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Sample records for 5-ht3a receptor knockout

  1. Impaired Social Behavior in 5-HT3A Receptor Knockout Mice

    PubMed Central

    Smit-Rigter, Laura A.; Wadman, Wytse J.; van Hooft, Johannes A.

    2010-01-01

    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 min 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. PMID:21103015

  2. Deletion of the 5-HT3A-receptor subunit blunts the induction of cocaine sensitization

    PubMed Central

    Hodge, C. W.; Bratt, A. M.; Kelley, S. P.

    2008-01-01

    Serotonin (5-HT) receptors are classified into seven groups (5-HT1–7), comprising at least 14 structurally and pharmacologically distinct receptor subtypes. Pharma-cological antagonism of ionotropic 5-HT3 receptors has been shown to modulate both behavioral and neuro-chemical aspects of the induction of sensitization to cocaine. It is not known, however, if specific molecular subunits of the 5-HT3 receptor influence the development of cocaine sensitization. To address this question, we studied the effects of acute and chronic intermittent cocaine administration in mice with a targeted deletion of the gene for the 5-HT3A-receptor subunit (5-HT3A −/−). 5-HT3A (−/−) mice showed blunted induction of cocaine-induced locomotor sensitization as compared with wild-type littermate controls. 5-HT3A (−/−) mice did not differ from wild-type littermate controls on measures of basal motor activity or response to acute cocaine treatment. Enhanced locomotor response to saline injection following cocaine sensitization was observed equally in 5-HT3A (−/−) and wild-type mice suggesting similar conditioned effects associated with chronic cocaine treatment. These data show a role for the 5-HT3A-receptor subunit in the induction of behavioral sensitization to cocaine and suggest that the 5-HT3A molecular subunit modulates neurobehavioral adaptations to cocaine, which may underlie aspects of addiction. PMID:17559417

  3. High yield and efficient expression and purification of the human 5-HT3A receptor

    PubMed Central

    Wu, Zhong-shan; Cui, Zhi-cheng; Cheng, Hao; Fan, Chen; Melcher, Karsten; Jiang, Yi; Zhang, Cheng-hai; Jiang, Hua-liang; Cong, Yao; Liu, Qian; Xu, H Eric

    2015-01-01

    Aim: To establish a method for efficient expression and purification of the human serotonin type 3A receptor (5-HT3A) that is suitable for structural studies. Methods: Codon-optimized cDNA of human 5-HT3A was inserted into a modified BacMam vector, which contained an IgG leader sequence, an 8×His tag linked with two-Maltose Binding Proteins (MBP), and a TEV protease cleavage site. The BacMam construct was used to generate baculoviruses for expression of 5-HT3A in HEK293F cells. The proteins were solubilized from the membrane with the detergent C12E 9, and purified using MBP affinity chromatography. The affinity tag was removed by TEV protease treatment and immobilized metal ion affinity chromatography. The receptors were further purified by size-exclusion chromatography (SEC). Western blot and SDS-PAGE were used to detect 5-HT3A during purification. The purified receptor was used in crystallization and analyzed with negative stain electron microscopy (EM). Results: The BacMam system yielded 0.5 milligram of the human 5-HT3A receptor per liter of cells. MBP affinity purification resulted in good yields with high purity and homogeneity. SEC profiles indicated that the purified receptors were pentameric. No protein crystals were obtained; however, a reconstructed 3D density map generated from the negative stain EM data fitted well with the mouse 5-HT3A structure. Conclusion: With the BacMam system, robust expression of the human 5-HT3A receptor is obtained, which is monodisperse, therefore enabling 3D reconstruction of an EM map. This method is suitable for high-throughput screening of different constructs, thus facilitating structural and biochemical studies of the 5-HT3A receptor. PMID:26073329

  4. Identification of ginsenoside interaction sites in 5-HT3A receptors.

    PubMed

    Lee, Byung-Hwan; Lee, Jun-Ho; Lee, Sang-Mok; Jeong, Sang Min; Yoon, In-Soo; Lee, Joon-Hee; Choi, Sun-Hye; Pyo, Mi Kyung; Rhim, Hyewhon; Kim, Hyoung-Chun; Jang, Choon-Gon; Lee, Byoung-Cheol; Park, Chul-Seung; Nah, Seung-Yeol

    2007-03-01

    We previously demonstrated that 20(S)-ginsenoside Rg(3) (Rg(3)), one of the active components of Panax ginseng, non-competitively inhibits 5-HT(3A) receptor channel activity on extracellular side of the cell. Here, we sought to elucidate the molecular mechanisms underlying Rg(3)-induced 5-HT(3A) receptor regulation. We used the two-microelectrode voltage-clamp technique to investigate the effect of Rg(3) on 5-HT-mediated ion currents (I(5-HT)) in Xenopus oocytes expressing wild-type or 5-HT(3A) receptors harboring mutations in the gating pore region of transmembrane domain 2 (TM2). In oocytes expressing wild-type 5-HT(3A) receptors, Rg(3) dose-dependently inhibited peak I(5-HT) with an IC(50) of 27.6+/-4.3microM. Mutations V291A, F292A, and I295A in TM2 greatly attenuated or abolished the Rg(3)-induced inhibition of peak I(5-HT). Mutation V291A but not F292A and I295A induced constitutively active ion currents with decrease of current decay rate. Rg(3) accelerated the rate of current decay with dose-dependent manner in the presence of 5-HT. Rg(3) and TMB-8, an open channel blocker, dose-dependently inhibited constitutively active ion currents. The IC(50) values of constitutively active ion currents in V291A mutant receptor were 72.4+/-23.1 and 6.5+/-0.7microM for Rg(3) and TMB-8, respectively. Diltiazem did not prevent Rg(3)-induced inhibition of constitutively active ion currents in occlusion experiments. These results indicate that Rg(3) inhibits 5-HT(3A) receptor channel activity through interactions with residues V291, F292, and I295 in the channel gating region of TM2 and further demonstrate that Rg(3) regulates 5-HT(3A) receptor channel activity in the open state at different site(s) from those of TMB-8 and diltiazem. PMID:17257631

  5. Pathways and Barriers for Ion Translocation through the 5-HT3A Receptor Channel

    PubMed Central

    Di Maio, Danilo; Chandramouli, Balasubramanian; Brancato, Giuseppe

    2015-01-01

    Pentameric ligand gated ion channels (pLGICs) are ionotropic receptors that mediate fast intercellular communications at synaptic level and include either cation selective (e.g., nAChR and 5-HT3) or anion selective (e.g., GlyR, GABAA and GluCl) membrane channels. Among others, 5-HT3 is one of the most studied members, since its first cloning back in 1991, and a large number of studies have successfully pinpointed protein residues critical for its activation and channel gating. In addition, 5-HT3 is also the target of a few pharmacological treatments due to the demonstrated benefits of its modulation in clinical trials. Nonetheless, a detailed molecular analysis of important protein features, such as the origin of its ion selectivity and the rather low conductance as compared to other channel homologues, has been unfeasible until the recent crystallization of the mouse 5-HT3A receptor. Here, we present extended molecular dynamics simulations and free energy calculations of the whole 5-HT3A protein with the aim of better understanding its ion transport properties, such as the pathways for ion permeation into the receptor body and the complex nature of the selectivity filter. Our investigation unravels previously unpredicted structural features of the 5-HT3A receptor, such as the existence of alternative intersubunit pathways for ion translocation at the interface between the extracellular and the transmembrane domains, in addition to the one along the channel main axis. Moreover, our study offers a molecular interpretation of the role played by an arginine triplet located in the intracellular domain on determining the characteristic low conductance of the 5-HT3A receptor, as evidenced in previous experiments. In view of these results, possible implications on other members of the superfamily are suggested. PMID:26465896

  6. Pathways and Barriers for Ion Translocation through the 5-HT3A Receptor Channel.

    PubMed

    Di Maio, Danilo; Chandramouli, Balasubramanian; Brancato, Giuseppe

    2015-01-01

    Pentameric ligand gated ion channels (pLGICs) are ionotropic receptors that mediate fast intercellular communications at synaptic level and include either cation selective (e.g., nAChR and 5-HT3) or anion selective (e.g., GlyR, GABAA and GluCl) membrane channels. Among others, 5-HT3 is one of the most studied members, since its first cloning back in 1991, and a large number of studies have successfully pinpointed protein residues critical for its activation and channel gating. In addition, 5-HT3 is also the target of a few pharmacological treatments due to the demonstrated benefits of its modulation in clinical trials. Nonetheless, a detailed molecular analysis of important protein features, such as the origin of its ion selectivity and the rather low conductance as compared to other channel homologues, has been unfeasible until the recent crystallization of the mouse 5-HT3A receptor. Here, we present extended molecular dynamics simulations and free energy calculations of the whole 5-HT3A protein with the aim of better understanding its ion transport properties, such as the pathways for ion permeation into the receptor body and the complex nature of the selectivity filter. Our investigation unravels previously unpredicted structural features of the 5-HT3A receptor, such as the existence of alternative intersubunit pathways for ion translocation at the interface between the extracellular and the transmembrane domains, in addition to the one along the channel main axis. Moreover, our study offers a molecular interpretation of the role played by an arginine triplet located in the intracellular domain on determining the characteristic low conductance of the 5-HT3A receptor, as evidenced in previous experiments. In view of these results, possible implications on other members of the superfamily are suggested. PMID:26465896

  7. Single-Channel Kinetic Analysis for Activation and Desensitization of Homomeric 5-HT3A Receptors

    PubMed Central

    Corradi, Jeremías; Gumilar, Fernanda; Bouzat, Cecilia

    2009-01-01

    Abstract The 5-HT3A receptor is a member of the Cys-loop family of ligand-gated ion channels. To perform kinetic analysis, we mutated the 5-HT3A subunit to obtain a high-conductance form so that single-channel currents can be detected. At all 5-HT concentrations (>0.1 μM), channel activity appears as openings in quick succession that form bursts, which coalesce into clusters. By combining single-channel and macroscopic data, we generated a kinetic model that perfectly describes activation, deactivation, and desensitization. The model shows that full activation arises from receptors with three molecules of agonist bound. It reveals an earlier conformational change of the fully liganded receptor that occurs while the channel is still closed. From this pre-open closed state, the receptor enters into an open-closed cycle involving three open states, which form the cluster whose duration parallels the time constant of desensitization. A similar model lacking the pre-open closed state can describe the data only if the opening rates are fixed to account for the slow activation rate. The application of the model to M4 mutant receptors shows that position 10′ contributes to channel opening and closing rates. Thus, our kinetic model provides a foundation for understanding structural bases of activation and drug action. PMID:19720021

  8. Fluorophore assisted light inactivation (FALI) of recombinant 5-HT3A receptor constitutive internalization and function

    PubMed Central

    Morton, Russell A.; Luo, Guoxiang; Davis, Margaret I.; Hales, Tim G.; Lovinger, David M.

    2011-01-01

    Fluorescent proteins and molecules are now widely used to tag and visualize proteins resulting in an improved understanding of protein trafficking, localization, and function. In addition, fluorescent tags have also been used to inactivate protein function in a spatially and temporally-defined manner, using a technique known as fluorophore-assisted light inactivation (FALI) or chromophore-assisted light inactivation (CALI). In this study we tagged the serotonin3 A subunit with the α-bungarotoxin binding sequence (BBS) and subsequently labeled 5-HT3A/BBS receptors with fluorescently conjugated α-bungarotoxin in live cells. We show that 5-HT3A/BBS receptors are constitutively internalized in the absence of an agonist and internalization as well as receptor function are inhibited by fluorescence. The fluorescence-induced disruption of function and internalization was reduced with oxygen radical scavengers suggesting the involvement of reactive oxygen species, implicating the FALI process. Furthermore, these data suggest that intense illumination during live-cell microscopy may result in inadvertent FALI and inhibition of protein trafficking. PMID:21338684

  9. Open probability of homomeric murine 5-HT3A serotonin receptors depends on subunit occupancy

    PubMed Central

    Mott, David D; Erreger, Kevin; Banke, Tue G; Traynelis, Stephen F

    2001-01-01

    The time course of macroscopic current responses of homomeric murine serotonin 5-HT3A receptors was studied in whole cells and excised membrane patches under voltage clamp in response to rapid application of serotonin. Serotonin activated whole cell currents with an EC50 value for the peak response of 2 μm and a Hill slope of 3.0 (n = 12), suggesting that the binding of at least three agonist molecules is required to open the channel. Homomeric 5-HT3A receptors in excised membrane patches had a slow activation time course (mean ±s.e.m. 10-90 % rise time 12.5 ± 1.6 ms; n = 9 patches) for 100 μm serotonin. The apparent activation rate was estimated by fitting an exponential function to the rising phase of responses to supramaximal serotonin to be 136 s−1. The 5-HT3A receptor response to 100 μm serotonin in outside-out patches (n = 19) and whole cells (n = 41) desensitized with a variable rate that accelerated throughout the experiment. The time course for desensitization was described by two exponential components (for patches τslow 1006 ± 139 ms, amplitude 31 % τfast 176 ± 25 ms, amplitude 69 %). Deactivation of the response following serotonin removal from excised membrane patches (n = 8) and whole cells (n = 29) was described by a dual exponential time course with time constants similar to those for desensitization (for patches τslow 838 ± 217 ms, 55 % amplitude; τfast 213 ± 44 ms, 45 % amplitude). In most patches (6 of 8), the deactivation time course in response to a brief 1-5 ms pulse of serotonin was similar to or slower than desensitization. This suggests that the continued presence of agonist can induce desensitization with a similar or more rapid time course than agonist unbinding. The difference between the time course for deactivation and desensitization was voltage independent over the range -100 to -40 mV in patches (n = 4) and -100 to +50 mV in whole cells (n = 4), suggesting desensitization of these receptors in the presence of

  10. Unraveling mechanisms underlying partial agonism in 5-HT3A receptors.

    PubMed

    Corradi, Jeremías; Bouzat, Cecilia

    2014-12-10

    Partial agonists have emerged as attractive therapeutic molecules. 2-Me-5HT and tryptamine have been defined as partial agonists of 5-HT3 receptors on the basis of macroscopic measurements. Because several mechanisms may limit maximal responses, we took advantage of the high-conductance form of the mouse serotonin type 3A (5-HT3A) receptor to understand their molecular actions. Individual 5-HT-bound receptors activate in long episodes of high open probability, consisting of groups of openings in quick succession. The activation pattern is similar for 2-Me-5HT only at very low concentrations since profound channel blockade takes place within the activating concentration range. In contrast, activation episodes are significantly briefer in the presence of tryptamine. Generation of a full activation scheme reveals that the fully occupied receptor overcomes transitions to closed preopen states (primed states) before opening. Reduced priming explains the partial agonism of tryptamine. In contrast, 2-Me-5HT is not a genuine partial agonist since priming is not dramatically affected and its low apparent efficacy is mainly due to channel blockade. The analysis also shows that the first priming step is the rate-limiting step and partial agonists require an increased number of priming steps for activation. Molecular docking suggests that interactions are similar for 5-HT and 2-Me-5HT but slightly different for tryptamine. Our study contributes to understanding 5-HT3A receptor activation, extends the novel concept of partial agonism within the Cys-loop family, reveals novel aspects of partial agonism, and unmasks molecular actions of classically defined partial agonists. Unraveling mechanisms underlying partial responses has implications in the design of therapeutic compounds. PMID:25505338

  11. Influence of the 5-HT3A Receptor Gene Polymorphism and Childhood Sexual Trauma on Central Serotonin Activity

    PubMed Central

    Huh, Hyu Jung; Chae, Jeong-Ho

    2015-01-01

    Background Gene-environment interactions are important for understanding alterations in human brain function. The loudness dependence of auditory evoked potential (LDAEP) is known to reflect central serotonergic activity. Single nucleotide polymorphisms (SNPs) in the 5-HT3A serotonin receptor gene are associated with psychiatric disorders. This study aimed to investigate the effect between 5-HT3A receptor gene polymorphisms and childhood sexual trauma on the LDAEP as an electrophysiological marker in healthy subjects. Methods A total of 206 healthy subjects were recruited and evaluated using the childhood trauma questionnaire (CTQ) and hospital anxiety and depression scale (HADS). Peak-to-peak N1/P2 was measured at five stimulus intensities, and the LDAEP was calculated as the linear-regression slope. In addition, the rs1062613 SNPs of 5-HT3A (CC, CT, and TT) were analyzed in healthy subjects. Results There was a significant interaction between scores on the CTQ-sexual abuse subscale and 5-HT3A genotype on the LDAEP. Subjects with the CC polymorphism had a significantly higher LDEAP than T carriers in the sexually abused group. In addition, CC genotype subjects in the sexually abused group showed a significantly higher LDAEP compared with CC genotype subjects in the non-sexually abused group. Conclusions Our findings suggest that people with the CC polymorphism of the 5-HT3A gene have a greater risk of developing mental health problems if they have experienced childhood sexual abuse, possibly due to low central serotonin activity. Conversely, the T polymorphism may be protective against any central serotonergic changes following childhood sexual trauma. PMID:26701104

  12. Kampo Medicine: Evaluation of the Pharmacological Activity of 121 Herbal Drugs on GABAA and 5-HT3A Receptors.

    PubMed

    Hoffmann, Katrin M; Herbrechter, Robin; Ziemba, Paul M; Lepke, Peter; Beltrán, Leopoldo; Hatt, Hanns; Werner, Markus; Gisselmann, Günter

    2016-01-01

    Kampo medicine is a form of Japanese phytotherapy originating from traditional Chinese medicine (TCM). During the last several decades, much attention has been paid to the pharmacological effects of these medical plants and their constituents. However, in many cases, a systematic screening of Kampo remedies to determine pharmacologically relevant targets is still lacking. In this study, a broad screening of Kampo remedies was performed to look for pharmacologically relevant 5-HT3A and GABAA receptor ligands. Several of the Kampo remedies are currently used for symptoms such as nausea, emesis, gastrointestinal motility disorders, anxiety, restlessness, or insomnia. Therefore, the pharmacological effects of 121 herbal drugs from Kampo medicine were analyzed as ethanol tinctures on heterologously expressed 5-HT3A and GABAA receptors, due to the involvement of these receptors in such pathophysiological processes. The tinctures of Lindera aggregata (radix) and Leonurus japonicus (herba) were the most effective inhibitory compounds on the 5-HT3A receptor. Further investigation of known ingredients in these compounds led to the identification of leonurine from Leonurus as a new natural 5-HT3A receptor antagonist. Several potentiating herbs (e.g., Magnolia officinalis (cortex), Syzygium aromaticum (flos), and Panax ginseng (radix)) were also identified for the GABAA receptor, which are all traditionally used for their sedative or anxiolytic effects. A variety of tinctures with antagonistic effects Salvia miltiorrhiza (radix) were also detected. Therefore, this study reveals new insights into the pharmacological action of a broad spectrum of herbal drugs from Kampo, allowing for a better understanding of their physiological effects and clinical applications. PMID:27524967

  13. Kampo Medicine: Evaluation of the Pharmacological Activity of 121 Herbal Drugs on GABAA and 5-HT3A Receptors

    PubMed Central

    Hoffmann, Katrin M.; Herbrechter, Robin; Ziemba, Paul M.; Lepke, Peter; Beltrán, Leopoldo; Hatt, Hanns; Werner, Markus; Gisselmann, Günter

    2016-01-01

    Kampo medicine is a form of Japanese phytotherapy originating from traditional Chinese medicine (TCM). During the last several decades, much attention has been paid to the pharmacological effects of these medical plants and their constituents. However, in many cases, a systematic screening of Kampo remedies to determine pharmacologically relevant targets is still lacking. In this study, a broad screening of Kampo remedies was performed to look for pharmacologically relevant 5-HT3A and GABAA receptor ligands. Several of the Kampo remedies are currently used for symptoms such as nausea, emesis, gastrointestinal motility disorders, anxiety, restlessness, or insomnia. Therefore, the pharmacological effects of 121 herbal drugs from Kampo medicine were analyzed as ethanol tinctures on heterologously expressed 5-HT3A and GABAA receptors, due to the involvement of these receptors in such pathophysiological processes. The tinctures of Lindera aggregata (radix) and Leonurus japonicus (herba) were the most effective inhibitory compounds on the 5-HT3A receptor. Further investigation of known ingredients in these compounds led to the identification of leonurine from Leonurus as a new natural 5-HT3A receptor antagonist. Several potentiating herbs (e.g., Magnolia officinalis (cortex), Syzygium aromaticum (flos), and Panax ginseng (radix)) were also identified for the GABAA receptor, which are all traditionally used for their sedative or anxiolytic effects. A variety of tinctures with antagonistic effects Salvia miltiorrhiza (radix) were also detected. Therefore, this study reveals new insights into the pharmacological action of a broad spectrum of herbal drugs from Kampo, allowing for a better understanding of their physiological effects and clinical applications. PMID:27524967

  14. Ion permeation and conduction in a human recombinant 5-HT3 receptor subunit (h5-HT3A)

    PubMed Central

    Brown, A M; Hope, A G; Lambert, J J; Peters, J A

    1998-01-01

    A human recombinant homo-oligomeric 5-HT3 receptor (h5-HT3A) expressed in a human embryonic kidney cell line (HEK 293) was characterized using the whole-cell recording configuration of the patch clamp technique. 5-HT evoked transient inward currents (EC50 = 3.4 μm; Hill coefficient = 1.8) that were blocked by the 5-HT3 receptor antagonist ondansetron (IC50 = 103 pm) and by the non-selective agents metoclopramide (IC50 = 69 nm), cocaine (IC50 = 459 nm) and (+)-tubocurarine (IC50 = 2.8 μm). 5-HT-induced currents rectified inwardly and reversed in sign (E5-HT) at a potential of −2.2 mV. N-Methyl-d-glucamine was finitely permeant. Permeability ratios PNa/PCs and PNMDG/PCs were 0.90 and 0.083, respectively. Permeability towards divalent cations was assessed from measurements of E5-HT in media where Ca2+ and Mg2+ replaced Na+. PCa/PCs and PMg/PCs were calculated to be 1.00 and 0.61, respectively. Single channel chord conductance (γ) estimated from fluctuation analysis of macroscopic currents increased with membrane hyperpolarization from 243 fS at −40 mV to 742 fS at −100 mV. Reducing [Ca2+]o from 2 to 0.1 mm caused an increase in the whole-cell current evoked by 5-HT. A concomitant reduction in [Mg2+]o produced further potentiation. Fluctuation analysis indicates that a voltage-independent augmentation of γ contributes to this phenomenon. The data indicate that homo-oligomeric receptors composed of h5-HT3A subunits form inwardly rectifying cation-selective ion channels of low conductance that are permeable to Ca2+ and Mg2+. PMID:9508827

  15. Length and Amino Acid Sequence of Peptides Substituted for the 5-HT3A Receptor M3M4 Loop May Affect Channel Expression and Desensitization

    PubMed Central

    McKinnon, Nicole K.; Bali, Moez; Akabas, Myles H.

    2012-01-01

    5-HT3A receptors are pentameric neurotransmitter-gated ion channels in the Cys-loop receptor family. Each subunit contains an extracellular domain, four transmembrane segments (M1, M2, M3, M4) and a 115 residue intracellular loop between M3 and M4. In contrast, the M3M4 loop in prokaryotic homologues is <15 residues. To investigate the limits of M3M4 loop length and composition on channel function we replaced the 5-HT3A M3M4 loop with two to seven alanine residues (5-HT3A-An = 2–7). Mutants were expressed in Xenopus laevis oocytes and characterized using two electrode voltage clamp recording. All mutants were functional. The 5-HT EC50's were at most 5-fold greater than wild-type (WT). The desensitization rate differed significantly among the mutants. Desensitization rates for 5-HT3A-A2, 5-HT3A-A4, 5-HT3A-A6, and 5-HT3A-A7 were similar to WT. In contrast, 5-HT3A-A3 and 5-HT3A-A5 had desensitization rates at least an order of magnitude faster than WT. The one Ala loop construct, 5-HT3A-A1, entered a non-functional state from which it did not recover after the first 5-HT application. These results suggest that the large M3M4 loop of eukaryotic Cys-loop channels is not required for receptor assembly or function. However, loop length and amino acid composition can effect channel expression and desensitization. We infer that the cytoplasmic ends of the M3 and M4 segments may undergo conformational changes during channel gating and desensitization and/or the loop may influence the position and mobility of these segments as they undergo gating-induced conformational changes. Altering structure or conformational mobility of the cytoplasmic ends of M3 and M4 may be the basis by which phosphorylation or protein binding to the cytoplasmic loop alters channel function. PMID:22539982

  16. The 4′lysine in the putative channel lining domain affects desensitization but not the single-channel conductance of recombinant homomeric 5-HT3A receptors

    PubMed Central

    Gunthorpe, Martin J; Peters, John A; Gill, Catherine H; Lambert, Jeremy J; Lummis, Sarah C R

    2000-01-01

    The 5-HT3 receptor is a transmitter-gated ion channel of the Cys-loop superfamily. Uniquely, 5-HT3 receptor subunits (5-HT3A and 5-HT3B) possess a positively charged lysine residue within the putative channel lining M2 domain (4′ position). Using whole cell recording techniques, we examined the role of this residue in receptor function using wild-type (WT) and mutant 5-HT3A receptor subunits of murine origin transiently expressed in human embryonic kidney (HEK 293) cells. WT 5-HT3A receptors mediated rapidly activating currents in response to 5-HT (10–90 % rise time, 103 ms; EC50, 2.34 μm; Hill coefficient, nH, 2.87). The currents rectified inwardly, reversed in sign at a potential of −9 mV and desensitized in the continuous presence of agonist (half-time of desensitization, t1/2, 2.13 s). 5-HT3A receptor subunits in which the 4′lysine was mutated to arginine, glutamine, serine or glycine formed functional receptors. 5-HT EC50 values were approximately 2-fold lower than for WT 5-HT3A receptors, but Hill coefficients, kinetics of current activation, rectification, and reversal potentials were unaltered. Each of the mutants desensitized more slowly than the WT 5-HT3A receptor, with the arginine and glycine mutations exhibiting the greatest effect (5-fold reduction). The rank order of effect was arginine > glycine > serine > glutamine. The single-channel conductance of the WT 5-HT3A receptor, as assessed by fluctuation analysis of macroscopic currents, was 390 fS. A similar value was obtained for the 4′lysine mutant receptors. Thus it appears unlikely that 4′lysine is exposed to the channel lumen. Mutation of residues immediately adjacent to 4′lysine to glutamate or lysine resulted in lack of receptor expression or function. We conclude that 4′lysine does not form part of the channel lining, but may play an important role in 5-HT3 receptor desensitization. PMID:10639097

  17. Identification of Glycyrrhiza as the rikkunshito constituent with the highest antagonistic potential on heterologously expressed 5-HT3A receptors due to the action of flavonoids

    PubMed Central

    Herbrechter, Robin; Ziemba, Paul M.; Hoffmann, Katrin M.; Hatt, Hanns; Werner, Markus; Gisselmann, Günter

    2015-01-01

    The traditional Japanese phytomedicine rikkunshito is traditionally used for the treatment of gastrointestinal motility disorders, cachexia and nausea. These effects indicate 5-HT3 receptor antagonism, due to the involvement of these receptors in such pathophysiological processes. E.g., setrons, specific 5-HT3 receptor antagonists are the strongest antiemetics, developed so far. Therefore, the antagonistic effects of the eight rikkunshito constituents at heterologously expressed 5-HT3Areceptors were analyzed using the two-electrode voltage-clamp technique. The results indicate that tinctures from Aurantii, Ginseng, Zingiberis, Atractylodis and Glycyrrhiza inhibited the 5-HT3A receptor response, whereas the tinctures of Poria cocos, Jujubae and Pinellia exhibited no effect. Surprisingly, the strongest antagonism was found for Glycyrrhiza, whereas the Zingiberis tincture, which is considered to be primarily responsible for the effect of rikkunshito, exhibited the weakest antagonism of 5-HT3A receptors. Rikkunshito contains various vanilloids, ginsenosides and flavonoids, a portion of which show an antagonistic effect on 5-HT3 receptors. A screening of the established ingredients of the active rikkunshito constituents and related substances lead to the identification of new antagonists within the class of flavonoids. The flavonoids (-)-liquiritigenin, glabridin and licochalcone A from Glycyrrhiza species were found to be the most effective inhibitors of the 5-HT-induced currents in the screening. The flavonoids (-)-liquiritigenin and hesperetin from Aurantii inhibited the receptor response in a non-competitive manner, whereas glabridin and licochalcone A exhibited a potential competitive antagonism. Furthermore, licochalcone A acts as a partial antagonist of 5-HT3A receptors. Thus, this study reveals new 5-HT3A receptor antagonists with the aid of increasing the comprehension of the complex effects of rikkunshito. PMID:26191003

  18. Regulation of the 5-HT3A receptor-mediated current by alkyl 4-hydroxybenzoates isolated from the seeds of Nelumbo nucifera.

    PubMed

    Youn, Ui Joung; Lee, Jun-Ho; Lee, Yoo Jin; Nam, Joo Won; Bae, Hyunsu; Seo, Eun-Kyoung

    2010-09-01

    Four known alkyl 4-hydroxybenzoates, i.e., methyl 4-hydroxybenzoate (1), ethyl 4-hydroxybenzoate (2), propyl 4-hydroxybenzoate (3), and butyl 4-hydroxybenzoate (4), were isolated from the seeds of Nelumbo nucifera Gaertner (Nymphaeaceae) for the first time. The structures of the isolates were identified by 1D- and 2D-NMR spectroscopy and comparison with published values. The compounds were evaluated for their effects on the 5-HT-stimulated inward current (I(5-HT)) mediated by the human 5-HT(3)A receptors expressed in Xenopus oocytes. Compounds 1 and 2 enhanced the I(5-HT), but 4 reduced it. These results indicate that 4 is an inhibitor of the 5-HT(3)A receptors expressed in Xenopus oocytes. PMID:20860031

  19. Conversion of the ion selectivity of the 5-HT(3a) receptor from cationic to anionic reveals a conserved feature of the ligand-gated ion channel superfamily.

    PubMed

    Gunthorpe, M J; Lummis, S C

    2001-06-15

    The 5-hydroxytryptamine(3) (5-HT(3)) receptor is a member of a superfamily of ligand-gated ion channels, which includes nicotinic acetylcholine, gamma-aminobutyric acid, and glycine receptors. The receptors are either cation or anion selective, leading to their distinctive involvement in either excitatory or inhibitory neurotransmission. Using a combination of site-directed mutagenesis and electrophysiological characterization of homomeric 5-HT(3A) receptors expressed in HEK293 cells, we have identified a set of mutations that convert the ion selectivity of the 5-HT(3A) receptor from cationic to anionic; these were substitution of V13'T in M2 together with neutralization of glutamate residues (E-1'A) and the adjacent insertion of a proline residue (P-1') in the M1-M2 loop. Mutant receptors showed significant chloride permeability (P(Cl)/P(Na) = 12.3, P(Na)/P(Cl) = 0.08), whereas WT receptors are predominantly permeable to sodium (P(Na)/P(Cl) > 20, P(Cl)/P(Na) < 0.05). Since the equivalent mutations have previously been shown to convert alpha7 nicotinic acetylcholine receptors from cationic to anionic (Galzi J.-L., Devillers-Thiery, A, Hussy, N., Bertrand, S. Changeux, J. P., and Bertrand, D. (1992) Nature 359, 500-505) and, recently, the converse mutations have allowed the construction of a cation selective glycine receptor (Keramidas, A., Moorhouse, A. J., French, C. R., Schofield, P. R., and Barry, P. H. (2000) Biophys. J. 78, 247-259), it appears that the determinants of ion selectivity represent a conserved feature of the ligand-gated ion channel superfamily. PMID:11439930

  20. Conversion of the ion selectivity of the 5-HT(3a) receptor from cationic to anionic reveals a conserved feature of the ligand-gated ion channel superfamily.

    PubMed

    Gunthorpe, M J; Lummis, S C

    2001-04-01

    The 5-hydroxytryptamine(3) (5-HT(3)) receptor is a member of a superfamily of ligand-gated ion channels, which includes nicotinic acetylcholine, gamma-aminobutyric acid, and glycine receptors. The receptors are either cation or anion selective, leading to their distinctive involvement in either excitatory or inhibitory neurotransmission. Using a combination of site-directed mutagenesis and electrophysiological characterization of homomeric 5-HT(3A) receptors expressed in HEK293 cells, we have identified a set of mutations that convert the ion selectivity of the 5-HT(3A) receptor from cationic to anionic; these were substitution of V13'T in M2 together with neutralization of glutamate residues (E-1'A) and the adjacent insertion of a proline residue (P-1') in the M1-M2 loop. Mutant receptors showed significant chloride permeability (P(Cl)/P(Na) = 12.3, P(Na)/P(Cl) = 0.08), whereas WT receptors are predominantly permeable to sodium (P(Na)/P(Cl) > 20, P(Cl)/P(Na) < 0.05). Since the equivalent mutations have previously been shown to convert alpha7 nicotinic acetylcholine receptors from cationic to anionic (Galzi J.-L., Devillers-Thiery, A, Hussy, N., Bertrand, S. Changeux, J. P., and Bertrand, D. (1992) Nature 359, 500-505) and, recently, the converse mutations have allowed the construction of a cation selective glycine receptor (Keramidas, A., Moorhouse, A. J., French, C. R., Schofield, P. R., and Barry, P. H. (2000) Biophys. J. 78, 247-259), it appears that the determinants of ion selectivity represent a conserved feature of the ligand-gated ion channel superfamily. PMID:11139582

  1. Serotonin homeostasis and serotonin receptors as actors of cortical construction: special attention to the 5-HT3A and 5-HT6 receptor subtypes

    PubMed Central

    Vitalis, Tania; Ansorge, Mark S.; Dayer, Alexandre G.

    2013-01-01

    Cortical circuits control higher-order cognitive processes and their function is highly dependent on their structure that emerges during development. The construction of cortical circuits involves the coordinated interplay between different types of cellular processes such as proliferation, migration, and differentiation of neural and glial cell subtypes. Among the multiple factors that regulate the assembly of cortical circuits, 5-HT is an important developmental signal that impacts on a broad diversity of cellular processes. 5-HT is detected at the onset of embryonic telencephalic formation and a variety of serotonergic receptors are dynamically expressed in the embryonic developing cortex in a region and cell-type specific manner. Among these receptors, the ionotropic 5-HT3A receptor and the metabotropic 5-HT6 receptor have recently been identified as novel serotonergic targets regulating different aspects of cortical construction including neuronal migration and dendritic differentiation. In this review, we focus on the developmental impact of serotonergic systems on the construction of cortical circuits and discuss their potential role in programming risk for human psychiatric disorders. PMID:23801939

  2. Pharmacological characterization of a rat 5-hydroxytryptamine type3 receptor subunit (r5-HT3A(b)) expressed in Xenopus laevis oocytes

    PubMed Central

    Mair, Ian D; Lambert, Jeremy J; Yang, Jay; Dempster, John; Peters, John A

    1998-01-01

    The present study has utilized the two electrode voltage-clamp technique to examine the pharmacological profile of a splice variant of the rat orthologue of the 5-hydroxytryptamine type 3A subunit (5-HT3A(b)) heterologously expressed in Xenopus laevis oocytes. At negative holding potentials, bath applied 5-HT (300 nM–10 μM) evoked a transient, concentration-dependent (EC50=1.1±0.1 μM), inward current. The response reversed in sign at a holding potential of −2.1±1.6 mV. The response to 5-HT was mimicked by the 5-HT3 receptor selective agonists 2-methyl-5-HT (EC50=4.1±0.2 μM), 1-phenylbiguanide (EC50=3.0±0.1 μM), 3-chlorophenylbiguanide (EC50=140± 10 nM), 3,5-dichlorophenylbiguanide (EC50=14.5±0.4 nM) and 2,5-dichlorophenylbiguanide (EC50= 10.2±0.6 nM). With the exception of 2-methyl-5-HT, all of the agonists tested elicited maximal current responses comparable to those produced by a saturating concentration (10 μM) of 5-HT. Responses evoked by 5-HT at EC50 were blocked by the 5-HT3 receptor selective antagonist ondansetron (IC50=231±22 pM) and by the less selective agents (+)-tubocurarine (IC50=31.9± 0.01 nM) and cocaine (IC50=2.1±0.2 μM). The data are discussed in the context of results previously obtained with the human and mouse orthologues of the 5-HT3A subunit. Overall, the study reinforces the conclusion that species differences detected for native 5-HT3 receptors extend to, and appear largely explained by, differences in the properties of homo-oligomeric receptors formed from 5-HT3A subunit orthologues. PMID:9756382

  3. Combinatorial Consensus Scoring for Ligand-Based Virtual Fragment Screening: A Comparative Case Study for Serotonin 5-HT(3)A, Histamine H(1), and Histamine H(4) Receptors.

    PubMed

    Schultes, Sabine; Kooistra, Albert J; Vischer, Henry F; Nijmeijer, Saskia; Haaksma, Eric E J; Leurs, Rob; de Esch, Iwan J P; de Graaf, Chris

    2015-05-26

    In the current study we have evaluated the applicability of ligand-based virtual screening (LBVS) methods for the identification of small fragment-like biologically active molecules using different similarity descriptors and different consensus scoring approaches. For this purpose, we have evaluated the performance of 14 chemical similarity descriptors in retrospective virtual screening studies to discriminate fragment-like ligands of three membrane-bound receptors from fragments that are experimentally determined to have no affinity for these proteins (true inactives). We used a complete fragment affinity data set of experimentally determined ligands and inactives for two G protein-coupled receptors (GPCRs), the histamine H1 receptor (H1R) and the histamine H4 receptor (H4R), and one ligand-gated ion channel (LGIC), the serotonin receptor (5-HT3AR), to validate our retrospective virtual screening studies. We have exhaustively tested consensus scoring strategies that combine the results of multiple actives (group fusion) or combine different similarity descriptors (similarity fusion), and for the first time systematically evaluated different combinations of group fusion and similarity fusion approaches. Our studies show that for these three case study protein targets both consensus scoring approaches can increase virtual screening enrichments compared to single chemical similarity search methods. Our cheminformatics analyses recommend to use a combination of both group fusion and similarity fusion for prospective ligand-based virtual fragment screening. PMID:25815783

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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 ...

  5. A Conditional Knockout Mouse Line of the Oxytocin Receptor

    PubMed Central

    Lee, Heon-Jin; Caldwell, Heather K.; Macbeth, Abbe H.; Tolu, Selen G.; Young, W. Scott

    2008-01-01

    Oxytocin plays important roles in reproductive physiology and various behaviors, including maternal behavior and social memory. Its receptor (Oxtr) is present in peripheral tissues and brain, so a conditional knockout (KO, −/−) would be useful to allow elimination of the receptor in specific sites at defined times. We created a line of mice in which loxP sites flank Oxtr coding sequence (floxed) enable Cre recombinase-mediated inactivation of the receptor. We expressed Cre recombinase in these mice either in all tissues (Oxtr−/−) or the forebrain (OxtrFB/FB) using the Ca2+/calmodulin-dependent protein kinase IIα promoter. The latter KO has reduced Oxtr binding beginning 21–28 d postnatally, leading to prominent reductions in the lateral septum, hippocampus, and ventral pallidum. The medial amygdala is spared, and there is significant retention of binding within the olfactory bulb and nucleus and neocortex. We did not observe any deficits in the general health, sensorimotor functions, anxiety-like behaviors, or sucrose intake in either Oxtr−/− or OxtrFB/FB mice. Females of both KO types deliver pups, but only the OxtrFB/FB mice are able to eject milk. Oxtr−/− males show impaired social memory for familiar females, whereas the OxtrFB/FB males appear to recognize their species but not individuals. Our results confirm the importance of oxytocin in social recognition and demonstrate that spatial and temporal inactivation of the Oxtr will enable finer understanding of the physiological, behavioral, and developmental roles of the receptor. PMID:18356275

  6. Social dominance in male vasopressin 1b receptor knockout mice.

    PubMed

    Caldwell, Heather K; Dike, Obianuju E; Stevenson, Erica L; Storck, Kathryn; Young, W Scott

    2010-07-01

    We have previously reported that mice with a targeted disruption of their vasopressin 1b receptor gene, Avpr1b, have mild impairments in social recognition and reduced aggression. The reductions in aggression are limited to social forms of aggression, i.e., maternal and inter-male aggression, while predatory aggression remains unaffected. To further clarify the role of the Avpr1b in the regulation of social behavior we first examined anxiety-like and depression-like behaviors in Avpr1b knockout (Avpr1b -/-) mice. We then went on to test the ability of Avpr1b -/- mice to form dominance hierarchies. No major differences were found between Avpr1b -/- and wildtype mice in anxiety-like behaviors, as measured using an elevated plus maze and an open field test, or depression-like behaviors, as measured using a forced swim test. In the social dominance study we found that Avpr1b -/- mice are able to form dominance hierarchies, though in early hierarchy formation dominant Avpr1b -/- mice display significantly more mounting behavior on Day 1 of testing compared to wildtype controls. Further, non-socially dominant Avpr1b -/- mice spend less time engaged in attack behavior than wildtype controls. These findings suggest that while Avpr1b -/- mice may be able to form dominance hierarchies they appear to employ alternate strategies. PMID:20298692

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

    SciTech Connect

    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.

  8. Bone Growth and Turnover in Progesterone Receptor Knockout Mice

    PubMed Central

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

    2008-01-01

    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 microcomputed tomography analyses of bone from homozygous female PR knockout (PRKO) mice at 6, 12, and 26 wk 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 were normal in PRKO mice. In contrast, total, cancellous, and cortical bone mass were increased in the humerus of 12-wk-old PRKO mice, whereas cortical and cancellous bone mass in the tibia was normal. At 26 wk of age, cancellous bone area in the proximal tibia metaphysis of PRKO mice was 153% greater than age matched wild-type mice. The improved cancellous bone balance in 6-month-old 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 is not essential for bone growth and turnover. However, at some skeletal sites, PR signaling attenuates the accumulation of cortical and cancellous bone mass during adolescence. PMID:18276762

  9. INDUCTION OF MAMMARY GLAND DEVELOPMENT IN ESTROGEN RECEPTOR-ALPHA KNOCKOUT MICE

    EPA Science Inventory

    Mammary glands from the estrogen receptor knockout ( ERKO) mouse do not undergo ductal morphogenesis or alveolar development. Disrupted Er signaling may result in reduced estrogen-responsive gene products in the mammary gland or reduced mammotropic hormones that contribute t...

  10. Effects of D1 receptor knockout on fear and reward learning.

    PubMed

    Abraham, Antony D; Neve, Kim A; Lattal, K Matthew

    2016-09-01

    Dopamine signaling is involved in a variety of neurobiological processes that contribute to learning and memory. D1-like dopamine receptors (including D1 and D5 receptors) are thought to be involved in memory and reward processes, but pharmacological approaches have been limited in their ability to distinguish between D1 and D5 receptors. Here, we examine the effects of a specific knockout of D1 receptors in associative learning tasks involving aversive (shock) or appetitive (cocaine) unconditioned stimuli. We find that D1 knockout mice show similar levels of cued and contextual fear conditioning to WT controls following conditioning protocols involving one, two, or four shocks. D1 knockout mice show increased generalization of fear conditioning and extinction across contexts, revealed as increased freezing to a novel context following conditioning and decreased freezing to an extinguished cue during a contextual renewal test. Further, D1 knockout mice show mild enhancements in extinction following an injection of SKF81297, a D1/D5 receptor agonist, suggesting a role for D5 receptors in extinction enhancements induced by nonspecific pharmacological agonists. Finally, although D1 knockout mice show decreased locomotion induced by cocaine, they are able to form a cocaine-induced conditioned place preference. We discuss these findings in terms of the role of dopamine D1 receptors in general learning and memory processes. PMID:27423521

  11. Comparison of nociceptive behavior in prostaglandin E, F, D, prostacyclin and thromboxane receptor knockout mice.

    PubMed

    Popp, Laura; Häussler, Annett; Olliges, Anke; Nüsing, Rolf; Narumiya, Shuh; Geisslinger, Gerd; Tegeder, Irmgard

    2009-08-01

    Antagonist at specific prostaglandin receptors might provide analgesia with a more favourable toxicity profile compared with cyclooxygenase inhibitors. We analyzed nociceptive responses in prostaglandin D, E, F, prostacyclin and thromboxane receptor knockout mice and mice deficient of cyclooxygenase 1 or 2 to evaluate the contribution of individual prostaglandin receptors for heat, mechanical and formalin-evoked pain. None of the knockouts was uniformly protected from all of these pain stimuli but COX-1 and EP4 receptor knockouts presented with reduced heat pain and EP3 receptor and COX-2 knockout mice had reduced licking responses in the 2nd phase of the formalin assay. This was accompanied with reduced c-Fos immunoreactivity in the spinal cord dorsal horn in EP3 knockouts. Oppositely, heat pain sensitivity was increased in FP, EP1 and EP1+3 double mutant mice possibly due to a loss of FP or EP1 receptor mediated central control of thermal pain sensitivity. Deficiency of either EP2 or DP1 was associated with increased formalin-evoked flinching responses and c-Fos IR in dorsal horn neurons suggesting facilitated spinal cord pain reflex circuity. Thromboxane and prostacyclin receptor knockout mice showed normal pain behavior in all tests. The results suggest a differential, pain-stimulus and site-specific contribution of specific PG-receptors for the processing of the nociceptive stimuli, a differential modulation of nociceptive responses by COX-1 and COX-2 derived prostaglandins and compensatory and/or developmental adaptations in mice lacking specific PG receptors. PMID:18938093

  12. Normal Maternal Behavior, But Increased Pup Mortality, in Conditional Oxytocin Receptor Knockout Females

    PubMed Central

    Macbeth, Abbe H.; Stepp, Jennifer E.; Lee, Heon-Jin; Young, W. Scott; Caldwell, Heather K.

    2011-01-01

    Oxytocin (Oxt) and the Oxt receptor (Oxtr) are implicated in the onset of maternal behavior in a variety of species. Recently, we developed two Oxtr knockout lines: a total body knockout (Oxtr−/−) and a conditional Oxtr knockout (OxtrFB/FB) in which the Oxtr is lacking only in regions of the forebrain, allowing knockout females to potentially nurse and care for their biological offspring. In the current study, we assessed maternal behavior of postpartum OxtrFB/FB females toward their own pups and maternal behavior of virgin Oxtr−/− females toward foster pups and compared knockouts of both lines to wildtype (Oxtr+/+) littermates. We found that both Oxtr−/− and OxtrFB/FB females appear to have largely normal maternal behaviors. However, with first litters, approximately 40% of the OxtrFB/FB knockout dams experienced high pup mortality, compared to fewer than 10% of the Oxtr+/+ dams. We then went on to test whether or not this phenotype occurred in subsequent litters or when the dams were exposed to an environmental disturbance. We found that regardless of the degree of external disturbance, OxtrFB/FB females lost more pups on their first and second litters compared to wildtype females. Possible reasons for higher pup mortality in OxtrFB/FB females are discussed. PMID:20939667

  13. Serotonin receptor 1A knockout: An animal model of anxiety-related disorder

    PubMed Central

    Ramboz, Sylvie; Oosting, Ronald; Amara, Djamel Aït; Kung, Hank F.; Blier, Pierre; Mendelsohn, Monica; Mann, J. John; Brunner, Dani; Hen, René

    1998-01-01

    To investigate the contribution of individual serotonin (5-hydroxytryptamine; 5-HT) receptors to mood control, we have used homologous recombination to generate mice lacking specific serotonergic receptor subtypes. In the present report, we demonstrate that mice without 5-HT1A receptors display decreased exploratory activity and increased fear of aversive environments (open or elevated spaces). 5-HT1A knockout mice also exhibited a decreased immobility in the forced swim test, an effect commonly associated with antidepressant treatment. Although 5-HT1A receptors are involved in controlling the activity of serotonergic neurons, 5-HT1A knockout mice had normal levels of 5-HT and 5-hydroxyindoleacetic acid, possibly because of an up-regulation of 5-HT1B autoreceptors. Heterozygote 5-HT1A mutants expressed approximately one-half of wild-type receptor density and displayed intermediate phenotypes in most behavioral tests. These results demonstrate that 5-HT1A receptors are involved in the modulation of exploratory and fear-related behaviors and suggest that reductions in 5-HT1A receptor density due to genetic defects or environmental stressors might result in heightened anxiety. PMID:9826725

  14. CB2 Cannabinoid Receptor Knockout in Mice Impairs Contextual Long-Term Memory and Enhances Spatial Working Memory

    PubMed Central

    Li, Yong; Kim, Jimok

    2016-01-01

    Neurocognitive effects of cannabinoids have been extensively studied with a focus on CB1 cannabinoid receptors because CB1 receptors have been considered the major cannabinoid receptor in the nervous system. However, recent discoveries of CB2 cannabinoid receptors in the brain demand accurate determination of whether and how CB2 receptors are involved in the cognitive effects of cannabinoids. CB2 cannabinoid receptors are primarily involved in immune functions, but also implicated in psychiatric disorders such as schizophrenia and depression. Here, we examined the effects of CB2 receptor knockout in mice on memory to determine the roles of CB2 receptors in modulating cognitive function. Behavioral assays revealed that hippocampus-dependent, long-term contextual fear memory was impaired whereas hippocampus-independent, cued fear memory was normal in CB2 receptor knockout mice. These mice also displayed enhanced spatial working memory when tested in a Y-maze. Motor activity and anxiety of CB2 receptor knockout mice were intact when assessed in an open field arena and an elevated zero maze. In contrast to the knockout of CB2 receptors, acute blockade of CB2 receptors by AM603 in C57BL/6J mice had no effect on memory, motor activity, or anxiety. Our results suggest that CB2 cannabinoid receptors play diverse roles in regulating memory depending on memory types and/or brain areas. PMID:26819779

  15. PROXIMAL GUT MUCOSAL EPITHELIAL HOMEOSTASIS IN AGED IL-1 TYPE I RECEPTOR KNOCKOUT MICE AFTER STARVATION

    PubMed Central

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

    2010-01-01

    Background 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. Materials and Methods 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. Results Aged IL-1R knockout mice were larger than aged-matched wild-type mice (p<0.05). Proximal gut mucosal height and mucosal cell number were not different between aged IL-1R knockout and wild-type groups. The apoptosis index in gut epithelial cells was higher in fed IL-1R knockout versus wild-type mice (p<0.05), while no significant difference in cell proliferation between both groups. Mucosal atrophy was induced in both aged IL-1R knockout and wild-type groups by starvation (p<0.05), however, aged IL-1R knockout mice experienced greater losses in proximal gut weight, mucosal length, and corresponding cell number than did wild-type mice at the 12-hour time point (p<0.05). The apoptosis index in gut epithelial cells significantly increased in both groups after starvation (p<0.05). Starvation decreased cell proliferation in IL-1R knockout mice (p<0.05), but not in wild-type mice. Conclusions The response in aged IL-1R knockout mice differs from wild-type mice in that starvation increases atrophy and is associated with decreased cell proliferation rather than increased apoptosis. PMID:20605606

  16. Cardiac-Specific Knockout of ETA Receptor Mitigates Paraquat-Induced Cardiac Contractile Dysfunction.

    PubMed

    Wang, Jiaxing; Lu, Songhe; Zheng, Qijun; Hu, Nan; Yu, Wenjun; Li, Na; Liu, Min; Gao, Beilei; Zhang, Guoyong; Zhang, Yingmei; Wang, Haichang

    2016-07-01

    Paraquat (1,1'-dim ethyl-4-4'-bipyridinium dichloride), a highly toxic quaternary ammonium herbicide widely used in agriculture, exerts potent toxic prooxidant effects resulting in multi-organ failure including the lung and heart although the underlying mechanism remains elusive. Recent evidence suggests possible involvement of endothelin system in paraquat-induced acute lung injury. This study was designed to examine the role of endothelin receptor A (ETA) in paraquat-induced cardiac contractile and mitochondrial injury. Wild-type (WT) and cardiac-specific ETA receptor knockout mice were challenged to paraquat (45 mg/kg, i.p.) for 48 h prior to the assessment of echocardiographic, cardiomyocyte contractile and intracellular Ca(2+) properties, as well as apoptosis and mitochondrial damage. Levels of the mitochondrial proteins for biogenesis and oxidative phosphorylation including UCP2, HSP90 and PGC1α were evaluated. Our results revealed that paraquat elicited cardiac enlargement, mechanical anomalies including compromised echocardiographic parameters (elevated left ventricular end-systolic and end-diastolic diameters as well as reduced factional shortening), suppressed cardiomyocyte contractile function, intracellular Ca(2+) handling, overt apoptosis and mitochondrial damage. ETA receptor knockout itself failed to affect myocardial function, apoptosis, mitochondrial integrity and mitochondrial protein expression. However, ETA receptor knockout ablated or significantly attenuated paraquat-induced cardiac contractile and intracellular Ca(2+) defect, apoptosis and mitochondrial damage. Taken together, these findings revealed that endothelin system in particular the ETA receptor may be involved in paraquat-induced toxic myocardial contractile anomalies possibly related to apoptosis and mitochondrial damage. PMID:26089164

  17. Prostanoids and inflammation: a new concept arising from receptor knockout mice.

    PubMed

    Narumiya, Shuh

    2009-10-01

    Prostanoids including various types of prostaglandins and thromboxanes are arachidonate metabolites produced and released in response to a variety of physiological and pathological stimuli and function to maintain the body homeostasis. Since cyclooxygenase, the enzyme initiating their biosynthesis, is inhibited by aspirin-like antipyretic, anti-inflammatory, and analgesic drugs, contribution of prostanoids to acute inflammation such as fever generation, pain sensitization, and inflammatory swelling has been recognized very early. On the other hand, since aspirin-like drugs generally show little effects on allergy and immunity, it has been believed that prostanoids play little roles in these processes. Prostanoids act on a family of G-protein-coupled receptors designated PGD receptor, PGE receptor subtypes EP1-EP4, PGF receptor, PGI receptor, and TX receptor to elicit their actions. Studies using mice deficient in each of these receptors have revealed that prostanoids indeed function in the above aspirin-sensitive processes. However, these studies have also revealed that prostanoids exert both pro-inflammatory and anti-inflammatory actions not only by acting as mediators of acute inflammation but also by regulating gene expression in mesenchymal and epithelial cells at inflammatory site. Such dual actions of prostanoids are frequently seen in immune and allergic reactions, where different type of prostanoids and their receptors often exert opposite actions in a single process. Thus, a new concept on the role of prostanoids in inflammation has arisen from studies using the receptor knockout mice. PMID:19609495

  18. Thyrotropin receptor knockout mice: studies on immunological tolerance to a major thyroid autoantigen.

    PubMed

    Pichurin, Pavel N; Pichurina, Oxana; Marians, Russell C; Chen, Chun-Rong; Davies, Terry F; Rapoport, Basil; McLachlan, Sandra M

    2004-03-01

    Graves' disease involves a breakdown in self-tolerance to the TSH receptor (TSHR). Central T cell tolerance is established by intrathymic deletion of immature T lymphocytes that bind with high affinity to peptides from autoantigens (like the TSHR) expressed ectopically in the thymus. In TSHR-knockout mice, tolerance cannot be induced to the TSHR, which should, therefore, be a foreign antigen for these animals. To test this hypothesis, TSHR-knockout mice and wild-type controls were vaccinated (three injections) with TSHR DNA or control DNA. TSHR antibodies, measured by ELISA, binding to TSHR-expressing eukaryotic cells, and TSH binding inhibition, developed in approximately 60% of TSHR-knockout mice, not significantly different from 80% in the wild-type mice. Antibody levels were also comparable in the two groups, and both strains recognized the same immunodominant linear antibody epitope at the amino terminus of the TSHR. Splenocyte responses to TSHR protein in culture, measured as interferon-gamma production, were similar in TSHR-knockout and wild-type mice. Moreover, T cells from both strains recognized the same two epitopes from a panel of 29 synthetic peptides encompassing the TSHR ectodomain and extracellular loops. This lack of difference in immune responses in TSHR-knockout and wild-type mice is unexpected and is contrary to observations in other induced animal models of autoimmunity. The importance of our finding is that the TSHR may not be similar to other model proteins used to define the concept of central immune tolerance. PMID:14630711

  19. Changes in the expression of neurotransmitter receptors in Parkin and DJ-1 knockout mice--A quantitative multireceptor study.

    PubMed

    Cremer, J N; Amunts, K; Schleicher, A; Palomero-Gallagher, N; Piel, M; Rösch, F; Zilles, K

    2015-12-17

    Parkinson's disease (PD) is a well-characterized neurological disorder with regard to its neuropathological and symptomatic appearance. At the genetic level, mutations of particular genes, e.g. Parkin and DJ-1, were found in human hereditary PD with early onset. Neurotransmitter receptors constitute decisive elements in neural signal transduction. Furthermore, since they are often altered in neurological and psychiatric diseases, receptors have been successful targets for pharmacological agents. However, the consequences of PD-associated gene mutations on the expression of transmitter receptors are largely unknown. Therefore, we studied the expression of 16 different receptor binding sites of the neurotransmitters glutamate, GABA, acetylcholine, adrenaline, serotonin, dopamine and adenosine by means of quantitative receptor autoradiography in Parkin and DJ-1 knockout mice. These knockout mice exhibit electrophysiological and behavioral deficits, but do not show the typical dopaminergic cell loss. We demonstrated differential changes of binding site densities in eleven brain regions. Most prominently, we found an up-regulation of GABA(B) and kainate receptor densities in numerous cortical areas of Parkin and DJ-1 knockout mice, as well as increased NMDA but decreased AMPA receptor densities in different brain regions of the Parkin knockout mice. The alterations of three different glutamate receptor types may indicate the potential relevance of the glutamatergic system in the pathogenesis of PD. Furthermore, the cholinergic M1, M2 and nicotinic receptors as well as the adrenergic α2 and the adenosine A(2A) receptors showed differentially increased densities in Parkin and DJ-1 knockout mice. Taken together, knockout of the PD-associated genes Parkin or DJ-1 results in differential changes of neurotransmitter receptor densities, highlighting a possible role of altered non-dopaminergic, and in particular of glutamatergic neurotransmission in PD pathogenesis. PMID

  20. Diacylglycerol Lipase α Knockout Mice Demonstrate Metabolic and Behavioral Phenotypes Similar to Those of Cannabinoid Receptor 1 Knockout Mice

    PubMed Central

    Powell, David R.; Gay, Jason P.; Wilganowski, Nathaniel; Doree, Deon; Savelieva, Katerina V.; Lanthorn, Thomas H.; Read, Robert; Vogel, Peter; Hansen, Gwenn M.; Brommage, Robert; Ding, Zhi-Ming; Desai, Urvi; Zambrowicz, Brian

    2015-01-01

    After creating >4,650 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 α or β (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. By contrast, neither Daglb nor Napepld KO mice were lean. Weanling Dagla KO mice ate less than WT mice and had body weight (BW) 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 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 BW and improve glycemic control in obese patients with type 2 diabetes, but may also induce undesirable neuropsychiatric side

  1. Diacylglycerol Lipase α Knockout Mice Demonstrate Metabolic and Behavioral Phenotypes Similar to Those of Cannabinoid Receptor 1 Knockout Mice.

    PubMed

    Powell, David R; Gay, Jason P; Wilganowski, Nathaniel; Doree, Deon; Savelieva, Katerina V; Lanthorn, Thomas H; Read, Robert; Vogel, Peter; Hansen, Gwenn M; Brommage, Robert; Ding, Zhi-Ming; Desai, Urvi; Zambrowicz, Brian

    2015-01-01

    After creating >4,650 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 α or β (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. By contrast, neither Daglb nor Napepld KO mice were lean. Weanling Dagla KO mice ate less than WT mice and had body weight (BW) 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 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 BW and improve glycemic control in obese patients with type 2 diabetes, but may also induce undesirable neuropsychiatric side

  2. Audiograms, gap detection thresholds, and frequency difference limens in cannabinoid receptor 1 knockout mice.

    PubMed

    Toal, Katrina L; Radziwon, Kelly E; Holfoth, David P; Xu-Friedman, Matthew A; Dent, Micheal L

    2016-02-01

    The cannabinoid receptor 1 (CB1R) is found at several stages in the auditory pathway, but its role in hearing is unknown. Hearing abilities were measured in CB1R knockout mice and compared to those of wild-type mice. Operant conditioning and the psychophysical Method of Constant Stimuli were used to measure audiograms, gap detection thresholds, and frequency difference limens in trained mice using the same methods and stimuli as in previous experiments. CB1R knockout mice showed deficits at frequencies above 8 kHz in their audiograms relative to wild-type mice. CB1R knockouts showed enhancements for detecting gaps in low-pass noisebursts relative to wild-type mice, but were similar for other noise conditions. Finally, the two groups of mice did not differ in their frequency discrimination abilities as measured by the frequency difference limens task. These experiments suggest that the CB1R is involved in auditory processing and lay the groundwork for future physiological experiments. PMID:26427583

  3. Compulsive behavior in the 5-HT2C receptor knockout mouse.

    PubMed

    Chou-Green, Jennifer M; Holscher, Todd D; Dallman, Mary F; Akana, Susan F

    2003-04-01

    The efficacy of serotonergic pharmacotherapy indicates that serotonin (5-HT) plays a role in the treatment, if not the etiology, of obsessive-compulsive disorder (OCD). While some clinical evidence implicates 5-HT(2C) receptors in this disorder, a definitive function has yet to be validated. We hypothesized that 5-HT(2C) receptor knockout (KO) mice may display compulsive-like behavior. This paper describes characterization of several distinct, highly organized behaviors in mice lacking functional 5-HT(2C) receptors, which supports a compulsive-like syndrome.Compulsive-like behavior was assessed in male 5-HT(2C) receptor KO and wildtype (WT) mice. Chewing of non-nutritive clay, chewing patterns on plastic-mesh screens, and the frequency of head dipping were measured. 5-HT(2C) receptor KO mice chewed more clay, produced a distinct pattern of "neat" chewing of plastic screens and exhibited reduced habituation of head dipping activity compared to WT mice. We conclude that the 5-HT(2C) receptor null mutant mouse provides a promising model of compulsive behavior and a means to further explore the role of 5-HT in OCD. PMID:12782219

  4. Knockout of insulin and IGF-1 receptors on vascular endothelial cells protects against retinal neovascularization

    PubMed Central

    Kondo, Tatsuya; Vicent, David; Suzuma, Kiyoshi; Yanagisawa, Masashi; King, George L.; Holzenberger, Martin; Kahn, C. Ronald

    2003-01-01

    Both insulin and IGF-1 have been implicated in control of retinal endothelial cell growth, neovascularization, and diabetic retinopathy. To precisely define the role of insulin and IGF-1 signaling in endothelium in these processes, we have used the oxygen-induced retinopathy model to study mice with a vascular endothelial cell–specific knockout of the insulin receptor (VENIRKO) or IGF-1 receptor (VENIFARKO). Following relative hypoxia, VENIRKO mice show a 57% decrease in retinal neovascularization as compared with controls. This is associated with a blunted rise in VEGF, eNOS, and endothelin-1. By contrast, VENIFARKO mice show only a 34% reduction in neovascularization and a very modest reduction in mediator generation. These data indicate that both insulin and IGF-1 signaling in endothelium play a role in retinal neovascularization through the expression of vascular mediators, with the effect of insulin being most important in this process. PMID:12813019

  5. GABAA-receptor modification in taurine transporter knockout mice causes striatal disinhibition

    PubMed Central

    Sergeeva, O A; Fleischer, W; Chepkova, A N; Warskulat, U; Häussinger, D; Siebler, M; Haas, H L

    2007-01-01

    The Striatum is involved in the regulation of movements and motor skills. We have shown previously, that the osmolyte and neuromodulator taurine plays a role in striatal plasticity. We demonstrate now that hereditary taurine deficiency in taurine-transporter knock-out (TAUT KO) mice results in disinhibition of striatal network activity, which can be corrected by taurine supplementation. Modification of GABAA but not glycine receptors (taurine is a ligand for both receptor types) underlies this disinhibition. Whole-cell recordings from acutely isolated as well as cultured striatal neurons revealed a decreased agonist sensitivity of the GABAA receptor in TAUT KO neurons in the absence of changes in the maximal GABA-evoked current amplitude. The striatal GABA level in TAUT KO mice was unchanged. The amplitude enhancement of spontaneous IPSCs by zolpidem was stronger in TAUT KO than in wild-type (WT) animals. Tonic inhibition was absent in striatal neurons under control conditions but was detected after incubation with the GABA-transaminase inhibitor vigabatrin: bicuculline induced a larger shift of baseline current in WT as compared to TAUT KO neurons. Lack of taurine leads to reduced sensitivity of synaptic and extrasynaptic GABAA receptors and consequently to disinhibition. These findings help in understanding neuropathologies accompanied by the loss of endogenous taurine, for instance in hepatic encephalopathy. PMID:17962336

  6. Distinct mixtures of muscarinic receptor subtypes mediate inhibition of noradrenaline release in different mouse peripheral tissues, as studied with receptor knockout mice

    PubMed Central

    Trendelenburg, Anne-Ulrike; Meyer, Angelika; Wess, Jürgen; Starke, Klaus

    2005-01-01

    The muscarinic heteroreceptors modulating noradrenaline release in atria, urinary bladder and vas deferens were previously studied in mice in which the M2 or the M4 muscarinic receptor genes had been disrupted. These experiments showed that these tissues possessed both M2 and non-M2 heteroreceptors. The analysis was now extended to mice in which either the M3, both the M2 and the M3, or both the M2 and the M4 genes had been disrupted (M3-knockout, M2/3-knockout and M2/4-knockout). Tissues were preincubated with 3H-noradrenaline and then stimulated electrically (20 pulses per 50 Hz). In wild-type atria, carbachol (0.01–100 μM) decreased the electrically evoked tritium overflow by maximally 60–78%. The maximum inhibition of carbachol was reduced to 57% in M3-knockout and to 23% in M2/4-knockout atria. Strikingly, the effect of carbachol was abolished in M2/3-knockout atria. In wild-type bladder, carbachol (0.01–100 μM) reduced the evoked tritium overflow by maximally 57–71%. This effect remained unchanged in the M3-knockout, but was abolished in the M2/4-knockout bladder. In wild-type vas deferens, carbachol (0.01–100 μM) reduced the evoked tritium overflow by maximally 34–48%. The maximum inhibition of carbachol was reduced to 40% in the M3-knockout and to 18% in the M2/4-knockout vas deferens. We conclude that the postganglionic sympathetic axons of mouse atria possess M2 and M3, those of the urinary bladder M2 and M4, and those of the vas deferens M2, M3 and M4 release-inhibiting muscarinic receptors. PMID:15965496

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

    SciTech Connect

    Baireddy, Praveena; Liu, Jing; Hinsdale, Myron; Pope, Carey

    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

  8. Effect of P2X7 receptor knockout on AQP-5 expression of type I alveolar epithelial cells.

    PubMed

    Ebeling, Georg; Bläsche, Robert; Hofmann, Falk; Augstein, Antje; Kasper, Michael; Barth, Kathrin

    2014-01-01

    P2X7 receptors, ATP-gated cation channels, are specifically expressed in alveolar epithelial cells. The pathophysiological function of this lung cell type, except a recently reported putative involvement in surfactant secretion, is unknown. In addition, P2X7 receptor-deficient mice show reduced inflammation and lung fibrosis after exposure with bleomycin. To elucidate the role of the P2X7 receptor in alveolar epithelial type I cells we characterized the pulmonary phenotype of P2X7 receptor knockout mice by using immunohistochemistry, western blot analysis and real-time RT PCR. No pathomorphological signs of fibrosis were found. Results revealed, however, a remarkable loss of aquaporin-5 protein and mRNA in young knockout animals. Additional in vitro experiments with bleomycin treated precision cut lung slices showed a greater sensitivity of the P2X7 receptor knockout mice in terms of aquaporin-5 reduction as wild type animals. Finally, P2X7 receptor function was examined by using the alveolar epithelial cell lines E10 and MLE-12 for stimulation experiments with bleomycin. The in vitro activation of P2X7 receptor was connected with an increase of aquaporin-5, whereas the inhibition of the receptor with oxidized ATP resulted in down regulation of aquaporin-5. The early loss of aquaporin-5 which can be found in different pulmonary fibrosis models does not implicate a specific pathogenetic role during fibrogenesis. PMID:24941004

  9. Effect of P2X7 Receptor Knockout on AQP-5 Expression of Type I Alveolar Epithelial Cells

    PubMed Central

    Ebeling, Georg; Bläsche, Robert; Hofmann, Falk; Augstein, Antje; Kasper, Michael; Barth, Kathrin

    2014-01-01

    P2X7 receptors, ATP-gated cation channels, are specifically expressed in alveolar epithelial cells. The pathophysiological function of this lung cell type, except a recently reported putative involvement in surfactant secretion, is unknown. In addition, P2X7 receptor-deficient mice show reduced inflammation and lung fibrosis after exposure with bleomycin. To elucidate the role of the P2X7 receptor in alveolar epithelial type I cells we characterized the pulmonary phenotype of P2X7 receptor knockout mice by using immunohistochemistry, western blot analysis and real-time RT PCR. No pathomorphological signs of fibrosis were found. Results revealed, however, a remarkable loss of aquaporin-5 protein and mRNA in young knockout animals. Additional in vitro experiments with bleomycin treated precision cut lung slices showed a greater sensitivity of the P2X7 receptor knockout mice in terms of aquaporin-5 reduction as wild type animals. Finally, P2X7 receptor function was examined by using the alveolar epithelial cell lines E10 and MLE-12 for stimulation experiments with bleomycin. The in vitro activation of P2X7 receptor was connected with an increase of aquaporin-5, whereas the inhibition of the receptor with oxidized ATP resulted in down regulation of aquaporin-5. The early loss of aquaporin-5 which can be found in different pulmonary fibrosis models does not implicate a specific pathogenetic role during fibrogenesis. PMID:24941004

  10. Food intake, tumor growth, and weight loss in EP2 receptor subtype knockout mice bearing PGE2-producing tumors

    PubMed Central

    Iresjö, Britt-Marie; Wang, Wenhua; Nilsberth, Camilla; Andersson, Marianne; Lönnroth, Christina; Smedh, Ulrika

    2015-01-01

    Previous studies in our laboratory have demonstrated that prostaglandin (PG) E2 is involved in anorexia/cachexia development in MCG 101 tumor-bearing mice. In the present study, we investigate the role of PGE receptor subtype EP2 in the development of anorexia after MCG 101 implantation in wild-type (EP2+/+) or EP2-receptor knockout (EP2−/−) mice. Our results showed that host absence of EP2 receptors attenuated tumor growth and development of anorexia in tumor-bearing EP2 knockout mice compared to tumor-bearing wild-type animals. Microarray profiling of the hypothalamus revealed a relative twofold change in expression of around 35 genes including mRNA transcripts coding for Phospholipase A2 and Prostaglandin D2 synthase (Ptgds) in EP2 receptor knockout mice compared to wild-type mice. Prostaglandin D2 synthase levels were increased significantly in EP2 receptor knockouts, suggesting that improved food intake may depend on altered balance of prostaglandin production in hypothalamus since PGE2 and PGD2 display opposing effects in feeding control. PMID:26197930

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

    PubMed

    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. PMID:27109771

  12. Sex-dependence of anxiety-like behavior in cannabinoid receptor 1 (Cnr1) knockout mice.

    PubMed

    Bowers, Mallory E; Ressler, Kerry J

    2016-03-01

    Epidemiological data suggest women are at increased risk for developing anxiety and depression, although the mechanisms for this sex/gender difference remain incompletely understood. Pre-clinical studies have begun to investigate sex-dependent emotional learning and behavior in rodents, particularly as it relates to psychopathology; however, information about how gonadal hormones interact with the central nervous system is limited. We observe greater anxiety-like behavior in male mice with global knockout of the cannabinoid 1 receptor (Cnr1) compared to male, wild-type controls as measured by percent open arm entries on an elevated plus maze test. A similar increase in anxiety-like behavior, however, is not observed when comparing female Cnr1 knockouts to female wild-type subjects. Although, ovariectomy in female mice did not reverse this effect, both male and female adult mice with normative development were sensitive to Cnr1 antagonist-mediated increases in anxiety-like behavior. Together, these data support an interaction between sex, potentially mediated by gonadal hormones, and the endocannabinoid system at an early stage of development that is critical for establishing adult anxiety-like behavior. PMID:26684509

  13. Immune malfunction in the GPR39 zinc receptor of knockout mice: Its relationship to depressive disorder.

    PubMed

    Młyniec, Katarzyna; Trojan, Ewa; Ślusarczyk, Joanna; Głombik, Katarzyna; Basta-Kaim, Agnieszka; Budziszewska, Bogusława; Skrzeszewski, Jakub; Siwek, Agata; Holst, Birgitte; Nowak, Gabriel

    2016-02-15

    Depression is a serious psychiatric disorder affecting not only the monaminergic, glutamatergic, and GABAergic neurosystems, but also the immune system. Patients suffering from depression show disturbance in the immune parameters as well as increased susceptibility to infections. Zinc is well known 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 investigated whether GPR39 knockout would cause depressive-like behavior as measured by the forced swim test, and whether these changes would coexist with immune malfunction. In GPR39 knockout mice versus a wild-type control we found: i) depressive-like behavior; ii) significantly reduced thymus weight; (iii) reduced cell viability of splenocytes; iv) reduced proliferative response of splenocytes; and v) increased IL-6 production of splenocytes after ConA stimulation and decreased IL-1b and IL-6 release after LPS stimulation. The results indicate depressive-like behavior in GPR39 KO animals with an immune response similar to that observed in depressive disorder. Here for the first time we show immunological changes under GPR39-deficient conditions. PMID:26857489

  14. Myeloid Deletion of α1AMPK Exacerbates Atherosclerosis in LDL Receptor Knockout (LDLRKO) Mice.

    PubMed

    Cao, Qiang; Cui, Xin; Wu, Rui; Zha, Lin; Wang, Xianfeng; Parks, John S; Yu, Liqing; Shi, Hang; Xue, Bingzhong

    2016-06-01

    Macrophage inflammation marks all stages of atherogenesis, and AMPK is a regulator of macrophage inflammation. We therefore generated myeloid α1AMPK knockout (MAKO) mice on the LDL receptor knockout (LDLRKO) background to investigate whether myeloid deletion of α1AMPK exacerbates atherosclerosis. When fed an atherogenic diet, MAKO/LDLRKO mice displayed exacerbated atherosclerosis compared with LDLRKO mice. To determine the underlying pathophysiological pathways, we characterized macrophage inflammation/chemotaxis and lipid/cholesterol metabolism in MAKO/LDLRKO mice. Myeloid deletion of α1AMPK increased macrophage inflammatory gene expression and enhanced macrophage migration and adhesion to endothelial cells. Remarkably, MAKO/LDLRKO mice also displayed higher composition of circulating chemotaxically active Ly-6C(high) monocytes, enhanced atherosclerotic plaque chemokine expression, and monocyte recruitment into plaques, leading to increased atherosclerotic plaque macrophage content and inflammation. MAKO/LDLRKO mice also exhibited higher plasma LDL and VLDL cholesterol content, increased circulating apolipoprotein B (apoB) levels, and higher liver apoB expression. We conclude that macrophage α1AMPK deficiency promotes atherogenesis in LDLRKO mice and is associated with enhanced macrophage inflammation and hypercholesterolemia and that macrophage α1AMPK may serve as a therapeutic target for prevention and treatment of atherosclerosis. PMID:26822081

  15. Enhanced self-administration of alcohol in muscarinic acetylcholine M4 receptor knockout mice.

    PubMed

    de la Cour, Cecilie; Sørensen, Gunnar; Wortwein, Gitta; Weikop, Pia; Dencker, Ditte; Fink-Jensen, Anders; Molander, Anna

    2015-01-01

    Modulation of cholinergic neurotransmission via nicotinic acetylcholine receptors is known to alter alcohol-drinking behavior. It is not known if muscarinic acetylcholine receptor subtypes have similar effects. The muscarinic M4 receptor is highly expressed in the brain reinforcement system and involved in regulation of cholinergic and dopaminergic transmission. Here we investigate, for the first time, the role of the M4 receptor in alcohol consumption using M4 knockout (M4(-/-)) and wild-type (M4(+/+)) mice. Experimentally naïve M4(-/-) and M4(+/+) mice were trained to orally self-administer 5%, 8% 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 M4(+/+) littermates. The highest alcohol concentration used (10%) did not immediately result in divergent drinking patterns, but after 4 weeks of 10% alcohol self-administration, baseline levels as well as a pattern of M4(-/-) mice consuming more alcohol than their M4(+/+) controls were re-established. Moreover, the M4(-/-) mice displayed a reduced capacity to extinguish their alcohol-seeking behavior. Taken together, alcohol consumption is elevated in M4(-/-) mice, indicating that the M4 receptor is involved in mediating the reinforcing effects of alcohol. The M4 receptor should be further explored as a potential target for pharmacological (positive allosteric modulators or future agonists) treatment of alcohol use disorders. PMID:25445043

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

    PubMed Central

    Cacciola, Giovanna; Chioccarelli, Teresa; Fasano, Silvia; Pierantoni, Riccardo; Cobellis, Gilda

    2013-01-01

    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. PMID:24324492

  17. Thyrotoropin receptor knockout changes monoaminergic neuronal system and produces methylphenidate-sensitive emotional and cognitive dysfunction.

    PubMed

    Mouri, Akihiro; Hoshino, Yuta; Narusawa, Shiho; Ikegami, Keisuke; Mizoguchi, Hiroyuki; Murata, Yoshiharu; Yoshimura, Takashi; Nabeshima, Toshitaka

    2014-10-01

    Attention deficit/hyperactivity disorder (ADHD) has been reported in association with resistance to thyroid hormone, a disease caused by a mutation in the thyroid hormone receptor β (TRβ) gene. TRβ is a key protein mediating down-regulation of thyrotropin (TSH) expression by 3,3',5-tri-iodothyronine (T3), an active form of thyroid hormone. Dysregulation of TSH and its receptor (TSHR) is implicated in the pathophysiology of ADHD but the role of TSHR remains elusive. Here, we clarified a novel role for TSHR in emotional and cognitive functions related to monoaminergic nervous systems. TSHR knockout mice showed phenotypes of ADHD such as hyperactivity, impulsiveness, a decrease in sociality and increase in aggression, and an impairment of short-term memory and object recognition memory. Administration of methylphenidate (1, 5 and 10mg/kg) reversed impulsiveness, aggression and object recognition memory impairment. In the knockout mice, monoaminergic changes including decrease in the ratio of 3-methoxy-4-hydroxyphenylglycol/noradrenaline and increase in the ratio of homovanillic acid/dopamine were observed in some brain regions, accompanied by increase in the expression of noradrenaline transporter in the frontal cortex. When TSH was completely suppressed by the supraphysiological administration of T3 to the adult mice, some behavioral and neurological changes in TSHR KO mice were also observed, suggesting that these changes were not due to developmental hypothyroidism induced by the inactivation of TSHR but to the loss of the TSH-TSHR pathway itself. Taken together, the present findings suggest a novel role for TSHR in behavioral and neurological phenotypes of ADHD. PMID:25016105

  18. Upregulation of hepatic LDL transport by n-3 fatty acids in LDL receptor knockout mice.

    PubMed

    Vasandani, Chandna; Kafrouni, Abdallah I; Caronna, Antonella; Bashmakov, Yuriy; Gotthardt, Michael; Horton, Jay D; Spady, David K

    2002-05-01

    We determined the effects of dietary n-6 and n-3 polyunsaturated fatty acids (PUFA) on parameters of plasma lipoprotein and hepatic lipid metabolism in LDL receptor (LDLr) knockout mice. Dietary n-3 PUFA decreased the rate of appearance and increased the hepatic clearance of IDL/LDL resulting in a marked decrease in the plasma concentration of these particles. Dietary n-3 PUFA increased the hepatic clearance of IDL/LDL through a mechanism that appears to involve apolipoprotein (apo)E but is independent of the LDLr, the LDLr related protein (LRP), the scavenger receptor B1, and the VLDLr. The decreased rate of appearance of IDL/VLDL in the plasma of animals fed n-3 PUFA could be attributed to a marked decrease in the plasma concentration of precursor VLDL. Decreased plasma VLDL concentrations were due in part to decreased hepatic secretion of VLDL triglyceride and cholesteryl esters, which in turn was associated with decreased concentrations of these lipids in liver. Decreased hepatic triglyceride concentrations in animals fed n-3 PUFA were due in part to suppression of fatty acid synthesis as a result of a decrease in sterol regulatory element binding protein-1 (SREBP-1) expression and processing. In conclusion, these studies indicate that n-3 PUFA can markedly decrease the plasma concentration of apoB-containing lipoproteins and enhance hepatic LDL clearance through a mechanism that does not involve the LDLr pathway or LRP. PMID:11971949

  19. Acceleration of atherogenesis by COX-1-dependent prostanoid formation in low density lipoprotein receptor knockout mice.

    PubMed

    Praticò, D; Tillmann, C; Zhang, Z B; Li, H; FitzGerald, G A

    2001-03-13

    The cyclooxygenase (COX) product, prostacyclin (PGI(2)), inhibits platelet activation and vascular smooth-muscle cell migration and proliferation. Biochemically selective inhibition of COX-2 reduces PGI(2) biosynthesis substantially in humans. Because deletion of the PGI(2) receptor accelerates atherogenesis in the fat-fed low density lipoprotein receptor knockout mouse, we wished to determine whether selective inhibition of COX-2 would accelerate atherogenesis in this model. To address this hypothesis, we used dosing with nimesulide, which inhibited COX-2 ex vivo, depressed urinary 2,3 dinor 6-keto PGF(1alpha) by approximately 60% but had no effect on thromboxane formation by platelets, which only express COX-1. By contrast, the isoform nonspecific inhibitor, indomethacin, suppressed platelet function and thromboxane formation ex vivo and in vivo, coincident with effects on PGI(2) biosynthesis indistinguishable from nimesulide. Indomethacin reduced the extent of atherosclerosis by 55 +/- 4%, whereas nimesulide failed to increase the rate of atherogenesis. Despite their divergent effects on atherogenesis, both drugs depressed two indices of systemic inflammation, soluble intracellular adhesion molecule-1, and monocyte chemoattractant protein-1 to a similar but incomplete degree. Neither drug altered serum lipids and the marked increase in vascular expression of COX-2 during atherogenesis. Accelerated progression of atherosclerosis is unlikely during chronic intake of specific COX-2 inhibitors. Furthermore, evidence that COX-1-derived prostanoids contribute to atherogenesis suggests that controlled evaluation of the effects of nonsteroidal anti-inflammatory drugs and/or aspirin on plaque progression in humans is timely. PMID:11248083

  20. Hypersomnolence and reduced activity in pan-leptin receptor knockout mice

    PubMed Central

    Wang, Yuping; He, Junyun; Kastin, Abba J.; Hsuchou, Hung; Pan, Weihong

    2013-01-01

    Excessive obesity correlates with hypersomnolence and impaired cognitive function, presumably induced by metabolic factors and cytokines. Production of the adipokine leptin correlates with the amount of adiposity, and leptin has been shown to promote sleep. To determine whether leptin plays a major role in the hypersomnolence of obesity, we measured sleep architecture in pan-leptin receptor knockout (POKO) mice that do not respond to leptin because of the production of a mutant, non-signaling receptor. The obese POKO mice had more non-rapid eye movement (NREM) sleep and less waking time than their littermate controls. This was mainly seen during the light span, although increased bouts of rapid eye movement (REM) sleep were also seen in the dark span. The increase of NREM sleep correlated with the extent of obesity. The POKO mice also had decreased locomotor activity and more immobility in the open field test, but there was no increase of forced immobility nor reduction of sucrose intake as would be seen in depression. The increased NREM sleep and reduced locomotor activity in the POKO mice suggest that it was obesity, rather than leptin signaling, that played a predominant role in altering sleep architecture and activity. PMID:23955775

  1. Orthotopic transplantation of LH receptor knockout and wild-type ovaries.

    PubMed

    Chudgar, Daksha; Lei, Zhenmin; Rao, Ch V

    2005-10-01

    Luteinizing hormone (LH) receptor knockout animals have an ovarian failure due to an arrest in folliculogenesis at the antral stage. As a result, the animals have an infertility phenotype. The present study was undertaken to determine whether this phenotype could be reversed by orthotopic transplantation of wild-type ovaries. The results revealed that transplanting wild-type ovaries into null animals did not result in resumption of estrus cycles. Although the number of different types of follicles increased, none progressed to ovulation. The serum hormone profiles improved, reflecting the ovarian changes. The wild-type animals with null ovaries also failed to cycle and their ovaries and serum hormone levels were more like null animals with their own ovaries. Although the lack of rescue of null ovaries placed into wild-type animals was predicted, the failure of wild-type ovaries placed in null animals was not, which could be due to chronic exposure of transplanted tissue to high circulating LH levels and also possibly due to altered internal milieu in null animals. These findings may have implications for potential future considerations of grafting normal donor ovaries into women who have an ovarian failure resulting from inactivating LH receptor mutations. PMID:15964032

  2. Memantine, an NMDA receptor antagonist, improves working memory deficits in DGKβ knockout mice.

    PubMed

    Kakefuda, Kenichi; Ishisaka, Mitsue; Tsuruma, Kazuhiro; Shimazawa, Masamitsu; Hara, Hideaki

    2016-09-01

    Diacylglycerol kinase (DGK) β is a type 1 isozyme of the DGK family. We previously reported that DGKβ was deeply involved in neurite spine formation, and DGKβ knockout (KO) mice exhibited behavioral abnormalities concerning spine formation, such as cognitive, emotional, and attentional impairment. Moreover, some of these abnormalities were ameliorated by the administration of a mood stabilizer. However, there is no data about how memory-improving drugs used in the treatment of Alzheimer's disease affect DGKβ KO mice. In the present study, we evaluated the effect of an anti-Alzheimer's drug, memantine on the working memory deficit observed in DGKβ KO mice. In the Y-maze test, the administration of memantine significantly improved working memory of DGKβ KO mice. We also found that the expression levels of the NR2A and NR2B N-methyl-d-aspartate (NMDA) receptor subunits were increased in the prefrontal cortex, but decreased in the hippocampus of DGKβ KO mice. These altered expression levels of NR2 subunits might be related to the effect of an NMDA receptor antagonist, memantine. Taken together, these findings may support the hypothesis that DGKβ has a pivotal role in cognitive function. PMID:27495014

  3. Suppression of diet-induced atherosclerosis in low density lipoprotein receptor knockout mice overexpressing lipoprotein lipase.

    PubMed Central

    Shimada, M; Ishibashi, S; Inaba, T; Yagyu, H; Harada, K; Osuga, J I; Ohashi, K; Yazaki, Y; Yamada, N

    1996-01-01

    Lipoprotein lipase (LPL) is a key enzyme in the hydrolysis of triglyceride-rich lipoproteins. Conflicting results have been reported concerning its role in atherogenesis. To determine the effects of the overexpressed LPL on diet-induced atherosclerosis, we have generated low density lipoprotein receptor (LDLR) knockout mice that overexpressed human LPL transgene (LPL/LDLRKO) and compared their plasma lipoproteins and atherosclerosis with those in nonexpressing LDLR-knockout mice (LDLRKO). On a normal chow diet, LPL/LDLRKO mice showed marked suppression of mean plasma triglyceride levels (32 versus 236 mg/dl) and modest decrease in mean cholesterol levels (300 versus 386 mg/dl) as compared with LDLRKO mice. Larger lipoprotein particles of intermediate density lipoprotein (IDL)/LDL were selectively reduced in LPL/LDLRKO mice. On an atherogenic diet, both mice exhibited severe hypercholesterolemia. But, mean plasma cholesterol levels in LPL/ LDLRKO mice were still suppressed as compared with that in LDLRKO mice (1357 versus 2187 mg/dl). Marked reduction in a larger subfraction of IDL/LDL, which conceivably corresponds to remnant lipoproteins, was observed in the LPL/LDLRKO mice. LDLRKO mice developed severe fatty streak lesions in the aortic sinus after feeding with the atherogenic diet for 8 weeks. In contrast, mean lesion area in the LPL/LDLRKO mice was 18-fold smaller than that in LDLRKO mice. We suggest that the altered lipoprotein profile, in particular the reduced level of remnant lipoproteins, is mainly responsible for the protection by LPL against atherosclerosis. Images Fig. 1 Fig. 3 PMID:8692976

  4. Hematopoietic G-protein-coupled receptor kinase 2 deficiency decreases atherosclerotic lesion formation in LDL receptor-knockout mice

    PubMed Central

    Otten, Jeroen J. T.; de Jager, Saskia C. A.; Kavelaars, Annemieke; Seijkens, Tom; Bot, Ilze; Wijnands, Erwin; Beckers, Linda; Westra, Marijke M.; Bot, Martine; Busch, Matthias; Bermudez, Beatriz; van Berkel, Theo J. C.; Heijnen, Cobi J.; Biessen, Erik A. L.

    2013-01-01

    Leukocyte chemotaxis is deemed instrumental in initiation and progression of atherosclerosis. It is mediated by G-protein-coupled receptors (e.g., CCR2 and CCR5), the activity of which is controlled by G-protein-coupled receptor kinases (GRKs). In this study, we analyzed the effect of hematopoietic deficiency of a potent regulator kinase of chemotaxis (GRK2) on atherogenesis. LDL receptor-deficient (LDLr−/−) mice with heterozygous hematopoietic GRK2 deficiency, generated by bone marrow transplantation (n=15), displayed a dramatic attenuation of plaque development, with 79% reduction in necrotic core and increased macrophage content. Circulating monocytes decreased and granulocytes increased in GRK2+/− chimeras, which could be attributed to diminished granulocyte colony-forming units in bone marrow. Collectively, these data pointed to myeloid cells as major mediators of the impaired atherogenic response in GRK2+/− chimeras. LDLr−/− mice with macrophage/granulocyte-specific GRK2 deficiency (LysM-Cre GRK2flox/flox; n=8) failed to mimic the aforementioned phenotype, acquitting these cells as major responsible subsets for GRK2 deficiency-associated atheroprotection. To conclude, even partial hematopoietic GRK2 deficiency prevents atherosclerotic lesion progression beyond the fatty streak stage, identifying hematopoietic GRK2 as a potential target for intervention in atherosclerosis.—Otten, J. J. T., de Jager, S. C. A., Kavelaars, A., Seijkens, T., Bot, I., Wijnands, E., Beckers, L., Westra, M. M., Bot, M., Busch, M., Bermudez, B., van Berkel, T. J. C., Heijnen, C. J., Biessen, E. A. L. Hematopoietic G-protein-coupled receptor kinase 2 deficiency decreases atherosclerotic lesion formation in LDL receptor-knockout mice. PMID:23047899

  5. The in vivo respiratory phenotype of the adenosine A1 receptor knockout mouse.

    PubMed

    Heitzmann, Dirk; Buehler, Philipp; Schweda, Frank; Georgieff, Michael; Warth, Richard; Thomas, Joerg

    2016-02-01

    The nucleoside adenosine has been implicated in the regulation of respiration, especially during hypoxia in the newborn. In this study the role of adenosine A1 receptors for the control of respiration was investigated in vivo. To this end, respiration of unrestrained adult and neonatal adenosine A1 receptor knockout mice (A1R(-/-)) was measured in a plethysmographic device. Under control conditions (21% O2) and mild hypoxia (12-15% O2) no difference of respiratory parameters was observed between adult wildtype (A1R(+/+)) and A1R(-/-) mice. Under more severe hypoxia (6-10% O2) A1R(+/+) mice showed, after a transient increase of respiration, a decrease of respiration frequency (fR) and tidal volume (VT) leading to a decrease of minute volume (MV). This depression of respiration during severe hypoxia was absent in A1R(-/-) mice which displayed a stimulated respiration as indicated by the enhancement of MV by some 50-60%. During hypercapnia-hyperoxia (3-10% CO2/97-90 % O2), no obvious differences in respiration of A1R(-/-) and A1R(+/+) was observed. In neonatal mice, the respiratory response to hypoxia was surprisingly similar in both genotypes. However, neonatal A1R(-/-) mice appeared to have more frequently periods of apnea during hypoxia and in the post-hypoxic control period. In conclusion, these data indicate that the adenosine A1 receptor is an important molecular component mediating hypoxic depression in adult mice and it appears to stabilize respiration of neonatal mice. PMID:26593641

  6. Delayed procedural learning in α7-nicotinic acetylcholine receptor knockout mice

    PubMed Central

    Young, J. W.; Meves, J. M.; Tarantino, I. S.; Caldwell, S.; Geyer, M. A.

    2014-01-01

    The α7-nicotinic acetylcholine receptor (nAChR) has long been a procognitive therapeutic target to treat schizophrenia. Evidence on the role of this receptor in cognition has been lacking, however, in part due to the limited availability of suitable ligands. The behavior of α7-nAChR knockout (KO) mice has been examined previously, but cognitive assessments using tests with cross-species translatability have been limited to date. Here, we assessed the cognitive performance of α7-nAChR KO and wild-type (WT) littermate mice in the attentional set-shifting task of executive functioning, the radial arm maze test of spatial working memory span capacity and the novel object recognition test of short-term memory. The reward motivation of these mutants was assessed using the progressive ratio breakpoint test. In addition, we assessed the exploratory behavior and sensorimotor gating using the behavioral pattern monitor and prepulse inhibition, respectively. α7-nAChR KO mice exhibited normal set-shifting, but impaired procedural learning (rule acquisition) in multiple paradigms. Spatial span capacity, short-term memory, motivation for food, exploration and sensorimotor gating were all comparable to WT littermates. The data presented here support the notion that this receptor is important for such procedural learning, when patterns in the environment become clear and a rule is learned. In combination with the impaired attention observed previously in these mice, this finding suggests that agonist treatments should be examined in clinical studies of attention and procedural learning, perhaps in combination with cognitive behavioral therapy. PMID:21679297

  7. Tissue- and cell-specific functions of the androgen receptor revealed through conditional knockout models in mice.

    PubMed

    De Gendt, Karel; Verhoeven, Guido

    2012-04-16

    This review aims to evaluate the contribution of individual cell-selective knockout models to our current understanding of androgen action. Cre/loxP technology has allowed the generation of cell-selective knockout models targeting the androgen receptor (AR) in distinct putative target cells in a wide variety of organs and tissues including: testis, ovary, accessory sex tissues, muscle, bone, fat, liver, skin and myeloid tissue. In some androgen-regulated processes such as spermatogenesis and folliculogenesis this approach has lead to the identification of a key cellular mediator of androgen action (Sertoli and granulosa cells, respectively). In many target tissues, however, the final response to androgens appears to be more complex. Here, cell-selective knockout technology offers a platform upon which we can begin to unravel the more complex interplay and signaling pathways of androgens. A prototypic example is the analysis of mesenchymal-epithelial interactions in many accessory sex glands. Furthermore, for some actions of testosterone, in which part of the effect is mediated by the active metabolite 17β-estradiol, conditional knockout technology offers a novel strategy to study the relative contribution of AR and estrogen receptor-mediated signaling. The latter approach has already resulted in a better understanding of androgen action in brain and bone. Finally, cell-selective knockout technology has generated valuable models to search for AR-controlled molecular mediators of androgen action, a strategy that has successfully been applied to the study of androgen action in the testis and in the epididymis. Although some conditional knockout models have provided clear answers to physiologic questions, it should be noted that others have pointed to unexpected complexities or technical limitations confounding interpretation of the results. PMID:21871526

  8. DENDRITIC SPINE ALTERATIONS IN THE HIPPOCAMPUS AND PARIETAL CORTEX OF ALPHA7 NICOTINIC ACETYLCHOLINE RECEPTOR KNOCKOUT MICE

    PubMed Central

    Morley, B. J.; Mervis, R. F.

    2013-01-01

    The α7 nicotinic acetylcholine receptor (nAChR) is involved in higher cognitive and memory functions, and is associated with the etiology of neurological diseases involving cognitive decline, including Alzheimer’s disease (AD). We hypothesized that spine changes in the α7 knockout might help to explain the behavioral deficits observed in α7 knockout mice and prodromal hippocampal changes in AD. We quantified several measures of dendritic morphology in the CA1 region of the mouse hippocampus in Golgi-stained material from wildtype and α7 knockout mice at P24. The most significant difference was a 64% increase in thin (L-type) dendritic spines on the CA1 basilar tree in knockout mice (p < .05). There were small decreases in the number of in N-type (−15%), M-type (−14%) and D-type (−4%) spine densities. The CA1 basilar dendritic tree of knockout mice had significantly less branching in the regions nearthesoma in comparison with wildtype animals (p < .01), but not in the more distal branching. Changes in the configuration of CA1 basilar dendritic spines have been observed in a number of experimental paradigms, suggesting that basilar dendritic spines are highly plastic. One component of cognitive dysfunction may be through α7-modulated GABAergic interneurons synapsing on CA1 basal dendrites. PMID:23270857

  9. Effects of dopamine D1-like and D2-like antagonists on cocaine discrimination in muscarinic receptor knockout mice.

    PubMed

    Thomsen, Morgane; Caine, Simon Barak

    2016-04-01

    Muscarinic and dopamine brain systems interact intimately, and muscarinic receptor ligands, like dopamine ligands, can modulate the reinforcing and discriminative stimulus (S(D)) effects of cocaine. To enlighten the dopamine/muscarinic interactions as they pertain to the S(D) effects of cocaine, we evaluated whether muscarinic M1, M2 or M4 receptors are necessary for dopamine D1 and/or D2 antagonist mediated modulation of the S(D) effects of cocaine. Knockout mice lacking M1, M2, or M4 receptors, as well as control wild-type mice and outbred Swiss-Webster mice, were trained to discriminate 10mg/kg cocaine from saline in a food-reinforced drug discrimination procedure. Effects of pretreatments with the dopamine D1 antagonist SCH 23390 and the dopamine D2 antagonist eticlopride were evaluated. In intact mice, both SCH 23390 and eticlopride attenuated the cocaine discriminative stimulus effect, as expected. SCH 23390 similarly attenuated the cocaine discriminative stimulus effect in M1 knockout mice, but not in mice lacking M2 or M4 receptors. The effects of eticlopride were comparable in each knockout strain. These findings demonstrate differences in the way that D1 and D2 antagonists modulate the S(D) effects of cocaine, D1 modulation being at least partially dependent upon activity at the inhibitory M2/M4 muscarinic subtypes, while D2 modulation appeared independent of these systems. PMID:26874213

  10. Combination effects of wild rice and phytosterols on prevention of atherosclerosis in LDL receptor knockout mice.

    PubMed

    Moghadasian, Mohammed H; Alsaif, Maha; Le, Khuong; Gangadaran, Surendiran; Masisi, Kabo; Beta, Trust; Shen, Garry X

    2016-07-01

    Dietary modifications including healthy eating constitute one of the first line strategies for prevention and treatment of atherosclerotic cardiovascular diseases (CVD), including atherosclerosis. In this study, we assessed anti-atherogenic effects of a combination of wild rice and phytosterols in low-density lipoprotein receptor knockout (LDL-r-KO) mice. Male LDL-r-KO mice were divided into four groups and fed with: (1) control diet; (2) the control diet containing 60% (w/w) wild rice; (3) the control diet containing 2% (w/w) phytosterols; or (4) the control diet containing both wild rice and phytosterols for 20weeks. All diets were supplemented with 0.06% (w/w) dietary cholesterol. Blood samples, hearts, and feces were collected and used for biochemical and histological examination. Consumption of 60% (w/w) wild rice in combination with 2% (w/w) phytosterols significantly reduced the size and severity of atherosclerotic lesions in the aortic roots as compared to those in the control group. This effect was associated with significant reductions in plasma total, LDL and VLDL cholesterol concentrations as well as an increase in fecal cholesterol excretion. In conclusion, the dietary combination of wild rice and phytosterols prevents atherogenesis in this animal model. Further investigations are needed to understand mechanisms of action and potential clinical outcome of such dietary intervention. PMID:27155919

  11. Lipodystrophy Due to Adipose Tissue-Specific Insulin Receptor Knockout Results in Progressive NAFLD.

    PubMed

    Softic, Samir; Boucher, Jeremie; Solheim, Marie H; Fujisaka, Shiho; Haering, Max-Felix; Homan, Erica P; Winnay, Jonathon; Perez-Atayde, Antonio R; Kahn, C Ronald

    2016-08-01

    Ectopic lipid accumulation in the liver is an almost universal feature of human and rodent models of generalized lipodystrophy and is also a common feature of type 2 diabetes, obesity, and metabolic syndrome. Here we explore the progression of fatty liver disease using a mouse model of lipodystrophy created by a fat-specific knockout of the insulin receptor (F-IRKO) or both IR and insulin-like growth factor 1 receptor (F-IR/IGFRKO). These mice develop severe lipodystrophy, diabetes, hyperlipidemia, and fatty liver disease within the first weeks of life. By 12 weeks of age, liver demonstrated increased reactive oxygen species, lipid peroxidation, histological evidence of balloon degeneration, and elevated serum alanine aminotransferase and aspartate aminotransferase levels. In these lipodystrophic mice, stored liver lipids can be used for energy production, as indicated by a marked decrease in liver weight with fasting and increased liver fibroblast growth factor 21 expression and intact ketogenesis. By 52 weeks of age, liver accounted for 25% of body weight and showed continued balloon degeneration in addition to inflammation, fibrosis, and highly dysplastic liver nodules. Progression of liver disease was associated with improvement in blood glucose levels, with evidence of altered expression of gluconeogenic and glycolytic enzymes. However, these mice were able to mobilize stored glycogen in response to glucagon. Feeding F-IRKO and F-IR/IGFRKO mice a high-fat diet for 12 weeks accelerated the liver injury and normalization of blood glucose levels. Thus, severe fatty liver disease develops early in lipodystrophic mice and progresses to advanced nonalcoholic steatohepatitis with highly dysplastic liver nodules. The liver injury is propagated by lipotoxicity and is associated with improved blood glucose levels. PMID:27207510

  12. TAp73 knockout mice show morphological and functional nervous system defects associated with loss of p75 neurotrophin receptor.

    PubMed

    Niklison-Chirou, Maria Victoria; Steinert, Joern R; Agostini, Massimiliano; Knight, Richard A; Dinsdale, David; Cattaneo, Antonio; Mak, Tak W; Melino, Gerry

    2013-11-19

    Total and N-terminal isoform selective p73 knockout mice show a variety of central nervous system defects. Here we show that TAp73 is a transcriptional activator of p75 neurotrophin receptor (p75(NTR)) and that p75(NTR) mRNA and protein levels are strongly reduced in the central and peripheral nervous systems of p73 knockout mice. In parallel, primary cortical neurons from p73 knockout mice showed a reduction in neurite outgrowth and in nerve growth factor-mediated neuronal differentiation, together with reduced miniature excitatory postsynaptic current frequencies and behavioral defects. p73 null mice also have impairments in the peripheral nervous system with reduced thermal sensitivity, axon number, and myelin thickness. At least some of these morphological and functional impairments in p73 null cells can be rescued by p75(NTR) re-expression. Together, these data demonstrate that loss of p75(NTR) contributes to the neurological phenotype of p73 knockout mice. PMID:24190996

  13. Molecular hydrogen stabilizes atherosclerotic plaque in low-density lipoprotein receptor-knockout mice.

    PubMed

    Song, Guohua; Zong, Chuanlong; Zhang, Zhaoqiang; Yu, Yang; Yao, Shutong; Jiao, Peng; Tian, Hua; Zhai, Lei; Zhao, Hui; Tian, Shuyan; Zhang, Xiangjian; Wu, Yun; Sun, Xuejun; Qin, Shucun

    2015-10-01

    Hydrogen (H(2)) attenuates the development of atherosclerosis in mouse models. We aimed to examine the effects of H(2) on atherosclerotic plaque stability. Low-density lipoprotein receptor-knockout (LDLR(-/-)) mice fed an atherogenic diet were dosed daily with H(2) and/or simvastatin. In vitro studies were carried out in an oxidized-LDL (ox-LDL)-stimulated macrophage-derived foam cell model treated with or without H(2). H(2) or simvastatin significantly enhanced plaque stability by increasing levels of collagen, as well as reducing macrophage and lipid levels in plaques. The decreased numbers of dendritic cells and increased numbers of regulatory T cells in plaques further supported the stabilizing effect of H(2) or simvastatin. Moreover, H(2) treatment decreased serum ox-LDL level and apoptosis in plaques with concomitant inhibition of endoplasmic reticulum stress (ERS) and reduction of reactive oxygen species (ROS) accumulation in the aorta. In vitro, like the ERS inhibitor 4-phenylbutyric acid, H(2) inhibited ox-LDL- or tunicamycin (an ERS inducer)-induced ERS response and cell apoptosis. In addition, like the ROS scavenger N-acetylcysteine, H(2) inhibited ox-LDL- or Cu(2+) (an ROS inducer)-induced reduction in cell viability and increase in cellular ROS. Also, H(2) increased Nrf2 (NF-E2-related factor-2, an important factor in antioxidant signaling) activation and Nrf2 small interfering RNA abolished the protective effect of H(2) on ox-LDL-induced cellular ROS production. The inhibitory effects of H(2) on the apoptosis of macrophage-derived foam cells, which take effect by suppressing the activation of the ERS pathway and by activating the Nrf2 antioxidant pathway, might lead to an improvement in atherosclerotic plaque stability. PMID:26117323

  14. Impact of food restriction and cocaine on locomotion in ghrelin- and ghrelin-receptor knockout mice.

    PubMed

    Clifford, Shane; Zeckler, Rosie Albarran; Buckman, Sam; Thompson, Jeff; Hart, Nigel; Wellman, Paul J; Smith, Roy G

    2011-07-01

    Food restriction (FR) augments the behavioral and reinforcing effects of psychomotor stimulants such as cocaine or amphetamine; effects that may be related to the capacity of FR to increase plasma levels of ghrelin (GHR), a 28-amino acid orexigenenic peptide linked to activation of brain dopamine systems. The present study used wild-type (WT) mice or mutant mice sustaining knockout of either GHR [GHR((-/-)) ] or of the growth hormone secretagogue receptor [GHS-R((-/-)) ] and subjected to FR or not to evaluate the role of GHR and GHS-R in cocaine-stimulated locomotion. WT, GHR((-/-)) , and GHS-R((-/-)) mice were either restricted to 60% of baseline caloric intake or allowed to free-feed (FF). Mice were treated with 0, 1.25, 2.5 and 5.0 mg/kg cocaine on separate test days (in random dose order) and forward locomotion was recorded on each drug day for 45 minutes after drug dosing. Food (and water) was available immediately after (but not during) each activity test. For FF mice, there was no interaction between cocaine and GHR status on locomotion. FR-WT mice treated with saline exhibited significant increases in anticipatory locomotion (relative to FF-WT mice), whereas FR-GHS-R((-/-)) mice did not. Cocaine significantly increased locomotion in FR-GHR((-/-)) and FR-GHS-R((-/-)) mice to the levels noted in FR-WT mice. These results suggest that GHS-R activity, but not GHR activity, is required for FR to augment food-associated anticipatory locomotion, but do not support the contention that GHR pathways are required for the capacity of FR to augment the acute effect of cocaine on locomotion. PMID:21054685

  15. [Effect of P2X7 receptor knock-out on bone cancer pain in mice].

    PubMed

    Zhao, Xin; Liu, Hui-Zhu; Zhang, Yu-Qiu

    2016-06-25

    Cancer pain is one of the most common symptoms in patients with late stage cancer. Lung, breast and prostate carcinoma are the most common causes of pain from osseous metastasis. P2X7 receptor (P2X7R) is one of the subtypes of ATP-gated purinergic ion channel family, predominately distributed in microglia in the spinal cord. Activation of P2X7Rs in the spinal dorsal horn has been associated with release of proinflammatory cytokines from glial cells, causing increased neuronal excitability and exaggerated nociception. Mounting evidence implies a critical role of P2X7R in inflammatory and neuropathic pain. However, whether P2X7R is involved in cancer pain remains controversial. Here we established a bone cancer pain model by injecting the Lewis lung carcinoma cells into the femur bone marrow cavity of C57BL/6J wild-type mice (C57 WT mice) and P2X7R knockout mice (P2rx7(-/-) mice) to explore the role of P2X7R in bone cancer pain. Following intrafemur carcinoma inoculation, robust mechanical allodynia and thermal hyperalgesia in C57 WT mice were developed on day 7 and 14, respectively, and persisted for at least 28 days in the ipsilateral hindpaw of the affected limb. CatWalk gait analysis showed significant decreases in the print area and stand phase, and a significant increase in swing phase in the ipsilateral hindpaw on day 21 and 28 after carcinoma cells inoculation. Histopathological sections (hematoxylin and eosin stain) showed that the bone marrow of the affected femur was largely replaced by invading tumor cells, and the femur displayed medullary bone loss and bone destruction on day 28 after inoculation. Unexpectedly, no significant changes in bone cancer-induced hypersensitivity of pain behaviors were found in P2rx7(-/-) mice, and the changes of pain-related values in CatWalk gait analysis even occurred earlier in P2rx7(-/-) mice, as compared with C57 WT mice. Together with our previous study in rats that blockade of P2X7R significantly alleviated bone cancer

  16. Functional consequences of hippocampal neuronal ectopia in the apolipoprotein E receptor-2 knockout mouse

    PubMed Central

    Fish, Kenneth. N.; Krucker, Thomas

    2008-01-01

    Little is known about the impact ectopically located neurons have on the functional connectivity of local circuits. The ApoER2 knockout mouse has subtle cytoarchitectural disruptions, altered prepulse inhibition, and memory abnormalities. We evaluated this mouse mutant as a model to study the role ectopic neurons play in the manifestation of symptoms associated with brain diseases. We found that ectopic CA1 pyramidal and inhibitory neurons in the ApoER2 knockout hippocampus are organized into two distinct stratum pyramidale layers. In vitro analyses found that ApoER2 is not required for neurons to reach maturity in regards to dendritic arborization and synaptic structure density, and electrophysiological testing determined that neurons in both strata pyramidale are integrated into the hippocampal network. However, the presence of these two layers alters the spatiotemporal pattern of hippocampal activity, which may explain why ApoER2 knockout mice have selective cognitive dysfunctions that are revealed only under challenging conditions. PMID:18778775

  17. The transcobalamin receptor knockout mouse: a model for vitamin B12 deficiency in the central nervous system

    PubMed Central

    Lai, Shao-Chiang; Nakayama, Yasumi; Sequeira, Jeffrey M.; Wlodarczyk, Bogdan J.; Cabrera, Robert M.; Finnell, Richard H.; Bottiglieri, Teodoro; Quadros, Edward V.

    2013-01-01

    The membrane receptor (TCblR/CD320) for transcobalamin (TC)-bound cobalamin (Cbl) facilitates the cellular uptake of Cbl. A genetically modified mouse model involving ablation of the CD320 gene was generated to study the effects on cobalamin homeostasis. The nonlethal nature of this knockout and the lack of systemic cobalamin deficiency point to other mechanisms for cellular Cbl uptake in the mouse. However, severe cobalamin depletion in the central nervous system (CNS) after birth (P<0.01) indicates that TCblR is the only receptor responsible for Cbl uptake in the CNS. Metabolic Cbl deficiency in the brain was evident from the increased methylmalonic acid (P<0.01–0.04), homocysteine (P<0.01), cystathionine (P<0.01), and the decreased S-adenosylmethionine/S-adenosyl homocysteine ratio (P<0.01). The CNS pathology of Cbl deficiency seen in humans may not manifest in this mouse model; however, it does provide a model with which to evaluate metabolic pathways and genes affected.—Lai, S.-C., Nakayama, Y., Sequeira, J. M., Wlodarczyk, B. J., Cabrera, R. M., Finnell, R. H., Bottiglieri, T., Quadros, E. V. The transcobalamin receptor knockout mouse: a model for vitamin B12 deficiency in the central nervous system. PMID:23430977

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

    PubMed Central

    Sanders, Jeff

    2016-01-01

    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]). PMID:26952134

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

    PubMed

    Sanders, Jeff

    2016-06-01

    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]). PMID:26952134

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

    NASA Astrophysics Data System (ADS)

    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

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

    PubMed Central

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

    2016-01-01

    Background 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. Results 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

  2. Kidney-specific reconstitution of the A1 adenosine receptor in A1 adenosine receptor knockout mice reduces renal ischemia–reperfusion injury

    PubMed Central

    Kim, Minjae; Chen, Sean W.C.; Park, Sang Won; Kim, Mihwa; D’Agati, Vivette D.; Yang, Jay; Lee, H. Thomas

    2009-01-01

    Genetic deletion of the adenosine A1 receptor (A1AR) increased renal injury following ischemia-reperfusion injury suggesting that receptor activation is protective in vivo. Here we tested this hypothesis by expressing the human-A1AR in A1AR knockout mice. Renal ischemia-reperfusion was induced in knockout mice 2 days after intrarenal injection of saline or a lentivirus encoding enhanced green fluorescent protein (EGFP) or EGFP-human-A1AR. We found that the latter procedure induced a robust expression of the reporter protein in the kidneys of knockout mice. Mice with kidney-specific human-A1AR reconstitution had significantly lower plasma creatinine, tubular necrosis, apoptosis, and tubular inflammation as evidenced by decreased leukocyte infiltration, pro-inflammatory cytokine, and intercellular adhesion molecule-1 expression in the kidney following injury compared to mice injected with saline or the control lentivirus. Additionally, there were marked disruptions of the proximal tubule epithelial filamentous (F)-actin cytoskeleton in both sets of control mice upon renal injury, whereas the reconstituted mice had better preservation of the renal tubule actin cytoskeleton, which co-localized with the human-A1ARs. Consistent with reduced renal injury, there was a significant increase in heat shock protein-27 expression, also co-localizing with the preserved F-actin cytoskeleton. Our findings suggest that selective expression of cytoprotective A1ARs in the kidney can attenuate renal injury. PMID:19190680

  3. (-)-Pentazocine induces visceral chemical antinociception, but not thermal, mechanical, or somatic chemical antinociception, in μ-opioid receptor knockout mice

    PubMed Central

    2011-01-01

    Background (-)-Pentazocine has been hypothesized to induce analgesia via the κ-opioid (KOP) receptor, although the involvement of other opioid receptor subtypes in the effects of pentazocine remains unknown. In this study, we investigated the role of the μ-opioid (MOP) receptor in thermal, mechanical, and chemical antinociception induced by (-)-pentazocine using MOP receptor knockout (MOP-KO) mice. Results (-)-Pentazocine-induced thermal antinociception, assessed by the hot-plate and tail-flick tests, was significantly reduced in heterozygous and abolished in homozygous MOP-KO mice compared with wildtype mice. The results obtained from the (-)-pentazocine-induced mechanical and somatic chemical antinociception experiments, which used the hind-paw pressure and formalin tests, were similar to the results obtained from the thermal antinociception experiments in these mice. However, (-)-pentazocine retained its ability to induce significant visceral chemical antinociception, assessed by the writhing test, in homozygous MOP-KO mice, an effect that was completely blocked by pretreatment with nor-binaltorphimine, a KOP receptor antagonist. In vitro binding and cyclic adenosine monophosphate assays showed that (-)-pentazocine possessed higher affinity for KOP and MOP receptors than for δ-opioid receptors. Conclusions The present study demonstrated the abolition of the thermal, mechanical, and somatic chemical antinociceptive effects of (-)-pentazocine and retention of the visceral chemical antinociceptive effects of (-)-pentazocine in MOP-KO mice. These results suggest that the MOP receptor plays a pivotal role in thermal, mechanical, and somatic chemical antinociception induced by (-)-pentazocine, whereas the KOP receptor is involved in visceral chemical antinociception induced by (-)-pentazocine. PMID:21477373

  4. 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.

    PubMed

    Sisay, Sofia; Pryce, Gareth; Jackson, Samuel J; Tanner, Carolyn; Ross, Ruth A; Michael, Gregory J; Selwood, David L; Giovannoni, Gavin; Baker, David

    2013-01-01

    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 value of some

  5. 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

    PubMed Central

    Jackson, Samuel J.; Tanner, Carolyn; Ross, Ruth A.; Michael, Gregory J.; Selwood, David L.; Giovannoni, Gavin; Baker, David

    2013-01-01

    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 (Cnr2tm1Zim) 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 (Cnr2Dgen) receptor knockout mice developed augmented severity of disease consistent with the genetically and pharmacologically-distinct, Cnr2tm1Zim 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 value of some

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

    PubMed Central

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

    2010-01-01

    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. PMID:20479251

  7. IL-1 receptor-antagonist (IL-1Ra) knockout mice show anxiety-like behavior by aging.

    PubMed

    Wakabayashi, Chisato; Numakawa, Tadahiro; Odaka, Haruki; Ooshima, Yoshiko; Kiyama, Yuji; Manabe, Toshiya; Kunugi, Hiroshi; Iwakura, Yoichiro

    2015-07-10

    Interleukin 1 (IL-1) plays a critical role in stress responses, and its mRNA is induced in the brain by restraint stress. Previously, we reported that IL-1 receptor antagonist (IL-1Ra) knockout (KO) mice, which lacked IL-1Ra molecules that antagonize the IL-1 receptor, showed anti-depression-like behavior via adrenergic modulation at the age of 8 weeks. Here, we report that IL-1Ra KO mice display an anxiety-like phenotype that is induced spontaneously by aging in the elevated plus-maze (EPM) test. This anxiety-like phenotype was improved by the administration of diazepam. The expression of the anxiety-related molecule glucocorticoid receptor (GR) was significantly reduced in 20-week-old but not in 11-week-old IL-1Ra KO mice compared to wild-type (WT) littermates. The expression of the mineralocorticoid receptor (MR) was not altered between IL-1Ra KO mice and WT littermates at either 11 or 20 weeks old. Analysis of monoamine concentration in the hippocampus revealed that tryptophan, the serotonin metabolite 5-hydroxyindole acetic acid (5-HIAA), and the dopamine metabolite homovanillic acid (HVA) were significantly increased in 20-week-old IL-1Ra KO mice compared to littermate WT mice. These findings strongly suggest that the anxiety-like behavior observed in older mice was caused by the complicated alteration of monoamine metabolism and/or GR expression in the hippocampus. PMID:26002078

  8. Interactive contribution of NK(1) and kinin receptors to the acute inflammatory oedema observed in response to noxious heat stimulation: studies in NK(1) receptor knockout mice.

    PubMed

    Rawlingson, A; Gerard, N P; Brain, S D

    2001-12-01

    1. Scald injury in Sv129+C57BL/6 mice induced a temperature and time dependent oedema formation as calculated by the extravascular accumulation of [(125)I]-albumin. Oedema formation was suppressed in NK(1) knockout mice compared to wildtypes at 10 (P<0.01) and 30 min (P<0.001). However, at 60 min a similar degree of extravasation was observed in the two groups. 2. Kinin B(1) (des-Arg(10) Hoe 140; 1 micromol kg(-1)) and B(2) (Hoe 140; 100 nmol kg(-1)) antagonists caused an inhibition of oedema in wildtype mice at 10 and 30 min (P<0.001), but not at 60 min or at 30 min in NK(1) receptor knockout mice. 3. The inhibition of thermic oedema by des-Arg(10) Hoe 140 was reversed by des-Arg(9) bradykinin (0.1 micromol kg(-1); P<0.01) and also observed with a second B(1) receptor antagonist (des-Arg(9) Leu(8) bradykinin; 3 micromol kg(-1); P<0.01). Furthermore des-Arg(10) Hoe 140 had no effect on capsaicin (200 microg ear(-1)) ear oedema, but this was significantly reduced with Hoe 140 (P<0.05). 4. Scalding induced a large neutrophil accumulation at 4 h, as assessed by myeloperoxidase assay (P<0.001). This was not suppressed by NK(1) receptor deletion or kinin antagonists. 5. These results confirm an essential role for the NK(1) receptor in mediating the early, but not the delayed phase of oedema formation or neutrophil accumulation in response to scalding. The results also demonstrate a pivotal link between the kinins and sensory nerves in the microvascular response to burn injury, and for the first time show a rapid involvement of the B(1) receptor in murine skin. PMID:11739258

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

    SciTech Connect

    Gerdin, Anna Karin; Surve, Vikas V.; Joensson, Marie; Bjursell, Mikael; Edenro, Anne; Schuelke, Meint; Saad, Alaa; Bjurstroem, Sivert; Lundgren, Elisabeth Jensen; Snaith, Michael; Fransson-Steen, Ronny; Toernell, Jan; Bohlooly-Y, Mohammad . E-mail: mohammad.bohlooly@astrazeneca.com

    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. 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}{sup +/+}), the HNF4-{gamma} knockout (HNF4-{gamma}{sup -/-}) 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}{sup -/-} mice were less inclined to build nest and were found to spend more time in a passive state during the forced swim test.

  10. Toll-like receptor 4 knockout alleviates paraquat-induced cardiomyocyte contractile dysfunction through an autophagy-dependent mechanism.

    PubMed

    Wang, Shuyi; Zhu, Xiaoling; Xiong, Lize; Zhang, Yingmei; Ren, Jun

    2016-08-22

    Paraquat, a quarternary nitrogen herbicide, is a toxic prooxidant leading to multi-organ failure including the heart although the underlying mechanism remains poorly understood. This study was designed to examine the role of the innate proinflammatory mediator toll-like receptor 4 (TLR4) in paraquat-induced cardiac contractile anomalies and the underlying mechanisms involved with a focus on autophagy, a conservative machinery governing protein and organelle degradation and recycling for cardiac homeostasis. Wild-type (WT) and TLR4 knockout (TLR4(-/-)) mice were challenged with paraquat (45mg/kg, i.p.) for 48h. Paraquat challenge did not affect mRNA levels of TLR2, TLR4 and TLR9 in WT mice nor did paraquat treatment alter TREM-1 levels. Paraquat challenge elicited cardiac mechanical defects including compromised cardiomyocyte contractile function, intracellular Ca(2+) handling, and overt autophagy as manifested by increased LC3BII-to-LC3BI ratio, Atg5, Atg7 and p62 levels. Interestingly, TLR4 knockout significantly attenuated paraquat-induced cardiac contractile and intracellular Ca(2+) derangement as well as alterations of autophagy markers. Paraquat-elicited changes in cardiac autophagy markers (LC3BII, LC3BII-to-LC3BI ratio and p62) were augmented by lysosomal inhibition using bafilomycin A1 in WT mice. TLR4 knockout significantly attenuated or negated paraquat-elicited increase in LC3BII, LC3BII-to-LC3BI ratio and p62 levels in the presence of lysosomal inhibition. In addition, paraquat challenge promoted phosphorylation of AMPK while suppressing the phosphorylation of mTOR and ULK1 (the autophagy inhibitory Ser(757)), the effects of which were significantly attenuated by TLR4 ablation. In vitro study revealed that AMPK activation using AICAR or mTOR inhibition using rapamycin effectively negated the beneficial cardiomyocyte mechanical effects of TLR4 inhibition (CLI-095) against paraquat toxicity, supporting a permissive role for AMPK-mTOR in TLR4 inhibition

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

    PubMed Central

    Kohlmeier, Kristi A.; Tyler, Christopher J.; Kalogiannis, Mike; Ishibashi, Masaru; Kristensen, Morten P.; Gumenchuk, Iryna; Chemelli, Richard M.; Kisanuki, Yaz Y.; Yanagisawa, Masashi; Leonard, Christopher S.

    2013-01-01

    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 OX−/−2 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 OX−/−1 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 OX−/−1 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 produced 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

  12. Abolished thermal and mechanical antinociception but retained visceral chemical antinociception induced by butorphanol in mu-opioid receptor knockout mice.

    PubMed

    Ide, Soichiro; Minami, Masabumi; Ishihara, Kumatoshi; Uhl, George R; Satoh, Masamichi; Sora, Ichiro; Ikeda, Kazutaka

    2008-06-01

    Butorphanol is hypothesized to induce analgesia via opioid pathways, although the precise mechanisms for its effects remain unknown. In this study, we investigated the role of the mu-opioid receptor (MOP) in thermal, mechanical, and visceral chemical antinociception induced by butorphanol using MOP knockout (KO) mice. Butorphanol-induced thermal antinociception, assessed by the hot-plate and tail-flick tests, was significantly reduced in heterozygous and abolished in homozygous MOP-KO mice compared with wildtype mice. The results obtained from our butorphanol-induced mechanical antinociception experiments, assessed by the Randall-Selitto test, were similar to the results obtained from the thermal antinociception experiments in these mice. Interestingly, however, butorphanol retained its ability to induce significant visceral chemical antinociception, assessed by the writhing test, in homozygous MOP-KO mice. The butorphanol-induced visceral chemical antinociception that was retained in homozygous MOP-KO mice was completely blocked by pretreatment with nor-binaltorphimine, a kappa-opioid receptor (KOP) antagonist. In vitro binding and cyclic adenosine monophosphate assays also showed that butorphanol possessed higher affinity for KOPs and MOPs than for delta-opioid receptors. These results molecular pharmacologically confirmed previous studies implicating MOPs, and partially KOPs, in mediating butorphanol-induced analgesia. PMID:18417173

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

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

    PubMed Central

    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. PMID:26506622

  15. Hippocampal place cell responses to distal and proximal cue manipulations in dopamine D2 receptor-knockout mice.

    PubMed

    Nguyen, Chien Le; Tran, Anh Hai; Matsumoto, Jumpei; Hori, Etsuro; Uwano, Teruko; Ono, Taketoshi; Nishijo, Hisao

    2014-06-01

    The human hippocampus is critical for learning and memory. In rodents, hippocampal pyramidal neurons fire in a location-specific manner and form relational representations of environmental cues. The important roles of dopaminergic D1 receptors in learning and in hippocampal neural synaptic plasticity in novel environments have been previously shown. However, the roles of D2 receptors in hippocampal neural plasticity in response to novel and familiar spatial stimuli remain unclear. In order to clarify this issue, we recorded from hippocampal neurons in dopamine D2 receptor-knockout (D2R-KO) mice and their wild-type (WT) littermates during manipulations of distinct spatial cues in familiar and novel environments. Here, we report that D2R-KO mice showed substantial deficits in place-cell properties (number of place cells, intra-field firing rates, spatial tuning, and spatial coherence). Furthermore, although place cells in D2R-KO mice responded to manipulations of distal and proximal cues in both familiar and novel environments in a manner that was similar to place cells in WT mice, place fields were less stable in the D . The axes represent the differences between the peak and the valley of each waveform of EL2 and EL3.2R-KO mice in the familiar environment, but not in the novel environment. The present results suggested that D2 receptors in the hippocampus are important for place response stability. The place-cell properties of D2R-KO mice were similar to aged animals, suggesting that the alterations of place-cell properties in aged animals might be ascribed partly to alterations in the D2R in the HF of aged animals. PMID:24747614

  16. Specific regions display altered grey matter volume in μ-opioid receptor knockout mice: MRI voxel-based morphometry

    PubMed Central

    Sasaki, Kazumasu; Sumiyoshi, Akira; Nonaka, Hiroi; Kasahara, Yoshiyuki; Ikeda, Kazutaka; Hall, F Scott; Uhl, George R; Watanabe, Masahiko; Kawashima, Ryuta; Sora, Ichiro

    2015-01-01

    BACKGROUND AND PURPOSE μ Opioid receptor knockout (MOP-KO) mice display several behavioural differences from wild-type (WT) littermates including differential responses to nociceptive stimuli. Brain structural changes have been tied to behavioural alterations noted in transgenic mice with targeting of different genes. Hence, we assess the brain structure of MOP-KO mice. EXPERIMENTAL APPROACH Magnetic resonance imaging (MRI) voxel-based morphometry (VBM) and histological methods were used to identify structural differences between extensively backcrossed MOP-KO mice and WT mice. KEY RESULTS MOP-KO mice displayed robust increases in regional grey matter volume in olfactory bulb, several hypothalamic nuclei, periaqueductal grey (PAG) and several cerebellar areas, most confirmed by VBM analysis. The largest increases in grey matter volume were detected in the glomerular layer of the olfactory bulb, arcuate nucleus of hypothalamus, ventrolateral PAG (VLPAG) and cerebellar regions including paramedian and cerebellar lobules. Histological analyses confirm several of these results, with increased VLPAG cell numbers and increased thickness of the olfactory bulb granule cell layer and cerebellar molecular and granular cell layers. CONCLUSIONS AND IMPLICATIONS MOP deletion causes previously undescribed structural changes in specific brain regions, but not in all regions with high MOP receptor densities (e.g. thalamus, nucleus accumbens) or that exhibit adult neurogenesis (e.g. hippocampus). Volume differences in hypothalamus and PAG may reflect behavioural changes including hyperalgesia. Although the precise relationship between volume change and MOP receptor deletion was not determined from this study alone, these findings suggest that levels of MOP receptor expression may influence a broader range of neural structure and function in humans than previously supposed. LINKED ARTICLES This article is part of a themed section on Opioids: New Pathways to Functional Selectivity

  17. The endogenous opioid system in cocaine addiction: what lessons have opioid peptide and receptor knockout mice taught us?

    PubMed Central

    Yoo, Ji Hoon; Kitchen, Ian; Bailey, Alexis

    2012-01-01

    Cocaine addiction has become a major concern in the UK as Britain tops the European ‘league table’ for cocaine abuse. Despite its devastating health and socio-economic consequences, no effective pharmacotherapy for treating cocaine addiction is available. Identifying neurochemical changes induced by repeated drug exposure is critical not only for understanding the transition from recreational drug use towards compulsive drug abuse but also for the development of novel targets for the treatment of the disease and especially for relapse prevention. This article focuses on the effects of chronic cocaine exposure and withdrawal on each of the endogenous opioid peptides and receptors in rodent models. In addition, we review the studies that utilized opioid peptide or receptor knockout mice in order to identify and/or clarify the role of different components of the opioid system in cocaine-addictive behaviours and in cocaine-induced alterations of brain neurochemistry. The review of these studies indicates a region-specific activation of the µ-opioid receptor system following chronic cocaine exposure, which may contribute towards the rewarding effect of the drug and possibly towards cocaine craving during withdrawal followed by relapse. Cocaine also causes a region-specific activation of the κ-opioid receptor/dynorphin system, which may antagonize the rewarding effect of the drug, and at the same time, contribute to the stress-inducing properties of the drug and the triggering of relapse. These conclusions have important implications for the development of effective pharmacotherapy for the treatment of cocaine addiction and the prevention of relapse. PMID:22428846

  18. Decreased Incentive Motivation Following Knockout or Acute Blockade of the Serotonin Transporter: Role of the 5-HT2C Receptor.

    PubMed

    Browne, Caleb J; Fletcher, Paul J

    2016-09-01

    Acute pharmacological elevation of serotonin (5-hydroxytryptamine; 5-HT) activity decreases operant responding for primary reinforcers, suggesting that 5-HT reduces incentive motivation. The mechanism by which 5-HT alters incentive motivation is unknown, but parallel evidence that 5-HT2C receptor agonists also reduce responding for primary reinforcers implicates this receptor as a potential candidate. These experiments examined whether chronic and acute disruptions of serotonin transporter (SERT) activity altered incentive motivation, and whether the 5-HT2C receptor mediated the effects of elevated 5-HT on behavior. To assess incentive motivation, we measured responding for three different reinforcers: a primary reinforcer (saccharin), a conditioned reinforcer (CRf), and an unconditioned sensory reinforcer (USRf). In the chronic condition, responding was compared between SERT knockout (SERT-KO) mice and their wild-type littermates. In the acute condition, responding was examined in wild-type mice following treatment with 10 or 20 mg/kg citalopram, or its vehicle. The ability of the selective 5-HT2C antagonist SB 242084 to prevent the effects of SERT-KO and citalopram on responding was subsequently examined. Both SERT-KO and citalopram reduced responding for saccharin, a CRf, and a USRf. Treatment with SB 242084 enhanced responding for a CRf and a USRf in SERT-KO mice and blocked the effects of citalopram on CRf and USRf responding. However, SB 242084 was unable to prevent the effects of SERT-KO or citalopram on responding for saccharin. These results support a powerful inhibitory function for 5-HT in the control of incentive motivation, and indicate that the 5-HT2C receptor mediates these effects of 5-HT in a reinforcer-dependent manner. PMID:27125304

  19. Role of endogenous prostacyclin in gastric ulcerogenic and healing responses--a study using IP-receptor knockout mice.

    PubMed

    Takeuchi, K; Kato, S; Ogawa, Y; Kanatsu, K; Umeda, M

    2001-01-01

    Endogenous prostaglandins (PGs) play an important role in the cytoprotective and healing responses in the stomach, by altering various functions, i.e., an increase of the mucosal blood flow, yet the role of prostacyclin (PGI(2)) and its receptor (IP-receptor) in these responses remains unclarified. In the present study, we used IP-receptor knockout mice [IP (-/-)] and examined the importance of IP-receptors in gastric ulcerogenic, cytoprotective and healing responses in these animals. The studies included the ulcerogenic response to cold-restraint stress, the cytoprotective response to a mild irritant (20 mM taurocholate: TC) and capsaicin, and the healing response of chronic gastric ulcers induced by thermo-cauterization. We first checked the absence of IP-receptors by examining the effect of cicaprost (a PGI(2) agonist, topical mucosal application) on gastric mucosal blood flow and found that this agent increased the mucosal blood flow in wild-type [WT (+/+)] mice but not in IP (+/-) mice. Cold-restraint stress (4 h) induced gastric lesions in both groups of mice, but the severity of damage was significantly greater in IP (-/-) mice. Prior p.o. administration of both TC and capsaicin exhibited a marked cytoprotection against HCl/ethanol-induced gastric damage in WT (+/+) mice, both responses being significantly mitigated in the presence of indomethacin. The adaptive cytoprotection induced by TC was similarly observed in IP (-/-) mice, while the capsaicin protection was totally attenuated in the animals lacking IP receptors. On the other hand, the healing of gastric ulcers was significantly delayed by daily administration of indomethacin in WT (+/+) mice. However, this process was not altered in IP (-/-) mice. These results suggest that endogenous PGI(2) is involved in the gastric ulcerogenic response to stress, but not in the healing of pre-existing gastric ulcers. In addition, PGI(2) and its receptors may play a crucial role in capsaicin-induced gastric

  20. The transcobalamin receptor knockout mouse: a model for vitamin B12 deficiency in the central nervous system.

    PubMed

    Lai, Shao-Chiang; Nakayama, Yasumi; Sequeira, Jeffrey M; Wlodarczyk, Bogdan J; Cabrera, Robert M; Finnell, Richard H; Bottiglieri, Teodoro; Quadros, Edward V

    2013-06-01

    The membrane receptor (TCblR/CD320) for transcobalamin (TC)-bound cobalamin (Cbl) facilitates the cellular uptake of Cbl. A genetically modified mouse model involving ablation of the CD320 gene was generated to study the effects on cobalamin homeostasis. The nonlethal nature of this knockout and the lack of systemic cobalamin deficiency point to other mechanisms for cellular Cbl uptake in the mouse. However, severe cobalamin depletion in the central nervous system (CNS) after birth (P<0.01) indicates that TCblR is the only receptor responsible for Cbl uptake in the CNS. Metabolic Cbl deficiency in the brain was evident from the increased methylmalonic acid (P<0.01-0.04), homocysteine (P<0.01), cystathionine (P<0.01), and the decreased S-adenosylmethionine/S-adenosyl homocysteine ratio (P<0.01). The CNS pathology of Cbl deficiency seen in humans may not manifest in this mouse model; however, it does provide a model with which to evaluate metabolic pathways and genes affected. PMID:23430977

  1. Genetic manipulation to analyze pheromone responses: knockouts of multiple receptor genes.

    PubMed

    Ishii, Tomohiro

    2013-01-01

    Gene targeting in the mouse is an essential technique to study gene function in vivo. Multigene families encoding vomeronasal receptor (VR) type 1 and type 2 consist of ~300 intact genes, which are clustered at multiple loci in the mouse genome. To understand the function of VRs and neurons expressing a particular VR in vivo, individual endogenous receptor genes can be manipulated by conventional gene targeting to create loss-of-function mutations or to visualize neurons and their axons expressing the VR. Multiple receptor genes in a cluster can also be deleted simultaneously by chromosome engineering, allowing analysis of function of a particular VR subfamily. Here, we describe protocols for conventional gene targeting and chromosome engineering for deleting a large genomic region in mouse embryonic stem (ES) cells. PMID:24014359

  2. Retention of NMDA receptor NR2 subunits in the lumen of endoplasmic reticulum in targeted NR1 knockout mice

    PubMed Central

    Fukaya, Masahiro; Kato, Akira; Lovett, Chanel; Tonegawa, Susumu; Watanabe, Masahiko

    2003-01-01

    Glutamate is a major excitatory neurotransmitter in the mammalian central nervous system, and the N-methyl-d-aspartate-selective glutamate receptor (NR) consisting of the NR1 subunit and an NR2 or NR3 subunit plays crucial roles in synaptic transmission, plasticity, and learning and memory. By using a knockout mouse strain, in which the NR1 gene deletion is primarily targeted to the CA1 pyramidal cells of the hippocampus, we investigated the in vivo effect of the loss of the NR1 subunit on the cellular expression and intracellular distribution of the NR2 subunits. The NR1 gene deletion had no apparent effect on the levels of NR2A or NR2B mRNA but led to severe reductions of NR2A and NR2B protein in dendrites of CA1 pyramidal cells. This reduced dendritic distribution of the NR2 subunits accompanied their robust accumulation in perikarya, where they were condensed in the lumen of the endoplasmic reticulum as electron-dense granules. These granules were also observed in CA1 pyramidal cells of the control mice but they were much fewer and contained no detectable levels of the NR2 subunit. The effect of the NR1 knockout on intracellular localization of the NR2 subunits was specific in that no such effect was observed for the GluR1 and PSD-95, two other major postsynaptic proteins. These results suggest that the NR1 subunit plays a crucial role in the release of the NR2 subunit from the endoplasmic reticulum in hippocampal pyramidal cells in vivo, and when the NR1 subunit is unavailable, the NR2 subunits are retained and aggregate into intracisternal granules. PMID:12676993

  3. Long-term effects of diazepam treatment of epileptic GABAA receptor beta3 subunit knockout mouse in early life.

    PubMed

    Liljelund, Patricia; Ferguson, Carolyn; Homanics, Gregg; Olsen, Richard W

    2005-01-01

    The knockout mouse for the beta3 subunit of the GABAA receptor exhibits spontaneous epilepsy and hyperactivity, and has been proposed as a model for the severe developmental disorder, Angelman's syndrome, which is known to be of genetic origin. We have used this mutant to test an approach of therapeutic intervention prior to seizure onset by daily injection with diazepam during either the first or second postnatal week. Results showed differences between postnatal week 1 and week 2 injections both acutely, with respect to sedative effects, and in long-term outcome, with respect to EEG and behavioral tests measured at 12-14 weeks of age. The EEG of control mice remained unaffected under all conditions, but the EEG of beta3 (-/-) injected with diazepam in week 1 was worsened, showing increased oscillatory activity at 5-6Hz, and more myoclonic jerks, particularly among males. For beta3 (-/-) injected with diazepam in week 2, the EEG was normalized in half the mice but worsened similarly to week 1 in the other half. Neonatal diazepam injection had a long-term normalizing effect on behavior of beta3 (-/-) mice injected in week 1, but diazepam treatment in week 2 did not affect the hyperactive and circling behavior characteristic of the beta3 knockout mouse. Diazepam treatment in postnatal week 2 significantly decreased anxiety in the adult beta3 group. Diazepam treatment in both postnatal weeks 1 and 2 improved the motor coordination of beta3 (-/-) on the rotarod, although performance of control mice injected with diazepam in postnatal week 2 was significantly impaired. The observed long-term outcome of neonatal diazepam injections may result from interference with developmental processes, and shows that enhancing GABAergic activity with diazepam during the period where GABA can be excitatory can produce narrow stage-related effects on brain development. PMID:16168624

  4. Altered mnemonic functions and resistance to NMDA receptor antagonism by forebrain conditional knockout of glycine transporter 1

    PubMed Central

    Singer, Philipp; Yee, Benjamin K.; Feldon, Joram; Iwasato, Takuji; Itohara, Shigeyoshi; Grampp, Thomas; Prenosil, George; Benke, Dietmar; Möhler, Hanns; Boison, Detlev

    2009-01-01

    Converging evidence from pharmacological and molecular studies has led to the suggestion that inhibition of glycine transporter 1 (GlyT1) constitutes an effective means to boost N-methyl-D-aspartate receptor (NMDAR) activity by increasing the extra-cellular concentration of glycine in the vicinity of glutamatergic synapses. However, the precise extent and limitation of this approach to alter cognitive function, and therefore its potential as a treatment strategy against psychiatric conditions marked by cognitive impairments, remains to be fully examined. Here, we generated mutant mice lacking GlyT1 in the entire forebrain including neurons and glia. This conditional knockout system allows a more precise examination of GlyT1 down-regulation in the brain on behaviour and cognition. The mutation was highly effective in attenuating the motor-stimulating effect of acute NMDAR blockade by phencyclidine, although no appreciable elevation in NMDAR-mediated EPSC was observed in the hippocampus. Enhanced cognitive performance was observed in spatial working memory and object recognition memory while spatial reference memory and associative learning remained unaltered. These findings provide further credence for the potential cognitive enhancing effects of brain GlyT1 inhibition. At the same time, they indicated potential phenotypic differences when compared with other constitutive and conditional GlyT1 knockout lines, and highlighted the possibility of a functional divergence between the neuronal and glia subpopulations of GlyT1 in the regulation of learning and memory processes. The relevance of this distinction to the design of future GlyT1 blockers as therapeutic tools in the treatment of cognitive disorders remains to be further investigated. PMID:19332109

  5. Peripheral benzodiazepine receptor/translocator protein global knock-out mice are viable with no effects on steroid hormone biosynthesis.

    PubMed

    Tu, Lan N; Morohaku, Kanako; Manna, Pulak R; Pelton, Susanne H; Butler, W Ronald; Stocco, Douglas M; Selvaraj, Vimal

    2014-10-01

    Translocator protein (TSPO), previously known as the peripheral benzodiazepine receptor, is a mitochondrial outer membrane protein implicated as essential for cholesterol import to the inner mitochondrial membrane, the rate-limiting step in steroid hormone biosynthesis. Previous research on TSPO was based entirely on in vitro experiments, and its critical role was reinforced by an early report that claimed TSPO knock-out mice were embryonic lethal. In a previous publication, we examined Leydig cell-specific TSPO conditional knock-out mice that suggested TSPO was not required for testosterone production in vivo. This raised controversy and several questions regarding TSPO function. To examine the definitive role of TSPO in steroidogenesis and embryo development, we generated global TSPO null (Tspo(-/-)) mice. Contrary to the early report, Tspo(-/-) mice survived with no apparent phenotypic abnormalities and were fertile. Examination of adrenal and gonadal steroidogenesis showed no defects in Tspo(-/-) mice. Adrenal transcriptome comparison of gene expression profiles showed that genes involved in steroid hormone biosynthesis (Star, Cyp11a1, and Hsd3b1) were unchanged in Tspo(-/-) mice. Adrenocortical ultrastructure illustrated no morphological alterations in Tspo(-/-) mice. In an attempt to correlate our in vivo findings to previously used in vitro models, we also determined that siRNA knockdown or the absence of TSPO in different mouse and human steroidogenic cell lines had no effect on steroidogenesis. These findings directly refute the dogma that TSPO is indispensable for steroid hormone biosynthesis and viability. By amending the current model, this study advances our understanding of steroidogenesis with broad implications in biology and medicine. PMID:24936060

  6. Presence of a truncated form of the vitamin D receptor (VDR) in a strain of VDR-knockout mice.

    PubMed

    Bula, Craig M; Huhtakangas, Johanna; Olivera, Christopher; Bishop, June E; Norman, Anthony W; Henry, Helen L

    2005-12-01

    As part of our studies on the membrane-initiated actions of 1alpha,25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)] and its localization in caveolae membrane fractions, we used a vitamin D receptor (VDR)-knockout (KO) mouse model to study the binding of [(3)H]-1alpha,25(OH)(2)D(3) in the presumed absence of the VDR. In this mouse model, known as the Tokyo strain, the second exon of the VDR gene, which encodes the first of the two zinc fingers responsible for DNA binding, was removed, and the resulting animals have been considered to be VDR-null mice. To our surprise, several tissues in these KO mice showed significant (5-50% of that seen in wild-type animals) specific binding of [(3)H]-1alpha,25(OH)(2)D(3) in nuclear and caveolae membrane fractions. The dissociation constants of this binding in samples from VDR-KO and wild-type mice were indistinguishable. RT-PCR analysis of intestinal mRNA from the VDR-KO animals revealed an mRNA that lacks exon 2 but contains exons 3-9 plus two 5'-untranslated exons. Western analysis of intestinal extracts from VDR-KO mice showed a protein of a size consistent with the use of Met52 as the translational start site. Transfection of a plasmid construct containing the sequence encoding the human analog of this truncated form of the receptor, VDR(52-C), into Cos-1 cells showed that this truncated form of the receptor retains full [(3)H]-1alpha,25(OH)(2)D(3) binding ability. This same construct was inactive in transactivation assays using the osteocalcin promoter in CV1 cells. Thus, we have determined that this widely used strain of the VDR-KO mouse can express a form of the VDR that can bind ligand but not activate gene transcription. PMID:16150907

  7. Knockout of Angiotensin AT2 receptors accelerates healing but impairs quality

    PubMed Central

    Faghih, Mahya; Hosseini, Sayed M.; Smith, Barbara; Ansari, Amir Mehdi.; Lay, Frank; Ahmed, Ali Karim; Inagami, Tedashi; Marti, Guy P.; Harmon, John W.; Walston, Jeremy D.; Abadir, Peter M.

    2015-01-01

    Wounds are among the most common, painful, debilitating and costly conditions in older adults. Disruption of the angiotensin type 1 receptors (AT1R), has been associated with impaired wound healing, suggesting a critical role for AT1R in this repair process. Biological functions of angiotensin type 2 receptors (AT2R) are less studied. We investigated effects of genetically disrupting AT2R on rate and quality of wound healing. Our results suggest that AT2R effects on rate of wound closure depends on the phase of wound healing. We observed delayed healing during early phase of wound healing (inflammation). An accelerated healing rate was seen during later stages (proliferation and remodeling). By day 12, fifty percent of AT2R−/− mice had complete wound closure as compared to none in either C57/BL6 or AT1R−/− mice. There was a significant increase in AT1R, TGFβ1 and TGFβ2 expression during the proliferative and remodeling phases in AT2R−/− mice. Despite the accelerated closure rate, AT2R−/− mice had more fragile healed skin. Our results suggest that in the absence of AT2R, wound healing rate is accelerated, but yielded worse skin quality. Elucidating the contribution of both of the angiotensin receptors may help fine tune future intervention aimed at wound repair in older individuals. PMID:26727887

  8. Deletion of the UT receptor gene results in the selective loss of urotensin-II contractile activity in aortae isolated from UT receptor knockout mice.

    PubMed

    Behm, David J; Harrison, Stephen M; Ao, Zhaohui; Maniscalco, Kristeen; Pickering, Susan J; Grau, Evelyn V; Woods, Tina N; Coatney, Robert W; Doe, Christopher P A; Willette, Robert N; Johns, Douglas G; Douglas, Stephen A

    2003-05-01

    1 Urotensin-II (U-II) is among the most potent mammalian vasoconstrictors identified and may play a role in the aetiology of essential hypertension. Currently, only one mouse U-II receptor (UT) gene has been cloned. It is postulated that this protein is solely responsible for mediating U-II-induced vasoconstriction. 2 This hypothesis has been investigated in the present study, which assessed basal haemodynamics and vascular reactivity to hU-II in wild-type (UT((+/+))) and UT receptor knockout (UT((-/-))) mice. 3 Basal left ventricular end-diastolic and end-systolic volumes/pressures, stroke volumes, mean arterial blood pressures, heart rates, cardiac outputs and ejection fractions in UT((+/+)) mice and in UT((-/-)) mice were similar. 4 Relative to UT((+/+)) mouse isolated thoracic aorta, where hU-II was a potent spasmogen (pEC(50)=8.26+/-0.08) that evoked relatively little vasoconstriction (17+/-2% 60 mM KCl), vessels isolated from UT((-/-)) mice did not respond to hU-II. However, in contrast, the superior mesenteric artery isolated from both the genotypes did not contract in the presence of hU-II. Reactivity to unrelated vasoconstrictors (phenylephrine, endothelin-1, KCl) and endothelium-dependent/independent vasodilator agents (carbachol, sodium nitroprusside) was similar in the aorta and superior mesenteric arteries isolated from both the genotypes. 5 The present study is the first to directly link hU-II-induced vasoconstriction with the UT receptor. Deletion of the UT receptor gene results in loss of hU-II contractile action with no 'nonspecific' alterations in vascular reactivity. However, as might be predicted based on the limited contractile efficacy recorded in vitro, the contribution that hU-II and its receptor make to basal systemic haemodynamics appears to be negligible in this species. PMID:12770952

  9. Serotonin 5-HT3 receptors in rat CA1 hippocampal interneurons: functional and molecular characterization

    PubMed Central

    Sudweeks, Sterling N; van Hooft, Johannes A; Yakel, Jerrel L

    2002-01-01

    The molecular makeup of the serotonin 5-HT3 receptor (5-HT3R) channel was investigated in rat hippocampal CA1 interneurons in slices using single-cell RT-PCR and patch-clamp recording techniques. We tested for the expression of the 5-HT3A (both short and long splice variants) and 5-HT3B subunits, as well as the expression of the α4 subunit of the neuronal nicotinic ACh receptors (nAChRs), the latter of which has been shown to co-assemble with the 5-HT3A subunit in heterologous expression systems. Both the 5-HT3A-short and α4-nAChR subunits were expressed in these interneurons, but we could not detect any expression of either the 5-HT3B or the 5-HT3A-long subunits. Furthermore, there was a strong tendency for the 5-HT3A-short and α4-nAChR subunits to be co-expressed in individual interneurons. To assess whether there was any functional evidence for co-assembly between the 5-HT3A-short and α4-nAChR subunits, we used the sulphydryl agent 2-aminoethyl methanethiosulphonate (MTSEA), which has previously been shown to modulate expressed 5-HT3Rs that contain the α4-nAChR subunit. In half of the interneurons examined, MTSEA significantly enhanced the amplitude of the 5-HT3R-mediated responses, which is consistent with the notion that the α4-nAChR subunit co-assembles with the 5-HT3A subunit to form a native heteromeric 5-HT3R channel in rat CA1 hippocampal interneurons in vivo. In addition, the single-channel properties of the 5-HT3R were investigated in outside-out patches. No resolvable single-channel currents were observed. Using non-stationary fluctuation analysis, we obtained an estimate of the single-channel conductance of 4 pS, which is well below that expected for channels containing both the 5-HT3A and 5-HT3B subunits. PMID:12411518

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

    SciTech Connect

    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.

  11. Cholesterol-Induced Hepatic Inflammation Does Not Underlie the Predisposition to Insulin Resistance in Dyslipidemic Female LDL Receptor Knockout Mice

    PubMed Central

    Gruben, Nanda; Funke, Anouk; Kloosterhuis, Niels J.; Schreurs, Marijke; Sheedfar, Fareeba; Havinga, Rick; Houten, Sander M.; 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 towards insulin resistance in low-density lipoprotein receptor knock-out (Ldlr−/−) mice. For this, wild type (WT) and Ldlr−/− mice were fed a chow diet, a high fat (HF) diet, or a high fat, high cholesterol (HFC) diet for 2 weeks. Plasma lipid levels were elevated in chow-fed Ldlr−/− mice compared to WT mice. Although short-term HF or HFC feeding did not result in body weight gain and adipose tissue inflammation, dyslipidemia was worsened in Ldlr−/− mice compared to WT mice. In addition, dyslipidemic HF-fed Ldlr−/− mice had a higher hepatic glucose production rate than HF-fed WT mice, while peripheral insulin resistance was unaffected. This suggests that HF-fed Ldlr−/− mice suffered from hepatic insulin resistance. While HFC-fed Ldlr−/− mice displayed the anticipated increased hepatic inflammation, this did neither exacerbate systemic nor hepatic insulin resistance. Therefore, our results show that hepatic insulin resistance is unrelated to cholesterol-induced hepatic inflammation in Ldlr−/− mice, indicating that hepatic inflammation may not contribute to metabolic dysfunction per se. PMID:25815343

  12. Metabolic alterations due to caloric restriction and every other day feeding in normal and growth hormone receptor knockout mice.

    PubMed

    Westbrook, Reyhan; Bonkowski, Michael S; Arum, Oge; Strader, April D; Bartke, Andrzej

    2014-01-01

    Mutations causing decreased somatotrophic signaling are known to increase insulin sensitivity and extend life span in mammals. Caloric restriction and every other day (EOD) dietary regimens are associated with similar improvements to insulin signaling and longevity in normal mice; however, these interventions fail to increase insulin sensitivity or life span in growth hormone receptor knockout (GHRKO) mice. To investigate the interactions of the GHRKO mutation with caloric restriction and EOD dietary interventions, we measured changes in the metabolic parameters oxygen consumption (VO2) and respiratory quotient produced by either long-term caloric restriction or EOD in male GHRKO and normal mice. GHRKO mice had increased VO2, which was unaltered by diet. In normal mice, EOD diet caused a significant reduction in VO2 compared with ad libitum (AL) mice during fed and fasted conditions. In normal mice, caloric restriction increased both the range of VO2 and the difference in minimum VO2 between fed and fasted states, whereas EOD diet caused a relatively static VO2 pattern under fed and fasted states. No diet significantly altered the range of VO2 of GHRKO mice under fed conditions. This provides further evidence that longevity-conferring diets cause major metabolic changes in normal mice, but not in GHRKO mice. PMID:23833202

  13. Oxytocin receptor and Mecp2 308/Y knockout mice exhibit altered expression of autism-related social behaviors.

    PubMed

    Pobbe, Roger L H; Pearson, Brandon L; Blanchard, D Caroline; Blanchard, Robert J

    2012-12-01

    The development of tasks measuring behaviors specific to the three major symptom categories for autism makes it possible to differentiate mouse models of autism spectrum disorders (ASD) in terms of changes in these specific categories. Prior studies indicate that BTBR T+tf/J mice, the strain that has been evaluated most extensively, show autism-relevant changes in all three symptom categories; reciprocal social interactions; communication; and repetitive, ritualized behaviors. This report reviews the behaviors of oxytocin receptor (Oxtr) and Mecp2(308/Y) wild-type (WT) and knockout (KO) mice, in a number of tests specifically designed to provide information on behaviors that may show functional parallels to the core symptoms of ASD. Oxtr KO mice show robust decreases in reciprocal social interactions, and reduced levels of communication, but no changes in repetitive, ritualized behaviors; whereas Mecp2(308/Y) KO mice show a slight but consistent enhancement of social behavior and communication, and no changes in repetitive, ritualized behaviors. This data base, although small, strongly indicates that mouse models can sort the diagnostic symptoms of autism, and suggests that biological and physiological analyses of these strains may be capable of providing differential information on the brain systems involved in particular symptoms of this disorder. Profiles of behavioral changes in other mouse models of ASD should provide additional specificity in the search for biomarkers associated with particular ASD symptoms and symptom clusters. PMID:22406388

  14. Knockout of fractalkine receptor Cx3cr1 does not alter disease or microglial activation in prion-infected mice.

    PubMed

    Striebel, James F; Race, Brent; Carroll, James A; Phillips, Katie; Chesebro, Bruce

    2016-06-01

    Microglial activation is a hallmark of the neuroimmunological response to Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis and prion disease. The CX3C chemokine axis consists of fractalkine (CX3CL1) and its receptor (CX3CR1); these are expressed by neurons and microglia respectively, and are known to modulate microglial activation. In prion-infected mice, both Cx3cr1 and Cx3cl1 are altered, suggesting a role in disease. To investigate the influence of CX3C axis signalling on prion disease, we infected Cx3cr1 knockout (Cx3cr1-KO) and control mice with scrapie strains 22L and RML. Deletion of Cx3cr1 had no effect on development of clinical signs or disease incubation period. In addition, comparison of brain tissue from Cx3cr1-KO and control mice revealed no significant differences in cytokine levels, spongiosis, deposition of disease-associated prion protein or microglial activation. Thus, microglial activation during prion infection did not require CX3C axis signalling. PMID:26935332

  15. Endothelial cell-specific aryl hydrocarbon receptor knockout mice exhibit hypotension mediated, in part, by an attenuated angiotensin II responsiveness

    PubMed Central

    Agbor, Larry N.; Elased, Khalid M.; Walker, Mary K.

    2011-01-01

    Hypotension in aryl hydrocarbon receptor knockout mice (ahr−/−) is mediated, in part, by a reduced contribution of angiotensin (Ang) II to basal blood pressure (BP). Since AHR is highly expressed in endothelial cells (EC), we hypothesized that EC-specific ahr−/− (ECahr−/−) mice would exhibit a similar phenotype. We generated ECahr−/− mice by crossing AHR floxed mice (ahrfx/fx) to mice expressing Cre recombinase driven by an EC-specific promoter. BP was assessed by radiotelemetry prior to and following an acute injection of Ang II or chronic treatment with an angiotensin converting enzyme inhibitor (ACEi). ECahr−/− mice were hypotensive (ECahr+/+: 116.1 ± 1.4; ECahr−/−: 107.4 ± 2.0 mmHg, n=11, p<0.05) and exhibited significantly different responses to Ang II and ACEi. While Ang II increased BP in both genotypes, the increase was sustained in ECahr+/+, whereas the increase in ECahr−/− mice steadily declined. Area under the curve analysis showed that Ang II-induced increase in diastolic BP (DBP) over 30 min was significantly lower in ECahr−/− mice (ECahr+/+ 1297 ± 223 mmHg/30 min; ECahr−/−AUC: 504 ± 138 mmHg/30 min, p<0.05). In contrast, while ACEi decreased BP in both genotypes, the subsequent rise in DBP after treatment was significantly delayed in the ECahr−/− mice. ECahr−/− mice also exhibited reduced vascular and adipose Ang II type 1 receptor (AT1R) expression, and reduced aortic Ang II-dependent vasoconstriction in the presence of vascular adipose. Taken together these data suggest that hypotension in ECahr−/− mice results from reduced vascular responsiveness to Ang II that is influenced by AT1R expression and adipose. PMID:21684261

  16. Increased Activation of the Wnt/β-Catenin Pathway in Spontaneous Hepatocellular Carcinoma Observed in Farnesoid X Receptor Knockout Mice

    PubMed Central

    Wolfe, Andy; Thomas, Ann; Edwards, Genea; Jaseja, Reshma; Guo, Grace L.

    2011-01-01

    Farnesoid X receptor (FXR), the primary bile acid-sensing nuclear receptor, also is known for its anticancer properties. It is known that FXR deficiency in mice results in spontaneous hepatocellular carcinoma (HCC), but the mechanisms are not completely understood. We report that sustained activation of the Wnt/β-catenin pathway is associated with spontaneous HCC in FXR-knockout (KO) mice. HCC development was studied in FXR-KO mice at 3, 8, and 14 months of age. No tumors were observed at either 3 or 8 months, but the presence of HCC was observed in 100% of the FXR-KO mice at the age of 14 months. Further analysis revealed no change in β-catenin activation in the livers of 3-month-old FXR-KO mice, but a moderate increase was observed in 8-month-old FXR-KO mice. β-Catenin activation further increased significantly in 14-month-old tumor-bearing mice. Further analysis revealed that two independent mechanisms might be involved in β-catenin activation in the livers of FXR-KO mice. Activation of canonical Wnt signaling was evident as indicated by increased Wnt4 and dishevelled expression along with glycogen synthase kinase-3β inactivation. We also observed decreased expression of E-cadherin, a known regulator of β-catenin, in FXR-KO mice. The decrease in E-cadherin expression was accompanied by increased expression of its transcriptional repressor, Snail. Consistent with the increased HCC in FXR-KO mice, we observed a significant decrease in FXR expression and activity in human HCC samples. Taken together, these data indicate that a temporal increase in the activation of Wnt/β-catenin is observed during spontaneous HCC development in FXR-KO mice and is potentially critical for tumor development. PMID:21430080

  17. The role of RXFP2 in mediating androgen-induced inguinoscrotal testis descent in LH receptor knockout mice

    PubMed Central

    Yuan, F P; Li, X; Lin, J; Schwabe, C; Büllesbach, E E; Rao, C V; Lei, Z M

    2011-01-01

    LH receptor knockout (LhrKO) male mice exhibit a bilateral cryptorchidism resulting from a developmental defect in the gubernaculum during the inguinoscrotal phase of testis descent, which is corrected by testosterone replacement therapy (TRT). In vivo and in vitro experiments were conducted to investigate the roles of the androgen receptor (AR) and RXFP2 signals in regulation of gubernacular development in LhrKO animals. This study demonstrated that AR and RXFP2 proteins were expressed in the gubernaculum during the entire postnatal period. TRT normalized gubernacular RXFP2 protein levels inLhrKO mice. Organ and primary cell cultures of gubernacula showed that 5α-dihydrotestosterone (DHT) upregulated the expression of Rxfp2 which was abolished by the addition of an AR antagonist, flutamide. A single s.c. testosterone injection also led to a significant increase in Rxfp2 mRNA levels in a time-dependent fashion in LhrKO animals. DHT, natural and synthetic insulin-like peptide 3 (INSL3), or relaxin alone did not affect proliferation of gubernacular mesenchymal cells, while co-treatments of DHT with either INSL3 or relaxin resulted in an increase in cell proliferation, and they also enhanced the mesenchymal cell differentiation toward the myogenic pathway, which included a decrease in a mesenchymal cell marker, CD44 and the expression of troponin. These effects were attenuated by the addition of flutamide, siRNA-mediated Rxfp2 knockdown, or by an INSL3 antagonist. Co-administration of an INSL3 antagonist curtailed TRT-induced inguinoscrotal testis descent in LhrKO mice. Our findings indicate that the RXFP2 signaling pathway plays an important role in mediating androgen action to stimulate gubernaculum development during inguinoscrotal testis descent. PMID:20154177

  18. Progesterone receptor knockout mice have an improved glucose homeostasis secondary to -cell proliferation

    NASA Astrophysics Data System (ADS)

    Picard, Frédéric; Wanatabe, Mitsuhiro; Schoonjans, Kristina; Lydon, John; O'Malley, Bert W.; Auwerx, Johan

    2002-11-01

    Gestational diabetes coincides with elevated circulating progesterone levels. We show that progesterone accelerates the progression of diabetes in female db/db mice. In contrast, RU486, an antagonist of the progesterone receptor (PR), reduces blood glucose levels in both female WT and db/db mice. Furthermore, female, but not male, PR-/- mice had lower fasting glycemia than PR+/+ mice and showed higher insulin levels on glucose injection. Pancreatic islets from female PR-/- mice were larger and secreted more insulin consequent to an increase in -cell mass due to an increase in -cell proliferation. These findings demonstrate an important role of progesterone signaling in insulin release and pancreatic function and suggest that it affects the susceptibility to diabetes.

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

    PubMed Central

    Xin, Wei; Li, Zhaoping; Chen, Liyong

    2016-01-01

    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. PMID:27077005

  20. Abnormal Mitochondrial Function and Impaired Granulosa Cell Differentiation in Androgen Receptor Knockout Mice

    PubMed Central

    Wang, Ruey-Sheng; Chang, Heng-Yu; Kao, Shu-Huei; Kao, Cheng-Heng; Wu, Yi-Chen; Yeh, Shuyuan; Tzeng, Chii-Reuy; Chang, Chawnshang

    2015-01-01

    In the ovary, the paracrine interactions between the oocyte and surrounded granulosa cells are critical for optimal oocyte quality and embryonic development. Mice lacking the androgen receptor (AR−/−) were noted to have reduced fertility with abnormal ovarian function that might involve the promotion of preantral follicle growth and prevention of follicular atresia. However, the detailed mechanism of how AR in granulosa cells exerts its effects on oocyte quality is poorly understood. Comparing in vitro maturation rate of oocytes, we found oocytes collected from AR−/− mice have a significantly poor maturating rate with 60% reached metaphase II and 30% remained in germinal vesicle breakdown stage, whereas 95% of wild-type AR (AR+/+) oocytes had reached metaphase II. Interestingly, we found these AR−/− female mice also had an increased frequency of morphological alterations in the mitochondria of granulosa cells with reduced ATP generation (0.18 ± 0.02 vs. 0.29 ± 0.02 µM/mg protein; p < 0.05) and aberrant mitochondrial biogenesis. Mechanism dissection found loss of AR led to a significant decrease in the expression of peroxisome proliferator-activated receptor γ (PPARγ) co-activator 1-β (PGC1-β) and its sequential downstream genes, nuclear respiratory factor 1 (NRF1) and mitochondrial transcription factor A (TFAM), in controlling mitochondrial biogenesis. These results indicate that AR may contribute to maintain oocyte quality and fertility via controlling the signals of PGC1-β-mediated mitochondrial biogenesis in granulosa cells. PMID:25941928

  1. Dopamine-dependent CB1 receptor dysfunction at corticostriatal synapses in homozygous PINK1 knockout mice.

    PubMed

    Madeo, G; Schirinzi, T; Maltese, M; Martella, G; Rapino, C; Fezza, F; Mastrangelo, N; Bonsi, P; Maccarrone, M; Pisani, A

    2016-02-01

    Recessive mutations in the PTEN-induced putative kinase 1 (PINK1) gene cause early-onset Parkinson's disease (PD). We investigated the interaction between endocannabinoid (eCB) and dopaminergic transmission at corticostriatal synapses in PINK1 deficient mice. Whole-cell patch-clamp and conventional recordings of striatal medium spiny neurons (MSNs) were made from slices of PINK1(-/-), heterozygous PINK1(+/-) mice and wild-type littermates (PINK1(+/+)). In PINK1(+/+) mice, CB1 receptor (CB1R) activation reduced spontaneous excitatory postsynaptic currents (sEPSCs). Likewise, CB1R agonists (ACEA, WIN55,212-3 and HU210) induced a dose-dependent reduction of cortically-evoked excitatory postsynaptic potential (eEPSP) amplitude. While CB1R agonists retained their inhibitory effect in heterozygous PINK1(+/-) mice, conversely, in PINK1(-/-) mice they failed to modulate sEPSC amplitude. Similarly, CB1R activation failed to reduce eEPSP amplitude in PINK1(-/-) mice. Parallel biochemical measurements revealed no significant difference in the levels of the two main eCBs, 2-arachidonoylglycerol (2-AG) and anandamide (AEA) in PINK1(-/-) striata. Similarly, no change was observed in the enzymatic activity of both fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), responsible for eCB hydrolysis. Instead, a significant reduction of binding ability of CB1R agonists was found in PINK1(-/-) mice. Notably, the CB1R-dependent inhibition of synaptic activity was restored either by amphetamine or after chronic treatment with the D2 dopamine receptor agonist quinpirole. Additionally, CB1R binding activity returned to control levels after chronic pretreatment with quinpirole. Consistent with the hypothesis of a close interplay with dopaminergic neurotransmission, our findings show a CB1R dysfunction at corticostriatal synapses in PINK1(-/-), but not in PINK1(+/-) mice, and provide a mechanistic link to the distinct plasticity deficits observed in both genotypes. PMID

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

    SciTech Connect

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

    2013-10-15

    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{sup −/−} and SHP{sup −/−} 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{sup −/−} mice and therefore, increased SHP expression in FXR{sup −/−} mice reduces liver tumorigenesis. To test this hypothesis, we generated FXR{sup −/−} mice with overexpression of SHP in hepatocytes (FXR{sup −/−}/SHP{sup Tg}) and determined the contribution of SHP in HCC development in FXR{sup −/−} mice. Hepatocyte-specific SHP overexpression did not affect liver tumor incidence or size in FXR{sup −/−} 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{sup −/−} 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.

  3. Knockout of the aryl hydrocarbon receptor results in distinct hepatic and renal phenotypes in rats and mice

    SciTech Connect

    Harrill, Joshua A.; Hukkanen, Renee R.; Lawson, Marie; Martin, Greg; Gilger, Brian; Soldatow, Valerie; LeCluyse, Edward L.; Budinsky, Robert A.; Rowlands, J. Craig; Thomas, Russell S.

    2013-10-15

    The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor which plays a role in the development of multiple tissues and is activated by a large number of ligands, including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). In order to examine the roles of the AHR in both normal biological development and response to environmental chemicals, an AHR knockout (AHR-KO) rat model was created and compared with an existing AHR-KO mouse. AHR-KO rats harboring either 2-bp or 29-bp deletion mutation in exon 2 of the AHR were created on the Sprague–Dawley genetic background using zinc-finger nuclease (ZFN) technology. Rats harboring either mutation type lacked expression of AHR protein in the liver. AHR-KO rats were also insensitive to thymic involution, increased hepatic weight and the induction of AHR-responsive genes (Cyp1a1, Cyp1a2, Cyp1b1, Ahrr) following acute exposure to 25 μg/kg TCDD. AHR-KO rats had lower basal expression of transcripts for these genes and also accumulated ∼ 30–45-fold less TCDD in the liver at 7 days post-exposure. In untreated animals, AHR-KO mice, but not AHR-KO rats, had alterations in serum analytes indicative of compromised hepatic function, patent ductus venosus of the liver and persistent hyaloid arteries in the eye. AHR-KO rats, but not AHR-KO mice, displayed pathological alterations to the urinary tract: bilateral renal dilation (hydronephrosis), secondary medullary tubular and uroepithelial degenerative changes and bilateral ureter dilation (hydroureter). The present data indicate that the AHR may play significantly different roles in tissue development and homeostasis and toxicity across rodent species. - Highlights: • An AHR knockout rat was generated on a Sprague–Dawley outbred background. • AHR-KO rats lack expression of AHR protein. • AHR-KO rats are insensitive to TCDD-mediated effects. • Data suggests difference in the role of AHR in tissue development of rats and mice. • Abnormalities in vascular

  4. Development of schemas revealed by prior experience and NMDA receptor knock-out

    PubMed Central

    Dragoi, George; Tonegawa, Susumu

    2013-01-01

    Prior experience accelerates acquisition of novel, related information through processes like assimilation into mental schemas, but the underlying neuronal mechanisms are poorly understood. We investigated the roles that prior experience and hippocampal CA3 N-Methyl-D-aspartate receptor (NMDAR)-dependent synaptic plasticity play in CA1 place cell sequence encoding and learning during novel spatial experiences. We found that specific representations of de novo experiences on linear environments were formed on a framework of pre configured network activity expressed in the preceding sleep and were rapidly, flexibly adjusted via NMDAR-dependent activity. This prior experience accelerated encoding of subsequent experiences on contiguous or isolated novel tracks, significantly decreasing their NMDAR-dependence. Similarly, de novo learning of an alternation task was facilitated by CA3 NMDARs; this experience accelerated subsequent learning of related tasks, independent of CA3 NMDARs, consistent with a schema-based learning. These results reveal the existence of distinct neuronal encoding schemes which could explain why hippocampal dysfunction results in anterograde amnesia while sparing recollection of old, schema-based memories. DOI: http://dx.doi.org/10.7554/eLife.01326.001 PMID:24327561

  5. Altered enteroendocrine cell expression in T cell receptor alpha chain knock-out mice.

    PubMed

    Rubin, D C; Zhang, H; Qian, P; Lorenz, R G; Hutton, K; Peters, M G

    2000-10-15

    Mice lacking T cell receptor alpha chain (TCRalpha(-/-)) develop inflammation of the colon. We have examined the effect of this inflammation on the colonic epithelium by studying markers of epithelial cuff, enteroendocrine, and immune cell differentiation. Using immunohistochemical techniques, colons were compared in normal C57/BL6 and murine TCR alpha(-/-) mice aged 2 and 3 weeks and 3-11 months. TCR alpha(-/-) mice aged 3-11 months had histologic evidence of inflammation with increased expression of CD45, CD4+, CD8+, and B220+ cells and a decrease in expression of IgA+ cells. There was a decrease in the number of cholecystokinin, serotonin, and neurotensin enteroendocrine expressing cells in the colon of TCR alpha(-/-) mice. These changes were not present in 2-3-week-old suckling/weaning mice. In contrast, peptide tyrosine tyrosine (PYY), glucagon-like peptide-1, and gastrin expression did not change and small intestinal enteroendocrine cells remained unaltered. The change in colonic enteroendocrine cell expression appears to be a specific response, since only a subset of these cells was altered, and the epithelium was intact by histologic analysis. The absence of functional T cells in TCR alpha(-/-) colon has a marked effect on differentiation of a specific subpopulation of enteroendocrine cells, prior to loss of integrity of the epithelium. PMID:11054861

  6. Quercetin inhibits the 5-hydroxytryptamine type 3 receptor-mediated ion current by interacting with pre-transmembrane domain I.

    PubMed

    Lee, Byung-Hwan; Jeong, Sang-Min; Jung, Sang-Min; Lee, Jun-Ho; Kim, Jong-Hoon; Yoon, In-Soo; Lee, Joon-Hee; Choi, Sun-Hye; Lee, Sang-Mok; Chang, Choon-Gon; Kim, Hyung-Chun; Han, YeSun; Paik, Hyun-Dong; Kim, Yangmee; Nah, Seung-Yeol

    2005-08-31

    The flavonoid, quercetin, is a low molecular weight substance found in apple, tomato and other fruit. Besides its antioxidative effect, quercetin, like other flavonoids, has a wide range of neuropharmacological actions including analgesia, and motility, sleep, anticonvulsant, sedative and anxiolytic effects. In the present study, we investigated its effect on mouse 5-hydroxytryptamine type 3 (5-HT3A) receptor channel activity, which is involved in pain transmission, analgesia, vomiting, and mood disorders. The 5-HT3A receptor was expressed in Xenopus oocytes, and the current was measured with the two-electrode voltage clamp technique. In oocytes injected with 5-HT3A receptor cRNA, quercetin inhibited the 5-HT-induced inward peak current (I(5-HT)) with an IC50 of 64.7 +/- 2.2 microM. Inhibition was competitive and voltage-independent. Point mutations of pre-transmembrane domain 1 (pre-TM1) such as R222T and R222A, but not R222D, R222E and R222K, abolished inhibition, indicating that quercetin interacts with the pre-TM1 of the 5-HT3A receptor. PMID:16258243

  7. 5-HT3 Receptors

    PubMed Central

    Thompson, A. J.; Lummis, S. C. R.

    2009-01-01

    The 5-HT3 receptor is a member of the Cys-loop family of ligand-gated ion channels. These receptors are located in both the peripheral and central nervous systems, where functional receptors are constructed from five subunits. These subunits may be the same (homopentameric 5-HT3A receptors) or different (heteropentameric receptors, usually comprising of 5-HT3A and 5-HT3B receptor subunits), with the latter having a number of distinct properties. The 5-HT3 receptor binding site is comprised of six loops from two adjacent subunits, and critical ligand binding amino acids in these loops have been largely identified. There are a range of selective agonists and antagonists for these receptors and the pharmacophore is reasonably well understood. There are also a wide range of compounds that can modulate receptor activity. Studies have suggested many diverse potential disease targets that might be amenable to alleviation by 5-HT3 receptor selective compounds but to date only two applications have been fully realised in the clinic: the treatment of emesis and irritable-bowel syndrome. PMID:17073663

  8. Knockout of the aryl hydrocarbon receptor results in distinct hepatic and renal phenotypes in rats and mice.

    PubMed

    Harrill, Joshua A; Hukkanen, Renee R; Lawson, Marie; Martin, Greg; Gilger, Brian; Soldatow, Valerie; Lecluyse, Edward L; Budinsky, Robert A; Rowlands, J Craig; Thomas, Russell S

    2013-10-15

    The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor which plays a role in the development of multiple tissues and is activated by a large number of ligands, including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). In order to examine the roles of the AHR in both normal biological development and response to environmental chemicals, an AHR knockout (AHR-KO) rat model was created and compared with an existing AHR-KO mouse. AHR-KO rats harboring either 2-bp or 29-bp deletion mutation in exon 2 of the AHR were created on the Sprague-Dawley genetic background using zinc-finger nuclease (ZFN) technology. Rats harboring either mutation type lacked expression of AHR protein in the liver. AHR-KO rats were also insensitive to thymic involution, increased hepatic weight and the induction of AHR-responsive genes (Cyp1a1, Cyp1a2, Cyp1b1, Ahrr) following acute exposure to 25 μg/kg TCDD. AHR-KO rats had lower basal expression of transcripts for these genes and also accumulated ~30-45-fold less TCDD in the liver at 7 days post-exposure. In untreated animals, AHR-KO mice, but not AHR-KO rats, had alterations in serum analytes indicative of compromised hepatic function, patent ductus venosus of the liver and persistent hyaloid arteries in the eye. AHR-KO rats, but not AHR-KO mice, displayed pathological alterations to the urinary tract: bilateral renal dilation (hydronephrosis), secondary medullary tubular and uroepithelial degenerative changes and bilateral ureter dilation (hydroureter). The present data indicate that the AHR may play significantly different roles in tissue development and homeostasis and toxicity across rodent species. PMID:23859880

  9. Knockout of NMDA-receptors from parvalbumin interneurons sensitizes to schizophrenia-related deficits induced by MK-801.

    PubMed

    Bygrave, A M; Masiulis, S; Nicholson, E; Berkemann, M; Barkus, C; Sprengel, R; Harrison, P J; Kullmann, D M; Bannerman, D M; Kätzel, D

    2016-01-01

    It has been suggested that a functional deficit in NMDA-receptors (NMDARs) on parvalbumin (PV)-positive interneurons (PV-NMDARs) is central to the pathophysiology of schizophrenia. Supportive evidence come from examination of genetically modified mice where the obligatory NMDAR-subunit GluN1 (also known as NR1) has been deleted from PV interneurons by Cre-mediated knockout of the corresponding gene Grin1 (Grin1(ΔPV) mice). Notably, such PV-specific GluN1 ablation has been reported to blunt the induction of hyperlocomotion (a surrogate for psychosis) by pharmacological NMDAR blockade with the non-competitive antagonist MK-801. This suggests PV-NMDARs as the site of the psychosis-inducing action of MK-801. In contrast to this hypothesis, we show here that Grin1(ΔPV) mice are not protected against the effects of MK-801, but are in fact sensitized to many of them. Compared with control animals, Grin1(ΔPV)mice injected with MK-801 show increased stereotypy and pronounced catalepsy, which confound the locomotor readout. Furthermore, in Grin1(ΔPV)mice, MK-801 induced medial-prefrontal delta (4 Hz) oscillations, and impaired performance on tests of motor coordination, working memory and sucrose preference, even at lower doses than in wild-type controls. We also found that untreated Grin1(ΔPV)mice are largely normal across a wide range of cognitive functions, including attention, cognitive flexibility and various forms of short-term memory. Taken together these results argue against PV-specific NMDAR hypofunction as a key starting point of schizophrenia pathophysiology, but support a model where NMDAR hypofunction in multiple cell types contribute to the disease. PMID:27070406

  10. Knockout of NMDA-receptors from parvalbumin interneurons sensitizes to schizophrenia-related deficits induced by MK-801

    PubMed Central

    Bygrave, A M; Masiulis, S; Nicholson, E; Berkemann, M; Barkus, C; Sprengel, R; Harrison, P J; Kullmann, D M; Bannerman, D M; Kätzel, D

    2016-01-01

    It has been suggested that a functional deficit in NMDA-receptors (NMDARs) on parvalbumin (PV)-positive interneurons (PV-NMDARs) is central to the pathophysiology of schizophrenia. Supportive evidence come from examination of genetically modified mice where the obligatory NMDAR-subunit GluN1 (also known as NR1) has been deleted from PV interneurons by Cre-mediated knockout of the corresponding gene Grin1 (Grin1ΔPV mice). Notably, such PV-specific GluN1 ablation has been reported to blunt the induction of hyperlocomotion (a surrogate for psychosis) by pharmacological NMDAR blockade with the non-competitive antagonist MK-801. This suggests PV-NMDARs as the site of the psychosis-inducing action of MK-801. In contrast to this hypothesis, we show here that Grin1ΔPV mice are not protected against the effects of MK-801, but are in fact sensitized to many of them. Compared with control animals, Grin1ΔPVmice injected with MK-801 show increased stereotypy and pronounced catalepsy, which confound the locomotor readout. Furthermore, in Grin1ΔPVmice, MK-801 induced medial-prefrontal delta (4 Hz) oscillations, and impaired performance on tests of motor coordination, working memory and sucrose preference, even at lower doses than in wild-type controls. We also found that untreated Grin1ΔPVmice are largely normal across a wide range of cognitive functions, including attention, cognitive flexibility and various forms of short-term memory. Taken together these results argue against PV-specific NMDAR hypofunction as a key starting point of schizophrenia pathophysiology, but support a model where NMDAR hypofunction in multiple cell types contribute to the disease. PMID:27070406

  11. Receptor-selective IL-4 mutein modulates inflammatory vascular cell phenotypes and attenuates atherogenesis in apolipoprotein E-knockout mice.

    PubMed

    Lin, Yanhui; Chen, Zhiheng; Kato, Seiya

    2015-08-01

    The therapeutic potential of interleukin-4-mediated immunomodulation has not been proven in atherogenesis. Type I IL-4 receptor consists of IL-4Rα and a common γ chain, whereas type II IL-4R is a heterodimer of IL-4Rα and IL-13Rα1. Reportedly, the human IL-4 mutein IL-4/R121E is able to act as an IL-4RI-specific agonist. Here, we investigated the effect of receptor-specific IL-4 mutein on vascular cell phenotypes and atherogenesis. Initially, a plasmid expressing murine IL-4/Q116E, analogous to human IL-4/R121E, was transfected to vascular lineage cells in-vitro. IL-4/Q116E induced the activation of STAT6 in b.End3 endothelial cells, Mm1 macrophages, and splenocytes isolated from C57BL6/J (B6) mice, but it failed to activate STAT6 in SMC and J774.1 macrophages. IL-4/Q116E induced the expression of vascular cell adhesion protein-1 in b.End3 cells but not in SMC. IL-4/Q116E did not exhibit pro-inflammatory actions in either macrophage cell line. Splenocytes were also infected with an adenovirus vector expressing IL-4/Q116E (AdIL-4/Q116E). Enzyme-linked immunosorbent assay for interferon-γ, IL-10 and IL-13 revealed that AdIL-4/Q116E-infected splenocytes showed Th2 deviation. Th2 deviation and M2 marker up-regulation were further revealed in ex-vivo assays using the splenocytes isolated from AdIL-4/Q116E-infected apolipoprotein-E knockout (ApoEKO) mice. Finally, adenoviral induction of IL-4/Q116E, but not wild type IL-4, double mutein IL-4/Q116D/Y119D or control β-galactosidase, significantly attenuated in-vivo atherogenesis of ApoEKO mice. Our data suggest that IL-4 signaling plays a pivotal role in the regulation of vascular cell phenotypes, and atherogenesis. The IL-4RI-selective mutein IL-4/Q116E may have therapeutic potential in vascular diseases. PMID:26093164

  12. Toll-like receptor 4 knockout ameliorates neuroinflammation due to lung-brain interaction in mechanically ventilated mice.

    PubMed

    Chen, Ting; Chen, Chang; Zhang, Zongze; Zou, Yufeng; Peng, Mian; Wang, Yanlin

    2016-08-01

    Toll-like receptor 4 (TLR4) is a crucial receptor in the innate immune system, and increasing evidence supports its role in inflammation, stress, and tissue injury, including injury to the lung and brain. We aimed to investigate the effects of TLR4 on neuroinflammation due to the lung-brain interaction in mechanically ventilated mice. Male wild-type (WT) C57BL/6 and TLR4 knockout (TLR4 KO) mice were divided into three groups: (1) control group (C): spontaneous breathing; (2) anesthesia group (A): spontaneous breathing under anesthesia; and (3) mechanical ventilation group (MV): 6h of MV under anesthesia. The behavioral responses of mice were tested with fear conditioning tests. The histological changes in the lung and brain were assessed using hematoxylin-eosin (HE) staining. The level of TLR4 mRNA in tissue was measured using reverse transcription-polymerase chain reaction (RT-PCR). The levels of inflammatory cytokines were measured with an enzyme-linked immunosorbent assay (ELISA). Microgliosis, astrocytosis, and the TLR4 immunoreactivity in the hippocampus were measured by double immunofluorescence. MV mice exhibited impaired cognition, and this impairment was less severe in TLR4 KO mice than in WT mice. In WT mice, MV increased TLR4 mRNA expression in the lung and brain. MV induced mild lung injury, which was prevented in TLR4 KO mice. MV mice exhibited increased levels of inflammatory cytokines, increased microglia and astrocyte activation. Microgliosis was alleviated in TLR4 KO mice. MV mice exhibited increased TLR4 immunoreactivity, which was expressed in microglia and astrocytes. These results demonstrate that TLR4 is involved in neuroinflammation due to the lung-brain interaction and that TLR4 KO ameliorates neuroinflammation due to lung-brain interaction after prolonged MV. In addition, Administration of a TLR4 antagonist (100μg/mice) to WT mice also significantly attenuated neuroinflammation of lung-brain interaction due to prolonged MV. TLR4 antagonism

  13. Estrogen receptor transcription and transactivation: Estrogen receptor knockout mice: what their phenotypes reveal about mechanisms of estrogen action.

    PubMed

    Curtis Hewitt, S; Couse, J F; Korach, K S

    2000-01-01

    Natural, synthetic and environmental estrogens have numerous effects on the development and physiology of mammals. Estrogen is primarily known for its role in the development and functioning of the female reproductive system. However, roles for estrogen in male fertility, bone, the circulatory system and immune system have been established by clinical observations regarding sex differences in pathologies, as well as observations following menopause or castration. The primary mechanism of estrogen action is via binding and modulation of activity of the estrogen receptors (ERs), which are ligand-dependent nuclear transcription factors. ERs are found in highest levels in female tissues critical to reproduction, including the ovaries, uterus, cervix, mammary glands and pituitary gland. Since other affected tissues have extremely low levels of ER, indirect effects of estrogen, for example induction of pituitary hormones that affect the bone, have been proposed. The development of transgenic mouse models that lack either estrogen or ER have proven to be valuable tools in defining the mechanisms by which estrogen exerts its effects in various systems. The aim of this article is to review the mouse models with disrupted estrogen signaling and describe the associated phenotypes. PMID:11250727

  14. Attenuated Stress Response to Acute Restraint and Forced Swimming Stress in Arginine Vasopressin 1b Receptor Subtype (Avpr1b) Receptor Knockout Mice and Wild-Type Mice Treated with a Novel Avpr1b Receptor Antagonist

    PubMed Central

    Roper, J A; Craighead, M; O’Carroll, A-M; Lolait, S J

    2010-01-01

    Arginine vasopressin (AVP) synthesised in the parvocellular region of the hypothalamic paraventricular nucleus and released into the pituitary portal vessels acts on the 1b receptor subtype (Avpr1b) present in anterior pituitary corticotrophs to modulate the release of adrenocorticotrophic hormone (ACTH). Corticotrophin-releasing hormone is considered the major drive behind ACTH release; however, its action is augmented synergistically by AVP. To determine the extent of vasopressinergic influence in the hypothalamic-pituitary-adrenal axis response to restraint and forced swimming stress, we compared the stress hormone levels [plasma ACTH in both stressors and corticosterone (CORT) in restraint stress only] following acute stress in mutant Avpr1b knockout (KO) mice compared to their wild-type controls following the administration of a novel Avpr1b antagonist. Restraint and forced swimming stress-induced increases in plasma ACTH were significantly diminished in mice lacking a functional Avpr1b and in wild-type mice that had been pre-treated with Avpr1b antagonist. A corresponding decrease in plasma CORT levels was also observed in acute restraint-stressed knockout male mice, and in Avpr1b-antagonist-treated male wild-type mice. By contrast, plasma CORT levels were not reduced in acutely restraint-stressed female knockout animals, or in female wild-type animals pre-treated with Avpr1b antagonist. These results demonstrate that pharmacological antagonism or inactivation of Avpr1b causes a reduction in the hypothalamic-pituitary-adrenal (HPA) axis response, particularly ACTH, to acute restraint and forced swimming stress, and show that Avpr1b knockout mice constitute a model by which to study the contribution of Avpr1b to the HPA axis response to acute stressors. PMID:20846299

  15. Modification of female and male social behaviors in estrogen receptor beta knockout mice by neonatal maternal separation

    PubMed Central

    Tsuda, Mumeko C.; Yamaguchi, Naoko; Nakata, Mariko; Ogawa, Sonoko

    2014-01-01

    Maternal separation (MS) is an animal model mimicking the effects of early life stress on the development of emotional and social behaviors. Recent studies revealed that MS stress increased social anxiety levels in female mice and reduced peri-pubertal aggression in male mice. Estrogen receptor (ER) β plays a pivotal role in the regulation of stress responses and anxiety-related and social behaviors. Behavioral studies using ERβ knockout (βERKO) mice reported increased social investigation and decreased social anxiety in βERKO females, and elevated aggression levels in βERKO males compared to wild-type (WT) mice. In the present study, using βERKO and WT mice, we examined whether ERβ contributes to MS effects on anxiety and social behaviors. βERKO and WT mice were separated from their dam daily (4 h) from postnatal day 1–14 and control groups were left undisturbed. First, MS and ERβ gene deletion individually increased anxiety-related behaviors in the open field test, but only in female mice. Anxiety levels were not further modified in βERKO female mice subjected to MS stress. Second, βERKO female mice showed higher levels of social investigation compared with WT in the social investigation test and long-term social preference test. However, MS greatly reduced social investigation duration and elevated number of stretched approaches in WT and βERKO females in the social investigation test, suggesting elevated levels of social anxiety in both genotypes. Third, peri-pubertal and adult βERKO male mice were more aggressive than WT mice as indicated by heightened aggression duration. On the other hand, MS significantly decreased aggression duration in both genotypes, but only in peri-pubertal male mice. Altogether, these results suggest that βERKO mice are sensitive to the adverse effects of MS stress on subsequent female and male social behaviors, which could then have overrode the ERβ effects on female social anxiety and male aggression. PMID:25228857

  16. Knockout of Toll-Like Receptors 2 and 4 Prevents Renal Ischemia-Reperfusion-Induced Cardiac Hypertrophy in Mice

    PubMed Central

    Trentin-Sonoda, Mayra; da Silva, Rogério Cirino; Kmit, Fernanda Vieira; Abrahão, Mariana Vieira; Monnerat Cahli, Gustavo; Brasil, Guilherme Visconde; Muzi-Filho, Humberto; Silva, Paulo André; Tovar-Moll, Fernanda Freire; Vieyra, Adalberto; Medei, Emiliano; Carneiro-Ramos, Marcela Sorelli

    2015-01-01

    We investigated whether the pathways linked to Toll-like receptors 2 and 4 (TLRs) are involved in renal ischemia-reperfusion (I/R)-induced cardiac hypertrophy. Wild type (WT) C57BL/6J, TLR2-/- and TLR4-/- mice were subjected to left kidney ischemia for 60 min followed by reperfusion for 5, 8, 12 and 15 days. Proton density magnetic resonance showed alterations in the injured kidney from WT mice, together with signs of parenchymal edema and higher levels of vimentin mRNA, accompanied by: (i) small, but significant, increase in serum urea after 24 h, (ii) 100% increase in serum creatinine at 24 h. A serum peak of inflammatory cytokines occurred after 5 days of reperfusion. Heart weight/body weight and heart weight/tibia length ratios increased after 12 and 15 days of reperfusion, respectively. Cardiac hypertrophy markers, B-type natriuretic peptide (BNP) and α-actin, left ventricle mass, cardiac wall thickness and myocyte width increased after 15 days of reperfusion, together with longer QTc and action potential duration. Cardiac TLRs, MyD88, HSP60 and HSP70 mRNA levels also increased. After 15 days of reperfusion, absence of TLRs prevented cardiac hypertrophy, as reflected by similar values of left ventricular cardiac mass and heart weight/body weight ratio compared to the transgenic Sham. Renal tissular injury also ameliorated in both knockout mice, as revealed by the comparison of their vimentin mRNA levels with those found in the WT on the same day after I/R. The I/R TLR2-/- group had TNF-α, IFN-γ and IL-1β levels similar to the non-I/R group, whereas the TLR4-/- group conserved the p-NF-κB/NF- κB ratio contrasting with that found in TLR2-/-. We conclude: (i) TLRs are involved in renal I/R-induced cardiac hypertrophy; (ii) absence of TLRs prevents I/R-induced cardiac hypertrophy, despite renal lesions seeming to evolve towards those of chronic disease; (iii) TLR2 and TLR4 selectively regulate the systemic inflammatory profile and NF- κB activation. PMID

  17. Differential action of methamphetamine on tyrosine hydroxylase and dopamine transport in the nigrostriatal pathway of μ-opioid receptor knockout mice.

    PubMed

    Park, Sang Won; He, Zhi; Shen, Xine; Roman, Richard J; Ma, Tangeng

    2012-06-01

    Extensive anatomical and functional interactions exist between central dopaminergic and opioidergic systems and both systems are proposed to be targets for amphetamine-like drugs. We have previously reported that μ-opioid receptor (μ-OR) knockout mice are resistant to the loss of dopamine in the striatum and the development of behavioral sensitization induced by repeated methamphetamine (METH) treatment. The present study assessed whether METH-treated μ-OR knockout mice exhibit a differential response of the expression of dopamine transporter and tyrosine hydroxylase (TH), the rate-limiting enzyme for dopamine synthesis and maintaining dopamine levels. Mice daily received intraperitoneal injection of METH (0, 0.6, 2.5, or 10 mg/kg) for 7 days and sacrificed on day 11 (4 days after the last injection). The expression of TH protein in the striatum and the levels of TH mRNA and number of TH positive neurons in the substantia nigra were reduced in wild-type mice treated with METH (2.5 and 10 mg/kg), but not in the μ-OR knockout mice. In contrast, METH exposure at the highest dose (10 mg/kg) reduced dopamine transporter levels in both strains of mice. These results suggest that the μ-OR contributes to METH-induced loss of dopamine and behavioral sensitization by decreasing the expression of TH. PMID:22329540

  18. GABA(A) receptor subunit alteration-dependent diazepam insensitivity in the cerebellum of phospholipase C-related inactive protein knockout mice.

    PubMed

    Mizokami, Akiko; Tanaka, Hiroto; Ishibashi, Hitoshi; Umebayashi, Hisanori; Fukami, Kiyoko; Takenawa, Tadaomi; Nakayama, Keiichi I; Yokoyama, Takeshi; Nabekura, Junichi; Kanematsu, Takashi; Hirata, Masato

    2010-07-01

    The GABA(A) receptor, a pentamer composed predominantly of alpha, beta, and gamma subunits, mediates fast inhibitory synaptic transmission. We have previously reported that phospholipase C-related inactive protein (PRIP) is a modulator of GABA(A) receptor trafficking and that knockout (KO) mice exhibit a diazepam-insensitive phenotype in the hippocampus. The alpha subunit affects diazepam sensitivity; alpha1, 2, 3, and 5 subunits assemble with any form of beta and the gamma2 subunits to produce diazepam-sensitive receptors, whereas alpha4 or alpha6/beta/gamma2 receptors are diazepam-insensitive. Here, we investigated how PRIP is implicated in the diazepam-insensitive phenotype using cerebellar granule cells in animals expressing predominantly the alpha6 subunit. The expression of alpha1/beta/gamma2 diazepam-sensitive receptors was decreased in the PRIP-1 and 2 double KO cerebellum without any change in the total number of benzodiazepine-binding sites as assessed by radioligand-binding assay. Since levels of the alpha6 subunit were increased, the alpha1/beta/gamma2 receptors might be replaced with alpha6 subunit-containing receptors. Then, we further performed autoradiographic and electrophysiologic analyses. These results suggest that the expression of alpha6/delta receptors was decreased in cerebellar granule neurons, while that of alpha6/gamma2 receptors was increased. PRIP-1 and 2 double KO mice exhibit a diazepam-insensitive phenotype because of a decrease in diazepam-sensitive (alpha1/gamma2) and increase in diazepam-insensitive (alpha6/gamma2) GABA(A) receptors in the cerebellar granule cells. PMID:20412381

  19. Conditional knockout of the leptin receptor in the colonic epithelium revealed the local effects of leptin receptor signaling in the progression of colonic tumors in mice.

    PubMed

    Higurashi, Takuma; Endo, Hiroki; Uchiyama, Takashi; Uchiyama, Shiori; Yamada, Eiji; Ohkubo, Hidenori; Sakai, Eiji; Takahashi, Hirokazu; Maeda, Shin; Wada, Koichiro; Natsumeda, Yutaka; Hippo, Yoshitaka; Nakajima, Atsushi; Nakagama, Hitoshi

    2014-09-01

    Leptin, secreted by the adipose tissue and known to be related to obesity, is considered to be involved in the onset and progression of colorectal cancer. However, the exact role of leptin in colorectal carcinogenesis is still unclear, as several controversial reports have been published on the various systemic effects of leptin. The aim of this study was to clarify the local and precise roles of leptin receptor (LEPR)-mediated signaling in colonic carcinogenesis using intestinal epithelium-specific LEPRb conditional knockout (cKO) mice. We produced and used colonic epithelium-specific LEPRb cKO mice to investigate the carcinogen-induced formation of aberrant crypt foci (ACF) and tumors in the colon, using their littermates as control. There were no differences in the body weight or systemic condition between the control and cKO mice. The tumor sizes and number of large-sized tumors were significantly lower in the cKO mice as compared with those in the control mice. On the other hand, there was no significant difference in the proliferative activity of the normal colonic epithelial cells or ACF formation between the control and cKO mice. In the control mice, marked increase of the LEPRb expression level was observed in the colonic tumors as compared with that in the normal epithelium; furthermore, signal transducer and activator of transcription (STAT3) was activated in the tumor cells. These findings suggest that STAT3 is one of the important molecules downstream of LEPRb, and LEPRb/STAT3 signaling controls tumor cell proliferation. We demonstrated the importance of local/regional LEPR-mediated signaling in colorectal carcinogenesis. PMID:24958593

  20. Activation and modulation of recombinantly expressed serotonin receptor type 3A by terpenes and pungent substances.

    PubMed

    Ziemba, Paul M; Schreiner, Benjamin S P; Flegel, Caroline; Herbrechter, Robin; Stark, Timo D; Hofmann, Thomas; Hatt, Hanns; Werner, Markus; Gisselmann, Günter

    2015-11-27

    Serotonin receptor type 3 (5-HT3 receptor) is a ligand-gated ion channel that is expressed in the central nervous system (CNS) as well as in the peripheral nervous system (PNS). The receptor plays an important role in regulating peristalsis of the gastrointestinal tract and in functions such as emesis, cognition and anxiety. Therefore, a variety of pharmacologically active substances target the 5-HT3 receptor to treat chemotherapy-induced nausea and vomiting. The 5-HT3 receptors are activated, antagonized, or modulated by a wide range of chemically different substances, such as 2-methyl-serotonin, phenylbiguanide, setrones, or cannabinoids. Whereas the action of all of these substances is well described, less is known about the effect of terpenoids or fragrances on 5-HT3A receptors. In this study, we screened a large number of natural odorous and pungent substances for their pharmacological action on recombinantly expressed human 5-HT3A receptors. The receptors were functionally expressed in Xenopus oocytes and characterized by electrophysiological recordings using the two-electrode voltage-clamp technique. A screening of two odorous mixes containing a total of 200 substances revealed that the monoterpenes, thymol and carvacrol, act as both weak partial agonists and positive modulators on the 5-HT3A receptor. In contrast, the most effective blockers were the terpenes, citronellol and geraniol, as well as the pungent substances gingerol, capsaicin and polygodial. In our study, we identified new modulators of 5-HT3A receptors out of the classes of monoterpenes and vanilloid substances that frequently occur in various plants. PMID:26456648

  1. Role of histamine H1-receptor on behavioral states and wake maintenance during deficiency of a brain activating system: A study using a knockout mouse model.

    PubMed

    Parmentier, Régis; Zhao, Yan; Perier, Magali; Akaoka, Hideo; Lintunen, Minnamaija; Hou, Yiping; Panula, Pertti; Watanabe, Takeshi; Franco, Patricia; Lin, Jian-Sheng

    2016-07-01

    Using knockout (KO) mice lacking the histamine (HA)-synthesizing enzyme (histidine decarboxylase, HDC), we have previously shown the importance of histaminergic neurons in maintaining wakefulness (W) under behavioral challenges. Since the central actions of HA are mediated by several receptor subtypes, it remains to be determined which one(s) could be responsible for such a role. We have therefore compared the cortical-EEG, sleep and W under baseline conditions or behavioral/pharmacological stimuli in littermate wild-type (WT) and H1-receptor KO (H1-/-) mice. We found that H1-/- mice shared several characteristics with HDC KO mice, i.e. 1) a decrease in W after lights-off despite its normal baseline daily amount; 2) a decreased EEG slow wave sleep (SWS)/W power ratio; 3) inability to maintain W in response to behavioral challenges demonstrated by a decreased sleep latency when facing various stimuli. These effects were mediated by central H1-receptors. Indeed, in WT mice, injection of triprolidine, a brain-penetrating H1-receptor antagonist increased SWS, whereas ciproxifan (H3-receptor antagonist/inverse agonist) elicited W; all these injections had no effect in H1-/- mice. Finally, H1-/- mice showed markedly greater changes in EEG power (notably in the 0.8-5 Hz band) and sleep-wake cycle than in WT mice after application of a cholinergic antagonist or an indirect agonist, i.e., scopolamine or physostigmine. Hence, the role of HA in wake-promotion is largely ensured by H1-receptors. An upregulated cholinergic system may account for a quasi-normal daily amount of W in HDC or H1-receptor KO mice and likely constitutes a major compensatory mechanism when the brain is facing deficiency of an activating system. This article is part of the Special Issue entitled 'Histamine Receptors'. PMID:26723880

  2. The 5-HT3B subunit affects high-potency inhibition of 5-HT3 receptors by morphine

    PubMed Central

    Baptista-Hon, Daniel T; Deeb, Tarek Z; Othman, Nidaa A; Sharp, Douglas; Hales, Tim G

    2012-01-01

    BACKGROUND AND PURPOSE Morphine is an antagonist at 5-HT3A receptors. 5-HT3 and opioid receptors are expressed in many of the same neuronal pathways where they modulate gut motility, pain and reinforcement. There is increasing interest in the 5-HT3B subunit, which confers altered pharmacology to 5-HT3 receptors. We investigated the mechanisms of inhibition by morphine of 5-HT3 receptors and the influence of the 5-HT3B subunit. EXPERIMENTAL APPROACH 5-HT-evoked currents were recorded from voltage-clamped HEK293 cells expressing human 5-HT3A subunits alone or in combination with 5-HT3B subunits. The affinity of morphine for the orthosteric site of 5-HT3A or 5-HT3AB receptors was assessed using radioligand binding with the antagonist [3H]GR65630. KEY RESULTS When pre-applied, morphine potently inhibited 5-HT-evoked currents mediated by 5-HT3A receptors. The 5-HT3B subunit reduced the potency of morphine fourfold and increased the rates of inhibition and recovery. Inhibition by pre-applied morphine was insurmountable by 5-HT, was voltage-independent and occurred through a site outside the second membrane-spanning domain. When applied simultaneously with 5-HT, morphine caused a lower potency, surmountable inhibition of 5-HT3A and 5-HT3AB receptors. Morphine also fully displaced [3H]GR65630 from 5-HT3A and 5-HT3AB receptors with similar potency. CONCLUSIONS AND IMPLICATIONS These findings suggest that morphine has two sites of action, a low-affinity, competitive site and a high-affinity, non-competitive site that is not available when the channel is activated. The affinity of morphine for the latter is reduced by the 5-HT3B subunit. Our results reveal that morphine causes a high-affinity, insurmountable and subunit-dependent inhibition of human 5-HT3 receptors. PMID:21740409

  3. Mu Opioid Receptors on Primary Afferent Nav1.8 Neurons Contribute to Opiate-Induced Analgesia: Insight from Conditional Knockout Mice

    PubMed Central

    Karchewski, Laurie; Gardon, Olivier; Matifas, Audrey; Filliol, Dominique; Becker, Jérôme A. J.; Wood, John N.; Kieffer, Brigitte L.; Gaveriaux-Ruff, Claire

    2013-01-01

    Opiates are powerful drugs to treat severe pain, and act via mu opioid receptors distributed throughout the nervous system. Their clinical use is hampered by centrally-mediated adverse effects, including nausea or respiratory depression. Here we used a genetic approach to investigate the potential of peripheral mu opioid receptors as targets for pain treatment. We generated conditional knockout (cKO) mice in which mu opioid receptors are deleted specifically in primary afferent Nav1.8-positive neurons. Mutant animals were compared to controls for acute nociception, inflammatory pain, opiate-induced analgesia and constipation. There was a 76% decrease of mu receptor-positive neurons and a 60% reduction of mu-receptor mRNA in dorsal root ganglia of cKO mice. Mutant mice showed normal responses to heat, mechanical, visceral and chemical stimuli, as well as unchanged morphine antinociception and tolerance to antinociception in models of acute pain. Inflammatory pain developed similarly in cKO and controls mice after Complete Freund’s Adjuvant. In the inflammation model, however, opiate-induced (morphine, fentanyl and loperamide) analgesia was reduced in mutant mice as compared to controls, and abolished at low doses. Morphine-induced constipation remained intact in cKO mice. We therefore genetically demonstrate for the first time that mu opioid receptors partly mediate opiate analgesia at the level of Nav1.8-positive sensory neurons. In our study, this mechanism operates under conditions of inflammatory pain, but not nociception. Previous pharmacology suggests that peripheral opiates may be clinically useful, and our data further demonstrate that Nav1.8 neuron-associated mu opioid receptors are feasible targets to alleviate some forms of persistent pain. PMID:24069332

  4. Knockout of the BK β4-subunit promotes a functional coupling of BK channels and ryanodine receptors that mediate a fAHP-induced increase in excitability.

    PubMed

    Wang, Bin; Bugay, Vladislav; Ling, Ling; Chuang, Hui-Hsui; Jaffe, David B; Brenner, Robert

    2016-08-01

    BK channels are large-conductance calcium- and voltage-activated potassium channels with diverse properties. Knockout of the accessory BK β4-subunit in hippocampus dentate gyrus granule neurons causes BK channels to change properties from slow-gated type II channels to fast-gated type I channels that sharpen the action potential, increase the fast afterhyperpolarization (fAHP) amplitude, and increase spike frequency. Here we studied the calcium channels that contribute to fast-gated BK channel activation and increased excitability of β4 knockout neurons. By using pharmacological blockers during current-clamp recording, we find that BK channel activation during the fAHP is dependent on ryanodine receptor activation. In contrast, L-type calcium channel blocker (nifedipine) affects the BK channel-dependent repolarization phase of the action potential but has no effect on the fAHP. Reducing BK channel activation during the repolarization phase with nifedipine, or during the fAHP with ryanodine, indicated that it is the BK-mediated increase of the fAHP that confers proexcitatory effects. The proexcitatory role of the fAHP was corroborated using dynamic current clamp. Increase or decrease of the fAHP amplitude during spiking revealed an inverse relationship between fAHP amplitude and interspike interval. Finally, we show that the seizure-prone ryanodine receptor gain-of-function (R2474S) knockin mice have an unaltered repolarization phase but larger fAHP and increased AP frequency compared with their control littermates. In summary, these results indicate that an important role of the β4-subunit is to reduce ryanodine receptor-BK channel functional coupling during the fAHP component of the action potential, thereby decreasing excitability of dentate gyrus neurons. PMID:27146987

  5. Enkephalin levels and the number of neuropeptide Y-containing interneurons in the hippocampus are decreased in female cannabinoid-receptor 1 knock-out mice.

    PubMed

    Rogers, Sophie A; Kempen, Tracey A Van; Pickel, Virginia M; Milner, Teresa A

    2016-05-01

    Drug addiction requires learning and memory processes that are facilitated by activation of cannabinoid-1 (CB1) and opioid receptors in the hippocampus. This involves activity-dependent synaptic plasticity that is partially regulated by endogenous opioid (enkephalin and dynorphin) and non-opioid peptides, specifically cholecystokinin, parvalbumin and neuropeptide Y, the neuropeptides present in inhibitory interneurons that co-express CB1 or selective opioid receptors. We tested the hypothesis that CB1 receptor expression is a determinant of the availability of one or more of these peptide modulators in the hippocampus. This was achieved by quantitatively analyzing the immunoperoxidase labeling for each of these neuropeptide in the dorsal hippocampus of female wild-type (CB1+/+) and cannabinoid receptor 1 knockout (CB1-/-) C57/BL6 mice. The levels of Leu(5)-enkephalin-immunoreactivity were significantly reduced in the hilus of the dentate gyrus and in stratum lucidum of CA3 in CB1-/- mice. Moreover, the numbers of neuropeptide Y-immunoreactive interneurons in the dentate hilus were significantly lower in the CB1-/- compared to wild-type mice. However, CB1+/+ and CB1-/- mice did not significantly differ in expression levels of either dynorphin or cholecystokinin, and showed no differences in numbers of parvalbumin-containing interneurons. These findings suggest that the cannabinoid and opioid systems have a nuanced, regulatory relationship that could affect the balance of excitation and inhibition in the hippocampus and thus processes such as learning that rely on this balance. PMID:27012427

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

    NASA Astrophysics Data System (ADS)

    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.

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

    PubMed

    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 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. PMID:26521965

  8. Secretin receptor-knockout mice are resistant to high-fat diet-induced obesity and exhibit impaired intestinal lipid absorption.

    PubMed

    Sekar, Revathi; Chow, Billy K C

    2014-08-01

    Secretin, a classical gastrointestinal hormone released from S cells in response to acid and dietary lipid, regulates pleiotropic physiological functions, such as exocrine pancreatic secretion and gastric motility. Subsequent to recently proposed revisit on secretin's metabolic effects, we have confirmed lipolytic actions of secretin during starvation and discovered a hormone-sensitive lipase-mediated mechanistic pathway behind. In this study, a 12 wk high-fat diet (HFD) feeding to secretin receptor-knockout (SCTR(-/-)) mice and their wild-type (SCTR(+/+)) littermates revealed that, despite similar food intake, SCTR(-/-) mice gained significantly less weight (SCTR(+/+): 49.6±0.9 g; SCTR(-/-): 44.7±1.4 g; P<0.05) and exhibited lower body fat content. These SCTR(-/-) mice have corresponding alleviated HFD-associated hyperleptinemia and improved glucose/insulin tolerance. Further analyses indicate that SCTR(-/-) have impaired intestinal fatty acid absorption while having similar energy expenditure and locomotor activity. Reduced fat absorption in the intestine is further supported by lowered postprandial triglyceride concentrations in circulation in SCTR(-/-) mice. In jejunal cells, transcript and protein levels of a key fat absorption regulator, cluster of differentiation 36 (CD36), was reduced in knockout mice, while transcript of Cd36 and fatty-acid uptake in isolated enterocytes was stimulated by secretin. Based on our findings, a novel positive feedback pathway involving secretin and CD36 to enhance intestinal lipid absorption is being proposed. PMID:24769669

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

    PubMed Central

    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 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. PMID:26521965

  10. Serotonin receptor diversity in the human colon: Expression of serotonin type 3 receptor subunits 5-HT3C, 5-HT3D, and 5-HT3E

    PubMed Central

    Kapeller, Johannes; Möller, Dorothee; Lasitschka, Felix; Autschbach, Frank; Hovius, Ruud; Rappold, Gudrun; Brüss, Michael; Gershon, Michael D.

    2011-01-01

    Since the first description of 5-HT3 receptors more than 50 years ago, there has been speculation about the molecular basis of their receptor heterogeneity. We have cloned the genes encoding novel 5-HT3 subunits 5-HT3C, 5-HT3D, and 5-HT3E and have shown that these subunits are able to form functional heteromeric receptors when coexpressed with the 5-HT3A subunit. However, whether these subunits are actually expressed in human tissue remained to be confirmed. In the current study, we performed immunocytochemistry to locate the 5-HT3A as well as the 5-HT3C, 5-HT3D, and 5-HT3E subunits within the human colon. Western blot analysis was used to confirm subunit expression, and RT-PCR was employed to detect transcripts encoding 5-HT3 receptor subunits in microdissected tissue samples. This investigation revealed, for the first time, that 5-HT3C, 5-HT3D, and 5-HT3E subunits are coexpressed with 5-HT3A in cell bodies of myenteric neurons. Furthermore, 5-HT3A and 5-HT3D were found to be expressed in submucosal plexus of the human large intestine. These data provide a strong basis for future studies of the roles that specific 5-HT3 receptor subtypes play in the function of the enteric and central nervous systems and the contribution that specific 5-HT3 receptors make to the pathophysiology of gastrointestinal disorders such as irritable bowel syndrome and dyspepsia. PMID:21192076

  11. Dysregulation of dopamine-dependent mechanisms as a determinant of hypertension: studies in dopamine receptor knockout mice

    PubMed Central

    Zeng, Chunyu; Armando, Ines; Luo, Yingjin; Eisner, Gilbert M.; Felder, Robin A.; Jose, Pedro A.

    2014-01-01

    Dopamine plays an important role in the pathogenesis of hypertension by regulating epithelial sodium transport and by interacting with vasoactive hormones/humoral factors, such as aldosterone, angiotensin, catecholamines, endothelin, oxytocin, prolactin pro-opiomelancortin, reactive oxygen species, renin, and vasopressin. Dopamine receptors are classified into D1-like (D1 and D5) and D2-like (D2, D3, and D4) subtypes based on their structure and pharmacology. In recent years, mice deficient in one or more of the five dopamine receptor subtypes have been generated, leading to a better understanding of the physiological role of each of the dopamine receptor subtypes. This review summarizes the results from studies of various dopamine receptor mutant mice on the role of individual dopamine receptor subtypes and their interactions with other G protein-coupled receptors in the regulation of blood pressure. PMID:18083900

  12. Rescue of Obesity-Induced Infertility in Female Mice due to a Pituitary-Specific Knockout of the Insulin Receptor (IR)

    PubMed Central

    Brothers, Kathryn J.; Wu, Sheng; DiVall, Sara A.; Messmer, Marcus R.; Kahn, C. Ronald; Miller, Ryan S.; Radovick, Sally; Wondisford, Fredric E.; Wolfe, Andrew

    2010-01-01

    Summary Obesity is associated with insulin resistance in metabolic tissues such as adipose, liver, and muscle, but it is unclear whether non-classical target tissues, such as those of the reproductive axis, are also insulin resistant. To determine if the reproductive axis maintains insulin sensitivity in obesity in vivo, murine models of diet-induced obesity with and without intact insulin signaling in pituitary gonadotrophs were created. Diet-induced obese wild type female mice (WT DIO) were infertile and experienced a robust increase in luteinizing hormone (LH) after gonadotropin releasing hormone (GnRH) or insulin stimulation. By contrast, both lean and obese mice with a pituitary-specific knockout of the insulin receptor (PitIRKO) exhibited reproductive competency, indicating that insulin signaling in the pituitary is required for the reproductive impairment seen in diet-induced obesity and that the gonadotroph maintains insulin sensitivity in a setting of peripheral insulin resistance. PMID:20816095

  13. Impact of Omega-6 Polyunsaturated Fatty Acid:Eicosapentaenoic acid (EPA)+Docosahexaenoic Acid (DHA) Ratios in LDL Receptor Knockout (LDLr-/-) Mice on Atherosclerotic Lesion Formation and Elicited Peritoneal Macrophage Inflamm

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Objective: Very long chain omega-3 fatty acids have been associated with decreased risk of CVD. LDL receptor knockout mice were used to assess the effect of different omega-6:EPA+DHA ratios on atherosclerotic lesion formation and elicited peritoneal macrophage inflammatory response. Methods and R...

  14. The low density lipoprotein receptor-related protein 1: Unique tissue-specific functions revealed by selective gene knockout studies

    PubMed Central

    Lillis, Anna P.; Van Duyn, Lauren B.; Murphy-Ullrich, Joanne E.; Strickland, Dudley K.

    2008-01-01

    The low-density lipoprotein (LDL) receptor-related protein (originally called LRP, but now referred to as LRP1) is a large endocytic receptor that is widely expressed in several tissues. LRP1 is a member of the LDL receptor family that plays diverse roles in various biological processes including lipoprotein metabolism, degradation of proteases, activation of lysosomal enzymes and cellular entry of bacterial toxins and viruses. Deletion of the LRP1 gene leads to lethality in mice, revealing a critical, but as of yet, undefined role in development. Tissue-specific gene deletion studies reveal an important contribution of LRP1 in the vasculature, central nervous system, in macrophages and in adipocytes. Three important properties of LRP1 dictate its diverse role in physiology: first, its ability to recognize more than thirty distinct ligands; second, its ability to bind a large number of cytoplasmic adaptor proteins via determinants located on its cytoplasmic domain in a phosphorylation-specific manner; and third, its ability to associate with and modulate the activity of other transmembrane receptors such as integrins and receptor tyrosine kinases. PMID:18626063

  15. High Affinity Dopamine D3 Receptor (D3R)-Selective Antagonists Attenuate Heroin Self-Administration in Wild-Type but not D3R Knockout Mice.

    PubMed

    Boateng, Comfort A; Bakare, Oluyomi M; Zhan, Jia; Banala, Ashwini K; Burzynski, Caitlin; Pommier, Elie; Keck, Thomas M; Donthamsetti, Prashant; Javitch, Jonathan A; Rais, Rana; Slusher, Barbara S; Xi, Zheng-Xiong; Newman, Amy Hauck

    2015-08-13

    The dopamine D3 receptor (D3R) is a promising target for the development of pharmacotherapeutics to treat substance use disorders. Several D3R-selective antagonists are effective in animal models of drug abuse, especially in models of relapse. Nevertheless, poor bioavailability, metabolic instability, and/or predicted toxicity have impeded success in translating these drug candidates to clinical use. Herein, we report a series of D3R-selective 4-phenylpiperazines with improved metabolic stability. A subset of these compounds was evaluated for D3R functional efficacy and off-target binding at selected 5-HT receptor subtypes, where significant overlap in SAR with D3R has been observed. Several high affinity D3R antagonists, including compounds 16 (Ki = 0.12 nM) and 32 (Ki = 0.35 nM), showed improved metabolic stability compared to the parent compound, PG648 (6). Notably, 16 and the classic D3R antagonist SB277011A (2) were effective in reducing self-administration of heroin in wild-type but not D3R knockout mice. PMID:26203768

  16. Expression of key regulators of mitochondrial biogenesis in growth hormone receptor knockout (GHRKO) mice is enhanced but is not further improved by other potential life-extending interventions.

    PubMed

    Gesing, Adam; Masternak, Michal M; Wang, Feiya; Joseph, Anna-Maria; Leeuwenburgh, Christiaan; Westbrook, Reyhan; Lewinski, Andrzej; Karbownik-Lewinska, Malgorzata; Bartke, Andrzej

    2011-10-01

    Mitochondrial biogenesis is essential for cell viability. Growth hormone receptor knockout (GHRKO), calorie restriction, and surgical visceral fat removal constitute experimental interventions to delay aging and increase life span. We examined the expression of known regulators of mitochondriogenesis: peroxisome proliferator-activated receptor γ co-activator 1α (PGC-1α), adenosine monophosphate (AMP)-activated protein kinase (AMPK), sirtuin-1 (SIRT-1) and sirtuin-3 (SIRT-3), endothelial nitric oxide synthase (eNOS), nuclear respiratory factor-1, mitochondrial transcription factor A (TFAM), and mitofusin-2 (MFN-2) in the skeletal muscles and hearts of control and calorie-restricted female GHRKO mice and in the kidneys of male GHRKOs after visceral fat removal or sham surgery. Expression of PGC-1α in skeletal muscles, AMPK, SIRT-1, SIRT-3, eNOS, and MFN-2 in the heart and PGC-1α, AMPK, SIRT-3, eNOS, and MFN-2 in kidneys was increased in GHRKO mice but was not affected by calorie restriction or visceral fat removal. GHRKO mice have increased expression of key regulators of mitochondriogenesis, which is not improved further by calorie restriction or visceral fat removal. PMID:21788651

  17. Toll-like receptor 4 (Tlr4) knockout rats produced by transcriptional activator-like effector nuclease- (TALEN)-mediated gene inactivation

    PubMed Central

    Ferguson, Carolyn; McKay, Matthew; Harris, R. Adron; Homanics, Gregg E.

    2013-01-01

    Genetically engineered mice are a valuable resource for studies of the behavioral effects of ethanol. However, for some behavioral tests of ethanol action, the rat is a superior model organism. Production of genetically engineered rats has been severely hampered due to technical limitations. Here we utilized a promising new technique for efficient site-specific gene modification to create a novel gene knockout rat line. This approach is based on Transcriptional Activator-Like Effector Nucleases (TALENs). TALENs function in pairs and bind DNA in a sequence-specific manner. Upon binding to the target sequence, a functional nuclease is reconstituted that creates double-stranded breaks in the DNA that are efficiently repaired by non-homologous end joining. This error-prone process often results in deletions of varying lengths at the targeted locus. The toll-like receptor 4 (Tlr4) gene was selected for TALEN-mediated gene inactivation. Tlr4 has been implicated in ethanol-induced neuroinflammation and neurodegeneration, as well as multiple ethanol-induced behavioral effects. To generate Tlr4 knockout rats, a pair of TALEN constructs was created that specifically target Exon 1 immediately downstream of the start of translation. TALEN mRNAs were microinjected into the cytoplasm of one-cell Wistar rat embryos. Of 13 live-born pups that resulted, one harbored a mutation in Exon 1 of Tlr4. The mutated allele consisted of a 13 base-pair deletion that was predicted to create a frameshift mutation after amino acid 25. This founder rat successfully transmitted the mutation to F1 offspring. Heterozygous F1 offspring were interbred to produce homozygous F2 animals. Homozygous mutants expressed the 13-bp deletion in Tlr4 mRNA. In contrast to control rats that produced a robust increase in plasma tumor necrosis factor alpha in response to a lipopolysaccharide challenge, homozygous rats had a markedly attenuated response. Thus, the mutant Tlr4 allele generated by TALEN-mediated gene

  18. Altered mnemonic functions and resistance to N-METHYL-d-Aspartate receptor antagonism by forebrain conditional knockout of glycine transporter 1.

    PubMed

    Singer, P; Yee, B K; Feldon, J; Iwasato, T; Itohara, S; Grampp, T; Prenosil, G; Benke, D; Möhler, H; Boison, D

    2009-06-30

    Converging evidence from pharmacological and molecular studies has led to the suggestion that inhibition of glycine transporter 1 (GlyT1) constitutes an effective means to boost N-methyl-d-aspartate receptor (NMDAR) activity by increasing the extra-cellular concentration of glycine in the vicinity of glutamatergic synapses. However, the precise extent and limitation of this approach to alter cognitive function, and therefore its potential as a treatment strategy against psychiatric conditions marked by cognitive impairments, remain to be fully examined. Here, we generated mutant mice lacking GlyT1 in the entire forebrain including neurons and glia. This conditional knockout system allows a more precise examination of GlyT1 downregulation in the brain on behavior and cognition. The mutation was highly effective in attenuating the motor-stimulating effect of acute NMDAR blockade by phencyclidine, although no appreciable elevation in NMDAR-mediated excitatory postsynaptic currents (EPSC) was observed in the hippocampus. Enhanced cognitive performance was observed in spatial working memory and object recognition memory while spatial reference memory and associative learning remained unaltered. These findings provide further credence for the potential cognitive enhancing effects of brain GlyT1 inhibition. At the same time, they indicated potential phenotypic differences when compared with other constitutive and conditional GlyT1 knockout lines, and highlighted the possibility of a functional divergence between the neuronal and glia subpopulations of GlyT1 in the regulation of learning and memory processes. The relevance of this distinction to the design of future GlyT1 blockers as therapeutic tools in the treatment of cognitive disorders remains to be further investigated. PMID:19332109

  19. Genetic knockout of the α7 nicotinic acetylcholine receptor gene alters hippocampal long-term potentiation in a background strain-dependent manner.

    PubMed

    Freund, Ronald K; Graw, Sharon; Choo, Kevin S; Stevens, Karen E; Leonard, Sherry; Dell'Acqua, Mark L

    2016-08-01

    Reduced α7 nicotinic acetylcholine receptor (nAChR) function is linked to impaired hippocampal-dependent sensory processing and learning and memory in schizophrenia. While knockout of the Chrna7 gene encoding the α7nAChR on a C57/Bl6 background results in changes in cognitive measures, prior studies found little impact on hippocampal synaptic plasticity in these mice. However, schizophrenia is a multi-genic disorder where complex interactions between specific genetic mutations and overall genetic background may play a prominent role in determining phenotypic penetrance. Thus, we compared the consequences of knocking out the α7nAChR on synaptic plasticity in C57/Bl6 and C3H mice, which differ in their basal α7nAChR expression levels. Homozygous α7 deletion in C3H mice, which normally express higher α7nAChR levels, resulted in impaired long-term potentiation (LTP) at hippocampal CA1 synapses, while C3H α7 heterozygous mice maintained robust LTP. In contrast, homozygous α7 deletion in C57 mice, which normally express lower α7nAChR levels, did not alter LTP, as had been previously reported for this strain. Thus, the threshold of Chrna7 expression required for LTP may be different in the two strains. Measurements of auditory gating, a hippocampal-dependent behavioral paradigm used to identify schizophrenia-associated sensory processing deficits, was abnormal in C3H α7 knockout mice confirming that auditory gating also requires α7nAChR expression. Our studies highlight the importance of genetic background on the regulation of synaptic plasticity and could be relevant for understanding genetic and cognitive heterogeneity in human studies of α7nAChR dysfunction in mental disorders. PMID:27233215

  20. Myeloid Cell-Specific ABCA1 Deletion Has Minimal Impact on Atherogenesis in Atherogenic Diet-Fed LDL Receptor Knockout Mice

    PubMed Central

    Bi, Xin; Zhu, Xuewei; Gao, Chuan; Shewale, Swapnil; Cao, Qiang; Liu, Mingxia; Boudyguina, Elena; Gebre, Abraham K.; Wilson, Martha D.; Brown, Amanda L.; Parks, John S.

    2014-01-01

    Objective Transplantation studies suggest that bone marrow (BM) cell ABCA1 protects against atherosclerosis development. However, the in vivo impact of macrophage ABCA1 expression on atherogenesis is not fully understood because BM contains other leukocytes and hematopoietic stem and progenitor cells. Myeloid-specific ABCA1 knockout (MSKO) mice in the LDL receptor knockout (LDLrKO) C57BL/6 background were developed to address this question. Approach and Results Chow-fed MSKO/LDLrKO (DKO) vs. LDLrKO (SKO) mice had similar plasma lipid concentrations, but atherogenic diet (AD)-fed DKO mice had reduced plasma VLDL/LDL concentrations resulting from decreased hepatic VLDL triglyceride secretion. Resident peritoneal macrophages from AD-fed DKO vs. SKO mice had significantly higher cholesterol content, but similar proinflammatory gene expression. Atherosclerosis extent was similar between genotypes after 10–16 wks of AD, but increased modestly in DKO mice by 24 wks of AD. Lesional macrophage content was similar, likely due to higher monocyte flux through aortic root lesions in DKO vs. SKO mice. After transplantation of DKO or SKO BM into SKO mice and 16 wk of AD feeding, atherosclerosis extent was similar and plasma apoB lipoproteins was reduced in mice receiving DKO BM. When differences in plasma VLDL/LDL concentrations were minimized by maintaining mice on chow for 24 wks, DKO mice had modest, but significantly more, atherosclerosis compared to SKO mice. Conclusions Myeloid cell ABCA1 increases hepatic VLDL triglyceride secretion and plasma VLDL/LDL concentrations in AD-fed LDLrKO mice, offsetting its atheroprotective role in decreasing macrophage cholesterol content, resulting in minimal increase in atherosclerosis. PMID:24833800

  1. Insulin and insulin-like growth factor-I (IGF-I) receptor phosphorylation in µ-calpain knockout mice

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Numerous cellular processes are controlled by insulin and IGF-I signaling pathways. Due to previous work in our laboratories, we hypothesized that insulin (IR) and type 1 IGF-I (IGF-IR) receptor signaling is decreased due to increased protein tyrosine phosphatase 1B (PTP1B) activity. C57BL/6J mice...

  2. Colonic mucosal DNA methylation, immune response, and microbiome patterns in Toll-like receptor 2-knockout mice

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The stunning complexity of the resident microbiota and the intricate pathways of microbial and host interactions provide a massive adaptive capacity for mammals. In this addendum we reflect on our recent publication on Toll-like receptor 2 deficiency related colonic mucosal epigenetic, immunologic a...

  3. Targeted overexpression of the human urotensin receptor transgene in smooth muscle cells: effect of UT antagonism in ApoE knockout mice fed with Western diet.

    PubMed

    Papadopoulos, Panayiota; Bousette, Nicolas; Al-Ramli, Wisam; You, Zhipeng; Behm, David J; Ohlstein, Eliot H; Harrison, Stephen M; Douglas, Stephen A; Giaid, Adel

    2009-06-01

    Urotensin II (UII) and its receptor UT are upregulated in the pathological setting of various cardiovascular diseases including atherosclerosis. However, their exact role in atherosclerosis remains to be determined. In the present study we used four strains of mice; wild-type (WT), UT(+) (a transgenic strain expressing human UT driven by the alpha-smooth muscle-specific, SM22, promoter), ApoE knockout (ko), and UT(+)/ApoE ko. All animals were fed high fat diet for 12 weeks. Western blot analysis revealed a significant increase in aortic UT expression in UT(+) relative to WT mice (P<0.05). Aortas of ApoE ko mice expressed comparable UT protein level to that of UT(+). Immunohistochemistry revealed the presence of strong expression of UT and UII proteins in the atheroma of UT(+), ApoE ko and UT(+)/ApoE ko mice, particularly in foam cells. Serum cholesterol and triglyceride levels were significantly increased in ApoE ko and in UT(+)/ApoE ko but not in UT(+) mice when compared to WT mice (P<0.0001). Analysis of aortas showed a significant increase in atherosclerotic lesion in the UT(+), ApoE ko and UT(+)/ApoE ko compared to WT mice (P<0.05). Oral administration of the UT receptor antagonist SB-657510A (30 microg/Kg/day gavage) for 10 weeks in a group of ApoE ko mice fed on high fat diet resulted in a significant reduction of lesion (P<0.001). SB-657510A also significantly reduced ACAT-1 protein expression in the atherosclerotic lesion of ApoE ko mice (P<0.05). The present findings demonstrate an important role for UT in the pathogenesis of atherosclerosis. The use of UT receptor antagonists may provide a beneficial tool in the management of this debilitating disease process. PMID:19111831

  4. Aggravation of Chronic Stress Effects on Hippocampal Neurogenesis and Spatial Memory in LPA1 Receptor Knockout Mice

    PubMed Central

    Castilla-Ortega, Estela; Hoyo-Becerra, Carolina; Pedraza, Carmen; Chun, Jerold; Rodríguez De Fonseca, Fernando; Estivill-Torrús, Guillermo; Santín, Luis J.

    2011-01-01

    Background 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. Methodology/Principal Findings 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. Conclusions/Significance 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. PMID:21980482

  5. Hyperammonaemia in V1a vasopressin receptor knockout mice caused by the promoted proteolysis and reduced intrahepatic blood volume.

    PubMed

    Hiroyama, Masami; Aoyagi, Toshinori; Fujiwara, Yoko; Oshikawa, Sayuri; Sanbe, Atsushi; Endo, Fumio; Tanoue, Akito

    2007-06-15

    An analysis of arginine-vasopressin (AVP) V1a receptor-deficient (V1aR-/-) mice revealed that glucose homeostasis and lipid metabolism were altered in the mutant mice. Here, we used V1aR-/- mice to investigate whether the deficiency of the V1a receptor, which led to altered insulin sensitivity, affected protein metabolism. The serum 3-methylhistidine levels were increased in V1aR-/- mice under feeding conditions, indicating that proteolysis was enhanced in muscle tissue from V1aR-/- mice. Furthermore, serum amino acid profiling revealed that the amino acid levels, including glycogenic and branched-chain amino acids, were reduced in V1aR-/- mice. In addition, an alanine-loading test showed that gluconeogenesis was enhanced in V1aR-/- mice. Blood ammonia, which is a by-product of amino acid catabolism, was two times higher in V1aR-/- mice without hepatopathy under the feeding and fasting conditions than in wild-type mice. Amino acid profiling also revealed that the amino acid pattern was not typical of a urea-cycle enzymatic disorder. An ammonia tolerance test and an indocyanine green elimination test showed that V1aR-/- mice had lower ammonia clearance due to a decreased intrahepatic circulating blood volume. Metabolic acidosis, including lactic- and keto-acidosis, was not observed in V1aR-/- mice. These results provide evidence that proteolysis promotes the production of glucose in the muscles of V1aR-/- mice and that hyperammonaemia is caused by promoted protein catabolism and reduced intrahepatic blood volume. Thus, our study with V1aR-/- mice indicates that AVP plays a physiological role via the V1a receptor in regulating both protein catabolism and glucose homeostasis. PMID:17379633

  6. Aberrant in Vivo T Helper Type 2 Cell Response and Impaired Eosinophil Recruitment in Cc Chemokine Receptor 8 Knockout Mice

    PubMed Central

    Chensue, Stephen W.; Lukacs, Nicholas W.; Yang, Tong-Yuan; Shang, Xiaozhou; Frait, Kirsten A.; Kunkel, Steven L.; Kung, Ted; Wiekowski, Maria T.; Hedrick, Joseph A.; Cook, Donald N.; Zingoni, Alessandra; Narula, Satwant K.; Zlotnik, Albert; Barrat, Franck J.; O'Garra, Anne; Napolitano, Monica; Lira, Sergio A.

    2001-01-01

    Chemokine receptors transduce signals important for the function and trafficking of leukocytes. Recently, it has been shown that CC chemokine receptor (CCR)8 is selectively expressed by Th2 subsets, but its functional relevance is unclear. To address the biological role of CCR8, we generated CCR8 deficient (−/−) mice. Here we report defective T helper type 2 (Th2) immune responses in vivo in CCR8−/− mice in models of Schistosoma mansoni soluble egg antigen (SEA)-induced granuloma formation as well as ovalbumin (OVA)- and cockroach antigen (CRA)-induced allergic airway inflammation. In these mice, the response to SEA, OVA, and CRA showed impaired Th2 cytokine production that was associated with aberrant type 2 inflammation displaying a 50 to 80% reduction in eosinophils. In contrast, a prototypical Th1 immune response, elicited by Mycobacteria bovis purified protein derivative (PPD) was unaffected by CCR8 deficiency. Mechanistic analyses indicated that Th2 cells developed normally and that the reduction in eosinophil recruitment was likely due to systemic reduction in interleukin 5. These results indicate an important role for CCR8 in Th2 functional responses in vivo. PMID:11238588

  7. Gene knockout of nuclear progesterone receptor provides insights into the regulation of ovulation by LH signaling in zebrafish

    PubMed Central

    Tang, Haipei; Liu, Yun; Li, Jianzhen; Yin, Yike; Li, Gaofei; Chen, Yu; Li, Shuisheng; Zhang, Yong; Lin, Haoran; Liu, Xiaochun; Cheng, Christopher H. K.

    2016-01-01

    It is well established that the luteinizing hormone surge triggers ovulation, a dynamic process leading to the release of the mature oocyte from the ovarian follicle. But how this process controlled by LH signaling remains largely unknown in non-mammalian species. In this study, we investigated the roles of nuclear progesterone receptor (npr) in LH-induced ovulation. Our results indicate that the nuclear progesterone receptor serves as an important mediator of LH action on ovulation. This conclusion is based on the following results: (1) the expression level of npr peaks at the full-grown stage of the follicles; (2) the expression of npr is stimulated by LH signaling in vitro and in vivo; and (3) the npr null females are infertile due to ovulation defects. Moreover, we further show that LH signaling could induce ptger4b expression in an npr-dependent manner, and blockage of Ptger4b could also block hCG-induced ovulation. Collectively, our results not only demonstrate that npr serves an indispensable role in mediating the action of LH on ovulation in zebrafish, but also provide insights into the molecular mechanisms of the regulation of ovulation in fish. PMID:27333837

  8. Gene knockout of nuclear progesterone receptor provides insights into the regulation of ovulation by LH signaling in zebrafish.

    PubMed

    Tang, Haipei; Liu, Yun; Li, Jianzhen; Yin, Yike; Li, Gaofei; Chen, Yu; Li, Shuisheng; Zhang, Yong; Lin, Haoran; Liu, Xiaochun; Cheng, Christopher H K

    2016-01-01

    It is well established that the luteinizing hormone surge triggers ovulation, a dynamic process leading to the release of the mature oocyte from the ovarian follicle. But how this process controlled by LH signaling remains largely unknown in non-mammalian species. In this study, we investigated the roles of nuclear progesterone receptor (npr) in LH-induced ovulation. Our results indicate that the nuclear progesterone receptor serves as an important mediator of LH action on ovulation. This conclusion is based on the following results: (1) the expression level of npr peaks at the full-grown stage of the follicles; (2) the expression of npr is stimulated by LH signaling in vitro and in vivo; and (3) the npr null females are infertile due to ovulation defects. Moreover, we further show that LH signaling could induce ptger4b expression in an npr-dependent manner, and blockage of Ptger4b could also block hCG-induced ovulation. Collectively, our results not only demonstrate that npr serves an indispensable role in mediating the action of LH on ovulation in zebrafish, but also provide insights into the molecular mechanisms of the regulation of ovulation in fish. PMID:27333837

  9. Androgen Receptor (AR) Physiological Roles in Male and Female Reproductive Systems: Lessons Learned from AR-Knockout Mice Lacking AR in Selective Cells1

    PubMed Central

    Chang, Chawnshang; Lee, Soo Ok; Wang, Ruey-Sheng; Yeh, Shuyuan; Chang, Ta-Min

    2013-01-01

    ABSTRACT Androgens/androgen receptor (AR) signaling is involved primarily in the development of male-specific phenotypes during embryogenesis, spermatogenesis, sexual behavior, and fertility during adult life. However, this signaling has also been shown to play an important role in development of female reproductive organs and their functions, such as ovarian folliculogenesis, embryonic implantation, and uterine and breast development. The establishment of the testicular feminization (Tfm) mouse model exploiting the X-linked Tfm mutation in mice has been a good in vivo tool for studying the human complete androgen insensitivity syndrome, but this mouse may not be the perfect in vivo model. Mouse models with various cell-specific AR knockout (ARKO) might allow us to study AR roles in individual types of cells in these male and female reproductive systems, although discrepancies are found in results between labs, probably due to using various Cre mice and/or knocking out AR in different AR domains. Nevertheless, no doubt exists that the continuous development of these ARKO mouse models and careful studies will provide information useful for understanding AR roles in reproductive systems of humans and may help us to develop more effective and more specific therapeutic approaches for reproductive system-related diseases. PMID:23782840

  10. Similarities in the behavior and molecular deficits in the frontal cortex between the neurotensin receptor subtype 1 knockout mice and chronic phencyclidine-treated mice: relevance to schizophrenia.

    PubMed

    Li, Zhimin; Boules, Mona; Williams, Katrina; Gordillo, Andres; Li, Shuhua; Richelson, Elliott

    2010-11-01

    Much evidence suggests that targeting the neurotensin (NT) system may provide a novel and promising treatment for schizophrenia. Our recent work shows that: NTS1 knockout (NTS1(-/-)) mice may provide a potential animal model for studying schizophrenia by investigating the effect of deletion NTS1 receptor on amphetamine-induced hyperactivity and neurochemical changes. The data indicate a hyper-dopaminergic state similar to the excessive striatal DA activity reported in schizophrenia. The present study was done to determine if NTS1(-/-) mice also have similar changes in behavior, in prefrontal neurotransmitters, and in protein expression, as observed in wild type (WT) mice treated with the psychotomimetic phencylclidine (PCP), an animal model for schizophrenia. Our results showed many similarities between untreated NTS1(-/-) mice and WT mice chronically treated with PCP (as compared with untreated WT mice): 1) lower PCP-induced locomotor activity; 2) similar avolition-like behavior in forced-swim test and tail suspension test; 3) lower prefrontal glutamate levels; 4) less PCP-induced dopamine release in medial prefrontal cortex (mPFC); and 5) down-regulation of mRNA and protein for DA D(1), DA D(2), and NMDAR2A in mPFC. Therefore, these data strengthen the hypothesis that the NTS1(-/-) mouse is an animal model of schizophrenia, particularly for the dysfunction of the prefrontal cortex. In addition, after chronic PCP administration, the DA D(1) receptor was up-regulated in NTS1(-/-) mice, results which suggest a possible interaction of NTS1/DA D(1) in mPFC contributing to chronic PCP-induced schizophrenia-like signs. PMID:20659557

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

    NASA Astrophysics Data System (ADS)

    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.

  12. Aryl hydrocarbon receptor knockout rats are insensitive to the pathological effects of repeated oral exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin.

    PubMed

    Harrill, Joshua A; Layko, Debra; Nyska, Abraham; Hukkanen, Renee R; Manno, Rosa Anna; Grassetti, Andrea; Lawson, Marie; Martin, Greg; Budinsky, Robert A; Rowlands, J Craig; Thomas, Russell S

    2016-06-01

    Sustained activation of the aryl hydrocarbon receptor (AHR) is believed to be the initial key event in AHR receptor-mediated tumorigenesis in the rat liver. The role of AHR in mediating pathological changes in the liver prior to tumor formation was investigated in a 4-week, repeated-dose study using adult female wild-type (WT) and AHR knockout (AHR-KO) rats treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Beginning at 8 weeks of age, AHR-KO and WT rats were dosed by oral gavage with varying concentrations of TCDD (0, 3, 22, 100, 300 and 1000 ng kg(-1)  day(-1) ). Lung, liver and thymus histopathology, hematology, serum chemistry and the distribution of TCDD in liver and adipose tissue were examined. Treatment-related increases in the severity of liver and thymus pathology were observed in WT, but not AHR-KO rats. In the liver, these included hepatocellular hypertrophy, bile duct hyperplasia, multinucleated hepatocytes and inflammatory cell foci. A loss of cellularity in the thymic cortex and thymic atrophy was observed. Treatment-related changes in serum chemistry parameters were also observed in WT, but not AHR-KO rats. Finally, dose-dependent accumulation of TCDD was observed primarily in the liver of WT rats and primarily in the adipose tissue of AHR-KO rats. The results suggest that AHR activation is the initial key event underlying the progression of histological effects leading to liver tumorigenesis following TCDD treatment. Copyright © 2015 John Wiley & Sons, Ltd. PMID:26278112

  13. Ultrasonic Vocalizations Induced by Sex and Amphetamine in M2, M4, M5 Muscarinic and D2 Dopamine Receptor Knockout Mice

    PubMed Central

    Wang, Haoran; Liang, Shuyin; Burgdorf, Jeffrey; Wess, Jurgen; Yeomans, John

    2008-01-01

    Adult mice communicate by emitting ultrasonic vocalizations (USVs) during the appetitive phases of sexual behavior. However, little is known about the genes important in controlling call production. Here, we study the induction and regulation of USVs in muscarinic and dopaminergic receptor knockout (KO) mice as well as wild-type controls during sexual behavior. Female mouse urine, but not female rat or human urine, induced USVs in male mice, whereas male urine did not induce USVs in females. Direct contact of males with females is required for eliciting high level of USVs in males. USVs (25 to120 kHz) were emitted only by males, suggesting positive state; however human-audible squeaks were produced only by females, implying negative state during male-female pairing. USVs were divided into flat and frequency-modulated calls. Male USVs often changed from continuous to broken frequency-modulated calls after initiation of mounting. In M2 KO mice, USVs were lost in about 70–80% of the mice, correlating with a loss of sexual interaction. In M5 KO mice, mean USVs were reduced by almost 80% even though sexual interaction was vigorous. In D2 KOs, the duration of USVs was extended by 20%. In M4 KOs, no significant differences were observed. Amphetamine dose-dependently induced USVs in wild-type males (most at 0.5 mg/kg i.p.), but did not elicit USVs in M5 KO or female mice. These studies suggest that M2 and M5 muscarinic receptors are needed for male USV production during male-female interactions, likely via their roles in dopamine activation. These findings are important for the understanding of the neural substrates for positive affect. PMID:18382674

  14. Altered ultrastructure, density and cathepsin K expression in bone of female muscarinic acetylcholine receptor M3 knockout mice.

    PubMed

    Lips, Katrin Susanne; Kneffel, Mathias; Willscheid, Fee; Mathies, Frank Martin; Kampschulte, Marian; Hartmann, Sonja; Panzer, Imke; Dürselen, Lutz; Heiss, Christian; Kauschke, Vivien

    2015-11-01

    High frequency of osteoporosis is found in postmenopausal women where several molecular components were identified to be involved in bone loss that subsequently leads to an increased fracture risk. Bone loss has already been determined in male mice with gene deficiency of muscarinic acetylcholine receptor M3 (M3R-KO). Here we asked whether bone properties of female 16-week old M3R-KO present similarities to osteoporotic bone loss by means of biomechanical, radiological, electron microscopic, cell- and molecular biological methods. Reduced biomechanical strength of M3R-KO correlated with cortical thickness and decreased bone mineral density (BMD). Femur and vertebrae of M3R-KO demonstrated a declined trabecular bone volume, surface, and a higher trabecular pattern factor and structure model index (SMI) compared to wild type (WT) mice. In M3R-KO, the number of osteoclasts as well as the cathepsin K mRNA expression was increased. Osteoclasts of M3R-KO showed an estimated increase in cytoplasmic vesicles. Further, histomorphometrical analysis revealed up-regulation of alkaline phosphatase. Osteoblasts and osteocytes showed a swollen cytoplasm with an estimated increase in the amount of rough endoplasmatic reticulum and in case of osteocytes a reduced pericellular space. Thus, current results on bone properties of 16-week old female M3R-KO are related to postmenopausal osteoporotic phenotype. Stimulation and up-regulation of muscarinic acetylcholine receptor subtype M3 expression in osteoblasts might be a possible new option for prevention and therapy of osteoporotic fractures. Pharmacological interventions and the risk of side effects have to be determined in upcoming studies. PMID:26002583

  15. Induction of Fatal Inflammation in LDL Receptor and ApoA-I Double-Knockout Mice Fed Dietary Fat and Cholesterol

    PubMed Central

    Zabalawi, Manal; Bhat, Shaila; Loughlin, Tara; Thomas, Michael J.; Alexander, Eric; Cline, Mark; Bullock, Bill; Willingham, Mark; Sorci-Thomas, Mary G.

    2003-01-01

    Atherogenic response to dietary fat and cholesterol challenge was evaluated in mice lacking both the LDL receptor (LDLr−/−) and apoA-I (apoA-I−/−) gene, LDLr−/−/apoA-I−/− or double-knockout mice. Gender- and age-matched LDLr−/−/apoA-I−/− mice were fed a diet consisting of 0.1% cholesterol and 10% palm oil for 16 weeks and compared to LDLr−/− mice or single-knockout mice. The LDLr−/− mice showed a 6- to 7-fold increase in total plasma cholesterol (TPC) compared to their chow-fed mice counterparts, while LDLr−/−/apoA-I−/− mice showed only a 2- to 3-fold increase in TPC compared to their chow-fed controls. This differential response to the atherogenic diet was unanticipated, since chow-fed LDLr−/− and LDLr−/−/apoA-I−/− mice began the study with similar LDL levels and differed primarily in their HDL concentration. The 6-fold diet-induced increase in TPC observed in the LDLr−/− mice occurred mainly in VLDL/LDL and not in HDL. Mid-study plasma samples taken after 8 weeks of diet feeding showed that LDLr−/− mice had TPC concentrations approximately 60% of their 16-week level, while the LDLr−/−/apoA-I−/− mice had reached 100% of their 16-week TPC concentration after only 8 weeks of diet. Male LDLr−/− mice showed similar aortic cholesterol levels to male LDLr−/−/apoA-I−/− mice despite a 4-fold higher VLDL/LDL concentration in the LDLr−/− mice. A direct comparison of the severity of aortic atherosclerosis between female LDLr−/− and LDLr−/−/apoA-I−/− mice was compromised due to the loss of female LDLr−/−/apoA-I−/− mice between 10 and 14 weeks into the study. Diet-fed female and, with time, male LDLr−/−/apoA-I−/− mice suffered from severe ulcerated cutaneous xanthomatosis. This condition, combined with a complete depletion of adrenal cholesterol, manifested in fatal wasting of the affected mice. In conclusion, LDLr−/− and LDLr−/−/apoA-I−/− mice showed

  16. Adipose tissue deficiency results in severe hyperlipidemia and atherosclerosis in the low-density lipoprotein receptor knockout mice.

    PubMed

    Wang, Mengyu; Gao, Mingming; Liao, Jiawei; Qi, Yanfei; Du, Ximing; Wang, Yuhui; Li, Ling; Liu, George; Yang, Hongyuan

    2016-05-01

    Adipose tissue can store over 50% of whole-body cholesterol; however, the physiological role of adipose tissue in cholesterol metabolism and atherogenesis has not been directly assessed. Here, we examined lipoprotein metabolism and atherogenesis in a unique mouse model of severe lipodystrophy: the Seipin(-/-) mice, and also in mice deficient in both low-density lipoprotein receptor (Ldlr) and Seipin: the Ldlr(-/-)Seipin(-/-) mice. Plasma cholesterol was moderately increased in the Seipin(-/-) mice when fed an atherogenic diet. Strikingly, plasma cholesterol reached ~6000 mg/dl in the Seipin(-/-)Ldlr(-/-) mice on an atherogenic diet, as compared to ~1000 mg/dl in the Ldlr(-/-) mice on the same diet. The Seipin(-/-)Ldlr(-/-) mice also developed spontaneous atherosclerosis on chow diet and severe atherosclerosis on an atherogenic diet. Rosiglitazone treatment significantly reduced the hypercholesterolemia of the Seipin(-/-)Ldlr(-/-) mice, and also alleviated the severity of atherosclerosis. Our results provide direct evidence, for the first time, that the adipose tissue plays a critical role in the clearance of plasma cholesterol. Our results also reveal a previously unappreciated strong link between adipose tissue and LDLR in plasma cholesterol metabolism. PMID:26921684

  17. Prenatal minocycline treatment alters synaptic protein expression, and rescues reduced mother call rate in oxytocin receptor-knockout mice.

    PubMed

    Miyazaki, Shinji; Hiraoka, Yuichi; Hidema, Shizu; Nishimori, Katsuhiko

    2016-04-01

    Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by impaired communication, difficulty in companionship, repetitive behaviors and restricted interests. Recent studies have shown amelioration of ASD symptoms by intranasal administration of oxytocin and demonstrated the association of polymorphisms in the oxytocin receptor (Oxtr) gene with ASD patients. Deficient pruning of synapses by microglial cells in the brain has been proposed as potential mechanism of ASD. Other researchers have shown specific activation of microglial cells in brain regions related to sociality in patients with ASD. Although the roles of Oxtr and microglia in ASD are in the spotlight, the relationship between them remains to be elucidated. In this study, we found abnormal activation of microglial cells and a reduction of postsynaptic density protein PSD95 expression in the Oxtr-deficient brain. Moreover, pharmacological inhibition of microglia during development can alter the expression of PSD95 and ameliorate abnormal mother-infant communication in Oxtr-deficient mice. Our results suggest that microglial abnormality is a potential mechanism of the development of Oxt/Oxtr mediated ASD-like phenotypes. PMID:26926566

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

    PubMed

    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. PMID:27013528

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

    PubMed Central

    Canelles, Sandra; Argente, Jesús; Barrios, Vicente

    2016-01-01

    ABSTRACT 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. PMID:27013528

  20. New knockout model confirms a role for androgen receptors in regulating anxiety-like behaviors and HPA response in mice.

    PubMed

    Chen, Chieh V; Brummet, Jennifer L; Lonstein, Joseph S; Jordan, Cynthia L; Breedlove, S Marc

    2014-03-01

    Men are less likely than women to suffer from anxiety disorders. Because gonadal hormones play a crucial role in many behavioral sex differences, they may underlie sex differences in human anxiety. In rodents, testosterone (T) exerts anxiolytic effects via the androgen receptor (AR): we found that male mice with a naturally-occurring mutation rendering the AR dysfunctional, referred to as spontaneous testicular feminization mutation (sTfm), showed more anxiety-like behaviors than wildtype (WT) males. Here, we used Cre-lox recombination technology to create another dysfunctional allele for AR. These induced Tfm (iTfm) animals also displayed more anxiety-like behaviors than WTs. We further found that AR-modulation of these behaviors interacts with circadian phase. When tested in the resting phase, iTfms appeared more anxious than WTs in the open field, novel object and elevated plus maze tests, but not the light/dark box. However, when tested during the active phase (lights off), iTfms showed more anxiety-related behavior than WTs in all four tests. Finally, we confirmed a role of T acting via AR in regulating HPA axis activity, as WT males with T showed a lower baseline and overall corticosterone response, and a faster return to baseline following mild stress than did WT males without T or iTfms. These findings demonstrate that this recombined AR allele is a valuable model for studying androgenic modulation of anxiety, that the anxiolytic effects of AR in mice are more prominent in the active phase, and that HPA axis modulation by T is AR dependent. PMID:24440052

  1. Epididymal Hypo-Osmolality Induces Abnormal Sperm Morphology and Function in the Estrogen Receptor Alpha Knockout Mouse1

    PubMed Central

    Joseph, Avenel; Shur, Barry D.; Ko, CheMyong; Chambon, Pierre; Hess, Rex A.

    2010-01-01

    Estrogen receptor-alpha (ESR1) is highly expressed in the efferent ductules of all species studied as well as in the epididymal epithelium in mice and other select species. Male mice lacking ESR1 (Esr1KO) are infertile, but transplantation studies demonstrated that Esr1KO germ cells are capable of fertilization when placed in a wild-type reproductive tract. These results suggest that extratesticular regions, such as the efferent ductules and epididymis, are the major source of pathological changes in Esr1KO males. Previous studies have shown alterations in ion and fluid transporters in the efferent duct and epididymal epithelia of Esr1KO males, leading to misregulation of luminal fluid pH. To determine the effect of an altered epididymal milieu on Esr1KO sperm, we assayed sperm morphology in the different regions of the epididymis. Sperm recovered from the epididymis exhibited abnormal flagellar coiling and increased incidence of spontaneous acrosome reactions, both of which are consistent with exposure to abnormal epididymal fluid. Analysis of the epididymal fluid revealed that the osmolality of the Esr1KO fluid was reduced relative to wild type, consistent with prior reports of inappropriate fluid absorption from the efferent ductules. This, along with the finding that morphological defects increased with transit through the epididymal duct, suggests that the anomalies in sperm are a consequence of the abnormal luminal environment. Consistent with this, incubating Esr1KO sperm in a more wild-type-like osmotic environment significantly rescued the abnormal flagellar coiling. This work demonstrates that Esr1KO mice exhibit an abnormal fluid environment in the lumen of the efferent ducts and epididymis, precluding normal sperm maturation and instead resulting in progressive deterioration of sperm that contributes to infertility. PMID:20130266

  2. Angiotensin receptor-1A knockout leads to hydronephrosis not associated with a loss of pyeloureteric peristalsis in the mouse renal pelvis.

    PubMed

    Nguyen, Michael J; Hashitani, Hikaru; Lang, Richard J

    2016-05-01

    The action of angiotensin II (AngII) on the Ca(2+) signals driving pyeloureteric peristalsis was investigated using both conventional and angiotensin receptor (ATr) ATr1A and ATr2 knockout ((-/-)) mice. Contractility in the renal pelvis of adult ATr1A(-/-) and ATr2(-/-) mice was compared to their respective wildtype (ATr1A(+/+) and ATr2(+/+)) controls of the same genetic background (FVB/N and C57Bl/6 respectively) using video microscopy. The effects of AngII on the Ca(2+) signals in typical and atypical smooth muscle cells (TSMCs and ASMCs, respectively) within the pelvic wall of conventional mice were recorded using Fluo-4 Ca(2+) imaging. Compared to ATr1A(+/+) , ATr2(+/+) and ATr2(-/-) mice, kidneys of the ATr1A(-/-) mouse were mildly-to-severely hydronephrotic, associated with an enlarged calyx, an atrophic papilla and a hypoplastic renal pelvis. Contraction frequencies in the renal pelvis of moderately hydronephrotic ATr1A(-/-) and unaffected ATr2(-/-) mice were not significantly different from their ATr1A(+/+), ATr2(+/+) controls. No contractions were observed in severely-hydronephrotic ATr1A(-/-) kidneys. AngII increased the spontaneous contraction frequency of the renal pelvis in ATr1A(+/+), ATr2(+/+) and ATr2(-/-) mice, but had little effect on the contractions in the mildly-hydronephrotic ATr1A(-/-) renal pelvis. The ATr1 blocker, candesartan prevented the positive chronotropic effects of AngII. AngII increased the frequency and synchronicity of Ca(2+) transients in both TSMCs and ASMCs. It was concluded that the hydronephrosis observed in ATr1A(-/-) mouse kidneys does not arise from a failure in the development of the essential pacemaker and contractile machinery driving pyeloureteric peristalsis. PMID:26876143

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

    PubMed Central

    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. PMID:26580567

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

    PubMed

    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. PMID:26580567

  5. The two-receptor model of lipoprotein clearance: tests of the hypothesis in "knockout" mice lacking the low density lipoprotein receptor, apolipoprotein E, or both proteins.

    PubMed Central

    Ishibashi, S; Herz, J; Maeda, N; Goldstein, J L; Brown, M S

    1994-01-01

    Apolipoprotein E (apoE) is hypothesized to mediate lipoprotein clearance by binding to two receptors: (i) the low density lipoprotein receptor (LDLR) and (ii) a chylomicron remnant receptor. To test this hypothesis, we have compared plasma lipoproteins in mice that are homozygous for targeted disruptions of the genes for apoE [apoE(-/-)], the LDLR [LDLR(-/-)], and both molecules [poE(-/-); LDLR(-/-)]. On a normal chow diet, apoE(-/-) mice had higher mean plasma cholesterol levels than LDLR(-/-) mice (579 vs. 268 mg/dl). Cholesterol levels in the apoE(-/-); LDLR(-/-) mice were not significantly different from those in the apoE(-/-) mice. LDLR(-/-) mice had a relatively isolated elevation in plasma LDL, whereas apoE(-/-) mice had a marked increase in larger lipoproteins corresponding to very low density lipoproteins and chylomicron remnants. The lipoprotein pattern in apoE(-/-); LDLR(-/-) mice resembled that of apoE(-/-) mice. The LDLR(-/-) mice had a marked elevation in apoB-100 and a modest increase in apoB-48. In contrast, the apoE(-/-) mice had a marked elevation in apoB-48 but not in apoB-100. The LDLR(-/-); apoE(-/-) double homozygotes had marked elevations of both apolipoproteins. The observation that apoB-48 increases more dramatically with apoE deficiency than with LDLR deficiency supports the notion that apoE binds to a second receptor in addition to the LDLR. This conclusion is also supported by the observation that superimposition of a LDLR deficiency onto an apoE deficiency [apoE(-/-); LDLR(-/-) double homozygotes] does not increase hypercholesterolemia beyond the level observed with apoE deficiency alone. Images PMID:8183926

  6. Pentameric quaternary structure of the intracellular domain of serotonin type 3A receptors

    PubMed Central

    Pandhare, Akash; Grozdanov, Petar N.; Jansen, Michaela

    2016-01-01

    In spite of extensive efforts over decades an experimentally-derived structure of full-length eukaryotic pentameric ligand-gated ion channels (pLGICs) is still lacking. These pharmaceutically highly-relevant channels contain structurally well-conserved and characterized extracellular and transmembrane domains. The intracellular domain (ICD), however, has been orphaned in structural studies based on the consensus assumption of being largely disordered. In the present study, we demonstrate for the first time that the serotonin type 3A (5-HT3A) ICD assembles into stable pentamers in solution in the absence of the other two domains, thought to be the drivers for oligomerization. Additionally, the soluble 5-HT3A-ICD construct interacted with the protein RIC-3 (resistance to inhibitors of cholinesterase). The interaction provides evidence that the 5-HT3A-ICD is not only required but also sufficient for interaction with RIC-3. Our results suggest the ICD constitutes an oligomerization domain. This novel role significantly adds to its known contributions in receptor trafficking, targeting, and functional fine-tuning. The innate diversity of the ICDs with sizes ranging from 50 to 280 amino acids indicates new methodologies need to be developed to determine the structures of these domains. The use of soluble ICD proteins that we report in the present study constitutes a useful approach to address this gap. PMID:27045630

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

    PubMed Central

    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. PMID:26441906

  8. Altered serotonin and dopamine metabolism in the CNS of serotonin 5-HT(1A) or 5-HT(1B) receptor knockout mice.

    PubMed

    Ase, A R; Reader, T A; Hen, R; Riad, M; Descarries, L

    2000-12-01

    Measurements of serotonin (5-HT), dopamine (DA), and noradrenaline, and of 5-HT and DA metabolites, were obtained by HPLC from 16 brain regions and the spinal cord of 5-HT(1A) or 5-HT(1B) knockout and wild-type mice of the 129/Sv strain. In 5-HT(1A) knockouts, 5-HT concentrations were unchanged throughout, but levels of 5-HT metabolites were higher than those of the wild type in dorsal/medial raphe nuclei, olfactory bulb, substantia nigra, and locus coeruleus. This was taken as an indication of increased 5-HT turnover, reflecting an augmented basal activity of midbrain raphe neurons and consequent increase in their somatodendritic and axon terminal release of 5-HT. It provided a likely explanation for the increased anxious-like behavior observed in 5-HT(1A) knockout mice. Concomitant increases in DA content and/or DA turnover were interpreted as the result of a disinhibition of DA, whereas increases in noradrenaline concentration in some territories of projection of the locus coeruleus could reflect a diminished activity of its neurons. In 5-HT(1B) knockouts, 5-HT concentrations were lower than those of the wild type in nucleus accumbens, locus coeruleus, spinal cord, and probably also several other territories of 5-HT innervation. A decrease in DA, associated with increased DA turnover, was measured in nucleus accumbens. These changes in 5-HT and DA metabolism were consistent with the increased aggressiveness and the supersensitivity to cocaine reported in 5-HT(1B) knockout mice. Thus, markedly different alterations in CNS monoamine metabolism may contribute to the opposite behavioral phenotypes of these two knockouts. PMID:11080193

  9. Role of ω-hydroxylase in adenosine-mediated aortic response through MAP kinase using A2A-receptor knockout mice.

    PubMed

    Ponnoth, Dovenia S; Nayeem, Mohammed A; Kunduri, Swati S; Tilley, Stephen L; Zeldin, Darryl C; Ledent, Catherine; Mustafa, S Jamal

    2012-02-15

    Previously, we have shown that A(2A) adenosine receptor (A(2A)AR) knockout mice (KO) have increased contraction to adenosine. The signaling mechanism(s) for A(2A)AR is still not fully understood. In this study, we hypothesize that, in the absence of A(2A)AR, ω-hydroxylase (Cyp4a) induces vasoconstriction through mitogen-activated protein kinase (MAPK) via upregulation of adenosine A(1) receptor (A(1)AR) and protein kinase C (PKC). Organ bath and Western blot experiments were done using isolated aorta from A(2A)KO and corresponding wild-type (WT) mice. Isolated aortic rings from WT and A(2A)KO mice were precontracted with submaximal dose of phenylephrine (10(-6) M), and concentration responses for selective A(1)AR, A(2A)AR agonists, angiotensin II and cytochrome P-450-epoxygenase, 20-hydroxyeicosatrienoic acid (20-HETE) PKC, PKC-α, and ERK1/2 inhibitors were obtained. 2-p-(2-Carboxyethyl)-phenethylamino-5'-N-ethylcarboxamidoadenosine hydrochloride (CGS-21680, A(2A)AR agonist) induced concentration-dependent relaxation in WT, which was blocked by methylsulfonyl-propargyloxyphenylhexanamide (cytochrome P-450-epoxygenase inhibitor; 10(-5) M) and also with removal of endothelium. A(1) agonist, 2-chloro-N(6)-cyclopentyladenosine (CCPA) produced higher contraction in A(2A)KO aorta than WT (49.2 ± 8.5 vs. 27 ± 5.9% at 10(-6) M, P < 0.05). 20-HETE produced higher contraction in A(2A)KO than WT (50.6 ± 8.8 vs. 21.1 ± 3.3% at 10(-7) M, P < 0.05). Contraction to CCPA in WT and A(2A)KO aorta was inhibited by PD-98059 (p42/p44 MAPK inhibitor; 10(-6) M), chelerythrine chloride (nonselective PKC blocker; 10(-6) M), Gö-6976 (selective PKC-α inhibitor; 10(-7) M), and HET0016 (20-HETE inhibitor; 10(-5) M). Also, contraction to 20-HETE in WT and A(2A)KO aorta was inhibited by PD-98059 and Gö-6976. Western blot analysis indicated the upregulation of A(1)AR, Cyp4a, PKC-α, and phosphorylated-ERK1/2 in A(2A)KO compared with WT (P < 0.05), while expression of Cyp2c29 was

  10. 5-HT3 Receptor Brain-Type B-Subunits are Differentially Expressed in Heterologous Systems

    PubMed Central

    2015-01-01

    Genes for five different 5-HT3 receptor subunits have been identified. Most of the subunits have multiple isoforms, but two isoforms of the B subunits, brain-type 1 (Br1) and brain-type 2 (Br2) are of particular interest as they appear to be abundantly expressed in human brain, where 5-HT3B subunit RNA consists of approximately 75% 5-HT3Br2, 24% 5-HT3Br1, and <1% 5-HT3B. Here we use two-electrode voltage-clamp, radioligand binding, fluorescence, whole cell, and single channel patch-clamp studies to characterize the roles of 5-HT3Br1 and 5-HT3Br2 subunits on function and pharmacology in heterologously expressed 5-HT3 receptors. The data show that the 5-HT3Br1 transcriptional variant, when coexpressed with 5-HT3A subunits, alters the EC50, nH, and single channel conductance of the 5-HT3 receptor, but has no effect on the potency of competitive antagonists; thus, 5-HT3ABr1 receptors have the same characteristics as 5-HT3AB receptors. There were some differences in the shapes of 5-HT3AB and 5-HT3ABr1 receptor responses, which were likely due to a greater proportion of homomeric 5-HT3A versus heteromeric 5-HT3ABr1 receptors in the latter, as expression of the 5-HT3Br1 compared to the 5-HT3B subunit is less efficient. Conversely, the 5-HT3Br2 subunit does not appear to form functional channels with the 5-HT3A subunit in either oocytes or HEK293 cells, and the role of this subunit is yet to be determined. PMID:25951416

  11. The Role of 5-HT3 Receptors in Signaling from Taste Buds to Nerves.

    PubMed

    Larson, Eric D; Vandenbeuch, Aurelie; Voigt, Anja; Meyerhof, Wolfgang; Kinnamon, Sue C; Finger, Thomas E

    2015-12-01

    Activation of taste buds triggers the release of several neurotransmitters, including ATP and serotonin (5-hydroxytryptamine; 5-HT). Type III taste cells release 5-HT directly in response to acidic (sour) stimuli and indirectly in response to bitter and sweet tasting stimuli. Although ATP is necessary for activation of nerve fibers for all taste stimuli, the role of 5-HT is unclear. We investigated whether gustatory afferents express functional 5-HT3 receptors and, if so, whether these receptors play a role in transmission of taste information from taste buds to nerves. In mice expressing GFP under the control of the 5-HT(3A) promoter, a subset of cells in the geniculate ganglion and nerve fibers in taste buds are GFP-positive. RT-PCR and in situ hybridization confirmed the presence of 5-HT(3A) mRNA in the geniculate ganglion. Functional studies show that only those geniculate ganglion cells expressing 5-HT3A-driven GFP respond to 10 μM 5-HT and this response is blocked by 1 μM ondansetron, a 5-HT3 antagonist, and mimicked by application of 10 μM m-chlorophenylbiguanide, a 5-HT3 agonist. Pharmacological blockade of 5-HT3 receptors in vivo or genetic deletion of the 5-HT3 receptors reduces taste nerve responses to acids and other taste stimuli compared with controls, but only when urethane was used as the anesthetic. We find that anesthetic levels of pentobarbital reduce taste nerve responses apparently by blocking the 5-HT3 receptors. Our results suggest that 5-HT released from type III cells activates gustatory nerve fibers via 5-HT3 receptors, accounting for a significant proportion of the neural taste response. PMID:26631478

  12. Impact of intracellular domain flexibility upon properties of activated human 5-HT3 receptors*

    PubMed Central

    Kozuska, J L; Paulsen, I M; Belfield, W J; Martin, I L; Cole, D J; Holt, A; Dunn, S M J

    2014-01-01

    Background and Purpose It has been proposed that arginine residues lining the intracellular portals of the homomeric 5-HT3A receptor cause electrostatic repulsion of cation flow, accounting for a single-channel conductance substantially lower than that of the 5-HT3AB heteromer. However, comparison of receptor homology models for wild-type pentamers suggests that salt bridges in the intracellular domain of the homomer may impart structural rigidity, and we hypothesized that this rigidity could account for the low conductance. Experimental Approach Mutations were introduced into the portal region of the human 5-HT3A homopentamer, such that putative salt bridges were broken by neutralizing anionic partners. Single-channel and whole cell currents were measured in transfected tsA201 cells and in Xenopus oocytes respectively. Computational simulations of protein flexibility facilitated comparison of wild-type and mutant receptors. Key Results Single-channel conductance was increased substantially, often to wild-type heteromeric receptor values, in most 5-HT3A mutants. Conversely, introduction of arginine residues to the portal region of the heteromer, conjecturally creating salt bridges, decreased conductance. Gating kinetics varied significantly between different mutant receptors. EC50 values for whole-cell responses to 5-HT remained largely unchanged, but Hill coefficients for responses to 5-HT were usually significantly smaller in mutants. Computational simulations suggested increased flexibility throughout the protein structure as a consequence of mutations in the intracellular domain. Conclusions and Implications These data support a role for intracellular salt bridges in maintaining the quaternary structure of the 5-HT3 receptor and suggest a role for the intracellular domain in allosteric modulation of cooperativity and agonist efficacy. Linked Article This article is commented on by Vardy and Kenakin, pp. 1614–1616 of volume 171 issue 7. To view this commentary

  13. Expression of 5-HT3 receptors and TTX resistant sodium channels (NaV1.8) on muscle nerve fibers in pain-free humans and patients with chronic myofascial temporomandibular disorders

    PubMed Central

    2014-01-01

    Background Previous studies have shown that 5-HT3-antagonists reduce muscle pain, but there are no studies that have investigated the expression of 5-HT3-receptors in human muscles. Also, tetrodotoxin resistant voltage gated sodium-channels (NaV) are involved in peripheral sensitization and found in trigeminal ganglion neurons innervating the rat masseter muscle. This study aimed to investigate the frequency of nerve fibers that express 5-HT3A-receptors alone and in combination with NaV1.8 sodium-channels in human muscles and to compare it between healthy pain-free men and women, the pain-free masseter and tibialis anterior muscles, and patients with myofascial temporomandibular disorders (TMD) and pain-free controls. Methods Three microbiopsies were obtained from the most bulky part of the tibialis and masseter muscles of seven and six healthy men and seven and six age-matched healthy women, respectively, while traditional open biopsies were obtained from the most painful spot of the masseter of five female patients and from a similar region of the masseter muscle of five healthy, age-matched women. The biopsies were processed by routine immunohistochemical methods. The biopsy sections were incubated with monoclonal antibodies against the specific axonal marker PGP 9.5, and polyclonal antibodies against the 5-HT3A-receptors and NaV1.8 sodium-channels. Results A similar percentage of nerve fibers in the healthy masseter (85.2%) and tibialis (88.7%) muscles expressed 5-HT3A-receptors. The expression of NaV1.8 by 5-HT3A positive nerve fibers associated with connective tissue was significantly higher than nerve fibers associated with myocytes (P < .001). In the patients, significantly more fibers per section were found with an average of 3.8 ± 3 fibers per section in the masseter muscle compared to 2.7 ± 0.2 in the healthy controls (P = .024). Further, the frequency of nerve fibers that co-expressed NaV1.8 and 5-HT3A receptors was significantly

  14. Effects of D-4F on Vasodilation and Vessel Wall Thickness in Hypercholesterolemic LDL Receptor Null and LDL receptor/ApoA-I Double Knockout Mice on Western Diet

    PubMed Central

    Ou, Jingsong; Wang, Jingli; Xu, Hao; Ou, Zhijun; Sorci-Thomas, Mary G.; Jones, Deron W.; Signorino, Paul; Densmore, John C.; Kaul, Sushma; Oldham, Keith T.; Pritchard, Kirkwood A.

    2005-01-01

    Previously we showed L-4F, a novel apolipoprotein A-I (apoA-I) mimetic, improved vasodilation in two dissimilar models of vascular disease; hypercholesterolemic low-density lipoprotein (LDL) receptor null (Ldlr −/−) mice and transgenic sickle cell disease mice. Here we determine the mechanisms by which D-4F improves vasodilation and arterial wall thickness in hypercholesterolemic Ldlr −/− mice and Ldlr −/−/apoA-I null (apoA-I −/−), double knockout mice. Ldlr −/− and Ldlr −/−/apoA-I −/− mice were fed western diet (WD) ± D-4F. Oral D-4F restored endothelium- and eNOS-dependent vasodilation in direct relationship to duration of treatments and reduced wall thickness in as little as 2 weeks in vessels with pre-existing disease in Ldlr −/− mice. D-4F had no effect on total or HDL cholesterol concentrations but reduced proinflammatory HDL levels. D-4F had no effect on plasma myeloperoxidase (MPO) concentrations but reduced MPO association with apoA-I as well as 3-nitrotyrosine in apoA-I. D-4F increased endothelium- and eNOS-dependent vasodilation in Ldlr −/−/apoA-I −/− mice but did not reduce wall thickness as it had in Ldlr −/− mice. Vascular endothelial cells were treated with 22-hydroxycholesterol (22-OHC) ± L-4F. 22-OHC decreased nitric oxide (•NO) and increased superoxide anion (O2 •−) production and increased ABCA-1 and collagen expression. L-4F restored •NO and O2 •− balance, had little effect on ABCA-1 expression but reduced collagen expression. These data demonstrate that although D-4F restores vascular endothelial cell and eNOS function to increase vasodilation, HDL containing apoA-I, or at least some critical concentration of the anti-atherogenic lipoprotein, is required for D-4F to decrease vessel wall thickness. PMID:16224061

  15. In vivo SPECT and ex vivo autoradiographic brain imaging of the novel selective CB1 receptor antagonist radioligand [125I]SD7015 in CB1 knock-out and wildtype mouse.

    PubMed

    Máthé, Domokos; Horváth, Ildikó; Szigeti, Krisztián; Donohue, Sean R; Pike, Victor W; Jia, Zisheng; Ledent, Catherine; Palkovits, Miklós; Freund, Tamás F; Halldin, Christer; Gulyás, Balázs

    2013-02-01

    We aimed to evaluate the novel high-affinity and relatively lipophilic CB(1) receptor (CB(1)R) antagonist radioligand [(125)I]SD7015 for SPECT imaging of CB(1)Rs in vivo using the multiplexed multipinhole dedicated small animal SPECT/CT system, NanoSPECT/CT(PLUS) (Mediso, Budapest, Hungary), in knock-out CB(1) receptor knock-out (CB(1)R-/-) and wildtype mice. In order to exclude possible differences in cerebral blood flow between the two types of animals, HMPAO SPECT scans were performed, whereas in order to confirm the brain uptake differences of the radioligand between knock-out mice and wildtype mice, in vivo scans were complemented with ex vivo autoradiographic measurements using the brains of the same animals. With SPECT/CT imaging, we measured the brain uptake of radioactivity, using %SUV (% standardised uptake values) in CB(1)R-/- mice (n=3) and C57BL6 wildtype mice (n=7) under urethane anaesthesia after injecting [(125)I]SD7015 intravenously or intraperitoneally. The Brookhaven Laboratory mouse MRI atlas was fused to the SPECT/CT images by using a combination of rigid and non-rigid algorithms in the Mediso Fusion™ (Mediso, Budapest, Hungary) and VivoQuant (inviCRO, Boston, MA, USA) softwares. Phosphor imager plate autoradiography (ARG) was performed on 4 μm-thin cryostat sections of the excised brains. %SUV was 8.6±3.6 (average±SD) in CB(1)R-/- mice and 22.1±12.4 in wildtype mice between 2 and 4 h after injection (p<0.05). ARG of identically taken sections from wildtype mouse brain showed moderate radioactivity uptake when compared with the in vivo images, with a clear difference between grey matter and white matter, whereas ARG in CB(1)R(-/-) mice showed practically no radioactivity uptake. [(125)I]SD7015 enters the mouse brain in sufficient amount to enable SPECT imaging. Brain radioactivity distribution largely coincides with that of the known CB(1)R expression pattern in rodent brain. We conclude that [(125)I]SD7015 should be a useful SPECT

  16. In vivo SPECT and ex vivo autoradiographic brain imaging of the novel selective CB1 receptor antagonist radioligand [125I]SD7015 in CB1 knock-out and wildtype mouse

    PubMed Central

    Máthé, Domokos; Horváth, Ildikó; Szigeti, Krisztián; Donohue, Sean R.; Pike, Victor W.; Jia, Zisheng; Ledent, Catherine; Palkovits, Miklós; Freund, Tamás F.; Halldin, Christer; Gulyás, Balázs

    2014-01-01

    We aimed to evaluate the novel high-affinity and relatively lipophilic CB1 receptor (CB1R) antagonist radioligand [125I]SD7015 for SPECT imaging of CB1Rs in vivo using the multiplexed multipinhole dedicated small animal SPECT/CT system, NanoSPECT/CTPLUS (Mediso, Budapest, Hungary), in knock-out CB1 receptor knock-out (CB1R-/-) and wildtype mice. In order to exclude possible differences in cerebral blood flow between the two types of animals, HMPAO SPECT scans were performed, whereas in order to confirm the brain uptake differences of the radioligand between knock-out mice and wildtype mice, in vivo scans were complemented with ex vivo autoradiographic measurements using the brains of the same animals. With SPECT/CT imaging, we measured the brain uptake of radioactivity, using %SUV (% standardised uptake values) in CB1R-/- mice (n = 3) and C57BL6 wildtype mice (n = 7) under urethane anaesthesia after injecting [125I]SD7015 intravenously or intraperitoneally. The Brookhaven Laboratory mouse MRI atlas was fused to the SPECT/CT images by using a combination of rigid and non-rigid algorithms in the Mediso Fusion™ (Mediso, Budapest, Hungary) and VivoQuant (inviCRO, Boston, MA, USA) softwares. Phosphor imager plate autoradiography (ARG) was performed on 4 μm-thin cryostat sections of the excised brains. %SUV was 8.6 ± 3.6 (average ± SD) in CB1R-/- mice and 22.1 ± 12.4 in wildtype mice between 2 and 4 h after injection (p < 0.05). ARG of identically taken sections from wildtype mouse brain showed moderate radioactivity uptake when compared with the in vivo images, with a clear difference between grey matter and white matter, whereas ARG in CB1R(-/-) mice showed practically no radioactivity uptake. [125I]SD7015 enters the mouse brain in sufficient amount to enable SPECT imaging. Brain radioactivity distribution largely coincides with that of the known CB1R expression pattern in rodent brain. We conclude that [125I]SD7015 should be a useful SPECT radioligand for

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

    PubMed Central

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

    2015-01-01

    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. PMID:25964300

  18. Delineation of the functional properties and the mechanism of action of TMPPAA, an allosteric agonist and positive allosteric modulator of 5-HT3 receptors.

    PubMed

    Gasiorek, Agnes; Trattnig, Sarah M; Ahring, Philip K; Kristiansen, Uffe; Frølund, Bente; Frederiksen, Kristen; Jensen, Anders A

    2016-06-15

    We have previously identified a novel class of 5-hydroxytryptamine type 3 receptor (5-HT3R) agonists sharing little structural similarity with orthosteric 5-HT3R ligands (Jørgensen et al., 2011). In the present study we have elucidated the functional characteristics and the mechanism of action of one of these compounds, trans-3-(4-methoxyphenyl)-N-(pentan-3-yl)acrylamide (TMPPAA). In electrophysiological recordings TMPPAA was found to be a highly-efficacious partial agonist equipotent with 5-HT at the 5-HT3A receptor (5-HT3AR) expressed in COS-7 cells and somewhat less potent at the receptor expressed in Xenopus oocytes. The desensitization kinetics of TMPPAA-evoked currents were very different from those mediated by 5-HT. Moreover, repeated TMPPAA applications resulted in progressive current run-down and persistent non-responsiveness of the receptor to TMPPAA, but not to 5-HT. In addition to its direct activation, TMPPAA potentiated 5-HT-mediated 5-HT3AR signalling, and the allosteric link between the two binding sites was corroborated by the analogous ability of 5-HT to potentiate TMPPAA-evoked responses. The agonism and potentiation exerted by TMPPAA at a chimeric α7-nACh/5-HT3A receptor suggested that the ligand acts through the transmembrane domain of 5-HT3AR, a notion further substantiated by its functional properties at chimeric and mutant human/murine 5-HT3ARs. A residue in the transmembrane helix 4 of 5-HT3A was identified as an important molecular determinant for the different agonist potencies exhibited by TMPPAA at human and murine 5-HT3ARs. In conclusion, TMPPAA is a novel allosteric agonist and positive allosteric modulator of 5-HT3Rs, and its aberrant signalling characteristics compared to 5-HT at the 5-HT3AR underline the potential in Cys-loop receptor modulation and activation through allosteric sites. PMID:27086281

  19. Mechanisms in Knockout Reactions

    NASA Astrophysics Data System (ADS)

    Bazin, D.; Charity, R. J.; de Souza, R. T.; Famiano, M. A.; Gade, A.; Henzl, V.; Henzlova, D.; Hudan, S.; Lee, J.; Lukyanov, S.; Lynch, W. G.; McDaniel, S.; Mocko, M.; Obertelli, A.; Rogers, A. M.; Sobotka, L. G.; Terry, J. R.; Tostevin, J. A.; Tsang, M. B.; Wallace, M. S.

    2009-06-01

    We report the first detailed study of the relative importance of the stripping and diffraction mechanisms involved in nucleon knockout reactions, by the use of a coincidence measurement of the residue and fast proton following one-proton knockout reactions. The measurements used the S800 spectrograph in combination with the HiRA detector array at the NSCL. Results for the reactions Be9(C9,B8+X)Y and Be9(B8,Be7+X)Y are presented and compared with theoretical predictions for the two reaction mechanisms calculated using the eikonal model. The data show a clear distinction between the stripping and diffraction mechanisms and the measured relative proportions are very well reproduced by the reaction theory. This agreement adds support to the results of knockout reaction analyses and their applications to the spectroscopy of rare isotopes.

  20. Specific Activation of A3, A2A and A1 Adenosine Receptors in CD73-Knockout Mice Affects B16F10 Melanoma Growth, Neovascularization, Angiogenesis and Macrophage Infiltration

    PubMed Central

    Koszałka, Patrycja; Gołuńska, Monika; Urban, Aleksandra; Stasiłojć, Grzegorz; Stanisławowski, Marcin; Majewski, Marceli; Składanowski, Andrzej C.; Bigda, Jacek

    2016-01-01

    CD73 (ecto-5'-nucleotidase), a cell surface enzyme hydrolyzing AMP to adenosine, was lately demonstrated to play a direct role in tumor progression including regulation of tumor vascularization. It was also shown to stimulate tumor macrophage infiltration. Interstitial adenosine, accumulating in solid tumors due to CD73 enzymatic activity, is recognized as a main mediator regulating the production of pro- and anti-angiogenic factors, but the engagement of specific adenosine receptors in tumor progression in vivo is still poorly researched. We have analyzed the role of high affinity adenosine receptors A1, A2A, and A3 in B16F10 melanoma progression using specific agonists (CCPA, CGS-21680 and IB-MECA, respectively). We limited endogenous extracellular adenosine background using CD73 knockout mice treated with CD73 chemical inhibitor, AOPCP (adenosine α,β-methylene 5’-diphosphate). Activation of any adenosine receptor significantly inhibited B16F10 melanoma growth but only at its early stage. At 14th day of growth, the decrease in tumor neovascularization and MAPK pathway activation induced by CD73 depletion was reversed by all agonists. Activation of A1AR primarily increased angiogenic activation measured by expression of VEGF-R2 on tumor blood vessels. However, mainly A3AR activation increased both the microvessel density and expression of pro-angiogenic factors. All agonists induced significant increase in macrophage tumor infiltration, with IB-MECA being most effective. This effect was accompanied by substantial changes in cytokines regulating macrophage polarization between pro-inflammatory and pro-angiogenic phenotype. Our results demonstrate an evidence that each of the analyzed receptors has a specific role in the stimulation of tumor angiogenesis and confirm significantly more multifaceted role of adenosine in its regulation than was already observed. They also reveal previously unexplored consequences to extracellular adenosine signaling depletion in

  1. 5-Chloroindole: a potent allosteric modulator of the 5-HT3 receptor

    PubMed Central

    Newman, Amy S; Batis, Nikolaos; Grafton, Gillian; Caputo, Francesca; Brady, Catherine A; Lambert, Jeremy J; Peters, John A; Gordon, John; Brain, Keith L; Powell, Andrew D; Barnes, Nicholas M

    2013-01-01

    Background and Purpose The 5-HT3 receptor is a ligand-gated ion channel that is modulated allosterically by various compounds including colchicine, alcohols and volatile anaesthetics. However the positive allosteric modulators (PAMs) identified to date have low affinity, which hinders investigation because of non-selective effects at pharmacologically active concentrations. The present study identifies 5-chloroindole (Cl-indole) as a potent PAM of the 5-HT3 receptor. Experimental Approach 5-HT3 receptor function was assessed by the increase in intracellular calcium and single-cell electrophysiological recordings in HEK293 cells stably expressing the h5-HT3A receptor and also the mouse native 5-HT3 receptor that increases neuronal contraction of bladder smooth muscle. Key Results Cl-indole (1–100 μM) potentiated agonist (5-HT) and particularly partial agonist [(S)-zacopride, DDP733, RR210, quipazine, dopamine, 2-methyl-5-HT, SR57227A, meta chlorophenyl biguanide] induced h5-HT3A receptor-mediated responses. This effect of Cl-indole was also apparent at the mouse native 5-HT3 receptor. Radioligand-binding studies identified that Cl-indole induced a small (∼twofold) increase in the apparent affinity of 5-HT for the h5-HT3A receptor, whereas there was no effect upon the affinity of the antagonist, tropisetron. Cl-indole was able to reactivate desensitized 5-HT3 receptors. In contrast to its effect on the 5-HT3 receptor, Cl-indole did not alter human nicotinic α7 receptor responses. Conclusions and Implications The present study identifies Cl-indole as a relatively potent and selective PAM of the 5-HT3 receptor; such compounds will aid investigation of the molecular basis for allosteric modulation of the 5-HT3 receptor and may assist the discovery of novel therapeutic drugs targeting this receptor. Linked Articles Recent reviews on allosteric modulation can be found at: Kenakin, T (2013). New concepts in pharmacological efficacy at 7TM receptors: IUPHAR Review 2

  2. GABA-A Receptor Inhibition of Local Calcium Signaling in Spines and Dendrites

    PubMed Central

    Marlin, Joseph J.

    2014-01-01

    Cortical interneurons activate GABA-A receptors to rapidly control electrical and biochemical signaling at pyramidal neurons. Different populations of interneurons are known to uniquely target the soma and dendrites of pyramidal neurons. However, the ability of these interneurons to inhibit Ca2+ signaling at spines and dendrites is largely unexplored. Here we use whole-cell recordings, two-photon microscopy, GABA uncaging and optogenetics to study dendritic inhibition at layer 5 (L5) pyramidal neurons in slices of mouse PFC. We first show that GABA-A receptors strongly inhibit action potential (AP)-evoked Ca2+ signals at both spines and dendrites. We find robust inhibition over tens of milliseconds that spreads along the dendritic branch. However, we observe no difference in the amount of inhibition at neighboring spines and dendrites. We then examine the influence of interneurons expressing parvalbumin (PV), somatostatin (SOM), or 5HT3a receptors. We determine that these populations of interneurons make unique contacts onto the apical and basal dendrites of L5 pyramidal neurons. We also show that SOM and 5HT3a but not PV interneurons potently inhibit AP Ca2+ signals via GABA-A receptors at both spines and dendrites. These findings reveal how multiple interneurons regulate local Ca2+ signaling in pyramidal neurons, with implications for cortical function and disease. PMID:25429132

  3. D2 dopamine receptors colocalize regulator of G-protein signaling 9-2 (RGS9-2) via the RGS9 DEP domain, and RGS9 knock-out mice develop dyskinesias associated with dopamine pathways.

    PubMed

    Kovoor, Abraham; Seyffarth, Petra; Ebert, Jana; Barghshoon, Sami; Chen, Ching-Kang; Schwarz, Sigrid; Axelrod, Jeffrey D; Cheyette, Benjamin N R; Simon, Melvin I; Lester, Henry A; Schwarz, Johannes

    2005-02-23

    Regulator of G-protein signaling 9-2 (RGS9-2), a member of the RGS family of G GTPase accelerating proteins, is expressed specifically in the striatum, which participates in antipsychotic-induced tardive dyskinesia and in levodopa-induced dyskinesia. We report that RGS9 knock-out mice develop abnormal involuntary movements when inhibition of dopaminergic transmission is followed by activation of D2-like dopamine receptors (DRs). These abnormal movements resemble drug-induced dyskinesia more closely than other rodent models. Recordings from striatal neurons of these mice establish that activation of D2-like DRs abnormally inhibits glutamate-elicited currents. We show that RGS9-2, via its DEP domain (for Disheveled, EGL-10, Pleckstrin homology), colocalizes with D2DRs when coexpressed in mammalian cells. Recordings from oocytes coexpressing D2DR or the m2 muscarinic receptor and G-protein-gated inward rectifier potassium channels show that RGS9-2, via its DEP domain, preferentially accelerates the termination of D2DR signals. Thus, alterations in RGS9-2 may be a key factor in the pathway leading from D2DRs to the side effects associated with the treatment both of psychoses and Parkinson's disease. PMID:15728856

  4. 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

    SciTech Connect

    Ryu, Min Sook; Woo, Min-Yeong; Kwon, Daeho; Hong, Allen E.; Song, Kye Yong; Park, Sun; Lim, In Kyoung

    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.

  5. Mechanisms in knockout reactions

    NASA Astrophysics Data System (ADS)

    Bazin, D.; Charity, R. J.; de Souza, R. T.; Famiano, M. A.; Gade, A.; Henzl, V.; Henzlova, D.; Hudan, S.; Lee, J.; Lukyanov, S.; Lynch, W. G.; McDaniel, S.; Mocko, M.; Obertelli, A.; Rogers, A. M.; Sobotka, L. G.; Terry, J. R.; Tostevin, J. A.; Tsang, M. B.; Wallace, M. S.

    2009-10-01

    We report on the first detailed study of the mechanisms involved in knockout reactions, via a coincidence measurement of the residue and fast proton in one-proton knockout reactions, using the S800 spectrograph in combination with the HiRA detector array at the NSCL. Results on the reactions ^9Be(^9C,^8B+X)Y and ^9Be(^8B,^7Be+X)Y are presented. They are compared with theoretical predictions for both the diffraction (elastic breakup) and stripping (inelastic breakup) reaction mechanisms, as calculated in the eikonal model. The data shows a clear distinction between the two reaction mechanisms, and the observed respective proportions are very well reproduced by the reaction theory. This agreement supports the results of knockout reaction analyses and their applications to the spectroscopy of rare isotopes. In particular, this add considerable support to the use of the eikonal model as a quantitative tool, able, for example, to determine single-particle spectroscopic strengths in rare isotopes.

  6. Prolonged Monoacylglycerol Lipase Blockade Causes Equivalent Cannabinoid Receptor Type 1 Receptor–Mediated Adaptations in Fatty Acid Amide Hydrolase Wild-Type and Knockout Mice

    PubMed Central

    Kinsey, Steven G.; Ignatowska-Jankowska, Bogna; Ramesh, Divya; Abdullah, Rehab A.; Tao, Qing; Booker, Lamont; Long, Jonathan Z.; Selley, Dana E.; Cravatt, Benjamin F.; Lichtman, Aron H.

    2014-01-01

    Complementary genetic and pharmacological approaches to inhibit monoacylglycerol lipase (MAGL) and fatty acid amide hydrolase (FAAH), the primary hydrolytic enzymes of the respective endogenous cannabinoids 2-arachidonoylglycerol (2-AG) and N-arachidonoylethanolamine, enable the exploration of potential therapeutic applications and physiologic roles of these enzymes. Complete and simultaneous inhibition of both FAAH and MAGL produces greatly enhanced cannabimimetic responses, including increased antinociception, and other cannabimimetic effects, far beyond those seen with inhibition of either enzyme alone. While cannabinoid receptor type 1 (CB1) function is maintained following chronic FAAH inactivation, prolonged excessive elevation of brain 2-AG levels, via MAGL inhibition, elicits both behavioral and molecular signs of cannabinoid tolerance and dependence. Here, we evaluated the consequences of a high dose of the MAGL inhibitor JZL184 [4-nitrophenyl 4-(dibenzo[d][1,3]dioxol-5-yl(hydroxy)methyl)piperidine-1-carboxylate; 40 mg/kg] given acutely or for 6 days in FAAH(−/−) and (+/+) mice. While acute administration of JZL184 to FAAH(−/−) mice enhanced the magnitude of a subset of cannabimimetic responses, repeated JZL184 treatment led to tolerance to its antinociceptive effects, cross-tolerance to the pharmacological effects of Δ9-tetrahydrocannabinol, decreases in CB1 receptor agonist–stimulated guanosine 5′-O-(3-[35S]thio)triphosphate binding, and dependence as indicated by rimonabant-precipitated withdrawal behaviors, regardless of genotype. Together, these data suggest that simultaneous elevation of both endocannabinoids elicits enhanced cannabimimetic activity but MAGL inhibition drives CB1 receptor functional tolerance and cannabinoid dependence. PMID:24849924

  7. Proestrous compared to diestrous wildtype, but not estrogen receptor beta knockout, mice have better performance in the spontaneous alternation and object recognition tasks and reduced anxiety-like behavior in the elevated plus and mirror maze

    PubMed Central

    Walf, Alicia A.; Koonce, Carolyn; Manley, Kevin; Frye, Cheryl A.

    2008-01-01

    17β-Estradiol (E2) may influence cognitive and/or affective behavior in part via the β isoform of the estrogen receptor (ERβ). Endocrine status and behavior in cognitive (object recognition, T-maze), anxiety (open field, elevated plus maze, mirror maze, emergence), and motor/coordination (rotarod, activity chamber) tasks of proestrous and diestrous wildtype (WT) and ERβ knockout (βERKO) mice was examined. Proestrous (WT or βERKO), versus diestrous, mice had higher E2 and progestin levels in plasma, hippocampus, and cortex. The only effect of genotype on hormone levels was for corticosterone, such that βERKO mice had higher concentrations of corticosterone than did WT mice. Proestrous WT, but not βERKO, mice had improved performance in the object recognition (greater percentage of time with novel object) and T-maze tasks (greater percentage of spontaneous alternations) and less anxiety-like behavior in the plus maze (increased duration on open arms) and mirror chamber task (increased duration in mirror) than did diestrous mice. This pattern was not seen in the rotarod, open field, or activity monitor, suggesting effects may be specific to affective and cognitive behavior, rather than motor behavior/coordination. Thus, enhanced performance in cognitive tasks and anti-anxiety-like behavior of proestrous mice may require actions of ERβ in the hippocampus and/or cortex. PMID:18926853

  8. Immunization of knock-out α/β interferon receptor mice against lethal bluetongue infection with a BoHV-4-based vector expressing BTV-8 VP2 antigen.

    PubMed

    Franceschi, Valentina; Capocefalo, Antonio; Calvo-Pinilla, Eva; Redaelli, Marco; Mucignat-Caretta, Carla; Mertens, Peter; Ortego, Javier; Donofrio, Gaetano

    2011-04-01

    New effective tools for vaccine strategies are necessary to limit the spread of bluetongue, an insect-transmitted viral disease of domestic and wild ruminants. In the present study, BoHV-4-based vector cloned as a bacterial artificial chromosome (BAC) was engineered to express the bluetongue virus (BTV) immune-dominant glycoprotein VP2 provided of a heterologous signal peptide to its amino terminal and a trans-membrane domain to its carboxyl terminal (IgK-VP2gDtm), to allow the VP2 expression targeting to the cell membrane fraction. Based on adult α/β interferon receptor knockout (IFNAR(-/-)) mice, a newly generated bluetongue laboratory animal model, a pre-challenge experiment was performed to test BoHV-4 safety on such immune-compromised animal. BoHV-4 infected IFNAR(-/-) mice did not show clinical signs even following the inoculation of BoHV-4 intra-cerebrally, although many areas of the brain got transduced. IFNAR(-/-) mice intraperitoneally inoculated twice with BoHV-4-A-IgK-VP2gDtm at different time points developed serum neutralizing antibodies against BTV and showed a strongly reduced viremia and a longer survival time when challenged with a lethal dose of BTV-8. The data acquired in this pilot study validate BoHV-4-based vector as a safe and effective heterologous antigen carrier/producer for the formulation of enhanced recombinant immunogens for the vaccination against lethal bluetongue. PMID:21320537

  9. Alterations of gene expression of sodium channels in dorsal root ganglion neurons of estrogen receptor knockout (ERKO) mice induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP).

    PubMed

    Ding, Haixia; Wang, Qiang; Liu, Jingli; Qian, Wenyi; Wang, Wenjuan; Wang, Jun; Gao, Rong; Xiao, Hang

    2012-08-01

    Estrogen receptors (ERα and ERβ) mediate the neuroprotection of estrogens against MPTP-induced striatal dopamine (DA) depletion. Pain is an important and distressing symptom in Parkinson's disease (PD). Voltage-gated sodium channels in sensory neurons are involved in the development of neuropathic pain. In this study, MPTP caused changes in nociception and alterations of gene expression of voltage-gated sodium channels in dorsal root ganglion (DRG) neurons in ER knockout (ERKO) mice were investigated. We found that administration of MPTP (11 mg/kg) to WT mice led to an extensive depletion of DA and its two metabolites, αERKO mice were observed to be more susceptible to MPTP toxicity than βERKO or WT mice. In addition, we found that the mRNA levels of TTX-S and TTX-R sodium channel subtypes were differentially affected in MPTP-treated WT animals. The MPTP-induced up-regulation of Nav1.1 and Nav1.9, down-regulation of Nav1.6 in DRG neurons may be through ERβ, up-regulation of Nav1.7 and down-regulation of Nav1.8 are dependent on both ERα and ERβ. Therefore, the MPTP-induced alterations of gene expression of sodium channels in DRG neurons could be an important mechanism to affect excitability and nociceptive thresholds, and the ERs appear to play a role in nociception in PD. PMID:22371119

  10. TRAIL-Death Receptor 4 Signaling via Lysosome Fusion and Membrane Raft Clustering In Coronary Arterial Endothelial Cells: Evidence from ASM Knockout Mice

    PubMed Central

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

    2012-01-01

    Tumor necrosis factor (TNF)-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 and 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 (MRs) clustering and 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 its co-localization with DR4 in coronary arterial endothelial cells (CAECs) isolated from wild-type (Smpd1+/+) mice. Further, 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 O2−· production in CAECs and abolished TRAIL-induced impairment on endothelium-dependent vasodilation in small resistance arteries. By measuring fluorescence resonance energy transfer (FRET), we found that Lamp-1 (lysosome membrane marker protein) and ganglioside GM1 (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 and fusion with membrane and formation of MR redox signaling platforms, which may

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

    PubMed Central

    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

  12. Activation of IKK/NF-κB provokes renal inflammatory responses in guanylyl cyclase/natriuretic peptide receptor-A gene-knockout mice

    PubMed Central

    Das, Subhankar; Periyasamy, Ramu

    2012-01-01

    The present study was aimed at determining the consequences of the disruption of guanylyl cyclase/natriuretic peptide receptor-A (GC-A/NPRA) gene (Npr1) on proinflammatory responses of nuclear factor kappa B, inhibitory kappa B kinase, and inhibitory kappa B alpha (NF-κB, IKK, IκBα) in the kidneys of mutant mice. The results showed that the disruption of Npr1 enhanced the renal NF-κB binding activity by 3.8-fold in 0-copy (−/−) mice compared with 2-copy (+/+) mice. In parallel, IKK activity and IκBα protein phosphorylation were increased by 8- and 11-fold, respectively, in the kidneys of 0-copy mice compared with wild-type mice. Interestingly, IκBα was reduced by 80% and the expression of proinflammatory cytokines and renal fibrosis were significantly enhanced in 0-copy mice than 2-copy mice. Treatment of 0-copy mice with NF-κB inhibitors andrographolide, pyrrolidine dithiocarbamate, and etanercept showed a substantial reduction in renal fibrosis, attenuation of proinflammatory cytokines gene expression, and significantly reduced IKK activity and IkBα phosphorylation. These findings indicate that the systemic disruption of Npr1 activates the renal NF-κB pathways in 0-copy mice, which transactivates the expression of various proinflammatory cytokines to initiate renal remodeling; however, inhibition of NF-κB pathway repairs the abnormal renal pathology in mutant mice. PMID:22318993

  13. Interactions between hepatic Mrp4 and Sult2a as revealed by the constitutive androstane receptor and Mrp4 knockout mice.

    PubMed

    Assem, Mahfoud; Schuetz, Erin G; Leggas, Markos; Sun, Daxi; Yasuda, Kazuto; Reid, Glen; Zelcer, Noam; Adachi, Masashi; Strom, Stephen; Evans, Ronald M; Moore, David D; Borst, Piet; Schuetz, John D

    2004-05-21

    The ABC transporter, Mrp4, transports the sulfated steroid DHEA-s, and sulfated bile acids interact with Mrp4 with high affinity. Hepatic Mrp4 levels are low, but increase under cholestatic conditions. We therefore inferred that up-regulation of Mrp4 during cholestasis is a compensatory mechanism to protect the liver from accumulation of hydrophobic bile acids. We determined that the nuclear receptor CAR is required to coordinately up-regulate hepatic expression of Mrp4 and an enzyme known to sulfate hydroxy-bile acids and steroids, Sult2a1. CAR activators increased Mrp4 and Sult2a1 expression in primary human hepatocytes and HepG2, a human liver cell line. Sult2a1 was down-regulated in Mrp4-null mice, further indicating an inter-relation between Mrp4 and Sult2a1 gene expression. Based on the hydrophilic nature of sulfated bile acids and the Mrp4 capability to transport sulfated steroids, our findings suggest that Mrp4 and Sult2a1 participate in an integrated pathway mediating elimination of sulfated steroid and bile acid metabolites from the liver. PMID:15004017

  14. Effect of long-term ingestion of weakly oxidised flaxseed oil on biomarkers of oxidative stress in LDL-receptor knockout mice.

    PubMed

    Nogueira, M S; Kessuane, M C; Lobo Ladd, A A B; Lobo Ladd, F V; Cogliati, B; Castro, I A

    2016-07-01

    The effect of oxidised fatty acids on atherosclerosis progression is controversial. Thus, our objective was to evaluate the effect of long-term consumption of weakly oxidised PUFA from flaxseed oil on oxidative stress biomarkers of LDL-receptor(-/-) mice. To test our hypothesis, mice were separated into three groups. The first group received a high-fat diet containing fresh flaxseed oil (CONT-), the second was fed the same diet prepared using heated flaxseed oil (OXID), and the third group received the same diet containing fresh flaxseed oil and had diabetes induced by streptozotocin (CONT+). Oxidative stress, aortic parameters and non-alcoholic fatty liver disease were assessed. After 3 months, plasma lipid profile, glucose levels, body weight, energy intake and dietary intake did not differ among groups. Likewise, oxidative stress, plasma malondialdehyde (MDA), hepatic MDA expressed as nmol/mg portion (ptn) and antioxidant enzymes did not differ among the groups. Hepatic linoleic acid, α-linolenic acid, arachidonic acid and EPA acid declined in the OXID and CONT+ groups. Aortic wall thickness, lumen and diameter increased only in the OXID group. OXID and CONT+ groups exhibited higher concentrations of MDA, expressed as μmol/mg ptn per %PUFA, when compared with the CONT- group. Our results suggest that ingestion of oxidised flaxseed oil increases hepatic MDA concentration and is potentially pro-atherogenic. In addition, the mean MDA value observed in all groups was similar to those reported in other studies that used xenobiotics as oxidative stress inducers. Thus, the diet applied in this study represents an interesting model for further research involving antioxidants. PMID:27197628

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

    PubMed

    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. PMID:24333375

  16. Gene-specific alterations of hepatic nuclear receptor regulated gene expression by ligand activation or hepatocyte-selective knockout inhibition of RXRα signaling during inflammation

    PubMed Central

    Kosters, Astrid; Tian, Feng; Wan, Yvonne Yu-Jie; Karpen, Saul J.

    2013-01-01

    Background Inflammation leads to transcriptional downregulation of many hepatic genes, particulary those activated by RXRα-heterodimers. Inflammation-mediated reduction of nuclear RXRα levels is a main factor in reduced nuclear receptor (NR)–regulated hepatic gene expression, eventually leading to cholestasis and liver damage. Aim To investigate roles for RXRα in hepatic gene expression during inflammation, using two complementary mouse models: ligand–activation of RXRα, and in mice expressing hepatocyte-specific expression of RXRα missing its DNA-binding-domain (DBD; hs-RxrαΔex4−/−) Methods To activate RXRα, mice were gavage-fed with LG268 or vehicle for 5 days. To inhibit RXRα function, hs-RxrαΔex4−/− were used. All mice were IP-injected with LPS or saline for 16 hrs prior to analysis of hepatic RNA, protein and NR-DNA binding. Results LG268-treatment attenuated the LPS-mediated reductions of several RXRα-regulated genes, coinciding with maintained RXRα occupancy in both Bsep and Ostβ promoters. Lacking full hepatocyte-RXRα function (hs-RxrαΔex4−/− mice) led to enhancement of LPS-mediated changes in gene expression, but surprisingly, maintenance of RNA levels of some RXRα-regulated genes. Investigations revealed that Hs-Rxrα−/− hepatocytes expressed an internally-truncated, ~44 kDa, RXRα-form. DNA-binding capacity of NR-heterodimers was equivalent in wt and hs-RxrαΔex4−/− livers, but reduced by LPS in both. ChIP-QPCR revealed reduced RXRα occupancy to the Bsep RXRα:FXR site was reduced, but not absent, in hs-RxrαΔex4−/− livers. Conclusions There are differential regulatory roles for hepatic RXRα, both in basal and inflammatory states, suggesting new and complex multi-domain roles for RXRα in regulating hepatic gene expression. Moreover, there is an unexpected non-obligate role for the DBD of RXRα. PMID:22098603

  17. The Role of 5-HT3 Receptors in Signaling from Taste Buds to Nerves

    PubMed Central

    Vandenbeuch, Aurelie; Voigt, Anja; Meyerhof, Wolfgang; Kinnamon, Sue C.; Finger, Thomas E.

    2015-01-01

    Activation of taste buds triggers the release of several neurotransmitters, including ATP and serotonin (5-hydroxytryptamine; 5-HT). Type III taste cells release 5-HT directly in response to acidic (sour) stimuli and indirectly in response to bitter and sweet tasting stimuli. Although ATP is necessary for activation of nerve fibers for all taste stimuli, the role of 5-HT is unclear. We investigated whether gustatory afferents express functional 5-HT3 receptors and, if so, whether these receptors play a role in transmission of taste information from taste buds to nerves. In mice expressing GFP under the control of the 5-HT3A promoter, a subset of cells in the geniculate ganglion and nerve fibers in taste buds are GFP-positive. RT-PCR and in situ hybridization confirmed the presence of 5-HT3A mRNA in the geniculate ganglion. Functional studies show that only those geniculate ganglion cells expressing 5-HT3A-driven GFP respond to 10 μm 5-HT and this response is blocked by 1 μm ondansetron, a 5-HT3 antagonist, and mimicked by application of 10 μm m-chlorophenylbiguanide, a 5-HT3 agonist. Pharmacological blockade of 5-HT3 receptors in vivo or genetic deletion of the 5-HT3 receptors reduces taste nerve responses to acids and other taste stimuli compared with controls, but only when urethane was used as the anesthetic. We find that anesthetic levels of pentobarbital reduce taste nerve responses apparently by blocking the 5-HT3 receptors. Our results suggest that 5-HT released from type III cells activates gustatory nerve fibers via 5-HT3 receptors, accounting for a significant proportion of the neural taste response. SIGNIFICANCE STATEMENT Historically, serotonin (5-hydroxytryptamine; 5-HT) has been described as a candidate neurotransmitter in the gustatory system and recent studies show that type III taste receptor cells release 5-HT in response to various taste stimuli. In the present study, we demonstrate that a subset of gustatory sensory neurons express functional

  18. 5-HT1A and 5-HT1B receptors control the firing of serotoninergic neurons in the dorsal raphe nucleus of the mouse: studies in 5-HT1B knock-out mice.

    PubMed

    Evrard, A; Laporte, A M; Chastanet, M; Hen, R; Hamon, M; Adrien, J

    1999-11-01

    The characteristics of the spontaneous firing of serotoninergic neurons in the dorsal raphe nucleus and its control by serotonin (5-hydroxytryptamine, 5-HT) receptors were investigated in wild-type and 5-HT1B knock-out (5-HT1B-/-) mice of the 129/Sv strain, anaesthetized with chloral hydrate. In both groups of mice, 5-HT neurons exhibited a regular activity with an identical firing rate of 0.5-4.5 spikes/s. Intravenous administration of the 5-HT reuptake inhibitor citalopram or the 5-HT1A agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) induced a dose-dependent inhibition of 5-HT neuronal firing which could be reversed by the selective 5-HT1A antagonist N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl)cyclohe xane carboxamide (WAY 100635). Both strains were equally sensitive to 8-OH-DPAT (ED50 approximately 6.3 microgram/kg i.v.), but the mutants were less sensitive than wild-type animals to citalopram (ED50 = 0.49 +/- 0.02 and 0.28 +/- 0.01 mg/kg i.v., respectively, P < 0.05). This difference could be reduced by pre-treatment of wild-type mice with the 5-HT1B/1D antagonist 2'-methyl-4'-(5-methyl-[1,2,4]oxadiazol-3-yl)-biphenyl-4-carbox yli c acid [4-methoxy-3-(4-methyl-piperazine-1-yl)-phenyl]amide (GR 127935), and might be accounted for by the lack of 5-HT1B receptors and a higher density of 5-HT reuptake sites (specifically labelled by [3H]citalopram) in 5-HT1B-/- mice. In wild-type but not 5-HT1B-/- mice, the 5-HT1B agonists 3-(1,2,5, 6-tetrahydro-4-pyridyl)-5-propoxypyrrolo[3,2-b]pyridine (CP 94253, 3 mg/kg i.v.) and 5-methoxy-3-(1,2,3, 6-tetrahydropyridin-4-yl)-1H-indole (RU 24969, 0.6 mg/kg i.v.) increased the firing rate of 5-HT neurons (+22.4 +/- 2.8% and +13.7 +/- 6.0%, respectively, P < 0.05), and this effect could be prevented by the 5-HT1B antagonist GR 127935 (1 mg/kg i.v.). Altogether, these data indicate that in the mouse, the firing of 5-HT neurons in the dorsal raphe nucleus is under both an inhibitory control through 5-HT1A

  19. Knockout beyond the dripline

    SciTech Connect

    Bonaccorso, A.; Charity, R. J.; Kumar, R.; Salvioni, G.

    2015-02-24

    In this contribution, we will describe neutron and proton removal from {sup 9}C and {sup 7}Be which are two particularly interesting nuclei entering the nucleo-synthesis pp-chain [1, 2]. Neutron and proton removal reactions have been used in the past twenty years to probe the single-particle structure of exotic nuclei. The core parallel-momentum distribution can give information on the angular momentum and spin of the nucleon initial state while the total removal cross section is sensitive to the asymptotic part of the initial wave function and also to the reaction mechanism. Because knockout is a peripheral reaction from which the Asymptotic Normalization Constant (ANC) of the single-particle wave function can be extracted, it has been used as an indirect method to obtain the rate of reactions like {sup 8}B(p,γ){sup 9}C or {sup 7}Be(p,γ){sup 8}B. Nucleon removal has recently been applied by the HiRA collaboration [3] to situations in which the remaining “core” is beyond the drip line, such as {sup 8}C and {sup 6}Be, unbound by one or more protons, and whose excitation-energy spectrum can be obtained by the invariant-mass method. By gating on the ground-state peak, “core” parallel-momentum distributions and total knockout cross sections have been obtained similar to previous studies with well-bound “cores”. In addition for each projectile, knock out to final bound states has also been obtained in several cases. We will report on the theoretical description and comparison to this experimental data for a few cases for which advances in the accuracy of the transfer-to-the continuum model [4, 5] have been made [6]. These include the use, when available, of “ab-initio” overlaps for the initial state [7] and in particular their ANC values [8]. Also, the construction of a nucleus-target folding potential for the treatment of the core-target S-matrix [9] using for the cores “ab-initio” densities [10] and state-of-the-art n−{sup 9}Be optical

  20. Identification of domains influencing assembly and ion channel properties in α7 nicotinic receptor and 5-HT3 receptor subunit chimaeras

    PubMed Central

    Gee, V J; Kracun, S; Cooper, S T; Gibb, A J; Millar, N S

    2007-01-01

    Background and purpose: Nicotinic acetylcholine receptors (nAChRs) and 5-hydroxytryptamine type 3 receptors (5-HT3Rs) are members of the superfamily of neurotransmitter-gated ion channels. Both contain five subunits which assemble to form either homomeric or heteromeric subunit complexes. With the aim of identifying the influence of subunit domains upon receptor assembly and function, a series of chimaeras have been constructed containing regions of the neuronal nAChR α7 subunit and the 5-HT3 receptor 3A subunit. Experimental approach: A series of subunit chimaeras containing α7 and 5-HT3A subunit domains have been constructed and expressed in cultured mammalian cells. Properties of the expressed receptors have been examined by means of radioligand binding, agonist-induced changes in intracellular calcium and patch-clamp electrophysiology. Key results: Subunit domains which influence properties such as rectification, desensitization and conductance have been identified. In addition, the influence of subunit domains upon subunit folding, receptor assembly and cell-surface expression has been identified. Co-expression studies with the nAChR-associated protein RIC-3 revealed that, in contrast to the potentiating effect of RIC-3 on α7 nAChRs, RIC-3 caused reduced levels of cell-surface expression of some α7/5-HT3A chimaeras. Conclusions and implications: Evidence has been obtained which demonstrates that subunit transmembrane domains are critical for efficient subunit folding and assembly. In addition, functional characterization of subunit chimaeras revealed that both extracellular and cytoplasmic domains exert a dramatic and significant influence upon single-channel conductance. These data support a role for regions other than hydrophobic transmembrane domains in determining ion channel properties. PMID:17721553

  1. Gal knockout and beyond.

    PubMed

    Zhong, R

    2007-01-01

    Recently, Galalpha1-3Galbeta1-4GlcNAc (Gal) knockout (k/o) pigs have been developed using genetic cloning technologies. This remarkable achievement has generated great enthusiasm in xenotransplantation studies. This review summarizes the current status of nonhuman primate experiments using Gal k/o pig organs. Briefly, when Gal k/o pig organs are transplanted into primates, hyperacute rejection does not occur. Although graft survival has been prolonged up to a few months in some cases, the overall results were not better than those using Gal-positive pig organs with human complement regulatory protein transgenes. Gal k/o pig kidneys rapidly developed rejection which was associated with increased anti-non-Gal antibodies. Although the precise mechanisms of Gal k/o pig organ rejection are not clear, it could result from incomplete deletion of Gal, up-regulation of new antigen (non-Gal antigen) and/or production of non-Gal antibodies. Future work in xenotransplantation should place emphasis on further modification of donors, such as combining human complement regulatory genes with Gal k/o, deleting non-Gal antigens and adding protective/surviving genes or a gene that inhibits coagulation. Induction of donor-specific T- and B-cell tolerance and promotion of accommodation are also warranted. PMID:17227553

  2. Ondansetron and Granisetron Binding Orientation in the 5-HT3 Receptor Determined by Unnatural Amino Acid Mutagenesis

    PubMed Central

    Duffy, Noah H.; Lester, Henry A.; Dougherty, Dennis A.

    2012-01-01

    The serotonin type 3 receptor (5-HT3R) is a ligand-gated ion channel that mediates fast synaptic transmission in the central and peripheral nervous systems. The 5-HT3R is a therapeutic target, and the clinically available drugs ondansetron and granisetron inhibit receptor activity. Their inhibitory action is through competitive binding to the native ligand binding site, although the binding orientation of the drugs at the receptor has been a matter of debate. Here we heterologously express mouse 5-HT3A receptors in Xenopus oocytes and use unnatural amino acid mutagenesis to establish a cation-π interaction for both ondansetron and granisetron to tryptophan 183 in the ligand binding pocket. This cation-π interaction establishes a binding orientation for both ondansetron and granisetron within the binding pocket. PMID:22873819

  3. On the voltage-dependent Ca2+ block of serotonin 5-HT3 receptors: a critical role of intracellular phosphates

    PubMed Central

    Noam, Yoav; Wadman, Wytse J; van Hooft, Johannes A

    2008-01-01

    Natively expressed serotonin 5-HT3 receptors typically possess a negative-slope conductance region in their I–V curve, due to a voltage-dependent block by external Ca2+ ions. However, in almost all studies performed with heterologously expressed 5-HT3 receptors, this feature was not observed. Here we show that mere addition of ATP to the pipette solution is sufficient to reliably observe a voltage-dependent block in homomeric (h5-HT3A) and heteromeric (h5-HT3AB) receptors expressed in HEK293 cells. A similar block was observed with a plethora of molecules containing a phosphate moiety, thus excluding a role of phosphorylation. A substitution of three arginines in the intracellular vestibule of 5-HT3A with their counterpart residues from the 5-HT3B subunit (RRR-QDA) was previously shown to dramatically increase single channel conductance. We find this mutant to have a linear I–V curve that is unaffected by the presence of ATP, with a fractional Ca2+ current (Pf%) that is reduced (1.8 ± 0.2%) compared to that of the homomeric receptor (4.1 ± 0.2%), and similar to that of the heteromeric form (2.0 ± 0.3%). Moreover, whereas ATP decreased the Pf% of the homomeric receptor, this was not observed with the RRR-QDA mutant. Finally, ATP was found to be critical for voltage-dependent channel block also in hippocampal interneurons that natively express 5-HT3 receptors. Taken together, our results indicate a novel mechanism by which ATP, and similar molecules, modulate 5-HT3 receptors via interactions with the intracellular vestibule of the receptor. PMID:18566001

  4. Effect of homologous serotonin receptor loop substitutions on the heterologous expression in Pichia of a chimeric acetylcholine-binding protein with alpha-bungarotoxin-binding activity.

    PubMed

    Paulo, Joao A; Hawrot, Edward

    2009-10-01

    The molluscan acetylcholine-binding protein (AChBP) is a soluble homopentameric homolog of the extracellular domain of various ligand-gated ion channels. Previous studies have reported that AChBP, when fused to the ion pore domain of the serotonin receptor (5HT(3A)R), can form a functional ligand-gated chimeric channel only if the AChBP loop regions between beta-strands beta1 and beta2 (beta1-beta2), beta6 and beta7 (beta6-beta7), and beta8 and beta9 (beta8-beta9) are replaced with those of the 5HT(3A)R. To investigate further the potential interactions among these three important loop regions in a membrane- and detergent-free system, we designed AChBP constructs in which loops beta1-beta2, beta6-beta7, and beta8-beta9 of the AChBP were individually and combinatorially substituted in all permutations with the analogous loops of the 5HT(3A)R. These chimeras were expressed as secreted proteins using the Pichia pastoris yeast expression system. [(125)I]-alpha-Bungarotoxin-binding was detected in the culture media obtained from homologous recombinant clones expressing the wild-type AChBP, the beta1-beta2 loop-only chimera, and the chimera containing all three 5HT(3A)R loop substitutions. The remaining chimeras failed to show [(125)I]-alpha-bungarotoxin binding, and further analysis of cellular extracts allowed us to determine that these binding-negative chimeric constructs accumulated intracellularly and were not secreted into the culture medium. Our results demonstrate that coordinated interactions among loops beta1-beta2, beta6-beta7, and beta8-beta9 are essential for the formation of a functional ligand-binding site, as evidenced by [(125)I]-alpha-bungarotoxin-binding, and for efficient protein secretion. In addition, the constructs described here demonstrate the feasibility of utilizing soluble scaffolds to explore functionally important interactions within the extracellular domain of membrane-bound proteins. PMID:19427904

  5. In Vivo Interaction of Steroid Receptor Coactivator (SRC)-1 and the Activation Function-2 Domain of the Thyroid Hormone Receptor (TR) β in TRβ E457A Knock-In and SRC-1 Knockout mice

    PubMed Central

    Alonso, Manuela; Goodwin, Charles; Liao, XiaoHui; Ortiga-Carvalho, Tania; Machado, Danielle S.; Wondisford, Fredric E.; Refetoff, Samuel; Weiss, Roy E.

    2009-01-01

    The activation function-2 (AF-2) domain of the thyroid hormone (TH) receptor (TR)-β is a TH-dependent binding site for nuclear coactivators (NCoA), which modulate TH-dependent gene transcription. In contrast, the putative AF-1 domain is a TH-independent region interacting with NCoA. We determined the specificity of the AF-2 domain and NCoA interaction by evaluating thyroid function in mice with combined disruption of the AF-2 domain in TRβ, due to a point mutation (E457A), and deletion of one of the NCoAs, steroid receptor coactivator (SRC)-1. The E457A mutation was chosen because it abolishes NCoA recruitment in vitro while preserving normal TH binding and corepressor interactions resulting in resistance to TH. At baseline, disruption of SRC-1 in the homozygous knock-in (TRβE457A/E457A) mice worsened the degree of resistance to TH, resulting in increased serum T4 and TSH. During TH deprivation, disruption of AF-2 and SRC-1 resulted in a TSH rise 50% of what was seen when AF-2 alone was removed, suggesting that SRC-1 was interacting outside of the AF-2 domain. Therefore, 1) during TH deprivation, SRC-1 is necessary for activating the hypothalamic-pituitary-thyroid axis; 2) ligand-dependent repression of TSH requires an intact AF-2; and 3) SRC-1 may interact with the another region of the TRβ or the TRα to regulate TH action in the pituitary. This report demonstrates the dual interaction of NCoA in vivo: the TH-independent up-regulation possibly through another domain and TH-dependent down-regulation through the AF-2 domain. PMID:19406944

  6. In vivo interaction of steroid receptor coactivator (SRC)-1 and the activation function-2 domain of the thyroid hormone receptor (TR) beta in TRbeta E457A knock-in and SRC-1 knockout mice.

    PubMed

    Alonso, Manuela; Goodwin, Charles; Liao, Xiaohui; Ortiga-Carvalho, Tania; Machado, Danielle S; Wondisford, Fredric E; Refetoff, Samuel; Weiss, Roy E

    2009-08-01

    The activation function-2 (AF-2) domain of the thyroid hormone (TH) receptor (TR)-beta is a TH-dependent binding site for nuclear coactivators (NCoA), which modulate TH-dependent gene transcription. In contrast, the putative AF-1 domain is a TH-independent region interacting with NCoA. We determined the specificity of the AF-2 domain and NCoA interaction by evaluating thyroid function in mice with combined disruption of the AF-2 domain in TRbeta, due to a point mutation (E457A), and deletion of one of the NCoAs, steroid receptor coactivator (SRC)-1. The E457A mutation was chosen because it abolishes NCoA recruitment in vitro while preserving normal TH binding and corepressor interactions resulting in resistance to TH. At baseline, disruption of SRC-1 in the homozygous knock-in (TRbeta(E457A/E457A)) mice worsened the degree of resistance to TH, resulting in increased serum T(4) and TSH. During TH deprivation, disruption of AF-2 and SRC-1 resulted in a TSH rise 50% of what was seen when AF-2 alone was removed, suggesting that SRC-1 was interacting outside of the AF-2 domain. Therefore, 1) during TH deprivation, SRC-1 is necessary for activating the hypothalamic-pituitary-thyroid axis; 2) ligand-dependent repression of TSH requires an intact AF-2; and 3) SRC-1 may interact with the another region of the TRbeta or the TRalpha to regulate TH action in the pituitary. This report demonstrates the dual interaction of NCoA in vivo: the TH-independent up-regulation possibly through another domain and TH-dependent down-regulation through the AF-2 domain. PMID:19406944

  7. Selectivity optimization of substituted 1,2,3-triazoles as α7 nicotinic acetylcholine receptor agonists.

    PubMed

    Arunrungvichian, Kuntarat; Fokin, Valery V; Vajragupta, Opa; Taylor, Palmer

    2015-08-19

    Three series of substituted anti-1,2,3-triazoles (IND, PPRD, and QND), synthesized by cycloaddition from azide and alkyne building blocks, were designed to enhance selectivity and potency profiles of a lead α7 nicotinic acetylcholine receptor (α7-nAChR) agonist, TTIn-1. Designed compounds were synthesized and screened for affinity by a radioligand binding assay. Their functional characterization as agonists and antagonists was performed by fluorescence resonance energy transfer assay using cell lines expressing transfected cDNAs, α7-nAChRs, α4β2-nAChRs, and 5HT3A receptors, and a fluorescence cell reporter. In the IND series, a tropane ring of TTIn-1, substituted at N1, was replaced by mono- and bicyclic amines to vary length and conformational flexibility of a carbon linker between nitrogen atom and N1 of the triazole. Compounds with a two-carbon atom linker optimized binding with Kd's at the submicromolar level. Further modification at the hydrophobic indole of TTIn-1 was made in PPRD and QND series by fixing the amine center with the highest affinity building blocks in the IND series. Compounds from IND and PPRD series are selective as agonists for the α7-nAChRs over α4β2-nAChRs and 5HT3A receptors. Lead compounds in the three series have EC50's between 28 and 260 nM. Based on the EC50, affinity, and selectivity determined from the binding and cellular responses, two of the leads have been advanced to behavioral studies described in the companion article (DOI: 10.1021/acschemneuro.5b00059). PMID:25932897

  8. Expression of serotonin receptor genes in cranial ganglia.

    PubMed

    Maeda, Naohiro; Ohmoto, Makoto; Yamamoto, Kurumi; Kurokawa, Azusa; Narukawa, Masataka; Ishimaru, Yoshiro; Misaka, Takumi; Matsumoto, Ichiro; Abe, Keiko

    2016-03-23

    Taste cells release neurotransmitters to gustatory neurons to transmit chemical information they received. Sweet, umami, and bitter taste cells use ATP as a neurotransmitter. However, ATP release from sour taste cells has not been observed so far. Instead, they release serotonin when they are activated by sour/acid stimuli. Thus it is still controversial whether sour taste cells use ATP, serotonin, or both. By reverse transcription-polymerase chain reaction and subsequent in situ hybridization (ISH) analyses, we revealed that of 14 serotonin receptor genes only 5-HT3A and 5-HT3B showed significant/clear signals in a subset of neurons of cranial sensory ganglia in which gustatory neurons reside. Double-fluorescent labeling analyses of ISH for serotonin receptor genes with wheat germ agglutinin (WGA) in cranial sensory ganglia of pkd1l3-WGA mice whose sour neural pathway is visualized by the distribution of WGA originating from sour taste cells in the posterior region of the tongue revealed that WGA-positive cranial sensory neurons rarely express either of serotonin receptor gene. These results suggest that serotonin receptors expressed in cranial sensory neurons do not play any role as neurotransmitter receptor from sour taste cells. PMID:26854841

  9. Developmental Divergence of Sleep-Wake Patterns in Orexin Knockout and Wild-Type Mice

    PubMed Central

    Coleman, Cassandra M.; Johnson, Eric D.; Shaw, Cynthia

    2008-01-01

    Narcolepsy, a disorder characterized by fragmented bouts of sleep and wakefulness during the day and night as well as cataplexy, has been linked in humans and non-human animals to the functional integrity of the orexinergic system. Adult orexin knockout mice and dogs with a mutation of the orexin receptor exhibit symptoms that mirror those seen in narcoleptic humans. As with narcolepsy, infant sleep-wake cycles in humans and rats are highly fragmented, with consolidated bouts of sleep and wakefulness developing gradually. Based on these common features of narcoleptics and infants, we hypothesized that the development of sleep-wake fragmentation in orexin knockout mice would be expressed as a developmental divergence between knockouts and wild-types, with the knockouts lagging behind the wild-types. We tested this hypothesis by recording the sleep-wake patterns of infant orexin knockout and wild-type mice across the first three postnatal weeks. Both knockouts and wild-types exhibited age-dependent, and therefore orexin-independent, quantitative and qualitative changes in sleep-wake patterning. At 3 weeks of age, however, by which time the sleep and wake bouts of the wild-types had consolidated further, the knockouts lagged behind the wild-types and exhibited significantly more bout fragmentation. These findings suggest the possibility that the fragmentation of behavioral states that characterizes narcolepsy in adults reflects reversion back toward the more fragmented sleep-wake patterns that characterize infancy. PMID:17284193

  10. Generation of conditional knockout mice.

    PubMed

    Sakamoto, Kazuhito; Gurumurthy, Channabasavaiah B; Wagner, Kay-Uwe

    2014-01-01

    Conditional knockout mouse models are powerful tools to examine the biological and molecular function(s) of genes in specific tissues. The general procedure to generate such genetically engineered mouse models consists of three main steps. The first step is to find the appropriate genomic clone of the gene of interest and to design the cloning and Southern blot strategies. The second step is the cloning of the gene-targeting vector with all its essential components including positive and negative selection cassettes and the insertion of LoxP sites. Although conventional methods are still being widely used for DNA cloning, we describe in this book chapter the use of λ Red phage-based homologous recombination in Escherichia coli to capture the genomic DNA of the gene of interest and to assemble the gene-targeting vector. This new method provides several advantages as it does not require the presence of restriction sites within the gene of interest to insert LoxP-flanked DNA fragments. In the final step, the gene-targeting vector is transferred into embryonic stem (ES) cells, and successfully targeted ES cell clones are injected into mouse blastocysts to generate conditional knockout mice. PMID:25064096

  11. Pharmacological characterisation of strychnine and brucine analogues at glycine and alpha7 nicotinic acetylcholine receptors.

    PubMed

    Jensen, Anders A; Gharagozloo, Parviz; Birdsall, Nigel J M; Zlotos, Darius P

    2006-06-01

    Strychnine and brucine from the plant Strychnos nux vomica have been shown to have interesting pharmacological effects on several neurotransmitter receptors, including some members of the superfamily of ligand-gated ion channels. In this study, we have characterised the pharmacological properties of tertiary and quaternary analogues as well as bisquaternary dimers of strychnine and brucine at human alpha1 and alpha1beta glycine receptors and at a chimera consisting of the amino-terminal domain of the alpha7 nicotinic receptor (containing the orthosteric ligand binding site) and the ion channel domain of the 5-HT3A serotonin receptor. Although the majority of the analogues displayed significantly increased Ki values at the glycine receptors compared to strychnine and brucine, a few retained the high antagonist potencies of the parent compounds. However, mirroring the pharmacological profiles of strychnine and brucine, none of the analogues displayed significant selectivity between the alpha1 and alpha1beta subtypes. The structure-activity relationships for the compounds at the alpha7/5-HT3 chimera were significantly different from those at the glycine receptors. Most strikingly, quaternization of strychnine and brucine with substituents possessing different steric and electronic properties completely eliminated the activity at the glycine receptors, whereas binding affinity to the alpha7/5-HT3 chimera was retained for the majority of the quaternary analogues. This study provides an insight into the structure-activity relationships for strychnine and brucine analogues at these ligand-gated ion channels. PMID:16687139

  12. Enhanced Long-Term and Impaired Short-Term Spatial Memory in GluA1 AMPA Receptor Subunit Knockout Mice: Evidence for a Dual-Process Memory Model

    ERIC Educational Resources Information Center

    Sanderson, David J.; Good, Mark A.; Skelton, Kathryn; Sprengel, Rolf; Seeburg, Peter H.; Rawlins, J. Nicholas P.; Bannerman, David M.

    2009-01-01

    The GluA1 AMPA receptor subunit is a key mediator of hippocampal synaptic plasticity and is especially important for a rapidly-induced, short-lasting form of potentiation. GluA1 gene deletion impairs hippocampus-dependent, spatial working memory, but spares hippocampus-dependent spatial reference memory. These findings may reflect the necessity of…

  13. Apolipoprotein E knockout as the basis for mouse models of dyslipidemia-induced neuropathy.

    PubMed

    Hinder, Lucy M; Vincent, Andrea M; Hayes, John M; McLean, Lisa L; Feldman, Eva L

    2013-01-01

    Dyslipidemia has been identified as an important pathogenic risk factor for diabetic neuropathy, but current animal models do not adequately reproduce the lipid profile observed in human diabetics (increased triglycerides with an elevated LDL-cholesterol and reduced HDL-cholesterol). High fat feeding of mice produces hyperlipidemia, but mice are resistant to increases in the LDL to HDL ratio, reducing the potential for peripheral lipid deposits to impact neuropathy, as is postulated to occur in human subjects. Genetic manipulations provide an alternative approach to reproducing a neuropathic plasma lipid profile. Based on findings from the atherosclerosis literature, we began with knockout of ApoE. Since knockout of ApoE alone only partially mimics the human diabetic lipid profile, we examined the impact of its combination with a well-characterized model of type 2 diabetes exhibiting neuropathy, the db/db mouse. We added further gene manipulations to increase hyperlipidemia by using mice with both ApoE and ApoB48 knockout on the ob/+ (leptin mutation) mice. In all of these models, we found that either the db/db or ob/ob genotypes had increased body weight, hyperlipidemia, hyperglycemia, and evidence of neuropathy compared with the control groups (db/+ or ob/+, respectively). We found that ApoE knockout combined with leptin receptor knockout produced a lipid profile most closely modeling human dyslipidemia that promotes neuropathy. ApoE knockout combined with additional ApoB48 and leptin knockout produced similar changes of smaller magnitude, but, notably, an increase in HDL-cholesterol. Our data suggest that the overall effects of ApoE knockout, either directly upon nerve structure and function or indirectly on lipid metabolism, are insufficient to significantly alter the course of diabetic neuropathy. Although these models ultimately do not deliver optimal lipid profiles for translational diabetic neuropathy research, they do present glycemic and lipid profile

  14. Studying TGF-beta superfamily signaling by knockouts and knockins.

    PubMed

    Chang, H; Lau, A L; Matzuk, M M

    2001-06-30

    The transforming growth factor beta (TGF-beta) superfamily has profound effects on many aspects of animal development. In the last decade, our laboratory and others have performed in vivo functional studies on multiple components of the TGF-beta superfamily signal transduction pathway, including upstream ligands, transmembrane receptors, receptor-associated proteins and downstream Smad proteins. We have taken gene knockout approaches to generate null alleles of the genes of interest, as well as a gene knockin approach to replace the mature region of one TGF-beta superfamily ligand with another. We found that activin betaB, expressed in the spatiotemporal pattern of activin betaA, can function as a hypomorphic allele of activin betaA and rescue the craniofacial defects and neonatal lethal phenotype of activin betaA-deficient mice. With the knockout approach, we have shown that the expression pattern of a component in the TGF-beta superfamily signal transduction cascade does not necessarily predict its in vivo function. Two liver-specific activins, activin betaC and activin betaE are dispensable for liver development, regeneration and function, whereas ubiquitously expressed Smad5 has specific roles in the development of multiple embryonic and extraembryonic tissues. PMID:11451570

  15. Microarray analysis of E9.5 reduced folate carrier (RFC1; Slc19a1) knockout embryos reveals altered expression of genes in the cubilin-megalin multiligand endocytic receptor complex

    PubMed Central

    Gelineau-van Waes, Janee; Maddox, Joyce R; Smith, Lynette M; van Waes, Michael; Wilberding, Justin; Eudy, James D; Bauer, Linda K; Finnell, Richard H

    2008-01-01

    Background The reduced folate carrier (RFC1) is an integral membrane protein and facilitative anion exchanger that mediates delivery of 5-methyltetrahydrofolate into mammalian cells. Adequate maternal-fetal transport of folate is necessary for normal embryogenesis. Targeted inactivation of the murine RFC1 gene results in post-implantation embryolethality, but daily folic acid supplementation of pregnant dams prolongs survival of homozygous embryos until mid-gestation. At E10.5 RFC1-/- embryos are developmentally delayed relative to wildtype littermates, have multiple malformations, including neural tube defects, and die due to failure of chorioallantoic fusion. The mesoderm is sparse and disorganized, and there is a marked absence of erythrocytes in yolk sac blood islands. The identification of alterations in gene expression and signaling pathways involved in the observed dysmorphology following inactivation of RFC1-mediated folate transport are the focus of this investigation. Results Affymetrix microarray analysis of the relative gene expression profiles in whole E9.5 RFC1-/- vs. RFC1+/+ embryos identified 200 known genes that were differentially expressed. Major ontology groups included transcription factors (13.04%), and genes involved in transport functions (ion, lipid, carbohydrate) (11.37%). Genes that code for receptors, ligands and interacting proteins in the cubilin-megalin multiligand endocytic receptor complex accounted for 9.36% of the total, followed closely by several genes involved in hematopoiesis (8.03%). The most highly significant gene network identified by Ingenuity™ Pathway analysis included 12 genes in the cubilin-megalin multiligand endocytic receptor complex. Altered expression of these genes was validated by quantitative RT-PCR, and immunohistochemical analysis demonstrated that megalin protein expression disappeared from the visceral yolk sac of RFC1-/- embryos, while cubilin protein was widely misexpressed. Conclusion Inactivation of

  16. Proton Knock-Out in Hall A

    SciTech Connect

    Kees de Jager

    2002-06-01

    Proton knock-out is studied in a broad program in Hall A at Jefferson Lab. The first experiment performed in Hall A studied the {sup 16}O(e,e'p) reaction. Since then proton knock-out experiments have studied a variety of aspects of that reaction, from single-nucleon properties to its mechanism, such as final-state interactions and two-body currents, in nuclei from {sup 2}H to {sup 16}O. In this review the results of this program will be summarized and an outlook given of future accomplishments.

  17. Delayed liver regeneration after partial hepatectomy in adiponectin knockout mice

    SciTech Connect

    Ezaki, Hisao; Yoshida, Yuichi; Saji, Yukiko; Takemura, Takayo; Fukushima, Juichi; Matsumoto, Hitoshi; Kamada, Yoshihiro; Wada, Akira; Igura, Takumi; Kihara, Shinji; Funahashi, Tohru; Shimomura, Iichiro; Tamura, Shinji; Kiso, Shinichi Hayashi, Norio

    2009-01-02

    We previously demonstrated that adiponectin has anti-fibrogenic and anti-inflammatory effects in the liver of mouse models of various liver diseases. However, its role in liver regeneration remains unclear. The aim of this study was to determine the role of adiponectin in liver regeneration. We assessed liver regeneration after partial hepatectomy in wild-type (WT) and adiponectin knockout (KO) mice. We analyzed DNA replication and various signaling pathways involved in cell proliferation and metabolism. Adiponectin KO mice exhibited delayed DNA replication and increased lipid accumulation in the regenerating liver. The expression levels of peroxisome proliferator-activated receptor (PPAR) {alpha} and carnitine palmitoyltransferase-1 (CPT-1), a key enzyme in mitochondrial fatty acid oxidation, were decreased in adiponectin KO mice, suggesting possible contribution of altered fat metabolism to these phenomena. Collectively, the present results highlight a new role for adiponectin in the process of liver regeneration.

  18. Pharmacogenomic study of the role of the nociceptin/orphanin FQ receptor and opioid receptors in diabetic hyperalgesia.

    PubMed

    Rutten, Kris; Tzschentke, Thomas M; Koch, Thomas; Schiene, Klaus; Christoph, Thomas

    2014-10-15

    Targeting functionally independent receptors may provide synergistic analgesic effects in neuropathic pain. To examine the interdependency between different opioid receptors (µ-opioid peptide [MOP], δ-opioid peptide [DOP] and κ-opioid peptide [KOP]) and the nociceptin/orphanin FQ peptide (NOP) receptor in streptozotocin (STZ)-induced diabetic polyneuropathy, nocifensive activity was measured using a hot plate test in wild-type and NOP, MOP, DOP and KOP receptor knockout mice in response to the selective receptor agonists Ro65-6570, morphine, SNC-80 and U50488H, or vehicle. Nocifensive activity was similar in non-diabetic wild-type and knockout mice at baseline, before agonist or vehicle administration. STZ-induced diabetes significantly increased heat sensitivity in all mouse strains, but MOP, DOP and KOP receptor knockouts showed a smaller degree of hyperalgesia than wild-type mice and NOP receptor knockouts. For each agonist, a significant antihyperalgesic effect was observed in wild-type diabetic mice (all P<0.05 versus vehicle); the effect was markedly attenuated in diabetic mice lacking the cognate receptor compared with wild-type diabetic mice. Morphine was the only agonist that demonstrated near-full antihyperalgesic efficacy across all non-cognate receptor knockouts. Partial or near-complete reductions in efficacy were observed with Ro65-6570 in DOP and KOP receptor knockouts, with SNC-80 in NOP, MOP and KOP receptor knockouts, and with U50488H in NOP and DOP receptor knockouts. There was no evidence of NOP and MOP receptor interdependency in response to selective agonists for these receptors. These findings suggest that concurrent activation of NOP and MOP receptors, which showed functional independence, may yield an effective and favorable therapeutic analgesic profile. PMID:25169429

  19. Antidepressants and antipsychotic drugs colocalize with 5-HT3 receptors in raft-like domains.

    PubMed

    Eisensamer, Brigitte; Uhr, Manfred; Meyr, Sabrina; Gimpl, Gerald; Deiml, Tobias; Rammes, Gerhard; Lambert, Jeremy J; Zieglgänsberger, Walter; Holsboer, Florian; Rupprecht, Rainer

    2005-11-01

    Despite different chemical structure and pharmacodynamic signaling pathways, a variety of antidepressants and antipsychotics inhibit ion fluxes through 5-HT3 receptors in a noncompetitive manner with the exception of the known competitive antagonists mirtazapine and clozapine. To further investigate the mechanisms underlying the noncompetitive inhibition of the serotonin-evoked cation current, we quantified the concentrations of different types of antidepressants and antipsychotics in fractions of sucrose flotation gradients isolated from HEK293 (human embryonic kidney 293) cells stably transfected with the 5-HT3A receptor and of N1E-115 neuroblastoma cells in relation to the localization of the 5-HT3 receptor protein within the cell membrane. Western blots revealed a localization of the 5-HT3 receptor protein exclusively in the low buoyant density (LBD) fractions compatible with a localization within raft-like domains. Also, the antidepressants desipramine, fluoxetine, and reboxetine and the antipsychotics fluphenazine, haloperidol, and clozapine were markedly enriched in LBD fractions, whereas no accumulation occurs for mirtazapine, carbamazepine, moclobemide, and risperidone. The concentrations of psychopharmacological drugs within LBD fractions was strongly associated with their inhibitory potency against serotonin-induced cation currents. The noncompetitive antagonism of antidepressants at the 5-HT3 receptor was not conferred by an enhancement of receptor internalization as shown by immunofluorescence studies, assessment of receptor density in clathrin-coated vesicles, and electrophysiological recordings after coexpression of a dominant-negative mutant of dynamin I, which inhibits receptor internalization. In conclusion, enrichment of antidepressants and antipsychotics in raft-like domains within the cell membrane appears to be crucial for their antagonistic effects at ligand-gated ion channels such as 5-HT3 receptors. PMID:16267227

  20. Nuclear receptors constitutive androstane receptor and pregnane X receptor ameliorate cholestatic liver injury

    PubMed Central

    Stedman, Catherine A. M.; Liddle, Christopher; Coulter, Sally A.; Sonoda, Junichiro; Alvarez, Jacqueline G. A.; Moore, David D.; Evans, Ronald M.; Downes, Michael

    2005-01-01

    Cholestasis is associated with accumulation of bile acids and lipids, and liver injury. The constitutive androstane receptor (CAR) and pregnane X receptor (PXR) are xenobiotic nuclear receptors that coordinate protective hepatic responses to potentially toxic stimuli, including bile acids. We investigated the role of these receptors in the regulation of bile acid and lipid metabolism in a bile duct ligation (BDL) model of cholestasis applied to receptor knockout mice. Hepatic damage from bile acid accumulation was increased in both CAR knockout (CARKO) and PXR knockout mice, but bile acid concentrations were lower in CARKO mice. High-density lipoprotein (HDL) cholesterol was elevated in CARKO mice, and serum total cholesterol increased less in CARKO or PXR knockout mice than WT mice after BDL. Gene expression analysis of the BDL knockout animals demonstrated that, in response to cholestasis, PXR and CAR both repressed and induced the specific hepatic membrane transporters Oatp-c (organic anion transporting polypeptide C) and Oatp2 (Na+-dependent organic anion transporter 2), respectively. Induction of the xenobiotic transporter multidrug resistance protein 1 in cholestasis was independent of either PXR or CAR, in contrast to the known pattern of induction of multidrug resistance protein 1 by xenobiotics. These results demonstrate that CAR and PXR influence cholesterol metabolism and bile acid synthesis, as well as multiple detoxification pathways, and suggest their potential role as therapeutic targets for the treatment of cholestasis and lipid disorders. PMID:15684063

  1. The muscarinic antagonists scopolamine and atropine are competitive antagonists at 5-HT3 receptors.

    PubMed

    Lochner, Martin; Thompson, Andrew J

    2016-09-01

    Scopolamine is a high affinity muscarinic antagonist that is used for the prevention of post-operative nausea and vomiting. 5-HT3 receptor antagonists are used for the same purpose and are structurally related to scopolamine. To examine whether 5-HT3 receptors are affected by scopolamine we examined the effects of this drug on the electrophysiological and ligand binding properties of 5-HT3A receptors expressed in Xenopus oocytes and HEK293 cells, respectively. 5-HT3 receptor-responses were reversibly inhibited by scopolamine with an IC50 of 2.09 μM. Competitive antagonism was shown by Schild plot (pA2 = 5.02) and by competition with the 5-HT3 receptor antagonists [(3)H]granisetron (Ki = 6.76 μM) and G-FL (Ki = 4.90 μM). The related molecule, atropine, similarly inhibited 5-HT evoked responses in oocytes with an IC50 of 1.74 μM, and competed with G-FL with a Ki of 7.94 μM. The reverse experiment revealed that granisetron also competitively bound to muscarinic receptors (Ki = 6.5 μM). In behavioural studies scopolamine is used to block muscarinic receptors and induce a cognitive deficit, and centrally administered concentrations can exceed the IC50 values found here. It is therefore possible that 5-HT3 receptors are also inhibited. Studies that utilise higher concentrations of scopolamine should be mindful of these potential off-target effects. PMID:27108935

  2. Cloning and expression of ligand-gated ion-channel receptor L2 in central nervous system

    SciTech Connect

    Houtani, Takeshi; Munemoto, Yumi; Kase, Masahiko; Sakuma, Satoru; Tsutsumi, Toshiyuki; Sugimoto, Tetsuo . E-mail: sugimoto@takii.kmu.ac.jp

    2005-09-23

    An orphan receptor of ligand-gated ion-channel type (L2, also termed ZAC according to the presence of zinc ion for channel activation) was identified by computer-assisted search programs on human genome database. The L2 protein shares partial homology with serotonin receptors 5HT3A and 5HT3B. We have cloned L2 cDNA derived from human caudate nucleus and characterized the exon-intron structure as follows: (1) The L2 protein has four transmembrane regions (M1-M4) and a long cytoplasmic loop between M3 and M4. (2) The sequence is conserved in species including chimpanzee, dog, cow, and opossum. (3) Nine exons form its protein-coding region and especially exon 5 corresponds to a disulfide bond region on the amino-terminal side. Our analysis using multiple tissue cDNA panels revealed that at least two splicing variants of L2 mRNA are present. The cDNA PCR amplification study revealed that L2 mRNA is expressed in tissues including brain, pancreas, liver, lung, heart, kidney, and skeletal muscle while 5HT3A mRNA could be detected in brain, heart, placenta, lung, kidney, pancreas, and skeletal muscle, and 5HT3B mRNA in brain, kidney, and skeletal muscle, suggesting different significance in tissue expression of these receptors. Regional expression of L2 mRNA and protein was examined in brain. The RT-PCR studies confirmed L2 mRNA expression in hippocampus, striatum, amygdala, and thalamus in adult brain. The L2 protein was immunolocalized by using antipeptide antibodies. Immunostained tissue sections revealed that L2-like immunoreactivity was dominantly expressed in the hippocampal CA3 pyramidal cells and in the polymorphic layer of the dentate gyrus. We analyzed the expression of L2 protein in HEK293 cells using GFP fusion protein reporter system. Western blots revealed that L2 protein confers sugar chains on the extracellular side. In transfected HEK293 cells, cellular membranes and intracellular puncta were densely labeled with GFP, suggesting selective dispatch to the

  3. Knockout Mice Challenge our Concepts of Glucose Homeostasis and the Pathogenesis of Diabetes

    PubMed Central

    2003-01-01

    A central component of type 2 diabetes and the metabolic syndrome is insulin resistance. Insulin exerts a multifaceted and highly integrated series of actions via its intracellular signaling systems. Generation of mice carrying null mutations of the genes encoding proteins in the insulin signaling pathway provides a unique approach to determining the role of individual proteins in the molecular mechanism of insulin action and the pathogenesis of insulin resistance and diabetes. The role of the four major insulin receptor substrates (IRS1-4) in insulin and IGF-1 signaling have been examined by creating mice with targeted gene knockouts. Each produces a unique phenotype, indicating the complementary role of these signaling components. Combined heterozygous defects often produce synergistic or epistatic effects, although the final severity of the phenotype depends on the genetic background of the mice. Conditional knockouts of the insulin receptor have also been created using the Cre-lox system. These tissue specific knockouts have provide unique insights into the control of glucose homeostasis and the pathogenesis of type 2 diabetes, and have led to development of new hypotheses about the nature of the insulin action and development of diabetes. PMID:15061645

  4. N-Benzylpiperidine Derivatives as α7 Nicotinic Receptor Antagonists.

    PubMed

    Criado, Manuel; Mulet, José; Sala, Francisco; Sala, Salvador; Colmena, Inés; Gandía, Luis; Bautista-Aguilera, Oscar M; Samadi, Abdelouahid; Chioua, Mourad; Marco-Contelles, José

    2016-08-17

    A series of multitarget directed propargylamines, as well as other differently susbstituted piperidines have been screened as potential modulators of neuronal nicotinic acetylcholine receptors (nAChRs). Most of them showed antagonist actions on α7 nAChRs. Especially, compounds 13, 26, and 38 displayed submicromolar IC50 values on homomeric α7 nAChRs, whereas they were less effective on heteromeric α3β4 and α4β2 nAChRs (up to 20-fold higher IC50 values in the case of 13). Antagonism was concentration dependent and noncompetitive, suggesting that these compounds behave as negative allosteric modulators of nAChRs. Upon the study of a series of less complex derivatives, the N-benzylpiperidine motif, common to these compounds, was found to be the main pharmacophoric group. Thus, 2-(1-benzylpiperidin-4-yl)-ethylamine (48) showed an inhibitory potency comparable to the one of the previous compounds and also a clear preference for α7 nAChRs. In a neuroblastoma cell line, representative compounds 13 and 48 also inhibited, in a concentration-dependent manner, cytosolic Ca(2+) signals mediated by nAChRs. Finally, compounds 38 and 13 inhibited 5-HT3A serotonin receptors whereas they had no effect on α1 glycine receptors. Given the multifactorial nature of many pathologies in which nAChRs are involved, these piperidine antagonists could have a therapeutic potential in cases where cholinergic activity has to be negatively modulated. PMID:27254782

  5. K Basins floor sludge retrieval system knockout pot basket fuel burn accident

    SciTech Connect

    HUNT, J.W.

    1998-11-11

    The K Basins Sludge Retrieval System Preliminary Hazard Analysis Report (HNF-2676) identified and categorized a series of potential accidents associated with K Basins Sludge Retrieval System design and operation. The fuel burn accident was of concern with respect to the potential release of contamination resulting from a runaway chemical reaction of the uranium fuel in a knockout pot basket suspended in the air. The unmitigated radiological dose to an offsite receptor from this fuel burn accident is calculated to be much less than the offsite risk evaluation guidelines for anticipated events. However, because of potential radiation exposure to the facility worker, this accident is precluded with a safety significant lifting device that will prevent the monorail hoist from lifting the knockout pot basket out of the K Basin water pool.

  6. Clues to VIP function from knockout mice.

    PubMed

    Hamidi, S A; Szema, A M; Lyubsky, S; Dickman, K G; Degene, A; Mathew, S M; Waschek, J A; Said, S I

    2006-07-01

    We have taken advantage of the availability of vasoactive intestinal polypeptide (VIP) knockout (KO) mice to examine the possible influence of deletion of the VIP gene on: (a) airway reactivity and airway inflammation, as indicators of bronchial asthma; (b) mortality from endotoxemia, a model of septic shock; and (c) the pulmonary circulation. VIP KO mice showed: (a) airway hyperresponsiveness to the cholinergic agonist methacholine, as well as peribronchial and perivascular inflammation; (b) a greater susceptibility to death from endotoxemia; and (c) evidence suggestive of pulmonary hypertension. PMID:16888146

  7. Universal statistics of the knockout tournament

    NASA Astrophysics Data System (ADS)

    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.

  8. Altered Reward Circuitry in the Norepinephrine Transporter Knockout Mouse

    PubMed Central

    Hall, F. Scott; Uhl, George R.; Bearer, Elaine L.; Jacobs, Russell E.

    2013-01-01

    Synaptic levels of the monoamine neurotransmitters dopamine, serotonin, and norepinephrine are modulated by their respective plasma membrane transporters, albeit with a few exceptions. Monoamine transporters remove monoamines from the synaptic cleft and thus influence the degree and duration of signaling. Abnormal concentrations of these neuronal transmitters are implicated in a number of neurological and psychiatric disorders, including addiction, depression, and attention deficit/hyperactivity disorder. This work concentrates on the norepinephrine transporter (NET), using a battery of in vivo magnetic resonance imaging techniques and histological correlates to probe the effects of genetic deletion of the norepinephrine transporter on brain metabolism, anatomy and functional connectivity. MRS recorded in the striatum of NET knockout mice indicated a lower concentration of NAA that correlates with histological observations of subtle dysmorphisms in the striatum and internal capsule. As with DAT and SERT knockout mice, we detected minimal structural alterations in NET knockout mice by tensor-based morphometric analysis. In contrast, longitudinal imaging after stereotaxic prefrontal cortical injection of manganese, an established neuronal circuitry tracer, revealed that the reward circuit in the NET knockout mouse is biased toward anterior portions of the brain. This is similar to previous results observed for the dopamine transporter (DAT) knockout mouse, but dissimilar from work with serotonin transporter (SERT) knockout mice where Mn2+ tracings extended to more posterior structures than in wildtype animals. These observations correlate with behavioral studies indicating that SERT knockout mice display anxiety-like phenotypes, while NET knockouts and to a lesser extent DAT knockout mice display antidepressant-like phenotypic features. Thus, the mainly anterior activity detected with manganese-enhanced MRI in the DAT and NET knockout mice is likely indicative of

  9. Proteomic Analysis of Loricrin Knockout Mouse Epidermis.

    PubMed

    Rice, Robert H; Durbin-Johnson, Blythe P; Ishitsuka, Yosuke; Salemi, Michelle; Phinney, Brett S; Rocke, David M; Roop, Dennis R

    2016-08-01

    The crosslinked envelope of the mammalian epidermal corneocyte serves as a scaffold for assembly of the lipid barrier of the epidermis. Thus, deficient envelope crosslinking by keratinocyte transglutaminase (TGM1) is a major cause of the human autosomal recessive congenital ichthyoses characterized by barrier defects. Expectations that loss of some envelope protein components would also confer an ichthyosis phenotype have been difficult to demonstrate. To help rationalize this observation, the protein profile of epidermis from loricrin knockout mice has been compared to that of wild type. Despite the mild phenotype of the knockout, some 40 proteins were incorporated into envelope material to significantly different extents compared to those of wild type. Nearly half were also incorporated to similarly altered extents into the disulfide bonded keratin network of the corneocyte. The results suggest that loss of loricrin alters their incorporation into envelopes as a consequence of protein-protein interactions during cell maturation. Mass spectrometric protein profiling revealed that keratin 1, keratin 10, and loricrin are prominent envelope components and that dozens of other proteins are also components. This finding helps rationalize the potential formation of functional envelopes, despite loss of a single component, due to the availability of many alternative transglutaminase substrates. PMID:27418529

  10. Environmental enrichment induces behavioural disturbances in neuropeptide Y knockout mice

    PubMed Central

    Reichmann, Florian; Wegerer, Vanessa; Jain, Piyush; Mayerhofer, Raphaela; Hassan, Ahmed M.; Fröhlich, Esther E.; Bock, Elisabeth; Pritz, Elisabeth; Herzog, Herbert; Holzer, Peter; Leitinger, Gerd

    2016-01-01

    Environmental enrichment (EE) refers to the provision of a complex and stimulating housing condition which improves well-being, behaviour and brain function of laboratory animals. The mechanisms behind these beneficial effects of EE are only partially understood. In the current report, we describe a link between EE and neuropeptide Y (NPY), based on findings from NPY knockout (KO) mice exposed to EE. Relative to EE-housed wildtype (WT) animals, NPY KO mice displayed altered behaviour as well as molecular and morphological changes in amygdala and hippocampus. Exposure of WT mice to EE reduced anxiety and decreased central glucocorticoid receptor expression, effects which were absent in NPY KO mice. In addition, NPY deletion altered the preference of EE items, and EE-housed NPY KO mice responded to stress with exaggerated hyperthermia, displayed impaired spatial memory, had higher hippocampal brain-derived neurotrophic factor mRNA levels and altered hippocampal synaptic plasticity, effects which were not seen in WT mice. Accordingly, these findings suggest that NPY contributes to the anxiolytic effect of EE and that NPY deletion reverses the beneficial effects of EE into a negative experience. The NPY system could thus be a target for “enviromimetics”, therapeutics which reproduce the beneficial effects of enhanced environmental stimulation. PMID:27305846

  11. Environmental enrichment induces behavioural disturbances in neuropeptide Y knockout mice.

    PubMed

    Reichmann, Florian; Wegerer, Vanessa; Jain, Piyush; Mayerhofer, Raphaela; Hassan, Ahmed M; Fröhlich, Esther E; Bock, Elisabeth; Pritz, Elisabeth; Herzog, Herbert; Holzer, Peter; Leitinger, Gerd

    2016-01-01

    Environmental enrichment (EE) refers to the provision of a complex and stimulating housing condition which improves well-being, behaviour and brain function of laboratory animals. The mechanisms behind these beneficial effects of EE are only partially understood. In the current report, we describe a link between EE and neuropeptide Y (NPY), based on findings from NPY knockout (KO) mice exposed to EE. Relative to EE-housed wildtype (WT) animals, NPY KO mice displayed altered behaviour as well as molecular and morphological changes in amygdala and hippocampus. Exposure of WT mice to EE reduced anxiety and decreased central glucocorticoid receptor expression, effects which were absent in NPY KO mice. In addition, NPY deletion altered the preference of EE items, and EE-housed NPY KO mice responded to stress with exaggerated hyperthermia, displayed impaired spatial memory, had higher hippocampal brain-derived neurotrophic factor mRNA levels and altered hippocampal synaptic plasticity, effects which were not seen in WT mice. Accordingly, these findings suggest that NPY contributes to the anxiolytic effect of EE and that NPY deletion reverses the beneficial effects of EE into a negative experience. The NPY system could thus be a target for "enviromimetics", therapeutics which reproduce the beneficial effects of enhanced environmental stimulation. PMID:27305846

  12. CRISPR-Mediated Triple Knockout of SLAMF1, SLAMF5 and SLAMF6 Supports Positive Signaling Roles in NKT Cell Development

    PubMed Central

    Huang, Bonnie; Gomez-Rodriguez, Julio; Preite, Silvia; Garrett, Lisa J.; Harper, Ursula L.; Schwartzberg, Pamela L.

    2016-01-01

    The SLAM family receptors contribute to diverse aspects of lymphocyte biology and signal via the small adaptor molecule SAP. Mutations affecting SAP lead to X-linked lymphoproliferative syndrome Type 1, a severe immunodysregulation characterized by fulminant mononucleosis, dysgammaglobulinemia, and lymphoproliferation/lymphomas. Patients and mice having mutations affecting SAP also lack germinal centers due to a defect in T:B cell interactions and are devoid of invariant NKT (iNKT) cells. However, which and how SLAM family members contribute to these phenotypes remains uncertain. Three SLAM family members: SLAMF1, SLAMF5 and SLAMF6, are highly expressed on T follicular helper cells and germinal center B cells. SLAMF1 and SLAMF6 are also implicated in iNKT development. Although individual receptor knockout mice have limited iNKT and germinal center phenotypes compared to SAP knockout mice, the generation of multi-receptor knockout mice has been challenging, due to the genomic linkage of the genes encoding SLAM family members. Here, we used Cas9/CRISPR-based mutagenesis to generate mutations simultaneously in Slamf1, Slamf5 and Slamf6. Genetic disruption of all three receptors in triple-knockout mice (TKO) did not grossly affect conventional T or B cell development and led to mild defects in germinal center formation post-immunization. However, the TKO worsened defects in iNKT cells development seen in SLAMF6 single gene-targeted mice, supporting data on positive signaling and potential redundancy between these receptors. PMID:27258160

  13. Erythropoiesis and Blood Pressure Are Regulated via AT1 Receptor by Distinctive Pathways

    PubMed Central

    Kato, Hideki; Ishida, Junji; Matsusaka, Taiji; Ishimaru, Tomohiro; Tanimoto, Keiji; Sugiyama, Fumihiro; Yagami, Ken-ichi; Nangaku, Masaomi; Fukamizu, Akiyoshi

    2015-01-01

    The renin–angiotensin system (RAS) plays a central role in blood pressure regulation. Although clinical and experimental studies have suggested that inhibition of RAS is associated with progression of anemia, little evidence is available to support this claim. Here we report that knockout mice that lack angiotensin II, including angiotensinogen and renin knockout mice, exhibit anemia. The anemia of angiotensinogen knockout mice was rescued by angiotensin II infusion, and rescue was completely blocked by simultaneous administration of AT1 receptor blocker. To genetically determine the responsible receptor subtype, we examined AT1a, AT1b, and AT2 knockout mice, but did not observe anemia in any of them. To investigate whether pharmacological AT1 receptor inhibition recapitulates the anemic phenotype, we administered AT1 receptor antagonist in hypotensive AT1a receptor knockout mice to inhibit the remaining AT1b receptor. In these animals, hematocrit levels barely decreased, but blood pressure further decreased to the level observed in angiotensinogen knockout mice. We then generated AT1a and AT1b double-knockout mice to completely ablate the AT1 receptors; the mice finally exhibited the anemic phenotype. These results provide clear evidence that although erythropoiesis and blood pressure are negatively controlled through the AT1 receptor inhibition in vivo, the pathways involved are complex and distinct, because erythropoiesis is more resistant to AT1 receptor inhibition than blood pressure control. PMID:26107632

  14. Universal statistics of the knockout tournament

    PubMed Central

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

    2013-01-01

    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. PMID:24217406

  15. Ppp2ca knockout in mice spermatogenesis.

    PubMed

    Pan, Xiaoyun; Chen, Xia; Tong, Xin; Tang, Chao; Li, Jianmin

    2015-04-01

    Protein phosphatase 2A (PP2A) is a ubiquitous serine/threonine phosphatase involved in meiosis, mitosis, sperm capacitation, and apoptosis. Abberant activity of PP2A has been associated with a number of diseases. The homolog PPP2CA and PPP2CB can each function as the phosphatase catalytic subunit generally referred to as PP2AC. We generated a Ppp2ca conditional knockout (CKO) in C57BL/6J mice. Exon 2 of Ppp2ca was knocked out in a spatial or temporal-specific manner in primordial germ cells at E12.5. This Ppp2ca-null mutation caused infertility in male C57BL/6J mice. These CKO mice provide a powerful tool to study the mechanisms of Ppp2ca in development and disease. PMID:25628439

  16. Normal gonadotropin production and fertility in gonadotrope-specific Bmpr1a knockout mice.

    PubMed

    Zhou, Xiang; Wang, Ying; Ongaro, Luisina; Boehm, Ulrich; Kaartinen, Vesa; Mishina, Yuji; Bernard, Daniel J

    2016-06-01

    Pituitary follicle-stimulating hormone (FSH) synthesis is regulated by transforming growth factorβsuperfamily ligands, most notably the activins and inhibins. Bone morphogenetic proteins (BMPs) also regulate FSHβ subunit (Fshb) expression in immortalized murine gonadotrope-like LβT2 cells and in primary murine or ovine primary pituitary cultures. BMP2 signals preferentially via the BMP type I receptor, BMPR1A, to stimulate murine Fshb transcription in vitro Here, we used a Cre-lox approach to assess BMPR1A's role in FSH synthesis in mice in vivo Gonadotrope-specific Bmpr1a knockout animals developed normally and had reproductive organ weights comparable with those of controls. Knockouts were fertile, with normal serum gonadotropins and pituitary gonadotropin subunit mRNA expression. Cre-mediated recombination of the floxed Bmpr1a allele was efficient and specific, as indicated by PCR analysis of diverse tissues and isolated gonadotrope cells. Furthermore, BMP2 stimulation of inhibitor of DNA binding 3 expression was impaired in gonadotropes isolated from Bmpr1a knockout mice, confirming the loss of functional receptor protein in these cells. Treatment of purified gonadotropes with small-molecule inhibitors of BMPR1A (and the related receptors BMPR1B and ACVR1) suppressed Fshb mRNA expression, suggesting that an autocrine BMP-like molecule might regulate FSH synthesis. However, deletion of Bmpr1a and Acvr1 in cultured pituitary cells did not alter Fshb expression, indicating that the inhibitors had off-target effects. In sum, BMPs or related ligands acting via BMPR1A or ACVR1 are unlikely to play direct physiological roles in FSH synthesis by murine gonadotrope cells. PMID:27029473

  17. Positron emission tomography and functional characterization of a complete PBR/TSPO knockout.

    PubMed

    Banati, Richard B; Middleton, Ryan J; Chan, Ronald; Hatty, Claire R; Kam, Winnie Wai-Ying; Quin, Candice; Graeber, Manuel B; Parmar, Arvind; Zahra, David; Callaghan, Paul; Fok, Sandra; Howell, Nicholas R; Gregoire, Marie; Szabo, Alexander; Pham, Tien; Davis, Emma; Liu, Guo-Jun

    2014-01-01

    The evolutionarily conserved peripheral benzodiazepine receptor (PBR), or 18-kDa translocator protein (TSPO), is thought to be essential for cholesterol transport and steroidogenesis, and thus life. TSPO has been proposed as a biomarker of neuroinflammation and a new drug target in neurological diseases ranging from Alzheimer's disease to anxiety. Here we show that global C57BL/6-Tspo(tm1GuWu(GuwiyangWurra))-knockout mice are viable with normal growth, lifespan, cholesterol transport, blood pregnenolone concentration, protoporphyrin IX metabolism, fertility and behaviour. However, while the activation of microglia after neuronal injury appears to be unimpaired, microglia from (GuwiyangWurra)TSPO knockouts produce significantly less ATP, suggesting reduced metabolic activity. Using the isoquinoline PK11195, the ligand originally used for the pharmacological and structural characterization of the PBR/TSPO, and the imidazopyridines CLINDE and PBR111, we demonstrate the utility of (GuwiyangWurra)TSPO knockouts to provide robust data on drug specificity and selectivity, both in vitro and in vivo, as well as the mechanism of action of putative TSPO-targeting drugs. PMID:25406832

  18. Positron emission tomography and functional characterization of a complete PBR/TSPO knockout

    PubMed Central

    Banati, Richard B.; Middleton, Ryan J.; Chan, Ronald; Hatty, Claire R.; Wai-Ying Kam, Winnie; Quin, Candice; Graeber, Manuel B.; Parmar, Arvind; Zahra, David; Callaghan, Paul; Fok, Sandra; Howell, Nicholas R.; Gregoire, Marie; Szabo, Alexander; Pham, Tien; Davis, Emma; Liu, Guo-Jun

    2014-01-01

    The evolutionarily conserved peripheral benzodiazepine receptor (PBR), or 18-kDa translocator protein (TSPO), is thought to be essential for cholesterol transport and steroidogenesis, and thus life. TSPO has been proposed as a biomarker of neuroinflammation and a new drug target in neurological diseases ranging from Alzheimer’s disease to anxiety. Here we show that global C57BL/6-Tspotm1GuWu(GuwiyangWurra)-knockout mice are viable with normal growth, lifespan, cholesterol transport, blood pregnenolone concentration, protoporphyrin IX metabolism, fertility and behaviour. However, while the activation of microglia after neuronal injury appears to be unimpaired, microglia from GuwiyangWurraTSPO knockouts produce significantly less ATP, suggesting reduced metabolic activity. Using the isoquinoline PK11195, the ligand originally used for the pharmacological and structural characterization of the PBR/TSPO, and the imidazopyridines CLINDE and PBR111, we demonstrate the utility of GuwiyangWurraTSPO knockouts to provide robust data on drug specificity and selectivity, both in vitro and in vivo, as well as the mechanism of action of putative TSPO-targeting drugs. PMID:25406832

  19. The alcohol-induced locomotor stimulation and accumbal dopamine release is suppressed in ghrelin knockout mice.

    PubMed

    Jerlhag, Elisabet; Landgren, Sara; Egecioglu, Emil; Dickson, Suzanne L; Engel, Jörgen A

    2011-06-01

    Ghrelin, the first endogenous ligand for the type 1A growth hormone secretagogue receptor (GHS-R1A), plays a role in energy balance, feeding behavior, and reward. Previously, we showed that pharmacologic and genetic suppression of the GHS-R1A attenuates the alcohol-induced stimulation, accumbal dopamine release, and conditioned place preference as well as alcohol consumption in mice, implying that the GHS-R1A is required for alcohol reward. The present study further elucidates the role of ghrelin for alcohol-induced dopamine release in nucleus accumbens and locomotor stimulation by means of ghrelin knockout mice. We found that the ability of alcohol to increase accumbal dopamine release in wild-type mice is not observed in ghrelin knockout mice. Furthermore, alcohol induced a locomotor stimulation in the wild-type mice and ghrelin knockout mice; however, the locomotor stimulation in homozygote mice was significantly lower than in the wild-type mice. The present series of experiments suggest that endogenous ghrelin may be required for the ability of alcohol to activate the mesolimbic dopamine system. PMID:21145690

  20. The effect of PDIA3 gene knockout on the mucosal immune function in IBS rats

    PubMed Central

    Zhuang, Zhao-Meng; Wang, Xiao-Teng; Zhang, Lu; Tao, Li-Yuan; Lv, Bin

    2015-01-01

    Objective: To observe the changes of intestinal inflammation on PDIA3 gene knockout IBS rats and its effect on immune function. Methods: 36 SD rats were randomly divided into four groups: the control group (n = 8); IBS- empty virus group (IBS-GFP, which); IBS-PDIA3 knockout group (n = 12); IBS- the control group (n = 12). After modeling, colon and ileocecal tissue pathology in each group were observed separately. Changes of immune and inflammatory markers were measured. At the same time, ultrastructural changes in each group were observed by electron microscopy. Results: Compared with the IBS control group, inflammation was reduced significantly in IBS-PDIA3 knockout group. IgE, IL-4 and IL-9 and the level of intestinal trypsin type were decreased significantly. Furthermore, mast cell degranulation and PAR 2 receptor reduced significantly. Conclusion: PDIA3 may play an important role in the development of IBS by mediating through immune responses of mucosal abnormalities. However, the mechanism needs to be confirmed in further study. PMID:26221224

  1. Human Knockout Carriers: Dead, Diseased, Healthy, or Improved?

    PubMed Central

    Narasimhan, Vagheesh M.; Xue, Yali; Tyler-Smith, Chris

    2016-01-01

    Whole-genome and whole-exome sequence data from large numbers of individuals reveal that we all carry many variants predicted to inactivate genes (knockouts). This discovery raises questions about the phenotypic consequences of these knockouts and potentially allows us to study human gene function through the investigation of homozygous loss-of-function carriers. Here, we discuss strategies, recent results, and future prospects for large-scale human knockout studies. We examine their relevance to studying gene function, population genetics, and importantly, the implications for accurate clinical interpretations. PMID:26988438

  2. Theoretical knock-outs on biological networks.

    PubMed

    Miranda, Pedro J; de S Pinto, Sandro E; Baptista, Murilo S; La Guardia, Giuliano G

    2016-08-21

    In this work we redefine the concept of biological importance and how to compute it, based on a model of complex networks and random walk. We call this new procedure, theoretical knock-out (KO). The proposed method generalizes the procedure presented in a recent study about Oral Tolerance. To devise this method, we make two approaches: algebraically and algorithmically. In both cases we compute a vector on an asymptotic state, called flux vector. The flux is given by a random walk on a directed graph that represents a biological phenomenon. This vector gives us the information about the relative flux of walkers on a vertex which represents a biological agent. With two vector of this kind, we can calculate the relative mean error between them by averaging over its coefficients. This quantity allows us to assess the degree of importance of each vertex of a complex network that evolves in time and has experimental background. We find out that this procedure can be applied in any sort of biological phenomena in which we can know the role and interrelationships of its agents. These results also provide experimental biologists to predict the order of importance of biological agents on a mounted complex network. PMID:27188251

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

    SciTech Connect

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

    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.

  4. Salty taste deficits in CALHM1 knockout mice.

    PubMed

    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. PMID:24846212

  5. mRNA expression profile of serotonin receptor subtypes and distribution of serotonergic terminations in marmoset brain

    PubMed Central

    Shukla, Rammohan; Watakabe, Akiya; Yamamori, Tetsuo

    2014-01-01

    To better understand serotonin function in the primate brain, we examined the mRNA expression patterns of all the 13 members of the serotonin receptor (5HTR) family, by in situ hybridization (ISH) and the distribution of serotonergic terminations by serotonin transporter (SERT) protein immunohistochemical analysis. Ten of the 13 5HTRs showed significant mRNA expressions in the marmoset brain. Our study shows several new features of the organization of serotonergic systems in the marmoset brain. (1) The thalamus expressed only a limited number of receptor subtypes compared with the cortex, hippocampus, and other subcortical regions. (2) In the cortex, there are layer-selective and area-selective mRNA expressions of 5HTRs. (3) Highly localized mRNA expressions of 5HT1F and 5HT3A were observed. (4) There was a conspicuous overlap of the mRNA expressions of receptor subtypes known to have somatodendritic localization of receptor proteins with dense serotonergic terminations in the visual cortex, the central lateral (CL) nucleus of the thalamus, the presubiculum, and the medial mammillary nucleus of the hypothalamus. This suggests a high correlation between serotonin availability and receptor expression at these locations. (5) The 5HTRs show differences in mRNA expression pattern between the marmoset and mouse cortices whereas the patterns of both the species were much similar in the hippocampus. We discuss the possible roles of 5HTRs in the marmoset brain revealed by the analysis of their overall mRNA expression patterns. PMID:24904298

  6. Combination of cell culture assays and knockout mouse analyses for the study of opioid partial agonism.

    PubMed

    Ide, Soichiro; Minami, Masabumi; Sora, Ichiro; Ikeda, Kazutaka

    2010-01-01

    Nonselective opioid partial agonists, such as buprenorphine, butorphanol, and pentazocine, have been widely used as analgesics and for anti-addiction therapy. However, the precise molecular mechanisms underlying the therapeutic and rewarding effects of these drugs have not been clearly delineated. Recent success in developing mu-opioid receptor knockout (MOP-KO) mice has elucidated the molecular mechanisms underlying the effects of morphine and other opioids. We have revealed the in vivo roles of MOPs in the effects of opioid partial agonists by using MOP-KO mice for behavioral tests (e.g., several kinds of antinociceptive tests for analgesic effects, conditioned place preference test for dependence). The combination of the cell culture assays using cDNA for mu, delta, and kappa opioid receptors and the behavioral tests using MOP-KO mice has provided novel theories on the molecular mechanisms underlying the effects of opioid ligands, especially opioid partial agonists. PMID:20336435

  7. Male contraception via simultaneous knockout of α1A-adrenoceptors and P2X1-purinoceptors in mice

    PubMed Central

    White, Carl W.; Choong, Yan-Ting; Short, Jennifer L.; Exintaris, Betty; Malone, Daniel T.; Allen, Andrew M.; Evans, Richard J.; Ventura, Sabatino

    2013-01-01

    Therapeutic targets for male contraception are associated with numerous problems due to their focus on disrupting spermatogenesis or hormonal mechanisms to produce dysfunctional sperm. Here we describe the dual genetic deletion of α1A-adrenergic G protein-coupled receptors (adrenoceptors) and P2X1-purinoceptor ligand gated ion channels in male mice, thereby blocking sympathetically mediated sperm transport through the vas deferens during the emission phase of ejaculation. This modification produced 100% infertility without effects on sexual behavior or function. Sperm taken from the cauda epididymides of double knockout mice were microscopically normal and motile. Furthermore, double knockout sperm were capable of producing normal offspring following intracytoplasmic sperm injection into wild-type ova and implantation of the fertilized eggs into foster mothers. Blood pressure and baroreflex function was reduced in double knockout mice, but no more than single knockout of α1A-adrenoceptors alone. These results suggest that this autonomic method of male contraception appears free of major physiological and behavioral side effects. In addition, they provide conclusive proof of concept that pharmacological antagonism of the P2X1-purinoceptor and α1A-adrenoceptor provides a safe and effective therapeutic target for a nonhormonal, readily reversible male contraceptive. PMID:24297884

  8. Male contraception via simultaneous knockout of α1A-adrenoceptors and P2X1-purinoceptors in mice.

    PubMed

    White, Carl W; Choong, Yan-Ting; Short, Jennifer L; Exintaris, Betty; Malone, Daniel T; Allen, Andrew M; Evans, Richard J; Ventura, Sabatino

    2013-12-17

    Therapeutic targets for male contraception are associated with numerous problems due to their focus on disrupting spermatogenesis or hormonal mechanisms to produce dysfunctional sperm. Here we describe the dual genetic deletion of α1A-adrenergic G protein-coupled receptors (adrenoceptors) and P2X1-purinoceptor ligand gated ion channels in male mice, thereby blocking sympathetically mediated sperm transport through the vas deferens during the emission phase of ejaculation. This modification produced 100% infertility without effects on sexual behavior or function. Sperm taken from the cauda epididymides of double knockout mice were microscopically normal and motile. Furthermore, double knockout sperm were capable of producing normal offspring following intracytoplasmic sperm injection into wild-type ova and implantation of the fertilized eggs into foster mothers. Blood pressure and baroreflex function was reduced in double knockout mice, but no more than single knockout of α1A-adrenoceptors alone. These results suggest that this autonomic method of male contraception appears free of major physiological and behavioral side effects. In addition, they provide conclusive proof of concept that pharmacological antagonism of the P2X1-purinoceptor and α1A-adrenoceptor provides a safe and effective therapeutic target for a nonhormonal, readily reversible male contraceptive. PMID:24297884

  9. Activation of human 5-hydroxytryptamine type 3 receptors via an allosteric transmembrane site.

    PubMed

    Lansdell, Stuart J; Sathyaprakash, Chaitra; Doward, Anne; Millar, Neil S

    2015-01-01

    In common with other members of the Cys-loop family of pentameric ligand-gated ion channels, 5-hydroxytryptamine type 3 receptors (5-HT3Rs) are activated by the binding of a neurotransmitter to an extracellular orthosteric site, located at the interface of two adjacent receptor subunits. In addition, a variety of compounds have been identified that modulate agonist-evoked responses of 5-HT3Rs, and other Cys-loop receptors, by binding to distinct allosteric sites. In this study, we examined the pharmacological effects of a group of monoterpene compounds on recombinant 5-HT3Rs expressed in Xenopus oocytes. Two phenolic monoterpenes (carvacrol and thymol) display allosteric agonist activity on human homomeric 5-HT3ARs (64 ± 7% and 80 ± 4% of the maximum response evoked by the endogenous orthosteric agonist 5-HT, respectively). In addition, at lower concentrations, where agonist effects are less apparent, carvacrol and thymol act as potentiators of responses evoked by submaximal concentrations of 5-HT. By contrast, carvacrol and thymol have no agonist or potentiating activity on the closely related mouse 5-HT3ARs. Using subunit chimeras containing regions of the human and mouse 5-HT3A subunits, and by use of site-directed mutagenesis, we have identified transmembrane amino acids that either abolish the agonist activity of carvacrol and thymol on human 5-HT3ARs or are able to confer this property on mouse 5-HT3ARs. By contrast, these mutations have no significant effect on orthosteric activation of 5-HT3ARs by 5-HT. We conclude that 5-HT3ARs can be activated by the binding of ligands to an allosteric transmembrane site, a conclusion that is supported by computer docking studies. PMID:25338672

  10. Establishment and phenotypic analysis of an Mstn knockout rat.

    PubMed

    Gu, Hao; Cao, Yong; Qiu, Bin; Zhou, Zhiqiang; Deng, Ran; Chen, Zhuang; Li, Rongfeng; Li, Xueling; Wei, Qiang; Xia, Xianzhu; Yong, Weidong

    2016-08-12

    Myostatin (Mstn) is an inhibitor of myogenesis, regulating the number and size of skeletal myocytes. In addition to its myogenic regulatory function, Mstn plays important roles in the development of adipose tissues and in metabolism. In the present study, an Mstn knockout rat model was generated using the zinc finger nuclease (ZFN) technique in order to further investigate the function and mechanism of Mstn in metabolism. The knockout possesses a frame shift mutation resulting in an early termination codon and a truncated peptide of 109 amino acids rather than the full 376 amino acids. The absence of detectable mRNA confirmed successful knockout of Mstn. Relative to wild-type (WT) littermates, Knockout (KO) rats exhibited significantly greater body weight, body circumference, and muscle mass. However, no significant differences in grip force was observed, indicating that Mstn deletion results in greater muscle mass but not greater muscle fiber strength. Additionally, KO rats were found to possess less body fat relative to WT littermates, which is consistent with previous studies in mice and cattle. The aforementioned results indicate that Mstn knockout increases muscle mass while decreasing fat content, leading to observed increases in body weight and body circumference. The Mstn knockout rat model provides a novel means to study the role of Mstn in metabolism and Mstn-related muscle hypertrophy. PMID:27289021

  11. Impaired conditioned taste aversion learning in spinophilin knockout mice.

    PubMed

    Stafstrom-Davis, C A; Ouimet, C C; Feng, J; Allen, P B; Greengard, P; Houpt, T A

    2001-01-01

    Plasticity in dendritic spines may underlie learning and memory. Spinophilin, a protein enriched in dendritic spines, has the properties of a scaffolding protein and is believed to regulate actin cytoskeletal dynamics affecting dendritic spine morphology. It also binds protein phosphatase-1 (PP-1), an enzyme that regulates dendritic spine physiology. In this study, we tested the role of spinophilin in conditioned taste aversion learning (CTA) using transgenic spinophilin knockout mice. CTA is a form of associative learning in which an animal rejects a food that has been paired previously with a toxic effect (e.g., a sucrose solution paired with a malaise-inducing injection of lithium chloride). Acquisition and extinction of CTA was tested in spinophilin knockout and wild-type mice using taste solutions (sucrose or sodium chloride) or flavors (Kool-Aid) paired with moderate or high doses of LiCl (0.15 M, 20 or 40 mL/kg). When sucrose or NaCl solutions were paired with a moderate dose of LiCl, spinophilin knockout mice were unable to learn a CTA. At the higher dose, knockout mice acquired a CTA but extinguished more rapidly than wild-type mice. A more salient flavor stimulus (taste plus odor) revealed similar CTA learning at both doses of LiCl in both knockouts and wild types. Sensory processing in the knockouts appeared normal because knockout mice and wild-type mice expressed identical unconditioned taste preferences in two-bottle tests, and identical lying-on-belly responses to acute LiCl. We conclude that spinophilin is a candidate molecule required for normal CTA learning. PMID:11584074

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

    PubMed Central

    Befort, Katia

    2015-01-01

    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. PMID:25698968

  13. Spatiotemporal Alterations in Primary Odorant Representations in Olfactory Marker Protein Knockout Mice

    PubMed Central

    Kass, Marley D.; Moberly, Andrew H.; McGann, John P.

    2013-01-01

    Olfactory marker protein (OMP) is highly and selectively expressed in primary olfactory sensory neurons (OSNs) across species, but its physiological function remains unclear. Previous studies in the olfactory epithelium suggest that it accelerates the neural response to odorants and may modulate the odorant-selectivity of OSNs. Here we used a line of gene-targeted mice that express the fluorescent exocytosis indicator synaptopHluorin in place of OMP to compare spatiotemporal patterns of odorant-evoked neurotransmitter release from OSNs in adult mice that were heterozygous for OMP or OMP-null. We found that these patterns, which constitute the primary neural representation of each odorant, developed more slowly during the odorant presentation in OMP knockout mice but eventually reached the same magnitude as in heterozygous mice. In the olfactory bulb, each glomerulus receives synaptic input from a subpopulation of OSNs that all express the same odor receptor and thus typically respond to a specific subset of odorants. We observed that in OMP knockout mice, OSNs innervating a given glomerulus typically responded to a broader range of odorants than in OMP heterozygous mice and thus each odorant evoked synaptic input to a larger number of glomeruli. In an olfactory habituation task, OMP knockout mice behaved differently than wild-type mice, exhibiting a delay in their onset to investigate an odor stimulus during its first presentation and less habituation to that stimulus over repeated presentations. These results suggest that the actions of OMP in olfactory transduction carry through to the primary sensory representations of olfactory stimuli in adult mice in vivo. PMID:23630588

  14. Cathepsin K knockout alleviates aging-induced cardiac dysfunction

    PubMed Central

    Hua, Yinan; Robinson, Timothy J; Cao, Yongtao; Shi, Guo-Ping; Ren, Jun; Nair, Sreejayan

    2015-01-01

    Aging is a major risk factor for cardiovascular disease. It has previously been shown that protein levels of cathepsin K, a lysosomal cysteine protease, are elevated in the failing heart and that genetic ablation of cathepsin K protects against pressure overload-induced cardiac hypertrophy and contractile dysfunction. Here we test the hypothesis that cathepsin K knockout alleviates age-dependent decline in cardiac function. Cardiac geometry, contractile function, intracellular Ca2+ properties, and cardiomyocyte apoptosis were evaluated using echocardiography, fura-2 technique, immunohistochemistry, Western blot and TUNEL staining, respectively. Aged (24-month-old) mice exhibited significant cardiac remodeling (enlarged chamber size, wall thickness, myocyte cross-sectional area, and fibrosis), decreased cardiac contractility, prolonged relengthening along with compromised intracellular Ca2+ release compared to young (6-month-old) mice, which were attenuated in the cathepsin K knockout mice. Cellular markers of senescence, including cardiac lipofuscin, p21 and p16, were lower in the aged-cathepsin K knockout mice compared to their wild-type counterpart. Mechanistically, cathepsin K knockout mice attenuated an age-induced increase in cardiomyocyte apoptosis and nuclear translocation of mitochondrial apoptosis-inducing factor (AIF). In cultured H9c2 cells, doxorubicin stimulated premature senescence and apoptosis. Silencing of cathepsin K blocked the doxorubicin-induced translocation of AIF from the mitochondria to the nuclei. Collectively, these results suggest that cathepsin K knockout attenuates age-related decline in cardiac function via suppressing caspase-dependent and caspase-independent apoptosis. PMID:25692548

  15. Knockout silkworms reveal a dispensable role for juvenile hormones in holometabolous life cycle.

    PubMed

    Daimon, Takaaki; Uchibori, Miwa; Nakao, Hajime; Sezutsu, Hideki; Shinoda, Tetsuro

    2015-08-01

    Insect juvenile hormones (JHs) prevent precocious metamorphosis and allow larvae to undergo multiple rounds of status quo molts. However, the roles of JHs during the embryonic and very early larval stages have not been fully understood. We generated and characterized knockout silkworms (Bombyx mori) with null mutations in JH biosynthesis or JH receptor genes using genome-editing tools. We found that embryonic growth and morphogenesis are largely independent of JHs in Bombyx and that, even in the absence of JHs or JH signaling, pupal characters are not formed in first- or second-instar larvae, and precocious metamorphosis is induced after the second instar at the earliest. We also show by mosaic analysis that a pupal specifier gene broad, which is dramatically up-regulated in the late stage of the last larval instar, is essential for pupal commitment in the epidermis. Importantly, the mRNA expression level of broad, which is thought to be repressed by JHs, remained at very low basal levels during the early larval instars of JH-deficient or JH signaling-deficient knockouts. Therefore, our study suggests that the long-accepted paradigm that JHs maintain the juvenile status throughout larval life should be revised because the larval status can be maintained by a JH-independent mechanism in very early larval instars. We propose that the lack of competence for metamorphosis during the early larval stages may result from the absence of an unidentified broad-inducing factor, i.e., a competence factor. PMID:26195792

  16. Hesr1 knockout mice exhibit behavioral alterations through the dopaminergic nervous system.

    PubMed

    Fuke, Satoshi; Minami, Natsumi; Kokubo, Hiroki; Yoshikawa, Ayumu; Yasumatsu, Hiroshi; Sasagawa, Noboru; Saga, Yumiko; Tsukahara, Toshifumi; Ishiura, Shoichi

    2006-11-15

    The basic helix-loop-helix (bHLH) transcriptional factor Hesr1 gene (hairy and enhancer of split-related 1, also called Hey1/HRT1/CHF2/HERP2) has been identified and characterized as a member of the subfamily of hairy/Enhancer of split, and shown to be involved in cardiovascular and neural development. We report that HESR1 binds directly to a part of the 3' non-coding region of the human dopamine transporter (DAT1) gene and represses the endogenous DAT1 gene in HEK293 cells. To investigate functions of the HESR1 gene in the dopaminergic nervous system in vivo, we analyzed the expressions of dopamine-related genes in the postnatal day 0 whole brains of Hesr1 knockout mice by real-time RT-PCR analysis. Several dopamine-related genes, such as DAT, dopamine receptors D1, D2, D4, and D5, were significantly upregulated. Moreover, young adults of Hesr1 knockout mice showed a decrease in spontaneous locomotor activity and a reduction in exploratory behavior or behavioral responses to novelty in the open-field, and elevated plus-maze tests. These results indicate that the HESR1 gene is related to neuropsychiatric disorders and behavioral traits through the dopaminergic nervous system. PMID:16998899

  17. Downregulation of Glutamate Transporter EAAT4 by Conditional Knockout of Rheb1 in Cerebellar Purkinje Cells.

    PubMed

    Jiang, Nan-Wei; Wang, De-Juan; Xie, Ya-Jun; Zhou, Liang; Su, Li-Da; Li, Huashun; Wang, Qin-Wen; Shen, Ying

    2016-06-01

    Excitatory amino acid transporter 4 (EAAT4) is believed to be critical to the synaptic activity of cerebellar Purkinje cells by limiting extracellular glutamate concentrations and facilitating the induction of long-term depression. However, the modulation of EAAT4 expression has not been elucidated. It has been shown that Ras homolog enriched in brain (Rheb)/mammalian target of rapamycin (mTOR) signaling plays essential roles in the regulation of protein translation, cell size, and cell growth. In addition, we previously found that a cascade including mTOR suppression and Akt activation induces increased expression of EAAT2 in astrocytes. In the present work, we explored whether Rheb/mTOR signaling is involved in the regulation of EAAT4 expression using conditional Rheb1 knockout mice. Our results demonstrated that Rheb1 deficiency resulted in the downregulation of EAAT4 expression, as well as decreased activity of mTOR and increased activity of Akt. The downregulation of EAAT4 was also confirmed by reduced EAAT4 currents and slowed kinetics of α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor-mediated currents. On the other hand, conditional knockout of Rheb1 did not alter the morphology of Purkinje cell layer and the number of Purkinje cells. Overall, our findings suggest that small GTPase Rheb1 is a modulator in the expression of EAAT4 in Purkinje cells. PMID:26194056

  18. Retinal Ganglion Cell Loss is Delayed Following Optic Nerve Crush in NLRP3 Knockout Mice

    PubMed Central

    Puyang, Zhen; Feng, Liang; Chen, Hui; Liang, Peiji; Troy, John B.; Liu, Xiaorong

    2016-01-01

    The NLRP3 inflammasome, a sensor for a variety of pathogen- and host-derived threats, consists of the adaptor ASC (Apoptosis-associated Speck-like protein containing a Caspase Activation and Recruitment Domain (CARD)), pro-caspase-1, and NLRP3 (NOD-Like Receptor family Pyrin domain containing 3). NLRP3-induced neuroinflammation is implicated in the pathogenesis and progression of eye diseases, but it remains unclear whether activation of NLRP3 inflammasome contributes to retinal ganglion cell (RGC) death. Here we examined NLRP3-induced neuroinflammation and RGC survival following partial optic nerve crush (pONC) injury. We showed that NLRP3 was up-regulated in retinal microglial cells following pONC, propagating from the injury site to the optic nerve head and finally the entire retina within one day. Activation of NLRP3-ASC inflammasome led to the up-regulation of caspase-1 and a proinflammatory cytokine, interleukin-1β (IL-1β). In NLRP3 knockout mice, up-regulation of ASC, caspase-1, and IL-1β were all reduced, and, importantly, RGC and axon loss was substantially delayed following pONC injury. The average survival time of RGCs in NLRP3 knockout mice was about one week longer than for control animals. Taken together, our study demonstrated that ablating the NLRP3 gene significantly reduced neuroinflammation and delayed RGC loss after optic nerve crush injury. PMID:26893104

  19. Beta2-adrenergic activity modulates vascular tone regulation in lecithin:cholesterol acyltransferase knockout mice

    PubMed Central

    Manzini, S.; Pinna, C.; Busnelli, M.; Cinquanta, P.; Rigamonti, E.; Ganzetti, G.S.; Dellera, F.; Sala, A.; Calabresi, L.; Franceschini, G.; Parolini, C.; Chiesa, G.

    2015-01-01

    Lecithin:cholesterol acyltransferase (LCAT) deficiency is associated with hypoalphalipoproteinemia, generally a predisposing factor for premature coronary heart disease. The evidence of accelerated atherosclerosis in LCAT-deficient subjects is however controversial. In this study, the effect of LCAT deficiency on vascular tone and endothelial function was investigated in LCAT knockout mice, which reproduce the human lipoprotein phenotype. Aortas from wild-type (Lcatwt) and LCAT knockout (LcatKO) mice exposed to noradrenaline showed reduced contractility in LcatKO mice (P < 0.005), whereas acetylcholine exposure showed a lower NO-dependent relaxation in LcatKO mice (P < 0.05). Quantitative PCR and Western blotting analyses suggested an adequate eNOS expression in LcatKO mouse aortas. Real-time PCR analysis indicated increased expression of β2-adrenergic receptors vs wild-type mice. Aorta stimulation with noradrenaline in the presence of propranolol, to abolish the β-mediated relaxation, showed the same contractile response in the two mouse lines. Furthermore, propranolol pretreatment of mouse aortas exposed to L-NAME prevented the difference in responses between Lcatwt and LcatKO mice. The results indicate that LCAT deficiency leads to increased β2-adrenergic relaxation and to a consequently decreased NO-mediated vasodilation that can be reversed to guarantee a correct vascular tone. The present study suggests that LCAT deficiency is not associated with an impaired vascular reactivity. PMID:26254103

  20. Beta2-adrenergic activity modulates vascular tone regulation in lecithin:cholesterol acyltransferase knockout mice.

    PubMed

    Manzini, S; Pinna, C; Busnelli, M; Cinquanta, P; Rigamonti, E; Ganzetti, G S; Dellera, F; Sala, A; Calabresi, L; Franceschini, G; Parolini, C; Chiesa, G

    2015-11-01

    Lecithin:cholesterol acyltransferase (LCAT) deficiency is associated with hypoalphalipoproteinemia, generally a predisposing factor for premature coronary heart disease. The evidence of accelerated atherosclerosis in LCAT-deficient subjects is however controversial. In this study, the effect of LCAT deficiency on vascular tone and endothelial function was investigated in LCAT knockout mice, which reproduce the human lipoprotein phenotype. Aortas from wild-type (Lcat(wt)) and LCAT knockout (Lcat(KO)) mice exposed to noradrenaline showed reduced contractility in Lcat(KO) mice (P<0.005), whereas acetylcholine exposure showed a lower NO-dependent relaxation in Lcat(KO) mice (P<0.05). Quantitative PCR and Western blotting analyses suggested an adequate eNOS expression in Lcat(KO) mouse aortas. Real-time PCR analysis indicated increased expression of β2-adrenergic receptors vs wild-type mice. Aorta stimulation with noradrenaline in the presence of propranolol, to abolish the β-mediated relaxation, showed the same contractile response in the two mouse lines. Furthermore, propranolol pretreatment of mouse aortas exposed to L-NAME prevented the difference in responses between Lcat(wt) and Lcat(KO) mice. The results indicate that LCAT deficiency leads to increased β2-adrenergic relaxation and to a consequently decreased NO-mediated vasodilation that can be reversed to guarantee a correct vascular tone. The present study suggests that LCAT deficiency is not associated with an impaired vascular reactivity. PMID:26254103

  1. Knockout silkworms reveal a dispensable role for juvenile hormones in holometabolous life cycle

    PubMed Central

    Daimon, Takaaki; Uchibori, Miwa; Nakao, Hajime; Sezutsu, Hideki; Shinoda, Tetsuro

    2015-01-01

    Insect juvenile hormones (JHs) prevent precocious metamorphosis and allow larvae to undergo multiple rounds of status quo molts. However, the roles of JHs during the embryonic and very early larval stages have not been fully understood. We generated and characterized knockout silkworms (Bombyx mori) with null mutations in JH biosynthesis or JH receptor genes using genome-editing tools. We found that embryonic growth and morphogenesis are largely independent of JHs in Bombyx and that, even in the absence of JHs or JH signaling, pupal characters are not formed in first- or second-instar larvae, and precocious metamorphosis is induced after the second instar at the earliest. We also show by mosaic analysis that a pupal specifier gene broad, which is dramatically up-regulated in the late stage of the last larval instar, is essential for pupal commitment in the epidermis. Importantly, the mRNA expression level of broad, which is thought to be repressed by JHs, remained at very low basal levels during the early larval instars of JH-deficient or JH signaling-deficient knockouts. Therefore, our study suggests that the long-accepted paradigm that JHs maintain the juvenile status throughout larval life should be revised because the larval status can be maintained by a JH-independent mechanism in very early larval instars. We propose that the lack of competence for metamorphosis during the early larval stages may result from the absence of an unidentified broad-inducing factor, i.e., a competence factor. PMID:26195792

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

    PubMed Central

    Belizário, José; Vieira-Cordeiro, Luiz; Enns, Sylvia

    2015-01-01

    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. PMID:26491219

  3. Appetitively motivated instrumental learning in SynGAP heterozygous knockout mice.

    PubMed

    Muhia, Mary; Feldon, Joram; Knuesel, Irene; Yee, Benjamin K

    2009-10-01

    The synaptic Ras/Rap-GTPase-activating protein (SynGAP) regulates specific intracellular events following N-methyl-d-aspartate receptor (NMDAR) activation. Here, the impact of SynGAP heterozygous knockout (SG+/-) on NMDAR-dependent functions was assessed using different positive reinforcement schedules in instrumental conditioning. The knockout did not affect the temporal control of operant responding under a fixed interval (FI) schedule, but led to a putative enhancement in response vigor and/or disinhibition. When examined on differential reinforcement of low rates of response (DRL) schedules, SG+/- mice showed increased responding under DRL-4s and DRL-8s, without impairing the response efficiency (total rewards/total lever presses) because both rewarded and nonrewarded presses were elevated. Motivation was unaffected as evaluated using a progressive ratio (PR) schedule. Yet, SG+/- mice persisted in responding during extinction at the end of PR training, although an equivalent phenotype was not evident in extinction learning following FI-20s training. This extinction phenotype is therefore schedule-specific and cannot be generalized to Pavlovian conditioning. In conclusion, constitutive SynGAP reduction increases vigor in the execution of learned operant behavior without compromising its temporal control, yielding effects readily distinguishable from NMDAR blockade. PMID:19824778

  4. Construction of Deletion-knockout Mutant Fowlpox Virus (FWPV)

    PubMed Central

    Laidlaw, Stephen M.; Skinner, Michael A.

    2016-01-01

    The construction of deletion-knockout poxviruses is a useful approach to determining the function of specific virus genes. This protocol is an adaptation of the transient dominant knockout selection protocol published by Falkner and Moss (1990) for use with vaccinia virus. The protocol makes use of the dominant selectable marker Escherichia coli guanine phosphoribosyltransferase (gpt) gene (Mulligan and Berg, 1981), under the control of an early/late poxvirus promoter. The deletion viruses that are produced no longer contain a selectable marker, which may be preferable for the production of vaccines.

  5. In vivo modulation of epidermal growth factor receptor phosphorylation in mice expressing different gangliosides.

    PubMed

    Daniotti, Jose L; Crespo, Pilar M; Yamashita, Tadashi

    2006-12-01

    We studied in this work the in vivo phosphorylation of the epidermal growth factor receptor (EGFr) in skin from knockout mice lacking different ganglioside glycosyltransferases. Results show an enhancement of EGFr phosphorylation, after EGF stimulation, in skin from Sial-T2 knockout and Sial-T2/GalNAc-T double knockout mice as compared with wild-type and Sial-T1 knockout mice. Qualitative analysis of ganglioside composition in mice skin suggest that the increase of EGFr phosphorylation observed in skin from Sial-T2 knockout and Sial-T2/GalNAc-T double knockout mice in response to EGF might not be primary attributed to the expression of GD3 or a-series gangliosides in mice skin. These studies provide, for the first time, an approach for studying the molecular mechanisms involved in the in vivo regulation of EGFr function by gangliosides. PMID:16817235

  6. Generation of myometrium-specific Bmal1 knockout mice for parturition analysis.

    PubMed

    Ratajczak, Christine K; Asada, Minoru; Allen, Gregg C; McMahon, Douglas G; Muglia, Lisa M; Smith, Donté; Bhattacharyya, Sandip; Muglia, Louis J

    2012-01-01

    Human and rodent studies indicate a role for circadian rhythmicity and associated clock gene expression in supporting normal parturition. The importance of clock gene expression in tissues besides the suprachiasmatic nucleus is emerging. Here, a Bmal1 conditional knockout mouse line and a novel Cre transgenic mouse line were used to examine the role of myometrial Bmal1 in parturition. Ninety-two percent (22/24) of control females but only 64% (14/22) of females with disrupted myometrial Bmal1 completed parturition during the expected time window of 5p.m. on Day 19 through to 9a.m. on Day 19.5 of gestation. However, neither serum progesterone levels nor uterine transcript expression of the contractile-associated proteins Connexin43 and Oxytocin receptor differed between females with disrupted myometrial Bmal1 and controls during late gestation. The data indicate a role for myometrial Bmal1 in maintaining normal time of day of parturition. PMID:22697126

  7. The 5-HT[subscript 3A] Receptor Is Essential for Fear Extinction

    ERIC Educational Resources Information Center

    Kondo, Makoto; Nakamura, Yukiko; Ishida, Yusuke; Yamada, Takahiro; Shimada, Shoichi

    2014-01-01

    The 5-HT [subscript 3] receptor, the only ionotropic 5-HT receptor, is expressed in limbic regions, including the hippocampus, amygdala, and cortex. However, it is not known whether it has a role in fear memory processes. Analysis of 5-HT [subscript 3A] receptor knockout mice in fear conditioning paradigms revealed that the 5-HT [subscript 3A]…

  8. Dopamine beta-hydroxylase knockout mice have alterations in dopamine signaling and are hypersensitive to cocaine.

    PubMed

    Schank, Jesse R; Ventura, Rossella; Puglisi-Allegra, Stefano; Alcaro, Antonio; Cole, Charlene D; Liles, L Cameron; Seeman, Philip; Weinshenker, David

    2006-10-01

    Multiple lines of evidence demonstrate that the noradrenergic system provides both direct and indirect excitatory drive onto midbrain dopamine (DA) neurons. We used DA beta-hydroxylase (DBH) knockout (Dbh-/-) mice that lack norepinephrine (NE) to determine the consequences of chronic NE deficiency on midbrain DA neuron function in vivo. Basal extracellular DA levels were significantly attenuated in the nucleus accumbens (NAc) and caudate putamen (CP), but not prefrontal cortex (PFC), of Dbh-/- mice, while amphetamine-induced DA release was absent in the NAc and attenuated in the CP and PFC. The decrease in dopaminergic tone was associated with a profound increase in the density of high-affinity state D1 and D2 DA receptors in the NAc and CP, while DA receptors in the PFC were relatively unaffected. As a behavioral consequence of these neurochemical changes, Dbh-/- mice were hypersensitive to the psychomotor, rewarding, and aversive effects of cocaine, as measured by locomotor activity and conditioned place preference. Antagonists of DA, but not 5-HT, receptors attenuated the locomotor hypersensitivity to cocaine in Dbh-/- mice. As DBH activity in humans is genetically controlled and the DBH inhibitor disulfiram has shown promise as a pharmacotherapy for cocaine dependence, these results have implications for the influence of genetic and pharmacological DBH inhibition on DA system function and drug addiction. PMID:16395294

  9. Trafficking defects in WASH-knockout fibroblasts originate from collapsed endosomal and lysosomal networks.

    PubMed

    Gomez, Timothy S; Gorman, Jacquelyn A; de Narvajas, Amaia Artal-Martinez; Koenig, Alexander O; Billadeau, Daniel D

    2012-08-01

    The Arp2/3-activator Wiskott-Aldrich syndrome protein and Scar homologue (WASH) is suggested to regulate actin-dependent membrane scission during endosomal sorting, but its cellular roles have not been fully elucidated. To investigate WASH function, we generated tamoxifen-inducible WASH-knockout mouse embryonic fibroblasts (WASHout MEFs). Of interest, although EEA1(+) endosomes were enlarged, collapsed, and devoid of filamentous-actin and Arp2/3 in WASHout MEFs, we did not observe elongated membrane tubules emanating from these disorganized endomembranes. However, collapsed WASHout endosomes harbored segregated subdomains, containing either retromer cargo recognition complex-associated proteins or EEA1. In addition, we observed global collapse of LAMP1(+) lysosomes, with some lysosomal membrane domains associated with endosomes. Both epidermal growth factor receptor (EGFR) and transferrin receptor (TfnR) exhibited changes in steady-state cellular localization. EGFR was directed to the lysosomal compartment and exhibited reduced basal levels in WASHout MEFs. However, although TfnR was accumulated with collapsed endosomes, it recycled normally. Moreover, EGF stimulation led to efficient EGFR degradation within enlarged lysosomal structures. These results are consistent with the idea that discrete receptors differentially traffic via WASH-dependent and WASH-independent mechanisms and demonstrate that WASH-mediated F-actin is requisite for the integrity of both endosomal and lysosomal networks in mammalian cells. PMID:22718907

  10. Basal and morphine-evoked dopaminergic neurotransmission in the nucleus accumbens of MOR- and DOR-knockout mice.

    PubMed

    Chefer, Vladimir I; Kieffer, Brigitte L; Shippenberg, Toni S

    2003-10-01

    Conventional and no net flux microdialysis were used to quantify basal and morphine-induced extracellular dopamine (DA) levels and the basal extraction fraction, which provides an estimate of the rate of DA uptake, in the nucleus accumbens (NAc) of wild-type mice and those with a constitutive deletion of mu (MOR)- or delta (DOR)-opioid receptors. Locomotor activity was assessed in these same animals. No difference between genotypes in basal dialysate DA levels was seen. No net flux studies revealed significant decreases in the DA extraction fraction in both MOR- and DOR-knockout mice, indicating decreased basal DA uptake in both genotypes. Extracellular DA, however, was unchanged. Because extracellular neurotransmitter levels are determined by the dynamics of both release and uptake, these findings provide suggestive evidence that basal DA release is decreased in mutant mice. Systemic administration of morphine significantly increased locomotor activity and dialysate DA levels in wild-type mice. MOR-knockout mice failed to exhibit a behavioural response to morphine. The ability of morphine to increase DA levels, however, was reduced but not prevented. No alteration in the effects of morphine was observed in DOR-knockout mice. These data provide genetic evidence for the existence of tonically active MOR and DOR systems that modulate basal DA neurotransmission in the NAc. Furthermore, they demonstrate that in contrast to the locomotor-activating effects of morphine, a small component of morphine-evoked DA release occurs independently of MOR activation. PMID:14622224

  11. Selective Photoreceptor Gene Knock-out Reveals a Regulatory Role for the Growth Behavior of Pseudomonas syringae.

    PubMed

    Shah, Rashmi; Pathak, Gopal; Drepper, Thomas; Gärtner, Wolfgang

    2016-07-01

    The plant pathogen Pseudomonas syringae (Ps) is a well-established model organism for bacterial infection of plants. The genome sequences of two pathovars, pv. syringae and pv. tomato, revealed one gene encoding a blue and two genes encoding red/far red light-sensing photoreceptors. Continuing former molecular characterization of the photoreceptor proteins, we here report selective photoreceptor gene disruption for pv. tomato aiming at identification of potentially regulatory functions of these photoreceptors. Transformation of Ps cells with linear DNA constructs yielded interposon mutations of the corresponding genes. Cell growth studies of the generated photoreceptor knock-out mutants revealed their role in light-dependent regulation of cell growth and motility. Disruption of the blue-light (BL) receptor gene caused a growth deregulation, in line with an observed increased virulence of this mutant (Moriconi et al., Plant J., 2013, 76, 322). Bacterial phytochrome-1 (BphP1) deletion mutant caused unaltered cell growth, but a stronger swarming capacity. Inactivation of its ortholog, BphP2, however, caused reduced growth and remarkably altered dendritic swarming behavior. Combined knock-out of both bacteriophytochromes reproduced the swarming pattern observed for the BphP2 mutant alone. A triple knock-out mutant showed a growth rate between that of the BL (deregulation) and the phytochrome-2 mutant (growth reduction). PMID:27289014

  12. Versatile strategy for isolating transcription activator-like effector nuclease-mediated knockout mutants in Caenorhabditis elegans.

    PubMed

    Sugi, Takuma; Sakuma, Tetsushi; Ohtani, Yasuko; Yamamoto, Takashi

    2014-01-01

    Targeted genome editing using transcription activator-like effector nuclease (TALEN) and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 systems has recently emerged as a potentially powerful method for creating locus-specific mutations in Caenorhabditis elegans. Due to the low mutation frequencies, one of the crucial steps in using these technologies is screening animals that harbor a targeted mutation. In previous studies, identifying targeted mutations in C. elegans usually depended on observations of fluorescent markers such as a green fluorescent protein or visible phenotypes such as dumpy and uncoordinated phenotypes. However, this strategy is limited in practice because the phenotypes caused by targeted mutations such as defects in sensory behaviors are often apparently invisible. Here, we describe a versatile strategy for isolating C. elegans knockout mutants by TALEN-mediated genome editing and a heteroduplex mobility assay. We applied TALENs to engineer the locus of the neural gene glr-1, which is a C. elegans AMPA-type receptor orthologue that is known to have crucial roles in various sensory behaviors. Knockout mutations in the glr-1 locus, which caused defective mechanosensory behaviors, were efficiently identified by the heteroduplex mobility assay. Thus, we demonstrated the utility of a TALEN-based knockout strategy for creating C. elegans with mutations that cause invisible phenotypes. PMID:24409999

  13. One-neutron knockout from 51-55 Sc

    NASA Astrophysics Data System (ADS)

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

    2012-12-01

    Results are presented from a one-neutron knockout experiment at relativistic energies of ≈ 420 A MeV on 51-55Sc 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 ν p_{1/2} , ν p_{3/2} , ( L = 1 and ν f_{7/2} , ν f_{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 ν f_{7/2} to the ν p_{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.

  14. A review of current large-scale mouse knockout efforts.

    PubMed

    Guan, Chunmei; Ye, Chao; Yang, Xiaomei; Gao, Jiangang

    2010-02-01

    After the successful completion of the human genome project (HGP), biological research in the postgenome era urgently needs an efficient approach for functional analysis of genes. Utilization of knockout mouse models has been powerful for elucidating the function of genes as well as finding new therapeutic interventions for human diseases. Gene trapping and gene targeting are two independent techniques for making knockout mice from embryonic stem (ES) cells. Gene trapping is high-throughput, random, and sequence-tagged while gene targeting enables the knockout of specific genes. It has been about 20 years since the first gene targeting and gene trapping mice were generated. In recent years, new tools have emerged for both gene targeting and gene trapping, and organizations have been formed to knock out genes in the mouse genome using either of the two methods. The knockout mouse project (KOMP) and the international gene trap consortium (IGTC) were initiated to create convenient resources for scientific research worldwide and knock out all the mouse genes. Organizers of KOMP regard it as important as the HGP. Gene targeting methods have changed from conventional gene targeting to high-throughput conditional gene targeting. The combined advantages of trapping and targeting elements are improving the gene trapping spectrum and gene targeting efficiency. As a newly-developed insertional mutation system, transposons have some advantages over retrovirus in trapping genes. Emergence of the international knockout mouse consortium (IKMP) is the beginning of a global collaboration to systematically knock out all the genes in the mouse genome for functional genomic research. PMID:20095055

  15. ROLE OF ESTROGEN RECEPTOR-α ON FOOD DEMAND ELASTICITY

    PubMed Central

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

    2016-01-01

    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. PMID:25869426

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

    PubMed

    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. PMID:25869426

  17. [From alcohol to liquid ecstasy (GHB)--a survey of old and modern knockout agents. Part 1: historic and classic knockout agents].

    PubMed

    Schütz, Harald; Jansen, Malin; Dettmeyer, Reinhard; Verhoff, Marcel A

    2011-01-01

    Alcohol has been the most important knockout drug in history and literature and continues to play an essential role up to now. Blunt force to the head in the form of a knockout punch is another mechanism leading to a transient loss of consciousness. Diethyl ether and chloroform are among the classical knockout substances. Although they have meanwhile been replaced by modern sedatives and hypnotics, their use is still observed in isolated cases. PMID:22039694

  18. Cortical Gene Expression After a Conditional Knockout of 67 kDa Glutamic Acid Decarboxylase in Parvalbumin Neurons.

    PubMed

    Georgiev, Danko; Yoshihara, Toru; Kawabata, Rika; Matsubara, Takurou; Tsubomoto, Makoto; Minabe, Yoshio; Lewis, David A; Hashimoto, Takanori

    2016-07-01

    In the cortex of subjects with schizophrenia, expression of glutamic acid decarboxylase 67 (GAD67), the enzyme primarily responsible for cortical GABA synthesis, is reduced in the subset of GABA neurons that express parvalbumin (PV). This GAD67 deficit is accompanied by lower cortical levels of other GABA-associated transcripts, including GABA transporter-1, PV, brain-derived neurotrophic factor (BDNF), tropomyosin receptor kinase B, somatostatin, GABAA receptor α1 subunit, and KCNS3 potassium channel subunit mRNAs. In contrast, messenger RNA (mRNA) levels for glutamic acid decarboxylase 65 (GAD65), another enzyme for GABA synthesis, are not altered. We tested the hypothesis that this pattern of GABA-associated transcript levels is secondary to the GAD67 deficit in PV neurons by analyzing cortical levels of these GABA-associated mRNAs in mice with a PV neuron-specific GAD67 knockout. Using in situ hybridization, we found that none of the examined GABA-associated transcripts had lower cortical expression in the knockout mice. In contrast, PV, BDNF, KCNS3, and GAD65 mRNA levels were higher in the homozygous mice. In addition, our behavioral test battery failed to detect a change in sensorimotor gating or working memory, although the homozygous mice exhibited increased spontaneous activities. These findings suggest that reduced GAD67 expression in PV neurons is not an upstream cause of the lower levels of GABA-associated transcripts, or of the characteristic behaviors, in schizophrenia. In PV neuron-specific GAD67 knockout mice, increased levels of PV, BDNF, and KCNS3 mRNAs might be the consequence of increased neuronal activity secondary to lower GABA synthesis, whereas increased GAD65 mRNA might represent a compensatory response to increase GABA synthesis. PMID:26980143

  19. What have we learned about GPER function in physiology and disease from knockout mice?

    PubMed

    Prossnitz, Eric R; Hathaway, Helen J

    2015-09-01

    Estrogens, predominantly 17β-estradiol, exert diverse effects throughout the body in both normal and pathophysiology, during development and in reproductive, metabolic, endocrine, cardiovascular, nervous, musculoskeletal and immune systems. Estrogen and its receptors also play important roles in carcinogenesis and therapy, particularly for breast cancer. In addition to the classical nuclear estrogen receptors (ERα and ERβ) that traditionally mediate predominantly genomic signaling, the G protein-coupled estrogen receptor GPER has become recognized as a critical mediator of rapid signaling in response to estrogen. Mouse models, and in particular knockout (KO) mice, represent an important approach to understand the functions of receptors in normal physiology and disease. Whereas ERα KO mice display multiple significant defects in reproduction and mammary gland development, ERβ KO phenotypes are more limited, and GPER KO exhibit no reproductive deficits. However, the study of GPER KO mice over the last six years has revealed that GPER deficiency results in multiple physiological alterations including obesity, cardiovascular dysfunction, insulin resistance and glucose intolerance. In addition, the lack of estrogen-mediated effects in numerous tissues of GPER KO mice, studied in vivo or ex vivo, including those of the cardiovascular, endocrine, nervous and immune systems, reveals GPER as a genuine mediator of estrogen action. Importantly, GPER KO mice have also demonstrated roles for GPER in breast carcinogenesis and metastasis. In combination with the supporting effects of GPER-selective ligands and GPER knockdown approaches, GPER KO mice demonstrate the therapeutic potential of targeting GPER activity in diseases as diverse as obesity, diabetes, multiple sclerosis, hypertension, atherosclerosis, myocardial infarction, stroke and cancer. PMID:26189910

  20. Pauli blocking and medium effects in nucleon knockout reactions

    SciTech Connect

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

    2010-06-15

    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{sub lab}=50-300 MeV/nucleon. The effect is more evident in reactions involving halo nuclei.

  1. Knockout driven reactions in complex molecules and their clusters

    NASA Astrophysics Data System (ADS)

    Gatchell, Michael; Zettergren, Henning

    2016-08-01

    Energetic ions lose some of their kinetic energy when interacting with electrons or nuclei in matter. Here, we discuss combined experimental and theoretical studies on such impulse driven reactions in polycyclic aromatic hydrocarbons (PAHs), fullerenes, and pure or mixed clusters of these molecules. These studies show that the nature of excitation is important for how complex molecular systems respond to ion/atom impact. Rutherford-like nuclear scattering processes may lead to prompt atom knockout and formation of highly reactive fragments, while heating of the molecular electron clouds in general lead to formation of more stable and less reactive fragments. In this topical review, we focus on recent studies of knockout driven reactions, and present new calculations of the angular dependent threshold (displacement) energies for such processes in PAHs. The so-formed fragments may efficiently form covalent bonds with neighboring molecules in clusters. These unique molecular growth processes may be important in astrophysical environments such as low velocity shock waves.

  2. Human Genetic Disorders and Knockout Mice Deficient in Glycosaminoglycan

    PubMed Central

    2014-01-01

    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. PMID:25126564

  3. Creation and Preliminary Characterization of a Leptin Knockout Rat

    PubMed Central

    Vaira, Sergio; Yang, Chang; McCoy, Aaron; Keys, Kelly; Xue, Shurong; Weinstein, Edward J.; Novack, Deborah V.

    2012-01-01

    Leptin, a cytokine-like hormone secreted mainly by adipocytes, regulates various pathways centered on food intake and energy expenditure, including insulin sensitivity, fertility, immune system, and bone metabolism. Here, using zinc finger nuclease technology, we created the first leptin knockout rat. Homozygous leptin null rats are obese with significantly higher serum cholesterol, triglyceride, and insulin levels than wild-type controls. Neither gender produced offspring despite of repeated attempts. The leptin knockout rats also have depressed immune system. In addition, examination by microcomputed tomography of the femurs of the leptin null rats shows a significant increase in both trabecular bone mineral density and bone volume of the femur compared with wild-type littermates. Our model should be useful for many different fields of studies, such as obesity, diabetes, and bone metabolism-related illnesses. PMID:22948215

  4. Targeted gene knockout in chickens mediated by TALENs.

    PubMed

    Park, Tae Sub; Lee, Hong Jo; Kim, Ki Hyun; Kim, Jin-Soo; Han, Jae Yong

    2014-09-01

    Genetically modified animals are used for industrial applications as well as scientific research, and studies on these animals contribute to a better understanding of biological mechanisms. Gene targeting techniques have been developed to edit specific gene loci in the genome, but the conventional strategy of homologous recombination with a gene-targeted vector has low efficiency and many technical complications. Here, we generated specific gene knockout chickens through the use of transcription activator-like effector nuclease (TALEN)-mediated gene targeting. In this study, we accomplished targeted knockout of the ovalbumin (OV) gene in the chicken primordial germ cells, and OV gene mutant offspring were generated through test-cross analysis. TALENs successfully induced nucleotide deletion mutations of ORF shifts, resulting in loss of chicken OV gene function. Our results demonstrate that the TALEN technique used in the chicken primordial germ cell line is a powerful strategy to create specific genome-edited chickens safely for practical applications. PMID:25139993

  5. Targeted gene knockout in chickens mediated by TALENs

    PubMed Central

    Park, Tae Sub; Lee, Hong Jo; Kim, Ki Hyun; Kim, Jin-Soo; Han, Jae Yong

    2014-01-01

    Genetically modified animals are used for industrial applications as well as scientific research, and studies on these animals contribute to a better understanding of biological mechanisms. Gene targeting techniques have been developed to edit specific gene loci in the genome, but the conventional strategy of homologous recombination with a gene-targeted vector has low efficiency and many technical complications. Here, we generated specific gene knockout chickens through the use of transcription activator-like effector nuclease (TALEN)-mediated gene targeting. In this study, we accomplished targeted knockout of the ovalbumin (OV) gene in the chicken primordial germ cells, and OV gene mutant offspring were generated through test-cross analysis. TALENs successfully induced nucleotide deletion mutations of ORF shifts, resulting in loss of chicken OV gene function. Our results demonstrate that the TALEN technique used in the chicken primordial germ cell line is a powerful strategy to create specific genome-edited chickens safely for practical applications. PMID:25139993

  6. Designer Nuclease-Mediated Generation of Knockout THP1 Cells.

    PubMed

    Schmidt, Tobias; Schmid-Burgk, Jonathan L; Ebert, Thomas S; Gaidt, Moritz M; Hornung, Veit

    2016-01-01

    Recent developments in the field of designer nucleases allow the efficient and specific manipulation of genomic architectures in eukaryotic cell lines. To this end, it has become possible to introduce DNA double strand breaks (DSBs) at user-defined genomic loci. If located in critical coding regions of genes, thus induced DSBs can lead to insertions or deletions (indels) that result in frameshift mutations and thereby the knockout of the target gene. In this chapter, we describe a step-by-step workflow for establishing knockout cell clones of the difficult-to-transfect suspension cell line THP1. The here described protocol encompasses electroporation, cell cloning, and a deep sequencing-based genotyping step that allows the in-parallel analysis of 96 cell clones per gene of interest. Furthermore, we describe the use of the analysis tool OutKnocker that allows rapid identification of cell clones with all-allelic frameshift mutations. PMID:26443227

  7. Murine mentors: transgenic and knockout models of surgical disease.

    PubMed Central

    Arbeit, J M; Hirose, R

    1999-01-01

    OBJECTIVE: Transgenic and knockout technologies have emerged from the "molecular biology revolution" as unprecedented techniques for manipulating gene function in intact mice. The goals of this review are to outline the techniques of creating transgenic and knockout mice, and to demonstrate their use in elucidation of the molecular mechanisms underlying common surgical diseases. SUMMARY BACKGROUND DATA: Gain of gene function is created by transgenic technology, whereas gene function is ablated using gene knockouts. Each technique has distinctive applications and drawbacks. A unique feature of genetically manipulated mice is that combinatorial genetic experiments can be executed that precisely define the functional contribution of a gene to disease progression. Transgenic and knockout mouse models of wound healing, cardiovascular disease, transplant immunology, gut motility and inflammatory bowel disease, and oncology are beginning to illuminate the precise molecular regulation of these diseases. Transgenic technology has also been extended to larger mammals such as pigs, with the goal of using genetic manipulation of the xenogenic immune response to increase the availability of transplant organs. Continual refinements in gene manipulation technology in mice offer the opportunity to turn genes on or off at precise time intervals and in particular tissues, according to the needs of the investigator. Ultimately, investigation of disease development and progression in genetically manipulated mammals may delineate new molecular targets for drug discovery and provide novel platforms for drug efficacy screens. CONCLUSIONS: Emulation of human disease and therapy using genetically manipulated mammals fulfills a promise of molecular medicine: fusion of molecular biochemistry with "classical" biology and physiology. Surgeons have unique skills spanning both worlds that can facilitate their success in this expanding arena. PMID:9923797

  8. Generation of Gene Knockout Mice by ES Cell Microinjection

    PubMed Central

    Longenecker, Glenn; Kulkarni, Ashok B

    2009-01-01

    This unit lists and describes protocols used in the production of chimeric mice leading to the generation of gene knockout mice. These protocols include the collection of blastocyst embryos, ES cell injection, and uterine transfer of injected blastocysts. Support protocols in the superovulation of blastocyst donor mice, generation of pseudopregnant recipients, fabrication of glass pipettes, and generation of germline mice are also included. Practical tips and solutions are mentioned to help troubleshoot problems that may occur. PMID:19731226

  9. Serotonin receptor expression is dynamic in the liver during the transition period in Holstein dairy cows.

    PubMed

    Laporta, J; Hernandez, L L

    2015-04-01

    Nonneuronal serotonin (5-HT) participates in glucose metabolism, but little is known regarding the actions of 5-HT in the liver during the transition period in dairy cattle. Here, we explore circulating patterns of 5-HT and characterize the hepatic 5-HT receptor and glucose transporter profiles around calving in multiparous Holstein dairy cows (n = 6, average lactation = 4 ± 1.9). Concentrations of serum 5-HT decreased on day -3 compared with -5 and -7 precalving (167.7 ± 80 vs 1511.1 ± 602 ng/mL). 5-HT nadir was on day -1 precalving and remained low postcalving (481.4 ± 49 ng/mL). Plasma glucose concentrations decreased precalving (P = 0.008) and were positively correlated with 5-HT during the precalving period (r = 0.55, P = 0.043). On day 1, postcalving hepatic messenger RNA expression of 5-HT1D, 2B, 3C, 6, and 7 receptors were decreased compared with day -7 (P < 0.048). The 5-HT3A and 5-HT3B decreased on day 7. The 5-HT2A increased on days 1 and 7 compared with -7 (P < 0.05). The 5-HT1F and 5-HT1A receptors were increased 2.5- and 3.8-fold on day 7, respectively, compared with days -7 and 1 (P < 0.046). The 5-HT5A was not detected, and 5-HT4 was detected on days -7 and 1 only. Expression of Glut-2,-5 and SGLT1 were decreased on days 1 and 7 compared with -7 (P < 0.05), whereas Glut-1 was increased on day 7 compared with -7 (P < 0.05). These results indicate that 5-HT could be important for liver glucose homeostasis possibly through receptor mediated signaling at specific times. Additional research is needed to further explore the functional role of these receptors in the liver during the transition from pregnancy to lactation. PMID:25528206

  10. The evolution of thymic lymphomas in p53 knockout mice.

    PubMed

    Dudgeon, Crissy; Chan, Chang; Kang, Wenfeng; Sun, Yvonne; Emerson, Ryan; Robins, Harlan; Levine, Arnold J

    2014-12-01

    Germline deletion of the p53 gene in mice gives rise to spontaneous thymic (T-cell) lymphomas. In this study, the p53 knockout mouse was employed as a model to study the mutational evolution of tumorigenesis. The clonality of the T-cell repertoire from p53 knockout and wild-type thymic cells was analyzed at various ages employing TCRβ sequencing. These data demonstrate that p53 knockout thymic lymphomas arose in an oligoclonal fashion, with tumors evolving dominant clones over time. Exon sequencing of tumor DNA revealed that all of the independently derived oligoclonal mouse tumors had a deletion in the Pten gene prior to the formation of the TCRβ rearrangement, produced early in development. This was followed in each independent clone of the thymic lymphoma by the amplification or overexpression of cyclin Ds and Cdk6. Alterations in the expression of Ikaros were common and blocked further development of CD-4/CD-8 T cells. While the frequency of point mutations in the genome of these lymphomas was one per megabase, there were a tremendous number of copy number variations producing the tumors' driver mutations. The initial inherited loss of p53 functions appeared to delineate an order of genetic alterations selected for during the evolution of these thymic lymphomas. PMID:25452272

  11. Knock-out models reveal new aquaporin functions.

    PubMed

    Verkman, Alan S

    2009-01-01

    Knockout mice have been informative in the discovery of unexpected biological functions of aquaporins. Knockout mice have confirmed the predicted roles of aquaporins in transepithelial fluid transport, as in the urinary concentrating mechanism and glandular fluid secretion. A less obvious, though predictable role of aquaporins is in tissue swelling under stress, as in the brain in stroke, tumor and infection. Phenotype analysis of aquaporin knockout mice has revealed several unexpected cellular roles of aquaporins whose mechanisms are being elucidated. Aquaporins facilitate cell migration, as seen in aquaporin-dependent tumor angiogenesis and tumor metastasis, by a mechanism that may involve facilitated water transport in lamellipodia of migrating cells. The ' aquaglyceroporins', aquaporins that transport both glycerol and water, regulate glycerol content in epidermis, fat and other tissues, and lead to a multiplicity of interesting consequences of gene disruption including dry skin, resistance to skin carcinogenesis, impaired cell proliferation and altered fat metabolism. An even more surprising role of a mammalian aquaporin is in neural signal transduction in the central nervous system. The many roles of aquaporins might be exploited for clinical benefit by modulation of aquaporin expression/function - as diuretics, and in the treatment of brain swelling, glaucoma, epilepsy, obesity and cancer. PMID:19096787

  12. The GABAA receptor is an FMRP target with therapeutic potential in fragile X syndrome

    PubMed Central

    Braat, Sien; D'Hulst, Charlotte; Heulens, Inge; De Rubeis, Silvia; Mientjes, Edwin; Nelson, David L; Willemsen, Rob; Bagni, Claudia; Van Dam, Debby; De Deyn, Peter P; Kooy, R Frank

    2015-01-01

    Previous research indicates that the GABAAergic system is involved in the pathophysiology of the fragile X syndrome, a frequent form of inherited intellectual disability and associated with autism spectrum disorder. However, the molecular mechanism underlying GABAAergic deficits has remained largely unknown. Here, we demonstrate reduced mRNA expression of GABAA receptor subunits in the cortex and cerebellum of young Fmr1 knockout mice. In addition, we show that the previously reported underexpression of specific subunits of the GABAA receptor can be corrected in YAC transgenic rescue mice, containing the full-length human FMR1 gene in an Fmr1 knockout background. Moreover, we demonstrate that FMRP directly binds several GABAA receptor mRNAs. Finally, positive allosteric modulation of GABAA receptors with the neurosteroid ganaxolone can modulate specific behaviors in Fmr1 knockout mice, emphasizing the therapeutic potential of the receptor. PMID:25790165

  13. The GABAA receptor is an FMRP target with therapeutic potential in fragile X syndrome.

    PubMed

    Braat, Sien; D'Hulst, Charlotte; Heulens, Inge; De Rubeis, Silvia; Mientjes, Edwin; Nelson, David L; Willemsen, Rob; Bagni, Claudia; Van Dam, Debby; De Deyn, Peter P; Kooy, R Frank

    2015-01-01

    Previous research indicates that the GABAAergic system is involved in the pathophysiology of the fragile X syndrome, a frequent form of inherited intellectual disability and associated with autism spectrum disorder. However, the molecular mechanism underlying GABAAergic deficits has remained largely unknown. Here, we demonstrate reduced mRNA expression of GABAA receptor subunits in the cortex and cerebellum of young Fmr1 knockout mice. In addition, we show that the previously reported underexpression of specific subunits of the GABAA receptor can be corrected in YAC transgenic rescue mice, containing the full-length human FMR1 gene in an Fmr1 knockout background. Moreover, we demonstrate that FMRP directly binds several GABAA receptor mRNAs. Finally, positive allosteric modulation of GABAA receptors with the neurosteroid ganaxolone can modulate specific behaviors in Fmr1 knockout mice, emphasizing the therapeutic potential of the receptor. PMID:25790165

  14. Direct interaction of the resistance to inhibitors of cholinesterase type 3 protein with the serotonin receptor type 3A intracellular domain.

    PubMed

    Nishtala, Sita Nirupama; Mnatsakanyan, Nelli; Pandhare, Akash; Leung, Chun; Jansen, Michaela

    2016-05-01

    Pentameric ligand-gated ion channels (pLGIC) are expressed in both excitable and non-excitable cells that are targeted by numerous clinically used drugs. Assembly from five identical or homologous subunits yields homo- or heteromeric pentamers, respectively. The protein known as Resistance to Inhibitors of Cholinesterase (RIC-3) was identified to interfere with assembly and functional maturation of pLGICs. We have shown previously for serotonin type 3A homopentamers (5-HT3A ) that the interaction with RIC-3 requires the intracellular domain (ICD) of this pLGIC. After expression in Xenopus laevis oocytes RIC-3 attenuated serotonin-induced currents in 5-HT3A wild-type channels, but not in functional 5-HT3A glvM3M4 channels that have the 115-amino acid ICD replaced by a heptapeptide. In complementary experiments we have shown that engineering the Gloeobacter violaceus ligand-gated ion channel (GLIC) to contain the 5-HT3A -ICD confers sensitivity to RIC-3 in oocytes to otherwise insensitive GLIC. In this study, we identify endogenous RIC-3 protein expression in X. laevis oocytes. We purified RIC-3 to homogeneity after expression in Echericia coli. By using heterologously over-expressed and purified RIC-3 and the chimera consisting of the 5-HT3A -ICD and the extracellular and transmembrane domains of GLIC in pull-down experiments, we demonstrate a direct and specific interaction between the two proteins. This result further underlines that the domain within 5-HT3 A R that mediates the interaction with RIC-3 is the ICD. Importantly, this is the first experimental evidence that the interaction between 5-HT3 A R-ICD and RIC-3 does not require other proteins. In addition, we demonstrate that the pentameric assembly of the GLIC-5-HT3A -ICD chimera interacts with RIC-3. We hypothesized that pentameric ligand-gated ion channels (pLGICs) associate directly with the chaperone protein RIC-3 (resistance to inhibitors of cholinesterase type 3), and that the interaction does not

  15. Impaired extinction of learned contextual fear memory in early growth response 1 knockout mice.

    PubMed

    Han, Seungrie; Hong, Soontaek; Mo, Jiwon; Lee, Dongmin; Choi, Eunju; Choi, June-seek; Sun, Woong; Lee, Hyun Woo; Kim, Hyun

    2014-01-01

    Inductive expression of early growth response 1 (Egr-1) in neurons is associated with many forms of neuronal activity. However, only a few Egr-1 target genes are known in the brain. The results of this study demonstrate that Egr-1 knockout (KO) mice display impaired contextual extinction learning and normal fear acquisition relative to wild-type (WT) control animals. Genome-wide microarray experiments revealed 368 differentially expressed genes in the hippocampus of Egr-1 WT exposed to different phases of a fear conditioning paradigm compared to gene expression profiles in the hippocampus of KO mice. Some of genes, such as serotonin receptor 2C (Htr2c), neuropeptide B (Npb), neuronal PAS domain protein 4 (Npas4), NPY receptor Y1 (Npy1r), fatty acid binding protein 7 (Fabp7), and neuropeptide Y (Npy) are known to regulate processing of fearful memories, and promoter analyses demonstrated that several of these genes contained Egr-1 binding sites. This study provides a useful list of potential Egr-1 target genes which may be regulated during fear memory processing. PMID:24552706

  16. The effects of cannabinoids on contextual conditioned fear in CB1 knockout and CD1 mice.

    PubMed

    Mikics, Eva; Dombi, Timea; Barsvári, Beáta; Varga, Balázs; Ledent, Catherine; Freund, Tamás F; Haller, József

    2006-05-01

    We studied the effects of cannabinoids on contextual conditioned fear responses. CB1 knockout and wild-type (CD1) mice were exposed to a brief session of electric shocks, and their behavior was studied in the same context 24 h later. In wild-type mice, shock exposure increased freezing and resting, and decreased locomotion and exploration. The genetic disruption of the CB1 receptor abolished the conditioned fear response. The CB1 antagonist AM-251 reduced the peak of the conditioned fear response when applied 30 min before behavioral testing (i.e. 24 h after shocks) in CD1 (wild-type) mice. The cannabinoid agonist WIN-55,212-2 markedly increased the conditioned fear response in CD1 mice, the effect of which was potently antagonized by AM-251. Thus, cannabinoid receptor activation appears to strongly promote the expression of contextual conditioned fear. In earlier experiments, cannabinoids did not interfere with the expression of cue-induced conditioned fear but strongly promoted its extinction. Considering the primordial role of the amygdala in simple associative learning (e.g. in cue-induced fear) and the role of the hippocampus in learning more complex stimulus relationships (e.g. in contextual fear), the present and earlier findings are not necessarily contradictory, but suggest that cannabinoid signaling plays different roles in the two structures. Data are interpreted in terms of the potential involvement of cannabinoids in trauma-induced behavioral changes. PMID:16572000

  17. Behavioural and neuroplastic properties of chronic lurasidone treatment in serotonin transporter knockout rats.

    PubMed

    Luoni, Alessia; Hulsken, Sjoerd; Cazzaniga, Greta; Racagni, Giorgio; Homberg, Judith R; Riva, Marco A

    2013-07-01

    Second-generation antipsychotics (SGA) are multi-target agents widely used for the treatment of schizophrenia and bipolar disorder that also hold potential for the treatment of impaired emotional control, thanks to their diverse receptor profiles as well as their potential in modulating neuroadaptive changes in key brain regions. The aim of this study was thus to establish the ability of lurasidone, a novel SGA characterized by a multi-receptor signature, to modulate behavioural and molecular defects associated with a genetic model of impaired emotional control, namely serotonin transporter knockout (SERT KO) rats. At behavioural level, we found that chronic lurasidone treatment significantly increased fear extinction in SERT KO rats, but not in wild-type control animals. Moreover, at molecular level, lurasidone was able to normalize the reduced expression of the neurotrophin brain-derived neurotrophic factor in the prefrontal cortex of SERT KO rats, an effect that occurred through the regulation of specific neurotrophin transcripts (primarily exon VI). Furthermore, chronic lurasidone treatment was also able to restore the reduced expression of different GABAergic markers that is present in these animals. Our results show that lurasidone can improve emotional control in SERT KO rats, with a primary impact on the prefrontal cortex. The adaptive changes set in motion by repeated treatment with lurasidone may in fact contribute to the amelioration of functional capacities, closely associated with neuronal plasticity, which are deteriorated in patients with schizophrenia, bipolar disease and major depression. PMID:23164505

  18. Impaired motor coordination and disrupted cerebellar architecture in Fgfr1 and Fgfr2 double knockout mice

    PubMed Central

    Smith, Karen Müller; Williamson, Theresa L.; Schwartz, Michael L.

    2012-01-01

    Fibroblast growth factor receptor (FGFR) signaling determines the size of the cerebral cortex by regulating the amplification of radial glial stem cells, and participates in the formation of midline glial structures. We show that Fgfr1 and Fgfr2 double knockouts (FGFR DKO) generated by Cre mediated recombination driven by the human GFAP promoter (hGFAP) have reduced cerebellar size due to reduced proliferation of radial glia and other glial precursors in late embryonic and neonatal FGFR DKO mice. The proliferation of granule cell progenitors (GCPs) in the EGL was also reduced, leading to reduced granule cell numbers. Furthermore, both inward migration of granule cells into the inner granule cell layer (IGL) and outward migration of GABA interneurons into the molecular layer (ML) were arrested, disrupting layer and lobular morphology. Purkinje neurons and their dendrites, which were not targeted by Cre mediated recombination of Fgf receptors, were also misplaced in FGFR DKO mice, possibly as a consequence of altered Bergmann glia orientation or reduced granule cell number. Our findings indicate a dual role for FGFR signaling in cerebellar morphogenesis. The first role is to amplify the number of granule neuron precursors in the external granular layer and glial precursor cells throughout the cerebellum. The second is to establish the correct Bergmann glia morphology, which is crucial for granule cell migration. The disrupted cerebellar size and laminar architecture resulting from loss of FGFR signaling impairs motor learning and coordination in FGFR DKO mice. PMID:22578469

  19. Age-dependent neonatal intracerebral hemorrhage in plasminogen activator inhibitor 1 knockout mice.

    PubMed

    Leroux, Philippe; Omouendze, Priscilla L; Roy, Vincent; Dourmap, Nathalie; Gonzalez, Bruno J; Brasse-Lagnel, Carole; Carmeliet, Peter; Leroux-Nicollet, Isabelle; Marret, Stéphane

    2014-05-01

    Intracerebral-intraventricular hemorrhages (ICH/IVH) in very preterm neonates are responsible for high mortality and subsequent disabilities. In humans, tissue plasminogen activator (t-PA) initiates fibrinolysis and activates endoluminal-endothelial receptors; dysfunction of the t-PA inhibitor (PAI-1) results in recurrent hemorrhages. We used PAI-1 knockout (PAI-1) mice to examine the role of t-PA in age-dependent intracranial hemorrhages as a possible model of preterm ICH/IVH. Intracortical injection of 2 μL of phosphate-buffered saline produced a small traumatic injury and a high rate of hemorrhage in PAI-1 pups at postnatal day 3 (P3) or P5, whereas it had no effect in wild-type neonates. This resulted in white matter and cortical lesions, ventricle enlargement, hyperlocomotion, and altered cortical levels of serotonin and dopamine in the adult PAI mice. N-methyl-D-aspartate receptor blockers, plasmin- and matrix metalloproteinases inhibitors reduced hemorrhage and tissue lesions. In contrast to P3 to P5, no significant hemorrhages were induced in P10 PAI-1 pups and there were no behavioral or neurochemical alterations in adulthood. These data suggest that microvascular immaturity up to P5 in mice is a determinant factor required for t-PA-dependent vascular rupture. Neonatal PAI-1 mice could be a useful ICH/IVH model for studying the ontogenic window of vascular immaturity and vascular protection against later neurodisabilities. PMID:24709679

  20. Hyperaldosteronism after decreased renal K+ excretion in KCNMB2 knockout mice.

    PubMed

    Larsen, Casper K; Jensen, Iben S; Sorensen, Mads V; de Bruijn, Pauline I; Bleich, Markus; Praetorius, Helle A; Leipziger, Jens

    2016-05-15

    The kidney is the primary organ ensuring K(+) homeostasis. K(+) is secreted into the urine in the distal tubule by two mechanisms: by the renal outer medullary K(+) channel (Kir1.1) and by the Ca(2+)-activated K(+) channel (KCa1.1). Here, we report a novel knockout mouse of the β2-subunit of the KCa1.1 channel (KCNMB2), which displays hyperaldosteronism after decreased renal K(+) excretion. KCNMB2(-/-) mice displayed hyperaldosteronism, normal plasma K(+) concentration, and produced dilute urine with decreased K(+) concentration. The normokalemia indicated that hyperaldosteronism did not result from primary aldosteronism. Activation of the renin-angiotensin-aldosterone system was also ruled out as renal renin mRNA expression was reduced in KCNMB2(-/-) mice. Renal K(+) excretion rates were similar in the two genotypes; however, KCNMB2(-/-) mice required elevated plasma aldosterone to achieve K(+) balance. Blockade of the mineralocorticoid receptor with eplerenone triggered mild hyperkalemia and unmasked reduced renal K(+) excretion in KCNMB2(-/-) mice. Knockout mice for the α-subunit of the KCa1.1 channel (KCNMA1(-/-) mice) have hyperaldosteronism, are hypertensive, and lack flow-induced K(+) secretion. KCNMB2(-/-) mice share the phenotypic traits of normokalemia and hyperaldosteronism with KCNMA1(-/-) mice but were normotensive and displayed intact flow-induced K(+) secretion. Despite elevated plasma aldosterone, KNCMB2(-/-) mice did not display salt-sensitive hypertension and were able to decrease plasma aldosterone on a high-Na(+) diet, although plasma aldosterone remained elevated in KCNMB2(-/-) mice. In summary, KCNMB2(-/-) mice have a reduced ability to excrete K(+) into the urine but achieve K(+) balance through an aldosterone-mediated, β2-independent mechanism. The phenotype of KCNMB2 mice was similar but milder than the phenotype of KCNMA1(-/-) mice. PMID:26962098

  1. trkA modulation of developing somatosensory neurons in oro-facial tissues: tooth pulp fibers are absent in trkA knockout mice.

    PubMed

    Matsuo, S; Ichikawa, H; Henderson, T A; Silos-Santiago, I; Barbacid, M; Arends, J J; Jacquin, M F

    2001-01-01

    To investigate the nerve growth factor requirement of developing oro-facial somatosensory afferents, we have studied the survival of sensory fibers subserving nociception, mechanoreception or proprioception in receptor tyrosine kinase (trkA) knockout mice using immunohistochemistry. trkA receptor null mutant mice lack nerve fibers in tooth pulp, including sympathetic fibers, and showed only sparse innervation of the periodontal ligament. Ruffini endings were formed definitively in the periodontal ligament of the trkA knockout mice, although calcitonin gene-related peptide- and substance P-immunoreactive fibers were reduced in number or had disappeared completely. trkA gene deletion had also no obvious effect on the formation of Meissner corpuscles in the palate. In the vibrissal follicle, however, some mechanoreceptive afferents were sensitive for trkA gene deletion, confirming a previous report [Fundin et al. (1997) Dev. Biol. 190, 94-116]. Moreover, calretinin-positive fibers innervating longitudinal lanceolate endings were completely lost in trkA knockout mice, as were the calretinin-containing parent cells in the trigeminal ganglion.These results indicate that trkA is indispensable for developing nociceptive neurons innervating oral tissues, but not for developing mechanoreceptive neurons innervating oral tissues (Ruffini endings and Meissner corpuscles), and that calretinin-containing, trkA dependent neurons in the trigeminal ganglion normally participate in mechanoreception through longitudinal lanceolate endings of the vibrissal follicle. PMID:11516838

  2. Alterations of Colonic Contractility in an Interleukin-10 Knockout Mouse Model of Inflammatory Bowel Disease

    PubMed Central

    Park, Jae Hyung; Kwon, Joong Goo; Kim, Sun Joo; Song, Dae Kyu; Lee, Seok Guen; Kim, Eun Su; Cho, Kwang Bum; Jang, Byung Ik; Kim, Dae Hwan; Sin, Jeong-Im; Kim, Tae Wan; Song, In Hwan; Park, Kyung Sik

    2015-01-01

    Background/Aims Inflammatory bowel disease is commonly accompanied by colonic dysmotility and causes changes in intestinal smooth muscle contractility. In this study, colonic smooth muscle contractility in a chronic inflammatory condition was investigated using smooth muscle tissues prepared from interleukin-10 knockout (IL-10−/−) mice. Methods Prepared smooth muscle sections were placed in an organ bath system. Cholinergic and nitrergic neuronal responses were observed using carbachol and electrical field stimulation with L-NG-nitroarginine methyl ester (L-NAME). The expression of interstitial cells of Cajal (ICC) networks, muscarinic receptors, neuronal nitric oxide synthase (nNOS) and inducible nitric oxide synthase (iNOS) was observed via immunofluorescent staining. Results The spontaneous contractility and expression of ICC networks in the proximal and distal colon was significantly decreased in IL-10−/− mice compared to IL-10+/+ mice. The contractility in response to carbachol was significantly decreased in the proximal colon of IL-10−/− mice compared to IL-10+/+ mice, but no significant difference was found in the distal colon. In addition, the expression of muscarinic receptor type 2 was reduced in the proximal colon of IL-10−/− mice. The nictric oxide-mediated relaxation after electrical field stimulation was significantly decreased in the proximal and distal colon of IL-10−/− mice. In inflamed colon, the expression of nNOS decreased, whereas the expression of iNOS increased. Conclusions These results suggest that damage to the ICC network and NOS system in the proximal and distal colon, as well as damage to the smooth muscle cholinergic receptor in the proximal colon may play an important role in the dysmotility of the inflamed colon. PMID:25537671

  3. Brain-derived neurotrophic factor signaling is altered in the forebrain of Engrailed-2 knockout mice.

    PubMed

    Zunino, G; Messina, A; Sgadò, P; Baj, G; Casarosa, S; Bozzi, Y

    2016-06-01

    Engrailed-2 (En2), a homeodomain transcription factor involved in regionalization and patterning of the midbrain and hindbrain regions has been associated to autism spectrum disorders (ASDs). En2 knockout (En2(-/-)) mice show ASD-like features accompanied by a significant loss of GABAergic subpopulations in the hippocampus and neocortex. Brain-derived neurotrophic factor (BDNF) is a crucial factor for the postnatal development of forebrain GABAergic neurons, and altered GABA signaling has been hypothesized to underlie the symptoms of ASD. Here we sought to determine whether interneuron loss in the En2(-/-) forebrain might be related to altered expression of BDNF and its signaling receptors. We first evaluated the expression of different BDNF mRNA isoforms in the neocortex and hippocampus of wild-type (WT) and En2(-/-) mice. Quantitative RT-PCR showed a marked down-regulation of several splicing variants of BDNF mRNA in the neocortex but not hippocampus of adult En2(-/-) mice, as compared to WT controls. Accordingly, levels of mature BDNF protein were lower in the neocortex but not hippocampus of En2(-/-) mice, as compared to WT. Increased levels of phosphorylated TrkB and decreased levels of p75 receptor were also detected in the neocortex of mutant mice. Accordingly, the expression of low density lipoprotein receptor (LDLR) and RhoA, two genes regulated via p75 was significantly altered in forebrain areas of mutant mice. These data indicate that BDNF signaling alterations might be involved in the anatomical changes observed in the En2(-/-) forebrain and suggest a pathogenic role of altered BDNF signaling in this mouse model of ASD. PMID:26987954

  4. Histamine H3 Receptor Regulates Sensorimotor Gating and Dopaminergic Signaling in the Striatum.

    PubMed

    Kononoff Vanhanen, Jenni; Nuutinen, Saara; Tuominen, Mervi; Panula, Pertti

    2016-05-01

    The brain histamine system has been implicated in regulation of sensorimotor gating deficits and in Gilles de la Tourette syndrome. Histamine also regulates alcohol reward and consumption via H3 receptor (H3R), possibly through an interaction with the brain dopaminergic system. Here, we identified the histaminergic mechanism of sensorimotor gating and the role of histamine H3R in the regulation of dopaminergic signaling. We found that H3R knockout mice displayed impaired prepulse inhibition (PPI), indicating deficiency in sensorimotor gating. Histamine H1 receptor knockout and histidine decarboxylase knockout mice had similar PPI as their controls. Dopaminergic drugs increased PPI of H3R knockout mice to the same level as in control mice, suggesting that changes in dopamine receptors might underlie deficient PPI response when H3R is lacking. Striatal dopamine D1 receptor mRNA level was lower, and D1 and D2 receptor-mediated activation of extracellular signal-regulated kinase 1/2 was absent in the striatum of H3R knockout mice, suggesting that H3R is essential for the dopamine receptor-mediated signaling. In conclusion, these findings demonstrate that H3R is an important regulator of sensorimotor gating, and the lack of H3R significantly modifies striatal dopaminergic signaling. These data support the usefulness of H3R ligands in neuropsychiatric disorders with preattentional deficits and disturbances in dopaminergic signaling. PMID:26945087

  5. Inter-residue coupling contributes to high-affinity subtype-selective binding of α-bungarotoxin to nicotinic receptors

    PubMed Central

    Sine, Steven M.; Huang, Sun; Li, Shu-Xing; daCOSTA, Corrie J. B.; Chen, Lin

    2014-01-01

    The crystal structure of a pentameric α7 ligand-binding domain chimaera with bound α-btx (α-bungarotoxin) showed that of the five conserved aromatic residues in α7, only Tyr184 in loop C of the ligand-binding site was required for high-affinity binding. To determine whether the contribution of Tyr184 depends on local residues, we generated mutations in an α7/5HT3A (5-hydroxytryptamine type 3A) receptor chimaera, individually and in pairs, and measured 125I-labelled α-btx binding. The results show that mutations of individual residues near Tyr184 do not affect α-btx affinity, but pairwise mutations decrease affinity in an energetically coupled manner. Kinetic measurements show that the affinity decreases arise through increases in the α-btx dissociation rate with little change in the association rate. Replacing loop C in α7 with loop C from the α-btx-insensitive α2 or α3 subunits abolishes high-affinity α-btx binding, but preserves acetylcholine-elicited single channel currents. However, in both the α2 and α3 construct, mutating either residue that flanks Tyr184 to its α7 counterpart restores high-affinity α-btx binding. Analogously, in α7, mutating both residues that flank Tyr184 to the α2 or α3 counterparts abolishes high-affinity α-btx binding. Thus interaction between Tyr184 and local residues contributes to high-affinity subtype-selective α-btx binding. PMID:23802200

  6. Guwiyang Wurra--'Fire Mouse': a global gene knockout model for TSPO/PBR drug development, loss-of-function and mechanisms of compensation studies.

    PubMed

    Middleton, Ryan J; Liu, Guo-Jun; Banati, Richard B

    2015-08-01

    The highly conserved 18-kDa translocator protein (TSPO) or peripheral benzodiazepine receptor (PBR), is being investigated as a diagnostic and therapeutic target for disease conditions ranging from inflammation to neurodegeneration and behavioural illnesses. Many functions have been attributed to TSPO/PBR including a role in the mitochondrial permeability transition pore (MPTP), steroidogenesis and energy metabolism. In this review, we detail the recent developments in determining the physiological role of TSPO/PBR, specifically based on data obtained from the recently generated Tspo knockout mouse models. In addition to defining the role of TSPO/PBR, we also describe the value of Tspo knockout mice in determining the selectivity, specificity and presence of any off-target effects of TSPO/PBR ligands. PMID:26551692

  7. Pre-Equilibrium Cluster Emission with Pickup and Knockout

    SciTech Connect

    Betak, E.

    2005-05-24

    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 {alpha}'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.

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

    PubMed Central

    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

  9. Transferrin Receptor Controls AMPA Receptor Trafficking Efficiency and Synaptic Plasticity

    PubMed Central

    Liu, Ke; Lei, Run; Li, Qiong; Wang, Xin-Xin; Wu, Qian; An, Peng; Zhang, Jianchao; Zhu, Minyan; Xu, Zhiheng; Hong, Yang; Wang, Fudi; Shen, Ying; Li, Hongchang; Li, Huashun

    2016-01-01

    Transferrin receptor (TFR) is an important iron transporter regulating iron homeostasis and has long been used as a marker for clathrin mediated endocytosis. However, little is known about its additional function other than iron transport in the development of central nervous system (CNS). Here we demonstrate that TFR functions as a regulator to control AMPA receptor trafficking efficiency and synaptic plasticity. The conditional knockout (KO) of TFR in neural progenitor cells causes mice to develop progressive epileptic seizure, and dramatically reduces basal synaptic transmission and long-term potentiation (LTP). We further demonstrate that TFR KO remarkably reduces the binding efficiency of GluR2 to AP2 and subsequently decreases AMPA receptor endocytosis and recycling. Thus, our study reveals that TFR functions as a novel regulator to control AMPA trafficking efficiency and synaptic plasticity. PMID:26880306

  10. Hair-Cell Mechanotransduction Persists in TRP Channel Knockout Mice

    PubMed Central

    Niksch, Paul D.; Webber, Roxanna M.; Garcia-Gonzalez, Miguel; Watnick, Terry; Zhou, Jing; Vollrath, Melissa A.; Corey, David P.

    2016-01-01

    Members of the TRP superfamily of ion channels mediate mechanosensation in some organisms, and have been suggested as candidates for the mechanotransduction channel in vertebrate hair cells. Some TRP channels can be ruled out based on lack of an inner ear phenotype in knockout animals or pore properties not similar to the hair-cell channel. Such studies have excluded Trpv4, Trpa1, Trpml3, Trpm1, Trpm3, Trpc1, Trpc3, Trpc5, and Trpc6. However, others remain reasonable candidates. We used data from an RNA-seq analysis of gene expression in hair cells as well as data on TRP channel conductance to narrow the candidate group. We then characterized mice lacking functional Trpm2, Pkd2, Pkd2l1, Pkd2l2 and Pkd1l3, using scanning electron microscopy, auditory brainstem response, permeant dye accumulation, and single-cell electrophysiology. In all of these TRP-deficient mice, and in double and triple knockouts, mechanotransduction persisted. Together with published studies, these results argue against the participation of any of the 33 mouse TRP channels in hair cell transduction. PMID:27196058

  11. Hair-Cell Mechanotransduction Persists in TRP Channel Knockout Mice.

    PubMed

    Wu, Xudong; Indzhykulian, Artur A; Niksch, Paul D; Webber, Roxanna M; Garcia-Gonzalez, Miguel; Watnick, Terry; Zhou, Jing; Vollrath, Melissa A; Corey, David P

    2016-01-01

    Members of the TRP superfamily of ion channels mediate mechanosensation in some organisms, and have been suggested as candidates for the mechanotransduction channel in vertebrate hair cells. Some TRP channels can be ruled out based on lack of an inner ear phenotype in knockout animals or pore properties not similar to the hair-cell channel. Such studies have excluded Trpv4, Trpa1, Trpml3, Trpm1, Trpm3, Trpc1, Trpc3, Trpc5, and Trpc6. However, others remain reasonable candidates. We used data from an RNA-seq analysis of gene expression in hair cells as well as data on TRP channel conductance to narrow the candidate group. We then characterized mice lacking functional Trpm2, Pkd2, Pkd2l1, Pkd2l2 and Pkd1l3, using scanning electron microscopy, auditory brainstem response, permeant dye accumulation, and single-cell electrophysiology. In all of these TRP-deficient mice, and in double and triple knockouts, mechanotransduction persisted. Together with published studies, these results argue against the participation of any of the 33 mouse TRP channels in hair cell transduction. PMID:27196058

  12. Screening methods to identify TALEN-mediated knockout mice.

    PubMed

    Nakagawa, Yoshiko; Yamamoto, Takashi; Suzuki, Ken-Ichi; Araki, Kimi; Takeda, Naoki; Ohmuraya, Masaki; Sakuma, Tetsushi

    2014-01-01

    Genome editing with site-specific nucleases, such as zinc-finger nucleases or transcription activator-like effector nucleases (TALENs), and RNA-guided nucleases, such as the CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated) system, is becoming the new standard for targeted genome modification in various organisms. Application of these techniques to the manufacture of knockout mice would be greatly aided by simple and easy methods for genotyping of mutant and wild-type pups among litters. However, there are no detailed or comparative reports concerning the identification of mutant mice generated using genome editing technologies. Here, we genotyped TALEN-derived enhanced green fluorescent protein (eGFP) knockout mice using a combination of approaches, including fluorescence observation, heteroduplex mobility assay, restriction fragment length polymorphism analysis and DNA sequencing. The detection sensitivities for TALEN-induced mutations differed among these methods, and we therefore concluded that combinatorial testing is necessary for the screening and determination of mutant genotypes. Since the analytical methods tested can be carried out without specialized equipment, costly reagents and/or sophisticated protocols, our report should be of interest to a broad range of researchers who are considering the application of genome editing technologies in various organisms. PMID:24521866

  13. BDNF restricted knockout mice as an animal model for aggression

    PubMed Central

    Ito, Wataru; Chehab, Mahmoud; Thakur, Siddarth; Li, Jiayang; Morozov, Alexei

    2011-01-01

    Mice with global deletion of one BDNF allele, or with forebrain-restricted deletion of both alleles show elevated aggression, but this phenotype is accompanied by other behavioral changes, including increases in anxiety and deficits in cognition. Here, we performed behavioral characterization of conditional BDNF knockout mice generated using a Cre recombinase driver line, KA1-Cre, which expresses Cre in few areas of brain: highly at hippocampal area CA3, moderately in dentate gyrus, cerebellum and facial nerve nucleus. The mutant animals exhibited elevated conspecific aggression and social dominance, but did not show changes in anxiety-like behaviors assessed using the elevated plus maze and open field test. There were no changes in depression like behaviors tested in the forced swim test, but small increase in immobility in the tail suspension test. In cognitive tasks, mutants showed normal social recognition and normal spatial and fear memory, but exhibited a deficit in object recognition. Thus, this knockout can serve as a robust model of BDNF-dependent aggression and object recognition deficiency. PMID:21255268

  14. Creation and preliminary characterization of a Tp53 knockout rat

    PubMed Central

    McCoy, Aaron; Besch-Williford, Cynthia L.; Franklin, Craig L.; Weinstein, Edward J.; Cui, Xiaoxia

    2013-01-01

    SUMMARY The tumor suppressor TP53 plays a crucial role in cancer biology, and the TP53 gene is the most mutated gene in human cancer. Trp53 knockout mouse models have been widely used in cancer etiology studies and in search for a cure of cancer with some limitations that other model organisms might help overcome. Via pronuclear microinjection of zinc finger nucleases (ZFNs), we created a Tp53 knockout rat that contains an 11-bp deletion in exon 3, resulting in a frameshift and premature terminations in the open reading frame. In cohorts of 25 homozygous (Tp53Δ11/Δ11), 37 heterozygous (Tp53Δ11/+) and 30 wild-type rats, the Tp53Δ11/Δ11 rats lived an average of 126 days before death or removal from study because of clinical signs of abnormality or formation of tumors. Half of Tp53Δ11/+ were removed from study by 1 year of age because of tumor formation. Both Tp53Δ11/+ and Tp53Δ11/Δ11 rats developed a wide spectrum of tumors, most commonly sarcomas. Interestingly, there was a strikingly high incidence of brain lesions, especially in Tp53Δ11/Δ11 animals. We believe that this mutant rat line will be useful in studying cancer types rarely observed in mice and in carcinogenicity assays for drug development. PMID:22917926

  15. Screening Methods to Identify TALEN-Mediated Knockout Mice

    PubMed Central

    Nakagawa, Yoshiko; Yamamoto, Takashi; Suzuki, Ken-Ichi; Araki, Kimi; Takeda, Naoki; Ohmuraya, Masaki; Sakuma, Tetsushi

    2014-01-01

    Genome editing with site-specific nucleases, such as zinc-finger nucleases or transcription activator-like effector nucleases (TALENs), and RNA-guided nucleases, such as the CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated) system, is becoming the new standard for targeted genome modification in various organisms. Application of these techniques to the manufacture of knockout mice would be greatly aided by simple and easy methods for genotyping of mutant and wild-type pups among litters. However, there are no detailed or comparative reports concerning the identification of mutant mice generated using genome editing technologies. Here, we genotyped TALEN-derived enhanced green fluorescent protein (eGFP) knockout mice using a combination of approaches, including fluorescence observation, heteroduplex mobility assay, restriction fragment length polymorphism analysis and DNA sequencing. The detection sensitivities for TALEN-induced mutations differed among these methods, and we therefore concluded that combinatorial testing is necessary for the screening and determination of mutant genotypes. Since the analytical methods tested can be carried out without specialized equipment, costly reagents and/or sophisticated protocols, our report should be of interest to a broad range of researchers who are considering the application of genome editing technologies in various organisms. PMID:24521866

  16. Norepinephrine Transporter Heterozygous Knockout Mice Exhibit Altered Transport and Behavior

    PubMed Central

    Fentress, HM; Klar, R; Krueger, JK; Sabb, T; Redmon, SN; Wallace, NM; Shirey-Rice, JK; Hahn, MK

    2013-01-01

    The norepinephrine (NE) transporter (NET) regulates synaptic NE availability for noradrenergic signaling in the brain and sympathetic nervous system. Although genetic variation leading to a loss of NET expression has been implicated in psychiatric and cardiovascular disorders, complete NET deficiency has not been found in people, limiting the utility of NET knockout mice as a model for genetically-driven NET dysfunction. Here, we investigate NET expression in NET heterozygous knockout male mice (NET+/−), demonstrating that they display an ~50% reduction in NET protein levels. Surprisingly, these mice display no significant deficit in NET activity, assessed in hippocampal and cortical synaptosomes. We found that this compensation in NET activity was due to enhanced activity of surface-resident transporters, as opposed to surface recruitment of NET protein or compensation through other transport mechanisms, including serotonin, dopamine or organic cation transporters. We hypothesize that loss of NET protein in the NET+/− mouse establishes an activated state of existing, surface NET proteins. NET+/− mice exhibit increased anxiety in the open field and light-dark box and display deficits in reversal learning in the Morris Water Maze. These data suggest recovery of near basal activity in NET+/− mice appears to be insufficient to limit anxiety responses or support cognitive performance that might involve noradrenergic neurotransmission. The NET+/− mice represent a unique model to study the loss and resultant compensatory changes in NET that may be relevant to behavior and physiology in human NET deficiency disorders. PMID:24102798

  17. RAG1/2 knockout pigs with severe combined immunodeficiency.

    PubMed

    Huang, Jiao; Guo, Xiaogang; Fan, Nana; Song, Jun; Zhao, Bentian; Ouyang, Zhen; Liu, Zhaoming; Zhao, Yu; Yan, Quanmei; Yi, Xiaoling; Schambach, Axel; Frampton, Jon; Esteban, Miguel A; Yang, Dongshan; Yang, Huaqiang; Lai, Liangxue

    2014-08-01

    Pigs share many physiological, biochemical, and anatomical similarities with humans and have emerged as valuable large animal models for biomedical research. Considering the advantages in immune system resemblance, suitable size, and longevity for clinical practical and monitoring purpose, SCID pigs bearing dysfunctional RAG could serve as important experimental tools for regenerative medicine, allograft and xenograft transplantation, and reconstitution experiments related to the immune system. In this study, we report the generation and phenotypic characterization of RAG1 and RAG2 knockout pigs using transcription activator-like effector nucleases. Porcine fetal fibroblasts were genetically engineered using transcription activator-like effector nucleases and then used to provide donor nuclei for somatic cell nuclear transfer. We obtained 27 live cloned piglets; among these piglets, 9 were targeted with biallelic mutations in RAG1, 3 were targeted with biallelic mutations in RAG2, and 10 were targeted with a monoallelic mutation in RAG2. Piglets with biallelic mutations in either RAG1 or RAG2 exhibited hypoplasia of immune organs, failed to perform V(D)J rearrangement, and lost mature B and T cells. These immunodeficient RAG1/2 knockout pigs are promising tools for biomedical and translational research. PMID:24973446

  18. Norepinephrine transporter heterozygous knockout mice exhibit altered transport and behavior.

    PubMed

    Fentress, H M; Klar, R; Krueger, J J; Sabb, T; Redmon, S N; Wallace, N M; Shirey-Rice, J K; Hahn, M K

    2013-11-01

    The norepinephrine (NE) transporter (NET) regulates synaptic NE availability for noradrenergic signaling in the brain and sympathetic nervous system. Although genetic variation leading to a loss of NET expression has been implicated in psychiatric and cardiovascular disorders, complete NET deficiency has not been found in people, limiting the utility of NET knockout mice as a model for genetically driven NET dysfunction. Here, we investigate NET expression in NET heterozygous knockout male mice (NET(+/-) ), demonstrating that they display an approximately 50% reduction in NET protein levels. Surprisingly, these mice display no significant deficit in NET activity assessed in hippocampal and cortical synaptosomes. We found that this compensation in NET activity was due to enhanced activity of surface-resident transporters, as opposed to surface recruitment of NET protein or compensation through other transport mechanisms, including serotonin, dopamine or organic cation transporters. We hypothesize that loss of NET protein in the NET(+/-) mouse establishes an activated state of existing surface NET proteins. The NET(+/-) mice exhibit increased anxiety in the open field and light-dark box and display deficits in reversal learning in the Morris water maze. These data suggest that recovery of near basal activity in NET(+/-) mice appears to be insufficient to limit anxiety responses or support cognitive performance that might involve noradrenergic neurotransmission. The NET(+/-) mice represent a unique model to study the loss and resultant compensatory changes in NET that may be relevant to behavior and physiology in human NET deficiency disorders. PMID:24102798

  19. Study of 19C by One-Neutron Knockout

    NASA Astrophysics Data System (ADS)

    Hwang, Jongwon; Kim, Sunji; Satou, Yoshiteru; Orr, Nigel A.; Nakamura, Takashi; Kondo, Yosuke; Gibelin, Julien; Achouri, N. Lynda; Aumann, Thomas; Baba, Hidetada; Delaunay, Franck; Doornenbal, Pieter; Fukuda, Naoki; Inabe, Naohito; Isobe, Tadaaki; Kameda, Daisuke; Kanno, Daiki; Kobayashi, Nobuyuki; Kobayashi, Toshio; Kubo, Toshiyuki; Leblond, Sylvain; Lee, Jenny; Marqués, F. Miguel; Minakata, Ryogo; Motobayashi, Tohru; Murai, Daichi; Murakami, Tetsuya; Muto, Kotomi; Nakashima, Tomohiro; Nakatsuka, Noritsugu; Navin, Alahari; Nishi, Seijiro; Ogoshi, Shun; Otsu, Hideaki; Sato, Hiromi; Shimizu, Yohei; Suzuki, Hiroshi; Takahashi, Kento; Takeda, Hiroyuki; Takeuchi, Satoshi; Tanaka, Ryuki; Togano, Yasuhiro; Tuff, Adam G.; Vandebrouck, Marine; Yoneda, Ken-ichiro

    2016-03-01

    The spectroscopic structure of 19C, a prominent one-neutron halo nucleus, has been studied with a 20C secondary beam at 290 MeV/nucleon and a carbon target. Neutron-unbound states populated by the one-neutron knockout reaction were investigated by means of the invariant mass method. The preliminary relative energy spectrum and parallel momentum distribution of the knockout residue, 19C*, were reconstructed from the measured four momenta of the 18C fragment, neutron, and beam. Three resonances were observed in the spectrum, which correspond to the states at Ex = 0.62(9), 1.42(10), and 2.89(10) MeV. The parallel momentum distributions for the 0.62-MeV and 2.89-MeV states suggest spin-parity assignments of 5/2+ and 1/2-, respectively. The 1.42-MeV state is in line with the reported 5/22+ state.

  20. The Novel, Nicotinic Alpha7 Receptor Partial Agonist, BMS-933043, Improves Cognition and Sensory Processing in Preclinical Models of Schizophrenia

    PubMed Central

    Bristow, Linda J.; Easton, Amy E.; Li, Yu-Wen; Sivarao, Digavalli V.; Lidge, Regina; Jones, Kelli M.; Post-Munson, Debra; Daly, Christopher; Lodge, Nicholas J.; Gallagher, Lizbeth; Molski, Thaddeus; Pieschl, Richard; Chen, Ping; Hendricson, Adam; Westphal, Ryan; Cook, James; Iwuagwu, Christiana; Morgan, Daniel; Benitex, Yulia; King, Dalton; Macor, John E.; Zaczek, Robert; Olson, Richard

    2016-01-01

    The development of alpha7 nicotinic acetylcholine receptor agonists is considered a promising approach for the treatment of cognitive symptoms in schizophrenia patients. In the present studies we characterized the novel agent, (2R)-N-(6-(1H-imidazol-1-yl)-4-pyrimidinyl)-4'H-spiro[4-azabicyclo[2.2.2]octane-2,5'-[1,3]oxazol]-2'-amine (BMS-933043), in vitro and in rodent models of schizophrenia-like deficits in cognition and sensory processing. BMS-933043 showed potent binding affinity to native rat (Ki = 3.3 nM) and recombinant human alpha7 nicotinic acetylcholine receptors (Ki = 8.1 nM) and agonist activity in a calcium fluorescence assay (EC50 = 23.4 nM) and whole cell voltage clamp electrophysiology (EC50 = 0.14 micromolar (rat) and 0.29 micromolar (human)). BMS-933043 exhibited a partial agonist profile relative to acetylcholine; the relative efficacy for net charge crossing the cell membrane was 67% and 78% at rat and human alpha7 nicotinic acetylcholine receptors respectively. BMS-933043 showed no agonist or antagonist activity at other nicotinic acetylcholine receptor subtypes and was at least 300 fold weaker at binding to and antagonizing human 5-HT3A receptors (Ki = 2,451 nM; IC50 = 8,066 nM). BMS-933043 treatment i) improved 24 hour novel object recognition memory in mice (0.1–10 mg/kg, sc), ii) reversed MK-801-induced deficits in Y maze performance in mice (1–10 mg/kg, sc) and set shift performance in rats (1–10 mg/kg, po) and iii) reduced the number of trials required to complete the extradimensional shift discrimination in neonatal PCP treated rats performing the intra-dimensional/extradimensional set shifting task (0.1–3 mg/kg, po). BMS-933043 also improved auditory gating (0.56–3 mg/kg, sc) and mismatch negativity (0.03–3 mg/kg, sc) in rats treated with S(+)ketamine or neonatal phencyclidine respectively. Given this favorable preclinical profile BMS-933043 was selected for further development to support clinical evaluation in humans. PMID

  1. The Novel, Nicotinic Alpha7 Receptor Partial Agonist, BMS-933043, Improves Cognition and Sensory Processing in Preclinical Models of Schizophrenia.

    PubMed

    Bristow, Linda J; Easton, Amy E; Li, Yu-Wen; Sivarao, Digavalli V; Lidge, Regina; Jones, Kelli M; Post-Munson, Debra; Daly, Christopher; Lodge, Nicholas J; Gallagher, Lizbeth; Molski, Thaddeus; Pieschl, Richard; Chen, Ping; Hendricson, Adam; Westphal, Ryan; Cook, James; Iwuagwu, Christiana; Morgan, Daniel; Benitex, Yulia; King, Dalton; Macor, John E; Zaczek, Robert; Olson, Richard

    2016-01-01

    The development of alpha7 nicotinic acetylcholine receptor agonists is considered a promising approach for the treatment of cognitive symptoms in schizophrenia patients. In the present studies we characterized the novel agent, (2R)-N-(6-(1H-imidazol-1-yl)-4-pyrimidinyl)-4'H-spiro[4-azabicyclo[2.2.2]octane-2,5'-[1,3]oxazol]-2'-amine (BMS-933043), in vitro and in rodent models of schizophrenia-like deficits in cognition and sensory processing. BMS-933043 showed potent binding affinity to native rat (Ki = 3.3 nM) and recombinant human alpha7 nicotinic acetylcholine receptors (Ki = 8.1 nM) and agonist activity in a calcium fluorescence assay (EC50 = 23.4 nM) and whole cell voltage clamp electrophysiology (EC50 = 0.14 micromolar (rat) and 0.29 micromolar (human)). BMS-933043 exhibited a partial agonist profile relative to acetylcholine; the relative efficacy for net charge crossing the cell membrane was 67% and 78% at rat and human alpha7 nicotinic acetylcholine receptors respectively. BMS-933043 showed no agonist or antagonist activity at other nicotinic acetylcholine receptor subtypes and was at least 300 fold weaker at binding to and antagonizing human 5-HT3A receptors (Ki = 2,451 nM; IC50 = 8,066 nM). BMS-933043 treatment i) improved 24 hour novel object recognition memory in mice (0.1-10 mg/kg, sc), ii) reversed MK-801-induced deficits in Y maze performance in mice (1-10 mg/kg, sc) and set shift performance in rats (1-10 mg/kg, po) and iii) reduced the number of trials required to complete the extradimensional shift discrimination in neonatal PCP treated rats performing the intra-dimensional/extradimensional set shifting task (0.1-3 mg/kg, po). BMS-933043 also improved auditory gating (0.56-3 mg/kg, sc) and mismatch negativity (0.03-3 mg/kg, sc) in rats treated with S(+)ketamine or neonatal phencyclidine respectively. Given this favorable preclinical profile BMS-933043 was selected for further development to support clinical evaluation in humans. PMID:27467081

  2. Time-lapse analysis of tangential migration in Sema6A and PlexinA2 knockouts.

    PubMed

    Renaud, Julie; Chédotal, Alain

    2014-11-01

    In the developing cerebellum, granule cells migrate tangentially in the external granule cell layer and then radially and inward, across the molecular layer and Purkinje cell layer. We showed previously that the transmembrane semaphorin Sema6A and its receptor PlexinA2 control the ability of migrating granule cells to switch from one mode of migration to the other. In both Sema6A and PlexinA2 knockouts, a large number of granule cells remain in the molecular layer, a defect that is most likely due to abnormal nuclear translocation. We show here that the lack of Sema6A or PlexinA2 preferentially much more severely perturbs the migration of later-born granule cells than early-born ones. We also use a cerebellum slice model system and electroporation to perform time-lapse analysis of granule cell migration in wild-type mice, Sema6A and PlexinA2 knockouts. This study reveals that defects of tangential migration can be detected in bipolar granule cells before the initiation of radial migration. Our results also directly confirm that the absence of Sema6A does not perturb radial migration. PMID:25284064

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

    PubMed Central

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

    2016-01-01

    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. PMID:26847765

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

    PubMed

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

    2016-01-01

    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. PMID:26847765

  5. GRK5-Knockout Mice Generated by TALEN-Mediated Gene Targeting.

    PubMed

    Nanjidsuren, Tsevelmaa; Park, Chae-Won; Sim, Bo-Woong; Kim, Sun-Uk; Chang, Kyu-Tae; Kang, Myung-Hwa; Min, Kwan-Sik

    2016-10-01

    Transcription activator-like effector nucleases (TALENs) are a new type of engineered nuclease that is very effective for directed gene disruption in any genome sequence. We investigated the generation of mice with genetic knockout (KO) of the G protein-coupled receptor kinase (GRK) 5 gene by microinjection of TALEN mRNA. TALEN vectors were designed to target exons 1, 3, and 5 of the mouse GRK5 gene. Flow cytometry showed that the activity of the TALEN mRNAs targeted to exons 1, 3, and 5 was 8.7%, 9.7%, and 12.7%, respectively. The TALEN mRNA for exon 5 was injected into the cytoplasm of 180 one-cell embryos. Of the 53 newborns, three (5.6%) were mutant founders (F0) with mutations. Two clones from F028 showed a 45-bp deletion and F039 showed the same biallelic non-frame-shifting 3-bp deletions. Three clones from F041 were shown to possess a combination of frame-shifting 2-bp deletions. All of the mutations were transmitted through the germline but not to all progenies (37.5%, 37.5%, and 57.1% for the F028, F039, and F041 lines, respectively). The homozygote GRK5-KO mice for 28 and 41 lines created on F3 progenies and the homozygous genotype was confirmed by PCR, T7E1 assay and sequencing. PMID:27565865

  6. Adult Conditional Knockout of PGC-1α Leads to Loss of Dopamine Neurons.

    PubMed

    Jiang, Haisong; Kang, Sung-Ung; Zhang, Shuran; Karuppagounder, Senthilkumar; Xu, Jinchong; Lee, Yong-Kyu; Kang, Bong-Gu; Lee, Yunjong; Zhang, Jianmin; Pletnikova, Olga; Troncoso, Juan C; Pirooznia, Shelia; Andrabi, Shaida A; Dawson, Valina L; Dawson, Ted M

    2016-01-01

    Parkinson's disease (PD) is a chronic progressive neurodegenerative disorder. Recent studies have implicated a role for peroxisome proliferator-activated receptor γ coactivator protein-1α (PGC-1α) in PD and in animal or cellular models of PD. The role of PGC-1α in the function and survival of substantia nigra pars compacta (SNpc) dopamine neurons is not clear. Here we find that there are four different PGC-1α isoforms expressed in SH-SY5Y cells, and these four isoforms are expressed across subregions of mouse brain. Adult conditional PGC-1α knock-out mice show a significant loss of dopaminergic neurons that is accompanied by a reduction of dopamine in the striatum. In human PD postmortem tissue from the SNpc, there is a reduction of PGC-1α isoforms and mitochondria markers. Our findings suggest that all four isoforms of PGC-1α are required for the proper expression of mitochondrial proteins in SNpc DA neurons and that PGC-1α is essential for SNpc DA neuronal survival, possibly through the maintenance of mitochondrial function. PMID:27622213

  7. Common mechanisms for calorie restriction and adenylyl cyclase type 5 knockout models of longevity.

    PubMed

    Yan, Lin; Park, Ji Yeon; Dillinger, Jean-Guillaume; De Lorenzo, Mariana S; Yuan, Chujun; Lai, Lo; Wang, Chunbo; Ho, David; Tian, Bin; Stanley, William C; Auwerx, Johan; Vatner, Dorothy E; Vatner, Stephen F

    2012-12-01

    Adenylyl cyclase type 5 knockout mice (AC5 KO) live longer and are stress resistant, similar to calorie restriction (CR). AC5 KO mice eat more, but actually weigh less and accumulate less fat compared with WT mice. CR applied to AC5 KO results in rapid decrease in body weight, metabolic deterioration, and death. These data suggest that despite restricted food intake in CR, but augmented food intake in AC5 KO, the two models affect longevity and metabolism similarly. To determine shared molecular mechanisms, mRNA expression was examined genome-wide for brain, heart, skeletal muscle, and liver. Significantly more genes were regulated commonly rather than oppositely in all the tissues in both models, indicating commonality between AC5 KO and CR. Gene ontology analysis identified many significantly regulated, tissue-specific pathways shared by the two models, including sensory perception in heart and brain, muscle function in skeletal muscle, and lipid metabolism in liver. Moreover, when comparing gene expression changes in the heart under stress, the glutathione regulatory pathway was consistently upregulated in the longevity models but downregulated with stress. In addition, AC5 and CR shared changes in genes and proteins involved in the regulation of longevity and stress resistance, including Sirt1, ApoD, and olfactory receptors in both young- and intermediate-age mice. Thus, the similarly regulated genes and pathways in AC5 KO and CR mice, particularly related to the metabolic phenotype, suggest a unified theory for longevity and stress resistance. PMID:23020244

  8. Adult Conditional Knockout of PGC-1α Leads to Loss of Dopamine Neurons

    PubMed Central

    Jiang, Haisong; Zhang, Shuran; Karuppagounder, Senthilkumar; Xu, Jinchong; Pletnikova, Olga; Troncoso, Juan C.; Pirooznia, Shelia; Andrabi, Shaida A.

    2016-01-01

    Parkinson’s disease (PD) is a chronic progressive neurodegenerative disorder. Recent studies have implicated a role for peroxisome proliferator-activated receptor γ coactivator protein-1α (PGC-1α) in PD and in animal or cellular models of PD. The role of PGC-1α in the function and survival of substantia nigra pars compacta (SNpc) dopamine neurons is not clear. Here we find that there are four different PGC-1α isoforms expressed in SH-SY5Y cells, and these four isoforms are expressed across subregions of mouse brain. Adult conditional PGC-1α knock-out mice show a significant loss of dopaminergic neurons that is accompanied by a reduction of dopamine in the striatum. In human PD postmortem tissue from the SNpc, there is a reduction of PGC-1α isoforms and mitochondria markers. Our findings suggest that all four isoforms of PGC-1α are required for the proper expression of mitochondrial proteins in SNpc DA neurons and that PGC-1α is essential for SNpc DA neuronal survival, possibly through the maintenance of mitochondrial function. PMID:27622213

  9. Pravastatin and Sarpogrelate Synergistically Ameliorate Atherosclerosis in LDLr-Knockout Mice.

    PubMed

    Park, Kyung-Yeon; Oh, Euichaul; Kwak, Mi-Kyoung; Jun, Hyun Sik; Heo, Tae-Hwe

    2016-01-01

    Pravastatin is a lipid-lowering agent that attenuates atherosclerosis. However, the multifactorial pathogenesis of atherosclerosis requires other drugs with different anti-atherogenic mechanisms. We chose sarpogrelate as an anti-platelet agent and a novel component of a complex drug with pravastatin due to its high potential but little information on its beneficial effects on atherosclerosis. Low-density lipoprotein receptor-knockout mice were fed a high-fat, high-cholesterol diet and treated with pravastatin alone, sarpogrelate alone, or a combination of both drugs. Although sarpogrelate alone did not significantly reduce atherosclerotic plaque areas, co-treatment with pravastatin significantly decreased aortic lesions compared to those of the pravastatin alone treated group. The combined therapy was markedly more effective than that of the single therapies in terms of foam cell formation, smooth muscle cell proliferation, and inflammatory cytokine levels. These results suggest that pravastatin and sarpogrelate combined therapy may provide a new therapeutic strategy for treating atherosclerosis. PMID:26950217

  10. Cognitive abnormalities and hippocampal alterations in monoamine oxidase A and B knockout mice

    PubMed Central

    Singh, Chanpreet; Bortolato, Marco; Bali, Namrata; Godar, Sean C.; Scott, Anna L.; Chen, Kevin; Thompson, Richard F.; Shih, Jean C.

    2013-01-01

    The monoamine oxidase isoenzymes (MAOs) A and B play important roles in the homeostasis of monoaminergic neurotransmitters. The combined deficiency of MAO A and B results in significantly elevated levels of serotonin (5-hydroxytryptamine), norepinephrine, dopamine, and β-phenylethylamine; in humans and mice, these neurochemical changes are accompanied by neurodevelopmental perturbations as well as autistic-like responses. Ample evidence indicates that normal levels of monoamines in the hippocampus, amygdala, frontal cortex, and cerebellum are required for the integrity of learning and memory. Thus, in the present study, the cognitive status of MAO A/B knockout (KO) mice was examined with a wide array of behavioral tests. In comparison with male wild-type littermates, MAO A/B KO mice exhibited abnormally high and overgeneralized fear conditioning and enhanced eye-blink conditioning. These alterations were accompanied by significant increases in hippocampal long-term potentiation and alterations in the relative expression of NMDA glutamate receptor subunits. Our data suggest that chronic elevations of monoamines, because of the absence of MAO A and MAO B, cause functional alterations that are accompanied with changes in the cellular mechanisms underlying learning and memory. The characteristics exhibited by MAO A/B KO mice highlight the potential of these animals as a useful tool to provide further insight into the molecular bases of disorders associated with abnormal monoaminergic profiles. PMID:23858446

  11. Pravastatin and Sarpogrelate Synergistically Ameliorate Atherosclerosis in LDLr-Knockout Mice

    PubMed Central

    Park, Kyung-Yeon; Oh, Euichaul; Kwak, Mi-Kyoung; Jun, Hyun Sik; Heo, Tae-Hwe

    2016-01-01

    Pravastatin is a lipid-lowering agent that attenuates atherosclerosis. However, the multifactorial pathogenesis of atherosclerosis requires other drugs with different anti-atherogenic mechanisms. We chose sarpogrelate as an anti-platelet agent and a novel component of a complex drug with pravastatin due to its high potential but little information on its beneficial effects on atherosclerosis. Low-density lipoprotein receptor-knockout mice were fed a high-fat, high-cholesterol diet and treated with pravastatin alone, sarpogrelate alone, or a combination of both drugs. Although sarpogrelate alone did not significantly reduce atherosclerotic plaque areas, co-treatment with pravastatin significantly decreased aortic lesions compared to those of the pravastatin alone treated group. The combined therapy was markedly more effective than that of the single therapies in terms of foam cell formation, smooth muscle cell proliferation, and inflammatory cytokine levels. These results suggest that pravastatin and sarpogrelate combined therapy may provide a new therapeutic strategy for treating atherosclerosis. PMID:26950217

  12. Improving expression of recombinant human IGF-1 using IGF-1R knockout CHO cell lines.

    PubMed

    Romand, Sandrine; Jostock, Thomas; Fornaro, Mara; Schmidt, Joerg; Ritter, Anett; Wilms, Burkhard; Laux, Holger

    2016-05-01

    Chinese Hamster Ovary (CHO) cells are widely used for the large-scale production of recombinant biopharmaceuticals. However, attempts to express IGF-1 (a mutated human Insulin-like growth factor 1 Ea peptide (hIGF-1Ea mut)) in CHO cells resulted in poor cell growth and low productivity (0.1-0.2 g/L). Human IGF-1 variants negatively impacted CHO cell growth via the IGF-1 receptor (IGF-1R). Therefore knockout (KO) of the IGF-1R gene in two different CHO cell lines as well as knockdown (KD) of IGF-1R in one CHO cell line were performed. These cell line engineering approaches decreased significantly the hIGF-1 mediated cell growth inhibition and increased productivity of both KO CHO cell lines as well as of the KD CHO cell line. A productivity increase of 10-fold at pool level and sevenfold at clone level was achieved, resulting in a titer of 1.3 g/L. This data illustrate that cell line engineering approaches are powerful tools to improve the yields of recombinant proteins which are difficult to produce in CHO cells. Biotechnol. Bioeng. 2016;113: 1094-1101. © 2015 Wiley Periodicals, Inc. PMID:26523469

  13. Hypoxia-Induced Endothelial Progenitor Cell Function Is Blunted in Angiotensinogen Knockout Mice

    PubMed Central

    Choi, Jin-Hwa; Nguyen, Minh-Phuong; Lee, Dongjin; Oh, Goo-Taeg; Lee, You-Mie

    2014-01-01

    Angiotensinogen (AGT), the precursor of angiotensin I, is known to be involved in tumor angiogenesis and associated with the pathogenesis of coronary atherosclerosis. This study was undertaken to determine the role played by AGT in endothelial progenitor cells (EPCs) in tumor progression and metastasis. It was found that the number of EPC colonies formed by AGT heterozygous knockout (AGT+/−) cells was less than that formed by wild-type (WT) cells, and that the migration and tube formation abilities of AGT+/− EPCs were significantly lower than those of WT EPCs. In addition, the gene expressions of vascular endothelial growth factor (VEGF), Flk1, angiopoietin (Ang)-1, Ang-2, Tie-2, stromal derived factor (SDF)-1, C-X-C chemokine receptor type 4 (CXCR4), and of endothelial nitric oxide synthase (eNOS) were suppressed in AGT+/− EPCs. Furthermore, the expressions of hypoxia-inducible factor (HIF)-1α and -2α were downregulated in AGT+/− early EPCs under hypoxic conditions, suggesting a blunting of response to hypoxia. Moreover, the activation of Akt/eNOS signaling pathways induced by VEGF, epithelial growth factor (EGF), or SDF-1α were suppressed in AGT+/− EPCs. In AGT+/− mice, the incorporation of EPCs into the tumor vasculature was significantly reduced, and lung tumor growth and melanoma metastasis were attenuated. In conclusion, AGT is required for hypoxia-induced vasculogenesis. PMID:24938229

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

    PubMed

    Lampert, Thomas J; Coleman, Kevin D; Hennessey, Todd M

    2011-01-01

    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. PMID:22140501

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

    PubMed

    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. PMID:25281279

  16. Gene Expression Profiles of Main Olfactory Epithelium in Adenylyl Cyclase 3 Knockout Mice

    PubMed Central

    Wang, Zhenshan; Zhou, Yanfen; Luo, Yingtao; Zhang, Jing; Zhai, Yunpeng; Yang, Dong; Zhang, Zhe; Li, Yongchao; Storm, Daniel R.; Ma, Runlin Z.

    2015-01-01

    Adenylyl Cyclase 3 (AC3) plays an important role in the olfactory sensation-signaling pathway in mice. AC3 deficiency leads to defects in olfaction. However, it is still unknown whether AC3 deficiency affects gene expression or olfactory signal transduction pathways within the main olfactory epithelium (MOE). In this study, gene microarrays were used to screen differentially expressed genes in MOE from AC3 knockout (AC3−/−) and wild-type (AC3+/+) mice. The differentially expressed genes identified were subjected to bioinformatic analysis and verified by qRT-PCR. Gene expression in the MOE from AC3−/− mice was significantly altered, compared to AC3+/+ mice. Of the 41266 gene probes, 3379 had greater than 2-fold fold change in expression levels between AC3−/− and AC3+/+ mice, accounting for 8% of the total gene probes. Of these genes, 1391 were up regulated, and 1988 were down regulated, including 425 olfactory receptor genes, 99 genes that are specifically expressed in the immature olfactory neurons, 305 genes that are specifically expressed in the mature olfactory neurons, and 155 genes that are involved in epigenetic regulation. Quantitative RT-PCR verification of the differentially expressed epigenetic regulation related genes, olfactory receptors, ion transporter related genes, neuron development and differentiation related genes, lipid metabolism and membrane protein transport etc. related genes showed that P75NTR, Hinfp, Gadd45b, and Tet3 were significantly up-regulated, while Olfr370, Olfr1414, Olfr1208, Golf, Faim2, Tsg101, Mapk10, Actl6b, H2BE, ATF5, Kirrrel2, OMP, Drd2 etc. were significantly down-regulated. In summary, AC3 may play a role in proximal olfactory signaling and play a role in the regulation of differentially expressed genes in mouse MOE. PMID:26633363

  17. Impaired striatum-dependent behavior in GASP-1-knock-out mice.

    PubMed

    Mathis, C; Bott, J-B; Candusso, M-P; Simonin, F; Cassel, J-C

    2011-04-01

    G protein-coupled receptor (GPCR) associated sorting protein-1 (GASP-1) is suspected to play a key role in recycling and degradation of several GPCRs. In a previous study, we have shown that GASP-1-knock-out (GASP-1-KO) mice displayed deficits in acquiring a cocaine self-administration task, associated with an exacerbated down-regulation of striatal dopaminergic and cholinergic receptors. Among several possibilities, GASP-1 deficiency could have impaired memory processes underlying the acquisition of the operant conditioning task. Therefore, the present study investigated cognitive performances of GASP-1-KO mice and their wild-type littermates (WT) in a broad variety of memory tasks. Consistent with a deficit in procedural memory, GASP-1-KO mice showed delayed acquisition of a food-reinforced bar-press task. During water-maze training in hidden- or visible-platform paradigms, mutant and WT mice acquired the tasks at the same rate. However, GASP-1 mice exhibited persistent thigmotaxic swimming, longer distance to the platform, and reduced swim speed. There was no deficit in several tasks requiring simple behavioral responses (Barnes maze, object recognition and passive avoidance tasks). Thus, the ability to acquire and/or express complex responses seems affected in GASP-1-deficient mice. Hippocampal functions were preserved, as the retention of an acquired memory in spatial tasks remained unaffected. The pattern of behavioral deficits observed in GASP-1-KO mice is coherent with current knowledge on the role of striatal GPCRs in acquisition/expression of skilled behavior and in motivation. Together with the previous findings, the so far established phenotype of GASP-1-KO mice makes them a potentially exciting tool to study striatal functions. PMID:21091868

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

    PubMed Central

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

    2014-01-01

    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. PMID:25281279

  19. Comprehensive behavioral study of mGluR3 knockout mice: implication in schizophrenia related endophenotypes

    PubMed Central

    2014-01-01

    Background We previously performed systematic association studies of glutamate receptor gene family members with schizophrenia, and found positive associations of polymorphisms in the GRM3 (a gene of metabotropic glutamate receptor 3: mGluR3) with the disorder. Physiological roles of GRM3 in brain functions and its functional roles in the pathogenesis of schizophrenia remain to be resolved. Results We generated mGluR3 knockout (KO) mice and conducted comprehensive behavioral analyses. KO mice showed hyperactivity in the open field, light/dark transition, and 24-hour home cage monitoring tests, impaired reference memory for stressful events in the Porsolt forced swim test, impaired contextual memory in cued and contextual fear conditioning test, and impaired working memory in the T-Maze forced alternation task test. Hyperactivity and impaired working memory are known as endophenotypes of schizophrenia. We examined long-term synaptic plasticity by assessing long-term potentiation (LTP) in the CA1 region in the hippocampi of KO and wild-type (WT) mice. We observed no differences in the amplitude of LTP between the two genotypes, suggesting that mGluR3 is not essential for LTP in the CA1 region of the mouse hippocampus. As hyperactivity is typically associated with increased dopaminergic transmission, we performed in vivo microdialysis measurements of extracellular dopamine in the nucleus accumbens of KO and WT mice. We observed enhancements in the methamphetamine (MAP)-induced release of dopamine in KO mice. Conclusions These results demonstrate that a disturbance in the glutamate-dopamine interaction may be involved in the pathophysiology of schizophrenia-like behavior, such as hyperactivity in mGluR3 KO mice. PMID:24758191

  20. Effects of trkB knockout on topography and ocular segregation of uncrossed retinal projections.

    PubMed

    Rodger, Jennifer; Frost, Douglas O

    2009-05-01

    TrkB is an important receptor for brain-derived neurotrophic factor and NT4, members of the neurotrophin family. TrkB signaling is crucial in many activity-dependent and activity-independent processes of neural development. Here, we investigate the role of trkB signaling in the development of two distinct, organizational features of retinal projections--the segregation of crossed and uncrossed retinal inputs along the "lines of projection" that represent a single point in the visual field and the "retinotopic" mapping of retinofugal axons within their cerebral targets. Using anterograde tracing, we obtained quantitative measures of the distribution of retinal projections in the dorsal nucleus of the lateral geniculate body (LGd) and superior colliculus (SC) of wild-type mice and mice homozygous for constitutive null mutation (knockout) of the full-length trkB receptor (trkB(FL)(-/-)). In trkB(FL)(-/-) mice, uncrossed retinal projections cluster normally but there is a topographic expansion in the distribution of these clusters across the SC. By contrast, the absence of trkB signaling has no significant effect on the segregation of crossed and uncrossed retinal projections along the lines of projection in LGd or SC. We conclude that the normal topographic organization of uncrossed retinal projections depends upon trkB signaling, whereas the segregation of crossed and uncrossed retinal projections is trkB-independent. We also found that in trkB(FL)(-/-) mice, neuronal number was reduced in the LGd and SC and in the caudate-putamen. Previous studies by ourselves and others have shown that the number of retinal ganglion cells (RGCs) is unchanged in trkB(FL)(-/-) mice. Together, these results demonstrate that there is no matching of the numbers of RGCs with neuronal numbers in the LGd or SC. PMID:19283373

  1. Substrate uptake and metabolism are preserved in hypertrophic caveolin-3 knockout hearts

    PubMed Central

    Augustus, Ayanna S.; Buchanan, Jonathan; Addya, Sankar; Rengo, Giuseppe; Pestell, Richard G.; Fortina, Paolo; Koch, Walter J.; Bensadoun, Andre; Abel, E. Dale; Lisanti, Michael P.

    2008-01-01

    Caveolin-3 (Cav3), the primary protein component of caveolae in muscle cells, regulates numerous signaling pathways including insulin receptor signaling and facilitates free fatty acid (FA) uptake by interacting with several FA transport proteins. We previously reported that Cav3 knockout mice (Cav3KO) develop cardiac hypertrophy with diminished contractile function; however, the effects of Cav3 gene ablation on cardiac substrate utilization are unknown. The present study revealed that the uptake and oxidation of FAs and glucose were normal in hypertrophic Cav3KO hearts. Real-time PCR analysis revealed normal expression of lipid metabolism genes including FA translocase (CD36) and carnitine palmitoyl transferase-1 in Cav3KO hearts. Interestingly, myocardial cAMP content was significantly increased by 42%; however, this had no effect on PKA activity in Cav3KO hearts. Microarray expression analysis revealed a marked increase in the expression of genes involved in receptor trafficking to the plasma membrane, including Rab4a and the expression of WD repeat/FYVE domain containing proteins. We observed a fourfold increase in the expression of cellular retinol binding protein-III and a 3.5-fold increase in 17β-hydroxysteroid dehydrogenase type 11, a member of the short-chain dehydrogenase/reductase family involved in the biosynthesis and inactivation of steroid hormones. In summary, a loss of Cav3 in the heart leads to cardiac hypertrophy with normal substrate utilization. Moreover, a loss of Cav3 mRNA altered the expression of several genes not previously linked to cardiac growth and function. Thus we have identified a number of new target genes associated with the pathogenesis of cardiac hypertrophy. PMID:18552160

  2. P2Y nucleotide receptors: Promise of therapeutic applications

    PubMed Central

    Jacobson, Kenneth A.; Boeynaems, Jean-Marie

    2010-01-01

    Extracellular nucleotides, such as ATP and UTP, have distinct signaling roles through a class of G protein-coupled receptors, termed P2Y. However, the receptor ligands are typically charged molecules of low bioavailability and stability in vivo. Recent progress in the development of selective agonists and antagonists for P2Y receptors and study of knockout mice have led to new drug concepts based on these receptors. The rapidly accelerating progress in this field has already resulted in drug candidates for cystic fibrosis, dry eye disease, and thrombosis. On the horizon are novel treatments of cardiovascular diseases, inflammatory diseases, and neurodegeneration. PMID:20594935

  3. Giant vesicles functionally expressing membrane receptors for an insect pheromone.

    PubMed

    Hamada, Satoshi; Tabuchi, Masashi; Toyota, Taro; Sakurai, Takeshi; Hosoi, Tomohiro; Nomoto, Tomonori; Nakatani, Kei; Fujinami, Masanori; Kanzaki, Ryohei

    2014-03-18

    To date, biochemical approaches to membrane receptors have been limited to the following methods: knockout or overexpression of membrane receptors by gene introduction and genome engineering or extraction of membrane receptor-surfactant complexes from innate cells and their introduction into model biomembranes. Here, we describe the development of a third method involving gene expression using cell-free in situ protein synthesis inside model biomembrane capsules. We verified this method by synthesizing olfactory receptors from the silkmoth Bombyx mori inside giant vesicles and found that they were excited in the presence of their ligand the Bombyx mori sex pheromone. PMID:24509495

  4. Dopamine D4 Receptors in Psychostimulant Addiction

    PubMed Central

    Di Ciano, Patricia; Grandy, David; Le Foll, Bernard

    2015-01-01

    Since the cloning of the D4 receptor in the 1990s, interest has been building in the role of this receptor in drug addiction, given the importance of dopamine in addiction. Like the D3 receptor, the D4 receptor has limited distribution within the brain suggesting it may have a unique role in drug abuse. However, compared to the D3 receptor, few studies have evaluated the importance of the D4 receptor. This may be due, in part, to the relative lack of compounds selective for the D4 receptor; the early studies were mainly conducted in mice lacking the D4 receptor. In this review, we summarize the literature on the structure and localization of the D4 receptor before reviewing the data from D4 knockout mice that used behavioral models relevant to the understanding of stimulant use. Next, we present evidence from more recent pharmacological studies using selective D4 agonists and antagonists and animal models of drug seeking and taking. The data summarized here suggest a role for D4 receptors in relapse to stimulant use. Therefore, treatments based on antagonism of the D4 receptor may be useful treatments for relapse to nicotine, cocaine and amphetamine use. PMID:24484981

  5. The rewarding properties of MDMA are preserved in mice lacking mu-opioid receptors.

    PubMed

    Robledo, Patricia; Mendizabal, Victoria; Ortuño, Jordi; de la Torre, Rafael; Kieffer, Brigitte L; Maldonado, Rafael

    2004-08-01

    The involvement of mu-opioid receptors in the rewarding properties of MDMA was explored in mu-opioid receptor knockout mice using the conditioning place preference paradigm. The associated release of dopamine in the nucleus accumbens was investigated by in vivo microdialysis. A significant rewarding effect of MDMA (10 mg/kg, i.p.) was observed in both wild-type and mu-opioid receptor knockout mice. MDMA (10 mg/kg, i.p.) also induced similar increases in dopamine and decreases in 3,4-dihydroxyphenylacetic acid and homovanillic acid in the nucleus accumbens dialysates of both wild-type and mu-opioid receptor knockout mice. No significant differences in basal levels of dopamine, 3,4-dihydroxyphenylacetic or homovanillic acids between wild-type and mu-opioid receptor knockout mice were observed. In summary, the present results suggest that, in contrast to what has been reported for other drugs of abuse such as opioids, ethanol, nicotine and Delta(9)-tetrahydrocannabinol, mu-opioid receptors do not play a major role in the rewarding properties of MDMA. These differences could be due to distinct mechanisms controlling dopamine release in the nucleus accumbens and suggest that the effects of MDMA on dopaminergic neurons are independent of micro -opioid receptors. PMID:15255997

  6. The Roles of P2Y2 Purinergic Receptors in Osteoblasts and Mechanotransduction

    PubMed Central

    Xing, Yanghui; Gu, Yan; Bresnahan, James J.; Paul, Emmanuel M.; Donahue, Henry J.; You, Jun

    2014-01-01

    We previously demonstrated, using osteoblastic MC3T3-E1 cells, that P2Y2 purinergic receptors are involved in osteoblast mechanotransduction. In this study, our objective was to further investigate, using a knockout mouse model, the roles of P2Y2 receptors in bone mechanobiology. We first examined bone structure with micro-CT and measured bone mechanical properties with three point bending experiments in both wild type mice and P2Y2 knockout mice. We found that bones from P2Y2 knockout mice have significantly decreased bone volume, bone thickness, bone stiffness and bone ultimate breaking force at 17 week old age. In order to elucidate the mechanisms by which P2Y2 receptors contribute to bone biology, we examined differentiation and mineralization of bone marrow cells from wild type and P2Y2 knockout mice. We found that P2Y2 receptor deficiency reduces the differentiation and mineralization of bone marrow cells. Next, we compared the response of primary osteoblasts, from both wild type and P2Y2 knockout mice, to ATP and mechanical stimulation (oscillatory fluid flow), and found that osteoblasts from wild type mice have a stronger response, in terms of ERK1/2 phosphorylation, to both ATP and fluid flow, relative to P2Y2 knockout mice. However, we did not detect any difference in ATP release in response to fluid flow between wild type and P2Y2 knock out osteoblasts. Our findings suggest that P2Y2 receptors play important roles in bone marrow cell differentiation and mineralization as well as in bone cell mechanotransduction, leading to an osteopenic phenotype in P2Y2 knockout mice. PMID:25268784

  7. Knockout of Foxp2 disrupts vocal development in mice.

    PubMed

    Castellucci, Gregg A; McGinley, Matthew J; McCormick, David A

    2016-01-01

    The FOXP2 gene is important for the development of proper speech motor control in humans. However, the role of the gene in general vocal behavior in other mammals, including mice, is unclear. Here, we track the vocal development of Foxp2 heterozygous knockout (Foxp2+/-) mice and their wildtype (WT) littermates from juvenile to adult ages, and observe severe abnormalities in the courtship song of Foxp2+/- mice. In comparison to their WT littermates, Foxp2+/- mice vocalized less, produced shorter syllable sequences, and possessed an abnormal syllable inventory. In addition, Foxp2+/- song also exhibited irregular rhythmic structure, and its development did not follow the consistent trajectories observed in WT vocalizations. These results demonstrate that the Foxp2 gene is critical for normal vocal behavior in juvenile and adult mice, and that Foxp2 mutant mice may provide a tractable model system for the study of the gene's role in general vocal motor control. PMID:26980647

  8. Drop tests of the Three Mile Island knockout canister

    SciTech Connect

    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.

  9. Neuregulin 3 Knockout Mice Exhibit Behaviors Consistent with Psychotic Disorders.

    PubMed

    Hayes, Lindsay N; Shevelkin, Alexey; Zeledon, Mariela; Steel, Gary; Chen, Pei-Lung; Obie, Cassandra; Pulver, Ann; Avramopoulos, Dimitrios; Valle, David; Sawa, Akira; Pletnikov, Mikhail V

    2016-07-01

    Neuregulin 3 (NRG3) is a paralog of NRG1. Genetic studies in schizophrenia demonstrate that risk variants in NRG3 are associated with cognitive and psychotic symptom severity, and several intronic single nucleotide polymorphisms in NRG3 are associated with delusions in patients with schizophrenia. In order to gain insights into the biological function of the gene, we generated a novel Nrg3 knockout (KO) mouse model and tested for neurobehavioral phenotypes relevant to psychotic disorders. KO mice displayed novelty-induced hyperactivity, impaired prepulse inhibition of the acoustic startle response, and deficient fear conditioning. No gross cytoarchitectonic or layer abnormalities were noted in the brain of KO mice. Our findings suggest that deletion of the Nrg3 gene leads to alterations consistent with aspects of schizophrenia. We propose that KO mice will provide a valuable animal model to determine the role of the NRG3 in the molecular pathogenesis of schizophrenia and other psychotic disorders. PMID:27606322

  10. Progranulin Knockout Accelerates Intervertebral Disc Degeneration in Aging Mice

    PubMed Central

    Zhao, Yun-peng; Tian, Qing-yun; Liu, Ben; Cuellar, Jason; Richbourgh, Brendon; Jia, Tang-hong; Liu, Chuan-ju

    2015-01-01

    Intervertebral disc (IVD) degeneration is a common degenerative disease, yet much is unknown about the mechanisms during its pathogenesis. Herein we investigated whether progranulin (PGRN), a chondroprotective growth factor, is associated with IVD degeneration. PGRN was detectable in both human and murine IVD. The levels of PGRN were upregulated in murine IVD tissue during aging process. Loss of PGRN resulted in an early onset of degenerative changes in the IVD tissue and altered expressions of the degeneration-associated molecules in the mouse IVD tissue. Moreover, PGRN knockout mice exhibited accelerated IVD matrix degeneration, abnormal bone formation and exaggerated bone resorption in vertebra with aging. The acceleration of IVD degeneration observed in PGRN null mice was probably due to the enhanced activation of NF-κB signaling and β-catenin signaling. Taken together, PGRN may play a critical role in homeostasis of IVD, and may serve as a potential molecular target for prevention and treatment of disc degenerative diseases. PMID:25777988

  11. Nonsudden limits of heavy-ion induced knockout reactions.

    PubMed

    Flavigny, F; Obertelli, A; Bonaccorso, A; Grinyer, G F; Louchart, C; Nalpas, L; Signoracci, A

    2012-06-22

    We report on the single neutron and proton removal reactions from unstable nuclei with large asymmetry ΔS = S(n)-S(p) at incident energies below 80 MeV/nucleon. Strong nonsudden effects are observed in the case of deeply-bound-nucleon removal. The corresponding parallel momentum distributions exhibit an abrupt cutoff at high momentum that corresponds to an energy threshold occurring when the incident energy per particle is of comparable magnitude to the nucleon separation energy. A large low-momentum tail is related to both dissipative processes and the dynamics of the nucleon removal process. New limits for the applicability of the sudden and eikonal approximations in nucleon knockout are given. PMID:23004591

  12. Spectroscopy of 17C via one-neutron knockout reaction

    NASA Astrophysics Data System (ADS)

    Kim, Sunji; Hwang, Jongwon; Satou, Yoshiteru; Orr, Nigel A.; Nakamura, Takashi; Kondo, Yosuke; Gibelin, Julien; Achouri, N. Lynda; Aumann, Thomas; Baba, Hidetada; Delaunay, Franck; Doornenbal, Pieter; Fukuda, Naoki; Inabe, Naohito; Isobe, Tadaaki; Kameda, Daisuke; Kanno, Daiki; Kobayashi, Nobuyuki; Kobayashi, Toshio; Kubo, Toshiyuki; Leblond, Sylvain; Lee, Jenny; Marqués, F. Miguel; Minakata, Ryogo; Motobayashi, Tohru; Murai, Daichi; Murakami, Tetsuya; Muto, Kotomi; Nakashima, Tomohiro; Nakatsuka, Noritsugu; Navin, Alahari; Nishi, Seijiro; Ogoshi, Shun; Otsu, Hideaki; Sato, Hiromi; Shimizu, Yohei; Suzuki, Hiroshi; Takahashi, Kento; Takeda, Hiroyuki; Takeuchi, Satoshi; Tanaka, Ryuki; Togano, Yasuhiro; Tuff, Adam G.; Vandebrouck, Marine; Yoneda, Ken-ichiro

    2016-03-01

    A spectroscopic study of 17C was performed via the one-neutron knockout reaction of 18C on a carbon target at RIKEN-RIBF. Three unbound states at excitation energies of 2.66(2), 3.16(5), and 3.97(3) MeV (preliminary) were observed. The energies are compared with shell-model calculations and existing measurements to deduce their spin-parities. From the comparison, the states at 2.66(2) and 3.97(3) MeV are suggested to be 1/2- and 3/2-, respectively. From its decay property, the state at 3.16(5) MeV is indicated to be 9/2+.

  13. Fission product release from nuclear fuel by recoil and knockout

    NASA Astrophysics Data System (ADS)

    Lewis, B. J.

    1987-03-01

    An analytical model has been developed to describe the fission product release from nuclear fuel arising from the surface-fission release mechanisms of recoil and knockout. Release expressions are evaluated and compared to the short-lived activity measurements from in-reactor experiments with intact operating fuel. Recoil is shown to be an important process for releasing fission products from free UO 2 surfaces into the fuel-to-sheath gap. The model is also applied to tramp uranium in a power reactor primary heat transport circuit where it is demonstrated that recoil is the dominant release mechanism for small particles of fuel which are deposited on in-core surfaces. A methodology is established whereby release from surface contamination can be distinguished from that of fuel pin failure.

  14. Increased desensitization of dopamine D₂ receptor-mediated response in the ventral tegmental area in the absence of adenosine A(2A) receptors.

    PubMed

    Al-Hasani, R; Foster, J D; Metaxas, A; Ledent, C; Hourani, S M O; Kitchen, I; Chen, Y

    2011-09-01

    G-protein coupled receptors interact to provide additional regulatory mechanisms for neurotransmitter signaling. Adenosine A(2A) receptors are expressed at a high density in striatal neurons, where they closely interact with dopamine D₂ receptors and modulate effects of dopamine and responses to psychostimulants. A(2A) receptors are expressed at much lower densities in other forebrain neurons but play a more prominent yet opposing role to striatal receptors in response to psychostimulants in mice. It is, therefore, possible that A(2A) receptors expressed at low levels elsewhere in the brain may also regulate neurotransmitter systems and modulate neuronal functions. Dopamine D₂ receptors play an important role in autoinhibition of neuronal firing in dopamine neurons of the ventral tegmental area (VTA) and dopamine release in other brain areas. Here, we examined the effect of A(2A) receptor deletion on D₂ receptor-mediated inhibition of neuronal firing in dopamine neurons in the VTA. Spontaneous activity of dopamine neurons was recorded in midbrain slices, and concentration-dependent effects of the dopamine D₂ receptor agonist, quinpirole, was compared between wild-type and A(2A) knockout mice. The potency of quinpirole applied in single concentrations and the expression of D₂ receptors were not altered in the VTA of the knockout mice. However, quinpirole applied in stepwise escalating concentrations caused significantly reduced maximal inhibition in A(2A) knockout mice, indicating an enhanced agonist-induced desensitization of D₂ receptors in the absence of A(2A) receptors. The A(2A) receptor agonist, CGS21680, did not exert any effect on dopamine neuron firing or response to quinpirole, revealing a novel non-pharmacological interaction between adenosine A(2A) receptors and dopaminergic neurotransmission in midbrain dopamine neurons. Altered D₂ receptor desensitization may result in changes in dopamine neuron firing rate and pattern and dopamine

  15. Bioelectric characterization of epithelia from neonatal CFTR knockout ferrets.

    PubMed

    Fisher, John T; Tyler, Scott R; Zhang, Yulong; Lee, Ben J; Liu, Xiaoming; Sun, Xingshen; Sui, Hongshu; Liang, Bo; Luo, Meihui; Xie, Weiliang; Yi, Yaling; Zhou, Weihong; Song, Yi; Keiser, Nicholas; Wang, Kai; de Jonge, Hugo R; Engelhardt, John F

    2013-11-01

    Cystic fibrosis (CF) is a life-shortening, recessive, multiorgan genetic disorder caused by the loss of CF transmembrane conductance regulator (CFTR) chloride channel function found in many types of epithelia. Animal models that recapitulate the human disease phenotype are critical to understanding pathophysiology in CF and developing therapies. CFTR knockout ferrets manifest many of the phenotypes observed in the human disease, including lung infections, pancreatic disease and diabetes, liver disease, malnutrition, and meconium ileus. In the present study, we have characterized abnormalities in the bioelectric properties of the trachea, stomach, intestine, and gallbladder of newborn CF ferrets. Short-circuit current (ISC) analysis of CF and wild-type (WT) tracheas revealed the following similarities and differences: (1) amiloride-sensitive sodium currents were similar between genotypes; (2) responses to 4,4'-diisothiocyano-2,2'-stilbene disulphonic acid were 3.3-fold greater in CF animals, suggesting elevated baseline chloride transport through non-CFTR channels in a subset of CF animals; and (3) a lack of 3-isobutyl-1-methylxanthine (IBMX)/forskolin-stimulated and N-(2-Naphthalenyl)-((3,5-dibromo-2,4-dihydroxyphenyl)methylene)glycine hydrazide (GlyH-101)-inhibited currents in CF animals due to the lack of CFTR. CFTR mRNA was present throughout all levels of the WT ferret and IBMX/forskolin-inducible ISC was only observed in WT animals. However, despite the lack of CFTR function in the knockout ferret, the luminal pH of the CF ferret gallbladder, stomach, and intestines was not significantly changed relative to WT. The WT stomach and gallbladder exhibited significantly enhanced IBMX/forskolin ISC responses and inhibition by GlyH-101 relative to CF samples. These findings demonstrate that multiple organs affected by disease in the CF ferret have bioelectric abnormalities consistent with the lack of cAMP-mediated chloride transport. PMID:23782101

  16. Preaxial Polydactyly in Sost/Sostdc1 Double Knockouts

    SciTech Connect

    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.

  17. Intercellular Adhesion Molecule 1 Knockout Abrogates Radiation Induced Pulmonary Inflammation

    NASA Astrophysics Data System (ADS)

    Hallahan, Dennis E.; Virudachalam, Subbulakshmi

    1997-06-01

    Increased expression of intercellular adhesion molecule 1 (ICAM-1; CD54) is induced by exposure to ionizing radiation. The lung was used as a model to study the role of ICAM-1 in the pathogenesis of the radiation-induced inflammation-like response. ICAM-1 expression increased in the pulmonary microvascular endothelium and not in the endothelium of larger pulmonary vessels following treatment of mice with thoracic irradiation. To quantify radiation-induced ICAM-1 expression, we utilized fluorescence-activated cell sorting analysis of anti-ICAM-1 antibody labeling of pulmonary microvascular endothelial cells from human cadaver donors (HMVEC-L cells). Fluorochrome conjugates and UV microscopy were used to quantify the fluorescence intensity of ICAM in the irradiated lung. These studies showed a dose- and time-dependent increase in ICAM-1 expression in the pulmonary microvascular endothelium. Peak expression occurred at 24 h, while threshold dose was as low as 2 Gy. To determine whether ICAM-1 is required for inflammatory cell infiltration into the irradiated lung, the anti-ICAM-1 blocking antibody was administered by tail vein injection to mice following thoracic irradiation. Inflammatory cells were quantified by immunofluorescence for leukocyte common antigen (CD45). Mice treated with the anti-ICAM-1 blocking antibody showed attenuation of inflammatory cell infiltration into the lung in response to ionizing radiation exposure. To verify the requirement of ICAM-1 in the inflammation-like radiation response, we utilized the ICAM-1 knockout mouse. ICAM-1 was not expressed in the lungs of ICAM-1-deficient mice following treatment with thoracic irradiation. ICAM-1 knockout mice had no increase in the inflammatory cell infiltration into the lung in response to thoracic irradiation. These studies demonstrate a radiation dose-dependent increase in ICAM-1 expression in the pulmonary microvascular endothelium, and show that ICAM-1 is required for inflammatory cell infiltration

  18. Intercellular adhesion molecule 1 knockout abrogates radiation induced pulmonary inflammation.

    PubMed

    Hallahan, D E; Virudachalam, S

    1997-06-10

    Increased expression of intercellular adhesion molecule 1 (ICAM-1; CD54) is induced by exposure to ionizing radiation. The lung was used as a model to study the role of ICAM-1 in the pathogenesis of the radiation-induced inflammation-like response. ICAM-1 expression increased in the pulmonary microvascular endothelium and not in the endothelium of larger pulmonary vessels following treatment of mice with thoracic irradiation. To quantify radiation-induced ICAM-1 expression, we utilized fluorescence-activated cell sorting analysis of anti-ICAM-1 antibody labeling of pulmonary microvascular endothelial cells from human cadaver donors (HMVEC-L cells). Fluorochrome conjugates and UV microscopy were used to quantify the fluorescence intensity of ICAM in the irradiated lung. These studies showed a dose- and time-dependent increase in ICAM-1 expression in the pulmonary microvascular endothelium. Peak expression occurred at 24 h, while threshold dose was as low as 2 Gy. To determine whether ICAM-1 is required for inflammatory cell infiltration into the irradiated lung, the anti-ICAM-1 blocking antibody was administered by tail vein injection to mice following thoracic irradiation. Inflammatory cells were quantified by immunofluorescence for leukocyte common antigen (CD45). Mice treated with the anti-ICAM-1 blocking antibody showed attenuation of inflammatory cell infiltration into the lung in response to ionizing radiation exposure. To verify the requirement of ICAM-1 in the inflammation-like radiation response, we utilized the ICAM-1 knockout mouse. ICAM-1 was not expressed in the lungs of ICAM-1-deficient mice following treatment with thoracic irradiation. ICAM-1 knockout mice had no increase in the inflammatory cell infiltration into the lung in response to thoracic irradiation. These studies demonstrate a radiation dose-dependent increase in ICAM-1 expression in the pulmonary microvascular endothelium, and show that ICAM-1 is required for inflammatory cell infiltration

  19. Bioelectric Characterization of Epithelia from Neonatal CFTR Knockout Ferrets

    PubMed Central

    Fisher, John T.; Tyler, Scott R.; Zhang, Yulong; Lee, Ben J.; Liu, Xiaoming; Sun, Xingshen; Sui, Hongshu; Liang, Bo; Luo, Meihui; Xie, Weiliang; Yi, Yaling; Zhou, Weihong; Song, Yi; Keiser, Nicholas; Wang, Kai; de Jonge, Hugo R.

    2013-01-01

    Cystic fibrosis (CF) is a life-shortening, recessive, multiorgan genetic disorder caused by the loss of CF transmembrane conductance regulator (CFTR) chloride channel function found in many types of epithelia. Animal models that recapitulate the human disease phenotype are critical to understanding pathophysiology in CF and developing therapies. CFTR knockout ferrets manifest many of the phenotypes observed in the human disease, including lung infections, pancreatic disease and diabetes, liver disease, malnutrition, and meconium ileus. In the present study, we have characterized abnormalities in the bioelectric properties of the trachea, stomach, intestine, and gallbladder of newborn CF ferrets. Short-circuit current (ISC) analysis of CF and wild-type (WT) tracheas revealed the following similarities and differences: (1) amiloride-sensitive sodium currents were similar between genotypes; (2) responses to 4,4′-diisothiocyano-2,2′-stilbene disulphonic acid were 3.3-fold greater in CF animals, suggesting elevated baseline chloride transport through non-CFTR channels in a subset of CF animals; and (3) a lack of 3-isobutyl-1-methylxanthine (IBMX)/forskolin–stimulated and N-(2-Naphthalenyl)-((3,5-dibromo-2,4-dihydroxyphenyl)methylene)glycine hydrazide (GlyH-101)–inhibited currents in CF animals due to the lack of CFTR. CFTR mRNA was present throughout all levels of the WT ferret and IBMX/forskolin–inducible ISC was only observed in WT animals. However, despite the lack of CFTR function in the knockout ferret, the luminal pH of the CF ferret gallbladder, stomach, and intestines was not significantly changed relative to WT. The WT stomach and gallbladder exhibited significantly enhanced IBMX/forskolin ISC responses and inhibition by GlyH-101 relative to CF samples. These findings demonstrate that multiple organs affected by disease in the CF ferret have bioelectric abnormalities consistent with the lack of cAMP-mediated chloride transport. PMID:23782101

  20. Reduced Extinction of Hippocampal-Dependent Memories in CPEB Knockout Mice

    ERIC Educational Resources Information Center

    Zearfoss, N. Ruth; Richter, Joel D.; Berger-Sweeney, Joanne

    2006-01-01

    CPEB is a sequence-specific RNA binding protein that regulates translation at synapses. In neurons of CPEB knockout mice, synaptic efficacy is reduced. Here, we have performed a battery of behavioral tests and find that relative to wild-type animals, CPEB knockout mice, although similar on many baseline behaviors, have reduced extinction of…

  1. Paigen Diet–Fed Apolipoprotein E Knockout Mice Develop Severe Pulmonary Hypertension in an Interleukin-1–Dependent Manner

    PubMed Central

    Lawrie, Allan; Hameed, Abdul G.; Chamberlain, Janet; Arnold, Nadine; Kennerley, Aneurin; Hopkinson, Kay; Pickworth, Josephine; Kiely, David G.; Crossman, David C.; Francis, Sheila E.

    2011-01-01

    Inflammatory mechanisms are proposed to play a significant role in the pathogenesis of pulmonary arterial hypertension (PAH). Previous studies have described PAH in fat-fed apolipoprotein E knockout (ApoE−/−) mice. We have reported that signaling in interleukin-1–receptor–knockout (IL-1R1−/−) mice leads to a reduction in diet-induced systemic atherosclerosis. We subsequently hypothesized that double-null (ApoE−/−/IL-1R1−/−) mice would show a reduced PAH phenotype compared with that of ApoE−/− mice. Male IL-1R1−/−, ApoE−/−, and ApoE−/−/IL-1R1−/− mice were fed regular chow or a high-fat diet (Paigen diet) for 8 weeks before phenotyping for PAH. No abnormal phenotype was observed in the IL-1R1−/− mice. Fat-fed ApoE−/− mice developed significantly increased right ventricular systolic pressure and substantial pulmonary vascular remodeling. Surprisingly, ApoE−/−/IL-1R1−/− mice showed an even more severe PAH phenotype. Further molecular investigation revealed the expression of a putative, alternatively primed IL-1R1 transcript expressed within the lungs but not aorta of ApoE−/−/IL-1R1−/− mice. Treatment of ApoE−/− and ApoE−/−/IL-1R1−/− mice with IL-1–receptor antagonist prevented progression of the PAH phenotype in both strains. Blocking IL-1 signaling may have beneficial effects in treating PAH, and alternative IL-1–receptor signaling in the lung may be important in driving PAH pathogenesis. PMID:21835155

  2. Kidney development and gene expression in the HIF2alpha knockout mouse.

    PubMed

    Steenhard, Brooke M; Freeburg, Paul B; Isom, Kathryn; Stroganova, Larysa; Borza, Dorin-Bogdan; Hudson, Billy G; St John, Patricia L; Zelenchuk, Adrian; Abrahamson, Dale R

    2007-04-01

    The hypoxia-inducible transcription factor-2 (HIF2), a heterodimer composed of HIF2alpha and HIF1beta subunits, drives expression of genes essential for vascularization, including vascular endothelial growth factor (VEGF) and VEGF receptor-2 (VEGFR-2, Flk-1). Here, we used a HIF2alpha/LacZ transgenic mouse to define patterns of HIF2alpha transcription during kidney development and maturation. Our results from embryonic heterozygotes showed HIF2alpha/LacZ expression by apparently all renal endothelial cells. At 4 weeks of age, glomerular mesangial and vascular smooth muscle cells were also positive together with endothelial cells. These expression patterns were confirmed by electron microscopy using Bluo-gal as a beta-galactosidase substrate. Small numbers of glomerular and tubular epithelial cells were also positive at all stages examined. Light and electron microscopic examination of kidneys from HIF2alpha null embryos showed no defects in renal vascular development or nephrogenesis. Similarly, the same amounts of Flk-1 protein were seen on Western blots of kidney extracts from homozygous and heterozygous HIF2alpha mutants. To examine responsiveness of HIF2alpha null kidneys to hypoxia, embryonic day 13.5 metanephroi were cultured in room air or in mild (5% O(2)) hypoxia. For both heterozygous and null samples, VEGF mRNA levels doubled when metanephroi were cultured in mild hypoxia. Anterior chamber grafts of embryonic HIF2alpha knockouts were morphologically indistinguishable from heterozygous grafts. Endothelial markers, platelet endothelial cell adhesion molecule and BsLB4, as well as glomerular epithelial markers, GLEPP1 and WT-1, were all expressed appropriately. Finally, we undertook quantitative real-time polymerase chain reaction of kidneys from HIF2alpha null embryos and wild-type siblings and found no compensatory up-regulation of HIF1alpha or -3alpha. Our results show that, although HIF2alpha was widely transcribed by kidney endothelium and vascular

  3. Protein tyrosine phosphatase alpha (PTP alpha) knockout mice show deficits in Morris water maze learning, decreased locomotor activity, and decreases in anxiety.

    PubMed

    Skelton, Matthew R; Ponniah, Sathivel; Wang, Dennis Z-M; Doetschman, Thomas; Vorhees, Charles V; Pallen, Catherine J

    2003-09-12

    Receptor PTPalpha is a widely expressed transmembrane enzyme enriched in brain. PTPalpha knockout (PTPalpha(-/-)) mice are viable and display no gross abnormalities. Brain and embryo derived fibroblast src and fyn activity is reduced to <50% in PTPalpha(-/-) mice. These protein kinases are implicated in multiple aspects of neuronal development and function. However, the effect of the loss of function of the PTPalpha gene on behavior has yet to be investigated. PTPalpha(-/-) and WT mice were tested for anxiety, swimming ability, spatial learning, cued learning, locomotor activity, and novel object recognition (NOR). PTPalpha(-/-) mice were indistinguishable from WT in swimming ability, cued learning and novel object recognition. Knockout mice showed decreased anxiety without an increase in head dips and stretch-attend movements. During Morris water maze (MWM) learning, PTPalpha(-/-) mice had increased latencies to reach the goal compared to WT on acquisition, but no memory deficit on probe trials. On reversal learning, knockout mice showed no significant effects. PTPalpha(-/-) mice showed decreased exploratory locomotor activity, but responded normally to a challenge dose of D-methamphetamine. The data suggest that PTPalpha serves a regulatory function in learning and other forms of neuroplasticity. PMID:12932834

  4. Health and population effects of rare gene knockouts in adult humans with related parents.

    PubMed

    Narasimhan, Vagheesh M; Hunt, Karen A; Mason, Dan; Baker, Christopher L; Karczewski, Konrad J; Barnes, Michael R; Barnett, Anthony H; Bates, Chris; Bellary, Srikanth; Bockett, Nicholas A; Giorda, Kristina; Griffiths, Christopher J; Hemingway, Harry; Jia, Zhilong; Kelly, M Ann; Khawaja, Hajrah A; Lek, Monkol; McCarthy, Shane; McEachan, Rosie; O'Donnell-Luria, Anne; Paigen, Kenneth; Parisinos, Constantinos A; Sheridan, Eamonn; Southgate, Laura; Tee, Louise; Thomas, Mark; Xue, Yali; Schnall-Levin, Michael; Petkov, Petko M; Tyler-Smith, Chris; Maher, Eamonn R; Trembath, Richard C; MacArthur, Daniel G; Wright, John; Durbin, Richard; van Heel, David A

    2016-04-22

    Examining complete gene knockouts within a viable organism can inform on gene function. We sequenced the exomes of 3222 British adults of Pakistani heritage with high parental relatedness, discovering 1111 rare-variant homozygous genotypes with predicted loss of function (knockouts) in 781 genes. We observed 13.7% fewer homozygous knockout genotypes than we expected, implying an average load of 1.6 recessive-lethal-equivalent loss-of-function (LOF) variants per adult. When genetic data were linked to the individuals' lifelong health records, we observed no significant relationship between gene knockouts and clinical consultation or prescription rate. In this data set, we identified a healthy PRDM9-knockout mother and performed phased genome sequencing on her, her child, and control individuals. Our results show that meiotic recombination sites are localized away from PRDM9-dependent hotspots. Thus, natural LOF variants inform on essential genetic loci and demonstrate PRDM9 redundancy in humans. PMID:26940866

  5. Health and population effects of rare gene knockouts in adult humans with related parents

    PubMed Central

    Narasimhan, Vagheesh M.; Hunt, Karen A.; Mason, Dan; Baker, Christopher L.; Karczewski, Konrad J.; Barnes, Michael R.; Barnett, Anthony H.; Bates, Chris; Bellary, Srikanth; Bockett, Nicholas A.; Giorda, Kristina; Griffiths, Christopher J.; Hemingway, Harry; Jia, Zhilong; Kelly, M. Ann; Khawaja, Hajrah A.; Lek, Monkol; McCarthy, Shane; McEachan, Rosie; O’Donnell-Luria, Anne; Paigen, Kenneth; Parisinos, Constantinos A.; Sheridan, Eamonn; Southgate, Laura; Tee, Louise; Thomas, Mark; Xue, Yali; Schnall-Levin, Michael; Petkov, Petko M.; Tyler-Smith, Chris; Maher, Eamonn R.; Trembath, Richard C.; MacArthur, Daniel G.; Wright, John; Durbin, Richard; van Heel, David A.

    2016-01-01

    Examining complete gene knockouts within a viable organism can inform on gene function. We sequenced the exomes of 3,222 British Pakistani-heritage adults with high parental relatedness, discovering 1,111 rare-variant homozygous genotypes with predicted loss of gene function (knockouts) in 781 genes. We observed 13.7% fewer than expected homozygous knockout genotypes, implying an average load of 1.6 recessive-lethal-equivalent LOF variants per adult. Linking genetic data to lifelong health records, knockouts were not associated with clinical consultation or prescription rate. In this dataset we identified a healthy PRDM9 knockout mother, and performed phased genome sequencing on her, her child and controls, which showed meiotic recombination sites localised away from PRDM9-dependent hotspots. Thus, natural LOF variants inform upon essential genetic loci, and demonstrate PRDM9 redundancy in humans. PMID:26940866

  6. Regulation of plasminogen receptors.

    PubMed

    Herren, Thomas; Swaisgood, Carmen; Plow, Edward F

    2003-01-01

    carboxypeptidase N and plasma carboxypeptidase B (TAFI) mediate such down-regulation. A non-protease dependent pathway for modulation of plasminogen receptors may be initiated by growth factors, chemokines or cytokines that alter the cell membrane and/or cytoskeleton architectures to expose plasminogen binding sites. Many examples of the modulation of plasminogen receptors have been demonstrated in vitro, and the development of knock-out mice may soon lead to incisive evaluations of the significance of the regulation of plasminogen receptors in vivo. PMID:12456318

  7. AMPKα1 knockout enhances nociceptive behaviors and spinal glutamatergic synaptic activities via production of reactive oxygen species in the spinal dorsal horn.

    PubMed

    Maixner, Dylan W; Yan, Xisheng; Hooks, Shelley B; Weng, Han-Rong

    2016-06-21

    Emerging studies have shown that pharmacological activation of adenosine monophosphate-activated protein kinase (AMPK) produces potent analgesic effects in different animal pain models. Currently, the spinal molecular and synaptic mechanism by which AMPK regulates the pain signaling system remains unclear. To address this issue, we utilized the Cre-LoxP system to conditionally knockout the AMPKα1 gene in the nervous system of mice. We demonstrated that AMPKα1 is imperative for maintaining normal nociception, and mice deficient for AMPKα1 exhibit mechanical allodynia. This is concomitantly associated with increased glutamatergic synaptic activities in neurons located in the superficial spinal dorsal horn, which results from the increased glutamate release from presynaptic terminals and function of ligand-gated glutamate receptors at the postsynaptic neurons. Additionally, AMPKα1 knockout mice have increased activities of extracellular signal-regulated kinases (ERK) and p38 mitogen-activated protein kinases (p38), as well as elevated levels of interleukin-1β (IL-1β), reactive oxygen species (ROS), and heme oxygenase 1 (HO-1) in the spinal dorsal horn. Systemic administration of a non-specific ROS scavenger (phenyl-N-tert-butylnitrone, PBN) or a HO-1 activator (Cobalt protoporphyrin IX, CoPP) attenuated allodynia in AMPKα1 knockout mice. Bath-perfusion of the ROS scavenger or HO-1 activator effectively attenuated the increased ROS levels and glutamatergic synaptic activities in the spinal dorsal horn. Our findings suggest that ROS are the key down-stream signaling molecules mediating the behavioral hypersensitivity in AMPKα1 knockout mice. Thus, targeting AMPKα1 may represent an effective approach for the treatment of pathological pain conditions associated with neuroinflammation at the spinal dorsal horn. PMID:27058143

  8. Update on melatonin receptors: IUPHAR Review 20.

    PubMed

    Jockers, Ralf; Delagrange, Philippe; Dubocovich, Margarita L; Markus, Regina P; Renault, Nicolas; Tosini, Gianluca; Cecon, Erika; Zlotos, Darius P

    2016-09-01

    Melatonin receptors are seven transmembrane-spanning proteins belonging to the GPCR superfamily. In mammals, two melatonin receptor subtypes exist - MT1 and MT2 - encoded by the MTNR1A and MTNR1B genes respectively. The current review provides an update on melatonin receptors by the corresponding subcommittee of the International Union of Basic and Clinical Pharmacology. We will highlight recent developments of melatonin receptor ligands, including radioligands, and give an update on the latest phenotyping results of melatonin receptor knockout mice. The current status and perspectives of the structure of melatonin receptor will be summarized. The physiological importance of melatonin receptor dimers and biologically important and type 2 diabetes-associated genetic variants of melatonin receptors will be discussed. The role of melatonin receptors in physiology and disease will be further exemplified by their functions in the immune system and the CNS. Finally, antioxidant and free radical scavenger properties of melatonin and its relation to melatonin receptors will be critically addressed. PMID:27314810

  9. Phenobarbital and Insulin Reciprocate Activation of the Nuclear Receptor Constitutive Androstane Receptor through the Insulin Receptor.

    PubMed

    Yasujima, Tomoya; Saito, Kosuke; Moore, Rick; Negishi, Masahiko

    2016-05-01

    Phenobarbital (PB) antagonized insulin to inactivate the insulin receptor and attenuated the insulin receptor downstream protein kinase B (AKT)-forkhead box protein O1 and extracellular signal-regulated kinase 1/2 signals in mouse primary hepatocytes and HepG2 cells. Hepatic AKT began dephosphorylation in an early stage of PB treatment, and blood glucose levels transiently increased in both wild-type and constitutive androstane receptor (CAR) knockout (KO) mice. On the other hand, blood glucose levels increased in wild-type mice, but not KO mice, in later stages of PB treatment. As a result, PB, acting as an insulin receptor antagonist, elicited CAR-independent increases and CAR-dependent decreases of blood glucose levels at these different stages of treatment, respectively. Reciprocally, insulin activation of the insulin receptor repressed CAR activation and induction of its target CYP2B6 gene in HepG2 cells. Thus, PB and insulin cross-talk through the insulin receptor to regulate glucose and drug metabolism reciprocally. PMID:26994072

  10. Phenobarbital and Insulin Reciprocate Activation of the Nuclear Receptor Constitutive Androstane Receptor through the Insulin Receptor

    PubMed Central

    Yasujima, Tomoya; Saito, Kosuke; Moore, Rick

    2016-01-01

    Phenobarbital (PB) antagonized insulin to inactivate the insulin receptor and attenuated the insulin receptor downstream protein kinase B (AKT)–forkhead box protein O1 and extracellular signal-regulated kinase 1/2 signals in mouse primary hepatocytes and HepG2 cells. Hepatic AKT began dephosphorylation in an early stage of PB treatment, and blood glucose levels transiently increased in both wild-type and constitutive androstane receptor (CAR) knockout (KO) mice. On the other hand, blood glucose levels increased in wild-type mice, but not KO mice, in later stages of PB treatment. As a result, PB, acting as an insulin receptor antagonist, elicited CAR-independent increases and CAR-dependent decreases of blood glucose levels at these different stages of treatment, respectively. Reciprocally, insulin activation of the insulin receptor repressed CAR activation and induction of its target CYP2B6 gene in HepG2 cells. Thus, PB and insulin cross-talk through the insulin receptor to regulate glucose and drug metabolism reciprocally. PMID:26994072

  11. Lipoprotein Receptors Redundantly Participate in Entry of Hepatitis C Virus

    PubMed Central

    Ono, Chikako; Uemura, Kentaro; Kawachi, Yukako; Shiokawa, Mai; Mori, Hiroyuki; Wada, Masami; Shima, Ryoichi; Okamoto, Toru; Hiraga, Nobuhiko; Suzuki, Ryosuke; Chayama, Kazuaki; Wakita, Takaji; Matsuura, Yoshiharu

    2016-01-01

    Scavenger receptor class B type 1 (SR-B1) and low-density lipoprotein receptor (LDLR) are known to be involved in entry of hepatitis C virus (HCV), but their precise roles and their interplay are not fully understood. In this study, deficiency of both SR-B1 and LDLR in Huh7 cells was shown to impair the entry of HCV more strongly than deficiency of either SR-B1 or LDLR alone. In addition, exogenous expression of not only SR-B1 and LDLR but also very low-density lipoprotein receptor (VLDLR) rescued HCV entry in the SR-B1 and LDLR double-knockout cells, suggesting that VLDLR has similar roles in HCV entry. VLDLR is a lipoprotein receptor, but the level of its hepatic expression was lower than those of SR-B1 and LDLR. Moreover, expression of mutant lipoprotein receptors incapable of binding to or uptake of lipid resulted in no or slight enhancement of HCV entry in the double-knockout cells, suggesting that binding and/or uptake activities of lipid by lipoprotein receptors are essential for HCV entry. In addition, rescue of infectivity in the double-knockout cells by the expression of the lipoprotein receptors was not observed following infection with pseudotype particles bearing HCV envelope proteins produced in non-hepatic cells, suggesting that lipoproteins associated with HCV particles participate in the entry through their interaction with lipoprotein receptors. Buoyant density gradient analysis revealed that HCV utilizes these lipoprotein receptors in a manner dependent on the lipoproteins associated with HCV particles. Collectively, these results suggest that lipoprotein receptors redundantly participate in the entry of HCV. PMID:27152966

  12. Roles of transferrin receptors in erythropoiesis.

    PubMed

    Kawabata, Hiroshi; Sakamoto, Soichiro; Masuda, Taro; Uchiyama, Tatsuki; Ohmori, Katsuyuki; Koeffler, H Phillip; Takaori-Kondo, Akifumi

    2016-07-01

    Erythropoiesis requires large amounts of iron for hemoglobin synthesis, which is mainly provided by macrophages and the intestines in a transferrin (Tf)-bound form. Bone marrow erythroblasts incorporate Tf through endocytosis, which is mediated by transferrin receptor 1 (TFR1). Recently, human TFR1, aside from its role as a Tf receptor, was also found to be a receptor for the H-subunit of ferritin (FTH). In humans, hematopoietic erythroid precursor cells express high levels of TFR1 and specifically take up the FTH homopolymer (H-ferritin). H-ferritin inhibits the formation of burst forming unit-erythroid colonies in vitro. TFR2, which is also a Tf receptor, is predominantly expressed in hepatocytes and erythroid precursor cells. In the liver, TFR2 forms a complex with HFE, a hereditary hemochromatosis-associated protein, and acts as an iron sensor. In mice, hepatocyte-specific knockout of the TFR2 gene has been shown to cause systemic iron-overload with decreased expression of hepcidin, the central regulator of iron homeostasis. In erythroid cells, TFR2 forms a complex with the erythropoietin receptor and facilitates its trafficking to the cell membrane. Moreover, hematopoietic cell-specific knockout of the TFR2 gene causes microcytic erythrocytosis in mice. This review focuses on the molecular evolution and functions of these TFRs and their ligands. PMID:27498743

  13. The impact of P-glycoprotein on the disposition of drugs targeted for indications of the central nervous system: evaluation using the MDR1A/1B knockout mouse model.

    PubMed

    Doran, Angela; Obach, R Scott; Smith, Bill J; Hosea, Natilie A; Becker, Stacey; Callegari, Ernesto; Chen, Cuiping; Chen, Xi; Choo, Edna; Cianfrogna, Julie; Cox, Loretta M; Gibbs, John P; Gibbs, Megan A; Hatch, Heather; Hop, Cornelis E C A; Kasman, Ilana N; Laperle, Jennifer; Liu, Jianhua; Liu, Xingrong; Logman, Michael; Maclin, Debra; Nedza, Frank M; Nelson, Frederick; Olson, Emily; Rahematpura, Sandhya; Raunig, David; Rogers, Sabrinia; Schmidt, Kari; Spracklin, Douglas K; Szewc, Mark; Troutman, Matthew; Tseng, Elaine; Tu, Meihua; Van Deusen, Jeffrey W; Venkatakrishnan, Karthik; Walens, Gary; Wang, Ellen Q; Wong, Diane; Yasgar, Adam S; Zhang, Chenghong

    2005-01-01

    Thirty-two structurally diverse drugs used for the treatment of various conditions of the central nervous system (CNS), along with two active metabolites, and eight non-CNS drugs were measured in brain, plasma, and cerebrospinal fluid in the P-glycoprotein (P-gp) knockout mouse model after subcutaneous administration, and the data were compared with corresponding data obtained in wild-type mice. Total brain-to-plasma (B/P) ratios for the CNS agents ranged from 0.060 to 24. Of the 34 CNS-active agents, only 7 demonstrated B/P area under the plasma concentration curve ratios between P-gp knockout and wild-type mice that did not differ significantly from unity. Most of the remaining drugs demonstrated 1.1- to 2.6-fold greater B/P ratios in P-gp knockout mice versus wild-type mice. Three, risperidone, its active metabolite 9-hydroxyrisperidone, and metoclopramide, showed marked differences in B/P ratios between knockout and wild-type mice (6.6- to 17-fold). Differences in B/P ratios and cerebrospinal fluid/plasma ratios between wild-type and knockout animals were correlated. Through the use of this model, it appears that most CNS-active agents demonstrate at least some P-gp-mediated transport that can affect brain concentrations. However, the impact for the majority of agents is probably minor. The example of risperidone illustrates that even good P-gp substrates can still be clinically useful CNS-active agents. However, for such agents, unbound plasma concentrations may need to be greater than values projected using receptor affinity data to achieve adequate receptor occupancy for effect. PMID:15502009

  14. Final-state interactions in two-nucleon knockout reactions

    NASA Astrophysics Data System (ADS)

    Colle, Camille; Cosyn, Wim; Ryckebusch, Jan

    2016-03-01

    Background: Exclusive two-nucleon knockout after electroexcitation of nuclei [A (e ,e'N N ) in brief] is considered to be a primary source of information about short-range correlations (SRCs) in nuclei. For a proper interpretation of the data, final-state interactions (FSIs) need to be theoretically controlled. Purpose: Our goal is to quantify the role of FSI effects in exclusive A (e ,e'p N ) reactions for four target nuclei representative of the whole mass region. Our focus is on processes that are SRC driven. We investigate the role of FSIs for two characteristic detector setups corresponding to "small" and "large" coverage of the available phase space. Method: Use is made of a factorized expression for the A (e ,e'p N ) cross section that is proportional to the two-body center-of-mass (c.m.) momentum distribution of close-proximity pairs. The A (e ,e'p p ) and A (e ,e'p n ) reactions for the target nuclei 12C,27Al,56Fe, and 208Pb are investigated. The elastic attenuation mechanisms in the FSIs are included using the relativistic multiple-scattering Glauber approximation (RMSGA). Single-charge exchange (SCX) reactions are also included. We introduce the nuclear transparency TAp N, defined as the ratio of exclusive (e ,e'p N ) cross sections on nuclei to those on "free" nucleon pairs, as a measure for the aggregated effect of FSIs in p N knockout reactions from nucleus A . A toy model is introduced in order to gain a better understanding of the A dependence of TAp N. Results: The transparency TAp N drops from 0.2 -0.3 for 12C to 0.04 -0.07 for 208Pb. For all considered kinematics, the mass dependence of TAp N can be captured by the power law TAp N∝A-λ with 0.4 ≲λ ≲0.5 . Apart from an overall reduction factor, we find that FSIs only modestly affect the distinct features of SRC-driven A (e ,e'p N ) which are dictated by the c.m. distribution of close-proximity pairs. Conclusion: The SCX mechanisms represent a relatively small (order of a few percent

  15. Complementation Test of Rpe65 Knockout and Tvrm148

    PubMed Central

    Wright, Charles B.; Chrenek, Micah A.; Foster, Stephanie L.; Duncan, Todd; Redmond, T. Michael; Pardue, Machelle T.; Boatright, Jeffrey H.; Nickerson, John M.

    2013-01-01

    Purpose. A mouse mutation, tvrm148, was previously reported as resulting in retinal degeneration. Tvrm148 and Rpe65 map between markers D3Mit147 and D3Mit19 on a genetic map, but the physical map places RPE65 outside the markers. We asked if Rpe65 or perhaps another nearby gene is mutated and if the mutant reduced 11-cis-retinal levels. We studied the impact of the tvrm148 mutation on visual function, morphology, and retinoid levels. Methods. Normal phase HPLC was used to measure retinoid levels. Rpe65+/+, tvrm148/+ (T+/−), tvrm148/tvrm148 (T−/−), RPE65KO/KO (Rpe65−/−), and Rpe65T/− mice visual function was measured by optokinetic tracking (OKT) and electroretinography (ERG). Morphology was assessed by light microscopy and transmission electron microscopy (TEM). qRT-PCR was used to measure Rpe65 mRNA levels. Immunoblotting measured the size and amount of RPE65 protein. Results. The knockout and tvrm148 alleles did not complement. No 11-cis-retinal was detected in T−/− or Rpe65−/− mice. Visual acuity in Rpe65+/+ and T+/− mouse was ∼0.382 c/d, but 0.037 c/d in T−/− mice at postnatal day 210 (P210). ERG response in T−/− mice was undetectable except at bright flash intensities. Outer nuclear layer (ONL) thickness in T−/− mice was ∼70% of Rpe65+/+ by P210. Rpe65 mRNA levels in T−/− mice were unchanged, yet 14.5% of Rpe65+/+ protein levels was detected. Protein size was unchanged. Conclusions. A complementation test revealed the RPE65 knockout and tvrm148 alleles do not complement, proving that the tvrm148 mutation is in Rpe65. Behavioral, physiological, molecular, biochemical, and histological approaches indicate that tvrm148 is a null allele of Rpe65. PMID:23778877

  16. Escherichia coli Strain Nissle 1917 Ameliorates Experimental Colitis via Toll-Like Receptor 2- and Toll-Like Receptor 4-Dependent Pathways

    PubMed Central

    Grabig, A.; Paclik, D.; Guzy, C.; Dankof, A.; Baumgart, D. C.; Erckenbrecht, J.; Raupach, B.; Sonnenborn, U.; Eckert, J.; Schumann, R. R.; Wiedenmann, B.; Dignass, A. U.; Sturm, A.

    2006-01-01

    Toll-like receptors (TLRs) are key components of the innate immune system that trigger antimicrobial host defense responses. The aim of the present study was to analyze the effects of probiotic Escherichia coli Nissle strain 1917 in experimental colitis induced in TLR-2 and TLR-4 knockout mice. Colitis was induced in wild-type (wt), TLR-2 knockout, and TLR-4 knockout mice via administration of 5% dextran sodium sulfate (DSS). Mice were treated with either 0.9% NaCl or 107 E. coli Nissle 1917 twice daily, followed by the determination of disease activity, mucosal damage, and cytokine secretion. wt and TLR-2 knockout mice exposed to DSS developed acute colitis, whereas TLR-4 knockout mice developed significantly less inflammation. In wt mice, but not TLR-2 or TLR-4 knockout mice, E. coli Nissle 1917 ameliorated colitis and decreased proinflammatory cytokine secretion. In TLR-2 knockout mice a selective reduction of gamma interferon secretion was observed after E. coli Nissle 1917 treatment. In TLR-4 knockout mice, cytokine secretion was almost undetectable and not modulated by E. coli Nissle 1917, indicating that TLR-4 knockout mice do not develop colitis similar to the wt mice. Coculture of E. coli Nissle 1917 and human T cells increased TLR-2 and TLR-4 protein expression in T cells and increased NF-κB activity via TLR-2 and TLR-4. In conclusion, our data provide evidence that E. coli Nissle 1917 ameliorates experimental induced colitis in mice via TLR-2- and TLR-4-dependent pathways. PMID:16790781

  17. Endothelial glucocorticoid receptor suppresses atherogenesis- Brief Report

    PubMed Central

    Zhang, Xinbo; Rotllan, Noemi; Feng, Yan; Zhou, Han; Fernández-Hernando, Carlos; Yu, Jun; Sessa, William C.

    2015-01-01

    Objective The purpose of this study was to determine the role of the endothelial glucocorticoid receptor in the pathogenesis of atherosclerosis. Approach and Results Control mice and mice lacking the endothelial glucocorticoid receptor were bred onto an Apoe knockout background and subjected to high-fat diet feeding for 12 weeks. Assessment of body weight and total cholesterol and triglycerides before and after the diet revealed no differences between the two groups of mice. However, mice lacking the endothelial glucocorticoid receptor developed more severe atherosclerotic lesions in the aorta, brachiocephalic artery and aortic sinus as well as a heightened inflammatory milieu as evidence by increased macrophage recruitment in the lesions. Conclusions These data suggest the endothelial glucocorticoid receptor is important for tonic inhibition of inflammation and limitation of atherosclerosis progression in this model. PMID:25810297

  18. Estrogen receptor signaling during vertebrate development

    PubMed Central

    Bondesson, Maria; Hao, Ruixin; Lin, Chin-Yo; Williams, Cecilia; Gustafsson, Jan-Åke

    2014-01-01

    Estrogen receptors are expressed and their cognate ligands produced in all vertebrates, indicative of important and conserved functions. Through evolution estrogen has been involved in controlling reproduction, affecting both the development of reproductive organs and reproductive behavior. This review broadly describes the synthesis of estrogens and the expression patterns of aromatase and the estrogen receptors, in relation to estrogen functions in the developing fetus and child. We focus on the role of estrogens for development of reproductive tissues, as well as non-reproductive effects on the developing brain. We collate data from human, rodent, bird and fish studies and highlight common and species-specific effects of estrogen signaling on fetal development. Morphological malformations originating from perturbed estrogen signaling in estrogen receptor and aromatase knockout mice are discussed, as well as the clinical manifestations of rare estrogen receptor alpha and aromatase gene mutations in humans. PMID:24954179

  19. Adaptation of the myoglobin knockout mouse to hypoxic stress.

    PubMed

    Schlieper, Georg; Kim, Jie-Hoon; Molojavyi, Andrei; Jacoby, Christoph; Laussmann, Tim; Flögel, Ulrich; Gödecke, Axel; Schrader, Jürgen

    2004-04-01

    Myoglobin knockout (myo-/-) mice were previously reported to show no obvious phenotype but revealed several compensatory mechanisms that include increases in cardiac capillary density, coronary flow, and hemoglobin. The aim of this study was to investigate whether severe hypoxic stress can exhaust these compensatory mechanisms and whether this can be monitored on the gene and protein level. Myo-/- and wild-type (WT) mice we e exposed to hypoxia (10% O(2)) fo 2 wk. Thereafter hemodynamic parameters were investigated by invasive measurement combined with magnetic resonance imaging. Cardiac gene and protein expression were analyzed using cDNA arrays and two-dimensional gel electrophoresis plus mass spectrometry, respectively. Hematocrit levels increased from 44% (WT) and 48% (myo-/-) to 72% in both groups. Similar to WT controls, hypoxic myo-/- animals maintained stable cardiovascular function (mean arterial blood pressure 82.4 mmHg, ejection fraction 72.5%). Cardiac gene expression of hypoxic myo-/- mice differed significantly from WT controls in 17 genes (e.g., keratinocyte lipid binding protein +202%, cytochrome c oxidase Vb +41%). Interestingly, hypoxia inducible factor-1alpha remained unchanged in both groups. Proteome analysis revealed reduced levels of heart fatty acid-binding protein and heat shock protein 27 both in hypoxic myo-/- and WT mice. Our data thus demonstrate that myo-/- mice do not decompensate du ing hypoxic st ess but a e surprisingly well adapted. Changes in ene gy metabolism of fatty acids may contribute to the robustness of myoglobin-deficient mice. PMID:14656764

  20. Drop tests of the Three Mile Island knockout canister

    SciTech Connect

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

    1986-09-01

    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 were designed to confirm 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. Work conducted at the Oak Ridge National Laboratory included (1) precise physical measurements of the internal poison rod configuration before assembly, (2) canister assembly and welding, (3) nondestructive examination (an initial hydrostatic pressure test and an x-ray profile of the internals before and after each drop test), (4) addition of a simulated fuel load, (5) instrumentation of the canister for each drop test, (6) fabrication of a cask simulation vessel with a developed and tested foam impact limiter, (7) use of refrigeration facilities to cool the canister to well below freezing prior to three of the drops, (8) recording the drop test with still, high-speed, and normal-speed photography, (9) recording the accelerometer measurements during impact, (10) disassembly and post-test examination with precise physical measurements, and (11) preparation of the final report.

  1. Characteristics of Skeletal Muscle Fibers of SOD1 Knockout Mice.

    PubMed

    Nagahisa, Hiroshi; Okabe, Kazuma; Iuchi, Yoshihito; Fujii, Junichi; Miyata, Hirofumi

    2016-01-01

    Cu/Zn superoxide dismutase (SOD1) knockout (KO) mice are known as an aging model in some aspects, but the damage and regeneration process of each fiber type have not been sufficiently studied. In this study, we investigated the damage and satellite cell state of the gastrocnemius muscle in SOD1 KO mice (6 months old) using immunohistochemical staining and real-time RT-PCR. The proportion of central nuclei-containing Type IIx/b fibers in the deep and superficial portions of the gastrocnemius muscle was significantly higher in SOD1 KO than control mice. The number of satellite cells per muscle fiber decreased in all muscle fiber types in the deep portion of the gastrocnemius muscle in SOD1 KO mice. In addition, the mRNA expression levels of Pax7 and myogenin, which are expressed in satellite cells in the activation, proliferation, and differentiation states, significantly increased in the gastrocnemius muscle of SOD1 KO mice. Furthermore, mRNA of myosin heavy chain-embryonic, which is expressed in the early phase of muscle regeneration, significantly increased in SOD1 KO mice. It was suggested that muscle is damaged by reactive oxygen species produced in the mitochondrial intermembrane space in Type IIxb fibers, accelerating the proliferation and differentiation of satellite cells through growth factors in SOD1 KO mice. PMID:26798428

  2. Knockout of Foxp2 disrupts vocal development in mice

    PubMed Central

    Castellucci, Gregg A.; McGinley, Matthew J.; McCormick, David A.

    2016-01-01

    The FOXP2 gene is important for the development of proper speech motor control in humans. However, the role of the gene in general vocal behavior in other mammals, including mice, is unclear. Here, we track the vocal development of Foxp2 heterozygous knockout (Foxp2+/−) mice and their wildtype (WT) littermates from juvenile to adult ages, and observe severe abnormalities in the courtship song of Foxp2+/− mice. In comparison to their WT littermates, Foxp2+/− mice vocalized less, produced shorter syllable sequences, and possessed an abnormal syllable inventory. In addition, Foxp2+/− song also exhibited irregular rhythmic structure, and its development did not follow the consistent trajectories observed in WT vocalizations. These results demonstrate that the Foxp2 gene is critical for normal vocal behavior in juvenile and adult mice, and that Foxp2 mutant mice may provide a tractable model system for the study of the gene’s role in general vocal motor control. PMID:26980647

  3. Improved microarray methods for profiling the yeast knockout strain collection

    PubMed Central

    Yuan, Daniel S.; Pan, Xuewen; Ooi, Siew Loon; Peyser, Brian D.; Spencer, Forrest A.; Irizarry, Rafael A.; Boeke, Jef D.

    2005-01-01

    A remarkable feature of the Yeast Knockout strain collection is the presence of two unique 20mer TAG sequences in almost every strain. In principle, the relative abundances of strains in a complex mixture can be profiled swiftly and quantitatively by amplifying these sequences and hybridizing them to microarrays, but TAG microarrays have not been widely used. Here, we introduce a TAG microarray design with sophisticated controls and describe a robust method for hybridizing high concentrations of dye-labeled TAGs in single-stranded form. We also highlight the importance of avoiding PCR contamination and provide procedures for detection and eradication. Validation experiments using these methods yielded false positive (FP) and false negative (FN) rates for individual TAG detection of 3–6% and 15–18%, respectively. Analysis demonstrated that cross-hybridization was the chief source of FPs, while TAG amplification defects were the main cause of FNs. The materials, protocols, data and associated software described here comprise a suite of experimental resources that should facilitate the use of TAG microarrays for a wide variety of genetic screens. PMID:15994458

  4. Immunosympathectomy as the first phenotypic knockout with antibodies

    PubMed Central

    Cattaneo, Antonino

    2013-01-01

    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. PMID:23515328

  5. The biology of novel animal genes: Mouse APEX gene knockout

    SciTech Connect

    MacInnes, M.; Altherr, M.R.; Ludwig, D.; Pedersen, R.; Mold, C.

    1997-07-01

    This is the final report of a one-year, Laboratory Directed Research and Development (LDRD) project at the Los Alamos National Laboratory (LANL). The controlled breeding of novel genes into mice, including the gene knockout (KO), or conversely by adding back transgenes provide powerful genetic technologies that together suffice to determine in large part the biological role(s) of novel genes. Inbred mouse remains the best understood and most useful mammalian experimental system available for tackling the biology of novel genes. The major mammalian apurinic/apyrimidinic (AP) endonuclease (APE), is involved in a key step in the repair of spontaneous and induced AP sites in DNA. Efficient repair of these lesions is imperative to prevent the stable incorporation of mutations into the cellular genome which may lead to cell death or transformation. Loss or modulation of base excison repair activity in vivo may elevate the spontaneous mutation rate in cells, and may lead to a substantial increase in the incidence of cancer. Despite extensive biochemical analysis, however, the significance of these individual APE functions in vivo has not been elucidated. Mouse embryonic stem (ES) cells heterozygous for a deletion mutation in APE have been generated and whole animals containing the APE mutation have been derived from these ES cells. Animals homozygous for the APE null mutation die early in gestation, underscoring the biological significance of this DNA repair gene.

  6. Gastrointestinal Pathology in Juvenile and Adult CFTR-Knockout Ferrets

    PubMed Central

    Sun, Xingshen; Olivier, Alicia K.; Yi, Yaling; Pope, Christopher E.; Hayden, Hillary S.; Liang, Bo; Sui, Hongshu; Zhou, Weihong; Hager, Kyle R.; Zhang, Yulong; Liu, Xiaoming; Yan, Ziying; Fisher, John T.; Keiser, Nicholas W.; Song, Yi; Tyler, Scott R.; Goeken, J. Adam; Kinyon, Joann M.; Radey, Matthew C.; Fligg, Danielle; Wang, Xiaoyan; Xie, Weiliang; Lynch, Thomas J.; Kaminsky, Paul M.; Brittnacher, Mitchell J.; Miller, Samuel I.; Parekh, Kalpaj; Meyerholz, David K.; Hoffman, Lucas R.; Frana, Timothy; Stewart, Zoe A.; Engelhardt, John F.

    2015-01-01

    Cystic fibrosis (CF) is a multiorgan disease caused by loss of a functional cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel in many epithelia of the body. Here we report the pathology observed in the gastrointestinal organs of juvenile to adult CFTR-knockout ferrets. CF gastrointestinal manifestations included gastric ulceration, intestinal bacterial overgrowth with villous atrophy, and rectal prolapse. Metagenomic phylogenetic analysis of fecal microbiota by deep sequencing revealed considerable genotype-independent microbial diversity between animals, with the majority of taxa overlapping between CF and non-CF pairs. CF hepatic manifestations were variable, but included steatosis, necrosis, biliary hyperplasia, and biliary fibrosis. Gallbladder cystic mucosal hyperplasia was commonly found in 67% of CF animals. The majority of CF animals (85%) had pancreatic abnormalities, including extensive fibrosis, loss of exocrine pancreas, and islet disorganization. Interestingly, 2 of 13 CF animals retained predominantly normal pancreatic histology (84% to 94%) at time of death. Fecal elastase-1 levels from these CF animals were similar to non-CF controls, whereas all other CF animals evaluated were pancreatic insufficient (<2 μg elastase-1 per gram of feces). These findings suggest that genetic factors likely influence the extent of exocrine pancreas disease in CF ferrets and have implications for the etiology of pancreatic sufficiency in CF patients. In summary, these studies demonstrate that the CF ferret model develops gastrointestinal pathology similar to CF patients. PMID:24637292

  7. Modeling fragile X syndrome in the Fmr1 knockout mouse

    PubMed Central

    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

  8. Technical knockout, a Drosophila model of mitochondrial deafness.

    PubMed Central

    Toivonen, J M; O'Dell, K M; Petit, N; Irvine, S C; Knight, G K; Lehtonen, M; Longmuir, M; Luoto, K; Touraille, S; Wang, Z; Alziari, S; Shah, Z H; Jacobs, H T

    2001-01-01

    Mutations in mtDNA-encoded components of the mitochondrial translational apparatus are associated with diverse pathological states in humans, notably sensorineural deafness. To develop animal models of such disorders, we have manipulated the nuclear gene for mitochondrial ribosomal protein S12 in Drosophila (technical knockout, tko). The prototypic mutant tko(25t) exhibits developmental delay, bang sensitivity, impaired male courtship, and defective response to sound. On the basis of a transgenic reversion test, these phenotypes are attributable to a single substitution (L85H) at a conserved residue of the tko protein. The mutant is hypersensitive to doxycyclin, an antibiotic that selectively inhibits mitochondrial protein synthesis, and mutant larvae have greatly diminished activities of mitochondrial redox enzymes and decreased levels of mitochondrial small-subunit rRNA. A second mutation in the tko gene, Q116K, which is predicted to impair the accuracy of mitochondrial translation, results in the completely different phenotype of recessive female sterility, based on three independent transgenic insertions. We infer that the tko(25t) mutant provides a model of mitochondrial hearing impairment resulting from a quantitative deficiency of mitochondrial translational capacity. PMID:11560901

  9. Autism spectrum disorder traits in Slc9a9 knock-out mice.

    PubMed

    Yang, Lina; Faraone, Stephen V; Zhang-James, Yanli

    2016-04-01

    Autism spectrum disorders (ASDs) are a group of neurodevelopmental disorders which begin in childhood and persist into adulthood. They cause lifelong impairments and are associated with substantial burdens to patients, families, and society. Genetic studies have implicated the sodium/proton exchanger (NHE) nine gene, Slc9a9, to ASDs and attention-deficit/hyperactivity disorder(ADHD). Slc9a9 encodes, NHE9, a membrane protein of the late recycling endosomes. The recycling endosome plays an important role in synapse development and plasticity by regulating the trafficking of membrane neurotransmitter receptors and transporters. Here we tested the hypothesis that Slc9a9 knock-out (KO) mice would show ADHD-like and ASD-like traits. Ultrasonic vocalization (USV) recording showed that Slc9a9 KO mice emitted fewer calls and had shorter call durations, which suggest communication impairment. Slc9a9 KO mice lacked a preference for social novelty, but did not show deficits in social approach; Slc9a9 KO mice spent more time self-grooming, an indicator for restricted and repetitive behavior. We did not observe hyperactivity or other behavior impairments which are commonly comorbid with ASDs in human, such as anxiety-like behavior. Our study is the first animal behavior study that links Slc9a9 to ASDs. By eliminatingNHE9 activity, it provides strong evidence that lack of Slc9a9leads to ASD-like behaviors in mice and provides the field with a new mouse model of ASDs. PMID:26755066

  10. Decreased BMP2 signal in GIT1 knockout mice slows bone healing

    PubMed Central

    Fan, Jin; Zhou, Hao; Zuscik, Michael J.; Xie, Chao; Yin, Guoyong; Berk, Bradford C.

    2015-01-01

    Endochondral ossification, an important stage of fracture healing, is regulated by a variety of signaling pathways. Transforming growth factor b (TGFb) superfamily plays important roles and comprises TGFbs, bone morphogenetic proteins (BMPs), and growth differentiation factors. TGFbs primarily regulate cartilage formation and endochondral ossification. BMP2 shows diverse efficacy, from the formation of skeleton and extraskeletal organs to the osteogenesis and remodeling of bone. G-protein-coupled receptor kinase 2-interacting protein-1 (GIT1), a shuttle protein in osteoblasts, facilitates fracture healing by promoting bone formation and increasing the secretion of vascular endothelial growth factor. Our study examined whether GIT1 regulates fracture healing through the BMP2 signaling pathway and/or through the TGFb signaling pathway. GIT1 knockout (KO) mice exhibited delayed fracture healing, chondrocyte accumulation in the fracture area, and reduced staining intensity of phosphorylated Smad1/5/8 (pSmad1/5/8) and Runx2. Endochondral mineralization diminished while the staining intensity of phosphorylated Smad2/3 (pSmad2/3) showed no significant change. Bone marrow mesenchymal stem cells extracted from GIT1 KO mice showed a decline of pSmad1/5/8 levels and of pSmad1/5/8 translocated into the cell nucleus after BMP2 stimulus. We detected no significant change in the pSmad2/3 level after TGFb1 stimulus. Data obtained from reporter gene analysis of C3H10T1/2 cells cultured in vitro confirmed these findings. GIT1-siRNA inhibited transcription in the cell nucleus via pSmad1/5/8 after BMP2 stimulus but had no significant effect on transcription via pSmad2/3 after TGFb1 stimulus. Our results indicate that GIT1 regulates Smad1/5/8 phosphorylation and mediates BMP2 regulation of Runx2 expression, thus affecting endochondral ossification at the fracture site. PMID:25138700

  11. Kv4.2 knockout mice have hippocampal-dependent learning and memory deficits.

    PubMed

    Lugo, Joaquin N; Brewster, Amy L; Spencer, Corinne M; Anderson, Anne E

    2012-05-01

    Kv4.2 channels contribute to the transient, outward K(+) current (A-type current) in hippocampal dendrites, and modulation of this current substantially alters dendritic excitability. Using Kv4.2 knockout (KO) mice, we examined the role of Kv4.2 in hippocampal-dependent learning and memory. We found that Kv4.2 KO mice showed a deficit in the learning phase of the Morris water maze (MWM) and significant impairment in the probe trial compared with wild type (WT). Kv4.2 KO mice also demonstrated a specific deficit in contextual learning in the fear-conditioning test, without impairment in the conditioned stimulus or new context condition. Kv4.2 KO mice had normal activity, anxiety levels, and prepulse inhibition compared with WT mice. A compensatory increase in tonic inhibition has been previously described in hippocampal slice recordings from Kv4.2 KO mice. In an attempt to decipher whether increased tonic inhibition contributed to the learning and memory deficits in Kv4.2 KO mice, we administered picrotoxin to block GABA(A) receptors (GABA(A)R), and thereby tonic inhibition. This manipulation had no effect on behavior in the WT or KO mice. Furthermore, total protein levels of the α5 or δ GABA(A)R subunits, which contribute to tonic inhibition, were unchanged in hippocampus. Overall, our findings add to the growing body of evidence, suggesting an important role for Kv4.2 channels in hippocampal-dependent learning and memory. PMID:22505720

  12. A Jacob/Nsmf Gene Knockout Results in Hippocampal Dysplasia and Impaired BDNF Signaling in Dendritogenesis

    PubMed Central

    Schumacher, Anne; Butnaru, Ioana; Macharadze, Tamar; Gomes, Guilherme M.; Yuanxiang, PingAn; Bayraktar, Gonca; Rodenstein, Carolin; Geiseler, Carolin; Kolodziej, Angela; Lopez-Rojas, Jeffrey; Montag, Dirk; Angenstein, Frank; Bär, Julia; D’Hanis, Wolfgang; Roskoden, Thomas; Mikhaylova, Marina; Budinger, Eike; Ohl, Frank W.; Stork, Oliver; Zenclussen, Ana C.; Karpova, Anna; Schwegler, Herbert; Kreutz, Michael R.

    2016-01-01

    Jacob, the protein encoded by the Nsmf gene, is involved in synapto-nuclear signaling and docks an N-Methyl-D-Aspartate receptor (NMDAR)-derived signalosome to nuclear target sites like the transcription factor cAMP-response-element-binding protein (CREB). Several reports indicate that mutations in NSMF are related to Kallmann syndrome (KS), a neurodevelopmental disorder characterized by idiopathic hypogonadotropic hypogonadism (IHH) associated with anosmia or hyposmia. It has also been reported that a protein knockdown results in migration deficits of Gonadotropin-releasing hormone (GnRH) positive neurons from the olfactory bulb to the hypothalamus during early neuronal development. Here we show that mice that are constitutively deficient for the Nsmf gene do not present phenotypic characteristics related to KS. Instead, these mice exhibit hippocampal dysplasia with a reduced number of synapses and simplification of dendrites, reduced hippocampal long-term potentiation (LTP) at CA1 synapses and deficits in hippocampus-dependent learning. Brain-derived neurotrophic factor (BDNF) activation of CREB-activated gene expression plays a documented role in hippocampal CA1 synapse and dendrite formation. We found that BDNF induces the nuclear translocation of Jacob in an NMDAR-dependent manner in early development, which results in increased phosphorylation of CREB and enhanced CREB-dependent Bdnf gene transcription. Nsmf knockout (ko) mice show reduced hippocampal Bdnf mRNA and protein levels as well as reduced pCREB levels during dendritogenesis. Moreover, BDNF application can rescue the morphological deficits in hippocampal pyramidal neurons devoid of Jacob. Taken together, the data suggest that the absence of Jacob in early development interrupts a positive feedback loop between BDNF signaling, subsequent nuclear import of Jacob, activation of CREB and enhanced Bdnf gene transcription, ultimately leading to hippocampal dysplasia. PMID:26977770

  13. Decreased Neointimal Extracellular Matrix Formation in RAGE-Knockout Mice After Microvascular Denudation

    SciTech Connect

    Groezinger, Gerd Schmehl, Joerg Bantleon, Ruediger Kehlbach, Rainer; Mehra, Tarun; Claussen, Claus Wiesinger, Benjamin

    2012-12-15

    Purpose: To evaluate in vivo the role of RAGE (receptor for advanced glycated end products) in the development of restenosis and neointimal proliferation in RAGE-deficient knockout (KO) mice compared with wild-type (WT) mice in an animal model. Materials and Methods: Sixteen WT and 15 RAGE-deficient mice underwent microvascular denudation of the common femoral artery under general anaesthesia. Contralateral arteries underwent a sham operation and served as controls. Four weeks after the intervention, all animals were killed, and paraformaldehyde-fixed specimens of the femoral artery were analysed with different stains (hematoxylin and eosin and Elastica van Gieson) and several different types of immunostaining (proliferating cell nuclear antigen, {alpha}-actin, collagen, von Willebrand factor, RAGE). Luminal area, area of the neointima, and area of the media were measured in all specimens. In addition, colony-formation assays were performed, and collagen production by WT smooth muscle cells (SMCs) and RAGE-KO SMCs was determined. For statistical analysis, P < 0.05 was considered statistically significant. Results: Four weeks after denudation, WT mice showed a 49.6% loss of luminal area compared with 14.9% loss of luminal area in RAGE-deficient mice (sham = 0% loss) (P < 0.001). The neointima was 18.2 (*1000 {mu}m{sup 2} [n = 15) in the WT group compared with only 8.4 (*1000 {mu}m{sup 2} [n = 16]) in the RAGE-KO group. RAGE-KO SMCs showed significantly decreased proliferation activity and production of extracellular matrix protein. Conclusion: RAGE may be shown to play a considerable role in the formation of neointima leading to restenosis after vascular injury.

  14. Changes in Spontaneous Firing Patterns of Cerebellar Purkinje Cells in p75 Knockout Mice

    PubMed Central

    Tian, Jinbin; Tep, Chhavy; Zhu, Michael X.; Yoon, Sung Ok

    2013-01-01

    The p75 neurotrophin receptor is highly expressed in the developing nervous system and is required for neuronal survival, growth and synaptic transmission. In young mice, p75 is present in both granular cells and Purkinje cells of the cerebellum. Although p75 has been implicated in modulation of neuronal excitability in several neuronal types, whether and how it affects the excitability of cerebellar Purkinje neurons remained unclear. Using extracellular recordings of spontaneous firing of Purkinje neurons in cerebellar slices prepared from wild type and p75 knockout mice, we measured intrinsic firing properties in the presence of fast synaptic blockers of more than 200 Purkinje cells, each for a period of 5 min, for each genotype. We detected a significant increase in the mean firing frequency in p75−/− neurons comparing to the wild type littermates. Upon separating tonically firing from phasically firing cells, i.e. cells with firing pauses of longer than 300 msec, we observed that the change mainly arose from phasic firing cells and can be explained by an increase in the firing/silence ratio and a decrease in the number of long pauses during the 5-min recording period. We conclude that p75 plays an important role in regulating the firing-to-silence transition during the phasic firing period of the spontaneous firing of Purkinje cells. Thus, p75 exerts a modulatory function on Purkinje cell firing patterns, through which it may act as a key player in motor coordination and other cerebellum-regulated activities since Purkinje cells represent the sole neuronal output of the cerebellar cortex. PMID:23307658

  15. Ceruloplasmin/Hephaestin Knockout Mice Model Morphologic and Molecular Features of AMD

    PubMed Central

    Hadziahmetovic, Majda; Dentchev, Tzvete; Song, Ying; Haddad, Nadine; He, Xining; Hahn, Paul; Pratico, Domenico; Wen, Rong; Harris, Z. Leah; Lambris, John D.; Beard, John; Dunaief, Joshua L.

    2008-01-01

    Purpose Iron is an essential element in human metabolism but also is a potent generator of oxidative damage with levels that increase with age. Several studies suggest that iron accumulation may be a factor in age-related macular degeneration (AMD). In prior studies, both iron overload and features of AMD were identified in mice deficient in the ferroxidase ceruloplasmin (Cp) and its homologue hephaestin (Heph) (double knockout, DKO). In this study, the location and timing of iron accumulation, the rate and reproducibility of retinal degeneration, and the roles of oxidative stress and complement activation were determined. Methods Morphologic analysis and histochemical iron detection by Perls' staining was performed on retina sections from DKO and control mice. Immunofluorescence and immunohistochemistry were performed with antibodies detecting activated complement factor C3, transferrin receptor, L-ferritin, and macrophages. Tissue iron levels were measured by atomic absorption spectrophotometry. Isoprostane F2α-VI, a specific marker of oxidative stress, was quantified in the tissue by gas chromatography/mass spectrometry. Results DKOs exhibited highly reproducible age-dependent iron overload, which plateaued at 6 months of age, with subsequent progressive retinal degeneration continuing to at least 12 months. The degeneration shared some features of AMD, including RPE hypertrophy and hyperplasia, photoreceptor degeneration, subretinal neovascularization, RPE lipofuscin accumulation, oxidative stress, and complement activation. Conclusions DKOs have age-dependent iron accumulation followed by retinal degeneration modeling some of the morphologic and molecular features of AMD. Therefore, these mice are a good platform on which to test therapeutic agents for AMD, such as antioxidants, iron chelators, and antiangiogenic agents. PMID:18326691

  16. A Jacob/Nsmf Gene Knockout Results in Hippocampal Dysplasia and Impaired BDNF Signaling in Dendritogenesis.

    PubMed

    Spilker, Christina; Nullmeier, Sven; Grochowska, Katarzyna M; Schumacher, Anne; Butnaru, Ioana; Macharadze, Tamar; Gomes, Guilherme M; Yuanxiang, PingAn; Bayraktar, Gonca; Rodenstein, Carolin; Geiseler, Carolin; Kolodziej, Angela; Lopez-Rojas, Jeffrey; Montag, Dirk; Angenstein, Frank; Bär, Julia; D'Hanis, Wolfgang; Roskoden, Thomas; Mikhaylova, Marina; Budinger, Eike; Ohl, Frank W; Stork, Oliver; Zenclussen, Ana C; Karpova, Anna; Schwegler, Herbert; Kreutz, Michael R

    2016-03-01

    Jacob, the protein encoded by the Nsmf gene, is involved in synapto-nuclear signaling and docks an N-Methyl-D-Aspartate receptor (NMDAR)-derived signalosome to nuclear target sites like the transcription factor cAMP-response-element-binding protein (CREB). Several reports indicate that mutations in NSMF are related to Kallmann syndrome (KS), a neurodevelopmental disorder characterized by idiopathic hypogonadotropic hypogonadism (IHH) associated with anosmia or hyposmia. It has also been reported that a protein knockdown results in migration deficits of Gonadotropin-releasing hormone (GnRH) positive neurons from the olfactory bulb to the hypothalamus during early neuronal development. Here we show that mice that are constitutively deficient for the Nsmf gene do not present phenotypic characteristics related to KS. Instead, these mice exhibit hippocampal dysplasia with a reduced number of synapses and simplification of dendrites, reduced hippocampal long-term potentiation (LTP) at CA1 synapses and deficits in hippocampus-dependent learning. Brain-derived neurotrophic factor (BDNF) activation of CREB-activated gene expression plays a documented role in hippocampal CA1 synapse and dendrite formation. We found that BDNF induces the nuclear translocation of Jacob in an NMDAR-dependent manner in early development, which results in increased phosphorylation of CREB and enhanced CREB-dependent Bdnf gene transcription. Nsmf knockout (ko) mice show reduced hippocampal Bdnf mRNA and protein levels as well as reduced pCREB levels during dendritogenesis. Moreover, BDNF application can rescue the morphological deficits in hippocampal pyramidal neurons devoid of Jacob. Taken together, the data suggest that the absence of Jacob in early development interrupts a positive feedback loop between BDNF signaling, subsequent nuclear import of Jacob, activation of CREB and enhanced Bdnf gene transcription, ultimately leading to hippocampal dysplasia. PMID:26977770

  17. Human adipose-derived stem cells attenuate inflammatory bowel disease in IL-10 knockout mice.

    PubMed

    Jung, Woo Yeun; Kang, Joo Hwan; Kim, Kyung Gon; Kim, Hee Snn; Jang, Byung Ik; Park, Yong Hoon; Song, In-Hwan

    2015-02-01

    Inflammatory bowel disease (IBD) is a complex immunological disorder characterized by chronic inflammation caused mainly by unknown factors. The interleukin-10 knockout (IL-10 KO) mouse is a well-established murine model of IBD which develops spontaneous intestinal inflammation that resembles Crohn's disease. In the present study, human adipose-derived mesenchymal stem cells (hAMSCs) were administrated to IL-10 KO mice to evaluate the anti-inflammatory effects of hAMSCs that may attenuate the progress of or treat IBD. After IBD was induced by feeding the IL-10 KO mouse a 125-250 ppm piroxicam mixed diet for 1 week, 2×10(6) hAMSCs were injected into the peritoneum and the mice were switched to a normal diet. After 1 week, the mice were sacrificed and tissue samples were harvested. Tissue scores for inflammation and inflammation-related genes expression were determined. The hAMSC-treated group showed significantly reduced inflammatory changes in histological analysis. Reverse transcription-PCR analysis showed that RANTES, Toll-like receptor 9, and IL-4 expression levels were not significantly different between the groups while IL-12, INF-γ, and TNF-α levels were significantly decreased in the hAMSC treated group. hAMSC attenuated IBD in the IL-10 KO mice by suppressing inflammatory cytokine expression, was mediated by the type 1 helper T cell pathway. Even though only a single injection of hAMSCs was delivered, the effect influenced chronic events associated with inflammatory changes and demonstrated that hAMSCs are a powerful candidate for IBD therapy. PMID:25544730

  18. Generation of Esr1-Knockout Rats Using Zinc Finger Nuclease-Mediated Genome Editing

    PubMed Central

    Dhakal, Pramod; Kubota, Kaiyu; Chakraborty, Damayanti; Lei, Tianhua; Larson, Melissa A.; Wolfe, Michael W.; Roby, Katherine F.; Vivian, Jay L.

    2014-01-01

    Estrogens play pivotal roles in development and function of many organ systems, including the reproductive system. We have generated estrogen receptor 1 (Esr1)-knockout rats using zinc finger nuclease (ZFN) genome targeting. mRNAs encoding ZFNs targeted to exon 3 of Esr1 were microinjected into single-cell rat embryos and transferred to pseudopregnant recipients. Of 17 live births, 5 had biallelic and 1 had monoallelic Esr1 mutations. A founder with monoallelic mutations was backcrossed to a wild-type rat. Offspring possessed only wild-type Esr1 alleles or wild-type alleles and Esr1 alleles containing either 482 bp (Δ482) or 223 bp (Δ223) deletions, indicating mosaicism in the founder. These heterozygous mutants were bred for colony expansion, generation of homozygous mutants, and phenotypic characterization. The Δ482 Esr1 allele yielded altered transcript processing, including the absence of exon 3, aberrant splicing of exon 2 and 4, and a frameshift that generated premature stop codons located immediately after the codon for Thr157. ESR1 protein was not detected in homozygous Δ482 mutant uteri. ESR1 disruption affected sexually dimorphic postnatal growth patterns and serum levels of gonadotropins and sex steroid hormones. Both male and female Esr1-null rats were infertile. Esr1-null males had small testes with distended and dysplastic seminiferous tubules, whereas Esr1-null females possessed large polycystic ovaries, thread-like uteri, and poorly developed mammary glands. In addition, uteri of Esr1-null rats did not effectively respond to 17β-estradiol treatment, further demonstrating that the Δ482 Esr1 mutation created a null allele. This rat model provides a new experimental tool for investigating the pathophysiology of estrogen action. PMID:24506075

  19. Blue-Green Algae Inhibit the Development of Atherosclerotic Lesions in Apolipoprotein E Knockout Mice.

    PubMed

    Ku, Chai Siah; Kim, Bohkyung; Pham, Tho X; Yang, Yue; Wegner, Casey J; Park, Young-Ki; Balunas, Marcy; Lee, Ji-Young

    2015-12-01

    Hyperlipidemia and inflammation contribute to the development of atherosclerotic lesions. Our objective was to determine antiatherogenic effect of edible blue-green algae (BGA) species, that is, Nostoc commune var. sphaeroides Kützing (NO) and Spirulina platensis (SP), in apolipoprotein E knockout (ApoE(-/-)) mice, a well-established mouse model of atherosclerosis. Male ApoE(-/-) mice were fed a high-fat/high-cholesterol (HF/HC, 15% fat and 0.2% cholesterol by wt) control diet or a HF/HC diet supplemented with 5% (w/w) of NO or SP powder for 12 weeks. Plasma total cholesterol (TC) and triglycerides (TG) were measured, and livers were analyzed for histology and gene expression. Morphometric analysis for lesions and immunohistochemical analysis for CD68 were conducted in the aorta and the aortic root. NO supplementation significantly decreased plasma TC and TG, and liver TC, compared to control and SP groups. In the livers of NO-fed mice, less lipid droplets were present with a concomitant decrease in fatty acid synthase protein levels than the other groups. There was a significant increase in hepatic low-density lipoprotein receptor protein levels in SP-supplemented mice than in control and NO groups. Quantification of aortic lesions by en face analysis demonstrated that both NO and SP decreased aortic lesion development to a similar degree compared with control. While lesions in the aortic root were not significantly different between groups, the CD68-stained area in the aortic root was significantly lowered in BGA-fed mice than controls. In conclusion, both NO and SP supplementation decreased the development of atherosclerotic lesions, suggesting that they may be used as a natural product for atheroprotection. PMID:26566121

  20. Alpha-asarone improves striatal cholinergic function and locomotor hyperactivity in Fmr1 knockout mice.

    PubMed

    Qiu, Guozhen; Chen, Shengqiang; Guo, Jialing; Wu, Jie; Yi, Yong-Hong

    2016-10-01

    Hyperactivity is a symptom found in several neurological and psychiatric disorders, including Fragile X syndrome (FXS). The animal model of FXS, fragile X mental retardation gene (Fmr1) knockout (KO) mouse, exhibits robust locomotor hyperactivity. Alpha (α)-asarone, a major bioactive component isolated from Acorus gramineus, has been shown in previous studies to improve various disease conditions including central nervous system disorders. In this study, we show that treatment with α-asarone alleviates locomotor hyperactivity in Fmr1 KO mice. To elucidate the mechanism underlying this improvement, we evaluated the expressions of various cholinergic markers, as well as acetylcholinesterase (AChE) activity and acetylcholine (ACh) levels, in the striatum of Fmr1 KO mice. We also analyzed the AChE-inhibitory activity of α-asarone. Striatal samples from Fmr1 KO mice showed decreased m1 muscarinic acetylcholine receptor (m1 mAChR) expression, increased AChE activity, and reduced ACh levels. Treatment with α-asarone improved m1 mAChR expression and ACh levels, and attenuated the increased AChE activity. In addition, α-asarone dose-dependently inhibited AChE activity in vitro. These results indicate that direct inhibition of AChE activity and up-regulation of m1 mAChR expression in the striatum might contribute to the beneficial effects of α-asarone on locomotor hyperactivity in Fmr1 KO mice. These findings might improve understanding of the neurobiological mechanisms responsible for locomotor hyperactivity. PMID:27316341

  1. Pharmacological ceramide reduction alleviates alcohol-induced steatosis and hepatomegaly in adiponectin knockout mice

    PubMed Central

    Correnti, Jason M.; Juskeviciute, Egle; Swarup, Aditi

    2014-01-01

    Hepatosteatosis, the ectopic accumulation of lipid in the liver, is one of the earliest clinical signs of alcoholic liver disease (ALD). Alcohol-dependent deregulation of liver ceramide levels as well as inhibition of AMP-activated protein kinase (AMPK) and peroxisome proliferator-activated receptor α (PPAR-α) activity are thought to contribute to hepatosteatosis development. Adiponectin can regulate lipid handling in the liver and has been shown to reduce ceramide levels and activate AMPK and PPAR-α. However, the mechanisms by which adiponectin prevents alcoholic hepatosteatosis remain incompletely characterized. To address this question, we assessed ALD progression in wild-type (WT) and adiponectin knockout (KO) mice fed an ethanol-containing liquid diet or isocaloric control diet. Adiponectin KO mice relative to WT had increased alcohol-induced hepatosteatosis and hepatomegaly, similar modest increases in serum alanine aminotransferase, and reduced liver TNF. Restoring circulating adiponectin levels using recombinant adiponectin ameliorated alcohol-induced hepatosteatosis and hepatomegaly in adiponectin KO mice. Alcohol-fed WT and adiponectin KO animals had equivalent reductions in AMPK protein and PPAR-α DNA binding activity compared with control-fed animals. No difference in P-AMPK/AMPK ratio was detected, suggesting that alcohol-dependent deregulation of AMPK and PPAR-α in the absence of adiponectin are not primary causes of the observed increase in hepatosteatosis in these animals. By contrast, alcohol treatment increased liver ceramide levels in adiponectin KO but not WT mice. Importantly, pharmacological inhibition of de novo ceramide synthesis in adiponectin KO mice abrogated alcohol-mediated increases in liver ceramides, steatosis, and hepatomegaly. These data suggest that adiponectin reduces alcohol-induced steatosis and hepatomegaly through regulation of liver ceramides, but its absence does not exacerbate alcohol-induced liver damage. PMID

  2. Hyodeoxycholic acid improves HDL function and inhibits atherosclerotic lesion formation in LDLR-knockout mice

    PubMed Central

    Shih, Diana M.; Shaposhnik, Zory; Meng, Yonghong; Rosales, Melenie; Wang, Xuping; Wu, Judy; Ratiner, Boris; Zadini, Filiberto; Zadini, Giorgio; Lusis, Aldons J.

    2013-01-01

    We examined the effects of a natural secondary bile acid, hyodeoxycholic acid (HDCA), on lipid metabolism and atherosclerosis in LDL receptor-null (LDLRKO) mice. Female LDLRKO mice were maintained on a Western diet for 8 wk and then divided into 2 groups that received chow, or chow + 1.25% HDCA, diets for 15 wk. We observed that mice fed the HDCA diet were leaner and exhibited a 37% (P<0.05) decrease in fasting plasma glucose level. HDCA supplementation significantly decreased atherosclerotic lesion size at the aortic root region, the entire aorta, and the innominate artery by 44% (P<0.0001), 48% (P<0.01), and 94% (P<0.01), respectively, as compared with the chow group. Plasma VLDL/IDL/LDL cholesterol levels were significantly decreased, by 61% (P<0.05), in the HDCA group as compared with the chow diet group. HDCA supplementation decreased intestinal cholesterol absorption by 76% (P<0.0001) as compared with the chow group. Furthermore, HDL isolated from the HDCA group exhibited significantly increased ability to mediate cholesterol efflux ex vivo as compared with HDL of the chow diet group. In addition, HDCA significantly increased the expression of genes involved in cholesterol efflux, such as Abca1, Abcg1, and Apoe, in a macrophage cell line. Thus, HDCA is a candidate for antiatherosclerotic drug therapy.—Shih, D. M., Shaposhnik, Z., Meng, Y., Rosales, M., Wang, X., Wu, J., Ratiner, B., Zadini, F., Zadini, G., Lusis, A. J. Hyodeoxycholic acid improves HDL function and inhibits atherosclerotic lesion formation in LDLR-knockout mice. PMID:23752203

  3. The hallucinogen N,N-dimethyltryptamine (DMT) is an endogenous sigma-1 receptor regulator.

    PubMed

    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. PMID:19213917

  4. The Hallucinogen N,N-Dimethyltryptamine (DMT) Is an Endogenous Sigma-1 Receptor Regulator

    PubMed Central

    Fontanilla, Dominique; Johannessen, Molly; Hajipour, Abdol R.; Cozzi, Nicholas V.; Jackson, Meyer B.; Ruoho, Arnold E.

    2010-01-01

    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. PMID:19213917

  5. Metabotropic glutamate receptor subtype 5 antagonism in learning and memory

    PubMed Central

    Simonyi, Agnes; Schachtman, Todd R.; Christoffersen, Gert R. J.

    2010-01-01

    Summary The role of the metabotropic glutamate receptor 5 (mGlu5 receptor) in learning and memory and other behaviors are reviewed by examining the influence of selective antagonists and genetic knockout on performance. This receptor is involved in spatial learning, contextual fear conditioning, inhibitory avoidance, fear potentiated startle, and conditioned taste aversion. However, mGlu5 receptor antagonists have proven to be ineffective in other learning tasks, such as the delayed-match-to-position test and a three-hole spatial learning task. Locomotion is often decreased by mGlu5 receptor antagonists; and other behaviors such as social interaction and consummatory responses can also be affected. In mGlu5 receptor knockout mice, performance in contextual fear conditioning and spatial water maze tasks is impaired. Although the available evidence is suggestive of an important contribution of mGlu5 receptors to cognitive functions, further studies are needed, particularly those with in vivo evaluation of the role of mGlu5 receptors in selective brain regions in different stages of memory formation. PMID:20363219

  6. Role for PPARγ in obesity-induced hepatic steatosis as determined by hepatocyte- and macrophage-specific conditional knockouts.

    PubMed

    Morán-Salvador, Eva; López-Parra, Marta; García-Alonso, Verónica; Titos, Esther; Martínez-Clemente, Marcos; González-Périz, Ana; López-Vicario, Cristina; Barak, Yaacov; Arroyo, Vicente; Clària, Joan

    2011-08-01

    Peroxisome proliferator-activated receptor (PPAR) γ is a nuclear receptor central to glucose and lipid homeostasis. PPARγ role in nonalcoholic fatty liver disease is controversial because PPARγ overexpression is a general property of steatotic livers, but its activation by thiazolidinediones reduces hepatic steatosis. Here, we investigated hepatic PPARγ function by using Cre-loxP technology to generate hepatocyte (PPARγ(Δhep))- and macrophage (PPARγ(Δmac))-specific PPARγ-knockout mice. Targeted deletion of PPARγ in hepatocytes, and to a lesser extent in macrophages, protected mice against high-fat diet-induced hepatic steatosis. Down-regulated expression of genes involved in lipogenesis (SCD1, SREBP-1c, and ACC), lipid transport (CD36/FAT, L-FABP, and MTP), and β-oxidation (PPARα and ACO) was observed in PPARγ(Δhep) mice. Moreover, PPARγ(Δhep) mice showed improved glucose tolerance and reduced PEPCK expression without changes in Pcx, Fbp1, and G6Pc expression and CREB and JNK phosphorylation. In precision-cut liver slices (PCLSs) and hepatocytes, rosiglitazone either alone or in combination with oleic acid increased triglyceride accumulation, an effect that was blocked by the PPARγ antagonist biphenol A diglycidyl ether (BADGE). PCLSs and hepatocytes from PPARγ(Δhep) mice showed blunted responses to rosiglitazone and oleic acid, whereas the response to these compounds remained intact in PCLSs from PPARγ(Δmac) mice. Collectively, these findings establish PPARγ expression in hepatocytes as a prosteatotic factor in fatty liver disease. PMID:21507897

  7. Lactobacillus acidophilus ATCC 4356 prevents atherosclerosis via inhibition of intestinal cholesterol absorption in apolipoprotein E-knockout mice.

    PubMed

    Huang, Ying; Wang, Jinfeng; Quan, Guihua; Wang, Xiaojun; Yang, Longfei; Zhong, Lili

    2014-12-01

    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 < 0.05) higher in the L. acidophilus ATCC 4356 treatment groups than in the control groups. Furthermore, L. acidophilus ATCC 4356 was detected in the rat small 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

  8. The vascular dysfunction in the α-galactosidase A knockout mouse is an endothelial cell, plasma membrane-based defect

    PubMed Central

    Park, James L; Whitesall, Steven E; D'Alecy, Louis G; Shu, Liming; Shayman, James A

    2009-01-01

    Summary Fabry disease results from an X-linked mutation in the lysosomal α-galactosidase A (Gla). Defective Gla results in multi-organ accumulation of neutral glycosphingolipids (GSLs), especially in the vascular endothelium, the major accumulating GSL being globotriaosylceramide (Gb3). Excessive endothelial Gb3 accumulation is associated with increased thrombosis, atherogenesis, and endothelial dysfunction. The mechanism(s) by which endothelial dysfunction occurs, however, is unclear. The purpose of this study was to further characterize the vasculopathy associated with a murine model of Fabry disease.Vascular reactivity was performed in vessels from wildtype (Gla +/0) and Gla knockout (Gla -/0) mice. Conscious blood pressure and heart rate were measured in the Gla +/0 and Gla -/0 mice by telemetry.The present study demonstrates that vascular smooth muscle (VSM) contractions were blunted in the Gla -/0 mice to phenylephrine and serotonin, but not to U46619. Endothelium-dependent contraction and receptor-mediated endothelium-dependent relaxation to acetylcholine were significantly attenuated in vessels from Gla -/0 mice. However, receptor-independent endothelium-dependent relaxation to the calcium ionophore, ionomycin, remained intact in vessels from Gla -/0 mice. Furthermore, VSM reactivity was normal in the aortas from Gla -/0 mice in the absence of endothelium. These changes in vascular function were observed without changes in whole-animal blood pressure or heart rate.These results suggest that the vasculopathy associated with Fabry disease is localized to the endothelium despite the accumulation of GSLs throughout the vasculature. PMID:18565198

  9. Lactobacillus acidophilus ATCC 4356 Prevents Atherosclerosis via Inhibition of Intestinal Cholesterol Absorption in Apolipoprotein E-Knockout Mice

    PubMed Central

    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 < 0.05) higher in the L. acidophilus ATCC 4356 treatment groups than in the control groups. Furthermore, L. acidophilus ATCC 4356 was detected in the rat small 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

  10. Rare complete knockouts in humans: population distribution and significant role in autism spectrum disorders

    PubMed Central

    Lim, Elaine T.; Raychaudhuri, Soumya; Sanders, Stephan J.; Stevens, Christine; Sabo, Aniko; MacArthur, Daniel G.; Neale, Benjamin M.; Kirby, Andrew; Ruderfer, Douglas M.; Fromer, Menachem; Lek, Monkol; Liu, Li; Flannick, Jason; Ripke, Stephan; Nagaswamy, Uma; Muzny, Donna; Reid, Jeffrey G.; Hawes, Alicia; Newsham, Irene; Wu, Yuanqing; Lewis, Lora; Dinh, Huyen; Gross, Shannon; Wang, Li-San; Lin, Chiao-Feng; Valladares, Otto; Gabriel, Stacey B.; dePristo, Mark; Altshuler, David M.; Purcell, Shaun M.; State, Matthew W.; Boerwinkle, Eric; Buxbaum, Joseph D.; Cook, Edwin H.; Gibbs, Richard A.; Schellenberg, Gerard D.; Sutcliffe, James S.; Devlin, Bernie; Roeder, Kathryn; Daly, Mark J.

    2013-01-01

    SUMMARY To characterize the role of rare complete human knockouts in autism spectrum disorders (ASD), we identify genes with homozygous or compound heterozygous loss-of-function (LoF) variants (defined as nonsense and essential splice sites) from exome sequencing of 933 cases and 869 controls. We identify a two-fold increase in complete knockouts of autosomal genes with low rates of LoF variation (≤5% frequency) in cases and estimate a 3% contribution to ASD risk by these events, confirming this observation in an independent set of 563 probands and 4,605 controls. Outside the pseudo-autosomal regions on the X-chromosome, we similarly observe a significant 1.5-fold increase in rare hemizygous knockouts in males, contributing to another 2% of ASDs in males. Taken together these results provide compelling evidence that rare autosomal and X-chromosome complete gene knockouts are important inherited risk factors for ASD. PMID:23352160

  11. Sweet Taste Receptor Deficient Mice Have Decreased Adiposity and Increased Bone Mass

    PubMed Central

    Simon, Becky R.; Learman, Brian S.; Parlee, Sebastian D.; Scheller, Erica L.; Mori, Hiroyuki; Cawthorn, William P.; Ning, Xiaomin; Krishnan, Venkatesh; Ma, Yanfei L.; Tyrberg, Björn; MacDougald, Ormond A.

    2014-01-01

    Functional expression of sweet taste receptors (T1R2 and T1R3) has been reported in numerous metabolic tissues, including the gut, pancreas, and, more recently, in adipose tissue. It has been suggested that sweet taste receptors in these non-gustatory tissues may play a role in systemic energy balance and metabolism. Smaller adipose depots have been reported in T1R3 knockout mice on a high carbohydrate diet, and sweet taste receptors have been reported to regulate adipogenesis in vitro. To assess the potential contribution of sweet taste receptors to adipose tissue biology, we investigated the adipose tissue phenotypes of T1R2 and T1R3 knockout mice. Here we provide data to demonstrate that when fed an obesogenic diet, both T1R2 and T1R3 knockout mice have reduced adiposity and smaller adipocytes. Although a mild glucose intolerance was observed with T1R3 deficiency, other metabolic variables analyzed were similar between genotypes. In addition, food intake, respiratory quotient, oxygen consumption, and physical activity were unchanged in T1R2 knockout mice. Although T1R2 deficiency did not affect adipocyte number in peripheral adipose depots, the number of bone marrow adipocytes is significantly reduced in these knockout animals. Finally, we present data demonstrating that T1R2 and T1R3 knockout mice have increased cortical bone mass and trabecular remodeling. This report identifies novel functions for sweet taste receptors in the regulation of adipose and bone biology, and suggests that in these contexts, T1R2 and T1R3 are either dependent on each other for activity or have common independent effects in vivo. PMID:24466105

  12. Insights from the Study of Animals Lacking Functional Estrogen Receptor

    NASA Astrophysics Data System (ADS)

    Korach, Kenneth S.

    1994-12-01

    Estrogen hormones produce physiological actions within a variety of target sites in the body and during development by activating a specific receptor protein. Hormone responsiveness for the estrogen receptor protein was investigated at different stages of development with the use of gene knockout techniques because no natural genetic mutants have been described. A mutant mouse line without a functional estrogen receptor was created and is being used to assess estrogen responsiveness. Both sexes of these mutant animals are infertile and show a variety of phenotypic changes, some of which are associated with the gonads, mammary glands, reproductive tracts, and skeletal tissues.

  13. Comparative Functional Genomics Analysis of NNK Tobacco-Carcinogen Induced Lung Adenocarcinoma Development in Gprc5a-Knockout Mice

    PubMed Central

    Men, Taoyan; van Pelt, Carolyn; Lotan, Dafna; Lotan, Reuben

    2010-01-01

    Background 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). Methodology/Principal Findings 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. Conclusions/Significance 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. PMID:20686609

  14. Completely Humanizing Prolactin Rescues Infertility in Prolactin Knockout Mice and Leads to Human Prolactin Expression in Extrapituitary Mouse Tissues

    PubMed Central

    Christensen, Heather R.; Murawsky, Michael K.; Horseman, Nelson D.; Willson, Tara A.

    2013-01-01

    A variety of fundamental differences have evolved in the physiology of the human and rodent prolactin (PRL) systems. The PRL gene in humans and other primates contains an alternative promoter, 5.8 kbp upstream of the pituitary transcription start site, which drives expression of PRL in “extrapituitary” tissues, where PRL is believed to exert local, or paracrine, actions. Several of these extrapituitary PRL tissues serve a reproductive function (eg, mammary gland, decidua, prostate, etc), consistent with the hypothesis that local PRL production may be involved in, and required for, normal reproductive physiology in primates. Rodent research models have generated significant findings regarding the role of PRL in reproduction. Specifically, disruption (knockout) of either the PRL gene or its receptor causes profound female reproductive defects at several levels (ovaries, preimplantation endometrium, mammary glands). However, the rodent PRL gene differs significantly from the human, most notably lacking the alternative promoter. Understanding of the physiological regulation and function of extrapituitary PRL has been limited by the absence of a readily accessible experimental model, because the rodent PRL gene does not contain the alternative promoter. To overcome these limitations, we have generated mice that have been “humanized” with regard to the structural gene and tissue expression of PRL. Here, we present the characterization of these animals, demonstrating that the human PRL transgene is responsive to known physiological regulators both in vitro and in vivo. More importantly, the expression of the human PRL transgene is able to rescue the reproductive defects observed in mouse PRL knockout (mPRL−) females, validating their usefulness in studying the function or regulation of this hormone in a manner that is relevant to human physiology. PMID:24029242

  15. β-Adrenoceptor-mediated Relaxation of Urinary Bladder Muscle in β2-Adrenoceptor Knockout Mice

    PubMed Central

    Propping, Stefan; Lorenz, Kristina; Michel, Martin C.; Wirth, Manfred P.; Ravens, Ursula

    2016-01-01

    Background and Objective: In order to characterize the β-adrenoceptor (AR) subtypes involved in agonist-stimulated relaxation of murine urinary bladder we studied the effects of (-)-isoprenaline and CL 316,243 on tonic contraction and spontaneous contractions in detrusor strips of wild-type (WT) and β2-AR knockout (β2-AR KO) mice. Materials and Methods: Urinary bladders were isolated from male WT and β2-AR KO mice. β-AR subtype expression was determined with quantitative real-time PCR. Intact muscle strips pre-contracted with KCl (40 mM) were exposed to cumulatively increasing concentrations of (-)-isoprenaline or β3-AR agonist CL 316,243 in the presence and absence of the subtype-selective β-AR blockers CGP 20712A (β1-ARs), ICI 118,551 (β2-ARs), and L748,337 (β3-ARs). Results: Quantitative real-time PCR confirmed lack of β2-AR expression in bladder tissue from β2-AR KO mice. In isolated detrusor strips, pre-contraction with KCl increased basal tone and enhanced spontaneous activity significantly more in β2-AR KO than in WT. (-)-Isoprenaline relaxed tonic tension and attenuated spontaneous activity with similar potency, but the concentrations required were two orders of magnitude higher in β2-AR KO than WT. The concentration-response curves (CRCs) for relaxation were not affected by CGP 20712A (300 nM), but were shifted to the right by ICI 118,551 (50 nM) and L748,337 (10 μM). The -logEC50 values for (-)-isoprenaline in WT and β2-AR KO tissue were 7.98 and 6.00, respectively, suggesting a large receptor reserve of β2-AR. (-)-CL 316,243 relaxed detrusor and attenuated spontaneous contractions from WT and β2-AR KO mice with a potency corresponding to the drug’s affinity for β3-AR. L743,337 shifted the CRCs to the right. Conclusion: Our findings in β2-AR KO mice suggest that there is a large receptor reserve for β2-AR in WT mice so that this β-AR subtype will mediate relaxation of tone and attenuation of spontaneous activity under physiological

  16. Involvement of the Tyro3 receptor and its intracellular partner Fyn signaling in Schwann cell myelination.

    PubMed

    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. PMID:26224309

  17. Knockout of leucine aminopeptidase in Toxoplasma gondii using CRISPR/Cas9.

    PubMed

    Zheng, Jun; Jia, Honglin; Zheng, Yonghui

    2015-02-01

    Leucine aminopeptidases of the M17 peptidase family represent ideal drug targets for therapies directed against the pathogens Plasmodium, Babesia a