased acetylcholine release. Copyright © 2017 Elsevier B.V. All rights reserved.

  6. No further loss of dorsal root ganglion cells after axotomy in p75 neurotrophin receptor knockout mice

    DEFF Research Database (Denmark)

    Sørensen, Bodil; Tandrup, Trine; Koltzenburg, Martin

    2003-01-01

    disector techniques. At birth, the total number of DRG neurons was 10,000 ±2,600 in control mice compared with 5,100 ±1,300 in p75 knockout mice. During postnatal development, 1,400 neuronal B-cell bodies were lost in p75 knockouts (2P ± 0.±05) and 1,100 in controls (NS), whereas the A-cell population......The role of the p75 neurotrophin receptor for neuronal survival after nerve crush was studied in L5 dorsal root ganglia (DRG) of knockout mice and controls with assumption-free stereological methods. Numbers of neuronal A- and B-cells were obtained using the optical fractionator and optical...... remained stable. After a sciatic nerve crush, the total neuron loss in controls was 15.4% ±3.5% (2P ±0.05) and 22.7% 5.1% (2P neurons after crush in p75 knockout mice. Neuronal A-cell number was unchanged after...

  7. Comparison of therapeutic effects of L-Thyroxin, apelin and a combination of both on antioxidant enzymes in the heart of PTU-induced hypothyroid rats

    Directory of Open Access Journals (Sweden)

    Farzaneh Faraji Shahrivar

    Full Text Available Atherosclerosis is one of the common disorders among hypothyroidism, which, increased the risk of cardiovascular diseases. Reactive oxygen species are associated with atherosclerosis development. Antioxidant defense systems are the scavenger for free radicals. Apelin is an endogenous ligand for the APJ receptor (apelin receptor that exists in most tissues, acts as an adiponectin. It has been identified that apelin administration, improve the antioxidant capacity (TAC. Therefore, this study was conducted to assess, therapeutic effects of apelin, T4 (L-Thyroxin or both on antioxidant capacity in 6-propyl-2-thiouracil (PTU-induced hypothyroid rats. Forty male Wistar rats were randomly assigned into five groups: C: control group; P group (hypothyroid: PTU (0.05 % administration for six weeks; P+A, P+T and P+A+T groups: after 4 weeks of PTU administration, animals treated with Apelin (200 μg/kg/day, ip T4 (0.02 µg/g/day, gavage and apelin+T4; for two weeks respectively accompanied by PTU administration. Aplein administration in P+A group and P+A+T group had beneficial effect to lowering of malondialdehyde (MDA content as compared to hypothyroid group (8.52±0.64 and 8.53±1 vs. 13.67±1.64 nmol/g tissue, P<0.05 and also had increasing effect on Superoxide dismutase (SOD and glutathion peroxidase (GPx activity and the total antioxidant capacity (TAC content compared to the hypothyroid group. This study showed that apelin was able to improve the oxidant-antioxidant balance in the heart tissue of the hypothyroid rats by elevating of antioxidant enzyme activity.

  8. Ameliorative effect of apelin on streptozotocin-induced diabetes and ...

    African Journals Online (AJOL)

    Islam Ibrahim Hegab

    Aim: Apelin, an adipocyte-derived factor, exhibited a number of cardioprotective properties; however, its effect in diabetes which is a major risk factor for cardiovascular disease (CVD) needs to be further studied. So this work was designed to evaluate the effect of apelin on diabetes and its associated cardiac hyper-.

  9. Contribution of 5-HT2 receptor subtypes to sleep-wakefulness and respiratory control, and functional adaptations in knock-out mice lacking 5-HT2A receptors.

    OpenAIRE

    Popa, Daniela; Léna, Clément; Fabre, Véronique; Prenat, Caroline; Gingrich, Jay; Escourrou, Pierre; Hamon, Michel; Adrien, Joëlle

    2005-01-01

    International audience; Serotonin (5-hydroxytryptamine; 5-HT) plays key roles in sleep-wakefulness regulation. Evidence indicates that 5-HT2 receptors are involved mainly in non-rapid eye movement sleep (NREMS) regulation and respiratory control. Here, we investigated the relative contribution of 5-HT(2A), 5-HT(2B), and 5-HT(2C) receptor subtypes to NREMS and breathing during sleep, using 5-HT2 subtype-selective ligands in wild-type (5-HT(2A)+/+) and knock-out (5-HT(2A)-/-) mice that do not e...

  10. Data on Arc and Zif268 expression in the brain of the α-2A adrenergic receptor knockout mouse

    Directory of Open Access Journals (Sweden)

    Jeff Sanders

    2016-06-01

    Full Text Available The α2-adrenergic receptor (α2-AR is widely distributed in the brain with distinct roles for α2-AR subtypes (A, B and C. In this article, data are provided on Activity Regulated Cytoskeleton Associated Protein (Arc and Zif268 expression in the brain of the α2A-AR knockout (α2A-AR KO mouse. These data are supplemental to an original research article examining Arc and Zif268 expression in rats injected with the α2-AR antagonist, RX821002 (http://dx.doi.org/10.1016/j.neulet.2015.12.002. [1].

  11. Characterization of the retina in the alpha7 nicotinic acetylcholine receptor knockout mouse

    Science.gov (United States)

    Smith, Marci L.

    Acetylcholine receptors (AChRs) are involved in visual processing and are expressed by inner retinal neurons in all species studied to date (Keyser et al., 2000; Dmitrieva et al., 2007; Liu et al., 2009), but their distribution in the mouse retina remains unknown. Reductions in alpha7 nicotinic AChRs (nAChRs) are thought to contribute to memory and visual deficits observed in Alzheimer's and schizophrenia (Coyle et al., 1983; Nordberg et al., 1999; Leonard et al., 2006). However, the alpha7 nAChR knockout (KO) mouse has a mild phenotype (Paylor et al., 1998; Fernandes et al., 2006; Young et al., 2007; Origlia et al., 2012). The purpose of this study was to determine the expression of AChRs in wildtype (WT) mouse retina and to assess whether up-regulation of other AChRs in the alpha7 nAChR KO retina may explain the minimal deficits described in the KO mouse. Reverse-transcriptase PCR (RT-PCR) showed that mRNA transcripts for alpha2-7, alpha 9, alpha10, beta2-4 nAChR subunits and m1-m5 muscarinic AChR (mAChR) subtypes were present in WT murine retina. Western blot analysis confirmed the presence of alpha3-5, alpha9, and m1-m5 AChR proteins and immunohistochemical analysis demonstrated nAChR and mAChR proteins expressed by subsets of bipolar, amacrine and ganglion cells. This is the first reported expression of alpha9 and alpha10 nAChR transcripts and alpha9 nAChR proteins in the retina of any species. Quantitative RT-PCR (qPCR) showed changes in AChR transcript expression in the alpha7 nAChR KO mouse retina relative to WT. Within whole retina alpha2, alpha9, alpha10, beta4, m1 and m4 AChR transcripts were up-regulated, while alpha5 nAChR transcripts were down-regulated. However, cell populations showed subtle differences; m4 mAChR transcripts were up-regulated in the ganglion cell layer and outer portion of the inner nuclear layer (oINL),while beta4 nAChR transcript up-regulation was limited to the oINL. Surprisingly, alpha2, alpha9, beta4, m2 and m4 transcripts were

  12. Cannabinoid 1 receptor knockout mice display cold allodynia, but enhanced recovery from spared-nerve injury-induced mechanical hypersensitivity.

    Science.gov (United States)

    Sideris, Alexandra; Piskoun, Boris; Russo, Lori; Norcini, Monica; Blanck, Thomas; Recio-Pinto, Esperanza

    2016-01-01

    The function of the Cannabinoid 1 receptor (CB1R) in the development of neuropathic pain is not clear. Mounting evidence suggest that CB1R expression and activation may contribute to pain. Cannabinoid 1 receptor knockout mice (CB1R-/-) generated on a C57Bl/6 background exhibit hypoalgesia in the hotplate assay and formalin test. These findings suggest that Cannabinoid 1 receptor expression mediates the responses to at least some types of painful stimuli. By using this mouse line, we sought to determine if the lack of Cannabinoid 1 receptor unveils a general hypoalgesic phenotype, including protection against the development of neuropathic pain. The acetone test was used to measure cold sensitivity, the electronic von Frey was used to measure mechanical thresholds before and after spared-nerve injury, and analysis of footprint patterns was conducted to determine if motor function is differentially affected after nerve-injury in mice with varying levels of Cannabinoid 1 receptor. At baseline, CB1R-/- mice were hypersensitive in the acetone test, and this phenotype was maintained after spared-nerve injury. Using calcium imaging of lumbar dorsal root ganglion (DRG) cultures, a higher percentage of neurons isolated from CB1R-/- mice were menthol sensitive relative to DRG isolated from wild-type (CB1R+/+) mice. Baseline mechanical thresholds did not differ among genotypes, and mechanical hypersensitivity developed similarly in the first two weeks following spared-nerve injury (SNI). At two weeks post-SNI, CB1R-/- mice recovered significantly from mechanical hypersensitivity, while the CB1R+/+ mice did not. Heterozygous knockouts (CB1R+/-) transiently developed cold allodynia only after injury, but recovered mechanical thresholds to a similar extent as the CB1R-/- mice. Sciatic functional indices, which reflect overall nerve health, and alternation coefficients, which indicate uniformity of strides, were not significantly different among genotypes. Cold allodynia and

  13. Apelin and nutritional status in children on dialysis.

    Science.gov (United States)

    Yavuz, Sevgi; Cetinkaya, Senay; Anarat, Ali; Bayazıt, Aysun Karabay

    2014-09-01

    We aimed to evaluate whether serum apelin could reflect the nutritional status of children on dialysis. Twelve patients on peritoneal dialysis (PD) and 20 patients on hemodialysis (HD) were enrolled. Patients received individualized diet for six months. Anthropometric and laboratory indices were measured at onset and the end of the study. The anthropometric indices were all significantly lower in patients than in controls whereas similar in PD and HD patients. The protein catabolic rate (nPCR), height, mid-arm circumference (MAC), triceps skinfold thickness (TSF), arm muscle area (AMA) and arm fat area (AFA) z scores were significantly increased in dialysis patients after nutritional intervention. Weight z scores statistically increased in HD group whereas did not statistically change in PD group. Serum albumin levels were significantly improved in PD and HD patients. Apelin levels were similar in PD, HD and control groups. Post nutritional apelin values did not differ in each dialysis groups. On multivariate analysis, apelin was independently associated with age, weight, ESR and TG. Apelin seems to be not a useful indicator for monitoring the nutritional status in children on dialysis. However, the close link of apelin with inflammatory and lipid parameters suggested that apelin might be a novel target for slowing the atherogenic process in pediatric dialysis patients.

  14. Increased amphetamine-induced locomotor activity, sensitization, and accumbal dopamine release in M5 muscarinic receptor knockout mice

    DEFF Research Database (Denmark)

    Schmidt, Lene S; Miller, Anthony D; Lester, Deranda B

    2010-01-01

    showed that M(5) receptor knockout (M (5) (-/-) ) mice are less sensitive to the reinforcing properties of addictive drugs. MATERIALS AND METHODS: Here, we investigate the role of M(5) receptors in the effects of amphetamine and cocaine on locomotor activity, locomotor sensitization, and dopamine release...... and locomotor sensitization were enhanced in M (5) (-/-) mice, while the effects of cocaine were similar in M (5) (-/-) and wild-type mice. RESULTS: Consistent with the behavioral results, amphetamine-, but not cocaine, -elicited dopamine release in nucleus accumbens was enhanced in M (5) (-/-) mice. DISCUSSION......: The different effects of amphetamine and cocaine in M (5) (-/-) mice may be due to the divergent pharmacological profile of the two drugs, where amphetamine, but not cocaine, is able to release intracellular stores of dopamine. In conclusion, we show here for the first time that amphetamine...

  15. Toward a molecular understanding of psychostimulant actions using genetically engineered dopamine receptor knockout mice as model systems.

    Science.gov (United States)

    Zhang, J; Xu, M

    2001-01-01

    A major focus in studying the progression and prevention of addictive diseases has been to understand the molecular and cellular mechanisms underlying drug addiction. The brain dopaminergic system plays a central role in reward and motivation and is thought to be the main neural substrate for the actions of abusive drugs. We have used the gene targeting technology to generate dopamine D1 and D3 receptor knockout mice and used these mice as model systems to gain a molecular understanding of acute effects of psychostimulants cocaine and amphetamine. The use of a combined approach involving behavioral, electrophysiological as well as molecular studies has allowed us to define initially the roles of dopamine D1 and D3 receptors in the acute effects of psychostimulants and will enable us to understand mechanisms underlying their chronic actions in the future.

  16. Genetic background can result in a marked or minimal effect of gene knockout (GPR55 and CB2 receptor in experimental autoimmune encephalomyelitis models of multiple sclerosis.

    Directory of Open Access Journals (Sweden)

    Sofia Sisay

    Full Text Available Endocannabinoids and some phytocannabinoids bind to CB1 and CB2 cannabinoid receptors, transient receptor potential vanilloid one (TRPV1 receptor and the orphan G protein receptor fifty-five (GPR55. Studies using C57BL/10 and C57BL/6 (Cnr2 (tm1Zim CB2 cannabinoid receptor knockout mice have demonstrated an immune-augmenting effect in experimental autoimmune encephalomyelitis (EAE models of multiple sclerosis. However, other EAE studies in Biozzi ABH mice often failed to show any treatment effect of either CB2 receptor agonism or antagonism on inhibition of T cell autoimmunity. The influence of genetic background on the induction of EAE in endocannabinoid system-related gene knockout mice was examined. It was found that C57BL/6.GPR55 knockout mice developed less severe disease, notably in female mice, following active induction with myelin oligodendrocyte glycoprotein 35-55 peptide. In contrast C57BL/6.CB2 (Cnr2 (Dgen receptor knockout mice developed augmented severity of disease consistent with the genetically and pharmacologically-distinct, Cnr2 (tm1Zim mice. However, when the knockout gene was bred into the ABH mouse background and EAE induced with spinal cord autoantigens the immune-enhancing effect of CB2 receptor deletion was lost. Likewise CB1 receptor and transient receptor potential vanilloid one knockout mice on the ABH background demonstrated no alteration in immune-susceptibility, in terms of disease incidence and severity of EAE, in contrast to that reported in some C57BL/6 mouse studies. Furthermore the immune-modulating influence of GPR55 was marginal on the ABH mouse background. Whilst sedative doses of tetrahydrocannabinol could induce immunosuppression, this was associated with a CB1 receptor rather than a CB2 receptor-mediated effect. These data support the fact that non-psychoactive doses of medicinal cannabis have a marginal influence on the immune response in MS. Importantly, it adds a note of caution for the translational

  17. A muscle-specific knockout implicates nuclear receptor coactivator MED1 in the regulation of glucose and energy metabolism.

    Science.gov (United States)

    Chen, Wei; Zhang, Xiaoting; Birsoy, Kivanc; Roeder, Robert G

    2010-06-01

    As conventional transcriptional factors that are activated in diverse signaling pathways, nuclear receptors play important roles in many physiological processes that include energy homeostasis. The MED1 subunit of the Mediator coactivator complex plays a broad role in nuclear receptor-mediated transcription by anchoring the Mediator complex to diverse promoter-bound nuclear receptors. Given the significant role of skeletal muscle, in part through the action of nuclear receptors, in glucose and fatty acid metabolism, we generated skeletal muscle-specific Med1 knockout mice. Importantly, these mice show enhanced insulin sensitivity and improved glucose tolerance as well as resistance to high-fat diet-induced obesity. Furthermore, the white muscle of these mice exhibits increased mitochondrial density and expression of genes specific to type I and type IIA fibers, indicating a fast-to-slow fiber switch, as well as markedly increased expression of the brown adipose tissue-specific UCP-1 and Cidea genes that are involved in respiratory uncoupling. These dramatic results implicate MED1 as a powerful suppressor in skeletal muscle of genetic programs implicated in energy expenditure and raise the significant possibility of therapeutical approaches for metabolic syndromes and muscle diseases through modulation of MED1-nuclear receptor interactions.

  18. Regional brain uptake of the muscarinic ligand, [18F]FP-TZTP, is greatly decreased in M2 receptor knockout mice but not in M1, M3 and M4 receptor knockout mice.

    Science.gov (United States)

    Jagoda, E M; Kiesewetter, D O; Shimoji, K; Ravasi, L; Yamada, M; Gomeza, J; Wess, J; Eckelman, W C

    2003-04-01

    A muscarinic receptor radioligand, 3-(3-(3-fluoropropyl)thio) -1,2,5,thiadiazol-4-yl)-1,2,5,6-tetrahydro-1-methylpyridine (fP-TZTP) radiolabeled with the positron emitting radionuclide (18)F ([(18)F]FP-TZTP) displayed regional brain distribution consistent with M2 receptor densities in rat brain. The purpose of the present study is to further elucidate the subtype selectivity of [(18)F]FP-TZTP using genetically engineered mice which lacked functional M1, M2, M3, or M4 muscarinic receptors. Using ex vivo autoradiography, the regional brain localization of [(18)F]FP-TZTP in M2 knockout (M2 KO) was significantly decreased (51.3 to 61.4%; Pcortex, hippocampus, hypothalamus, superior colliculus, and thalamus. In similar studies with M1KO, M3KO and M4KO compared to their WT mice, [(18)F]FP-TZTP uptakes in the same brain regions were not significantly decreased at P<0.01. However, in amygdala and hippocampus small decreases of 19.5% and 22.7%, respectively, were observed for M1KO vs WT mice at P<0.05. Given the fact that large decreases in [(18)F]FP-TZTP brain uptakes were seen only in M2 KO vs. WT mice, we conclude that [(18)F]FP-TZTP preferentially labels M2 receptors in vivo.

  19. Tumor Endothelial Cell-Specific Drug Delivery System Using Apelin-Conjugated Liposomes

    Science.gov (United States)

    Kawahara, Hiroki; Naito, Hisamichi; Takara, Kazuhiro; Wakabayashi, Taku; Kidoya, Hiroyasu; Takakura, Nobuyuki

    2013-01-01

    Background A drug delivery system specifically targeting endothelial cells (ECs) in tumors is required to prevent normal blood vessels from being damaged by angiogenesis inhibitors. The purpose of this study was to investigate whether apelin, a ligand for APJ expressed in ECs when angiogenesis is taking place, can be used for targeting drug delivery to ECs in tumors. Methods and Results Uptake of apelin via APJ stably expressed in NIH-3T3 cells was investigated using TAMRA (fluorescent probe)-conjugated apelin. Both long and short forms of apelin (apelin 36 and apelin 13) were taken up, the latter more effectively. To improve efficacy of apelin- liposome conjugates, we introduced cysteine, with its sulfhydryl group, to the C terminus of apelin 13, resulting in the generation of apelin 14. In turn, apelin 14 was conjugated to rhodamine-encapsulating liposomes and administered to tumor-bearing mice. In the tumor microenvironment, we confirmed that liposomes were incorporated into the cytoplasm of ECs. In contrast, apelin non-conjugated liposomes were rarely found in the cytoplasm of ECs. Moreover, non-specific uptake of apelin-conjugated liposomes was rarely detected in other normal organs. Conclusions ECs in normal organs express little APJ; however, upon hypoxic stimulation, such as in tumors, ECs start to express APJ. The present study suggests that apelin could represent a suitable tool to effectively deliver drugs specifically to ECs within tumors. PMID:23799018

  20. Tumor endothelial cell-specific drug delivery system using apelin-conjugated liposomes.

    Directory of Open Access Journals (Sweden)

    Hiroki Kawahara

    Full Text Available BACKGROUND: A drug delivery system specifically targeting endothelial cells (ECs in tumors is required to prevent normal blood vessels from being damaged by angiogenesis inhibitors. The purpose of this study was to investigate whether apelin, a ligand for APJ expressed in ECs when angiogenesis is taking place, can be used for targeting drug delivery to ECs in tumors. METHODS AND RESULTS: Uptake of apelin via APJ stably expressed in NIH-3T3 cells was investigated using TAMRA (fluorescent probe-conjugated apelin. Both long and short forms of apelin (apelin 36 and apelin 13 were taken up, the latter more effectively. To improve efficacy of apelin- liposome conjugates, we introduced cysteine, with its sulfhydryl group, to the C terminus of apelin 13, resulting in the generation of apelin 14. In turn, apelin 14 was conjugated to rhodamine-encapsulating liposomes and administered to tumor-bearing mice. In the tumor microenvironment, we confirmed that liposomes were incorporated into the cytoplasm of ECs. In contrast, apelin non-conjugated liposomes were rarely found in the cytoplasm of ECs. Moreover, non-specific uptake of apelin-conjugated liposomes was rarely detected in other normal organs. CONCLUSIONS: ECs in normal organs express little APJ; however, upon hypoxic stimulation, such as in tumors, ECs start to express APJ. The present study suggests that apelin could represent a suitable tool to effectively deliver drugs specifically to ECs within tumors.

  1. Characterization of central inhibitory muscarinic autoreceptors by the use of muscarinic acetylcholine receptor knock-out mice.

    Science.gov (United States)

    Zhang, Weilie; Basile, Anthony S; Gomeza, Jesus; Volpicelli, Laura A; Levey, Allan I; Wess, Jürgen

    2002-03-01

    Forebrain muscarinic acetylcholine (ACh) receptors (mAChRs; M1-M5) are predicted to play important roles in many fundamental central functions, including higher cognitive processes and modulation of extrapyramidal motor activity. Synaptic ACh levels are known to be regulated by the activity of presynaptic muscarinic autoreceptors mediating inhibition of ACh release. Primarily because of the use of ligands with limited receptor subtype selectivity, classical pharmacological studies have led to conflicting results regarding the identity of the mAChR subtypes mediating this activity in different areas of the brain. To investigate the molecular identity of hippocampal, cortical, and striatal inhibitory muscarinic autoreceptors in a more direct manner, we used genetically altered mice lacking functional M2 and/or M4 mAChRs [knock-out (KO) mice]. After labeling of cellular ACh pools with [3H]choline, potassium-stimulated [3H]ACh release was measured in superfused brain slices, either in the absence or the presence of muscarinic drugs. The nonsubtype-selective muscarinic agonist, oxotremorine (0.1-10 microm), inhibited potassium-stimulated [3H]ACh release in hippocampal, cortical, and striatal slices prepared from wild-type mice by up to 80%. This activity was totally abolished in tissues prepared from M2-M4 receptor double KO mice. Strikingly, release studies with brain slices from M2 and M4 receptor single KO mice indicated that autoinhibition of ACh release is mediated primarily by the M2 receptor in hippocampus and cerebral cortex, but predominantly by the M4 receptor in the striatum. These results, together with additional receptor localization studies, support the novel concept that autoinhibition of ACh release involves different mAChRs in different regions of the brain.

  2. Increased susceptibility to diet-induced obesity in GPRC6A receptor knockout mice

    DEFF Research Database (Denmark)

    Clemmensen, Christoffer; Smajilovic, Sanela; Madsen, Andreas N

    2013-01-01

    locomotor activity. Moreover, diet-induced obese Gprc6a KO mice had increased circulating insulin and leptin levels relative to WT animals, thereby demonstrating that endocrine abnormalities associate with the reported disturbances in energy balance. The phenotype was further accompanied by disruptions...... complications is still elusive. In the present study, we investigated the impact of GPRC6A deficiency in a murine model of diet-induced obesity (DIO). Male Gprc6a knockout (KO) mice and WT littermates were subjected to a high-fat diet (HFD) for 25 weeks and exposed to comprehensive metabolic phenotyping...

  3. Phenotypic screening of hepatocyte nuclear factor (HNF) 4-γ receptor knockout mice

    International Nuclear Information System (INIS)

    Gerdin, Anna Karin; Surve, Vikas V.; Joensson, Marie; Bjursell, Mikael; Bjoerkman, Maria; Edenro, Anne; Schuelke, Meint; Saad, Alaa; Bjurstroem, Sivert; Lundgren, Elisabeth Jensen; Snaith, Michael; Fransson-Steen, Ronny; Toernell, Jan; Berg, Anna-Lena; Bohlooly-Y, Mohammad

    2006-01-01

    Using the mouse as a model organism in pharmaceutical research presents unique advantages as its physiology in many ways resembles the human physiology, it also has a relatively short generation time, low breeding and maintenance costs, and is available in a wide variety of inbred strains. The ability to genetically modify mouse embryonic stem cells to generate mouse models that better mimic human disease is another advantage. In the present study, a comprehensive phenotypic screening protocol is applied to elucidate the phenotype of a novel mouse knockout model of hepatocyte nuclear factor (HNF) 4-γ. HNF4-γ is expressed in the kidneys, gut, pancreas, and testis. First level of the screen is aimed at general health, morphologic appearance, normal cage behaviour, and gross neurological functions. The second level of the screen looks at metabolic characteristics and lung function. The third level of the screen investigates behaviour more in-depth and the fourth level consists of a thorough pathological characterisation, blood chemistry, haematology, and bone marrow analysis. When compared with littermate wild-type mice (HNF4-γ +/+ ), the HNF4-γ knockout (HNF4-γ -/- ) mice had lowered energy expenditure and locomotor activity during night time that resulted in a higher body weight despite having reduced intake of food and water. HNF4-γ -/- mice were less inclined to build nest and were found to spend more time in a passive state during the forced swim test

  4. Phenotypic screening of hepatocyte nuclear factor (HNF) 4-gamma receptor knockout mice.

    Science.gov (United States)

    Gerdin, Anna Karin; Surve, Vikas V; Jönsson, Marie; Bjursell, Mikael; Björkman, Maria; Edenro, Anne; Schuelke, Meint; Saad, Alaa; Bjurström, Sivert; Lundgren, Elisabeth Jensen; Snaith, Michael; Fransson-Steen, Ronny; Törnell, Jan; Berg, Anna-Lena; Bohlooly-Y, Mohammad

    2006-10-20

    Using the mouse as a model organism in pharmaceutical research presents unique advantages as its physiology in many ways resembles the human physiology, it also has a relatively short generation time, low breeding and maintenance costs, and is available in a wide variety of inbred strains. The ability to genetically modify mouse embryonic stem cells to generate mouse models that better mimic human disease is another advantage. In the present study, a comprehensive phenotypic screening protocol is applied to elucidate the phenotype of a novel mouse knockout model of hepatocyte nuclear factor (HNF) 4-gamma. HNF4-gamma is expressed in the kidneys, gut, pancreas, and testis. The first level of the screen is aimed at general health, morphologic appearance, normal cage behaviour, and gross neurological functions. The second level of the screen looks at metabolic characteristics and lung function. The third level of the screen investigates behaviour more in-depth and the fourth level consists of a thorough pathological characterisation, blood chemistry, haematology, and bone marrow analysis. When compared with littermate wild-type mice (HNF4-gamma(+/+)), the HNF4-gamma knockout (HNF4-gamma(-/-)) mice had lowered energy expenditure and locomotor activity during night time that resulted in a higher body weight despite having reduced intake of food and water. HNF4-gamma(-/-) mice were less inclined to build nest and were found to spend more time in a passive state during the forced swim test.

  5. GPR39 (zinc receptor) knockout mice exhibit depression-like behavior and CREB/BDNF down-regulation in the hippocampus

    DEFF Research Database (Denmark)

    Młyniec, Katarzyna; Budziszewska, Bogusława; Holst, Birgitte

    2015-01-01

    Background: Zinc may act as a neurotransmitter in the central nervous system by activation of the GPR39 metabotropic receptors. Methods: In the present study, we investigated whether GPR39 knockout would cause depressive-like and/or anxiety-like behavior, as measured by the forced swim test, tail...... to the forced swim test, as measured by Western-blot analysis. Results: In this study, GPR39 knockout mice showed an increased immobility time in both the forced swim test and tail suspension test, indicating depressive-like behavior and displayed anxiety-like phenotype. GPR39 knockout mice had lower CREB...... mice in comparison with the wild-type control mice, which does not support a role of GPR39 in hypothalamus-pituitary-adrenal axis regulation. The results of this study indicate the involvement of the GPR39 Zn2+-sensing receptor in the pathophysiology of depression with component of anxiety....

  6. Minimal Effects of Age and Exposure to a Noisy Environment on Hearing in Alpha9 Nicotinic Receptor Knockout Mice

    Directory of Open Access Journals (Sweden)

    Amanda M. Lauer

    2017-06-01

    Full Text Available Studies have suggested a role of weakened medial olivocochlear (OC efferent feedback in accelerated hearing loss and increased susceptibility to noise. The present study investigated the progression of hearing loss with age and exposure to a noisy environment in medial OC-deficient mice. Alpha9 nicotinic acetylcholine receptor knockout (α9KO and wild types were screened for hearing loss using auditory brainstem responses. α9KO mice housed in a quiet environment did not show increased hearing loss compared to wild types in young adulthood and middle age. Challenging the medial OC system by housing in a noisy environment did not increase hearing loss in α9KO mice compared to wild types. ABR wave 1 amplitudes also did not show differences between α9KO mice and wild types. These data suggest that deficient medial OC feedback does not result in early onset of hearing loss.

  7. Isolation and characterization of coronary endothelial and smooth muscle cells from A1 adenosine receptor-knockout mice.

    Science.gov (United States)

    Teng, Bunyen; Ansari, Habib R; Oldenburg, Peter J; Schnermann, J; Mustafa, S Jamal

    2006-04-01

    Mice have been used widely in in vivo and in vitro cardiovascular research. The availability of knockout mice provides further clues to the physiological significance of specific receptor subtypes. Adenosine A(1) receptor (A(1)AR)-knockout (A(1)KO) mice and their wild-type (A(1)WT) controls were employed in this investigation. The heart and aortic arch were carefully removed and retroinfused with enzyme solution (1 mg/ml collagenase type I, 0.5 mg/ml soybean trypsin inhibitor, 3% BSA, and 2% antibiotics) through the aortic arch. The efflux was collected at 30-, 60-, and 90-min intervals. The cells were centrifuged, and the pellets were mixed with medium [medium 199-F-12 medium with 10% FBS and 2% antibiotics (for endothelial cells) and advanced DMEM with 10% FBS, 10% mouse serum, 2% GlutaMax, and 2% antibiotics (for smooth muscle cells)] and plated. Endothelial cells were characterized by a cobblestone appearance and positive staining with acetylated LDL labeled with 1,1'-dioctadecyl-3,3,3',3-tetramethylindocarbocyanine perchlorate. Smooth muscle cells were characterized by positive staining of smooth muscle alpha-actin and smooth muscle myosin heavy chain. Homogeneity of the smooth muscle cells was approximately 91%. Western blot analysis showed expression of smoothelin in the cells from passages 3, 7, and 11 in A(1)WT and A(1)KO mice. Furthermore, the A(1)AR was characterized by Western blot analysis using an A(1)AR-specific antibody. To our knowledge, this is the first isolation and successful characterization of smooth muscle cells from the mouse coronary system.

  8. Differential actions of orexin receptors in brainstem cholinergic and monoaminergic neurons revealed by receptor knockouts: implications for orexinergic signaling in arousal and narcolepsy

    Directory of Open Access Journals (Sweden)

    Kristi A Kohlmeier

    2013-12-01

    Full Text Available Orexin neuropeptides influence multiple homeostatic functions and play an essential role in the expression of normal sleep-wake behavior. While their two known receptors (OX1 and OX2 are targets for novel pharmacotherapeutics, the actions mediated by each receptor remain largely unexplored. Using brain slices from mice constitutively lacking either receptor, we used whole-cell and Ca2+ imaging methods to delineate the cellular actions of each receptor within cholinergic (laterodorsal tegmental nucleus; LDT and monoaminergic (dorsal raphe; DR and locus coeruleus; LC brainstem nuclei – where orexins promote arousal and suppress REM sleep. In slices from OX2-/- mice, orexin-A (300 nM elicited wild-type responses in LDT, DR and LC neurons consisting of a depolarizing current and augmented voltage-dependent Ca2+ transients. In slices from OX1-/- mice, the depolarizing current was absent in LDT and LC neurons and was attenuated in DR neurons, although Ca2+-transients were still augmented. Since orexin-A produced neither of these actions in slices lacking both receptors, our findings suggest that orexin-mediated depolarization is mediated by both receptors in DR, but is exclusively mediated by OX1 in LDT and LC neurons, even though OX2 is present and OX2 mRNA appears elevated in brainstems from OX1-/- mice. Considering published behavioral data, these findings support a model in which orexin-mediated excitation of mesopontine cholinergic and monoaminergic neurons contributes little to stabilizing spontaneous waking and sleep bouts, but functions in context-dependent arousal and helps restrict muscle atonia to REM sleep. The augmented Ca2± transients mediated by both receptors appeared mediated by influx via L-type Ca2+ channels, which is often linked to transcriptional signaling. This could provide an adaptive signal to compensate for receptor loss or prolonged antagonism and may contribute to the reduced severity of narcolepsy in single receptor

  9. Characterization of adult ghrelin and ghrelin receptor knockout mice under positive and negative energy balance

    Science.gov (United States)

    Ghrelin and the ghrelin receptor (GH secretagogue receptor, GHS-R) are believed to have important roles in energy homeostasis. We describe results from the first studies to be conducted in congenic (N10) adult ghrelin(-/-) and Ghsr(-/-) mice under conditions of both positive (high-fat diet) and nega...

  10. Is serum apelin related to portal hemodynamics in patients with liver cirrhosis?

    Directory of Open Access Journals (Sweden)

    Ashraf G Dala

    2018-01-01

    Conclusion Serum apelin is elevated in patients with cirrhosis and PH, and a positive correlation is found between serum apelin and the degree of hepatic fibrosis. Measurement of serum apelin represents a rapid, noninvasive method for the prediction of PH in cirrhotics and can assess the degree of hepatic fibrosis.

  11. Serotonin/dopamine interactions in a hyperactive mouse: reduced serotonin receptor 1B activity reverses effects of dopamine transporter knockout.

    Directory of Open Access Journals (Sweden)

    Frank Scott Hall

    Full Text Available Knockout (KO mice that lack the dopamine transporter (SL6A3; DAT display increased locomotion that can be attenuated, under some circumstances, by administration of drugs that normally produce psychostimulant-like effects, such as amphetamine and methylphenidate. These results have led to suggestions that DAT KO mice may model features of attention deficit hyperactivity disorder (ADHD and that these drugs may act upon serotonin (5-HT systems to produce these unusual locomotor decreasing effects. Evidence from patterns of brain expression and initial pharmacologic studies led us to use genetic and pharmacologic approaches to examine the influence of altered 5-HT1B receptor activity on hyperactivity in DAT KO mice. Heterozygous 5-HT1B KO and pharmacologic 5-HT1B antagonism both attenuated locomotor hyperactivity in DAT KO mice. Furthermore, DAT KO mice with reduced, but not eliminated, 5-HT1B receptor expression regained cocaine-stimulated locomotion, which was absent in DAT KO mice with normal levels of 5-HT1B receptor expression. Further experiments demonstrated that the degree of habituation to the testing apparatus determined whether cocaine had no effect on locomotion in DAT KO or reduced locomotion, helping to resolve differences among prior reports. These findings of complementation of the locomotor effects of DAT KO by reducing 5-HT1B receptor activity underscore roles for interactions between specific 5-HT receptors and dopamine (DA systems in basal and cocaine-stimulated locomotion and support evaluation of 5-HT1B antagonists as potential, non-stimulant ADHD therapeutics.

  12. Selective Attention to Visual Stimuli Using Auditory Distractors Is Altered in Alpha-9 Nicotinic Receptor Subunit Knock-Out Mice.

    Science.gov (United States)

    Terreros, Gonzalo; Jorratt, Pascal; Aedo, Cristian; Elgoyhen, Ana Belén; Delano, Paul H

    2016-07-06

    During selective attention, subjects voluntarily focus their cognitive resources on a specific stimulus while ignoring others. Top-down filtering of peripheral sensory responses by higher structures of the brain has been proposed as one of the mechanisms responsible for selective attention. A prerequisite to accomplish top-down modulation of the activity of peripheral structures is the presence of corticofugal pathways. The mammalian auditory efferent system is a unique neural network that originates in the auditory cortex and projects to the cochlear receptor through the olivocochlear bundle, and it has been proposed to function as a top-down filter of peripheral auditory responses during attention to cross-modal stimuli. However, to date, there is no conclusive evidence of the involvement of olivocochlear neurons in selective attention paradigms. Here, we trained wild-type and α-9 nicotinic receptor subunit knock-out (KO) mice, which lack cholinergic transmission between medial olivocochlear neurons and outer hair cells, in a two-choice visual discrimination task and studied the behavioral consequences of adding different types of auditory distractors. In addition, we evaluated the effects of contralateral noise on auditory nerve responses as a measure of the individual strength of the olivocochlear reflex. We demonstrate that KO mice have a reduced olivocochlear reflex strength and perform poorly in a visual selective attention paradigm. These results confirm that an intact medial olivocochlear transmission aids in ignoring auditory distraction during selective attention to visual stimuli. The auditory efferent system is a neural network that originates in the auditory cortex and projects to the cochlear receptor through the olivocochlear system. It has been proposed to function as a top-down filter of peripheral auditory responses during attention to cross-modal stimuli. However, to date, there is no conclusive evidence of the involvement of olivocochlear

  13. Dysfunctional Presynaptic M2 Receptors in the Presence of Chronically High Acetylcholine Levels: Data from the PRiMA Knockout Mouse.

    Science.gov (United States)

    Mohr, Franziska; Krejci, Eric; Zimmermann, Martina; Klein, Jochen

    2015-01-01

    The muscarinic M2 receptor (M2R) acts as a negative feedback regulator in central cholinergic systems. Activation of the M2 receptor limits acetylcholine (ACh) release, especially when ACh levels are increased because acetylcholinesterase (AChE) activity is acutely inhibited. Chronically high ACh levels in the extracellular space, however, were reported to down-regulate M2R to various degrees. In the present study, we used the PRiMA knockout mouse which develops severely reduced AChE activity postnatally to investigate ACh release, and we used microdialysis to investigate whether the function of M2R to reduce ACh release in vivo was impaired in adult PRiMA knockout mice. We first show that striatal and hippocampal ACh levels, while strongly increased, still respond to AChE inhibitors. Infusion or injection of oxotremorine, a muscarinic M2 agonist, reduced ACh levels in wild-type mice but did not significantly affect ACh levels in PRiMA knockout mice or in wild-type mice in which ACh levels were artificially increased by infusion of neostigmine. Scopolamine, a muscarinic antagonist, increased ACh levels in wild-type mice receiving neostigmine, but not in wild-type mice or in PRiMA knockout mice. These results demonstrate that M2R are dysfunctional and do not affect ACh levels in PRiMA knockout mice, likely because of down-regulation and/or loss of receptor-effector coupling. Remarkably, this loss of function does not affect cognitive functions in PRiMA knockout mice. Our results are discussed in the context of AChE inhibitor therapy as used in dementia.

  14. Spatial learning in the 5-HT1B receptor knockout mouse: selective facilitation/impairment depending on the cognitive demand.

    Science.gov (United States)

    Buhot, Marie-Christine; Wolff, Mathieu; Benhassine, Narimane; Costet, Pierre; Hen, René; Segu, Louis

    2003-01-01

    Age-related memory decline is associated with a combined dysfunction of the cholinergic and serotonergic systems in the hippocampus and frontal cortex, in particular. The 5-HT1B receptor occupies strategic cellular and subcellular locations in these structures, where it plays a role in the modulation of ACh release. In an attempt to characterize the contribution of this receptor to memory functions, 5-HT1B receptor knockout (KO) mice were submitted to various behavioral paradigms carried out in the same experimental context (water maze), which were aimed at exposing mice to various levels of memory demand. 5-HT1BKO mice exhibited a facilitation in the acquisition of a hippocampal-dependent spatial reference memory task in the Morris water maze. This facilitation was selective of task difficulty, showing thus that the genetic inactivation of the 5-HT1B receptor is associated with facilitation when the complexity of the task is increased, and reveals a protective effect on age-related hippocampal-dependent memory decline. Young-adult and aged KO and wild-type (WT) mice were equally able to learn a delayed spatial matching-to-sample working memory task in a radial-arm water maze with short (0 or 5 min) delays. However, 5-HT1BKO mice, only, exhibited a selective memory impairment at intermediate and long (15, 30, and 60 min) delays. Treatment by scopolamine induced the same pattern of performance in wild type as did the mutation for short (5 min, no impairment) and long (60 min, impairment) delays. Taken together, these studies revealed a beneficial effect of the mutation on the acquisition of a spatial reference memory task, but a deleterious effect on a working memory task for long delays. This 5-HT1BKO mouse story highlights the problem of the potential existence of "global memory enhancers."

  15. Transgenic Expression of the Vitamin D Receptor Restricted to the Ileum, Cecum, and Colon of Vitamin D Receptor Knockout Mice Rescues Vitamin D Receptor-Dependent Rickets.

    Science.gov (United States)

    Dhawan, Puneet; Veldurthy, Vaishali; Yehia, Ghassan; Hsaio, Connie; Porta, Angela; Kim, Ki-In; Patel, Nishant; Lieben, Liesbet; Verlinden, Lieve; Carmeliet, Geert; Christakos, Sylvia

    2017-11-01

    Although the intestine plays the major role in 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] action on calcium homeostasis, the mechanisms involved remain incompletely understood. The established model of 1,25(OH)2D3-regulated intestinal calcium absorption postulates a critical role for the duodenum. However, the distal intestine is where 70% to 80% of ingested calcium is absorbed. To test directly the role of 1,25(OH)2D3 and the vitamin D receptor (VDR) in the distal intestine, three independent knockout (KO)/transgenic (TG) lines expressing VDR exclusively in the ileum, cecum, and colon were generated by breeding VDR KO mice with TG mice expressing human VDR (hVDR) under the control of the 9.5-kb caudal type homeobox 2 promoter. Mice from one TG line (KO/TG3) showed low VDR expression in the distal intestine (<50% of the levels observed in KO/TG1, KO/TG2, and wild-type mice). In the KO/TG mice, hVDR was not expressed in the duodenum, jejunum, kidney, or other tissues. Growth arrest, elevated parathyroid hormone level, and hypocalcemia of the VDR KO mice were prevented in mice from KO/TG lines 1 and 2. Microcomputed tomography analysis revealed that the expression of hVDR in the distal intestine of KO/TG1 and KO/TG2 mice rescued the bone defects associated with systemic VDR deficiency, including growth plate abnormalities and altered trabecular and cortical parameters. KO/TG3 mice showed rickets, but less severely than VDR KO mice. These findings show that expression of VDR exclusively in the distal intestine can prevent abnormalities in calcium homeostasis and bone mineralization associated with systemic VDR deficiency. Copyright © 2017 Endocrine Society.

  16. Enhanced voluntary wheel running in GPRC6A receptor knockout mice

    DEFF Research Database (Denmark)

    Clemmensen, Christoffer; Pehmøller, Christian; Klein, Anders B

    2013-01-01

    in energy metabolism are largely unexplored. Based on GPRC6A's expression pattern and ligand preferences, we hypothesize that the receptor may impact energy metabolism via regulating physical activity levels. Thus, in the present study, we exposed GPRC6A receptor KO mice and their wild-type (WT) littermates...... to voluntary wheel running and forced treadmill exercise. Moreover, we assessed energy expenditure in the basal state, and evaluated the effects of wheel running on food intake, body composition, and a range of exercise-induced central and peripheral biomarkers. We found that adaptation to voluntary wheel...... running is affected by GPRC6A, as ablation of the receptor significantly enhances wheel running in KO relative to WT mice. Both genotypes responded to voluntary exercise by increasing food intake and improving body composition to a similar degree. In conclusion, these data demonstrate that the GPRC6A...

  17. Orp8 deficiency in bone marrow-derived cells reduces atherosclerotic lesion progression in LDL receptor knockout mice.

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    Erik van Kampen

    Full Text Available INTRODUCTION: Oxysterol binding protein Related Proteins (ORPs mediate intracellular lipid transport and homeostatic regulation. ORP8 downregulates ABCA1 expression in macrophages and cellular cholesterol efflux to apolipoprotein A-I. In line, ORP8 knockout mice display increased amounts of HDL cholesterol in blood. However, the role of macrophage ORP8 in atherosclerotic lesion development is unknown. METHODS AND RESULTS: LDL receptor knockout (KO mice were transplanted with bone marrow (BM from ORP8 KO mice and C57Bl/6 wild type mice. Subsequently, the animals were challenged with a high fat/high cholesterol Western-type diet to induce atherosclerosis. After 9 weeks of Western-Type diet feeding, serum levels of VLDL cholesterol were increased by 50% in ORP8 KO BM recipients compared to the wild-type recipients. However, no differences were observed in HDL cholesterol. Despite the increase in VLDL cholesterol, lesions in mice transplanted with ORP8 KO bone marrow were 20% smaller compared to WT transplanted controls. In addition, ORP8 KO transplanted mice displayed a modest increase in the percentage of macrophages in the lesion as compared to the wild-type transplanted group. ORP8 deficient macrophages displayed decreased production of pro-inflammatory factors IL-6 and TNFα, decreased expression of differentiation markers and showed a reduced capacity to form foam cells in the peritoneal cavity. CONCLUSIONS: Deletion of ORP8 in bone marrow-derived cells, including macrophages, reduces lesion progression after 9 weeks of WTD challenge, despite increased amounts of circulating pro-atherogenic VLDL. Reduced macrophage foam cell formation and lower macrophage inflammatory potential are plausible mechanisms contributing to the observed reduction in atherosclerosis.

  18. Duodenal calcium absorption in vitamin D receptor-knockout mice: functional and molecular aspects.

    NARCIS (Netherlands)

    Cromphaut, S.J. van; Dewerchin, M.; Hoenderop, J.G.J.; Stockmans, I.; Herck, E. van; Kato, S.; Bindels, R.J.M.; Collen, D.; Carmeliet, P.; Bouillon, R.; Carmeliet, G.

    2001-01-01

    Rickets and hyperparathyroidism caused by a defective vitamin D receptor (VDR) can be prevented in humans and animals by high calcium intake, suggesting that intestinal calcium absorption is critical for 1,25(OH)(2) vitamin D [1,25(OH)(2)D(3)] action on calcium homeostasis. We assessed the rate of

  19. Immune malfunction in the GPR39 zinc receptor of knockout mice

    DEFF Research Database (Denmark)

    Młyniec, Katarzyna; Trojan, Ewa; Ślusarczyk, Joanna

    2016-01-01

    as an anti-inflammatory agent, and its link with depression has been proved, zinc deficiency causing depression- and anxiety-like behavior with immune malfunction. It has been discovered that trace-element zinc acts as a neurotransmitter in the central nervous system via zinc receptor GPR39. In this study we...

  20. Attenuated renovascular constrictor responses to angiotensin II in adenosine 1 receptor knockout mice

    DEFF Research Database (Denmark)

    Hansen, Pernille B; Hashimoto, Seiji; Briggs, Josie

    2003-01-01

    In the present experiments we examined the renovascular constrictor effects of ANG II in the chronic and complete absence of A1 adenosine receptors (A1AR) using mice with targeted deletion of the A1AR gene. Glomerular filtration rate (GFR) was not different between A1AR +/+ and A1AR -/- mice unde...

  1. Aggravation of chronic stress effects on hippocampal neurogenesis and spatial memory in LPA₁ receptor knockout mice.

    Directory of Open Access Journals (Sweden)

    Estela Castilla-Ortega

    Full Text Available The lysophosphatidic acid LPA₁ receptor regulates plasticity and neurogenesis in the adult hippocampus. Here, we studied whether absence of the LPA₁ receptor modulated the detrimental effects of chronic stress on hippocampal neurogenesis and spatial memory.Male LPA₁-null (NULL and wild-type (WT mice were assigned to control or chronic stress conditions (21 days of restraint, 3 h/day. Immunohistochemistry for bromodeoxyuridine and endogenous markers was performed to examine hippocampal cell proliferation, survival, number and maturation of young neurons, hippocampal structure and apoptosis in the hippocampus. Corticosterone levels were measured in another a separate cohort of mice. Finally, the hole-board test assessed spatial reference and working memory. Under control conditions, NULL mice showed reduced cell proliferation, a defective population of young neurons, reduced hippocampal volume and moderate spatial memory deficits. However, the primary result is that chronic stress impaired hippocampal neurogenesis in NULLs more severely than in WT mice in terms of cell proliferation; apoptosis; the number and maturation of young neurons; and both the volume and neuronal density in the granular zone. Only stressed NULLs presented hypocortisolemia. Moreover, a dramatic deficit in spatial reference memory consolidation was observed in chronically stressed NULL mice, which was in contrast to the minor effect observed in stressed WT mice.These results reveal that the absence of the LPA₁ receptor aggravates the chronic stress-induced impairment to hippocampal neurogenesis and its dependent functions. Thus, modulation of the LPA₁ receptor pathway may be of interest with respect to the treatment of stress-induced hippocampal pathology.

  2. Upregulation of Cannabinoid Type 1 Receptors in Dopamine D2 Receptor Knockout Mice Is Reversed by Chronic Forced Ethanol Consumption

    Energy Technology Data Exchange (ETDEWEB)

    Thanos, P.K.; Wang, G.; Thanos, P.K.; Gopez, V.; Delis, F.; Michaelides, M.; Grand, D.K.; Wang, G.-J.; Kunos, G.; Volkow, N.D.

    2011-01-01

    The anatomical proximity of the cannabinoid type 1 (CNR1/CB1R) and the dopamine D2 receptors (DRD2), their ability to form CB1R-DRD2 heteromers, their opposing roles in locomotion, and their involvement in ethanol's reinforcing and addictive properties prompted us to study the levels and distribution of CB1R after chronic ethanol intake, in the presence and absence of DRD2. We monitored the drinking patterns and locomotor activity of Drd2+/+ and Drd2-/- mice consuming either water or a 20% (v/v) ethanol solution (forced ethanol intake) for 6 months and used the selective CB1 receptor antagonist [{sup 3}H]SR141716A to quantify CB1R levels in different brain regions with in vitro receptor autoradiography. We found that the lack of DRD2 leads to a marked upregulation (approximately 2-fold increase) of CB1R in the cerebral cortex, the caudate-putamen, and the nucleus accumbens, which was reversed by chronic ethanol intake. The results suggest that DRD2-mediated dopaminergic neurotransmission and chronic ethanol intake exert an inhibitory effect on cannabinoid receptor expression in cortical and striatal regions implicated in the reinforcing and addictive properties of ethanol.

  3. Weekly Treatment of 2-Hydroxypropyl-β-cyclodextrin Improves Intracellular Cholesterol Levels in LDL Receptor Knockout Mice

    Directory of Open Access Journals (Sweden)

    Sofie M. A. Walenbergh

    2015-09-01

    Full Text Available Recently, the importance of lysosomes in the context of the metabolic syndrome has received increased attention. Increased lysosomal cholesterol storage and cholesterol crystallization inside macrophages have been linked to several metabolic diseases, such as atherosclerosis and non-alcoholic fatty liver disease (NAFLD. Two-hydroxypropyl-β-cyclodextrin (HP-B-CD is able to redirect lysosomal cholesterol to the cytoplasm in Niemann-Pick type C1 disease, a lysosomal storage disorder. We hypothesize that HP-B-CD ameliorates liver cholesterol and intracellular cholesterol levels inside Kupffer cells (KCs. Hyperlipidemic low-density lipoprotein receptor knockout (Ldlr−/− mice were given weekly, subcutaneous injections with HP-B-CD or control PBS. In contrast to control injections, hyperlipidemic mice treated with HP-B-CD demonstrated a shift in intracellular cholesterol distribution towards cytoplasmic cholesteryl ester (CE storage and a decrease in cholesterol crystallization inside KCs. Compared to untreated hyperlipidemic mice, the foamy KC appearance and liver cholesterol remained similar upon HP-B-CD administration, while hepatic campesterol and 7α-hydroxycholesterol levels were back increased. Thus, HP-B-CD could be a useful tool to improve intracellular cholesterol levels in the context of the metabolic syndrome, possibly through modulation of phyto- and oxysterols, and should be tested in the future. Additionally, these data underline the existence of a shared etiology between lysosomal storage diseases and NAFLD.

  4. Layer- and column-specific knockout of NMDA receptors in pyramidal neurons of the mouse barrel cortex.

    Directory of Open Access Journals (Sweden)

    Rachel Aronoff

    2007-11-01

    Full Text Available Viral vectors injected into the mouse brain offer the possibility for localized genetic modifications in a highly controlled manner. Lentivector injection into mouse neocortex transduces cells within a diameter of approximately 200µm, which closely matches the lateral scale of a column in barrel cortex. The depth and volume of the injection determines which cortical layer is transduced. Furthermore, transduced gene expression from the lentivector can be limited to predominantly pyramidal neurons by using a 1.3kb fragment of the αCaMKII promoter. This technique therefore allows genetic manipulation of a specific cell type in defined columns and layers of the neocortex. By expressing Cre recombinase from such a lentivector in gene-targeted mice carrying a floxed gene, highly specific genetic lesions can be induced. Here, we demonstrate the utility of this approach by specifically knocking out NMDA receptors (NMDARs in pyramidal neurons in the somatosensory barrel cortex of gene-targeted mice carrying floxed NMDAR 1 genes. Neurons transduced with lentivector encoding GFP and Cre recombinase exhibit not only reductions in NMDAR 1 mRNA levels, but reduced NMDAR-dependent currents and pairing-induced synaptic potentiation. This technique for knockout of NMDARs in a cell type, column- and layer-specific manner in the mouse somatosensory cortex may help further our understanding of the functional roles of NMDARs in vivo during sensory perception and learning.

  5. Curcumin Protects against Atherosclerosis in Apolipoprotein E-Knockout Mice by Inhibiting Toll-like Receptor 4 Expression.

    Science.gov (United States)

    Zhang, Shanshan; Zou, Jun; Li, Peiyang; Zheng, Xiumei; Feng, Dan

    2018-01-17

    Toll-like receptor 4 (TLR4) has been reported to play a critical role in the pathogenesis of atherosclerosis, the current study aimed to investigate whether curcumin suppresses atherosclerosis development in ApoE-knockout (ApoE -/- ) mice by inhibiting TLR4 expression. ApoE -/- mice were fed a high-fat diet supplemented with or without curcumin (0.1% w/w) for 16 weeks. Curcumin supplementation significantly reduced TLR4 expression and macrophage infiltration in atherosclerotic plaques. Curcumin also reduced aortic interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) expression, nuclear factor-κB (NF-κB) activity, and plasma IL-1β, TNF-α, soluble VCAM-1 and ICAM-1 levels. In addition, aortic sinus sections revealed that curcumin treatment reduced the extent of atherosclerotic lesions and inhibited atherosclerosis development. In vitro, curcumin inhibited NF-κB activation in macrophages and reduced TLR4 expression induced by lipopolysaccharide. Our results indicate that curcumin protects against atherosclerosis at least partially by inhibiting TLR4 expression and its related inflammatory reaction.

  6. Autophagy downregulation contributes to insulin resistance mediated injury in insulin receptor knockout podocytes in vitro

    Directory of Open Access Journals (Sweden)

    Ying Xu

    2016-04-01

    Full Text Available It is unknown whether autophagy activity is altered in insulin resistant podocytes and whether autophagy could be a therapeutic target for diabetic nephropathy (DN. Here we used shRNA transfection to knockdown the insulin receptor (IR gene in cultured human immortalized podocytes as an in vitro insulin resistant model. Autophagy related proteins LC3, Beclin, and p62 as well as nephrin, a podocyte injury marker, were assessed using western blot and immunofluorescence staining. Our results show that autophagy is suppressed when podocytes lose insulin sensitivity and that treatment of rapamycin, an mTOR specific inhibitor, could attenuate insulin resistance induced podocytes injury via autophagy activation. The present study deepens our understanding of the role of autophagy in the pathogenesis of DN.

  7. Small heterodimer partner overexpression partially protects against liver tumor development in farnesoid X receptor knockout mice

    International Nuclear Information System (INIS)

    Li, Guodong; Kong, Bo; Zhu, Yan; Zhan, Le; Williams, Jessica A.; Tawfik, Ossama; Kassel, Karen M.; Luyendyk, James P.; Wang, Li; Guo, Grace L.

    2013-01-01

    Farnesoid X receptor (FXR, Nr1h4) and small heterodimer partner (SHP, Nr0b2) are nuclear receptors that are critical to liver homeostasis. Induction of SHP serves as a major mechanism of FXR in suppressing gene expression. Both FXR −/− and SHP −/− mice develop spontaneous hepatocellular carcinoma (HCC). SHP is one of the most strongly induced genes by FXR in the liver and is a tumor suppressor, therefore, we hypothesized that deficiency of SHP contributes to HCC development in the livers of FXR −/− mice and therefore, increased SHP expression in FXR −/− mice reduces liver tumorigenesis. To test this hypothesis, we generated FXR −/− mice with overexpression of SHP in hepatocytes (FXR −/− /SHP Tg ) and determined the contribution of SHP in HCC development in FXR −/− mice. Hepatocyte-specific SHP overexpression did not affect liver tumor incidence or size in FXR −/− mice. However, SHP overexpression led to a lower grade of dysplasia, reduced indicator cell proliferation and increased apoptosis. All tumor-bearing mice had increased serum bile acid levels and IL-6 levels, which was associated with activation of hepatic STAT3. In conclusion, SHP partially protects FXR −/− mice from HCC formation by reducing tumor malignancy. However, disrupted bile acid homeostasis by FXR deficiency leads to inflammation and injury, which ultimately results in uncontrolled cell proliferation and tumorigenesis in the liver. - Highlights: • SHP does not prevent HCC incidence nor size in FXR KO mice but reduces malignancy. • Increased SHP promotes apoptosis. • Bile acids and inflammation maybe critical for HCC formation with FXR deficiency

  8. Polyomic profiling reveals significant hepatic metabolic alterations in glucagon-receptor (GCGR knockout mice: implications on anti-glucagon therapies for diabetes

    Directory of Open Access Journals (Sweden)

    Molloy Mark P

    2011-06-01

    Full Text Available Abstract Background Glucagon is an important hormone in the regulation of glucose homeostasis, particularly in the maintenance of euglycemia and prevention of hypoglycemia. In type 2 Diabetes Mellitus (T2DM, glucagon levels are elevated in both the fasted and postprandial states, which contributes to inappropriate hyperglycemia through excessive hepatic glucose production. Efforts to discover and evaluate glucagon receptor antagonists for the treatment of T2DM have been ongoing for approximately two decades, with the challenge being to identify an agent with appropriate pharmaceutical properties and efficacy relative to potential side effects. We sought to determine the hepatic & systemic consequence of full glucagon receptor antagonism through the study of the glucagon receptor knock-out mouse (Gcgr-/- compared to wild-type littermates. Results Liver transcriptomics was performed using Affymetric expression array profiling, and liver proteomics was performed by iTRAQ global protein analysis. To complement the transcriptomic and proteomic analyses, we also conducted metabolite profiling (~200 analytes using mass spectrometry in plasma. Overall, there was excellent concordance (R = 0.88 for changes associated with receptor knock-out between the transcript and protein analysis. Pathway analysis tools were used to map the metabolic processes in liver altered by glucagon receptor ablation, the most notable being significant down-regulation of gluconeogenesis, amino acid catabolism, and fatty acid oxidation processes, with significant up-regulation of glycolysis, fatty acid synthesis, and cholesterol biosynthetic processes. These changes at the level of the liver were manifested through an altered plasma metabolite profile in the receptor knock-out mice, e.g. decreased glucose and glucose-derived metabolites, and increased amino acids, cholesterol, and bile acid levels. Conclusions In sum, the results of this study suggest that the complete ablation

  9. The Sleep–Wake Cycle in the Nicotinic Alpha-9 Acetylcholine Receptor Subunit Knock-Out Mice

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    Natalia Madrid-López

    2017-10-01

    Full Text Available There is a neural matrix controlling the sleep–wake cycle (SWC embedded within high ranking integrative mechanisms in the central nervous system. Nicotinic alpha-9 acetylcholine receptor subunit (alpha-9 nAChR participate in physiological processes occurring in sensory, endocrine and immune systems. There is a relationship between the SWC architecture, body homeostasis and sensory afferents so that disruption of afferent signaling is expected to affect the temporal organization of sleep and wake states. The analysis of the SWC of 9 nAChR knock-out animals may help to reveal the contribution of alpha-9 nAChR to sleep chronobiological determinants. Here we explore the polysomnogram in chronically implanted alpha-9 nAChR knock-out (KO and wild-type (WT individuals of the hybrid CBA/Sv129 mouse strain. Records were obtained in isolation chambers under a stable 12:12 light:dark cycle (LD. To unmask the 24-h modulation of the SWC a skeleton photoperiod (SP protocol was performed. Under LD the daily quota (in % of wakefulness (W, NREM sleep and REM sleep obtained in KO and WT animals were 45, 48 and 7, and 46, 46 and 8 respectively. Both groups exhibit nocturnal phase preference of W as well as diurnal and unimodal phase preference of NREM and REM sleep. The acrophase mean angles of KO vs. WT genotypes were not different (Zeitgeber Time: 6.5 vs. 14.9 for W, 4.3 vs. 2.8 for NREM sleep and 5.3 vs. 3.4 for REM sleep, respectively. Transference to SP do not affect daily state quotas, phase preferences and acrophases among genotypes. Unmasking phenomena of the SWC such as wake increment during the rest phase under SP was evident only among WT mice suggesting the involvement of retinal structures containing alpha-9 nAChR in masking processes. Furthermore, KO animals exhibit longer NREM and REM sleep episodes that is independent of illumination conditions. Consolidated diurnal NREM sleep contributed to obtain higher values of NREM sleep delta-EEG activity

  10. Sleep architecture of the melanin-concentrating hormone receptor 1-knockout mice.

    Science.gov (United States)

    Adamantidis, Antoine; Salvert, Denise; Goutagny, Romain; Lakaye, Bernard; Gervasoni, Damien; Grisar, Thierry; Luppi, Pierre-Hervé; Fort, Patrice

    2008-04-01

    Growing amounts of data indicate involvement of the posterior hypothalamus in the regulation of sleep, especially paradoxical sleep (PS). Accordingly, we previously showed that the melanin-concentrating hormone (MCH)-producing neurons of the rat hypothalamus are selectively activated during a PS rebound. In addition, intracerebroventricular infusion of MCH increases total sleep duration, suggesting a new role for MCH in sleep regulation. To determine whether activation of the MCH system promotes sleep, we studied spontaneous sleep and its homeostatic regulation in mice with deletion of the MCH-receptor 1 gene (MCH-R1-/- vs. MCH-R1+/+) and their behavioural response to modafinil, a powerful antinarcoleptic drug. Here, we show that the lack of functional MCH-R1 results in a hypersomniac-like phenotype, both in basal conditions and after total sleep deprivation, compared to wild-type mice. Further, we found that modafinil was less potent at inducing wakefulness in MCH-R1-/- than in MCH-R1+/+ mice. We report for the first time that animals with genetically inactivated MCH signaling exhibit altered vigilance state architecture and sleep homeostasis. This study also suggests that the MCH system may modulate central pathways involved in the wake-promoting effect of modafinil.

  11. Comparison of the hepatic and thyroid gland effects of sodium phenobarbital in wild type and constitutive androstane receptor (CAR) knockout rats and pregnenolone-16α-carbonitrile in wild type and pregnane X receptor (PXR) knockout rats.

    Science.gov (United States)

    Haines, Corinne; Chatham, Lynsey R; Vardy, Audrey; Elcombe, Clifford R; Foster, John R; Lake, Brian G

    2018-03-13

    A number of chemicals produce liver and thyroid gland tumours in rodents by nongenotoxic modes of action (MOAs). In this study the hepatic and thyroid gland effects of the constitutive androstane receptor (CAR) activator sodium phenobarbital (NaPB) were examined in male Sprague-Dawley wild type (WT) rats and in CAR knockout (CAR KO) rats and the effects of the pregnane X receptor (PXR) activator pregnenolone-16α-carbonitrile (PCN) were examined in WT and PXR knockout (PXR KO) rats. Rats were either fed diets containing 0 (control) or 500 ppm NaPB or were dosed with 0 (control) or 100 mg/kg/day PCN orally for 7 days. The treatment of WT rats with NaPB and PCN for 7 days resulted in increased relative liver weight, increased hepatocyte replicative DNA synthesis (RDS) and the induction of cytochrome P450 CYP2B and CYP3A subfamily enzyme, mRNA and protein levels. In marked contrast, the treatment of CAR KO rats with NaPB and PXR KO rats with PCN did not result in any increases in liver weight and induction of CYP2B and CYP3A enzymes. The treatment of CAR KO rats with NaPB had no effect on hepatocyte RDS, while PCN produced only a small increase in hepatocyte RDS in PXR KO rats. Treatment with NaPB had no effect on thyroid gland weight in WT and CAR KO rats, whereas treatment with PCN resulted in an increase in relative thyroid gland weight in WT, but not in PXR KO, rats. Thyroid gland follicular cell RDS was increased by the treatment of WT rats with NaPB and PCN, with NaPB also producing a small increase in thyroid gland follicular cell RDS in CAR KO rats. Overall, the present study with CAR KO rats demonstrates that a functional CAR is required for NaPB-mediated increases in liver weight, stimulation of hepatocyte RDS and induction of hepatic CYP enzymes. The studies with PXR KO rats demonstrate that a functional PXR is required for PCN-mediated increases in liver weight and induction of hepatic CYP enzymes; with induction of hepatocyte RDS also being

  12. Wild rice (Zizania palustris L.) prevents atherogenesis in LDL receptor knockout mice.

    Science.gov (United States)

    Surendiran, Gangadaran; Goh, Chunyan; Le, Khuong; Zhao, Zhaohui; Askarian, Fatemeh; Othman, Rgia; Nicholson, Tiffany; Moghadasian, Paymahn; Wang, Ya-Jane; Aliani, Michel; Shen, Garry; Beta, Trust; Moghadasian, Mohammed H

    2013-10-01

    Dietary modifications including healthy eating constitute one of the first line strategies for prevention and treatment of cardiovascular disease (CVD) risk factors including high cholesterol and atherosclerosis. The purpose of the present study was to investigate the potential cardiovascular benefits of wild rice in male and female LDL-receptor-deficient (LDLr-KO) mice. Wild rice was used to create a semi-synthetic diet containing approximately 60% of total energy from carbohydrate. Two other experimental diets were similar in macronutrient composition, but containing either white rice or commercial carbohydrate sources. All diets were supplemented with 0.06% (w/w) dietary cholesterol. The mice were divided into six experimental groups and fed with these diets over 24 weeks. Consumption of wild rice significantly reduced the size and severity of atherosclerotic lesions in the aortic roots of male and female mice by 71 and 61% respectively, compared to the control group of the same gender. This effect was associated with significant reductions of plasma cholesterol levels by 15 and 40%, low density lipoprotein (LDL) levels by 12 and 42%, and very low density lipoprotein (VLDL) levels by 35 and 75% respectively, in male and female mice compared to the control group of the same gender. Increased fecal cholesterol excretion of up to 34% was also noted, compared to the control group of the same gender. However, the antiatherogenic effect of wild rice was not associated with increased superoxide dismutase (SOD) and catalase (CAT) activities. Current data suggest that cholesterol-lowering effects of wild rice may be the main factor for the prevention of atherogenesis in LDLr-KO mice. Additional studies are needed to understand the mechanism of action. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  13. Acylated apelin-13 amide analogues exhibit enzyme resistance and prolonged insulin releasing, glucose lowering and anorexic properties.

    Science.gov (United States)

    O'Harte, Finbarr P M; Parthsarathy, Vadivel; Hogg, Christopher; Flatt, Peter R

    2017-12-15

    The adipokine, apelin has many biological functions but its activity is curtailed by rapid plasma degradation. Fatty acid derived apelin analogues represent a new and exciting avenue for the treatment of obesity-diabetes. This study explores four novel fatty acid modified apelin-13 analogues, namely, (Lys 8 GluPAL)apelin-13 amide, pGlu(Lys 8 GluPAL)apelin-13 amide, Lys 8 GluPAL(Tyr 13 )apelin-13 and Lys 8 GluPAL(Val 13 )apelin-13. Fatty acid modification extended the half-life of native apelin-13 to >24 h in vitro. pGlu(Lys 8 GluPAL)apelin-13 amide was the most potent insulinotropic analogue in BRIN-BD11 cells and isolated islets with maximal stimulatory effects of up to 2.7-fold (p glucose uptake in 3T3-L1 adipocytes (2.3-fold, p glucose (39-43%, p glucose tolerance tests in diet-induced obese mice. pGlu(Lys 8 GluPAL)apelin-13 amide and (Lys 8 GluPAL)apelin-13 amide also inhibited feeding (28-40%, p < .001), whereas Lys 8 GluPAL(Val 13 )apelin-13 increased food intake (8%, p < .05) in mice. These data indicate that novel enzymatically stable analogues of apelin-13 may be suitable for future development as therapeutic agents for obesity-diabetes. Copyright © 2017 Elsevier Inc. All rights reserved.

  14. Catalase can protect spermatozoa of FSH receptor knock-out mice against oxidant-induced DNA damage in vitro.

    Science.gov (United States)

    Libman, J; Gabriel, M S; Sairam, M R; Zini, A

    2010-12-01

    The aetiology of sperm DNA damage is likely multi-factorial with abnormal compaction of nuclear DNA, abortive apoptosis and oxidative stress implicated as potential causes of DNA damage. The objective of this study was to evaluate DNA damage in spermatozoa from wild-type (WT) and FSH receptor knock-out (FORKO) mice, compare the relative susceptibility of spermatozoa from these animals to oxidative DNA damage, and examine the protective effect of the antioxidant catalase on sperm DNA damage. Epididymal spermatozoa from FORKO mice (n = 5) and WT controls (n = 5) were extracted and incubated with or without catalase. Sperm DNA damage was assessed immediately after epididymal extraction (time 0 control) and following 2-h incubation at 37 °C. DNA damage was measured by the sperm chromatin structure assay and the results expressed as the %DNA fragmentation index or %DFI. Freshly retrieved epididymal spermatozoa from WT mice had a significantly lower mean (±SD) %DFI than that of FORKO mice (2.7 ± 1.8 vs. 6.4 ± 2.9%, p catalase protected these spermatozoa from DNA damage (9.8 ± 4.1 vs. 17.9 ± 9.2%, respectively, p 0.05) and the addition of catalase (vs. no catalase) did not result in a significant reduction in %DFI (5.8 ± 5.0 vs. 7.7 ± 6.5%, respectively, p > 0.05). These data indicate that catalase may protect sperm nuclear DNA from oxidative stress in vitro. The data also demonstrate the differential susceptibility of WT and FORKO mice spermatozoa to oxidative stress. © 2010 The Authors. International Journal of Andrology © 2010 European Academy of Andrology.

  15. Liver protein profiles in insulin receptor-knockout mice reveal novel molecules involved in the diabetes pathophysiology.

    Science.gov (United States)

    Capuani, Barbara; Della-Morte, David; Donadel, Giulia; Caratelli, Sara; Bova, Luca; Pastore, Donatella; De Canio, Michele; D'Aguanno, Simona; Coppola, Andrea; Pacifici, Francesca; Arriga, Roberto; Bellia, Alfonso; Ferrelli, Francesca; Tesauro, Manfredi; Federici, Massimo; Neri, Anna; Bernardini, Sergio; Sbraccia, Paolo; Di Daniele, Nicola; Sconocchia, Giuseppe; Orlandi, Augusto; Urbani, Andrea; Lauro, Davide

    2015-05-01

    Liver has a principal role in glucose regulation and lipids homeostasis. It is under a complex control by substrates such as hormones, nutrients, and neuronal impulses. Insulin promotes glycogen synthesis, lipogenesis, and lipoprotein synthesis and inhibits gluconeogenesis, glycogenolysis, and VLDL secretion by modifying the expression and enzymatic activity of specific molecules. To understand the pathophysiological mechanisms leading to metabolic liver disease, we analyzed liver protein patterns expressed in a mouse model of diabetes by proteomic approaches. We used insulin receptor-knockout (IR(-/-)) and heterozygous (IR(+/-)) mice as a murine model of liver metabolic dysfunction associated with diabetic ketoacidosis and insulin resistance. We evaluated liver fatty acid levels by microscopic examination and protein expression profiles by orthogonal experimental strategies using protein 2-DE MALDI-TOF/TOF and peptic nLC-MS/MS shotgun profiling. Identified proteins were then loaded into Ingenuity Pathways Analysis to find possible molecular networks. Twenty-eight proteins identified by 2-DE analysis and 24 identified by nLC-MS/MS shotgun were differentially expressed among the three genotypes. Bioinformatic analysis revealed a central role of high-mobility group box 1/2 and huntigtin never reported before in association with metabolic and related liver disease. A different modulation of these proteins in both blood and hepatic tissue further suggests their role in these processes. These results provide new insight into pathophysiology of insulin resistance and hepatic steatosis and could be useful in identifying novel biomarkers to predict risk for diabetes and its complications. Copyright © 2015 the American Physiological Society.

  16. Macrophage ABCA2 deletion modulates intracellular cholesterol deposition, affects macrophage apoptosis, and decreases early atherosclerosis in LDL receptor knockout mice.

    Science.gov (United States)

    Calpe-Berdiel, Laura; Zhao, Ying; de Graauw, Marjo; Ye, Dan; van Santbrink, Peter J; Mommaas, A Mieke; Foks, Amanda; Bot, Martine; Meurs, Illiana; Kuiper, Johan; Mack, Jody T; Van Eck, Miranda; Tew, Kenneth D; van Berkel, Theo J C

    2012-08-01

    The ABCA2 transporter shares high structural homology to ABCA1, which is crucial for the removal of excess cholesterol from macrophages and, by extension, in atherosclerosis. It has been suggested that ABCA2 sequesters cholesterol inside the lysosomes, however, little is known of the macrophage-specific role of ABCA2 in regulating lipid homeostasis in vivo and in modulating susceptibility to atherosclerosis. Chimeras with dysfunctional macrophage ABCA2 were generated by transplantation of bone marrow from ABCA2 knockout (KO) mice into irradiated LDL receptor (LDLr) KO mice. Interestingly, lack of ABCA2 in macrophages resulted in a diminished lesion size in the aortic root (-24.5%) and descending thoracic aorta (-36.6%) associated with a 3-fold increase in apoptotic cells, as measured by both caspase 3 and TUNEL. Upon oxidized LDL exposure, macrophages from wildtype (WT) transplanted animals developed filipin-positive droplets in lysosomal-like compartments, corresponding to free cholesterol (FC) accumulation. In contrast, ABCA2-deficient macrophages displayed an abnormal diffuse distribution of FC over peripheral regions. The accumulation of neutral sterols in lipid droplets was increased in ABCA2-deficient macrophages, but primarily in cytoplasmic clusters and not in lysosomes. Importantly, apoptosis of oxLDL loaded macrophages lacking ABCA2 was increased 2.7-fold, probably as a consequence of the broad cellular distribution of FC. Lack of functional ABCA2 generates abnormalities in intracellular lipid distribution/trafficking in macrophages consistent with its lysosomal sequestering role, leading to an increased susceptibility to apoptosis in response to oxidized lipids and reduced atherosclerotic lesion development. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

  17. Yellow wine polyphenolic compounds inhibit matrix metalloproteinase-2, -9 expression and improve atherosclerotic plaque in LDL-receptor-knockout mice.

    Science.gov (United States)

    Zhai, Xiaoya; Chi, Jufang; Tang, Weiliang; Ji, Zheng; Zhao, Fei; Jiang, Chengjian; Lv, Haitao; Guo, Hangyuan

    2014-01-01

    Many epidemiological studies have strongly suggested an inverse correlation between dietary polyphenol consumption and reduced risks of cardiovascular diseases. Yellow rice wine is a Chinese specialty and one of the three most ancient wines in the world (Shaoxing rice wine, beer, and grape wine). There is a large amount of polyphenol substances in yellow rice wine. This experiment was designed to study the potential beneficial effects of yellow wine polyphenolic compounds (YWPC) from yellow rice wine on progression of atherosclerosis in vivo and to further explore its underlying mechanisms. Six-week-old male LDL-receptor-knockout mice were treated with high-fat diet to establish the mouse model with atherosclerosis. Animals received 10, 30, or 50 mg/kg per day of YWPC or 10 mg/kg per day rosuvastatin or water (vehicle) for 14 weeks. The results indicated that YWPC and rosuvastatin significantly decreased circulating total cholesterol and low-density lipoprotein cholesterol. Compared to the control group, the atherosclerosis lesion area in the rosuvastatin-intervention group and YWPC at doses of 10, 30, and 50 mg/kg per day intervention groups decreased by 74.14%, 18.51%, 40.09%, and 38.42%, respectively. YWPC and rosuvastatin decreased the expression and activity of matrix metalloproteinases (MMP)-2, 9, whereas the expression of the endogenous inhibitors of these proteins, namely, tissue inhibitors of matrix metalloproteinases (TIMP)-1, 2, increased when compared to the control group. It can be concluded that the YWPC is similar to the benefic effects of rosuvastatin on cardiovascular system. These effects may be attributed to their anti-atherosclerotic actions by lowering lipid and modulating the activity and expression of MMP-2, 9 and TIMP-1, 2.

  18. Trace eyeblink conditioning is impaired in α7 but not in β2 nicotinic acetylcholine receptor knock-out mice

    Directory of Open Access Journals (Sweden)

    Kevin L Brown

    2010-10-01

    Full Text Available Nicotinic acetylcholine receptors (nAChRs are essentially involved in learning and memory. A neurobiologically and behaviorally well-characterized measure of learning and memory, eyeblink classical conditioning, is sensitive to disruptions in acetylcholine neurotransmission. The two most common forms of eyeblink classical conditioning – the delay and trace paradigms - differentially engage forebrain areas densely-populated with nAChRs. The present study used genetically modified mice to investigate the effects of selective nAChR subunit deletion on delay and trace eyeblink classical conditioning. α7 and β2 nAChR subunit knockout (KO mice and their wild-type littermates were trained for 10 daily sessions in a 500 ms delay or 500 ms trace eyeblink conditioning task, matched for the interstimulus interval (ISI between conditioned stimulus (CS and unconditioned stimulus (US onset. Impairments in conditioned responding were found in α7 KO mice trained in trace – but not delay – eyeblink conditioning. Relative to littermate controls, β2 KO mice were unimpaired in the trace task but displayed higher levels of conditioned responding in delay eyeblink conditioning. Elevated conditioned response levels in delay-conditioned β2 KOs corresponded to elevated levels of alpha responding in this group. These findings suggest that α7 nAChRs play a role in normal acquisition of 500 ms trace eyeblink classical conditioning in mice. The prominent distribution of α7 nAChRs in the hippocampus and other forebrain regions may account for these genotype-specific acquisition effects in this hippocampus-dependent trace paradigm.

  19. Sex differences in the effects of adolescent social deprivation on alcohol consumption in μ-opioid receptor knockout mice.

    Science.gov (United States)

    Moriya, Yuki; Kasahara, Yoshiyuki; Hall, F Scott; Sakakibara, Yasufumi; Uhl, George R; Tomita, Hiroaki; Sora, Ichiro

    2015-04-01

    Evidence based on clinical and experimental animal studies indicates that adolescent social deprivation influences alcohol consumption in a sex-dependent manner, perhaps by influencing stress responses. However, the mechanisms underlying the interaction between these phenomena remain to be elucidated. Since the μ-opioid receptor (MOP) has been reported to have key roles in social stress responses as well as the reinforcing/addictive effects of ethanol, MOP is a candidate molecule that may link adolescent social deprivation and subsequent alterations in alcohol consumption. To evaluate the involvement of MOP and social isolation-induced changes in alcohol consumption, as well as the effect of sex differences on responses to social isolation, alcohol consumption was assessed using a two-bottle home-cage consumption procedure (8 % ethanol vs. water) in MOP knockout (MOP-KO) and wild type (WT) mice of both sexes exposed to adolescent social deprivation or reared socially. Isolation rearing had no effects upon alcohol consumption of WT mice, whereas it significantly altered alcohol consumption in both male and female MOP-KO mice. Interestingly, social isolation affected ethanol consumption differently in male and female mice. Ethanol consumption was increased in male MOP-KO mice, but decreased in female MOP-KO mice, by isolation rearing. These results indicate that disturbances of MOP function influence the effects of isolation rearing on ethanol consumption in a sex-dependent manner. Consequently, this suggests the possibility that genetic variation that influences MOP function may have differential roles in alcoholism in men and women, and alcoholism treatments that target MOP function may be differentially effective in males and females.

  20. The role of serum apelin in retinopathy of prematurity

    Directory of Open Access Journals (Sweden)

    Ali YF

    2017-02-01

    Full Text Available Yasser F Ali,1 Salah El-Morshedy,1 Abdulbasit Abdulhalim Imam,2 Nasser Ismai A Abdelrahman,1 Riad M Elsayed,3 Usama M Alkholy,1 Nermin Abdalmonem,1 Mohammed M Shehab1 1Department of Pediatrics, Faculty of Medicine, Zagazig University, Zagazig, 2Department of Pediatrics, Al-Azhar Faculty of Medicine-Girls, Cairo, 3Pediatric Neurology Unit, Pediatric Department, Mansoura University, Mansoura, Egypt Objective: To evaluate the role of serum apelin as a diagnostic tool in retinopathy of prematurity (ROP disease.Patients and methods: Thirty-eight preterm infants (60% male with gestational age ranging from 30 to 36 weeks admitted to the neonatal intensive care unit, KJO Hospital, Saudi Arabia with proven diagnosis of ROP were included in the study. In addition, 27 preterm infants without ROP served as controls. All newborn infants in the study were subjected to adequate history taking, full clinical examination, and fundus examination by indirect ophthalmoscope (at 4–6 weeks as well as determination of serum apelin at birth and at 4–6 weeks of age.Results: The study revealed that oxygen therapy longer than 7 days’ duration, cesarean section (as a mode of delivery, sepsis, mechanical ventilation, blood transfusion, premature rupture of membranes, pneumothorax, perinatal asphyxia, cardiac problems, and neonatal jaundice were considered as risk factors related to development of ROP. Serum apelin levels were significantly lower in patients than controls (P<0.001 at time of diagnosis of the disease (4–6 weeks while no significant differences were observed in levels at birth.Conclusion: Serum apelin was found to be of significant diagnostic value in the occurrence of ROP. Keywords: retinopathy of prematurity, preterm infants, serum apelin

  1. Adaptations in pre- and postsynaptic 5-HT(1A) receptor function and cocaine supersensitivity in serotonin transporter knockout rats

    NARCIS (Netherlands)

    Homberg, Judith R; De Boer, Sietse F; Raasø, Halfdan S; Olivier, Jocelien D A; Verheul, Mark; Ronken, Eric; Cools, Alexander R; Ellenbroek, Bart A; Schoffelmeer, Anton N M; Vanderschuren, Louk J M J; De Vries, Taco J; Cuppen, Edwin

    2008-01-01

    RATIONALE: While individual differences in vulnerability to psychostimulants have been largely attributed to dopaminergic neurotransmission, the role of serotonin is not fully understood. OBJECTIVES: To study the rewarding and motivational properties of cocaine in the serotonin transporter knockout

  2. Effects of apelin on reproductive functions: relationship with feeding behavior and energy metabolism.

    Science.gov (United States)

    Tekin, Suat; Erden, Yavuz; Sandal, Suleyman; Etem Onalan, Ebru; Ozyalin, Fatma; Ozen, Hasan; Yilmaz, Bayram

    2017-02-01

    Apelin is an adipose tissue derived peptidergic hormone. In this study, 40 male Sprague-Dawley rats were used (four groups; n = 10). Apelin-13 at three different dosages (1, 5 and 50 μg/kg) was given intraperitoneally while the control group received vehicle the same route for a period of 14 days. In results, apelin-13 caused significant decreases in serum testosterone, luteinizing hormone and follicle-stimulating hormone levels (p hormones.

  3. Biochemical and genetic role of apelin in essential hypertension and acute coronary syndrome.

    Science.gov (United States)

    Gupta, Mohit D; Girish, M P; Shah, Dhaval; Rain, Manjari; Mehta, Vimal; Tyagi, Sanjay; Trehan, Vijay; Pasha, Qadar

    2016-11-15

    Apelin-APJ pathway has emerged as a potent regulator of blood pressure (BP) and blood flow in vasculature and heart. Variants in apelin gene may affect the vascular tone in peripheral circulation or heart, thereby predisposing to cardiovascular diseases. The aim of our study was to investigate the association of two apelin gene polymorphisms rs3761581 and rs2235312, and apelin levels in patients with essential hypertension (EH) and acute coronary syndrome (ACS). The study comprised of three groups namely, (1) 118 healthy control subjects, (2) 92 EH patients, and (3) 60 ACS patients. DNA was extracted from peripheral blood leukocytes and genotyping was performed by SNaPshot method. Plasma apelin 13 levels were estimated using ELISA. EH and ACS patients had a significantly lower level of apelin 13, regardless of gender (p=0.003, p=0.017, respectively). Interestingly, the female EH and ACS patients had lower levels of apelin 13 than their male counterparts. The G allele of rs3761581 was more apparent in patients especially in ACS than the controls. Reduced apelin levels may enhance vasoconstriction to influence high BP and heart's workload in EH and ACS. Genetic involvement of apelin needs to be established in well-defined larger sample size. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  4. β-Arrestin-2 knockout prevents development of cellular μ-opioid receptor tolerance but does not affect opioid-withdrawal-related adaptations in single PAG neurons.

    Science.gov (United States)

    Connor, M; Bagley, E E; Chieng, B C; Christie, M J

    2015-01-01

    Tolerance to the behavioural effects of morphine is blunted in β-arrestin-2 knockout mice, but opioid withdrawal is largely unaffected. The cellular mechanisms of tolerance have been studied in some neurons from β-arrestin-2 knockouts, but tolerance and withdrawal mechanisms have not been examined at the cellular level in periaqueductal grey (PAG) neurons, which are crucial for central tolerance and withdrawal phenomena. μ-Opioid receptor (MOPr) inhibition of voltage-gated calcium channel currents (ICa ) was examined by patch-clamp recordings from acutely dissociated PAG neurons from wild-type and β-arrestin-2 knockout mice treated chronically with morphine (CMT) or vehicle. Opioid withdrawal-induced activation of GABA transporter type 1 (GAT-1) currents was determined using perforated patch recordings from PAG neurons in brain slices. MOPr inhibition of ICa in PAG neurons was unaffected by β-arrestin-2 deletion. CMT impaired coupling of MOPrs to ICa in PAG neurons from wild-type mice, but this cellular tolerance was not observed in neurons from CMT β-arrestin-2 knockouts. However, β-arrestin-2 knockouts displayed similar opioid-withdrawal-induced activation of GAT-1 currents as wild-type PAG neurons. In β-arrestin-2 knockout mice, the central neurons involved in the anti-nociceptive actions of opioids also fail to develop cellular tolerance to opioids following chronic morphine. The results also provide the first cellular physiological evidence that opioid withdrawal is not disrupted by β-arrestin-2 deletion. However, the unaffected basal sensitivity to opioids in PAG neurons provides further evidence that changes in basal MOPr sensitivity cannot account for the enhanced acute nociceptive response to morphine reported in β-arrestin-2 knockouts. This article is part of a themed section on Opioids: New Pathways to Functional Selectivity. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-2. © 2014 The British

  5. Antinociceptive effects of morphine and naloxone in mu-opioid receptor knockout mice transfected with the MORS196A gene

    Directory of Open Access Journals (Sweden)

    Tao Pao-Luh

    2010-04-01

    Full Text Available Abstract Background Opioid analgesics such as morphine and meperidine have been used to control moderate to severe pain for many years. However, these opioids have many side effects, including the development of tolerance and dependence after long-term use, which has limited their clinical use. We previously reported that mutations in the mu-opioid receptors (MOR S196L and S196A rendered them responsive to the opioid antagonist naloxone without altering the agonist phenotype. In MORS196A knock-in mice, naloxone and naltrexone were antinociceptive but did not cause tolerance or physical dependence. In this study we delivery this mutated MOR gene into pain related pathway to confirm the possibility of in vivo transfecting MORS196A gene and using naloxone as a new analgesic agent. Methods The MOR-knockout (MOR-KO mice were used to investigate whether morphine and naloxone could show antinociceptive effects when MORS196A gene was transfected into the spinal cords of MOR-KO mice. Double-stranded adeno-associated virus type 2 (dsAAV2 was used to deliver the MORS196A-enhanced green fluorescence protein (EGFP gene by microinjected the virus into the spinal cord (S2/S3 dorsal horn region. Tail-flick test was used to measure the antinociceptive effect of drugs. Results Morphine (10 mg/kg, s.c. and naloxone (10 mg/kg, s.c. had no antinociceptive effects in MOR-KO mice before gene transfection. However, two or three weeks after the MOR-S196A gene had been injected locally into the spinal cord of MOR-KO mice, significant antinociceptive effects could be induced by naloxone or morphine. On the other hand, only morphine but not naloxone induced significant tolerance after sub-chronic treatment. Conclusion Transfecting the MORS196A gene into the spinal cord and systemically administering naloxone in MOR-KO mice activated the exogenously delivered mutant MOR and provided antinociceptive effect without causing tolerance. Since naloxone will not activate natural

  6. The evidence of apelin has the bidirectional effects on feeding regulation in Siberian sturgeon (Acipenser baerii).

    Science.gov (United States)

    Hao, Jin; Liu, Qing; Zhang, Xin; Wu, Yuanbing; Zhu, Jieyao; Qi, Jinwen; Tang, Ni; Wang, Shuyao; Wang, Hong; Chen, Defang; Li, Zhiqiong

    2017-08-01

    Apelin is a peptide, mainly produced in the brain, which participates in several physiologic effects. However, knowledge about the mechanism of appetite regulation in teleosts, including the role of apelin is not well understood. The aim of this study is to explore the effect of feeding status on the expression of apelin mRNA in the whole brain and the effects of injection of apelin on food intake in Siberian sturgeon (Acipenser baerii). In this study, we first cloned the apelin cDNA sequence of the Siberian sturgeon. We obtained a 1046-bp cDNA fragment, including a 237-bp open reading frame (ORF) that encoded 78 amino acids. Apelin was widely distributed in 11 tissues related to feeding regulation, with the highest expression in thewhole brain, followed by the spleen and trunk kidney. In addition, we measured the effects of periprandial (preprandial and postprandial) change, fasting and re-feeding on apelin mRNA expression in whole brain. The level of apelin mRNA was significantly decreased 1h after feeding. The results of the fasting experiment showed that the expression of apelin mRNA in the brain was significantly reduced after 1day of fasting but consistently increased throughout the 15-day food deprivation period. When the 15-day fasted fish were re-fed, apelin mRNA expression in the brain was significantly increased as compared to that of the control. These results suggest that apelin may play a bidirectional role in the regulation of food intake in the Siberian sturgeon. In order to further examine the effect of apelin on feeding regulation in Siberian sturgeons, acute and chronic intraperitoneal (i.p.) injection experiments were performed and food intakes were recorded. Results showed that acute i.p. injection of apelin-13 reduced food intake, however, chronic i.p. injection apelin-13 increased the food intake for 7days in Siberian sturgeons. In conclusion, our results show that apelin has a bidirectional effect on feeding regulation in Siberian sturgeons

  7. Enhanced effects of amphetamine but reduced effects of the hallucinogen, 5-MeO-DMT, on locomotor activity in 5-HT1A receptor knockout mice: Implications for schizophrenia

    Science.gov (United States)

    van den Buuse, Maarten; Ruimschotel, Emma; Martin, Sally; Risbrough, Victoria B.; Halberstadt, Adam L.

    2012-01-01

    Serotonin-1A (5-HT1A) receptors may play a role in schizophrenia and the effects of certain antipsychotic drugs. However, the mechanism of interaction of 5-HT1A receptors with brain systems involved in schizophrenia, remains unclear. Here we show that 5-HT1A receptor knockout mice display enhanced locomotor hyperactivity to acute treatment with amphetamine, a widely used animal model of hyperdopaminergic mechanisms in psychosis. In contrast, the effect of MK-801 on locomotor activity, modeling NMDA receptor hypoactivity, was unchanged in the knockouts. The effect of the hallucinogen 5-methoxy-N,N-dimethyltryptamine (5-MeO-DMT) was markedly reduced in 5-HT1A receptor knockout mice. There were no changes in apomorphine-induced disruption of PPI, a model of sensory gating deficits seen in schizophrenia. Similarly, there were no major changes in density of dopamine transporters (DAT) or dopamine D1 or D2 receptors which could explain the behavioural changes observed in 5-HT1A receptor knockout mice. These results extend our insight into the possible role of these receptors in aspects of schizophrenia. As also suggested by previous studies using agonist and antagonist drugs, 5-HT1A receptors may play an important role in hallucinations and to modulate dopaminergic activity in the brain. PMID:21501627

  8. Enhanced effects of amphetamine but reduced effects of the hallucinogen, 5-MeO-DMT, on locomotor activity in 5-HT(1A) receptor knockout mice: implications for schizophrenia.

    Science.gov (United States)

    van den Buuse, Maarten; Ruimschotel, Emma; Martin, Sally; Risbrough, Victoria B; Halberstadt, Adam L

    2011-01-01

    Serotonin-1A (5-HT(1A)) receptors may play a role in schizophrenia and the effects of certain antipsychotic drugs. However, the mechanism of interaction of 5-HT(1A) receptors with brain systems involved in schizophrenia, remains unclear. Here we show that 5-HT(1A) receptor knockout mice display enhanced locomotor hyperactivity to acute treatment with amphetamine, a widely used animal model of hyperdopaminergic mechanisms in psychosis. In contrast, the effect of MK-801 on locomotor activity, modeling NMDA receptor hypoactivity, was unchanged in the knockouts. The effect of the hallucinogen 5-methoxy-N,N-dimethyltryptamine (5-MeO-DMT) was markedly reduced in 5-HT(1A) receptor knockout mice. There were no changes in apomorphine-induced disruption of PPI, a model of sensory gating deficits seen in schizophrenia. Similarly, there were no major changes in density of dopamine transporters (DAT) or dopamine D(1) or D(2) receptors which could explain the behavioural changes observed in 5-HT(1A) receptor knockout mice. These results extend our insight into the possible role of these receptors in aspects of schizophrenia. As also suggested by previous studies using agonist and antagonist drugs, 5-HT(1A) receptors may play an important role in hallucinations and to modulate dopaminergic activity in the brain. Copyright © 2011 Elsevier Ltd. All rights reserved.

  9. Effects of muscarinic receptor antagonists on cocaine discrimination in wild-type mice and in muscarinic receptor M1, M2, and M4 receptor knockout mice

    DEFF Research Database (Denmark)

    Joseph, Lauren; Thomsen, Morgane

    2017-01-01

    Muscarinic M1/M4 receptor stimulation can reduce abuse-related effects of cocaine and may represent avenues for treating cocaine addiction. Muscarinic antagonists can mimic and enhance effects of cocaine, including discriminative stimulus (SD) effects, but the receptor subtypes mediating those...

  10. The time point of β-catenin knockout in hepatocytes determines their response to xenobiotic activation of the constitutive androstane receptor

    International Nuclear Information System (INIS)

    Ganzenberg, Katrin; Singh, Yasmin; Braeuning, Albert

    2013-01-01

    The constitutive androstane receptor (CAR) controls the expression of drug-metabolizing enzymes and regulates hepatocyte proliferation. Studies with transgenic mice with an early postnatal conditional hepatocyte-specific knockout of the β-catenin gene Ctnnb1 revealed that β-catenin deficiency decreases the magnitude of induction of drug-metabolizing enzymes by CAR activators, abrogates zonal differences in the hepatocytes’ susceptibility to these compounds, and impacts on hepatocyte proliferation. These data, however, do not allow distinguishing between effects caused by β-catenin deficiency during postnatal liver development and acute effects of β-catenin deficiency in the adult animal at the time point of CAR activation. Therefore, CAR activation was now studied in a different mouse model allowing for the hepatocyte-specific knockout of β-catenin in adult mice. Treatment of these mice with 3 mg/kg body weight of the model CAR activator 1,4-bis-[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP) confirmed previous findings related to the coordinate regulation of drug metabolism by β-catenin and CAR. More importantly, the present study clarified that the impact of β-catenin signaling on CAR-mediated enzyme induction in the liver is not merely due to developmental defects caused by a postnatal lack of β-catenin, but depends on the presence of β-catenin at the time point of xenobiotic treatment. The study also revealed interesting differences between the two mouse models: hepatic zonation of TCPOBOP-dependent induction of drug-metabolizing enzymes was restored in mice with late knockout of β-catenin, and the strong proliferative response of female mice was exclusively abolished when using animals with a late β-catenin knockout. This suggests a β-catenin-dependent postnatal priming of hepatocytes during postnatal liver development, later affecting the proliferative response of adult animals to CAR-activating xenobiotics

  11. The role of serum apelin in retinopathy of prematurity

    OpenAIRE

    Ali, Yasser F; El-Morshedy, Salah; Imam, Abdulbasit Abdulhalim; Abdelrahman, Nasser Ismai A; Elsayed, Riad M; Alkholy, Usama M; Abdalmonem, Nermin; Shehab, Mohammed M

    2017-01-01

    Yasser F Ali,1 Salah El-Morshedy,1 Abdulbasit Abdulhalim Imam,2 Nasser Ismai A Abdelrahman,1 Riad M Elsayed,3 Usama M Alkholy,1 Nermin Abdalmonem,1 Mohammed M Shehab1 1Department of Pediatrics, Faculty of Medicine, Zagazig University, Zagazig, 2Department of Pediatrics, Al-Azhar Faculty of Medicine-Girls, Cairo, 3Pediatric Neurology Unit, Pediatric Department, Mansoura University, Mansoura, Egypt Objective: To evaluate the role of serum apelin as a diagnostic tool in retinopathy of...

  12. Lateral intracerebroventricular injection of Apelin-13 inhibits apoptosis after cerebral ischemia/reperfusion injury

    Directory of Open Access Journals (Sweden)

    Xiao-ge Yan

    2015-01-01

    Full Text Available Apelin-13 inhibits neuronal apoptosis caused by hydrogen peroxide, yet apoptosis following cerebral ischemia-reperfusion injury has rarely been studied. In this study, Apelin-13 (0.1 µg/g was injected into the lateral ventricle of middle cerebral artery occlusion model rats. TTC, TUNEL, and immunohistochemical staining showed that compared with the cerebral ischemia/reperfusion group, infarct volume and apoptotic cell number at the ischemic penumbra region were decreased in the Apelin-13 treatment group. Additionally, Apelin-13 treatment increased Bcl-2 immunoreactivity and decreased caspase-3 immunoreactivity. Our findings suggest that Apelin-13 is neuroprotective against cerebral ischemia/reperfusion injury through inhibition of neuronal apoptosis.

  13. Evaluation of organ-specific glucose metabolism by 18F-FDG in insulin receptor substrate-1 (IRS-1) knockout mice as a model of insulin resistance

    International Nuclear Information System (INIS)

    Cheng, Chao; Nakamura, Akinobu; Minamimoto, Ryogo; Shinoda, Kazuaki; Tateishi, Ukihide; Terauchi, Yasuo; Inoue, Tomio; Goto, Atsuhi; Kadowaki, Takashi

    2011-01-01

    Insulin resistance (IR) is a physiological condition in which the body produces insulin but does not result in a sufficient biological effect. Insulin resistance is usually asymptomatic but is associated with health problems and is a factor in the metabolic syndrome. The aim of the present study is to clarify organ-specific insulin resistance in normal daily conditions using [ 18 F]-2-fluoro-2-deoxy-D-glucose ([ 18 F]-FDG). The biodistribution of [ 18 F]-FDG was examined in insulin receptor substrate-1 (IRS-1) knockout mice, an animal model of skeletal muscle insulin resistance, and C57BL/6J (wild-type) mice with and without insulin loading. Mice received 0.5 MBq of [ 18 F]-FDG injected into the tail vein, immediately followed by nothing (control cohorts) or an intraperitoneal injection of 1.5 mU/g body weight of human insulin as an insulin loading test. Blood glucose concentrations for all of the experimental animals were assessed at 0, 20, 40, and 60 min post-injection. The mice were subsequently killed, and tissue was collected for evaluation of [ 18 F]-FDG biodistribution. The radioactivity of each organ was measured using a gamma counter. In the absence of insulin, the blood glucose concentrations of wild-type mice (132±26 mg/dl) and IRS-1 knockout mice (134±18 mg/dl) were not significantly different. Blood glucose concentrations decreased following insulin administration, with lower concentrations in wild-type mice than in knockout mice at 20, 40, and 60 min. A statistically significant difference in [ 18 F]-FDG uptake between wild-type mice and IRS-1 knockout mice was confirmed in the heart, abdominal muscle, and femoral muscle. With insulin loading, [ 18 F]-FDG uptake in the heart, back muscle, and abdominal muscle was significantly increased compared to without insulin loading in both wild-type mice and knockout mice. Our results showed that IR significantly affected [ 18 F]-FDG uptake in the heart in normal daily conditions. IR was associated with

  14. Fibroblast growth factor 21, fibroblast growth factor receptor 1, and β-Klotho expression in bovine growth hormone transgenic and growth hormone receptor knockout mice.

    Science.gov (United States)

    Brooks, Nicole E; Hjortebjerg, Rikke; Henry, Brooke E; List, Edward O; Kopchick, John J; Berryman, Darlene E

    Although growth hormone (GH) and fibroblast growth factor 21 (FGF21) have a reported relationship, FGF21 and its receptor, fibroblast growth factor receptor 1 (FGFR1) and cofactor β-Klotho (KLB), have not been analyzed in chronic states of altered GH action. The objective of this study was to quantify circulating FGF21 and tissue specific expression of Fgf21, Fgfr1, and Klb in mice with modified GH action. Based on previous studies, we hypothesized that bovine GH transgenic (bGH) mice will be FGF21 resistant and GH receptor knockout (GHR-/-) mice will have normal FGF21 action. Seven-month-old male bGH mice (n=9) and wild type (WT) controls (n=10), and GHR-/- mice (n=8) and WT controls (n=8) were used for all measurements. Body composition was determined before dissection, and tissue weights were measured at the time of dissection. Serum FGF21 levels were evaluated by ELISA. Expression of Fgf21, Fgfr1, and Klb mRNA in white adipose tissue (AT), brown AT, and liver were evaluated by reverse transcription quantitative PCR. As expected, bGH mice had increased body weight (p=3.70E -8 ) but decreased percent fat mass (p=4.87E -4 ). Likewise, GHR-/- mice had decreased body weight (p=1.78E -10 ) but increased percent fat mass (p=1.52E -9 ), due to increased size of the subcutaneous AT depot when normalized to body weight (p=1.60E -10 ). Serum FGF21 levels were significantly elevated in bGH mice (p=0.041) and unchanged in GHR-/- mice (p=0.88). Expression of Fgf21, Fgfr1, and Klb mRNA in white AT and liver were downregulated or unchanged in both bGH and GHR-/- mice. The only exception was Fgf21 expression in brown AT of GHR-/-, which trended toward increased expression (p=0.075). In accordance with our hypothesis, we provide evidence that circulating FGF21 is increased in bGH animals, but remains unchanged in GHR-/- mice. Downregulation or no change in Fgf21, Fgfr1, and Klb expression are seen in white AT, brown AT, and liver of bGH and GHR-/- mice when compared to their

  15. Absence seizures in C3H/HeJ and knockout mice caused by mutation of the AMPA receptor subunit Gria4.

    Science.gov (United States)

    Beyer, Barbara; Deleuze, Charlotte; Letts, Verity A; Mahaffey, Connie L; Boumil, Rebecca M; Lew, Timothy A; Huguenard, John R; Frankel, Wayne N

    2008-06-15

    Absence epilepsy, characterized by spike-wave discharges (SWD) in the electroencephalogram, arises from aberrations within the circuitry of the cerebral cortex and thalamus that regulates awareness. The inbred mouse strain C3H/HeJ is prone to absence seizures, with a major susceptibility locus, spkw1, accounting for most of the phenotype. Here we find that spkw1 is associated with a hypomorphic retroviral-like insertion mutation in the Gria4 gene, encoding one of the four amino-3-hydroxy-5-methyl-4isoxazolepropionic acid (AMPA) receptor subunits in the brain. Consistent with this, Gria4 knockout mice also have frequent SWD and do not complement spkw1. In contrast, null mutants for the related gene Gria3 do not have SWD, and Gria3 loss actually lowers SWD of spkw1 homozygotes. Gria3 and Gria4 encode the predominant AMPA receptor subunits in the reticular thalamus, which is thought to play a central role in seizure genesis by inhibiting thalamic relay cells and promoting rebound burst firing responses. In Gria4 mutants, synaptic excitation of inhibitory reticular thalamic neurons is enhanced, with increased duration of synaptic responses-consistent with what might be expected from reduction of the kinetically faster subunit of AMPA receptors encoded by Gria4. These results demonstrate for the first time an essential role for Gria4 in the brain, and suggest that abnormal AMPA receptor-dependent synaptic activity can be involved in the network hypersynchrony that underlies absence seizures.

  16. Knockout crickets for the study of learning and memory: Dopamine receptor Dop1 mediates aversive but not appetitive reinforcement in crickets.

    Science.gov (United States)

    Awata, Hiroko; Watanabe, Takahito; Hamanaka, Yoshitaka; Mito, Taro; Noji, Sumihare; Mizunami, Makoto

    2015-11-02

    Elucidation of reinforcement mechanisms in associative learning is an important subject in neuroscience. In mammals, dopamine neurons are thought to play critical roles in mediating both appetitive and aversive reinforcement. Our pharmacological studies suggested that octopamine and dopamine neurons mediate reward and punishment, respectively, in crickets, but recent studies in fruit-flies concluded that dopamine neurons mediates both reward and punishment, via the type 1 dopamine receptor Dop1. To resolve the discrepancy between studies in different insect species, we produced Dop1 knockout crickets using the CRISPR/Cas9 system and found that they are defective in aversive learning with sodium chloride punishment but not appetitive learning with water or sucrose reward. The results suggest that dopamine and octopamine neurons mediate aversive and appetitive reinforcement, respectively, in crickets. We suggest unexpected diversity in neurotransmitters mediating appetitive reinforcement between crickets and fruit-flies, although the neurotransmitter mediating aversive reinforcement is conserved. This study demonstrates usefulness of the CRISPR/Cas9 system for producing knockout animals for the study of learning and memory.

  17. Blood pressures, heart rate and locomotor activity during salt loading and angiotensin II infusion in protease-activated receptor 2 (PAR2 knockout mice

    Directory of Open Access Journals (Sweden)

    Halfyard Sarah J

    2008-10-01

    Full Text Available Abstract Background In this study we used radiotelemetry to measure hemodynamic variables and locomotor activity in conscious unrestrained male Protease-Activated Receptor 2 (PAR-2 knockout mice in order to provide a detailed assessment of their blood pressure phenotype. In addition we tested for an influence of PAR-2 on salt-sensitivity (8% versus 0.5% NaCl diet, 2.5 weeks and angiotensin II-induced hypertension (1 μg Ile5-angiotensin II/kg/min versus 0.25 μl/h saline, 2 weeks. Results Systolic arterial pressures of PAR-2 -/- (129 ± 1 mmHg, n = 21, P P Conclusion The data indicated gene knockout of PAR-2 was associated with a modest change in blood pressure phenotype. PAR-2 -/- mice exhibited moderate elevation of systolic arterial and pulse pressures, yet no increased diastolic arterial pressure, no increased blood pressure responses to high salt diet and a subtle difference in the time course of the blood pressure responses to angiotensin II infusion.

  18. Effect of AVE 0991 angiotensin-(1-7) receptor agonist treatment on elemental and biomolecular content and distribution in atherosclerotic plaques of apoE-knockout mice

    Science.gov (United States)

    Kowalska, J.; Gajda, M.; Jawień, J.; Kwiatek, W. M.; Appel, K.; Dumas, P.

    2013-12-01

    Gene-targeted apolipoprotein E-knockout (apoE-KO) mice display early and highly progressive vascular lesions containing lipid deposits and they became a reliable animal model to study atherosclerosis. The aim of the present study was to investigate the effect of AVE 0991 angiotensin-(1-7) receptor agonist on the distribution of selected pro- and anti- inflammatory elements as well as biomolecules in atherosclerotic plaques of apoE-knockout mice. Synchrotron radiation-based X-ray fluorescence (micro-XRF) and Fourier Transform Infrared (micro-FTIR) microspectroscopies were applied. Two-month-old apoE-KO mice were fed for following four months diet supplemented with AVE 0991 (0.58 μmol/kg b.w. per day). Histological sections of ascending aortas were analyzed spectroscopically. The distribution of P, Ca, Fe and Zn were found to correspond with histological structure of the lesion. Significantly lower contents of P, Ca, Zn and significantly higher content of Fe were observed in animals treated with AVE 0991. Biomolecular analysis showed lower lipids saturation level and lower lipid to protein ratio in AVE 0991 treated group. Protein secondary structure was studied according to the composition of amide I band (1660 cm-1) and it demonstrated higher proportion of β-sheet structure as compared to α-helix in both studied groups.

  19. Narcolepsy-like sleep disturbance in orexin knockout mice are normalized by the 5-HT1A receptor agonist 8-OH-DPAT.

    Science.gov (United States)

    Mori, Tomohisa; Uzawa, Naoki; Iwase, Yoshiyuki; Masukawa, Daiki; Rahmadi, Mahardian; Hirayama, Shigeto; Hokazono, Mayuna; Higashiyama, Kimio; Shioda, Seiji; Suzuki, Tsutomu

    2016-06-01

    Orexin knockout (KO) mice exhibit a phenotype that is similar to human narcolepsy, and monoamine-related compounds, such as psychostimulants and 5-HT uptake inhibitors, have been used for the treatment of narcoleptic disorders. However, little information is available regarding the pathophysiological features of orexin KO mice, particularly with respect to their narcoleptic-like disorder and how it is affected by monoamine-related compounds. The present study was designed to investigate both the nature of the neuronal changes in orexin KO mice and the therapeutic effects of monoamine-related compounds on the sleep disorder in orexin KO mice. A decrease in locomotor activity in the dark phase was observed in orexin KO mice, and psychostimulants and 5-HT-related compounds, such as 8-OH-DPAT (5-HT1A receptor agonist) and DOI (5-HT2 receptor agonist), inhibited this hypolocomotion. We also found that 5-HT1A receptor mRNA levels, but not those for 5-HT2 or dopamine receptors, were significantly decreased in the prefrontal cortex of orexin KO mice in the dark period and were accompanied by compromising the increase in 5-HT metabolite levels. In addition, the sleep disorder in orexin KO mice, as analyzed by a polysomnography during the dark period, was completely normalized by 8-OH-DPAT. These results suggest that a dysfunction of 5-HT1A receptors is involved in the narcoleptic-like sleep dysfunction in orexin KO mice, and such dysfunction may participate in orexin deficiency-induced sleep disorders. Further, the use of 5-HT1A receptor agonist could be useful for treating the sleep disorder under a deficiency of orexin.

  20. Effect of P2X(7) receptor knockout on exocrine secretion of pancreas, salivary glands and lacrimal glands

    DEFF Research Database (Denmark)

    Novak, Ivana; Jans, Ida M; Wohlfahrt, Louise

    2010-01-01

    The purinergic P2X(7) receptors are expressed in different cell types where they have varied functions, including regulation of cell survival. The P2X(7) receptors are also expressed in exocrine glands, but their integrated role in secretion is unclear. The aim of our study was to determine whether...... between purinergic and cholinergic receptor signalling and that function of the P2X(7) receptor is suppressed in females. We conclude that the P2X(7) receptors are important in short-term physiological regulation of exocrine gland secretion....... the P2X(7) receptors affect fluid secretion in pancreas, salivary glands and tear glands. We monitored gland secretions in in vivo preparations of wild-type and P2X(7)(-/-) (Pfizer) mice stimulated with pilocarpine. In cell preparations from pancreas, parotid and lacrimal glands we measured ATP release...

  1. P2Y2 receptor knock-out mice display normal NaCl absorption in medullary thick ascending limb

    DEFF Research Database (Denmark)

    Marques, Rita D; Praetorius, Helle A; Leipziger, Jens

    2013-01-01

    Local purinergic signals modulate renal tubular transport. Acute activation of renal epithelial P2 receptors causes inhibition of epithelial transport and thus, should favor increased water and salt excretion by the kidney. So far only a few studies have addressed the effects of extracellular...... nucleotides on ion transport in the thick ascending limb (TAL). In the medullary thick ascending limb (mTAL), basolateral P2X receptors markedly (~25%) inhibit NaCl absorption. Although this segment does express both apical and basolateral P2Y2 receptors, acute activation of the basolateral P2Y2 receptors had...... no apparent effect on transepithelial ion transport. Here we studied, if the absence of the P2Y2 receptor causes chronic alterations in mTAL NaCl absorption by comparing basal and AVP-stimulated transepithelial transport rates. We used perfused mouse mTALs to electrically measure NaCl absorption in juvenile...

  2. Effect of P2X(7) receptor knockout on exocrine secretion of pancreas, salivary glands and lacrimal glands.

    Science.gov (United States)

    Novak, Ivana; Jans, Ida M; Wohlfahrt, Louise

    2010-09-15

    The purinergic P2X(7) receptors are expressed in different cell types where they have varied functions, including regulation of cell survival. The P2X(7) receptors are also expressed in exocrine glands, but their integrated role in secretion is unclear. The aim of our study was to determine whether the P2X(7) receptors affect fluid secretion in pancreas, salivary glands and tear glands. We monitored gland secretions in in vivo preparations of wild-type and P2X(7)(-/-) (Pfizer) mice stimulated with pilocarpine. In cell preparations from pancreas, parotid and lacrimal glands we measured ATP release and intracellular Ca(2+) activity using Fura-2. The data showed that pancreatic secretion and salivary secretions were reduced in P2X(7)(-/-) mice, and in contrast, tear secretion was increased in P2X(7)(-/-) mice. The secretory phenotype was also dependent on the sex of the animal, such that males were more dependent on the P2X(7) receptor expression. ATP release in all cell preparations could be elicited by carbachol and other agonists, and this was independent of the P2X(7) receptor expression. ATP and carbachol increased intracellular Ca(2+) activity, but responses depended on the gland type, presence of the P2X(7) receptor and the sex of the animal. Together, these results demonstrate that cholinergic stimulation leads to release of ATP that can via P2X(7) receptors up-regulate pancreatic and salivary secretion but down-regulate tear secretion. Our data also indicate that there is an interaction between purinergic and cholinergic receptor signalling and that function of the P2X(7) receptor is suppressed in females. We conclude that the P2X(7) receptors are important in short-term physiological regulation of exocrine gland secretion.

  3. Effect of P2X7 receptor knockout on exocrine secretion of pancreas, salivary glands and lacrimal glands

    Science.gov (United States)

    Novak, Ivana; Jans, Ida M; Wohlfahrt, Louise

    2010-01-01

    The purinergic P2X7 receptors are expressed in different cell types where they have varied functions, including regulation of cell survival. The P2X7 receptors are also expressed in exocrine glands, but their integrated role in secretion is unclear. The aim of our study was to determine whether the P2X7 receptors affect fluid secretion in pancreas, salivary glands and tear glands. We monitored gland secretions in in vivo preparations of wild-type and P2X7−/− (Pfizer) mice stimulated with pilocarpine. In cell preparations from pancreas, parotid and lacrimal glands we measured ATP release and intracellular Ca2+ activity using Fura-2. The data showed that pancreatic secretion and salivary secretions were reduced in P2X7−/− mice, and in contrast, tear secretion was increased in P2X7−/− mice. The secretory phenotype was also dependent on the sex of the animal, such that males were more dependent on the P2X7 receptor expression. ATP release in all cell preparations could be elicited by carbachol and other agonists, and this was independent of the P2X7 receptor expression. ATP and carbachol increased intracellular Ca2+ activity, but responses depended on the gland type, presence of the P2X7 receptor and the sex of the animal. Together, these results demonstrate that cholinergic stimulation leads to release of ATP that can via P2X7 receptors up-regulate pancreatic and salivary secretion but down-regulate tear secretion. Our data also indicate that there is an interaction between purinergic and cholinergic receptor signalling and that function of the P2X7 receptor is suppressed in females. We conclude that the P2X7 receptors are important in short-term physiological regulation of exocrine gland secretion. PMID:20643770

  4. Altered learning, memory, and social behavior in type 1 taste receptor subunit 3 knock-out mice are associated with neuronal dysfunction.

    Science.gov (United States)

    Martin, Bronwen; Wang, Rui; Cong, Wei-Na; Daimon, Caitlin M; Wu, Wells W; Ni, Bin; Becker, Kevin G; Lehrmann, Elin; Wood, William H; Zhang, Yongqing; Etienne, Harmonie; van Gastel, Jaana; Azmi, Abdelkrim; Janssens, Jonathan; Maudsley, Stuart

    2017-07-07

    The type 1 taste receptor member 3 (T1R3) is a G protein-coupled receptor involved in sweet-taste perception. Besides the tongue, the T1R3 receptor is highly expressed in brain areas implicated in cognition, including the hippocampus and cortex. As cognitive decline is often preceded by significant metabolic or endocrinological dysfunctions regulated by the sweet-taste perception system, we hypothesized that a disruption of the sweet-taste perception in the brain could have a key role in the development of cognitive dysfunction. To assess the importance of the sweet-taste receptors in the brain, we conducted transcriptomic and proteomic analyses of cortical and hippocampal tissues isolated from T1R3 knock-out (T1R3KO) mice. The effect of an impaired sweet-taste perception system on cognition functions were examined by analyzing synaptic integrity and performing animal behavior on T1R3KO mice. Although T1R3KO mice did not present a metabolically disrupted phenotype, bioinformatic interpretation of the high-dimensionality data indicated a strong neurodegenerative signature associated with significant alterations in pathways involved in neuritogenesis, dendritic growth, and synaptogenesis. Furthermore, a significantly reduced dendritic spine density was observed in T1R3KO mice together with alterations in learning and memory functions as well as sociability deficits. Taken together our data suggest that the sweet-taste receptor system plays an important neurotrophic role in the extralingual central nervous tissue that underpins synaptic function, memory acquisition, and social behavior. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  5. Growth Hormone Secretagogue Receptor (GHS-R1a) Knockout Mice Exhibit Improved Spatial Memory and Deficits in Contextual Memory

    OpenAIRE

    Albarran-Zeckler, Rosie G.; Brantley, Alicia Faruzzi; Smith, Roy G.

    2012-01-01

    Although the hormone ghrelin is best known for its stimulatory effect on appetite and regulation of growth hormone release, it is also reported to have beneficial effects on learning and memory formation in mice. Nevertheless, controversy exists about whether endogenous ghrelin acts on its receptors in extra-hypothalamic areas of the brain. The ghrelin receptor (GHS-R1a) is co-expressed in neurons that express dopamine receptor type-1 (DRD1a) and type-2 (DRD2), and we have shown that a subset...

  6. TIMP3 interplays with apelin to regulate cardiovascular metabolism in hypercholesterolemic mice

    Directory of Open Access Journals (Sweden)

    Robert Stöhr

    2015-10-01

    Conclusion: TIMP3 regulates lipid metabolism as well as oxidative stress response via apelin. These findings therefore suggest that TIMP3 maintains metabolic flexibility in the heart, particularly during episodes of increased cardiac stress.

  7. Apelin acts in the subfornical organ to influence neuronal excitability and cardiovascular function.

    Science.gov (United States)

    Dai, Li; Smith, Pauline M; Kuksis, Markus; Ferguson, Alastair V

    2013-07-01

    Apelin is an adipocyte-derived hormone involved in the regulation of water balance, food intake and the cardiovascular system partially through actions in the CNS. The subfornical organ (SFO) is a circumventricular organ with identified roles in body fluid homeostasis, cardiovascular control and energy balance. The SFO lacks a normal blood-brain barrier, and is thus able to detect circulating signalling molecules such as angiotensin II and leptin. In this study, we investigated actions of apelin-13, the predominant apelin isoform in brain and circulatory system, on the excitability of dissociated SFO neurons using electrophysiological approaches, and determined the cardiovascular consequences of direct administration into the SFO of anaesthetized rats. Whole cell current clamp recording revealed that bath-applied 100 nm apelin-13 directly influences the excitability of the majority of SFO neurons by eliciting either depolarizing (31.8%, mean 7.0 ± 0.8 mV) or hyperpolarizing (28.6%, mean -10.4 ± 1.8 mV) responses. Using voltage-clamp techniques, we also identified modulatory actions of apelin-13 on specific ion channels, demonstrating that apelin-13 activates a non-selective cationic conductance to depolarize SFO neurons while activation of the delayed rectifier potassium conductance underlies hyperpolarizing effects. In anaesthetized rats, microinjection of apelin into SFO decreased both blood pressure (BP) (mean area under the curve -1492.3 ± 357.1 mmHg.s, n = 5) and heart rate (HR) (-32.4 ± 10.39 beats, n = 5). Our data suggest that circulating apelin can directly affect BP and HR as a consequence of the ability of this peptide to modulate the excitability of SFO neurons.

  8. Difference in Perseverative Errors during a Visual Attention Task with Auditory Distractors in Alpha-9 Nicotinic Receptor Subunit Wild Type and Knock-Out Mice

    Directory of Open Access Journals (Sweden)

    Pascal Jorratt

    2017-11-01

    Full Text Available The auditory efferent system is a neural network that originates in the auditory cortex and projects to the cochlear receptor through olivocochlear (OC neurons. Medial OC neurons make cholinergic synapses with outer hair cells (OHCs through nicotinic receptors constituted by α9 and α10 subunits. One of the physiological functions of the α9 nicotinic receptor subunit (α9-nAChR is the suppression of auditory distractors during selective attention to visual stimuli. In a recent study we demonstrated that the behavioral performance of alpha-9 nicotinic receptor knock-out (KO mice is altered during selective attention to visual stimuli with auditory distractors since they made less correct responses and more omissions than wild type (WT mice. As the inhibition of the behavioral responses to irrelevant stimuli is an important mechanism of the selective attention processes, behavioral errors are relevant measures that can reflect altered inhibitory control. Errors produced during a cued attention task can be classified as premature, target and perseverative errors. Perseverative responses can be considered as an inability to inhibit the repetition of an action already planned, while premature responses can be considered as an index of the ability to wait or retain an action. Here, we studied premature, target and perseverative errors during a visual attention task with auditory distractors in WT and KO mice. We found that α9-KO mice make fewer perseverative errors with longer latencies than WT mice in the presence of auditory distractors. In addition, although we found no significant difference in the number of target error between genotypes, KO mice made more short-latency target errors than WT mice during the presentation of auditory distractors. The fewer perseverative error made by α9-KO mice could be explained by a reduced motivation for reward and an increased impulsivity during decision making with auditory distraction in KO mice.

  9. Difference in Perseverative Errors during a Visual Attention Task with Auditory Distractors in Alpha-9 Nicotinic Receptor Subunit Wild Type and Knock-Out Mice.

    Science.gov (United States)

    Jorratt, Pascal; Delano, Paul H; Delgado, Carolina; Dagnino-Subiabre, Alexies; Terreros, Gonzalo

    2017-01-01

    The auditory efferent system is a neural network that originates in the auditory cortex and projects to the cochlear receptor through olivocochlear (OC) neurons. Medial OC neurons make cholinergic synapses with outer hair cells (OHCs) through nicotinic receptors constituted by α9 and α10 subunits. One of the physiological functions of the α9 nicotinic receptor subunit (α9-nAChR) is the suppression of auditory distractors during selective attention to visual stimuli. In a recent study we demonstrated that the behavioral performance of alpha-9 nicotinic receptor knock-out (KO) mice is altered during selective attention to visual stimuli with auditory distractors since they made less correct responses and more omissions than wild type (WT) mice. As the inhibition of the behavioral responses to irrelevant stimuli is an important mechanism of the selective attention processes, behavioral errors are relevant measures that can reflect altered inhibitory control. Errors produced during a cued attention task can be classified as premature, target and perseverative errors. Perseverative responses can be considered as an inability to inhibit the repetition of an action already planned, while premature responses can be considered as an index of the ability to wait or retain an action. Here, we studied premature, target and perseverative errors during a visual attention task with auditory distractors in WT and KO mice. We found that α9-KO mice make fewer perseverative errors with longer latencies than WT mice in the presence of auditory distractors. In addition, although we found no significant difference in the number of target error between genotypes, KO mice made more short-latency target errors than WT mice during the presentation of auditory distractors. The fewer perseverative error made by α9-KO mice could be explained by a reduced motivation for reward and an increased impulsivity during decision making with auditory distraction in KO mice.

  10. Apelin could reduce risk of contrast-induced nephropathy in patients with congestive heart failure.

    Science.gov (United States)

    Seifirad, Soroush; Masoudkabir, Farzad

    2013-11-01

    Compared to the normal population, patients with congestive heart failure are at higher risk for contrast-induced nephropathy. A variety of interventions are suggested to reduce the risk for contrast-induced nephropathy. Unfortunately results of none of current protective treatments are satisfactory. Apelin a vasodilator adipocytokine, positively inotropic agent, and free radical scavenger has been recently introduced. It has been shown that endogenous apelin levels are decreased in patients with congestive heart failure. Two major mechanisms have been suggested for pathophysiology of contrast induced nephropathy including reactive oxygen species production and impaired renal perfusion due to vasoconstriction. Pretreatment with recombinant apelin (exogenous apelin-13), could compensate decreased endogenous apelin serum levels of congestive heart failure patients. Its antioxidant and cell-protective properties, decrease nephrotoxicity of contrast agent; additionally impaired renal perfusion due to malfunction of cardiac pump will refurbish, because of positively inotropic property of apelin plus its vasodilatation effect in renal arteries. We believe that the triangle of increased contractility, decreased vascular resistance and decreased contrast agent nephrotoxicity could significantly reduce risk of contrast-induced nephropathy in patients with congestive heart failure. Copyright © 2013 Elsevier Ltd. All rights reserved.

  11. Dose-related neuroprotective effects of chronic nicotine in 6-hydroxydopamine treated rats, and loss of neuroprotection in α4 nicotinic receptor subunit knockout mice

    Science.gov (United States)

    Ryan, R E; Ross, S A; Drago, J; Loiacono, R E

    2001-01-01

    The present study examined the effect of a range of doses of chronic nicotine (0.75, 1.5, 3.0 and 30.0 mg kg−1 day−1, s.c., 14 days) upon striatal dopaminergic nerve terminal survival following 6-hydroxydopamine (6-OHDA; 10 μg intrastriatal unilaterally) in rats; and the effects of acute nicotine (1 mg kg−1, s.c.) pretreatment upon striatal neurodegeneration induced by methamphetamine (5 mg kg−1, i.p., three doses at 2 h intervals) in wild-type and α4 nicotinic receptor (nAChR) subunit knockout mice.In both models of Parkinsonian-like damage, loss of striatal dopaminergic nerve terminals was assessed by [3H]-mazindol autoradiography.In rats, chronic nicotine infusion delivered by osmotic minipump implanted subcutaneously 7 days prior to intrastriatal 6-OHDA injection produced significant and dose-related protection against 6-OHDA-induced neurodegeneration. Low (0.75 and 1.5 mg kg−1 day−1) but not high (3.0 and 30.0 mg kg−1 day−1) nicotine doses significantly inhibited 6-OHDA-induced degeneration.In wild-type mice, acute nicotine treatment produced significant inhibition of methamphetamine-induced neurodegeneration. In α4 nAChR subunit knockout mice, acute nicotine treatment failed to inhibit methamphetamine-induced neurodegeneration.Nicotine is capable of protecting dopaminergic neurons against Parkinsonian-like neurodegeneration in vivo. In rats, this neuroprotective effect is critically dependent upon nicotine dose and is consistent with the activation of nAChRs, as high, desensitizing doses of nicotine fail to be neuroprotective. Further, neuroprotection is absent in α4 nAChR subunit knockout mice. The current results therefore suggest that activation of α4 subunit containing nAChRs constitutes a major component of the neuroprotective effect of nicotine upon Parkinsonian-like damage in vivo. PMID:11309235

  12. Growth hormone secretagogue receptor (GHS-R1a) knockout mice exhibit improved spatial memory and deficits in contextual memory.

    Science.gov (United States)

    Albarran-Zeckler, Rosie G; Brantley, Alicia Faruzzi; Smith, Roy G

    2012-06-15

    Although the hormone ghrelin is best known for its stimulatory effect on appetite and regulation of growth hormone release, it is also reported to have beneficial effects on learning and memory formation in mice. Nevertheless, controversy exists about whether endogenous ghrelin acts on its receptors in extra-hypothalamic areas of the brain. The ghrelin receptor (GHS-R1a) is co-expressed in neurons that express dopamine receptor type-1 (DRD1a) and type-2 (DRD2), and we have shown that a subset of GHS-R1a, which are not occupied by the agonist (apo-GHSR1a), heterodimerize with these two receptors to regulate dopamine signaling in vitro and in vivo. To determine the consequences of ghsr ablation on brain function, congenic ghsr -/- mice on the C57BL6/J background were subjected to a battery of behavioral tests. We show that the ghsr -/- mice exhibit normal balance, movement, coordination, and pain sensation, outperform ghsr +/+ mice in the Morris water maze, but show deficits in contextual fear conditioning. Copyright © 2012 Elsevier B.V. All rights reserved.

  13. Lack of Genotype Effect on D1, D2 Receptors and Dopamine Transporter Binding in Triple MOP-, DOP-, and KOP-Opioid Receptor Knockout Mice of Three Different Genetic Backgrounds

    Science.gov (United States)

    Yoo, Ji-Hoon; Bailey, Alexis; Ansonoff, Micheal; Pintar, John E.; Matifas, Audrey; Kieffer, Brigitte L.; Kitchen, Ian

    2015-01-01

    We investigated D1, D2 receptors and dopamine transporter (DAT) binding levels in mice lacking all three opioid receptors and wild-type (WT) mice on three different genetic backgrounds. Quantitative autoradiography was used to determine the level of radioligand binding to the D1 and D2 receptors and DAT labeled with [3H]SCH23390, [3H]raclopride, and [3H]mazindol, respectively in triple-opioid receptor knockout (KO) and WT maintained on C57BL/6 (B6) and 129/SvEvTac (129) as well as C57BL/6 × 129/SvPas (B6 × 129) strains. No significant genotype effect was observed in D1, D2 receptors and DAT binding in any regions analyzed in any of the strains studied, suggesting that a lack of all three opioid receptors does not influence D1, D2 receptors and DAT expression, irrespective of their genetic strain background. However, strain differences were observed in D1 binding between the three strains of mice studied. Lower levels of D1 binding were observed in the substantia nigra of B6 × 129 WT mice compared with the 129 WT mice and in the olfactory tubercle of B6 × 129 WT compared with B6 WT and 129 WT mice. Lower levels of D1 binding were observed in the caudate putamen of B6 × 129 KO mice compared with 129 KO mice. In contrast, no significant strain differences were observed in D2 and DAT binding between the three strains of mice in any regions analyzed. Overall, these results indicate a lack of modulation of the dopaminergic system by the deletion of all three opioid receptors regardless of different background strains. PMID:20196137

  14. Impairment of the natriuretic peptide system in follitropin receptor knockout mice and reversal by estradiol: implications for obesity-associated hypertension in menopause.

    Science.gov (United States)

    Belo, Najara O; Sairam, M Ram; Dos Reis, Adelina M

    2008-03-01

    Estrogen is considered a major regulator of adipose tissue in females. Estrogen increases circulating levels of atrial natriuretic peptide (ANP), a hormone with renal and cardiovascular effects. The aim of this study was to determine the status of the natriuretic peptide system in female follitropin-receptor knockout (FORKO) mice that could be associated with obesity and hypertension observed in these mutants. Furthermore, estradiol treatment was used to reverse alterations observed. FORKO and wild-type (WT) mice received daily injections of estradiol for 4 d. On the fifth day, blood was collected for determination of plasma ANP levels, and selected tissues were collected for determination of ANP, natriuretic peptide receptor type-A (NPR-A) and type-C (NPR-C) gene expression by RT-PCR and binding of [(125)I]ANP by autoradiography. At 5 months of age, FORKO mice were heavier and had more adipose tissue than WT mice. FORKO mice had lower plasma ANP levels and atrial ANP gene expression and higher renal and adipocyte NPR-C gene expression than WT mice. Estradiol treatment reduced weight gain and increased atrial ANP synthesis as well as decreased ANP clearance NPR-C receptors, resulting in elevation of circulating ANP level. In conclusion, this study shows that FORKO females have an impaired natriuretic peptide system, which may contribute to the susceptibility of FORKO mice to developing age-related hypertension previously shown in these animals. This study establishes a relation between estrogen, adipose tissue, and ANP, which may have important implications in menopausal women.

  15. Brain activation by short-term nicotine exposure in anesthetized wild-type and beta2-nicotinic receptors knockout mice: a BOLD fMRI study

    Energy Technology Data Exchange (ETDEWEB)

    Suarez, S.V.; Changeux, J.P.; Granon, S. [Unite de Neurobiologie Integrative du Systeme Cholinergique, URA CNRS 2182, Institut Pasteur, Departement de Neuroscience, 25 rue du Dr Roux, 75015 Paris (France); Amadon, A.; Giacomini, E.; Le Bihan, D. [Service Hospitalier Frederic Joliot, 4 place du general Leclerc, 91400 Orsay (France); Wiklund, A. [Section of Anaesthesiology and Intensive Care Medicine, Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm (Sweden)

    2009-07-01

    Rationale: The behavioral effects of nicotine and the role of the beta2-containing nicotinic receptors in these behaviors are well documented. However, the behaviors altered by nicotine rely on the functioning on multiple brain circuits where the high-affinity {beta}2-containing nicotinic receptors ({beta}2*nAChRs) are located. Objectives We intend to see which brain circuits are activated when nicotine is given in animals naive for nicotine and whether the {beta}2*nAChRs are needed for its activation of the blood oxygen level dependent (BOLD) signal in all brain areas. Materials and methods: We used functional magnetic resonance imaging (fMRI) to measure the brain activation evoked by nicotine (1 mg/kg delivered at a slow rate for 45 min) in anesthetized C57BL/6J mice and {beta}2 knockout (KO) mice. Results: Acute nicotine injection results in a significant increased activation in anterior frontal, motor, and somatosensory cortices and in the ventral tegmental area and the substantia nigra. Anesthetized mice receiving no nicotine injection exhibited a major decreased activation in all cortical and subcortical structures, likely due to prolonged anesthesia. At a global level, {beta}2 KO mice were not rescued from the globally declining BOLD signal. However, nicotine still activated regions of a meso-cortico-limbic circuit likely via {alpha}7 nicotinic receptors. Conclusions: Acute nicotine exposure compensates for the drop in brain activation due to anesthesia through the meso-cortico-limbic network via the action of nicotine on {beta}2*nAChRs. The developed fMRI method is suitable for comparing responses in wild-type and mutant mice. (authors)

  16. Brain activation by short-term nicotine exposure in anesthetized wild-type and beta2-nicotinic receptors knockout mice: a BOLD fMRI study

    International Nuclear Information System (INIS)

    Suarez, S.V.; Changeux, J.P.; Granon, S.; Amadon, A.; Giacomini, E.; Le Bihan, D.; Wiklund, A.

    2009-01-01

    Rationale: The behavioral effects of nicotine and the role of the beta2-containing nicotinic receptors in these behaviors are well documented. However, the behaviors altered by nicotine rely on the functioning on multiple brain circuits where the high-affinity β2-containing nicotinic receptors (β2*nAChRs) are located. Objectives We intend to see which brain circuits are activated when nicotine is given in animals naive for nicotine and whether the β2*nAChRs are needed for its activation of the blood oxygen level dependent (BOLD) signal in all brain areas. Materials and methods: We used functional magnetic resonance imaging (fMRI) to measure the brain activation evoked by nicotine (1 mg/kg delivered at a slow rate for 45 min) in anesthetized C57BL/6J mice and β2 knockout (KO) mice. Results: Acute nicotine injection results in a significant increased activation in anterior frontal, motor, and somatosensory cortices and in the ventral tegmental area and the substantia nigra. Anesthetized mice receiving no nicotine injection exhibited a major decreased activation in all cortical and subcortical structures, likely due to prolonged anesthesia. At a global level, β2 KO mice were not rescued from the globally declining BOLD signal. However, nicotine still activated regions of a meso-cortico-limbic circuit likely via α7 nicotinic receptors. Conclusions: Acute nicotine exposure compensates for the drop in brain activation due to anesthesia through the meso-cortico-limbic network via the action of nicotine on β2*nAChRs. The developed fMRI method is suitable for comparing responses in wild-type and mutant mice. (authors)

  17. Participation of 5-HT1B receptors in the inhibitory actions of serotonin on masculine sexual behaviour of mice: pharmacological analysis in 5-HT1B receptor knockout mice

    Science.gov (United States)

    Rodríguez-Manzo, Gabriela; López-Rubalcava, Carolina; Hen, Rene; Fernández-Guasti, Alonso

    2002-01-01

    The role of the 5-Hydroxytryptamine1B (5-HT1B) receptor subtype in masculine sexual behaviour in mice was analysed in both 5-HT1B receptor knockout (KO1B) and wild-type (WT) animals. Comparison of male copulatory behaviour of WT and KO1B strains revealed that KO1B mice become interested earlier in sexual behaviour, but require more stimulation to achieve ejaculation than its corresponding WT strain. The pharmacological manipulation of male sexual activity in the WT strain showed that the serotonin precursor 5-Hydroxytryptophan (5-HTP), the 5-HT1B agonist (1-(m-trifluoromethylphenyl) piperazine (TFMPP) and the 5-Hydroxytryptamine1A (5-HT1A) receptor agonist 8-hydroxy-2-di-n-propylamino-tetralin (8-OH-DPAT) all inhibited male copulatory behaviour in mice. In KO1B mice, TFMPP lacked an effect, 5-HTP exerted a mild inhibitory effect while 8-OH-DPAT provoked only a tendency towards a reduction in the percentage of animals that achieved ejaculation. In general, KO1B mice were less sensitive to the inhibitory actions of 5-HTP and 8-OH-DPAT than the WT strain. Based on these results, we can suggest that serotonin plays a general inhibitory role in the sexual behaviour of male mice and that both 5-HT1B and 5-HT1A receptor subtypes participate in the inhibitory actions of this neurotransmitter. The absence of the 5-HT1B receptor subtype affected both components of mouse masculine sexual behaviour, motivation and execution, further confirming the involvement of this receptor subtype in the control of this behaviour. In addition, the diminished sensitivity to serotonergic stimulation exhibited by KO1B mice suggests the occurrence of compensatory changes as a consequence of the absence of the 5-HT1B receptor subtype. PMID:12163345

  18. Treatment with anti-interferon-gamma monoclonal antibodies modifies experimental autoimmune encephalomyelitis in interferon-gamma receptor knockout mice

    DEFF Research Database (Denmark)

    Espejo, C; Penkowa, M; Sáez-Torres, I

    2001-01-01

    antibodies (mAb) on day 8 postimmunization. Clinical scoring and both histological and immunohistochemical studies were undertaken for all groups. We hereby show that treatment with anti-IFN-gamma mAb worsened the disease course of 129Sv wild-type mice. However, it decreased the mean daily score in IFN......-gamma R(-/-) 129Sv and the incidence of the disease down to 50% in C57Bl/6x129Sv IFN-gamma R(-/-) mice. Moreover, after anti-IFN-gamma mAb treatment, oxidative stress levels, metallothionein I and II antioxidant protein expression, and apoptoticneuronal death were increased in wild-type mice while......(-/-)) mouse types: C57Bl/6x129Sv, with a disruption of the IFN-gamma receptor cytoplasmic domain, and 129Sv, homozygous for a disrupted IFN-gamma receptor gene. Mice were immunized with peptide 40-55 from rat myelin oligodendrocyte glycoprotein. A subgroup of mice was treated with anti-IFN-gamma monoclonal...

  19. Anti-atherogenic effects of a phenol-rich fraction from Brazilian red wine (Vitis labrusca L.) in hypercholesterolemic low-density lipoprotein receptor knockout mice.

    Science.gov (United States)

    Hort, Mariana Appel; Schuldt, Elke Zuleika; Bet, Angela Cristina; DalBó, Silvia; Siqueira, Jarbas Mota; Ianssen, Carla; Abatepaulo, Fátima; de Souza, Heraldo Possolo; Veleirinho, Beatriz; Maraschin, Marcelo; Ribeiro-do-Valle, Rosa Maria

    2012-10-01

    Moderate wine intake (i.e., 1-2 glasses of wine a day) is associated with a reduced risk of morbidity and mortality from cardiovascular disease. The aim of this study was to evaluate the anti-atherosclerotic effects of a nonalcoholic ethyl acetate fraction (EAF) from a South Brazilian red wine obtained from Vitis labrusca grapes. Experiments were carried out on low-density lipoprotein (LDL) receptor knockout (LDLr⁻/⁻) mice, which were subjected to a hypercholesterolemic diet and treated with doses of EAF (3, 10, and 30 mg/kg) for 12 weeks. At the end of the treatment, the level of plasma lipids, the vascular reactivity, and the atherosclerotic lesions were evaluated. Our results demonstrated that the treatment with EAF at 3 mg/kg significantly decreased total cholesterol, triglycerides, and LDL plus very low-density lipoprotein levels compared with control hypercholesterolemic mice. The treatment of mice with EAF at 3 mg/kg also preserved the vasodilatation induced by acetylcholine on isolated thoracic aorta from hypercholesterolemic LDLr⁻/⁻ mice. This result is in agreement with the degree of lipid deposit on arteries. Taken together, the results show for the first time that the lowest concentration of an EAF obtained from a red wine produced in southern Brazil significantly reduced the progression of atherosclerosis in mice.

  20. Tempol improves lipid profile and prevents left ventricular hypertrophy in LDL receptor gene knockout (LDLr-/-) mice on a high-fat diet.

    Science.gov (United States)

    Viana Gonçalves, Igor Cândido; Cerdeira, Cláudio Daniel; Poletti Camara, Eduardo; Dias Garcia, José Antônio; Ribeiro Pereira Lima Brigagão, Maísa; Bessa Veloso Silva, Roberta; Bitencourt Dos Santos, Gérsika

    2017-09-01

    Dyslipidemia is associated with increased risk of cardiovascular disease and atherosclerosis, and hence with high morbidity and mortality. This study investigated the effects of the nitroxide 4-hydroxy-2,2,6,6-tetramethylpiperidine 1-oxyl (Tempol) on lipid profile and cardiac morphology in low-density lipoprotein (LDL) receptor gene knockout (LDLr-/-) mice. Male LDLr-/- mice (three months old, approximately 22 g weight) were divided into the following groups: controls, including (1) standard chow (SC, n=8) and (2) high-fat diet (HFD, n=8); and treatment, including (3) standard chow + Tempol (SC+T, n=8) (30 mg/kg administered by gavage, once daily) and (4) high-fat diet + Tempol (HFD+T, n=8) (30 mg/kg). After 30 days of the diet/treatment, whole blood was collected for analysis of biochemical parameters (total cholesterol, triglycerides [TG], high-density lipoprotein [HDL], LDL, and very low-density lipoprotein [VLDL]). The heart was removed through thoracotomy and histological analysis of the left ventricle was performed. A significant increase in TG, LDL, and VLDL and marked left ventricular hypertrophy (LVH) were demonstrated in the HFD group relative to the SC group (pTempol treatment (HFD+T group) significantly (pTempol showed potential for the prevention of events related to serious diseases of the cardiovascular system. Copyright © 2017 Sociedade Portuguesa de Cardiologia. Publicado por Elsevier España, S.L.U. All rights reserved.

  1. Circulating apelin level in relation to nutritional status in polycystic ovary syndrome and its association with metabolic and hormonal disturbances.

    Science.gov (United States)

    Olszanecka-Glinianowicz, Magdalena; Madej, Paweł; Nylec, Marcin; Owczarek, Aleksander; Szanecki, Wojciech; Skałba, Piotr; Chudek, Jerzy

    2013-08-01

    The aim of the study was to analyse relationships between plasma apelin-36 and apelin-12 levels, nutritional status, insulin resistance and hormonal disturbances, as well as plasma adiponectin, leptin and resistin concentrations in PCOS women. A cross-sectional study involving 87 PCOS (48 obese) and 67 non-PCOS women (36 obese). Anthropometric parameters and body composition were determined. Serum glucose, androgens, FSH, LH, SHBG, insulin, apelin-36, apelin-12, adiponectin, leptin and resistin were measured in the fasting state. Plasma apelin-36 and apelin-12 levels were significantly higher in normal weight women than in the obese women with PCOS (3·1 ± 2·2 vs 1·2 ± 0·7 μg/l, P Nutritional status seems to have different effects on apelin release, particularly, its active isoform, in women with PCOS compared with women without PCOS. This may be partially caused by changes in leptin and resistin secretion and may enhance pituitary-ovarian axis disturbances. The association between both isoforms of apelin and insulin resistance seems to be bidirectional. © 2012 John Wiley & Sons Ltd.

  2. Relationship between Angiotensin Converting Enzyme, Apelin, and New-Onset Atrial Fibrillation after Off-Pump Coronary Artery Bypass Grafting

    Directory of Open Access Journals (Sweden)

    Shu Xu

    2017-01-01

    Full Text Available It has been shown that inflammation and oxidative stress are important factors in postoperative atrial fibrillation (POAF. Angiotensin converting enzyme (ACE and apelin have a close relationship with inflammation and oxidative stress. The effect of ACE and apelin on POAF after off-pump coronary artery bypass grafting (OPCABG remains a question. The concentrations of serum ACE, angiotensin II (Ang II, apelin, bradykinin (BK, malondialdehyde (MDA, and C reactive protein (CRP were measured in the perioperative period of OPCABG. The levels of serum ACE in the POAF group were higher than in the no POAF group both preoperatively and postoperatively. Apelin in the POAF group was lower than in the no POAF group. There was a correlation between serum ACE and apelin. Postoperatively, CRP and MDA in the POAF group were higher than in the no POAF group; however, there was no difference before the operation. Preoperative ACE and apelin were both significant and independent risk factors for POAF. In conclusion, the high ACE and low apelin preoperatively led to CRP and MDA being increased postoperatively, which was probably associated with POAF after OPCABG. Apelin may be a new predictor for POAF.

  3. Automated pipeline to analyze non-contact infrared images of the paraventricular nucleus specific leptin receptor knock-out mouse model

    Science.gov (United States)

    Diaz Martinez, Myriam; Ghamari-Langroudi, Masoud; Gifford, Aliya; Cone, Roger; Welch, E. B.

    2015-03-01

    Evidence of leptin resistance is indicated by elevated leptin levels together with other hallmarks of obesity such as a defect in energy homeostasis.1 As obesity is an increasing epidemic in the US, the investigation of mechanisms by which leptin resistance has a pathophysiological impact on energy is an intensive field of research.2 However, the manner in which leptin resistance contributes to the dysregulation of energy, specifically thermoregulation,3 is not known. The aim of this study was to investigate whether the leptin receptor expressed in paraventricular nucleus (PVN) neurons plays a role in thermoregulation at different temperatures. Non-contact infrared (NCIR) thermometry was employed to measure surface body temperature (SBT) of nonanesthetized mice with a specific deletion of the leptin receptor in the PVN after exposure to room (25 °C) and cold (4 °C) temperature. Dorsal side infrared images of wild type (LepRwtwt/sim1-Cre), heterozygous (LepRfloxwt/sim1-Cre) and knock-out (LepRfloxflox/sim1-Cre) mice were collected. Images were input to an automated post-processing pipeline developed in MATLAB to calculate average and maximum SBTs. Linear regression was used to evaluate the relationship between sex, cold exposure and leptin genotype with SBT measurements. Findings indicate that average SBT has a negative relationship to the LepRfloxflox/sim1-Cre genotype, the female sex and cold exposure. However, max SBT is affected by the LepRfloxflox/sim1-Cre genotype and the female sex. In conclusion this data suggests that leptin within the PVN may have a neuroendocrine role in thermoregulation and that NCIR thermometry combined with an automated imaging-processing pipeline is a promising approach to determine SBT in non-anesthetized mice.

  4. O sistema apelinérgico: papel na fisiologia e patologia humanas e potenciais aplicações terapêuticas The apelinergic system: the role played in human physiology and pathology and potential therapeutic applications

    Directory of Open Access Journals (Sweden)

    Ricardo Ladeiras-Lopes

    2008-05-01

    Full Text Available A apelina é um peptídeo recentemente descoberto e identificado como o ligando endógeno do receptor APJ. A apelina e o receptor APJ são expressos numa grande variedade de tecidos, tais como coração, cérebro, rins e pulmões, onde a sua interação pode ter importantes efeitos fisiopatológicos. Com efeito, a última década foi fértil no esclarecimento de possíveis papéis desempenhados pela apelina na fisiologia humana, nomeadamente como peptídeo regulador dos sistemas cardiovascular, hipotálamo-hipófisário, gastrointestinal e imunitário. Um possível envolvimento da apelina na patogênese de doenças com elevada prevalência e co-morbilidades, como a hipertensão arterial, a insuficiência cardíaca e o diabete melito tipo 2, perspectivam-na como um possível alvo terapêutico a explorar no futuro. Este trabalho fornece uma visão geral dos efeitos fisiológicos da apelina e apresenta o possível papel desse peptídeo na patogênese de várias doenças, associado a implicações terapêuticas que poderão vir a ser, assim, exploradas.Apelin is a recently discovered peptide, identified as an endogenous ligand of receptor APJ. Apelin and receptor APJ are expressed in a wide variety of tissues including heart, brain, kidneys and lungs. Their interaction may have relevant pathophysiologic effects in those tissues. In fact, the last decade has been rich in illustrating the possible roles played by apelin in human physiology, namely as a regulating peptide of cardiovascular, hypothalamus-hypophysis, gastrointestinal, and immune systems. The possible involvement of apelin in the pathogenesis of high prevalence conditions and comorbidities - such as hypertension, heart failure, and Diabetes Mellitus Type 2 (T2DM - rank it as a likely therapeutic target to be investigated in the future. The present paper is an overview of apelin physiologic effects and presents the possible role played by this peptide in the pathogenesis of a number of

  5. Effects of stimulation of soluble guanylate cyclase on diabetic nephropathy in diabetic eNOS knockout mice on top of angiotensin II receptor blockade.

    Directory of Open Access Journals (Sweden)

    Ina M Ott

    Full Text Available The prevalence of diabetes mellitus and its complications, such as diabetic nephropathy (DN, is rising worldwide and prevention and treatment are therefore becoming increasingly important. Therapy of DN is particularly important for patients who do not adequately respond to angiotensin receptor blocker (ARB treatment. Novel approaches include the stimulation of soluble guanylate cyclase (sGC as it is reported to have beneficial effects on cardiac and renal damage. We aimed to investigate the effects of the sGC stimulator riociguat and ARB telmisartan on kidney function and structure in a hypertensive model of diabetic nephropathy. Seventy-six diabetic male eNOS knockout C57BL/6J mice were randomly divided after having received streptozotocin: telmisartan (1 mg/kg/d, riociguat (3 mg/kg/d, riociguat+telmisartan (3+1 mg/kg/d, and vehicle. Fourteen mice were used as non-diabetic controls. Treatment duration was 11 weeks. Glucose concentrations were increased and similar in all diabetic groups. Telmisartan insignificantly reduced blood pressure by 5.9 mmHg compared with diabetic controls (111.2±2.3 mmHg vs. 117.1±2.2 mmHg; p = 0.071. Treatment with riociguat both alone and in combination with telmisartan led to a significant reduction of blood pressure towards diabetic vehicle (105.2±2.5 mmHg and 105.0±3.2 mmHg, respectively, vs. 117.1±2.2 mmHg. Combined treatment also significantly decreased albuminuria compared with diabetic controls (47.3±9.6 µg/24 h vs. 170.8±34.2 µg/24 h; p = 0.002 reaching levels similar to those of non-diabetic controls (34.4±10.6 µg/24 h, whereas the reduction by single treatment with either telmisartan (97.8±26.4 µg/24 h or riociguat (97.1±15.7 µg/24 h was not statistically significant. The combination treatment led to a significant (p<0.01 decrease of tissue immunoreactivity of malondialdehyde, as consequence of reduced oxidative stress. In conclusion, stimulation of sGC significantly reduced urinary

  6. Comparison of the hepatic and thyroid gland effects of sodium phenobarbital and pregnenolone-16α-carbonitrile in wild type and constitutive androstane receptor (CAR)/pregnane X receptor (PXR) knockout rats.

    Science.gov (United States)

    Haines, Corinne; Chatham, Lynsey R; Vardy, Audrey; Elcombe, Clifford R; Foster, John R; Lake, Brian G

    2018-02-09

    1. The hepatic and thyroid gland effects of the constitutive androstane receptor (CAR) activator sodium phenobarbital (NaPB) and the pregnane X receptor (PXR) activator pregnenolone-16α-carbonitrile (PCN) were examined in male Sprague-Dawley wild type (WT) and knockout (KO) rats lacking both hepatic CAR and PXR receptors (CAR KO/PXR KO rats). 2. The treatment of WT rats for 7 days with 500 ppm NaPB in the diet and 100 mg/kg/day PCN by gavage resulted in increased relative liver weight, hepatocyte hypertrophy, increased hepatocyte replicative DNA synthesis (RDS) and induction of cytochrome P450 CYP2B and CYP3A subfamily enzymes. NaPB and PCN also induced thyroid gland follicular cell RDS and hepatic microsomal UDPglucuronosyltransferase activity towards thyroxine as substrate. These effects were not observed in the liver and thyroid gland of CAR KO/PXR KO rats. 3. Male C57BL/6J (WT) and CAR KO/PXR KO mice were given 1000 ppm NaPB in the diet for 7 days. In WT, but not in CAR KO/PXR KO, mice NaPB treatment resulted in liver hypertrophy and induction of hepatocyte RDS and Cyp2b enzymes. 4. These results suggest that the CAR KO/PXR KO rat and mouse models are useful experimental models for mode of action studies with rodent CAR activators.

  7. Research on the promoting role of apelin-13 in proliferation, migration and capillary-like tube formation of RF/6A cells

    Directory of Open Access Journals (Sweden)

    Kun-Peng Xie

    2017-05-01

    Full Text Available AIM: To investigate the effects of apelin-13 on proliferation, migration and capillary-like tube formation of a monkey choroid / retinal endothelial cell line, RF/6A, to clarify whether apelin-13 could promote retinal angiogenesis in vitro.METHODS: RF/6A cells in good conditions were administrated with DMSO(the control group, apelin-13 at 0.1μmol/L(low dose groupor apelin-13 at 1μmol/L(high dose group. Cell proliferation, migration and capillary-like tube formation were detected by using the MTT assay, scratch assay and matrigel assay, respectively, at 24h after plating the cells. RESULTS: Cell proliferation was promoted in both low and high dose apelin-13 groups compared to the control cells(PPPCONCLUSION: Apelin-13 could obviously promote the angiogenesis capacity of RF/6A cells, suggesting that apelin-13 was an important pro-angiogenic factor in retinal endothelial cells.

  8. Cholesterol-induced hepatic inflammation does not contribute to the development of insulin resistance in male LDL receptor knockout mice.

    Science.gov (United States)

    Funke, Anouk; Schreurs, Marijke; Aparicio-Vergara, Marcela; Sheedfar, Fareeba; Gruben, Nanda; Kloosterhuis, Niels J; Shiri-Sverdlov, Ronit; Groen, Albert K; van de Sluis, Bart; Hofker, Marten H; Koonen, Debby P Y

    2014-02-01

    It is generally assumed that hepatic inflammation in obesity is linked to the pathogenesis of insulin resistance. Several recent studies have shed doubt on this view, which questions the causality of this association. This study focuses on Kupffer cell-mediated hepatic inflammation as a possible driver of insulin resistance in the absence and presence of obesity. We used male mice deficient for the low-density lipoprotein receptor (Ldlr(-/-)) and susceptible to cholesterol-induced hepatic inflammation. Whole body and hepatic insulin resistance was measured in mice fed 4 diets for 2 and 15 weeks, i.e., chow, high-fat (HF), HF-cholesterol (HFC; 0.2% cholesterol) and HF without cholesterol (HFnC). Biochemical parameters in plasma and liver were measured and inflammation was determined using immunohistochemistry and RT-PCR. At 2 weeks, we did not find significant metabolic effects in either diet group, except for the mice fed a HFC diet which showed pronounced hepatic inflammation (p insulin sensitivity. At 15 weeks, a significant increase in insulin levels, HOMA-IR, and hepatic insulin resistance was observed in mice fed a HFC, HFnC, and HF diet compared to chow-fed mice (p HF, HFnC; p insulin resistance in mice fed HFC was no worse compared to mice on a HFnC and HF diet. These data show that cholesterol-induced hepatic inflammation does not contribute to the development of insulin resistance in male Ldlr(-/-) mice. This study suggests that Kupffer cell-driven hepatic inflammation is a consequence, not a cause, of metabolic dysfunction in obesity. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  9. Increased oxidative stress and apoptosis in the hypothalamus of diabetic male mice in the insulin receptor substrate-2 knockout model.

    Science.gov (United States)

    Baquedano, Eva; Burgos-Ramos, Emma; Canelles, Sandra; González-Rodríguez, Agueda; Chowen, Julie A; Argente, Jesús; Barrios, Vicente; Valverde, Angela M; Frago, Laura M

    2016-05-01

    Insulin receptor substrate-2-deficient (IRS2(-/-)) mice are considered a good model to study the development of diabetes because IRS proteins mediate the pleiotropic effects of insulin-like growth factor-I (IGF-I) and insulin on metabolism, mitogenesis and cell survival. The hypothalamus might play a key role in the early onset of diabetes, owing to its involvement in the control of glucose homeostasis and energy balance. Because some inflammatory markers are elevated in the hypothalamus of diabetic IRS2(-/-) mice, our aim was to analyze whether the diabetes associated with the absence of IRS2 results in hypothalamic injury and to analyze the intracellular mechanisms involved. Only diabetic IRS2(-/-) mice showed increased cell death and activation of caspase-8 and -3 in the hypothalamus. Regulators of apoptosis such as FADD, Bcl-2, Bcl-xL and p53 were also increased, whereas p-IκB and c-FLIPL were decreased. This was accompanied by increased levels of Nox-4 and catalase, enzymes involved in oxidative stress. In summary, the hypothalamus of diabetic IRS2(-/-) mice showed an increase in oxidative stress and inflammatory markers that finally resulted in cell death via substantial activation of the extrinsic apoptotic pathway. Conversely, non-diabetic IRS2(-/-) mice did not show cell death in the hypothalamus, possibly owing to an increase in the levels of circulating IGF-I and in the enhanced hypothalamic IGF-IR phosphorylation that would lead to the stimulation of survival pathways. In conclusion, diabetes in IRS2-deficient male mice is associated with increased oxidative stress and apoptosis in the hypothalamus. © 2016. Published by The Company of Biologists Ltd.

  10. Effect of human scavenger receptor class A overexpression in bone marrow-derived cells on lipoprotein metabolism and atherosclerosis in low density lipoprotein receptor knockout mice

    NARCIS (Netherlands)

    Herijgers, N.; de Winther, M. P.; van Eck, M.; Havekes, L. M.; Hofker, M. H.; Hoogerbrugge, P. M.; van Berkel, T. J.

    2000-01-01

    Scavenger receptors, which include various classes, play an important role in atherogenesis by mediating the unrestricted uptake of modified lipoproteins, resulting in the massive accumulation of cholesteryl esters. Because macrophage-derived foam cells are considered to be an important feature in

  11. Subunit compensation and plasticity of synaptic GABAA receptors induced by ethanol in α4 subunit knockout mice

    Directory of Open Access Journals (Sweden)

    Asha eSuryanarayanan

    2011-09-01

    Full Text Available There is considerable evidence that ethanol (EtOH potentiates γ-aminobutyric acid type A receptor (GABAAR action, but only GABAARs containing δ subunits appear sensitive to low mM EtOH. The α4 and δ subunits co-assemble into GABAARs which are relatively highly expressed at extrasynaptic locations in the dentate gyrus where they mediate tonic inhibition. We previously demonstrated reversible- and time-dependent changes in GABAAR function and subunit composition in rats after single-dose EtOH intoxication. We concluded that early tolerance to EtOH occurs by over-activation and subsequent internalization of EtOH-sensitive extrasynaptic α4βδ-GABAARs. Based on this hypothesis, any highly EtOH-sensitive GABAARs should be subject to internalization following exposure to suitably high EtOH doses. To test this, we studied the GABAARs in mice with a global deletion of the α4 subunit (KO. The dentate granule cells (DGCs of these mice exhibited greatly reduced tonic currents and greatly reduced potentiation by acutely applied EtOH, whereas synaptic currents showed heightened sensitivity to low EtOH concentrations. The hippocampus of naive KO mice showed reduced δ subunit protein levels, but increased α2, and γ2 levels compared to wild-type (WT controls, suggesting at least partial compensation by these subunits in synaptic, highly EtOH-sensitive GABAARs of KO mice. In WT mice, cross-linking and Western blot analysis at 1 h after an EtOH challenge (3.5 g/kg, i.p. revealed increased intracellular fraction of the α1, α4 and δ, but not α2, α5 or γ2 subunits. By contrast, we observed significant internalization of α1, α2, δ, and γ2 subunits after a similar EtOH challenge in KO mice. Synaptic currents from naïve KO mice were more sensitive to potentiation by zolpidem (0.3 μM, requiring α1/α2, inactive at α4/5 GABAARs than those from naïve WT mice. At 1 h after EtOH, synaptic currents of WT mice were unchanged, whereas those of KO mice

  12. Experimental transmission of AA amyloidosis by injecting the AA amyloid protein into interleukin-1 receptor antagonist knockout (IL-1raKO) mice.

    Science.gov (United States)

    Watanabe, K; Uchida, K; Chambers, J K; Tei, M; Shoji, A; Ushio, N; Nakayama, H

    2015-05-01

    The incidence of AA amyloidosis is high in humans with rheumatoid arthritis and several animal species, including cats and cattle with prolonged inflammation. AA amyloidosis can be experimentally induced in mice using severe inflammatory stimuli and a coinjection of AA amyloid; however, difficulties have been associated with transmitting AA amyloidosis to a different animal species, and this has been attributed to the "species barrier." The interleukin-1 receptor antagonist knockout (IL-1raKO) mouse, a rodent model of human rheumatoid arthritis, has been used in the transmission of AA amyloid. When IL-1raKO and BALB/c mice were intraperitoneally injected with mouse AA amyloid together with a subcutaneous pretreatment of 2% AgNO3, all mice from both strains that were injected with crude or purified murine AA amyloid developed AA amyloidosis. However, the amyloid index, which was determined by the intensity of AA amyloid deposition, was significantly higher in IL-1raKO mice than in BALB/c mice. When IL-1raKO and BALB/c mice were injected with crude or purified bovine AA amyloid together with the pretreatment, 83% (5/6 cases) and 38% (3/8 cases) of IL-1raKO mice and 17% (1/6 cases) and 0% (0/6 cases) of BALB/c mice, respectively, developed AA amyloidosis. Similarly, when IL-1raKO and BALB/c mice were injected with crude or purified feline AA amyloid, 33% (2/6 cases) and 88% (7/8 cases) of IL-1raKO mice and 0% (0/6 cases) and 29% (2/6 cases) of BALB/c mice, respectively, developed AA amyloidosis. These results indicated that IL-1raKO mice are a useful animal model for investigating AA amyloidogenesis. © The Author(s) 2014.

  13. High Fat High Cholesterol Diet (Western Diet) Aggravates Atherosclerosis, Hyperglycemia and Renal Failure in Nephrectomized LDL Receptor Knockout Mice: Role of Intestine Derived Lipopolysaccharide.

    Science.gov (United States)

    Ghosh, Siddhartha S; Righi, Samuel; Krieg, Richard; Kang, Le; Carl, Daniel; Wang, Jing; Massey, H Davis; Sica, Domenic A; Gehr, Todd W B; Ghosh, Shobha

    2015-01-01

    A high fat meal, frequently known as western diet (WD), exacerbates atherosclerosis and diabetes. Both these diseases are frequently associated with renal failure. Recent studies have shown that lipopolysaccharide (LPS) leaks into the circulation from the intestine in the setting of renal failure and after WD. However, it is not clear how renal function and associated disorders are affected by LPS. This study demonstrates that circulatory LPS exacerbates renal insufficiency, atherosclerosis and glucose intolerance. Renal insufficiency was induced by 2/3 nephrectomy in LDL receptor knockout mice. Nx animals were given normal diet (Nx) or WD (Nx+WD). The controls were sham operated animals on normal diet (control) and WD (WD). To verify if LPS plays a role in exaggerating renal insufficiency, polymyxin (PM), a known LPS antagonist, and curcumin (CU), a compound known to ameliorate chronic kidney disease (CKD), was given to Nx animals on western diet (Nx+WD+PM and Nx+WD+CU, respectively). Compared to control, all other groups displayed increased circulatory LPS. The Nx+WD cohort had the highest levels of LPS. Nx group had significant renal insufficiency and glucose intolerance but not atherosclerosis. WD had intense atherosclerosis and glucose intolerance but it did not show signs of renal insufficiency. Compared to other groups, Nx+WD had significantly higher cytokine expression, macrophage infiltration in the kidney, renal insufficiency, glucose intolerance and atherosclerosis. PM treatment blunted the expression of cytokines, deterioration of renal function and associated disorders, albeit not to the levels of Nx, and was significantly inferior to CU. PM is a non-absorbable antibiotic with LPS binding properties, hence its beneficial effect can only be due to its effect within the GI tract. We conclude that LPS may not cause renal insufficiency but can exaggerate kidney failure and associated disorders following renal insufficiency.

  14. Eight-week aerobic training effects on Apelin-13 and insulin resistance in overweight men

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    Rahman Soori

    2017-06-01

    Full Text Available Background & aims: Obesity as a pandemic disease is the high accumulation of adipose tissue which secrets different hormones such as apelin. Apelin as an adipocytokine increases in obesity. Aerobic training induced apelin responses are not clarify well. So, we aimed to determine the effect of eight weeks aerobic training on Apelin-13 and insulin resistance in overweight men. Methods: Current study was quasi-experiment design. Twenty-six overweight men with BMI between 27-30 kg/m2 were randomly enrolled in the present study following public call announcement and match to inclusion criteria. They accidently divided into submaximal-aerobic and control groups. The submaximal-aerobic group carried out exercise training for 24 continuous sessions (with 50-70% of maximum heart rate and 3 sessions/per-week for eight weeks. The anthropometrical, VO2max and blood sampling assessments performed and later assessments were completed 24 hours after last training period. Then, whole of data were analyzed by Stata software at P0.05. In addition, there were direct and significant relationship between anthropometrical indices and HbA1c with Apelin-13 (p<0.05. Conclusions: Obesity increases the possibility of metabolic diseases and insulin resistance. In the current study we represented that the internal factors of exercise, such as intensity, had meaningful effects on anthropometric features of overweight individuals but it was not enough for exercise induced-apelin-13 and insulin resistance changes.  According to this record, longer and higher intense exercise compare with the current study's protocol were recommended to beneficially decrease and control the incidence and catch the type II diabetes and cardiovascular diseases.

  15. Endogenous Peptide Apelin and Pathological Cardiac Remodeling in Hypertensive Patients with Diabetes Mellitus Type 2

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    S.M. Koval

    2015-08-01

    Full Text Available The study involved 63 patients with essential hypertension and diabetes mellitus type 2. The control group consisted of 16 apparently healthy individuals. Complex of examination included conventional clinical-laboratory and instrumental methods, ultrasound of the heart with Doppler sonography, determining apelin blood concentration by ELISA. Patients with essential hypertension and diabetes mellitus type 2 compared with the control group had a probable reduction in apelin level associated with the development of pathological cardiac remodeling, increasing the size of the left atrium and diastolic dysfunction of the heart.

  16. Serum concentrations of apelin-17 isoform vary in accordance to blood pressure categories in individuals with obesity class 3.

    Science.gov (United States)

    Cano Martínez, Luis Javier; Coral Vázquez, Ramón Mauricio; Méndez, Juan Pablo; Trejo, Silvia; Pérez Razo, Juan Carlos; Canto, Patricia

    2018-04-13

    The aim of this study was to investigate if serum concentrations of apelin-36, apelin-17, apelin-13 or apelin-12 were different in obesity class 3 individuals with hypertension, when compared to those without hypertension (normal or high-normal). Twenty six individuals with obesity class 3-related hypertension and thirty three individuals without hypertension, who were divided in individuals with normal (n = 23) or with high-normal (n = 10) blood pressure (BP) were analyzed. All individuals presented obesity class 3, without diabetes mellitus. Measurements of all apelin isoforms were performed using enzyme-linked immunosorbent assay kits. Analysis of differences between groups of Apelin isoform concentrations was performed by a One-way ANOVA, with a Tukey test post hoc. The individuals of the hypertensive group presented a slightly lower serum concentration of all apelin isoforms, but these differences were not statistically significant. These results were more evident when the group of patients without hypertension were divided based in normal and high-normal BP, observing that apelin-17 isoform were higher in individuals with high-normal BP in comparison to subjects with normal BP (P = 0.018); concentrations were also higher when compared to subjects with hypertension (P = 0.004). To our knowledge, this is the first study regarding the differences of apelin-17 isoform concentrations in individuals pertaining to different categories of BP, who presented obesity class 3. The group of patients that presented hypertension showed a lower concentration of all isoforms. This observation could be due to the fact that these patients were taking antihypertensive medication.

  17. Kimchi attenuates fatty streak formation in the aorta of low-density lipoprotein receptor knockout mice via inhibition of endoplasmic reticulum stress and apoptosis.

    Science.gov (United States)

    Woo, Minji; Kim, Mijeong; Noh, Jeong Sook; Park, Chan Hum; Song, Yeong Ok

    2017-12-01

    Endoplasmic reticulum (ER) stress is positively associated with atherosclerosis via elevating macrophage cell death and plaque formation, in which oxidative stress plays a pivotal role. Antioxidative, lipid-lowering, and anti-atherogenic effects of kimchi, a Korean fermented vegetable, have been established, wherein capsaicin, ascorbic acid, quercetin, 3-(4'-hydroxyl-3',5'-dimethoxyphenyl)propionic acid, and lactic acids were identified. In this study, mechanisms of action of kimchi methanol extracts (KME) on fatty streak formation via suppression of ER stress and apoptosis in aorta were examined in low-density lipoprotein receptor knockout mice. Mice fed a high cholesterol diet with an oral administration of KME (KME group, 200 mg·kg-bw -1 ·day -1 ) or distilled water (control group) for 8 weeks (n = 20 for group). Plasma lipid and oxidative stress levels were evaluated. Protein expression was measured by western blot assay. Fatty streak lesion size and the degree of apoptosis were examined in the aorta. Compared to the control group, in the KME group, plasma lipids levels were decreased and oxidative stress was alleviated ( P < 0.05). Protein expression levels of nuclear factor (erythroid-derived 2)-like 2-mediated antioxidants in aorta were increased whereas those for ER stress markers, glucose regulated protein 78, phospho-protein kinase RNA-like ER kinase, phospho-eukaryotic initiation factor 2 subunit α, X-box binding protein 1, and C/EBP homologous protein were decreased in the KME group ( P < 0.05). Moreover, apoptosis was suppressed via downregulation of phospho-c-Jun N-terminal kinase, bcl-2-associated X protein, caspases-9, and -3 with a concomitant upregulation of anti-apoptotic protein, B-cell lymphoma 2 ( P < 0.05). Fatty streak lesion size was reduced and the degree of apoptosis was less severe in the KME group ( P < 0.05). In conclusion, antioxidant activity of KME might prevent fatty streak formation through, in part, inhibition of ER stress and

  18. Oleylphosphocholine (OlPC) arrests Cryptosporidium parvum growth in vitro and prevents lethal infection in interferon gamma receptor knock-out mice.

    Science.gov (United States)

    Sonzogni-Desautels, Karine; Renteria, Axel E; Camargo, Fabio V; Di Lenardo, Thomas Z; Mikhail, Alexandre; Arrowood, Michael J; Fortin, Anny; Ndao, Momar

    2015-01-01

    Cryptosporidium parvum is a species of protozoa that causes cryptosporidiosis, an intestinal disease affecting many mammals including humans. Typically, in healthy individuals, cryptosporidiosis is a self-limiting disease. However, C. parvum can cause a severe and persistent infection that can be life-threatening for immunocompromised individuals, such as AIDS patients. As there are no available treatments for these patients that can cure the disease, there is an urgent need to identify treatment options. We tested the anti-parasitic activity of the alkylphosphocholine oleylphosphocholine (OlPC), an analog of miltefosine, against C. parvum in in vitro and in vivo studies. In vitro experiments using C. parvum infected human ileocecal adenocarcinoma cells (HCT-8 cells) showed that OlPC has an EC50 of 18.84 nM. Moreover, no cell toxicity has been seen at concentrations ≤50 μM. C57BL/6 interferon gamma receptor knock-out mice, were infected by gavage with 4000 C. parvum oocysts on Day 0. Oral treatments, with OlPC, miltefosine, paromomycin or PBS, began on Day 3 post-infection for 10 days. Treatment with OlPC, at 40 mg/kg/day resulted in 100% survival, complete clearance of parasite in stools and a 99.9% parasite burden reduction in the intestines at Day 30. Doses of 30 and 20 mg/kg/day also demonstrated an increased survival rate and a dose-dependent parasite burden reduction. Mice treated with 10 mg/kg/day of miltefosine resulted in 50% survival at Day 30. In contrast, control mice, treated with PBS or 100 mg/kg/day of paromomycin, died or had to be euthanized between Days 6 and 13 due to severe illness. Results of parasite burden were obtained by qPCR and cross-validated by both flow cytometry of stool oocysts and histological sections of the ileum. Together, our results strongly support that OlPC represents a potential candidate for the treatment of C. parvum infections in immunocompromised patients.

  19. Oleylphosphocholine (OlPC arrests Cryptosporidium parvum growth in vitro and prevents lethal infection in interferon gamma receptor knock-out mice

    Directory of Open Access Journals (Sweden)

    Karine eSonzogni-Desautels

    2015-09-01

    Full Text Available Cryptosporidium parvum is a species of protozoa that causes cryptosporidiosis, an intestinal disease affecting many mammals including humans. Typically, in healthy individuals, cryptosporidiosis is a self-limiting disease. However, C. parvum can cause a severe and persistent infection that can be life-threatening for immunocompromised individuals, such as AIDS patients. As there are no available treatments for these patients that can cure the disease, there is an urgent need to identify treatment options. We tested the anti-parasitic activity of the alkylphosphocholine oleylphosphocholine (OlPC, an analog of miltefosine, against C. parvum in in vitro and in vivo studies. In vitro experiments using C. parvum infected human ileocecal adenocarcinoma cells (HCT-8 cells showed that OlPC has an EC50 of 18.84 nM. Moreover, no cell toxicity has been seen at concentrations ≤50 µM. C57BL/6 interferon gamma receptor knock-out mice, were infected by gavage with 4000 C. parvum oocysts on Day 0. Oral treatments, with OlPC, miltefosine, paromomycin or PBS, began on Day 3 post-infection for 10 days. Treatment with OlPC, at 40 mg/kg/day resulted in 100% survival, complete clearance of parasite in stools and a 99.9% parasite burden reduction in the intestines at Day 30. Doses of 30 mg/kg/day and 20 mg/kg/day also demonstrated an increased survival rate and a dose-dependent parasite burden reduction. Mice treated with 10 mg/kg/day of miltefosine resulted in 50% survival at Day 30. In contrast, control mice, treated with PBS or 100 mg/kg/day of paromomycin, died or had to be euthanized between Days 6 and 13 due to severe illness. Results of parasite burden were obtained by qPCR and cross-validated by both flow cytometry of stool oocysts and histological sections of the ileum. Together, our results strongly support that OlPC represents a potential candidate for the treatment of C. parvum infections in immunocompromised patients.

  20. [Plasma level of apelin and carotid atherosclerosis in maintenance hemodialysis patients].

    Science.gov (United States)

    Tang, Rong; Wang, Ruiling; Ao, Xiang; Cheng, Xiaomiao

    2017-06-28

    To determine the plasma level of apelin in patients of maintenance hemodialysis (MHD) and to explore the relationship between apelin level and carotid atherosclerosis (AS) in MHD patients.
 Methods: A total of 92 MHD patients and 36 sex- and age-matched healthy subjects were enrolled in this study. The plasma level of apelin was evaluated by radiation immunoassay; serum endothelial injury markers including thrombomodulin, von Willebrand factor (vWF), and CD146, and inflammatory factors including high sensitive C-reactive protein (hsCRP), IL-6 and TNF-α were determined by ELISA. Common Carotid arteries intima media thickness (CCA-IMT), cross-sectional calculated intima-media area (cIM area) area and atherosclerotic plaque were measured by non-invasive high-resolution B-mode ultrasonography.
 Results: The plasma levels of apelin was significantly decreased in MHD patients compared with healthy subjects (P<0.01), accompanied with elevated plasma levels of thrombomodulin, vWF, CD146, hsCRP, IL-6 and TNF-α (all P<0.01). The plasma levels of apelinin in MHD patients with carotid artery plaques were obviously lower than those without plaques [(43.16±10.12) pg/mL vs (61.43±16.25) pg/mL, P<0.01]. Plasma level of apelin was inversely related with CCA-IMT and cIM area (r=-0.355 and r=-0.297 respectively, all P<0.01). Multiple stepwise regression analysis showed that plasma level of apelin was an independent risk factor for CCA-IMT and cIM area. 
 Conclusion: The plasma apelin in MHD patients might take part in vascular endothelial injury and the progress of atherosclerosis. It plays an important role in the initiation and development of uremia associated atherosclerosis through elevating inflammatory factors including hsCRP, IL-6 and TNF-α levels.

  1. Apelin as a marker for monitoring the tumor vessel normalization window during antiangiogenic therapy.

    Science.gov (United States)

    Zhang, Li; Takara, Kazuhiro; Yamakawa, Daishi; Kidoya, Hiroyasu; Takakura, Nobuyuki

    2016-01-01

    Antiangiogenic agents transiently normalize tumor vessel structure and improve vessel function, thereby providing a window of opportunity for enhancing the efficacy of chemotherapy or radiotherapy. Currently, there are no reliable predictors or markers reflecting this vessel normalization window during antiangiogenic therapy. Apelin, the expression of which is regulated by hypoxia, and which has well-described roles in tumor progression, is an easily measured secreted protein. Here, we show that apelin can be used as a marker for the vessel normalization window during antiangiogenic therapy. Mice bearing s.c. tumors resulting from inoculation of the colon adenocarcinoma cell line HT29 were treated with a single injection of bevacizumab, a mAb neutralizing vascular endothelial growth factor. Tumor growth, vessel density, pericyte coverage, tumor hypoxia, and small molecule delivery were determined at four different times after treatment with bevacizumab (days 1, 3, 5, and 8). Tumor growth and vessel density were significantly reduced after bevacizumab treatment, which also significantly increased tumor vessel maturity, and improved tumor hypoxia and small molecule delivery between days 3 and 5. These effects abated by day 8, suggesting that a time window for vessel normalization was opened between days 3 and 5 during bevacizumab treatment in this model. Apelin mRNA expression and plasma apelin levels decreased transiently at day 5 post-treatment, coinciding with vessel normalization. Thus, apelin is a potential indicator of the vessel normalization window during antiangiogenic therapy. © 2015 The Authors. Cancer Science published by Wiley Publishing Asia Pty Ltd on behalf of Japanese Cancer Association.

  2. Villin promoter-mediated transgenic expression of transient receptor potential cation channel, subfamily V, member 6 (TRPV6) increases intestinal calcium absorption in wild-type and vitamin D receptor knockout mice.

    Science.gov (United States)

    Cui, Min; Li, Qiang; Johnson, Robert; Fleet, James C

    2012-10-01

    Transient receptor potential cation channel, subfamily V, member 6 (TRPV6) is an apical membrane calcium (Ca) channel in the small intestine proposed to be essential for vitamin D-regulated intestinal Ca absorption. Recent studies have challenged the proposed role for TRPV6 in Ca absorption. We directly tested intestinal TRPV6 function in Ca and bone metabolism in wild-type (WT) and vitamin D receptor knockout (VDRKO) mice. TRPV6 transgenic mice (TG) were made with intestinal epithelium-specific expression of a 3X Flag-tagged human TRPV6 protein. TG and VDRKO mice were crossed to make TG-VDRKO mice. Ca and bone metabolism was examined in WT, TG, VDRKO, and TG-VDRKO mice. TG mice developed hypercalcemia and soft tissue calcification on a chow diet. In TG mice fed a 0.25% Ca diet, Ca absorption was more than three-fold higher and femur bone mineral density (BMD) was 26% higher than WT. Renal 1α hydroxylase (CYP27B1) mRNA and intestinal expression of the natural mouse TRPV6 gene were reduced to intestine calbindin-D(9k) expression was elevated >15 times in TG mice. TG-VDRKO mice had high Ca absorption that prevented the low serum Ca, high renal CYP27B1 mRNA, low BMD, and abnormal bone microarchitecture seen in VDRKO mice. In addition, small intestinal calbindin D(9K) mRNA and protein levels were elevated in TG-VDRKO. Transgenic TRPV6 expression in intestine is sufficient to increase Ca absorption and bone density, even in VDRKO mice. VDR-independent upregulation of intestinal calbindin D(9k) in TG-VDRKO suggests this protein may buffer intracellular Ca during Ca absorption. © 2012 American Society for Bone and Mineral Research. Copyright © 2012 American Society for Bone and Mineral Research.

  3. Scavenger Receptor Class B Type 1 Deletion Led to Coronary Atherosclerosis and Ischemic Heart Disease in Low-density Lipoprotein Receptor Knockout Mice on Modified Western-type Diet.

    Science.gov (United States)

    Liao, Jiawei; Guo, Xin; Wang, Mengyu; Dong, Chengyan; Gao, Mingming; Wang, Huan; Kayoumu, Abudurexiti; Shen, Qiang; Wang, Yuhui; Wang, Fan; Huang, Wei; Liu, George

    2017-02-01

    Atherosclerosis-prone apolipoprotein E (apoE) or low-density lipoprotein receptor (LDL-R) knockout (KO) mice are generally resistant to developing coronary atherosclerosis (CA) and ischemic heart disease (IHD). However, studies have demonstrated the occurrence of spontaneous CA and IHD in scavenger receptor class B type 1 (SR-BI)/apoE double KO (dKO) mice, which suggests that SR-BI could be a potential target for the prevention and therapy of CA and IHD. This possibility was later investigated in SR-BI/LDL-R dKO mice, but no signs of CA or IHD was identified when mice were fed a normal western-type diet. Here we explored whether SR-BI deletion could result in CA and IHD in LDL-R KO mice when fed a modified western-type diet containing higher (0.5%) cholesterol. Cardiac functions were detected by electrocardiography, single photon emission computed tomography (SPECT), echocardiography (Echo) and 2,3,5-triphenyltetrazolium chloride staining. CA was visualized by hematoxylin-eosin staining. After 12 weeks on the modified diet, SR-BI/LDL-R dKO mice developed cardiac ischemia/infarction, together with systolic dysfunction and left ventricular dilatation. CA was most severe at the aortic sinus level to an extent that no dKO mice survived to 20 weeks on the modified diet. None of control mice, however, developed CA or IHD. SR-BI deletion led to CA and IHD in LDL-R KO mice when fed the modified western-type diet. We established SR-BI/LDL-R dKO mice as a diet-induced murine model of human IHD and developed detection methods, using a combination of SPECT and Echo, for effective in vivo evaluation of cardiac functions.

  4. The Knockout Mouse Project

    Science.gov (United States)

    Austin, Christopher P; Battey, James F; Bradley, Allan; Bucan, Maja; Capecchi, Mario; Collins, Francis S; Dove, William F; Duyk, Geoffrey; Dymecki, Susan; Eppig, Janan T; Grieder, Franziska B; Heintz, Nathaniel; Hicks, Geoff; Insel, Thomas R; Joyner, Alexandra; Koller, Beverly H; Lloyd, K C Kent; Magnuson, Terry; Moore, Mark W; Nagy, Andras; Pollock, Jonathan D; Roses, Allen D; Sands, Arthur T; Seed, Brian; Skarnes, William C; Snoddy, Jay; Soriano, Philippe; Stewart, David J; Stewart, Francis; Stillman, Bruce; Varmus, Harold; Varticovski, Lyuba; Verma, Inder M; Vogt, Thomas F; von Melchner, Harald; Witkowski, Jan; Woychik, Richard P; Wurst, Wolfgang; Yancopoulos, George D; Young, Stephen G; Zambrowicz, Brian

    2009-01-01

    Mouse knockout technology provides a powerful means of elucidating gene function in vivo, and a publicly available genome-wide collection of mouse knockouts would be significantly enabling for biomedical discovery. To date, published knockouts exist for only about 10% of mouse genes. Furthermore, many of these are limited in utility because they have not been made or phenotyped in standardized ways, and many are not freely available to researchers. It is time to harness new technologies and efficiencies of production to mount a high-throughput international effort to produce and phenotype knockouts for all mouse genes, and place these resources into the public domain. PMID:15340423

  5. THE RELATIONSHIP OF SERUM LEPTIN AND PLASMA APELIN LEVELS IN MEN WITH METABOLIC SYNDROME

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    A. V. Fedotova

    2016-01-01

    Full Text Available Background: The metabolic syndrome is seen as a  cluster of high cardiovascular risk factors. New hormone-like substances, such as adipokines leptin and apelin, produced by fat tissue, are important for the pathophysiology of the metabolic syndrome.Aim: To evaluate levels of plasma apelin and serum leptin in patients with metabolic syndrome.Materials and methods: We examined 122 male patients with metabolic syndrome and 30 healthy males aged from 25 to 60 years. All patients were assessed accordingly to confirm the diagnosis of the metabolic syndrome, with additional measurements of serum leptin and plasma apelin.Results: Serum leptin levels were 10-fold higher and plasma apelin levels 3-fold higher in patients with metabolic syndrome (n=122, compared to the controls (n=30: 25.43 vs. 3.99 ng/mL (p<0.05 and 1.13 vs. 0.66 ng/mL (p<0.05, respectively. Serum leptin levels correlated with all parameters of the excess body weight, such as body mass (r=0.79, р<0.05, body mass index (r=0.93, р<0.05, waist circumference (r=0.61, р<0.05, hip circumference (r=0.57, р<0.05, and waist-to-hip ratio (WHR; r=0.4, р<0.05. Plasma apelin levels correlated with the waist circumference (r=0.27, р<0.05 and WHR (r=0.29, р<0.05. There was a significant increase of serum leptin dependent on the bodyweight category: 19.8 ng/mL in patients with obesity Grade I  (n=49 and 28.7  ng/mL in those with obesity Grade II (n=46 (р<0.05. There was a  non-significant trend towards an increase in plasma apelin depending on bodyweight. As far as abdominal obesity is concerned, in patients with WHR<1 (n=35, plasma apelin level was 0.36  ng/mL, whereas in those with WHR of ≥1 (n=87, it was 3-fold higher (1.09 ng/mL, р<0.05; the increase in serum leptin levels was non-significant. There was no association between plasma apelin and serum leptin in patients with metabolic syndrome (r=0.1, р>0.05.Conclusion: The adipokine levels in men with metabolic syndrome are

  6. Cluster knockout reactions

    Indian Academy of Sciences (India)

    2014-04-07

    Apr 7, 2014 ... advancements in the area of (α, 2α) reactions and heavy cluster knockout reactions are discussed. Importance of the finite-range vertex and the final-state interactions are brought out. Keywords. Cluster knockout reactions; FR-DWIA calculations; t-matrix effective interaction. PACS Nos 14.20.Pt; 24.10.

  7. Positron Emission Tomographic Imaging of the Cannabinoid Type 1 Receptor System with [11C]OMAR ([11C]JHU75528: Improvements in Image Quantification Using Wild-Type and Knockout Mice

    Directory of Open Access Journals (Sweden)

    Raúl Herance

    2011-11-01

    Full Text Available In this study, we assessed the feasibility of using positron emission tomography (PET and the tracer [11C]OMAR ([11C]JHU75528, an analogue of rimonabant, to study the brain cannabinoid type 1 (CB1 receptor system. Wild-type (WT andCB1 knockout (KO animals were imaged at baseline and after pretreatment with blocking doses of rimonabant. Brain uptake in WT animals was higher (50% than in KO animals in baseline conditions. After pretreatment with rimonabant, WT uptake lowered to the level of KO animals. The results of this study support the feasibility of using PET with the radiotracer [11C]JHU75528 to image the brain CB1 receptor system in mice. In addition, this methodology can be used to assess the effect of new drugs in preclinical studies using genetically manipulated animals.

  8. High-intensity interval training lowers blood pressure and improves apelin and NOx plasma levels in older treated hypertensive individuals.

    Science.gov (United States)

    Izadi, Mohammad Reza; Ghardashi Afousi, Alireza; Asvadi Fard, Maryam; Babaee Bigi, Mohammad Ali

    2018-02-01

    Hypertension is the major risk factor for cardiovascular diseases and is one of the primary causes of morbidity and mortality worldwide. Apelin levels and NO bioavailability are impaired in older hypertensive patients. Exercise is an effective intervention for treating hypertension. Our purpose was to evaluate the effect of high-intensity interval training on blood pressure, apelin, and NOx plasma levels in older treated hypertensive individuals. Thirty treated hypertensive subjects (61.70 ± 5.78 years, 17 males, 13 females) were randomly divided into 6 weeks of high-intensity interval training (n = 15) and control (n = 15). The exercise training was conducted for three 35-min sessions a week (1.5-min interval at 85-90% of heart rate reserve [HRR] and 2 min active phase at 50-55% of HRR). Assessment of plasma apelin, nitrite/nitrate (NOx), and endothelin-1 (ET-1) was performed before and after the intervention. At the end of the study, apelin, and NOx plasma levels increased significantly in the high-intensity interval training (HIIT) group (P = 0.021, P = 0.003, respectively). Conversely, ET-1 plasma levels significantly decreased in the training group after the intervention (P = 0.015). Moreover, there was a positive correlation between the change of plasma apelin and change of plasma NOx (r = 0. 771, P = 0.0008). In addition, there was a negative correlation between the change of plasma ET-1, change of plasma apelin (r = - 0.595, P = 0.019), and variation of NOx (r = - 0.572, P = 0.025). This study indicates that, by increasing of apelin and NOx plasma levels, HIIT may be effective in reducing blood pressure.

  9. What is the role of apelin regarding cardiovascular risk and progression of renal disease in type 2 diabetic patients with diabetic nephropathy?

    Science.gov (United States)

    Silva, Ana Paula; Fragoso, André; Silva, Claudia; Viegas, Carla; Tavares, Nelson; Guilherme, Patrícia; Santos, Nélio; Rato, Fátima; Camacho, Ana; Cavaco, Cidália; Pereira, Victor; Faísca, Marilia; Ataíde, João; Jesus, Ilídio; Neves, Pedro

    2013-01-01

    To evaluate the association of different apelin levels with cardiovascular mortality, hospitalization, renal function, and cardiovascular risk factors in type 2 diabetic patients with mild to moderate CKD. An observational, prospective study involving 150 patients divided into groups according to baseline apelin levels: 1 ≤ 98 pg/mL, 2 = 98-328 pg/mL, and 3 ≥ 329 pg/mL. Baseline characteristics were analyzed and compared. Multivariate Cox regression was used to find out predictors of cardiovascular mortality, and multivariate logistic regression was used to find out predictors of hospitalization and disease progression. Simple linear regressions and Pearson correlations were used to investigate correlations between apelin and renal disease and cardiovascular risk factors. Patients' survival at 83 months in groups 1, 2, and 3 was 39%, 40%, and 71.2%, respectively (P = 0.046). Apelin, age, and eGFR were independent predictors of mortality, and apelin, creatinine, eGFR, resistin, and visfatin were independent predictors of hospitalization. Apelin levels were negatively correlated with cardiovascular risk factors and positively correlated with eGFR. Patients with lower apelin levels were more likely to start a depurative technique. Apelin levels might have a significant clinical use as a marker/predictor of cardiovascular mortality and hospitalization or even as a therapeutic agent for CKD patients with cardiovascular disease.

  10. mRNA transfection of a novel TAL effector nuclease (TALEN) facilitates efficient knockout of HIV co-receptor CCR5.

    Science.gov (United States)

    Mock, Ulrike; Machowicz, Rafał; Hauber, Ilona; Horn, Stefan; Abramowski, Pierre; Berdien, Belinda; Hauber, Joachim; Fehse, Boris

    2015-06-23

    Homozygosity for a natural deletion variant of the HIV-coreceptor molecule CCR5, CCR5Δ32, confers resistance toward HIV infection. Allogeneic stem cell transplantation from a CCR5Δ32-homozygous donor has resulted in the first cure from HIV ('Berlin patient'). Based thereon, genetic disruption of CCR5 using designer nucleases was proposed as a promising HIV gene-therapy approach. Here we introduce a novel TAL-effector nuclease, CCR5-Uco-TALEN that can be efficiently delivered into T cells by mRNA electroporation, a gentle and truly transient gene-transfer technique. CCR5-Uco-TALEN mediated high-rate CCR5 knockout (>90% in PM1 and >50% in primary T cells) combined with low off-target activity, as assessed by flow cytometry, next-generation sequencing and a newly devised, very convenient gene-editing frequency digital-PCR (GEF-dPCR). GEF-dPCR facilitates simultaneous detection of wild-type and gene-edited alleles with remarkable sensitivity and accuracy as shown for the CCR5 on-target and CCR2 off-target loci. CCR5-edited cells were protected from infection with HIV-derived lentiviral vectors, but also with the wild-type CCR5-tropic HIV-1BaL strain. Long-term exposure to HIV-1BaL resulted in almost complete suppression of viral replication and selection of CCR5-gene edited T cells. In conclusion, we have developed a novel TALEN for the targeted, high-efficiency knockout of CCR5 and a useful dPCR-based gene-editing detection method. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  11. Apelin-13 upregulates Egr-1 expression in rat vascular smooth muscle cells through the PI3K/Akt and PKC signaling pathways

    International Nuclear Information System (INIS)

    Liu, Qi-Feng; Yu, Hong-Wei; Sun, Li-Li; You, Lu; Tao, Gui-Zhou; Qu, Bao-Ze

    2015-01-01

    Previous studies have shown that Apelin-13 upregulates early growth response factor-1 (Egr-1) via the extracellular signal-regulated protein kinase (ERK) signaling pathway. Apelin-13 induces proliferation and migration of vascular smooth muscle cells (VSMCs) as well as the upregulation of osteopontin (OPN) via the upregulation of Egr-1. This study was designed to further explore the activity of Apelin-13 in VSMCs by investigating members of the mitogen-activated protein kinase (MAPK) family, in particular Jun kinase (JNK) and p38 mitogen-activated protein kinase (P38). We also examined whether the phosphatidylinositol 3 kinase (PI3K)/protein kinase B (Akt) and protein kinase C (PKC) signaling pathways were involved in the regulation of Egr-1 by Apelin-13. We treated rat aortic VSMCs with Apelin-13 and examined the expression of JNK, p-JNK, P38, and p-P38 to investigate whether Apelin-13-mediated increases in Egr-1 occurred through the JNK and P38 signaling pathways. We then pretreated VSMCs with the Gi protein inhibitor pertussis toxin (PTX) and the Gq inhibitor YM254890, added Apelin-13 and looked for changes in Egr-1 expression. Finally, we pretreated with the PI3K inhibitor LY294002 and the PKC inhibitor GF109203X, and treated with Apelin-13. Our results showed that JNK and P38 did not participate in Apelin-13-mediated increase in Egr-1. Instead, Apelin-13 upregulation of Egr-1 was mediated by a PTX-sensitive Gi protein. Apelin-13 did increase ERK phosphorylation through the PI3K/Akt and PKC signaling pathways, resulting in changes in Egr-1 expression. These data provide important targets for future studies to modulate vascular remodeling. - Highlights: • Apelin-13 mediates Egr-1 upregulation in vascular smooth muscle cells via ERK1/2. • The underlying mechanisms are unknown, but exclude Jnk or p38 pathway activation. • Apelin-13 binds to Gi, activating the PI3K/Akt and PKC signaling cascades. • Consequent ERK phosphorylation results in increased Egr-1

  12. Apelin-13 upregulates Egr-1 expression in rat vascular smooth muscle cells through the PI3K/Akt and PKC signaling pathways

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Qi-Feng [Department of Cardiology, The First Affiliated Hospital of Liaoning Medical University, Jinzhou 121001 (China); Yu, Hong-Wei [Department of Cardiology, Jinzhou Central Hospital, Jinzhou 121001 (China); Sun, Li-Li [Department of Ophthalmology, The Third Affiliated Hospital of Liaoning Medical University, Jinzhou 121001 (China); You, Lu; Tao, Gui-Zhou [Department of Cardiology, The First Affiliated Hospital of Liaoning Medical University, Jinzhou 121001 (China); Qu, Bao-Ze, E-mail: qubaoze1971@hotmail.com [Department of Cardiology, The First Affiliated Hospital of Liaoning Medical University, Jinzhou 121001 (China)

    2015-12-25

    Previous studies have shown that Apelin-13 upregulates early growth response factor-1 (Egr-1) via the extracellular signal-regulated protein kinase (ERK) signaling pathway. Apelin-13 induces proliferation and migration of vascular smooth muscle cells (VSMCs) as well as the upregulation of osteopontin (OPN) via the upregulation of Egr-1. This study was designed to further explore the activity of Apelin-13 in VSMCs by investigating members of the mitogen-activated protein kinase (MAPK) family, in particular Jun kinase (JNK) and p38 mitogen-activated protein kinase (P38). We also examined whether the phosphatidylinositol 3 kinase (PI3K)/protein kinase B (Akt) and protein kinase C (PKC) signaling pathways were involved in the regulation of Egr-1 by Apelin-13. We treated rat aortic VSMCs with Apelin-13 and examined the expression of JNK, p-JNK, P38, and p-P38 to investigate whether Apelin-13-mediated increases in Egr-1 occurred through the JNK and P38 signaling pathways. We then pretreated VSMCs with the Gi protein inhibitor pertussis toxin (PTX) and the Gq inhibitor YM254890, added Apelin-13 and looked for changes in Egr-1 expression. Finally, we pretreated with the PI3K inhibitor LY294002 and the PKC inhibitor GF109203X, and treated with Apelin-13. Our results showed that JNK and P38 did not participate in Apelin-13-mediated increase in Egr-1. Instead, Apelin-13 upregulation of Egr-1 was mediated by a PTX-sensitive Gi protein. Apelin-13 did increase ERK phosphorylation through the PI3K/Akt and PKC signaling pathways, resulting in changes in Egr-1 expression. These data provide important targets for future studies to modulate vascular remodeling. - Highlights: • Apelin-13 mediates Egr-1 upregulation in vascular smooth muscle cells via ERK1/2. • The underlying mechanisms are unknown, but exclude Jnk or p38 pathway activation. • Apelin-13 binds to Gi, activating the PI3K/Akt and PKC signaling cascades. • Consequent ERK phosphorylation results in increased Egr-1

  13. Co-administration of Apelin and T4 Protects Inotropic and Chronotropic Changes Occurring in Hypothyroid Rats

    Directory of Open Access Journals (Sweden)

    Zahra Akhondali

    2015-01-01

    Full Text Available Abstract Background: One of the most important thyroid hormone targets is the cardiovascular system. Hemodynamic changes, such as decreased resting heart rate (HR, myocardial contractility, and cardiac output, and increased diastolic pressure and systemic vascular resistance, have been observed in hypothyroid patients. Moreover, in these patients, ECG changes include sinus bradycardia and low voltage complexes (P waves or QRS complexes. Objective: This study aimed at evaluating the prophylactic effect of apelin on HR changes and QRS voltage that occur in propylthiouracil (PTU-induced hypothyroid rats. Method: In this study, 48 adult male Wistar rats weighing 170-235g were randomly divided into 6 groups: Control group (normal saline ip injection + tap water gavage; P group (PTU 0.05%, in drinking water; A group (apelin 200 µg.kg-1.day-1, ip; PA group [co-administration of PTU and apelin]; PT group [co-administration of PTU + T4 (0.2 mg/g per day, gavage]; and PAT group (co-administration of PTU, apelin and T4. All experiments were performed for 28 consecutive days, and then the animals were anesthetized with an ip injection of ketamine (80 mg/kg and xylazine (12 mg/kg. Lead II electrocardiogram was recorded to calculate HR and QRS voltage. Results: Heart rate and QRS voltage increased more significantly in the hypothyroid group that consumed both apelin and T4 (201 ± 4 beat/min, 0.71 ± 0.02 mv vs. hypothyroid 145 ± 9 beat/min, 0.563 ± 0.015 mv; respectively. Conclusion: The co-administration of apelin and T4 showed a protective effect on QRS voltage and HR in PTU‑induced hypothyroid rats.

  14. Co-administration of Apelin and T4 Protects Inotropic and Chronotropic Changes Occurring in Hypothyroid Rats

    Energy Technology Data Exchange (ETDEWEB)

    Akhondali, Zahra; Badavi, Mohammad; Dianat, Mahin, E-mail: dianat@ajums.ac.ir; Faraji, Farzaneh [Physiology Research Center and Department of Physiology, Faculty of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz (Iran, Islamic Republic of)

    2015-09-15

    One of the most important thyroid hormone targets is the cardiovascular system. Hemodynamic changes, such as decreased resting heart rate (HR), myocardial contractility, and cardiac output, and increased diastolic pressure and systemic vascular resistance, have been observed in hypothyroid patients. Moreover, in these patients, ECG changes include sinus bradycardia and low voltage complexes (P waves or QRS complexes). This study aimed at evaluating the prophylactic effect of apelin on HR changes and QRS voltage that occur in propylthiouracil (PTU)-induced hypothyroid rats. In this study, 48 adult male Wistar rats weighing 170-235g were randomly divided into 6 groups: Control group (normal saline ip injection + tap water gavage); P group (PTU 0.05%, in drinking water); A group (apelin 200 µg.kg{sup -1}.day{sup -1}, ip); PA group [co-administration of PTU and apelin]; PT group [co-administration of PTU + T4 (0.2 mg/g per day, gavage)]; and PAT group (co-administration of PTU, apelin and T4). All experiments were performed for 28 consecutive days, and then the animals were anesthetized with an ip injection of ketamine (80 mg/kg) and xylazine (12 mg/kg). Lead II electrocardiogram was recorded to calculate HR and QRS voltage. Heart rate and QRS voltage increased more significantly in the hypothyroid group that consumed both apelin and T4 (201 ± 4 beat/min, 0.71 ± 0.02 mv vs. hypothyroid 145 ± 9 beat/min, 0.563 ± 0.015 mv; respectively). The co-administration of apelin and T4 showed a protective effect on QRS voltage and HR in PTU‑induced hypothyroid rats.

  15. Effect of 4 Weeks of Detraining After a Period of Resistance Training on Plasma Apelin Levels in Overweight and Obese Girls

    Directory of Open Access Journals (Sweden)

    Rezvaneh Galdavi

    2017-02-01

    Full Text Available Background and Objectives: Apelin is secreted from visceral adipose tissue. However, the effect of resistance training and consequent detraining on the apelin level in obesity, has not yet been clearly determined. In the current research, the effect of 4 weeks of detraining after a period of resistance training, was investigated on the plasma apelin levels in overweight and obese girls. Methods: In this quasi-experimental study, 22 overweight and obese girls were purposefully selected and randomly divided into two groups of experimental (n=12 and control (n=10. The experimental group exercised in an 8-week training program (4 sessions per week according to a training program with an intensity of 65-80% of one maximum repetition, and then experienced 4 weeks of detraining. Blood sampling was performed after a 12-h fasting in various phases and the level of apelin was measured. Data were analyzed by Kolmogorov-Smirnov, repeated measure ANOVA, post-hoc LSD, and independent t-tests at a significance level of α 0.05. Also, after 4 weeks of detraining a slight increase was seen in the levels of apelin, but was not significant (p>0.05. Conclusion: According to the results of this study, resistance training as a new non-drug therapy can be effective in reducing the levels of apelin. Also, apelin levels increases with discontinuation of exercise, which may lead to the emergence of inflammatory features in the cardiovascular system.

  16. Knockout of Vasohibin-1 Gene in Mice Results in Healthy Longevity with Reduced Expression of Insulin Receptor, Insulin Receptor Substrate 1, and Insulin Receptor Substrate 2 in Their White Adipose Tissue

    Directory of Open Access Journals (Sweden)

    Eichi Takeda

    2017-01-01

    Full Text Available Vasohibin-1 (Vash1, originally isolated as an endothelium-derived angiogenesis inhibitor, has a characteristic of promoting stress tolerance in endothelial cells (ECs. We therefore speculated that the lack of the vash1 gene would result in a short lifespan. However, to our surprise, vash1−/− mice lived significantly longer with a milder senescence phenotype than wild-type (WT mice. We sought the cause of this healthy longevity and found that vash1−/− mice exhibited mild insulin resistance along with reduced expression of the insulin receptor (insr, insulin receptor substrate 1 (irs-1, and insulin receptor substrate 2 (irs-2 in their white adipose tissue (WAT but not in their liver or skeletal muscle. The expression of vash1 dominated in the WAT among those 3 organs. Importantly, vash1−/− mice did not develop diabetes even when fed a high-fat diet. These results indicate that the expression of vash1 was required for the normal insulin sensitivity of the WAT and that the target molecules for this activity were insr, irs1, and irs2. The lack of vash1 caused mild insulin resistance without the outbreak of overt diabetes and might contribute to healthy longevity.

  17. Cluster knockout reactions

    Indian Academy of Sciences (India)

    2014-04-07

    Apr 7, 2014 ... Cluster knockout reactions are expected to reveal the amount of clustering (such as that of , d and even of heavier clusters such as 12C, 16O etc.) in the target nucleus. In simple terms, incident medium high-energy nuclear projectile interacts strongly with the cluster (present in the target nucleus) as if it ...

  18. KnockoutJS blueprints

    CERN Document Server

    Russo, Carlo

    2015-01-01

    If you are a JavaScript developer and already know the basics of KnockoutJS and you want to get the most out of it, then this book is for you. This book will help in your transition from a small site to a large web application that is easily maintainable.

  19. Double incretin receptor knockout (DIRKO) mice reveal an essential role for the enteroinsular axis in transducing the glucoregulatory actions of DPP-IV inhibitors

    DEFF Research Database (Denmark)

    Hansotia, Tanya; Baggio, Laurie L; Delmeire, Dominique

    2004-01-01

    Glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide 1 (GLP-1) are gut-derived incretins that potentiate glucose clearance following nutrient ingestion. Elimination of incretin receptor action in GIPR(-/-) or GLP-1R(-/-) mice produces only modest impairment in glucose home...

  20. Role of the cholinergic nervous system in rheumatoid arthritis: aggravation of arthritis in nicotinic acetylcholine receptor alpha7 subunit gene knockout mice

    NARCIS (Netherlands)

    van Maanen, Marjolein A.; Stoof, Susanne P.; Larosa, Gregory J.; Vervoordeldonk, Margriet J.; Tak, Paul P.

    2010-01-01

    BACKGROUND: The alpha7 subunit of nicotinic acetylcholine receptors (alpha7nAChR) can negatively regulate the synthesis and release of proinflammatory cytokines by macrophages and fibroblast-like synoviocytes in vitro. In addition, stimulation of the alpha7nAChR can reduce the severity of arthritis

  1. Accumulation of cytolytic CD8{sup +} T cells in B16-melanoma and proliferation of mature T cells in TIS21-knockout mice after T cell receptor stimulation

    Energy Technology Data Exchange (ETDEWEB)

    Ryu, Min Sook [Department of Biochemistry and Molecular Biology, Ajou University School of Medicine, 164, World cul-ro, Yeongtong-gu, Suwon, Gyeonggi-do 443-380 (Korea, Republic of); Woo, Min-Yeong [Department of Microbiology, Ajou University School of Medicine, 164, World cul-ro, Yeongtong-gu, Suwon, Gyeonggi-do 443-380 (Korea, Republic of); Department of Biomedical Sciences, The Graduate School, Ajou University (Korea, Republic of); Kwon, Daeho [Department of Microbiology, Kwandong University College of Medicine, Gangneung, Gangwon-do 210-701 (Korea, Republic of); Hong, Allen E. [Department of Biochemistry and Molecular Biology, Ajou University School of Medicine, 164, World cul-ro, Yeongtong-gu, Suwon, Gyeonggi-do 443-380 (Korea, Republic of); Song, Kye Yong [Department of Pathology, Chung-Ang University College of Medicine, Dongjak-gu, Seoul 156-756 (Korea, Republic of); Park, Sun [Department of Microbiology, Ajou University School of Medicine, 164, World cul-ro, Yeongtong-gu, Suwon, Gyeonggi-do 443-380 (Korea, Republic of); Lim, In Kyoung [Department of Biochemistry and Molecular Biology, Ajou University School of Medicine, 164, World cul-ro, Yeongtong-gu, Suwon, Gyeonggi-do 443-380 (Korea, Republic of)

    2014-10-01

    In vivo and in vitro effects of TIS21 gene on the mature T cell activation and antitumor activities were explored by employing MO5 melanoma orthograft and splenocytes isolated from the TIS21-knockout (KO) mice. Proliferation and survival of mature T cells were significantly increased in the KO than the wild type (WT) cells, indicating that TIS21 inhibits the rate of mature T cell proliferation and its survival. In MO5 melanoma orthograft model, the KO mice recruited much more CD8{sup +} T cells into the tumors at around day 14 after tumor cell injection along with reduced tumor volumes compared with the WT. The increased frequency of granzyme B{sup +} CD8{sup +} T cells in splenocytes of the KO mice compared with the WT may account for antitumor-immunity of TIS21 gene in the melanoma orthograft. In contrast, reduced frequencies of CD107a{sup +} CD8{sup +} T cells in the splenocytes of KO mice may affect the loss of CD8{sup +} T cell infiltration in the orthograft at around day 19. These results indicate that TIS21 exhibits antiproliferative and proapoptotic effects in mature T cells, and differentially affects the frequencies of granzyme B{sup +} CD8{sup +} T-cells and CD107a{sup +} CD8{sup +} T-cells, thus transiently regulating in vivo anti-tumor immunity. - Highlights: • Constitutive expression of TIS21 in splenocytes and upregulation by TCR stimulation. • Proliferation of mature T-cells in spleen of TIS21KO mice after TCR stimulation. • Inhibition of cell death in mature T-cells of TIS21KO mice compared with the wild type. • Inhibition of melanoma growth in TIS21KO mice and CD8{sup +} T cell infiltration in tumor. • Reduction of CD 107{sup +}CD8{sup +} T cells, but increased granzyme B{sup +} CD8{sup +} T cells in TIS21KO mice.

  2. The influence of angiotensin-(1-7) Mas receptor agonist (AVE 0991) on mitochondrial proteome in kidneys of apoE knockout mice.

    Science.gov (United States)

    Suski, Maciej; Olszanecki, Rafał; Stachowicz, Aneta; Madej, Józef; Bujak-Giżycka, Beata; Okoń, Krzysztof; Korbut, Ryszard

    2013-12-01

    Excessive action of angiotensin II on mitochondria has been shown to play an important role in mitochondrial dysfunction, a common feature of atherogenesis and kidney injury. Angiotensin-(1-7)/Mas receptor axis constitutes a countermeasure to the detrimental effects of angiotensin II on AT1 receptors. The aim of the study was to assess the effects of angiotensin-(1-7) peptidomimetic AVE0991 on the kidney mitochondrial proteome in widely used animal model of atherosclerosis (apoE(-/-) mice). Proteins changed in apoE(-/-) mice belonged to the groups of antioxidant enzymes, apoptosis regulators, inflammatory factors and metabolic enzymes. Importantly, AVE0991 partially reversed atherosclerosis-related changes in apoE(-/-) mice. © 2013.

  3. Knockout crickets for the study of learning and memory: Dopamine receptor Dop1 mediates aversive but not appetitive reinforcement in crickets

    OpenAIRE

    Awata, Hiroko; Watanabe, Takahito; Hamanaka, Yoshitaka; Mito, Taro; Noji, Sumihare; Mizunami, Makoto

    2015-01-01

    Elucidation of reinforcement mechanisms in associative learning is an important subject in neuroscience. In mammals, dopamine neurons are thought to play critical roles in mediating both appetitive and aversive reinforcement. Our pharmacological studies suggested that octopamine and dopamine neurons mediate reward and punishment, respectively, in crickets, but recent studies in fruit-flies concluded that dopamine neurons mediates both reward and punishment, via the type 1 dopamine receptor Do...

  4. KnockoutJS essentials

    CERN Document Server

    Ferrando, Jorge

    2015-01-01

    If you are a JavaScript developer who has been using DOM manipulation libraries such as Mootools or Scriptaculous, and you want go further in modern JavaScript development with a simple and well-documented library, then this book is for you. Learning how to use Knockout will be perfect as your next step towards building JavaScript applications that respond to user interaction.

  5. Cluster knockout reactions

    Indian Academy of Sciences (India)

    2014-04-07

    Apr 7, 2014 ... (figure 2), the corresponding α–α t-matrix effective interactions are seen to be widely different (figure 3). 2. Formalism. The finite-range transition matrix element T n,l. BA in the triple differential cross-section [13], d3σ d 1d 2dE1. = Fkin × Sl × |T n,l. BA (kf 1, kf 2, ki)|2 for the (α, 2α) cluster knockout reactions is ...

  6. The effects of nonlinear resistance and aerobic interval training on serum levels of apelin and insulin resistance in middle-aged obese men

    Directory of Open Access Journals (Sweden)

    Mahmoud Nikseresht

    2015-08-01

    Conclusion: The practical applications indicate that obese men can use both AIT and NRT exercise programs to reduce insulin resistance. However, the AIT may have better beneficial effects (as indicated by apelin-13 compared to NRT.

  7. The Effect of Fenugreek (Trigonella foenum-graecum Seed and 17-β Estradiol on Serum Apelin, Glucose, Lipids, and Insulin in Ovariectomized Rats

    Directory of Open Access Journals (Sweden)

    Abedinzade

    2015-08-01

    Full Text Available Background Menopause, a natural phenomenon, is defined by the fall of ovarian hormones mainly estrogens causing major problems such as insulin resistance. Fenugreek (Trigonella foenum-graecum is known to have some useful properties such as insulin sensitizing effect. Apelin is an adipokine, which has several roles such as regulation of insulin secretion. Objectives The objective of the present study was to evaluate the effect of fenugreek seed and 17-β estradiol on serum Apelin along with glucose, lipids and insulin in ovariectomized rats. Materials and Methods Forty-nine adult female Wistar rats were randomly divided to seven groups: normal control, ovariectomized control, ovariectomized treated with ethanolic and hexanic extract of fenugreek seed (50 and 150 mg/kg/daily for each, and ovariectomized treated with 17-β estradiol (10 µg/kg/daily for 42 days. Serum Apelin, glucose, lipids and insulin were measured. Results Serum Apelin, glucose, lipids and insulin significantly increased in ovariectomized controls in comparison with normal controls (P < 0.05. Serum glucose, lipids and insulin in ovariectomized rats treated with fenugreek seed extract and 17-β estradiol were remarkably lower than ovariectomized controls (P < 0.05. Furthermore, 17-β estradiol caused a significant decrease (P < 0.05 in serum Apelin in ovariectomized rats. Conclusions It appears that fenugreek seed might be effective against hyperglycemia, hyperlipidemia and insulin resistance in ovariectomized rats without impact on serum Apelin. Furthermore, 17-β estradiol could have similar effects along with possible inhibitory effects on serum Apelin. The complicated role of Apelin in menopause needs to be further explored.

  8. Apelin-13 attenuates cisplatin-induced cardiotoxicity through inhibition of ROS-mediated DNA damage and regulation of MAPKs and AKT pathways.

    Science.gov (United States)

    Zhang, Pu; Yi, Lu-Hua; Meng, Guang-Yuan; Zhang, Huan-Yi; Sun, Hai-Hui; Cui, Lian-Qun

    2017-05-01

    Platinum-based chemotherapy represents one of the most effective ways in combating human cancers. However, the cardiotoxicity subsequent severely limited its clinical application. Increased evidences indicate that oxidative stress plays a crucial role in the pathological process of platinum-induced cardiotoxicity. It is reported that apelin-13 a bioactive peptide has the scavenging capacity of free radical, and it has the potential to regulate the cardiovascular system. Hence, the potential of apelin-13 to antagonize cisplatin-induced cardiotoxicity was evaluated in H9c2 rat myocardial cells in vitro and in C57 mice in vivo. The results showed that cisplatin indeed caused DNA damage in H9c2 cells by promoting the accumulation of intracellular reactive oxygen species (ROS) and superoxide anion, which led to cell apoptosis and resulted in overt cardiotoxicity. However, apelin-13 pre-treatment effectively attenuated the cisplatin-induced ROS and superoxide anion generation, inhibited DNA damage, and suppressed the PARP cleavage and caspases activation. Further investigation revealed that apelin-13 blocked cisplatin-induced H9c2 cells apoptosis involving the regulation of MAPKs and PI3K/Akt signaling pathway. Importantly, apelin-13 co-treatment also significantly attenuated cisplatin-induced cardiotoxicity in vivo by inhibiting myocardial cells apoptosis and improving angiogenesis in mice heart. Taken together, our results suggest that the use of apelin-13 may be an effective strategy for antagonizing the cardiotoxicity-induced by platinum-based chemotherapy.

  9. TRAIL death receptor 4 signaling via lysosome fusion and membrane raft clustering in coronary arterial endothelial cells: evidence from ASM knockout mice.

    Science.gov (United States)

    Li, Xiang; Han, Wei-Qing; Boini, Krishna M; Xia, Min; Zhang, Yang; Li, Pin-Lan

    2013-01-01

    Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its receptor, death receptor 4 (DR4), have been implicated in the development of endothelial dysfunction and atherosclerosis. However, the signaling mechanism mediating DR4 activation leading to endothelial injury remains unclear. We recently demonstrated that ceramide production via hydrolysis of membrane sphingomyelin by acid sphingomyelinase (ASM) results in membrane raft (MR) clustering and the formation of important redox signaling platforms, which play a crucial role in amplifying redox signaling in endothelial cells leading to endothelial dysfunction. The present study aims to investigate whether TRAIL triggers MR clustering via lysosome fusion and ASM activation, thereby conducting transmembrane redox signaling and changing endothelial function. Using confocal microscopy, we found that TRAIL induced MR clustering and co-localized with DR4 in coronary arterial endothelial cells (CAECs) isolated from wild-type (Smpd1 (+/+)) mice. Furthermore, TRAIL triggered ASM translocation, ceramide production, and NADPH oxidase aggregation in MR clusters in Smpd1 ( +/+ ) CAECs, whereas these observations were not found in Smpd1 (-/-) CAECs. Moreover, ASM deficiency reduced TRAIL-induced O(2) (-[Symbol: see text]) production in CAECs and abolished TRAIL-induced impairment on endothelium-dependent vasodilation in small resistance arteries. By measuring fluorescence resonance energy transfer, we found that Lamp-1 (lysosome membrane marker protein) and ganglioside G(M1) (MR marker) were trafficking together in Smpd1 (+/+) CAECs, which was absent in Smpd1 (-/-) CAECs. Consistently, fluorescence imaging of living cells with specific lysosome probes demonstrated that TRAIL-induced lysosome fusion with membrane was also absent in Smpd1 (-/-) CAECs. Taken together, these results suggest that ASM is essential for TRAIL-induced lysosomal trafficking, membrane fusion and formation of MR redox signaling platforms

  10. Double P2X2/P2X3 Purinergic Receptor Knockout Mice Do Not Taste NaCl or the Artificial Sweetener SC45647

    Science.gov (United States)

    Eddy, Meghan C.; Eschle, Benjamin K.; Barrows, Jennell; Hallock, Robert M.; Finger, Thomas E.

    2009-01-01

    The P2X ionotropic purinergic receptors, P2X2 and P2X3, are essential for transmission of taste information from taste buds to the gustatory nerves. Mice lacking both P2X2 and P2X3 purinergic receptors (P2X2/P2X3Dbl−/−) exhibit no taste-evoked activity in the chorda tympani and glossopharyngeal nerves when stimulated with taste stimuli from any of the 5 classical taste quality groups (salt, sweet, sour, bitter, and umami) nor do the mice show taste preferences for sweet or umami, or avoidance of bitter substances (Finger et al. 2005. ATP signaling is crucial for communication from taste buds to gustatory nerves. Science. 310[5753]:1495–1499). Here, we compare the ability of P2X2/P2X3Dbl−/− mice and P2X2/P2X3Dbl+/+ wild-type (WT) mice to detect NaCl in brief-access tests and conditioned aversion paradigms. Brief-access testing with NaCl revealed that whereas WT mice decrease licking at 300 mM and above, the P2X2/P2X3Dbl−/− mice do not show any change in lick rates. In conditioned aversion tests, P2X2/P2X3Dbl−/− mice did not develop a learned aversion to NaCl or the artificial sweetener SC45647, both of which are easily avoided by conditioned WT mice. The inability of P2X2/P2X3Dbl−/− mice to show avoidance of these taste stimuli was not due to an inability to learn the task because both WT and P2X2/P2X3Dbl−/− mice learned to avoid a combination of SC45647 and amyl acetate (an odor cue). These data suggest that P2X2/P2X3Dbl−/− mice are unable to respond to NaCl or SC45647 as taste stimuli, mirroring the lack of gustatory nerve responses to these substances. PMID:19833661

  11. DNA Damage-Inducible Transcript 4 Is an Innate Surveillant of Hair Follicular Stress in Vitamin D Receptor Knockout Mice and a Regulator of Wound Re-Epithelialization

    Directory of Open Access Journals (Sweden)

    Hengguang Zhao

    2016-11-01

    Full Text Available Mice and human patients with impaired vitamin D receptor (VDR signaling have normal developmental hair growth but display aberrant post-morphogenic hair cycle progression associated with alopecia. In addition, VDR–/– mice exhibit impaired cutaneous wound healing. We undertook experiments to determine whether the stress-inducible regulator of energy homeostasis, DNA damage-inducible transcript 4 (Ddit4, is involved in these processes. By analyzing hair cycle activation in vivo, we show that VDR−/− mice at day 14 exhibit increased Ddit4 expression within follicular stress compartments. At day 29, degenerating VDR−/− follicular keratinocytes, but not bulge stem cells, continue to exhibit an increase in Ddit4 expression. At day 47, when normal follicles and epidermis are quiescent and enriched for Ddit4, VDR−/− skin lacks Ddit4 expression. In a skin wound healing assay, the re-epithelialized epidermis in wildtype (WT but not VDR−/− animals harbor a population of Ddit4- and Krt10-positive cells. Our study suggests that VDR regulates Ddit4 expression during epidermal homeostasis and the wound healing process, while elevated Ddit4 represents an early growth-arresting stress response within VDR−/− follicles.

  12. DNA Damage-Inducible Transcript 4 Is an Innate Surveillant of Hair Follicular Stress in Vitamin D Receptor Knockout Mice and a Regulator of Wound Re-Epithelialization.

    Science.gov (United States)

    Zhao, Hengguang; Rieger, Sandra; Abe, Koichiro; Hewison, Martin; Lisse, Thomas S

    2016-11-26

    Mice and human patients with impaired vitamin D receptor (VDR) signaling have normal developmental hair growth but display aberrant post-morphogenic hair cycle progression associated with alopecia. In addition, VDR -/- mice exhibit impaired cutaneous wound healing. We undertook experiments to determine whether the stress-inducible regulator of energy homeostasis, DNA damage-inducible transcript 4 (Ddit4), is involved in these processes. By analyzing hair cycle activation in vivo, we show that VDR -/- mice at day 14 exhibit increased Ddit4 expression within follicular stress compartments. At day 29, degenerating VDR -/- follicular keratinocytes, but not bulge stem cells, continue to exhibit an increase in Ddit4 expression. At day 47, when normal follicles and epidermis are quiescent and enriched for Ddit4, VDR -/- skin lacks Ddit4 expression. In a skin wound healing assay, the re-epithelialized epidermis in wildtype (WT) but not VDR -/- animals harbor a population of Ddit4- and Krt10-positive cells. Our study suggests that VDR regulates Ddit4 expression during epidermal homeostasis and the wound healing process, while elevated Ddit4 represents an early growth-arresting stress response within VDR -/- follicles.

  13. Gene knockout of the alpha6 subunit of the gamma-aminobutyric acid type A receptor: lack of effect on responses to ethanol, pentobarbital, and general anesthetics.

    Science.gov (United States)

    Homanics, G E; Ferguson, C; Quinlan, J J; Daggett, J; Snyder, K; Lagenaur, C; Mi, Z P; Wang, X H; Grayson, D R; Firestone, L L

    1997-04-01

    The alpha6 subunit of the gamma-aminobutyric acid type A receptor (GABA(A)-R) has been implicated in mediating the intoxicating effects of ethanol and the motor ataxic effects of general anesthetics. To test this hypothesis, we used gene targeting in embryonic stem cells to create mice lacking a functional alpha6 gene. Homozygous mice are viable and fertile and have grossly normal cerebellar cytoarchitecture. Northern blot and reverse transcriptase-polymerase chain reaction analyses demonstrated that the targeting event disrupted production of functional alpha6 mRNA. Autoradiography of histological sections of adult brains demonstrated that diazepam-insensitive binding of [3H]Ro15-4513 to the cerebellar granule cell layer of wild-type mice was completely absent in homozygous mice. Cerebellar GABA(A)-R density was unchanged in the mutant mice; however, the apparent affinity for muscimol was markedly reduced. Sleep time response to injection of ethanol after pretreatment with vehicle or Ro15-4513 did not differ between genotypes. Sleep time response to injection of pentobarbital and loss of righting reflex and response to tail clamp stimulus in mice anesthetized with volatile anesthetics also did not differ between genotypes. Thus, the alpha6 subunit of the GABA(A)-R is not required for normal development, viability, and fertility and does not seem to be a critical or unique component of the neuronal pathway mediating the hypnotic effect of ethanol and its antagonism by Ro15-4513 in mice. Similarly, the alpha6 subunit does not seem to be involved in the behavioral responses to general anesthetics or pentobarbital.

  14. Effects of chemokine receptor signalling on cognition-like, emotion-like and sociability behaviours of CCR6 and CCR7 knockout mice.

    Science.gov (United States)

    Jaehne, E J; Baune, B T

    2014-03-15

    Inflammation is regarded as an important mechanism of neuropsychiatric disorders. Chemokines, which are a part of the immune system, have effects on various aspects of brain function, but little is known about their effects on behaviour. We have compared the cognition-like behaviour (learning and spatial memory) of CCR6(-/-) and CCR7(-/-) mice with wild type (WT) C57BL/6 mice, in the Barnes maze, as well as a range of other behaviours, including exploratory, anxiety and depression-like behaviour, using a battery of tests. Levels of cytokines TNF-α, IL-1β and IL-6 were also measured. In the Barnes maze, CCR7(-/-) mice were shown to take longer to learn the location of the escape box on the 1st of 4 days of training. In the behavioural battery, CCR6(-/-) mice showed higher locomotor activity and lower anxiety in the open field test, and a lack of preference for social novelty in a sociability test. CCR7(-/-) mice behaved much like WT mice, although showed higher anxiety in Elevated Zero Maze. While baseline saccharin preference in a 2-bottle choice test, a test for anhedonia depression-like behaviour, was equal in all strains at baseline, weekly tests showed that both CCR6(-/-) and CCR7(-/-) mice developed a decreased preference for saccharin compared to WT over time. There were no differences between strains in any of the cytokines measured. These results suggest that chemokine receptors may play a role in cognition and learning behaviour, as well as anxiety and other behaviours, although the biological mechanisms are still unclear. Copyright © 2013 Elsevier B.V. All rights reserved.

  15. Immunological characterization of the aryl hydrocarbon receptor (AHR) knockout rat in the presence and absence of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)

    International Nuclear Information System (INIS)

    Phadnis-Moghe, Ashwini S.; Chen, Weimin; Li, Jinpeng; Crawford, Robert B.; Bach, Anthony; D’Ingillo, Shawna; Kovalova, Natalia; Suarez-Martinez, Jose E.; Kaplan, Barbara L.F.; Harrill, Joshua A.; Budinsky, Robert; Rowlands, J. Craig; Thomas, Russell S.

    2016-01-01

    The aryl hydrocarbon receptor (AHR) has been extensively characterized for the essential role it plays in mediating the toxic responses elicited by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Despite similarities across animal species, species-specific differences exist in the profile of toxicity and sensitivity to TCDD owing, in part, to differences in the AHR. Newer reports have implicated the importance of AHR in the development and regulation of the immune system. Our present studies seek to further explore the essential role of AHR in lymphoid tissue composition, B cell function and the immunological responses after TCDD administration using the recently established AHR KO rats. Comprehensive immune cell phenotyping showed a decrease in the CD8 + T cell, CD11c + populations and an increase in NKT cells in 3-week-old AHR KO rats compared to the WT controls. The lipopolysaccharide-induced IgM response and proliferation was markedly suppressed in the WT but not in the AHR KO B cells in the presence of TCDD. However, the percentage of LPS-activated IgM + B cells was significantly higher in the AHR KO B cells as compared to that of WT suggesting the role of AHR in regulating the IgM response. The use of an AHR antagonist further alluded to the endogenous role of AHR in regulating B cell responses in the rat. Overall, the studies report for the first time, comprehensive immune cell phenotyping of the AHR KO rat and the endogenous role of AHR in the regulation of B cell function in the rat.

  16. Akt2/LDLr double knockout mice display impaired glucose tolerance and develop more complex atherosclerotic plaques than LDLr knockout mice

    NARCIS (Netherlands)

    Rensing, Katrijn L.; de Jager, Saskia C. A.; Stroes, Erik S.; Vos, Mariska; Twickler, Marcel Th B.; Dallinga-Thie, Geesje M.; de Vries, Carlie J. M.; Kuiper, Johan; Bot, Ilze; von der Thüsen, Jan H.

    2014-01-01

    To characterize the phenotype of Akt2/low-density-lipoprotein receptor double knockout (dKO) (Akt2/LDLr dKO) mice with respect to insulin resistance and features of atherosclerotic plaque progression. Metabolic profile and atherosclerotic plaque progression were compared between LDLr KO mice and

  17. Inducible knockout of the interleukin-2 receptor alpha chain: expression of the high-affinity IL-2 receptor is not required for the in vitro growth of HTLV-I-transformed cell lines.

    Science.gov (United States)

    Richardson, J H; Waldmann, T A; Sodroski, J G; Marasco, W A

    1997-10-27

    Adult T cell leukemia (ATL) is an aggressive malignancy that is associated with HTLV-I infection and characterized by constitutive expression of the high-affinity interleukin-2 receptor. The alpha subunit of the high-affinity receptor (IL-2Ralpha), which is normally present only on activated T cells, is specifically upregulated by HTLV-I and constitutively expressed on fresh leukemic cells from ATL patients as well as cell lines transformed by HTLV-I in vitro. Here we directly address the functional significance of IL-2Ralpha expression in HTLV-I transformed cell lines by using an endoplasmic reticulum-targeted single-chain antibody to inhibit the cell surface expression of IL-2Ralpha. Using constitutive and tetracycline-repressible systems to express the ER-targeted antibody against IL-2Ralpha, we have reduced cell surface expression of IL-2Ralpha by more that 2 logs of mean fluorescence intensity to virtually undetectable levels in the IL-2-independent HTLV-I-transformed cell lines C8166-45 and HUT102. No toxicity was associated with the intracellular retention of IL-2Ralpha, and the growth rate of the IL-2Ralpha-negative cells was in each case comparable to that of the parental cell line. We conclude that cell surface expression of IL-2Ralpha is dispensable for the in vitro growth of these HTLV-I-transformed cells. Copyright 1997 Academic Press.

  18. Pharmacological treatment of fragile X syndrome with GABAergic drugs in a knockout mouse model

    NARCIS (Netherlands)

    Heulens, Inge; D'Hulst, Charlotte; Van Dam, Debby; De Deyn, Peter P.; Kooy, R. Frank

    2012-01-01

    Molecular and electrophysiological studies have provided evidence for a general downregulation of the GABAergic system in the Fmr1 knockout mouse. GABA(A) receptors are the main inhibitory receptors in the brain and the GABA(A) receptor was proposed as a novel target for treatment of the fragile X

  19. KnockoutJS web development

    CERN Document Server

    Farrar, John

    2015-01-01

    This book is for web developers and designers who work with HTML and JavaScript to help them manage data and interactivity with data using KnockoutJS. Knowledge about jQuery will be useful but is not necessary.

  20. Regulation of oxidative stress and somatostatin, cholecystokinin, apelin gene expressions by ghrelin in stomach of newborn diabetic rats.

    Science.gov (United States)

    Coskun, Zeynep Mine; Sacan, Ozlem; Karatug, Ayse; Turk, Neslihan; Yanardag, Refiye; Bolkent, Sehnaz; Bolkent, Sema

    2013-09-01

    The aim of the study was to determine whether ghrelin treatment has a protective effect on gene expression and biochemical changes in the stomach of newborn streptozotocin (STZ) induced diabetic rats. In this study, four groups of Wistar rats were used: control, ghrelin control, diabetic and diabetic+ghrelin. The rats were sacrificed after four weeks of treatment for diabetes. The gene expressions of: somatostatin, cholecystokinin, apelin and the altered active caspase-3, active caspase-8, proliferating cell nuclear antigen, were investigated in the pyloric region of the stomach and antioxidant parameters were measured in all the stomach. Although ghrelin treatment to diabetic rats lowered the stomach lipid peroxidation levels, the stomach glutathione levels were increased. Exogenous ghrelin caused an increased activities of stomach catalase, superoxide dismutase, glutathione reductase and glutathione peroxidase in diabetic rats. Numbers of somatostatin, cholecystokinin and proliferating cell nuclear antigen immunoreactive cells decreased in the diabetic+ghrelin group compared to the diabetic group. Apelin mRNA expressions were remarkably less in the diabetic+ghrelin rats than in diabetic rats. The results may indicate that ghrelin treatment has a protective effect to some extent on the diabetic rats. This protection is possibly accomplished through the antioxidant activity of ghrelin observed in type 2 diabetes. Consequently exogenous ghrelin may be a candidate for therapeutic treatment of diabetes. Copyright © 2013 Elsevier GmbH. All rights reserved.

  1. Changes in 5-HT2A-mediated behavior and 5-HT2A- and 5-HT1A receptor binding and expression in conditional brain-derived neurotrophic factor knock-out mice

    DEFF Research Database (Denmark)

    Klein, A B; Santini, M A; Aznar, S

    2010-01-01

    specific for the serotonin 2A receptor (5-HT(2A)R) in prefrontal cortex was described previously in these mice. This is of much interest, as 5-HT(2A)Rs have been linked to neuropsychiatric disorders and anxiety-related behavior. Here we further characterized the serotonin receptor alterations triggered...

  2. Somatostatin receptors

    DEFF Research Database (Denmark)

    Møller, Lars Neisig; Stidsen, Carsten Enggaard; Hartmann, Bolette

    2003-01-01

    therefore been acknowledged to be a third endogenous ligand at SRIF receptors. This review goes through mechanisms of signal transduction, pharmacology, and anatomical distribution of SRIF receptors. Structurally, SRIF receptors belong to the superfamily of G protein-coupled (GPC) receptors, sharing....... The generation of knock-out (KO) mice, intended as a means to define the contributions made by individual receptor subtypes, necessarily marks but an approximation. Furthermore, we must now take into account the stunning complexity of receptor co-operation indicated by the observation of receptor homo......-peptides, receptor agonists and antagonists. Relatively long half lives, as compared to those of the endogenous ligands, have been paramount from the outset. Motivated by theoretical puzzles or the shortcomings of present-day diagnostics and therapy, investigators have also aimed to produce subtype...

  3. Molecular cloning, genomic organization, developmental regulation, and a knock-out mutant of a novel leu-rich repeats-containing G protein-coupled receptor (DLGR-2) from Drosophila melanogaster

    DEFF Research Database (Denmark)

    Eriksen, Kathrine Krageskov; Hauser, Frank; Schiøtt, Morten

    2000-01-01

    After screening the Berkeley Drosophila Genome Project database with sequences from a recently characterized Leu-rich repeats-containing G protein-coupled receptor (LGR) fromDrosophila (DLGR-1), we identified a second gene for a different LGR (DLGR-2) and cloned its cDNA. DLGR-2 is 1360 amino acid...... LGRs (LGR-4 and LGR-5). This homology includes the seven transmembrane region (e.g., 49% amino acid identity with the human TSH receptor) and the very large extracellular amino terminus. This amino terminus contains 18 Leu-rich repeats-in contrast with the 3 mammalian glycoprotein hormone receptors...

  4. Changes in 5-HT2A-mediated behavior and 5-HT2A- and 5-HT1A receptor binding and expression in conditional BDNF knock-out mice

    OpenAIRE

    Klein, AB; Santini, MA; Aznar, S; Knudsen, GM; Rios, M

    2010-01-01

    Changes in brain-derived neurotrophic factor (BDNF) expression have been implicated in the etiology of psychiatric disorders. To investigate pathological mechanisms elicited by perturbed BDNF signaling, we examined mutant mice with central depletion of BDNF (BDNF2L/2LCk-cre). A severe impairment specific for the serotonin 2A receptor (5-HT2A) in prefrontal cortex was described previously in these mice. This is of much interest, as 5-HT2A receptors have been linked to neuropsychiatric disorder...

  5. The serotonin transporter knockout rat : A review

    NARCIS (Netherlands)

    Olivier, Jocelien; Cools, Alexander; Ellenbroek, Bart A.; Cuppen, E.; Homberg, Judith; Kalueff, Allan V.; LaPorte, Justin L.

    2010-01-01

    This chapter dicusses the most recent data on the serotonin transporter knock-out rat, a unique rat model that has been generated by target-selected N-ethyl-N-nitrosourea (ENU) driven mutagenesis. The knock-out rat is the result of a premature stopcodon in the serotonin transporter gene, and the

  6. Changes in 5-HT2A-mediated behavior and 5-HT2A- and 5-HT1A receptor binding and expression in conditional brain-derived neurotrophic factor knock-out mice

    DEFF Research Database (Denmark)

    Klein, A B; Santini, M A; Aznar, S

    2010-01-01

    Changes in brain-derived neurotrophic factor (BDNF) expression have been implicated in the etiology of psychiatric disorders. To investigate pathological mechanisms elicited by perturbed BDNF signaling, we examined mutant mice with central depletion of BDNF (BDNF(2L/2LCk-cre)). A severe impairment...... was decreased in hippocampus of BDNF mutants, but unchanged in frontal cortex. Molecular analysis indicated corresponding changes in 5-HT(2A) and 5-HT(1A) mRNA expression but normal 5-HT(2C) content in these brain regions in BDNF(2L/2LCk-cre) mice. We investigated whether the reduction in frontal 5-HT(2A......)R binding was reflected in reduced functional output in two 5-HT(2A)-receptor mediated behavioral tests, the head-twitch response (HTR) and the ear-scratch response (ESR). BDNF(2L/2LCk-cre) mutants treated with the 5-HT(2A) receptor agonist (+/-)-2,5-dimethoxy-4-iodoamphetamine (DOI) showed a clearly...

  7. The N-terminal domain of APJ, a CNS-based coreceptor for HIV-1, is essential for its receptor function and coreceptor activity

    International Nuclear Information System (INIS)

    Zhou Naiming; Zhang Xiaoling; Fan Xuejun; Argyris, Elias; Fang Jianhua; Acheampong, Edward; DuBois, Garrett C.; Pomerantz, Roger J.

    2003-01-01

    The human APJ, a G protein-coupled seven-transmembrane receptor, has been found to be dramatically expressed in the human central nervous system (CNS) and also to serve as a coreceptor for the entry of human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV). Studies with animal models suggested that APJ and its natural ligand, apelin, play an important role in the central control of body fluid homeostasis, and in regulation of blood pressure and cardiac contractility. In this study, we characterize the structural and functional determinants of the N-terminal domain of APJ in interactions with its natural ligand and HIV-1 envelope glycoprotein. We demonstrate that the second 10 residues of the N-terminal domain of APJ are critical for association with apelin, while the first 20 amino acids play an important role in supporting cell-cell fusion mediated by HIV-1 gp120. With site-directed mutagenesis, we have identified that the negatively charged amino acid residues Glu20 and Asp23 are involved in receptor and coreceptor functions, but residues Tyr10 and Tyr11 substantially contribute to coreceptor function for both T-tropic (CXCR4) and dual-tropic (CXCR4 and CCR5) HIV-1 isolates. Thus, this study provides potentially important information for further characterizing APJ-apelin functions in vitro and in vivo and designing small molecules for treatment of HIV-1 infection in the CNS

  8. The angiotensin converting enzyme inhibitor, captopril, prevents the hyperactivity and impulsivity of neurokinin-1 receptor gene 'knockout' mice: sex differences and implications for the treatment of attention deficit hyperactivity disorder.

    Science.gov (United States)

    Porter, Ashley J; Pillidge, Katharine; Grabowska, Ewelina M; Stanford, S Clare

    2015-04-01

    Mice lacking functional neurokinin-1 receptors (NK1R-/-) display behavioural abnormalities resembling attention deficit hyperactivity disorder (ADHD): locomotor hyperactivity, impulsivity and inattentiveness. The preferred ligand for NK1R, substance P, is metabolised by angiotensin converting enzyme (ACE), which forms part of the brain renin angiotensin system (BRAS). In view of evidence that the BRAS modulates locomotor activity and cognitive performance, we tested the effects of drugs that target the BRAS on these behaviours in NK1R-/- and wildtype mice. We first tested the effects of the ACE inhibitor, captopril, on locomotor activity. Because there are well-established sex differences in both ADHD and ACE activity, we compared the effects of captopril in both male and female mice. Locomotor hyperactivity was evident in male NK1R-/- mice, only, and this was abolished by treatment with captopril. By contrast, male wildtypes and females of both genotypes were unaffected by ACE inhibition. We then investigated the effects of angiotensin AT1 (losartan) and AT2 (PD 123319) receptor antagonists on the locomotor activity of male NK1R-/- and wildtype mice. Both antagonists increased the locomotor activity of NK1R-/- mice, but neither affected the wildtypes. Finally, we tested the effects of captopril on the performance of male NK1R-/- and wildtype mice in the 5-choice serial reaction-time task (5-CSRTT) and found that ACE inhibition prevented the impulsivity of NK1R-/- mice. These results indicate that certain behaviours, disrupted in ADHD, are influenced by an interaction between the BRAS and NK1R, and suggest that ACE inhibitors could provide a novel treatment for this disorder. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

  9. Phenotypic assessment of THC discriminative stimulus properties in fatty acid amide hydrolase knockout and wildtype mice.

    Science.gov (United States)

    Walentiny, D Matthew; Vann, Robert E; Wiley, Jenny L

    2015-06-01

    A number of studies have examined the ability of the endogenous cannabinoid anandamide to elicit Δ(9)-tetrahydrocannabinol (THC)-like subjective effects, as modeled through the THC discrimination paradigm. In the present study, we compared transgenic mice lacking fatty acid amide hydrolase (FAAH), the enzyme primarily responsible for anandamide catabolism, to wildtype counterparts in a THC discrimination procedure. THC (5.6 mg/kg) served as a discriminative stimulus in both genotypes, with similar THC dose-response curves between groups. Anandamide fully substituted for THC in FAAH knockout, but not wildtype, mice. Conversely, the metabolically stable anandamide analog O-1812 fully substituted in both groups, but was more potent in knockouts. The CB1 receptor antagonist rimonabant dose-dependently attenuated THC generalization in both groups and anandamide substitution in FAAH knockouts. Pharmacological inhibition of monoacylglycerol lipase (MAGL), the primary catabolic enzyme for the endocannabinoid 2-arachidonoylglycerol (2-AG), with JZL184 resulted in full substitution for THC in FAAH knockout mice and nearly full substitution in wildtypes. Quantification of brain endocannabinoid levels revealed expected elevations in anandamide in FAAH knockout mice compared to wildtypes and equipotent dose-dependent elevations in 2-AG following JZL184 administration. Dual inhibition of FAAH and MAGL with JZL195 resulted in roughly equipotent increases in THC-appropriate responding in both groups. While the notable similarity in THC's discriminative stimulus effects across genotype suggests that the increased baseline brain anandamide levels (as seen in FAAH knockout mice) do not alter THC's subjective effects, FAAH knockout mice are more sensitive to the THC-like effects of pharmacologically induced increases in anandamide and MAGL inhibition (e.g., JZL184). Copyright © 2015 Elsevier Ltd. All rights reserved.

  10. Knockout of endothelial cell-derived endothelin-1 attenuates skin fibrosis but accelerates cutaneous wound healing.

    Science.gov (United States)

    Makino, Katsunari; Jinnin, Masatoshi; Aoi, Jun; Kajihara, Ikko; Makino, Takamitsu; Fukushima, Satoshi; Sakai, Keisuke; Nakayama, Kazuhiko; Emoto, Noriaki; Yanagisawa, Masashi; Ihn, Hironobu

    2014-01-01

    Endothelin (ET)-1 is known for the most potent vasoconstrictive peptide that is released mainly from endothelial cells. Several studies have reported ET-1 signaling is involved in the process of wound healing or fibrosis as well as vasodilation. However, little is known about the role of ET-1 in these processes. To clarify its mechanism, we compared skin fibrogenesis and wound repair between vascular endothelial cell-specific ET-1 knockout mice and their wild-type littermates. Bleomycin-injected fibrotic skin of the knockout mice showed significantly decreased skin thickness and collagen content compared to that of wild-type mice, indicating that bleomycin-induced skin fibrosis is attenuated in the knockout mice. The mRNA levels of transforming growth factor (TGF)-β were decreased in the bleomycin-treated skin of ET-1 knockout mice. On the other hand, skin wound healing was accelerated in ET-1 knockout mice, which was indicated by earlier granulation tissue reduction and re-epithelialization in these mice. The mRNA levels of TGF-β, tumor necrosis factor (TNF)-α and connective tissue growth factor (CTGF) were reduced in the wound of ET-1 knockout mice. In endothelial ET-1 knockout mouse, the expression of TNF-α, CTGF and TGF-β was down-regulated. Bosentan, an antagonist of dual ET receptors, is known to attenuate skin fibrosis and accelerate wound healing in systemic sclerosis, and such contradictory effect may be mediated by above molecules. The endothelial cell-derived ET-1 is the potent therapeutic target in fibrosis or wound healing, and investigations of the overall regulatory mechanisms of these pathological conditions by ET-1 may lead to a new therapeutic approach.

  11. Knockout of endothelial cell-derived endothelin-1 attenuates skin fibrosis but accelerates cutaneous wound healing.

    Directory of Open Access Journals (Sweden)

    Katsunari Makino

    Full Text Available Endothelin (ET-1 is known for the most potent vasoconstrictive peptide that is released mainly from endothelial cells. Several studies have reported ET-1 signaling is involved in the process of wound healing or fibrosis as well as vasodilation. However, little is known about the role of ET-1 in these processes. To clarify its mechanism, we compared skin fibrogenesis and wound repair between vascular endothelial cell-specific ET-1 knockout mice and their wild-type littermates. Bleomycin-injected fibrotic skin of the knockout mice showed significantly decreased skin thickness and collagen content compared to that of wild-type mice, indicating that bleomycin-induced skin fibrosis is attenuated in the knockout mice. The mRNA levels of transforming growth factor (TGF-β were decreased in the bleomycin-treated skin of ET-1 knockout mice. On the other hand, skin wound healing was accelerated in ET-1 knockout mice, which was indicated by earlier granulation tissue reduction and re-epithelialization in these mice. The mRNA levels of TGF-β, tumor necrosis factor (TNF-α and connective tissue growth factor (CTGF were reduced in the wound of ET-1 knockout mice. In endothelial ET-1 knockout mouse, the expression of TNF-α, CTGF and TGF-β was down-regulated. Bosentan, an antagonist of dual ET receptors, is known to attenuate skin fibrosis and accelerate wound healing in systemic sclerosis, and such contradictory effect may be mediated by above molecules. The endothelial cell-derived ET-1 is the potent therapeutic target in fibrosis or wound healing, and investigations of the overall regulatory mechanisms of these pathological conditions by ET-1 may lead to a new therapeutic approach.

  12. The Knockout of Secretin in Cerebellar Purkinje Cells Impairs Mouse Motor Coordination and Motor Learning

    Science.gov (United States)

    Zhang, Li; Chung, Sookja Kim; Chow, Billy Kwok Chong

    2014-01-01

    Secretin (SCT) was first considered to be a gut hormone regulating gastrointestinal functions when discovered. Recently, however, central actions of SCT have drawn intense research interest and are supported by the broad distribution of SCT in specific neuronal populations and by in vivo physiological studies regarding its role in water homeostasis and food intake. The direct action of SCT on a central neuron was first discovered in cerebellar Purkinje cells in which SCT from cerebellar Purkinje cells was found to potentiate GABAergic inhibitory transmission from presynaptic basket cells. Because Purkinje neurons have a major role in motor coordination and learning functions, we hypothesize a behavioral modulatory function for SCT. In this study, we successfully generated a mouse model in which the SCT gene was deleted specifically in Purkinje cells. This mouse line was tested together with SCT knockout and SCT receptor knockout mice in a full battery of behavioral tasks. We found that the knockout of SCT in Purkinje neurons did not affect general motor ability or the anxiety level in open field tests. However, knockout mice did exhibit impairments in neuromuscular strength, motor coordination, and motor learning abilities, as shown by wire hanging, vertical climbing, and rotarod tests. In addition, SCT knockout in Purkinje cells possibly led to the delayed development of motor neurons, as supported by the later occurrence of key neural reflexes. In summary, our data suggest a role in motor coordination and motor learning for SCT expressed in cerebellar Purkinje cells. PMID:24356714

  13. receptores

    Directory of Open Access Journals (Sweden)

    Salete Regina Daronco Benetti

    2006-01-01

    Full Text Available Se trata de un estudio etnográfico, que tuvo lo objetivo de interpretar el sistema de conocimiento y del significado atribuidos a la sangre referente a la transfusión sanguínea por los donadores y receptores de un banco de sangre. Para la colecta de las informaciones se observaron los participantes y la entrevista etnográfica se realizó el análisis de dominio, taxonómicos y temáticos. Los dominios culturales fueron: la sangre es vida: fuente de vida y alimento valioso; creencias religiosas: fuentes simbólicas de apoyos; donación sanguínea: un gesto colaborador que exige cuidarse, gratifica y trae felicidad; donación sanguínea: fuente simbólica de inseguridad; estar enfermo es una condición para realizar transfusión sanguínea; transfusión sanguínea: esperanza de vida; Creencias populares: transfusión sanguínea como riesgo para la salud; donadores de sangre: personas benditas; donar y recibir sangre: como significado de felicidad. Temática: “líquido precioso que origina, sostiene, modifica la vida, provoca miedo e inseguridad”.

  14. Patched Knockout Mouse Models of Basal Cell Carcinoma

    Directory of Open Access Journals (Sweden)

    Frauke Nitzki

    2012-01-01

    Full Text Available Basal cell carcinoma (BCC is the most common human tumor. Mutations in the hedgehog (HH receptor Patched (PTCH are the main cause of BCC. Due to their high and increasing incidence, BCC are becoming all the more important for the health care system. Adequate animal models are required for the improvement of current treatment strategies. A good model should reflect the situation in humans (i.e., BCC initiation due to Ptch mutations on an immunocompetent background and should allow for (i BCC induction at a defined time point, (ii analysis of defined BCC stages, and (iii induction of BCC in 100% of animals. In addition, it should be easy to handle. Here, we compare several currently existing conventional and conditional Ptch knockout mouse models for BCC and their potential use in preclinical research. In addition, we provide new data using conditional Ptchflox/flox mice and the K5-Cre-ERT+/− driver.

  15. Nucleon knockout: off-shell effects

    International Nuclear Information System (INIS)

    Stephenson, G.J. Jr.

    1977-01-01

    The effect of the off-energy-shell extrapolation of the proton-proton scattering amplitude on the analysis of (p,2p) reactions is discussed. In particular, the range of expected variations in this extrapolation is explored and the possibility of using knock-out reactions to limit models of the p-p amplitude is studied

  16. Hydrocarbon Receptor Pathway in Dogs

    NARCIS (Netherlands)

    Steenbeek, F.G. van; Spee, B.; Penning, L.C.; Kummeling, A.; Gils, I.H.M.; Grinwis, G.C.M.; Leenen, D. van; Holstege, F.C.P.; Vos-Loohuis, M.; Rothuizen, J.; Leegwater, P.A.J.

    The aryl hydrocarbon receptor (AHR) mediates biological responses to toxic chemicals. An unexpected role for AHR in vascularization was suggested when mice lacking AHR displayed impaired closure of the ductus venosus after birth, as did knockout mice for aryl hydrocarbon receptor interacting

  17. Acute secondhand smoke-induced pulmonary inflammation is diminished in RAGE knockout mice.

    Science.gov (United States)

    Wood, Tyler T; Winden, Duane R; Marlor, Derek R; Wright, Alex J; Jones, Cameron M; Chavarria, Michael; Rogers, Geraldine D; Reynolds, Paul R

    2014-11-15

    The receptor for advanced glycation end-products (RAGE) has increasingly been demonstrated to be an important modulator of inflammation in cases of pulmonary disease. Published reports involving tobacco smoke exposure have demonstrated increased expression of RAGE, its participation in proinflammatory signaling, and its role in irreversible pulmonary remodeling. The current research evaluated the in vivo effects of short-term secondhand smoke (SHS) exposure in RAGE knockout and control mice compared with identical animals exposed to room air only. Quantitative PCR, immunoblotting, and immunohistochemistry revealed elevated RAGE expression in controls after 4 wk of SHS exposure and an anticipated absence of RAGE expression in RAGE knockout mice regardless of smoke exposure. Ras activation, NF-κB activity, and cytokine elaboration were assessed to characterize the molecular basis of SHS-induced inflammation in the mouse lung. Furthermore, bronchoalveolar lavage fluid was procured from RAGE knockout and control animals for the assessment of inflammatory cells and molecules. As a general theme, inflammation coincident with leukocyte recruitment was induced by SHS exposure and significantly influenced by the availability of RAGE. These data reveal captivating information suggesting a role for RAGE signaling in lungs exposed to SHS. However, ongoing research is still warranted to fully explain roles for RAGE and other receptors in cells coping with involuntary smoke exposure for prolonged periods of time. Copyright © 2014 the American Physiological Society.

  18. Generation of ERα-floxed and knockout mice using the Cre/LoxP system

    International Nuclear Information System (INIS)

    Antonson, P.; Omoto, Y.; Humire, P.; Gustafsson, J.-Å.

    2012-01-01

    Highlights: ► ERα floxed and knockout mice were generated. ► Disruption of the ERα gene results in sterility in both male and female mice. ► ERα −/− mice have ovaries with hemorrhagic follicles and hypoplastic uterus. ► Female ERα −/− mice develop obesity. -- Abstract: Estrogen receptor alpha (ERα) is a nuclear receptor that regulates a range of physiological processes in response to estrogens. In order to study its biological role, we generated a floxed ERα mouse line that can be used to knock out ERα in selected tissues by using the Cre/LoxP system. In this study, we established a new ERα knockout mouse line by crossing the floxed ERα mice with Cre deleter mice. Here we show that genetic disruption of the ERα gene in all tissues results in sterility in both male and female mice. Histological examination of uterus and ovaries revealed a dramatically atrophic uterus and hemorrhagic cysts in the ovary. These results suggest that infertility in female mice is the result of functional defects of the reproductive tract. Moreover, female knockout mice are hyperglycemic, develop obesity and at the age of 4 months the body weight of these mice was more than 20% higher compared to wild type littermates and this difference increased over time. Our results demonstrate that ERα is necessary for reproductive tract development and has important functions as a regulator of metabolism in females.

  19. Universal statistics of the knockout tournament

    Science.gov (United States)

    Baek, Seung Ki; Yi, Il Gu; Park, Hye Jin; Kim, Beom Jun

    2013-11-01

    We study statistics of the knockout tournament, where only the winner of a fixture progresses to the next. We assign a real number called competitiveness to each contestant and find that the resulting distribution of prize money follows a power law with an exponent close to unity if the competitiveness is a stable quantity and a decisive factor to win a match. Otherwise, the distribution is found narrow. The existing observation of power law distributions in various kinds of real sports tournaments therefore suggests that the rules of those games are constructed in such a way that it is possible to understand the games in terms of the contestants' inherent characteristics of competitiveness.

  20. High-throughput knockout screen in fission yeast

    OpenAIRE

    Gregan, Juraj; Rabitsch, Peter K; Rumpf, Cornelia; Novatchkova, Maria; Schleiffer, Alexander; Nasmyth, Kim

    2006-01-01

    We have designed the most efficient strategy to knock out genes in fission yeast Schizosaccharomyces pombe on a large scale. Our technique is based on knockout constructs that contain regions homologous to the target gene cloned into vectors carrying dominant drug-resistance markers. Most of the steps are carried out in a 96-well format, allowing simultaneous deletion of 96 genes in one batch. Based on our knockout technique, we designed a strategy for cloning knockout constructs for all pred...

  1. New insight into the role of the β3 subunit of the GABAA-R in development, behavior, body weight regulation, and anesthesia revealed by conditional gene knockout

    Directory of Open Access Journals (Sweden)

    Hileman Stanley M

    2007-10-01

    Full Text Available Abstract Background The β3 subunit of the γ-aminobutyric acid type A receptor (GABAA-R has been reported to be important for palate formation, anesthetic action, and normal nervous system function. This subunit has also been implicated in the pathogenesis of Angelman syndrome and autism spectrum disorder. To further investigate involvement of this subunit, we previously produced mice with a global knockout of β3. However, developmental abnormalities, compensation, reduced viability, and numerous behavioral abnormalities limited the usefulness of that murine model. To overcome many of these limitations, a mouse line with a conditionally inactivated β3 gene was engineered. Results Gene targeting and embryonic stem cell technologies were used to create mice in which exon 3 of the β3 subunit was flanked by loxP sites (i.e., floxed. Crossing the floxed β3 mice to a cre general deleter mouse line reproduced the phenotype of the previously described global knockout. Pan-neuronal knockout of β3 was achieved by crossing floxed β3 mice to Synapsin I-cre transgenic mice. Palate development was normal in pan-neuronal β3 knockouts but ~61% died as neonates. Survivors were overtly normal, fertile, and were less sensitive to etomidate. Forebrain selective knockout of β3 was achieved using α CamKII-cre transgenic mice. Palate development was normal in forebrain selective β3 knockout mice. These knockouts survived the neonatal period, but ~30% died between 15–25 days of age. Survivors had reduced reproductive fitness, reduced sensitivity to etomidate, were hyperactive, and some became obese. Conclusion Conditional inactivation of the β3 gene revealed novel insight into the function of this GABAA-R subunit. The floxed β3 knockout mice described here will be very useful for conditional knockout studies to further investigate the role of the β3 subunit in development, ethanol and anesthetic action, normal physiology, and pathophysiologic processes.

  2. Pion-induced knock-out reactions

    International Nuclear Information System (INIS)

    Jain, B.K.; Phatak, S.C.

    1977-01-01

    A strong absorption model for pion-induced Knock-out reactions is proposed. The distortion of the in-coming and out-going pions has been included by (1) computing pion wave number in nuclear medium (dispersive effect) and (2) excluding the central region of the nucleus where the real pion-absorption is dominant (absorption effect). In order to study the dependence of the (π + π + p) reaction on the off-shell pion-nucleon t-matrix, different off-shell extrapolations are used. The magnitude of the cross-sections seems to be sensitive to the type of off-shell extrapolation; their shapes, however, are similar. The theoretical results are compared with experimental data. The agreement between the theoretical results for separable off-shell extrapolation and the data is good. (author)

  3. Altered Sleep Homeostasis in Rev-erbα Knockout Mice

    Science.gov (United States)

    Mang, Géraldine M.; La Spada, Francesco; Emmenegger, Yann; Chappuis, Sylvie; Ripperger, Jürgen A.; Albrecht, Urs; Franken, Paul

    2016-01-01

    Study Objectives: The nuclear receptor REV-ERBα is a potent, constitutive transcriptional repressor critical for the regulation of key circadian and metabolic genes. Recently, REV-ERBα's involvement in learning, neurogenesis, mood, and dopamine turnover was demonstrated suggesting a specific role in central nervous system functioning. We have previously shown that the brain expression of several core clock genes, including Rev-erbα, is modulated by sleep loss. We here test the consequences of a loss of REV-ERBα on the homeostatic regulation of sleep. Methods: EEG/EMG signals were recorded in Rev-erbα knockout (KO) mice and their wild type (WT) littermates during baseline, sleep deprivation, and recovery. Cortical gene expression measurements after sleep deprivation were contrasted to baseline. Results: Although baseline sleep/wake duration was remarkably similar, KO mice showed an advance of the sleep/wake distribution relative to the light-dark cycle. After sleep onset in baseline and after sleep deprivation, both EEG delta power (1–4 Hz) and sleep consolidation were reduced in KO mice indicating a slower increase of homeostatic sleep need during wakefulness. This slower increase might relate to the smaller increase in theta and gamma power observed in the waking EEG prior to sleep onset under both conditions. Indeed, the increased theta activity during wakefulness predicted delta power in subsequent NREM sleep. Lack of Rev-erbα increased Bmal1, Npas2, Clock, and Fabp7 expression, confirming the direct regulation of these genes by REV-ERBα also in the brain. Conclusions: Our results add further proof to the notion that clock genes are involved in sleep homeostasis. Because accumulating evidence directly links REV-ERBα to dopamine signaling the altered homeostatic regulation of sleep reported here are discussed in that context. Citation: Mang GM, La Spada F, Emmenegger Y, Chappuis S, Ripperger JA, Albrecht U, Franken P. Altered sleep homeostasis in Rev

  4. Interrater agreement of an observational tool to code knockouts and technical knockouts in mixed martial arts.

    Science.gov (United States)

    Lawrence, David W; Hutchison, Michael G; Cusimano, Michael D; Singh, Tanveer; Li, Luke

    2014-09-01

    Interrater agreement evaluation of a tool to document and code the situational factors and mechanisms of knockouts (KOs) and technical knockouts (TKOs) in mixed martial arts (MMA). Retrospective case series. Professional MMA matches from the Ultimate Fighting Championship-2006-2012. Two nonmedically trained independent raters. The MMA Knockout Tool (MMA-KT) consists of 20 factors and captures and codes information on match characteristics, situational context preceding KOs and TKOs, as well as describing competitor states during these outcomes. The MMA-KT also evaluates the mechanism of action and subsequent events surrounding a KO. The 2 raters coded 125 unique events for a total of 250 events. The 8 factors of Part A had an average κ of 0.87 (SD = 0.10; range = 0.65-0.98); 7 were considered "substantial" agreement and 1 "moderate." Part B consists of 12 factors with an average κ of 0.84 (SD = 0.16; range = 0.59-1.0); 7 classified as "substantial" agreement, 4 "moderate," and 1 "fair." The majority of the factors in the MMA-KT demonstrated substantial interrater agreement, with an average κ of 0.86 (SD = 0.13; range = 0.59-1.0). The MMA-KT is a reliable tool to extract and code relevant information to investigate the situational factors and mechanism of KOs and TKOs in MMA competitions.

  5. Knockout of targeted gene in porcine somatic cells using zinc-finger nuclease.

    Science.gov (United States)

    Hisamatsu, Shin; Sakaue, Motoharu; Takizawa, Akiko; Kato, Tsubasa; Kamoshita, Maki; Ito, Junya; Kashiwazaki, Naomi

    2015-02-01

    Targeted genome editing is a widely applicable approach for efficiently modifying any sequence of interest in animals. It is very difficult to generate knock-out and knock-in animals except for mice up to now. Very recently, a method of genome editing using zinc-finger nucleases (ZFNs) has been developed to produce knockout rats. Since only injection of ZFNs into the pronuclear (PN) embryo is required, it seems to be useful for generating gene-targeted animals, including domestic species. However, no one has reported the successful production of knockout pigs by direct injection of ZFNs into PN embryos. We examined whether ZFN works on editing the genome of porcine growth hormone receptor in two kinds of cell lines (ST and PT-K75) derived from the pig as a preliminary study. Our data showed that pZFN1/2 vectors were efficiently transfected into both ST and PT-K75 cells. In both cell lines, results from Cel-I assay showed that modification of the targeted gene was confirmed. We injected ZFN1/2 mRNAs into the nucleus of PN stage embryos and then they were transferred to the recipients. However, pups were not delivered. Taken together, ZFN can be an available technology of genome editing even in the pig but further improvement will be required for generating genome-modified pigs. © 2014 Japanese Society of Animal Science.

  6. ATP Synthase β-Chain Overexpression in SR-BI Knockout Mice Increases HDL Uptake and Reduces Plasma HDL Level

    Directory of Open Access Journals (Sweden)

    Kexiu Song

    2014-01-01

    Full Text Available HDL cholesterol is known to be inversely correlated with cardiovascular disease due to its diverse antiatherogenic functions. SR-BI mediates the selective uptake of HDL-C. SR-BI knockout diminishes but does not completely block the transport of HDL; other receptors may be involved. Ectopic ATP synthase β-chain in hepatocytes has been previously characterized as an apoA-I receptor, triggering HDL internalization. This study was undertaken to identify the overexpression of ectopic ATP synthase β-chain on DIL-HDL uptake in primary hepatocytes in vitro and on plasma HDL levels in SR-BI knockout mice. Human ATP synthase β-chain cDNA was delivered to the mouse liver by adenovirus and GFP adenovirus as control. The adenovirus-mediated overexpression of β-chain was identified at both mRNA and protein levels on mice liver and validated by its increasing of DiL-HDL uptake in primary hepatocytes. In response to hepatic overexpression of β-chain, plasma HDL-C levels and cholesterol were reduced in SR-BI knockout mice, compared with the control. The present data suggest that ATP synthase β-chain can serve as the endocytic receptor of HDL, and its overexpression can reduce plasma HDL-C.

  7. The Effect of 8 Weeks of Aerobic Training and Consumption of Hydro-alcoholic Extract of Nettle on Apelin and hs-CRP plasma Levels of Overweight and Obese Women

    Directory of Open Access Journals (Sweden)

    M Madadi Jaberi

    2016-12-01

    Full Text Available Background and aim: The use of exercise along with herbal supplements is one method proposed for controlling obesity and its complications. The aim of this study was to investigate the effect of 8 weeks aerobic training and use of hydro-alcoholic extract of nettle on levels apelin and hs-CRP plasma in overweight and obese women. Methods: The present quasi-experimental study was conducted with blind randomized clinical trial. 46 overweight and obese women (body mass index greater than 25 kilograms per square millimeter two, aged 25-45 years were selected purposefully and randomly divided into four groups of: aerobic training + hydro alcoholic extract of nettle, aerobic exercise + placebo extract of nettle and placebo. The intervention group and placebo received 8 mg of hydro alcoholic extract of nettle 8 ml of water-soluble daily for 8 weeks respectively. Aerobic exercise ergometer for 8 weeks, 3 sessions of 16 to 30 minutes with the intensity of 60-75% heart rate was reserved. In two pre and post-test after 14 hours of fasting at the same conditions, blood samples were collected. The ELISA method was use to assess levels of plasma apelin and hs-CRP d. Data obtained were analyzed using the Kolmogorov-Smirnov test, ANOVA, t-test and LSD test. Results: The results showed that the levels of hs-CRP were significantly different in comparison among the groups as well as in groups of aerobic exercise + hydro alcoholic extract of nettle, nettle and hydro-alcholic aerobic exercise + placebo significant reduction was observed (p>0.05. Conclusion: It seems that consumption of Nettle extract along with aerobic exercise through Weight loss, body fat percentage and BMI, play an effective role in control of obesity and reducing of inflammatory Apelin markers and hs-CRP in obese women

  8. Common arterial trunk and ventricular non-compaction in Lrp2 knockout mice indicate a crucial role of LRP2 in cardiac development

    NARCIS (Netherlands)

    T. Baardman (Taco); M.V. Zwier (Mathijs V.); L.J. Wisse (Lambertus); A.C. Gittenberger-De Groot (Adriana); W.S. Kerstjens-Frederikse (Wilhelmina); R.M.W. Hofstra (Robert); A. Jurdzinski (Angelika); B.P. Hierck (Beerend); M.R.M. Jongbloed (Monique); R.M.F. Berger (Rolf); T. Plösch (Torsten); M.C. DeRuiter (Marco)

    2016-01-01

    textabstractLipoprotein-related receptor protein 2 (LRP2) is important for development of the embryonic neural crest and brain in both mice and humans. Although a role in cardiovascular development can be expected, the hearts of Lrp2 knockout (KO) mice have not yet been investigated. We studied the

  9. Common arterial trunk and ventricular non-compaction in Lrp2 knockout mice indicate a crucial role of LRP2 in cardiac development

    NARCIS (Netherlands)

    Baardman, Maria E.; Zwier, Mathijs V.; Wisse, Lambertus J.; Gittenberger-de Groot, Adriana C.; Kerstjens-Frederikse, Wilhelmina S.; Hofstra, Robert M. W.; Jurdzinski, Angelika; Hierck, Beerend P.; Jongbloed, Monique R. M.; Berger, Rolf M. F.; Plosch, Torsten; DeRuiter, Marco C.

    2016-01-01

    Lipoprotein-related receptor protein 2 (LRP2) is important for development of the embryonic neural crest and brain in both mice and humans. Although a role in cardiovascular development can be expected, the hearts of Lrp2 knockout (KO) mice have not yet been investigated. We studied the

  10. Differential suppression of seizures via Y2 and Y5 neuropeptide Y receptors

    DEFF Research Database (Denmark)

    Woldbye, David P D; Nanobashvili, Avtandil; Sørensen, Andreas Vehus

    2005-01-01

    Neuropeptide Y (NPY) prominently inhibits epileptic seizures in different animal models. The NPY receptors mediating this effect remain controversial partially due to lack of highly selective agonists and antagonists. To circumvent this problem, we used various NPY receptor knockout mice with the......, while activation of Y5 receptors in extra-hippocampal regions reduces generalized seizures in vivo.......Neuropeptide Y (NPY) prominently inhibits epileptic seizures in different animal models. The NPY receptors mediating this effect remain controversial partially due to lack of highly selective agonists and antagonists. To circumvent this problem, we used various NPY receptor knockout mice...... with the same genetic background and explored anti-epileptic action of NPY in vitro and in vivo. In Y2 (Y2-/-) and Y5 (Y5-/-) receptor knockouts, NPY partially inhibited 0 Mg2+-induced epileptiform activity in hippocampal slices. In contrast, in double knockouts (Y2Y5-/-), NPY had no effect, suggesting...

  11. Sleep in Kcna2 knockout mice

    Directory of Open Access Journals (Sweden)

    Messing Albee

    2007-10-01

    Full Text Available Abstract Background Shaker codes for a Drosophila voltage-dependent potassium channel. Flies carrying Shaker null or hypomorphic mutations sleep 3–4 h/day instead of 8–14 h/day as their wild-type siblings do. Shaker-like channels are conserved across species but it is unknown whether they affect sleep in mammals. To address this issue, we studied sleep in Kcna2 knockout (KO mice. Kcna2 codes for Kv1.2, the alpha subunit of a Shaker-like voltage-dependent potassium channel with high expression in the mammalian thalamocortical system. Results Continuous (24 h electroencephalograph (EEG, electromyogram (EMG, and video recordings were used to measure sleep and waking in Kcna2 KO, heterozygous (HZ and wild-type (WT pups (P17 and HZ and WT adult mice (P67. Sleep stages were scored visually based on 4-s epochs. EEG power spectra (0–20 Hz were calculated on consecutive 4-s epochs. KO pups die by P28 due to generalized seizures. At P17 seizures are either absent or very rare in KO pups ( Conclusion Kv1.2, a mammalian homologue of Shaker, regulates neuronal excitability and affects NREM sleep.

  12. Generation of ER{alpha}-floxed and knockout mice using the Cre/LoxP system

    Energy Technology Data Exchange (ETDEWEB)

    Antonson, P., E-mail: per.antonson@ki.se [Department of Biosciences and Nutrition, Karolinska Institutet, Novum, SE-141 83 Huddinge (Sweden); Omoto, Y.; Humire, P. [Department of Biosciences and Nutrition, Karolinska Institutet, Novum, SE-141 83 Huddinge (Sweden); Gustafsson, J.-A. [Department of Biosciences and Nutrition, Karolinska Institutet, Novum, SE-141 83 Huddinge (Sweden); Center for Nuclear Receptors and Cell Signaling, Department of Biology and Biochemistry, University of Houston, Houston, TX 77204 (United States)

    2012-08-10

    Highlights: Black-Right-Pointing-Pointer ER{alpha} floxed and knockout mice were generated. Black-Right-Pointing-Pointer Disruption of the ER{alpha} gene results in sterility in both male and female mice. Black-Right-Pointing-Pointer ER{alpha}{sup -/-} mice have ovaries with hemorrhagic follicles and hypoplastic uterus. Black-Right-Pointing-Pointer Female ER{alpha}{sup -/-} mice develop obesity. -- Abstract: Estrogen receptor alpha (ER{alpha}) is a nuclear receptor that regulates a range of physiological processes in response to estrogens. In order to study its biological role, we generated a floxed ER{alpha} mouse line that can be used to knock out ER{alpha} in selected tissues by using the Cre/LoxP system. In this study, we established a new ER{alpha} knockout mouse line by crossing the floxed ER{alpha} mice with Cre deleter mice. Here we show that genetic disruption of the ER{alpha} gene in all tissues results in sterility in both male and female mice. Histological examination of uterus and ovaries revealed a dramatically atrophic uterus and hemorrhagic cysts in the ovary. These results suggest that infertility in female mice is the result of functional defects of the reproductive tract. Moreover, female knockout mice are hyperglycemic, develop obesity and at the age of 4 months the body weight of these mice was more than 20% higher compared to wild type littermates and this difference increased over time. Our results demonstrate that ER{alpha} is necessary for reproductive tract development and has important functions as a regulator of metabolism in females.

  13. Altered Sleep Homeostasis in Rev-erbα Knockout Mice.

    Science.gov (United States)

    Mang, Géraldine M; La Spada, Francesco; Emmenegger, Yann; Chappuis, Sylvie; Ripperger, Jürgen A; Albrecht, Urs; Franken, Paul

    2016-03-01

    The nuclear receptor REV-ERBα is a potent, constitutive transcriptional repressor critical for the regulation of key circadian and metabolic genes. Recently, REV-ERBα's involvement in learning, neurogenesis, mood, and dopamine turnover was demonstrated suggesting a specific role in central nervous system functioning. We have previously shown that the brain expression of several core clock genes, including Rev-erbα, is modulated by sleep loss. We here test the consequences of a loss of REV-ERBα on the homeostatic regulation of sleep. EEG/EMG signals were recorded in Rev-erbα knockout (KO) mice and their wild type (WT) littermates during baseline, sleep deprivation, and recovery. Cortical gene expression measurements after sleep deprivation were contrasted to baseline. Although baseline sleep/wake duration was remarkably similar, KO mice showed an advance of the sleep/wake distribution relative to the light-dark cycle. After sleep onset in baseline and after sleep deprivation, both EEG delta power (1-4 Hz) and sleep consolidation were reduced in KO mice indicating a slower increase of homeostatic sleep need during wakefulness. This slower increase might relate to the smaller increase in theta and gamma power observed in the waking EEG prior to sleep onset under both conditions. Indeed, the increased theta activity during wakefulness predicted delta power in subsequent NREM sleep. Lack of Rev-erbα increased Bmal1, Npas2, Clock, and Fabp7 expression, confirming the direct regulation of these genes by REV-ERBα also in the brain. Our results add further proof to the notion that clock genes are involved in sleep homeostasis. Because accumulating evidence directly links REV-ERBα to dopamine signaling the altered homeostatic regulation of sleep reported here are discussed in that context. © 2016 Associated Professional Sleep Societies, LLC.

  14. Salty taste deficits in CALHM1 knockout mice.

    Science.gov (United States)

    Tordoff, Michael G; Ellis, Hillary T; Aleman, Tiffany R; Downing, Arnelle; Marambaud, Philippe; Foskett, J Kevin; Dana, Rachel M; McCaughey, Stuart A

    2014-07-01

    Genetic ablation of calcium homeostasis modulator 1 (CALHM1), which releases adenosine triphosphate from Type 2 taste cells, severely compromises the behavioral and electrophysiological responses to tastes detected by G protein-coupled receptors, such as sweet and bitter. However, the contribution of CALHM1 to salty taste perception is less clear. Here, we evaluated several salty taste-related phenotypes of CALHM1 knockout (KO) mice and their wild-type (WT) controls: 1) In a conditioned aversion test, CALHM1 WT and KO mice had similar NaCl avoidance thresholds. 2) In two-bottle choice tests, CALHM1 WT mice showed the classic inverted U-shaped NaCl concentration-preference function but CALHM1 KO mice had a blunted peak response. 3) In brief-access tests, CALHM1 KO mice showed less avoidance than did WT mice of high concentrations of NaCl, KCl, NH(4)Cl, and sodium lactate (NaLac). Amiloride further ameliorated the NaCl avoidance of CALHM1 KO mice, so that lick rates to a mixture of 1000 mM NaCl + 10 µM amiloride were statistically indistinguishable from those to water. 4) Relative to WT mice, CALHM1 KO mice had reduced chorda tympani nerve activity elicited by oral application of NaCl, NaLac, and sucrose but normal responses to HCl and NH(4)Cl. Chorda tympani responses to NaCl and NaLac were amiloride sensitive in WT but not KO mice. These results reinforce others demonstrating that multiple transduction pathways make complex, concentration-dependent contributions to salty taste perception. One of these pathways depends on CALHM1 to detect hypertonic NaCl in the mouth and signal the aversive taste of concentrated salt. © The Author 2014. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  15. Behavioral analysis of NR2C knockout mouse reveals deficit in acquisition of conditioned fear and working memory.

    Science.gov (United States)

    Hillman, Brandon G; Gupta, Subhash C; Stairs, Dustin J; Buonanno, Andres; Dravid, Shashank M

    2011-05-01

    N-methyl-D-aspartate (NMDA) receptors play an important role in excitatory neurotransmission and mediate synaptic plasticity associated with learning and memory. NMDA receptors are composed of two NR1 and two NR2 subunits and the identity of the NR2 subunit confers unique electrophysiologic and pharmacologic properties to the receptor. The precise role of NR2C-containing receptors in vivo is poorly understood. We have performed a battery of behavioral tests on NR2C knockout/nβ-galactosidase knock-in mice and found no difference in spontaneous activity, basal anxiety, forced-swim immobility, novel object recognition, pain sensitivity and reference memory in comparison to wildtype counterparts. However, NR2C knockout mice were found to exhibit deficits in fear acquisition and working memory compared to wildtype mice. Deficit in fear acquisition correlated with lack of fear conditioning-induced plasticity at the thalamo-amygdala synapse. These findings suggest a unique role of NR2C-containing receptors in associative and executive learning representing a novel therapeutic target for deficits in cognition. Copyright © 2011 Elsevier Inc. All rights reserved.

  16. Interactions of the opioid and cannabinoid systems in reward: Insights from knockout studies

    Directory of Open Access Journals (Sweden)

    Katia eBefort

    2015-02-01

    Full Text Available The opioid system consists of three receptors, mu, delta, and kappa, which are activated by endogenous opioid peptides (enkephalins, endorphins and dynorphins. The endogenous cannabinoid system comprises lipid neuromodulators (endocannabinoids, enzymes for their synthesis and their degradation and two well-characterized receptors, cannabinoid receptors CB1 and CB2. These systems play a major role in the control of pain as well as in mood regulation, reward processing and the development of addiction. Both opioid and cannabinoid receptors are coupled to G proteins and are expressed throughout the brain reinforcement circuitry. Extending classical pharmacology, research using genetically modified mice has provided important progress in the identification of the specific contribution of each component of these endogenous systems in vivo on reward process. This review will summarize available genetic tools and our present knowledge on the consequences of gene knockout on reinforced behaviors in both systems, with a focus on their potential interactions. A better understanding of opioid-cannabinoid interactions may provide novel strategies for therapies in addicted individuals.

  17. High-throughput knockout screen in fission yeast.

    Science.gov (United States)

    Gregan, Juraj; Rabitsch, Peter K; Rumpf, Cornelia; Novatchkova, Maria; Schleiffer, Alexander; Nasmyth, Kim

    2006-01-01

    We have designed the most efficient strategy to knock out genes in fission yeast Schizosaccharomyces pombe on a large scale. Our technique is based on knockout constructs that contain regions homologous to the target gene cloned into vectors carrying dominant drug-resistance markers. Most of the steps are carried out in a 96-well format, allowing simultaneous deletion of 96 genes in one batch. Based on our knockout technique, we designed a strategy for cloning knockout constructs for all predicted fission yeast genes, which is available in a form of a searchable database http://mendel.imp.ac.at/Pombe_deletion/. We validated this technique in a screen where we identified novel genes required for chromosome segregation during meiosis. Here, we present our protocol with detailed instructions. Using this protocol, one person can knock out 96 S. pombe genes in 8 days.

  18. Necroptotic Cell Death Signaling and Execution Pathway: Lessons from Knockout Mice

    Directory of Open Access Journals (Sweden)

    José Belizário

    2015-01-01

    Full Text Available Under stress conditions, cells in living tissue die by apoptosis or necrosis depending on the activation of the key molecules within a dying cell that either transduce cell survival or death signals that actively destroy the sentenced cell. Multiple extracellular (pH, heat, oxidants, and detergents or intracellular (DNA damage and Ca2+ overload stress conditions trigger various types of the nuclear, endoplasmic reticulum (ER, cytoplasmatic, and mitochondrion-centered signaling events that allow cells to preserve the DNA integrity, protein folding, energetic, ionic and redox homeostasis, thus escaping from injury. Along the transition from reversible to irreversible injury, death signaling is highly heterogeneous and damaged cells may engage autophagy, apoptotic, or necrotic cell death programs. Studies on multiple double- and triple- knockout mice identified caspase-8, flip, and fadd genes as key regulators of embryonic lethality and inflammation. Caspase-8 has a critical role in pro- and antinecrotic signaling pathways leading to the activation of receptor interacting protein kinase 1 (RIPK1, RIPK3, and the mixed kinase domain-like (MLKL for a convergent execution pathway of necroptosis or regulated necrosis. Here we outline the recent discoveries into how the necrotic cell death execution pathway is engaged in many physiological and pathological outcome based on genetic analysis of knockout mice.

  19. Obese Neuronal PPARγ Knockout Mice Are Leptin Sensitive but Show Impaired Glucose Tolerance and Fertility.

    Science.gov (United States)

    Fernandez, Marina O; Sharma, Shweta; Kim, Sun; Rickert, Emily; Hsueh, Katherine; Hwang, Vicky; Olefsky, Jerrold M; Webster, Nicholas J G

    2017-01-01

    The peroxisome-proliferator activated receptor γ (PPARγ) is expressed in the hypothalamus in areas involved in energy homeostasis and glucose metabolism. In this study, we created a deletion of PPARγ brain-knockout (BKO) in mature neurons in female mice to investigate its involvement in metabolism and reproduction. We observed that there was no difference in age at puberty onset between female BKOs and littermate controls, but the BKOs gave smaller litters when mated and fewer oocytes when ovulated. The female BKO mice had regular cycles but showed an increase in the number of cycles with prolonged estrus. The mice also had increased luteinizing hormone (LH) levels during the LH surge and histological examination showed hemorrhagic corpora lutea. The mice were challenged with a 60% high-fat diet (HFD). Metabolically, the female BKO mice showed normal body weight, glucose and insulin tolerance, and leptin levels but were protected from obesity-induced leptin resistance. The neuronal knockout also prevented the reduction in estrous cycles due to the HFD. Examination of ovarian histology showed a decrease in the number of primary and secondary follicles in both genotypes due to the HFD, but the BKO ovaries showed an increase in the number of hemorrhagic follicles. In summary, our results show that neuronal PPARγ is required for optimal female fertility but is also involved in the adverse effects of diet-induced obesity by creating leptin resistance potentially through induction of the repressor Socs3. Copyright © 2017 by the Endocrine Society.

  20. Obesity occurring in apolipoprotein E-knockout mice has mild effects on fertility.

    Science.gov (United States)

    Zhang, Ting; Dai, Pengyuan; Cheng, Dong; Zhang, Liang; Chen, Zijiang; Meng, Xiaoqian; Zhang, Fumiao; Han, Xiaoying; Liu, Jianwei; Pan, Jie; Yang, Guiwen; Zhang, Cong

    2014-02-01

    The Apolipoprotein (Apo) family is implicated in lipid metabolism. There are five types of Apo: Apoa, Apob, Apoc, Apod, and Apoe. Apoe has been demonstrated to play a central role in lipoprotein metabolism and to be essential for efficient receptor-mediated plasma clearance of chylomicron remnants and VLDL remnant particles by the liver. Apoe-deficient (Apoe(-/-)) mice develop atherosclerotic plaques spontaneously, followed by obesity. In this study, we investigated whether lipid deposition caused by Apoe knockout affects reproduction in female mice. The results demonstrated that Apoe(-/-) mice were severely hypercholesterolemic, with their cholesterol metabolism disordered, and lipid accumulating in the ovaries causing the ovaries to be heavier compared with the WT counterparts. In addition, estrogen and progesterone decreased significantly at D 100. Quantitative PCR analysis demonstrated that at D 100 the expression of cytochromeP450 aromatase (Cyp19a1), 3β-hydroxysteroid dehydrogenase (Hsd3b), mechanistic target of rapamycin (Mtor), and nuclear factor-κB (Nfkb) decreased significantly, while that of BCL2-associated agonist of cell death (Bad) and tuberous sclerosis complex 2 (Tsc2) increased significantly in the Apoe(-/-) mice. However, there was no difference in the fertility rates of the Apoe(-/-) and WT mice; that is, obesity induced by Apoe knockout has no significant effect on reproduction. However, the deletion of Apoe increased the number of ovarian follicles and the ratio of ovarian follicle atresia and apoptosis. We believe that this work will augment our understanding of the role of Apoe in reproduction.

  1. Increased radiosensitivity and radiation-induced apoptosis in SRC-3 knockout mice

    International Nuclear Information System (INIS)

    Jin Jie; Wang Yu; Xu Yang; Chen Shilei; Wang Junping; Ran Xinze; Su Yongping; Wang Jin

    2014-01-01

    Steroid receptor coactivator-3 (SRC-3), a multifunctional transcriptional coactivator, plays an important role in regulation of cell apoptosis in chemoresistant cancer cells. However, its role in radiation-induced apoptosis in hematopoietic cells is still unclear. In this study, we used SRC-3 knockout (SRC-3 -/- ) mice to assess the role of SRC-3 in radiation-induced hematopoietic injury in vivo. After a range of doses of irradiation, SRC-3 -/- mice exhibited lower counts of peripheral blood cells and bone marrow (BM) mononuclear cells and excessive BM depression, which resulted in a significantly higher mortality compared with wildtype mice. Moreover, BM mononuclear cells obtained from SRC-3 -/- mice showed a remarkable increase in radiation-induced apoptosis. Collectively, our data demonstrate that SRC-3 plays a role in radiation-induced apoptosis of BM hematopoietic cells. Regulation of SRC-3 might influence the radiosensitivity of hematopoietic cells, which highlights a potential therapeutic target for radiation-induced hematopoietic injury. (author)

  2. Sortilin 1 knockout alters basal adipose glucose metabolism but not diet-induced obesity in mice.

    Science.gov (United States)

    Li, Jibiao; Matye, David J; Wang, Yifeng; Li, Tiangang

    2017-04-01

    Sortilin 1 (Sort1) is a trafficking receptor that has been implicated in the regulation of plasma cholesterol in humans and mice. Here, we use metabolomics and hyperinsulinemic-euglycemic clamp approaches to obtain further understanding of the in vivo effects of Sort1 deletion on diet-induced obesity as well as on adipose lipid and glucose metabolism. Results show that Sort1 knockout (KO) does not affect Western diet-induced obesity nor adipose fatty acid and ceramide concentrations. Under the basal fasting state, chow-fed Sort1 KO mice have decreased adipose glycolytic metabolites, but Sort1 deletion does not affect insulin-stimulated tissue glucose uptake during the insulin clamp. These results suggest that Sort1 loss-of-function in vivo does not affect obesity development, but differentially modulates adipose glucose metabolism under fasting and insulin-stimulated states. © 2017 Federation of European Biochemical Societies.

  3. A critical look at the function of the P2Y11 receptor

    DEFF Research Database (Denmark)

    Dreisig, Karin; Kornum, Birgitte Rahbek

    2016-01-01

    The P2Y11 receptor is a member of the purinergic receptor family. It has been overlooked, somewhat due to the lack of a P2ry11 gene orthologue in the murine genome, which prevents the generation of knockout mice, which have been so helpful for defining the roles of other P2Y receptors. Furthermor...

  4. Molecular pharmacology of promiscuous seven transmembrane receptors sensing organic nutrients

    DEFF Research Database (Denmark)

    Wellendorph, Petrine; Johansen, Lars Dan; Bräuner-Osborne, Hans

    2009-01-01

    A number of highly promiscuous seven transmembrane (7TM) receptors have been cloned and characterized within the last few years. It is noteworthy that many of these receptors are activated broadly by amino acids, proteolytic degradation products, carbohydrates, or free fatty acids and are expressed...... receptors FFA1, FFA2, FFA3, GPR84, and GPR120. The involvement of the individual receptors in sensing of food intake has been validated to different degrees because of limited availability of specific pharmacological tools and/or receptor knockout mice. However, as a group, the receptors represent potential...

  5. Adenosine receptor neurobiology: overview.

    Science.gov (United States)

    Chen, Jiang-Fan; Lee, Chien-fei; Chern, Yijuang

    2014-01-01

    Adenosine is a naturally occurring nucleoside that is distributed ubiquitously throughout the body as a metabolic intermediary. In the brain, adenosine functions as an important upstream neuromodulator of a broad spectrum of neurotransmitters, receptors, and signaling pathways. By acting through four G-protein-coupled receptors, adenosine contributes critically to homeostasis and neuromodulatory control of a variety of normal and abnormal brain functions, ranging from synaptic plasticity, to cognition, to sleep, to motor activity to neuroinflammation, and cell death. This review begun with an overview of the gene and genome structure and the expression pattern of adenosine receptors (ARs). We feature several new developments over the past decade in our understanding of AR functions in the brain, with special focus on the identification and characterization of canonical and noncanonical signaling pathways of ARs. We provide an update on functional insights from complementary genetic-knockout and pharmacological studies on the AR control of various brain functions. We also highlight several novel and recent developments of AR neurobiology, including (i) recent breakthrough in high resolution of three-dimension structure of adenosine A2A receptors (A2ARs) in several functional status, (ii) receptor-receptor heterodimerization, (iii) AR function in glial cells, and (iv) the druggability of AR. We concluded the review with the contention that these new developments extend and strengthen the support for A1 and A2ARs in brain as therapeutic targets for neurologic and psychiatric diseases. © 2014 Elsevier Inc. All rights reserved.

  6. Role of estrogen receptor-α on food demand elasticity.

    Science.gov (United States)

    Minervini, Vanessa; Rowland, Neil E; Robertson, Kimberly L; Foster, Thomas C

    2015-05-01

    Estrogens have been shown to have an inhibitory effect on food intake under free-feeding conditions, yet the effects of estrogens on food-maintained operant responding have been studied to a much lesser extent and, thus, are not well understood. Therefore, the purpose of the present experiment was to use a behavioral economics paradigm to assess differences in demand elasticity between mice with knockout of the estrogen receptor subtype α, knockout of subtype β, and their wild type controls. The mice responded in a closed economy, and the price of food was increased by increasing the fixed-ratio response requirement every four sessions. Overall, we found that mice with the knockout of receptor subtype α had the most elastic demand functions. Therefore, under these conditions, estrogens increased food seeking via activation of the receptor subtype α. The results were inconsistent with those reported by previous studies that employed free-feeding conditions. © Society for the Experimental Analysis of Behavior.

  7. Étude des fonctions du récepteur opioïde delta exprimé dans le cerveau antérieur grâce à une approche de knockout conditionnel

    OpenAIRE

    Chu Sin Chung , Paul

    2013-01-01

    Delta opioid receptors (DORs) are G-protein coupled receptors belonging to the opioid system, which play a central role in chronic pain and emotional responses. DORs are strongly expressed in olfactory bulb, cortex, striatum, basolateral nucleus of the amygdala and pons nuclei. Using constitutive gene knockout, we have previously demonstrated the role of DORs in reducingchronic pain (Gaveriaux-Ruff, Nozaki et al. 2011), anxiety-related behaviors and impulsivity(Olmstead,Ouagazzal et al. 2009)...

  8. Human Genetic Disorders and Knockout Mice Deficient in Glycosaminoglycan

    Directory of Open Access Journals (Sweden)

    Shuji Mizumoto

    2014-01-01

    Full Text Available Glycosaminoglycans (GAGs are constructed through the stepwise addition of respective monosaccharides by various glycosyltransferases and maturated by epimerases and sulfotransferases. The structural diversity of GAG polysaccharides, including their sulfation patterns and sequential arrangements, is essential for a wide range of biological activities such as cell signaling, cell proliferation, tissue morphogenesis, and interactions with various growth factors. Studies using knockout mice of enzymes responsible for the biosynthesis of the GAG side chains of proteoglycans have revealed their physiological functions. Furthermore, mutations in the human genes encoding glycosyltransferases, sulfotransferases, and related enzymes responsible for the biosynthesis of GAGs cause a number of genetic disorders including chondrodysplasia, spondyloepiphyseal dysplasia, and Ehlers-Danlos syndromes. This review focused on the increasing number of glycobiological studies on knockout mice and genetic diseases caused by disturbances in the biosynthetic enzymes for GAGs.

  9. Rapid curation of gene disruption collections using Knockout Sudoku.

    Science.gov (United States)

    Anzai, Isao A; Shaket, Lev; Adesina, Oluwakemi; Baym, Michael; Barstow, Buz

    2017-10-01

    Knockout Sudoku is a method for the construction of whole-genome knockout collections for a wide range of microorganisms with as little as 3 weeks of dedicated labor and at a cost of ∼$10,000 for a collection for a single organism. The method uses manual 4D combinatorial pooling, next-generation sequencing, and a Bayesian inference algorithm to rapidly process and then accurately annotate the extremely large progenitor transposon insertion mutant collections needed to achieve saturating coverage of complex microbial genomes. This method is ∼100× faster and 30× lower in cost than the next comparable method (In-seq) for annotating transposon mutant collections by combinatorial pooling and next-generation sequencing. This method facilitates the rapid, algorithmically guided condensation and curation of the progenitor collection into a high-quality, nonredundant collection that is suitable for rapid genetic screening and gene discovery.

  10. Pauli blocking and medium effects in nucleon knockout reactions

    International Nuclear Information System (INIS)

    Bertulani, C. A.; De Conti, C.

    2010-01-01

    We study medium modifications of the nucleon-nucleon (NN) cross sections and their influence on the nucleon knockout reactions. Using the eikonal approximation, we compare the results obtained with free NN cross sections with those obtained with a purely geometrical treatment of Pauli blocking and with NN obtained with more elaborated Dirac-Bruecker methods. The medium effects are parametrized in terms of the baryon density. We focus on symmetric nuclear matter, although the geometrical Pauli blocking also allows for the treatment of asymmetric nuclear matter. It is shown that medium effects can change the nucleon knockout cross sections and momentum distributions up to 10% in the energy range E lab =50-300 MeV/nucleon. The effect is more evident in reactions involving halo nuclei.

  11. One-neutron knockout from 51-55Sc

    International Nuclear Information System (INIS)

    Schwertel, S.; Maierbeck, P.; Gernhaeuser, R.; Bildstein, V.; Boehmer, M.; Eppinger, K.; Faestermann, T.; Friese, J.; Fabbietti, L.; Maier, L.; Winkler, S.; Kruecken, R.; Kroell, T.; Alvarez-Pol, H.; Benjamim, E.A.; Benlliure, J.; Caamano, M.; Cortina-Gil, D.; Gascon, M.; Kurtukian, T.; Perez, D.; Rodriguez-Tajes, C.; Aksouh, F.; Aumann, T.; Behr, K.; Boretzky, K.; Bruenle, A.; Chatillon, A.; Chulkov, L.V.; Geissel, H.; Gerl, J.; Gorska, M.; Kojouharov, I.; Klimkiewicz, A.; Kurz, N.; Nociforo, C.; Schaffner, H.; Simon, H.; Stanoiu, M.; Suemmerer, K.; Weick, H.; Borge, M.J.G.; Pascual-Izarra, C.; Perea, A.; Tengblad, O.; Buerger, A.; Casarejos, E.; Brown, B.A.; Enders, J.; Schrieder, G.; Hansen, P.G.; Jonson, B.; Nyman, G.; Kanungo, R.; Kiselev, O.; Larsson, K.; Le Bleis, T.; Mahata, K.; Nilsson, T.; Prochazka, A.; Rossi, D.; Sitar, B.; Otsuka, T.; Tostevin, J.A.; Rae, W.D.M.

    2012-01-01

    Results are presented from a one-neutron knockout experiment at relativistic energies of ∼ 420 A MeV on 51-55 Sc using the GSI Fragment Separator as a two-stage magnetic spectrometer and the MINIBALL array for gamma-ray detection. Inclusive longitudinal momentum distributions and cross-sections were measured enabling the determination of the contributions corresponding to knockout from the νp 1/2 , νp 3/2 , (L = 1) and νf 7/2 , νf 5/2 (L = 3) neutron orbitals. The observed L = 1 and L = 3 contributions are compared with theoretical cross-sections using eikonal knockout theory and spectroscopic factors from shell model calculations using the GXPF1A interaction. The measured inclusive knockout cross-sections generally follow the trends expected theoretically and given by the spectroscopic strength predicted from the shell model calculations. However, the deduced L = 1 cross-sections are generally 30-40% higher while the L = 3 contributions are about a factor of two smaller than predicted. This points to a promotion of neutrons from the νf 7/2 to the νp 3/2 orbital indicating a weakening of the N = 28 shell gap in these nuclei. While this is not predicted for the phenomenological GXPF1A interaction such a weakening is predicted by recent calculations using realistic low-momentum interactions V low k obtained by evolving a chiral N3LO nucleon-nucleon potential. (orig.)

  12. Single proton knock-out from 24F

    International Nuclear Information System (INIS)

    Thoennessen, M.; Baumann, T.; Brown, B.A.; Enders, J.; Frank, N.H.; Hansen, P.G.; Heckman, P.; Luther, B.A.; Seitz, J.P.; Stolz, A.; Tryggestad, E.

    2004-01-01

    The measurement of the single proton knock-out reaction from 24 F on a 12 C target at 46.7 MeV/nucleon yielded a 23 O ground state population of (6.6+/-1.0) mb. The data were compared to calculations based on the many-body shell model and the eikonal theory. The results are consistent with a [0d5/26]-bar 1s1/2 configuration of 23 O

  13. Evidence that the angiotensin at 2-receptor agonist compound 21 is also a low affinity thromboxane TXA2-receptor antagonist

    DEFF Research Database (Denmark)

    Fredgart, M.; Leurgans, T.; Stenelo, M.

    2015-01-01

    AT2-receptor specificity, arteries were pre-incubated with the AT2-receptor antagonist PD123319 (10muM), or mesenteric arteries from AT2-receptor knock-out (AT2R-/y) mice were used. An inhibitory effect of C21 (100nM - 10muM) on U46619 (0,3muM) induced platelet aggregation was examined in whole human...

  14. Protease activity of legumain is inhibited by an increase of cystatin E/M in the DJ-1-knockout mouse spleen, cerebrum and heart

    Directory of Open Access Journals (Sweden)

    Takuya Yamane

    2017-03-01

    Full Text Available Legumain (EC 3.4.22.34 is an asparaginyl endopeptidase. Legumain activity has been detected in various mouse tissues including the kidney, spleen and epididymis. Legumain is overexpressed in the majority of human solid tumors and transcription of the legumain gene is regulated by the p53 tumor suppressor in HCT116 cells. The legumain activity is also increased under acid conditions in Alzheimer's disease brains. DJ-1/PARK7, a cancer- and Parkinson's disease-associated protein, works as a coactivator to various transcription factors, including the androgen receptor, p53, PSF, Nrf2, SREBP and RREB1. Recently, we found that legumain expression, activation and cleavage of annexin A2 are regulated by DJ-1 through p53. In this study, we found that the expression levels of legumain mRNA were increased in the cerebrum, kidney, spleen, heart, lung, epididymis, stomach, small intestine and pancreas from DJ-1-knockout mice, although legumain activity levels were decreased in the cerebrum, spleen and heart from DJ-1-knockout mice. Furthermore, we found that cystatin E/M expression was increased in the spleen, cerebrum and heart from DJ-1-knockout mice. These results suggest that reduction of legumain activity is caused by an increase of cystatin E/M expression in the spleen, cerebrum and heart from DJ-1-knockout mice.

  15. Comparison of the Tastes of L-Alanine and Monosodium Glutamate in C57BL/6J Wild Type and T1r3 Knockout Mice.

    Science.gov (United States)

    Eddy, Meghan C; Eschle, Benjamin K; Delay, Eugene R

    2017-09-01

    Previous research showed that L-alanine and monosodium L-glutamate elicit similar taste sensations in rats. This study reports the results of behavioral experiments designed to compare the taste capacity of C57BL/6J wild type and T1r3- mice for these 2 amino acids. In conditioned taste aversion (CTA) experiments, wild-type mice exhibited greater sensitivity than knockout mice for both L-amino acids, although knockout mice were clearly able to detect both amino acids at 50 mM and higher concentrations. Generalization of CTA between L-alanine and L-glutamate was bidirectionally equivalent for both mouse genotypes, indicating that both substances elicited similar tastes in both genotypes. This was verified by the discrimination experiments in which both mouse genotypes performed at or near chance levels at 75 and 150 mM. Above 150 mM, discrimination performance improved, suggesting the taste qualities of the 2 L-amino acids are not identical. No differences between knockout and wild-type mice in discrimination ability were detected. These results indicate that while the T1r3 receptor is important for tasting L-alanine and L-glutamate, other receptors are also important for tasting these amino acids. © The Author 2017. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  16. Serotonin and urocortin 1 in the dorsal raphe and Edinger-Westphal nuclei after early life stress in serotonin transporter knockout rats.

    Science.gov (United States)

    van der Doelen, Rick H A; Robroch, Berit; Arnoldussen, Ilse A; Schulpen, Maya; Homberg, Judith R; Kozicz, Tamás

    2017-01-06

    The interaction of early life stress (ELS) and the serotonin transporter (5-HTT) gene-linked polymorphic region (5-HTTLPR) has been associated with increased risk to develop depression in later life. We have used the maternal separation paradigm as a model for ELS exposure in homozygous and heterozygous 5-HTT knockout rats and measured urocortin 1 (Ucn1) mRNA and/or protein levels, Ucn1 DNA methylation, as well as 5-HT innervation in the centrally projecting Edinger-Westphal (EWcp) and dorsal raphe (DR) nuclei, both implicated in the regulation of stress response. We found that ELS and 5-HTT genotype increased the number of 5-HT neurons in specific DR subdivisions, and that 5-HTT knockout rats showed decreased 5-HT innervation of EWcp-Ucn1 neurons. Furthermore, ELS was associated with increased DNA methylation of the promoter region of the Ucn1 gene and increased expression of 5-HT receptor 1A in the EWcp. In contrast, 5-HTT deficiency was associated with site-specific alterations in DNA methylation of the Ucn1 promoter, and heterozygous 5-HTT knockout rats showed decreased expression of CRF receptor 1 in the EWcp. Together, our findings extend the existing literature on the relationship between EWcp-Ucn1 and DR-5-HT neurons. These observations will further our understanding on their potential contribution to mediate affect as a function of ELS interacting with 5-HTTLPR. Copyright © 2016 IBRO. Published by Elsevier Ltd. All rights reserved.

  17. Ghrelin knockout mice show decreased voluntary alcohol consumption and reduced ethanol-induced conditioned place preference.

    Science.gov (United States)

    Bahi, Amine; Tolle, Virginie; Fehrentz, Jean-Alain; Brunel, Luc; Martinez, Jean; Tomasetto, Catherine-Laure; Karam, Sherif M

    2013-05-01

    Recent work suggests that stomach-derived hormone ghrelin receptor (GHS-R1A) antagonism may reduce motivational aspects of ethanol intake. In the current study we hypothesized that the endogenous GHS-R1A agonist ghrelin modulates alcohol reward mechanisms. For this purpose ethanol-induced conditioned place preference (CPP), ethanol-induced locomotor stimulation and voluntary ethanol consumption in a two-bottle choice drinking paradigm were examined under conditions where ghrelin and its receptor were blocked, either using ghrelin knockout (KO) mice or the specific ghrelin receptor (GHS-R1A) antagonist "JMV2959". We showed that ghrelin KO mice displayed lower ethanol-induced CPP than their wild-type (WT) littermates. Consistently, when injected during CPP-acquisition, JMV2959 reduced CPP-expression in C57BL/6 mice. In addition, ethanol-induced locomotor stimulation was lower in ghrelin KO mice. Moreover, GHS-R1A blockade, using JMV2959, reduced alcohol-stimulated locomotion only in WT but not in ghrelin KO mice. When alcohol consumption and preference were assessed using the two-bottle choice test, both genetic deletion of ghrelin and pharmacological antagonism of the GHS-R1A (JMV2959) reduced voluntary alcohol consumption and preference. Finally, JMV2959-induced reduction of alcohol intake was only observed in WT but not in ghrelin KO mice. Taken together, these results suggest that ghrelin neurotransmission is necessary for the stimulatory effect of ethanol to occur, whereas lack of ghrelin leads to changes that reduce the voluntary intake as well as conditioned reward by ethanol. Our findings reveal a major, novel role for ghrelin in mediating ethanol behavior, and add to growing evidence that ghrelin is a key mediator of the effects of multiple abused drugs. Copyright © 2013 Elsevier Inc. All rights reserved.

  18. Robust and sensitive analysis of mouse knockout phenotypes.

    Directory of Open Access Journals (Sweden)

    Natasha A Karp

    Full Text Available A significant challenge of in-vivo studies is the identification of phenotypes with a method that is robust and reliable. The challenge arises from practical issues that lead to experimental designs which are not ideal. Breeding issues, particularly in the presence of fertility or fecundity problems, frequently lead to data being collected in multiple batches. This problem is acute in high throughput phenotyping programs. In addition, in a high throughput environment operational issues lead to controls not being measured on the same day as knockouts. We highlight how application of traditional methods, such as a Student's t-Test or a 2-way ANOVA, in these situations give flawed results and should not be used. We explore the use of mixed models using worked examples from Sanger Mouse Genome Project focusing on Dual-Energy X-Ray Absorptiometry data for the analysis of mouse knockout data and compare to a reference range approach. We show that mixed model analysis is more sensitive and less prone to artefacts allowing the discovery of subtle quantitative phenotypes essential for correlating a gene's function to human disease. We demonstrate how a mixed model approach has the additional advantage of being able to include covariates, such as body weight, to separate effect of genotype from these covariates. This is a particular issue in knockout studies, where body weight is a common phenotype and will enhance the precision of assigning phenotypes and the subsequent selection of lines for secondary phenotyping. The use of mixed models with in-vivo studies has value not only in improving the quality and sensitivity of the data analysis but also ethically as a method suitable for small batches which reduces the breeding burden of a colony. This will reduce the use of animals, increase throughput, and decrease cost whilst improving the quality and depth of knowledge gained.

  19. Pre-Equilibrium Cluster Emission with Pickup and Knockout

    International Nuclear Information System (INIS)

    Betak, E.

    2005-01-01

    We present a generalization of the Iwamoto-Harada-Bisplinghoff pre-equilibrium model of light cluster formation and emission, which is enhanced by allowing for possible admixtures of knockout for strongly coupled ejectiles, like α's. The model is able to attain the Weisskopf-Ewing formula for compound-nucleus decay at long-time limit; it keeps the philosophy of pre-equilibrium decay during the equilibration stage and it describes the initial phase of a reaction as direct process(es) expressed using the language of the exciton model

  20. Neurobehavioral phenotyping of Gaq knockout mice reveals impairments in motor functions and spatial working memory without changes in anxiety or behavioral despair

    Directory of Open Access Journals (Sweden)

    Aliya L Frederick

    2012-06-01

    Full Text Available Many neurotransmitters, hormones and sensory stimuli elicit their cellular responses through the targeted activation of receptors coupled to Gq family heterotrimeric G proteins. Nevertheless, we still understand little about the consequences of loss of this signaling activity on brain function. We therefore examined the effects of genetic inactivation of Gnaq on responsiveness in a battery of behavioral tests in order to assess the contribution of Gaq signaling capacity in the brain circuits mediating expression of affective behaviors (anxiety and behavioral despair, spatial working memory and locomotor output (coordination, strength, spontaneous activity and drug-induced responses. First, we replicated and extended findings showing clear motor deficits in Gaq knockout mice as assessed on an accelerating rotarod and the inverted screen test. We then assessed the contribution of the basal ganglia motor loops to these impairments, using open field testing and analysis of drug-induced locomotor responses to the psychostimulant cocaine, the benzazepine D1 receptor agonists SKF83822 and SKF83959, and the NMDA receptor antagonist MK-801. We observed significant increases in drug-induced locomotor activity in Gaq knockout mice from the dopaminergic agonists but not MK-801, indicating that basal ganglia locomotor circuitry is largely intact in the absence of Gaq. Additionally, we observed normal phenotypes in both the elevated zero maze and the forced swim test indicating that anxiety and depression-related circuitry appears to be largely intact after loss of Gnaq expression. Lastly, use of the Y-maze revealed spatial memory deficits in Gaq knockout mice, indicating that receptors signaling through Gaq are necessary in these circuits for proficiency in this task.

  1. Neurobehavioral phenotyping of Gαq knockout mice reveals impairments in motor functions and spatial working memory without changes in anxiety or behavioral despair

    Science.gov (United States)

    Frederick, Aliya L.; Saborido, Tommy P.; Stanwood, Gregg D.

    2012-01-01

    Many neurotransmitters, hormones, and sensory stimuli elicit their cellular responses through the targeted activation of receptors coupled to the Gαq family of heterotrimeric G proteins. Nevertheless, we still understand little about the consequences of loss of this signaling activity on brain function. We therefore examined the effects of genetic inactivation of Gnaq, the gene that encode for Gαq, on responsiveness in a battery of behavioral tests in order to assess the contribution of Gαq signaling capacity in the brain circuits mediating expression of affective behaviors (anxiety and behavioral despair), spatial working memory, and locomotor output (coordination, strength, spontaneous activity, and drug-induced responses). First, we replicated and extended findings showing clear motor deficits in Gαq knockout mice as assessed on an accelerating rotarod and the inverted screen test. We then assessed the contribution of the basal ganglia motor loops to these impairments, using open field testing and analysis of drug-induced locomotor responses to the psychostimulant cocaine, the benzazepine D1 receptor agonists SKF83822 and SKF83959, and the NMDA receptor antagonist MK-801. We observed significant increases in drug-induced locomotor activity in Gαq knockout mice from the dopaminergic agonists but not MK-801, indicating that basal ganglia locomotor circuitry is largely intact in the absence of Gαq. Additionally, we observed normal phenotypes in both the elevated zero maze and the forced swim test indicating that anxiety and depression-related circuitry appears to be largely intact after loss of Gnaq expression. Lastly, use of the Y-maze revealed spatial memory deficits in Gαq knockout mice, indicating that receptors signaling through Gαq are necessary in these circuits for proficiency in this task. PMID:22723772

  2. CRISPR/Cas9 knockouts reveal genetic interaction between strain-transcendent erythrocyte determinants ofPlasmodium falciparuminvasion.

    Science.gov (United States)

    Kanjee, Usheer; Grüring, Christof; Chaand, Mudit; Lin, Kai-Min; Egan, Elizabeth; Manzo, Jale; Jones, Patrick L; Yu, Tiffany; Barker, Robert; Weekes, Michael P; Duraisingh, Manoj T

    2017-10-31

    During malaria blood-stage infections, Plasmodium parasites interact with the RBC surface to enable invasion followed by intracellular proliferation. Critical factors involved in invasion have been identified using biochemical and genetic approaches including specific knockdowns of genes of interest from primary CD34 + hematopoietic stem cells (cRBCs). Here we report the development of a robust in vitro culture system to produce RBCs that allow the generation of gene knockouts via CRISPR/Cas9 using the immortal JK-1 erythroleukemia line. JK-1 cells spontaneously differentiate, generating cells at different stages of erythropoiesis, including terminally differentiated nucleated RBCs that we term "jkRBCs." A screen of small-molecule epigenetic regulators identified several bromodomain-specific inhibitors that promote differentiation and enable production of synchronous populations of jkRBCs. Global surface proteomic profiling revealed that jkRBCs express all known P falciparum host receptors in a similar fashion to cRBCs and that multiple P falciparum strains invade jkRBCs at comparable levels to cRBCs and RBCs. Using CRISPR/Cas9, we deleted two host factors, basigin (BSG) and CD44, for which no natural nulls exist. BSG interacts with the parasite ligand Rh5, a prominent vaccine candidate. A BSG knockout was completely refractory to parasite invasion in a strain-transcendent manner, confirming the essential role for BSG during invasion. CD44 was recently identified in an RNAi screen of blood group genes as a host factor for invasion, and we show that CD44 knockout results in strain-transcendent reduction in invasion. Furthermore, we demonstrate a functional interaction between these two determinants in mediating P falciparum erythrocyte invasion. Published under the PNAS license.

  3. A STAT-1 knockout mouse model for Machupo virus pathogenesis

    Directory of Open Access Journals (Sweden)

    Shurtleff Amy C

    2011-06-01

    Full Text Available Abstract Background Machupo virus (MACV, a member of the Arenaviridae, causes Bolivian hemorrhagic fever, with ~20% lethality in humans. The pathogenesis of MACV infection is poorly understood, and there are no clinically proven treatments for disease. This is due, in part, to a paucity of small animal models for MACV infection in which to discover and explore candidate therapeutics. Methods Mice lacking signal transducer and activator of transcription 1 (STAT-1 were infected with MACV. Lethality, viral replication, metabolic changes, hematology, histopathology, and systemic cytokine expression were analyzed throughout the course of infection. Results We report here that STAT-1 knockout mice succumbed to MACV infection within 7-8 days, and presented some relevant clinical and histopathological manifestations of disease. Furthermore, the model was used to validate the efficacy of ribavirin in protection against infection. Conclusions The STAT-1 knockout mouse model can be a useful small animal model for drug testing and preliminary immunological analysis of lethal MACV infection.

  4. Efficient CRISPR/Cas9-based gene knockout in watermelon.

    Science.gov (United States)

    Tian, Shouwei; Jiang, Linjian; Gao, Qiang; Zhang, Jie; Zong, Mei; Zhang, Haiying; Ren, Yi; Guo, Shaogui; Gong, Guoyi; Liu, Fan; Xu, Yong

    2017-03-01

    CRISPR/Cas9 system can precisely edit genomic sequence and effectively create knockout mutations in T0 generation watermelon plants. Genome editing offers great advantage to reveal gene function and generate agronomically important mutations to crops. Recently, RNA-guided genome editing system using the type II clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) has been applied to several plant species, achieving successful targeted mutagenesis. Here, we report the genome of watermelon, an important fruit crop, can also be precisely edited by CRISPR/Cas9 system. ClPDS, phytoene desaturase in watermelon, was selected as the target gene because its mutant bears evident albino phenotype. CRISPR/Cas9 system performed genome editing, such as insertions or deletions at the expected position, in transfected watermelon protoplast cells. More importantly, all transgenic watermelon plants harbored ClPDS mutations and showed clear or mosaic albino phenotype, indicating that CRISPR/Cas9 system has technically 100% of genome editing efficiency in transgenic watermelon lines. Furthermore, there were very likely no off-target mutations, indicated by examining regions that were highly homologous to sgRNA sequences. Our results show that CRISPR/Cas9 system is a powerful tool to effectively create knockout mutations in watermelon.

  5. Proton-induced knockout reactions with polarized and unpolarized beams

    Science.gov (United States)

    Wakasa, T.; Ogata, K.; Noro, T.

    2017-09-01

    Proton-induced knockout reactions provide a direct means of studying the single particle or cluster structures of target nuclei. In addition, these knockout reactions are expected to play a unique role in investigations of the effects of the nuclear medium on nucleon-nucleon interactions as well as the properties of nucleons and mesons. However, due to the nature of hadron probes, these reactions can suffer significant disturbances from the nuclear surroundings and the quantitative theoretical treatment of such processes can also be challenging. In this article, we review the experimental and theoretical progress in this field, particularly focusing on the use of these reactions as a spectroscopic tool and as a way to examine the medium modification of nucleon-nucleon interactions. With regard to the former aspect, the review presents a semi-quantitative evaluation of these reactions based on existing experimental data. In terms of the latter point, we introduce a significant body of evidence that suggests, although does not conclusively prove, the existence of medium effects. In addition, this paper also provides information and comments on other related subjects.

  6. Lack of adenosine A(3) receptors causes defects in mouse peripheral blood parameters

    Czech Academy of Sciences Publication Activity Database

    Hofer, Michal; Pospíšil, Milan; Dušek, L.; Hoferová, Zuzana; Komůrková, Denisa

    2014-01-01

    Roč. 10, č. 3 (2014), s. 509-514 ISSN 1573-9538 R&D Projects: GA ČR(CZ) GAP303/11/0128 Institutional support: RVO:68081707 Keywords : Adenosine A(3) receptor * Adenosine A(3) receptor knockout mice * Hematopoiesis Subject RIV: BO - Biophysics Impact factor: 3.886, year: 2014

  7. Shorter duration of non-rapid eye movement sleep slow waves in EphA4 knockout mice.

    Science.gov (United States)

    Freyburger, Marlène; Poirier, Gaétan; Carrier, Julie; Mongrain, Valérie

    2017-10-01

    Slow waves occurring during non-rapid eye movement sleep have been associated with neurobehavioural performance and memory. In addition, the duration of previous wakefulness and sleep impacts characteristics of these slow waves. However, molecular mechanisms regulating the dynamics of slow-wave characteristics remain poorly understood. The EphA4 receptor regulates glutamatergic transmission and synaptic plasticity, which have both been linked to sleep slow waves. To investigate if EphA4 regulates slow-wave characteristics during non-rapid eye movement sleep, we compared individual parameters of slow waves between EphA4 knockout mice and wild-type littermates under baseline conditions and after a 6-h sleep deprivation. We observed that, compared with wild-type mice, knockout mice display a shorter duration of positive and negative phases of slow waves under baseline conditions and after sleep deprivation. However, the mutation did not change slow-wave density, amplitude and slope, and did not affect the sleep deprivation-dependent changes in slow-wave characteristics, suggesting that EphA4 is not involved in the response to elevated sleep pressure. Our present findings suggest a role for EphA4 in shaping cortical oscillations during sleep that is independent from sleep need. © 2017 European Sleep Research Society.

  8. Polyhydramnios in Lrp4 knockout mice with bilateral kidney agenesis: Defects in the pathways of amniotic fluid clearance.

    Science.gov (United States)

    Tanahashi, Hiroshi; Tian, Qing-Bao; Hara, Yoshinobu; Sakagami, Hiroyuki; Endo, Shogo; Suzuki, Tatsuo

    2016-02-05

    Amniotic fluid volume during mid-to-late gestation depends mainly on the urine excretion from the foetal kidneys and partly on the fluid secretion from the foetal lungs during foetal breathing-like movements. Urine is necessary for foetal breathing-like movements, which is critical for foetal lung development. Bilateral renal agenesis and/or obstruction of the urinary tract lead to oligohydramnios, which causes infant death within a short period after birth due to pulmonary hypoplasia. Lrp4, which functions as an agrin receptor, is essential for the formation of neuromuscular junctions. Herein, we report novel phenotypes of Lrp4 knockout (Lrp4(-/-)) mice. Most Lrp4(-/-) foetuses showed unilateral or bilateral kidney agenesis, and Lrp4 knockout resulted in polyhydramnios. The loss of Lrp4 compromised foetal swallowing and breathing-like movements and downregulated the expression of aquaporin-9 in the foetal membrane and aquaporin-1 in the placenta, which possibly affected the amniotic fluid clearance. These results suggest that amniotic fluid removal was compromised in Lrp4(-/-) foetuses, resulting in polyhydramnios despite the impairment of urine production. Our findings indicate that amniotic fluid removal plays an essential role in regulating the amniotic fluid volume.

  9. The role of P2X receptors in bone biology

    DEFF Research Database (Denmark)

    Jørgensen, N R; Syberg, S; Ellegaard, M

    2015-01-01

    come from studies on murine knockout models and from pharmacologic studies on cells and animals. More recently, the role of P2X receptors in human bone diseases has been documented. Loss-of-functions polymorphisms in the P2X7 receptorare associated with bone loss and increased fracture risk. Very...

  10. Genetic Variant of the Scavenger Receptor BI in Humans

    NARCIS (Netherlands)

    Vergeer, Menno; Korporaal, Suzanne J. A.; Franssen, Remco; Meurs, Illiana; Out, Ruud; Hovingh, G. Kees; Hoekstra, Menno; Sierts, Jeroen A.; Dallinga-Thie, Geesje M.; Motazacker, Mohammad Mahdi; Holleboom, Adriaan G.; van Berkel, Theo J. C.; Kastelein, John J. P.; van Eck, Miranda; Kuivenhoven, Jan Albert

    2011-01-01

    BACKGROUND In mice, the scavenger receptor class B type I (SR-BI) is essential for the delivery of high-density lipoprotein (HDL) cholesterol to the liver and steroidogenic organs. Paradoxically, elevated HDL cholesterol levels are associated with increased atherosclerosis in SR-BI-knockout mice. It

  11. Genetic variant of the scavenger receptor BI in humans

    NARCIS (Netherlands)

    Vergeer, Menno; Korporaal, Suzanne J A; Franssen, Remco; Meurs, Illiana; Out, Ruud; Hovingh, G Kees; Hoekstra, Menno; Sierts, Jeroen A; Dallinga-Thie, Geesje M; Motazacker, Mohammad Mahdi; Holleboom, Adriaan G; Van Berkel, Theo J C; Kastelein, John J P; Van Eck, Miranda; Kuivenhoven, Jan Albert

    2011-01-01

    BACKGROUND: In mice, the scavenger receptor class B type I (SR-BI) is essential for the delivery of high-density lipoprotein (HDL) cholesterol to the liver and steroidogenic organs. Paradoxically, elevated HDL cholesterol levels are associated with increased atherosclerosis in SR-BI-knockout mice.

  12. The role of CB1 receptors in psychostimulant addiction

    NARCIS (Netherlands)

    Wiskerke, J.; Pattij, T.; Schoffelmeer, A.N.M.; de Vries, T.J.

    2008-01-01

    Recent studies have implicated the endocannabinoid (eCB) system in the neuronal mechanisms underlying substance dependence. Here, we review results of studies using cannabinoid receptor subtype 1 (CB1) knockout mice as well as CB1 antagonists to elucidate the role of this neurotransmitter system in

  13. Examination of MARCO activity on dendritic cell phenotype and function using a gene knockout mouse.

    Directory of Open Access Journals (Sweden)

    Hiroshi Komine

    Full Text Available We have reported the upregulation of MARCO, a member of the class A scavenger receptor family, on the surface of murine and human dendritic cells (DCs pulsed with tumor lysates. Exposure of murine tumor lysate-pulsed DCs to an anti-MARCO antibody led to loss of dendritic-like processes and enhanced migratory capacity. In this study, we have further examined the biological and therapeutic implications of MARCO expression by DCs. DCs generated from the bone marrow (bm of MARCO knockout (MARCO⁻/⁻ mice were phenotypically similar to DCs generated from the bm of wild-type mice and produced normal levels of IL-12 and TNF-α when exposed to LPS. MARCO⁻/⁻ DCs demonstrated enhanced migratory capacity in response to CCL-21 in vitro. After subcutaneous injection into mice, MARCO⁻/⁻ TP-DCs migrated more efficiently to the draining lymph node leading to enhanced generation of tumor-specific IFN-γ producing T cells and improved tumor regression and survival in B16 melanoma-bearing mice. These results support targeting MARCO on the surface of DCs to improve trafficking and induction of anti-tumor immunity.

  14. Toll-like receptor 4 in atherosclerosis

    OpenAIRE

    Li, Hongli; Sun, Baogui

    2007-01-01

    Abstract Toll-like receptor 4 (TLR4) is key regulators of both innate and adaptive immune responses. TLR4 recognizes pathogen-associated molecular patterns (PAMPs) and activates the inflammatory cells. The function of TLR4 in atherosclerosis has been investigated in mouse knockout studies and epidemiological studies of human TLR4 polymorphisms. These studies have shown that TLR4 function affects the initiation and progression of atherosclerosis. This article reviews the biological functions a...

  15. Histamine H4 receptor antagonists are superior to traditional antihistamines in the attenuation of experimental pruritus.

    Science.gov (United States)

    Dunford, Paul J; Williams, Kacy N; Desai, Pragnya J; Karlsson, Lars; McQueen, Daniel; Thurmond, Robin L

    2007-01-01

    Histamine is a potent mediator of itch in humans, yet histamine H(1) receptor antagonists have been shown to be of limited use in the treatment of certain chronic pruritic diseases. The histamine H(4) receptor is a recently described histamine receptor, expressed on hematopoietic cells, linked to the pathology of allergy and asthma. The contribution of the novel histamine H(4) receptor to histaminergic and allergic pruritus was investigated. Histamine and a selective histamine H(4) receptor agonist caused scratching responses in mice, which were almost completely attenuated in histamine H(4) receptor knockout mice or by pretreatment with the selective histamine H(4) receptor antagonist, JNJ 7777120. Pruritus induced by allergic mechanisms was also potently inhibited with histamine H(4) receptor antagonist treatment or in histamine H(4) receptor knockout mice. In all cases, the inhibitory effect of histamine H(4) receptor antagonist was greater than those observed with histamine H(1) receptor antagonists. The histamine H(4) receptor-mediated pruritus was shown to be independent of mast cells or other hematopoietic cells and may result from actions on peripheral neurons. These results demonstrate that the histamine H(4) receptor is involved in pruritic responses in mice to a greater extent than the histamine H(1) receptor. Histamine H(4) receptor antagonists may have therapeutic utility for treating chronic pruritic diseases in humans where histamine H(1) receptor antagonists are not effective.

  16. Insulin receptors

    International Nuclear Information System (INIS)

    Kahn, C.R.; Harrison, L.C.

    1988-01-01

    This book contains the proceedings on insulin receptors. Part A: Methods for the study of structure and function. Topics covered include: Method for purification and labeling of insulin receptors, the insulin receptor kinase, and insulin receptors on special tissues

  17. A knockout mutation of a constitutive GPCR in Tetrahymena decreases both G-protein activity and chemoattraction.

    Directory of Open Access Journals (Sweden)

    Thomas J Lampert

    Full Text Available Although G-protein coupled receptors (GPCRs are a common element in many chemosensory transduction pathways in eukaryotic cells, no GPCR or regulated G-protein activity has yet been shown in any ciliate. To study the possible role for a GPCR in the chemoresponses of the ciliate Tetrahymena, we have generated a number of macronuclear gene knockouts of putative GPCRs found in the Tetrahymena Genome database. One of these knockout mutants, called G6, is a complete knockout of a gene that we call GPCR6 (TTHERM_00925490. Based on sequence comparisons, the Gpcr6p protein belongs to the Rhodopsin Family of GPCRs. Notably, Gpcr6p shares highest amino acid sequence homologies to GPCRs from Paramecium and several plants. One of the phenotypes of the G6 mutant is a decreased responsiveness to the depolarizing ions Ba²⁺ and K⁺, suggesting a decrease in basal excitability (decrease in Ca²⁺ channel activity. The other major phenotype of G6 is a loss of chemoattraction to lysophosphatidic acid (LPA and proteose peptone (PP, two known chemoattractants in Tetrahymena. Using microsomal [³⁵S]GTPγS binding assays, we found that wild-type (CU427 have a prominent basal G-protein activity. This activity is decreased to the same level by pertussis toxin (a G-protein inhibitor, addition of chemoattractants, or the G6 mutant. Since the basal G-protein activity is decreased by the GPCR6 knockout, it is likely that this gene codes for a constitutively active GPCR in Tetrahymena. We propose that chemoattractants like LPA and PP cause attraction in Tetrahymena by decreasing the basal G-protein stimulating activity of Gpcr6p. This leads to decreased excitability in wild-type and longer runs of smooth forward swimming (less interrupted by direction changes towards the attractant. Therefore, these attractants may work as inverse agonists through the constitutively active Gpcr6p coupled to a pertussis-sensitive G-protein.

  18. Behavioral Phenotyping of Dopamine Transporter Knockout Rats: Compulsive Traits, Motor Stereotypies, and Anhedonia

    Directory of Open Access Journals (Sweden)

    Stefano Cinque

    2018-02-01

    Full Text Available Alterations in dopamine neurotransmission are generally associated with diseases such as attention-deficit/hyperactivity disorder (ADHD and obsessive-compulsive disorder (OCD. Such diseases typically feature poor decision making and lack of control on executive functions and have been studied through the years using many animal models. Dopamine transporter (DAT knockout (KO and heterozygous (HET mice, in particular, have been widely used to study ADHD. Recently, a strain of DAT KO rats has been developed (1. Here, we provide a phenotypic characterization of reward sensitivity and compulsive choice by adult rats born from DAT–HET dams bred with DAT–HET males, in order to further validate DAT KO rats as an animal model for preclinical research. We first tested DAT KO rats’ sensitivity to rewarding stimuli, provided by highly appetitive food or sweet water; then, we tested their choice behavior with an Intolerance-to-Delay Task (IDT. During these tests, DAT KO rats appeared less sensitive to rewarding stimuli than wild-type (WT and HET rats: they also showed a prominent hyperactive behavior with a rigid choice pattern and a wide number of compulsive stereotypies. Moreover, during the IDT, we tested the effects of amphetamine (AMPH and RO-5203648, a trace amine-associated receptor 1 (TAAR1 partial agonist. AMPH accentuated impulsive behaviors in WT and HET rats, while it had no effect in DAT KO rats. Finally, we measured the levels of tyrosine hydroxylase, dopamine receptor 2 (D2, serotonin transporter, and TAAR1 mRNA transcripts in samples of ventral striatum, finding no significant differences between WT and KO genotypes. Throughout this study, DAT KO rats showed alterations in decision-making processes and in motivational states, as well as prominent motor and oral stereotypies: more studies are warranted to fully characterize and efficiently use them in preclinical research.

  19. Repetitive grooming and sensorimotor abnormalities in an ephrin-A knockout model for Autism Spectrum Disorders.

    Science.gov (United States)

    Wurzman, Rachel; Forcelli, Patrick A; Griffey, Christopher J; Kromer, Lawrence F

    2015-02-01

    EphA receptors and ephrin-A ligands play important roles in neural development and synaptic plasticity in brain regions where expression persists into adulthood. Recently, EPHA3 and EPHA7 gene mutations were linked with Autism Spectrum Disorders (ASDs) and developmental neurological delays, respectively. Furthermore, deletions of ephrin-A2 or ephrin-A3, which exhibit high binding affinity for EphA3 and EphA7 receptors, are associated with subtle deficits in learning and memory behavior and abnormalities in dendritic spine morphology in the cortex and hippocampus in mice. To better characterize a potential role for these ligands in ASDs, we performed a comprehensive behavioral characterization of anxiety-like, sensorimotor, learning, and social behaviors in ephrin-A2/-A3 double knockout (DKO) mice. The predominant phenotype in DKO mice was repetitive and self-injurious grooming behaviors such as have been associated with corticostriatal circuit abnormalities in other rodent models of neuropsychiatric disorders. Consistent with ASDs specifically, DKO mice exhibited decreased preference for social interaction in the social approach assay, decreased locomotor activity in the open field, increased prepulse inhibition of acoustic startle, and a shift towards self-directed activity (e.g., grooming) in novel environments, such as marble burying. Although there were no gross deficits in cognitive assays, subtle differences in performance on fear conditioning and in the Morris water maze resembled traits observed in other rodent models of ASD. We therefore conclude that ephrin-A2/-A3 DKO mice have utility as a novel ASD model with an emphasis on sensory abnormalities and restricted, repetitive behavioral symptoms. Copyright © 2014 Elsevier B.V. All rights reserved.

  20. Antiatherogenic effects of oleanolic acid in apolipoprotein E knockout mice

    DEFF Research Database (Denmark)

    Buus, Niels Henrik; Hansson, Nicolaj Christopher; Rodriguez-Rodriguez, Rosalia

    2011-01-01

    Oleanolic acid (OA) is a plant triterpenoid steroid with potentially antiatherogenic properties. We investigated whether OA affected atherosclerosis development and vascular function in apolipoprotein E knockout (ApoE(-/-)) mice. ApoE(-/-) mice were fed a high cholesterol Western-type diet...... in combination with OA (100 mg/kg/day), fluvastatin (5 mg/kg/day) or vehicle, with wild type (WT) mice serving as controls. After 8 weeks of treatment atherosclerotic plaque areas in the aortic arch and plasma lipid concentrations were determined. Vasoconstriction and relaxation of the proximal part of aorta...... were investigated in vitro. Inducible nitric oxide synthase (iNOS) was visualized using immunoblotting. As opposed to WT and fluvastatin- and vehicle-treated mice, OA-fed ApoE(-/-) mice gained no weight during the treatment period. Plasma concentrations of total-cholesterol and triglyceride were...

  1. Drop tests of the Three Mile Island knockout canister

    International Nuclear Information System (INIS)

    Box, W.D.; Aaron, W.S.; Shappert, L.B.; Childress, P.C.; Quinn, G.J.; Smith, J.V.

    1987-01-01

    A type of Three Mile Island Unit 2 (TMI-2) defueling canister, called a ''knockout'' canister, was subjected to a series of drop tests at the Oak Ridge National Laboratory's Drop Test Facility. These tests confirmed the structural integrity of internal fixed neutron poisons in support of a request for NRC licensing of this type of canister for the shipment of TMI-2 reactor fuel debris to the Idaho National Engineering Laboratory (INEL) for the Core Examination R and D Program. This report presents the data generated and the results obtained from a series of four drop tests that included two drops with the test assembly in the vertical position and two drops with the assembly in the horizontal position

  2. Medium effects on spin observables of proton knockout reactions

    Energy Technology Data Exchange (ETDEWEB)

    Krein, G. [Instituto de Fisica Teorica (IFT), Sao Paulo, SP (Brazil); Maris, T.A.J.; Rodrigues, B.B.; Veit, E.A. [Rio Grande do Sul Univ., Porto Alegre, RS (Brazil). Inst. de Fisica

    1994-07-01

    Medium modifications of the properties of bound nucleons and mesons are investigated by means of medium energy quasi free proton knockout reactions with polarized incident protons. The sensitivity of the spin observables of these reactions to modifications of the nucleon and meson properties is studied using the Bonn one-boson exchange model of the nucleon-nucleon interaction. A method proposed to extract the pp analysing power in medium from the (p, 2 p) asymmetries indicates a reduction of this quantity compared to its free space value. This reduction is linked to modifications of masses and coupling constants of the nucleons and mesons in the nucleus. The implications of these modifications for another spin observable to be measured in the future are discussed. (author). 39 refs, 9 figs.

  3. Piroxicam treatment augments bone abnormalities in interleukin-10 knockout mice

    DEFF Research Database (Denmark)

    Holgersen, Kristine; Dobie, Ross; Farquharson, Colin

    2015-01-01

    BACKGROUND: Osteoporosis and fractures are common complications of inflammatory bowel disease. The pathogenesis is multifactorial and has been partly attributed to intestinal inflammation. The aim of this study was to evaluate bone status and assess the association between bone loss and gut...... inflammation in an experimental colitis model. METHODS: Colitis was induced in interleukin-10 knockout mice (PAC IL-10 k.o.) by peroral administration of piroxicam for 12 days. The degree of colitis was assessed by clinical, macroscopic, and microscopic evaluation. Trabecular and cortical bone...... microarchitecture of tibia were determined using micro-computed tomography. Moreover, the serum levels of bone formation and bone resorption biomarkers were measured, and inflammatory protein profiling was performed on colons. RESULTS: PAC IL-10 k.o. mice developed severe colitis, characterized by hyperplasia...

  4. Preaxial Polydactyly in Sost/Sostdc1 Double Knockouts

    Energy Technology Data Exchange (ETDEWEB)

    Yee, C M; Collette, N M; Loots, G G

    2011-07-29

    In the United States, {approx}5% are born with congenital birth defects due to abnormal function of cellular processes and interactions. Sclerosteosis, a rare autosomal recessive disease, causes hyperostosis of the axial and appendicular skeleton, and patients present radial deviation, digit syndactyly, nail dysplasia, and overall high bone mineral density. Sclerosteosis is due to a loss of function of sclerostin (Sost). Sost is a Wnt (abbrev.) antagonist; when mutated, nonfunctional Sost results in hyperactive osteoblast activity which leads to abnormal high bone mass. Previous studies have shown that Sost overexpression in transgenic mice causes reduced bone mineral density and a variety of limb phenotypes ranging from lost, fused, and split phalanges. Consistent with clinical manifestations of Sclerosteosis, Sost knockout mice exhibit increased generalized bone mineral density and syndactyly of the digits. Sostdc1 is a paralog of Sost that has also been described as an antagonist of Wnt signaling, in developing tooth buds. Unlike Sost knockouts, Sostdc1 null mice do not display any limb abnormalities. To determine if Sost and Sostdc1 have redundant functions during limb patterning, we examined Sost; Sostdc1 mice determined that they exhibit a novel preaxial polydactyly phenotype with a low penetrance. LacZ staining, skeletal preparations, and in situ hybridization experiments were used to help characterize this novel phenotype and understand how this phenotype develops. We find Sost and Sostdc1 to have complementary expression patterns during limb development, and the loss of their expression alters the transcription of several key limb regulators, such as Fgf8, Shh and Grem.

  5. Unique nuclear vacuoles in the motor neurons of conditional ADAR2-knockout mice.

    Science.gov (United States)

    Sasaki, Shoichi; Takenari Yamashita; Hideyama, Takuto; Kwak, Shin

    2014-03-06

    A reduction in adenosine deaminase acting on RNA 2 (ADAR2) activity causes the death of spinal motor neurons specifically via the GluA2 Q/R site-RNA editing failure in sporadic amyotrophic lateral sclerosis (ALS). We studied, over time, the spinal cords of ADAR2-knockout mice, which are the mechanistic model mice for sporadic ALS, using homozygous ADAR2(flox/flox)/VAChT-Cre.Fast (AR2), homozygous ADAR2(flox/flox)/VAChT-Cre.Slow (AR2Slow), and heterozygous ADAR2(flox/+)/VAChT-Cre.Fast (AR2H) mice. The conditional ADAR2-knockout mice were divided into 3 groups by stage: presymptomatic (AR2H mice), early symptomatic (AR2 mice, AR2H mice) and late symptomatic (AR2Slow mice). Light-microscopically, some motor neurons in AR2 and AR2H mice (presymptomatic) showed simple neuronal atrophy and astrogliosis, and AR2H (early symptomatic) and AR2Slow mice often showed vacuoles predominantly in motor neurons. The number of vacuole-bearing anterior horn neurons decreased with the loss of anterior horn neurons in AR2H mice after 40 weeks of age. Electron-microscopically, in AR2 mice, while the cytoplasm of normal-looking motor neurons was almost always normal-appearing, the interior of dendrites was frequently loose and disorganized. In AR2H and AR2Slow mice, large vacuoles without a limiting membrane were observed in the anterior horns, preferentially in the nuclei of motor neurons, astrocytes and oligodendrocytes. Nuclear vacuoles were not observed in AR2res (ADAR2(flox/flox)/VAChT-Cre.Fast/GluR-B(R/R)) mice, in which motor neurons express edited GluA2 in the absence of ADAR2. These findings suggest that ADAR2-reduction is associated with progressive deterioration of nuclear architecture, resulting in vacuolated nuclei due to a Ca(2+)-permeable AMPA receptor-mediated mechanism. Copyright © 2014 Elsevier B.V. All rights reserved.

  6. Erythropoiesis- and Thrombopoiesis-Characterizing Parameters in Adenosine A(3) Receptor Knock-Out Mice

    Czech Academy of Sciences Publication Activity Database

    Hofer, Michal; Pospíšil, Milan; Dušek, L.; Hoferová, Zuzana; Weiterová, Lenka

    2013-01-01

    Roč. 62, č. 3 (2013), s. 305-311 ISSN 0862-8408 R&D Projects: GA ČR(CZ) GAP303/11/0128 Institutional support: RVO:68081707 Keywords : ELEVATING EXTRACELLULAR ADENOSINE * COLONY-STIMULATING FACTOR * HEMATOPOIETIC PROGENITOR CELLS Subject RIV: BO - Biophysics Impact factor: 1.487, year: 2013

  7. Atherosclerosis in low density lipoprotein receptor knockout mice fed cholesterol and soybean oil

    DEFF Research Database (Denmark)

    Mortensen, Alicja; Olsen, P.; Frandsen, H.

    1999-01-01

    In order to study aortic atherosclerosis and atherosclerotic response to dietary cholesterol and soybean oil in homozygous LDLR-/- mice, the 16 weeks old animals were randomized in 4 groups either fed standard diet (no cholesterol added, group I, 12 male and 12 female), standard diet added 0.......5% cholesterol (group II, 12 male and 12 female), standard diet added 10% soybean oil (group Iii, 7 male) or standard diet added 0.5% cholesterol and 10% soybean oil (group IV, 7 male) for 14 weeks. At termination, the plasma cholesterol of males was: 9.4 mmol/I +/- 0.3 (SD) (group I), 34.4 +/- 6.2 (group II), 9...

  8. Proximal Gut Mucosal Epithelial Homeostasis in Aged IL-1 Type I Receptor Knockout Mice After Starvation

    Science.gov (United States)

    2011-08-01

    L , Loach D, Tannock G, Haller D. Lactobacillus reuteri 100-23 transiently activates intestinal epithelial cells of mice that have a complex...Rondeau V, Dequae-Merchadou L , Salles-Montaudon N, Emeriau JP, Manciet G, Dartigues JF. A multi-center trial of the effects of oral nutritional...supplementation during acute illness. Am J Med. 2006; 119:693. [PubMed: 16887416] 9. Howard L , Malone M. Clinical outcome of geriatric patients in the

  9. Triheteromeric NMDA Receptors at Hippocampal Synapses

    Science.gov (United States)

    Tovar, Kenneth R.; McGinley, Matthew J.; Westbrook, Gary L.

    2013-01-01

    NMDA receptors are composed of two GluN1 (N1) and two GluN2 (N2) subunits. Constituent N2 subunits control the pharmacological and kinetic characteristics of the receptor. NMDA receptors in hippocampal or cortical neurons are often thought of as diheteromeric, i.e., containing only one type of N2 subunit. However, triheteromeric receptors with more than one type of N2 subunit also have been reported and the relative contribution of di- and triheteromeric NMDA receptors at synapses has been difficult to assess. Because wild-type hippocampal principal neurons express N1, N2A and N2B, we used cultured hippocampal principal neurons from N2A and N2B-knockout mice as templates for diheteromeric synaptic receptors. Summation of N1/N2B and N1/N2A excitatory postsynaptic currents could not account for the deactivation kinetics of wild-type excitatory postsynaptic currents (EPSCs) however. To make a quantitative estimate of NMDA receptor subtypes at wild-type synapses, we used the deactivation kinetics, as well as the effects of the competitive antagonist NVP-AAM077. Our results indicate that three types of NMDA receptors contribute to the wild-type EPSC, with at least two-thirds being triheteromeric receptors. Functional isolation of synaptic triheteromeric receptors revealed deactivation kinetics and pharmacology distinct from either diheteromeric receptor subtype. Because of differences in open probability, synaptic triheteromeric receptors outnumbered N1/N2A receptors by 5.8 to 1 and N1/N2B receptors by 3.2 to 1. Our results suggest that triheteromeric NMDA receptors must be either preferentially assembled or preferentially localized at synapses. PMID:23699525

  10. Immunosympathectomy as the first phenotypic knockout with antibodies.

    Science.gov (United States)

    Cattaneo, Antonino

    2013-03-26

    In a PNAS Classic Article published in 1960, Rita Levi-Montalcini offered formal and conclusive proof that endogenous NGF was responsible for the survival of sympathetic neurons in vivo. Thus ended an experimental tour de force lasting a decade, starting with the demonstration that a humoral factor, produced from a tumor transplanted in a chicken embryo, was responsible for stimulating outgrowth of nerve fibers from sympathetic and sensory neurons. From a more general methodological point of view, this work provided a breakthrough in the quest to achieve targeted loss of function and experimentally validate the function of biological molecules. Finally, this work provided an example of the ablation of a specific neuronal subpopulation in an otherwise intact nervous system, an immunological knife of unsurpassed effectiveness and precision. The novelty and the importance of the PNAS Classic Article is discussed here, collocating it within the context of the particular moment of the NGF discovery saga, of Rita Levi-Montalcini's scientific and academic career, and of the general scientific context of those years. This seminal work, involving the use of antibodies for phenotypic knockout in vivo, planted seeds that were to bear new fruit many years later with the advent of monoclonal antibodies and recombinant antibody technologies.

  11. Arterial injury promotes medial chondrogenesis in Sm22 knockout mice.

    Science.gov (United States)

    Shen, Jianbin; Yang, Maozhou; Jiang, Hong; Ju, Donghong; Zheng, Jian-Pu; Xu, Zhonghui; Liao, Tang-Dong; Li, Li

    2011-04-01

    Expression of SM22 (also known as SM22alpha and transgelin), a vascular smooth muscle cells (VSMCs) marker, is down-regulated in arterial diseases involving medial osteochondrogenesis. We investigated the effect of SM22 deficiency in a mouse artery injury model to determine the role of SM22 in arterial chondrogenesis. Sm22 knockout (Sm22(-/-)) mice developed prominent medial chondrogenesis 2 weeks after carotid denudation as evidenced by the enhanced expression of chondrogenic markers including type II collagen, aggrecan, osteopontin, bone morphogenetic protein 2, and SRY-box containing gene 9 (SOX9). This was concomitant with suppression of VSMC key transcription factor myocardin and of VSMC markers such as SM α-actin and myosin heavy chain. The conversion tendency from myogenesis to chondrogenesis was also observed in primary Sm22(-/-) VSMCs and in a VSMC line after Sm22 knockdown: SM22 deficiency altered VSMC morphology with compromised stress fibre formation and increased actin dynamics. Meanwhile, the expression level of Sox9 mRNA was up-regulated while the mRNA levels of myocardin and VSMC markers were down-regulated, indicating a pro-chondrogenic transcriptional switch in SM22-deficient VSMCs. Furthermore, the increased expression of SOX9 was mediated by enhanced reactive oxygen species production and nuclear factor-κB pathway activation. These findings suggest that disruption of SM22 alters the actin cytoskeleton and promotes chondrogenic conversion of VSMCs.

  12. Modeling fragile X syndrome in the Fmr1 knockout mouse

    Science.gov (United States)

    Kazdoba, Tatiana M.; Leach, Prescott T.; Silverman, Jill L.; Crawley, Jacqueline N.

    2014-01-01

    Summary Fragile X Syndrome (FXS) is a commonly inherited form of intellectual disability and one of the leading genetic causes for autism spectrum disorder. Clinical symptoms of FXS can include impaired cognition, anxiety, hyperactivity, social phobia, and repetitive behaviors. FXS is caused by a CGG repeat mutation which expands a region on the X chromosome containing the FMR1 gene. In FXS, a full mutation (> 200 repeats) leads to hypermethylation of FMR1, an epigenetic mechanism that effectively silences FMR1 gene expression and reduces levels of the FMR1 gene product, fragile X mental retardation protein (FMRP). FMRP is an RNA-binding protein that is important for the regulation of protein expression. In an effort to further understand how loss of FMR1 and FMRP contribute to FXS symptomology, several FXS animal models have been created. The most well characterized rodent model is the Fmr1 knockout (KO) mouse, which lacks FMRP protein due to a disruption in its Fmr1 gene. Here, we review the behavioral phenotyping of the Fmr1 KO mouse to date, and discuss the clinical relevance of this mouse model to the human FXS condition. While much remains to be learned about FXS, the Fmr1 KO mouse is a valuable tool for understanding the repercussions of functional loss of FMRP and assessing the efficacy of pharmacological compounds in ameliorating the molecular and behavioral phenotypes relevant to FXS. PMID:25606362

  13. Pheochromocytoma cell lines from heterozygous neurofibromatosis knockout mice.

    Science.gov (United States)

    Powers, J F; Evinger, M J; Tsokas, P; Bedri, S; Alroy, J; Shahsavari, M; Tischler, A S

    2000-12-01

    Transplantable tumors and cell lines have been developed from pheochromocytomas arising in mice with a heterozygous knockout mutation of the neurofibromatosis gene, Nf1. Nf1 encodes a ras-GTPase-activating protein, neurofibromin, and mouse pheochromocytoma (MPC) cells in primary cultures typically show extensive spontaneous neuronal differentiation that may result from the loss of the remaining wild-type allele and defective regulation of ras signaling. However, all MPC cell lines express neurofibromin, suggesting that preservation of the wild-type allele may be required to permit the propagation of MPC cells in vitro. MPC lines differ from PC12 cells in that they express both endogenous phenylethanolamine N-methyltransferase (PNMT) and full-length PNMT reporter constructs. PNMT expression is increased by dexamethasone and by cell-cell contact in suspension cultures. Mouse pheochromocytomas are a new tool for studying genes and signaling pathways that regulate cell growth and differentiation in adrenal medullary neoplasms and are a unique model for studying the regulation of PNMT expression.

  14. Orexin knockout mice exhibit impaired spatial working memory.

    Science.gov (United States)

    Dang, Ruozhi; Chen, Qiuhan; Song, Jie; He, Chao; Zhang, Jun; Xia, Jianxia; Hu, Zhian

    2018-03-06

    Orexins play a crucial role in the maintenance of arousal and are involved in the modulation of diverse physiological process, including cognitive function. Recent data have suggested that orexins are involved in learning and memory processes. The purpose of this study was to assess the effects of orexin deficiency on working memory. A delayed non-matching-to-place T-maze task was used to evaluate spatial working memory in mice lacking orexin prepro-peptide (orexin knockout; KO) and wild-type controls. We demonstrated that the number of correct choices in the orexin KO mice became lower than that of the controls over training. In an object exploration task, the controls explored the displaced object more than the mutants did, whereas this difference was not observed for the nondisplaced objects in either group. The orexin KO mice showed locomotor activity comparable to the control mice in terms of total distance traveled across training in both the object exploration task and the open field test. These findings indicate that the orexin system plays an important role in working memory of spatial cues. Copyright © 2018 Elsevier B.V. All rights reserved.

  15. Identification of a large set of rare complete human knockouts.

    Science.gov (United States)

    Sulem, Patrick; Helgason, Hannes; Oddson, Asmundur; Stefansson, Hreinn; Gudjonsson, Sigurjon A; Zink, Florian; Hjartarson, Eirikur; Sigurdsson, Gunnar Th; Jonasdottir, Adalbjorg; Jonasdottir, Aslaug; Sigurdsson, Asgeir; Magnusson, Olafur Th; Kong, Augustine; Helgason, Agnar; Holm, Hilma; Thorsteinsdottir, Unnur; Masson, Gisli; Gudbjartsson, Daniel F; Stefansson, Kari

    2015-05-01

    Loss-of-function mutations cause many mendelian diseases. Here we aimed to create a catalog of autosomal genes that are completely knocked out in humans by rare loss-of-function mutations. We sequenced the whole genomes of 2,636 Icelanders and imputed the sequence variants identified in this set into 101,584 additional chip-genotyped and phased Icelanders. We found a total of 6,795 autosomal loss-of-function SNPs and indels in 4,924 genes. Of the genotyped Icelanders, 7.7% are homozygotes or compound heterozygotes for loss-of-function mutations with a minor allele frequency (MAF) below 2% in 1,171 genes (complete knockouts). Genes that are highly expressed in the brain are less often completely knocked out than other genes. Homozygous loss-of-function offspring of two heterozygous parents occurred less frequently than expected (deficit of 136 per 10,000 transmissions for variants with MAF <2%, 95% confidence interval (CI) = 10-261).

  16. Sortilin Associates with Trk Receptors to Enhance Anterograde Transport and Signaling by Neurotrophins

    DEFF Research Database (Denmark)

    Vægter, Christian Bjerggaard

      Neurotrophins (NT) are neuronal growth factors essential for development and maintenance of the nervous system. They are released in two forms with opposing biological functions. The proforms induce apoptosis by engaging a death signaling complex comprising the p75NTR neurotrophin receptor...... and survival has been extensively studied in knockout mouse models. We recently generated a Sortilin knockout mouse model (Sort1-/-) and showed that the receptor is essential for p75NTR to induce death of neurons during certain stages of development and aging and upon brain injury (Jansen et al, Nat. Neurosci...

  17. GLUT2 and the incretin receptors are involved in glucose-induced incretin secretion

    DEFF Research Database (Denmark)

    Cani, Patrice D; Holst, Jens Juul; Drucker, Daniel J

    2007-01-01

    to those described for beta-cells, brain and hepatoportal sensors. We determined the role of GLUT2, GLP-1 or GIP receptors in glucose-induced incretins secretion, in the corresponding knockout mice. GLP-1 secretion was reduced in all mutant mice, while GIP secretion did not require GLUT2. Intestinal GLP-1...... content was reduced only in GIP and GLUT2 receptors knockout mice suggesting that this impairment could contribute to the phenotype. Intestinal GIP content was similar in all mice studied. Furthermore, the impaired incretins secretion was associated with a reduced glucose-stimulated insulin secretion...

  18. Prenatal nicotine exposure increases hyperventilation in α4-knock-out mice during mild asphyxia.

    Science.gov (United States)

    Avraam, Joanne; Cohen, Gary; Drago, John; Frappell, Peter B

    2015-03-01

    Prenatal nicotine exposure alters breathing and ventilatory responses to stress through stimulation of nicotine acetylcholine receptors (nAChRs). We tested the hypothesis that α4-containing nAChRs are involved in mediating the effects of prenatal nicotine exposure on ventilatory and metabolic responses to intermittent mild asphyxia (MA). Using open-flow plethysmography, we measured ventilation (V̇(E)) and rate of O2 consumption ( V̇(O2)) of wild-type (WT) and α4-knock-out (KO) mice, at postnatal (P) days 1-2 and 7-8, with and without prenatal nicotine exposure (6 mg kg(-1) day(-1) beginning on embryonic day 14). Mice were exposed to seven 2 min cycles of mild asphyxia (10% O2 and 5% CO2), each interspersed with 2 min of air. Compared to WT, α4 KO mice had increased air V̇(E) and V̇(O2) at P7-8, but not P1-2. Irrespective of age, genotype had no effect on the hyperventilatory response (increase in V̇(E)/V̇(O2)) to MA. At P1-2, nicotine suppressed air V̇(E) and V̇(O2) in both genotypes but did not affect the hyperventilatory response to MA. At P7-8 nicotine suppressed air V̇(E) and V̇(O2) of only α4 KO's but also significantly enhanced V̇(E) during MA (nearly double that of WT; pprenatal nicotine exposure on ventilatory and metabolic interactions and responses to stress. Copyright © 2015 Elsevier B.V. All rights reserved.

  19. Pregnenolone rescues schizophrenia-like behavior in dopamine transporter knockout mice.

    Directory of Open Access Journals (Sweden)

    Peiyan Wong

    Full Text Available Pregnenolone belongs to a class of endogenous neurosteroids in the central nervous system (CNS, which has been suggested to enhance cognitive functions through GABA(A receptor signaling by its metabolites. It has been shown that the level of pregnenolone is altered in certain brain areas of schizophrenic patients, and clozapine enhances pregnenolone in the CNS in rats, suggesting that pregnenolone could be used to treat certain symptoms of schizophrenia. In addition, early phase proof-of-concept clinical trials have indicated that pregnenolone is effective in reducing the negative symptoms and cognitive deficits of schizophrenia patients. Here, we evaluate the actions of pregnenolone on a mouse model for schizophrenia, the dopamine transporter knockout mouse (DAT KO. DAT KO mice mirror certain symptoms evident in patients with schizophrenia, such as the psychomotor agitation, stereotypy, deficits of prepulse inhibition and cognitive impairments. Following acute treatment, pregnenolone was found to reduce the hyperlocomotion, stereotypic bouts and pre-pulse inhibition (PPI deficits in DAT KO mice in a dose-dependent manner. At 60 mg/kg of pregnenolone, there were no significant differences in locomotor activities and stereotypy between wild-type and DAT KO mice. Similarly, acute treatment of 60 mg/kg of pregnenolone fully rescued PPI deficits of DAT KO mice. Following chronic treatment with pregnenolone at 60 mg/kg, the cognitive deficits of DAT KO mice were rescued in the paradigms of novel object recognition test and social transmission of food preference test. Pregnenolone thus holds promise as a therapeutic candidate in schizophrenia.

  20. Tissue inhibitor of metalloproteinase-3 knockout mice exhibit enhanced energy expenditure through thermogenesis.

    Directory of Open Access Journals (Sweden)

    Yohsuke Hanaoka

    Full Text Available Tissue inhibitors of metalloproteinases (TIMPs regulate matrix metalloproteinase activity and maintain extracellular matrix homeostasis. Although TIMP-3 has multiple functions (e.g., apoptosis, inhibition of VEGF binding to VEGF receptor, and inhibition of TNFα converting enzyme, its roles in thermogenesis and metabolism, which influence energy expenditure and can lead to the development of metabolic disorders when dysregulated, are poorly understood. This study aimed to determine whether TIMP-3 is implicated in metabolism by analyzing TIMP-3 knockout (KO mice. TIMP-3 KO mice had higher body temperature, oxygen consumption, and carbon dioxide production than wild-type (WT mice, although there were no differences in food intake and locomotor activity. These results suggest that metabolism is enhanced in TIMP-3 KO mice. Real-time PCR analysis showed that the expression of PPAR-δ, UCP-2, NRF-1 and NRF-2 in soleus muscle, and PGC-1α and UCP-2 in gastrocnemius muscle, was higher in TIMP-3 KO mice than in WT mice, suggesting that TIMP-3 deficiency may increase mitochondrial activity. When exposed to cold for 8 hours to induce thermogenesis, TIMP-3 KO mice had a higher body temperature than WT mice. In the treadmill test, oxygen consumption and carbon dioxide production were higher in TIMP-3 KO mice both before and after starting exercise, and the difference was more pronounced after starting exercise. Our findings suggest that TIMP-3 KO mice exhibit enhanced metabolism, as reflected by a higher body temperature than WT mice, possibly due to increased mitochondrial activity. Given that TIMP-3 deficiency increases energy expenditure, TIMP-3 may present a novel therapeutic target for preventing metabolic disorders.

  1. Features of emotional and social behavioral phenotypes of calsyntenin2 knockout mice.

    Science.gov (United States)

    Ranneva, S V; Pavlov, K S; Gromova, A V; Amstislavskaya, T G; Lipina, T V

    2017-08-14

    Calsyntenin-2 (Clstn2) is the synaptic protein that belongs to the super family of cadherins, playing an important role in learning and memory. We recently reported that Clstn2 knockout mice (Clstn2-KO) have a deficit of GABAergic interneurons coupled with hyperactivity and deficient spatial memory. Given, that impaired functioning of GABA receptors is linked to several psychopathologies, including anxiety and autism, we sought to further characterize Clstn2-KO mice with respect to emotional and social behavior. Clstn2-KO males and females were tested in the elevated plus-maze (EPM), open field (OF), forced swim test, social affiliation and recognition test, social transmission of food preference (STFP), dyadic social interactions and marble burying test. Clstn2-KO mice demonstrated high exploration and hyperactivity in the dimly lit EPM that affect anxiety parameters. In contrast, in a more adverse situation in the OF have increased emotionality in Clstn2-KO males, not females. Assessment of hyperactivity for prolong period in the OF showed that Clstn2-KO animals were able to decline their hyperactivity, but their ambulation still remained higher than in WT littermates. Additionally, Clstn2-KO mice expressed stereotyped behavior. Strikingly, analysis of social behavior identified deficient social motivation and social recognition only in Clstn2-KO males, but not in females. Further analysis of social communication in the STFP and direct observation of agonistic interactions confirmed the reduced social behavior in Clstn2-KO males. Altogether, current results showed Clstn2 gene and sex interactions on socio-emotional performance in mice, suggesting a possible role of calsyntenin2 in psychopathological mechanisms of autism. Copyright © 2017 Elsevier B.V. All rights reserved.

  2. Comprehensive behavioral analysis of pituitary adenylate cyclase-activating polypeptide (PACAP knockout mice

    Directory of Open Access Journals (Sweden)

    Satoko eHattori

    2012-10-01

    Full Text Available Pituitary adenylate cyclase-activating polypeptide (PACAP is a neuropeptide acting as a neurotransmitter, neuromodulator, or neurotrophic factor. PACAP is widely expressed throughout the brain and exerts its functions through the PACAP-specific receptor (PAC1. Recent studies reveal that genetic variants of the PACAP and PAC1 genes are associated with mental disorders, and several behavioral abnormalities of PACAP knockout (KO mice are reported. However, an insufficient number of backcrosses was made using PACAP KO mice on the C57BL/6J background due to their postnatal mortality. To elucidate the effects of PACAP on neuropsychiatric function, the PACAP gene was knocked out in F1 hybrid mice (C57BL/6J x 129SvEv for appropriate control of the genetic background. The PACAP KO mice were then subjected to a behavioral test battery. PACAP deficiency had no significant effects on neurological screen. As shown previously, the mice exhibited significantly increased locomotor activity in a novel environment and abnormal anxiety-like behavior, while no obvious differences between genotypes were shown in home cage activity. In contrast to previous reports, the PACAP KO mice showed normal prepulse inhibition and slightly decreased depression-like behavior. Previous study demonstrates that the social interaction in a resident-intruder test was decreased in PACAP KO mice. On the other hand, we showed that PACAP KO mice exhibited increased social interaction in Crawley’s three-chamber social approach test, although PACAP KO had no significant impact on social interaction in a home cage. PACAP KO mice also exhibited mild performance deficit in working memory in an eight-arm radial maze and the T-maze, while they did not show any significant abnormalities in the left-right discrimination task in the T-maze. These results suggest that PACAP has an important role in the regulation of locomotor activity, social behavior, anxiety-like behavior and, potentially

  3. The hallucinogen N,N-dimethyltryptamine (DMT) is an endogenous sigma-1 receptor regulator.

    Science.gov (United States)

    Fontanilla, Dominique; Johannessen, Molly; Hajipour, Abdol R; Cozzi, Nicholas V; Jackson, Meyer B; Ruoho, Arnold E

    2009-02-13

    The sigma-1 receptor is widely distributed in the central nervous system and periphery. Originally mischaracterized as an opioid receptor, the sigma-1 receptor binds a vast number of synthetic compounds but does not bind opioid peptides; it is currently considered an orphan receptor. The sigma-1 receptor pharmacophore includes an alkylamine core, also found in the endogenous compound N,N-dimethyltryptamine (DMT). DMT acts as a hallucinogen, but its receptor target has been unclear. DMT bound to sigma-1 receptors and inhibited voltage-gated sodium ion (Na+) channels in both native cardiac myocytes and heterologous cells that express sigma-1 receptors. DMT induced hypermobility in wild-type mice but not in sigma-1 receptor knockout mice. These biochemical, physiological, and behavioral experiments indicate that DMT is an endogenous agonist for the sigma-1 receptor.

  4. CRISPR/Cas9-based knockouts reveal that CpRLP1 is a negative regulator of the sex pheromone PR-IP in the Closterium peracerosum-strigosum-littorale complex.

    Science.gov (United States)

    Kanda, Naho; Ichikawa, Machiko; Ono, Ayaka; Toyoda, Atsushi; Fujiyama, Asao; Abe, Jun; Tsuchikane, Yuki; Nishiyama, Tomoaki; Sekimoto, Hiroyuki

    2017-12-19

    Heterothallic strains of the Closterium peracerosum-strigosum-littorale (C. psl.) complex have two sexes, mating-type plus (mt + ) and mating-type minus (mt - ). Conjugation between these two sexes is regulated by two sex pheromones, protoplast-release-inducing protein (PR-IP) and PR-IP Inducer, which are produced by mt + and mt - cells, respectively. PR-IP mediates the release of protoplasts from mt - cells during mating. In this study, we examined the mechanism of action of CpRLP1 (receptor-like protein 1), which was previously identified in a cDNA microarray analysis as one of the PR-IP-inducible genes. Using CRISPR/Cas9 technology, we generated CpRLP1 knockout mutants in mt - cells of the C. psl. complex. When the knockout mt - cells were mixed with wild-type mt + cells, conjugation was severely reduced. Many cells released protoplasts without pairing, suggesting a loss of synchronization between the two mating partners. Furthermore, the knockout mutants were hypersensitive to PR-IP. We conclude that CpRLP1 is a negative regulator of PR-IP that regulates the timing of protoplast release in conjugating C. psl. cells. As the first report of successful gene knockout in the class Charophyceae, this study provides a basis for research aimed at understanding the ancestral roles of genes that are indispensable for the development of land plants.

  5. Improved cognitive flexibility in serotonin transporter knockout rats is unchanged following chronic cocaine self-administration

    NARCIS (Netherlands)

    Nonkes, L.J.; Maes, J.H.R.; Homberg, J.R.

    2013-01-01

    Cocaine dependence is associated with orbitofrontal cortex (OFC)-dependent cognitive inflexibility in both humans and laboratory animals. A critical question is whether cocaine self-administration affects pre-existing individual differences in cognitive flexibility. Serotonin transporter knockout

  6. Serotonin transporter knockout rats show improved strategy set-shifting and reduced latent inhibition

    NARCIS (Netherlands)

    Nonkes, L.J.P.; Vondervoort, I.I.G.M. van de; Leeuw, M.J.C. de; Wijlaars, L.P.; Maes, J.H.R.; Homberg, J.R.

    2012-01-01

    Behavioral flexibility is a cognitive process depending on prefrontal areas allowing adaptive responses to environmental changes. Serotonin transporter knockout (5-HTT−/−) rodents show improved reversal learning in addition to orbitofrontal cortex changes. Another form of behavioral flexibility,

  7. Serotonin transporter knockout rats show improved strategy set-shifting and reduced latent inhibition.

    NARCIS (Netherlands)

    Nonkes, L.J.P.; Vondervoort, I.I. van de; Leeuw, M.J. de; Wijlaars, L.P.; Maes, J.H.; Homberg, J.R.

    2012-01-01

    Behavioral flexibility is a cognitive process depending on prefrontal areas allowing adaptive responses to environmental changes. Serotonin transporter knockout (5-HTT(-/-)) rodents show improved reversal learning in addition to orbitofrontal cortex changes. Another form of behavioral flexibility,

  8. Lactobacillus acidophilus ATCC 4356 Prevents Atherosclerosis via Inhibition of Intestinal Cholesterol Absorption in Apolipoprotein E-Knockout Mice

    Science.gov (United States)

    Wang, Jinfeng; Quan, Guihua; Wang, Xiaojun; Yang, Longfei; Zhong, Lili

    2014-01-01

    The objective of this study was to investigate the effect of Lactobacillus acidophilus ATCC 4356 on the development of atherosclerosis in apolipoprotein E-knockout (ApoE−/−) mice. Eight-week-old ApoE−/− mice were fed a Western diet with or without L. acidophilus ATCC 4356 daily for 16 weeks. L. acidophilus ATCC 4356 protected ApoE−/− mice from atherosclerosis by reducing their plasma cholesterol levels from 923 ± 44 to 581 ± 18 mg/dl, likely via a marked decrease in cholesterol absorption caused by modulation of Niemann-Pick C1-like 1 (NPC1L1). In addition, suppression of cholesterol absorption induced reverse cholesterol transport (RCT) in macrophages through the peroxisome proliferator-activated receptor/liver X receptor (PPAR/LXR) pathway. Fecal lactobacillus and bifidobacterium counts were significantly (P intestine, colon, and feces during the feeding trial. The bacterial levels remained high even after the administration of lactic acid bacteria had been stopped for 2 weeks. These results suggest that administration of L. acidophilus ATCC 4356 can protect against atherosclerosis through the inhibition of intestinal cholesterol absorption. Therefore, L. acidophilus ATCC 4356 may be a potential therapeutic material for preventing the progression of atherosclerosis. PMID:25261526

  9. Central nervous system-specific knockout of steroidogenic factor 1 results in increased anxiety-like behavior.

    Science.gov (United States)

    Zhao, Liping; Kim, Ki Woo; Ikeda, Yayoi; Anderson, Kimberly K; Beck, Laurel; Chase, Stephanie; Tobet, Stuart A; Parker, Keith L

    2008-06-01

    Steroidogenic factor 1 (SF-1) plays key roles in adrenal and gonadal development, expression of pituitary gonadotropins, and development of the ventromedial hypothalamic nucleus (VMH). If kept alive by adrenal transplants, global knockout (KO) mice lacking SF-1 exhibit delayed-onset obesity and decreased locomotor activity. To define specific roles of SF-1 in the VMH, we used the Cre-loxP system to inactivate SF-1 in a central nervous system (CNS)-specific manner. These mice largely recapitulated the VMH structural defect seen in mice lacking SF-1 in all tissues. In multiple behavioral tests, mice with CNS-specific KO of SF-1 had significantly more anxiety-like behavior than wild-type littermates. The CNS-specific SF-1 KO mice had diminished expression or altered distribution in the mediobasal hypothalamus of several genes whose expression has been linked to stress and anxiety-like behavior, including brain-derived neurotrophic factor, the type 2 receptor for CRH (Crhr2), and Ucn 3. Moreover, transfection and EMSAs support a direct role of SF-1 in Crhr2 regulation. These findings reveal important roles of SF-1 in the hypothalamic expression of key regulators of anxiety-like behavior, providing a plausible molecular basis for the behavioral effect of CNS-specific KO of this nuclear receptor.

  10. Abnormal lipid/lipoprotein metabolism and high plasma testosterone levels in male but not female aromatase-knockout mice.

    Science.gov (United States)

    Amano, Akiko; Kondo, Yoshitaka; Noda, Yoshihiro; Ohta, Mitsuhiro; Kawanishi, Noriaki; Machida, Shuichi; Mitsuhashi, Kazuteru; Senmaru, Takafumi; Fukui, Michiaki; Takaoka, Osamu; Mori, Taisuke; Kitawaki, Jo; Ono, Masafumi; Saibara, Toshiji; Obayashi, Hiroshi; Ishigami, Akihito

    2017-05-15

    Sex steroid hormones, such as estrogen and testosterone, are believed to play important roles in lipid metabolism. To elucidate the effects of estrogen depletion on lipid metabolism in male and female mice, we used aromatase-knockout (ArKO) mice, in which Cyp19 gene disruption prevented estrogen synthesis in vivo. These mice were divided into the following 4 groups: male and female ArKO mice and male and female wild-type (WT) mice. These mice were fed a normal-fat diet (13.6% fat) ad libitum. At 159 days after birth, the mice were tested for liver and plasma lipid content and hepatic hormone receptor- and lipid/lipoprotein metabolism-related gene expression. Interestingly, we found that hepatic steatosis was accompanied by markedly elevated plasma testosterone levels in male ArKO mice but not in female ArKO mice. Plasma lipoprotein profiles exhibited concurrent decreases in LDL- and small dense LDL-triglyceride (TG) levels in male ArKO mice. Moreover, male mice, but not female mice, exhibited marked elevations in androgen receptor (AR), sterol regulatory element-binding protein 1 (SREBP1), and CD36 expression. These results strongly suggest that Cyp19 gene disruption, which induces a sexually dimorphic response and high plasma testosterone levels in male mice, also induces hepatic steatosis. Copyright © 2017 Elsevier Inc. All rights reserved.

  11. PKCδ Knockout Mice Are Protected from Dextromethorphan-Induced Serotonergic Behaviors in Mice: Involvements of Downregulation of 5-HT1AReceptor and Upregulation of Nrf2-Dependent GSH Synthesis.

    Science.gov (United States)

    Tran, Hai-Quyen; Lee, Youngho; Shin, Eun-Joo; Jang, Choon-Gon; Jeong, Ji Hoon; Mouri, Akihiro; Saito, Kuniaki; Nabeshima, Toshitaka; Kim, Hyoung-Chun

    2018-02-22

    We investigated whether a specific serotonin (5-HT) receptor-mediated mechanism was involved in dextromethorphan (DM)-induced serotonergic behaviors. We firstly observed that the activation of 5-HT 1A receptor, but not 5-HT 2A receptor, contributed to DM-induced serotonergic behaviors in mice. We aimed to determine whether the upregulation of 5-HT 1A receptor induced by DM facilitates the specific induction of certain PKC isoform, because previous reports suggested that 5-HT 1A receptor activates protein kinase C (PKC). A high dose of DM (80 mg/kg, i.p.) induced a selective induction of PKCδ out of PKCα, PKCβI, PKCβII, PKCξ, and PKCδ in the hypothalamus of wild-type (WT) mice. More importantly, 5-HT 1A receptor co-immunoprecipitated PKCδ in the presence of DM. Consistently, rottlerin, a pharmacological inhibitor of PKCδ, or PKCδ knockout significantly protected against increases in 5-HT 1A receptor gene expression, 5-HT turnover rate, and serotonergic behaviors induced by DM. Treatment with DM resulted in an initial increase in nuclear factor erythroid-2-related factor 2 (Nrf2) nuclear translocation and DNA-binding activity, γ-glutamylcysteine (GCL) mRNA expression, and glutathione (GSH) level. This compensative induction was further potentiated by rottlerin or PKCδ knockout. However, GCL mRNA and GSH/GSSG levels were decreased 6 and 12 h post-DM. These decreases were attenuated by PKCδ inhibition. Our results suggest that interaction between 5-HT 1A receptor and PKCδ is critical for inducing DM-induced serotonergic behaviors and that inhibition of PKCδ attenuates the serotonergic behaviors via downregulation of 5-HT 1A receptor and upregulation of Nrf2-dependent GSH synthesis.

  12. Adipocyte SIRT1 knockout promotes PPARγ activity, adipogenesis and insulin sensitivity in chronic-HFD and obesity.

    Science.gov (United States)

    Mayoral, Rafael; Osborn, Olivia; McNelis, Joanne; Johnson, Andrew M; Oh, Da Young; Izquierdo, Cristina Llorente; Chung, Heekyung; Li, Pingping; Traves, Paqui G; Bandyopadhyay, Gautam; Pessentheiner, Ariane R; Ofrecio, Jachelle M; Cook, Joshua R; Qiang, Li; Accili, Domenico; Olefsky, Jerrold M

    2015-05-01

    Adipose tissue is the primary site for lipid deposition that protects the organisms in cases of nutrient excess during obesogenic diets. The histone deacetylase Sirtuin 1 (SIRT1) inhibits adipocyte differentiation by targeting the transcription factor peroxisome proliferator activated-receptor gamma (PPARγ). To assess the specific role of SIRT1 in adipocytes, we generated Sirt1 adipocyte-specific knockout mice (ATKO) driven by aP2 promoter onto C57BL/6 background. Sirt1 (flx/flx) aP2Cre (+) (ATKO) and Sirt1 (flx/flx) aP2Cre (-) (WT) mice were fed high-fat diet for 5 weeks (short-term) or 15 weeks (chronic-term). Metabolic studies were combined with gene expression analysis and phosphorylation/acetylation patterns in adipose tissue. On standard chow, ATKO mice exhibit low-grade chronic inflammation in adipose tissue, along with glucose intolerance and insulin resistance compared with control fed mice. On short-term HFD, ATKO mice become more glucose intolerant, hyperinsulinemic, insulin resistant and display increased inflammation. During chronic HFD, WT mice developed a metabolic dysfunction, higher than ATKO mice, and thereby, knockout mice are more glucose tolerant, insulin sensitive and less inflamed relative to control mice. SIRT1 attenuates adipogenesis through PPARγ repressive acetylation and, in the ATKO mice adipocyte PPARγ was hyperacetylated. This high acetylation was associated with a decrease in Ser273-PPARγ phosphorylation. Dephosphorylated PPARγ is constitutively active and results in higher expression of genes associated with increased insulin sensitivity. Together, these data establish that SIRT1 downregulation in adipose tissue plays a previously unknown role in long-term inflammation resolution mediated by PPARγ activation. Therefore, in the context of obesity, the development of new therapeutics that activate PPARγ by targeting SIRT1 may provide novel approaches to the treatment of T2DM.

  13. Comparative functional genomics analysis of NNK tobacco-carcinogen induced lung adenocarcinoma development in Gprc5a-knockout mice.

    Directory of Open Access Journals (Sweden)

    Junya Fujimoto

    2010-07-01

    Full Text Available Improved understanding of lung cancer development and progression, including insights from studies of animal models, are needed to combat this fatal disease. Previously, we found that mice with a knockout (KO of G-protein coupled receptor 5A (Gprc5a develop lung tumors after a long latent period (12 to 24 months.To determine whether a tobacco carcinogen will enhance tumorigenesis in this model, we administered 4-(methylnitrosamino-1-(3-pyridyl-1-butanone (NNK i.p. to 2-months old Gprc5a-KO mice and sacrificed groups (n=5 of mice at 6, 9, 12, and 18 months later. Compared to control Gprc5a-KO mice, NNK-treated mice developed lung tumors at least 6 months earlier, exhibited 2- to 4-fold increased tumor incidence and multiplicity, and showed a dramatic increase in lesion size. A gene expression signature, NNK-ADC, of differentially expressed genes derived by transcriptome analysis of epithelial cell lines from normal lungs of Gprc5a-KO mice and from NNK-induced adenocarcinoma was highly similar to differential expression patterns observed between normal and tumorigenic human lung cells. The NNK-ADC expression signature also separated both mouse and human adenocarcinomas from adjacent normal lung tissues based on publicly available microarray datasets. A key feature of the signature, up-regulation of Ube2c, Mcm2, and Fen1, was validated in mouse normal lung and adenocarcinoma tissues and cells by immunohistochemistry and western blotting, respectively.Our findings demonstrate that lung tumorigenesis in the Gprc5a-KO mouse model is augmented by NNK and that gene expression changes induced by tobacco carcinogen(s may be conserved between mouse and human lung epithelial cells. Further experimentation to prove the reliability of the Gprc5a knockout mouse model for the study of tobacco-induced lung carcinogenesis is warranted.

  14. Piroxicam treatment augments bone abnormalities in interleukin-10 knockout mice.

    Science.gov (United States)

    Holgersen, Kristine; Dobie, Ross; Farquharson, Colin; vanʼt Hof, Rob; Ahmed, Syed Faisal; Hansen, Axel Kornerup; Holm, Thomas L

    2015-02-01

    Osteoporosis and fractures are common complications of inflammatory bowel disease. The pathogenesis is multifactorial and has been partly attributed to intestinal inflammation. The aim of this study was to evaluate bone status and assess the association between bone loss and gut inflammation in an experimental colitis model. Colitis was induced in interleukin-10 knockout mice (PAC IL-10 k.o.) by peroral administration of piroxicam for 12 days. The degree of colitis was assessed by clinical, macroscopic, and microscopic evaluation. Trabecular and cortical bone microarchitecture of tibia were determined using micro-computed tomography. Moreover, the serum levels of bone formation and bone resorption biomarkers were measured, and inflammatory protein profiling was performed on colons. PAC IL-10 k.o. mice developed severe colitis, characterized by hyperplasia and focal transmural inflammation, which was consistent with Crohn's disease-like pathology. The gut inflammation was accompanied by a 14% and 12% reduction in trabecular thickness relative to piroxicam-treated wild type and untreated wild type mice, respectively (P < 0.001). The trabecular bone structure was also changed in PAC IL-10 k.o. mice, whereas no differences in cortical bone geometry were observed. The trabecular thickness was inversely correlated with serum levels of CTX (r = -0.93, P = 0.006). Moreover, numerous inflammatory mediators, including RANKL and osteoprotegerin, were significantly increased in the colon of PAC IL-10 k.o. mice. PAC IL-10 k.o. mice develop bone loss and changed trabecular structure, as a result of increased bone resorption. Thus, the PAC IL-10 k.o. model could be a useful experimental model in preclinical research of inflammatory bowel disease-associated bone loss.

  15. Analysis of microsatellite polymorphism in inbred knockout mice.

    Directory of Open Access Journals (Sweden)

    Baofen Zuo

    Full Text Available Previously, we found that the genotype of 42 out of 198 mouse microsatellite loci, which are distributed among all chromosomes except the Y chromosome, changed from monomorphism to polymorphism (CMP in a genetically modified inbred mouse strain. In this study, we further examined whether CMP also relates to the homologous recombination in gene knockout (KO mouse strains. The same 42 microsatellite loci were analyzed by polymerase chain reaction (PCR in 29 KO inbred mouse strains via short tandem sequence repeat (STR scanning and direct sequence cloning to justify microsatellite polymorphisms. The C57BL/6J and 129 mouse strains, from which these 29 KO mice were derived, were chosen as the background controls. The results indicated that 10 out of 42 (23.8% loci showed CMP in some of these mouse strains. Except for the trinucleotide repeat locus of D3Mit22, which had microsatellite CMP in strain number 9, the core sequences of the remaining 41 loci were dinucleotide repeats, and 9 out of 41 (21.95% showed CMPs among detected mouse strains. However, 11 out of 29 (37.9% KO mice strains were recognized as having CMPs. The popular dinucleotide motifs in CMP were (TG(n (50%, 2/4, followed by (GT(n (27.27%, 3/11 and (CA(n (23.08%, 3/13. The microsatellite CMP in (CT(n and (AG(n repeats were 20% (1/5. According to cloning sequencing results, 6 KO mouse strains showed insertions of nucleotides whereas 1 showed a deletion. Furthermore, 2 loci (D13Mit3 and D14Mit102 revealed CMP in 2 strains, and mouse strain number 9 showed CMPs in two loci (D3Mit22 and D13Mit3 simultaneously. Collectively, these results indicated that microsatellite polymorphisms were present in the examined inbred KO mice.

  16. Osteoprotegerin-Knockout Mice Developed Early Onset Root Resorption.

    Science.gov (United States)

    Liu, Yi; Du, Haiming; Wang, Yunfei; Liu, Mengmeng; Deng, Shijian; Fan, Linlin; Zhang, Lili; Sun, Yao; Zhang, Qi

    2016-10-01

    Recent studies indicate that the osteoprotegerin (OPG)/RANKL/RANK pathway takes part in root resorption. However, the relationship between OPG and root resorption is vague. The purpose of our study was to investigate the role of OPG in root resorption. The first molars of the mandibles of osteoprotegerin-knockout (Opg-KO) mice and wild-type (WT) mice were evaluated by micro-computed tomography, histology, and immunohistochemistry at 4, 6, 26, and 52 weeks. To detect the activity of the osteoclasts, we induced bone marrow macrophages into osteoclast-like cells from Opg-KO mice and wild-type mice in vitro and then compared their osteoclast activities. To evaluate the cementum quality, an osteoclast-cementum co-culture model was established in vitro. In Opg-KO mice, root resorption began at the age of 4 weeks. At 6 weeks the cementum damage extended to the coronal and apical regions, and at 52 weeks the damage reached the predentin. At all observed stages, more tartrate-resistant acid phosphatase (TRAP)-positive cells were found on the surface of cementum in Opg-KO mice. In vitro, the mRNA levels of cathepsin K, TRAP, matrix metalloproteinase-9, and matrix metalloproteinase-1, as well as the protein expression of nuclear factor of activated T cell 1 and TRAP, increased significantly in osteoclast-like cells from Opg-KO mice. In addition, the cementum resorption pits of Opg-KO mice were larger when co-cultured with osteoclast-like cells. Our study demonstrated that loss of OPG led to root resorption via increasing activation of osteoclasts and reducing mineralization of cementum. Copyright © 2016. Published by Elsevier Inc.

  17. Characterization of Heterogeneous Prostate Tumors in Targeted Pten Knockout Mice.

    Directory of Open Access Journals (Sweden)

    Hanneke Korsten

    Full Text Available Previously, we generated a preclinical mouse prostate tumor model based on PSA-Cre driven inactivation of Pten. In this model homogeneous hyperplastic prostates (4-5m developed at older age (>10m into tumors. Here, we describe the molecular and histological characterization of the tumors in order to better understand the processes that are associated with prostate tumorigenesis in this targeted mouse Pten knockout model. The morphologies of the tumors that developed were very heterogeneous. Different histopathological growth patterns could be identified, including intraductal carcinoma (IDC, adenocarcinoma and undifferentiated carcinoma, all strongly positive for the epithelial cell marker Cytokeratin (CK, and carcinosarcomas, which were negative for CK. IDC pattern was already detected in prostates of 7-8 month old mice, indicating that it could be a precursor stage. At more than 10 months IDC and carcinosarcoma were most frequently observed. Gene expression profiling discriminated essentially two molecular subtypes, denoted tumor class 1 (TC1 and tumor class 2 (TC2. TC1 tumors were characterized by high expression of epithelial markers like Cytokeratin 8 and E-Cadherin whereas TC2 tumors showed high expression of mesenchyme/stroma markers such as Snail and Fibronectin. These molecular subtypes corresponded with histological growth patterns: where TC1 tumors mainly represented adenocarcinoma/intraductal carcinoma, in TC2 tumors carcinosarcoma was the dominant growth pattern. Further molecular characterization of the prostate tumors revealed an increased expression of genes associated with the inflammatory response. Moreover, functional markers for senescence, proliferation, angiogenesis and apoptosis were higher expressed in tumors compared to hyperplasia. The highest expression of proliferation and angiogenesis markers was detected in TC2 tumors. Our data clearly showed that in the genetically well-defined PSA-Cre;Pten-loxP/loxP prostate tumor

  18. Maximal Oxygen Consumption is Reduced in Aquaporin-1 Knockout Mice

    Directory of Open Access Journals (Sweden)

    Samer Al-Samir

    2016-08-01

    Full Text Available We have measured maximal oxygen consumption (V’O2,max of mice lacking one or two of the established mouse red-cell CO2 channels AQP1, AQP9 and Rhag. We intended to study whether these proteins, by acting as channels for O2, determine O2 exchange in the lung and in the periphery. We found that V’O2,max as determined by the Helox technique is reduced by ~ 16%, when AQP1 is knocked out, but not when AQP9 or Rhag are lacking. This figure holds for animals respiring normoxic as well as hypoxic gas mixtures. To see whether the reduction of V’O2,max is due to impaired O2 uptake in the lung, we measured carotid arterial O2 saturation (SO2 by pulse oximetry. Neither under normoxic (inspiratory O2 21% nor under hypoxic conditions (11% O2 is there a difference in SO2 between AQP1null and WT mice, suggesting that AQP1 is not critical for O2 uptake in the lung. The fact that the % reduction of V’O2,max is identical in normoxia and hypoxia indicates moreover that the limitation of V’O2,max is not due to an O2 diffusion problem, neither in the lung nor in the periphery. Instead, it appears likely that AQP1null animals exhibit a reduced V’O2,max due to the reduced wall thickness and muscle mass of the left ventricles of their hearts, as reported previously. We conclude that very likely the properties of the hearts of AQP1 knockout mice cause a reduced maximal cardiac output and thus cause a reduced V’O2,max, which constitutes a new phenotype of these mice.

  19. Elevated body temperature during sleep in orexin knockout mice

    Science.gov (United States)

    Mochizuki, Takatoshi; Klerman, Elizabeth B.; Sakurai, Takeshi; Scammell, Thomas E.

    2008-01-01

    Core body temperature (Tb) is influenced by many physiological factors, including behavioral state, locomotor activity, and biological rhythms. To determine the relative roles of these factors, we examined Tb in orexin knockout (KO) mice, which have a narcolepsy-like phenotype with severe sleep-wake fragmentation. Because orexin is thought to promote heat production during wakefulness, we hypothesized that orexin KO mice would have lower Tb while awake. Surprisingly, the Tb of orexin KO mice was 0.4°C higher than wild-type (WT) littermates during the dark period. Orexin KO mice had normal diurnal variations in Tb, but the ultradian rhythms of Tb, locomotor activity, and wakefulness were markedly reduced. During sustained wakefulness, Tb was the same in both groups. During the first 15 min of spontaneous sleep, the Tb of WT mice decreased by 1.0°C, but Tb in orexin KO mice decreased only 0.4°C. Even during intense recovery sleep after 8 hr of sleep deprivation, the Tb of orexin KO mice remained 0.7°C higher than in WT mice. This blunted fall in Tb during sleep may be due to inadequate activation of heat loss mechanisms or sustained activity in heat-generating systems. These observations reveal an unexpected role for orexin in thermoregulation. In addition, because heat loss is an essential aspect of sleep, the blunted fall in Tb of orexin KO mice may provide an explanation for the fragmented sleep of narcolepsy. PMID:16556901

  20. β1/2 or M2/3 Receptors Are Required for Different Gastrointestinal Motility Responses Induced by Acupuncture at Heterotopic or Homotopic Acupoints

    Science.gov (United States)

    Yu, Xiaochun; Cui, Changxiang; Yang, Zhaokun; Shi, Hong; Jing, Xianghong; Zhu, Bing

    2016-01-01

    Acupuncture at homotopic acupoints or heterotopic acupoints is known to either inhibit or facilitate gastrointestinal motility, depending on the acupoint location. However, little effort has been made to investigate the roles of specific receptors (such as adrenergic and muscarinic acetylcholine receptors) in mediating the effects of acupuncture at heterotopic and homotopic acupoints. Different adrenergic receptor subtypes or cholinergic receptor subtypes are predominantly expressed in various sections of the gut, resulting in variations between the effects of acupuncture at heterotopic or homotopic acupoints on gastrointestinal motility. Here, we investigated the role of β1/β2 receptors and M2/M3 receptors in gastrointestinal motility regulated by acupuncture at ST37, a heterotopic acupoint, and ST25, a homotopic acupoint, by simultaneously recording intraluminal pressures in the distal colon and stomach or jejunum and examining fecal phenol red excretion in β1/2 receptor-knockout mice and M2/3 receptor-knockout mice. We found that knockout of the M2/3 receptor significantly inhibited ST37 acupuncture-induced enhancement of gastric motility, jejunal motility, and colonic motility. Additionally, knocking out of the β1/2 receptor significantly diminished the ST25 acupuncture-induced inhibition of gastric motility and jejunal motility without significantly altering the enhancement of colonic motility induced by acupuncture at ST25. Acupuncture at ST37 significantly accelerated gastrointestinal transition in β1/2 receptor-knockout mice and their wild-type littermates. However, this acceleration of gastrointestinal transition was markedly diminished in M2/3 receptor-knockout mice relative to their wild-type littermates. Acupuncture at ST25 significantly increased gastrointestinal transition in β1/2 receptor-knockout mice and significantly decreased gastrointestinal transition in M2/3 receptor-knockout mice without altering gastrointestinal transition in wild

  1. Sildenafil restores endothelial function in the apolipoprotein E knockout mouse

    Directory of Open Access Journals (Sweden)

    Balarini Camille M

    2013-01-01

    Full Text Available Abstract Background Atherosclerosis is an inflammatory process of the arterial walls and is initiated by endothelial dysfunction accompanied by an imbalance in the production of reactive oxygen species (ROS and nitric oxide (NO. Sildenafil, a selective phosphodiesterase-5 (PDE5 inhibitor used for erectile dysfunction, exerts its cardiovascular effects by enhancing the effects of NO. The aim of this study was to investigate the influence of sildenafil on endothelial function and atherosclerosis progression in apolipoprotein E knockout (apoE−/− mice. Methods ApoE−/− mice treated with sildenafil (Viagra®, 40 mg/kg/day, for 3 weeks, by oral gavage were compared to the untreated apoE−/− and the wild-type (WT mice. Aortic rings were used to evaluate the relaxation responses to acetylcholine (ACh in all of the groups. In a separate set of experiments, the roles of NO and ROS in the relaxation response to ACh were evaluated by incubating the aortic rings with L-NAME (NO synthase inhibitor or apocynin (NADPH oxidase inhibitor. In addition, the atherosclerotic lesions were quantified and superoxide production was assessed. Results Sildenafil restored the vasodilator response to acetylcholine (ACh in the aortic rings of the apoE−/− mice. Treatment with L-NAME abolished the vasodilator responses to ACh in all three groups of mice and revealed an augmented participation of NO in the endothelium-dependent vasodilation in the sildenafil-treated animals. The normalized endothelial function in sildenafil-treated apoE−/− mice was unaffected by apocynin highlighting the low levels of ROS production in these animals. Moreover, morphological analysis showed that sildenafil treatment caused approximately a 40% decrease in plaque deposition in the aorta. Conclusion This is the first study demonstrating the beneficial effects of chronic treatment with sildenafil on endothelial dysfunction and atherosclerosis in a model of spontaneous

  2. Repressive Epigenetic Changes at the mGlu2 Promoter in Frontal Cortex of 5-HT2A Knockout Mice

    Science.gov (United States)

    Kurita, Mitsumasa; Moreno, José L.; Holloway, Terrell; Kozlenkov, Alexey; Mocci, Giuseppe; García-Bea, Aintzane; Hanks, James B.; Neve, Rachael; Nestler, Eric J.; Russo, Scott J.

    2013-01-01

    Serotonin 5-HT2A and metabotropic glutamate 2 (mGlu2) are G protein–coupled receptors suspected in the pathophysiology of psychiatric disorders, such as schizophrenia, depression, and suicide. Previous findings demonstrate that mGlu2 mRNA expression is down-regulated in brain cortical regions of 5-HT2A knockout (KO) mice. However, the molecular mechanism responsible for this alteration remains unknown. We show here repressive epigenetic changes at the promoter region of the mGlu2 gene in frontal cortex of 5-HT2A-KO mice. Disruption of 5-HT2A receptor-dependent signaling in mice was associated with decreased acetylation of histone H3 (H3ac) and H4 (H4ac) and increased tri-methylation of histone H3 at lysine 27 (H3K27me3) at the mGlu2 promoter, epigenetic changes that correlate with transcriptional repression. Neither methylation of histone H3 at lysine 4 (H3K4me1/2/3) nor tri-methylation of histone H3 at lysine 9 (H3K9me3) was affected. We found that Egr1, a transcription factor in which promoter activity was positively regulated by the 5-HT2A receptor agonist 4-bromo-3,6-dimethoxybenzocyclobuten-1-yl)methylamine hydrobromide, binds less to the mGlu2 promoter in frontal cortex of 5-HT2A-KO, compared with wild-type mice. Furthermore, expression of mGlu2 was increased by viral-mediated gene transfer of FLAG-tagged Egr1 in mouse frontal cortex. Together, these observations suggest that 5-HT2A receptor–dependent signaling epigenetically affects mGlu2 transcription in mouse frontal cortex. PMID:23508685

  3. Pharmacological or genetic blockade of the dopamine D3 receptor increases cell proliferation in the hippocampus of adult mice.

    Science.gov (United States)

    Egeland, Martin; Zhang, Xiaoqun; Millan, Mark J; Mocaer, Elisabeth; Svenningsson, Per

    2012-12-01

    Dopamine plays an important role in cellular processes controlling the functional and structural plasticity of neurons, as well as their generation and proliferation, both in the developing and the adult brain. The precise roles of individual dopamine receptors subtypes in adult neurogenesis remain poorly defined, although D3 receptors are known to be involved in neurogenesis in the subventricular zone. By contrast, very few studies have addressed the influence of dopamine and D3 receptors upon neurogenesis in the subgranular zone of the hippocampus, an issue addressed herein employing constitutive D3 receptor knockout mice, or chronic exposure to the preferential D3 receptor antagonist, S33138. D3 receptor knockout mice revealed increased baseline levels of cell proliferation and ongoing neurogenesis, as measured both using Ki-67 and doublecortin, whereas there was no difference in cell survival as measured by BrdU (5-bromo-2'-deoxyuridine). Chronic administration of S33138 was shown to be functionally active in enhancing levels of the plasticity-related molecule, delta-FosB, in the D3 receptor-rich nucleus accumbens. In accordance with the stimulated neurogenesis seen in D3 receptor knockout mice, S33138 increased proliferation in wild-type mice. These observations suggest that D3 receptors exert a tonic, constitutive inhibitory influence upon adult hippocampal neurogenesis. © 2012 The Authors Journal of Neurochemistry © 2012 International Society for Neurochemistry.

  4. Knockouts of high-ranking males have limited impact on baboon social networks.

    Science.gov (United States)

    Franz, Mathias; Altmann, Jeanne; Alberts, Susan C

    Social network structures can crucially impact complex social processes such as collective behaviour or the transmission of information and diseases. However, currently it is poorly understood how social networks change over time. Previous studies on primates suggest that `knockouts' (due to death or dispersal) of high-ranking individuals might be important drivers for structural changes in animal social networks. Here we test this hypothesis using long-term data on a natural population of baboons, examining the effects of 29 natural knockouts of alpha or beta males on adult female social networks. We investigated whether and how knockouts affected (1) changes in grooming and association rates among adult females, and (2) changes in mean degree and global clustering coefficient in these networks. The only significant effect that we found was a decrease in mean degree in grooming networks in the first month after knockouts, but this decrease was rather small, and grooming networks rebounded to baseline levels by the second month after knockouts. Taken together our results indicate that the removal of high-ranking males has only limited or no lasting effects on social networks of adult female baboons. This finding calls into question the hypothesis that the removal of high-ranking individuals has a destabilizing effect on social network structures in social animals.

  5. Stabilization of tooth movement by administration of reveromycin A to osteoprotegerin-deficient knockout mice.

    Science.gov (United States)

    Yabumoto, Takahiro; Miyazawa, Ken; Tabuchi, Masako; Shoji, Satsuki; Tanaka, Miyuki; Kadota, Manami; Yoshizako, Mamoru; Kawatani, Makoto; Osada, Hiroyuki; Maeda, Hatsuhiko; Goto, Shigemi

    2013-09-01

    In this study, mechanical stress in the form of tooth movement was applied to osteoprotegerin-deficient knockout mice, which served as an animal model for juvenile Paget's disease. To compare and evaluate bone turnover and response of the surrounding bony tissue, we administered reveromycin A. We also investigated the ability of reveromycin A to control osteoclastic activity in juvenile Paget's disease. Eight-week-old male osteoprotegerin-deficient knockout and wild-type mice were injected with reveromycin A (15 mg/kg of body weight) intraperitoneally twice daily. An elastic module was inserted interproximally between the maxillary left first and second molars. Administration of reveromycin A to osteoprotegerin-deficient knockout mice reduced tooth movement distances, increased bone volumes at the interradicular septum, decreased osteoclast counts, and reduced serum alkaline phosphatase and tartrate resistant acid phosphatase. Reveromycin A administration also caused a temporal shift in peak Runx2 staining in osteoprotegerin-deficient knockout mice so that the overall staining time course was similar to that observed for wild-type mice. Reveromycin A administration in osteoprotegerin-deficient knockout mice inhibited bone resorption and normalized bone formation. As a result, normal bone turnover was obtained. Copyright © 2013 American Association of Orthodontists. Published by Mosby, Inc. All rights reserved.

  6. Gene Knockout Identification Using an Extension of Bees Hill Flux Balance Analysis

    Directory of Open Access Journals (Sweden)

    Yee Wen Choon

    2015-01-01

    Full Text Available Microbial strain optimisation for the overproduction of a desired phenotype has been a popular topic in recent years. Gene knockout is a genetic engineering technique that can modify the metabolism of microbial cells to obtain desirable phenotypes. Optimisation algorithms have been developed to identify the effects of gene knockout. However, the complexities of metabolic networks have made the process of identifying the effects of genetic modification on desirable phenotypes challenging. Furthermore, a vast number of reactions in cellular metabolism often lead to a combinatorial problem in obtaining optimal gene knockout. The computational time increases exponentially as the size of the problem increases. This work reports an extension of Bees Hill Flux Balance Analysis (BHFBA to identify optimal gene knockouts to maximise the production yield of desired phenotypes while sustaining the growth rate. This proposed method functions by integrating OptKnock into BHFBA for validating the results automatically. The results show that the extension of BHFBA is suitable, reliable, and applicable in predicting gene knockout. Through several experiments conducted on Escherichia coli, Bacillus subtilis, and Clostridium thermocellum as model organisms, extension of BHFBA has shown better performance in terms of computational time, stability, growth rate, and production yield of desired phenotypes.

  7. Glycogen synthase kinase-3beta heterozygote knockout mice as a model of findings in postmortem schizophrenia brain or as a model of behaviors mimicking lithium action: negative results.

    Science.gov (United States)

    Bersudsky, Yuly; Shaldubina, Alona; Kozlovsky, Nitzan; Woodgett, James R; Agam, Galila; Belmaker, R H

    2008-05-01

    In mice glycogen synthase kinase (GSK)-3beta heterozygote knockout status was reported to cause reduced immobility in the Porsolt forced swim test and reduced amphetamine-induced hyperactivity, behaviors that mimic the effects of lithium. GSK-3beta protein and mRNA level and activity have been reported to be reduced in the postmortem brain of schizophrenia patients and this could suggest the involvement of GSK-3beta in the etiology of schizophrenia. However, apomorphine-induced stereotyping was reported to be unchanged in GSK-3beta heterozygote (HZ) knockout (KO) mice. As such behaviors are not always robust, study in another laboratory seemed indicated. Motor activity and coordination were assessed in the rotarod test. Behavior was studied in the following tests: pilocarpine-induced seizures model for lithium action, Porsolt forced swim test, tail suspension test, elevated plus-maze, large open field, startle response and prepulse inhibition of acoustic startle response, amphetamine-induced hyperactivity, and apomorphine-induced stereotypic climbing. We could not confirm the report that GSK-3beta HZ KO mice exhibit reduced immobility in the Porsolt forced swim or reduced amphetamine-induced hyperactivity in a manner mimicking the behavioral effects of lithium. We did not find increased apomorphine-induced stereotypic climbing or disruption of prepulse inhibition, suggesting that human postmortem findings regarding GSK-3beta in schizophrenia are not mediated by changes in dopamine receptors and are not the cause of prepulse inhibition deficits in schizophrenia. These data do not support the role of GSK-3beta in schizophrenia or in the mechanism of therapeutic action of lithium. Although differences in the genetic background of the GSK-3beta HZ KOs used in the present study compared with that of the previous study could be responsible, such results could suggest that the previously reported effects of GSK-3beta knockout on behavior are not robust.

  8. Disruption of the GH Receptor Gene in Adult Mice Increases Maximal Lifespan in Females

    DEFF Research Database (Denmark)

    Junnila, Riia K.; Duran-Ortiz, Silvana; Suer, Ozan

    2016-01-01

    GH and IGF-1 are important for a variety of physiological processes including growth, development, and aging. Mice with reduced levels of GH and IGF-1 have been shown to live longer than wild-type controls. Our laboratory has previously found that mice with a GH receptor gene knockout (GHRKO) fro...

  9. Vitamin D receptor and vitamin D metabolizing enzymes are expressed in the human male reproductive tract

    DEFF Research Database (Denmark)

    Blomberg Jensen, Martin; Nielsen, John E; Jørgensen, Anne

    2010-01-01

    The vitamin D receptor (VDR) is expressed in human testis, and vitamin D (VD) has been suggested to affect survival and function of mature spermatozoa. Indeed, VDR knockout mice and VD deficient rats show decreased sperm counts and low fertility. However, the cellular response to VD is complex...

  10. Transgenic mouse models to study the role of the macrophage scavenger receptor class A in atherosclerosis

    NARCIS (Netherlands)

    de Winther, M. P.; Gijbels, M. J.; van Dijk, K. W.; Havekes, L. M.; Hofker, M. H.

    2000-01-01

    Several in vivo studies have been performed on the role of the macrophage scavenger receptor class A (SR-A) in atherosclerosis using SR-A knockout mice. The results indicate both an antiatherogenic and a proatherogenic role of SR-A, depending on the nature of the animal model serving as the

  11. Deficits in Sensory-Specific Devaluation Task Performance Following Genetic Deletions of Cannabinoid (CB1) Receptor

    Science.gov (United States)

    Crombag, Hans S.; Johnson, Alexander W.; Zimmer, Anne M.; Zimmer, Andreas; Holland, Peter C.

    2010-01-01

    Cannabinoid CB1 receptor is abundantly expressed throughout the CNS and is implicated in numerous physiological and behavioral functions, including appetite and feeding. In the present study, wild-type and CB1 heterozygous and homozygous knockout mice were tested on an instrumental outcome-selective devaluation task to assess changes in acquired…

  12. Impaired Discrimination Learning in Mice Lacking the NMDA Receptor NR2A Subunit

    Science.gov (United States)

    Brigman, Jonathan L.; Feyder, Michael; Saksida, Lisa M.; Bussey, Timothy J.; Mishina, Masayoshi; Holmes, Andrew

    2008-01-01

    N-Methyl-D-aspartate receptors (NMDARs) mediate certain forms of synaptic plasticity and learning. We used a touchscreen system to assess NR2A subunit knockout mice (KO) for (1) pairwise visual discrimination and reversal learning and (2) acquisition and extinction of an instrumental response requiring no pairwise discrimination. NR2A KO mice…

  13. Partial functional complementation between human and mouse cytomegalovirus chemokine receptor homologues

    DEFF Research Database (Denmark)

    Farrell, Helen E; Abraham, Alexander M; Cardin, Rhonda D

    2011-01-01

    The human cytomegalovirus (CMV) proteins US28 and UL33 are homologous to chemokine receptors (CKRs). Knockout of the mouse CMV M33 protein (UL33 homologue) results in substantial attenuation of salivary gland infection/replication and reduced efficiency of reactivation from tissue explants. M33...

  14. Evaluation of [{sup 18}F]fluoroxanomeline {l_brace}5-{l_brace}4-[(6-[{sup 18}F]fluorohexyl)oxy]-1,2,5-thiadiazol-3-yl{r_brace}-1-methyl-1,2,3,6 -tetrahydropyridine{r_brace} in muscarinic knockout mice

    Energy Technology Data Exchange (ETDEWEB)

    Kiesewetter, Dale O. [Positron Emission Tomography Radiochemistry Group, NIBIB, NIH, Bethesda, MD 20892 (United States)]. E-mail: dk7k@nih.gov; Jagoda, Elaine M. [Positron Emission Tomography Radiochemistry Group, NIBIB, NIH, Bethesda, MD 20892 (United States); Shimoji, Kazuaki [Positron Emission Tomography Department, Clinical Center, NIH, Bethesda, MD 20892 (United States); Ma, Ying [Positron Emission Tomography Radiochemistry Group, NIBIB, NIH, Bethesda, MD 20892 (United States); Eckelman, William C. [Positron Emission Tomography Department, Clinical Center, NIH, Bethesda, MD 20892 (United States); Molecular Tracer LLC, Bethesda, MD 20814 (United States)

    2007-02-15

    Introduction: We set out to develop a muscarinic M1-selective agonist (based on the structure of the functionally M1-selective xanomeline) that could be radiolabeled with fluorine-18 for use as an imaging agent for positron emission tomography. Methods: The radiochemical synthesis was achieved, employing the arts of organic and radiochemical syntheses. Binding selectivity studies employed biodistribution studies, using autoradiography and/or tissue dissection, in wild-type or muscarinic receptor knockout mice. Results: [{sup 18}F]Fluoroxanomeline shows rather uniform uptake in all mouse brain regions and high specific binding, with a brain-to-blood ratio of 32 at 60 min postinjection. In addition, the specific binding is demonstrated by a 58% to 75% decrease in brain uptake upon coinjection with 5 nmol of unlabeled fluoroxanomeline or xanomeline. Brain uptake studies with [{sup 3}H]xanomeline in muscarinic knockout mice show decreased uptake in M1 (17-34%) and M2 (2-20%) knockout mice compared with control. However, statistical significance was observed in only a few regions. Comparison of [{sup 18}F]fluoroxanomeline in knockout mice showed no difference in M1 or M4 knockout mice but a general decrease in M2 (2-24%) knockout mice. The decrease of [{sup 18}F]fluoroxanomeline uptake in M2 knockout mice reached statistical significance in brain stem, cerebellum, frontal cortex, hippocampus, inferior colliculus and superior colliculus. Conclusion: Although xanomeline displays highly selective M1 agonist activity in functional assays, little selectivity for muscarinic subtype binding was observed for xanomeline or its fluorine-containing analogue, fluoroxanomeline. This emphasizes the lack of correlation between functional selectivity and binding selectivity.

  15. Postsynaptic P2X3-containing receptors in gustatory nerve fibres mediate responses to all taste qualities in mice.

    Science.gov (United States)

    Vandenbeuch, Aurelie; Larson, Eric D; Anderson, Catherine B; Smith, Steven A; Ford, Anthony P; Finger, Thomas E; Kinnamon, Sue C

    2015-03-01

    Taste buds release ATP to activate ionotropic purinoceptors composed of P2X2 and P2X3 subunits, present on the taste nerves. Mice with genetic deletion of P2X2 and P2X3 receptors (double knockout mice) lack responses to all taste stimuli presumably due to the absence of ATP-gated receptors on the afferent nerves. Recent experiments on the double knockout mice showed, however, that their taste buds fail to release ATP, suggesting the possibility of pleiotropic deficits in these global knockouts. To test further the role of postsynaptic P2X receptors in afferent signalling, we used AF-353, a selective antagonist of P2X3-containing receptors to inhibit the receptors acutely during taste nerve recording and behaviour. The specificity of AF-353 for P2X3-containing receptors was tested by recording Ca(2+) transients to exogenously applied ATP in fura-2 loaded isolated geniculate ganglion neurons from wild-type and P2X3 knockout mice. ATP responses were completely inhibited by 10 μm or 100 μm AF-353, but neither concentration blocked responses in P2X3 single knockout mice wherein the ganglion cells express only P2X2-containing receptors. Furthermore, AF-353 had no effect on taste-evoked ATP release from taste buds. In wild-type mice, i.p. injection of AF-353 or simple application of the drug directly to the tongue, inhibited taste nerve responses to all taste qualities in a dose-dependent fashion. A brief access behavioural assay confirmed the electrophysiological results and showed that preference for a synthetic sweetener, SC-45647, was abolished following i.p. injection of AF-353. These data indicate that activation of P2X3-containing receptors is required for transmission of all taste qualities. © 2015 The Authors. The Journal of Physiology © 2015 The Physiological Society.

  16. Influence of δ-Opioid Receptors in the Behavioral Effects of Nicotine

    Science.gov (United States)

    Berrendero, Fernando; Plaza-Zabala, Ainhoa; Galeote, Lola; Flores, África; Bura, S Andreea; Kieffer, Brigitte L; Maldonado, Rafael

    2012-01-01

    Multiple studies in animal models and humans suggest that the endogenous opioid system is an important neurobiological substrate for nicotine addictive properties. In this study, we evaluated the participation of δ-opioid receptors in different behavioral responses of nicotine by using δ-opioid receptor knockout mice. Acute nicotine administration induced hypolocomotion and antinociception in wild-type mice, which were similar in knockout animals. The development of tolerance to nicotine-induced antinociception was also similar in both genotypes. In agreement, the expression and functional activity of δ-opioid receptors were not modified in the different layers of the spinal cord and brain areas evaluated after chronic nicotine treatment. The somatic manifestation of the nicotine withdrawal syndrome precipitated by mecamylamine was also similar in wild-type and δ-opioid receptor knockout mice. In contrast, nicotine induced a conditioned place preference in wild-type animals that was abolished in knockout mice. Moreover, a lower percentage of acquisition of intravenous nicotine self-administration was observed in mice lacking δ-opioid receptors as well as in wild-type mice treated with the selective δ-opioid receptor antagonist naltrindole. Accordingly, in-vivo microdialysis studies revealed that the enhancement in dopamine extracellular levels induced by nicotine in the nucleus accumbens was reduced in mutant mice. In summary, the present results show that δ-opioid receptors are involved in the modulation of nicotine rewarding effects. However, this opioid receptor does not participate either in several acute effects of nicotine or in the development of tolerance and physical dependence induced by chronic nicotine administration. PMID:22669166

  17. High-temperature expansion and knock-out properties of moulding sands with water glass

    Directory of Open Access Journals (Sweden)

    Major-Gabryś K.

    2007-01-01

    Full Text Available The article focuses on the topic of improving the knock-out properties of moulding sand with water glass and ester hardener. It is settled that the cause of worse knock-out properties of moulding sand can be brought by their thermal expansion in increased temperatures. There is a presentation of the influence of different additives, containing Al2O3, on moulding sands’ expansion in increased temperatures. Within the frames of research, there was an elaboration of the influence of authors own additive- Glassex, on the expansion phenomenon of moulding sands with water glass and ester hardener. It is concluded, that the new additive stops the expansion of moulding sands and as well it improves their knock-out properties.

  18. The emerging role of promiscuous 7TM receptors as chemosensors for food intake

    DEFF Research Database (Denmark)

    Wellendorph, Petrine; Johansen, Lars Dan; Bräuner-Osborne, Hans

    2010-01-01

    In recent years, several highly promiscuous seven transmembrane (7TM) receptors have been cloned and characterized of which many are activated broadly by amino acids, proteolytic degradation products, carbohydrates, or free fatty acids (FFAs) and are expressed in taste tissue, the gastrointestina...... and therapeutic prospect of their receptors. However, an increasing number of subtype-selective ligands and/or receptor knockout mice are being developed which at least for some of the receptors have validated them as promising drug targets in, for example, type II diabetes....

  19. Genetic variation in 5-hydroxytryptamine transporter expression causes adaptive changes in 5-HT4 receptor levels

    DEFF Research Database (Denmark)

    Jennings, Katie Ann; Licht, Cecilie Löe; Bruce, Aynsley

    2012-01-01

    Genetic variation in 5-HT transporter (5-HTT) expression is a key risk factor for psychiatric disorder and has been linked to changes in the expression of certain 5-HT receptor subtypes. This study investigated the effect of variation in 5-HTT expression on 5-HT4 receptor levels in both 5-HTT...... knockout (KO) and overexpressing (OE) mice using autoradiography with the selective 5-HT4 receptor radioligand, [3H]SB207145. Compared to wild-type (5-HTT+/+) controls, homozygous 5-HTT KO mice (5-HTT-/-) had reduced 5-HT4 receptor binding site density in all brain regions examined (35-65% of 5-HTT...

  20. Evaluation and Design of Genome-Wide CRISPR/SpCas9 Knockout Screens

    Directory of Open Access Journals (Sweden)

    Traver Hart

    2017-08-01

    Full Text Available The adaptation of CRISPR/SpCas9 technology to mammalian cell lines is transforming the study of human functional genomics. Pooled libraries of CRISPR guide RNAs (gRNAs targeting human protein-coding genes and encoded in viral vectors have been used to systematically create gene knockouts in a variety of human cancer and immortalized cell lines, in an effort to identify whether these knockouts cause cellular fitness defects. Previous work has shown that CRISPR screens are more sensitive and specific than pooled-library shRNA screens in similar assays, but currently there exists significant variability across CRISPR library designs and experimental protocols. In this study, we reanalyze 17 genome-scale knockout screens in human cell lines from three research groups, using three different genome-scale gRNA libraries. Using the Bayesian Analysis of Gene Essentiality algorithm to identify essential genes, we refine and expand our previously defined set of human core essential genes from 360 to 684 genes. We use this expanded set of reference core essential genes, CEG2, plus empirical data from six CRISPR knockout screens to guide the design of a sequence-optimized gRNA library, the Toronto KnockOut version 3.0 (TKOv3 library. We then demonstrate the high effectiveness of the library relative to reference sets of essential and nonessential genes, as well as other screens using similar approaches. The optimized TKOv3 library, combined with the CEG2 reference set, provide an efficient, highly optimized platform for performing and assessing gene knockout screens in human cell lines.

  1. Hyperactivity of Newborn Pten Knock-out Neurons Results from Increased Excitatory Synaptic Drive

    Science.gov (United States)

    Williams, Michael R.; DeSpenza, Tyrone; Li, Meijie; Gulledge, Allan T.

    2015-01-01

    Developing neurons must regulate morphology, intrinsic excitability, and synaptogenesis to form neural circuits. When these processes go awry, disorders, including autism spectrum disorder (ASD) or epilepsy, may result. The phosphatase Pten is mutated in some patients having ASD and seizures, suggesting that its mutation disrupts neurological function in part through increasing neuronal activity. Supporting this idea, neuronal knock-out of Pten in mice can cause macrocephaly, behavioral changes similar to ASD, and seizures. However, the mechanisms through which excitability is enhanced following Pten depletion are unclear. Previous studies have separately shown that Pten-depleted neurons can drive seizures, receive elevated excitatory synaptic input, and have abnormal dendrites. We therefore tested the hypothesis that developing Pten-depleted neurons are hyperactive due to increased excitatory synaptogenesis using electrophysiology, calcium imaging, morphological analyses, and modeling. This was accomplished by coinjecting retroviruses to either “birthdate” or birthdate and knock-out Pten in granule neurons of the murine neonatal dentate gyrus. We found that Pten knock-out neurons, despite a rapid onset of hypertrophy, were more active in vivo. Pten knock-out neurons fired at more hyperpolarized membrane potentials, displayed greater peak spike rates, and were more sensitive to depolarizing synaptic input. The increased sensitivity of Pten knock-out neurons was due, in part, to a higher density of synapses located more proximal to the soma. We determined that increased synaptic drive was sufficient to drive hypertrophic Pten knock-out neurons beyond their altered action potential threshold. Thus, our work contributes a developmental mechanism for the increased activity of Pten-depleted neurons. PMID:25609613

  2. Evaluation and Design of Genome-Wide CRISPR/SpCas9 Knockout Screens

    Science.gov (United States)

    Hart, Traver; Tong, Amy Hin Yan; Chan, Katie; Van Leeuwen, Jolanda; Seetharaman, Ashwin; Aregger, Michael; Chandrashekhar, Megha; Hustedt, Nicole; Seth, Sahil; Noonan, Avery; Habsid, Andrea; Sizova, Olga; Nedyalkova, Lyudmila; Climie, Ryan; Tworzyanski, Leanne; Lawson, Keith; Sartori, Maria Augusta; Alibeh, Sabriyeh; Tieu, David; Masud, Sanna; Mero, Patricia; Weiss, Alexander; Brown, Kevin R.; Usaj, Matej; Billmann, Maximilian; Rahman, Mahfuzur; Costanzo, Michael; Myers, Chad L.; Andrews, Brenda J.; Boone, Charles; Durocher, Daniel; Moffat, Jason

    2017-01-01

    The adaptation of CRISPR/SpCas9 technology to mammalian cell lines is transforming the study of human functional genomics. Pooled libraries of CRISPR guide RNAs (gRNAs) targeting human protein-coding genes and encoded in viral vectors have been used to systematically create gene knockouts in a variety of human cancer and immortalized cell lines, in an effort to identify whether these knockouts cause cellular fitness defects. Previous work has shown that CRISPR screens are more sensitive and specific than pooled-library shRNA screens in similar assays, but currently there exists significant variability across CRISPR library designs and experimental protocols. In this study, we reanalyze 17 genome-scale knockout screens in human cell lines from three research groups, using three different genome-scale gRNA libraries. Using the Bayesian Analysis of Gene Essentiality algorithm to identify essential genes, we refine and expand our previously defined set of human core essential genes from 360 to 684 genes. We use this expanded set of reference core essential genes, CEG2, plus empirical data from six CRISPR knockout screens to guide the design of a sequence-optimized gRNA library, the Toronto KnockOut version 3.0 (TKOv3) library. We then demonstrate the high effectiveness of the library relative to reference sets of essential and nonessential genes, as well as other screens using similar approaches. The optimized TKOv3 library, combined with the CEG2 reference set, provide an efficient, highly optimized platform for performing and assessing gene knockout screens in human cell lines. PMID:28655737

  3. Has the sun set on κ3-opioid receptors?

    Science.gov (United States)

    Connor, Mark; Kitchen, Ian

    2006-01-01

    μ-Opioid receptor agonists are a mainstay of clinical analgesia, despite the significant unwanted effects and dependence liability associated with drugs like morphine. The quest for opioids that produce analgesia with fewer undesirable effects has lead to the putative identification of multiple opioid receptor subtypes, despite the identification of only four opioid-related receptor genes. One such putative receptor subtype is the κ3 receptor, activation of which supposedly produces analgesia in animals. In the present issue of this Journal, Olianas and co-workers have demonstrated that the prototypic κ3 agonist naloxone benzoylhydrazone is actually a partial agonist at the cloned μ, δ, and κ opioid receptors and an antagonist at opioid-like NOP receptors. Together with a recent study that showed that high-affinity naloxone benzoylhydrazone binding is abolished in triple μ/δ/κ receptor knockout mice, the present study provides strong evidence that in vivo effects attributed to κ3 receptor activation probably just reflect the combined actions of a particularly nonselective opioid drug. Indeed, molecular identification of any of the proposed subtypes of μ, δ, and κ opioid receptors has proven elusive, suggesting that it is perhaps time to retire the notion of opioid receptor subtypes until definitive evidence for their existence is provided. PMID:16402044

  4. Has the sun set on kappa3-opioid receptors?

    Science.gov (United States)

    Connor, Mark; Kitchen, Ian

    2006-02-01

    Mu-opioid receptor agonists are a mainstay of clinical analgesia, despite the significant unwanted effects and dependence liability associated with drugs like morphine. The quest for opioids that produce analgesia with fewer undesirable effects has lead to the putative identification of multiple opioid receptor subtypes, despite the identification of only four opioid-related receptor genes. One such putative receptor subtype is the kappa3 receptor, activation of which supposedly produces analgesia in animals. In the present issue of this Journal, Olianas and co-workers have demonstrated that the prototypic kappa3 agonist naloxone benzoylhydrazone is actually a partial agonist at the cloned mu, delta, and kappa opioid receptors and an antagonist at opioid-like NOP receptors. Together with a recent study that showed that high-affinity naloxone benzoylhydrazone binding is abolished in triple mu/delta/kappa receptor knockout mice, the present study provides strong evidence that in vivo effects attributed to kappa3 receptor activation probably just reflect the combined actions of a particularly nonselective opioid drug. Indeed, molecular identification of any of the proposed subtypes of mu, delta, and kappa opioid receptors has proven elusive, suggesting that it is perhaps time to retire the notion of opioid receptor subtypes until definitive evidence for their existence is provided.

  5. Data on the effect of knockout of cytohesin-1 in myelination-related protein kinase signaling.

    Science.gov (United States)

    Tsuneishi, Ruri; Matsumoto, Naoto; Itaoka, Misa; Urai, Yuri; Kaneko, Minami; Watanabe, Natsumi; Takashima, Shou; Seki, Yoichi; Morimoto, Takako; Sakagami, Hiroyuki; Miyamoto, Yuki; Yamauchi, Junji

    2017-12-01

    Cytohesin-1 is the guanine-nucleotide exchange factor of Arf6, a small GTPase of Arf family, and participates in cellular morphological changes. Knockout mice of cytohesin-1 exhibit decreased myelination of neuronal axons in the peripheral nervous system (PNS) "Phosphorylation of cytohesin-1 by Fyn is required for initiation of myelination and the extent of myelination during development (Yamauchi et al., 2012) [1]". Herein we provide the data regarding decreased phosphorylation levels of protein kinases involved in two major myelination-related kinase cascades in cytohesin-1 knockout mice.

  6. Generation of knockout rats with X-linked severe combined immunodeficiency (X-SCID) using zinc-finger nucleases.

    Science.gov (United States)

    Mashimo, Tomoji; Takizawa, Akiko; Voigt, Birger; Yoshimi, Kazuto; Hiai, Hiroshi; Kuramoto, Takashi; Serikawa, Tadao

    2010-01-25

    Although the rat is extensively used as a laboratory model, the inability to utilize germ line-competent rat embryonic stem (ES) cells has been a major drawback for studies that aim to elucidate gene functions. Recently, zinc-finger nucleases (ZFNs) were successfully used to create genome-specific double-stranded breaks and thereby induce targeted gene mutations in a wide variety of organisms including plants, drosophila, zebrafish, etc. We report here on ZFN-induced gene targeting of the rat interleukin 2 receptor gamma (Il2rg) locus, where orthologous human and mouse mutations cause X-linked severe combined immune deficiency (X-SCID). Co-injection of mRNAs encoding custom-designed ZFNs into the pronucleus of fertilized oocytes yielded genetically modified offspring at rates greater than 20%, which possessed a wide variety of deletion/insertion mutations. ZFN-modified founders faithfully transmitted their genetic changes to the next generation along with the severe combined immune deficiency phenotype. The efficient and rapid generation of gene knockout rats shows that using ZFN technology is a new strategy for creating gene-targeted rat models of human diseases. In addition, the X-SCID rats that were established in this study will be valuable in vivo tools for evaluating drug treatment or gene therapy as well as model systems for examining the treatment of xenotransplanted malignancies.

  7. Contribution of PPARγ in modulation of acrolein-induced inflammatory signaling in gp91phox knock-out mice.

    Science.gov (United States)

    Yousefipour, Zivar; Chug, Neha; Marek, Katarzyna; Nesbary, Alicia; Mathew, Joseph; Ranganna, Kasturi; Newaz, Mohammad A

    2017-08-01

    Oxidative stress and inflammation are major contributors to acrolein toxicity. Peroxisome proliferator activated receptor gamma (PPARγ) has antioxidant and anti-inflammatory effects. We investigated the contribution of PPARγ ligand GW1929 to the attenuation of oxidative stress in acrolein-induced insult. Male gp91 phox knock-out (KO) mice were treated with acrolein (0.5 mg·(kg body mass) -1 by intraperitoneal injection for 7 days) with or without GW1929 (GW; 0.5 mg·(kg body mass) -1 ·day -1 , orally, for 10 days). The livers were processed for further analyses. Acrolein significantly increased 8-isoprostane and reduced PPARγ activity (P acrolein-treated WT mice, and was reduced by GW1929 (by 65%). KO mice exhibited higher xanthine oxidase (XO). Acrolein increased XO and COX in WT mice and XO in KO mice. GW1929 significantly reduced COX in WT and KO mice and reduced XO in KO mice. Acrolein significantly reduced the total antioxidant status in WT and KO mice (P acrolein-treated WT mice. GW1929 reduced NF-κB levels (by 51%) in KO mice. Acrolein increased CD36 in KO mice (by 43%), which was blunted with GW1929. Data confirms that the generation of free radicals by acrolein is mainly through NAD(P)H, but other oxygenates play a role too. GW1929 may alleviate the toxicity of acrolein by attenuating NF-κB, COX, and CD36.

  8. Generation of knockout rats with X-linked severe combined immunodeficiency (X-SCID using zinc-finger nucleases.

    Directory of Open Access Journals (Sweden)

    Tomoji Mashimo

    Full Text Available BACKGROUND: Although the rat is extensively used as a laboratory model, the inability to utilize germ line-competent rat embryonic stem (ES cells has been a major drawback for studies that aim to elucidate gene functions. Recently, zinc-finger nucleases (ZFNs were successfully used to create genome-specific double-stranded breaks and thereby induce targeted gene mutations in a wide variety of organisms including plants, drosophila, zebrafish, etc. METHODOLOGY/PRINCIPAL FINDINGS: We report here on ZFN-induced gene targeting of the rat interleukin 2 receptor gamma (Il2rg locus, where orthologous human and mouse mutations cause X-linked severe combined immune deficiency (X-SCID. Co-injection of mRNAs encoding custom-designed ZFNs into the pronucleus of fertilized oocytes yielded genetically modified offspring at rates greater than 20%, which possessed a wide variety of deletion/insertion mutations. ZFN-modified founders faithfully transmitted their genetic changes to the next generation along with the severe combined immune deficiency phenotype. CONCLUSIONS AND SIGNIFICANCE: The efficient and rapid generation of gene knockout rats shows that using ZFN technology is a new strategy for creating gene-targeted rat models of human diseases. In addition, the X-SCID rats that were established in this study will be valuable in vivo tools for evaluating drug treatment or gene therapy as well as model systems for examining the treatment of xenotransplanted malignancies.

  9. Somatostatin receptors

    DEFF Research Database (Denmark)

    Møller, Lars Neisig; Stidsen, Carsten Enggaard; Hartmann, Bolette

    2003-01-01

    therefore been acknowledged to be a third endogenous ligand at SRIF receptors. This review goes through mechanisms of signal transduction, pharmacology, and anatomical distribution of SRIF receptors. Structurally, SRIF receptors belong to the superfamily of G protein-coupled (GPC) receptors, sharing......- and heterodimerisation, let alone oligomerisation. Theoretically, this phenomenon adds a novel series of functional megareceptors/super-receptors, with varied pharmacological profiles, to the catalogue of monomeric receptor subtypes isolated and cloned in the past. SRIF analogues include both peptides and non......-peptides, receptor agonists and antagonists. Relatively long half lives, as compared to those of the endogenous ligands, have been paramount from the outset. Motivated by theoretical puzzles or the shortcomings of present-day diagnostics and therapy, investigators have also aimed to produce subtype...

  10. Smooth Muscle Endothelin B Receptors Regulate Blood Pressure but Not Vascular Function or Neointimal Remodeling.

    Science.gov (United States)

    Miller, Eileen; Czopek, Alicja; Duthie, Karolina M; Kirkby, Nicholas S; van de Putte, Elisabeth E Fransen; Christen, Sibylle; Kimmitt, Robert A; Moorhouse, Rebecca; Castellan, Raphael F P; Kotelevtsev, Yuri V; Kuc, Rhoda E; Davenport, Anthony P; Dhaun, Neeraj; Webb, David J; Hadoke, Patrick W F

    2017-02-01

    The role of smooth muscle endothelin B (ET B ) receptors in regulating vascular function, blood pressure (BP), and neointimal remodeling has not been established. Selective knockout mice were generated to address the hypothesis that loss of smooth muscle ET B receptors would reduce BP, alter vascular contractility, and inhibit neointimal remodeling. ET B receptors were selectively deleted from smooth muscle by crossing floxed ET B mice with those expressing cre-recombinase controlled by the transgelin promoter. Functional consequences of ET B deletion were assessed using myography. BP was measured by telemetry, and neointimal lesion formation induced by femoral artery injury. Lesion size and composition (day 28) were analyzed using optical projection tomography, histology, and immunohistochemistry. Selective deletion of ET B was confirmed by genotyping, autoradiography, polymerase chain reaction, and immunohistochemistry. ET B -mediated contraction was reduced in trachea, but abolished from mesenteric veins, of knockout mice. Induction of ET B -mediated contraction in mesenteric arteries was also abolished in these mice. Femoral artery function was unaltered, and baseline BP modestly elevated in smooth muscle ET B knockout compared with controls (+4.2±0.2 mm Hg; P<0.0001), but salt-induced and ET B blockade-mediated hypertension were unaltered. Circulating endothelin-1 was not altered in knockout mice. ET B -mediated contraction was not induced in femoral arteries by incubation in culture medium or lesion formation, and lesion size was not altered in smooth muscle ET B knockout mice. In the absence of other pathology, ET B receptors in vascular smooth muscle make a small but significant contribution to ET B -dependent regulation of BP. These ET B receptors have no effect on vascular contraction or neointimal remodeling. © 2016 The Authors.

  11. Characteristics of Allergic Pulmonary Inflammation in CXCR3Knockout Mice Sensitized and Challenged with House Dust Mite Protein.

    Science.gov (United States)

    Liu, Zhongjuan; Chen, Huaxia; Chen, Xiaolan; Gao, Jinming; Guo, Zijian

    2016-01-01

    Chemokine C-X-C motif receptor 3 (CXCR3) is a chemokine receptor that is mainly expressed by activated T lymphocytes. T cells play important roles in allergic pulmonary inflammation, which is a hallmark of asthma and elicits the localized accumulation of activated T cells in the lung. In China, a marked increase in the incidence rate of chronic allergic pulmonary inflammation has made it a major public health threat. In the present study, we investigated the role of CXCR3 and its ligands in airway inflammation induced by house dust mite protein (HDMP) in a CXCR3 knockout (CXCR3KO) asthma mouse model. Pathological manifestations in the lung, cell counts and bronchoalveolar lavage fluid (BALF) classifications were studied using hematoxylin and eosin (H&E) staining. The levels of IL-4 and IFN-γ in the BALF and splenocyte supernatants were measured using ELISA. CD4+ and CD8+ T cells in the lung and spleen were analyzed by flow cytometry. RT-PCR was applied to measure the mRNA transcript levels of monokines induced by IFN-γ(CXCL9) and IFN-γ inducible protein 10(CXCL10). The total cell counts, eosinophil counts, and IL-4 levels in the BALF and cultured splenocyte supernatants were significantly increased, while the levels of IFN-γ were reduced in the HDMP groups(Pmice than in C57BL/6 wild-type mice. Furthermore, the expression levels of CXCL9 and CXCL10 mRNA transcripts in the lungs of CXCR3KO mice were lower than those in C57BL/6 wild-type mice (P<0.05). CXCR3 and its ligands (i.e., CXCL9 and CXCL10) may play anti-inflammatory roles in this animal model. Promoting the expression of CXCR3 and its ligands may represent a novel therapeutic approach for preventing and curing asthma.

  12. Knockout of SRC-1 and SRC-3 in Mice Decreases Cardiomyocyte Proliferation and Causes a Noncompaction Cardiomyopathy Phenotype

    Science.gov (United States)

    Chen, Xian; Qin, Li; Liu, Zhaoliang; Liao, Lan; Martin, James F.; Xu, Jianming

    2015-01-01

    Noncompaction cardiomyopathy (NCC) is a congenital heart disease that causes ventricular dysfunction and high mortality rate in children. The mechanisms responsible for NCC are still unknown. The steroid receptor coactivator-1 (SRC-1) and SRC-3 are transcriptional coactivators for nuclear hormone receptors and certain other transcription factors that regulate many genes in development and organ function. However, the roles of SRC-1/3 in heart morphogenesis, function and NCC occurrence are unknown. This study aims to examine the spatial and temporal expression patterns of SRC-1/3 in the heart and investigate the specific roles of SRC-1/3 in heart development, function and NCC occurrence. Immunochemical analysis detected SRC-1/3 expressions in the proliferating cardiomyocytes of mouse heart at prenatal and neonatal stages, while these expressions disappeared within two weeks after birth. Through generating and characterizing mouse lines with global or cardiomyocyte-specific knockouts of SRC-1/3, we found ablation of SRC-1/3 in the myocardial lineage resulted in prominent trabeculae, deep intertrabecular recesses and thin ventricular wall and septum. These developmental defects caused a failure of trabecular compaction, decreased internal ventricular dimension, reduced cardiac ejection fraction and output and led to a high rate of postnatal mortality. Collectively, these structural and functional abnormalities closely simulate the phenotype of NCC patients. Further molecular analysis of cardiomyocytes in vivo and in vitro revealed that SRC-1/3 directly up-regulate cyclin E2, cyclin B1 and myocardin to promote cardiomyocyte proliferation and differentiation. In conclusion, SRC-1/3 are required for cardiomyocyte proliferation and differentiation at earlier developmental stages, and their dysfunction causes NCC-like abnormalities in the hearts of newborn and adult mice. PMID:26221073

  13. MicroRNA-134 Promotes the Development of Atherosclerosis Via the ANGPTL4/LPL Pathway in Apolipoprotein E Knockout Mice.

    Science.gov (United States)

    Ye, Qiong; Tian, Guo-Ping; Cheng, Hai-Peng; Zhang, Xin; Ou, Xiang; Yu, Xiao-Hua; Tan, Ru-Qi; Yang, Feng-Yun; Gong, Duo; Huang, Chong; Pan, Yan-Jun; Zhang, Jie; Chen, Ling-Yan; Zhao, Zhen-Wang; Xie, Wei; Li, Liang; Zhang, Min; Xia, Xiao-Dan; Zheng, Xi-Long; Tang, Chao-Ke

    2018-03-01

    Atherosclerosis is the most common cause of cardiovascular disease, such as myocardial infarction and stroke. Previous study revealed that microRNA (miR)-134 promotes lipid accumulation and proinflammatory cytokine secretion through angiopoietin-like 4 (ANGPTL4)/lipid lipoprotein (LPL) signaling in THP-1 macrophages. ApoE KO male mice on a C57BL/6 background were fed a high-fat/high-cholesterol Western diet, from 8 to 16 weeks of age. Mice were divided into four groups, and received a tail vein injection of miR-134 agomir, miR-134 antagomir, or one of the corresponding controls, respectively, once every 2 weeks after starting the Western diet. After 8 weeks we measured aortic atherosclerosis, LPL Activity, mRNA and protein levels of ANGPTL4 and LPL, LPL/ low-density lipoprotein receptor related protein 1 Complex Formation, proinflammatory cytokine secretion and lipid levels. Despite this finding, the influence of miR-134 on atherosclerosis in vivo remains to be determined. Using the well-characterized mouse atherosclerosis model of apolipoprotein E knockout, we found that systemic delivery of miR-134 agomir markedly enhanced the atherosclerotic lesion size, together with a significant increase in proinflammatory cytokine secretion and peritoneal macrophages lipid contents. Moreover, overexpression of miR-134 decreased ANGPTL4 expression but increased LPL expression and activity in both aortic tissues and peritoneal macrophages, which was accompanied by increased formation of LPL/low-density lipoprotein receptor-related protein 1 complexes in peritoneal macrophages. However, an opposite effect was observed in response to miR-134 antagomir. These findings suggest that miR-134 accelerates atherogenesis by promoting lipid accumulation and proinflammatory cytokine secretion via the ANGPTL4/LPL pathway. Therefore, targeting miR-134 may offer a promising strategy for the prevention and treatment of atherosclerotic cardiovascular disease.

  14. Nicotinic acetylcholine receptors control acetylcholine and noradrenaline release in the rodent habenulo-interpeduncular complex.

    Science.gov (United States)

    Beiranvand, F; Zlabinger, C; Orr-Urtreger, A; Ristl, R; Huck, S; Scholze, P

    2014-12-01

    Nicotinic acetylcholine receptors (nACh receptors) play a central role in the habenulo-interpeduncular system. We studied nicotine-induced release of NA and ACh in the habenula and interpeduncular nucleus (IPN). The habenula and IPN were loaded with [(3) H]-choline or [(3) H]-NA and placed in superfusion chambers. [(3) H]-ACh release was also stimulated using nicotinic agonists, electrical pulses and elevated [KCl]o in hippocampal and cortical slices from rats, wild-type mice and mice lacking α5, α7, β2, or β4 nACh receptor subunits. Finally, we analysed nACh receptor subtypes in the IPN using immunoprecipitation. Nicotine induced release of [(3) H]-ACh in the IPN of rats and mice. This release was calcium-dependent but not blocked by tetrodotoxin (TTX); moreover, [(3) H]-ACh release was abolished in β4-knockout mice but was unaffected in β2- and α5-knockout mice. In contrast, nicotine-induced release of [(3) H]-NA in the IPN and habenula was blocked by TTX and reduced in both β2-knockout and β4-knockout mice, and dose-response curves were right-shifted in α5-knockout mice. Although electrical stimuli triggered the release of both transmitters, [(3) H]-ACh release required more pulses delivered at a higher frequency. Our results confirm previous findings that β4-containing nACh receptors are critical for [(3) H]-ACh release in the mouse IPN. Experiments using α5-knockout mice also revealed that unlike in the hippocampus, nicotine-induced [(3) H]-NA release in the habenulo-interpeduncular system is altered in this knockout model. As α5-containing nACh receptors play a key role in nicotine intake, our results add NA to the list of transmitters involved in this mechanism. © 2014 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of The British Pharmacological Society.

  15. Uterine artery dysfunction in pregnant ACE2 knockout mice is associated with placental hypoxia and reduced umbilical blood flow velocity.

    Science.gov (United States)

    Yamaleyeva, Liliya M; Pulgar, Victor M; Lindsey, Sarah H; Yamane, Larissa; Varagic, Jasmina; McGee, Carolynne; daSilva, Mauro; Lopes Bonfa, Paula; Gurley, Susan B; Brosnihan, K Bridget

    2015-07-01

    Angiotensin-converting enzyme 2 (ACE2) knockout is associated with reduced fetal weight at late gestation; however, whether uteroplacental vascular and/or hemodynamic disturbances underlie this growth-restricted phenotype is unknown. Uterine artery reactivity and flow velocities, umbilical flow velocities, trophoblast invasion, and placental hypoxia were determined in ACE2 knockout (KO) and C57Bl/6 wild-type (WT) mice at day 14 of gestation. Although systolic blood pressure was higher in pregnant ACE2 KO vs. WT mice (102.3 ± 5.1 vs. 85.1 ± 1.9 mmHg, n = 5-6), the magnitude of difference was similar to that observed in nonpregnant ACE2 KO vs. WT mice. Maternal urinary protein excretion, serum creatinine, and kidney or heart weights were not different in ACE2 KO vs. WT. Fetal weight and pup-to-placental weight ratio were lower in ACE2 KO vs. WT mice. A higher sensitivity to Ang II [pD2 8.64 ± 0.04 vs. 8.5 ± 0.03 (-log EC50)] and greater maximal contraction to phenylephrine (169.0 ± 9.0 vs. 139.0 ± 7.0% KMAX), were associated with lower immunostaining for Ang II receptor 2 and fibrinoid content of the uterine artery in ACE2 KO mice. Uterine artery flow velocities and trophoblast invasion were similar between study groups. In contrast, umbilical artery peak systolic velocities (60.2 ± 4.5 vs. 75.1 ± 4.5 mm/s) and the resistance index measured using VEVO 2100 ultrasound were lower in the ACE2 KO vs. WT mice. Immunostaining for pimonidazole, a marker of hypoxia, and hypoxia-inducible factor-2α were higher in the trophospongium and placental labyrinth of the ACE2 KO vs. WT. In summary, placental hypoxia and uterine artery dysfunction develop before major growth of the fetus occurs and may explain the fetal growth restricted phenotype. Copyright © 2015 the American Physiological Society.

  16. Lentivirus-ABCG1 instillation reduces lipid accumulation and improves lung compliance in GM-CSF knock-out mice

    Energy Technology Data Exchange (ETDEWEB)

    Malur, Anagha; Huizar, Isham [Program in Lung Cell Biology and Translational Research, Division of Pulmonary, Critical Care and Sleep Medicine, East Carolina University, Greenville, NC (United States); Wells, Greg [Department of Microbiology and Immunology, East Carolina University, Greenville, NC (United States); Barna, Barbara P. [Program in Lung Cell Biology and Translational Research, Division of Pulmonary, Critical Care and Sleep Medicine, East Carolina University, Greenville, NC (United States); Malur, Achut G. [Department of Microbiology and Immunology, East Carolina University, Greenville, NC (United States); Thomassen, Mary Jane, E-mail: thomassenm@ecu.edu [Program in Lung Cell Biology and Translational Research, Division of Pulmonary, Critical Care and Sleep Medicine, East Carolina University, Greenville, NC (United States); Department of Microbiology and Immunology, East Carolina University, Greenville, NC (United States)

    2011-11-18

    Highlights: Black-Right-Pointing-Pointer Lentivirus-ABCG1 reduces lipid accumulation in lungs of GM-CSF knock-out mice. Black-Right-Pointing-Pointer Up-regulation of ABCG1 improves lung function. Black-Right-Pointing-Pointer Upregulation of ABCG1 improves surfactant metabolism. -- Abstract: We have shown decreased expression of the nuclear transcription factor, peroxisome proliferator-activated receptor-gamma (PPAR{gamma}) and the PPAR{gamma}-regulated ATP-binding cassette transporter G1 (ABCG1) in alveolar macrophages from patients with pulmonary alveolar proteinosis (PAP). PAP patients also exhibit neutralizing antibodies to granulocyte-macrophage colony stimulating factor (GM-CSF), an upregulator of PPAR{gamma}. In association with functional GM-CSF deficiency, PAP lung is characterized by surfactant-filled alveolar spaces and lipid-filled alveolar macrophages. Similar pathology characterizes GM-CSF knock-out (KO) mice. We reported previously that intratracheal instillation of a lentivirus (lenti)-PPAR{gamma} plasmid into GM-CSF KO animals elevated ABCG1 and reduced alveolar macrophage lipid accumulation. Here, we hypothesized that instillation of lenti-ABCG1 might be sufficient to decrease lipid accumulation and improve pulmonary function in GM-CSF KO mice. Animals received intratracheal instillation of lenti-ABCG1 or control lenti-enhanced Green Fluorescent Protein (eGFP) plasmids and alveolar macrophages were harvested 10 days later. Alveolar macrophage transduction efficiency was 79% as shown by lenti-eGFP fluorescence. Quantitative PCR analyses indicated a threefold (p = 0.0005) increase in ABCG1 expression with no change of PPAR{gamma} or ABCA1 in alveolar macrophages of lenti-ABCG1 treated mice. ABCG1 was unchanged in control lenti-eGFP and PBS-instilled groups. Oil Red O staining detected reduced intracellular neutral lipid in alveolar macrophages from lenti-ABCG1 treated mice. Extracellular cholesterol and phospholipids were also decreased as shown by

  17. Efficient TALEN-mediated gene knockout in livestock.

    Science.gov (United States)

    Carlson, Daniel F; Tan, Wenfang; Lillico, Simon G; Stverakova, Dana; Proudfoot, Chris; Christian, Michelle; Voytas, Daniel F; Long, Charles R; Whitelaw, C Bruce A; Fahrenkrug, Scott C

    2012-10-23

    Transcription activator-like effector nucleases (TALENs) are programmable nucleases that join FokI endonuclease with the modular DNA-binding domain of TALEs. Although zinc-finger nucleases enable a variety of genome modifications, their application to genetic engineering of livestock has been slowed by technical limitations of embryo-injection, culture of primary cells, and difficulty in producing reliable reagents with a limited budget. In contrast, we found that TALENs could easily be manufactured and that over half (23/36, 64%) demonstrate high activity in primary cells. Cytoplasmic injections of TALEN mRNAs into livestock zygotes were capable of inducing gene KO in up to 75% of embryos analyzed, a portion of which harbored biallelic modification. We also developed a simple transposon coselection strategy for TALEN-mediated gene modification in primary fibroblasts that enabled both enrichment for modified cells and efficient isolation of modified colonies. Coselection after treatment with a single TALEN-pair enabled isolation of colonies with mono- and biallelic modification in up to 54% and 17% of colonies, respectively. Coselection after treatment with two TALEN-pairs directed against the same chromosome enabled the isolation of colonies harboring large chromosomal deletions and inversions (10% and 4% of colonies, respectively). TALEN-modified Ossabaw swine fetal fibroblasts were effective nuclear donors for cloning, resulting in the creation of miniature swine containing mono- and biallelic mutations of the LDL receptor gene as models of familial hypercholesterolemia. TALENs thus appear to represent a highly facile platform for the modification of livestock genomes for both biomedical and agricultural applications.

  18. Phytosterol Feeding Causes Toxicity in ABCG5/G8 Knockout Mice

    Science.gov (United States)

    McDaniel, Allison L.; Alger, Heather M.; Sawyer, Janet K.; Kelley, Kathryn L.; Kock, Nancy D.; Brown, J. Mark; Temel, Ryan E.; Rudel, Lawrence L.

    2014-01-01

    Plant sterols, or phytosterols, are very similar in structure to cholesterol and are abundant in typical diets. The reason for poor absorption of plant sterols by the body is still unknown. Mutations in the ABC transporters G5 and G8 are known to cause an accumulation of plant sterols in blood and tissues (sitosterolemia). To determine the significance of phytosterol exclusion from the body, we fed wild-type and ABCG5/G8 knockout mice a diet enriched with plant sterols. The high-phytosterol diet was extremely toxic to the ABCG5/G8 knockout mice but had no adverse effects on wild-type mice. ABCG5/G8 knockout mice died prematurely and developed a phenotype that included high levels of plant sterols in many tissues, liver abnormalities, and severe cardiac lesions. This study is the first to report such toxic effects of phytosterol accumulation in ABCG5/G8 knockout mice. We believe these new data support the conclusion that plant sterols are excluded from the body because they are toxic when present at high levels. PMID:23380580

  19. Age-Related Changes in the Behavior of Apolipoprotein E Knockout Mice.

    Science.gov (United States)

    Fuentes, Dasha; Fernández, Nidia; García, Yenela; García, Teidy; Morales, Ana Ruth; Menéndez, Roberto

    2018-03-03

    The knockout mouse model, B6.129P2-Apoe tm1Unc is homozygotic for the Apolipoprotein E (ApoE) deletion; thus, it is capable of developing hyperlipidemia and atherosclerosis but ApoE is also a lipid-transport protein abundantly expressed in most neurons in the central nervous system, so these animals could also be models of neurodegenerative diseases. The aim of this study was to determine age-related changes in spontaneous behavior and in learning and memory of Apolipoprotein E knockout mice. Spontaneous behavioral measurements included sleeping pattern, motor coordination and balance by rotarod and open field activity, whereas learning and memory tests included forced alternation in Y-maze, novel object recognition and passive avoidance conditioning. Significant behavioral differences between aged knockout mice and age-matched wild type strain, C57Bl/6 were found in all the behavioral tests, except for the rotarod test. Genetically' modified mice exhibited less huddling contact during sleeping, decreased locomotor activity in novel environments and in learning and memory deficits. These results are consistent with the cognitive impairment and memory loss seen as the earliest clinical symptoms in neurodegenerative disorders such as Alzheimer's disease. The ApoE knockout mice might therefore be an appropriate model for studying the underlying mechanisms involved in behavioral changes caused by neurodegenerative diseases as well as for evaluating new therapies for these pathologies.

  20. Generation and basic characterization of glutamate carboxypeptidase II knock-out mice

    Czech Academy of Sciences Publication Activity Database

    Vorlová, Barbora; Kašpárek, Petr; Šácha, Pavel; Sedláček, Radislav; Konvalinka, Jan

    2016-01-01

    Roč. 25, č. 2 (2016), s. 267 ISSN 0962-8819. [Transgenic Technology Meeting (TT2016) /13./. 20.03.2016-23.03.2016, Praha] Institutional support: RVO:61388963 ; RVO:68378050 Keywords : GCPII * PSMA * FolhI * knock-out mice Subject RIV: CE - Biochemistry

  1. Revealing novel functions of glutamate carboxypeptidase II using knock-out mice

    Czech Academy of Sciences Publication Activity Database

    Vorlová, Barbora; Kašpárek, P.; Šácha, Pavel; Sedláček, R.; Konvalinka, Jan

    2017-01-01

    Roč. 284, Suppl 1 (2017), s. 358 ISSN 1742-464X. [FEBS Congress /42./ From Molecules to Cells and Back. 10.09.2017-14.09.2017, Jerusalem] Institutional support: RVO:61388963 Keywords : glutamate carboxypeptidase II * knock-out mice Subject RIV: CE - Biochemistry

  2. Efficient target-selected mutagenesis in Caenorhabditis elegans : toward a knockout for every gene

    NARCIS (Netherlands)

    Cuppen, Edwin; Gort, Eelke; Hazendonk, Esther; Mudde, Josine; van de Belt, José; Nijman, Isaäc J; Guryev, Victor; Plasterk, Ronald H A

    Reverse genetic or gene-driven knockout approaches have contributed significantly to the success of model organisms for fundamental and biomedical research. Although various technologies are available for C. elegans, none of them scale very well for genome-wide application. To address this, we

  3. Mig-6 Gene Knockout Induces Neointimal Hyperplasia in the Vascular Smooth Muscle Cell

    Directory of Open Access Journals (Sweden)

    Ju Hee Lee

    2014-01-01

    Full Text Available Although advances in vascular interventions can reduce the mortality associated with cardiovascular disease, neointimal hyperplasia remains a clinically significant obstacle limiting the success of current interventions. Identification of signaling pathways involved in migration and proliferation of vascular smooth muscle cells (SMCs is an important approach for the development of modalities to combat this disease. Herein we investigate the role of an immediate early response gene, mitogen-inducible gene-6 (Mig-6, in the development of neointimal hyperplasia using vascular smooth muscle specific Mig-6 knockout mice. We induced endoluminal injury to one side of femoral artery by balloon dilatation in both Mig-6 knockout and control mice. Four weeks following injury, the artery of Mig-6 knockout mice demonstrated a 5.3-fold increase in the neointima/media ratio compared with control mice (P=0.04. In addition, Mig-6 knockout vascular SMCs displayed an increase in both cell migration and proliferation compared with wild-type SMCs. Taken together, our data suggest that Mig-6 plays a critical role in the development of atherosclerosis. This finding provides new insight into the development of more effective ways to treat and prevent neointimal hyperplasia, particularly in-stent restenosis after percutaneous vascular intervention.

  4. Probing the structure of unstable nuclei through the recoiled proton tagged knockout reaction

    International Nuclear Information System (INIS)

    Ye, Y.; Cao, Z.; Jiang, D.

    2010-01-01

    Recoiled proton tagged knockout reaction experiments were carried-out for 8 He at 82,5 MeV/u in RIKEN and for 6 He at 65 MeV/u in Lanzhou. The very preliminary results for the distinguish of the reaction mechanism are presented and compared to the kinematics calculation. (authors)

  5. Hyperactivity and lack of social discrimination in the adolescent Fmr1 knockout mouse

    DEFF Research Database (Denmark)

    Sørensen, Emilie M; Bertelsen, Freja; Weikop, Pia

    2015-01-01

    The aims of this study were to investigate behaviour relevant to human autism spectrum disorder (ASD) and the fragile X syndrome in adolescent Fmr1 knockout (KO) mice and to evaluate the tissue levels of striatal monoamines. Fmr1 KO mice were evaluated in the open field, marble burying and three...

  6. Myocardial ischemia and reperfusion injury: Studies using transgenic and knockout mice

    NARCIS (Netherlands)

    Jong, W. M. C.; ten Cate, H.; Reitsma, P. H.; de Winter, R. J.

    2005-01-01

    Transgenic and knockout mice are created and used for a large variety of research objectives. This overview describes the (genetically modified) mouse models that have been used to study the development of myocardial ischemia and reperfusion injury. The role of cytokines, chemokines, leukocytes,

  7. A baculovirus alkaline nuclease knockout construct produces fragmented DNA and aberrant capsids

    International Nuclear Information System (INIS)

    Okano, Kazuhiro; Vanarsdall, Adam L.; Rohrmann, George F.

    2007-01-01

    DNA replication of bacmid-derived constructs of the Autographa californica multiple nucleocapsid nucleopolyhedrovirus (AcMNPV) was analyzed by field inversion gel electrophoresis (FIGE) in combination with digestion at a unique Eco81I restriction enzyme site. Three constructs were characterized: a parental bacmid, a bacmid deleted for the alkaline nuclease gene, and a bacmid from which the gp64 gene had been deleted. The latter was employed as a control for comparison with the alkaline nuclease knockout because neither yields infectious virus and their replication is limited to the initially transfected cells. The major difference between DNA replicated by the different constructs was the presence in the alkaline nuclease knockout of high concentrations of relatively small, subgenome length DNA in preparations not treated with Eco81I. Furthermore, upon Eco81I digestion, the alkaline nuclease knockout bacmid also yielded substantially more subgenome size DNA than the other constructs. Electron microscopic examination of cells transfected with the alkaline nuclease knockout indicated that, in addition to a limited number of normal-appearing electron-dense nucleocapsids, numerous aberrant capsid-like structures were observed indicating a defect in nucleocapsid maturation or in a DNA processing step that is necessary for encapsidation. Because of the documented role of the baculovirus alkaline nuclease and its homologs from other viruses in homologous recombination, these data suggest that DNA recombination may play a major role in the production of baculovirus genomes

  8. Serotonin Transporter Knockout Rats Show Improved Strategy Set-Shifting and Reduced Latent Inhibition

    Science.gov (United States)

    Nonkes, Lourens J. P.; van de Vondervoort, Ilse I. G. M.; de Leeuw, Mark J. C.; Wijlaars, Linda P.; Maes, Joseph H. R.; Homberg, Judith R.

    2012-01-01

    Behavioral flexibility is a cognitive process depending on prefrontal areas allowing adaptive responses to environmental changes. Serotonin transporter knockout (5-HTT[superscript -/-]) rodents show improved reversal learning in addition to orbitofrontal cortex changes. Another form of behavioral flexibility, extradimensional strategy set-shifting…

  9. Crucial role of alkaline sphingomyelinase in sphingomyelin digestion: a study on enzyme knockout mice

    DEFF Research Database (Denmark)

    Zhang, Yao; Cheng, Yajun; Hansen, Gert H

    2011-01-01

    Alkaline sphingomyelinase (alk-SMase) hydrolyses sphingomyelin (SM) to ceramide in the gut. To evaluate the physiological importance of the enzyme, we generated alk-SMase knockout (KO) mice by the Cre-recombinase-Locus of X-over P1(Cre-LoxP) system and studied SM digestion. Both wild-type (WT...

  10. Cross sections for one-neutron knock-out from Ca-37 at intermediate energy

    Czech Academy of Sciences Publication Activity Database

    Burger, A.; Azaiez, F.; Algora, A.; Al-Khatib, A.; Bastin, B.; Benzoni, G.; Borcea, R.; Bourgeois, C.; Bringel, P.; Clement, E.; Dalouzy, J.-C.; Dlouhý, Zdeněk; Dombradi, Z.; Drouart, A.; Engelhardt, C.; Franchoo, S.; Fülöp, Zs.; Gorgen, A.; Grévy, S.; Hubel, H.; Ibrahim, F.; Korten, W.; Mrázek, Jaromír; Navin, A.; Rotaru, F.; Chomaz, P. R.; Saint-Laurent, M. G.; Sletten, G.; Sohler, D.; Sorlin, O.; Stanoiu, M.; Stefan, I.; Theisen, C.; Timis, C.; Verney, D.; Williams, S.

    2012-01-01

    Roč. 86, č. 6 (2012), 064609/1-064609/5 ISSN 0556-2813 Institutional support: RVO:61389005 Keywords : nuclear structure * knock-out reactions * shell model Subject RIV: BG - Nuclear, Atomic and Molecular Physics, Colliders Impact factor: 3.715, year: 2012 http://prc.aps.org/pdf/PRC/v86/i6/e064609

  11. Behavior training reverses asymmetry in hippocampal transcriptome of the cav3.2 knockout mice.

    Directory of Open Access Journals (Sweden)

    Ni-Chun Chung

    Full Text Available Homozygous Cav3.2 knockout mice, which are defective in the pore-forming subunit of a low voltage activated T-type calcium channel, have been documented to show impaired maintenance of late-phase long-term potentiation (L-LTP and defective retrieval of context-associated fear memory. To investigate the role of Cav3.2 in global gene expression, we performed a microarray transcriptome study on the hippocampi of the Cav3.2-/- mice and their wild-type littermates, either naïve (untrained or trace fear conditioned. We found a significant left-right asymmetric effect on the hippocampal transcriptome caused by the Cav3.2 knockout. Between the naive Cav3.2-/- and the naive wild-type mice, 3522 differentially expressed genes (DEGs were found in the left hippocampus, but only 4 DEGs were found in the right hippocampus. Remarkably, the effect of Cav3.2 knockout was partially reversed by trace fear conditioning. The number of DEGs in the left hippocampus was reduced to 6 in the Cav3.2 knockout mice after trace fear conditioning, compared with the wild-type naïve mice. To our knowledge, these results demonstrate for the first time the asymmetric effects of the Cav3.2 and its partial reversal by behavior training on the hippocampal transcriptome.

  12. Histidine decarboxylase knockout mice, a genetic model of Tourette syndrome, show repetitive grooming after induced fear.

    Science.gov (United States)

    Xu, Meiyu; Li, Lina; Ohtsu, Hiroshi; Pittenger, Christopher

    2015-05-19

    Tics, such as are seen in Tourette syndrome (TS), are common and can cause profound morbidity, but they are poorly understood. Tics are potentiated by psychostimulants, stress, and sleep deprivation. Mutations in the gene histidine decarboxylase (Hdc) have been implicated as a rare genetic cause of TS, and Hdc knockout mice have been validated as a genetic model that recapitulates phenomenological and pathophysiological aspects of the disorder. Tic-like stereotypies in this model have not been observed at baseline but emerge after acute challenge with the psychostimulant d-amphetamine. We tested the ability of an acute stressor to stimulate stereotypies in this model, using tone fear conditioning. Hdc knockout mice acquired conditioned fear normally, as manifested by freezing during the presentation of a tone 48h after it had been paired with a shock. During the 30min following tone presentation, knockout mice showed increased grooming. Heterozygotes exhibited normal freezing and intermediate grooming. These data validate a new paradigm for the examination of tic-like stereotypies in animals without pharmacological challenge and enhance the face validity of the Hdc knockout mouse as a pathophysiologically grounded model of tic disorders. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  13. cGMP-dependent protein kinase type II knockout mice exhibit working memory impairments, decreased repetitive behavior, and increased anxiety-like traits.

    Science.gov (United States)

    Wincott, Charlotte M; Abera, Sinedu; Vunck, Sarah A; Tirko, Natasha; Choi, Yoon; Titcombe, Roseann F; Antoine, Shannon O; Tukey, David S; DeVito, Loren M; Hofmann, Franz; Hoeffer, Charles A; Ziff, Edward B

    2014-10-01

    Neuronal activity regulates AMPA receptor trafficking, a process that mediates changes in synaptic strength, a key component of learning and memory. This form of plasticity may be induced by stimulation of the NMDA receptor which, among its activities, increases cyclic guanosine monophosphate (cGMP) through the nitric oxide synthase pathway. cGMP-dependent protein kinase type II (cGKII) is ultimately activated via this mechanism and AMPA receptor subunit GluA1 is phosphorylated at serine 845. This phosphorylation contributes to the delivery of GluA1 to the synapse, a step that increases synaptic strength. Previous studies have shown that cGKII-deficient mice display striking spatial learning deficits in the Morris Water Maze compared to wild-type littermates as well as lowered GluA1 phosphorylation in the postsynaptic density of the prefrontal cortex (Serulle et al., 2007; Wincott et al., 2013). In the current study, we show that cGKII knockout mice exhibit impaired working memory as determined using the prefrontal cortex-dependent Radial Arm Maze (RAM). Additionally, we report reduced repetitive behavior in the Marble Burying task (MB), and heightened anxiety-like traits in the Novelty Suppressed Feeding Test (NSFT). These data suggest that cGKII may play a role in the integration of information that conveys both anxiety-provoking stimuli as well as the spatial and environmental cues that facilitate functional memory processes and appropriate behavioral response. Published by Elsevier Inc.

  14. CB2 cannabinoid receptors contribute to bacterial invasion and mortality in polymicrobial sepsis.

    Directory of Open Access Journals (Sweden)

    Balázs Csóka

    2009-07-01

    Full Text Available Sepsis is a major healthcare problem and current estimates suggest that the incidence of sepsis is approximately 750,000 annually. Sepsis is caused by an inability of the immune system to eliminate invading pathogens. It was recently proposed that endogenous mediators produced during sepsis can contribute to the immune dysfunction that is observed in sepsis. Endocannabinoids that are produced excessively in sepsis are potential factors leading to immune dysfunction, because they suppress immune cell function by binding to G-protein-coupled CB(2 receptors on immune cells. Here we examined the role of CB(2 receptors in regulating the host's response to sepsis.The role of CB(2 receptors was studied by subjecting CB(2 receptor wild-type and knockout mice to bacterial sepsis induced by cecal ligation and puncture. We report that CB(2 receptor inactivation by knockout decreases sepsis-induced mortality, and bacterial translocation into the bloodstream of septic animals. Furthermore, CB(2 receptor inactivation decreases kidney and muscle injury, suppresses splenic nuclear factor (NF-kappaB activation, and diminishes the production of IL-10, IL-6 and MIP-2. Finally, CB(2 receptor deficiency prevents apoptosis in lymphoid organs and augments the number of CD11b(+ and CD19(+ cells during CLP.Taken together, our results establish for the first time that CB(2 receptors are important contributors to septic immune dysfunction and mortality, indicating that CB(2 receptors may be therapeutically targeted for the benefit of patients suffering from sepsis.

  15. The antipsychotic-like effects in rodents of YQA31 involve dopamine D3 and 5-HT1A receptor.

    Science.gov (United States)

    Gou, Hong-Yan; Sun, Xue; Li, Fei; Wang, Zhi-Yuan; Wu, Ning; Su, Rui-Bin; Cong, Bin; Li, Jin

    2017-12-01

    We previously reported that YQA31 is a dopamine D3 receptor antagonist with modest 5-HT1A receptor affinity and that it exhibits antipsychotic properties in animal models of schizophrenia. However, the contributions of D3 and 5-HT1A receptors in the anti-psychotic effects of YQA31 are not clear. The current study evaluated the role of these two receptors in the effect of YQA31 on the hyperactivity and novel object recognition deficit in mice. We used dopamine D3 receptor knockout mice and 5-HT1A receptor antagonist WAY100635 pretreatment, respectively, to investigate the involvement of these receptors in the effects of YQA31. The anti-psychotic effects were tested by inducing hyperlocomotion with methamphetamine or MK-801 and by inducing novel object recognition deficit with MK-801, which are the animal models to represent a positive symptom and a cognitive disorder. YQA31 significantly inhibited MK-801-induced hyperlocomotion and novel object recognition deficit in WT mice, which was significantly inhibited by dopamine D3 receptor knockout. The 5-HT1A receptor antagonist, WAY100635, also blocked the effect of YQA31 in MK-801-induced novel object recognition deficit but not hyperlocomotion. The effect of YQA31 on methamphetamine-induced hyperlocomotion was not reversed by either dopamine D3 receptor knockout or WAY100635 pretreatment. These results demonstrate the different roles of dopamine D3 and 5-HT1A receptors in the anti-psychotic effects of YQA31. Both dopamine D3 and 5-HT1A receptors contributed to the effects of YQA31 on the inhibition of MK-801-induced novel object recognition deficit, and the dopamine D3 receptor mediated the inhibiting effect of YQA31 on hyperlocomotion induced by MK-801. Copyright © 2017. Published by Elsevier Urban & Partner Sp. z o.o.

  16. Sdhd and SDHD/H19 knockout mice do not develop paraganglioma or pheochromocytoma.

    Directory of Open Access Journals (Sweden)

    Jean-Pierre Bayley

    Full Text Available BACKGROUND: Mitochondrial succinate dehydrogenase (SDH is a component of both the tricarboxylic acid cycle and the electron transport chain. Mutations of SDHD, the first protein of intermediary metabolism shown to be involved in tumorigenesis, lead to the human tumors paraganglioma (PGL and pheochromocytoma (PC. SDHD is remarkable in showing an 'imprinted' tumor suppressor phenotype. Mutations of SDHD show a very high penetrance in man and we postulated that knockout of Sdhd would lead to the development of PGL/PC, probably in aged mice. METHODOLOGY/PRINCIPAL FINDINGS: We generated a conventional knockout of Sdhd in the mouse, removing the entire third exon. We also crossed this mouse with a knockout of H19, a postulated imprinted modifier gene of Sdhd tumorigenesis, to evaluate if loss of these genes together would lead to the initiation or enhancement of tumor development. Homozygous knockout of Sdhd results in embryonic lethality. No paraganglioma or other tumor development was seen in Sdhd KO mice followed for their entire lifespan, in sharp contrast to the highly penetrant phenotype in humans. Heterozygous Sdhd KO mice did not show hyperplasia of paraganglioma-related tissues such as the carotid body or of the adrenal medulla, or any genotype-related pathology, with similar body and organ weights to wildtype mice. A cohort of Sdhd/H19 KO mice developed several cases of profound cardiac hypertrophy, but showed no evidence of PGL/PC. CONCLUSIONS: Knockout of Sdhd in the mouse does not result in a disease phenotype. H19 may not be an initiator of PGL/PC tumorigenesis.

  17. P-glycoprotein interaction with risperidone and 9-OH-risperidone studied in vitro, in knock-out mice and in drug-drug interaction experiments

    DEFF Research Database (Denmark)

    Ejsing, Thomas B.; Pedersen, Anne D.; Linnet, Kristian

    2005-01-01

    P-glycoprotein, risperidone, nortriptyline, cyclosporine A, drug-drug interaction, blood-brain barrier, knock-out mice......P-glycoprotein, risperidone, nortriptyline, cyclosporine A, drug-drug interaction, blood-brain barrier, knock-out mice...

  18. Targeting of the retroviral envelope protein SL3-2 towards the human G-Protein coupled receptor hAPJ

    DEFF Research Database (Denmark)

    Pagh, Kristina

    enzym, der så kobler biotin til virus partiklen, mens peptid 2 tidligere har vist sig at binde sig til biotin. Det første peptid er også blevet sat ind i apelin receptoren. Gennem disse forsøg kan vi konstatere, at det er muligt at binde biotin til både virus og apelin receptoren, uden at dette påvirker...

  19. Highly segmented CVD diamond detectors and high-resolution momentum measurements in knockout reactions; Hochsegmentierte CVD Diamant Detektoren und hochaufloesende Impulsmessungen in Knockout Reaktionen

    Energy Technology Data Exchange (ETDEWEB)

    Schwertel, Sabine

    2009-11-26

    In recent years knockout reactions have proven to be important tools for investigations of the structure of light exotic nuclei. In spring 2006 an experiment was performed with the fragment separator at GSI in Darmstadt to extend this method to medium-mass nuclei with energies of about 400 AMeV. An experiment with a stable and well-known {sup 48}Ca primary beam was performed as a reference. The FRS was set for the reaction {sup 56}Ti{yields}{sup 55}Ti. Because of the high acceptance of the FRS, mother and daughter nuclei of one-neutron knockout reactions in the Sc isotopes {sup 51,52,53,54,55}Sc were also transported with high efficiency. These are investigated in the first part of this thesis. Inclusive cross sections of 77(10) mbarn for one-neutron knockout from {sup 48}Ca and 78(12) mbarn, 99(15) mbarn, 101(15) mbarn, 113(17) mbarn and 72(14) mbarn for knockout from {sup 51,52,53,54,55}Sc, respectively, were measured for the first time. For the Sc isotopes the reduction factors are close to 1. For the one-neutron knockout reactions in {sup 48}Ca and the Sc isotopes, respectively, the momentum distributions could be measured with a relative resolution of 0.17-0.27 %. From the momentum distributions spectroscopic factors of the involved orbitals could be extracted. In the future, further knockout experiments should be performed using the R{sup 3}B setup at FAIR. The available beam intensity will be up to four orders of magnitude higher. As the beam has to be tracked from the dispersive plane of the Super-FRS up to the R{sup 3}B target, radiation hard detectors are needed. In the framework of this thesis extensive measurements were performed at the tandem accelerator in Munich with numerous small (10 x 10 mm{sup 2}) test detectors. Samples using new manufacturing methods were characterized. A dose of some 10{sup 11} ions/mm{sup 2} was determined as a limit for the exposure of the material with heavy ions of high ionisation density. It could be shown that even

  20. Exploring the Behavioral and Metabolic Phenotype Generated by Re-Introduction of the Ghrelin Receptor in the Ventral Tegmental Area

    DEFF Research Database (Denmark)

    Skov, Louise J; Jensen, Morten; Christiansen, Søren H

    2017-01-01

    Ghrelin receptor (Ghr-R) signaling in neurons of the ventral tegmental area (VTA) can modulate dopaminergic function and the reward-related effects of both palatable foods and drugs of abuse. In this study, we re-introduced the Ghr-R in VTA neurons in Ghr-R knockout mice (Ghr-R(VTA) mice...

  1. Host defence during Klebsiella pneumonia relies on haematopoietic-expressed Toll-like receptors 4 and 2

    NARCIS (Netherlands)

    Wieland, C. W.; van Lieshout, M. H. P.; Hoogendijk, A. J.; van der Poll, T.

    2011-01-01

    In this study, the relative roles of Toll-like receptor (TLR)2 and TLR4 were investigated independently and together. Moreover, we studied the role of haematopoietic compartment in anti-Klebsiella host defence. We infected TLR2 and TLR4 single-, and TLR2×4 double knockout (KO) animals with different

  2. Deletion of the GluA1 AMPA Receptor Subunit Alters the Expression of Short-Term Memory

    Science.gov (United States)

    Sanderson, David J.; Sprengel, Rolf; Seeburg, Peter H.; Bannerman, David M.

    2011-01-01

    Deletion of the GluA1 AMPA receptor subunit selectively impairs short-term memory for spatial locations. We further investigated this deficit by examining memory for discrete nonspatial visual stimuli in an operant chamber. Unconditioned suppression of magazine responding to visual stimuli was measured in wild-type and GluA1 knockout mice.…

  3. Describing the role of Drosophila melanogaster ABC transporters in insecticide biology using CRISPR-Cas9 knockouts.

    Science.gov (United States)

    Denecke, Shane; Fusetto, Roberto; Batterham, Philip

    2017-12-01

    ABC transporters have a well-established role in drug resistance, effluxing xenobiotics from cells and tissues within the organism. More recently, research has been dedicated to understanding the role insect ABC transporters play in insecticide toxicity, but progress in understanding the contribution of specific transporters has been hampered by the lack of functional genetic tools. Here, we report knockouts of three Drosophila melanogaster ABC transporter genes, Mdr49, Mdr50, and Mdr65, that are homologous to the well-studied mammalian ABCB1 (P-glycoprotein). Each knockout mutant was created in the same wild type background and tested against a panel of insecticides representing different chemical classes. Mdr65 knockouts were more susceptible to all neuroactive insecticides tested, but Mdr49 and Mdr50 knockouts showed increased susceptibility or resistance depending on the insecticide used. Mdr65 was chosen for further analysis. Calculation of LC 50 values for the Mdr65 knockout allowed the substrate specificity of this transporter to be examined. No obvious distinguishing structural features were shared among MDR65 substrates. A role for Mdr65 in insecticide transport was confirmed by testing the capacity of the knockout to synergize with the ABC inhibitor verapamil and by measuring the levels of insecticide retained in the body of knockout flies. These data unambiguously establish the influence of ABC transporters on the capacity of wild type D. melanogaster to tolerate insecticide exposure and suggest that both tissue and substrate specificity underpin this capacity. Copyright © 2017 Elsevier Ltd. All rights reserved.

  4. Metabotropic glutamate 5 receptor antagonism is associated with antidepressant-like effects in mice.

    Science.gov (United States)

    Li, Xia; Need, Anne B; Baez, Melvyn; Witkin, Jeffrey M

    2006-10-01

    Antidepressant-like effects of metabotropic glutamate (mGlu)5 receptor antagonists have been reported previously. We now provide definitive identification of mGlu5 receptors as a target for these effects through the combined use of selective antagonists and mice with targeted deletion of the mGlu5 protein. In these experiments, the mGlu5 receptor antagonists 2-methyl-6-(phenylethynyl)-pyridine (MPEP) and the more selective and metabolically stable analog 3-[(2-methyl-1,3-thiazol-4-yl)ethynyl]-pyridine (MTEP) decreased immobility in the mouse forced swim test, a test predictive of antidepressant efficacy in humans. mGlu5 receptor knockout mice had a phenotype in the forced swim test that was congruent with the effects of receptor blockade; mGlu5 receptor knockout mice were significantly less immobile than their wild-type counterparts. Consistent with mGlu5 receptor mediation of the antidepressant-like effects of MPEP, the effects of MPEP were not observed in mGlu5 receptor knockout mice, whereas comparable effects of the tricyclic antidepressant imiprimine remained active in the mutant mice. MPEP and imiprimine resulted in a synergistic antidepressant-like effect in the forced swim test. The drug interaction was not likely because of increased levels of drugs in the brain, suggesting a pharmacodynamic interaction of mGlu5 and monoaminergic systems in this effect. Thus, the present findings substantiate the hypothesis that mGlu5 receptor antagonism is associated with antidepressant-like effects. This mechanism may not only provide a novel approach to the therapeutic management of depressive disorders but also may be useful in the augmentation of effects of traditional antidepressant agents.

  5. Characterisation of iunH gene knockout strain from Mycobacterium tuberculosis

    Directory of Open Access Journals (Sweden)

    Anne Drumond Villela

    Full Text Available BACKGROUND Tuberculosis (TB is an infectious disease caused mainly by the bacillus Mycobacterium tuberculosis. The better understanding of important metabolic pathways from M. tuberculosis can contribute to the development of novel therapeutic and prophylactic strategies to combat TB. Nucleoside hydrolase (MtIAGU-NH, encoded by iunH gene (Rv3393, is an enzyme from purine salvage pathway in M. tuberculosis. MtIAGU-NH accepts inosine, adenosine, guanosine, and uridine as substrates, which may point to a pivotal metabolic role. OBJECTIVES Our aim was to construct a M. tuberculosis knockout strain for iunH gene, to evaluate in vitro growth and the effect of iunH deletion in M. tuberculosis in non-activated and activated macrophages models of infection. METHODS A M. tuberculosis knockout strain for iunH gene was obtained by allelic replacement, using pPR27xylE plasmid. The complemented strain was constructed by the transformation of the knockout strain with pNIP40::iunH. MtIAGU-NH expression was analysed by Western blot and LC-MS/MS. In vitro growth was evaluated in Sauton’s medium. Bacterial load of non-activated and interferon-γ activated RAW 264.7 cells infected with knockout strain was compared with wild-type and complemented strains. FINDINGS Western blot and LC-MS/MS validated iunH deletion at protein level. The iunH knockout led to a delay in M. tuberculosis growth kinetics in Sauton’s medium during log phase, but did not affect bases and nucleosides pool in vitro. No significant difference in bacterial load of knockout strain was observed when compared with both wild-type and complemented strains after infection of non-activated and interferon-γ activated RAW 264.7 cells. MAIN CONCLUSION The disruption of iunH gene does not influence M. tuberculosis growth in both non-activated and activated RAW 264.7 cells, which show that iunH gene is not important for macrophage invasion and virulence. Our results indicated that MtIAGU-NH is not a

  6. Construction of Mycobacterium tuberculosis cdd knockout and evaluation of invasion and growth in macrophages

    Directory of Open Access Journals (Sweden)

    Anne Drumond Villela

    Full Text Available Cytidine deaminase (MtCDA, encoded by cdd gene (Rv3315c, is the only enzyme identified in nucleotide biosynthesis pathway of Mycobacterium tuberculosis that is able to recycle cytidine and deoxycytidine. An M. tuberculosis knockout strain for cdd gene was obtained by allelic replacement. Evaluation of mRNA expression validated cdd deletion and showed the absence of polar effect. MudPIT LC-MS/MS data indicated thymidine phosphorylase expression was decreased in knockout and complemented strains. The cdd disruption does not affect M. tuberculosis growth both in Mid- dlebrook 7H9 and in RAW 264.7 cells, which indicates that cdd is not important for macrophage invasion and virulence.

  7. Knockout of GAD65 has major impact on synaptic GABA synthesized from astrocyte-derived glutamine

    DEFF Research Database (Denmark)

    Walls, Anne Byriel; Eyjolfsson, Elvar M.; Smeland, Olav B.

    2011-01-01

    γ-Aminobutyric acid (GABA) synthesis from glutamate is catalyzed by glutamate decarboxylase (GAD) of which two isoforms, GAD65 and GAD67, have been identified. The GAD65 has repeatedly been shown to be important during intensified synaptic activity. To specifically elucidate the significance of GAD......65 for maintenance of the highly compartmentalized intracellular and intercellular GABA homeostasis, GAD65 knockout and corresponding wild-type mice were injected with [1-(13)C]glucose and the astrocyte-specific substrate [1,2-(13)C]acetate. Synthesis of GABA from glutamine in the GABAergic synapses...... was further investigated in GAD65 knockout and wild-type mice using [1,2-(13)C]acetate and in some cases γ-vinylGABA (GVG, Vigabatrin), an inhibitor of GABA degradation. A detailed metabolic mapping was obtained by nuclear magnetic resonance (NMR) spectroscopic analysis of tissue extracts of cerebral cortex...

  8. Generation of knockout rabbits using transcription activator-like effector nucleases

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    Yu Wang

    2014-01-01

    Full Text Available Zinc-finger nucleases and transcription activator-like effector nucleases are novel gene-editing platforms contributing to redefine the boundaries of modern biological research. They are composed of a non-specific cleavage domain and a tailor made DNA-binding module, which enables a broad range of genetic modifications by inducing efficient DNA double-strand breaks at desired loci. Among other remarkable uses, these nucleases have been employed to produce gene knockouts in mid-size and large animals, such as rabbits and pigs, respectively. This approach is cost effective, relatively quick, and can produce invaluable models for human disease studies, biotechnology or agricultural purposes. Here we describe a protocol for the efficient generation of knockout rabbits using transcription activator-like effector nucleases, and a perspective of the field.

  9. Investigating the Role of Serotonin in Methamphetamine Psychosis: Unaltered Behavioral Effects of Chronic Methamphetamine in 5-HT1A Knockout Mice

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    Maarten van den Buuse

    2017-04-01

    Full Text Available Methamphetamine (Meth is a widely abused stimulant drug, but this abuse is associated with an increased risk of developing psychosis. In addition to its well-known action on brain dopamine, Meth also affects serotonergic (5-HT neurons. The aim of this study was to investigate this role in mice, which lack one of the main serotonin receptors, the 5-HT1A receptor, which has been implicated in both schizophrenia and Meth-induced psychosis. Male and female wild-type or 5-HT1A knockout (KO mice received daily treatment with increasing doses of methamphetamine from 6 to 9 weeks of age (1–4 mg/kg/day twice a day. At least 2 weeks after the last injection, the mice underwent a battery of behavioral tests focusing on psychosis-related behaviors, including Meth-induced hyperactivity, prepulse inhibition (PPI, social interaction, elevated plus maze (EPM, and Y-maze. Meth pretreatment resulted in significantly increased hyperlocomotion in response to an acute Meth challenge, but this effect was independent of genotype. Chronic Meth treatment resulted in decreased levels of anxiety in the EPM in both sexes, as well as increased startle responses in female mice only, again independent of genotype. 5-HT1A KO mice showed an increased locomotor response to acute Meth in both sexes, as well as increased PPI and decreased startle responses in female mice only, independent of Meth pretreatment. In conclusion, the effects of chronic Meth appear unaffected by the absence of the 5-HT1A receptor. These results do not support a role of the 5-HT1A receptor in Meth-induced psychosis.

  10. Effects of High-Fat Diet on Stress Response in Male and Female Wildtype and Prolactin Knockout Mice.

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    Kalyani, Manu; Hasselfeld, Kathryn; Janik, James M; Callahan, Phyllis; Shi, Haifei

    2016-01-01

    Prolactin (PRL) is well characterized for its roles in initiation and maintenance of lactation, and it also suppresses stress-induced responses. Feeding a high-fat diet (HFD) disrupts activity of the hypothalamic-pituitary-adrenal (HPA) axis. Whether PRL regulates HPA axis activation under HFD feeding is not clear. Male and female wildtype (WT) and PRL knockout (KO) mice were fed either a standard low-fat diet (LFD) or HFD for 12 weeks. Circulating corticosterone (CORT) levels were measured before, during, and after mice were subjected to an acute restraint stress or remained in their home cages as no stress controls. HFD feeding increased leptin levels, but the increase was lower in KO than in WT mice. All stressed female groups and only LFD-fed stressed males had elevated CORT levels compared to their no stress same-sex counterparts regardless of genotype. These results indicated that HFD consumption blunted the HPA axis response to acute stress in males but not females. Additionally, basal hypothalamic CRH content was lower in HFD than LFD males, but was similar among female groups. Furthermore, although basal CORT levels were similar among KO and WT groups, CORT levels were higher in KO mice than their WT counterparts during stress, suggesting that loss of PRL led to greater HPA axis activation. Basal PRL receptor mRNA levels in the choroid plexus were higher in HFD than LFD same-sex counterparts, suggesting activation of central PRL's action by HFD feeding in both males and females. Current results confirmed PRL's roles in suppression of the stress-induced HPA axis activation. Although HFD feeding activated central PRL's action in both sexes, only the male HPA axis was dampened by HFD feeding.

  11. Orexin/Hypocretin and Histamine: Distinct Roles in the Control of Wakefulness Demonstrated Using Knock-Out Mouse Models

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    Anaclet, Christelle; Parmentier, Régis; Ouk, Koliane; Guidon, Gérard; Buda, Colette; Sastre, Jean-Pierre; Akaoka, Hidéo; Sergeeva, Olga A.; Yanagisawa, Masashi; Ohtsu, Hiroshi; Franco, Patricia; Haas, Helmut L.; Lin, Jian Sheng

    2009-01-01

    To determine the respective role played by orexin/hypocretin and histamine (HA) neurons in maintaining wakefulness (W), we characterized the behavioral and sleep-wake phenotypes of orexin(Ox) knockout(−/−) mice and compared them with those of histidine-decarboxylase(HDC, HA-synthesizing enzyme)−/−mice. While both mouse strains displayed sleep fragmentation and increased paradoxical sleep(PS), they presented a number of marked differences: 1) The PS-increase in HDC−/−mice was seen during lightness, whereas that in Ox−/−mice occurred during darkness; 2) Contrary to HDC−/−, Ox−/−mice had no W deficiency around lights-off, nor an abnormal EEG and responded to a new environment with increased W; 3) Only Ox−/−, but not HDC−/−mice, displayed narcolepsy and deficient W when faced with motor challenge. Thus, when placed on a wheel, WT, but not littermate Ox−/−mice, voluntarily spent their time in turning it and as a result, remained highly awake; this was accompanied by dense c-fos expression in many areas of their brains, including Ox-neurons in the dorsolateral hypothalamus. The W and motor deficiency of Ox−/−mice was due to the absence of Ox because intraventricular dosing of Ox-A restored their W amount and motor performance whereas SB-334867 (Ox1-receptor antagonist, i.p.) impaired W and locomotion of WT mice during the test. These data indicate that Ox, but not HA, promotes W through enhanced locomotion and suggest that HA and Ox neurons exert a distinct, but complementary and synergistic control of W: the neuropeptide being more involved in its behavioral aspects, whereas the amine is mainly responsible for its qualitative cognitive aspects and cortical-EEG activation. PMID:19923277

  12. Predictive validity and immune cell involvement in the pathogenesis of piroxicam-accelerated colitis in interleukin-10 knockout mice.

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    Holgersen, Kristine; Kvist, Peter Helding; Hansen, Axel Kornerup; Holm, Thomas Lindebo

    2014-07-01

    Piroxicam administration is a method for induction of enterocolitis in interleukin-10 knockout (IL-10 k.o.) mice. The piroxicam-accelerated colitis (PAC) IL-10 k.o. model combines a dysregulated immune response against the gut microbiota with a decreased mucosal integrity. The predictive validity and pathogenic mechanisms of the model have not been thoroughly investigated. In this study, IL-10 k.o. mice received piroxicam in the chow, and model qualification was performed by examining the efficacy of prophylactic anti-IL-12/23p40 monoclonal antibody (mAb), anti-TNFα mAb, cyclosporine A (CsA) and oral prednisolone treatment. To evaluate cell involvement in the disease pathogenesis, specific cell subsets were depleted by treatment with anti-CD4 mAb, anti-CD8 mAb or clodronate-encapsulated liposomes. T cell receptor co-stimulation was blocked by CTLA4-Ig. Cytokine profiling ELISAs and calprotectin immunohistochemistry were performed on colon tissue. Treatments with anti-IL-12/23p40 mAb and CsA prevented disease in PAC IL-10 k.o. mice and reduced IFNγ, IL-17A, MPO and calprotectin levels in colon. Anti-TNFα mAb treatment caused amelioration of selected clinical parameters. No effect of prednisolone was detected. Depletion of CD8(+) cells tended to increase mortality, whereas treatment with anti-CD4 mAb or CTLA4-Ig had no significant effect on disease development. Clodronate liposome treatment induced a loss of body weight; nevertheless macrophage depletion was associated with a significant reduction in colonic pathology. In conclusion, reference drugs with known efficacy in severe inflammatory bowel disease were efficacious in the PAC IL-10 k.o. model. Our data indicate that in this model macrophages are a main driver of colitis, whereas CD4(+) cells are not. Copyright © 2014 Elsevier B.V. All rights reserved.

  13. The FKBP5 Gene Affects Alcohol Drinking in Knockout Mice and Is Implicated in Alcohol Drinking in Humans

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    Bin Qiu

    2016-08-01

    Full Text Available FKBP5 encodes FK506-binding protein 5, a glucocorticoid receptor (GR-binding protein implicated in various psychiatric disorders and alcohol withdrawal severity. The purpose of this study is to characterize alcohol preference and related phenotypes in Fkbp5 knockout (KO mice and to examine the role of FKBP5 in human alcohol consumption. The following experiments were performed to characterize Fkpb5 KO mice. (1 Fkbp5 KO and wild-type (WT EtOH consumption was tested using a two-bottle choice paradigm; (2 The EtOH elimination rate was measured after intraperitoneal (IP injection of 2.0 g/kg EtOH; (3 Blood alcohol concentration (BAC was measured after 3 h limited access of alcohol; (4 Brain region expression of Fkbp5 was identified using LacZ staining; (5 Baseline corticosterone (CORT was assessed. Additionally, two SNPs, rs1360780 (C/T and rs3800373 (T/G, were selected to study the association of FKBP5 with alcohol consumption in humans. Participants were college students (n = 1162 from 21–26 years of age with Chinese, Korean or Caucasian ethnicity. The results, compared to WT mice, for KO mice exhibited an increase in alcohol consumption that was not due to differences in taste sensitivity or alcohol metabolism. Higher BAC was found in KO mice after 3 h of EtOH access. Fkbp5 was highly expressed in brain regions involved in the regulation of the stress response, such as the hippocampus, amygdala, dorsal raphe and locus coeruleus. Both genotypes exhibited similar basal levels of plasma corticosterone (CORT. Finally, single nucleotide polymorphisms (SNPs in FKBP5 were found to be associated with alcohol drinking in humans. These results suggest that the association between FKBP5 and alcohol consumption is conserved in both mice and humans.

  14. Nicotine reward and affective nicotine withdrawal signs are attenuated in calcium/calmodulin-dependent protein kinase IV knockout mice.

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    Kia J Jackson

    Full Text Available The influx of Ca(2+ through calcium-permeable nicotinic acetylcholine receptors (nAChRs leads to activation of various downstream processes that may be relevant to nicotine-mediated behaviors. The calcium activated protein, calcium/calmodulin-dependent protein kinase IV (CaMKIV phosphorylates the downstream transcription factor cyclic AMP response element binding protein (CREB, which mediates nicotine responses; however the role of CaMKIV in nicotine dependence is unknown. Given the proposed role of CaMKIV in CREB activation, we hypothesized that CaMKIV might be a crucial molecular component in the development of nicotine dependence. Using male CaMKIV genetically modified mice, we found that nicotine reward is attenuated in CaMKIV knockout (-/- mice, but cocaine reward is enhanced in these mice. CaMKIV protein levels were also increased in the nucleus accumbens of C57Bl/6 mice after nicotine reward. In a nicotine withdrawal assessment, anxiety-related behavior, but not somatic signs or the hyperalgesia response are attenuated in CaMKIV -/- mice. To complement our animal studies, we also conducted a human genetic association analysis and found that variants in the CaMKIV gene are associated with a protective effect against nicotine dependence. Taken together, our results support an important role for CaMKIV in nicotine reward, and suggest that CaMKIV has opposing roles in nicotine and cocaine reward. Further, CaMKIV mediates affective, but not physical nicotine withdrawal signs, and has a protective effect against nicotine dependence in human genetic association studies. These findings further indicate the importance of calcium-dependent mechanisms in mediating behaviors associated with drugs of abuse.

  15. Hypocretin-2 saporin lesions of the ventrolateral periaquaductal gray (vlPAG increase REM sleep in hypocretin knockout mice.

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    Satvinder Kaur

    2009-07-01

    Full Text Available Ten years ago the sleep disorder narcolepsy was linked to the neuropeptide hypocretin (HCRT, also known as orexin. This disorder is characterized by excessive day time sleepiness, inappropriate triggering of rapid-eye movement (REM sleep and cataplexy, which is a sudden loss of muscle tone during waking. It is still not known how HCRT regulates REM sleep or muscle tone since HCRT neurons are localized only in the lateral hypothalamus while REM sleep and muscle atonia are generated from the brainstem. To identify a potential neuronal circuit, the neurotoxin hypocretin-2-saporin (HCRT2-SAP was used to lesion neurons in the ventral lateral periaquaductal gray (vlPAG. The first experiment utilized hypocretin knock-out (HCRT-ko mice with the expectation that deletion of both HCRT and its target neurons would exacerbate narcoleptic symptoms. Indeed, HCRT-ko mice (n = 8 given the neurotoxin HCRT2-SAP (16.5 ng/23nl/sec each side in the vlPAG had levels of REM sleep and sleep fragmentation that were considerably higher compared to HCRT-ko given saline (+39%; n = 7 or wildtype mice (+177%; n = 9. However, cataplexy attacks did not increase, nor were levels of wake or non-REM sleep changed. Experiment 2 determined the effects in mice where HCRT was present but the downstream target neurons in the vlPAG were deleted by the neurotoxin. This experiment utilized an FVB-transgenic strain of mice where eGFP identifies GABA neurons. We verified this and also determined that eGFP neurons were immunopositive for the HCRT-2 receptor. vlPAG lesions in these mice increased REM sleep (+79% versus saline controls and it was significantly correlated (r = 0.89 with loss of eGFP neurons. These results identify the vlPAG as one site that loses its inhibitory control over REM sleep, but does not cause cataplexy, as a result of hypocretin deficiency.

  16. Panax Notoginseng Saponins Promote Endothelial Progenitor Cell Mobilization and Attenuate Atherosclerotic Lesions in Apolipoprotein E Knockout Mice

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    Ya Liu

    2013-09-01

    Full Text Available Background: Endothelial progenitor cells (EPCs derived from the bone marrow (BM play a key role in the homeostasis of vascular repair by enhanced reendothelialization. Panax notoginseng saponins (PNS, a highly valued traditional Chinese medicine, has been shown to reduce morbidity and mortality from coronary artery disease. The present research was designed to explore the contribution of progenitor cells to the progression of atherosclerotic plaques and the possible modulatory role of PNS in this process. Methods: PNS (60 or 120 mg/kg via intraperitoneal injection was administered over 8 weeks in apolipoprotein E knockout mice on an atherogenic diet. The sizes and histochemical alteration of atherosclerotic lesions and numbers of EPCs in BM and peripheral blood were analyzed. The expression of chemokine stromal cell-derived factor 1α (SDF-1α and its receptor, CXCR4, was monitored as well. Results: PNS significantly reduced the lesion area and intima-to-media ratio compared to vehicle treatment. PNS also augmented endothelialization and reduced the smooth muscle cell (SMCs content of the lesions. The number of c-kit and sca-1 double-positive progenitor cells and flk-1 and sca-1 double-positive progenitor cells were significantly increased in the BM and the peripheral blood of the PNS-treated groups. PNS treatment increased the plasma levels of SDF-1α and SCF as well as the BM levels of matrix metalloproteinase-9 (MMP-9. Moreover, the mRNA levels of SDF-1α and protein levels of CXCR4 were both increased in the BM of mice treated with PNS, while SDF-1α expression decreased. Conclusion: PNS reduce the size of atherosclerotic plaques, and this effect appears to involve progenitor cell mobilization. SDF-1α-CXCR4 interactions and the possible modulatory role of PNS in this process may contribute to the increased progenitor cell mobilization.

  17. Selection-independent generation of gene knockout mouse embryonic stem cells using zinc-finger nucleases.

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    Anna Osiak

    Full Text Available Gene knockout in murine embryonic stem cells (ESCs has been an invaluable tool to study gene function in vitro or to generate animal models with altered phenotypes. Gene targeting using standard techniques, however, is rather inefficient and typically does not exceed frequencies of 10(-6. In consequence, the usage of complex positive/negative selection strategies to isolate targeted clones has been necessary. Here, we present a rapid single-step approach to generate a gene knockout in mouse ESCs using engineered zinc-finger nucleases (ZFNs. Upon transient expression of ZFNs, the target gene is cleaved by the designer nucleases and then repaired by non-homologous end-joining, an error-prone DNA repair process that introduces insertions/deletions at the break site and therefore leads to functional null mutations. To explore and quantify the potential of ZFNs to generate a gene knockout in pluripotent stem cells, we generated a mouse ESC line containing an X-chromosomally integrated EGFP marker gene. Applying optimized conditions, the EGFP locus was disrupted in up to 8% of ESCs after transfection of the ZFN expression vectors, thus obviating the need of selection markers to identify targeted cells, which may impede or complicate downstream applications. Both activity and ZFN-associated cytotoxicity was dependent on vector dose and the architecture of the nuclease domain. Importantly, teratoma formation assays of selected ESC clones confirmed that ZFN-treated ESCs maintained pluripotency. In conclusion, the described ZFN-based approach represents a fast strategy for generating gene knockouts in ESCs in a selection-independent fashion that should be easily transferrable to other pluripotent stem cells.

  18. [Propagation of prdm1 gene knockout mouse and its genotype identification].

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    Lu, Xiao-Yun; Chen, Chong; Pan, Xiu-Ying; Zeng, Ling-Yu; Li, Zhen-Yu; Song, Xu-Guang; Xu, Kai-Lin

    2012-08-01

    This study was aimed to propagate and identify the prdm1 gene-knockout mice, so as to lay the foundation for studying Blimp-1 protein. Two kinds of transgenic homozygous mice with B6.prdm1(flox/flox) and B6.Lck-Cre were feed and propagated; after successful propagating, the first passage mice were obtained; after the first passage mice were copulated once again, the genotypes were obtained as follows: B6. prdm1(wild/wild). Lck-Cre, B6. prdm1(wild/wild), B6.prdm1(flox/flox). Lck-Cre, B6.prdm1(flox/wild). Lck-Cre, B6.prdm1(flox/flox), B6. prdm1(flox/wild). The genomic DNA of second passage mice was extracted, the Cre and loxp gene fragments were amplified by PCR, then the size of Cre and loxp genomic DNA were detected by agarose gel electrophoresis. The mice with B6.prdm1(flow/flox). Lek-Cre were used as conditionally prdm1-knockout mice, B6.prdm1(flox/wild). Lck-Cre mice, B6.prdm1(flox/flox) and B6 mice were used as controls. The spleen T lymphocytes and B lymphocytes were sorted by using magnetic beads, the blimp-1 target protein was identified by Western blot. The results showed that the two transgenic homozygous mice had the ability to reproduce, and the separation ratio of second passage mice generated from propagation of their offspring cach other meet Mendelian laws, and the prdm1 gene-knockout mice also could successfully obtained. It is concluded that the application of Cre-loxp system may successfully obtain plentiful prdm1 gene-knockout mice.

  19. Dual gene activation and knockout screen reveals directional dependencies in genetic networks. | Office of Cancer Genomics

    Science.gov (United States)

    Understanding the direction of information flow is essential for characterizing how genetic networks affect phenotypes. However, methods to find genetic interactions largely fail to reveal directional dependencies. We combine two orthogonal Cas9 proteins from Streptococcus pyogenes and Staphylococcus aureus to carry out a dual screen in which one gene is activated while a second gene is deleted in the same cell. We analyze the quantitative effects of activation and knockout to calculate genetic interaction and directionality scores for each gene pair.

  20. Fetal growth retardation and lack of hypotaurine in ezrin knockout mice.

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    Tomohiro Nishimura

    Full Text Available Ezrin is a membrane-associated cytoplasmic protein that serves to link cell-membrane proteins with the actin-based cytoskeleton, and also plays a role in regulation of the functional activities of some transmembrane proteins. It is expressed in placental trophoblasts. We hypothesized that placental ezrin is involved in the supply of nutrients from mother to fetus, thereby influencing fetal growth. The aim of this study was firstly to clarify the effect of ezrin on fetal growth and secondly to determine whether knockout of ezrin is associated with decreased concentrations of serum and placental nutrients. Ezrin knockout mice (Ez(-/- were confirmed to exhibit fetal growth retardation. Metabolome analysis of fetal serum and placental extract of ezrin knockout mice by means of capillary electrophoresis-time-of-flight mass spectrometry revealed a markedly decreased concentration of hypotaurine, a precursor of taurine. However, placental levels of cysteine and cysteine sulfinic acid (precursors of hypotaurine and taurine were not affected. Lack of hypotaurine in Ez(-/- mice was confirmed by liquid chromatography with tandem mass spectrometry. Administration of hypotaurine to heterogenous dams significantly decreased the placenta-to-maternal plasma ratio of hypotaurine in wild-type fetuses but only slightly decreased it in ezrin knockout fetuses, indicating that the uptake of hypotaurine from mother to placenta is saturable and that disruption of ezrin impairs the uptake of hypotaurine by placental trophoblasts. These results indicate that ezrin is required for uptake of hypotaurine from maternal serum by placental trophoblasts, and plays an important role in fetal growth.

  1. Effects of ptb knockout on butyric acid fermentation by Clostridium tyrobutyricum.

    Science.gov (United States)

    Zhang, Yali; Yu, Mingrui; Yang, Shang-Tian

    2012-01-01

    Clostridium tyrobutyricum ATCC 25755 is an anaerobic, rod-shaped, gram-positive bacterium that produces butyrate, acetate, hydrogen, and carbon dioxide from various saccharides, including glucose and xylose. Phosphotransbutyrylase (PTB) is a key enzyme in the butyric acid synthesis pathway. In this work, effects of ptb knockout by homologous recombination on metabolic flux and product distribution were investigated. When compared with the wild type, the activities of PTB and butyrate kinase in ptb knockout mutant decreased 76 and 42%, respectively; meanwhile, phosphotransacetylase and acetate kinase increased 7 and 29%, respectively. However, ptb knockout did not significantly reduce butyric acid production from glucose or xylose in batch fermentations. Instead, it increased acetic acid and hydrogen production 33.3-53.8% and ≈ 11%, respectively. Thus, the ptb knockout did increase the carbon flux toward acetate synthesis, resulting in a significant decrease (28-35% reduction) in the butyrate/acetate ratio in ptb mutant fermentations. In addition, the mutant displayed a higher specific growth rate (0.20 h(-1) vs. 0.15 h(-1) on glucose and 0.14 h(-1) vs. 0.10 h(-1) on xylose) and tolerance to butyric acid. Consequently, batch fermentation with the mutant gave higher fermentation rate and productivities (26-48% increase for butyrate, 81-100% increase for acetate, and 38-46% increase for hydrogen). This mutant thus can be used more efficiently than the parental strain in fermentations to produce butyrate, acetate, and hydrogen from glucose and xylose. Copyright © 2011 American Institute of Chemical Engineers (AIChE).

  2. Impact of temporal variation on design and analysis of mouse knockout phenotyping studies.

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    Natasha A Karp

    Full Text Available A significant challenge facing high-throughput phenotyping of in-vivo knockout mice is ensuring phenotype calls are robust and reliable. Central to this problem is selecting an appropriate statistical analysis that models both the experimental design (the workflow and the way control mice are selected for comparison with knockout animals and the sources of variation. Recently we proposed a mixed model suitable for small batch-oriented studies, where controls are not phenotyped concurrently with mutants. Here we evaluate this method both for its sensitivity to detect phenotypic effects and to control false positives, across a range of workflows used at mouse phenotyping centers. We found the sensitivity and control of false positives depend on the workflow. We show that the phenotypes in control mice fluctuate unexpectedly between batches and this can cause the false positive rate of phenotype calls to be inflated when only a small number of batches are tested, when the effect of knockout becomes confounded with temporal fluctuations in control mice. This effect was observed in both behavioural and physiological assays. Based on this analysis, we recommend two approaches (workflow and accompanying control strategy and associated analyses, which would be robust, for use in high-throughput phenotyping pipelines. Our results show the importance in modelling all sources of variability in high-throughput phenotyping studies.

  3. FANCA knockout in human embryonic stem cells causes a severe growth disadvantage.

    Science.gov (United States)

    Vanuytsel, Kim; Cai, Qing; Nair, Nisha; Khurana, Satish; Shetty, Swati; Vermeesch, Joris R; Ordovas, Laura; Verfaillie, Catherine M

    2014-09-01

    Fanconi anemia (FA) is an autosomal recessive disorder characterized by progressive bone marrow failure (BMF) during childhood, aside from numerous congenital abnormalities. FA mouse models have been generated; however, they do not fully mimic the hematopoietic phenotype. As there is mounting evidence that the hematopoietic impairment starts already in utero, a human pluripotent stem cell model would constitute a more appropriate system to investigate the mechanisms underlying BMF in FA and its developmental basis. Using zinc finger nuclease (ZFN) technology, we have created a knockout of FANCA in human embryonic stem cells (hESC). We introduced a selection cassette into exon 2 thereby disrupting the FANCA coding sequence and found that whereas mono-allelically targeted cells retain an unaltered proliferation potential, disruption of the second allele causes a severe growth disadvantage. As a result, heterogeneous cultures arise due to the presence of cells still carrying an unaffected FANCA allele, quickly outgrowing the knockout cells. When pure cultures of FANCA knockout hESC are pursued either through selection or single cell cloning, this rapidly results in growth arrest and such cultures cannot be maintained. These data highlight the importance of a functional FA pathway at the pluripotent stem cell stage. Copyright © 2014. Published by Elsevier B.V.

  4. Myo5b knockout mice as a model of microvillus inclusion disease

    Science.gov (United States)

    Cartón-García, Fernando; Overeem, Arend W.; Nieto, Rocio; Bazzocco, Sarah; Dopeso, Higinio; Macaya, Irati; Bilic, Josipa; Landolfi, Stefania; Hernandez-Losa, Javier; Schwartz, Simo; Ramon y Cajal, Santiago; van Ijzendoorn, Sven C. D.; Arango, Diego

    2015-01-01

    Inherited MYO5B mutations have recently been associated with microvillus inclusion disease (MVID), an autosomal recessive syndrome characterized by intractable, life-threatening, watery diarrhea appearing shortly after birth. Characterization of the molecular mechanisms underlying this disease and development of novel therapeutic approaches is hampered by the lack of animal models. In this study we describe the phenotype of a novel mouse model with targeted inactivation of Myo5b. Myo5b knockout mice show perinatal mortality, diarrhea and the characteristic mislocalization of apical and basolateral plasma membrane markers in enterocytes. Moreover, in transmission electron preparations, we observed microvillus atrophy and the presence of microvillus inclusion bodies. Importantly, Myo5b knockout embryos at day 20 of gestation already display all these structural defects, indicating that they are tissue autonomous rather than secondary to environmental cues, such as the long-term absence of nutrients in the intestine. Myo5b knockout mice closely resemble the phenotype of MVID patients and constitute a useful model to further investigate the underlying molecular mechanism of this disease and to preclinically assess the efficacy of novel therapeutic approaches. PMID:26201991

  5. Knockout of the Nogo-B Gene Attenuates Tumor Growth and Metastasis in Hepatocellular Carcinoma

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    Bo Zhu

    2017-07-01

    Full Text Available Human hepatocellular carcinoma (HCC is a malignant cancer. It is a challenge to develop anti-HCC drugs due to HCC's extreme aggressiveness and with the sensitivity of the liver to show severe adverse effects. More importantly, the precise mechanisms causing HCC pathogenicity are not known. Our previous study disclosed Nogo-B as a reticulon 4 (Rtn4 family member. In the present study, we first identified that Nogo-B played a critical role in HCC progression. We found, via in vitro and in vivo assays, that Nogo-B was expressed aberrantly in primary HCC tumor tissues and immortal HCC cells but was relatively scarce in the normal liver tissues or cells. Nogo-B knockout, via the CRISPR-Cas9 technique, resulted in significant suppression of HCC cell proliferation and tumor growth. Next-generation sequencing analysis showed that Nogo-B knockout have effects on interleukin-6 (IL-6 signaling pathway. Furthermore, we observed that IL-6 induced phosphorylation of STAT3 (pSTAT3 in wild-type HCC cells, but Nogo-B knockout could reduce IL-6–induced increase of pSTAT3, supporting that Nogo-B affects HCC tumor progression possibly via regulating the IL-6/STAT3 signaling pathway. In conclusion, Nogo-B is expressed aberrantly in HCCs and plays an oncogenic role. These findings support that Nogo-B may be a novel anti-HCC therapeutic target.

  6. Characterization of Phototransduction Gene Knockouts Revealed Important Signaling Networks in the Light-Induced Retinal Degeneration

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    Jayalakshmi Krishnan

    2008-01-01

    Full Text Available Understanding the molecular pathways mediating neuronal function in retinas can be greatly facilitated by the identification of genes regulated in the retinas of different mutants under various light conditions. We attempted to conduct a gene chip analysis study on the genes regulated during rhodopsin kinase (Rhok-/- and arrestin (Sag-/- knockout and double knockouts in mice retina. Hence, mice were exposed to constant illumination of 450 lux or 6,000 lux on dilated pupils for indicated periods. The retinas were removed after the exposure and processed for microarray analysis. Double knockout was associated with immense changes in gene expression regulating a number of apoptosis inducing transcription factors. Subsequently, network analysis revealed that during early exposure the transcription factors, p53, c-MYC, c-FOS, JUN, and, in late phase, NF-B, appeared to be essential for the initiation of light-induced retinal rod loss, and some other classical pro- and antipoptotic genes appeared to be significantly important as well.

  7. Rapid construction of a whole-genome transposon insertion collection for Shewanella oneidensis by Knockout Sudoku.

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    Baym, Michael; Shaket, Lev; Anzai, Isao A; Adesina, Oluwakemi; Barstow, Buz

    2016-11-10

    Whole-genome knockout collections are invaluable for connecting gene sequence to function, yet traditionally, their construction has required an extraordinary technical effort. Here we report a method for the construction and purification of a curated whole-genome collection of single-gene transposon disruption mutants termed Knockout Sudoku. Using simple combinatorial pooling, a highly oversampled collection of mutants is condensed into a next-generation sequencing library in a single day, a 30- to 100-fold improvement over prior methods. The identities of the mutants in the collection are then solved by a probabilistic algorithm that uses internal self-consistency within the sequencing data set, followed by rapid algorithmically guided condensation to a minimal representative set of mutants, validation, and curation. Starting from a progenitor collection of 39,918 mutants, we compile a quality-controlled knockout collection of the electroactive microbe Shewanella oneidensis MR-1 containing representatives for 3,667 genes that is functionally validated by high-throughput kinetic measurements of quinone reduction.

  8. Probing short-range correlations in asymmetric nuclei with quasi-free pair knockout reactions

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    Stevens, Sam; Ryckebusch, Jan; Cosyn, Wim; Waets, Andreas

    2018-02-01

    Short-range correlations (SRC) in asymmetric nuclei with an unusual neutron-to-proton ratio can be studied with quasi-free two-nucleon knockout processes following the collision between accelerated ions and a proton target. We derive an approximate factorized cross section for those SRC-driven p (A ,p‧N1N2) reactions. Our reaction model hinges on the factorization properties of SRC-driven A (e ,e‧N1N2) reactions for which strong indications are found in theory-experiment comparisons. In order to put our model to the test we compare its predictions with results of 12C (p ,p‧ pn) measurements conducted at Brookhaven National Laboratory (BNL) and find a fair agreement. The model can also reproduce characteristic features of SRC-driven two-nucleon knockout reactions, like back-to-back emission of the correlated nucleons. We study the asymmetry dependence of nuclear SRC by providing predictions for the ratio of proton-proton to proton-neutron knockout cross sections for the carbon isotopes 9-15C thereby covering neutron excess values (N - Z) / Z between -0.5 and +0.5.

  9. Txnip ablation reduces vascular smooth muscle cell inflammation and ameliorates atherosclerosis in apolipoprotein E knockout mice.

    Science.gov (United States)

    Byon, Chang Hyun; Han, Tieyan; Wu, Judy; Hui, Simon T

    2015-08-01

    Inflammation of vascular smooth muscle cells (VSMC) is intimately linked to atherosclerosis and other vascular inflammatory disease. Thioredoxin interacting protein (Txnip) is a key regulator of cellular sulfhydryl redox and a mediator of inflammasome activation. The goals of the present study were to examine the impact of Txnip ablation on inflammatory response to oxidative stress in VSMC and to determine the effect of Txnip ablation on atherosclerosis in vivo. Using cultured VSMC, we showed that ablation of Txnip reduced cellular oxidative stress and increased protection from oxidative stress when challenged with oxidized phospholipids and hydrogen peroxide. Correspondingly, expression of inflammatory markers and adhesion molecules were diminished in both VSMC and macrophages from Txnip knockout mice. The blunted inflammatory response was associated with a decrease in NF-ĸB nuclear translocation. Loss of Txnip in VSMC also led to a dramatic reduction in macrophage adhesion to VSMC. In vivo data from Txnip-ApoE double knockout mice showed that Txnip ablation led to 49% reduction in atherosclerotic lesion in the aortic root and 71% reduction in the abdominal aorta, compared to control ApoE knockout mice. Our data show that Txnip plays an important role in oxidative inflammatory response and atherosclerotic lesion development in mice. The atheroprotective effect of Txnip ablation implicates that modulation of Txnip expression may serve as a potential target for intervention of atherosclerosis and inflammatory vascular disease. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  10. Generation of RUNX3 knockout pigs using CRISPR/Cas9-mediated gene targeting.

    Science.gov (United States)

    Kang, J-T; Ryu, J; Cho, B; Lee, E-J; Yun, Y-J; Ahn, S; Lee, J; Ji, D-Y; Lee, K; Park, K-W

    2016-12-01

    Pigs are an attractive animal model to study the progression of cancer because of their anatomical and physiological similarities to human. However, the use of pig models for cancer research has been limited by availability of genetically engineered pigs which can recapitulate human cancer progression. Utilizing genome editing technologies such as CRISPR/Cas9 system allows us to generate genetically engineered pigs at a higher efficiency. In this study, specific CRISPR/Cas9 systems were used to target RUNX3, a known tumour suppressor gene, to generate a pig model that can induce gastric cancer in human. First, RUNX3 knockout cell lines carrying genetic modification (monoallelic or biallelic) of RUNX3 were generated by introducing engineered CRISPR/Cas9 system specific to RUNX3 into foetal fibroblast cells. Then, the genetically modified foetal fibroblast cells were used as donor cells for somatic cell nuclear transfer, followed by embryo transfer. We successfully obtained four live RUNX3 knockout piglets from two surrogates. The piglets showed the lack of RUNX3 protein in their internal organ system. Our results demonstrate that the CRISPR/Cas9 system is effective in inducing mutations on a specific locus of genome and the RUNX3 knockout pigs can be useful resources for human cancer research and to develop novel cancer therapies. © 2016 Blackwell Verlag GmbH.

  11. Less is More: unveiling the functional core of hematopoietic stem cells through knockout mice

    Science.gov (United States)

    Rossi, Lara; Lin, Kuanyin K.; Boles, Nathan C.; Yang, Liubin; King, Katherine Y.; Jeong, Mira; Mayle, Allison; Goodell, Margaret A.

    2012-01-01

    Summary Hematopoietic stem cells (HSCs) represent one of the first recognized somatic stem cells. As such, nearly 200 genes have been examined for roles in HSC function in knockout mice. In this review, we compile the majority of these reports to provide a broad overview of the functional modules revealed by these genetic analyses and highlight some key regulatory pathways involved, including cell cycle control, TGF-β signaling, Pten/AKT signaling, Wnt signaling, and cytokine signaling. Finally, we propose recommendations for characterization of HSC function in knockout mice to facilitate cross-study comparisons that would generate a more cohesive picture of HSC biology. In the field of design, the minimalist movement stripped down buildings and objects to their most basic features, a sentiment that architect Ludwig Mies van der Rohe summarized in his motto “less is more”. By depleting HSCs of specific genes, knockout studies transpose the minimalist approach into research biology, providing insights into the essential core of genetic features that is indispensable for a well-functioning hematopoietic system. PMID:22958929

  12. Hippocampal long-term potentiation is enhanced in urethane-anesthetized RGS2 knockout mice.

    Science.gov (United States)

    Hutchison, R Matthew; Chidiac, Peter; Leung, L Stan

    2009-08-01

    RGS2 is a member of the regulator of G-protein signaling (RGS) family and has been implicated in cellular mechanisms associated with neuronal plasticity. Long-term potentiation (LTP) of RGS2 knockout and wild-type mice was examined at the Schaffer collaterals to CA1 pathway in urethane-anesthetized mice in vivo to examine RGS2's possible role in the regulation of potentiation. As compared to wild-type mice, RGS2 knockouts demonstrated much stronger LTP of the extracellular population spikes at the somatic and dendritic layers in CA1 region and more pronounced LTP of the population excitatory postsynaptic current sink. Under baseline conditions, RGS2 knockouts showed lower paired-pulse facilitation of the excitatory postsynaptic potentials and associated current sinks in vivo as compared with wild-type mice. The data show for the first time that RGS2 deficient mice in vivo differ from wild-type mice in both short-term and long-term synaptic plasticity suggesting that RGS2 serves as a negative regulator of long-term synaptic plasticity. Copyright (c) 2009 Wiley-Liss, Inc.

  13. Evidence for Heterodimerization and Functional Interaction of the Angiotensin Type 2 Receptor and the Receptor MAS.

    Science.gov (United States)

    Leonhardt, Julia; Villela, Daniel C; Teichmann, Anke; Münter, Lisa-Marie; Mayer, Magnus C; Mardahl, Maibritt; Kirsch, Sebastian; Namsolleck, Pawel; Lucht, Kristin; Benz, Verena; Alenina, Natalia; Daniell, Nicholas; Horiuchi, Masatsugu; Iwai, Masaru; Multhaup, Gerhard; Schülein, Ralf; Bader, Michael; Santos, Robson A; Unger, Thomas; Steckelings, Ulrike Muscha

    2017-06-01

    The angiotensin type 2 receptor (AT2R) and the receptor MAS are receptors of the protective arm of the renin-angiotensin system. They mediate strikingly similar actions. Moreover, in various studies, AT2R antagonists blocked the effects of MAS agonists and vice versa. Such cross-inhibition may indicate heterodimerization of these receptors. Therefore, this study investigated the molecular and functional interplay between MAS and the AT2R. Molecular interactions were assessed by fluorescence resonance energy transfer and by cross correlation spectroscopy in human embryonic kidney-293 cells transfected with vectors encoding fluorophore-tagged MAS or AT2R. Functional interaction of AT2R and MAS was studied in astrocytes with CX3C chemokine receptor-1 messenger RNA expression as readout. Coexpression of fluorophore-tagged AT2R and MAS resulted in a fluorescence resonance energy transfer efficiency of 10.8 ± 0.8%, indicating that AT2R and MAS are capable to form heterodimers. Heterodimerization was verified by competition experiments using untagged AT2R and MAS. Specificity of dimerization of AT2R and MAS was supported by lack of dimerization with the transient receptor potential cation channel, subfamily C-member 6. Dimerization of the AT2R was abolished when it was mutated at cysteine residue 35. AT2R and MAS stimulation with the respective agonists, Compound 21 or angiotensin-(1-7), significantly induced CX3C chemokine receptor-1 messenger RNA expression. Effects of each agonist were blocked by an AT2R antagonist (PD123319) and also by a MAS antagonist (A-779). Knockout of a single of these receptors made astrocytes unresponsive for both agonists. Our results suggest that MAS and the AT2R form heterodimers and that-at least in astrocytes-both receptors functionally depend on each other. © 2017 American Heart Association, Inc.

  14. Involvement of the Tyro3 receptor and its intracellular partner Fyn signaling in Schwann cell myelination.

    Science.gov (United States)

    Miyamoto, Yuki; Torii, Tomohiro; Takada, Shuji; Ohno, Nobuhiko; Saitoh, Yurika; Nakamura, Kazuaki; Ito, Akihito; Ogata, Toru; Terada, Nobuo; Tanoue, Akito; Yamauchi, Junji

    2015-10-01

    During early development of the peripheral nervous system, Schwann cell precursors proliferate, migrate, and differentiate into premyelinating Schwann cells. After birth, Schwann cells envelop neuronal axons with myelin sheaths. Although some molecular mechanisms underlying myelination by Schwann cells have been identified, the whole picture remains unclear. Here we show that signaling through Tyro3 receptor tyrosine kinase and its binding partner, Fyn nonreceptor cytoplasmic tyrosine kinase, is involved in myelination by Schwann cells. Impaired formation of myelin segments is observed in Schwann cell neuronal cultures established from Tyro3-knockout mouse dorsal root ganglia (DRG). Indeed, Tyro3-knockout mice exhibit reduced myelin thickness. By affinity chromatography, Fyn was identified as the binding partner of the Tyro3 intracellular domain, and activity of Fyn is down-regulated in Tyro3-knockout mice, suggesting that Tyro3, acting through Fyn, regulates myelination. Ablating Fyn in mice results in reduced myelin thickness. Decreased myelin formation is observed in cultures established from Fyn-knockout mouse DRG. Furthermore, decreased kinase activity levels and altered expression of myelination-associated transcription factors are observed in these knockout mice. These results suggest the involvement of Tyro3 receptor and its binding partner Fyn in Schwann cell myelination. This constitutes a newly recognized receptor-linked signaling mechanism that can control Schwann cell myelination. © 2015 Miyamoto et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

  15. The serotonin transporter plays an important role in male sexual behavior: a study in serotonin transporter knockout rats

    NARCIS (Netherlands)

    Chan, J.Y.; Snoeren, E.; Cuppen, E.; Waldinger, M.; Olivier, B.; Oosting, R.

    2011-01-01

    INTRODUCTION: Serotonin (5-HT) is an important neurotransmitter for sexual behaviors. Heterozygous (+/-) serotonin transporter (SERT) rats and SERT knockout rats (-/-) have serotonergic disturbances with significant elevations of basal extracellular 5-HT levels. AIM: To investigate the putative role

  16. An analysis of IL-36 signature genes and individuals with IL1RL2 knockout mutations validates IL-36 as a psoriasis therapeutic target.

    Science.gov (United States)

    Mahil, Satveer K; Catapano, Marika; Di Meglio, Paola; Dand, Nick; Ahlfors, Helena; Carr, Ian M; Smith, Catherine H; Trembath, Richard C; Peakman, Mark; Wright, John; Ciccarelli, Francesca D; Barker, Jonathan N; Capon, Francesca

    2017-10-11

    Interleukin (IL)-36α, IL-36β, and IL-36γ are innate mediators of acute epithelial inflammation. We sought to demonstrate that these cytokines are also required for the pathogenesis of plaque psoriasis, a common and chronic skin disorder, caused by abnormal T helper 17 (T H 17) cell activation. To investigate this possibility, we first defined the genes that are induced by IL-36 cytokines in primary human keratinocytes. This enabled us to demonstrate a significant IL-36 signature among the transcripts that are up-regulated in plaque psoriasis and the susceptibility loci associated with the disease in genome-wide studies. Next, we investigated the impact of in vivo and ex vivo IL-36 receptor blockade using a neutralizing antibody or a recombinant antagonist. Both inhibitors had marked anti-inflammatory effects on psoriatic skin, demonstrated by statistically significant reductions in IL-17 expression, keratinocyte activation, and leukocyte infiltration. Finally, we explored the potential safety profile associated with IL-36 blockade by phenotyping 12 individuals carrying knockout mutations of the IL-36 receptor gene. We found that normal immune function was broadly preserved in these individuals, suggesting that IL-36 signaling inhibition would not substantially compromise host defenses. These observations, which integrate the results of transcriptomics and model system analysis, pave the way for early-stage clinical trials of IL-36 antagonists. Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

  17. Age-Dependent Long-Term Potentiation Deficits in the Prefrontal Cortex of the Fmr1 Knockout Mouse Model of Fragile X Syndrome.

    Science.gov (United States)

    Martin, Henry G S; Lassalle, Olivier; Brown, Jonathan T; Manzoni, Olivier J

    2016-05-01

    The most common inherited monogenetic cause of intellectual disability is Fragile X syndrome (FXS). The clinical symptoms of FXS evolve with age during adulthood; however, neurophysiological data exploring this phenomenon are limited. The Fmr1 knockout (Fmr1KO) mouse models FXS, but studies in these mice of prefrontal cortex (PFC) function are underrepresented, and aging linked data are absent. We studied synaptic physiology and activity-dependent synaptic plasticity in the medial PFC of Fmr1KO mice from 2 to 12 months. In young adult Fmr1KO mice, NMDA receptor (NMDAR)-mediated long-term potentiation (LTP) is intact; however, in 12-month-old mice this LTP is impaired. In parallel, there was an increase in the AMPAR/NMDAR ratio and a concomitant decrease of synaptic NMDAR currents in 12-month-old Fmr1KO mice. We found that acute pharmacological blockade of mGlu5 receptor in 12-month-old Fmr1KO mice restored a normal AMPAR/NMDAR ratio and LTP. Taken together, the data reveal an age-dependent deficit in LTP in Fmr1KO mice, which may correlate to some of the complex age-related deficits in FXS. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  18. The Effect of Acute and Repeated Stress on CRH-R1 and CRH-R2 mRNA Expression in Pituitaries of Wild Type and CRH Knock-Out Mice.

    Science.gov (United States)

    Klenerova, Vera; Kvetnansky, Richard; Hynie, Sixtus

    2018-01-01

    The activation of the HPA axis is the endocrine measure of stress responsiveness that is initiated by corticotropin-releasing hormone (CRH). CRH exerts its effects via CRHR1 and CRH-R2 receptors coupled to the cAMP signaling system and this process involves transcription factor cAMP-responsive element-binding protein (CREB).This study investigated the role of CRH and the possible involvement of CREB in gene regulation of CRH receptor, under basal conditions and after stress application in the pituitary. We used wild type (wt +/+) controls and CRH knock-out (CRH-KO -/-) male mice. Using CRH-deficient mice, we were able to investigate the consequences of the lack of the CRH on the expression of CRH receptors and transcriptional regulation mediated by CREB. We estimated the effect of acute (IMO 1×) and repeated (IMO 7×) restraint stressors lasting 30 and 120 min on the expression of mRNA CREB, CRH-R1, and CRH-R2 by qPCR. We found very significant difference in the expression of these peptides under the effect of single and repeated stress in control and CRH-KO mice. Our results indicate that both CRH receptors and CREB might be involved in the regulation of stress response in the pituitary of mice. We propose that regulation of the stress response may be better understood if more were known about the mechanisms of CRH receptor signal transduction and involvement of CREB system.

  19. An In Silico Knockout Model for Gastrointestinal Absorption Using a Systems Pharmacology Approach - Development and Application for Ketones.

    Directory of Open Access Journals (Sweden)

    Vittal Shivva

    Full Text Available Gastrointestinal absorption and disposition of ketones is complex. Recent work describing the pharmacokinetics (PK of d-β-hydroxybutyrate (BHB following oral ingestion of a ketone monoester ((R-3-hydroxybutyl (R-3-hydroxybutyrate found multiple input sites, nonlinear disposition and feedback on endogenous production. In the current work, a human systems pharmacology model for gastrointestinal absorption and subsequent disposition of small molecules (monocarboxylic acids with molecular weight < 200 Da was developed with an application to a ketone monoester. The systems model was developed by collating the information from the literature and knowledge gained from empirical population modelling of the clinical data. In silico knockout variants of this systems model were used to explore the mechanism of gastrointestinal absorption of ketones. The knockouts included active absorption across different regions in the gut and also a passive diffusion knockout, giving 10 gut knockouts in total. Exploration of knockout variants has suggested that there are at least three distinct regions in the gut that contribute to absorption of ketones. Passive diffusion predominates in the proximal gut and active processes contribute to the absorption of ketones in the distal gut. Low doses are predominantly absorbed from the proximal gut by passive diffusion whereas high doses are absorbed across all sites in the gut. This work has provided mechanistic insight into the absorption process of ketones, in the form of unique in silico knockouts that have potential for application with other therapeutics. Future studies on absorption process of ketones are suggested to substantiate findings in this study.

  20. Development of the Multiple Gene Knockout System with One-Step PCR in Thermoacidophilic Crenarchaeon Sulfolobus acidocaldarius

    Directory of Open Access Journals (Sweden)

    Shoji Suzuki

    2017-01-01

    Full Text Available Multiple gene knockout systems developed in the thermoacidophilic crenarchaeon Sulfolobus acidocaldarius are powerful genetic tools. However, plasmid construction typically requires several steps. Alternatively, PCR tailing for high-throughput gene disruption was also developed in S. acidocaldarius, but repeated gene knockout based on PCR tailing has been limited due to lack of a genetic marker system. In this study, we demonstrated efficient homologous recombination frequency (2.8 × 104 ± 6.9 × 103 colonies/μg DNA by optimizing the transformation conditions. This optimized protocol allowed to develop reliable gene knockout via double crossover using short homologous arms and to establish the multiple gene knockout system with one-step PCR (MONSTER. In the MONSTER, a multiple gene knockout cassette was simply and rapidly constructed by one-step PCR without plasmid construction, and the PCR product can be immediately used for target gene deletion. As an example of the applications of this strategy, we successfully made a DNA photolyase- (phr- and arginine decarboxylase- (argD- deficient strain of S. acidocaldarius. In addition, an agmatine selection system consisting of an agmatine-auxotrophic strain and argD marker was also established. The MONSTER provides an alternative strategy that enables the very simple construction of multiple gene knockout cassettes for genetic studies in S. acidocaldarius.

  1. Reduced Acetylcholine Receptor Density, Morphological Remodeling, and Butyrylcholinesterase Activity Can Sustain Muscle Function in Acetylcholinesterase Knockout Mice

    National Research Council Canada - National Science Library

    Adler, Michael; Manley, Heather A; Deshpande, Sharad S; Hamilton, Tracey A; Kan, Robert K; Oyler, George; Lockridge, Oksana; Duysen, Ellen G; Sheridan, Robert E

    2004-01-01

    ...) and can survive into adulthood. Twitch tensions elicited in diaphragms of AChE-/- mice by single supramaximal stimuli had larger amplitudes and slower rise and decay times than did those in wild-type animals...

  2. Enhanced self-administration of alcohol in muscarinic acetylcholine M4 receptor knockout mice

    DEFF Research Database (Denmark)

    de la Cour, Cecilie; Sørensen, Gunnar; Wörtwein, Gitta

    2015-01-01

    % and 10% alcohol in 60min sessions, 6 days/week, after having undergone a standard sucrose fading training procedure on a fixed ratio schedule. The mice were further subjected to an extinction period followed by a 1 day reinstatement trial. M4-/- mice consumed more alcohol at 5% and 8% compared to their M...

  3. Effect of apolipoprotein M on high density lipoprotein metabolism and atherosclerosis in low density lipoprotein receptor knock-out mice

    DEFF Research Database (Denmark)

    Christoffersen, Christina; Jauhiainen, Matti; Moser, Markus

    2008-01-01

    To investigate the role of apoM in high density lipoprotein (HDL) metabolism and atherogenesis, we generated human apoM transgenic (apoM-Tg) and apoM-deficient (apoM(-/-)) mice. Plasma apoM was predominantly associated with 10-12-nm alpha-migrating HDL particles. Human apoM overexpression (11-fold......) increased plasma cholesterol concentration by 13-22%, whereas apoM deficiency decreased it by 17-21%. The size and charge of apoA-I-containing HDL in plasma were not changed in apoM-Tg or apoM(-/-) mice. However, in plasma incubated at 37 degrees C, lecithin:cholesterol acyltransferase-dependent conversion...... of alpha- to pre-alpha-migrating HDL was delayed in apoM-Tg mice. Moreover, lecithin: cholesterol acyltransferase-independent generation of pre-beta-migrating apoA-I-containing particles in plasma was increased in apoM-Tg mice (4.2 +/- 1.1%, p = 0.06) and decreased in apoM(-/-) mice (0.5 +/- 0.3%, p = 0...

  4. Evolution of hepatic steatosis to fibrosis and adenoma formation in liver specific growth hormone receptor knockout (GHRLD mice

    Directory of Open Access Journals (Sweden)

    Yong eFan

    2014-12-01

    Full Text Available Background: Non-alcoholic fatty liver disease (NAFLD is one of the most common forms of chronic liver diseases closely associated with obesity and insulin resistance; deficient growth hormone (GH action in liver has been implicated as a mechanism. Here, we investigated the evolution of NAFLD in aged mice with liver-specific GHR deletion. Methods: We examined glucose tolerance, insulin responsiveness and lipid profiles in aged male mice (44-50 weeks with GHRLD. We performed proteomics analysis, pathway-based Superarray assay, as well as quantitative RT-PCR to gain molecular insight into the mechanism(s of GHR-deficiency mediated NAFLD. In addition, we examined the pathological changes of livers of aged GHRLD male mice. Results: The biochemical profile was consistent with that of the metabolic syndrome: abnormal glucose tolerance, impaired insulin secretion, and hyperlipidemia. RT-qPCR analysis of key markers of inflammation revealed a 3-5 fold increase in TNFα and CCL3, confirming the presence of inflammation. Expression of fibrotic markers (e.g., Col1A2 and Col3A1 was significantly increased, together with a 2-3 fold increase in TGFβ transcripts. Proteomics analyses showed a marked decrease of Mup1 and Selenbp2. In addition, pathway-analysis showed that the expression of cell cycle and growth relevant genes (i.e., Ccnd1, Socs2, Socs3 and Egfr were markedly affected in GHRLD liver. Microscopic analyses (H&E of GHRLD livers revealed the presence of hepatic adenomas of different stages of malignancy. Conclusion: Abrogation of GH-signaling in male liver leads to metabolic syndrome, hepatic steatosis, increased inflammation and fibrosis, and development of hepatic tumor. Since obesity, a common precursor of NAFLD, is a state of deficient GH secretion and action, the GHRLD model could be used to unravel the contribution of compromised hepatic GH-signaling in these pathological processes, and help to identify potential targets for intervention.

  5. Genetically modified Medicago truncatula lacking calcium oxalate has increased calcium bioavailability and partially rescues vitamin D receptor knockout mice phenotypes

    Science.gov (United States)

    How the distribution and sequestered form of plant macro/micro-nutrients influence their bioavailability, and ultimately impact human health, is poorly understood. The legume Medicago truncatula has a portion of its tissue calcium sequestered in the form of the calcium oxalate crystal, which reduces...

  6. Cholesterol-Induced Hepatic Inflammation Does Not Underlie the Predisposition to Insulin Resistance in Dyslipidemic Female LDL Receptor Knockout Mice

    NARCIS (Netherlands)

    Gruben, Nanda; Funke, Anouk; Kloosterhuis, Niels J.; Schreurs, Marijke; Sheedfar, Fareeba; Havinga, Rick; Houten, Sander M.; Shiri-Sverdlov, Ronit; van de Sluis, Bart; Kuivenhoven, Jan Albert; Koonen, Debby P. Y.; Hofker, Marten H.

    2015-01-01

    Chronic inflammation is considered a causal risk factor predisposing to insulin resistance. However, evidence is accumulating that inflammation confined to the liver may not be causal to metabolic dysfunction. To investigate this, we assessed if hepatic inflammation explains the predisposition

  7. Enhanced survival of lethally irradiated adenosine A(3) receptor knockout mice. A role for hematopoietic growth factors?

    Czech Academy of Sciences Publication Activity Database

    Hofer, Michal; Pospíšil, Milan; Dušek, L.; Hoferová, Zuzana; Komůrková, Denisa

    2015-01-01

    Roč. 11, č. 1 (2015), s. 79-85 ISSN 1573-9538 Institutional support: RVO:68081707 Keywords : G-CSF PRODUCTION * CANCER-THERAPY * CELL-GROWTH Subject RIV: BO - Biophysics Impact factor: 3.196, year: 2015

  8. The receptor RAGE: Bridging inflammation and cancer

    Directory of Open Access Journals (Sweden)

    Hess Jochen

    2009-05-01

    Full Text Available Abstract The receptor for advanced glycation end products (RAGE is a single transmembrane receptor of the immunoglobulin superfamily that is mainly expressed on immune cells, neurons, activated endothelial and vascular smooth muscle cells, bone forming cells, and a variety of cancer cells. RAGE is a multifunctional receptor that binds a broad repertoire of ligands and mediates responses to cell damage and stress conditions. It activates programs responsible for acute and chronic inflammation, and is implicated in a number of pathological diseases, including diabetic complications, stroke, atheriosclerosis, arthritis, and neurodegenerative disorders. The availability of Rage knockout mice has not only advanced our knowledge on signalling pathways within these pathophysiological conditions, but also on the functional importance of the receptor in processes of cancer. Here, we will summarize molecular mechanisms through which RAGE signalling contributes to the establishment of a pro-tumourigenic microenvironment. Moreover, we will review recent findings that provide genetic evidence for an important role of RAGE in bridging inflammation and cancer.

  9. Frequency-dependent cannabinoid receptor-independent modulation of glycine receptors by endocannabinoid 2-AG

    Directory of Open Access Journals (Sweden)

    Natalia eLozovaya

    2011-07-01

    Full Text Available Endocannabinoids are known as retrograde messengers, being released from the postsynaptic neuron and acting on specific presynaptic G-protein-coupled cannabinoid (CB receptors to decrease neurotransmitter release. Also, at physiologically relevant concentrations cannabinoids can directly modulate the function of voltage-gated and receptor-operated ion channels. Using patch-clamp recording we analyzed the consequences of the direct action of an endocannabinoid, 2-arachidonoylglycerol (2-AG, on the functional properties of glycine receptor channels (GlyRs and ionic currents in glycinergic synapses. At physiologically relevant concentrations (0.1-1 µM, 2-AG directly affected the functions of recombinant homomeric alpha1H GlyR: it inhibited peak amplitude and dramatically enhanced desensitization. The action of 2-AG on GlyR-mediated currents developed rapidly, within ~300 milliseconds. Addition of 1 µM 2-AG strongly facilitated the depression of glycine-induced currents during repetitive (4-10 Hz application of short (2-ms duration pulses of glycine to outside-out patches. In brainstem slices from CB1 receptor-knockout mice, 2-AG significantly decreased the extent of facilitation of synaptic currents in hypoglossal motoneurons during repetitive (10-20 Hz stimulation. These observations suggest that endocannabinoids can modulate postsynaptic metaplasticity of glycinergic synaptic currents in a CB1 receptor-independent manner.

  10. Validation of dopamine receptor DRD1 and DRD2 antibodies using receptor deficient mice.

    Science.gov (United States)

    Stojanovic, Tamara; Orlova, Michaela; Sialana, Fernando J; Höger, Harald; Stuchlik, Stanislav; Milenkovic, Ivan; Aradska, Jana; Lubec, Gert

    2017-06-01

    Dopamine receptors 1 and 2 (DRD1, DRD2) are essential for signaling in the brain for a multitude of brain functions. Previous work using several antibodies against these receptors is abundant but only the minority of antibodies used have been validated and, therefore, the results of these studies remain uncertain. Herein, antibodies against DRD1 (Merck Millipore AB1765P, Santa Cruz Biotechnology sc-14001, Sigma Aldrich D2944, Alomone Labs ADR-001) and DRD2 (Abcam ab21218, Merck Millipore AB5084P, Santa Cruz Biotechnology sc-5303) have been tested using western blotting and immunohistochemistry on mouse striatum (wild type and corresponding knock-out mice) and when specific, they were further evaluated on rat and human striatum. Moreover, a DRD1 antibody and a DRD2 antibody that were found specific in our tests were used for immunoprecipitation with subsequent mass spectrometrical identification of the immunoprecipitate. Two out of nine antibodies (anti DRD1 Sigma Aldrich D2944 and anti DRD2 Merck Millipore AB5084P) against the abovementioned dopamine receptors were specific for DRD1 and DRD2 as evaluated by western blotting and immunohistochemistry and the immunoprecipitate indeed contained DRD1 and DRD2 as revealed by mass spectrometry. The observed findings may question the use of so far non-validated antibodies against the abovementioned dopamine receptors. Own observations may be valuable for the interpretation of previous results and the design of future studies using dopamine receptors DRD1 or DRD2.

  11. Attachment and Postattachment Receptors Important for Hepatitis C Virus Infection and Cell-to-Cell Transmission.

    Science.gov (United States)

    Fan, Huahao; Qiao, Luhua; Kang, Kyung-Don; Fan, Junfen; Wei, Wensheng; Luo, Guangxiang

    2017-07-01

    Hepatitis C virus (HCV) requires multiple receptors for its attachment to and entry into cells. Our previous studies found that human syndecan-1 (SDC-1), SDC-2, and T cell immunoglobulin and mucin domain-containing protein 1 (TIM-1) are HCV attachment receptors. Other cell surface molecules, such as CD81, Claudin-1 (CLDN1), Occludin (OCLN), SR-BI, and low-density lipoprotein receptor (LDLR), function mainly at postattachment steps and are considered postattachment receptors. The underlying molecular mechanisms of different receptors in HCV cell-free and cell-to-cell transmission remain elusive. In the present study, we used a clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 technology, gene-specific small interfering RNAs, and a newly developed luciferase-based reporter system to quantitatively determine the importance of individual receptors in HCV cell-free and cell-to-cell transmission. Knockouts of SDC-1 and SDC-2 resulted in remarkable reductions of HCV infection and cell attachment, whereas SDC-3 and SDC-4 knockouts did not affect HCV infection. Defective HCV attachment to SDC-1 and/or SDC-2 knockout cells was completely restored by SDC-1 and SDC-2 but not SDC-4 expression. Knockout of the attachment receptors SDC-1, SDC-2, and TIM-1 also modestly decreased HCV cell-to-cell transmission. In contrast, silencing and knockout of the postattachment receptors CD81, CLDN1, OCLN, SR-BI, and LDLR greatly impaired both HCV cell-free and cell-to-cell transmission. Additionally, apolipoprotein E was found to be important for HCV cell-to-cell spread, but very-low-density lipoprotein (VLDL)-containing mouse serum did not affect HCV cell-to-cell transmission, although it inhibited cell-free infection. These findings demonstrate that attachment receptors are essential for initial HCV binding and that postattachment receptors are important for both HCV cell-free and cell-to-cell transmission. IMPORTANCE The importance and underlying molecular mechanisms

  12. The role of P2X receptors in bone biology

    DEFF Research Database (Denmark)

    Jørgensen, N R; Syberg, S; Ellegaard, M

    2015-01-01

    Bone is a highly dynamic organ, being constantly modeled and remodeled in order to adapt to the changing need throughout life. Bone turnover involves the coordinated actions of bone formation and bone degradation. Over the past decade great effort has been put into the examination of how P2X...... receptors regulate bone metabolism and especially for the P2X7 receptor an impressive amount of evidence has now documented its expression in osteoblasts, osteoclasts, and osteocytes as well as important functional roles in proliferation, differentiation, and function of the cells of bone. Key evidence has...... come from studies on murine knockout models and from pharmacologic studies on cells and animals. More recently, the role of P2X receptors in human bone diseases has been documented. Loss-of-functions polymorphisms in the P2X7 receptorare associated with bone loss and increased fracture risk. Very...

  13. Importance of GluA1 subunit-containing AMPA glutamate receptors for morphine state-dependency.

    Directory of Open Access Journals (Sweden)

    Teemu Aitta-aho

    Full Text Available In state-dependency, information retrieval is most efficient when the animal is in the same state as it was during the information acquisition. State-dependency has been implicated in a variety of learning and memory processes, but its mechanisms remain to be resolved. Here, mice deficient in AMPA-type glutamate receptor GluA1 subunits were first conditioned to morphine (10 or 20 mg/kg s.c. during eight sessions over four days using an unbiased procedure, followed by testing for conditioned place preference at morphine states that were the same as or different from the one the mice were conditioned to. In GluA1 wildtype littermate mice the same-state morphine dose produced the greatest expression of place preference, while in the knockout mice no place preference was then detected. Both wildtype and knockout mice expressed moderate morphine-induced place preference when not at the morphine state (saline treatment at the test; in this case, place preference was weaker than that in the same-state test in wildtype mice. No correlation between place preference scores and locomotor activity during testing was found. Additionally, as compared to the controls, the knockout mice showed unchanged sensitization to morphine, morphine drug discrimination and brain regional μ-opioid receptor signal transduction at the G-protein level. However, the knockout mice failed to show increased AMPA/NMDA receptor current ratios in the ventral tegmental area dopamine neurons of midbrain slices after a single injection of morphine (10 mg/kg, s.c., sliced prepared 24 h afterwards, in contrast to the wildtype mice. The results indicate impaired drug-induced state-dependency in GluA1 knockout mice, correlating with impaired opioid-induced glutamate receptor neuroplasticity.

  14. Vitamin D receptor and vitamin D metabolizing enzymes are expressed in the human male reproductive tract

    DEFF Research Database (Denmark)

    Blomberg Jensen, Martin; Nielsen, John E; Jørgensen, Anne

    2010-01-01

    The vitamin D receptor (VDR) is expressed in human testis, and vitamin D (VD) has been suggested to affect survival and function of mature spermatozoa. Indeed, VDR knockout mice and VD deficient rats show decreased sperm counts and low fertility. However, the cellular response to VD is complex, s...... a comprehensive analysis of the expression of VDR, VD activating (CYP2R1, CYP27A1, CYP27B1) and inactivating (CYP24A1) enzymes in the testis, epididymis, seminal vesicle (SV), prostate and spermatozoa.......The vitamin D receptor (VDR) is expressed in human testis, and vitamin D (VD) has been suggested to affect survival and function of mature spermatozoa. Indeed, VDR knockout mice and VD deficient rats show decreased sperm counts and low fertility. However, the cellular response to VD is complex...

  15. Peroxisome Proliferator-Activated Receptor-alpha-Null Mice Have Increased White Adipose Tissue Glucose Utilization, GLUT4, and Fat Mass: Role in Liver and Brain

    NARCIS (Netherlands)

    Knauf, C.; Rieusset, J.; Foretz, M.; Cani, P.D.; Uldry, M.; Hosokawa, M.; Martinez, E.; Bringart, M.; Waget, A.; Kersten, A.H.; Desvergne, B.; Gremlich, S.; Wahli, W.; Seydoux, J.; Delzenne, N.M.; Thorens, B.; Burcelin, R.

    2006-01-01

    Activation of the peroxisome proliferator-activated receptor (PPAR)-¿ increases lipid catabolism and lowers the concentration of circulating lipid, but its role in the control of glucose metabolism is not as clearly established. Here we compared PPAR¿ knockout mice with wild type and confirmed that

  16. Generation of Hoxc13 knockout pigs recapitulates human ectodermal dysplasia-9.

    Science.gov (United States)

    Han, Kai; Liang, Liuping; Li, Li; Ouyang, Zhen; Zhao, Bentian; Wang, Qi; Liu, Zhaoming; Zhao, Yu; Ren, Xiaoshuai; Jiang, Fei; Lai, Chengdan; Wang, Kepin; Yan, Sen; Huang, Liang; Guo, Lin; Zeng, Kang; Lai, Liangxue; Fan, Nana

    2017-01-01

    Atrichia and sparse hair phenotype cause distress to many patients. Ectodermal dysplasia-9 (ED-9) is a congenital condition characterized by hypotrichosis and nail dystrophy without other disorders, and Hoxc13 is a pathogenic gene for ED-9. However, mice carrying Hoxc13 mutation present several other serious disorders, such as skeletal defects, progressive weight loss and low viability. Mouse models cannot faithfully mimic human ED-9. In this study, we generated an ED-9 pig model via Hoxc13 gene knockout through single-stranded oligonucleotides (c.396C > A) combined with CRISPR/Cas9 and somatic cell nuclear transfer. Eight cloned piglets with three types of biallelic mutations (five piglets with Hoxc13c.396C > A/c.396C > A, two piglets with Hoxc13c.396C > A/c.396C > A + 1 and one piglet with Hoxc13Δ40/Δ40) were obtained. Hoxc13 was not expressed in pigs with all three mutation types, and the expression levels of Hoxc13-regulated genes, namely, Foxn1, Krt85 and Krt35, were decreased. The hair follicles displayed various abnormal phenotypes, such as reduced number of follicles and disarrayed hair follicle cable without normal hair all over the body. By contrast, the skin structure, skeleton phenotype, body weight gain and growth of Hoxc13 knockout pigs were apparently normal. The phenotypes of Hoxc13 mutation in pigs were similar to those in ED-9 patients. Therefore, Hoxc13 knockout pigs could be utilized as a model for ED-9 pathogenesis and as a hairless model for hair regeneration research. Moreover, the hairless pigs without other major abnormal phenotypes generated in this study could be effective models for other dermatological research because of the similarity between pig and human skins. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  17. Unintentional miRNA ablation is a risk factor in gene knockout studies: a short report.

    Directory of Open Access Journals (Sweden)

    Ivan Osokine

    2008-02-01

    Full Text Available One of the most powerful techniques for studying the function of a gene is to disrupt the expression of that gene using genetic engineering strategies such as targeted recombination or viral integration of gene trap cassettes. The tremendous utility of these tools was recognized this year with the awarding of the Nobel Prize in Physiology or Medicine to Capecchi, Evans, and Smithies for their pioneering work in targeted recombination mutagenesis in mammals. Another noteworthy discovery made nearly a decade ago was the identification of a novel class of non-coding genes called microRNAs. MicroRNAs are among the largest known classes of regulatory elements with more than 1000 predicted to exist in the mouse genome. Over 50% of known microRNAs are located within introns of coding genes. Given that currently about half of the genes in mouse have been knocked out, we investigated the possibility that intronic microRNAs may have been coincidentally deleted or disrupted in some of these mouse models. We searched published murine knockout studies and gene trap embryonic stem cell line databases for cases where a microRNA was located within or near the manipulated genomic loci, finding almost 200 cases where microRNA expression may have been disrupted along with another gene. Our results draw attention to the need for careful planning in future knockout studies to minimize the unintentional disruption of microRNAs. These data also raise the possibility that many knockout studies may need to be reexamined to determine if loss of a microRNA contributes to the phenotypic consequences attributed to loss of a protein-encoding gene.

  18. Generation of beta-lactoglobulin knock-out goats using CRISPR/Cas9

    Science.gov (United States)

    Zhou, Wenjun; Wan, Yongjie; Guo, Rihong; Deng, Mingtian; Deng, Kaiping; Wang, Zhen; Zhang, Yanli; Wang, Feng

    2017-01-01

    Goat’s milk, considered a substitute for cow’s milk, has a high nutritional value. However, goat’s milk contains various allergens, predominantly β-lactoglobulin (BLG). In this study, we employed the CRISPR/Cas9 system to target the BLG locus in goat fibroblasts for sgRNA optimization and generate BLG knock-out goats through co-injection of Cas9 mRNA and small guide RNAs (sgRNAs) into goat embryos at the one-cell stage. We firstly tested sgRNA editing efficiencies in goat fibroblast cells, and approximately 8.00%–9.09% of the cells were modified in single sgRNA-guided targeting experiment. Among the kids, the genome-targeting efficiencies of single sgRNA were 12.5% (10 ng/μL sg1) and 0% (10 ng/μL sg2) and efficiencies of dual sgRNAs were 25.0% (25 ng/μL sg2+sg3 group) and 28.6% (50 ng/μL sg2+sg3 group). Relative expression of BLG in BLG knock-out goat mammary glands significantly (p milk protein coding genes, such as CSN1S1, CSN1S2, CSN2, CSN3 and LALBA (p milk of the BLG knock-out goat. In addition, most of the targeted kids were chimeric (3/4), and their various body tissues were edited simultaneously. Our study thus provides a basis for optimizing the quality of goat milk, which can be applied to biomedical and agricultural research. PMID:29016691

  19. Generation of beta-lactoglobulin knock-out goats using CRISPR/Cas9.

    Science.gov (United States)

    Zhou, Wenjun; Wan, Yongjie; Guo, Rihong; Deng, Mingtian; Deng, Kaiping; Wang, Zhen; Zhang, Yanli; Wang, Feng

    2017-01-01

    Goat's milk, considered a substitute for cow's milk, has a high nutritional value. However, goat's milk contains various allergens, predominantly β-lactoglobulin (BLG). In this study, we employed the CRISPR/Cas9 system to target the BLG locus in goat fibroblasts for sgRNA optimization and generate BLG knock-out goats through co-injection of Cas9 mRNA and small guide RNAs (sgRNAs) into goat embryos at the one-cell stage. We firstly tested sgRNA editing efficiencies in goat fibroblast cells, and approximately 8.00%-9.09% of the cells were modified in single sgRNA-guided targeting experiment. Among the kids, the genome-targeting efficiencies of single sgRNA were 12.5% (10 ng/μL sg1) and 0% (10 ng/μL sg2) and efficiencies of dual sgRNAs were 25.0% (25 ng/μL sg2+sg3 group) and 28.6% (50 ng/μL sg2+sg3 group). Relative expression of BLG in BLG knock-out goat mammary glands significantly (p milk protein coding genes, such as CSN1S1, CSN1S2, CSN2, CSN3 and LALBA (p milk of the BLG knock-out goat. In addition, most of the targeted kids were chimeric (3/4), and their various body tissues were edited simultaneously. Our study thus provides a basis for optimizing the quality of goat milk, which can be applied to biomedical and agricultural research.

  20. Generation of beta-lactoglobulin knock-out goats using CRISPR/Cas9.

    Directory of Open Access Journals (Sweden)

    Wenjun Zhou

    Full Text Available Goat's milk, considered a substitute for cow's milk, has a high nutritional value. However, goat's milk contains various allergens, predominantly β-lactoglobulin (BLG. In this study, we employed the CRISPR/Cas9 system to target the BLG locus in goat fibroblasts for sgRNA optimization and generate BLG knock-out goats through co-injection of Cas9 mRNA and small guide RNAs (sgRNAs into goat embryos at the one-cell stage. We firstly tested sgRNA editing efficiencies in goat fibroblast cells, and approximately 8.00%-9.09% of the cells were modified in single sgRNA-guided targeting experiment. Among the kids, the genome-targeting efficiencies of single sgRNA were 12.5% (10 ng/μL sg1 and 0% (10 ng/μL sg2 and efficiencies of dual sgRNAs were 25.0% (25 ng/μL sg2+sg3 group and 28.6% (50 ng/μL sg2+sg3 group. Relative expression of BLG in BLG knock-out goat mammary glands significantly (p < 0.01 decreased as well as other milk protein coding genes, such as CSN1S1, CSN1S2, CSN2, CSN3 and LALBA (p < 0.05. As expected, BLG protein had been abolished in the milk of the BLG knock-out goat. In addition, most of the targeted kids were chimeric (3/4, and their various body tissues were edited simultaneously. Our study thus provides a basis for optimizing the quality of goat milk, which can be applied to biomedical and agricultural research.

  1. IKKε knockout prevents high fat diet induced arterial atherosclerosis and NF-κB signaling in mice.

    Directory of Open Access Journals (Sweden)

    Changchun Cao

    Full Text Available AIMS: Atherosclerosis is a public health concern affecting many worldwide, but its pathogenesis remains unclear. In this study we investigated the role of IKKε during the formation of atherosclerosis and its molecular mechanism in the mouse aortic vessel wall. METHODS AND RESULTS: C57BL/6 wild-type or IKKε knockout mice bred into the ApoE knockout genetic background were divided into 4 groups: (1 wild-type (WT, (2 ApoE knockout (AK, (3 IKKε knockout (IK, (4 or both ApoE and IKKε knockout (DK. Each group of mice were fed with a high fat diet (HFD for 12 weeks from 8 weeks of age. Immunohistochemistry and Western blotting analysis demonstrated obvious increases in the expression of IKKε in the AK group compared with the WT group, especially in the intima. Serum lipid levels were significantly higher in the AK and DK groups than in the other two groups. Staining with hematoxylin-eosin and Oil Red, as well as scanning electron microscopy revealed less severe atherosclerotic lesions in the DK group than in the AK group. Immunofluorescence and Western blot analysis demonstrated obvious increases in the expression of NF-κB pathway components and downstream factors in the AK group, especially in the intima, while these increases were blocked in the DK group. CONCLUSION: The knockout of IKKε prevented significant atherosclerosis lesions in the mouse aorta from in both wild-type and ApoE knockout mice fed a HFD, suggesting that IKKε may play a vital role in HFD-induced atherosclerosis and would be an important target for the treatment of atherosclerosis.

  2. Glutamate receptors

    DEFF Research Database (Denmark)

    Kristensen, Anders S; Geballe, Matthew T; Snyder, James P

    2006-01-01

    Fast excitatory synaptic transmission in the CNS relies almost entirely on the neurotransmitter glutamate and its family of ion channel receptors. An appreciation of the coupling between agonist binding and channel opening has advanced rapidly during the past five years, largely as a result of ne...

  3. Distinct Function of Estrogen Receptor α in Smooth Muscle and Fibroblast Cells in Prostate Development

    OpenAIRE

    Vitkus, Spencer; Yeh, Chiuan-Ren; Lin, Hsiu-Hsia; Hsu, Iawen; Yu, Jiangzhou; Chen, Ming; Yeh, Shuyuan

    2012-01-01

    Estrogen signaling, through estrogen receptor (ER)α, has been shown to cause hypertrophy in the prostate. Our recent report has shown that epithelial ERα knockout (KO) will not affect the normal prostate development or homeostasis. However, it remains unclear whether ERα in different types of stromal cells has distinct roles in prostate development. This study proposed to elucidate how KO of ERα in the stromal smooth muscle or fibroblast cells may interrupt cross talk between prostate stromal...

  4. Astrocytic β2 Adrenergic Receptor Gene Deletion Affects Memory in Aged Mice.

    Directory of Open Access Journals (Sweden)

    Cathy Joanna Jensen

    Full Text Available In vitro and in vivo studies suggest that the astrocytic adrenergic signalling enhances glycogenolysis which provides energy to be transported to nearby cells and in the form of lactate. This energy source is important for motor and cognitive functioning. While it is suspected that the β2-adrenergic receptor on astrocytes might contribute to this energy balance, it has not yet been shown conclusively in vivo. Inducible astrocyte specific β2-adrenergic receptor knock-out mice were generated by crossing homozygous β2-adrenergic receptor floxed mice (Adrb2flox and mice with heterozygous tamoxifen-inducible Cre recombinase-expression driven by the astrocyte specific L-glutamate/L-aspartate transporter promoter (GLAST-CreERT2. Assessments using the modified SHIRPA (SmithKline/Harwell/Imperial College/Royal Hospital/Phenotype Assessment test battery, swimming ability test, and accelerating rotarod test, performed at 1, 2 and 4 weeks, 6 and 12 months after tamoxifen (or vehicle administration did not reveal any differences in physical health or motor functions between the knock-out mice and controls. However deficits were found in the cognitive ability of aged, but not young adult mice, reflected in impaired learning in the Morris Water Maze. Similarly, long-term potentiation (LTP was impaired in hippocampal brain slices of aged knock-out mice maintained in low glucose media. Using microdialysis in cerebellar white matter we found no significant differences in extracellular lactate or glucose between the young adult knock-out mice and controls, although trends were detected. Our results suggest that β2-adrenergic receptor expression on astrocytes in mice may be important for maintaining cognitive health at advanced age, but is dispensable for motor function.

  5. Multiple Phenotypes in Adult Mice following Inactivation of the Coxsackievirus and Adenovirus Receptor (Car) Gene

    OpenAIRE

    Pazirandeh, Ahmad; Sultana, Taranum; Mirza, Momina; Rozell, Björn; Hultenby, Kjell; Wallis, Karin; Vennström, Björn; Davis, Ben; Arner, Anders; Heuchel, Rainer; Löhr, Matthias; Philipson, Lennart; Sollerbrant, Kerstin

    2011-01-01

    To determine the normal function of the Coxsackievirus and Adenovirus Receptor (CAR), a protein found in tight junctions and other intercellular complexes, we constructed a mouse line in which the CAR gene could be disrupted at any chosen time point in a broad spectrum of cell types and tissues. All knockouts examined displayed a dilated intestinal tract and atrophy of the exocrine pancreas with appearance of tubular complexes characteristic of acinar-to-ductal metaplasia. The mice also exhib...

  6. Increased Salt-Sensitivity in Endothelial Nitric Oxide Synthase-Knockout Mice

    OpenAIRE

    Leonard, Allison M.; Chafe, Linda L.; Montani, Jean-Pierre; Van Vliet, Bruce N.

    2017-01-01

    Background: Although impaired nitric oxide production contributes importantly to salt-sensitivity, the role of the endothelial isoform of nitric oxide synthase (eNOS) has received little attention. In the present study we compared the effects of a high-salt diet on the blood pressure response of eNOS knockout (eNOS−/−) and control (eNOS+/+) mice. Methods: Mean arterial pressure (MAP), heart rate, pulse pressure, and activity levels were recorded by telemetry in mice fed a regular-salt diet (0...

  7. Transcriptomic profiling comparison of YAP over-expression and conditional knockout mouse tooth germs

    Directory of Open Access Journals (Sweden)

    Ming Liu

    2015-09-01

    Full Text Available To identify the downstream target genes of YAP, we used RNA-Seq technology to compare the transcriptomic profilings of Yap conditional knockout (Yap CKO and YAP over-expression mouse tooth germs. Our results showed that some Hox, Wnt and Laminin family genes had concurrent changes with YAP transcripts, indicating that the expression of these genes may be regulated by YAP. Here, we provide the detailed experimental procedure for the transcriptomic profiling results (NCBI GEO accession number GSE65524. The associated study on the regulation of Hoxa1 and Hoxc13 genes by YAP was published in Molecular Cellular Biology in 2015 [Liu et al., 2015].

  8. Transcriptomic profiling comparison of YAP over-expression and conditional knockout mouse tooth germs.

    Science.gov (United States)

    Liu, Ming; Wang, Xiu-Ping

    2015-09-01

    To identify the downstream target genes of YAP, we used RNA-Seq technology to compare the transcriptomic profilings of Yap conditional knockout (Yap CKO) and YAP over-expression mouse tooth germs. Our results showed that some Hox, Wnt and Laminin family genes had concurrent changes with YAP transcripts, indicating that the expression of these genes may be regulated by YAP. Here, we provide the detailed experimental procedure for the transcriptomic profiling results (NCBI GEO accession number GSE65524). The associated study on the regulation of Hoxa1 and Hoxc13 genes by YAP was published in Molecular Cellular Biology in 2015 [Liu et al., 2015].

  9. Prohormone convertase 2 activity is increased in the hippocampus of Wfs1 knockout mice

    Directory of Open Access Journals (Sweden)

    Karin eTein

    2015-08-01

    Full Text Available BackgroundMutations in WFS1 gene cause Wolfram syndrome, which is a rare autosomal recessive disorder, characterized by diabetes insipidus, diabetes mellitus, optic nerve atrophy and deafness (DIDMOAD. The WFS1 gene product wolframin is located in the endoplasmic reticulum. Mice lacking this gene exhibit disturbances in the processing and secretion of peptides, such as vasopressin and insulin. In the brain, high levels of the wolframin protein have been observed in the hippocampus, amygdala and limbic structures. The aim of this study was to investigate the effect of Wfs1 knockout on peptide processing in mouse hippocampus. A peptidomic approach was used to characterize individual peptides in the hippocampus of wild-type and Wfs1 knockout mice. ResultsWe identified 126 peptides in hippocampal extracts and the levels of 10 peptides differed between Wfs1 KO and wild-type mice at P<0.05. The peptide with the largest alteration was little-LEN, which level was 25 times higher in the hippocampus of Wfs1 KO mice compared to wild-type mice. Processing (cleavage of little-LEN from the Pcsk1n gene product proSAAS involves prohormone convertase 2 (PC2. Thus, PC2 activity was measured in extracts prepared from the hippocampus of Wfs1 knockout mice. The activity of PC2 in Wfs1 mutant mice was significantly higher (149.9±2.3%, p<0.0001, n=8 than in wild-type mice (100.0±7.0%, n=8. However, Western blot analysis showed that protein levels of 7B2, proPC2 and PC2 were same in both groups, and so were gene expression levels.ConclusionsProcessing of proSAAS is altered in the hippocampus of Wfs1-KO mice, which is caused by increased activity of PC2. Increased activity of PC2 in Wfs1 knockout mice is not caused by alteration in the levels of PC2 protein. Our results suggest a functional link between Wfs1 and PC2. Thus, the detailed molecular mechanism of the role of Wfs1 in the regulation of PC2 activity needs further investigation.

  10. Wip1 knockout inhibits the proliferation and enhances the migration of bone marrow mesenchymal stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Tang, Yiting [College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095 (China); State Key Laboratory of Animal Nutrition and Key Laboratory of Farm Animal Genetic Resources and Germplasm Innovation of Ministry of Agriculture, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193 (China); Liu, Lan [State Key Laboratory of Animal Nutrition and Key Laboratory of Farm Animal Genetic Resources and Germplasm Innovation of Ministry of Agriculture, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193 (China); Sheng, Ming [College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095 (China); Xiong, Kai [Department of Veterinary Clinical and Animal Sciences, University of Copenhagen, Grønnegårdsvej 7, 1870 Frederiksberg C (Denmark); Huang, Lei; Gao, Qian; Wei, Jingliang; Wu, Tianwen; Yang, Shulin [State Key Laboratory of Animal Nutrition and Key Laboratory of Farm Animal Genetic Resources and Germplasm Innovation of Ministry of Agriculture, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193 (China); Liu, Honglin, E-mail: liuhonglinnjau@163.com [College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095 (China); Mu, Yulian, E-mail: muyulian76@iascaas.net.cn [State Key Laboratory of Animal Nutrition and Key Laboratory of Farm Animal Genetic Resources and Germplasm Innovation of Ministry of Agriculture, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193 (China); Li, Kui [State Key Laboratory of Animal Nutrition and Key Laboratory of Farm Animal Genetic Resources and Germplasm Innovation of Ministry of Agriculture, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193 (China)

    2015-06-10

    Mesenchymal stem cells (MSCs), a unique population of multipotent adult progenitor cells originally found in bone marrow (BM), are extremely useful for multifunctional therapeutic approaches. However, the growth arrest and premature senescence of MSCs in vitro prevent the in-depth characterization of these cells. In addition, the regulatory factors involved in MSCs migration remain largely unknown. Given that protein phosphorylation is associated with the processes of MSCs proliferation and migration, we focused on wild-type p53-inducible phosphatase-1 (Wip1), a well-studied modulator of phosphorylation, in this study. Our results showed that Wip1 knockout significantly inhibited MSCs proliferation and induced G2-phase cell-cycle arrest by reducing cyclinB1 expression. Compared with WT-MSCs, Wip1{sup −/−} MSCs displayed premature growth arrest after six passages in culture. Transwell and scratch assays revealed that Wip1{sup −/−} MSCs migrate more effectively than WT-MSCs. Moreover, the enhanced migratory response of Wip1{sup −/−} MSCs may be attributed to increases in the induction of Rac1-GTP activity, the pAKT/AKT ratio, the rearrangement of filamentous-actin (f-actin), and filopodia formation. Based on these results, we then examined the effect of treatment with a PI3K/AKT and Rac1 inhibitor, both of which impaired the migratory activity of MSCs. Therefore, we propose that the PI3K/AKT/Rac1 signaling axis mediates the Wip1 knockout-induced migration of MSCs. Our findings indicate that the principal function of Wip1 in MSCs transformation is the maintenance of proliferative capacity. Nevertheless, knocking out Wip1 increases the migratory capacity of MSCs. This dual effect of Wip1 provides the potential for purposeful routing of MSCs. - Highlights: • Wip1 knockout inhibited MSCs proliferation through reducing cyclinB1 expression. • Wip1{sup −/−} MSCs displayed premature growth arrest in vitro after six passages. • Knocking out Wip1

  11. Knockout of Tmem70 alters biogenesis of ATP synthase and leads to embryonal lethality in mice

    Czech Academy of Sciences Publication Activity Database

    Vrbacký, Marek; Kovalčíková, Jana; Chawengsaksophak, Kallayanee; Beck, Inken; Mráček, Tomáš; Nůsková, Hana; Sedmera, David; Papoušek, František; Kolář, František; Sobol, Margaryta; Hozák, Pavel; Sedláček, Radislav; Houštěk, Josef

    2016-01-01

    Roč. 25, č. 21 (2016), s. 4674-4685 ISSN 0964-6906 R&D Projects: GA ČR(CZ) GB14-36804G; GA MŠk(CZ) LL1204; GA MŠk(CZ) ED1.1.00/02.0109; GA TA ČR(CZ) TE01020118; GA MŠk(CZ) LM2015062; GA MZd(CZ) NV16-33018A; GA MŠk(CZ) LM2015040 Institutional support: RVO:67985823 ; RVO:68378050 Keywords : mouse knockout * mitochondria * ATP synthase * TMEM70 * biogenesis * mitochondrial disease s Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 5.340, year: 2016

  12. Wip1 knockout inhibits the proliferation and enhances the migration of bone marrow mesenchymal stem cells

    International Nuclear Information System (INIS)

    Tang, Yiting; Liu, Lan; Sheng, Ming; Xiong, Kai; Huang, Lei; Gao, Qian; Wei, Jingliang; Wu, Tianwen; Yang, Shulin; Liu, Honglin; Mu, Yulian; Li, Kui

    2015-01-01

    Mesenchymal stem cells (MSCs), a unique population of multipotent adult progenitor cells originally found in bone marrow (BM), are extremely useful for multifunctional therapeutic approaches. However, the growth arrest and premature senescence of MSCs in vitro prevent the in-depth characterization of these cells. In addition, the regulatory factors involved in MSCs migration remain largely unknown. Given that protein phosphorylation is associated with the processes of MSCs proliferation and migration, we focused on wild-type p53-inducible phosphatase-1 (Wip1), a well-studied modulator of phosphorylation, in this study. Our results showed that Wip1 knockout significantly inhibited MSCs proliferation and induced G2-phase cell-cycle arrest by reducing cyclinB1 expression. Compared with WT-MSCs, Wip1 −/− MSCs displayed premature growth arrest after six passages in culture. Transwell and scratch assays revealed that Wip1 −/− MSCs migrate more effectively than WT-MSCs. Moreover, the enhanced migratory response of Wip1 −/− MSCs may be attributed to increases in the induction of Rac1-GTP activity, the pAKT/AKT ratio, the rearrangement of filamentous-actin (f-actin), and filopodia formation. Based on these results, we then examined the effect of treatment with a PI3K/AKT and Rac1 inhibitor, both of which impaired the migratory activity of MSCs. Therefore, we propose that the PI3K/AKT/Rac1 signaling axis mediates the Wip1 knockout-induced migration of MSCs. Our findings indicate that the principal function of Wip1 in MSCs transformation is the maintenance of proliferative capacity. Nevertheless, knocking out Wip1 increases the migratory capacity of MSCs. This dual effect of Wip1 provides the potential for purposeful routing of MSCs. - Highlights: • Wip1 knockout inhibited MSCs proliferation through reducing cyclinB1 expression. • Wip1 −/− MSCs displayed premature growth arrest in vitro after six passages. • Knocking out Wip1 increases the migratory

  13. Hepatic caveolin-1 is enhanced in Cyp27a1/ApoE double knockout mice

    OpenAIRE

    Escher, Geneviève; Zurkinden, Line; Mistry, H; Mansour, Y; Rohrbach, B; Vogt, Bruno

    2016-01-01

    Sterol 27?hydroxylase (CYP27A1) is involved in bile acid synthesis and cholesterol homoeostasis. Cyp27a1 (?/?)/Apolipoprotein E (?/?) double knockout mice (DKO) fed a western diet failed to develop atherosclerosis. Caveolin?1 (CAV?1), the main component of caveolae, is associated with lipid homoeostasis and has regulatory roles in vascular diseases. We hypothesized that liver CAV?1 would contribute to the athero?protective mechanism in DKO mice. Cyp27a1 (+/+)/ApoE (?/?) (ApoE KO), Cyp27a1 (+/...

  14. Somatostatin sst(2) receptors inhibit peristalsis in the rat and mouse jejunum.

    Science.gov (United States)

    Abdu, Faiza; Hicks, Gareth A; Hennig, Grant; Allen, Jeremy P; Grundy, David

    2002-04-01

    Somatostatin [somatotropin release-inhibitory factor (SRIF)] has widespread actions throughout the gastrointestinal tract, but the receptor mechanisms involved are not fully characterized. We have examined the effect of selective SRIF-receptor ligands on intestinal peristalsis by studying migrating motor complexes (MMCs) in isolated segments of jejunum from rats, mice, and sst(2)-receptor knockout mice. MMCs were recorded in 4- to 5-cm segments of jejunum mounted horizontally in vitro. MMCs occurred in rat and mouse jejunum with intervals of 104.4 +/- 10 and 131.2 +/- 8 s, respectively. SRIF, octreotide, and BIM-23027 increased the interval between MMCs, an effect fully or partially antagonized by the sst(2)-receptor antagonist Cyanamid154806. A non-sst(2) receptor-mediated component was evident in mouse as confirmed by the observation of an inhibitory action of SRIF in sst(2) knockout tissue. Blocking nitric oxide generation abolished the response to SRIF in rat but not mouse jejunum. sst(2) Receptors mediate inhibition of peristalsis in both rat and mouse jejunum, but a non-sst(2) component also exists in the mouse. Nitrergic mechanisms are differentially involved in rat and mouse jejunum.

  15. Gene expression profiling of gastric mucosa in mice lacking CCK and gastrin receptors

    DEFF Research Database (Denmark)

    Zhao, Chun-Mei; Kodama, Yosuke; Flatberg, Arnar

    2014-01-01

    The stomach produces acid, which may play an important role in the regulation of bone homeostasis. The aim of this study was to reveal signaling pathways in the gastric mucosa that involve the acid secretion and possibly the bone metabolism in CCK1 and/or CCK2 receptor knockout (KO) mice. Gastric...... acid secretion was impaired and the ECL cell signaling pathway was inhibited in CCK2 receptor KO mice but not in CCK1 receptor KO mice. However, in CCK1+2 receptor double KO mice the acid secretion in response to pylorus ligation-induced vagal stimulation and the ECL cell pathway were partially...... hydroxylase probably in trefoil peptide2-immunoreactive cells. In conclusion, mice lacking CCK receptors exhibited a functional shift from the gastrin-CCK pathways to the neuronal pathway in control of the ECL cells and eventually the acid secretion. Taking the present data together with previous findings, we...

  16. P2X7 receptor-deficient mice are susceptible to bone cancer pain

    DEFF Research Database (Denmark)

    Hansen, RR; Nielsen, CK; Nasser, A

    2011-01-01

    The purinergic P2X7 receptor is implicated in both neuropathic and inflammatory pain, and has been suggested as a possible target in pain treatment. However, the specific role of the P2X7 receptor in bone cancer pain is unknown. We demonstrated that BALB/cJ P2X7 receptor knockout (P2X7R KO) mice...... of the P2X7R KO mouse. Further experiments are needed to elucidate the exact role of the P2X7 receptors in bone cancer pain. Pain-related behaviours had an earlier onset in bone cancer-bearing, P2X7 receptor-deficient mice, and treatment with A-438079 failed to alleviate pain-related behaviours....

  17. α7 nicotinic ACh receptor-deficient mice exhibit sustained attention impairments that are reversed by β2 nicotinic ACh receptor activation.

    Science.gov (United States)

    Kolisnyk, Benjamin; Al-Onaizi, Mohammed A; Prado, Vania F; Prado, Marco A M

    2015-10-01

    Disruptions of executive function, including attentional deficits, are a hallmark of a number of diseases. ACh in the prefrontal cortex regulates attentive behaviour; however, the role of α7 nicotinic ACh receptor (α7nAChR) in attention is contentious. In order to probe attention, we trained both wild-type and α7nAChR knockout mice on a touch screen-based five-choice serial reaction time task (5-CSRT). Following training procedures, we then tested sustained attention using a probe trial experiment. To further differentiate the role of specific nicotinic receptors in attention, we then tested the effects of both α7nAChR and β2nAChR agonists on the performance of both wild-type and knockout mice on the 5-CSRT task. At low doses, α7nAChR agonists improved attentional performance of wild-type mice, while high doses had deleterious effects on attention. α7nAChR knockout mice displayed deficits in sustained attention that were not ameliorated by α7nAChR agonists. However, these deficits were completely reversed by the administration of a β2nAChR agonist. Furthermore, administration of a β2nAChR agonist in α7nAChR knockout mice elicited similar biochemical response in the prefrontal cortex as the administration of α7nAChR agonists in wild-type mice. Our experiments reveal an intricate relationship between distinct nicotinic receptors to regulate attentional performance and provide the basis for targeting β2nAChRs pharmacologically to decrease attentional deficits due to a dysfunction in α7nAChRs. © 2015 The British Pharmacological Society.

  18. Quantitative changes of main components of erythrocyte membranes which define architectonics of cells under pttg gene knockout

    Directory of Open Access Journals (Sweden)

    О. P. Kanyuka

    2014-04-01

    Full Text Available A pttg gene knockout affects the functional state of erythron in mice which could be associated with structural changes in the structure of erythrocyte membranes. The pttg gene knockout causes a significant modification of fatty acids composition of erythrocyte membrane lipids by reducing the content of palmitic acid and incre